i
• a •
i
1 jffi. ; • •
M m
m - *
jp^l
;•'
Pentland's Students Manuals.
MANUAL OF BACTERIOLOGY.
NUNQU*M ALIUD NATURA, ALIUD SAPIENT.A
HI
MANUAL
OF
BACTERIOLOGY
BY
EGBERT MUIR, M.A., M.D., F.R.C.P.ED.
PROFESSOR OF PATHOLOGY, UNIVERSITY OF GLASGOW
AND
JAMES RITCHIE, M.A., M.D., B.Sc.
SUPERINTENDENT OF THE ROYAL COLLEGE OF PHYSICIANS' LABORATORY, EDINBURGH
FORMERLY PROFESSOR OF PATHOLOGY IN THE UNIVERSITY OF OXFORD
FOURTH EDITION
WITH ONE HUNDRED & SEVENTY-ONE ILLUSTRATIONS
EDINBURGH AND LONDON
YOUNG J. PENTLAND
1907
vi PREFACE TO THE FOURTH EDITION
the diseases in question in the original arrangement. In the
appendix will be found an additional chapter dealing with
trypanosomiasis and allied affections. A number of new
illustrations have been added throughout the book, and the
bibliography has been brought up to date.
0,-fnber 1907.
PREFACE TO THE FIEST EDITION.
THE science of Bacteriology has, within recent years, become
so extensive, that in treating the subject in a book of this size
we are necessarily restricted to some special departments, unless
the description is to be of a superficial character. Accordingly,
as this work is intended primarily for students and practitioners
of medicine, only those bacteria which are associated with
disease in the human subject have been considered. We have
made it a chief endeavour to render the work of practical utility
for beginners, and, in the account of the more important
methods, have given elementary details which our experience in
the practical teaching of the subject has shown to be necessary.
In the systematic description of the various bacteria, an
attempt has been made to bring into prominence the evidence of
their having an etiological relationship to the corresponding
diseases, to point out the general laws governing their action as
producers of disease, and to consider the effects in particular
instances of various modifying circumstances. Much research
on certain subjects is so recent that conclusions on many points
must necessarily be of a tentative character. We have, therefore,
in our statement of results aimed at drawing a distinction
between what is proved and what is only probable.
In an Appendix we have treated of four diseases ; in two of
these the causal organism is not a bacterium, whilst in the other
two its nature is not yet determined. These diseases have been
vii
viii PREFACE TO THE FIRST EDITION
included on account of their own importance and that of the
pathological processes which they illustrate.
Our best thanks are due to Professor Greenfield for his kind
advice in connection with certain parts of the work. We have
also great pleasure in acknowledging our indebtedness to
Dr. Patrick Manson, who kindly lent us the negatives or pre-
parations from which Figs. 160-165 have been executed.
As we are convinced that to any one engaged in practical
study, photographs and photomicrographs supply the most useful
and exact information, we have used these almost exclusively in
illustration of the systematic description. These have been
executed in the Pathological Laboratory of the University of
Edinburgh by Mr. Richard Muir. The line drawings were
prepared for us by Mr. Alfred Robinson, of the University
Museum, Oxford.
To the volume is appended a short Bibliography, which,
while having no pretension to completeness, will, we hope, be of
use in putting those who desire further information on the track
of the principal papers which have been published on each of
the subjects considered
June 1897.
CONTENTS.
CHAPTER I.
GENERAL MORPHOLOGY AND BIOLOGY.
PAGE
INTRODUCTORY — Terminology — Structure of the bacterial cell —
Reproduction of bacteria — Spore formation — Motility — -
Minuter structure of the bacterial protoplasm — Chemical
composition of bacteria — Classification — Food supply — Re-
lation of bacteria to moisture, gaseous environment, tempera-
ture, and light — Conditions affecting bacterial motility —
Effects of bacteria in nature — Methods of bacterial action —
Variability among bacteria . . . . .1
CHAPTER II.
METHODS OF CULTIVATION OP BACTERIA.
Introductory— Methods of sterilisation— Preparation of culture
media — Use of the culture media — Methods of the separation
of aerobic organisms — Principles of the culture of anaerobic
organisms — Miscellaneous methods — General laboratory
rules ........ 25
CHAPTER III.
MICROSCOPIC METHODS — GENERAL BACTERIOLOGICAL
DIAGNOSIS — INOCULATION OF ANIMALS.
The microscope — Examination of hanging-drop cultures — Film pre-
parations— Examination of bacteria in tissues — The cutting
I ix
CONTENTS
f'ACK
of sections — Staining principles — Mordants and decolorisers
— Formulae of stains — Gram's method and its modifications
— Stain for tubercle and other acid-fast bacilli — Staining of
spores and flagella — The Romanowsky stains — Observation of
agglutination and sedimentation — Method of measuring the
phagocytic capacity of the leucocytes — Routine bacteriological
examination — Methods of inoculation — Autopsies on animals 85
CHAPTER IV.
BACTERIA IN AIR, SOIL, AND WATER. ANTISEPTICS.
Air : Methods of examination — Results. Soil : Methods of
Examination — Varieties of bacteria in soil. Water : Methods
of examination — Bacteria in water — Bacterial treatment of
sewage. Antiseptics : Methods of investigation — The action
of antiseptics — Certain particular antiseptics . . . 126
CHAPTER V.
RELATIONS OF BACTERIA TO DISEASE — THE PRODUCTION
OF TOXINS BY BACTERIA.
Introductory — Conditions modifying pathogenicity — Modes of
bacterial action — Tissue changes produced by bacteria— Local
lesions — General lesions — Disturbance of metabolism by
bacterial action — The production of toxins by bacteria, and
the nature of these — Allied vegetable and animal poisons —
The theory of toxic action ..... 149
CHAPTER VI.
INFLAMMATORY AND SUPPURATIVE CONDITIONS.
The relations of inflammation and suppuration — The bacteria of
inflammation and suppuration — Experimental inoculation —
Lesions in the human subject — Mode of entrance and spread
of pyogenic bacteria — Ulcerative endocarditis — Acute suppur-
ative periostitis — Erysipelas — Conjunctivitis — Acute rheu-
matism— Vaccination treatment of infections by the pyogenic
cocci — Methods of examination in inflammatory and suppur-
ative conditions ...... 172
CONTENTS xi
CHAPTER VII.
INFLAMMATORY AND SUPPURATIVE CONDITIONS, CONTINUED :
THE ACUTE PNEUMONIAS, EPIDEMIC CEREBRO-SPINAL
MENINGITIS.
PAGE
Introductory — Historical — Bacteria in pneumonia — Fraenkel's
pneumococcus — Friedlaender 's pneumococcus — Distribution of
pneumobacteria — Experimental inoculation — Pathology of
pneumococcus — Methods of examination. Epidemic cerebro-
spinal meningitis ...... 196
CHAPTER VIII.
GONORRHCEA, SOFT SORE, SYPHILIS.
The gonococcus — Microscopical characters — Cultivation — Rela-
tions to the disease — Its toxin — Distribution — Gonococcus in
joint affections — Methods of diagnosis — Soft sore — Syphilis —
Spirochsete pallida — Transmission of the disease to animals . 219
CHAPTER IX.
TUBERCULOSIS.
Historical — Tuberculosis in animals — Tubercle bacillus — Staining
reactions — Cultivation of tubercle bacillus — Powers of resist-
ance— Action on the tissues — Histology of tuberculous nodules
— Distribution of bacilli — Bacilli in tuberculous discharges —
Experimental inoculation — Varieties of tuberculosis — Other
acid-fast bacilli — Action of dead tubercle bacilli — Sources of
human tuberculosis — Toxins of the tubercle bacillus — Koch's
tuberculin — Active immunisation against the tubercle bacillus
— Koch's Tuberculin-R — Agglutinative phenomena — Methods
of examination ....... 235
CHAPTER X.
LEPROSY.
Pathological changes — Bacillus of leprosy — Position of the bacilli
— Relations to the disease — Methods of diagnosis . . 267
xii CONTENTS
CHAPTER XL
GLANDERS AND RHINOSCLEROMA.
PAGE
Glanders : The natural disease — The glanders bacillus — Cultiva-
tion of glanders bacillus — Powers of resistance — Experimental
inoculation — Action on the tissues — Mode of spread — Mallein
and its preparation — Methods of examination. Rhinoscleroma 275
CHAPTER XII.
ACTINOMYCOSIS AND ALLIED DISEASES.
Characters of the actinomyces — Tissue lesions — Distribution of
lesions — Cultivation of actinomyces — Varieties of actinomyces
and allied forms — Experimental inoculation — Methods of
examination and diagnosis — Madura disease . . .286
CHAPTER XIII.
ANTHRAX.
Historical summary — Bacillus anthracis — Appearances of cultures
— Biology — Sporulation — Natural anthrax in animals — Ex-
perimental anthrax — Anthrax in man — Pathology — Toxins of
the bacillus anthracis — Mode of spread in nature — Immunisa-
tion of animals against anthrax — Methods of examination . 300
CHAPTER XIV.
TYPHOID FEVER — BACILLI ALLIED TO THE TYPHOID
BACILLUS.
Bacillus typhosus — Morphological characters — Characters of cul-
tures— Bacillus coli communis — Reactions of b. typhosus and
b. coli — Pathological changes in typhoid fever — Suppuration
in typhoid fever — Pathogenic effects produced in animals —
The toxic products of typhoid bacillus — Immunisation of
animals — Relations of bacilli to the disease — Paratyphoid
bacillus — Bacillus enteritidis (Gaertner) — Psittacosis bacillus
— Serum diagnosis — Vaccination against typhoid— 'Methods of
examination — Bacteria in dysentery — Bacillus enteritidis
sporogenes — Summer diarrhoea . . .319
CONTENTS xiii
CHAPTER XV.
DIPHTHERIA.
PAGE
Historical — General facts — Bacillus diphtherias — Microscopical
characters — Distribution — Cultivation — Inoculation experi-
ments— The toxins of diphtheria — Variations in virulence of
bacilli — Bacilli allied to the diphtheria bacillus — Summary of
pathogenic action — Methods of diagnosis . . . 352
CHAPTER XVI.
TETANUS.
Introductory — Historical — Bacillus tetani — Isolation of bacillus
tetani — Characters of cultures — Conditions of growth — Patho-
genic effects — Experimental inoculation — Tetanus toxins —
Antitetanic serum — Methods of examination — Malignant
oedema — Characters of bacjllus — Experimental inoculation —
Methods of diagnosis — Bacillus botulinus — Quarter - evil —
Bacillus serogenes capsulatus ... . . . 371
CHAPTER XVII.
CHOLERA.
^ Introductory — The cholera spirillum — Distribution of the spirilla —
Cultivation — Powers of resistance — Experimental inoculation
Toxins of cholera spirillum — Inoculation of human subject —
Immunity — Methods of diagnosis — General summary — Other
spirilla resembling the cholera organism — MetchnikofFs
spirillum — Finkler and Prior's spirillum — Deneke's spirillum 399
CHAPTER XVIII.
INFLUENZA, PLAGUE, RELAPSING FEVER, MALTA FEVER,
YELLOW FEVER.
Influenza bacillus — Microscopical characters — Cultivation — Dis-
tribution— Experimental inoculation — Methods of examina-
xiv CONTENTS
PAGE
tion — Bacillus of plague — Microscopical characters — Cultiva-
tion— Anatomical changes produced and distribution of
bacilli — Experimental inoculation — Paths and . mode of in-
fection — Toxins, immunity, etc. — Methods of diagnosis —
Relapsing fever and African tick fever — Characters of the
spirillum — Relations to the disease — Immunity — African
tick fever — Malta fever — Micrococcus melitensis — Relations to
the disease — Mode of spread of the disease — Methods of
diagnosis — Yellow fever — Etiology of yellow fever . . 420
CHAPTER XIX.
IMMUNITY.
Introductory — Acquired immunity — Artificial immunity —
Varieties — Active immunity — Methods of production — At-
tenuation and exaltation of virulence — Passive immunity —
Action of the serum — Antitoxic serum — Standardising of
toxins and of antisera — Nature of antitoxic action — Ehrlich's
theory of the constitution of toxins — Antibacterial serum —
Bactericidal and lysogenic action — Haemolytic and other
sera — Methods of hsemolytic tests — Opsonic action — Ag-
glutination— Precipitins — Therapeutic effects of anti-sera —
Theories as to acquired immunity — Ehrlich's side-chain theory
L- — Serum anaphylaxis — Theory of phagocytosis — Natural
immunity — Natural bactericidal powers — Natural suscepti-
bility to toxins ....... 456
APPENDIX A.
SMALLPOX AND VACCINATION.
Jennerian vaccination — Relationship of smallpox to cowpox —
Micro-organisms associated with smallpox — The nature of
vaccination ....... 503
APPENDIX B.
HYDROPHOBIA.
Introductory — Pathology — The virus of hydrophobia — Prophylaxis
— Antirabic serum — Methods . . 510
CONTENTS xv
APPENDIX C.
MALARIAL FEVER.
PAGE
The malarial parasite — The cycle of the malarial parasite in man
— The cycle in the mosquito — Varieties of the malarial para-
site— General considerations — The pathology of malaria —
Methods of examination ..... 521
APPENDIX D.
AMCEBIC DYSENTERY.
Amoebic dysentery — Characters of the amoeba — Distribution of the
amoebae — Experimental inoculation — Methods of examination 537
APPENDIX E.
TRYPANOSOMIASIS — KALA-AZAR — PIROPLASMOSIS.
The pathogenic trypanosomes — General morphology of the trypano-
somata — Trypanosoma Lewisi — Nagana or tse-tse fly disease
— Trypanosoma of sleeping sickness — Trypanosoma gambiense
— Kdla-dzar — Dehli sore — Piroplasmosis . . . 544
BIBLIOGRAPHY . . .571
INDEX 593
LIST OF ILLUSTRATIONS.
FIG. PAGE
1. Forms of bacteria . . . . . .13
2. Hot-air steriliser ... 27
3. Koch's steam steriliser ...... 27
4. Autoclave . . ... . . .29
5. Steriliser for blood serum . . . . .30
6. Meat press . . . . . . .31
7. Hot-water funnel ...... 35
8. Blood serum inspissator . . . . .40
9. Potato jar ....... 45
10. Cylinder of potato cut obliquely . . . .45
11. Ehrlich's tube containing piece of potato . . .45
12. Apparatus for filling tubes . . . . .48
13. Tubes of media ...... 48
14. Platinum wires in glass handles . . . .49
15. Method of inoculating solid tubes . . . .50
16. Rack for platinum needles . . . . .50
17. Petri's capsule . . . . . . .51
18. Koch's levelling apparatus for use in preparing plates . 54
19. Koch's levelling apparatus ..... 54
20. Esmarch's tube for roll culture . . . .55
21. Apparatus for supplying hydrogen for anaerobic cultures . 58
22. Esmarch's roll-tube adapted for culture containingi'anaerobes 59
23. Bulloch's apparatus for anaerobic plate cultures . . 59
24. Flask for anaerobes in liquid media . . . .61
25. Flask arranged for culture of anaerobes which develop gas . 62
26. Tubes for anaerobic cultures on the surface of solid media . 62
27. Slides for hanging-drop cultures . . . . 63
28. Graham Brown's chamber for anaerobic hanging-drops . 64
29. Apparatus for counting colonies . . . .65
30. Wright's 250 c.mm. pipette fitted with nipple . . 66
31. Geissler's vacuum pump for filtering cultures . . .70
32. Chamberland's candle and flask arranged for filtration . 70
xvii
.\viii LIST OK [ILLUSTRATIONS
no,
33. Chamberland's bou^i'1 with Ump funnel . 71
34. Bougie inserted througli rubber .st(>i>]»ci . . 71
35. Muenck^'s inodilication of Ch;Liiil>ci!ainr ; liltrr . . 72
36. Flask fitted \\ it b poroelain bougie for fllterii [uantitie
of fluid ....... 7-'*
37. Flask for filtering smaU quantities of fluid . . . 73
38. Tubes for demonstrating gas-formation by bacteri.i . 70
39. Goryk air-pump for drying in vacua . . .79
40. Reichert's gas regulator . . . . .80
41. Hearson's incubator for use at 37° C. . . . .81
42. Cornet's forceps for holding rover-glasses . . .87
43. Needle with square of paper on end for manipulating puniflin
sections ....... 92
44. Syphon wash-bottle for distilled water . . .96
45. Wright's 5 c.mm. pipette . 108
46. Tubes used in testing agglutinating and sedimenting properties
of serum ....... 110
47. Wright's blood -capsi i !•• . . . . .114
48. Test-tube and pipette arranged for obtaining fluids containing
bactnit . . . . . 116
49. Hollow needle for intraperitoneal inoculation . .121
50. Hesse's tube . ..... 127
61. Petri's sand filter . . . . . .128
52. Staphylococcus pyogenes aureus, young culture 011 agar.
xlOOO . . . . . . .176
53. Two stab cultures of staphylococcus pyogenes aureus in gelatin 175
54. Streptococcus pyogenes, young culture on agar. x 1000 . 176
55. Culture of the streptococcus pyogenes on an agar plate . 177
56. Bacillus pyocyaneus ; young culture on agar. xlOOO . 177
57. Micrococcus tetragenus. x 1000 . . .181
58. Streptococci in acute suppuration, x 1000 . . 184
59. Minute focus of commencing suppuration in brain, x 50 . 1 86
60. Secondary infection of a glomerulus of kidney by the staphylo-
coccus aureus. x 300 . . . .187
61. Section of a vegetation in ulcerative endocarditis, x 600 . 189
62. Film preparation from a case of acute conjunctivitis, showing
the Koch-Weeks bacilli, x 1000 . . 192
63. Film preparation of conjunctival secretion showing the diplo-
bacillus of conjunctivitis, x 1000 . .192
64. Film preparation of pneumonic sputum, showing imineroii*
pneumococci (Fraenkel's). x 1000 . . . 199
65. Friedlander's pneumobacillus, from exudate in a <;as<- of
pneumonia, x 1000 ... . 200
66. Fraenkel's pneumococcus in serous exudation, x 1000 . 200
67. Stroke culture of Fraenkel's pneumococcus on blood agar . 201
LIST OK ILLCSTKATIONS
i i«. I-AI;K
68. Kraenkel's pneumococcus from a pure culture on blood agar.
x 1000 . 202
69. Stab culture at' 1'Yiefllaii'ler • |.in:iiiii<>Li.<:i I Ills . . 203
70. Kric'llaii'lei' (.neiiiiiobaeillu.,, from a young culture on agar.
xlOOO . .203
71. CapHulated pneumococci in blood taken from the heart of a
r;ibl,it. XlOOO . . 206
72. FilmjWparatjonofY^MlationfVomacasconiieningitis. .< 1000 21tf
7-'>. I'ure culture of diploeoceus infraccllularis . . . 214
74. Portion of lilrn of ^onorrho-al pus. xlOOO . . . 220
75. Gonococoi, from a pva cnltiire oo blood agar. xlOOO . 221
76. Film preparations of pus from .soft chancre, showing Ducrey's
bacillus. x!500 ... .227
77. Ducrcy's bacillus, x 1500 . 228
78 and 7S>. Film preparations from juice of hard chancre showing
piroehBtc paiiid;i. xlOOO . . 230
80. Section of spleen from a case of congenital syphilis, showing
spiroeh:etc pallida. x 1000 .... iWl
81. SpiiochfEterofringeiiH. x 1000 .... 231
82. Tubercle bacilli, from a pure culture onglycerinagar. x 1000 2:57
«:;. Tubercle bacilli in phthisical sputum, x 1000 . . 238
84. Cultures of tubercle bacilli on glycerin agar . . . 240
86. Tubercle bacilli in section of human lun<j in acute phthisis.
xlOOO ....... 244
86. Tubercle bacilli in giant-cells, x 1000 . . 21 f.
87. Tubercle bacilli in urine, x 1000 . 246
88. Moeller's Timothy-grass bacillus, x 1000 . . . 2f»:j
89. Cultures of acid-fast bacilli grown at, mom l« ni|Miature . W>
90. Smegma bacilli, x 1000 ..... 2f>4
91. Section l.linnitfh leprous skin, sho \vin- I In- m:i •• ..f i-.-llul;ir
granulation tissue in the cutis. x 80 . . 268
92. Superficial part of leprous skin. x f)<Mi . . . 270
!»".. Ili^h power view of portion of leprous nodule showing the
arraii;,'«!iiii-nt, of the bacilli within the eells of tlie granula-
tion tissue. xllOO ..... 271
'.»!. (i landers bacilli amongst broken-down cells, x 1000 . 277
!•:.. Clauders bacilli. x 1000 . . . . .278
96. Actinomyeosis of human liviir. x 500 . . . 288
97. Aetinomyees in human kidney. x f>0<) . . . 289
98. Colonies of actinomyces. x 60 . . . . 290
99. Cultures of the actinomyces on glycerin agar . . 293
100. Actinomyees, from a ml! m-e on ^lyeri in agar. x 1000 . 294
101. Shake en If ures of actinomyces in glucose agar . . 295
102. Seel inn ,,j' ,, ,-olony of ael; from a culture, in blood
x!500 295
xx LIST OF ILLUSTRATIONS
FIG. PAGE
103. Streptothrix Madura, x 1000 . . . .298
104. Surface colony of the anthrax bacillus on an agar plate.
x30 . . . . . . . 302
105. Anthrax bacilli, arranged in chains, from a twenty-four
hours' culture on agar at 37° C. x 1000 . . . 303
106. Stab culture of the anthrax bacillus in peptone-gelatin . 303
107. Anthrax bacilli containing spores, x 1000 . . . 305
108. Scraping from spleen of guinea-pig dead of anthrax, x 1000 307
109. Portion of kidney of a guinea-pig dead of anthrax, x 300 . 309
110. A large clump of typhoid bacilli in a spleen. x 500 . 320
111. Typhoid bacilli, from a young culture on agar, showing some
filamentous forms, x 1000 . . . .321
112. Typhoid bacilli, from a young culture on agar, showing
flagella. x 1000 ...... 322
113. Culture of the typhoid bacillus and of the bacillus coli . 323
114. Colonies of the typhoid bacillus in a gelatin plate, x 15 . 324
115. Bacillus coli communis. x 1000 .... 325
116. Film preparation from diphtheria membrane ; showing
numerous diphtheria bacilli, x 1000 . . . 354
117. Section through a diphtheritic membrane in trachea, show-
ing diphtheria bacilli, x 1000 .... 355
118. Cultures of the diphtheria bacillus on an agar plate . 357
119. Diphtheria bacilli from a twenty-four hours' culture on
agar. x 1000 ...... 357
120. Diphtheria bacilli, from a three days' agar culture. x 1000 358
121. Involution forms of the diphtheria bacillus, x 1000 . 358
122. Pseudo-diphtheria bacillus (Hofmann's). x 1000 . . 366
123. Xerosis bacillus from a young agar culture, x 1000 . 367
124. Film preparation of discharge from wound in a case of
tetanus, showing several tetanus bacilli of "drumstick "
form. xlOOO . . . . . .373
125. Tetanus bacilli, showing flagella. x 1000 . • . . 374
126. Spiral composed of numerous twisted flagella of the tetanus
bacillus, x 1000 . . . . . 375
127. Tetanus bacilli, some of which possess spores, x 1000 . 375
128. Stab culture of the tetanus bacillus in glucose gelatin . 376
129. Film preparation from the affected tissues in a case of
malignant cedema. x 1000 .... 389
130. Bacillus of malignant cedema, showing spores. x 1000 . 390
131. Stab cultures in agar — tetanus bacillus, bacillus of malignant
cedema, and bacillus of quarter-evil . . . 391
132. Bacillus of quarter-evil, showing spores. x 1000 . . 397
133. Bacillus serogenes capsulatus .... 398
134. Cholera spirilla, from a culture on agar of twenty- four hours'
growth. xlOOO . ' . . . . . 400
LIST OF ILLUSTRATIONS xxi
FIG. PAGE
135. Cholera spirilla stained to show the terminal flagella.
xlOOO ....... 401
136. Cholera spirilla from an old agar culture. x 1000 . . 401
137. Puncture culture of the cholera spirillum . . . 403
138. Colonies of the cholera spirillum on a gelatin plate . . 404
139. MetchnikofFs spirillum. x 1000 . . . . ' 417
140. Puncture cultures in peptone-gelatin . . .418
141. Finkler and Prior's spirillum, x 1000 . . . 419
142. Influenza bacilli from a culture on blood agar. x 1000 . 420
143. Film preparation from a plague bubo. x 1000 . . 426
144. Bacillus of plague from a young culture on agar. x 1000 . 427
145. Bacillus of plague in chains, x 1000 . . . 427
146. Culture of the bacillus of plague on 4 per cent salt agar.
xlOOO ....... 428
147. Section of a human lymphatic gland in plague, x 50 . 430
148. Film preparation of spleen of rat after inoculation with the
bacillus of plague, x 1000 , . . . 432
149. Spirilla of relapsing fever in human blood, x about 1000 . 439
150. Spirillum Obermeieri in blood of infected mouse, x 1000 . 441
151. Film of human blood containing spirillum of tick fever.
xlOOO ....... 444
152. Spirillum of human tick fever (Spirillum Duttonij in blood
of infected mouse. x 1000 .... 445
153. Micrococcus melitensis. x 1000 .... 448
154-159. Various phases of the benign tertian parasite . . 525
160-165. Exemplifying phases of the malignant parasite . . 526
166. Amoebae of dysentery ...... 538
167. Section of wall of liver abscess, showing an amoeba of spherical
form with vacuolated protoplasm. x 1000 . . 540
168. Trypanosoma Brucei from blood of infected rat. Note in two
of the organisms commencing division of micronucleus and
undulating membrane, x 1000 .... 554
169. Trypanosoma gambiense from blood of guinea-pig. x 1000 . 557
170. Leishman-Donovan bodies from spleen smear, x 1000 . 564
171. Leishman-Donovan bodies within endothelial cell in spleen.
x 1000 565
MANUAL OF BACTERIOLOGY
MANUAL OF BACTEBIOLOGY.
CHAPTER I.
GENERAL MORPHOLOGY AND BIOLOGY.
Introductory. — At the bottom of the scale of living things there
exists a group of organisms to which the name of bacteria is
usually applied. These are apparently of very simple structure
and may be subdivided into two sub-groups, a lower and simpler
and a higher and better developed.
The lower forms are the more numerous, and consist of
minute unicellular masses of protoplasm devoid of chlorophyll,
which multiply by simple fission. Some are motile, others non-
motile. Their minuteness may be judged of by the fact that in
one direction at least they usually do not measure more than
1 /z (35Soo incn)- These forms can be classified according to
their shapes into three main groups — (1) A group in which the
shape is globular. The members of this are called cocci. (2) A
group in which the shape is that of a straight rod — the pro-
portion of the length to the breadth of the rod varying greatly
among the different members. These are called bacilli. (3) A
group in which the shape is that of a curved or spiral rod.
These are called spirilla. The full description of the characters
of these groups will be more conveniently taken later (p. 11).
In some cases, especially among the bacilli, there may occur
under certain circumstances changes in the protoplasm whereby
a resting stage or spore is formed.
The higher forms show advance on the lower along two lines.
(1) On the one hand they consist of filaments made up of
simple elements such as occur in the lower forms. These
1
GENERAL MORPHOLOGY AXD BIOLOGY
filaments may be more or less septate, may be provided with a
sheath, and may show branching either true or false. The
minute structure of the elements comprising these filaments is
analogous to that of the lower forms. Their size, however, is
often somewhat greater. The lower forms sometimes occur in
filaments, but here every member of the filament is independent,
while in the higher forms there seems to be a certain inter-
dependence among the individual elements. For instance,
growth may occur only at one end of a filament, the other
forming an attachment to some fixed object. (2) The higher
forms, moreover, present this further development that in certain
cases some of the elements may be set apart for the reproduction
of new individuals.
Terminology. — The term bacterium of course in strictness
only refers to the rod-shaped varieties of the group, but as it
has given the name bacteriology to the science which deals with
the whole group, it is convenient to apply it to all the members
of the latter, and to reserve the term bacillus for the rod-shaped
varieties. Other general words, such as germ, microbe, micro-
organism, are often used as synonymous with bacterium, though,
strictly, they include the smallest organisms of the animal
kingdom.
While no living organisms lower than the bacteria are known
(though the occurrence of such is now suspected), the upper
limits of the group are difficult to define, and it is further
impossible in the present state of our knowledge to give other
than a provisional classification of the forms which all recognise
to be bacteria. The division into lower and higher forms,
however, is fairly well marked, and we shall therefore refer to
the former as the lower bacteria, and to the latter as the higher
bacteria.
Morphological Relations. — Whe relations of the bacteria to the animal
kingdom on the one hand and to the vegetable on the other constitute a
somewhat difficult question. It is best to think of there being a group
of small, unicellular organisms, which may represent the most primitive
forms of life before differentiation into animal and vegetable types had
occurred. This would include the flagellata and infusoria, the myxomy-
cetes, the lower algae, and the bacteria. To the lower algae the bacteria
possess many similarities. These algae are unicellular masses of proto-
plasm, having generally the same shapes as the bacteria, and largely
multiply by fission. Endogenous sporulation, however, does not occur,
nor is motility associated with the possession of flagella. Also their
protoplasm differs from that of the bacteria in containing chlorophyll and
another blue-green pigment called phyfigpyan. From the morphological
resemblances, however, between these algae and the bacteria, and from
the fact that fission plays a predominant part in the multiplication of
STRUCTURE OF THE BACTERIAL CELL 3
both, they have been grouped together in one class as the Schizophyta
or splitting plants (German, Spaltpflanzen). And of the two divisions
forming these Schizophyta the splitting algae are denominated the
schizophycese (German, Spaltalgen), while the bacteria or splitting fungi
are called the schizomycetes (German, Spaltpilzen). The bacteria are,
therefore, often spoken of as the schizomycetes. Certain bacteria which
have been described as containing chlorophyll ought probably to be
grouped among the schizophycese.
GENERAL MORPHOLOGY OF THE BACTERIA.
The Structure of the Bacterial Cell. — On account of the
minuteness of bacteria the investigation of their structure is
attended with great difficulty. When examined under the
microscope, in their natural condition, e.g. in water, they appeal-
merely as colourless refractile bodies of the different shapes
named. Spore formation and motility, when these exist, can
also be observed, but little else can be made out. For their
proper investigation advantage is always taken of the fact of
their affinities for various dyes, especially those which are usually
chosen as good stains for the nuclei of animal cells. Certain
points have thus been determined. The bacterial cell consists
of a sharply contoured mass of protoplasm which reacts to,
especially basic, aniline dyes like the nucleus of an animal cell
— though from this fact we cannot deduce that the two are
identical in composition. A healthy bacterium when thus
stained presents the appearance of a finely granular or almost
homogeneous structure. The protoplasm is surrounded by an
envelope which can in some cases be demonstrated by over-
staining a specimen with a strong aniline dye, when it will appear
as a halo round the bacterium. This envelope may sometimes
be seen to be of considerable thickness. Its innermost layer is
probably of a denser consistence, and sharply contours the
contained protoplasm, giving the latter the appearance of being
surrounded by a membrane. It is only, however, in some|0i
the higher forms that a true membrane occurs. Sometimes the
outer margin of the envelope is sharply defined, in which case
the bacterium appears to have a distinct capsule, and iap:nown
as a capsulated bacterium (vide Fig. 1, No. 3 ; and Fig. 64).
The cohesion of bacteria into masses depends largely on the
character of the envelope. If the latter is glutinous, tlien a
large mass of the same species may occur, formed of individual
bacteria embedded in what appears to be a mass of jelly. When
this occurs, it is knownas a zoogloea mass. On the other hand,
if the envelope lias not this cohesive property the separation of
*
4 GENERAL MORPHOLOGY AND BIOLOGY
individuals may easily take place, especially in a fluid medium
in which they may float entirely free from one another. Many
of the higher bacteria possess a sheath which has a much more
definite structure than is found among the lower forms. It
resists external influences, possesses elasticity, and serves to bind
the elements of the organism together.
Reproduction among the Lower Bacteria. — When a bacterial
cell is placed in favourable surroundings it multiplies ; as has
been said, this, in the great majority of cases, takes place by
simple fission. In the process a constriction appears in the
middle and a transverse . unstained line develops across the
protoplasm at that point. The process goes on till two
individuals can be recognised, which may remain for a time
attached to one another, or become separate, according to the
character of the envelope, as already explained. In most
bacteria growth and multiplication go on with great rapidity.
A bacterium may reach maturity and divide in from twenty
minutes to half an hour. If division takes place only every
hour, from one individual after twenty-four hours 17,000,000
similar individuals will be produced. As shown by the results
of artificial cultivation, others, such as the tubercle bacillus,
multiply much more slowly. Sometimes division proceeds so
rapidly that the young individuals do not reach the adult size
before multiplication again occurs. This may give rise to
anomalous appearances. When bacteria are placed in unfavour-
able conditions as regards food, etc., growth and multiplication
take place with difficulty. In the great majority of cases this^is
evidenced by changes in the appearance of the protoplasm.
Instead of its maintaining the regularity of shape seen in healthy
bacteria, various aberrant appearances are presented. This occurs
especially in the rod -shaped varieties, where flask- shaped or
dumb-bell-shaped individuals may be seen. The regularity in
structure and size is quite lost. The appearance of the protoplasm
also is often altered. Instead of, as formerly, staining well, it
does not stain readily, and may have a uniformly pale, homo-
geneous appearance, while in an old culture only a small
proportion of the bacteria may stain at all. Sometimes, on the
other hand, a .degenerated bacterium contains intensely stained
granules or globules which may be of large size. Such aberrant
and degenerate appearances are referred to as involution forms.
That these forms really betoken degenerative changes is shown
by the fact that, on their being again transferred to favourable
conditions, only slight growth at first takes place. Many
individuals have undoubtedly died, and the remainder which
SPORE FORMATION 5
live and develop into typical forms may sometimes have lost
some of their properties.
Reproduction among the Higher Bacteria. — Most of the higher bacteria
consist of thread-like structures more or less septate and often surrounded
by a sheath. The organism is frequently attached at one end to some
object or to another individual. It grows to a certain length and then
at the free end certain cells called gonidia are cast off from which new
individuals are formed. These gonidia may be formed by a division
taking place in the terminal element of the filament such as has occurred
in the growth of the latter. In some cases, however, division takes
place in three dimensions of space. The gonidia have a free existence
for a certain time before becoming attached, and in this stage are
sometimes motile. They are usually rod-like in shape, sometimes
pyriform. They do not possess any special powers of resistance.
Spore Formation. — In certain species of the lower bacteria,
under certain circumstances, changes take place in the protoplasm
which result in the formation of bodies called spores, to which
the vital activities of the original bacteria are transferred.
Spore formation occurs chiefly among the bacilli and in some
spirilla. Its commencement in a bacterium is indicated by the
appearance in the protoplasm of a minute highly refractile
granule unstained by the ordinary methods. This increases in
size, and assumes a round, oval, or short rod-shaped form, always
shorter but often . broader than the original bacterium. In the
process of spore formation the rest of the bacterial protoplasm
may remain unchanged in appearance and staining power for a
considerable time (e.g. b. tetani), or, on the other hand, it may
soon lose its power of staining and ultimately disappear, leaving
the spore in the remains of the envelope (e.g. b. anthracis).
This method of spore formation is called endogenous. Bacterial
spores are always non-motile. The spore may appear in the
centre of the bacterium, or it may be at one extremity, or a
short distance from one extremity (Fig. 1, No. 11). In structure
the spore consists of a mass of protoplasm surrounded by a dense
membrane. This can be demonstrated by methods which will
be described, the underlying principle of which is the prolonged
application of a powerful stain. The membrane is supposed to
confer on the spore its characteristic feature, namely, great
capacity of resistance to external influences such as heat or
noxious chemicals. Koch, for instance, in one series of experi-
ments, found that while the bacillus anthracis in the unspored ,
form was killed by a two minutes' exposure to 1 per cent carbolic!
acid, spores of the same organism resisted an exposure of from*
one to fifteen days.
When a spore is placed in suitable surroundings for growth
6 GENERAL MORPHOLOGY AND BIOLOGY
it again assumes the original bacillary or spiral form. Thei
capsule dehisces either longitudinally, or terminally, or trans-
versely. In the last case the dehiscence may be partial, and the
new individual may remain for a time attached by its ends to
the hinged spore-case, or the dehiscence may be complete and
the bacillus grow with a cap at each end consisting of half the
spore-case. Sometimes the spore-case does not dehisce, but is
simply absorbed by the developing bacterium.
It is important to note that in the bacteria spore formation
is rarely, if ever, to be considered as a method of multiplication.
In at least the great majority of cases only one spore is formed
from one bacterium, and only one bacterium in the first instance
from one spore. Sporulation is to be looked upon as a resting
stage of a bacterium, and is to be contrasted with the stage
wThen active multiplication takes place. The latter is usually
referred to as the vegetative stage of the bacterium. Regarding
the signification of spore formation in bacteria there has been
some difference of opinion. According to one view it may be
regarded as representing the highest stage in the vital activity
of a bacterium. There is thus an alternation between the
vegetative and spore stage, the occurrence of the latter being
necessary to the maintenance of the species in its greatest
vitality. Such a rejuvenescence, as it were, through sporulation,
is known in many algae. In support of this view there are
certain facts. In many cases, for instance, spore formation only
occurs at temperatures specially favourable for growth and
multiplication. There is often a temperature below which,
while vegetative growth still takes place, sporulation will not
occur ; and in the case of b. anthracis, if the organism be kept
at a temperature above the limit at which it grows best, not
only are no spores formed, but the species may lose the power
of sporulation. Furthermore, in the case of bacteria preferring
the presence of oxygen for their growth, an abundant supply of
this gas may favour sporulation. It is probable that even among
bacteria preferring the absence of oxygen for vegetative growth,
the presence of this gas favours sporulation. Most bacteriologists
are, however, of opinion that when a bacterium forms a spore,
it only does so when its surroundings, especially its food supply,
become unfavourable for vegetative growth ; it then remains in
this condition until it is placed in more suitable surroundings.
Such an occurrence would be analogous to what takes place
under similar conditions in many of the protozoa. Often
sporulation can be prevented from taking place for an indefinite
time if a bacterium is constantly supplied with fresh food (the
SPORE FORMATION 7
other conditions of life being equal). The presence of substances
excreted by the bacteria themselves plays, however, a more
important part in making the surroundings unfavourable than
the mere exhaustion of the food supply. A living spore will
always develop into a vegetative form if placed in a fresh food
supply. With regard to the rapid formation of spores when
the conditions are favourable for vegetative growth, it must be
borne in mind that in such circumstances the conditions may
really very quickly become unfavourable for a continuance of
growth, since not only will the food supply around the growing
bacteria be rapidly exhausted, but the excretion of effete and
inimical matters will be all the more rapid.
We must note that the usually applied tests of a body
developed within a bacterium being a spore are (1) its staining
reaction, namely, resistance to ordinary staining fluids, but
capacity of being stained by the special methods devised for
the purpose (vide p. 102) ; (2) the fact that the bacterium
containing the spore has higher powers of resistance against
inimical conditions than a vegetative form. It is important to
bear these tests in mind, as in some of the smaller bacteria
especially, it is very difficult to say whether they spore or not.
There may appear in such organisms small unstained spots the
significance of which it is very difficult to determine.
The Question of Arthrosporo.us Bacteria.— It is stated by Hueppe that
among certain organisms, e.g. some streptococci, certain individuals may,
without endogenous sporulation, take on a resting stage. These become
swollen, stain well with ordinary stains, and they are stated to have
higher power of resistance than the other forms ; further, when vegetative
life again occurs it is from them that multiplication is said to take place.
From the fact that there is no new formation within the protoplasm,
but that it is the whole of the latter which participates in the change,
these individuals have been called arthrospores. The existence of such
special individuals amongst the lower bacteria is extremely problematical.
They have no distinct capsule, and they present no special staining
reactions, nor any microscopic features by which they can be certainly
recognised, while their alleged increased powers of resistance are very
doubtful. All the phenomena noted can be explained by the undoubted
fact that in an ordinary growth there is very great variation among the
individual organisms in their powers of resistance to external conditions.
Motility. — As has been stated, many bacteria are motile.
Motility can be studied by means of hanging-drop preparations
(vide p. 63). The movements are of a darting, rolling, or
vibratile character. The degree of motility depends on the
species, the temperature, the age of the growth, and on the
medium in which the bacteria are growing. Sometimes the
movements are most active just after the cell has multiplied,
8 GENERAL MORPHOLOGY AND BIOLOGY
sometimes it goes on all through the life of the bacterium,
sometimes it ceases when sporulation is about to occur. Motility
is associated with the possession of fine wavy thread-like
appendages called flagella, which for their demonstration require
the application of special staining methods (vide Fig. 1, No. 12 ;
and Fig. 112). They have been shown to occur in many bacilli
and spirilla, but only in a few species of cocci. They vary in
length, but may be several times the length of the bacterium,
and may be at one or both extremities or all round. When
terminal they may occur singly or there may be several. The
nature of these flagella has been much disputed. Some have
held that, unlike what occurs in many algae, they are not actual
prolongations of the bacterial protoplasm, but merely appendages
of the envelope, and have doubted whether they are really organs
of locomotion. There is now, however, little doubt that they
belong to the protoplasm. By appropriate means the central
parts of the latter can be made to shrink away from the peripheral
(vide infra, " plasmolysis "). In such a case movement goes on
as before, and in stained preparations the flagella can be seen
to be attached to the peripheral zone. It is to be noted that
flagella have never been demonstrated in non- motile bacteria,
while, on the other hand, they have been observed in nearly all
motile forms. There is little doubt, however, that all cases of
motility among the bacteria are not dependent on the possession
of flagella, for in some of the special spiral forms, and in most
of the higher bacteria, motility is probably due to contractility
of the protoplasm itself.
The Minuter Structure of the Bacterial Protoplasm.— Many attempts
have been made to obtain deeper information as to the structure of the
bacterial cell, and especially as to its behaviour in division. These
have largely turned on the interpretation to be put on certain appear-
ances which have been observed. These appearances are of two kinds.
First, under certain circumstances irregular deeply-stained granules are
observed in the protoplasm, often, when they occur in a bacillus, giving
the latter the appearance of a short chain of cocci. They are often
called metachromatic granules (vide Fig. 1, No. 16) from the fact that
by appropriate procedure they can be stained with one dye, and the
protoplasm in which they lie with another ; sometimes, when a single
stain is used, such as methylene blue, they assume a slightly different
tint from the protoplasm.
For the demonstration of the metachromatic granules two methods
have been advanced. Ernst recommends that a few drops of Loffler's
methylene blue (vide p. 98) be placed on a cover-glass preparation and
the latter passed backwards and forwards over a Bunsen flame for half
a minute after steam begins to rise. The preparation is then washed
in water and counter-stained for one to two minutes in watery Bismarck-
brown. The granules are here stained blue, the protoplasm brown.
STRUCTURE OF BACTERIAL PROTOPLASM 9
Neisser stains a similar preparation in warm carbol-fuchsin, washes
with 1 per cent sulphuric acid, and counter-stains with Loffler's blue.
Here the granules are magenta, the protoplasm blue. The general
character of the granules thus is that they retain the first stain more
intensely than the rest of the protoplasm does.
A second appearance which can sometimes be seen in specimens
stained in ordinary ways is the occurrence of a concentration of the
protoplasm at each end of a bacterium, indicated by these parts being
deeply stained. These deeply stained parts are sometimes called polar
franules (vide Fig. 1, No. 16, the bacillus most to the right), (German,
olkornchen or Polkorner).
With regard to the significance that is to be attached to such
appearances, much depends on whether they are constantly present
under all circumstances, or only occasionally, when the organism is
grown in special media or under special growth conditions. Some
bacteria, however stained, show evidence of having the protoplasm
somewhat granular, e.g. the diphtheria bacillus. In other cases this
granular condition is only seen when the organism has been grown under
bad conditions, or where the food supply is becoming exhausted. Some
have thought that the appearances might be due to a process allied to
mitosis and might signify approaching division, but of this there is no
evidence.
In perfectly healthy and young bacteria, appearances of granule
formation and of vacuolation may be accidentally produced by physical
means in the occurrence of wha*t is known as plasmolysis. To speak
generally, when a mass of protoplasm surrounded by a fairly firm
envelope of a colloidal nature is placed in a solution containing salts in
greater concentration than that in which it has previously been living,
then by a process of osmosis the water held in the protoplasm passes
out through the membrane, and, the protoplasm retracting from the
latter, the appearance of vacuolation is presented. Now in making a
dried film for the microscopic examination of bacteria the conditions
necessary for the occurrence of this process may be produced, and the
appearances of vacuolation and, in certain cases, of Polkorner may thus
be brought about. Plasmolysis in bacteria has been extensively
investigated,1 and has been found to occur in some species more readily
than in others. Furthermore it is often most readily observed in old or
otherwise enfeebled cultures.
Btitschli, from a study of some large sulphur-containing forms, con-
cludes that the greater part of the bacterial cell may correspond to a
nucleus, and that this is surrounded by a thin layer of protoplasm which
in the smaller bacteria escapes notice, unless when, as in the bacilli, it
can be made out at the ends of the cells. Fischer, it may be said, looks
on the appearances seen in Biitschli's preparations as due to plasmolysis.
The Chemical Composition of Bacteria. — In the bodies of
bacteria many definite substances occur. Some bacteria have
been described as containing chlorophyll, but these are properly
to be classed with the schizophyceaB. Sulphur is found in some
of the higher forms, and starch granules are also described as
occurring. Many species of bacteria, when growing in masses,
1 Consult Fischer, " Untersuchuugen iiber Bakterien," Berlin, 1894;
"Ueber den Bau der Cyanophyceeu und Bakterien," Jena, 1897.
10 GENERAL MORPHOLOGY AND BIOLOGY
are brilliantly coloured, though few bacteria associated with the
production of disease give rise to pigments. In some of the
organisms classed as bacteria a pigment named bacterio-purpurin
has been observed in the protoplasm, and similar intracellular
pigments probably occur in some of the larger forms of the
lower bacteria and may occur in the smaller ; but it is usually
impossible to determine whether the pigment occurs inside or
outside the protoplasm. In many cases, for the free production
of pigment abundant oxygen supply is necessary; but sometimes,
as in the case of spirillum rubrum, the pigment is best formed
in the absence of oxygen. Sometimes the faculty of forming it
may be lost by an organism for a time, if not permanently, by
the conditions of its growth being altered. Thus, for example,
if the b. pyocyaneus be exposed to the temperature of 42° C.
for a certain time, it loses its power of producing its bluish
pigment. Pigments formed by bacteria often diffuse out into,
and colour, the medium for a considerable distance around.
Comparatively little is known of the nature of bacterial pigments.
Zopf, however, has found that many of them belong to a group of
colouring matters which occur widely in the vegetable and animal
kingdoms, viz. the lipochromes. These lipochromes, which get their
name from the colouring matter of animal fat, include the colouring
matter in the petals of Ranunculaceae, the yellow pigments of serum and
of the yolks of eggs, and many bacterial pigments. The lipochromes are
characterised by their solubility in chloroform, alcohol, ether, and
petroleum, and by their giving indigo-blue crystals with strong sulphuric
acid, and a green colour with iodine dissolved in potassium iodide.
Though crystalline compounds of these have been obtained, their
chemical constitution is entirely unknown and even their percentage
composition is disputed.
Some observations have been made on the chemical structure
of bacterial protoplasm. Nencki isolated from the bodies of
certain putrefactive bacteria proteid bodies which, according to
Ruppel, appear to have been allied to peptone, and which
differed from nucleo- proteids in not containing phosphorus,
but many of the proteids isolated by other chemists have
been allied in their nature to the protoplasm of the nuclei
of cells. Buchner in certain researches obtained bodies of this
nature allied to the vegetable caseins, and he adduces evide
to show that it is to these that the characteristic staini
properties are due. Various observers have isolated similar
phosphorus-containing proteids from different bacteria. Besides
proteids, however, substances of a different nature have been
isolated. Thus cellulose, fatty material, chitin, wax-like bodies,
and other substances have been observed. There are also found
THE CLASSIFICATION OF BACTERIA 11
various mineral salts, especially those of sodium, potassium, and
magnesium. The amount of different constituents varies ac-
cording to the age of the culture and the medium used for
growth, and certainly great variation takes place in the com-
position of different species.
The Classification of Bacteria. — There have been numerous
schemes set forth for the classification of bacteria, the funda-
mental principle running through all of which has been the
recognition of the two sub-groups and the type forms mentioned
in the opening paragraph above. In the attempts to still
further subdivide the group, scarcely two systematists are agreed
as to the characters on which sub-classes are to be based. Our
present knowledge of the essential morphology and relations of
bacteria is as yet too limited for a really natural classification to
be attempted. To prepare for the elaboration of the latter,
Marshall Ward suggested that in every species there should be
studied the habitat, best food supply, condition as to gaseous
environment, range of growth, temperature, morphology, life
history, special properties and pathogenicity.
We must thus be content with a provisional and incomplete
classification. We have said that the division into lower and
higher bacteria is recognised by all, though, as in every other
classification, there occur transitional forms. In subdividing
the bacteria further, the forms they assume constitute at present
the only practicable basis of classification. The lower bacteria
thus naturally fall into the three groups mentioned, the cocci,
bacilli, and spirilla, though the higher are more difficult to deal
with. Subsidiary, though important, points in still further sub-
division are the planes in which fission takes place and the
presence or absence of spores. The recognition of actual species
is often a matter of great difficulty. The points to be observed
in this will be discussed later (p. 115).
I. The Lower Bacteria.1 — These, as we have seen, are
minute unicellular masses of protoplasm surrounded by an
envelope, the total vital capacities of a species being represented
in every cell. They present three distinct type forms, the
coccus, the bacillus, and the spirillum ; endogenous sporulation
may occur. They may also be motile.
1. The Cocci. — In this group the cells range in different
species from '5 //, to 2 //, in diameter, but most measure about 1 p.
Before division they may increase in size in all directions. The
species are usually classified according to the method of division.
1 For the illustration of this aud the succeeding systematic paragraphs,
vide Fig. 1.
12 GENERAL MORPHOLOGY AND BIOLOGY
If the cells divide only in one axis, and through the consistency
of their envelopes remain attached, then a chain of cocci will be
formed. A species in which this occurs is known as a strepto-
coccus. If division takes place irregularly the resultant mass may
be compared to a bunch of grapes, and the species is often called
a staphylococcus. Division may take place in two axes at right
angles to one another, in which case cocci adherent to each other
in packets of four (called tetrads) or sixteen may be found,
the former number being the more frequent. To all these forms
the word micrococcus is often generally applied. The individuals
in a growth of mfcrbcocci often- show a tendency to remain
united in twos. These are spoken of as diplococci, but this is
not a distinctive character, since every coccus as a result of
division becomes a diplococcus, though in some species the
tendency to remain in pairs is well marked. The adhesion of
cocci to one another depends on the character of the capsule.
Often this has a well-marked outer limit (micrococcus tetragenus),
sometimes it is of great extent, its diameter being many times
that of the coccus (streptococcus mesenteriodes). ' It is especially
among the streptococci and staphylococci that the phenomenon
of the formation of arthrospores is said to occur. In none of
the cocci have endogenous spores been certainly observed. The
number of species of the streptococci and staphylococci probably
exceeds 150. Usually included in this group are coccus-like
organisms which divide in three axes at right angles to one
another. These are usually referred to as sarcinm. If the cells
are lying single they are round, but usually they are seen in
cubes of eight with the sides which are in contact slightly
flattened. Large numbers of such cubes may be lying together.
The sarcinae are, as a rule, rather larger than the other members
of the group. Most of the cocci are non-motile, but a few motile
species possessing flagella have been described.
2. Bacilli. — These consist of long or short cylindrical cells,
with rounded or sharply rectangular ends, usually not more than
1 //. broad, but varying very greatly in length. They may be
motile or non-motile. Where flagella occur, these may be
distributed all round the organism, or only at one or both of
the poles (pseudomonas). Several species are provided with
sharply -marked capsules (b. pneumoniae). In many species
endogenous sporulation occurs. The spores may be central or
terminal, round, oval, or spindle-shaped.
Great confusion in nomenclature has arisen in this group in con-
sequence of the different artilicial meanings assigned to the essentially
synonymous terms bacterium and bacillus. Migula, for instance, applies
THE LOWER BACTERIA
13
15&
IS
,
FIG. 1. — 1. Coccus. 2. Streptococcus. 3. Staghylococcus. 4. Capsulated cliplococcus.
5. " Biscuit "-shaped coccus.. 6. Tetrads.. ?. Sarcina form. 8. Types of bacilli
(1-8 are diagrammatic). 9. Non-septate spirillum xlOOO. 10. Ordinary spirillum —
(a) comma-shaped element; (b) formation of spiral by comma-shaped elements
XlOOO. 11. Types of spore formation. 12. Flagellated bacteria. 13. Changes in
bacteria produced by plasmolysis (after Fischer). 14. Bacilli with terminal proto-
plasm (Biitschli). 15. (a) Bacillus composed of five protoplasmic meshes ; (b) proto-
plasmic network in npcrococcus (Biitschli). 16. Bacteria containing nietachromatic
granules (Ernst, Neisser) — some contain polar granules. 17. Beggiatoa alba. Both
filaments contain sulphur granules — one is septate. 18. Thiothrix tenuis (Wino-
gradski). 19. Leptothrix innominata (Miller). 20. Cladothiix dichotoma (Zopt).
21. Streptothrixactinomyces'(Bostrom), (a) colony under low power ; (b) filament
showing true branching ; (c) filament containing coccus-like bodies ; (d) filament
with club at end.
14 GENERAL MORPHOLOGY AND BIOLOGY
the former term to non-motile species, the latter to the motile. Hueppe,
on the other hand, calls those in which endogenous sporulation does
not occur, bacteria, and those where it does, bacilli. In the ordinary
terminology of systematic bacteriology the word bacterium has been
almost dropped, and is reserved, as we have done, as a general term for
the whole group. It is usual to call all the rod-shaped varieties bacilli.
3. Spirilla. — These consist of cylindrical cells more or less
spiral or wavy. Of such there are two main types. In one there
is a long non-septate, usually slender, wavy or spiral thread
(Fig. 1, No. 9). In the other type the unit is a short curved
rod (often referred to as of a " comma" shape). When two
or more of the latter occur, as they often do, end to end
with their curves alternating, then a wavy or spiral thread
results. An example of this is the chojej^-microbe (Fig. 1,
No. 10). This latter type is of much more frequent occurrence,
and contains the more important species. Among the first group
motility is often not associated, as far as is known, with the
possession of flagella. The cells here apparently move by an
undulating or screw-like contraction of the protoplasm. Most of
the motile spirilla, however, possess flagella. Of the latter there
may be one or two, or a bunch containing as many as twenty, at
one or both poles. Division takes place as among the bacilli,
and in some species endogenous sporulation has been observed.
Three terms are used in dividing this group, to which different authors
have given different meanings. These terms are spirillum, spirochaete,
vibrio. Migula makes " vibjyo " synonymous with "microspira," which
he applies to members of the group which possess only one or two polar
flagella ; "spirillum" he applies to similar species which have bunches
of polar flagella, while " spirochfete " is reserved for the long unflagellated
spiral cells. Hueppe applies the term " spirochfete " to forms without
endospores, "vibrio" to those with endospores in which during sporula-
tion the organism changes 'its form, and "spirillum" to the latter
when no change of form takes place in sporulation. Flugge, another
systematist, applies " spirochsete " and "spirillum" indiscriminately to
any wavy or corkscrew form, and "vibrio" to forms where the undula-
tions are not so well marked. It is thus necessary, in denominating such
a bacterium by a specific name, to give the authority from whom the
name is taken.
Quite recently great doubt has arisen as to whether many of
the non-septate spirillary forms are to be looked on as bacteria
at all, — the view being taken that in, it may be, many cases
they represent a stage in the life history of what are really
protozoa of the nature of trypanosomes. The ultimate classifica-
tion of the spirilla must thus be left an open question.
II. The Higher Bacteria. — These show advance on the lower
in consisting of definite filaments branched or unbranched. In
THE HIGHER BACTERIA 15
most cases the filaments at more or less regular intervals are cut
by septa into short rod-shaped or curved elements. Such
elements are more or less interdependent on one another, and
special staining methods are often necessary to demonstrate the
septa which demarcate the individuals of a filament. There is
further often a definite membrane or sheath common to all the
elements in a filament. Not only, however, is there this close
organic relationship between the elements of the higher bacteria,
but there is also interdependence of function ; for example, one
end of a filament is frequently concerned merely in attaching
the organism to some other object. The greatest advance,
however, consists in the setting apart among most of the higher
bacteria of the free terminations of the filaments for the produc-
tion of new individuals, as has been described (p. 2). There
are various classes under which the species of the higher bacteria
are grouped ; but our knowledge of them is still somewhat
limited, as many of the members have not yet been artificially
cultivated. The hggcizatoa group consists of free swimming
forms, motile by undulating contractions of their protoplasm.
For the demonstration of the rod-like elements of the filaments
special staining is necessary. The filaments have no special
sheath, and the protoplasm contains sulphur granules. The
method of reproduction is doubtful. The thiothrix group re-
sembles the last in structure, and the protoplasm also contains
sulphur granules; but the filaments are attached at one end,
and at the other form gonidia. The leptothrix group resembles
closely the thiothrix group, but the protoplasm does not contain
sulphur granules. In the cladothrix group there is the appearance
of branching, which, however, is of a false kind. What happens
is that a terminal cell divides, and on dividing again, it pushes
the -product of its first division to one side. There are thus two
terminal cells lying side by side, and as each goes on dividing,
the appearance of branching is given. Here, again, there is
gonidium formation ; and while the parent organism is in some
of its elements motile, the gonidia move by means of flagella.
The highest development is in the streptothrix group, to which
belongs the streptothrix actinomyces, or the actinomyces bovis, and
several other important pathogenic agents. Here the organism
consists of a felted mass of non-septate filaments, in which true
dichotomous branching occurs. Under certain circumstances
threads grow out, and produce chains of coccus-like bodies from
which new individuals can be reproduced. Such bodies are often
referred to as spores, but they have not the same staining reactions
nor resisting powers of so high a degree as ordinary bacterial
16 GENERAL MORPHOLOGY AND BIOLOGY
spores. Sometimes too the protoplasm of the filaments breaks
up into bacillus-like elements, which may also have the capacity
of originating new individuals. In the streptothrix actinomyces
there may appear a club-shaped swelling of the membrane at the
end of the filament, which has by some been looked on as an
organ of fructification, but which is most probably a product of
a degenerative change. The streptothrix group, though its
morphology and relationships are much disputed, may be looked
on as a link between the bacteria on the one hand, and the
lower fungi on the other. Like the latter, the streptothrix forms
show the felted mass of non-septate branching filaments, which
is usually called a mycelium. On the other hand, the breaking
up of the protoplasm of the streptothrix into coccus- and bacillus-
like forms, links it to the other bacteria.
GENERAL BIOLOGY OF THE BACTERIA.
There are five prime factors which must be considered in
the growth of bacteria, namely, food supply, moisture, relation to
gaseous environment, temperature, and light.
Food Supply. — The bacteria are chiefly found living on the
complicated organic substances which form the bodies of dead
plants and animals, or which are excreted by the latter while
they are yet alive. Seeing that, as a general rule, many bacteria
grow side by side, the food supply of any particular variety is,
relatively to it, altered by the growth of the other varieties
present. It is thus impossible to imitate the complexity of the
natural food environment of any species. The artificial media
used in bacteriological work may therefore be poor substitutes
for the natural supply. In certain cases, however, the conditions
under which we grow cultures may be better than the natural
conditions. For while one of two species of bacteria growing
side by side may favour the growth of the other, it may also
in certain cases hinder it, and, therefore, when the latter is
grown alone it may grow better. Most bacteria seem to
produce excretions which are unfavourable to their own
vitality, for, when a species is sown on a mass of artificial
food medium, it does not in the great majority of cases go on
growing till the food supply is exhausted, but soon ceases to
grow. Effete products diffuse out into the medium and prevent
growth. Such diffusion may be seen when the organism pro-
duces pigment, e.g. b. pyocyaneus growing on gelatin. In
supplying artificial food for bacterial growth, the general principle
ought to be to imitate as nearly as possible the natural surround-
RELATION TO GASEOUS ENVIRONMENT 17
ings, though it is found that there exists a considerable adapta-
bility among organisms. With the pathogenic varieties it is
usually found expedient to use media derived from the fluids of
the animal body, and in cases where bacteria growing on plants
are being studied, infusions of the plants on which they grow
are frequently used. Some bacteria can exist on inorganic food,
but most require organic material to be supplied. Of the latter,
some require for their proper nourishment proteid to be present,
while others can derive their nitrogen from such a non-proteid
as asparagin. All bacteria require nitrogen to be present in
some form, and many require to derive their carbon from
carbohydrates. Mineral salts, especially sulphates, chlorides, and
phosphates, and also salts of iron are necessary. Occasionally
special substances are needed to support life. Thus some
species, in the protoplasm of which sulphur granules occur,
require sulphuretted hydrogen to be present. In nature the
latter is usually provided by the growth of other bacteria. When
the food supply of a bacterium fails, it degenerates and dies.
The proof of death lies in the fact that when it is transferred to
fresh and good food supply it does not multiply. If the
bacterium spores, it may then survive the want of food for a
very long time. It may here be stated that the reaction of the
food medium is a matter of great importance. Most bacteria
prefer a slightly alkaline medium, and some, e.g. the cholera
spirillum, will not grow in the presence of the smallest amount
of free acid.
Moisture. — The presence of water is necessary for the con-
tinued growth of all bacteria. The amount of drying which
bacteria in the vegetative stage will resist varies very much in
different species. Thus the cholera spirillum is killed by two or
three hours' drying, while the staphylococcus pyogenes aureus
will survive ten days' drying, and the bacillus diphtherise still
more. In the case of spores the periods are much longer.
Anthrax spores will survive drying for several years, but here
again moisture enables them to resist longer than when they are
quite dry. When organisms have been subjected to such hostile
influences, even though they survive, it by no means follows that
they retain all their vital properties.
Relation to Gaseous Environment. — The relation of bacteria
to the oxygen of the air is such an important factor in the life
of bacteria that it enables a biological division to be made among
them. Some bacteria will only live and grow when oxygen is
present. To these the title of obligatory aerobes is given. Other
bacteria will only grow when no oxygen is present. These are
2
18 GENERAL MORPHOLOGY AND BIOLOGY
called obligatory anaerobes. In still other bacteria the presence
or absence of oxygen is a matter of indifference. This group
might theoretically be divided into those which are preferably
aerobes, but can be anaerobes, and those which are preferably
anaerobes, but can be aerobes. As a matter of fact such
(littVivnces are manifested to a slight degree, but all such
organisms are usually grouped as facultative anaerobes, i.e. pre-
ferably aerobic but capable of existing without oxygen. Examples
of obligatory aerobes are b. proteus vulgaris, b. subtilis ; of
obligatory anaerobes, b. tetani, b. oedematis maligni, while the
great majority of pathogenic bacteria are facultative anaerobes.
With regard to anaerobes, hydrogen and nitrogen are indifferent
gases. Many anaerobes, however, do not flourish well in an
atmosphere of carbon dioxide. Very few experiments have
been made to investigate the action on bacteria of gas under
pressure. A great pressure of carbon dioxide is said to make
the b. anthracis lose its power of sporing, but it seems to have
no effect on its vitality or on that of the b. typhosus. With
the bacillus pyocyaneus, however, it is said to destroy life.
Temperature. — For every species of bacterium there is a
temperature at which it grows best. This is called the " optimum
temperature." There is also in each case a maximum tempera-
ture above which growth does not take place, and a minimum
temperature below which growth does not take place. As a
general rule the optimum temperature is about the temperature
of the natural habitat of the organism. For organisms taking
part in the ordinary processes of putrefaction the temperature of
warm summer weather (20° to 24° C.) may be taken as the
average optimum, while for organisms normally inhabiting animal
tissues 35° to 39° C is a fair average. The lowest limit of
ordinary growth is from 12° to 14° C., and the upper is from
42° to 44° C. In exceptional cases growth may take place as
low as 5° C., and as high as 70° C. Some organisms which
grow best at a temperature of from 60° to 70° C. have been
isolated from dung, the intestinal tract, etc. These have been
called thermophilic bacteria. It is to be noted that while growth
does not take place below or above a certain limit it by
no means follows that death takes place outside such limits.
Organisms can resist cooling below their minimum or heating
beyond their maximum without being killed. Their- vital activity
is merely paralysed. Especially is this true of the effect of cold
on bacteria. The results of different observers vary ; but if we
take as an example the cholera vibrio, Koch found that while
the minimum temperature of growth was 16° C., a culture might
CONDITIONS AFFECTING BACTERIAL MOTILITY 19
be cooled to -32° C. without being killed. With regard to the
upper limit, few ordinary organisms in a spore-free condition will
survive a temperature of 57° C., if long enough applied. Many
organisms lose some of their properties when grown at unnatural
temperatures. Thus many pathogenic organisms lose their
virulence if grown above their optimum temperature, and some
chromogenic forms, most of which prefer rather low tempera-
tures, lose their capacity of producing pigment, e.g. spirillum
rubrum.
Effect of Light. — Of recent years much attention has been
paid to this factor in the life of bacteria. Direct sunlight is
found to have a very inimical effect. It has been found that
an exposure of dry anthrax spores for one and a half hours to
sunlight kills them. When they are moist, a much longer
exposure is necessary. Typhoid bacilli are killed in about
one and a half hours, and similar results have been obtained
with many other organisms. In such experiments the thickness
of the medium surrounding the growth is an important point.
Death takes place more readily if the medium is scanty or if the
organisms are suspended in water. Any fallacy which might
arise from the effect of the heat rays of the sun has been ex-
cluded, though light plus heat is more fatal than light alone.
In direct sunlight it is chiefly the green, violet, and, it may be,
the ultra-violet rays which are fatal. Diffuse daylight has also a
bad effect upon bacteria, though it takes a much longer exposure
to do serious harm. A powerful electric light is as fatal as sun-
light. Here, as with other factors, the results vary very much
with the species under observation, and a distinction must be
drawn between a mere cessation of growth and the condition of
actual death. Some bacteria especially occurring on the dead
bodies of fresh fish are phosphorescent.
Conditions affecting the Movements of Bacteria. — In some
cases differences are observed in the behaviour of motile bacteria,
contemporaneous with changes in their life history. Thus, in
the case of bacillus subtilis, movement ceases when sporulation
is about to take place. On the other hand, in the bacillus of
symptomatic anthrax, movement continues while sporulation is
progressing. Under ordinary circumstances motile bacteria
appear not to be constantly moving but occasionally to rest.
In every case the movements become more active if the
temperature be raised. Most interest, however, attaches to the
fact that bacilli may be attracted to certain substances and
repelled by others. Schenk, for instance, observed that motile"
bacteria were attracted to a warm point in a way which did not
20 • GENERAL MORPHOLOGY AND BIOLOGY
occur when *the bacteria were dead and therefore only subject
to physical conditions. Most important observations have been
made on the attraction and repulsion exercised on bacteria by
chemical agents, which have been denominated respectively
positive and negative chemiotaxis. Pfeffer investigated this
subject in many lowly organisms, including bacterium termo
and spirillum undula. The method was to fill with the agent
a fine capillary tube, closed at one end, to introduce this into a
drop of fluid containing the bacteria under a cover-glass, and
to watch the effect through the microscope. The general result
was to indicate that motile bacteria may be either attracted or
repelled by the fluid in the tube. The effect of a given fluid
differs in different organisms, and a fluid chemiotactic for one
organism may not act on another. Degree of concentration is
important, but the nature of the fluid is more so. Of inorganic
bodies salts of potassium are the most powerfully attracting
bodies, and in comparing organic bodies the important factor
is the molecular constitution. These observations have been
confirmed by Ali-Cohen, who found that while the vibrio of
cholera and the typhoid bacillus were scarcely attracted by
chloride of potassium, they were powerfully influenced by
potato juice. Further, the filtered products of the growth of
many bacteria have been found to have powerful chemiotactic
properties. It is evident that all these observations have a
most important bearing on the action of bacteria, though we do
not yet know their true significance. Corresponding chemio-
tactic phenomena are shown also by certain animal cells, e.g.
leucocytes, to which reference is made below.
The Parts played by Bacteria in Nature. — As has been said,
the chief effect of bacterial action in nature is to break up into
more simple combinations the complex molecules of the organic
substances which form the bodies of plants and animals, or
which are excreted by them. In some cases we kno*v some of
the stages of disintegration, but in most cases we know only
general principles and sometimes only results. In the case of
milk, for instance, we know that lactic acid is produced from
the lactose by the action of the bacillus acidi lactici and of
other bacteria, and that from urea ammonium carbonate
is produced by the micrococcus ureae. That the very compli-
cated process of putrefaction is due to bacteria is absolutely
proved, for any organic substance can be preserved indefinitely
from ordinary putrefaction by the adoption of some method of
killing all bacteria present in it, as will be afterwards described.
This statement, however, does not exclude the fact that molecular
ACTION OF BACTERIAL FERMENTS 21
changes take place spontaneously in the passing of the organic
body from life to death. Many processes not usually referred to
as putrefactive are also bacterial in their origin. The souring
of milk, already referred to, the becoming rancid of butter, the
ripening of cream and of cheese, are all due to bacteria.
A certain comparatively small number of bacteria have been
proved to be the causal agents in some disease processes.,
occurring in man, animals, and plants. This means that the
fluids and tissues of living bodies are, under certain circum-
stances, a suitable pabulum for the bacteria involved. The
effects of the action of these bacteria are analogous to those
taking place in the action of the same or other bacteria on dead
animal or vegetable matter. The complex organic molecules
are broken up into simpler products. We shall study these
processes more in detail later. Meantime we may note that
the disease -producing effects of bacteria form the basis of
another biological division of the group. Some bacteria are
harmless to animals and plants, and apparently under no
circumstances give rise to disease in either. These are known
saprophytes. They are normally engaged in breaking up
dead animal and vegetable matter. Others normally live on
or in the bodies of plants and animals and produce disease.
_These are known as parasitic bacteria. Sometimes an attempt
is made to draw a hard and fast line between the saprophytes
and the parasites, and obligatory saprophytes or parasites are
spoken of. This is an erroneous distinction. Some bacteria
which are normally saprophytes can produce pathogenic effects.
(e.g. bacillus oedematis maligni), and it is consistent with our
knowledge that the best-known parasites may have been derived
_from saprophytes. On the other hand, the fact that most
bacteria associated with disease processes, and proved to be
the cause of the latter, can be grown in artificial media, shows
that for a time at least such parasites can be saprophytic^ As
to how far such a saprophytic existence of disease-producing
bacteria occurs in nature, we are in many instances still ignorant.
The Methods of Bacterial Action. — The processes which
bodies undergo in being split up by bacteria depend, first, on
the chemical nature of the bodies involved and, secondly, on the
varieties of the bacteria which are acting. The destruction of
albuminous bodies which is mostly involved in the wide and
varied process of putrefaction can be undertaken by whole
groups of different varieties of bacteria. The action of the
latter on such substances is analogous to what takes place when
albumins are subjected to ordinary gastric and intestinal digestion.
22 GENERAL MORPHOLOGY AND BIOLOGY
In these circumstances, therefore, the production of albumoses,
peptones, etc., similar to those of ordinary digestion, can be
recognised in putrefying solutions, though the process of destruc-
tion always goes further, and still simpler substances, e.g. indol,
and, it may be, crystalline bodies of an alkaloidal nature, are
the ultimate results. The process is an exceedingly complicated
one when it takes place in nature, and different bacteria are
probably concerned in the different stages. Many other bacteria,
e.g. some pathogenic forms, though not concerned in ordinary
putrefactive processes, have a similar digestive capacity. When
carbohydrates are being split up, then various alcohols, ethers,
and acids are produced. During bacterial growth there is
not infrequently the abundant production of such gases as
sulphuretted hydrogen, carbon dioxide, methane, etc. For an
exact knowledge of the destructive capacities of any particular
bacterium there must be an accurate chemical examination of its
effects when it has been grown in artificial media the nature of
which is known. The precise substances it is capable of forming
can thus be found out. Many substances, however, are produced
by bacteria, of the exact nature of which we are still ignorant,
for example, the toxic bodies which play such an important part
in the action of many pathogenic species.
Many of the actions of bacteria depend on the production by
them of ferments of a very varied nature and complicated action.
Thus the digestive action on albumins probably depends on the
production of a peptic ferment analogous to that produced in
the animal stomach. Ferments which invert sugar, which split
sugars up into alcohols or acids, which coagulate casein, which
split up urea into ammonium carbonate, also occur.
Such ferments may be diffused into the surrounding fluid, or
be retained in the cells where they are formed. Sometimes the
breaking down of the organic matter appears to take place
within, or in the immediate proximity of, the bacteria, sometimes
wherever the soluble ferments reach the organic substances.
And in certain cases the ferment diffused out into the sur-
rounding medium probably break down the constituents of
the latter to some extent, and prepare them for a further,
probably intracellular, disintegration. Thus in certain putre-
factions of fibrin, if the process be allowed to go on naturally,
the fibrin dissolves and ultimately great gaseous evolution
of carbon dioxide and ammonia takes place, but if the
bacteria, shortly after the process has begun, are killed or
paralysed by chloroform, then only a peptonisation of the
fibrin occurs, without the further splitting up and gaseous pro-
VARIABILITY AMONG BACTERIA 23
duction being observed. That a purely intracellular digestion
may take place is illustrated by what has been shown to occur
in the case of the micrococcus ureae, which from urea forms
ammonium carbonate by adding water to the urea molecule.
Here, if after the action has commenced, the bacteria are filtered
off, no further production of ammonium carbonate takes place,
which shows that no ferment has been dissolved out into the
urine. If now the bodies of the bacteria be extracted with
absolute alcohol or ether, which of course destroy their vitality,
a substance is obtained of the nature of a ferment, which, when
added to sterile urine, rapidly causes the production of ammonium
carbonate. This ferment has evidently been contained within
the bacterial cells.
In considering the effects of bacteria in nature it must be recognised
that some species are capable of building up complex substances out of
simple chemical compounds. Examples of these are found in the
bacteria which in the soil make nitrogen more available for plant nutri-
tion by converting ammonia into nitrites and nitrates. Winogradski, by
using media containing non-nitrogenous salts of magnesium, potassium,
and ammonium, and free of organic matter, has demonstrated the
existence of forms which convert, by oxidation, ammonia into nitrites,
and of other forms which convert these nitrites into nitrates. Both can
derive their necessary carbon from alkaline carbonates. Other bacteria,
or organisms allied to the bacteria, exist which can actually take up and
combine into new compounds the free nitrogen of the air. These are
found in the tubercles which develop on the rootlets of the leguminosae.
Without such organisms the tubercles do not develop, and without the
development of the tubercles the plants are poor and stunted. Bacteria
thus play an important part in the enrichment and fertilisation of the
soil.
The Occurrence of Variability among Bacteria. — The question of the
division of the group of bacteria into definite species has given rise to
much discussion among vegetable and animal morphologists, and at
one time very divergent views were held. Some even thought that the
same species might at one time give rise to one disease, — at another
time to another. There is, however, now practical unanimity that
bacteria show as distinct species as the other lower plants and animals,
though, of course, the difficulty of defining the concept of a species is as
great in them as it is in the latter. Still, we can say that among the
bacteria we see exhibited (to use the words of De Bary) "the same
periodically repeated course of development within certain empirically
determined limits of variation " which justifies, among higher forms of
life, a species to be recognised. What at first raised doubts as to the
occurrence of species among the bacteria was the observation in certain
cases of what is known as pleomorphism. By this is meant that one
species may assume at different times different forms, e.g. appear as a
coccus, a bacillus, or a leptothrix. Undoubtedly, many of the cases
where this was alleged to have been observed occurred before the
elaboration of the modern technique for the obtaining of pure cultures,
but at the present day there are cases where evidence appears to exist
24 GENERAL MORPHOLOGY AND BIOLOGY
of the occurrence of pleomorphisra. This is especially the case with
certain bacilli, and it may lead to such forms being classed among the
higher bacteria. Pleomorphism is, however, a rare condition, and with
regard to the bacteria as a whole we may say that each variety tends to
conform- to a definite type of structure and function which is peculiar to
it and to it alone. On the other hand, slight variations from such type
can occur in each. The size may vary a little with the medium in
which the organism is growing, and under certain similar conditions the
adhesion of bacteria to each other may also vary. Thus cocci, which are
ordinarily seen in short chains, may grow in long chains. The capacity
to form spores may be altered, and such properties as the elaboration
of certain ferments or of certain pigments may be impaired. Also the
characters of the growths on various media .may undergo variations.
As has been remarked, variation as observed consists largely in a tendency
in a bacterium to lose properties ordinarily possessed, and all attempts
to transform one bacterium into an apparently closely allied variety
(such as the b. coli into the b. typhosus) have failed. This of course
does not preclude the possibility of one species having been originally
derived from another or of both having descended from a common
ancestor, but we can say that only variations of an unimportant order
have been observed to take place, and here it must be remembered that
in many cases we can have forty-eight or more generations under obser-
vation within twenty- four hours.
CHAPTEE II.
METHODS OF CULTIVATION OF BACTERIA.
Introductory. — In order to study the characters of any species
of bacterium it is necessary to have it growing apart from every
other species. In the great majority of cases where bacteria
occur in nature, this condition is not fulfilled. Only in the
blood and tissues in some diseases do particular species occur
singly and alone. We usually have, therefore, to remove a
bacterium from its natural surroundings and grow it on an
artificial food medium. When we have succeeded in separating
it, and have got it to grow on a medium which suits it, we are
said to have obtained a pure culture. The recognition of
different species of bacteria depends, in fact, far more on the
characters presented by pure cultures and their behaviour in
different food media, than on microscopic examination. The
latter in most cases only enables us to refer a given bacterium
to its class. Again, in inquiring as to the possible possession
of pathogenic properties by a bacterium, the obtaining of pure
cultures is absolutely essential.
To obtain pure cultures, then, is the first requisite of
bacteriological research. Now, as bacteria are practically
omnipresent, we must first of all have means of destroying all
extraneous organisms which may be present in the food media
to be used in the vessels in which the food media are contained,
and on all instruments which are to come in contact with our
cultures. The technique of this destructive process is called
sterilisation. We must therefore study the methods of sterilisation.
The growth of bacteria in other than their natural surroundings
involves further the preparation of sterile artificial food media,
and when we have such media 'prepared we have still to look at
the technique of the separation of micro-organisms from mixtures
of these^ and the maintaining of pure cultures when the latter
have been obtained. We shall here find that different methods
25
26 METHODS OF CULTIVATION OF BACTERIA
are necessary according as we are dealing with aerobes or atiaerobes.
Each of these methods will be considered in turn.
THE METHODS OF STERILISATION.
To exclude extraneous organisms, all food materials, glass
vessels containing them, wires used in transferring bacteria from
one culture medium to another, instruments used in making
autopsies, etc., must be sterilised. These objects being so
different, various methods are necessary, but underlying these
methods is the general principle that all bacteria are destroyed
by heat. The temperature necessary varies with different
bacteria, and the vehicle of heat is also of great importance.
The two vehicles employed are hot air and hot water or steam.
The former is usually referred to as "dry heat," the latter as
"moist heat." As showing the different effects of the two
vehicles, Koch found, for instance, that the spores of bacillus
anthracis, which were killed by moist heat at 100° C., in one
hour, required three hours' dry heat at 140° C. to effect death.
Both forms of heat may be applied at different temperatures —
in the case of moist heat above 100° C., a pressure higher than
that of the atmosphere must of course be present.
A. Sterilisation by Dry Heat.
A. (1) Red Heat or Dull Red Heat.— Red heat is used for
the sterilisation of the platinum needles which, it will be found,
are so constantly in use. • A dull heat is used for cauteries, the
points of forceps, and may be used for the incidental sterilisation
of small glass objects (cover-slips, slides, occasionally when
necessary even test-tubes), care of course being taken not to
melt the glass. The heat is obtained by an ordinary Bunsen
burner.
A. (2) Sterilisation by Dry Heat in a Hot- Air Chamber.—
The chamber (Fig. 2) consists of an outer and inner case of
sheet iron. In the bottom of the outer there is a large hole.
A Bunsen is lit beneath this, and thus plays on the bottom of
the inner case, round all of the sides of which the hot air rises
and escapes through holes in the top of the outer case. A
thermometer passes down into the interior of the chamber, half-
way up which its bulb should be situated. It is found as a
matter of experience, that an exposure in such a chamber for
STERILISATION BY MOIST HEAT
27
one hour to a temperature of 170° C., is sufficient to kill all the
organisms which usually pollute
articles in a bacteriological
laboratory, though circum-
stances might arise where this
would be insufficient. This
means of sterilisation is used
for the glass flasks, test-tubes,
plates, Petri's dishes, the use of
which will be described. Such
pieces of apparatus are thus
obtained sterile and dry. It is
advisable to put glass vessels
into the chamber before heat-
ing it, and to allow them to
stand in it after sterilisation
till the temperature falls. Sud-
den heating or cooling is apt
to cause glass to crack. The
method is manifestly unsuitable
for food media.
FIG. 2. — Hot-air steriliser.
B. Sterilisation by Moist Heat.
B. (1) By Boiling. — The boiling of a liquid for five minutes
is sufficient to kill ordinary germs if no
spores be present, and this method is useful
for sterilising distilled or tap water which
may be required in various manipulations.
It is best to sterilise knives and instruments
used in autopsies by boiling in water to
which a little sodium carbonate has been
added t9 prevent rusting. ' Twenty minutes'
boiling will here be sufficient. The boiling
of any fluid at 100° C. for one and a half
hours will ensure sterilisation under almost
any circumstances.
B. (2) By Steam at 100° C.— This is by
far the most useful means of sterilisation.
It may be accomplished in an ordinary
potato steamer placed on a kitchen pot.
The apparatus ordinarily used is " Koch's
steam steriliser" (Fig. 3). This consists
FIG. 3.— Koch's steam of a tal1 metal cylinder on legs, provided
steriliser. with a lid, and covered externally by
28 METHODS OF CULTIVATION OF BACTERIA
some bad conductor of heat, such as felt or asbestos. A
perforated tin diaphragm is fitted in the interior at a little
distance above the bottom, and there is a tap at the bottom
by which water may be supplied or withdrawn. If water
to the depth of 3 inches be placed in the interior and heat
applied, it will quickly boil, and the steam streaming up will
surround any flask or other object standing on the diaphragm.
Here no evaporation takes place from any medium as it is
surrounded during sterilisation by an atmosphere saturated with
water vapour. It is convenient to have the cylinder tall enough
to hold a litre flask with a funnel 7 inches in diameter standing
in its neck. The funnel may be supported by passing its tube
through a second perforated diaphragm placed in the upper part
of the steam chamber. With such a " Koch " in the laboratory
a hot-water filter is not needed. As has been said, one and a
half hour's steaming will sterilise any medium, but in the case
of media containing gelatin such an exposure is not practic-
able, as with long boiling, gelatin tends to lose its physical
property of solidification. The method adopted in this case
is to steam for a quarter of an hour on each of three succeeding
days.
This is a modification of what is known as " Tyndall's intermittent
sterilisation." The fundamental principle of this method is that all
bacteria in a non-spored form are killed by the temperature of boiling
water, while if in a spored form they may not be thus killed. Thus by
the sterilisation on the first day all the non-spored forms are destroyed —
the spores remaining alive. During the twenty-four hours which
intervene before the next heating, these spores, being in a favourable
medium, are likely to assume the non-spored form. The next heating
kills these. In case any may still not have changed their spored form,
the process is repeated on a third day. Experience shows that usually
the medium can now be kept indefinitely in a sterile condition.
.Steam at 100° C. is therefore available for the sterilisation of
all^ ordinary media. In using the Koch's steriliser, especially
when a large bulk of medium is to be sterilised, it is best to
put the media in while the apparatus is cold, in order to make
certain that the whole of the food mass reaches the temperature
of 100° C. The period of exposure is reckoned from the time
boiling commences in the water in the steriliser. At any rate
allowance must always be made for the time required to raise
the temperature of the medium to that of the steam surrounding it.
If we wish to use such a substance as blood serum as a
medium, the albumin would be coagulated by a temperature of
100° C. Therefore other means have to be adopted in this case.
STERILISATION" BY HIGH-PRESSURE STEAM 29
°o o o o o o
B. (3) Sterilisation by Steam at High Pressure. — This is
the most rapid and effective means of sterilisation. It is effected
in an autoclave (Fig. 4). This is a gun-metal cylinder supported
in a cylindrical sheet-iron case ; its top is fastened down with
screws and nuts and is furnished with a safety valve, pressure-
gauge, and a hole for thermometer. As in the Koch's steriliser,
the contents are supported on a perforated diaphragm. The
source of heat is a large Bunsen beneath. The temperature
employed is usually 115° C. or 120° C.
To boil at 115° C., water requires a pres-
sure of about 23 Ibs. to the square inch (i.e.
8 Ibs. plus the 15 Ibs. of ordinary atmo-
spheric pressure). To boil at 120° C., a
pressure of about 30 Ibs. (i.e. 15 Ibs. plus
the usual pressure) is necessary. In such an
apparatus the desired temperature is main-
tained by adjusting the safety valve so as
to blow off at the corresponding pressure.
One exposure of media to such temperatures
for a quarter of an hour is amply sufficient
to kill all organisms or spores. Here, again,
care must be taken when gelatin is to be
sterilised. It must not be exposed to a
temperature above 105° C., and is best
sterilised by the intermittent method. Cer-
tain precautions are necessary in Using the
autoclave. In all cases it is necessary to Fia 4 Autoclave.
allow the apparatus to cool well below 1 00°
C., before opening it or allowing steam to
blow off, otherwise there will be a sudden
development of steam when the pressure is removed, and fluid
media will be blown out of the flasks. Sometimes the instrument
is not fitted with a thermometer. In this case care must be
taken to expel all the air initially present, otherwise a mixture
of air and steam being present, the pressure read off the gauge
cannot be accepted as an indication of the temperature. Further,
care must be taken to ensure the presence of a residuum of
water when steam is fully up, otherwise the steam is super-
heated, and the pressure on the gauge again does not indicate
the temperature correctly.
B. (4) Sterilisation at Low Temperatures. — Most organisms
in a non-spored form are killed, by a prolonged exposure to a
temperature of 57° C. This fact has been taken advantage of for
the sterilisation of blood serum, which will coagulate if exposed to a
rt. Safety-valve.
6. Blow-off pipe,
c. Gauge.
30 METHODS OF CULTIVATION OF BACTERIA
FIG. o. — Steriliser for blood
serum.
temperature above that point. Such a
medium is sterilised on Tyndall's prin-
ciple by exposing it for an hour at 57°
C. for eight consecutive days, it being
allowed to cool in the interval to the
room temperature. The apparatus shown
in Fig. 5 is a small hot- water jacket
heated by a Bunsen placed beneath
it, the temperature being controlled
by a gas regulator. To ensure that
the temperature all around shall be
the same, the lid also is hollow and
filled with water, and there is a special
gas burner at the side to heat it.
This is the form originally used, but
serum sterilisers are now constructed
in which the test-tubes are placed in
the sloped position, and in which
inspissation (vide p. 40) can after-
wards be performed at a higher
temperature.
THE PREPARATION OF CULTURE MEDIA.
The general principle to be observed in the artificial culture
of bacteria is that the medium used should approximate as
closely as possible to that on which the bacterium grows naturally.
In the case of pathogenic bacteria the medium therefore should
resemble the juices of the body. The serum of the blood
satisfies this condition and is often used, but its application is
limited by the difficulties in its preparation and preservation.
Other media have been found which can support the life of all
the pathogenic bacteria isolated. These consist of proteids or
carbohydrates in a fluid, semi-solid, or solid form, in a trans-
parent or opaque condition. The advantage of having a variety
of media lies in the fact that growth characters on particular
media, non-growth on some and growth on others, etc., constitute
specific differences which are valuable in the identification of
bacteria. The most commonly used media have as their basis
a watery extract of meat. Most bacteria in growing in such an
extract cause only a grey turbidity. A great advance resulted
when Koch, by adding to it gelatin, provided a transparent solid
medium in which growth characteristics of particular bacteria
PREPARATION OF MEAT EXTRACt
31
become evident. Many organisms, however, grow best at a
temperature at which this nutrient gelatin is fluid, and there-
fore another gelatinous substance called agar, which does not
melt below 98° C., was substituted. Bouillon made from meat
extract, gelatin, and agar media, and the modifications of
these, constitute the chief materials in which bacteria are
grown.
Preparation of Meat Extract.
The flesh of the ox, calf, or horse is usually employed.
Horse-flesh has the advantage of being cheaper and containing
less fat than the others ; though generally quite suitable, it has
the disadvantage for certain purposes of containing a larger
proportion of fermentable sugar. The
flesh must be freed from fat, and finely
minced. To a pound of mince add 1000
c.c. distilled water, and mix thoroughly
in a shallow dish. Set aside in a cool
place for twenty-four hours. Skim off
any fat present, removing the last traces
by stroking the surface of the fluid with
pieces of filter paper. Place a clean linen
cloth over the mouth of a large filter
funnel, and strain the fluid through it
into a flask. Pour the minced meat into
the cloth, and gathering up the edges
of the latter in the left hand, squeeze
out the juice still held back in the con-
tained meat. Finish this expression by putting the cloth and its
contents into a meat press (Fig. 6), similar to that used by
pharmacists in preparing extracts ; thus squeeze out the last drops.
The resulting sanguineous fluid contains the soluble albumins of
the meat, the soluble salts, extractives, and colouring matter,
chiefly haemoglobin. It is now boiled thoroughly for two hours,
by which process the albumins coagulable by heat are coagulated.
Strain now through a clean cloth, boil for another half hour, and
filter through white Swedish filter paper (best, C. Schleicher u.
Schull, No. 595). Make up to 1000 c.c. with distilled water.
The resulting fluid ought to be quite transparent, of a yellowish
colour without any red tint. If there is any redness, the fluid
must be reboiled and filtered till this colour disappears, other-
wise in the later stages it will become opalescent. A large
quantity of the extract may be made at a time, and what is not
FIG. 6. — Meat press.
32 METHODS OF CULTIVATION OF BACTERIA
immediately required is put into a large flask, the neck plugged
with cotton wool, and the whole sterilised by methods B
(2) or (3). This extract contains very little albuminous matter,
and consists chiefly of the soluble salts of the muscle, certain
extractives, and altered colouring matters, along with any slight
traces of soluble proteid not coagulated by heat.. It is of acid
reaction. We have now to see how, by the addition of proteid
and other matter, it may be transformed into proper culture
media.
1. Bouillon Media. — These consist of meat extract with the
addition of certain substances to render them suitable for the
growth of bacteria.
(1) (a). Peptone Broth or Bouillon. — This has the com-
position : —
Meat extract .... 1000 c.c.
Sodium chloride ... 5 grms.
Peptone albumin . . . 10 „
Boil till the ingredients are quite dissolved, and neutralise
with a saturated solution of sodium hydrate. Add the latter
drop by drop, shaking thoroughly between each drop and testing
the reaction by means of litmus paper. Go on till the reaction
is slightly but distinctly alkaline. Neutralisation must be
practised with great care, as under certain circumstances,
depending on the relative proportions of the different phosphates
of sodium and potassium, what is known as the amphoteric
reaction is obtained, i.e. red litmus is turned blue, and blue red,
by the same solution. The sodium hydrate must be added till
red litmus is turned slightly but distinctly blue, and blue litmus
is not at all tinted red. After alkalinisation, allow the fluid to
become cold, filter through- Swedish filter paper into flasks, make
up to original volume with distilled water, plug the flasks with
cotton wool, and sterilise by methods B (2) or (3), (pp. 27, 29).
This method of neutralisation is to be recommended for all
ordinary work.
In this medium the place of the original albumins of the meat is
taken by peptone, a soluble proteid not coagulated by heat. Here it
may be remarked that the commercial peptone albumin is not pure
peptone, but a mixture of albumoses (see footnote, p. 165) with a variable
amount of pure peptone. The addition of the sodium chloride is
necessitated by the fact that alkalinisation precipitates some of the
phosphates and carbonates present. Experience has shown that sodium
chloride can quite well be substituted. The reason for the alkalinisation
is that it is found that most bacteria grow best on a medium slightly
alkaline to litmus. Some, e.g. the cholera vibrio, will- not grow at all
on even a slightly acid medium.
STANDARDISING THE REACTION OF MEDIA 33
Standardisation of Reaction of Media. — While the above
procedure of dealing with the reaction of a medium is sufficient
for ordinary work, it has been thought advisable to have a more
exact method for making media to be used in growing organisms,
the growth characteristics of which are to be described for
systematic purposes. Such a method should also be used in
studying the changes in reaction produced in a medium by the
growth of bacteria. It, however, involves considerable difficulty,
and should not be undertaken by the beginner. It entails the
preparation of solutions of acid and alkali which may be used
for determining the original reaction of the medium, and for
accurately making it of a definite degree of alkalinity. Normal J
and decinormal solutions of sodium hydrate and hydrochloric
acid are used.
Preparation of Standard Solutions. — The first requisites here are
normal solutions of acid and alkali. The latter is prepared as follows :
— 85 grammes of pure sodium bicarbonate are heated to dull redness
for ten minutes in a platinum vessel and allowed to cool in an exsiccator ;
just over 54 grammes of sodium carbonate should now be present. Any
excess is quickly removed, and the rest being dissolved in one litre of
distilled water, abnormal solution is obtained. A measured quantity is
placed in a porcelain dish, and a few drops of a '5 per cent solution of
phenol- phthaleine in neutral methylated spirit is added to act as indicator.
The alkali produces in the latter a brilliant rose pink which, however,
disappears on the least excess of acid being present. The mixture is
boiled and a solution of hydrochloric acid of unknown strength is run
into the dish from a burette till the colour goes and does not return
after very thorough stirring. The strength of the acid can then be
calculated, and a normal solution can be obtained. From these two
solutions any strength of acid or alkali (such as the decinormal solution
of NaOH mentioned below) may be derived.
As Eyre has suggested, the reaction "of a medium may be
conveniently expressed by the sign + or - to indicate acid or
alkaline respectively, and a number to indicate the number of
cubic centimetres of normal acid or alkaline solution necessary
to make a litre of the medium neutral to phenol-phthaleine.
Thus, for example, "reaction = - 15," will mean that the medium
is alkaline, and requires 15 c.c. of normal HC1 to make a litre
1 A "normal" solution of any salt is prepared by dissolving an "equivalent"
weight in grammes of that salt in a litre of distilled water. If the metal of
the salt be monovalent, i.e. if it be replaceable in a compound by one atom
of hydrogen (e.g. sodium), an equivalent is the molecular weight in
grammes. In the case of NaCl, it would be 58'5 grammes (atomic weight of
Na = 23, of Cl = 35'5). If the metal be bivalent, i.e. requiring two atoms of H
for its replacement in a compound (e.g. calcium), an equivalent is the
molecular weight in grammes divided by two. Thus iu the case of CaClo an
equivalent would be 55 '5 grammes (atomic weight of Ca — 40, of Clo — 71).
3
34 METHODS OF CULTIVATION OF BACTERIA
neutral. It has been found that when a medium such as bouillon
reacts neutral to litmus, its reaction to phenol-phthaleine, accord-
ing to the above standard, is on the average + 25. Now as
litmus was originally introduced by Koch, and as nearly all
bacterial research has been done with media tested by litmus,
it is evidently difficult to say exactly what precise degree of
alkalinity is the optimum for bacterial growth. It is probably
safe to say, however, that when a medium has been rendered
neutral to phenol-phthaleine by the addition of NaOH, the
optimum degree is generally attained by the addition of from
10 to 15 c.c. of normal HC1 per litre, i.e. the optimum reaction
is •+ 10 to + 15. In other words, the optimum reaction for
bacterial growth lies, as Fuller has pointed out, about midway
between the neutral point indicated by phenol-phthaleine and
the neutral point indicated by litmus.
The only objection to the use of phenol-phthaleine is that its
action is somewhat vitiated if free CO2 be present. This can
be completely obviated as follows. Before testing any medium
it is boiled in the porcelain dish into which titration takes place.
The soda solutions are best stored in bottles such as that shown
in Fig. 44, having on the air inlet a little bottle filled with soda
lime with tubes fitted as in the large one. The C02 of the air
which passes through is thus removed.
Method. — The following procedure includes most of the
improvements introduced by Eyre. The medium with all its
constituents dissolved is filtered and then heated for about 45
minutes in the steamer, the maximum acidity being reached
after this time. Of the warm medium take 25 c.c. and put in
a porcelain dish, add 25 c.c. distilled water, and 1 c.c. phenol-
phthaleine solution. Run in decinormal soda till neutral point
is reached, indicated by the first trace of pink colour, the mixture
being kept hot.1 Repeat process thrice, and take the mean ;
this divided by 10 will give the amount (x) of normal soda
required to neutralise 25 c.c. of medium ; then 40 x = amount
necessary to neutralise a litre; and 40 #-10 = amount of
normal soda necessary to give a litre its optimum reaction.
Then measure the amount of medium to be dealt with, and add
the requisite amount of soda solution.
1 The beginner may find considerable difficulty in recognising the first
tint of pink in the yellow bouillon. A good way of getting over this is to
take two samples of the medium, adding the indicator to one only ; then to
run the soda into these from separate burettes ; for each few drops run into
the medium containing the indicator the same amount is run into the other.
Thus the recognition of the first permanent change in tint will be at once
recognised by comparing the two lots of solution.
GELATIN MEDIA
35
Eyre uses a soda solution of ten times normal strength, which
is delivered out of a 1 c.c. pipette divided into hundred ths ; this
obviates, to a large extent, the error introduced by increasing
the bulk of the medium on the addition of the neutralising
solution.
1 (b). Glucose Broth. — To the other constituents of 1 (a)
there is added 1 or 2 per cent of grape sugar. The steps in the
preparation are the same. Glucose being a reducing agent,
no free oxygen can exist in a medium containing it, and there-
fore glucose broth is used as a culture fluid for anaerobic
organisms.
1 (c). Glycerin Broth. — The initial steps are the same as in
1 (a), but after filtration 6 to 8 per cent of glycerin (sp. grav. 1 *25)
is added. This medium is especially used for growing the
tubercle bacillus when the soluble products of the growth of the
latter are required.
2. Gelatin Media. — These are simply the above broths, with
gelatin added as a solidifying body.
2 (a). Peptone Gelatin : —
Meat extract
Sodium chloride
Peptone albumin
Gelatin
. 1000 c.c.
5 grms.
10 „
100-150
(the "gold label" gelatin of Coignet et Cie, Paris, is the best).
The gelatin is cut into small pieces, and added with the other
constituents to the extract ; they are
then thoroughly melted on a sand bath,
or in the " Koch." The fluid medium
is then rendered slightly alkaline, as
in 1 (a), and filtered through filter
paper. As the medium must not be
allowed to solidify during the process,
it must be kept warm. This is effected
by putting the flask and funnel into a
tall Koch's steriliser, in which case
the funnel must be supported on a
tripod or diaphragm, as there is great
danger of the neck of the flask breaking
if it has to support the funnel and
its contents. The filtration may also
be carried out in a funnel with water-
jacket which is heated, as shown in Fig. 7. Whichever instrument
be used, before filtering shake up the melted medium, as it is apt
FIG. 7. — Hot-water funnel.
36 METHODS OF CULTIVATION OF BACTERIA
while melting to have settled into layers of different density.
Sometimes what first comes through is turbid. If so, replace it
in the unfiltered part : often the subsequent filtrate in such cir-
cumstances is quite clear. A litre flask of the finished product
ought to be quite transparent. If, however, it is partially opaque,
add the white of an egg, shake up well, and boil thoroughly over
the sand bath. The consequent coagulation of the albumin carries
down the opalescent material, and on making up with distilled
water to the original quantity and refiltering, it will be found to
be clear. The flask containing it is then plugged with cotton
wool and sterilised, best by method B (2), p. 27. If the
autoclave be used the temperature employed must not be above
105° C., and exposure not more than a quarter of an hour on
three successive days. Too much boiling, or boiling at too high
a temperature, as has been said, causes a gelatin medium to
lose its property of solidification. The exact percentage of
gelatin used in its preparation depends on the temperature at
which growth is to take place. Its firmness is its most valuable
characteristic, and to maintain this in summer weather, 15 parts
per 100 are necessary. A limit is placed on higher percentages
by the fact that, if the gelatin be too stiff, it will split on the
perforation of its substance by the platinum needle used in
inoculating it with a bacterial growth ; 15 per cent gelatin melts
at about 24° C.
2 (&). Glucose Gelatin. — The constituents are the same as
2 (a), with the addition of 1 to 2 per cent of grape sugar. The
method of preparation is identical. This medium is used for
growing anaerobic organisms at the ordinary temperatures.
3. Agar Media (French, "gelose"). — The disadvantage of
gelatin is that at the blood temperature (38° C.), at which most
pathogenic organisms grow best, it is liquid. To get a medium
which will be solid at this temperature, agar is used as the
stiffening agent instead of gelatin. Unlike the latter, which is
a proteid, agar is a carbohydrate. It is derived from the stems of
various sea-weeds growing in the Chinese seas, popularly classed
together as " Ceylon Moss." For bacteriological purposes the dried
stems of the seaweed may be used, but there is in the market a
purified product in the form of a powder ; this is preferable.
3 (a). " Ordinary " Agar. — This has the following composi-
tion : —
Meat extract . . . . . . 1000 c.c.
Sodium chloride . . . . * 5 grms.
Peptone albumin . l. . . 10 ,,
Agar 15 „
AGAR MEDIA 37
Cut up the agar into very fine fragments (in fact till it is as
nearly as possible dust), add to the meat extract with the other
ingredients, and preferably allow to stand all night. Then boil
gently in a water bath for two or three hours, till the agar is
thoroughly melted. The process of melting may be hastened
by boiling the medium in a sand bath and passing through it a
stream of steam generated in another flask ; the steam is led
from the second flask by a bent glass tube passing from just
beneath the cork to beneath the surface of the medium (Eyre).
After melting render slightly alkaline with sodium hydrate
solution, make up to original volume with distilled water, and
filter. Filtration here is a very slow process and is best carried
out in a tall Koch's steriliser. In doing this, it is well to put a
glass plate over the filter funnel to prevent condensation water
from dropping off the roof of the steriliser into the medium. If
a slight degree of turbidity may be tolerated, it is sufficient to
filter through a felt bag or jelly strainer. Plug the flask con-
taining the filtrate, and sterilise either in autoclave for fifteen
minute's or in Koch's steriliser for one and a half hours. Agar
melts just below 100° C., and on cooling solidifies about 39° C.
3 (6). Glycerin Agar.— To 3 (a) after filtration add 6 to 8
per cent of glycerin and sterilise as above. This is used
especially for growing the tubercle bacillus.
3 (c). Glucose Agar. — Prepare as in 3 (a), but add 1 to 2
per cent of grape sugar along with agar. This medium is used
for the culture of anaerobic organisms at temperatures above the
melting-point of gelatin. It is also a superior culture medium
for some aerobes, e,g. the b. diphtherise.
These bouillon, gelatin, and agar preparations constitute
the most frequently used media. Growths in bouillon do not
usually show any characteristic appearances which facilitate
classification, but such a medium is of great use in investigating
the soluble toxic products of bacteria. The most characteristic
developments of organisms take place on the gelatin media.
These have, however, the disadvantage of not being available
when growth is to take place at any temperature above 24° C.
For higher temperatures agar must be employed. Agar is, how-
ever, never so transparent. Though quite clear when fluid, on
solidifying it always becomes slightly opaque. Further, growths
upon it are never so characteristic as those on gelatin. It is,
for instance, never liquefied, whereas some organisms, by their
growth, liquefy gelatin and others do not — a fact of prime
importance.
Litmus Media. — To any of the above media litmus (French,
38 METHODS OF CULTIVATION OF BACTERIA
tournesol) may be added to show change in reaction during
bacterial growth. The litmus is added, before sterilisation, as
a strong watery solution (e.g. the Kubel-Tiemann solution, vide
p. 42) in sufficient quantity to give the medium a distinctly
bluish tint. During the development of an acid reaction the
colour changes to a pink and may subsequently be discharged.
Use of neutral red. — This dye has been introduced as an aid
in determining the presence or absence of members of the b. coli
group, especially in the examination of water. The media found
most suitable are agar or bouillon containing '5 per cent of
glucose, to which *5 per cent of a one per cent watery solution
of neutral red is added. The use of these media and their
probable value are described below (vide Typhoid Fever).
Blood Agar : Serum Agar. — The former medium was intro-
duced by Pfeiffer for growing the influenza bacillus, and it
has been used for the organisms which are not easily grown on
the ordinary media, e.g. the gonococcus and the pneumococcus.
Human blood or the blood of animals may be used. " Sloped
tubes " (vide p. 48) of agar are employed (glycerin agar is' not so
suitable). Purify a finger first with 1-1000 corrosive sublimate,
dry, and then wash with absolute alcohol to remove the sublimate.
Allow the alcohol to evaporate. Prick with a needle sterilised
by heat, and, catching a drop of blood in the loop of a sterile
platinum wire (vide p. 49), smear it on the surface of the agar.
The excess of the blood runs down and leaves a film on the
surface. Cover the tubes with india-rubber caps, and incubate
them for one or two days at 38° C. before use, to make certain
that they are sterile. Agar poured out in a thin layer in a
Petri dish may be smeared with blood in the same way and
used for cultures. A medium composed of one part of fresh
blood (drawn aseptically) and two parts of fluid agar at 40° C.,
has been used for the cultivation of the bacillus of soft sore.
Serum agar is prepared in a similar way by smearing the
surface of the agar with blood serum, or by adding a few drops
of serum to the tube and then allowing it to flow over the
surface.
Peptone Solution.
V A simple solution of peptone (Witte) constitutes a suitable
culture medium for many bacteria, The peptone in the propor-
tion of 1 to 2 per cent, along with '5 per cent NaCl, is dissolved
in distilled water by heating. The fluid is then filtered, placed
in tubes and sterilised. The reaction is usually distinctly
alkaline, which condition is suitable for most purposes. For
BLOOD SERUM 39
special purposes the reaction may be standardised. In such a
solution the cholera vibrio grows with remarkable rapidity. It
is also much used for testing the formation of indol by a
particular bacterium ; and by the addition of one of the sugars
to it the fermentative powers of an organism may be tested
(p. 75). Litmus may be added to show any change in reaction.
Blood Serum.
Koch introduced this medium, and it is prepared as follows :
Plug the mouth of a tall cylindrical glass vessel (say of 1000 c.c.
capacity) with cotton wool, and sterilise by steaming it in a
Koch's steriliser for one and a half hours. Take it to the place
where a horse, ox, or sheep is to be killed. When the artery
or vein of the animal is opened, allow the first blood which
flows, and which may be contaminated from the hair, etc., to
escape ; fill the vessel with the blood subsequently shed. Carry
carefully back to the laboratory without shaking, and place for
twenty-four hours in a cool place, preferably an ice chest. The
clear serum will separate from the clotted blood. If a centrifuge
is available, a large yield of serum may be obtained by centri-
fugalising the freshly drawn blood. If coagulation has occurred,
the clot must first be thoroughly broken up. With a sterile 10
c.c. pipette, transfer this quantity of serum to each of a series
of test-tubes which must previously have been sterilised by dry
heat. The serum may, with all precautions, have been con-
taminated during the manipulations, and must be sterilised.
As it will coagulate if heated above 68° C., advantage must be
taken of the intermittent process of sterilisation at 57° C.
[method B (4)]. It is therefore kept for one hour at this
temperature on each of eight successive days. It is always
well to incubate it for a day at 37° C. before use, to see that
the result is successful. After sterilisation it is "inspissated,"
by which process a clear solid medium is obtained. " Inspissa-
tion " is probably an initial stage of coagulation, and is effected
by keeping the serum at 65° C. till it stiffens. This temperature
is just below the coagulation point of the serum. The more
slowly the operation is performed the clearer will be the serum.
The apparatus used for the purpose is one of the various forms
of serum steriliser (e.g. Fig. 8), generally a chamber with water-
jacket heated with a Bunsen below. The temperature is con-
trolled by a gas regulator, and such an apparatus can, by altering
the temperature, be used either for sterilisation or inspissation.
As is evident, the preparation of this medium is tedious, but its
40 METHODS OF CULTIVATION OF BACTERIA
use is necessary for the observation of particular characteristics
in several pathogenic bacteria, notably the tubercle bacillus.
Pleuritic and other effusions may be prepared in the same way,
and used as media, but care must be taken in their use, as we
have no right to say that pathological effusions have the same
chemical composition as
normal serum.
If blood be collected
with strict aseptic precau-
tions, then sterilisation of
the serum is unnecessary.
To this end the mouth of
the cylinder used for col-
lecting the blood, instead
of being plugged with wool,
has an india-rubber bung
inserted in it through
which two bent glass tubes
pass. The outer end of
one of these is of conveni-
ent length, and, before
sterilisation, a large cap of
cotton wool is tied over it ;
the other tube is plugged
with a piece of cotton wool.
In the slaughter-house the
cap is removed and the
tube is inserted into the
blood-vessel as a cannula.
The cylinder is thus easily
filled. Another method is
to conduct the blood to
the cylinder by means of
a sterilised cannula and
india-rubber tube, the
former being inserted in
the blood-vessel. In every case the serum must be incubated
before use, to make sure that it is sterile.
Coagulated Blood Serum. — If fresh serum be placed in
sterile tubes and be steamed in the sloped position for an hour
it coagulates, and there is thus obtained a solid medium very
useful for the growth of the diphtheria bacillus for diagnostic
purposes.
Loffler's Blood Serum. — This is the best medium for the
FIG. 8. — Blood serum inspissator.
BLOOD SERUM 41
growth of the b. diphtheriae and may be used for other
organisms. It has the following composition. Three parts of
calf's or lamb's blood serum are mixed with one part ordinary
neutral peptone bouillon made from veal with 1 per cent of grape
sugar added to it. Though this is the original formula it can
be made from ox or sheep serum and beef bouillon without its
qualities being markedly impaired. Sterilise by method B (4)
as above (p. 29).
Alkaline Blood Serum (Lorrain Smith's Method). — To each
100 c.c. of the serum obtained as before, add 1 to 1*5 c.c. of a
10 per cent solution of sodium hydrate and shake gently. Put
sufficient of the mixture into each of a series of test-tubes, and
laying them on their sides, sterilise by method B (2). If the
process of sterilisation be carried out too quickly, bubbles of gas
are apt to form before the serum is solid, and these interfere with
the usefulness of the medium. Dr. Lorrain Smith informs us
that this can be obviated if the serum be solidified high up in the
Koch's steriliser, in which the water is allowed only to simmer.
In this case sterilisation ought to go on for one and a half hours.
A clear solid medium (consisting practically of alkali-albumin) is
thus obtained, and he has found it of value for the growth of the
organisms for which Koch's serum is used, and especially for the
growth of the b. diphtherise. Its great advantage is that aseptic
precautions in obtaining blood from the animal are not necessary,
and it is easily sterilised.
Marmorek's Serum Media. — There has always been a diffi-
culty in maintaining the virulence of cultures of the pyogenic
streptococci, but Marmorek has succeeded in doing so by growing
them on the following media, which are arranged in the order of
their utility : — ,
1. Human serum 2 parts, bouillon 1 part.
2. Pleuritic or ascitic serum 1 part, bouillon 2 parts.
3. Asses' or mules' serum 2 parts, bouillon 1 part.
4. Horse serum 2 parts, bouillon 1 part.
Human serum can be obtained from the blood shed in
venesection, the usual aseptic precautions" being taken. In the
case of these media, sterilisation is effected by method B (4), and
they are used fluid.
Hiss's Serum Water Media. — These are composed of one
part of ox's serum and three parts of distilled water with 1
per cent litmus ; various sugars in a pure condition are added in
the proportion of 1 per cent. The development of acid by
fermentation is shown by the alteration of the colour and by
42 METHODS OF CULTIVATION OF BACTERIA
coagulation of the medium. These media do not coagulate at
100° C. and thus can be sterilised in the steam steriliser.
They have been extensively used by American workers in
studying the fermentative properties of the b. dysenteriae,
b. coli, etc.
Drigalski and Conradi's Medium. — This is one of the media used for
the study of intestinal bacteria and especially for the isolation of the
typhoid group of organisms, (a) Three pounds of meat are treated with
two litres of water overnight ; the fluid is separated as usual, boiled
for an hour, filtered, and there are added 20 grammes Witte's peptone,
20 grammes nutrose,1 10 grammes sodium chloride ; the mixture is then
boiled for an hour, 60 grammes finest agar are added, and it is placed in
the autoclave till melted (usually one hour) ; it is then rendered slightly
alkaline to litmus, filtered, and boiled for half an hour, (b) 260 c.c. Kubel-
Tiemann litmus 2 solution is boiled for ten minutes, 30 grammes milk
sugar (chemically pure) are added, and the mixture is boiled for fifteen
minutes ; (a) and (b) are then mixed hot, well shaken, and, if necessary,
the slightly alkaline reaction restored. There are then added 4 c.c. of
a 10 per cent sterile solution of water-free sodium hydrate and 20 c.c. of
a freshly prepared solution made by dissolving '1 gramme crystal- violet
B, Hoechst, in 100 c.c. hot sterile distilled water. This is the finished
medium, and great care must be taken not to overheat it or to heat it too
long, "as changes in the lactose may be originated. It is convenient to
distribute the medium in 80 c.c. flasks.
The principle of the medium is that while there is a food supply very
favourable to the b. typhosus and the b. coli the antiseptic action of the
crystal-violet tends to inhibit the growth of other bacteria likely to
occur in material which has been subjected to intestinal contamination.
In examining faeces a little is rubbed up in from ten to twenty times its
volume of sterile normal salt solution ; in the case of urine or water the
fluid is centrifugalised and the deposit or lower portion is used for the
inoculation procedures.
For use the medium is distributed in Petri capsules in a rather thicker
layer than is customary in an ordinary plate. The sheet of medium
must be transparent, but must not be less than 2 mm. in thickness —
in fact, ought to be about 4 mhi. After being poured, the capsules are
left with the covers off for an hour or so, to allow the superficial
layers of the medium to become set hard. The effect of this is
that during incubation no water of condensation forms on the lid
of the capsule, and thus the danger of this fluid dropping on to the
developing colonies is avoided. The antiseptic nature of the crystal-
violet is sufficient to prevent the growth of any aerial organisms falling
1 Nutrose is an alkaline preparation of casein.
2 The litmus solution is made as follows. Solid commercial litmus is
digested with pure spirit at 30° C. till on adding fresh alcohol the latter
becomes only of a light violet colour. A saturated solution of the residue is
then made in distilled water and filtered. When this is diluted with a little
distilled water it is of a violet colour, which further dilution turns to a pure
blue. To such a blue solution very weak -sulphuric acid (made by adding
two drops of dilute sulphuric acid to 200 c.c. water) is added till the blue
colour is turned to a wine-red. Then the saturated solution of the dye is added
till the blue colour returns.
MACCONKEY'S BILE-SALT MEDIA 43
on the agar during its exposure to the air. The plates are usually
inoculated by means of a glass spatula made by bending three inches of a
piece of glass rod at right angles to the rest of the rod. This part is
dipped in the infective material and smeared in all directions over the
surfaces of three or four plates successively without any intervening
sterilisation. The plates are again exposed to the air after inoculation
for half an hour and then incubated for twenty-four hours. At the end
of such a period b. coli colonies are 2 to 6 mm. in diameter, stained
distinctly red, and are non-transparent. Colonies of the b. typhosus are
seldom larger "than 2 mm., they are blue or bluish-violet in colour, are
glassy and dew-like in character, and have a single contour. Sometimes
in the plates b. subtilis and its congeners appear, and colonies of these
organisms have a blue colour. Their growth is, however, more exuberant
than that of the typhoid bacillus, — being often heaped up in the centre,
— and the contour of the colony is often double.
MacConkey's Bile-Salt Media. — These media were introduced for the
purpose of differentiating the intestinal bacteria, and have been exten-
sively used for the study of the b. coli, b. typhosus, b. dysenterise, etc.
The characteristic ingredients are bile salts and various sugars. The
stock solution is the following : — Commercial sodium taurocholate, 0 '5
gramme ; Witte's peptone, 2'0 grammes ; distilled water, 100 c.c.
For a liquid medium there is added to this "5 per cent of a freshly
prepared 1 per cent solution of neutral red l and the sugar, — when
glucose is used 0'5 per cent is added, in the case of other sugars 1
per cent. The fluid is distributed in Durham's fermentation tubes and
sterilised in the steamer for ten minutes on two successive days, care
being taken not to overheat the medium.
For bile-salt agar 1*5 to 2 per cent agar is dissolved in the stock
solution in the autoclave, if necessary cleared with white of egg and
filtered. Neutral red and a sugar are added, as in the case of the liquid
medium. As with Drigalski's medium, it is well to sterilise it in flasks
containing 80 c.c., this being an amount sufficient for three Petri
capsules. When this medium is used for examining urine or feces,
plates are inoculated as with Drigalski's medium (supra} ; for its use in
water examinations see p. 136.
In the bile-salt bouillon the formation of both acid and gas is
observed if such formation occurs, and in the bile-salt agar acid produc-
tion is recognised by the red colour of the colonies of the acid-producing
organisms.
MacConkey's original medium was a 1 per cent bile-salt lactose agar
with no indicator, and was used for the detection of intestinal bacteria
in water. Such a medium is unfavourable to all the common spore-
bearing organisms found in water, and by incubating at 42° C. tubes, in
which there is probably a mixed infection from such a source, the growth
of most other water bacteria is inhibited. B. coli and b. typhosus, on
the other hand, grow readily. "With the former the surface colonies are
broad, irregular, and flat, of opaque colour, and with a small spot of
yellow or orange in the centre, and the colony is surrounded by a haze ;
the deep colonies are lens-shaped, of orange colour, and are likewise
surrounded by a haze. With the typhoid organism at the end of forty-eight
hours the surface colonies are small, round, raised, and semi-transparent,
while the deep colonies are lens-shaped, white, and have no surrounding
1 Neutral red gives a deep crimson with acids and a yellow -red with
alkalies.
44 METHODS OF CULTIVATION OF BACTERIA
haze. The haze in the case of b. coli is due to the ready production of
acid from the lactose causing a precipitate of the taurocholate. Any other
organism capable of producing acid from lactose will give a similar
reaction, and the haze can be readily cleared up by floating a drop of
ammonia on the surface of the medium. MacConkey also used with a
similar object a 5 per cent glucose bile-salt bouillon tinted with neutral
litmus as in Drigalski's medium.
With reference to MacConkey's fluid media, organisms are divided into
(1) those which produce both acid and gas ; (2) those producing acid only ;
(3) those growing but not producing either acid or gas ; (4) those
incapable of growing. B. coli belongs to the first group and b. typhosus
to the second, and to these groups also belong most ordinary organisms
growing in faeces, practically none of which are found in the third and
fourth classes. Thus if any growth takes place on this medium when
inoculated with, say, water, the probability is that the bacteria have been
derived from faeces, but of course their identification might present some
difficulty. With the neutral-red solid media the colonies of any organism
giving rise to acid will be of a beautiful rose red colour.
Petruschky's Litmus Whey. — The preparation of this medium, which
is somewhat difficult, is as follows : — Fresh milk is slightly warmed,
and sufficient very dilute hydrochloric acid is added to cause precipita-
tion of the casein, which is now filtered off. Dilute sodium hydrate
solution is added up to, but not beyond, the point of neutralisation, and
the fluid steamed for one to two hours, by which procedure any casein
which has been converted into acid albumin by the hydrochloric acid
is precipitated. This is filtered off, and a clear, colourless, perfectly
neutral fluid should result. Its chief constituent, of course, will be
lactose. To this sufficient Kubel-Tiemann solution of litmus is added,
the medium is put into tubes and then sterilised. After growth has
taken place, the amount of acid formed can be estimated by dropping
in standardised soda solution till the tint of an uninoculated tube is
reached.
Media for growing Trichophyta, Moulds, etc. — 1. Beer Wort Agar.
Take beer wort as obtainable from the brewery and dilute it till it has an
s.g. of 1100. Add 1'5 per cent of powdered agar and heat in the Koch
till it is dissolved (usually about two hours are necessary). Filter
rapidly and fill into tubes. Sterilise in the Koch for twenty minutes on
three successive days. If the medium is heated too long it loses the
capacity of solidifying.
2. Sabouraud's medium (modified). Take 40 grammes maltose and
10 grammes Witte's peptone and dissolve these in one litre of water, then
add 13 grammes of powdered agar. Heat in the Koch till the agar is
dissolved, filter and fill into tubes, sterilise in the autoclave for twenty
minutes at 120° C.
To use these for isolating, say, the Tinea tonsurans, pick out an
infected hair, wash in absolute alcohol for a few seconds, then wash in
changes of sterile water and stab the hair into the surface of the medium
in a number of places ; incubate at 24° C. Usually it is sufficient to
stab the hair as it is picked from the skin into the medium.
Potatoes as Culture Material.
(a) In Potato Jars. — The jar consists of a round, shallow,
glass vessel with a similar cover (vide Fig. 9). It is washed
POTATOES AS CULTURE MATERIAL
45
with 1-1000 corrosive sublimate, and a piece of circular filter
paper, moistened with the same, is laid in its bottom. On this
latter are placed four sterile watch
glasses. Two firm, healthy, small,
round potatoes, as free from eyes
as possible, and with the skin whole,
are scrubbed well with a brush
under the tap and steeped for two
or three hours in 1-1000 corrosive
sublimate. They are steamed in
the Koch's steriliser for thirty
minutes or longer, or in the auto-
clave for a quarter of an hour. When
cold, each is grasped between the
FIG. 9. — Potato jar.
FIG. 10.— Cylinder of potato
cut obliquely.
left thumb and forefinger
(which have been sterilised with sub-
limate) and cut through the middle with
a sterile knife. It is best to have the
cover of the jar raised by an assistant,
and to perform the cutting beneath it.
Each half is put in one of the watch
glasses, the cut surfaces,
which are then ready for /#>-,_
inoculation with a bacterial growth, being upper- m*
most. Smaller jars, each of which holds half of a
potato, are also used in the same way and are very
convenient.
(6) By Slices in Tubes. — -This method, intro-
duced by Ehrlich, is the best means of utilising
potatoes as a medium. A large, long potato is well
washed and scrubbed, and peeled with a clean knife.
A cylinder is then bored from its interior with an
apple corer or a large cork borer, and is cut obliquely,
as in Fig. 10. Two wedges are thus obtained, each
of which is placed broad end downward in a test-tube
of special form (see Fig. 11). In the wide part at
the bottom of this tube is placed a piece of cotton
wool, which catches any condensation water which
may form. The wedge rests on the constriction
above this bulbous portion. The tubes, washed,
dried, and with cotton wool in the bottom and in the
mouth, are sterilised before the slices of potato are potato.
introduced. After the latter are inserted, the tubes
are sterilised in the Koch steam steriliser for one hour, or in
the autoclave for fifteen minutes, at 115° C. An ordinary test-
FIG. 11.
— Ehrlich's
tube contain-
ing piece of
46 METHODS OF CULTIVATION OF BACTERIA
tube may be used with a piece of sterile absorbent wool in its
bottom, on which the potato may rest.
Glycerin potato, suitable for the growth of the tubercle
bacillus, may be prepared by covering the slices in the tubes
with 6 per cent solution of glycerin in water and steaming for
half an hour. The fluid is then poured off and the sterilisation
continued for another half hour.
Potatoes ought not to be prepared long before being used, as
the surface is apt to become dry and discoloured. It is well to
take the reaction of the potato with litmus before sterilisation,
as this varies ; normally in young potatoes it is weakly acid.
The reaction of the potato may be more accurately estimated by
steaming the potato slices for a quarter of an hour in a known
quantity of distilled water and then estimating the reaction of
the water by phenol-phthaleine. The required degree of acidity
or alkalinity is obtained by adding the necessary quantity of
HC1 or NaOH solution (p. 34) and steaming for other fifteen
minutes. The water is then poured off and sterilisation
continued for another half hour. Potatoes before being
inoculated ought always to be incubated at 37° C. for a night,
to make sure that their sterilisation has been successful.
Eisner's Medium. — This is one of the media introduced in the study
of the comparative reactions of the typhoid bacillus and the B. coli.
The preparation is as follows : 500 grammes potato are grated up in a
litre of water, allowed to stand over night, then strained, and added to
an equal quantity ol' ordinary 15 per cent peptone gelatin which has not
been neutralised. Normal sodium hydrate solution is added till the
reaction is feebly acid to litmus, the whole boiled together, filtered, and
sterilised. Just before use potassium iodide is added so as to constitute
one per cent of the medium. Moore has used a similar agar preparation.
Here 500 grammes potato are scraped up in one litre of water, allowed to
stand for three hours, strained, and put aside over night. The clear
fluid is poured off, made up to one litre, rendered slightly alkaline, 20
grammes agar are added, and the whole is treated as in making ordinary
agar. The medium is distributed in test-tubes — 10 c.c. to each — and
immediately before use, to each is added '5 c.c. of a solution of 10 grammes
potassium iodide to 50 c.c. water.
Milk as a Culture Medium.
This is a convenient medium for observing the effects of
bacterial growth in changing the reaction, in coagulating the
soluble albumin, and in fermenting the lactose. It is prepared
as follows : fresh milk is taken, preferably after having had the
cream " separated " by centrifugalisation, as is practised in the
best dairies, and is steamed for fifteen minutes in the Koch, it
is then set aside in an ice chest or cool place over night to
THE USE OF THE CULTURE MEDIA 47
facilitate further separation of cream. The milk is siphoned off
from beneath the cream. The reaction of fresh milk is alkaline.
If great accuracy is necessary any required degree of reaction
may be obtained by the titration method. It is then placed in
tubes and sterilised by methods B (2) or B (3).
Bread Paste.
This is useful for growing torulse, moulds, etc. Some
ordinary bread is cut into slices, and then dried in an oven till
it is so dry that it can be pounded to a fine powder in a mortar,
or rubbed down with the fingers and passed through a sieve.
Some 100 c.c. flasks are washed, dried, and sterilised, and a
layer of the powder half an inch thick placed on the bottom.
Distilled water, sufficient to cover the whole of it, is then run in
with a pipette held close to the surface of the bread, and, the
cotton-wool plugs being replaced, the flasks are sterilised in the
Koch's steriliser by method B (2). The reaction is slightly acid.
THE USE OF THE CULTURE MEDIA.
The culture of bacteria is usually carried on in test-tubes
conveniently 6 x f in. These ought to be very thoroughly
washed and dripped, and their mouths plugged with plain
cotton wool. They are then sterilised for one hour at 170° C.
If the tubes be new, the glass, being usually packed in straw,
may be contaminated with the extremely resisting spores of
the b. subtilis. Cotton -wool plugs are universally used for
protecting the sterile contents of flasks and tubes from con-
tamination with the bacteria of the air. A medium thus
protected will remain sterile for years. Whenever a protecting
plug is removed for even a short time, the sterility of the
contents may be endangered. It is well to place the bouillon,
gelatin, and agar media in the test-tubes directly after filtration.
The media can then be sterilised in the test-tubes.
In filling tubes, care must be taken to run the liquid down
the centre, so that none of it drops on the inside of the upper
part of the tube with which the cotton-wool plug will be in
contact, otherwise the latter will subsequently stick to
the glass and its removal will be difficult. In the case of
liquid media, test-tubes are filled about one-third full. With
the solid media the amount varies. In the case of gelatin
media, tubes filled one -third full and allowed to solidify
while standing upright, are those commonly used. With
48 METHODS OF CULTIVATION OF BACTERIA
organisms needing an abundant supply of oxygen the best
growth takes place on the surface of the medium, and for
practical purposes the surface ought thus to be as large as
possible. To this end "sloped" agar and gelatin tubes are
used. To prepare these, tubes are filled only about one-sixth
full, and after sterilisation are allowed to solidify, lying on their
sides with their necks supported so that the contents extend 3
b c
FIG. 13.— Tubes of media.
- tube-
maybe used for filling tubes. c< « Deep " tube for cultures of anaerobes.
The apparatus explains itself.
The india-rubber stopper with
its tubes ought to be sterilised
before use.
to 4 inches up, giving an oblique surface after solidification.
Thus agar is commonly used in such tubes (less frequently
gelatin is also " sloped "), and this is the position in which blood
serum is inspissated. Tubes, especially those of the less commonly
used media, should be placed in large jars provided with stoppers,
otherwise the contents are apt to evaporate. A tube of medium
which has been inoculated with a bacterium, and on which
growth has taken place, is called a " culture." A " pure culture "
is one in which only one orgc\nism is present. The methods of
USE OF CULTURE MEDIA 49
\
obtaining pure cultures will presently be described. When a
fresh tube of medium is inoculated from an already existing
culture, the resulting growth is said to be a " sub-culture " of the
, first. All manipulations involving the transference of small
portions of growth either from one medium to another, as in the
inoculation of tubes, or, as will be seen later, to cover-glasses for
microscopic examination, are effected by pieces of platinum wire
(Nos. 24 or 27 Birmingham wire gauge — the former being the
thicker) fixed in glass rods 8 inches long.1 Every worker should
have three such wires. Two are 2^ inches long, one of these
being straight (Fig. 14, a), and the other having a loop turned
upon it (Fig. 14, 6). The latter is referred to as the platinum
"loop." or platinum " eyelet," and is used for many purposes.
i
FIG. 14. — Platinum wires in glass handles.
die for ordinary puncture inoculations, b. " I
c. Long needle for inoculating " deep " tubes.
a. Straight needle for ordinary puncture inoculations, b. " Platinum loop."
c. Long needle for
" Taking a loopf ul " is a phrase constantly used. The third wire
(Fig. 14, c) ought to be 4| inches long and straight. It is used
for making anaerobic cultures. It is also very useful to have
at hand a platinum-indium spud. This- consists of a piece of
platinum-iridium about 1^ inches long, 2 mm. broad, and of
sufficient thickness to give it a firm consistence ; its distal end is
expanded into a diamond shape, and its proximal is screwed
into an aluminium rod. It is very useful for making scrapings
from organs and for disintegrating felted bacterial cultures ; in
such manipulations the ordinary platinum wire is awkward to
work with as it bends so easily. Cultures on a solid medium
are referred to (1) as "puncture" or "stab" cultures (German,
Stichkultur), or (2) as "stroke" or "slant" cultures (Strichkultur),
according as they are made (1) on tubes solidified in the upright
position, or (2) on sloped tubes.
1 Aluminium rods are made which are very convenient. The end is split
with a knife, the platinum wire is inserted and fixed by pinching the
aluminium on it in a vice.
50 METHODS OF CULTIVATION OF BACTERIA
FIG. 15. — Another method of inoculating
solid tubes.
To inoculate, say, one ordinary upright gelatin tube from
another, the two tubes are held in an inverted position between
the forefinger and thumb of the left hand with their mouths
towards the person holding them ; the plugs are twisted round
once or twice, to make
sure they are not adher-
ing to the glass. The
short, straight platinum
wire is then heated to
redness from point to
insertion, and 2 to 3
inches of the glass rod
are also passed two or
three times through the
Bunsen flame. It is held
between the right fore
and middle fingers, with
the needle projecting
backwards, i.e. away
from the right palm.
Remove plug from cul-
ture tube with right forefinger and thumb, and continue to hold
it between the same fingers, by the part which projected beyond
the mouth of the tube. Now touch the culture with the platinum
needle, and, withdrawing it, replace plug. In the same way
remove plug from tube to be inoculated, and plunge platinum
wire down the centre of the
gelatin to within half an inch
of the bottom. It must on
no account touch the glass
above the medium. The wire
is then immediately sterilised.
A variation in detail of this
method is to hold the plug
of the tube next the thumb
between the fore and middle
fingers, and the plug of the FJG- 16.— Rack for platinum needles,
other between the middle and
ring fingers, then to make the inoculation (Fig. 15). If a tube
contain a liquid medium, it must be held in a sloping position
between the same fingers, as above. For a stroke culture the
platinum loop is used, and a little of the culture is smeared in
a line along the surface of the medium from below upwards. In
inoculating tubes, it is always well, on removing the plugs, to
SEPARATION OF BACTERIA 51
make sure that no strands of cotton fibre are adhering to the inside
of the necks. As these might be touched with the charged needle
and the plug thus be contaminated, they must be removed by heat-
ing the inoculating needle red-hot and scorching them off with it.
When the platinum wires are not in use they may be laid in a
rack made by bending up the ends of a piece of tin, as in
Fig. 16. To prevent contamination of cultures by bacteria
falling on the plugs while these are exposed to the air during
inoculation manipulations, some bacteriologists singe the plugs
in the flame before replacing. This is, however, in most cases a
needless precaution. If the top of a plug be dusty it is best to
singe it before extraction.
THE METHODS OF THE SEPARATION OF AEROBIC ORGANISMS.
PLATE CULTURES.
The general principle underlying the methods of separation
is the distribution of the bacteria in one of the solid media
liquefied by heat and the dilution of the mixture so that the
growths produced by the individual bacteria — called colonies —
shall be suitably apart. In order to render the colonies easily
accessible, the medium is made to solidify in as thin a layer as
possible, by being poured out on glass plates — hence the term
"plate cultures."
As the optimum temperature varies with different bacteria,
it is necessary to use both gelatin and agar media. Many
pathogenic organisms, e.g. pneumococcus, b. diphtheriae, etc.,
grow too slowly on gelatin to allow its ready use. On the other
hand, many organisms, e.g. some occurring in water, do not
develop on agar incubated at 37° C.
Separation by Gelatin Media. — As the naked-eye and micro-
scopic appearances of colonies are often very characteristic,
plate cultures, besides use in separ-
ation, are often taken advantage of
in the description of individual
organisms. The plate - culture
method can also be used to test
whether a tube culture is or is not
pure. The suspected culture is
plated (three plates being pre-
pared, as will be described). If FIG. 17.— Petri's capsule,
all the colonies are the same, then (Cover shown partially raised.)
the cultures may be held to be pure.
Either simple plates of glass 4 inches by 3 inches are used,
52 METHODS OF CULTIVATION OF BACTERIA
or, what are more convenient, circular glass cells with similar
overlapping covers. The latter are known as Petri's dishes or
capsules (Fig. 17). They are usually 3 inches in diameter and
half an inch deep. The advantage of these is that they do not
require to be kept level by a special apparatus while the medium
is solidifying, and can be readily handled afterwards without
admitting impurities. Whether plates or capsules are used,
they are washed, dried with a clean cloth, and sterilised for one
hour in dry air at 170° C., the plates being packed in sheet-iron
boxes made for the purpose see (Fig. 18).
1. Glass Capsules. — While in certain circumstances, as when
the number of colonies has to be counted, it is best to use plates
of glass ; in the usual laboratory routine Petri's capsules are to
be preferred for the above reasons.
The contents of three gelatin tubes, marked a, &, c,1 are
liquefied by placing in a beaker of water at any temperature
between 25° C. and 38° C. Inoculate a with the bacterial
mixture. The amount of the latter to be taken varies, and can
only be regulated by experience. If the microscope shows
enormous numbers of different kinds of bacteria present, just as
much as adheres to the point of a straight platinum needle is
sufficient. If the number of bacilli is small, one to three loops
of the mixture may be transferred to the medium. Shake a
well, but not so as to cause many fine air-bubbles to form.
Transfer two loops of gelatin from a to b. Shake b and transfer
five loops to c. The plugs of the tubes are in each case replaced
and the tubes returned to the beaker. The contents of the
three tubes are then poured out into three capsules. In doing
so the plug of each tube is removed and the mouth of the tube
passed two or three times' through the Bunsen flame, the tube
being meantime rotated round a longitudinal axis. -Any organ-
isms on its rim are thus killed. The capsules are labelled and
set aside till growth takes place.
For accurate work it will be found convenient to carry out
the dilutions in definite proportions. The following is the pro-
cedure which we have found very serviceable. In a number of
small sterile test-tubes '95 c.c. sterile water is put. To the first
tube we add '05 c.c. of the bacterial mixture. The contents of
the tube are well shaken up, and the pipette is sterilised by
being washed out with boiling water. It is allowed to cool, and
•05 c.c. of fluid is transferred from the first tube to the second.
By a similar procedure '05 c.c. is transferred from the second to
1 For marking glass vessels it is convenient to use the red, blue, or yellow
oil pencils made for the purpose by Faber.
KOCH'S METHOD OF PLATE CULTURES 53
the third and so on. There is thus effected a twenty-fold
dilution in each successive tube. After these steps have been
carried out a definite amount, say, '05 c.c. is transferred from
each tube 'to a tube of melted gelatine, — the gelatine being after-
wards plated and the colonies counted when growth occurs.
The number of tubes required will vary according to the
number of bacteria in the original mixture, but usually four or
five will be sufficient. It is quite evident that this method not
only enables us to separate bacteria, but if necessary gives us a
means of estimating exactly the number in the original mixture.
The colonies appear as minute rounded points, whitish or
variously coloured. Their characters can be more minutely
studied by means of a hand-lens or by inverting the capsule on
the stage of a microscope and examining with a low power
through the bottom. From their characters, colour, shape,
contour, appearance of surface, liquefaction or non-liquefaction
of the gelatin, etc., the colonies can be classified into groups.
Further aid in the grouping of the varieties is obtained by
making film preparations and examining them microscopically.
Gelatin or agar tubes may then be inoculated from a colony of
each variety, and the growths obtained are then examined both
as to their purity and as to their special characters, with a view
to their identification (p. 115).
2. Glass Plates (Koch). — When plates of glass are to be used, an
apparatus on which they may be kept level while the medium is solidi-
fying is, as has been said, necessary. An apparatus devised by Koch is
used (Figs. 18, 19). This consists of a circular plate of glass (with the
upper surface ground, the lower polished) on which the plate used for
pouring out the medium is placed. The latter is protected from the air
during solidification by a bell jar. The circular plate and bell jar rest
on the flat rim of a circular glass trough, which is filled quite full with
a mixture of ice and water, to facilitate the lowering of the temperature
of whatever is placed beneath the bell jar. The glass trough rests on
corks on the bottom of a large circular trough, which catches any water
that may be spilled. This trough in turn rests on a wooden triangle
with a foot at each corner, the height of which can be adjusted, and
which 'thus constitutes the levelling apparatus. A spirit level is placed
where the plate is to go, and the level of the ground glass plate thus
assured. There is also prepared a "damp chamber," in which the
plates are to be stored after being made. This consists of a circular
glass trough with a similar cover. It is sterilised by being washed out-
side and inside with perchloride of mercury 1-1000, and a circle of filter-
paper moistened with the same is laid on its bottom. Glass benches on
which the plates may be laid are similarly purified.
To separate organisms by this method three tubes, a, b, c, are inocu-
lated as in using Petri's capsules (p. 52). The hands having been
washed in perchloride of mercury 1-1000 and dried, the plate box is
opened, and a plate lifted by its opposite edges and transferred to the
54 METHODS OF CULTIVATION OF BACTERIA
levelled ground glass (as in Figs. 18, 19). The bell jar of the levelle
being now lifted a little, the gelatin in tube a is poured out on thi
surface of the sterile plate, and while still fluid, is spread by stroking
FIG. 18. — Koch's levelling apparatus for use in preparing plates.
Hands shown in first position for transferring sterile plate from iron
• box to beneath bell jar, where it subsequently has the medium poured
out upon it.
with the rim of the tube. After the medium solidifies, the plate is
transferred to the moist chamber as rapidly as possible, so as to avoid
FIG. 19. — Koch's levelling apparatus. Hands shown in second
position just as the plate is lowered on to the ground glass surface.
By executing the transference of the plate from the box in this way,
the surface which was undermost in the latter is uppermost in the
leveller, and thus never meets a current of air which might con-
taminate it.
atmospheric contamination. In doing this, it is advisable to have an
assistant to raise the glass covers. Tubes b and c are similarly treated,
SEPARATION BY AGAR MEDIA
55
and the resulting plates stacked in series on the top of a. The chamber
is labelled and set aside for a few days till the colonies appear on the
gelatin plates. The further procedure is of the same nature as with
Petri's capsules.
3. EsmarcKs Roll Tubes. — Here the principle is that of
dilution as before. In each of three test-tubes 1J or 1J inch in
diameter, gelatin to the depth of f of an inch is placed. These
are sterilised. The gelatin is melted and in-
oculated in series with the bacterial mixture as
in making plate cultures, but instead of being
poured out it is rolled in a nearly horizontal
position under a cold tap or on a block of ice till
it solidifies as a uniformly thin layer on the inside
of the tube. Practically we deal with a cylindrical
sheet of gelatin instead of a flat one. A con-
venient form of tube for this method is one with
a constriction a short distance below the plug of
cotton wool (Fig. 20). The great disadvantage of
the method is, that if organisms liquefying the
gelatin be present, the liquefied gelatin contamin-
ates the rest of the medium.
Separation by Agar Media. — 1. Agar Plates.
— The only difference between the technique here
and that with gelatin depends on the difference
in the melting-points of the two media. Agar,
we have said, melts at 98° C., and becomes again
solid a little under 40° C. As it is dangerous
to expose organisms to a temperature much
above 42° C., it is necessary in preparing tubes
of agar to be used in plate cultures to first
melt the agar, by boiling in a vessel of water for a few minutes,
and then to cool them to about 42° C. before inoculating. The
manipulation must be rapidly carried out, as the margin of
time, before solidification occurs, is narrow; otherwise the
details are the same as for gelatin. Esmarch's tubes are not
suitable for use here, as the agar does not adhere well to the
sides. If to the agar 2 per cent of a strong watery solution of
pure gum arabic is added, Esmarch's tubes may, however, be
used.
2. Separation by Stroking Mixture on Surface of Agar
Media. — The bacterial mixture, instead of being mixed in the
medium, is spread out on its surface. The method may be used
both when the bacteria to be separated are in a fluid, and when
contained in a fairly solid -tissue or substance, such as a piece
FIG. 20.
Esmarch's tube
for roll culture.
56 METHODS OF CULTIVATION OF BACTERIA
of diphtheritic membrane. In the case of a tissue, for example,
a small portion entangled in the loop of a platinum needle is
stroked in successive parallel longitudinal strokes on sloped
agar, the same aspect being brought in contact with the agar in
all the strokes. Three strokes may be made on each tube, and
three tubes are usually sufficient. In this process the organisms
on the surface of the tissue are gradually rubbed off, and when
growth has taken place it will be found that in the later
strokes the colonies are less numerous than in the earlier, and
sufficiently far apart to enable parts of them to be picked off
without the needle touching any but one colony. When, as in
the case of diphtheritic membrane, putrefactive organisms may
be present on the surface of the tissue, these can be in great
part removed by washing it well in cold water previously
sterilised (vide Diphtheria). In the case of liquids, the loop is
charged and similarly stroked. Tubes thus inoculated must be
put in the incubator in the upright position and must be
handled carefully so that the condensation water, which always
is present in incubated agar tubes, may not run over the surface.
Agar, poured out in a Petri's capsule and allowed to stand till
firm, may be used instead of successive tubes. Here a sufficient
number of strokes can be made in one capsule. Sloped blood-
serum tubes may be used instead of agar. The method is rapid
and easy, and gives good results.
Separation of Pathogenic Bacteria by Inoculation of
Animals. — It is found difficult and often impossible to separate
by ordinary plate methods certain pathogenic organisms, such
as b. tuberculosis, b. mallei, and the pneumococcus, when such
occur in conjunction with other bacteria. These grow best on
special media, and the first two (especially the tubercle bacillus)
grow so slowly that the other organisms present outgrow them,
cover the whole plates, and make separation impossible. The
method adopted in such cases is to inoculate an animal with
the mixture of bacilli, wait until the particular disease develops,
kill the animal, and with all aseptic precautions (vide p. 123)
inoculate tubes of suitable media from characteristic lesions
situated away from the seat of inoculation, e.g. from spleen in
the case of b. tuberculosis, spleen or liver in the case of b.
mallei, and heart blood in the case of pneumococcus.
Separation by killing Non-spored Forms by Heat. — This is
a method which has a limited application. As has been said,
the spores of a bacterium resist heat more than the vegetative
forms. When a mixture contains spores of one bacterium and
vegetative forms of this and other bacteria, then if the mixture
SEPARATION OF ANAEROBES 57
be boiled for a few minutes all the vegetative forms will
be killed, while the spores will remain alive and will develop
subsequently. This method can be easily tested in the case of
cultivating b. subtilis from hay infusion. A little chopped-up
hay is placed in a flask of water, which is boiled for about ten
minutes. On this being allowed to cool and stand, in a day or
two a scum forms on the surface, which is found to be a pure
culture of the bacillus subtilis. The method is also often used
to aid in the separation of b. tetani, vide infra.
THE PRINCIPLES OF THE CULTURE OF ANAEROBIC
ORGANISMS.
All ordinary media, after preparation, may contain traces of
free oxygen, and will absorb more from the air on standing. (1)
For the growth of anaerobes this oxygen may be expelled by
the prolonged passing of an inert gasT such as hydrogen, through
the medium (liquefied if necessary). Further, the medium must
be kept in an atmosphere of the same gas, while growth is going
on. (2) Media for anaerobes may be kept in contact with the
air, if they contain a reducing agent which does not interfere
with bacterial growth. Such an agent takes up any oxygen
which may already be in the medium, and prevents further
absorption. The reducing body used is generally glucose, though
formate of sodium may be similarly employed. The preparation
of such media has already been described (pp. 36, 37). In this
case the medium ought to be of considerable thickness.
The Supply of Hydrogen for Anaerobic Cultures. — The gas is generated
in a large Kipp's apparatus from pure sulphuric acid and pure zinc. It
is passed through three wash-bottles, as in Fig. 21. In the first is
placed a solution of lead acetate (1 in 10 of water) to remove any traces
of sulphuretted hydrogen. In the second is placed a 1 in 10 solution of
silver nitrate to remove any arsenietted hydrogen which may be present
if the zinc is not quite pure. In the third is a 10 per cent solution
of pyrogallic acid in caustic potash solution (1 : 10) to remove any
traces of oxygen. The tube leading from the last bottle to the vessel
containing the medium ought to be sterilised by passing through a
Bunsen flame, and should have a small plug of cotton wool in it to filter
the hydrogen germ-free.
Separation of Anaerobic Organisms. — (a) By Roll-tubes. —
A 1J inch test-tube has as much gelatin put into it as would be
used in the Esmarch roll-tube method. It is corked with an
india-rubber stopper having two tubes passing through it, as in
Fig. 22. The ends of the tubes are partly drawn out as shown,
58 METHODS OF CULTIVATION OF BACTERIA
and covered with plugs of cotton wool. Three such test-tubes
are prepared, and they are sterilised in the steam steriliser (p. 27).
After sterilisation the gelatin is melted and one tube inoculated
with the mixture containing the anaerobes ; the second is inocu-
lated from the first, and the third from the second, as in making
ordinary gelatin plates. After inoculation the gelatin is kept
liquid by the lower ends of the tubes being placed in water at
about 30° C., and hydrogen is passed in through tube x for
twenty minutes. The gas -supply tubes are then completely
sealed off at x and it and each test-tube is rolled as in Esmarch's
method till the gelatin solidifies as a thin layer on the internal
FIG. 21. — Apparatus for supplying hydrogen for anaerobic cultures.
a. Kipp's apparatus for manufacture of hydrogen. 6. Wash-bottle containing
1-10 solution of lead acetate, c. Wash-bottle containing 1-10 solution of silver
nitrate, d. Wash-bottle containing 1-10 solution of pyrogallic acid. (&, c, and d
are intentionally drawn to a larger scale than a to show details.)
surface. A little hard paraffin may be run between the rim of the
test-tube and the stopper, and round the perforations for the gas-
supply tubes, to ensure that the apparatus is air-tight. The
gelatin is thus in an atmosphere of hydrogen in which the
colonies may develop. The latter may be examined and isolated
in a way which will be presently described. The method is
admirably suited for all anaerobes which grow at the ordinary
temperature.
(b) Bulloch's Apparatus for Anaerobic Culture. — This can
be recommended for plating out mixtures containing anaerobes,
and for obtaining growths (especially surface growths) of the
latter. It consists (Fig. 23) of a glass plate as base on which a
bell jar can be firmly luted down with unguentum resinae. In
CULTURE OF ANAEROBES
59
22.— Esmarch's roll-
tube adapted for culture
containing anaerobes.
the upper part of the bell jar are two apertures furnished with
ground stoppers, and through each of the latter passes a glass tube
on which is a stop-cock. One tube, bent slightly just after
passing through the stopper, extends
nearly to the bottom of the chamber; x L
the other terminates immediately below
the stopper. In using the apparatus
there is set on the base-plate a shallow
dish, of slightly less diameter than that
of the bell jar, and having a little heap
of from two to four grammes of dry
pyrogallic acid placed in it towards one
side. Culture plates made in the usual
way can be stacked on a frame of glass
rods resting on the edges of the dish,
or a beaker containing culture tubes can
be placed in it. The bell jar is then
placed in position so that the longer glass
tube is situated over that part of the
bottom of the shallow dish farthest away
from the pyrogallic acid, and the bottom
and stoppers are luted. The air in the bell jar is now expelled
by passing a current of hydrogen through the short glass-tube,
and both stoppers are.closed. A
partial vacuum is then effected in
the jar by connecting up the short
tube with an air-pump, opening
the tap, and giving a few strokes
of the latter. A solution of 109
grms. solid caustic potash dissolved
in 145 c.c. water is made, and
into the vessel containing it a
rubber tube connected with the
long glass tube is made to dip,
and the stopper of the latter
being opened, the fluid is forced
into the chamber and spreads over
the bottom of the shallow dish ;
potassium pyrogallate is thus
formed, which absorbs any free
oxygen still present. Before the
whole of the fluid is forced in,
FIG. 23. — Bulloch's apparatus for
anaerobic plate cultures.
the rubber tube is placed in a little boiled water, and this, passing
through the glass tubes, washes out the potash and prevents
60 METHODS OF CULTIVATION OF BACTERIA
erosion of the glass. The whole apparatus may be placed in the
incubator till growth occurs.
It is often advisable in dealing with material suspected to
contain anaerobes to inoculate an ordinary deep glucose agar
tube with it, and incubating for 24 or 48 hours, to then apply an
anaerobic separation method to the resultant growth. Sometimes
the high powers of resistance of spores to heat may be taken
advantage of in aiding the separation (vide Tetanus).
Cultures of Anaerobes. — When by one or other of the above
methods separate colonies have been obtained, growth may be
maintained on media in contact with ordinary air. The media
generally used are those which contain reducing agents, and the
test-tubes containing the medium must be filled to a depth of 4
inches. They are sterilised as usual and are called " deep "
tubes. The long straight platinum wire is used for inoculating,
and it is plunged well down into the " deep " tube. A little air
gets into the upper part of the needle track, and no growth takes
place there, but in the lower part of the needle track growth
occurs. From such " deep " cultures growths may be maintained
indefinitely by successive sub-cultures in similar tubes. Even
ordinary gelatin and agar can be used in the same way if the
medium is heated to boiling-point before use to expel any
absorbed oxygen.
Carroll's Method for Anaerobic Cultures. — This may be used
with culture tubes containing any of the media suitable for
anaerobes, with Esmarch's roll-tubes, or with fermentation tubes.
There is required a dry tube of the same diameter as the culture
tube, a short U-shaped glass tube, and two pieces of rubber tub-
ing all of like diameter. The culture tube having been inoculated,
the plug is pushed home below the lip of the tube. The ends
of the U-tube are smeared with vaseline and a rubber tube
slipped over each ; the end of the culture tube being similarly
treated, the free end of one of the rubber tubes is pushed over it
till the glass of the U-tube is in contact with the glass of the
culture tube. In the dry tube one or two grammes of pyrogallic
acid are placed and the powder is packed down with a layer of
filter paper. Ten or twenty cubic centimetres of a ten per cent
solution of sodium hydrate are then poured in and the tube is
quickly connected up by the rubber tubing with the other end
of the U-tube. In this apparatus the oxygen is absorbed by the
sodium pyrogallate and the conditions for anaerobic growth
are fulfilled.
Cultures of Anaerobes in Liquid Media. — It is necessary to
employ such in order to obtain the toxic products of the growth
CULTURE OF ANAEROBES IN LIQUID MEDIA 61
of anaerobes. Glucose broth is most convenient. It is placed
either (1) in a conical flask with a lateral opening and a perfor-
ated india-rubber stopper, through which a bent glass tube passes,
as in Fig. 24, a, by which hydrogen may be delivered, or (2) in
a conical flask with a rubber stopper furnished with two holes,
as in Fig. 24, 6, through a tube in one of which hydrogen is
delivered, while through the tube in the other the gas escapes.
The inner end of the gas delivery tube must in either case be
below the surface of the liquid ; the inner end of the lateral
nozzle in the one case, and the inner end of the escape tube in
the other, must of course be above the surface of the liquid.
The single tube in the one case and the two tubes in the other
FIG. 24.
a. Flask for anaerobes in liquid media. Lateral nozzle and stopper fitted for
hydrogen supply, b. A stopper arranged for a flask without lateral nozzle.
ought to be partially drawn out in a flame to facilitate subsequent
complete sealing. The ends of the tubes through which the gas
is to pass are previously protected by pieces of cotton wool tied
on them. It is well previously to place in the tube, through
which the hydrogen is to be delivered, a little plug of cotton
wool. The flask being thus prepared, it is sterilised by methods
B (2) or B (3). On cooling it is ready for inoculation. In the
case of the flask with the lateral nozzle, the cotton-wool covering
having been momentarily removed, a wire charged with the
organism is passed down to the bouillon. In the other kind of
flask the stopper must be removed for an instant to admit the
wire. The flask is then connected with the hydrogen apparatus
by means of a short piece of sterile india-rubber tubing, and
hydrogen is passed through for half an hour. In the case of
62 METHODS OF CULTIVATION OF BACTERIA
flask (1), the lateral nozzle is plugged with molten paraffin
covered with alternate layers of cotton wool and paraffin, the
whole being tightly bound on with string. The entrance tube
is now completely drawn off in the flame before being dis-
connected from the hydrogen
apparatus. In the case of
flask (2), first the exit tube
and then the entrance tube
are sealed off in the flame
before the flask is discon-
nected from the hydrogen
apparatus. It is well in the
case of both flasks to run
some melted paraffin all over
the rubber stopper. Some-
times much gas is evolved by
anaerobes, and in dealing
with an organism where this
FIG. 25.-Flask arranged for culture of will occur provision must be
made for its escape. This
is conveniently done by lead-
ing down the exit tube, and
letting the end just dip into a trough of mercury (Fig. 25),
or into mercury in a little bottle tied on to the end of the
exit tube. The pressure of gas within causes an escape at the
mercury contact, which at the same time acts as an efficient
valve. The method of culture in fluid media is used to obtain
the soluble products of such anaerobes as the tetanus bacillus.
6 is a trough of mercury into which exit
tube dips.
FIG. 26. — Tubes for auaerobic cultures on the surface of solid media.
When it is desired to grow anaerobes on the surface of a
solid medium such as agar, tubes of the form shown in Fig. 26,
a and b, may be used. A stroke culture having been made, the
air is replaced by hydrogen as just described, and the tubes are
fused at the constrictions. Such a method is of great value
HANGING-DROP PREPARATIONS 63
when it is required to get the bacteria free from admixture of
medium, as in the case of staining flagella.
MISCELLANEOUS METHODS.
Hanging-drop Cultures. — It is often necessary to observe
micro-organisms alive, either to watch the method and rate of
their multiplication, or to investigate whether or not they are
motile. This is effected by making hanging-drop cultures. The
FIG. 27.
A. Hollow-ground slide for hanging-drop cultures shown in plan and section.
B. Another form of slide for similar cultures.
method in the form to be described is only suitable for aerobes.
For this special slides are necessary. Two forms are in use and
are shown in Fig. 27. In A there is ground out on one surface
a hollow having a diameter of about half an inch. That shown
in B explains itself. The slide to be used and a cover-glass are
sterilised by hot air in a Petri's dish, or simply by being heated
in a Bunsen and laid in a sterile Petri to cool. In the case of
A, one or other of two manipulation methods may be employed.
(1) If the organism be growing in a liquid culture, a loop of the
liquid is placed on the middle of the under surface of the sterile
cover-glass, which is held in forceps, the points of which have
been sterilised in a Bunsen flame. If the organism be growing
in a solid medium, a loopful of sterile bouillon is placed on the
cover-glass in the same position, and a very small quantity of
the culture (picked up with a platinum needle) is rubbed up in
64 METHODS OF CULTIVATION OF BACTERIA
the bouillon. The cover is then carefully lowered over the cell
on the slide, the drop not being allowed to touch the wall or the
edge of the cell. The edge of the cover-glass is covered with
vaseline, and the preparation is then complete and may be
placed under the microscope. If necessary, it may be first in-
cubated and then examined on a warm stage. (2) The sterile
cover-glass is placed on a sterile plate (an ordinary glass plate
used for plate cultures is convenient). The drop is then placed
on its upper surface, the- details being the same as in the last
case. The edge of the cell in the slide is then painted with
vaseline, and the slide, held with the hollow surface downwards,
is lowered on to the cover-glass, to the rim of which it of course
adheres. The slide with the cover attached is then quickly
turned right side up, and the preparation is complete.
In the case of B the drop of fluid is placed on the centre of
the table x. The drop must be thick enough to come in contact
with the cover-glass when the latter is lowered on the slide, and
not large enough to run over into the surrounding trench y.
The cover-glass is then lowered on to the drop, and vaseline is
painted along the margin of the cover-glass. The method of
microscopic examination is described on page 85.
Anaerobic Hanging-drop Cultures. — The growth and examination of
bacteria in hanging-drops under anaerobic conditions involve consider-
FIG. 28. — Graham Brown's chamber for anaerobic hanging-drops.
(A portion of one edge of upper plate is shown cut away.)
able difficulty, but may be carried out in an apparatus devised by
Graham Brown (Fig. 28). It consists of two brass plates (a and a')
which can be approximated by screws, and which have rounded
apertures in their middles f in. in diameter. These support two rubber
rings, an upper thinner one (b) and a lower thick one (d), the inner
diameters being the same as that of the apertures in the plates. Between
THE COUNTING OF COLONIES
65
b and d is placed a stout cover-glass of suitable size (c) ; d is separated
from the plate of by a square plate of glass (e) (a portion of an ordinary
glass-slide for microscopical purposes does well). Two small metal
tubes (/) are inserted through the rubber d. Method of use : — Fix up
the apparatus as shown above, the screws being just tight enough to
keep the parts in position, and sterilise in the steam steriliser. Screw
up more firmly so as to make the rubber bulge slightly. Fill a
hypodermic syringe with some sterile glucose bouillon, push the needle
through the rubber d, and, tilting the point of the needle against the
glass c, slowly inject enough to form a drop on the under surface of c.
Withdraw the syringe and inoculate its point with the bacterium, again
introduce and inoculate the drop. Pass hydrogen through one of the
tubes for fifteen minutes, close the ends of the tubes, and incubate at the
required temperature. The apparatus can be put on the stage of a
microscope and examined from time to time.
The Counting of Colonies. — An approximate estimate of the
number of bacteria present in a given amount of a fluid (say,
water) can be arrived at
FIG. 29. — Apparatus for counting colonies.
of colonies which develop
7vlieii_ that amount is
added to a tube of suit-
Tttlle medium, and the
latter plated and incu-
bated. An ordinary plate
should be used in such a
case, and the medium
poured out in as rect-
angular a shape as pos-
sible. For the counting,
an apparatus such as is
shown in Fig. 29 is employed. This consists of a sheet of glass
ruled into squares as indicated, and supported by its corners on
wooden blocks. The table to which these blocks are attached
has a dark surface. The plate-culture containing the colonies
is laid on the top of the ruled glass. The numbers of colonies
in, say, twenty of the smaller squares, are then counted, and an
average struck. The total number of squares covered by the
medium is then taken, and by a simple calculation the total
number of colonies present can be obtained. Plate-cultures in
Petri's dishes are sometimes employed for purposes of count-
ing. The bottoms of such dishes are, however, never flat, and
the thickness of the medium thus varies in different parts.
If these dishes are to be used, a circle of the same size as the
dish can be drawn with Chinese white on a black card, the
circumference divided into equal arcs, and radii drawn. The
5
66 METHODS OF CULTIVATION OF BACTERIA
dish is then laid on the card, the number of colonies in a
few of the sectors counted, and an average struck as before.
In counting colonies it is always best to aid the eye with a small
hand-lens.
Method of counting Living Bacteria in a Culture. — This
is accomplished by putting into practice a dilution method
such as that described on p. 52.
Measured amounts of high dilutions
are plated, and the numbers of
colonies which subsequently develop
are counted. In applying such a
method it is necessary to have pipettes capable
of measuring small quantities of fluid. Those
discharging -05 and '1 c.c. will be found con-
venient, and such pipettes can have sub-
divisions which enable them to be used for
measuring still smaller fractions of a cubic
centimetre. Pipettes of this kind can be
obtained at the instrument makers. Wright
has described a method by which a pipette
(Fig. 30) for measuring small quantities of
fluid can be made from ordinary quill tubing.
The method is as follows : — A .piece of quill
tubing about 15 cm. long is drawn out to a
capillary stem. A standard 5 c.mm. pipette
(such as that of the Gower's haemocytometer),
or the pipette described later on p. 108, is
filled with mercury and the metal transferred
to the capillary stem and run down to near
its extremity ; the upper and lower limits of
the mercury are marked with an oil pencil ;
Fl°- 30-— w^h*>sthe mercury is then displaced up the tube
250 c.mm. pipette ,.„ ., . , , ,. , , N .
fitted with nipple *"* ^s previously distal end is at the proximal
of the two marks, and a third mark is made
at the new position of the upper end of the droplet ; the mani-
pulation is repeated three more times, and finally the tip of
the tube beyond the lowest mark is broken off. Thus on the
capillary part of the pipette we have five divisions, each cap-
able of holding 5 c.mm. of fluid. The rest of the pipette is now
calibrated so as to determine that part capable of containing
225 c.mm. and 250 c.mm. This is done by placing a rubber
nipple on the wide end of the pipette and sucking up some
water tinted with, say, methylene-blue till the 25 c.mm. mark
is reached ; a small air-bubble is then allowed to enter the
250 c. mm,
225
20
15
10
2-5
METHOD OF COUNTING BACTERIA 67
pipette, then other 25 c.mm. of fluid, then another bubble, and
so on till nine volumes each of 25 c.mm. have been sucked up.
A mark is then made on the tube at the upper level of this
amount, other 25 c.mm. are sucked up, and another mark made.
The fluid is expelled, the tube dried, and that part containing
the 225 and 250 marks is drawn out into an almost capillary
diameter, the manipulation by which the marks were originally
arrived at is repeated, and thus in the new marks made a more
accurate calibration for these amounts is attained. In order to
form a safety chamber a second bulb is formed by drawing out
the tube a little higher up as in the figure, and finally the upper
inch or two are bent at right angles to the calibrated limb. In
doing this a loop may be thrown on the plastic melted capillary
tube exactly in the way in which a similar loop may be thrown
on a piece of cord. With such a pipette any required dilution
of a culture can be made on the principles already described.
Wright's Method of counting the Bacteria in Dead
Cultures. — In the making of vaccines for use in Wright's pro-
cedures it is necessary to know the total number of bacterial
cells, whether dead or living, present in a culture, for the dead
as well as the living contain the toxins which may stimulate
the therapeutic capacities of the body. The method consists
in making a mixture of blood (whose content in red blood
corpuscles is known) with the bacterial culture and comparing
the number of bacteria with the number of corpuscles. The
observer first estimates the red cells in his blood ; a capillary
pipette with a rubber nipple and with a mark near its capillary
extremity is then taken, blood is sucked up to the mark, then
an air-bubble, then (according to the empirical estimate the
observer forms of the strength of his bacterial emulsion) either
one volume of culture and three volumes of diluting fluid
(e.g. !85 per cent sodium chloride) or two of culture and two
of fluid, and so' on ; the five volumes are thoroughly mixed by
being drawn backwards and forwards in the wide part of the
pipette, a drop is then blown out on to a slide, and a blood
film is spread which may be stained by Irishman's method.
The bacteria and blood-corpuscles are now separately enumerated
in a series of fields in different parts of the preparation. If
a dilution has been taken in which a large number of bacteria
are present, an artificial field may be used, made by drawing
with the oil pencil a small square on a circular cover-glass and
dropping the latter on to the diaphragm of the microscope
eye-piece. Suppose, now, that the observer's blood contained
5,000,000 red cells per c.mm., that one volume of bacterial
68 METHODS OF CULTIVATION OF BACTERIA
emulsion and three of diluent had been present in the mixture,
and that in the fields examined there were 500 red cells and
600 bacteria. It is evident that in the undiluted culture for
500 red cells there would have been 2400 bacteria. Now
500: 2400:: 5,000,000: 24,000,000, which last figure is the
number of bacteria per c.mm. of the emulsion.
The Bacteriological Examination of the Blood. — (a) This
may be done by taking a small drop from the skin surface, e.g. the
lobe of the ear. The part should be thoroughly washed with
1-1000 corrosive sublimate and dried with sterile cotton wool.
It is then washed with absolute alcohol to remove the antiseptic,
drying being allowed to take place by evaporation. A prick is
then made with a sterile surgical needle ; the drop of blood is
caught with a sterile platinum loop and smeared on the surface
of agar or blood serum. Film preparations for microscopic
examination may be made at the same time. It is rare to obtain
growths from the blood of the human subject by this method
(vide special chapters), and if colonies appear the procedure
should be repeated to exclude the possibility of accidental con-
tamination.
(6) A larger quantity of blood may be obtained by puncture
of a vein ; this is the only satisfactory method, and should be
the one followed whenever practicable. The skin over a vein
in the forearm or on the dorsum of the foot having been sterilised,
the vein is made turgid by pressure, and the needle of a syringe
of 10-15 c.c. capacity, carefully sterilised, is then plunged
obliquely through the skin into the lumen of the vessel. Several
cubic centimetres of blood can thus be withdrawn into the
syringe. Some of the blood (e.g. 1 c.c.) should be added to small
flasks containing 50 c.c. of bouillon ; the rest may be used for
smearing the surface of agar tubes or may be added to melted
agar at 42° C., which is then plated. The flasks, etc., are then
incubated. By this method cultures can often be obtained where
the former method fails, especially in severe conditions such as
ulcerative endocarditis, streptococcus infection, etc. Part of the
blood may be incubated by itself for twenty-four hours and cultures
then made.
In examining the blood of the spleen a portion of the skin
over the organ is sterilised in the same way, a few drops are
withdrawn from the organ by a sterile hypodermic syringe and
cultures made. (For microscopic methods, vide p. 87.)
Bacteriological Examination of the Cerebro-spinal Fluid-
Lumbar Puncture. — This diagnostic procedure, which is some-
times called for in .cases of meningitis, can be carried out with
EXAMINATION OF CEREBRO-SPINAL FLUID 69
a sterilised " antitoxin needle" as follows. The patient should
lie on the right side, with knees somewhat drawn up and left
shoulder tilted somewhat forward, so that the back is fully
exposed. The skin over the lumbar region is then carefully
sterilised, as above described, and the hands of the operator should
be similarly treated. The spines of the lumbar vertebrae having
been counted, the left thumb or forefinger is pressed into the
space between the 3rd and 4th spines in the middle line ; the
jgfidlft JiLthp.Ti in^rted al>OUt bftlf an inr-h t.n tliA riprht rf thft
middle line atthis leveLand pushed through the tissues, its
ourse being directed slightlyinwrdad^upwards, ffll it.
hP nHVrfll ^nrfL wh"n
m*
the needle, sometimes actually spurting out, and should be
received in a sterile test-tube. Several cubic centimetres of
fluid can thus usually be obtained, no suction being required ;
thereafter it can be examined bacteriologically by the usual
methods. The depth of the subdural space from the surface
varies from a little over an inch in children to three inches, or
even more, in adults — the length of the needle must be suited
accordingly. In making the puncture it is convenient to have
either a sterile syringe attached, or to have the thick end of
the needle covered with a pad of sterile wool, which is of course
removed at once when the fluid begins to flow. It is advisable
to use the platinum needles which are specially made for the
purpose, ;is a .sudden movement of the patient may snap an
'ordinary steel needle.
The Bacteriological Examination of Urine. — In such an
examination care must be taken to prevent the contamination
of the urine by extraneous organisms. In the male it is usually
sufficient to wash thoroughly the glans penis and the meatus
with 1-1000 corrosive sublimate — the lips of the meatus being
everted for more thorough cleansing. The urine is then passed
into a series of s,terile flasks, the first of which is rejected in
case contamination has occurred. In ' the female, after similar
precautions as regards external cleansing, the catheter must be
used. The latter must be boiled for half an hour, and anointed
with olive oil sterilised by half au hour's exposure in a plugged
flask to a temperature of 120° 0. Here, again, it is well to
reject the urine first passed. It is often advisable to allow the
urine to stand in a cool place for some hours, to then withdraw
the lower portion with a sterile pipette, to centrifugalise this,
and to use the urine in the lower parts of the centrifuge tubes
for microscopic examination or culture.
Filtration of Cultures. — For many purposes it is necessary
70 METHODS OF CULTIVATION OF BACTERIA
to filter all the organisms from fluids in which they may have
been growing. This is done especially in obtaining the soluble
toxic products of bac-
teria. The only filter
capable of keeping back
such minute bodies as
bacteria is that formed
from a tube of unglazed
porcelain as introduced
by Chainberland. The
efficiency of such a filter
depends on the fineness
of the grain of the clay
from which it is made ;
the finest is the Kitasato
filter and the Chamber-
land " B " pattern ; the
next finest is the Cham-
berland "F" pattern,
which is quite good
enough for ordinary
work. There are several
filters, differing slightly
FIG. 31. — Geissler's vacuum pump arranged with
manometer for filtering cultures. (The tap
and pump are intentionally drawn to a larger .
scale than the manometer board to show 1 *"» a11 P01
details.) the common principle.
Sometimes the fluid is
forced through the porcelain tube. In one form the filter
consists practically of an ordinary tap screwed into the top
of a porcelain tube. Through the latter the fluid is forced and
passes into a chamber formed by a
metal cylinder which surrounds the
porcelain tube. The fluid escapes
by an aperture at the bottom. Such
a filter is very suitable for domestic
use, or for use in surgical operating-
theatres. As considerable pressure
is necessary, it is evident it must be
put on a pipe leading directly from
the main. Sometimes, when fluids
to be filtered are very albuminous, FlG 32._Chamberland's candle
they are forced through a porcelain and flask arranged for filtration,
cylinder by compressed carbonic
acid gas. For ordinary bacteriological work, filters of various
kinds are in the market (such as those of Klein and
THE FILTEATION OF CULTURES
71
others), but the most generally convenient is that in which
the fluid is sucked through the porcelain by exhausting the
air in the receptacle into which it is to flow. This is con-
veniently done by means of a Geissler's water-exhaust pump
(Fig. 31, 0), which must .be fixed to a tap leading directly from
the main. The connection with the tap must be effected by
means of a piece of thick- walled rubber-tubing as short as
possible, wired on to tap and pump, and firmly lashed externally
with many turns of strong tape. Before lashing with the tape
the tube may be strengthened by fixing round it with rubber
solution strips of the rubbered canvas used for mending punctures
FIG. 33. — Chamberland's bougie
arranged with lamp funnel for
filtering a small quantity of
fluid.
FIG. 34. — bougie in-
serted through
rubber stopper
for same purpose
as in Fig. 33.
in the outer case of a bicycle tyre. A manometer tube (6) and
a receptacle (c) (the latter to catch any back flow of water from
the pump if the filter accidentally breaks) are intercepted between
the filter and the pump. These are usually arranged on a
board a, as in Fig. 31. Between the tube /and the pump g,
and between the tube d and the filter, it is convenient to insert
lengths of flexible lead-tubing connected up at each end with
short, stout-walled rubber-tubing.
Filters are arranged in various ways, (a) An apparatus is
arranged as in Fig. 32. The fluid to be filtered is placed in the
cylindrical vessel a. Into this a "candle" or "bougie" of
porcelain dips. From the upper end of the bougie a glass tube
with thick rubber connections, as in Fig. 32, proceeds to flask b
72 METHODS OF CULTIVATION OF BACTERIA
and passes through one of the two perforations with which the
rubber stopper of the flask is furnished. Through the other
opening a similar tube proceeds to the exhaust-pump. When
the latter is put into action the fluid is sucked through the
porcelain and passes over into flask b. This apparatus is very
good, but not suitable for small quantities of fluid.
(6) A very good apparatus can be arranged with a lamp
funnel and the porcelain bougie. These may be fitted up in
two ways. (1) An india-rubber washer is placed round the
bougie c at its glazed end (vide Fig. 33). On this the narrow
end of the funnel d, which must, of course, be of an appropriate
size, rests. A broad band of sheet rubber is then wrapped
round the lower end of the
4 funnel, and the projecting
..-'' part of the bougie. It is
firmly wired to the funnel
above and to the bougie
below. The extreme point
of the latter is left exposed,
and the whole apparatus,
being supported on a stand,
FIG. 35. — Muencke's modification of
Chamberland's filter.
is connected by a glass tube
with the lateral tube of the
flask b } the tube a is con-
nected with the exhaust-
pump. The fluid to be
filtered is placed between
the funnel and the bougie
in the space e, and is sucked
through into the flask b.
(2) This modification is shown in Fig. 34. Into the narrow part
of the funnel an india-rubber bung is fitted, with a perforation
in it sufficiently large to receive the candle, which it should grasp
tightly.
(c) Muencke's modification of the Chamberland filter is
seen in Fig. 35. It consists of a thick-walled flask a, the lower
part conical, the upper cylindrical, with a strong flange on the
lip. There are two lateral tubes, one horizontal to connect with
exhaust-pipe, and one sloping, by which the contents may be
poured out. Passing into the upper cylindrical part of the flask
is a hollow porcelain cylinder b, of less diameter than the
cylindrical part of flask a. It is closed below, open above, and
rests by a projecting rim on the flange of the flask, an asbestos
washer, c, being interposed. The fluid to be filtered is placed
THE FILTRATION OF CULTURES
73
in the porcelain cylinder, and the whole top covered, as shown
at /, with an india-rubber cap with a central perforation ; the
tube d is connected with the exhaust-pump
and the tube e plugged with a rubber stopper.
When a large quantity of fluid is to be filtered,
. a receptacle such as that shown in
Fig. 36 may be used. The tap in
its bottom enables the filtrate to be
removed without the apparatus
being unshipped, but it is difficult
to get the tap to fit so accurately as not to
allow air to pass into the vacuum chamber.
For filtering small quantities of fluid the
apparatus shown in Fig. 37 may be used.
It consists of a small Chamberland bougie
fitted by a rubber tube to a funnel, the stem
of which has been passed
through a rubber cork ;
this cork fits into a tri-
angular flask with side
arm for connection with
exhaust.
Before any one of
the above apparatus is
used, it ought to be con-
nected up as far as pos-
sible and sterilised in
the Koch's steriliser.
The ends of any im-
portant unconnected
parts ought to have pieces of cotton wool
tied over them. After use the bougie is
to be sterilised in the autoclave, and after
being dried is to be passed carefully through
a Bunsen flame, to burn oft7 all organic
matter. If the latter is allowed to accumu-
late the pores become filled up.
The success of filtration must be tested
by inoculating tubes of media from the
filtrate, and observing if growth takes place,
as there may be minute perforations in the
candies sufficiently large to allow bacteria
to pass through. Filtered fluids keep for a long time if the
openings of the glass vessels in which they are placed are kept
FIG. 36.— Flask fitted
with porcelain
bougie for filtering-
large quantities of
fluid.
FIG. 37. — Flask for
filtering small quanti-
ties of fluid.
74 METHODS OF CULTIVATION OF BACTERIA
thoroughly closed, and if these vessels be kept in a cool place in
the dark. A layer of sterile toluol about half an inch thick
ought to be run on to the top of the filtered fluid to protect the
latter from the atmospheric oxygen.
Instead of being filtered off, the bacteria may be killed by
various antiseptics, chiefly volatile oils, such as oil of mustard
(Roux). These oils are stated to have no injurious effect on the
chemical substances in the fluid, and they may be subsequently
removed by evaporation. It is not practicable to kill the bacteria,
by heat when their soluble products are to be studied, as many
of the latter are destroyed by a lower temperature than is required
to kill the bacteria themselves.
Bacteria can be almost entirely removed from fluid cultures
by spinning the latter in a centrifuge of very high speed (e.g.
C. J. Martin's turbine centrifuge), and this method is sometimes
adopted in practice.
The Observation of Bacterial Fermentation of Sugars, etc.
— The capacity of certain species of bacteria to originate fermenta-
tions in sugars constitutes an important biological factor. It
is well to consider this factor in relation to the chemical con-
stitution of the sugars. These bodies are now known to be (to
use the definition of Holleman) aldehyde or ketone alcohols
containing one or more hydroxyl groups, one of which is directly
linked to a carbon atom in union with carbonyl. The group
characteristic of a sugar is thus - CHOH - CO - . The sugars are
divided into monosaccharides or monoses, disaccharides (dioses),
and polysaccharides (polyoses). The members of the last two
groups may be looked on as derived from the combination of two
or more molecules of a rnonosaccharide with the elimination of
water (e.g. 2C6H12O6 = C12H22On + H2O).
Monosaccharides. — These are classified according to the
number of C atoms they contain. The pentoses ordinarily used
are arabinose (obtained from gum arable), rhamnose and xylose
(from wood). Among the hexoses are glucose (dextrose) with
dextro-rotatory properties. Glucose is an aldehyde alcohol
(aldose). In fruit there is also a ketone alcohol (ketose) called
fructose, which from its laevo-rotatory properties is also known
as laevulose. Other hexoses are mannose (from the vegetable
ivory nut) and galactose (a hydrolytic derivative of lactose).
Disaccharides (C]2H22On). — The ordinary members of this
group are maltose (derived from starch), lactose, and cane sugar
(sucrose, saccharose).
Polysaccharides. — Examples are starch, raffinose, inulin (from
dahlia roots), dextrin, arabin, glycogen, cellulose.
BACTERIAL FERMENTATION OF SUGARS 75
If we consider sugars generally from the point of view of
the capacity of yeast to originate alcoholic fermentation in them,
we may say that the simpler the constitution of the sugar the
more easily is it fermented. Thus the monosaccharides are
more easily acted on by yeast than the di- or polysaccharides.
Usually an independent process resulting in the splitting of the
higher into the lower is preliminary to the alcoholic fermentation.
Thus yeast first inverts cane sugar into glucose and fructose and
then acts on these products. From what is known it is probable
that similar facts hold with regard to the action of bacteria.
Besides sugars other alcohols with large molecules may be
broken down by bacterial action, and these bodies have been
used for differentiating the properties of allied bacteria. Among
these substances may be mentioned the trihydric alcohol glycerol
(glycerin), the tetrahydric erythritol and the hexahydric dulcitol
(dulcite), mannitol (mannite), and sorbitol (sorbite).
Similarly certain glucosides, such as salicin, coniferin, etc.,
have been used for testing the fermentative properties of
bacteria. Other substances allied to sugars (e.g. inosite) have
also been used.
The end products of bacterial fermentations may be various.
They differ according to the sugar employed and according to
the species of bacterium under observation, and frequently a species
which will ferment one sugar has no effect on another. The sub-
stances finally produced, speaking roughly, may be alcohols, acids,
or gaseous bodies (chiefly carbon dioxide, hydrogen, and methane).
For the estimation of the first groups complicated chemical
procedure may be necessary. The tests usually employed for
the detection of ordinary fermentative processes depend on two
kinds of changes, namely (a) the evolution of gases and (b) the
formation of acids. Generally speaking, we may say that such
tests are reliable and the methods to be pursued are simple.
Besides such gases as those named 'some organisms give rise
to sulphuretted hydrogen by breaking up the proteid. The
formation of this gas can be detected by the blackening of lead
acetate when it is added to the g'as-containing medium.
In testing the effect of a bacterium on a given sugar it is
essential that this sugar alone be present ; the basis of the
medium ought therefore to be either peptone solution (v. p. 38)
or a dextrose-free bouillon (v. infra). The sugar or other
substance is added in the proportion of from a half to one
per cent, and care is taken not to overheat during sterilisation.
To obtain a "dextrose-free " bouillon it is usual to inoculate ordinary
bouillon with some organism, such as b. coli, which is known to ferment
76 METHODS OF CULTIVATION OF BACTERIA
dextrose, and allow the latter to act for forty-eight hours. The bouillon
is then filtered and re-sterilised. A sample is tested for another period of
forty-eight hours with b. coli, to make certain that all the dextrose has
been removed. If no fresh gas - formation is observed, then to the
remainder of the bouillon the sugar to be investigated may be added.
It is preferable that the addition should be made in the form of a sterile
solution. If the sugar in solid form be placed in the bouillon and this
then sterilised, there is danger that chemical changes may take place
in the sugar, in consequence of its bei-ng heated in the presence of
substances (such as the alkali) which may act deleteriously upon it ; in
any case sterilisation should not be at a temperature above 100° C.
For the observation of gas-formation either of the following
methods may be employed : —
(1) Durham's Tubes (Fig. 38, 6).— The plug of a tube which
contains about one-third more than usual of a liquid medium is
It a c
FIG. 38. — Tubes for demonstrating gas-formation by bacteria.
«, tube with "shake" culture.
5, Durham's fermentation tube,
c, ordinary form of fermentation tube.
removed, and a small test-tube is slipped into the latter mouth
downwards. The plug is replaced and the tube sterilised thrice
for ten minutes at 100° C. The air remaining in the smaller
tube is thereby expelled. The tube is then inoculated with the
bacterium to be tested. Any gas developed collects in the upper
part of the inner tube.
(2) The Fermentation Tube (Fig. 38, c).— This consists of a
tube of the form shown, and the figure also indicates the extent
BACTERIAL FERMENTATION OF SUGARS 77
to which it ought to be filled. It is inoculated in the bend with
the gas -forming organism, and when growth occurs the gas
collects in the upper part of the closed limit, the medium being
displaced into the bulb.
For the observation of the effect of an organism on glucose
the following method may be employed : —
Gelatin Shake Cultures (Fig. 38, a). — The gelatin in the tube
is melted as for making plates ; while liquid it is inoculated
with the growth to be observed, and shaken to distribute the
organisms throughout the jelly. It is then allowed to solidify,
and is set aside at a suitable temperature. If the bacterium used
is a gas-forming one, then, as growth occurs, little bubbles appear
round the colonies.
In this method the gas -formation results from fermenta-
tion of the glucose naturally present in the medium from
transformation of the glycogen of muscle. The amount of
glucose naturally present, however, varies much, and therefore
glucose should be added to the medium if the effects on this
sugar are to be observed with certainty. The shake culture
method may be utilised for observing fermentation in other
sugars by adding to peptone solution containing the sugar
10-15 per cent of gelatin.
The development of an acid reaction is demonstrated by the
addition of an indicator to the medium, litmus being generally
used. The details of composition of such media have already
been given. In Hiss's serum water media the production of
acid also leads to coagulation of the medium. Sometimes acid
is formed very slowly from sugars, so that it is well to keep the
cultures under observation for several days.
Acid and gas-formation may be simultaneously tested for, by
placing the fluid medium containing the indicator in Durham's
tubes.
In all tests in which sugars are used a control uninoculated
tube ought to be incubated with the bacterial cultures, as changes
in reaction sometimes spontaneously occur in media containing
unstable sugars.
The capacity of an organism to produce acid may be measured
by taking a standard amount of a fluid medium and allowing
growth to take place for a standard time, and then adding an
amount of, say, decinormal soda solution sufficient to bring the
litmus back to the tint of the original medium.
The Observation of Indol-formation by Bacteria. — The
formation of indol from albumin by a bacterium sometimes con-
stitutes an important specific characteristic. To observe indol
78 METHODS OF CULTIVATION OF BACTERIA
production the bacterium is grown, preferably at incubation
temperature, in a fluid medium containing peptone. The latter
may either be ordinary bouillon or preferably peptone solution
(see p. 38). Indol production is recognised by the fact that when
acted on by nitric acid in the presence of nitrites, a nitroso-indol
compound is produced, which has a rosy red colour. Some
bacteria (e.g. the cholera vibrio) produce nitrites as well as indol,
but usually in making the test (e.g. in the case of b. coli) the
nitrites must be added. This is effected by adding to an ordinary
tube of medium 1 c.c. of a '02 per cent solution of potassium
nitrite, and testing with pure nitric or sulphuric acid. In any
case only a drop of the acid need be added to, say, 10 c.c. of
medium. If no result be obtained at once it is well to allow
the tube to stand for an hour, as sometimes the reaction is very
slowly produced. In many instances incubation at 37° C. for
several days may be necessary before the presence of indol is
demonstrable. The amount of indol produced by a bacterium
seems to vary very much with certain unknown qualities of the
peptone. It is well therefore to test a series of peptones with
an organism (such as the b. coli) known to produce indol, and
noting the sample with which the best reaction is obtained, to
reserve it for making media to be used for the detection of this
product.
The Drying of Substances in vacuo. — As many substances,
for example toxins and antitoxins, with which bacteriology is
concerned would be destroyed by drying with heat as is done in
ordinary chemical work, it is necessary to remove the water at
the ordinary room temperature. This is most quickly effected
by drying in vacuo in the presence of some substance such as
strong sulphuric acid which readily takes up water vapour. The
vacuum produced by a water-pump is here not available, as in
such a vacuum there must always be water vapour present. An
air-pump is therefore to be employed. Here we have found the
Geryk pump most efficient, and it has this further advantage,
that its internal parts are lubricated with an oil of very low
vapour density so that almost a perfect vacuum is obtainable.
The apparatus is shown in Fig. 39. The vacuum chamber
consists of a bell-jar set on a brass plate. A perforation in the
centre of the latter leads into the pipe a, which can be connected
by strong- walled rubber-tubing with the air-pump, and which
can be cut off from the latter by a stop-cock 6. In using the
apparatus the substance to be dried is poured out in flat dishes
(one-half of a Petri capsule does very well), and these are stacked
alternately with similar dishes of strong sulphuric acid on a
STORING AND INCUBATION OF CULTURES 79
stand which rests on the brass plate. The edge of the bell-jar
is well luted with unguentum resinse and placed in position and
the chamber exhausted. In a few hours, if, as is always advis-
able, each dish have contained only a thin layer of fluid, the
drying will be complete. The vacuum is then broken by
admitting air very slowly through a bye-pass c, and the bell-jar
is removed. In such an apparatus it is always advisable, as is
shown in the figure, to have interposed between the pump and
the vacuum chamber a Wolffs bottle containing sulphuric acid.
This protects the oil of the pump from contamination with
FIG. 39. — Geryk air-pump for drying in vacuo.
water vapour. Whenever the vacuum is produced the rubber-
tube should be at once disconnected from a, the cock b being
shut. It is advisable when the apparatus is exhausted to cover
the vacuum chamber and the Wolff's bottle with wire guards
covered with strong cloth, in case, under the external pressure,
the glass vessels give way.
The Storing and Incubation of Cultures. — Gelatin cultures
must be grown at a temperature below their melting-point, i.e.
for 10 per cent gelatin, below 22° C. They are usually kept in
ordinary rooms, which vary, of course, in temperature at different
times, but which have usually a range of from about 12° C. to
18° C. Agar and serum media are usually employed to grow
bacteria at a higher temperature, corresponding to that at which
80 METHODS OF CULTIVATION OF BACTERIA
the organisms grow best, usually 37° C. in the case of
pathogenic organisms. For the purpose of maintaining a uniform
temperature incubators are used. These vary much in the
details of their structure, but all consist of a chamber with
double walls between which some nuid (water or glycerin and
water) is placed, which, when raised to a certain temperature,
ensures a fairly constant distribution of the heat round the
chamber. The latter is also furnished with double doors, the
inner being usually of glass. Heat is supplied from a burner
fixed below. These burners vary much in
y design. Sometimes a mechanism devised in
.-•--.... Koch's laboratory is affixed, which auto-
a matically turns off the gas if the light be
accidentally extinguished. Between the tap
supplying the gas, and the burner, is inter-
posed a gas regulator. Such regulators
vary in design, but for ordinary chambers
which require to be kept at a constant tem-
perature, Reichert's is as good and simple
as any and is not expensive. It is shown
in Fig. 40.
It consists of a long tube /closed at the lower
eiid, open at the upper, and furnished with two
lateral tubes. The lower part is filled with
mercury up to a point above the level of the lower
lateral tube. The end of the latter is closed by a
brass cap through which a screw d passes, the
inner end of which lies free in the mercury. The
_ height of the latter in the perpendicular tube can
J thus be varied by increasing or decreasing the
capacity of the lateral tube by turning the screw
a few turns out of or into it. Into the upper
open end of the perpendicular tube fits accurately
FIG. 40. — Reichert's a bent tube g, drawn out below to a comparatively
gas regulator. small open point c, and having in its side a
little above the point a minute needle-hole
called the peephole or bye-pass e. To fix the apparatus the long
mercury bulb is placed in the jacket of the chamber to be controlled,
tube a is connected to gas supply, tube b with the burner. The upper
level of the mercury should be some distance below the lower open end
of tube c. The burner is now lit. The gas passes in at a through c
and e and out at b to the burner. When the thermometer in the
interior of the chamber indicates that the desired temperature has been
reached, the screw d is turned till the mercury reaches the end of the
tube c. Gas can only now pass through the peephole e, and the flame
goes down. The contents of the jacket cool, the mercury contracts off
the end of tube c, and the flame rises. This alternation going on, the
temperature of the chamber is kept very nearly constant. If the mercury
cuts off the gas supply before the desired temperature is reached, and
STORING AND INCUBATION OF CULTURES 81
the screw d is as far out as it will go, then some of the mercury must be
removed. Similarly, if when the desired temperature is reached and the
screw d is as far in as it can go, the mercury does not reach c, some more
must be introduced. If the amount of gas which passes through the
peephole is sufficient still to raise the temperature of the chamber when
c is closed by the rise of the mercury, then the peephole is too large. Tube
c must be unshipped and e plastered over with sealing-wax, which is
pricked, while still soft, with a very fine needle. The gas flame, when
only the peephole is supplying gas, ought to be sufficiently large not to
be blown out by small currents of air. If the pressure of gas supplied to a
regulator varies much in the 24 hours a pressure regulator ought to be
interposed between the gas tap and the instrument. Several varieties of
these can be obtained. In all cases g ought to be fixed to b with a turn
of wire.
FIG. 41. — Hearson's incubator for use at 37° C.
The varieties of incubators are, as -we have said, numerous.
The most complicated and expensive are made by German
manufacturers. Many of these are unsatisfactory. They easily
get out of order and are difficult to repair. We have found
those of Hearson of London extremely good, and in proportion
to their size much cheaper than the German articles. They are
fitted with an admirable regulator. It is preferable in using an
incubator to connect the regulator with the gas supply and with
the Bunsen by flexible metal tubing. It is necessary to see that
there is not too much evaporation from the surface of cultures
placed within incubators, otherwise they may quickly dry up.
It is thus advisable to raise the amount of water vapour in the
interior by having in the bottom of the incubator a flat dish full
82 METHODS OF CULTIVATION OF BACTERIA
of water from which evaporation may take place. Tubes which
will require to be long in the incubator should have their plugs
covered either by india-rubber caps or by pieces of sheet rubber
tied over them. These caps should be previously sterilised in
1-1000 corrosive sublimate and then dried. Before they are
placed on the tubes the cotton-wool plug ought to be well singed
in a flame. " Cool " incubators are often used for incubating
gelatin at 21° to 22° C. An incubator of this kind fitted with a
low-temperature Hearson's regulator is in the market.
Method of mounting Bacterial Cultures as Permanent
Museum Specimens. (Richard Muir). — (a) Stab or stroke
cultures in nutrient gelatin or agar media. — When the culture
shows typical characters, further growth is arrested by placing
tube in a formol vapour chamber, or by saturating the cotton-
wool plug with strong formalin. Then leave for a day or two.
Make up following : —
(1) Thymol Water (saturated in cold) ... 100 c.c.
Glycerin . . . . . . . 20 c.c.
Acetate of Potash ...... 5 grams.
Coignet's (gold label) Gelatin . . . 10 grams.
Render the mixture acid to litmus with acetic acid ; clear with white
of egg and filter.
Warm to about 40° C., and removing cotton-wool plug from
culture take a little of the preserving fluid in a pipette and
allow to run gently over surface of medium in tube. Place in
such a position that a thin layer of the preserving medium
remains completely covering the growth and the surface of
culture medium. The gelatin is now allowed to solidify. Add
three or four drops of strong formalin to the tube and fill up to
within a quarter of an inch of the top of the tube with the
following fluid : —
(2) Thymol Water (saturated in cold) . . . 100 c.c.
Glycerin . 20 c.c.
Acetate of Potash ...... 5 grams.
Cover top of tube with a small piece of paper so as to keep
out dust, allow to stand for a day or two so that small air-bells
may rise to the surface.
To seal tube, pour melted paraffin gently on to the surface
of fluid to near the top of tube ; allow to solidify. Cover paraffin
with layer of alcoholic orange shellac cement ; allow this to set
and repeat until the cement becomes level with top of test tube.
When set, a few drops of black lacquer are put on and a circular
cover-glass of about the same diameter as the mouth of tube is
placed so as completely to seal it.
GENERAL LABORATORY RULES 83
(b) The following method is useful for preserving plate cultures.
Instead of making the cultures in Petri's capsules, use ordinary
watch-glasses. The watch-glass is sterilised in a Petri's capsule,
and the inoculated medium is poured out into the watch-glass,
allowed to solidify in the usual way, and left in the Petri's cap-
sule until the colonies of growth have developed. The watch-
glass is now removed from capsule and a layer of the preserving
gelatin medium (1), to which have been added a few drops of
strong formalin, is allowed to spread over the surface of the culture
medium. When the layer is solidified the watch-glass is filled up
with the same, and a clean square or oblong piece of glass
(which of course should be of slightly larger diameter than the
watch-glass) is now carefully placed over watch-glass, care being
taken that no air-bells are formed. The edge of watch-glass
should be closely applied to the glass cover and left in position
until the gelatin has solidified. The superfluous gelatin is now
removed, and the glasses sealed first with the orange shellac
cement, then with black lacquer. It is now finished off by
using a circular mask of suitable size.
The various kinds of solid media used in the cultivation of
bacteria, such as blood serum, potato, bread paste, etc., can be
treated in the same manner with excellent results.
General Laboratory Rules.— On the working bench of every
bacteriologist there should be a large dish of 1-1000 solution of
mercuric chloride in water. Into this all tubes, vessels, plates,
hanging-drop cultures, etc., which have contained bacteria and
with which he has finished, ought to be at once plunged (in the
case of tubes the tube and plug should be put in separately).
On no account whatever are such infected articles to be left
lying about the laboratory. The basin is to be repeatedly
cleaned out. All the glass is carefully washed in repeated
changes of tap water to remove the last trace of perchloride of
mercury, a very minute quantity of which is sufficient to inhibit
growth. Old cultures which have been stored for a time and
from which fresh sub-cultures have been made ought to be
steamed in the Koch's steriliser for two or three hours, or in the
autoclave for a shorter period, and the tubes thoroughly washed
out. Besides a basin of mercuric chloride solution for infected
apparatus, etc., there ought to be a second reserved for the
worker's hands in case of any accidental contamination. When,
as in public-health work, a large number of tubes are being
daily put out of use, they may be placed in an enamelled slop-
pail and this when full is placed in the steam s'teriliser.
A white glazed tile on which a bell-jar can be set is very
84 METHODS OF CULTIVATION OF BACTERIA
convenient to have on a bench. Infective material in watch-
glasses can be placed thus under cover while investigation is
going on, and if anything is spilled the whole can be easily
disinfected. In making examinations of organs containing
virulent bacteria, the hands should be previously dipped in
1-1000 mercuric chloride and allowed to remain wet with this
solution. No food ought to be partaken of in the laboratory,
and pipes, etc., are not to be laid with their mouth-pieces on
the bench. No label is to be licked with the tongue. Before
leaving the laboratory the bacteriologist ought to wash the
hands and forearms with 1-1000 mercuric chloride and then
with yellow soap. In the case of any fluid containing bacteria
being accidentally spilt on the bench or floor, 1-1000 mercuric
chloride is to be at once poured on the spot. The air of the
laboratory ought to be kept as quiet as possible.
CHAPTER III.
MICROSCOPIC METHODS— GENERAL BACTERIO-
LOGICAL DIAGNOSIS— INOCULATION OF ANIMALS.
The Microscope. — For ordinary bacteriological work a good
microscope is essential. It ought to have a heavy stand, with
rack and pinion and fine adjustment, a double mirror (flat on
one side, concave on the other), a good condenser, with an iris
diaphragm, and a triple nose-piece. It is best to have three
objectives, either Zeiss A, D, and -/^-inch oil immersion, or the
lenses of other makers corresponding to these. The oil immer--
sion lens is essential. It is well to have two eye-pieces, say
Nos. 2 and 4 of Zeiss or lenses of corresponding strengths.
The student must be thoroughly familiar with the focussing of
the light on the lens by means of the condenser, and also with
the use of the immersion lens. It may here be remarked that
when it is desired to bring out in sharp relief the margins of
unstained objects, e.g. living bacteria in a fluid, a narrow
aperture of the diaphragm should be used, whereas, in the case
of stained bacteria, when a pure colour picture is desired, the
diaphragm ought to be widely opened. The flat side of the
mirror ought to be used along with tlofc condenser. When the
observer has finished for the time being with the immersion lens
he ought to wipe off the oil with a piece of silk or very fine
washed linen. If the oil has dried on the lens it may be
moistened with xylol — never with alcohol, which will dissolve
the material by which the lens is fixed in its metal carrier.
Microscopic Examination of Bacteria. 1. Hanging -drop
Preparations. — Micro-organisms may be examined : (1) alive or
dead in fluids ; (2) in film preparations ; (3) in sections of
tissues. In the two last cases advantage is always taken of the
affinity of bacteria for certain stains. When they are to be
examined in fluids a drop of the liquid may be placed on a slide
85
86 MICROSCOPIC METHODS
and covered with a cover-glass.1 It is more usual, however, to
employ hanging-drop preparations. The technique of making
these has already been described (p. 63). In examining them
microscopically, it is necessary to use a very small diaphragm.
It is best to focus the edge of the drop with a low -power
objective, and, arranging the slide so that part of the edge
crosses the centre of the field, to clamp the preparation in this
position. A high-power lens is then turned into position and
lowered by the coarse adjustment to a short distance above its
focal distance ; it is now carefully screwed down by the fine
adjustment, the eye being kept at the tube meanwhile. The
shadow of the edge will be first recognised, and then the bacteria
must be carefully looked for. Often a dry lens is sufficient, but
for some purposes the oil immersion is required. If the bacteria
are small and motile a beginner may have great difficulty in
seeing them, and it is well to practise at first on .some large non-
motile form such as anthrax. In fluid preparations the natural
appearance of bacteria may be studied, and their rate of growth
determined. The great use of such preparations, however, is to
find whether or not the bacteria are motile, and for determining
this point it is advisable to use either broth or agar cultures not
more than twenty-four hours old. In the latter case a small
fragment of growth is broken down in broth or in sterile water.
Sometimes it is an advantage to colour the solution in which
the hanging-drop is made up with a minute quantity of an
aniline dye, say a small crystal of gentian violet to 100 c.c. of
bouillon. Such a degree of dilution will not have any effect on
the vitality of the bacteria. Ordinarily, living bacteria will not
take up a stain, but even though they do not, the contrast
between the unstained bacteria and the tinted fluid will enable
the observer more easily to recognise them.
2. Film Preparations, (a) Dry Method. — This is the most
extensively applicable method of microscopically examining
bacteria. Fluids containing bacteria, such as blood, pus,
scrapings of organs, can be thus investigated, as also cultures
in fluid and solid media. The first requisite is a perfectly clean
cover-glass. Many methods are recommended for obtaining
such. The test of this being accomplished is that, when the
drop of fluid containing the bacteria is placed upon the glass, it
can be uniformly spread with the platinum needle all over the
surface without showing any tendency to retract into droplets.
1 In bacteriological work it is essential that cover-glasses of No. 1 thick-
ness (i.e. '14 ram. thick) should he used, as those of greater thickness are not
suitable for a ^-in. lens.
FILM PREPARATIONS 87
The best method is that recommended by Van Ermengem. The
cover-glasses are placed for some time in a mixture of con-
centrated sulphuric acid 6 parts, potassium bichromate 6 parts,
water 100 parts, then washed thoroughly in water and stored in
absolute alcohol. For use, a cover-glass is either dried by
wiping with a clean duster or is simply allowed to dry. This
method will amply repay the trouble, and really saves time in
the end. A clean cover having been obtained, the film pre-
paration can now be made. If a fluid is to be examined a
loopful may be placed on the cover -glass, and either spread
out over the surface with the needle, or another clean cover
may be placed on the top of the first, the drop thus spread
out between them and the two then drawn apart. When
a culture on a solid medium is to be examined a loopful of
distilled water is placed on the
cover-glass and a minute par-
ticle of growth rubbed up in it
and spread over the glass. The
great mistake made by begin-
. . \ 4,, FIG. 42. — Cornet s forceps for holding
ners is to take too much of the cover-classes,
growth. The point of the
straight needle should just touch the surface of the culture, and
when this is rubbed up in the droplet of water and the film dried,
there should be an opaque cloud just visible on the cover-glass.
When the film has been spread it must next be dried by being
waved backwards and forwards at arm's-length above a Bunsen
flame. The film must then be fixed on the glass by being
passed three or four times slowly through the flame. In doing
this a good plan is to hold the cover-glass between the right
forefinger and thumb; if the fingers just escape being burned
no harm will accrue to the bacteria in tjie film.
In making films of a thick fluid such as pus it is best to
spread it out on one cover with the needle. The result will be
a film of irregular thickness, but sufficiently thin at many parts
for proper examination. Scrapings of organs may be smeared
directly on the cover-glasses.
In the case of blood, a fairly large drop should be allowed to
spread itself between two clean cover-glasses, which are then to
be slipped apart, and being held between the forefinger and
thumb are to be dried by a rapid to-and-fro movement in the
air. A film prepared in this way may be too thick at one edge,
but at the other is beautifully thin. If it is desired to preserve
the red blood corpuscles in such a film it may be fixed by one
of the following methods : by being placed (a) in a hot-air
88 MICROSCOPIC METHODS
chamber at 120° C. for half an hour, (b) in a mixture of equal
parts of alcohol and ether for half an hour, then washed ^ and
dried, (c) in formol-alcohol (Gulland) (formalin 1 part, absolute
alcohol 9 parts) for five minutes, then washed and dried, or (d)
in a saturated solution of corrosive sublimate for two or three
minutes, then washed well in running water and dried. (Fig. 71
shows a film prepared by the last method.) In using the
Romanowsky stains no previous fixation is necessary (vide infra).
In the case of urine, the specimen must be allowed to stand, and
films made from any deposit which occurs ; or, what is still
better, the urine is centrifugalised, and films made from the
deposit which forms. After dried films are thus made from
urine it is an advantage to place a drop of distilled water on the
film and heat gently to dissolve the deposit of salts ; then wash
in water and dry. In this way a much clearer picture is
obtained when the preparation is stained.
Within recent years it has become common to make blood
films on ordinary microscopic slides instead of upon cover-
glasses. Here the slides must be clean. This can be effected by
washing thoroughly first with weak alkali and then with water
and storing in alcohol. For use, a slide is taken from the
alcohol and the fluid adhering to it set on fire and allowed to
burn off, a dry clean slide being thus obtained. To make a film
on such, a small drop of blood is placed near one end, the edge
of a second clean slide is lowered through the drop on to the
surface of the glass on which the blood has been placed. This
second slide is held at an angle to the first, on which it rests by
its edge. The droplet of blood by capillarity spreads itself in
the angle between the two slides. The edge of the second slide is
then stroked along the surface of the first slide, and in this
procedure the blood is spread out in a film whose thickness can
be regulated by the angle formed by the second slide. Large-
sized films can thus be obtained, and when these are stained they
are often examined without any cover-glass being placed upon
them. A drop of cedar oil is placed on the preparation, and
after use this can be removed by the careful application of
xylol.
Films dried and fixed by the above methods are now ready to
be stained by the methods to be described below.
(b) Wet Method. — If it is desired to examine the fine
histological structure of the cells of a discharge as well as to
investigate the bacteria present, it is advisable to substitute
" wet " films for the " dried " films, the preparation of which has
been described. The nuclear structure, mitotic figures, etc., are
EXAMINATION OF BACTERIA IN TISSUES 89
by this method well preserved, whereas these are considerably
distorted in dried films. The initial stages in the preparation of
wet films are the same as above, but instead of being dried in
air they are placed, while still wet, film downwards in the
fixative. The following are some of the best fixing methods : —
(a) A saturated solution of perchloride of mercury in '75 per cent
sodium chloride ; fix for five minutes. Then place the films for half an
hour, with occasional gentle shaking, in 75 per cent sodium chloride
solution to wash out the corrosive sublimate ; they are thereafter washed
in successive strengths of methylated spirit. After this treatment the
films are stained and treated as if they were sections.
(b) Formol-alcohol — formalin 1 part, absolute alcohol 9. Fix films
for three minutes ; then wash well in methylated spirit. This is an
excellent and very rapid method.
(c) Another excellent method of fixing has been devised by Gulland.
The fixing solution has the composition — absolute alcohol 25 c.c., pure
ether 25 c.c., alcoholic solution of corrosive sublimate (2 grm. in 10 c.c.
of alcohol) about 5 drops. The films are placed in this solution for five
minutes or longer. They are then washed well in water, and are ready
for staining. A contrast stain can be applied at the same time as the
fixing solution, by saturating the 25 c.c. of alcohol with eosin before
mixing. Thereafter the bacteria, etc., may be stained with methylene-
blue or other stain, as described below. This method has the advantage
over (a) that, as a small amount of corrosive sublimate is used, less
washing is necessary to remove it from the preparation, and deposits are
less liable to occur.
3. Examination of Bacteria in Tissues. — For the examina-
tion of bacteria in the tissues, the latter must be fixed and
hardened, in preparation for being cut with a microtome.
Fixation consists in so treating a tissue that it shall permanently
maintain, as far as possible, the condition it was in when re-
moved from the body. Hardening consists in giving such a
fixed tissue sufficient consistence to enable a thin section of it
to be cut. A tissue, after being hardened, may be cut in a
freezing microtome (e.g. Cathcart's or one of the newer instru-
ments in which the freezing is accomplished by compressed
carbonic acid gas), but far finer results can be obtained by
embedding the tissue in solid paraffin and cutting with some of
the more delicate microtomes of which, for pathological purposes,
the small Cambridge rocker is by far the best. For bacterio-
logical purposes embedding in celloidin is not advisable, as the
celloidin takes on the aniline dyes which are used for staining
bacteria, and is apt thus to spoil the preparation, and besides
thinner sections can be obtained by the paraffin method.
The Fixation and Hardening of Tissues. — The following
are amongst the best methods for bacteriological purposes : —
(a) Absolute alcohol may be used for the double purpose of fixing and
hardening. If the piece of tissue is not more than ^ inch in thickness it
90 « MICROSCOPIC METHODS
is sufficient to keep it in this reagent for a few hours. If the pieces
are thicker a longer exposure is necessary, and in such cases it is better
to change the alcohol at the end of the first twenty-four hours. The
tissue must be tough without being hard, and the necessary consistence,
as estimated by feeling with the fingers, can only be judged of after
some experience. If the tissues are not to be cut at once, they may be
preserved in 50 per cent spirit.
(b) Formol-alcohol — formalin 1, absolute alcohol 9. Fix for not more
than twenty-four hours ; then place in absolute alcohol if the tissue is
to be embedded at once, in 50 per cent spirit if it is to be kept for some
time. For small pieces of tissue fixation for twelve hours or even less is
sufficient. The method is a rapid and very satisfactory one.
(c) Corrosive sublimate is an excellent fixing agent. It is best used
as a saturated solution in '75 per cent sodium chloride solution. Dis-
solve the sublimate in the salt solution by heat ; the separation of
crystals on cooling shows that the solution is saturated. For small
pieces of tissue ^ inch in thickness, twelve hours' immersion is sufficient.
If the pieces are larger, twenty-four hours is necessary. They should
then be tied up in a piece of gauze, and placed in a stream of running
water for from twelve to twenty-four hours, according to the size of the
pieces, to wash out the excess of sublimate. They are then placed for
twenty-four hours in each of the following strengths of methylated
spirit (free from naphtha l) : 30 per cent, 60 per cent, and 90 per cent.
Finally they are placed in absolute alcohol for twenty-four hours and
are then ready to be prepared for cutting.
If the tissue is very small, as in the case of minute pieces removed
for diagnosis, the stages may be all compressed into twenty-four hours.
In fact after fixation in corrosive the tissue may be transferred directly
to absolute alcohol, the perchloride of mercury being removed after the
sections are cut, as will be afterwards described.
(d) Methylated Spirit. — Small pieces of tissue may be placed in
methylated spirit, which is to be changed after the first day. In from
six to seven days they will be hardened. If the pieces are large, a
longer time is necessary.
The Cutting of Sections. — 1. By Means of ttte Freezing
Microtome. — Pieces of tissue hardened by any of the above
methods must have all the alcohol removed from them by wash-
ing in running water for twenty-four hours. They are then
placed for from twelve to twenty-four hours (according to their
size) in a thick syrupy solution containing two parts of gum
arabic and one part of sugar. They are then cut on a freezing
microtome and placed for a few hours in a bowl of water so that
the gum and syrup may dissolve out. They are then stained or
they may be stored in methylated spirit.
1 In Britain ordinary commercial methylated spirit has wood naphtha
added to it to discourage its being used as a beverage. The naphtha being
insoluble in water a milky fluid results from the dilution of the spirit. By
law, chemists can only sell 8 ounces of pure spirit at a time. Most patho-
logical laboratories are, however, licensed by the Excise to buy "industrial
spirit," which contains only one -nineteenth of wood naphtha.
THE CUTTING OF SECTIONS 91
2. Embedding and Cutting in Solid Paraffin. — This method
gives by far the finest results, and should always be adopted
when practicable. The principle is the impregnation of the
tissue with paraffin in the melted state. This paraffin when it
solidifies gives support to all the tissue elements. The method
involves that, after hardening, the tissue shall be thoroughly
dehydrated, and then thoroughly permeated by some solvent
of paraffin which will expel the dehydrating fluid and prepare
for the entrance of the paraffin. The solvents most in use are
chloroform, cedar oil, xylol, and turpentine ; of these chloroform
and cedar oil are the best, the former being preferred as it per-
meates the tissue more rapidly. The more gradually the tissues
are changed from reagent to ceagent in the processes to be gone
through, the more successful is the result. A necessity of the
process is an oven with hot-water jacket, in which the paraffin
can be kept at a constant temperature just above its melting-
point, a gas regulator, e.g. Beichert's, being of course necessary.
The tissues occurring in pathological work have a tendency to
"become brittle if overheated, and therefore the best results are
not obtained by using paraffin melting about 58° C., such as is
employed in most biological laboratories. We have used for
some years a mixture of one part of paraffin, melting at 48°, and
two parts of .paraffin melting at 54° C. This mixture has a
melting-point between 52° and 53° C., and it serves all ordinary
purposes well. An excellent quality of paraffin is that known
as the " Cambridge paraffin," but many scientific-instrument
makers supply paraffins which, for ordinary purposes, are quite
as good, and much cheaper. The successive steps in the process
of paraffin embedding are as follows : —
1. Pieces of tissue, however hardened, are placed in fresh absolute
alcohol for twenty-four hours in order to their complete dehydration.
2. Transfer now to a mixture of equal parts of absolute alcohol and
chloroform for twenty-four hours.
3. Transfer to pure chloroform for twenty-four hours or longer. At
the end of this time the tissues should sink or float heavily.
4. Transfer now to a mixture of equal parts of chloroform and paraffin
and place on the top of the oven for from twelve to twenty-four hours.
If the temperature there is not sufficient to keep the mixture melted
then they must be put inside.
5. Place in pure melted paraffin in the oven for twenty-four hours.
For holding the paraffin containing the tissues, small tin dishes such as are
used by pastry-cooks will be found very suitable. There must be a
considerable excess of paraffin over the bulk of tissue present, otherwise
sufficient chloroform will be present to vitiate the final result and not
give the perfectly hard block obtained with pure paraffin. With ex-
perience, the persistence of the slightest trace of chloroform can be
recognised by smell.
92 MICROSCOPIC METHODS
In the case of very small pieces of tissue the time given for eacli stage •
may be much shortened, and where haste is desirable Nos. 2 and 4 may
be omitted. Otherwise it is better to carry out the process as de-
scribed. When it is advisable to avoid all shrinkage it is well to change
the paraffin every few hours during the embedding process.
6. Cast the tissues in blocks of paraffin as follows : Pairs of L- shaped
pieces of metal made for the purpose by instrument makers must be at
hand. By laying two of these together on a glass plate, a rectangular
trough is formed. This is filled with melted paraffin taken from a stock
in a separate dish. In it is immersed the piece of tissue, which is lifted
out of its pure paraffin bath with heated forceps. The direction in
which it is to be cut must be noted before the paraffin becomes opaque.
When the paraffin has begun to set, the glass plate and trough have
cold water run over them. When the block is cold, the metal L's are
broken off, and, its edges having been pared, it is stored in a pill-box.
The Cutting of Paraffin Sections. — Sections must be cut as
thin as possible, the Cambridge rocking microtome being, on
the whole, most suitable. They should not exceed 8 /x, in thick-
FIG. 43. — Needle with square of paper on end for manipulating paraffin
sections.
ness, and ought, if possible, to be about 4 p. For their mani-
pulation it is best to have two needles on handles, two camel's-
hair brushes on handles, and a needle with a rectangle of stiff
writing paper fixed on it as in the diagram (Fig. 43). When
cut, sections are floated on the surface of a beaker of water kept
at a temperature about 10° C. below the melting-point of the
paraffin. On the surface of the warm water they become perfectly
flat.
Fixation on Ordinary Slides, (a) Gulland's Method.— A. supply of
slides well cleaned being at hand, one of them is thrust obliquely "into
the water below the section, a corner of the section is fixed on it with a
needle and the slide withdrawn. The surplus of water being wiped off
with a cloth, the slide is placed on a support, with the section down-
wards, and allowed to remain on the top of the paraffin oven or in a
bacteriological incubator for from twelve to twenty-four hours. It will
then be sufficiently fixed on the slide to withstand all the manipulations
necessary during staining and mounting.
(b) Fixation by Mann's Method. — This has the advantage of being
more rapid than the previous one. A solution of albumin is prepared
by mixing the white of a fresh egg with ten parts of distilled water and
filtering. Slides are made perfectly clean with alcohol. One is dipped
into the solution and its edge is then drawn over one surface of another
slide so as to leave on it a thin film of albumin. This is repeated with
the others. As each is thus coated, it is leant, with the film down-
DEHYDRATION AND CLEARING 93
wards, on a ledge till dry, and then the slides are stored in a wide
stoppered jar till needed. The floating out is performed as before.
The albuminised side of the slide is easily recognised by the fact that if
it is breathed on, the breath does not condense on it. The great
advantage of this method is that the section is fixed after twenty to
thirty minutes' drying at 37° C. If the tissue has been hardened in any
of the bichromate solutions and embedded in paraffin, this or some
corresponding method of fixing the sections on the slide must be used.
Preparation of Paraffin Sections for Staining. — Before stain-
ing, the paraffin must be removed from the section. This is
best done by dropping on xylol out of a drop bottle. When the
paraffin is dissolved out, the superfluous xylol is wiped off with
a cloth and a little absolute alcohol dropped on. When the
xylol is removed the superfluous alcohol is wiped off and a
little 50 per cent methylated spirit dropped on. During these
procedures sections must on no account be allowed to dry.
The sections are now ready to be stained. Deposits of crystals
of corrosive sublimate often occur in sections which have been
fixed by this reagent. These can be removed by placing the
sections, before staining, for a few minutes in equal parts of
Gram's iodine solution (p. 99) and water, and then washing out
the iodine with methylated spirit.
To save repetition we shall in treating of stains suppose that,
with paraffin sections, the above preliminary steps have already
been taken, and further that sections cut by a freezing microtome
are also in spirit and water.
Dehydration and Clearing. — It is convenient, first of all, to
indicate the final steps to be taken after a specimen is stained.
Dry films after being stained are washed in water, dried and
mounted in xylol balsam • ivet Jilms and sections must be
dehydrated, cleared, and then mounted in xylol balsam.
Dehydration is most commonly effected with absolute alcohol.
Alcohol, however, sometimes decolorises the stained organisms
more than is desirable, and therefore Weigert devised the
following method of dehydrating and clearing by aniline oil,
which, though it may decolorise somewhat, does not do so to the
same extent as alcohol. As much as possible of the water being
removed, the section placed on a slide is partially dried by
draining with fine blotting-paper. Some aniline oil is placed on
the section and the slide moved to and fro. The section is
dehydrated and becomes clear. The process may be accelerated
by heating gently. The preparation is then treated with a
mixture of two parts of aniline oil and one part of xylol, and
then with xylol alone, after which it is mounted in xylol balsam.
Balsam as ordinarily supplied has often an acid reaction, and
94 MICROSCOPIC METHODS
preparations stained with aniline dyes are apt to fade when
mounted in it. It is accordingly a great advantage to use the
acid-free balsam supplied by Griibler. Paraffin sections can
usually be dehydrated and cleared by the mixture of aniline oil
and xylol alone.
Sections stained for bacteria should always be cleared, at
least finally, in xylol, for the same reason that xylol balsam is
to be used for mounting films, viz. that it dissolves out aniline
dyes less readily than such clearing reagents as clove oil, etc.
Xylol, however, requires the previous dehydration to have been
more complete than clove oil, which will clear a section readily
when the dehydration has been only partially effected by, say,
methylated spirit. If a little decolorisation of a section is still
required before mounting, clove oil may be used to commence
the clearing, the process being finished with xylol. With a little
experience the progress, not only of these processes but also of
staining, can be very accurately judged of by observing the
appearances under a low objective.
THE STAINING OF BACTERIA.
Staining Principles. — To speak generally, the protoplasm of
bacteria reacts to ^stains in a manner similar to the nuclear
chromatin, though sometimes more and sometimes less actively.
The bacterial stains par excellence are the basic aniline dyes.
These dyes are more or less complicated compounds derived
from the coal-tar product aniline (C(5H5 . NH2). Many of them
have the constitution of salts. Such compounds are divided
into two groups according as the staining action depends on the
basic or the acid portion of the molecule. Thus the acetate of
rosaniline derives its staining action from the rosaniline. It is
therefore called a basic aniline dye. On the other hand,
ammonium picrate owes its action to the picric acid part of the
molecule. It is therefore termed an acid aniline dye. These
two groups have affinities for different parts of the animal cell.
The basic stains have a special affinity for the nuclear chromatin,
the acid for the protoplasm and various formed elements. Thus
it is that the former — the basic aniline dyes — are especially the
bacterial stains.
The number of basic aniline stains is very large. The following are
the most commonly used : —
Violet Stains. — Methyl- violet, R-5R (synonyms : Hoffmann's violet,
dahlia).
Gentian-violet (synonyms : benzyl-violet, Pyoktanin).
THE STAINING OF BACTERIA 95
Crystal violet.
Blue Stains. — Methylene-blue1 (synonym : phenylene-blue).
Victoria- blue.
Thionin-blue.
Red Stains.— -Basic fuchsin (synonyms : basic rubin, magenta).
Safranin (synonyms : fuchsia, Girofle).
Brown Stain. — Bismarck- brown (synonyms: vesuvin, phenylene-
brown).
It is of the greatest importance that the stains used by the
bacteriologist should be good, and therefore it is advisable to
obtain those prepared by Griibler of Leipzig. One is then
perfectly sure that one has got the right stain.
Of the stains specified, the violets and reds are the most
intense in action, especially the former. It is thus easy in using
them to overstain a specimen. Of the blues, methylene-blue
probably gives the best differentiation of structure, and it is
difficult to overstain with it. Thionin-blue also gives good
differentiation and does not readily overstain. Its tone is deeper
than that of methylene-blue and it approaches the violets in tint.
Bismarck- brown is a weak stain, but is useful for some purposes.
Formerly it was much used in photomicrographic work, as it
was less actinic than the other stains. It is not, however,
needed now, on account of the improved sensitiveness of plates.
It is most convenient to keep saturated alcoholic solutions
of the stains made up, and for use to filter a little into about
ten times its bulk of distilled water in a watch-glass. A solution
of good body is thus obtained. Most bacteria (except those of
tubercle, leprosy, and a few others) will stain in a short time in
such a fluid. Watery solutions may also be made up, e.g. a
saturated watery solution of methylene-blue or a 1 per cent
solution of gentian- violet. Stains must always be filtered before
use ; otherwise there may be deposited on the preparation
granules which it is impossible to wash off. The violet stains in
solution in water have a great tendency to decompose. Only
small quantities should therefore be prepared at a time.
The Staining of Cover-glass Films. — Films are made from
cultures as described above. The cover-glass may be floated on
the surface of the stain in a watch-glass, or the cover-glass held in
forceps with film side uppermost may have as much stain poured
on it as it will hold. When the preparation has been exposed for
the requisite time, usually a few minutes, it is well washed in
tap water in a bowl, or with distilled water with such a simple
contrivance as that figured (Fig. 44). The figure explains itself.
1 Tliis is to be distinguished from methyl-blue, which is a different com-
pound.
96
MICROSCOPIC METHODS
When the film has been washed the surplus of water is drawn
off with a piece of filter-paper, the preparation is carefully dried
high over a flame, a drop of xylol balsam is applied, and the
cover-glass mounted on a slide. It is sometimes advantageous
to examine films in a drop of water in place of balsam. The.
films can be subsequently dried and mounted permanently. In
the case of tubercle, special stains are necessary (p. 100), but with
this exception, practically all bacterial films made from cultures
can be stained in this way. Some bac-
teria, e.g. typhoid, glanders, take up the
stains rather slowly, and for these the
more intensive stains, red or violet, are
to be preferred.
Films of fluids from the body (blood,
pus, etc.) can be generally stained in the
same way, and this is often quite suffi-
cient for diagnostic purposes. The blue
dyes are here preferable, as they do not
readily overstain. In the case of such
fluids, if the histological elements also
claim attention it is best first to stain
the cellular protoplasm with a one to
two per cent watery solution of eosin
(which is an acid dye), and then to use
a blue which will stain the bacteria and
the nuclei of the cells. The Romanowsky
stains (v. p. 105) are here most useful, as
by these the preparations are fixed as
well as stained. Fixation by heat which
is apt to injure delicate cellular structures
is thus avoided. In the case of films
made from urine, where there is little
or no albuminous matter present, the
bacteria may be imperfectly fixed on
the slide, and are thus apt to be washed off. In such a case it
is well to modify the staining method. A drop of stain is
placed on a slide, and the cover-glass, film-side down, lowered
upon it. After the lapse of the time necessary for staining,
a drop of water is placed at one side of the cover-glass and a
little piece of filter-paper at the other side. The result is that
the stain is sucked out by the filter-paper. By adding fresh
drops of water and using fresh pieces of filter-paper, the speci-
men is washed without any violent application of water, and
the bacteria are not displaced.
FIG. 44. — Syphon wash-
bottle for distilled water
used in washing prepara-
tions.
MORDANTS AND DECOLORISING AGENTS 97
For the general staining of films a saturated watery solution
of methylene-blue will be found to be the best stain to com-
mence with, the Gram method (v. infra) is then applied, and
subsequently any special stains which may appear advisable.
The Use of Mordants and Decolorising Agents. — In films of
blood and pus, and still more so in sections of tissues, if the above
methods are used, the tissue elements may be stained to such an
extent as to quite obscure the bacteria. Hence many methods
have been devised in which the general principle may be said to
be (a) the use of substances which, while increasing the staining
.power, tend to fix the stain in the bacteria, and (b) the subse-
quent treatment by substances which decolorise the overstained
tissues to a greater or less extent, while they leave the bacteria
coloured. The staining capacity of a solution may be increased —
(a) By the addition of substances such as carbolic acid,
aniline oil, or metallic salts.
(6) By the addition of alkalies, such as caustic potash or
ammonium carbonate, in weak solution.
(c) By the employment of heat.
(d) By long duration of the staining process.
As decolorising agents we use chiefly mineral acids (hydro-
chloric, nitric, sulphuric), vegetable acids (especially acetic acid),
alcohol (either methylated spirit or absolute alcohol), or a com-
bination of spirit and acid, e.g. methylated spirit with a drop or
two of hydrochloric acid added, also various oils, e.g. aniline,
clove, etc. In most cases about thirty drops of acetic acid in a
bowl of water will be sufficient to remove the excess of stain
from over-stained films and sections. More of the acid may, of
course, be added if necessary.
Hot water also decolorises to a certain extent ; over-stained
films can be readily decolorised by placing a drop of water on
the film and heating gently over a flame.
When preparations have been sufficiently decolorised by an
acid, they should be well washed in tap water, or in distilled
water with a little lithium carbonate added.
The methods embracing the use of a stain with a mordant,
and a decoloriser, are very numerous, and we can only enumerate
the best of them.
Different organisms take up and retain the stains with
various degrees of intensity, and thus duration of staining and
decolorising must be modified accordingly. We sometimes have
to deal with bacteria which show a special tendency to be
decolorised. This tendency can be obviated by adding a little
98 MICROSCOPIC METHODS
of the stain to the alcohol, or aniline oil, employed in dehydra-
tion. In the latter case a little of the stain is rubbed down in
the oil. The mixture is allowed to stand. After a little time a
clear layer forms on the top with stain in solution, and this can
be drawn off with a pipette.
When methylene-blue, methyl-violet, or gentian-violet is used,
the stain can, after the proper degree of decolorisation has been
reached, be fixed in the tissues by treating for a minute with
ammonium molybdate (2£ per cent in water).
The Formulae of some of the more commonly used Stain Combinations.
1. Ldfflers Methylene-blue.
Saturated solution of methylene-blue in alcohol . . 30 c.c.
Solution of potassium hydrate in distilled water (1-10,000) . 100 ,,
(This dilute solution may be conveniently made by adding 1 c.c. of a
1 per cent solution to 99 c.c. of water.)
Sections may be stained in this mixture for from a quarter of an hour
to several hours. They do not readily overstain. The tissue containing
the bacteria is then decolorised if necessary with £-1 per cent acetic acid,
till it is a pale blue-green. The section is washed in water, rapidly
dehydrated with alcohol or aniline oil, cleared in xylol, and mounted.
The tissue may be contrast stained with eosin. If this is desired,
after decolorisation wash with water, place for a few seconds in 1 per cent
solution of eosin in absolute alcohol, rapidly complete dehydration with
pure absolute alcohol, and proceed as before.
Films may be stained with Lbffler's blue by five minutes' exposure or
longer in the cold. They usually do not require decolorisation, as the
tissue elements are not overstained.
2. Kiihne's Methylene-blue.
Methylene-blue . . . . 1*5 gr.
Absolute alcohol . . . . 10 c.c.
Carbolic acid solution (1-20) . . 100 ,,
Stain and decolorise as with Loffler's blue, or decolorise with very
weak hydrochloric acid (a few drops in a bowl of water).
3. Carbol-Thionin-blue. — Make up a stock solution consisting of 1
gramme of thionin-blue dissolved in 100 c.c. carbolic acid solution (1-40).
For use, dilute 1 volume with 3 of water and filter. Stain sections for
five minutes or upwards. Wash very thoroughly with water, otherwise
a deposit of crystals may occur in the subsequent stages. Decolorise
with very weak acetic acid. A few drops of the acid added to a bowl
of water are quite sufficient. Wash again thoroughly with water.
Dehydrate with absolute alcohol. Thionin-blue stains more deeply
than methylene-blue, and gives equally good differentiation. It is very
suitable for staining typhoid and glanders bacilli in sections. Cover-
glass preparations stained by this method do not usually require
decolorisation. As a contrast stain, 1 per cent watery solution of eosin
may be used before staining with the thionin.
4. Gentian-violet in Aniline Oil Water. — Two solutions have here to
be made up. (a) Aniline oil water. Add about 5 c.c. aniline oil to
100 c.c. distilled water in a flask, and shake violently till as much as
possible of the oil has dissolved. Filter and keep in a covered bottle
GRAM'S STAIN 99
to prevent access of light. (&) Make a saturated solution of gentian-
violet in alcohol. When the stain is to be used, 1 part of (6) is
added to 10 parts of (a), and the mixture filtered. The mixture should
be made nob more than twenty-four hours before use. Stain sections
for a few minutes ; then decolorise with methylated spirit. Sometimes
it is advantageous to add to the methylated spirit a little hydrochloric
acid (2-3 minims to 100 c.c. ). This staining solution is not so much
used by itself, as in Gram's method, which is presently to be described:
5. Carrol- Gentian-Violet. — 1 part of saturated alcoholic solution of
gentian-violet is mixed with 10 parts of 5 per cent solution of carbolic
acid. It is used as No. 4.
6. Carbol-Fuchsin (see p. 101). — This is a very powerful stain, and,
when used in the undiluted condition, ^-1 minute's staining is usually
sufficient. It is better, however, to dilute with from five to ten times its
volume of water and stain for a few minutes. In this form it has a very
wide application. Methylated spirit with or without a few drops of
acetic acid is the most convenient decolorising agent. Then dehydrate
thoroughly, clear, and mount.
Gram's Method and its Modifications. — In the methods
already described the tissues, and more especially the nuclei,
retain some stain when decolorisation has reached the point to
which it can safely go without the bacteria themselves being
affected. In the method of Gram, now to be detailed, this does
not occur, for the stain can here be removed completely from
the ordinary tissues, and left only in the bacteria. All kinds of
bacteria, however, do not retain the stain in this method, and
therefore in the systematic description of any species it is
customary to state whether it is, or is not, stained by Gram's
method — by this is meant, as will be understood from what has
been said, whether the particular organism retains the colour
after the latter has been completely removed from the tissues. It
must, however, be remarked that some tissue elements may retain
the stain as firmly as any bacteria, e.g. keratinised epithelium,
calcified particles, the granules of mast cells, and sometimes
altered red blood corpuscles, etc.
In Gram's method the essential feature is the treating of the
tissue, after staining, with a solution of iodine. This solution is
spoken of as Gram's solution, and has the following composition : —
Iodine ..... 1 part
Potassium iodide ... 2 parts
Distilled water . . . 300 „
The following is the method : —
1. Stain in aniline oil gentian- violet or in carbol-gentian- violet (vide
supra) for about five minutes, and wash in water.
2. Treat the section or film with Gram's solution till its colour
becomes a purplish black — generally about half a minute or a minute is
sufficient for the action to take place.
100 MICROSCOPIC METHODS
3. Decolorise with absolute alcohol or methylated spirit till the
colour has almost entirely disappeared, the tissues having only a faint
violet tint.
4. Dehydrate completely, clear with xylol and mount. In the case
of film preparations, the specimen is simply washed in water, dried and
mounted.
In stage (3) the process of decolorisation is more satisfactorily
performed by using clove oil after sufficient dehydration with alcohol,
the clove oil being afterwards removed by xylol.
As a contrast stain for the tissues carmalum or lithia carmine is used
before staining with gentian-violet (1). As a contrast stain for other
bacteria which are decolorised by Gram's method carbol-fuchsin diluted
with ten volumes of water or a saturated watery solution of Bismarck -
brown may be used before stage (4).
The following modifications of Gram's method may be given : —
1. Weigerfs Modification. — The contrast staining of the tissues and
stages (1) and (2) are performed as above.
(3) After using the iodine solution the preparation is dried by
blotting and then decolorised by aniline-xylol (aniline-oil 2, xylol 1).
(4) Wash well in xylol and mount in xylol balsam. Film preparations
after being washed in xylol may be dried and thereafter dilute carbol-
fuchsin may be used to stain bacteria which have been decolorised.
This modification probably gives the most uniformly successful results.
2. Nicolles Modification. — Carbol-gentian-violet is used as the stain.
Treatment with iodine is carried out as above and decolorisation is
effected with a mixture of acetone (1 part) and alcohol ^2 parts).
3. Kuhnes Modification. — (1) Stain for five minutes in a solution
made up of equal parts of saturated alcoholic solution of crystal-violet
(" Kry stall- vi olet ") and 1 per cent solution of ammonium carbonate.
(2) Wash in water.
(3) Place for two to three minutes in Gram's iodine solution, or in
the following modification by Kuhne : —
Iodine ...... 2 parts
Potassium iodide . . . . 4 ,,
Distilled water . ' . . . . 100 ,,
For use, dilute with water to make a sherry-coloured solution.
(4) Wash in water.
(5) Decolorise in a saturated alcoholic solution of fluorescein (a
saturated solution in methylated spirit does equally well).
(6) Dehydrate, clear and mount.
There is great variability in the avidity with which organisms stained
by Gram retain the dye when washed with alcohol, and sometimes
difficulty is experienced in saying whether an organism does or does not
stain by this method.
Stain for Tubercle and other Acid -fast Bacilli. — These
bacilli cannot be well stained with a simple watery solution of a
basic aniline dye. This fact can easily be tested by attempting
to stain a film of a tubercle culture with such a solution. They
require a powerful stain containing a mordant, and must be
exposed to the stain for a long time, or its action may be aided
TUBERCLE STAINS 101
by a short application of heat. When once stained, however,
they resist decolorising even with very powerful acids ; they are
therefore called " acid-fast." The smegma bacillus also resists
decolorising with strong acids (p. 254), and a number of other
acid-fast bacilli have recently been discovered (p. 252). Any
combination of gentian-violet or fuchsin with aniline oil or
carbolic acid or other mordant will stain the bacilli named, but
the following methods are most commonly used : —
Ziehl-Neelsen Carbol-Fuchsin Stain.
t
Basic fuchsin . . . 1 part
Absolute alcohol . . . .10 parts
Solution of carbolic acid (1 : 20) . 100 „
1. Place the specimen in this fluid, and having heated it till steam
rises, allo\v it to remain there for five minutes, or allow it to remain in
the cold stain for from twelve to twenty-four hours. (Films and paraffin
sections are usually stained with hot stain, loose sections with cold ; in
hot stain the latter shrink.)
2. Decolorise with 20 per cent solution of strong sulphuric acid, nitric
acid, or hydrochloric acid, in water. In this the tissues become yellow.
3. Wash well with water. The tissues will regain a faint pink tint.
If the colour is distinctly red, the decolorisation is insufficient, and the
specimen must be returned to the acid. As a matter of practice, it is
best to remove the preparation from the acid every few seconds ani
wash in water, replacing the specimen in the acid and re- washing till
the proper pale pink tint is obtained. Then wash in alcohol for half a
minute and replace in water.
4. Contrast stain with a saturated watery solution of rnethylene-blue
for half a minute, or with saturated watery Bismarck-brown for from
two to three minutes.
5. Wash well with water. In the case of films, dry and mount. In
the case of sections, dehydrate, clear and mount.
Fraenkel's Modification of the Ziehl-Neelsen Stain.
Here the process is shortened by using a mixture containing
both the decolorising agent and the contrast stain.
The sections or films are stained with the carbol- fuchsin as above
described, and then placed in the following solution : —
Distilled water . .. . . i 50 parts
Absolute alcohol . . '. . . 30 ,,
Nitric acid 20 ,,
Methylene-blue in crystals to saturation.
They are treated with this till the red colour has quite disappeared and
been replaced by blue. The subsequent stages are the same as in No.
5, supra.
102 MICROSCOPIC METHODS
Leprosy bacilli are stained in the same way, but are rather
more easily decolorised than tubercle bacilli, and it is better to
use only 5 per cent sulphuric acid in decolorising.
In the case of specimens stained either by the original Ziehl-
Neelsen method, or by Fraenkel's modification, the tubercle or
leprosy bacilli ought to be bright red, and the tissue blue or
brown, according to the contrast stain used. Other bacteria
which may be present are also coloured with the contrast stain.
The Staining of Spores. — If bacilli containing spores are
stained with a watery solution of a basic aniline dye the spores
remain unstained. The spores either take up the stain less
readily than the protoplasm of the bacilli or they have a resisting
envelope which prevents the stain penetrating to the protoplasm.
Like the tubercle bacilli, when once stained they retain the colour
with considerable tenacity. The following is the simplest method
for staining spores : —
1. Stain cover-glass films as for tubercle bacilli.
2. Decolorise with 1 per cent sulphuric acid in water or with methyl-
ated spirit. This removes the stain from the bacilli.
3. Wash in water.
4. Stain with saturated watery methylene-blue for half a minute.
5. Wash in water, dry, and mount in balsam.
The result is that the spores are stained red, the protoplasm of the
bacilli blue.
The spores of some organisms lose the stain more readily than those
of others, and for some, methylated spirit is a sufficiently strong
decolorising agent for use. If sulphuric acid stronger than 1 per cent
is used the spores of many bacilli are readily decolorised.
Moller's Method. — The following method, recommended by Mbller, is
much more satisfactory than the previous. Before being stained, the films
are placed in chloroform for 2 minutes, and then in a 5 per cent solution
of chromic acid for £-2 minutes, the preparation being well washed
after each reagent. Thereafter they are stained and decolorised as above.
The Staining of Capsules. — The two following methods may
be recommended in the case of capsulated bacteria : —
(a) Welch's Method. — This depends on the fact that in many cases
the capsules can be fixed with glacial acetic acid.
Films when still wet are placed in this acid for a few seconds.
The superfluous acid is removed with filter-paper and the preparation
is treated with gentian-violet in aniline oil water repeatedly till all the
acetic acid is removed.
Then wash with 1-2 per cent solution of sodium chloride and examine
in the same solution.
The capsule appears as a pale violet halo around the deeply stained
bacterium.
(b) Richard Muirs Method (as recently modified).
1. The film containing the bacteria must be very thin. It is dried
and stained in filtered carbol-fuchsin for half a minute, the preparation
being gently heated.
STAINING OF FLAGELLA 103
2. Wash slightly with spirit and then well in water.
3. Place in following mordant for a few seconds : —
Saturated solution of corrosive sublimate . . .2 parts
Tannic acid solution — 20 per cent . . . 2 ,,
Saturated solution of potash alum . . . . 5 , ,
4. Wash well in water.
5. Treat with methylated spirit for about a minute.
The preparation has a pale reddish appearance.
6. Wash well in water.
7. Counterstain with watery solution of ordinary methylene-blue for
half a minute.
8. Dehydrate in alcohol, clear in xylol, and mount in balsam.
The bacteria are a deep crimson and the capsules of a blue tint. The
capsules of bacteria in cultures may sometimes be demonstrated by this
method.
The Staining of Flagella. — The staining of the flagella of
bacteria is the most difficult of all bacteriological procedures,
and it requires considerable practice to ensure that good results
shall be obtained. Many methods have been introduced, of
which the two following are the most satisfactory.
Preparation of Films. — In all the methods of staining
flagella, young cultures on agar should be used, say a culture
incubated for from ten to eighteen hours at 37° C. A very
small portion of the growth is taken on the point of a platinum
needle and carefully mixed in a little water in a watch-glass ;
the amount should be such as to produce scarcely any turbidity
in the water. A film is then made by placing a drop on a
clean cover-glass and carefully spreading it out with the needle.
It is allowed to dry in the air and is then passed twice or thrice
through a flame, care being taken not to over-heat it. The
cover-glasses used should always be cleaned in the mixture of
sulphuric acid and potassium bichromate described on page 87.
1. Pitfield's Method as modified by Richard Muir.
Prepare the following solutions : —
A. The Mordant.
Tannic acid, 10 per cent watery solution, filtered . 10 c.c.
Corrosive sublimate, saturated watery solution
Alum, saturated watery solution . . . 5 ,,
Carbol-fuchsin (vide p. 101) . . . . 5 ,,
Mix thoroughly. A precipitate forms, which must be allowed to
deposit, either by centrifugalising 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 ,,
104 MICROSCOPIC METHODS
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 mordant 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, allowing to steam for about a minute,,
wash well in water, dry and mount in a drop of xylol balsam.
This method has yielded the best results in our hands.
2. Van Ermengems Method for Staining Flagella.
The films are prepared as above described. Three solutions are here
necessary : —
Solution A. (Bain fixateur) —
Osmic acid, 2 per cent solution .... 1 part
Tannin, 10-25 per cent solution .... 2 parts
Place the films in this for one hour at room temperature, or heat over
a flame till steam rises and keep in the hot stain for five minutes.
Wash with distilled water, then with absolute alcohol for three to four
minutes, and again in distilled water, and treat with
Solution B. (Bain sensibilisateur] —
*5 per cent solution of nitrate of silver in distilled water. Allow
films to be in this a few seconds. Then without washing transfer to
Solution C. (Bain reducteur et reinforqateur) —
Gallic acid 5 grm.
Tannin 3 ,,
Fused potassium acetate . . . . . 10 ,,
Distilled water . . . . . . . 350 c.c.
Keep in this for a few seconds. Then treat again with Solution B till
the preparation begins to turn black. Wash, dry, and mount.
It is better, as Mervyn Gordon recommends, to leave the specimen in
B for two minutes and then to transfer to C for one and a half to two
minutes, and not to transfer again to B. It will also be found an
advantage to use a fresh supply of C for each preparation, a small
quantity being sufficient. The beginner will find the typhoid bacillus
or the bacillus coli communis very suitable organisms to stain by this
method.
Although the results obtained by this method are sometimes excellent,
they vary considerably. Frequently both the organisms and flagella
appear of abnormal thickness. This is due to the fact that the process
on which the method depends, is a precipitation rather than a true
staining. The pictures on the whole are less faithful than in the first
method.
Staining of Spirochsete in Sections. — The following im-
pregnation method, which is practically that of Ramon-y-Cajal
THE ROMANOWSKY STAINS 105
for nerve fibrillse, has been applied for this purpose by Levaditi
and gives excellent results.
(1) The tissues, which ought to be in thin slices, about 1 mm.
in thickness, are best fixed in 10 per cent formalin solution for
twenty-four hours.
(2) They are washed for an hour in water and then brought
into 96 per cent alcohol for twenty-four hours.
(3) They are then placed in 1'5 per cent solution of nitrate
of silver in a dark bottle, and are kept in an incubator at 37° C.
for three days.
(4) They are washed in water for about twenty minutes, and
are thereafter placed in the following mixture, namely : —
Pyrogallic acid, 4 parts.
Formalin, 5 parts.
Distilled water up to 100 parts.
They are kept in this mixture in a dark bottle for forty-eight
hours at room temperature.
(5) They are then washed in water for a few minutes, taken
through increasing strengths of alcohol, and embedded in
paraffin in the usual way. The sections ought to be as thin as
possible. In satisfactory preparations the spirochaetes appear of
an almost black colour against the pale yellow background of
the tissues. The latter can be contrast stained by weak carbol-
fuchsin or by toluidin blue.
(For the staining of spirocheetes in films see p. 107.)
The Romanowsky Stains. — Within recent years the numerous
modifications of the Romanowsky stain have been extensively
used. The dye concerned is the compound which is formed
when watery solutions of medicinal methylene-blue and water-
soluble eosin are brought together. This compound is insoluble
in water but soluble in alcohol — the alcohol employed being
methyl alcohol. The stain was originally used by Romanowsky
for the malarial parasite, and its special quality is that it
imparts to certain elements, such as the chromatin of this
organism, a reddish-purple hue. This was at first thought to be
simply due to the combination of the methylene-blue and the
eosin, but it is now recognised that certain changes, such as
occur in methylene-blue solutions with age, are necessary. In
the modern formulae these changes are brought about by
treatment with alkalies, especially alkaline carbonates, as was
first practised by Unna in the preparation of his polychrome
methylene-blue. It is not certainly known to what particular
106 MICROSCOPIC METHODS
new body the reddish hue is due, but it may be to methyl-violet
or to methyl-azure, both of which result from the action of alkali
on methylene-blue. The stains are much used in staining blood-
films (in which the characters of both nucleus and cytoplasm are
beautifully brought out), in staining bacteria in tissues or
exudates, the malaria parasite, trypanosomes, the pathogenic
spirochsetes (such as the spirochaete pallida), and protozoa
generally.
The following are the chief formulae in use : —
1. Jenners Stain. — This is an excellent blood stain, but is not so good
for the study of parasites as the others to be mentioned. In its
preparation no alkali is used. It is made by mixing equal parts of (a)
a 1 '2 to 1 '25 per cent solution of Griibler's water soluble eosin (yellow
shade) in distilled water and (b) 1 per cent Griibler's medicinal methy-
lene-blue (also a watery solution). The mixture is allowed to stand
twenty-four hours, is filtered, and the residue is dried at 55° C. ; the
powder is shaken up in distilled water, filtered, washed with distilled
water and dried. Of the powder, 5 grms. are dissolved in 100 c.c. Merck's
methyl alcohol. For use a few drops are placed on the dried unfixed
film for one to three minutes, the dye is poured off, and the preparation
washed with distilled water till it presents a pink colour ; it is then
dried between filter-paper and mounted in xylol balsam.
2. Leishmans Stain. — The following solutions are prepared : (a) to
a 1 per cent solution of medicinal methylene-blue is added '5 per cent
sodium carbonate ; the mixture is kept at 65° C. for twelve hours and
then for ten days at room temperature ('25 per cent formalin may be
added as a preservative) ; (b) 1-1000 solution of eosin, extra B.A., in
distilled water. Equal volumes of the two solutions are mixed and
allowed to stand for six to twelve hours with occasional stirring, the
precipitate is collected, filtered, washed with distilled water and dried.
For use '15 per cent is dissolved in Merck's methyl alcohol ("for
analysis, acetone free ") as follows : the powder is placed in a clean
mortar, a little of the alcohol is added and well rubbed up with a
pestle ; the undissolved powder is allowed to settle and the fluid
decanted into a dry bottle ; the process is repeated with fresh fractions
of the solvent till practically all the stain is dissolved, and the bottle
is well stoppered ; the stain will keep for a long period. For the
staining of films a few drops of the stain are placed on the unfixed
preparation for fifteen to thirty seconds so as to cover it with a
shallow layer (the stain may be conveniently spread over the film
with a glass rod) and the film is tilted to and fro so as to prevent
drying. This treatment efficiently fixes the film by the action of the
methyl alcohol. About double the quantity of distilled water is now
dropped on the film, and the stain and diluent are quickly mixed with
the rod. Five minutes are now allowed for staining, and the stain is
then gently washed off with distilled water. A little of the water is
kept on the film for half a minute to intensify the colour contrasts in
the various cells. For certain special structures such as Schiiffner's dots
or Maurer's dots in the malarial parasite a longer staining (up to one
hour) may be necessary, and in any case it is well to practise being able
to control the depth of the staining effect by observation with a low
power objective. If a preparation is to be stained for a long time it
THE ROMANOWSKY STAINS 107
must be kept covered, and if in such cases a granular deposit is formed
this may be got rid of by a quick wash with absolute alcohol. If in blood
films the red corpuscles appear bluish instead of pink the colour may
be restored by washing the tilm with acetic acid, 1-1500. The film is
dried between filter-paper and mounted.
For staining sections a little modification is necessary. A paraffin
section is taken into distilled water as usual, the excess of water is drained
off, and a mixture of one part of stain' and two parts of distilled water
is placed on it. The stain is allowed to act for five to ten minutes till the
tissue appears a deep Oxford blue ; it is then decolorised with 1-1500
acetic acid — the effect being watched under a low-power lens. The blue
begins to come out, and the process is allowed to go on till only the
nuclei remain blue. The section is then washed with distilled water,
rapidly dehydrated with alcohol, cleared and mounted. If, as some-
times happens, the eosin tint be too well marked it can be lightened
by the action of 1-7000 solution of caustic soda, this being washed off
whenever the desired colour has been attained.
3. J. H. Wright's Stain. — In this modification 1 per cent methylene-
blue (BX or Ehrlich's rectified) and \ per cent sodium carbonate (both
in water) are mixed and placed in a Koch's steriliser for an hour. When
the fluid is cold, 1-1000 solution of extra B. A. eosin is added till the mixture
becomes purplish and a finely granular black precipitate appears in
suspension (about 500 c.c. eosin to 100 c.c. methylene-blue solution are
required) ; the precipitate is filtered off and dried without being washed.
A saturated solution of this is made in the pure methyl alcohol ; this is
filtered and diluted by adding to 80 c.c. of the saturated solution 20 c.c.
of methyl alcohol. The application of the stain is almost the same as
with Leishman's. A few drops are placed on the preparation for a
minute for fixation ; water is then dropped on till a green iridescent
scum appears on the top of the fluid and staining goes on for about
two minutes ; the stain is then washed off with distilled water and a
little is allowed to remain on the film till differentiation is complete ;
the preparation is carefully dried with filter-paper and mounted.
4. Giemsa's Stain. — Giemsa believes that the reddish-blue hue
characteristic of the Romanowsky stain is due to the formation of
methyl-azure, and he has prepared this by a method of his own under
the name " Azur I." From this, by the addition of equal parts of
medicinal methylene-blue, he prepares what he calls "Azur II.," and
from this again by the addition of eosin he prepares " Azur II. -eosin."
The latest formula for the finished stain is as follows : — Azur II. -eosin 3
gr., Azur II. 8 gr., Glycerin (Merck, chemically pure) 250 gr., Methyl
alcohol (Kahlbaum, I.) 250 gr. This stain has been extensively used
for demonstrating the Spirochsete pallida, but it can be used for any
other purpose to which the Romanowsky stains are applicable. For the
spirochsete the following are Giemsa's directions: —
(1) Fix films in absolute alcohol for fifteen to twenty minutes, dry
with I filter-paper. (2) Dilute stain with distilled water — one drop of
stain to 1 c.c. water (the mixture being well shaken). (Sometimes the
water is made alkaline by the addition of one drop of 1 per cent potassium
carbonate to 10 c.c. water). (3) Stain for fifteen minutes. (4) Wash in
brisk stream of distilled water. (5) Drain with filter-paper, dry, and
mount in Canada balsam.
With regard to the Jenner and Giemsa stains it is best to obtain the
solutions from Griibler ready for use ; the powder for Leishman's stain
may be obtained from the same source and the solution made up by
108
MICROSCOPIC METHODS
the worker himself. Cabot states that Wright's stain can be obtained
from the Harvard Co-operative Society, Boylston Street, Boston, U.S.A.
Neisser's Stain. — Neisser introduced the following stain as an aid to
the diagnosis of the diphtheria bacillus. Two solutions are used as
follows : (a) 1 grm. methylene-blue (Griibler) is dissolved in 20 c.c. of
96 per cent alcohol, and to the solution are added 950 c.c. of distilled
water and 50 c.c. of glacial acetic acid ; (&) 2 grins. Bismarck-brown
(vesuvin) dissolved in a litre of distilled water. Films are stained in
(a) for 1-3 seconds or a little longer, washed in water, stained for 3-5
seconds in (b), dried, and mounted. The protoplasm of the diphtheria
bacillus is stained a faint brown colour, the granules a blue colour.
Neisser considers that this reaction is characteristic of the organism,
provided that cultures on Loffler's serum are used and examined after
9-24 hours' incubation at 34-35° C. Satisfactory results are not always
obtained in the case of films prepared from
membrane, etc., but there is no doubt that here
also the method is one of considerable value.
SPECIAL BACTEKIOLOGICAL METHODS.
Wright's Methods of measuring small
amounts of Fluids. — In ordinary work fine
calibrated pipettes may be used for measur-
ing small quantities of fluids, but such
pipettes are not always available, and by
Wright's technique if a Gower's 5 c.mm.
haemocytometer pipette be at hand any
measurements may be undertaken, — in fact,
once the pipette now to be described (see
Fig. 45) is made we are independent of
other means of measurement. A piece of
quill tubing is drawn out to capillary
dimensions, and the extreme tip of it is
heated in a peep flame and then drawn out
till it is of the thickness of a hair though
FIG 45 —Wright's 5 st^ Possessing a bore. If the point be
c» nun. pipGttG, Aj ^ i i •
Casing of quill tubing ; into the tube the metal will be caught
B, rubber nipple ; C, where the tube narrows and will pass no
wax luting ; E to F, f urther — in fact, though air will pass,
-E
— -A
W
C
— F
E, hair capillary. this tube 5 c.mm. of mercury, measured
from a Gower's pipette, is run down till
it will go no further. A mark is made on the tube at the
proximal end of the mercury, which is now allowed to run out,
and the tube is carefully cut through at the mark. A piece
of ordinary quill tubing is drawrn out and broken off just
below where its narrowing has begun, the capillary tube has
TESTING OF PROPERTIES OF SERUM 109
some wax moulded round its middle, the hair end is slipped
through the broken-off end just mentioned, and the tube is fixed
in position as shown in the figure. A rubber nipple placed on
the end of the pipette completes the apparatus. If by pressing
the nipple the air be expelled from the pipette and the end
dipped under mercury, exactly 5 c.mm. will be taken up. Thus
when pressure on the nipple is relaxed, other tubes can be very
readily calibrated by the mercury being expelled into them and
its limits marked on their bores.
For measuring equal parts of different fluids the pipette
described in connection with agglutination is very useful
(see Fig. 46 d).
The Testing of Agglutinative and Sedimenting Properties
of Serum.
liv ti'i'jl lit! nation is meant the aggregation into clumps of
uniformly disposed bacteria in a Huid ; by sedimentation the"
of n deposit composed of such clumps, when the fluid
is allowed to stand. Sedimentation is thus the naked-eye evidence
of agglutination". The blood serum may acquire this clumping
power towards a particular organism under certain conditions ;
these being chiefly met with when the individual is suffering
from the disease produced by the organism, or has recovered from
it, or when a certain degree of immunity has been produced
artificially by injections of the organism. The nature of this
property will be discussed later. Here we shall only give the
technique by which the presence or absence of the property may
be tested. There are two chief methods, a microscopic and a
naked eye, corresponding to the effects mentioned above. In
both, the essential process is the bringing of the diluted serum
into contact with the bacteria uniformly disposed in a fluid. In
the former this is done on a glass slide, and the result is watched
under the microscope ; the occurrence of the phenomenon is
shown by the aggregation of the bacteria into clumps, and if the
organism is motile this change is preceded or accompanied by
more or less complete loss of motility. In the latter method
the mixture is placed in an upright thin glass tube ; sediment-
ation is shown by the formation within a given time (say 12 or
24 hours) of a somewhat flocculent layer at the bottom, the fluid
above being clear. Two points should be attended to : (a)
controls should always be made with normal serum, and (b) the
serum to be tested should never be brought in the undiluted
110
MICROSCOPIC METHODS
condition into contact with the bacteria. The stages of pro-
cedure are the following : —
1. Blood is conveniently obtained by pricking the lobe of the ear,
which ..should previously have been washed with a mixture of alcohol
and ether and allowed to
dry. The blood is drawn
up into a Wright's blood
capsule (Fig. 47) or into
the bulbous portion of a
capillary pipette, such as
in Fig. 46, a. (These pip-
ettes can be readily made
by drawing out quill glass
tubing in a flame. It is
convenient always to have
several ready for use.)
The pipette is kept in the
upright position, one end
being closed. For purposes
of transit, break off the
bulb at the constriction
and seal the ends. After
the serum has separated
from the coagulum the
bulb is broken through
near its upper end and the
serum removed by means
of another capillary pip-
ette. The serum is then
to be diluted.
2. The serum may be
diluted (a) by means of a
grad aated pipette — either
a leucocytometer pipette
(Fig. 46, b) or some corre-
sponding form. In this
way successive dilutions
of 1 : 10, 1 : 20, 1 : 100,
etc., can be rapidly made.
This is the best method.
, (b) By means of a capillary
\ / -§- n pipette with a mark on the
tube, the serum is drawn
b d up to the mark and then
. blown out into a glass
FIG. 46.-Tubes used in testing agglutinating and capsule . equal quantities
segmenting properties of serum. of bouillon are successively
measured in the same way
and added till the requisite dilution is obtained, (c) By means of a
platinum needle with a loop at the end (Delepine's method). A loopful
of serum is placed on a slide and the desired number of similar loopfuls
of bouillon are separately placed around on the slide. The drops are
then mixed.
A very convenient and rapid method of combining the steps 1 and 2
THE OPSONTC TECHNIQUE 111
is to draw a drop of Hood up to the mark 1-or "5 on a leucocytometer
pipette and draw the bouillon after it till the bulb is filled. A dilution
of 10 or 20 times is thus obtained. Then blow the mixture into a
U-shaped tube (Fig. 46, c] and centrifugalise or simply allow the red
corpuscles to separate by standing. (In this method of course the
dilution is really greater than if pure serum were used, and allowance
must therefore be made in comparing results.) The presence of red
corpuscles is no drawback in the case of the microscopic method, but wrhen
sedimentation tubes are used the corpuscles should be separated first.
3. The bacteria to be tested should be taken from young cultures,
preferably not more than twenty-four hours old, inciibated at 37° C.
They may be used either as a bouillon culture or as an emulsion made
by adding a small portion of an agar culture to bouillon. In the latter
case the mass of bacteria on a platinum loop should be gently broken
down at the margin of the fluid in a watch-glass. When a thick
turbidity is thus obtained, any remaining fragments should first be
removed and then the organisms should be uniformly mixed with the
rest of the fluid. The bacterial emulsion ought to have a faint but
distinct turbidity. (When the exact degree of sedimenting power of a
serum is to be tested — expressed as the highest dilution in which it
produces complete sedimentation within twenty-four hours — a standard
quantity (by weight) of bacteria must be added to a given quantity of
bouillon. This is not necessary for clinical diagnosis.)
4. To test microscopically, mix equal quantities (measured by a
marked capillary pipette) of the diluted serum and the bacterial
emulsion on a glass slide, cover with a cover-glass, and examine under
the microscope. The form of glass slide used for hang-drop cultures
(Fig. 27) will be found very suitable. The ultimate dilution of the
serum will, of course, be double the original dilution.
To observe sedimentation mix equal parts of diluted serum and of
bacterial emulsion and place in a thin glass tube — a simple tube with
closed end or a U-tube. Keep in upright position for twenty-four
hours. One of Wright's sedimentation tubes is shown in Fig. 46, d.
Diluted serum is drawn up to fill the space mn, a small quantity of air
is sucked up after it to separate it from the bacterial emulsion, which
is then drawn up in the same quantity ; the diluted serum will then
occupy the position Jcl. The fluids are then drawn several times up
into the bulb and returned to the capillary tube so as to mix, and finally
blown carefully down close to the lower end, which is then sealed off'.
The sediment collects at the lower extremity.
It may be said that.it is often important to observe not only the
•strongest concentration of a serum which will produce agglutination but
also the weakest.
Method of measuring the Phagocytic Capacity of the
Leucocytes — the Opsonic Technique. — This was first done
by Leishman by a very simple method as follows : A piece
of quill tubing is drawn out to a capillary diameter so as
to make a pipette about six inches long. The point is
broken off and a rubber nipple adjusted to the wide end, a
mark is made with an oil pencil about three-quarters of an
inch above the orifice. Blood is drawn from the finger up
to the mark, then an air-bubble is allowed to pass in. A thin
112 MICROSCOPIC METHODS
emulsion of the bacterium to be tested having been prepared, a
quantity of this is also drawn up to the mark. The two fluids
are then thoroughly mixed by being first blown out on to a
sterile slide and then being drawn back into the pipette and
expelled, — this being repeated several times. A cover-glass is
placed over the drop, and the slide is placed in the incubator at
37° C. for fifteen minutes. The cover-glass is then slipped off
so as to make a film preparation which in the case of ordinary
bacteria may be stained by Leishman's method. The number
of bacteria present in, say, 50 polymorphonuclear cells succes-
sively examined is determined and an average struck. The
method was first used for showing that in cases of staphylococcus
infection the average number of bacteria taken up was less than
in a control in which the same bacterial emulsion was exposed
to the blood of a healthy individual. In making such an
observation drops from the two mixtures are placed on the same
slide under separate cover-glasses and the preparation incubated.
One cover is then slipped to one end of the slide and the other to
the other, — the two films being then stained as one.
According to Wright's view the process of phagocytosis in
blood outside the body is not a simple one, and^e^pj^.^,
leucocyte takes up a bacterium the latter most be acted on_
in some way by substances present in the serum, which Wrignt
'c&lls^ogsoniiis^ (see Immunity). The technique by which the
actions of these opsonins is studied has been elaborated by
Wright and his co-workers in connection with his work on
bacterial vaccines, especially in relation to infection by the
pyogenic cocci and the tubercle bacillus. This technique
involves (1) the preparation of the bacterial emulsion, (2) the
preparation of the leucocytes, (3) the preparation of samples of
(a) serum from a normal person, (6) serum from the infected
person.
(1) Preparation of bacterial emulsion. In the case of the
pyogenic cocci a little of a twenty-four hour living culture off a
sloped agar tube is taken and rubbed up in a watch-glass with
•85 per cent saline. The mixture is placed in a tube and centri-
fugalised so as to deposit any masses of bacteria which may be
present. Only by experience can a knowledge be gained of the
amount of culture to be used in the first instance, but the
resultant emulsion usually should exhibit only the merest trace of
cloudiness to the naked eye. Wright states it will then contain
from 7000 to 10,000 million bacteria per c.cm. If too strong an
emulsion be used the leucocytes may take up so many organisms
that these cannot be accurately enumerated. In the case of
PREPARATION OF THE SERA 113
the tubercle bacillus as short a variety of the organism as
possible should be selected, and a mass of growth off a solid
medium is taken (bacilli in mass can be obtained in the market
from wholesale chemists) and is well washed with changes of
distilled water, dried on filter paper in a Petri dish and
thoroughly rubbed up with a little 1*5 per cent saline in an
agate mortar so as to disintegrate the bacterial masses and
get an emulsion composed as far as possible of individual bacilli.
This must be controlled by microscopic examination. A thick
cream should be obtained, and this should be sterilised by
steaming for half an hour on three successive days. Before
sterilisation it is convenient to seal up the stock emulsion in
small quantities in a number of pieces of quill tubing so that in
the subsequent procedures only small portions of the emulsion
are exposed to aerial contamination at one time. For actual
use, one of those tubes is opened, a little is withdrawn with a
sterile pipette, and a weak emulsion made in the same way as
with the staphylococcus except that 1/5 per cent saline is used.
The stock tube may be sealed with wax and kept for use again.
A fresh emulsion ought to be made up for each day's work.
(2) Preparation of leucocytes. Here the observer uses his
own blood cells. A 1*5 per cent solution of sodium citrate in
•85 per cent sodium chloride is prepared. This is placed in a
glass tube three inches long made by drawing out a piece of
half -inch tubing to a point, the tube being filled nearly to the
brim. A handkerchief being bound round the finger, this is
now pricked and the blood allowed to flow directly into the
fluid, to the bottom of which it sinks. The tube ought to be
inverted between the addition of every few drops of blood so as
to bring the blood in contact with the citrate and prevent
coagulation. The equivalent of about ten to twenty drops of
blood should be obtained. The diluted blood is then centri-
fugalised, and when the corpuscles are separated the super-
natant fluid is removed, '85 per cent saline is substituted and
the centrifugalisation repeated. The fluid is again removed,
care being taken not to disturb the layer of white cells lying on
the top of the red corpuscles. This layer is then pipetted off
into a watch-glass or tube and the leucocytes required are thus
obtained.
(3) Preparation of the sera. The serum whose sensitising
effect on the bacteria it is desired to test is obtained by Wright
as follows. A "blood capsule" is made by drawing a piece of
No. 3 quill tubing into the shape shown in Fig. 47, the part not
drawn out being about one inch in length. It is convenient to
8
114
MICROSCOPIC METHODS
make a number of these capsules at one time and to draw off
their extremities and seal them in the flame. For use the tips of
both extremities are broken off, the finger is pricked, and blood
allowed to pass into the capsule through the bent limb till
the capsule is about half full. The air remaining in the capsule
is rarefied by passing the straight end through a flame and
then sealing it off. By this manipulation the blood is sucked
over the bend into the straight part of the tube, and the bent
end is now also sealed off or closed with wax. It is well to
shake the blood down towards the closed straight end, care being
taken to previously allow the glass to cool sufficiently. The
capsule is now hung by the bend on the edge of a centrifuge
tube 'and the serum separated by spinning the instrument. In
any particular case a capsule of serum
from the infected person and one from
a normal individual are prepared.
The emulsion, corpuscles, and serum
being thus prepared, the next step is
to mix them. This is done by taking
a piece of quill tubing and drawing it
out to a capillary point so as to make
a pipette about eight inches long ; on
the thick end of this a rubber teat is
fixed, and about one inch from the
capillary point a mark is made with
FTO. 47.-Wright'8 Blood-cap- an oi! Penci}' From the ™tch-glass
sule and method of filling containing the separated leucocytes a
same. portion is sucked up to the mark and
then an air-bubble is allowed to pass
in. A similar portion of the serum is drawn up, and then another
air-bubble, and finally a similar portion of the bacterial emulsion.
The three droplets are carefully blown on to a slide and are
thoroughly mixed with one another by being alternately
drawn up into the tube and expelled several times. The
mixture is then drawn into the tube and the end sealed off in the
flame. The rubber nipple is removed and the tube placed in the
incubator at 37° for fifteen minutes. A slide is now prepared
by rubbing it once or twice with very fine emery paper
(No. 000) and thoroughly wiping it. This is a procedure
adopted by Wright to cause an evenly distributed film to be
made. The tube being removed from the incubator and the
end broken off, its contents are again mixed by expelling
and drawing up into the tube. A minute droplet is placed
on the prepared slide, and by means of the edge of the end
GENERAL BACTERIOLOGICAL DIAGNOSIS 115
of another slide a film is made which is then dried and is ready
for staining. Films containing staphylococci are stained either
by Leishman's stain (q.v.) or with carbol-thionin blue. In the
former case no fixation is necessary, in the latter it is usual to
fix in saturated perchloride of mercury for 1J minutes, wash in
water and then stain. With tubercle films the following is the
procedure : the film is fixed for two minutes in perchloride of
mercury, washed thoroughly, stained with carbol-fuchsin as
usual, decolorised with 2 '5 per cent sulphuric acid, cleared with
4 per cent acetic acid, counterstained with watery solution of
methylene-blue, and dried.
In applying the technique two preparations are made, in both
of which the same emulsion and the same leucocytes are
employed, but in one of which the bacteria have been exposed
to the serum of the infected individual under observation, and
in the other to that of a normal person — usually the observer
himself. Each of these is now examined microscopically with
a movable stage, the number of bacteria in the protoplasm of
at least 50 polymorphonucleated leucocytes is counted and an
average per leucocyte struck ; the proportion which this average
in the case of the abnormal serum bears to the average in the
preparation in which the healthy serum was used, constitutes the
opsonic index, — that of the healthy serum being reckoned as
unity. The reliability of the method of course depends on the
phagocytic activity of the 50 cells counted representing the
phagocytic activity of all the cells in the preparation.
GENERAL BACTERIOLOGICAL DIAGNOSIS.
Under this heading we have to consider the general routine
which is to be observed by the bacteriologist when any material
is submitted to him for examination. The object of such
examination may be to determine whether any organisms are
present, and if so, what organisms ; or the bacteriologist may
simply be asked whether a particular organism is or is not
present. In any case his inquiry must consist (\\ of a micro- ^
scopic examination r\\ fH ™"*"™"i .I.T. «.;**„ a . (2) oran attemni^
To isolate tfie organisms present ; and (3) of the identification of ,
ili ' ii'ganisms isolated.. \Vo must, however, before considering
these points look at a matter often neglected by those who seek
a bacteriological opinion, viz. : the proper methods of obtaining
and transferring to the bacteriologist the material which he is to
be asked to examine. The general principles here are (1) that
every precaution must be adopted to prevent the material from
116 GENERAL BACTERIOLOGICAL DIAGNOSIS
being contaminated with extraneous organisms ; (2) that nothing
be done which may kill any organisms which may be proper to
the inquiry ; and (3) that the bacteriologist obtain the material
as soon as possible after it has been removed from its "natural
surroundings.
The sources of materials to be examined, even in patho-
logical bacteriology alone, are of course so varied that we can
but mention a few examples. It is, for instance, often necessary
to examine the contents of an abscess. Here the skin must be
carefully purified by the usual surgical methods ; the knife used
for the incision is preferably to be sterilised by boiling, the first
part of the pus which escapes allowed to flow away (as it might
be spoiled by containing some of the antiseptics used in the
purification) and a little of what subsequently escapes allowed
to flow into a sterile test-tube. If test-tubes
sterilised in a laboratory are not at hand, an
ordinary test-tube may be a quarter filled with
water, which is then well boiled over a spirit-
lamp. The tube is then emptied and plugged
with a plug of cotton wool, the outside of which
has been singed in a flame. Small stoppered
bottles may be sterilised and used in the same
way. A discharge to be examined may be so
small in quantity as to make the procedure
described impracticable. It may be caught on
a piece of sterile plain gauze, or of plain ab-
sorbent wool, which is then placed in a sterile
vessel. Wool or gauze used for this purpose,
or for swobbing out, say the throat, to obtain
shreds 'of suspicious matter, must have no
antiseptic impregnated in it, as the latter may
kill the bacteria present and make the obtaining
of cultures impossible.
Fluids from the body cavities, urine, etc.,
may ^e secured with sterile pipettes. To make
T of *ese> 'ake nine inches °/ ordinary quill
glass-tubing, draw out one end to a capillary
diameter, and place a little plug of cotton wool
Insert this tube through the cotton plug of an
ordinary test-tube and sterilise by heat. To use it, remove
test-tube plug with the quill tube in its centre, suck up some
of the fluid into the latter, and replace in its former position
in the test-tube (Fig. 48). Another method very convenient for
transport is to' make two constrictions on the glass tube at
FIG. 48.— Test-tube
and pipette ar-
ing bacteria.
in the other end.
ROUTINE EXAMINATION OF MATERIAL 117
suitable distances, according to the amount of fluid to be taken.
The fluid is drawn up into the part between the constrictions,
but so as not to fill it completely. The tube is then broken
through at both constrictions and • the thin ends are sealed by
heating in a flame.
Solid organs to be examined should, if possible, be obtained
whole. They may be treated in one of two ways. 1. The
surface over one part about an inch broad is seared with a
cautery heated to dull red heat. All superficial organisms are
thus killed. An incision is made in this seared zone with a
sterile scalpel, and small quantities of the juice are removed by
a platinum spud to make cover-glass preparations and plate or
smear cultures. 2. An alternative method is as follows : — The
surface is sterilised by soaking it well with 1 to 1000 corrosive
sublimate for half an hour. It is then dried, and the capsule of
the organ is cut through with a sterile knife, the incision being
further deepened by tearing. In this way a perfectly uncon-
taminated surface is obtained. Hints are often obtained from
the clinical history of the case as to what the procedure ought
to be in examination. Thus, as a matter of practice, cultures
of tubercle and often of glanders bacilli can be easily obtained
only by inoculation experiments. Typhoid bacilli need hardly
be looked for in the faeces after the first ten days of the disease,
and so on.
Routine Procedure in Bacteriological Examination of
Material. — -In the case of a discharge regarding which nothing is
known the following procedure should be adopted : — (1) Several
cover-glass preparations should V>P( |pa.rlp ^Onp. ought to HDC
stamea with saturated watery methy1pT1p-hl"pi one with a stain
containing a mordant such as Ziehl-Neelsen p.a.rhnl-fijfljisjfl. one
_by Gram's method ^2} (a} Gelatin plates should be made and^
^kgpi at room temperature, (b] a series of agar p1a.t,ps or successive
^trolves on agiir tuLcs_(p. 55) should be made and incubated at
37° C. Method (b) of course gives results more quickly. If
microscopic investigation reveals the presence of bacteria, it is
well to keep the material in a cool place till next day when, if
no growth has appeared in the incubated agar, some other culture
medium (e.g. blood serum or agar smeared with blood) may be
employed. If growth has taken place, say in the agar plates,
one with about 200 or fewer colonies should be made the chief
basis for research. In such a plate the first question to be
cleared up is; Do all the colonies present consist of the same
__ ba^tejium ? flie shape of the colony, its size, the appearance of
the margin, the graining of the substance, its colour, etc., are all
118 GENERAL BACTERIOLOGICAL DIAGNOSIS
to be noted. One precaution is necessary, viz., it must be noted
whether the colony is on the surface of the medium or in its
substance, as colonies of the same bacterium may -exhibit
differences according to their position. The arrangement of the
bacteria in a surface colony may be still more minutely studied
by means of impression preparations. A cover-glass is carefully '
cleaned and sterilised by passing quickly several times through
a Bunsen flame. It is then placed on the surface of the medium
and gently pressed down on the colony. The edge is then raised
by a sterile needle, it is seized with forceps, dried high over the
flame, and treated as an ordinary cover-glass preparation. In
this way very characteristic appearances may sometimes be noted
and preserved, as in the case of the anthrax bacillus. The
colonies on a plate having been classified, a microscopic examina-
tion of each group may be made by means of cover-glass
preparations, and tubes of gelatin and agar are inoculated from
each representative colony. Each of the colonies used must be
marked for future reference, preferably by drawing a circle
round it on the under surface of the plate or capsule with one
of Faber's pencils for marking on glass, a number or letter
being added for easy reference.
The general lines along which observation is to be made in the
case of a particular bacterium may be indicated as follows : —
1. Microscopic Appearances. — For ordinary descriptive pur-
poses young cultures, say of 24 hours' growth, on agar should
be used, though appearances in older cultures, such as involution
forms, etc., may also require attention. Note (1) the form, (2)
the size, (3) the appearance of the protoplasmic contents,
especially as regards uniformity or irregularity of staining, (4)
the method of grouping, (5) the staining reactions. Has it
a capsule? Does the bacterium stain with simple watery
solutions? Does it require the use of stains containing
mordants 1 How does it behave towards Gram's method 1 It is
important to investigate the first four points both when the
organism is in the fluids or tissues of the body and when growing
in artificial media, as slight variations occur. It must also
be borne in mind that slight variations are observed according
to the kind and consistence of the medium in which the organism
is growing. (6) Is it motile and has it flagella ? If so, how are
they arranged 1 (7) Does it form spores, and if so, under what
conditions as to temperature, etc. ?
2. Growth Characteristics. — Here the most important points
on which information is to be asked are, What are the
characters of growth and what are the relations of growth (1) to
GROWTH CHARACTERISTICS 119
temperature, (2) to oxygen 1 These can be answered from some
of the following experiments : —
A. Growth on gelatin. (1) Stab culture. Note (a) rate of
growth ; (6) form of growth, (a) on surface, (/?) in substance ; (c)
presence or absence of liquefaction ; (d) colour ; (e) presence or
absence of gas formation and of characteristic smell ; (/) relation
to reaction of medium. (2) Streak culture. (3) Shake culture.
(4) Plate cultures. Note appearances of colonies (a) superficial,
(6) deep. (5) Growth in fluid gelatin at 37° C.
B. Growth on agar at 37° C. (1) Stab. (2) Streak. Also
on glycerin agar, blood agar, etc. Appearances of colonies in
agar plates.
C. Growth in bouillon, (a) character of growth, (b) smell, (c)
reaction.
D. Growth on special media. (1) Solidified blood serum.
(2) Potatoes. (3) Lactose and other sugar media. Does fermenta-
tion occur and is gas formed ? (4) Milk. Is it curdled or turned
sour 1 (5) Litmus media. Note changes in colour. (6) Peptone
solution. Is indol formed 1
E. What is the viability of organism on artificial media 1
3. Results of inoculation experiments on animals.
By attention to such points as these a considerable knowledge
is attained regarding the bacterium, which will lead to its
identification. In the case of many well-known organisms, how-
ever, a few of the above points taken together will often be
sufficient for the recognition of the species, and experience
teaches what are the essential points as regards any individual
organism. In the course of the systematic description of the
pathogenic organisms, it«will be found that all the above points
will be referred to, though not in every case.
The methods by which the morphological and biological characteristics
of any growth may be observed have already been fully described. It
need only be pointed out here that in giving descriptions of bacteria the
greatest care must be taken to state every detail of investigation. Thus
in any description of microscopic appearances the age of the growth from
which the preparation was made, the medium employed, the temperature
at which development took place must be noted, along with the stain
which was used ; and with regard to the latter it is always preferable to
employ one of the well-known staining combinations, such as Loffler's
methylene-blue. Especial care is necessary in stating the size of a
bacterium. The apparent size often shows slight variations dependent
on the stain used and the growth conditions of the culture. Accurate
measurements of bacteria can only be made by preparing microphoto-
graphs of a definite magnification and measuring the sizes on the
negatives. From these the actual sizes can easily be calculated. In
describing bacterial cultures it must be borne in mind that the appearances
120 INOCULATION OF ANIMALS
often vary with the age. It is suggested that in the case of cultures
grown at from 36° to 37° C. the appearances between 24 and 48 hours
should be made the basis of description, and in the case of cultures
grown between 18° and 22° C. the appearances between 48 and 72 hours
should be employed. The culture fluids used must be made up and
neutralised by the precise methods already described. The investigator
must give every detail of the' methods he has employed in order that his
observations may be capable of repetition.
INOCULATION OF ANIMALS. l
The animals generally chosen for inoculation are the mouse,
the rat, the guinea-pig, the rabbit, and the pigeon. Great caution
must be shown in drawing conclusions from isolated experiments
on rabbits, as these animals often manifest exceptional symptoms,
and are very easily killed. Dogs are, as a rule, rather insusceptible
to microbic disease, and the larger animals are too expensive for
ordinary laboratory purposes. In the case of the mouse and rat
the variety must be carefully noted, as there are differences in
susceptibility between the wild and tame varieties, and between
the white and brown varieties of the latter. In the case of the
wild varieties, these must be kept in the laboratory for a week or
two before use, as in captivity they are apt to die from very slight
causes, and, further, each individual should be kept in a separate
cage, as they show great tendencies to cannibalism. Of all the
ordinary animals the most susceptible to microbic disease is the
guinea-pig. Practically all inoculations are performed by means
of the hypodermic syringe. The best variety is made on the
ordinary model with metal mountings, asbestos washers, and
preferably furnished with platmum-iridium needles. Before use
the syringe and the needle are sterilised by boiling for five minutes.
The materials used for inoculation are cultures, animal exudations,
or the juice of organs. If the bacteria already exist in a fluid
there is no difficulty. The syringe is most conveniently filled out
of a shallow conical test-glass which ought previously to have
been covered with a cover of filter paper and sterilised. If an
inoculation is to be made from organisms growing on the surface
of a solid medium, either a little ought to be scraped off and
shaken up in sterile distilled water or '85 per cent salt solution
to make an emulsion, or a little sterile fluid is poured on the growth
and the latter scraped off into it. This fluid is then filtered into
the test-glass through a plug of sterile glass wool. This is easily
effected by taking a piece of f in. glass-tubing 3 in. long, drawing
1 Experiments on animals, of course, caunot be performed in this country
without a license granted by the Home Secretary.
INOCULATION OF ANIMALS
121
one end out to a fairly narrow point, plugging the tube with
glass wool above the point where the narrowing commences, and
sterilising by heat. By filtering an emulsion through such a
pipette, flocculi which might block the needle are removed. If
a solid organ or an old culture is used for inoculation it ought
to be rubbed up in a sterile porcelain or metal crucible with a
little sterile distilled water, by means of a sterile glass rod, and
the emulsion filtered as in the last case.
The methods of inoculation generally used are : (1) by
scarification of the skin ; (2) by subcutaneous injection ; (3) by
intraperitoneal injection ; (4) by intravenous injection ; (5) by
injections into special regions, such as the anterior chamber of the
eye, the substance of the lung, etc. Of these (2) and (3) are
most frequently used. When an anaesthetic is to be administered,
this is conveniently done by placing the animal, along with a
piece of cotton-wool or sponge soaked in chloroform, under a
bell-jar or inverted glass beaker of suitable size.
1. Scarification. — A few parallel scratches are made in the
skin of the abdomen previously cleansed, just sufficiently deep
to draw blood, and the infective material is rubbed in with a
platinum eyelet. The disadvantage of this method is that the
inoculation is easily contaminated. The method is only
occasionally used.
2. Subcutaneous Injection. — A hypodermic syringe is charged
with the fluid to be inoculated. The hair is cut
off the part to be inoculated, and the skin
purified with 1 to 1000 corrosive sublimate.
The skin is then pinched up and, the needle
being inserted, the requisite dose is administered.
The wound is then sealed with a little collodion.
3. Intraperitoneal Injection. — This may be
performed by means of a special form of needle.
The needle is curved, and has its opening not at
the point, but in the side in the middle of the
arch (Fig. 49). The hair over the lower part
of the abdomen is cut, and the skin purified with
an antiseptic. The whole thickness of the ab-
dominal walls is then pinched up by an assistant,
between the forefingers and thumbs of the two
hands. The needle is then plunged through
the fold thus formed. The result is that the
hole in the side of the needle is within the
abdominal cavity, and the inoculation can thus be made.
Intraperitoneal inoculation can also be practised with an
FIG. 49.— Hollow
needle with
lateral aperture
(at a) for intra-
peritoneal in-
oculations.
122 INOCULATION OF ANIMALS
ordinary needle. The mode of procedure is similar, but after
the needle is plunged through the abdominal fold, it is partially
•withdrawn till the point is felt to be free in the peritoneal
cavity when the injection is made. There is little risk of
injuring the intestines by either method.
4. Intravenous Injection. — The vein most usually chosen is
one of the auricular veins. The part has the hair removed, the
skin is purified, and the vein made prominent by pressing on it
between the point of inoculation and the heart. The needle is
then plunged into the vein arid the fluid injected. That it has
perforated the vessel will be shown by the escape of a little blood;
and that the injection has taken place into the lumen of the
vessel will be known by the absence of the small swelling which
occurs in subcutaneous injections. If preferred, the vein may be
first laid bare by snipping the skin over it. The needle is then
introduced.
5. Inoculation into the Anterior Chamber of the Eye. — Local
anaesthesia is established by applying a few drops of 2 per cent
solution of hydrochlorate of cocaine. The eye is fixed by
pinching up the orbital conjunctiva with a pair of fine forceps,
and the edge of the cornea being perforated by the hypodermic
needle, the injection is easily accomplished.
Sometimes inoculations are made by planting small pieces of
pathological tissues in the subcutaneous tissue. This is especially
done in the case of glanders and tubercle. The skin over the
back is purified, and the hair cut. A small incision is made with
a sterile knife, and the skin being separated from the subjacent
tissues by means of the ends of a blunt pair of forceps, a little
pocket is formed into which a piece of the suspected tissue is
inserted. The wound is then closed with a suture, and collodion
is applied. In the case of guinea-pigs, the abdominal wall is to
be preferred as the site of inoculation, as the skin over the back
is extremely thick.
Injections are sometimes made into other parts of the body,
e.g. the pleurae and the cranium. It is unnecessary to describe
these, as the application of the general principles employed above,
together with those of modern aseptic surgery, will sufficiently
guide the investigator as to the technique which is requisite.
After inoculation, the animals ought to be kept in comfortable
cages, which must be capable of easy and thorough disinfection
subsequently. For this purpose galvanised iron wire cages are the
best. They can easily be sterilised by boiling them in the large
fish-kettle which it is useful to have in a bacteriological laboratory
for such a purpose. It is preferable to have the cages opening
COLLODION CAPSULES 123
from above. Otherwise material which may be infective may be
scratched out of the cage by the animal. The general condition
of the animal is to be observed, how far it differs from the normal, -
whether there is increased rapidity of breathing, etc. The
temperature is usually to be taken. This is generally done per
rectum. The thermometer (the ordinary 5 min. clinical variety)
is smeared with vaselin, and the bulb inserted just within the
sphincter, where it is allowed to remain for a minute ; it is then
pushed well into the rectum for five minutes. If this precaution
be not adopted, a reflex contraction of the vessels may take place,
which is likely to vitiate the result by giving too low a reading.
Collodion Capsules.— These have been used to allow the
sojourn of bacteria within the animal body without their coming
into contact with the cells of the tissues. Various substances
in solution can pass in either direction through the wall by
diffusion, but the wall is impermeable alike to bacteria and
leucocytes. The following method of preparing such capsules is
that of M'Rae modified by Harris. A gelatine capsule, such as
is used by veterinary surgeons, is taken, and in one end there
is fixed a small piece of thin glass tubing by gently heating the
glass and inserting it. The tube becomes fixed when quite cold,
and the junction is then painted round with collodion, which
is allowed to dry thoroughly. The bore of the tubing is cleared
of any obstructing gelatine, and the whole capsule is dipped into
a solution of collodion so as to coat it completely. The collodion
is allowed to dry and the coating is repeated ; it is also advis-
able to strengthen the layer by further painting it at the
extremity and at the junction. The interior of the capsule is
then filled with water by a fine capillary pipette, and the capsule
is placed in hot water in order to liquefy the gelatine, which can
be removed from the interior by means of the fine pipette. The
sac is filled with bouillon and is placed in a tube of bouillon. It
is then sterilised in the autoclave. A small quantity of the
bouillon is removed, and the contents are inoculated with the
particular bacterium to be studied or an emulsion of the bacterium
is added. The glass tubing is seized in sterile forceps and is
sealed off in a small flame a short distance above the junction.
The closed sac ought then to be placed in a tube of sterile
bouillon to test its impermeability. The result is satisfactory if
no growth occurs in the surrounding medium. The sac with its
contents can now be transferred to the peritoneal cavity of an
animal.
Autopsies on Animals dead or killed after Inoculation.—
These should be made as soon as possible after death. It is
124 INOCULATION OF ANIMALS
necessary to have some shallow troughs, constructed either of
metal or of wood covered with metal, convenientfy with sheet
lead, and having a perforation at each corner to admit a tape or
strong cord. The animal is tightly stretched out in the trough and
tied in position. The size of the trough will therefore have to vary
with the size of the outstretched body of the animal to be examined.
In certain cases it is well to soak the surface of the animal in
carbolic acid solution (1 to 20) or in corrosive sublimate (1 to
1000) before it is tied out. This not only to a certain extent
disinfects the skin, but, what is more important, prevents hairs
which might be affected with pathogenic products from getting
into the air of the laboratory. The instruments necessary are
scalpels (preferably with metal handles), dissecting forceps, and
scissors. They are to be sterilised by boiling for five minutes.
This is conveniently done in one of the small portable sterilisers
used by surgeons. Two sets at least ought to be used in an
autopsy, and they may be placed, after boiling, on a sterile glass
plate covered by a bell -jar. It is also necessary to have a medium-
sized hatchet-shaped cautery, or other similar piece of metal. It
is well to have prepared a few freshly-drawn-out capillary tubes
stored in a sterile cylindrical glass vessel, and also some larger
sterile glass pipettes. The hair of the abdomen of the animal is
removed. If some of the peritoneal fluid is wanted, a band
should be cauterised down the linea alba from the sternum to the
pubes, and another at right angles to the upper end of this ; an
incision should be made in the middle of these bands, and the
abdominal walls thrown to each side. One or more capillary
tubes should then be filled with the fluid collected in the flanks,
the fluid being allowed to run up the tube and the point sealed
off; or a larger quantity, if desired, is taken in a sterile pipette.
If peritoneal fluid be not wanted, then an incision may be made
from the episternum to the pubes, and the thorax and abdomen
opened in the usual way. The organs ought to be removed with
another set of instruments, and it is convenient to place them
pending examination in deep Petri's capsules (sterile). It is
generally advisable to make cultures and film preparations from
the heart's blood. To do this, open the pericardium, sear the
front of the right ventricle with a cautery, make an incision in the
middle of the part seared, and remove some of the blood with a
capillary tube for future examination, or, introducing a platinum
eyelet, inoculate tubes and make cover-glass preparations at once.
To examine any organ, sear the surface with a cautery, cut into it,
and inoculate tubes and make film preparations with a platinum
loop. For removing small parts of organs for making inoculations
AUTOPSIES ON ANIMALS 125
on tubes, a small platinum spud is very useful, as the ordinary
wires are apt to become bent. Place pieces of the organs in
some preservative fluid for microscopic examination. The organs
ought not to be touched with the fingers. When the examination
is concluded the body should have corrosive sublimate or carbolic
acid solution poured over it, and be forthwith burned. The
dissecting trough and all the instruments ought to be boiled for
half an hour. The amount of precaution to be taken will, of
course, depend on the character of the bacterium under investiga-
tion, but as a general rule every care should be used.
CHAPTER IV.
BACTERIA IN AIR, SOIL, AND WATER.
ANTISEPTICS.
IT is impossible here to do more than indicate the chief methods
which are employed by bacteriologists in the investigation of the
bacteria present in air, soil, and water, and to add an outline of
the chief results obtained. In dealing with the latter the subject
has been approached mainly from the standpoint of the bearings
which the results have towards human pathology. In dealing
with antiseptics, so far as possible the effects of the various
agents on the chief pathogenic bacteria have been given, though
in many cases our information is very imperfect.
AIR.
Very little information of value can be obtained from the
examination of the air, but the following are the chief methods
used, along with the results obtained. More can be learned
from the examination of atmospheres experimentally contamin-
ated than by the investigation of the air as it exists under
natural conditions.
Methods of Examination. — The methods employed vary with the
objects in view. If it be sought to compare the relative richness of
different atmospheres in organisms, and if the atmospheres in question
be fairly quiescent, then it is sufficient to expose gelatin plates for
definite times in the rooms to be examined. Bacteria, or the particles of
dust carrying them, fall on the plates, and from the number of colonies
which develop a rough idea of the richness of the air in bacteria can be
obtained. Petri states that in five minutes the bacteria present in
10 litres of air are deposited on 100 square centimetres of a gelatin
plate.
126
METHODS OF EXAMINATION
127
More complete results are available when some method is employed by
which the bacteria in a given quantity of air are examined. The oldest
method employed, and one which is still used, is that of Hesse. The
apparatus is shown in Fig. 50. It consists of a cylindrical tube a about
20 inches long and 2 inches in diameter. At one end this is closed by a
rubber cork having a piece of quill tubing, /, passing through it and
projecting some distance into the interior. For use the tube is sterilised
in a tall "Koch," and then a quantity of peptone gelatin, sufficient to
cover the whole interior to the thickness of an ordinary gelatin plate, is
poured in. This gelatin
is kept from escaping by ./
the projection of the quill
tubing into the lumen of
the large tube. A plug
of cotton wool is now
placed in the outer end
of the quill tubing. Over
the other end of the large
tube is tied a sheet of
rubber having a hole
about a quarter of an inch
in diameter in its centre,
and over this again is tied
a piece of similar but un-
perforated sheet rubber.
The tube is then steri-
lised in the tall " Koch.'
On removal from this it
is rolled, after the manner
of an Esmarch's tube
(q.v.), till the gelatin
is set as a layer over
its interior, and it is then
placed horizontally on
the tripod as shown. The
other part of the appar-
atus is an aspirator by
means of which a known
quantity • of air can be
brought in contact with
the gelatin. It consists
FIG. 50. — Hesse's tube, mounted for use.
of two conical glass flasks connected by means of a tube which passes
through the cork of each down to the bottom of the flask. When this
tube is filled with water, it, of course, can act as a syphon tube between
volumes of water in the flasks. Such a syphon system being established,
the levels of the water are marked on the flasks, and to one a litre of
water is added, and by depressing flask b the whole litre can be got into
it and the connecting tube c is then clamped. The two flasks are then
connected by a rubber tube with the tube/, the clamp on c is opened,
and the passing of a litre of water into d will draw a litre of air through
the gelatin tube, when the outer rubber sheet is removed from the end
and the clamp li opened. By disconnecting at g and reversing the syphon
flasks, another litre can be sucked through, and so any desired quantity
of air can be brought in contact with the gelatin. The speed ought not
to be more than one litre in two minutes, and in such a case practically
128
BACTERIA IN AIR
all the organisms will be found to have fallen out of the air on to the
gelatin in the course of their transit. This fact can be tested by
interposing between the tube a and the aspirator a second tube prepared
in the same way, which ought, of course, to show no growth. When
forty-eight hours at 20° C. or four days at lower temperature have
elapsed, the colonies which develop in a may be counted. The dis-
advantage of the method is that if particles of dust carrying more than
one bacterium alight on the gelatin, these bacteria develop in one colony,
and thus the enumeration results may be too low ; difficulties may also
arise from liquefying colonies developing in the upper parts of the tubS
and running over the gelatin.
Petri's Sand-Filter Method. — A glass tube open at both ends, and
about 3| inches long and half an inch wide, is taken, and in its centre is
placed a transverse diaphragm of very fine iron
gauze (Fig. 51, e) ; on each side of this is placed
some fine quartz sand which has been well washed,
dried, and burned to remove all impurities, and this
is kept in position by cotton plugs. The whole is
sterilised by dry heat. One plug is removed and a
sterile rubber cork, c, inserted, through which a tube,
d, passes to an exhausting apparatus. The tube is
then clamped in an upright position in the atmo-
sphere to be examined, with the remaining plug,
/, uppermost. The latter is removed and the air
sucked through. Difficulty may be experienced
from the resistance of the sand if quick filtration be
attempted. The best means to adopt is to use an
air-pump — the amount of air drawn per stroke of
which is accurately known — and have a manometer
(as in Fig. 31) interposed between the tube and the
pump. Between each two strokes of the air-pump
the mercury is allowed to return to zero. After
the required amount of air has passed, the sand a
is removed, and is distributed among a number of
sterile gelatin tubes which are well shaken ; plate
cultures are then made, and when growth has occurred
the colonies are enumerated ; the sand b is similarly
treated and acts as a control.
When it is necessary to examine air for particular
organisms, special methods must often be adopted.
Thus in the case of the suspected presence of tubercle bacilli a given
quantity of air is drawn through a small quantity of water and then
injected" into a guinea-pig.
It must be admitted that comparatively little information
bearing on the harmlessness or harmfulness of the air is obtain-
able by the mere enumeration of the living organisms present,
for under certain conditions the number may be increased by
the presence of many individuals of a purely non-pathogenic
character. The organisms found in the air belong to two
groups — firstly, a great variety of bacteria ; secondly, yeasts and
the spores of moulds and of the lower fungi. With regard to
the spores, the organisms from which they are derived often
FIG. 51. — Petri's
sand filter.
PETRI'S SAND-FILTER METHOD 129
consist of felted masses of threads, from which are thrust into the
air special filaments, and in connection with these the spores are
formed. By currents of air these latter can easily be detached,
and may float about in a free condition. With the bacteria, on
the other hand, the case is different. Usually these are growing
together in little masses on organic materials, or in fluids, and
it is very much by the detachment of minute particles of the
substratum that the organisms become free. The entrance of
bacteria into the air, therefore, is associated with conditions
which favour the presence of dust, minute droplets of fluid, etc.
The presence of dust in particular would specially favour a large
number of bacteria being observed, and this is the case with the
air in many industrial conditions, where the bacteria, though
numerous, may be quite innocuous. Great numbers of bacteria
thus may not indicate any condition likely to injure health, and
this may be true also even when the bacteria come from the
crowding together of a number of healthy human beings. On
the other hand, there is no doubt that disease germs can be
disseminated by means of the air. The possibility of this
has been shown experimentally by infecting the mouth with the
b. prodigiosus, which is easily recognised by its brilliantly
coloured colonies, and then studying its subsequent distribution.
Most important here is the infection of the air from sick persons.
The actions of coughing, sneezing, speaking, and even of deep
breathing, distribute, often to a considerable distance, minute
droplets of secretions from the mouth, throat, and nose, and these
may float in the air for a considerable time. Even five hours
after an atmosphere has been thus infected evidence may be
found of bacteria still floating free. Before this time, however,
most of the bacteria have settled upon various objects, where
they rapidly dry, and are no longer displaceable by ordinary air
currents. The diseases of known etiology where infection can
thus take place are diphtheria, influenza, pneumonia, and phthisis ;
and here also probably whooping-cough, typhus fever, and measles
are to be added, though the morbific agents are unknown. In
the case of phthisis, the alighting of tubercle bacilli has been
demonstrated on cover-glasses held before the mouths of patients
while talking, and animals made to breathe directly in front of
the mouths of such patients have become infected with
tuberculosis. Apart from direct infection from individuals,
however, pathogenic bacteria may be spread in some cases from
the splashing of infected water, as from a sewage outfall.
This possibility has to be recognised especially in the cases of
typhoid and cholera. Besides infection through fluid particles,
130 BACTERIA IN AIR
infection can be caused in the air by 'dust coming, say, from
infected skin or clothes, etc. Fliigge, in dealing with this
subject in an experimental inquiry, distinguishes between large
particles of dust which require an air current moving at the rate
of 1 centimetre per second to keep them suspended, and the finer
dust which can be kept in suspension by currents moving at from
1 to 4 millimetres per second. In the former case, when once
the particles alight they cannot be displaced by currents of air
except when these are moving at, at least, 5 metres per second,
but the brushing, shaking, or beating of objects may, of course,
distribute them. In the case of the finer dust the particles will
remain for long suspended, and when they have settled can be
more easily displaced, as by the waving of an arm, breathing,
etc. With regard to infection by dust, a most important factor,
however, is whether or not the infecting agent can preserve its
vitality in a dry condition. In the case of a sporing organism
such as anthrax, vitality is preserved for long periods of time,
and great resistance to drying is also possessed by the tubercle
and diphtheria bacilli ; but apart from such cases there is little
doubt that infection is usually necessarily associated with the
transport of moist particles, and is thus confined to a limited
area around a sick person. Among diseases which may
occasionally be thus spread cholera and typhoid have been classed.
Considerable controversy has arisen with regard to certain out-
breaks of the latter disease, which have apparently been spread
by dusty winds, although we have the fact that the typhoid
bacillus does not survive being dried even for a short time.
It appears, however, that in such epidemics the transport of
infection by means of insects carried by the wind has not been
entirely excluded.
As in the cases of the soil and of water, presently to be described,
attempts have been made to obtain indirect evidence of the contamination
of the air by man. Thus Gordon has shown that certain streptococci
are common in the saliva ; these resemble the streptococcus pyogenes, but
are relatively non-pathogenic, grow well at 37° C. and under anaerobic
conditions, cause clotting and acid-formation in litmus milk at 37°, and
in neutral-red media have an action resembling that of b. coli. These
characters serve, according to Gordon, to differentiate organisms of
human origin from ordinary streptococci occurring in the air and which
he states grow better at about 22° C., are facultative anaerobes and
do not produce the changes in milk and in neutral-red media. Thus
the finding of streptococci of the first group in plates exposed to air
would indicate that a human source was probable, and, if the observation
were made on air from the neighbourhood of a sick person, that risk of
the dissemination of disease germs was present. The value of this as a
practical method has yet to be determined.
BACTERIA IN SOIL 131*
SOIL.
The investigation of the bacteria which may be found in the
soil is undertaken from various points of view. Information
may be desired as to the change its composition undergoes by a
bacterial action, the result of which may be an increase in
fertility and thus in economic value. Under this head may be
grouped inquiries relating to the bacteria which convert
ammonia and its salts into nitrates and nitrites, and to the
organisms concerned in the fixation of the free nitrogen of the
air. The discussion of the questions involved in such inquiries
is outside the scope of the present chapter, which is more con-
cerned with the relation of the bacteriology of the soil to questions
of public health. So far as this narrower view is concerned, soil
bacteria are chiefly of importance in so far as they can be washed
out of the soils into potable water supplies. An important aspect
of this question thus is as to the significance of certain bacterio-
logical appearances in a water in relation to the soil from which
it has come or over which it has flowed. In this country these
questions have been chiefly investigated by Houston, and it is
from his papers that the following account is largely taken.
Methods of Examination. — For examination of soil on surface or not
far from surface, Houston recommends tin troughs 10 in. by 3 in. and
pointed at one extremity, to be wrapped in layers of paper and sterilised
by dry heat. If several of these be provided, then the soil can be well
rubbed up and a sample secured and placed in a sterile test-tube for
examination as soon as convenient after collection. If samples are to
be taken at some depth beneath the surface, then a special instrument
of which many varieties have been devised must be used. The general
form of these is that of a gigantic gimlet stoutly made of steel. Just
above the point of the instrument the shaft has in it a hollow chamber,
and a sliding lateral door in this can be opened and shut by a mechanism
controlled at the handle. The chamber being sterilised and closed, the
instrument is bored to the required depth, the door is slid back, and by
varying devices it is effected that the chamber is filled with earth ; the
door is reclosed and the instrument withdrawn.
In any soil the two important lines of inquiry are first as to the total
number of organisms (usually reckoned per gramme of the fresh sample),
and secondly as to the varieties of organisms present. The number of
organisms present in a soil is often, however, so enormous that it is con-
venient to submit only a fraction of a gramme to examination. The
method employed is to weigh the tube containing the soil, shake out an
amount of about the size of a bean into a litre of distilled water, and re-
weigh the tube. The amount placed in the water is distributed as
thoroughly as possible by shaking, and, if necessary, by rubbing down
with a sterile glass rod, and small quantities measured from a graduated
pipette are used for the investigation. For estimating the total number of
organisms present in the portion of soil used, small quantities, say *1 c.c.
and 1 c.c., of the fluid are added to melted tubes of ordinary alkaline
peptone gelatin ; after being shaken, the gelatin is plated, incubated at
132 BACTERIA IN SOIL
22° C., and the colonies are counted as late'as the liquefaction, which
always occurs round some of them, will allow. From these numbers the
total number of organisms present in the amount of soil originally present
can be calculated.
The numbers of bacteria in the soil vary very much. Accord-
ing to Houston's results, fewest occur in uncultivated sandy soils,
these containing on an average 100,000 per gramme. Peaty soils,
though rich in organic matter, also give low results, it being
possible that the acidity of such soils inhibits free bacterial
growth. Garden soils yield usually about 1,500,000 bacteria
per gramme, but the greatest numbers are found in soils which
have been polluted by sewage, when the figures may rise to
115,000,000. In addition to the enumeration of the numbers
of bacteria present, it is a question whether something may not
be gained from a knowledge of the number of spores present in
a soil relative to the total number of bacteria. This is a point
which demands further inquiry, especially by the periodic investi-
gation of examples of different classes of soils. The method is to
take 1 c.c. of such a soil emulsion as that just described, add it
to 10 c.c. of gelatin, heat for ten minutes at 80° C. to destroy
the non-spored bacteria, plate, incubate, and count as before.
Besides the enumeration of the numbers of bacteria present in
a soil, an important question in its bacteriological examination
lies in inquiring what kinds of bacteria are present in any par-
ticular case. Practically this resolves itself into studying the
most common bacteria present, for the complete examination of
the bacterial flora of any one sample would occupy far too much
time. Of these common bacteria the most important are those
from whose presence indications can be gathered of the con-
tamination of the soil -by sewage, for from the public health
standpoint this is by far the most important question on which
bacteriology can shed light.
Bacillus mycoides. — This bacillus is 1'6 to 2*4 /j, in length and about *9
in breadth. It grows in long threads which often show motility. It
can be readily stained by such a combination as carbol-thionin, and re-
tains the dye in Gram's method. All ordinary media will support its
growth, and, in surface growths on agar or potato spore, formation is
readily produced. Its optimum temperature is about 18° C. On gelatin
plates it shows a very characteristic appearance. At first under a low
power it shows a felted mass of filaments throwing out irregular shoots
from the centre, and later to the naked eye these appear to be in the
form of thick threads like the growth of a mould. They rapidly spread
over the surface of the medium, and the whole resembles a piece of wet
teased-out cotton wool. The gelatin is liquefied.
Cladothrices. — Of these several kinds are common in the soil. The
ordinary dadothrix dichotoma is among them. This organism appears
BACTERIA IN SOIL 133
as colourless flocculent growth with an opaque centre, and can be seen
under the microscope to send out into the medium apparently branched
threads which vary in thickness, being sometimes 2 p across. They
consist of rods enclosed in a sheath. These rods may divide at any
point, and thus the terminal elements may be pushed along the sheath.
Sometimes the sheath ruptures, and thus by the extrusion of these
dividing cells and their further division the branching appearance is
originated. Reproduction takes place by the formation of gonidia in the
interior of the terminal cells. These gonidia acquire at one end a bundle
of flagella, and for some time swim free before becoming attached and
forming a new colony. Houston describes as occurring in the soil another
variety, which with similar microscopic characters appears as a brownish
growth with a pitted surface, and diffusing a bismarck-brown pigment
into the gelatin which it liquefies.
A few experiments made with an ordinary field soil will, however,
familiarise the worker with the non-pathogenic bacteria usually present.
We have referred to these two because of their importance. In regard
to pathogenic organisms, especially in relation to possible sewage con-
tamination, attention is to be directed to three groups of organisms,
those resembling the b. coli, the bacillus enteritidis sporogenes, and the
streptococcus pyogenes. The characters of the first two of these will
be found in the chapter on Typhoid Fever ; of the third in Chap. VI.
For the detection of these bacteria Houston recommends the following
procedure.
(a) The B. coli group. A third of a gramme of soil is added to 10 c.c.
phenol broth (vide chapter on Typhoid Fever) and incubated at 37° C.
In this medium very few if any other bacteria except those of the b. coli
group will grow, so that if after twenty-four hours a turbidity appears,
some of the latter may be suspected to be present. In such a case a
loopful of the broth is shaken up in 5 c.c. sterile distilled water, and of
this one or two loopfuls are spread over the surface of a solid plate of
phenol gelatin in a Petri capsule either by means of the loop or of
a small platinum spatula, and the plate is incubated at 20° C. Any
colonies which resemble b. coli are then examined by the culture
methods detailed under that organism. Further, all organisms having
the microscopic appearances of b. coli, and which generally conform to
its culture reactions, are to be reckoned in the coli group. The media
of MacConkey and Drigalski are very useful in connection with the separa-
tion of such soil organisms (v. pp. 42, 43).
(b) The Bacillus enteritidis sporogenes. To search for this organism 1
gramme of the soil is thoroughly distributed in 100 c.c. sterile distilled
water, and of this 1 c.c., "1 c.c., and '01 c.c. is added to each of three sterile
milk tubes. These are heated to 80° C. for ten minutes and then cultivated
anaerobically at 37° C. for twenty-four hours. If the characteristic appear-
ances seen in such cultures of the b. enteritidis (q.v.) are developed,
then it may fairly safely be deduced that it is this organism which has
produced them.
(c) Fcecal Streptococci. The method here is to pour out a tube of agar
into a Petri capsule, and when it has solidified to spread out "1 c.c. of
the emulsion of soil over it and incubate at 37° C. for twenty-four hours.
At this temperature many of the non-pathogenic bacteria grow with
difficulty, and thus the number of colonies which develop is relatively
small. Colonies having appearances resembling those of the streptococcus
pyogenes (q.v.} can thus be investigated. Much work has been devoted
to the question of these feecal streptococci presenting specific characters by
134 BACTERIA IN SOIL
which they could be differentiated from other streptococci. No definite
results have as yet been obtained. Houston gives as the general
characters of these organisms that they usually grow in short chains,
that they produce uniform turbidity in broth, that they give rise to acid
and clot in litmus milk at 37° C., and that they are non-pathogenic to
mice. The important point is to recognise that streptococci of fairly
ordinary types exist in great numbers in human fseces, and that when
in any circumstances faecal contamination is suspected the isolation of
streptococci strengthens the suspicion.
We may now give in brief the results at which Houston has
arrived by the application of these methods. First of all, un-
cultivated soils contain very few, if any, representatives of the
b. mycoides, and this is also true to a less extent of the
cladothrices. Cultivated soils, on the other hand, do practically
always contain these organisms. With regard to the b. coli its
presence in a soil must be looked on as indicative of recent
pollution with excremental matter. The presence of b.
enteritidis is also evidence of such pollution, but from the
fact that it is a sporing organism this pollution may not have
been recent. With regard to the streptococci, on the other
hand, the opinion is advanced that their presence is, on account
of their want of viability outside the animal body, to be looked
on as evidence of extremely recent excremental pollution. The
very great importance of these results in relation to the
bacteriological examination of water supplies will be at once
apparent, and will be referred to again in connection with the
subject of water.
While such means have been advanced for the obtaining of
indirect evidence of excremental pollution of soil, and therefore
of a pollution dangerous to health from the possible presence of
pathogenic organisms in excreta, investigations have also been
conducted with regard to the viability in the soil of pathogenic
bacteria, especially of those likely to be present in excreta, namely,
the typhoid and cholera organisms. The solution of this problem
is attended with difficulty, as it is not easy to identify these
organisms when they are present in such bacterial mixtures as
naturally occur in the soil. Now there is evidence that bacteria
when growing together often influence each other's growth in an
unfavourable way, so that it is only by studying the organisms in
question when growing in unsterilised soils that information can
be obtained as to what occurs in nature. For instance, it has
been found that the b. typhosus, when grown in an organically
polluted soil which has been sterilised, can maintain its vitality
for fifteen weeks, but if the conditions occurring naturally be so
far imitated by growing it in soil in the presence of a pure culture
BACTERIA IN WATER 135
of one or other certain soil bacteria, it is found that sometimes the
typhoid bacillus, sometimes the soil bacterium in the course of a
few weeks, or even in a few days, disappears. Further, the char-
acter of the soil exercises an important effect on what happens ;
for instance, the typhoid bacillus soon dies out in a virgin sandy
soil, even when it is the only organism present. In experiments
made by sowing cultures of cholera and diphtheria in plots in a
field it was found that after, at the longest, forty days they were
no longer recognisable. Further, it is a question whether
ordinary disease organisms, even if they remain alive, can
multiply to any great extent in soil under natural conditions.
If we are dealing with a sporing organism such as the b.
anthracis, the capacity for remaining in a quiescent condition of
potential pathogenicity is, of course, much greater. The most
important principle to be deduced from these experiments is that
the ordinary conditions of soil rather tend to be unfavourable
to the continued existence of pathogenic bacteria, so that by
natural processes soil tends to purify itself. It must, however,
be noted that such an organism as the typhoid bacillus can exist
long enough in soil to be a serious source of danger.
WATER.
In the bacteriological jexamination of water three lines of
inquiry may have to be followed. First, the number of bacteria
per cubic centimetre may be estimated. Second, the kinds of
bacteria present may be investigated. Third, it may be necessary
to ask if a particular organism is present, and, if so, in what
number per c.c. it occurs.
Methods. — In the two first cases a small quantity ("5-1 c.c.) is taken
in a sterile pipette and added to a tube of gelatin, which is then plated
and incubated at the room temperature. In the case of water taken
from a house tap the water should be allowed to run for several hours
before the sample is taken, as water standing in pipes in a house is under
very favourable conditions for multiplication of bacteria taking place,
and if this precaution be not adopted an altogether erroneous idea of the
number present may be obtained. In the case of the examination of
river water the gelatin plates ought to be prepared on the spot ; at any
rate, the time clasping between the sample being taken and the plates
being prepared must be as short as possible, otherwise the bacteria will
multiply, and again an erroneous idea of their number be obtained.
When samples have to be taken for transport to the laboratory, these
are best collected in four-ounce, wide-mouthed stoppered bottles, which
are to be sterilised by dry heat (the stopper must be sterilised separately
from the bottle and not inserted in the latter till both are cold, otherwise
it will be so tightly held as to make removal very difficult). In using
such a bottle it is best to immerse it in the water and then remove the
136 BACTERIA IN WATEK
stopper with forceps. Care must be taken not to touch the water-bed,
as the vegetable matter covering it contains a large number of organisms.
The bottles ought to be packed in ice and sawdust, and plates must be
prepared from the samples as soon as possible. When the object in
view is to determine the number of bacteria per cubic centimetre, it is
important to note that water bacteria grow at very varied rates, and
therefore it is well that the same time should always elapse before the
colonies are counted. The period of growing usually allowed is forty-
eight hours at 20° C.
Several points may be here noted. It has been found, for instance,
that slight variations in the reaction of the medium affect the number
of colonies which develop. A slightly greater degree of alkalinity than
peptone gelatin, as ordinarily prepared, possesses — such an increased
degree as that caused by the addition of '01 grm. Na2C03 to 10 c.c.
peptone gelatin — will give a greater yield of colonies than the ordinary
gelatin. Again, the natural temperature of the growth of water bacteria
in temperate climates is comparatively low, being not often above 18° C.,
and, on account of this, gelatin suggests itself as the most suitable
medium. This can be seen by comparing the growth on an agar plate
inoculated with a given quantity of water, and incubated at 37° C., with
the growth on a precisely similar gelatin plate incubated at 20° C. , as it
will be found that many more colonies have developed on the latter.
This fact may be taken advantage of when pathogenic bacteria are being
sought for in a water. The latter usually grow well at 37° C., and thus
if agar plates be used the search may be facilitated. Apart from the
difference of incubation temperatures, however, in such a case as that
cited, it is probable that agar is a less suitable medium than gelatin for
the growth of water bacteria, for in plates incubated at the same
temperature the colonies which grow on the agar are often fewer than
those on the gelatin. Probably no one medium will support the growth
of all the organisms present in a given sample of water, and under
certain circumstances special media must therefore be used. Thus
Hansen found that in testing waters to be used in brewing it was
advisable to have in the medium employed some sterile wort or beer, so
that the organisms in the test experiments should be provided with the
food materials which would be present in the commercial use of the water.
Manifestly this principle applies generally in the bacteriological examina-
tion of waters to be used for industrial purposes.
In ordinary public health work it may be taken that the most
frequent and important inquiry is directed towards the presence or
absence of the b. coli and its congeners. Many methods are here used
but we consider that in which MacConkey's bile-salt media are employed
the most convenient. For small quantities of water, — up to 1 c.c., — the
sample is simply added to a Durham's tube of bile-salt glucose neutral-
red broth and incubated for 48 hours. When it is necessary to examine
larger samples it is convenient, as Savage recommends, to have the bile-
salt broth made of double, treble, or quadruple its usual strength.
The water to be examined is used as the diluent by which the medium
is brought down to the ordinary concentration. If gas forms, some of
the coli group are almost certainly present. The organisms may be
plated out by smearing a little of the broth on bile-salt agar for further
isolation and examination.
With regard to the objects with which the bacteriological
BACTERIA IN WATER 137
examination of water may be undertaken, though these may
be of a purely scientific character, they usually aim at contribut-
ing to the settlement of questions relating to the potability of
waters, to their use in commerce, and to the efficiency of pro-
cesses undertaken for the purification of waters which have
undergone pollution. The last of these objects is often closely
associated with the first two, as the question so often arises
whether a purification process is so efficient as to make the
water again fit for use.
Water derived from any natural source contains bacteria,
though, as in the case of some artesian wells and some springs,
the numbers may be very small, e.g. 4 to 100 per c.c. In rain,
snow, and ice there are often great numbers, those in the first two
being derived from the air. Great attention has been paid to
the bacterial content of wells and rivers. With regard to the
former, precautions are necessary in arriving at a judgment.
If the water in a well has been standing for some time,
multiplication of bacteria may give a high value. To meet this
difficulty, if practicable,- the well ought to be pumped dry and
then allowed to fill, in order to get at what is really the im-
portant point, namely, the bacterial content of the water entering
the well. Again, if the sediment of the well has been stirred
up a high value is obtained. Ordinary wells of medium depth
contain from 100 to 2000 per c.c. With regard to rivers very
varied results are obtained. Moorland streams are usually very
pure. In an ordinary river the numbers present vary at
different seasons of the year, whilst the prevailing temperature,
the presence or absence of decaying vegetation, or of washings
from land, and dilution with large quantities of pure spring
water, are other important features. Thus the Franklands
found the rivers Thames and Lea purest in summer, and this
they attributed to the fact that in this season there is most
spring water entering, and very little water as washings off land.
In the case of other rivers the bacteria have been found to be
fewest in winter. A great many circumstances must therefore
be taken into account in dealing with mere enumerations of
water bacteria, and such enumerations are only useful w7hen
they are taken simultaneously over a stretch of river, with
special reference to the sources of the water entering the river.
Thus it is usually found that immediately below a sewage
effluent the bacterial content rises, though in a comparatively
short distance the numbers may markedly decrease, and it may
be that the river as far as numbers are concerned may appear
to return to its previous bacterial content. The numbers of
138 BACTERIA IN WATER
bacteria present in rivers vary so greatly that there is little use
in quoting figures, most information being obtainable by
comparative enumerations before and after a given event has
occurred to a particular water. Such a method is thus of great
use in estimating the efficacy of the filter beds of a town water
supply. These usually remove from 95 to 98 per cent of the
bacteria present, and a town supply as it issues from the filter
beds should not contain more than 100 bacteria per c.c. Again,
is it found that the storage of water diminishes the number .of
bacteria present. The highest counts of bacteria per c.c. are
observed with sewage ; for example, in the London sewage the
numbers range from six to twelve millions.
Much more important than the mere enumeration of the
bacteria present in a water is the question whether these include
forms pathogenic to man. The chief interest here, so far as
Europe is concerned, lies in the fact that typhoid fever is so
frequently water-borne, but cholera and certain other intestinal
diseases have a similar source. The search in waters for the
organisms concerned in these diseases is a matter of the greatest
difficulty, for each belongs to a group of organisms morpho-
logically similar, very widespread in nature, and many of which
have little or no pathogenic action. The biological characters
of these organisms will be given in the chapters devoted to
the diseases in question, but here it may be said that from
the public health standpoint the making of their being
found a criterion for the condemning of a water is impractic-
able. There is no doubt that the typhoid and cholera
bacteria can exist for some time in water — at least this
has been found to be the case when sterile water has been
inoculated with these bacteria. But to what extent the same is
true when they are placed in natural conditions, which involve
their living in the presence of other organisms, is unknown, for
it may be safely said that by no known method can the presence
of either be demonstrated in the complex mixtures which occur
in nature. With regard to the typhoid bacillus, of late the
tendency has been to seek for the presence of indirect bacterio-
logical evidence which might point in the direction of the
possibility of the presence of this organism. The methods
employed and the lines along which such investigations have
gone have already been alluded to in connection with soil.
The whole question turns on the possibility of recognising
bacteriologically the contamination of water with sewage.
Klein and Houston here insist on the fact that in crude sewage
the b. coli or the members of the coli group are practically
BACTERIAL TREATMENT OF SEWAGE 139
never fewer than 100,000 per c.c., and therefore if in a water
this organism forms a considerable proportion of the total
number of organisms present, then there is grave reason for
suspecting sewage pollution. Houston holds that, the nearer the
majority of the coli organisms in a water approach to the typical
reactions of coli the more likely is sewage contamination to be
present. The reactions regarded by him as typical are, gas
production in gelatin shake culture, production of indol,
clotting of milk, production of fluorescence in neutral-red broth,
acid and gas production in lactose peptone solution (v. b. coli).
The presence of b. coli in 100 c.c. of deep well water or in
10 c.c. of river or shallow well water is sufficient to condemn
that water. As the b. coli is fairly widespread in nature, Klein
and Houston hold that valuable supporting evidence is
found in the presence of the b. enteritidis sporogenes and of
the streptococci, both of which are probably constant inhabit-
ants of the human intestine. The spores of the former usually
number 100 per c.c. in sewage, and the presence of the latter
can always be recognised in *001 grm. of human faeces. The
deductions to be drawn from the presence of these in water
are the same as those to be drawn from their presence in soil.
A further point here is that it is well, wherever practicable, that
the indirect evidence as to the potability of a water which is
usually derived from chemical analysis should be supplemented
by a bacteriological search for the three groups of organisms
mentioned. It has been found that in water artificially polluted
with sewage containing them, they can be detected by bacterio-
logical methods in mixtures from ten to a hundred times more
dilute than those in which the pollution can be detected by
purely chemical methods.
Bacterial Treatment of Sewage. — Of late years the opinion
has been growing that the most appropriate method of dealing
with the disposal of sewage is to imitate as far as possible the
processes which occur in nature for the breaking up of organic
material. These practically depend entirely on bacterial action.
Hence the rationale of the most approved methods of sewage
disposal is to encourage the growth of bacteria which naturally
exist in sewage, and which are capable of breaking up organic
compounds and of converting the nitrogen into nitrates and
nitrites. The technique by which this is accomplished is very
varied and sometimes rather empirical, but probably the general
principles underlying the different methods are comparatively
simple. It is probable that for the complete destruction of the
organic matter of sewage both aerobic and anaerobic bacteria
140 BACTERIA IN WATER
are required, though on this point there may be some difference
of opinion. Certainly very fair results are obtained when
apparently the conditions chiefly favour aerobic organisms alone.
This is usually effected by running the sewage on to beds of sand,
or preferably of coke, allowing it to stand for some hours, slowly
running the effluent out through the bottom of the bed, and
leaving the bed to rest for some hours before recharging. The
final result is better if the effluent be afterwards run over another
similar coke-bed. According to some authorities the sewage, as
it runs into the first bed, takes up from the air considerable free
oxygen, which, however, soon disappears during the stationary
period, so that on leaving the first bed the sewage contains little
oxygen. In the latter part of its stay it has thus been submitted
to anaerobic conditions. Further, while by the passage of the
effluent out of the first bed oxygen is sucked in, this rapidly dis-
appears, and during the greater part of the resting stage the
interstices of the bed are filled with carbonic acid gas, with
nitrogen partly derived from the air, partly from putrefactive
processes, and thus in the filter anaerobic conditions prevail,
under which the bacteria can act on the deposit left on the coke.
On this latter point there is difference of opinion, for, in examin-
ing London sewage, Clowes has found oxygen present in
abundance from four to forty hours after the sewage has been
run off. Sometimes the treatment of the sewage consists in
allowing it continuously to trickle through sand or gravel or coke
beds. Probably the best results in sewage treatment are obtained
when it is practicable to introduce a step where there can be no
doubt that the conditions are anaerobic. This involves as a pre-
liminary stage the treatment of the sewage in what is called a
septic tank, and the method has been adopted at Exeter, Button,
and Yeovil in this country, and very fully worked at in America
by the State Board of Health of Massachusetts. In the explana-
tion given of the rationale of this process, sewage is looked on as
existing in three stages. (1) First of all, fresh sewage — the newly
mixed and very varied material as it enters the main sewers.
(2) Secondly, stale sewage — the ordinary contents of the main
sewers. Here there is abundant oxygen, and as the sewage flows
along there occurs by bacterial action a certain formation of
carbon dioxide and ammonia which combine to form ammonium
carbonate. This is the sewage as it reaches the purification works.
Here a preliminary mechanical screening may be adopted, after
which it is run into an air-tight tank — the septic tank. (3)
It remains there for from twenty-four to thirty-six hours, and
becomes a foul-smelling fluid — the septic sewage. The chemical
ANTISEPTICS 141
changes which take place in the septic tank are of a most complex
nature. The sewage entering it contains little free oxygen, and
therefore the bacteria in the tank are probably largely anaerobic,
and the changes which they originate consist of the formation of
comparatively simple compounds of hydrogen with carbon,
sulphur, and phosphorus. As a result there is a great reduction
in the amount of organic nitrogen, of albuminoid ammonia, and
of carbonaceous matter. The latter fact is important, as the
clogging of ordinary filter beds is largely due to the accumulation
of such material, and of matters generally consisting of cellulose.
One further important effect is that the size of the deposited
matter is decreased, and therefore it is more easily broken up
in the next stage of the process. This consists of running the
effluent from the septic tank on to filter beds, preferably of coke,
where a further purification process takes place. By this method
there is first an anaerobic treatment succeeded by an aerobic ;
in the latter the process of nitrification occurs by means of the
special bacteria concerned. The results are of a satisfactory nature,
there being often a marked diminution in the number of coli
organisms present.
Often the effluent from a sewage purification system contains
as many bacteria as the sewage entering, but, especially by means
of the septic tank method, there is often a marked diminution.
It is said by some that pathogenic bacteria do not live in sewage.
The typhoid bacillus has been found to die out when placed in
sewage, but it certainly can live in this fluid for a much longer
period than that embraced by any purification method. Thus
the constant presence of b. coli, b. enteritidis, and streptococci
which has been observed in sewage effluents must here still be
looked on as indicating a possible infection with the typhoid
bacillus, and it is only by great dilution and prolonged exposure
to the conditions present in running water that such an effluent
can be again a part of a potable water.
ANTISEPTICS.
The death of bacteria is judged of by the fact that when
they are placed on a suitable food medium no development
takes place. Microscopically it would be observed that division
no longer occurred, and that in the case of motile species move-
ment would have ceased, but such an observation has only
scientific interest. From the importance of being able to kill
bacteria an enormous amount of work has been done in the way
of investigating the means of doing so by chemical means,
142 ANTISEPTICS
and the bodies having such a capacity are called antiseptics. It
is now known that the activity of these agents is limited to
the killing of bacteria outside the animal body, but still even
this is of high importance.
Methods. — These vary very much. In early inquiries a great point
was made of the prevention of putrefaction, and work was done in the
way of finding how much of an agent must be added to a given solution
such as beef extract, urine, etc., in order that the bacteria accidentally
present might not develop ; but as bacteria vary in their powers of re-
sistance, the method was unsatisfactory, and now an antiseptic is usually
judged of by its effects on pure cultures of definite pathogenic microbes,
and in the case of a sporing bacterium the effect on both the vegetative
and spore forms is investigated. The organisms most used are the
staphylococcus pyogenes, streptococcus pyogenes, and the organisms of
typhoid, cholera, diphtheria, and anthrax — the latter being most used
for testing the action on spores. The best method to employ is to take
sloped agar cultures of the test organism, scrape off the growth, and mix
it up with a small amount of distilled water and filter this emulsion
through a plug of sterile glass wool held in a small sterile glass funnel,
add a measured quantity of this fluid to a given quantity of a solution
of the antiseptic in distilled water, then alter the lapse of the period of
observation to remove one or two loopfuls of the mixture and place them
in a great excess of culture medium. Here it is preferable to use fluid
agar, which is then plated and incubated ; such a procedure is prefer-
able to the use of bouillon tubes, as any colonies developing can easily
be recognised as belonging to the species of bacterium used. In dealing
with strong solutions of chemical agents it is necessary to be sure that
the culture fluid is in great excess, so that the small amount of the
antisej.tic which is transferred with the bacteria may be diluted far
beyond the strength at which it still can have any noxious influence.
Sometimes it is possible at the end of the period of observation to
change the antiseptic into inert bodies by the addition of some other
substance and then test the condition of the bacteria, and if the inert
substances are fluid there is no objection to this proceeding, but if in
the process a precipitate results, then it is better not to have recourse
to such a method, as sometimes the bacteria are carried down with the
precipitate and may escape the culture test. The advisability of, when
possible, thus chemically changing the antiseptic was first brought to
notice by the criticism of Koch's statements as to the efficacy of
mercuric chloride in killing the spores of the b. anthracis. The method
he employed in his experiments was to soak silk threads in an emulsion
of anthrax spores and dry them. These were then subjected to the
action of the antiseptic, well washed in water, and laid on the surface of
agar. It was found, however, that with threads exposed to a far higher
concentration of the corrosive sublimate than Koch had stated was
sufficient to prevent growth, if the salt were broken up by the action of
ammonium sulphide and this washed off, growth of anthrax still occurred
when the threads were laid on agar. The explanation given was that
the antiseptic had formed an albuminate with the case of each spore, and
that this prevented the antiseptic from acting upon the contained
protoplasm. Such an occurrence only takes place with spores, and the
method given above, in which the small amount of antiseptic adhering
THE ACTION OF ANTISEPTICS 143
to the bacteria is swamped in an excess of culture fluid, can safely be
followed, especially when a series of antiseptics is being compared.
Much attention has been paid to the standardisation of antiseptics,
and a watery solution of carbolic acid is now generally taken as the
standard with which other antiseptics are compared. Rideal and
Walker point out that 110 parts by weight of B. P. carbolic acid equal
100 parts by weight of phenol, and they recommend the following method
of standardising. To 5 c.c. of a particular dilution of the di.-infectant
add 5 drops of a 24-hour-old bouillon culture of the organism (usually
b. typhosus) which has been incubated at 87° C. Shake the mixture and
make subcultures every 2^ minutes to 15 minutes. Perform a parallel
series of experiments with carbolic acid and express the comparative
result iu multiples of the carbolic acid doing the same work.
The Action of Antiseptics. — In inquiries into the actions of
antiseptic^ attention to a great variety of factors is necessary,
especially when the object is not to compare different antiseptics
with one another, but when the absolute value of any body is
being investigated. Thus the medium in which the bacteria to
be killed are situated, is important ; the more albuminous the
surroundings are, the greater degree of concentration is required.
Again, the higher the temperature at which the action is to take
place, the more dilute may the antiseptic be, or the shorter the
exposure necessary for a given effect to take place. The most
important factor, however, to be considered is the chemical
nature of the substances employed. Though nearly every sub-
stance which is not a food to the animal or vegetable body is
more or less harmful to bacterial life, yet certain bodies have
a more marked action than others. Thus it may be said that
the most important antiseptics are the salts of the heavy metals,
certain acids, especially mineral acids, certain oxidising and re-
ducing agents, a great variety of substances belonging to the
aromatic series, and volatile oils generally. In comparing
different bodies belonging to any one of these groups the
chemical composition or constitution is very important, and if
such comparisons are to be made, the solutions compared must
be equimolecular ; in other words, the action of a molecule of
one body must be compared with the action of a molecule of
another body. This can be done by dissolving the molecular
weight in grammes in say a litre of water (see p. 33). When
this is done important facts emerge. Thus, generally speaking,
the compounds of a metal of high atomic weight are more
powerful antiseptics than those of one belonging to the same
series, but of a lower atomic weight. Among organic bodies
again substances with high molecular weight are more powerful
than those of low molecular weight — thus butyric alcohol is more
powerful than e thy lie alcohol — and important differences among
144 ANTISEPTICS
the aromatic bodies are associated with their chemical constitu-
tion. Thus among the cresols the ortho- and para-bodies re-
semble each other in general chemical properties, and stand apart
from metacresol ; they also are similar in antiseptic action, and
are much stronger than the meta-body. The same may be
observed in the other groups of ortho-, meta-, and para-bodies.
Again, such a property as acidity is important in the action of a
substance, and, generally speaking, the greater the avidity of an
acid to combine with an alkali, the more powerful an antiseptic
it is. With regard to oxidising agents and reducing agents,
probably the possession of such properties has been overrated as
increasing bactericidal potency. Thus in the case of such re-
ducers as sulphurous acid and formic acid, the effect is apparently
chiefly due to the fact that these substances are acids. Formic
acid is much more efficient than formate of sodium. In the case
of permanganate of potassium, which is usually taken as the
type of oxidising agents in this connection, it can be shown that
the greater amount of the oxidation which takes place when this
agent is brought into contact with bacteria occurs after the
organisms are killed. Such an observation is, however, not
conclusive as to the non-efficiency of the oxidation process, for
the death of the bacteria might be due to the oxidation of a
very small part of the bacterial protoplasm. Apart from the
chemical nature of antiseptic agents, the physical factors con-
cerned in their solution, especially when they are electrolytes,
probably play a part in their action. The part played by such
factors is exemplified in the important fact that a strong solution
acting for a short time will have the same effect as a weaker
solution acting for a longer time. From what has been said it
will be realised that the real causes of a material being an
antiseptic are very obscure, and at present we can only have a
remote idea of the factors at work.
The Actions of certain Antiseptics. — Here we can only
briefly indicate certain results obtained with the more common
members of the group.
Chlorine. — All the halogens have been found to be powerful
antiseptics, but from the cheapness with which it can be produced
chlorine has been most used ; not only is it the chief active
agent in the somewhat complex action of bleaching powder, but
it is also the chief constituent of several proprietary substances,
of which " Electrozone " is a good example. This last substance
is made from electrolysing sea -water, when magnesia and
chlorine being liberated, magnesium hypochlorite and magnesium
chloride are formed. In the action of this substance free hypo-
ACTIONS OF CERTAIN ANTISEPTICS 145
chlorous acid is formed, and the effect produced is thus similar
to that of bleaching powder. Nissen, investigating the action of
the latter, found that 1^ per cent killed typhoid bacilli in faeces ;
and Rideal found that 1 part to 400-500 disinfected sewage in
fourteen minutes, and Delepine's results show that 1 part to 50
(equal to '66 per cent of chlorine) rapidly kills the tubercle
bacillus, and 1 part to 10 (equal to 3 '3 per cent) killed anthrax
spores. Klein found that *05 per cent of chlorine killed most
bacterial spores in five minutes.
Iodine Terchloride. — This is a very unstable compound of
iodine and chlorine, and though it has been much used as an
antiseptic, seeing that the- substance only remains as IC13 in
an atmosphere of chlorine gas, it is open to doubt whether the
effects described are not due to a very complicated action of
free hydrochloric acid, hydriodic acid, of oxyacids of chlorine
and iodine produced by its decomposition, and also, in certain
cases, of organic iodine compounds formed from its contact with
albuminous material. It is stated that the action is very potent :
a 1 per cent solution is said instantly to kill even anthrax spores,
but if the spores be in bouillon, death occurs after from ten to
twelve minutes. In serum the necessary exposure is from thirty
to forty minutes. A solution of 1-1000 will kill the typhoid,
cholera, and diphtheria organisms in five minutes.
Nascent Oxygen. — This is chiefly available in two ways — firstly,
when in the breaking up of ozone the free third atom of the
ozone molecule is seeking to unite with another similar atom ;
secondly, when peroxide of hydrogen is broken up into water
and an oxygen atom is thereby liberated. In commerce the
activity of " Sanitas " compounds is due to the formation of
ozone by the slow oxidation of the resin, camphor, and thymol
they contain.
Per chloride of Mercury. — Of all the salts of the heavy metals
this has been most widely employed, and must be regarded as
one of the most powerful and useful of known antiseptics. In
testing its action on anthrax spores there is no doubt that in the
earlier results its potency was overrated from a neglect of the
fact already alluded to, that in the spore-case an albuminate of
mercury was formed which prevented the contained protoplasm
from developing, while not depriving it of life. It has been
found, however, that this salt in a strength of 1-100 will kill the
spores in twenty minutes, although an hour's exposure to 1-1000
has no effect. The best results are obtained by the addition to
the corrosive sublimate solution of -5 per cent of sulphuric acid
or hydrochloric acid ; the spores will then be killed by a seventy-
10
146 ANTISEPTICS'
minute exposure to a 1-200 solution. When, however, organisms
in the vegetative condition are being dealt with, much weaker
solutions are sufficient; thus anthrax bacilli in blood will be
killed in a few minutes by 1-2000, in bouillon by 1-40,000, and
in water by 1-500,000. Plague bacilli are killed by one to two
minutes' exposure to 1-3000. Generally speaking, it may be said
that a 1-2000 solution must be used for the practically instan-
taneous killing of vegetative organisms.
Perchloride of mercury is one of the substances which have
been used for disinfecting rooms by distributing it from a spray
producer, of which the Equifex may be taken as a type. With
such a machine it is calculated that 1 oz. of perchloride of
mercury used in a solution of 1-1000 will probably disinfect 3000
square feet of surface. Such a procedure has been extensively
used in the. disinfection of plague houses, but the use of a stronger
solution (1-500 acidulated) is probably preferable.
Formalin as a commercial article is a 40 per cent solution of
formaldehyde in water. This is a substance which of late years
has come much into vogue, and it is undoubtedly a valuable
antiseptic. A disadvantage, however, to its use is that, when
diluted and exposed to air, amongst other changes which it
undergoes it may be transformed, under little understood
conditions, into trioxymethylene and paraform aldehyde, these
being polymers of formaldehyde. The bactericidal values of these
mixtures are thus indefinite. Formalin may be used either by
applying it in its liquid form or as a spray, or the gas which
evaporates at ordinary temperatures from the solution may be
utilised. To disinfect such an organic mixture as pus containing
pyogenic organisms a 10 per cent solution acting for half an
hour is necessary. In the case of pure cultures, a 5 per cent
solution will kill the cholera organism in three minutes, anthrax
bacilli in a quarter of an hour, and the spores in five hours.
When such organisms as pyogenic cocci, cholera spirillum, and
anthrax bacillus infect clothing, an exposure to the full strength
of formalin for two hours is necessary, and in the case of anthrax
spores, for twenty-four hours. Silk threads impregnated with
the plague bacillus were found to be sterile after two minutes'
exposure to formalin.
The action of formalin vapour has been much studied, as its
use constitutes a cheap method of treating infected rooms, in
which case some spray-producing machine is employed. It is
stated that a mixture of 8 c.c. of formalin with 48 c.c. of water
is sufficient when vapourised to disinfect one cubic metre, so far
as non-sporing organisms are concerned. It is stated that 1 part
ACTIONS OF CERTAIN ANTISEPTICS 147
formalin in 10,000 of air will kill the cholera vibrio in one hour,
diphtheria bacillus in three hours, the staphylococcus pyogenes
in six hours, and anthrax spores in thirteen hours. In the case
of organisms which have become dry it is probable, however,
that much longer exposures are necessary, but on this point we
have not definite information.
Formalin gas has only a limited application ; it has little
effect on dry organisms, and in the case of wet organisms, in
order to be effective, probably must become dissolved so as to
give the moisture a proportion analogous to the strengths stated
above with regard to the vapour.
Sulphurous Acid. — This substance has long been in use,
largely from the cheapness with which it can be produced by
burning sulphur in the air. An atmosphere containing "98 per
cent will kill the pyogenic cocci in two minutes if they are wet,
and in twenty minutes if they are dry ; and anthrax bacilli are
killed by thirty minutes' exposure, but to kill anthrax spores an
exposure of from one to two hours to an atmosphere containing
1 1 per cent is necessary. For a small room the burning of about
a pound and a half (most easily accomplished by moistening the
sulphur with methylated spirit) is usually considered sufficient.
It has been found that if bacteria are protected, e.g. when
they, are in the middle of small bundles of clothes, no effect is
produced even by an atmosphere containing a large proportion
of the sulphurous acid gas. The practical applications of this
agent are therefore limited.
Potassium Permanganate. — The action of this agent very
much depends on whether it can obtain free access to the
bacteria to be killed or whether these are present in a solution
containing much organic matter. In the latter case the oxidation
of the organic material throws so much of the salt out of action
that there may be little left to attack the organisms. Koch
found that to kill anthrax spores a 5 per cent solution required
to act for about a day ; for most organisms a similar solution
acting for shorter periods has been found sufficient, and in the
case of the pyogenic cocci a 1 per cent solution will kill in ten
minutes. There is little doubt that such weaker solutions are of
value in disinfecting the throat on account of their non-irritating
properties, and good results in this connection have been obtained
in cases of diphtheria. A solution of 1 in 10,000 has been
found to kill plague bacilli in five minutes.
Carbolic Acid. — Of all the aromatic series this is the most
extensively employed antiseptic. All ordinary bacteria in the
vegetative condition, and of these the staphylococcus pyogenes
148 ANTISEPTICS
is the most resistant, are killed in less than five minutes by a
2-3 per cent solution in water, so that the 5 per cent solution
usually employed in surgery leaves a margin of safety. But for
the killing of such organisms as anthrax spores a very much
longer exposure is necessary ; thus Koch found it necessary to
expose these spores for four days to ensure disinfection. The
risk of such spores being present in ordinary surgical procedure
may be overlooked, but there might be risk of tetanus spores
not being killed, as these will withstand fifteen hours' exposure
to a 5 per cent solution.
In the products of the distillation of coal there occur, besides
carbolic acid, many bodies of a similar chemical constitution, and
many mixtures of these are in the market — the chief being creolin,
izal, and lysol, all of which are agents of value. Of these lysol
is perhaps the most noticeable, as from its nature it acts as a soap
and thus can remove fat and dirt from the hands. A one-third
per cent solution is said to destroy the typhoid and cholera
organisms in twenty minutes. A 1 per cent solution is sufficient
for ordinary surgical procedures.
lodoform. — This is an agent regarding the efficacy of which
there has been much dispute. There is little doubt that it owes
its efficiency to its capacity for being broken up by bacterial
action in such a way as to set free iodine, which acts as a powerful
disinfectant. . The substance is therefore of value in the treatment
of foul wounds, such as those of the mouth and Tectum, where
reducing bacteria are abundantly present. It acts more slightly
where there are only pyogenic cocci, and it seems to have a
specially beneficial effect in tubercular affections. In certain
cases its action may apparently be aided by the presence of the
products of tissue degeneration.
From the results which have been given it will easily be
recognised that the choice of an antiseptic and the precise
manner in which it is to be employed depend entirely on the
environment of the bacteria which are to be killed. In many
cases it will be quite impossible, without original inquiry, to say
what course is likely to be attended with most success.
CHAPTER V.
RELATIONS OF BACTERIA TO DISEASE— THE
PRODUCTION OF TOXINS BY BACTERIA.
Introductory. — It has already been stated that a strict division
of micro-organisms into saprophytes and true parasites cannot be
made. No doubt there are organisms such as the bacillus of
leprosy which as yet have not been cultivated outside the animal
body, and others, such as the gonococcus, which are in natural
conditions always parasites associated with disease. But these
latter can lead a saprophytic existence in specially prepared
conditions, and there are many of the disease-producing organisms,
such as the organisms of typhoid and cholera, which can nourish
readily outside the body, even in ordinary conditions. The
conditions of growth are, however, of very great importance in
the study of the modes of infection in the various diseases,
though they do not form the basis of a scientific division.
A similar statement applies to the terms pathogenic and
saprophytic, and even to the terms pathogenic and non-pathogenic.
By the term pathogenic is meant the power which an organism
has of producing morbid changes or effects in the animal
-body, either under natural conditions or in conditions artificially
arranged as in direct experiment. Now we know of no organ-
isms which will in all circumstances produce disease in all
animals, and, on the other hand, many bacteria described as
harmless saprophytes will produce pathological changes if intro-
duced in sufficient quantity. When, therefore, we speak of a
pathogenic organism, the term is merely a relative one, and
indicates that in certain circumstances the organism will produce
disease, though in the science of human pathology it is often
used for convenience as implying that the organism produces
disease in man in natural conditions.
Modifying Conditions. — In studying the pathogenic effects in
149
150 RELATIONS OF BACTERIA TO DISEASE
any instance, both the micro-organisms and the animal affected
must be considered, and not only the species of each, but also
its exact condition at the time of infection. In other words,
the resulting disease is the product of the sum total of the
characters of the infecting agent, on the one hand, and of the
subject of infection, on the other. We may, therefore, state some
of the chief circumstances which modify each of these two factors
involved and, consequently, the diseased condition produced.
1. The Infecting Agent. — In the case of a particular species
of bacterium its effect will depend chiefly upon (a) its virulence,
and (6) the number introduced into the body. To these may
be added (c) the path of infection.
The virulence, i.e. the power of multiplying in the body and
producing disease, varies greatly in different conditions, and the
methods by which it can be diminished or increased will be
afterwards described (vide Chapter XIX.). One important
point is that when a bacterium has been enabled to invade
and multiply in the tissues of an animal, its virulence for that
species is often increased. This is well seen in the case of
certain bacteria which are normally present on the skin or
mucous surfaces. Thus it has been repeatedly proved that the
bacillus coli cultivated from a septic peritonitis is much more
virulent than that taken from the bowel of the same animal.
The virulence may be still more increased by inoculating from
one animal to another in series — the method of passage. Widely
different effects are, of course, produced on the virulence being
altered. For example, a streptococcus which produces merely
a local inflammation or suppuration, may produce a rapidly fatal
septicaemia when its virulence is raised. Virulence also has a
relation to the animal employed, as occasionally on being in-
creased for one species of animal it is diminished for another.
For example, streptococci, on being inoculated in series through
a number of mice, acquire increased virulence for these animals,
but become less virulent for rabbits. (Knorr.) The theoretical
consideration of virulence must be reserved for a later
chapter (see Immunity).
The number of the organisms introduced, i.e. the dose of the
infecting agent, is another point of importance. The healthy
tissues can usually resist a certain number of pathogenic organisms
of given virulence, and it is only in a few instances that one or
two organisms introduced will produce a fatal disease, e.g. the
case of anthrax in white mice. The healthy peritoneum of a
rabbit can resist and destroy a considerable number of pyogenic
micrococci without any serious result, but if a larger dose be
CONDITIONS MODIFYING PATHOGENICITY 151
introduced, a fatal peritonitis may follow. Again, a certain
quantity of a particular organism injected subcutaneously may
produce only a local inflammatory change, but in the case of a
larger dose the organisms may gain entrance to the blood stream
and produce septicaemia. There is, therefore, for a particular
animal, a minimum lethal dose which can be determined by
experiment only ; a dose, moreover, which is modified by various
circumstances difficult to control.
The path of infection may alter the result, serious effects often
following a direct entrance into the blood stream. Staphylococci
injected subcutaneously in a rabbit may produce only a local
abscess, whilst on intravenous injection multiple abscesses in ,
certain organs may result and death may follow. Local inflam-
matory reaction with subsequent destruction of the organisms
may be restricted to the site of infection or may occur also in
the lymphatic glands in relation. The latter therefore act as a
second barrier of defence, or as a filtering mechanism which aids
in protecting against blood infection. This is well illustrated in
the case of " poisoned wounds." In some other cases, however,
the organisms are very rapidly destroyed in the blood stream,
and Klemperer has found that in the dog, subcutaneous injection
of the pneumococcus produces death more readily than intra-
venous injection.
2. The Subject of Infection. — Amongst healthy individuals
susceptibility and, in inverse ratio, resistance to a particular
microbe may vary according to (a) species, (b) race and individual
peculiarities, (c) age. Different species of the lower animals
show the widest variation in this respect,' some being extremely
susceptible, others highly resistant. Then there are diseases,
such as leprosy, gonorrhoea, etc., which appear to be peculiar to
the human subject and have not yet been transmitted to animals.
And further, there are others, such as cholera and typhoid, which
do not naturally affect animals, and the typical lesions of which
cannot be experimentally reproduced in them, or appear only
imperfectly, although pathogenic effects follow inoculation with
the organisms. In the case of the human subject, differences in
susceptibility to a certain disease are found amongst different
races and also amongst individuals of the same race, as is well
seen in the case of tubercle and other diseases. Age also plays
an important part, young subjects being more liable to certain
diseases, e.g. to diphtheria. Further, at different periods of life
certain parts of the body are more susceptible, for example, in
early life, the bones and joints to tubercular and acute suppura-
tive affections.
152 RELATIONS OF BACTERIA TO DISEASE
In increasing the susceptibility of a given individual, condi-
tions of local or general diminished vitality play the most
important part. It has been experimentally proved that
conditions such as exposure to cold, fatigue, starvation, etc.,
all diminish the natural resistance to bacterial infection. Rats
naturally immune can be rendered susceptible to glanders by
being fed with phloridzin, which produces a sort of diabetes, a
large amount of sugar being excreted in the urine (Leo).
Guinea-pigs may resist subcutaneous injection of a certain dose
of the typhoid bacillus, but if at the same time a sterilised
culture of the bacillus coli be injected into the peritoneum, they
quickly die of a general infection. Also a local susceptibility
may be produced by injuring or diminishing the vitality of a
part. If, for example, previous to an intravenous injection of
staphylococci, the aortic cusps of a rabbit be injured, the
organisms may settle there and set up an ulcerative endocarditis ;
or if a bone be injured, they may produce suppuration at the
part, whereas in ordinary circumstances these lesions would not
take place. The action of one species of bacterium is also often
aided by the simultaneous presence of other species. In this
case the latter may act simply as additional irritants which
lessen the vitality of the tissues, but in some cases their presence
also appears to favour the development of a higher degree of
virulence of the former.
These facts, established by experiment (and many others
might be given), illustrate the important part which local or
general conditions of diminished vitality may play in the pro-
duction of disease in the human subject. This has long been
known by clinical observation. In normal conditions the blood
and tissues of the body, with the exception of the skin and
certain of the mucous surfaces, are bacterium-free, and if a few
organisms gain entrance, they are destroyed. But if the vitality
becomes lowered their entrance becomes easier and the possibility
of their multiplying and producing disease greatly increased. In
this way the favouring part played by fatigue, cold, etc., in the
production of diseases of which the direct cause is a bacterium,
may be understood. It is important to keep in view in this con-
nection that many of the inflammation-producing and pyogenic
organisms are normally present on the skin and various mucous
surfaces. The action of a certain organism may devitalise the
tissues to such an extent as to pave the way for the entrance of
other bacteria ; we may mention the liability of the occurrence
of pneumonia, erysipelas, and various suppurative conditions in
the course of or following infective fevers. In some cases the
MODES OF BACTERIAL ACTION 153
specific organism may produce lesions through which the other
organisms gain entrance, e.g. in typhoid, diphtheria, etc. A
notable example of diminished resistance to bacterial infection is
seen in the case of diabetes7; tuberculosis and infection with
pyogenic organisms are prone to occur in this disease and are of
a severe character. It is not uncommon to find in the bodies of
those who have died from chronic wasting disease, collections of
micrococci or bacilli in the capillaries of various organs, which
have entered in the later hours of life ; that is to say, the
bacterium-free condition of the blood has been lost in the period
of prostration preceding death.
The methods by which the natural resistance may be specific-
ally increased belong to the subject of immunity, and are
decribed in the chapter on that subject.
Modes of Bacterial Action. — In the production of disease
by micro-organisms there are two main factors involved, namely,
(a) the multiplication of the living organisms after they have
entered the body, and (b) the production by them of poisons
which may act both upon the tissues around and upon the body
generally. The former corresponds to infection, the latter is of
the nature of intoxication or poisoning. In different diseases one
of these is usually the more prominent feature, but both are
always more or less concerned.
1 . Infection and Distribution of the Bacteria in the Body. —
After pathogenic bacteria have invaded the tissues, or in other
words after infection by bacteria has taken place, their further
behaviour varies greatly in different cases. In certain cases
they may reach and multiply in the blood stream, producing a
fatal septicaemia. In the lower animals this multiplication of the
organisms in the blood throughout the body may be very exten-
sive (for example, the septicaemia produced by the pneumococcus
in rabbits) ; but in septicaemia in man, it very seldom, if ever,
occurs to so great a degree, the organisms rarely remain in large
numbers in the circulating blood, and their detection in it during
life by microscopic examination is rare, and even culture methods
may give negative results unless a large amount of blood is
used. In such cases, however, the organisms may be found
post mortem lying in large numbers within the capillaries of
various organs, e.g. in cases of septicaemia produced by strepto-
cocci. In the human subject more frequently one of two things
happens. In the first place, the organisms may remain local,
producing little reaction around them, as in tetanus, or a well-
marked lesion, as in diphtheria, pneumonia, etc. Or in the
second place, they may pass by the lymph or blood stream to
154 RELATIONS OF BACTERIA TO DISEASE
other parts or organs in which they settle, multiply, and produce
lesions, as in tubercle.
2. Production of Chemical Poisons. — In all these cases the
growth of the organisms is accompanied by the formation of
chemical products, which act generally or locally in varying degree
as toxic substances. The toxic substances become diffused
throughout the system, and their effects are manifested chiefly
by symptoms such as the occurrence of fever, disturbances of
the circulatory, respiratory, and nervous systems, etc. In some
cases corresponding changes in the tissues are found, for example,
the changes in the nervous system in diphtheria, to be after-
wards described. The general toxic effects may be so slight as
to be of no importance, as in the case of a local suppuration,
or they may be very intense as in tetanus, or again, less severe
but producing cachexia by their long continuance, as in tuber-
culosis.
The occurrence of local tissue changes or lesions produced in
the neighbourhood of the bacteria, as already mentioned, is one
of the most striking results of bacterial action, but these also
must be traced to chemical substances formed in or around the
bacteria, and either directly or through the medium of ferments.
In this case it is more difficult to demonstrate the mode of
action, for, in the tissues the chemical products are formed by
the bacteria slowly, continuously, and in a certain degree of
concentration, and these conditions cannot be exactly reproduced
by experiment. It is also to be noted that more than one poison
may be produced by a given bacterium, e.g. the tetanus
bacillus (p. 380). Further, it is very doubtful whether all the
chemical substances formed by a certain bacillus growing in the
tissues are also formed by it in cultures outside the body. The
separated toxin of diphtheria, like various vegetable and animal
toxins (vide infra), however, possesses a local toxic action of
very intense character, evidenced often by extensive necrotic
change.
The injection of large quantities of many different pathogenic
organisms in the dead conditions results in the production of a
local inflammatory change which may be followed by suppura-
tion, this effect being possibly brought about by certain sub-
stances in the bacterial protoplasm common to various species,
or at least possessing a common physiological action (Buchner
and others). When dead tubercle bacilli, however, are intro-
duced into the blood stream, nodules do result in certain parts
which have a resemblance to ordinary tubercles. In this case
the bodies of the bacilli evidently contain a highly resistant and
TISSUE CHANGES PRODUCED BY BACTERIA 155
slowly acting substance which gradually diffuses around and
produces effects (vide Tuberculosis).
Summary. — We may say then that the action of bacteria as
disease-producers, as in fact their power to exist and multiply in
the living body, depends upon the chemical products formed
directly or indirectly by them. This action is shown by tissue
changes produced in the vicinity of the bacteria or throughout
the system, and by toxic symptoms of great variety of degree and
character.
We shall first consider the effects of bacteria on the body
generally, and afterwards the nature of the chemical products.
EFFECTS OF BACTERIAL ACTION.
These may be for convenience arranged in a tabular form as
follows : —
A. Tissue Changes.
(1) Local changes, i.e. changes produced in the neigh-
bourhood of the bacteria.
Position (a) At primary lesion.
(6) At secondary foci.
Character (a) Tissue reactions ) Acute or
(b) Degeneration and necrosis j Chronic.
(2) Produced at a distance from the bacteria, directly or
indirectly, by the absorption of toxins.
(a) In special tissues —
(a) as the result of damage, e.g. nerve cells and
fibres, secreting cells, vessel walls, or
(/3) changes of a reactive nature in the blood-
forming organs.
(&) General anatomical changes, the effects of mal-
nutrition or of increased waste.
B. Changes in Metabolism.
The occurrence of fever, of errors of assimilation and
elimination, etc.
A. Tissue Changes produced by Bacteria. — The effects of
bacterial action are so various as to include almost all known
pathological changes. However varied in character, they may
be classified under two main headings : — (a) those of a degenera-
tive or necrotic nature, the direct result of damage, and (6) those
156 RELATIONS OF BACTERIA TO DISEASE
of reactive nature, defensive or reparative. The former are the
expression of the necessary vulnerability of the tissues, the latter
of protective powers evolved for the benefit of the organism. In
the means of defence both leucocytes and the fixed cells of the
tissues are concerned. Both show phagocytic properties, i.e.
have the power of taking up bacteria into their protoplasm. The
cells are guided towards the focus of infection by chemiotaxis,
and thus we find that different bacteria attract different cells.
The most rapid and abundant supply of phagocytes is seen in
the case of suppurative conditions where the neutrophile leuco-
cytes of the blood are chiefly concerned. When the local lesion
is of some extent there is usually an increase of these cells in
the blood — a neutrophile leucocytosis. And further, recent
observations have shown that associated with this there is in the
bone-marrow an increased number of the mother-cells of these
leucocytes — the neutrophile myelocytes. The passage of the
neutrophile leucocytes from the marrow into the blood, with the
resulting leucocytosis, is also apparently due to the absorbed
bacterial toxins acting chemiotactically on the marrow. These
facts abundantly show that the means of defence is not a mere
local mechanism, but that increased proliferative activity in
distant tissues is called into play. In addition to direct phagocyt-
osis by these leucocytes, there is now abundant evidence that
an important function is the production in the body of bactericidal
and other antagonistic substances. In other cases the cells
chiefly involved are the mononuclear hyaline leucocytes, and
with them the endothelial cells, e.g. of serous membranes, often
play an important part in the defence ; this is well seen in
typhoid fever, where the specific bacillus appears to have little
or no action on the neutrophile leucocytes. In other cases,
again, the reaction is chiefly on the part of the connective
cells, though their proliferation is always associated with some
variety of leucocytic infiltration and usually also with the forma-
tion of new blood vessels. Such a connective tissue reaction
occurs especially in slow infections or in the later stages of an
acute infection. The tissue changes resulting from cellular
activity in the presence of bacterial invasion are naturally very
varied — examples of this will be found in subsequent chapters —
but they may be said to be manifestations of the two funda-
mental processes of (a) increased functional activity — movement,
phagocytosis, secretion, etc. — and (b) increased formative activity
— cell growth and division. The exudation from the blood
vessels has been variously interpreted. There is no doubt that
the exudate has bactericidal properties and also acts as a diluting
LOCAL LESIONS 157
agent, but it must still be held as uncertain whether the process
of exudation ought to be regarded as primarily defensive or as
the direct result of damage to the endothelium of the vessels.
It may also be pointed out that the various changes referred to
are none of them peculiar to bacterial invasion ; they are examples
of the general laws of tissue change under abnormal conditions,
and they can all be reproduced by chemical substances in solution
or in a particulate state. What constitutes their special feature
is their progressive or spreading nature, due to the bacterial
multiplication.
(1) Local Lesions. — In some diseases the lesion has a special
site ; for example, the lesion of typhoid fever and, to a less
extent, that of diphtheria. In other cases it depends entirely
upon the point of entrance, e.g. malignant pustule and the
conditions known as wound infections. In others again, there is
a special tendency for certain parts to be affected, as the upper
parts of the lungs in tubercle. In some cases the site has a
mechanical explanation.
When organisms gain an entrance to the blood from a primary
lesion, the organs specially liable to be affected vary greatly in
different diseases. Pyogenic cocci show a special tendency to
settle in the capillaries of the kidneys and produce miliary
abscesses, whilst these lesions rarely occur in the spleen. On
the other hand, the nodules in disseminated tubercle or glanders
are much more numerous in the spleen than in the kidneys,
which in the latter disease are usually free from them. The
important point is that the position of the disseminated lesions
is not to be explained by a mechanical process, such as embolism,
but depends upon a special relation between the organisms and
the tissues, which may be spoken of either as a selective power
on the part of the organisms or a special susceptibility of tissues,
possibly in part due to their affording to the organisms more
suitable conditions of nutriment. Even in the case of the
lesions produced by dead tubercle bacilli, a certain selective
character is observed.
Acute Local Lesions. — The local inflammatory reaction
presents different characters in different conditions. It may be
accompanied by abundant fibrinous exudation, or by great
catarrh (in the case of an epithelial surface), or by haemorrhage,
or by oedema ; it may be localised or spreading in character ; it
may be followed by suppuration, and may be accompanied or
lead up to necrosis of the tissues of the part, a good example of
the latter event being found in a boil. Examples will be given
in subsequent chapters. The necrotic or degenerative changes
158 RELATIONS OF BACTEKIA TO DISEASE
affecting especially the more highly developed elements of tissues
are chiefly produced by the direct action of the bacterial poisons,
though aided by the disturbances of nutrition involved in the
vascular phenomena. It may here be pointed out that a well-
marked inflammatory reaction is often found in animals which
occupy a medium position in the scale of susceptibility, and that
an organism which causes a general infection in a certain animal
may produce only a local inflammation when its virulence is
lessened.
Chronic Local Lesions. — In a considerable number of diseases
produced by bacteria the local tissue reaction is a more chronic
process than those described. In other words, the specific
irritant is less intense, so that vhere is less vascular disturbance
and a greater preponderance of the proliferative processes,
leading to new formation of connective tissue or a modified
connective tissue. This formation may occur in foci here and
there, so that nodules of greater or less consistence result, or it
may be more diffuse. Such changes especially occur in the
diseases often known as the infective granulomata, of which
tubercle, leprosy, glanders, actinomycosis, syphilis, etc., are
examples. A hard and fast lin£, however, cannot be drawn
between such conditions and those described above as acute.
In glanders, for example, especially in the human subject, the
lesion often approaches very nearly to an acute suppurative
change, and sometimes actually is of this nature. Whilst in
these diseases the fundamental change is the same — viz. a re-
action to an irritant of minor intensity — the exact structural
characters and arrangement vary in different diseases. In some
cases the disease may be identified by the histological changes
alone, bnt on the other hand, this is often impossible. These
changes often include the occurren3e of degenerations or of
actual necrosis in the newly formed tissue. In the granulomata,
infection of other parts from the primary lesion takes place
chiefly by the blood vessels and lymphatics, though sometimes
along natural tubes such as the bronchi, intestine, etc.
(2) General Lesions produced by Toxins. — In the various in-
fective conditions produced by bactaria, changes commonly
occur in certain organs unassociated vrith the presence of the
bacteria ; these are produced by the 'action of bacterial pro-
ducts circulating in the blood. Many such lesions can be pro-
duced experimentally. The secreting cells of various organs,
especially the kidney and liver, are specially liable to change
of this kind. Cloudy swelling, which may be followed by fatty
change or by actual necrosis with granular disintegration, is
DISTURBANCES OF METABOLISM, ETC. 159
common. Hyaline change in the walls of arterioles may occur,
and in certain chronic conditions waxy change is brought about
in a similar manner. The latter has been produced in animals
by the repeated injection of the staphylococcus aureus.
Capillary haemorrhages are not uncommon, and are in many
cases due to an increased permeability of the vessel walls, aided
by changes in the blood plasma, as evidenced sometimes by
diminished coagulability. Similar haemorrhages may follow the
injection of some bacterial toxins, e.g. of diphtheria, and also of
vegetable poisons, e.g. ricin and abrin. Skin eruptions occurring
in the exanthemata are probably produced in the same way,
though in many of these diseases the causal organism has not
yet been isolated. We have, however, the important fact that
corresponding skin eruptions may be produced by poisoning with
certain drugs. In the nervous system degenerative changes
have been found in diphtheria, both in the spinal cord and in
the peripheral nerves, and have been reproduced experimentally
by the products of the diphtheria bacilli. There is also experi-
mental evidence that the bacillus coli communis and the strepto-
coccus pyogenes may, by means of their products, produce areas
of softening in the spinal cord, and this may furnish an explana-
tion of some of the lesions found clinically. It is also possible
that some serous inflammations may be produced in the same
way.
B. Disturbances of Metabolism, etc. — It will easily be
realised that such profound tissue changes as have been detailed
cannot occur without great interference with the normal bodily
metabolism. General malnutrition and cachexia are of common
occurrence, and it is a striking fact found by experiment that
after injection of bacterial products, e.g. of the diphtheria bacillus,
a marked loss of body weight often occurs which may be pro-
gressive, leading to the death of the animal. In bacterial
disease assimilation is often imperfect, for the digestive glands
are affected, it may be, by actual poisoning by bacterial products,
it may be by the occurrence of fever. The fatty degenerations
which are so common are indicative of a breaking down of the
proteid molecules, and are associated with increased urea produc-
tion, while the degeneration of the kidney epithelium renders
the excretion of waste products deficient or impossible, and this
is not infrequently the immediate cause of death. But of all
the changes in metabolism the most difficult to understand is
the occurrence of that interference with the heat-regulating
mechanism which results in fever. The degree and course of the
latter vary, sometimes conforming to a more or less definite type,
160 RELATIONS OF BACTERIA TO DISEASE
where the bacilli are selective in their field of operation, as in
croupous pneumonia or typhoid, sometimes being of a very ir-
regular kind, especially when the bacteria from time to time
invade fresh areas of the body, as in pysemic affections. The
main point of interest regarding the development of fever is as
to whether it is a direct effect of the circulation of bacterial
toxins, or if it is to be looked on as part of the reaction of the
body against the irritant. This question has still to be settled,
and all that we can do is to adduce certain facts bearing on it.
Thus in diphtheria and tetanus, where toxic action leading to
degeneration plays such an important part, fever may be a very
subsidiary feature, except in the terminal stage of the latter
disease ; and in fact in diphtheria profoundly toxic effects may
be produced with little or no interference with heat regulation.
On the other hand, in bacterial disease, where defensive and re-
parative processes predominate, fever is rarely absent, and it is
nearly always present when an active leucocytosis is going on.
In this connection it may be remarked that several observers
have found that, when a relatively small amount of the dead
bodies of certain bacteria are injected into an animal, fever
occurs; while the injection of a large amount of the same is
followed by subnormal temperatures and rapidly fatal collapse.
It might appear as if this indicated that the occurrence of fever
had a beneficial effect, but this is one of the points at issue.
Certainly such an effect is not due to the bacteria being unable
to multiply at the higher degrees of temperature occurring in
fever, for this has been shown not to be the case. Whether the
increase of bodily temperature indicates the occurrence of
changes resulting in the production of bactericidal bodies, etc.,
is very doubtful ; a production of antagonistic- substances may
be effected without the occurrence of fever or of any appar-
ent disturbance of health. If we consider the site of the heat
production in fever we again are in difficulties. It might appear
as if the tissue destruction, indicated by the occurrence of fatty
degeneration, would lead to heat development, but frequently
excessive heat production with increased proteid metabolism
occurs without any discoverable changes in the tissues ; and
further, in phosphorus poisoning there is little fever with great
tissue destruction. The increased work performed by the heart
in most bacterial infections no doubt contributes to the rise of
bodily temperature. But we must bear in mind that in fever
there is more than mere increase of heat production — there is
also a diminished loss of heat from interference with the nervous
mechanism of the sweat apparatus. The known facts would
THE- TOXINS PRODUCED BY BACTERIA 161
indicate that in fever there is a factor involving the nervous
system to be taken into account. The whole subject is thus
very obscure.
Symptoms. — Many of the symptoms occurring in bacterial
affections are produced by the histological changes mentioned,
as can be readily understood ; whilst in the case of others, corre-
sponding changes have not yet been discovered. Of the latter
those associated with fever, with its disturbances of metabolism
and manifold affections of the various systems, are the most
important. The nervous system is especially liable to be
affected — convulsions, spasms, coma, paralysis, etc., being
common. The symptoms due to disturbance or abolition of the
functions of secretory glands also constitute an important group,
forming, as they do, a striking analogy to what is found in the
action of various drugs.
These tissue changes and symptoms are given only as illus-
trative examples, and the list might easily be greatly amplified.
The important fact, however, is that necwly all, if not quite all,
the changes found throughout the organs {without the actuaLl
presence of bacteria), and also the symptoms occurring in infectvti£[
diseases, can either be exyerimeu tally revrnduMd by tfi* *.i
~~bf bacterial /><>is<>n.* or ka.v<- an, analogy in the action of drugs.
THE TOXINS PKODUCED BY BACTERIA.
Early Work on Toxins. — We know that bacteria are capable
of giving rise to poisonous bodies within the animal body and
also in artificial media. We know, however, comparatively little
of the actual nature of such bodies, and therefore we apply to
them as a class the general term toxins. The necessity for
accounting for the general pathogenic effects of certain bacteria,
which in the corresponding diseases were not distributed
throughout the body, directed attention to the probable exist-
ence of such toxins ; and the first to systematically study the
production of such poisonous bodies was Brieger. This observer
isolated from putrefying substances, and also from bacterial
cultures, nitrogen-containing bodies, which he called ptomaines.
Similar bodies occurring in the ordinary metabolic processes of
the body had previously been described and called leucomaines.
Ptomaines isolated from pathogenic bacteria in no case repro-
duced the symptoms of the disease, except perhaps in tetanus
and this only owing to their impurity. The methods by
which they were isolated were faulty, and they have therefore
only a historic interest.
11
162 THE TOXINS PRODUCED BY BACTERIA
The introduction of the principle of rendering fluid cultures
bacteria-free by nitration through unglazed porcelain, and its
application by Roux and Yersin to obtain, in the case of the
b. diphtherias, a solution containing a toxin which reproduced
the symptoms of this disease (vide Chap. XV.), encouraged the
further inquiry as to the nature of this toxin. An attempt on
the part of Brieger and Fraenkel to obtain a purified diphtheria
toxin by precipitating bouillon cultures by alcohol (the product
being denominated a toxalbumin) did not greatly advance know-
ledge on the subject, and further investigation soon showed that
characteristic toxins can be isolated from but few bacteria.
General Facts regarding Bacterial Toxins. — The following
may be regarded as the chief facts regarding bacterial toxins
which have been revealed by the study, partly of the bodily
tissues of animals infected by the bacteria concerned, partly of
artificial cultures of : these bacteria. The dead bodies of certain
bacteria have been found to be very toxic. When, for instance,
tubercle bacilli are killed by heat and injected into the body
tissues of a susceptible animal tubercular nodules are found
to develop round the sites where they have lodged. From
this it is inferred that they must have contained characteristic
toxins, seeing that characteristic lesions result. The bodies
of the cholera vibrio are likewise toxic. Such intracellular
toxins, as they have been called, may appear in the nuids
in which the bacteria are living (1) by excretion in an un-
or alto/rod condition, (2) by the disintegration of the
which we know are always
in any bacterial growth/ The death of bacteria occurs also
rn the body of an infected animal, and the disintegration of
these dead bacteria constitutes an important means by which
the poisons they contain are absorbed. There is some evidence
that often bacteria produce during growth poisons which
are hurtful to their own vitality, and also that ferments
are produced by them which have a solvent effect on the
poisoned members of the colony. Such a process of autolysis^
as it has been called, may have an important effect in
liberating intracellular toxins. We do not, however, under-
stand all that takes place under such circumstances ; for the
dead bodies of many bacteria, such as those of anthrax and
diphtheria, are relatively non-toxic. As it is impossible, at
present, to obtain intracellular toxins apart from other deriva-
tives of the bacterial protoplasm, all our knowledge concerning
their effects is derived from the study of what happens when the
bodies of bacteria killed by chloroform vapour or by heat are
FACTS REGARDING BACTERIAL TOXINS 163
injected into animals. When effects are produced by such
injections they do not present in any particular case specific
characters. They are of the nature of general disturbances of
metabolism, as manifested by fever, loss of weight, etc., often of
such serious degree as to result in death. It is important to
note that when pathogenic effects are produced these usually
appear very soon, it may be in a few hours after injection of the
toxic material; there is not the definite period of incubation
which with other toxins often elapses before symptoms appear.
Sometimes the media in which bacteria are growing become
extremely toxic. This is more marked in some cases than in
others. The two best examples of bacteria thus producing
soluble toxins are the diphtheria and tetanus bacilli. In these
and similar cases when bouillon cultures are filtered bacterium-
free by means of a porcelain filter, toxic fluids are obtained,
which on injection into animals reproduce the highly character-
istic symptoms of the corresponding diseases. In the case of
the b. anthracis and of many others, at any rate when grow-
ing in artificial media, such toxin production is much less
marked, a filtered bouillon culture being relatively non-toxic.
Pnianna nppftan'ncr in nnltnrq mftHio. m
cellular toxins, but we cannot as yet say whether they are
excreted by the bacteria or whether they are produced by
the bacteria acting on the constituents of the media. We
therefore cannot as yet draw a hard and fast line between
intra- and extracellular toxins, but the terms are convenient,
and may apply to two actually different sets of bodies. That
the poisonous capacities of a bacterium may be very compli-
cated is shown by what is known in the case of the cholera
vibrio, where the poisons which dissolve out into the culture
fluid are probably different in their nature from those which act
when the dead bacteria are injected into an animal. The extra-
cellular toxins are the more easily obtainable in large quantities,
and it is their nature and effects which are best known. No
method, however, has been discovered of obtaining them in a
pure form, and our knowledge of their properties is exclusively
derived from the study of the toxic filtrates of bouillon cultures
— these filtrates being usually referred to simply as the toxins.
These toxins differ in their effects from the intracellular poisons
in that specific actions on certain tissues are often manifested.
Thus the toxins of the diphtheria, the tetanus, and the botu-
lismus bacilli all act on the nervous system ; with some of the
pyogenic bacteria, on the other hand, poisons, probably of
similar nature, produce solution of red blood corpuscles (this
164 THE TOXINS PRODUCED BY BACTERIA
last may explain, in part at least, the anaemias so common in
the associated diseases). In the action of many of these toxins
the occurrence of a period of incubation between the introduction
of the poison into the animal tissues and the appearance of
symptoms is often a feature.
We have seen that in certain cases there is difficulty in under-
standing the action of bacteria which do not form toxins in fluid
media, especially as in the cases of some of these the bacterial
protoplasm does not seem very toxic. Yet we often see effects
produced at a distance from the focus of infection, e.g. in
anthrax. To explain such occurrences it has long been put
forward as a possibility that some bacteria are only capable of
producing toxins within the animal tissues, and it has further
been thought possible that bacteria, such as, for example, the
typhoid bacillus, which do in media give rise to intracellular
toxins, might either produce these toxins more readily in the
tissues or might produce in addition other toxins of a different
nature. Recently such toxins have been much studied, and the
name aggressins has been given to them. The evidence adduced
for the existence of these aggressins as a separate group of bacterial
poisons is of the following kind. An animal is killed by a dose
of the typhoid, dysentery, cholera, or tubercle bacillus, or by a
staphylococcus, the organism being introduced into one of the
serous cavities. After death the serous exudation, which in all
these cases is present, is removed, and centrifugalised to remove
the bacteria so far as this can be done by such a procedure ;
the bacteria which are left are killed by shaking the fluid up with
toluol and leaving it to stand for some days. It is stated that
such a fluid is of itself without pathogenic effect, but has the
property of transforming a non-lethal dose of the bacterium used
into one having fatal effect. Further, the effects of the combined
actions of the bacteria and aggressins are often of a much more
acute character than can be obtained with toxic products
developed in vitro. Thus, in the case of the action of a non-
lethal dose of the tubercle bacillus plus its aggressin, death may
occur in twenty hours, a result never obtained with artificial
cultures of the organism. The results obtained are attributed
to. a paralysing action which the aggressin is supposed to have
on the phagocytic functions of the leucocytes. The subject is
full of difficulties, and in the case of certain of the organisms
employed, it is stated that results similar to those attributed to
aggressin action have been observed with macerated cultures, —
the deduction being that in the aggressins we are merely deal-
ing with concentrated intracellular toxins. On the other hand,
THE NATURE OF TOXINS 165
as evidence of the existence of a special group of toxins, it has
been stated that a special type of immunity against the
aggressins can be originated. Perhaps the most important
aspect of the controversy is the recognition of the existence of
toxins having an action on the leucocytes. A poison causing
death of these cells in connection with the pus-forming action of
the pyogenic cocci has been described under the name of
leucocidin. The investigation of such poisons must be of the
highest importance in view of the part played by the blood-
cells in the protection of the body against infection, and
it is possible that toxins having a fatal effect in strong con-
centrations, may, when dilute, be responsible for the phenomena
of attraction or repulsion of leucocytes which we know occur
round a focus of bacterial growth in the body.
It is to be noted that in the case of any particular bacterium
several different toxins may be at work, and it is also possible
that one toxin may have different effects on different tissues
of the body. Intracellular toxins of an organism may cause
general metabolic disturbances, and its special toxins may act
on special tissues. Thus the staphylococcus pyogenes aureus
may cause fever, wasting, etc., by its intracellular poisons, a
special action on the leucocytes by a leucocidin toxin, and
anaemia by its hsemolytic properties. The phenomena of any
bacterial disease may thus in reality be due to very different and
complex causes.
The Nature of Toxins. — There is still comparatively little
known regarding this subject, and it chiefly relates to the extra-
cellular toxins. The earlier investigations upon toxins suggested
that analogies exist between the modes of bacterial action and what
takes place in ordinary gastric digestion, and the idea was worked
out for anthrax, diphtheria, tetanus, and ulcerative endocarditis by
Sidney Martin. This observer took, not solutions artificially made
up with albumoses,1 but the natural fluids of the body or definite
1 In the digestion of albumins by the gastric and pancreatic juices the
albumoses are a group of bodies formed preliminarily to the production of
peptone. Like the latter they differ from the albumins in their not being
coagulated by heat, and in being slightly dialysable. They differ from the
peptones in being precipitated by dilute acetic acid in presence of much
sodium chloride, and also by neutral saturated sulphate of ammonia. Both
are precipitated by alcohol. The first albumoses formed in digestion are
proto-albumose and hetero-albumose, which differ in the insolubility of the
latter in hot and cold water (insolubility and coagulability are quite different
properties). They have been called the primary albumoses. By further
digestion both pass into the secondary albumose, deutero-albumose, which
differs slightly in chemical reactions from the parent bodies, e.g. it cannot be
precipitated from watery solutions by saturated sodium chloride unless a
166 THE TOXINS PEODUCED BY BACTERIA
solutions of albumins, and, further, never subjected the results
of the bacterial growth to heat above 40° C., or to any stronger
agent than absolute alcohol. He found that albumoses and
sometimes peptones were formed by the action of the patho-
genic bacteria studied, and further, that the precipitate contain-
ing these albumoses was toxic. In certain cases the process of
splitting up of the albumins went further than in peptic diges-
tion, and organic bases or acids might be formed. According to
Martin, the characteristic symptoms of the diseases could be
explained by compound actions, in which the albumoses were
responsible for some of the effects, the remaining bodies for
others. A similar digestive action has been traced in the case
of the tubercle bacillus by Kiihne.
Further evidence that bacterial toxins are either albumoses
or bodies having a still smaller molecule is furnished by C. J.
Martin. This worker, by filling the pores of a Chamberland
bougie with gelatin, has obtained what is practically a strongly
supported colloid membrane through which dialysis can be made
to take place under great pressure, say, of compressed oxygen.
He finds that in such an apparatus toxins, — at least two kinds
tried, — will pass through just as an albumose will.
Brieger and Boer, working with bouillon cultures of diphtheria
and tetanus, have, by precipitation with zinc chloride, separated
bodies which show characteristic toxic properties, but which have
the reactions neither of peptone, albumose, nor albuminate, and
the nature of which is unknown. It has also been found that
the bacteria of tubercle, tetanus, diphtheria, and cholera can
produce toxins when growing in proteid-free fluids. In the case
of diphtheria when the toxin is produced in such a fluid a proteid
reaction appears. Of course this need not necessarily be caused
by the toxin. Further investigation is here required, for
Uschinsky, applying Brieger and Boer's method to a toxin so
produced, states that the toxic body is not precipitated by zinc
salts, but remains free in the medium. If the toxins are really
non-proteid they may, on the one hand, be the final product of
a digestive action, or they may be the manifestation of a separate
vital activity on the part of the bacteria. On the latter theory
the toxicity of the toxic albumoses of Sidney Martin may be due to
the precipitation of the true toxins along with these other bodies.
From the chemical standpoint this is quite possible. When we
take into account the extraordinary potency of these poisons (in
trace of acetic acid be present. Dysalbumose is probably merely a temporary
modification of hetero-albumose. Further digestion of deutero-albumose
results in the formation of peptone.
THE NATURE OF TOXINS 167
the case of tetanus the fatal dose of the pure poison for a
guinea-pig must often be less than '000001 gr.), we can under-
stand how attempts by present chemical methods to isolate them
in a pure condition are not likely to be successful, and of their
real nature we know nothing. In a recent research Friedberger
and Moreschi have shown that the intravenous injection in the
human subject of a fraction of a loopful of a dead typhoid
culture gives rise to toxic symptoms, including marked febrile
reaction. Such injections are followed by the appearance of
agglutinating and bacteriolytic substances in the serum. These
results show that intracellular toxins may be comparable with
extra-cellular toxins so far as concerns the extremely small dose
sufficient to produce toxic effects.
Amongst the properties of the extracellular toxins are
the following. They are certainly all uncrvstallisable • they
are soluble in water and they are dialysable ; they are pre-
• •ipitated along \yith proteids by concentrated alcohol, and als<3
by ammonium sulphate; if they are proteids they are either
alEumoses or allied to the albumoses ; they are often relatively un-
stable, having their toxicity HiminiiAed or destroyed bv heat (the
'degree of heat wmcn is destructive varies much in different cases),
light, and by certain chemical agents. Their potency is often
altered in the precipitations practised to obtain them in a pure
or concentrated condition, but among the precipitants ammonium
sulphate has little if any harmful effect. Regarding the toxins
which are more intimately associated with the bacterial proto-
plasm we know much less, but it is probable that their nature is
similar, though some of them at least are not so easily injured by
heat, e.g. those of the tubercle bacillus, already mentioned. In
the case of all toxins the fatal dose for an animaJLjsajdfia-jdtlL,
species, body weight, age, a.nrl 'prevTona' p.n™titinnff y trT
food, temperature, etc. In estimating the minimal lethal dose
of a toxin tiiese factors must be carefully considered.
The following is the best method of obtaining concentrated extra-
cellular toxins. The toxic fluid is placed in a shallow dish, and ammonium
sulphate crystals are well stirred in till no more dissolve. Fresh crystals
to form a bulk nearly equal to that of the whole fluid are added, and the
dish set in an incubator at 37° C. over night. Next day a brown scum
of precipitate will be found floating on the surface. This contains the
toxin. It is skimmed off with a spoon, placed in watch glasses, and
these are dried in vacuo and stored in the dark, also in vacuo, or in an
exsiccator containing strong sulphuric acid. For use the contents of one
are dissolved up in a little normal saline solution.
The comparison of the action of bacteria in the tissues in
the production of these toxins to what takes place in the gastric
168 THE TOXINS PRODUCED BY BACTERIA
digestion, has raised the question of the possibility of the elabora-
tion by these bacteria of ferments by which the process may be
started. Thus Sidney Martin puts forward the view that
ferments may be produced, which we may look on as the
primary toxic agents, and which act by digesting surrounding
material and producing albumoses, — these poisons being, as it
were, secondary poisons. Hitherto all attempts at the isolation
of bacterial ferments of such a nature have failed.
But apart from the fact that with such bacteria as these of
tetanus and diphtheria, a digestive action may occur, analogies have
been drawn between ferment and toxic action. The chief facts
upon which such analogies have been founded are as follows.
Thus the toxic products of these and other bacteria lose their
toxicity by exposure to a temperature which puts an end to the
activity of such an undoubted ferment as that of the gastric
juice. If a bouillon containing diphtheria toxin be heated at
65° C. for one hour, it is found to have lost much of its toxic
effect, and in the case of b. tetani all the toxicity is lost by
exposure at this temperature. In both diseases there is a still
further fact which is adduced in favour of the toxic substances
being of the nature of ferments, namely, the existence of a
definite period of incubation between the injection of the toxic
bodies and the appearance of symptoms. This may be inter-
preted as showing that after the introduction of, say, a filtered
bouillon culture, further chemical substances are formed in the
body before the actual toxic effect is produced. Too much
reliance must not be placed on such an argument, for in the
case of tetanus, at least, the delay may be explained by the fact
that the poison apparently has to travel up the nerve trunks
before the real poisonous action is developed. Further, with
some poisons presently to be mentioned which are closely allied
to the bacterial toxins an incubation period may not exist.
It would not be prudent to dogmatise as to whether the toxins
do or do not belong to such an ill-defined group of substances
as the ferments. It may be pointed out, however, that the
essential concept of a ferment is that of a body which can
originate change without itself being changed, and no evidence
has been adduced that toxins fulfil this condition. Another
property of ferments is that so long as the products of fermenta-
tion are removed, the action of a given amount of ferment is
indefinite. Again, in the case of toxins no evidence of such an
occurrence has been found. A certain amount of a toxin is
always associated with a given amount of disease effect, though
a process of elimination of waste products must be all the time
VEGETABLE AND ANIMAL POISONS 169
going on in the animal's body. Again, too much importance
must not be attached to loss of toxicity by toxins at relatively
low temperatures. This is not true of all toxins, and further-
more many proteids show a tendency to change at such
temperatures ; for instance, if egg albumin be kept long
enough at 55° C. nearly the whole of it will be coagulated.
We must therefore maintain an open mind on this subject.
Similar Vegetable and Animal Poisons. — Within recent years it has
been found that the bacterial poisons belong to a group of toxic bodies
all presenting very similar properties, other members of which occur
widely in the vegetable and animal kingdoms. Among plants the best-
known examples are the ricin and abrin poisons obtained by making
watery emulsions of the seeds of the Ricinus communis and the Abrus
precatorius (jequirity) respectively. From the Robinia pseudacacia
another poison — robin — belonging to the same group is obtained. The
chemical reactions of ricin and abrin correspond to those of the bacterial
toxins. They are soluble in water, they are precipitable by alcohol, but
being less easily dialysable than the alburnoses they have been called
toxalbumins. Their toxicity is seriously impaired by boiling, and they
also gradually become less toxic on being kept. Both are among the
most active poisons known — ricin being the more powerful. When they
are injected subcutaneously a period of twenty-four hours usually elapses
— whatever be the dose — before symptoms set in. Both tend to produce
great inflammation at the seat of inoculation, which in the case of ricin
may end in an acute necrosis ; in fatal cases hsemorrhagic enteritis and
nephritis may be found. Both act as irritants to mucous membranes,
abrin especially being capable of setting up most acute conjunctivitis.
It is also certain that the poisons of scorpions and of poisonous snakes
belong to the same group. The poisons derived from the latter are
usually called venins, and a very representative group of such venins
derived from different species has been studied. To speak generally
there is derivable from the natural secretions of the poison glands a
series of venins which have all the reactions of the bodies previously
considered. Like ricin and abrin, they are not so easily dialysable as
bacterial toxins, and therefore have also been classed as toxalbumins.
Their properties are also similar ; many of them are destroyed by heat,
but the degree necessary here also varies much, and some will stand
boiling. There is also evidence that in a crude venin there may be several
poisons differently sensitive to heat. All the venins are very powerful
poisons, but here there is practically no period of incubation — the effects
are almost immediate. An outstanding feature of the venins is the
complexity of the crude poison secreted by any particular species of
snake. C. J. Martin in summing up the results of many observers has
pointed out that different venoms have been found to contain one or
more of the following poisons : a neurotoxin acting on the respiratory
centre, a neurotoxiu acting on the nerve-endings in muscle, a toxin
causing haemolysis, toxins acting on other cells, e.g. the endothelium of
blood-vessels (this from its effects has been named haemerrhagin),
leucocytes, nerve-cells, a toxin causing thrombosis, a toxin haying an
opposite effect and preventing coagulation, a toxin neutralising the
bactericidal qualities of the body fluids and thus favouring putrefaction,
a toxin causing agglutination of the red blood corpuscles, a proteolytic
170 THE TOXINS PRODUCED BY BACTERIA
ferment, a toxin causing systolic standstill of the excised heart. Any
particular venom contains a mixture in varying proportions of such
toxins, and the different effects produced by the bites of different snakes
largely depend on this variability of composition. The neurotoxic, the
thrombotic, and the hsemolytic toxins are very important constituents
of any venom. The toxicity of different venoms varies much, and no
general statement can be made with regard to the toxicity of different
poisons towards man. Lamb has calculated that the fatal dose of crude
cobra venom for man is probably about -015 of a gramme, and that if
such a snake bites with full glands many times this dose would
probably be injected, but, of course, the amount emitted depends largely
on the period which has elapsed since the animal last emptied its glands.
When a dose of a venom not sufficient to cause immediate death from
general effects be given, very rapid and widespread necrosis often may
occur in a few hours round the site of inoculation.
An extremely important fact was discovered by Flexner and Noguchi,
namely, that the hsemolytic toxin of cobra venom in certain cases has no
action by itself, but produces rapid solution of red corpuscles when some
normal serum is added, the latter containing a labile complement-like
body, which activates the venom. In this there is a close analogy to
what holds in the case of a hsemolytic serum deprived of complement by
heat at 55° C. (p. 479). Kyes and Sachs further showed that in addition
to serum-complement a substance with definitely known constitution,
namely lecithin, had the property of activating the hsemoly tic substance
in cobra venom, the two apparently uniting to form an actively toxic
substance. Later still, Kyes succeeded in demonstrating the union of
the two substances to form a cobra-lecithid, and in separating the
latter as a practically pure compound, which is, unlike lecithin,
insoluble in ether, but soluble in chloroform. So far no example of
activating a bacterial toxin is known, but the results mentioned point to
the possibility of this occurring in some cases in the tissues of the body.
The Theory of Toxic Action. — While we know little of the
chemical nature of any toxins we may, from our knowledge of
their properties, group together the tetanus and diphtheria
poisons, ricin, abrin, snake poisons, and scorpion poisons.
Besides the points of agreement already noted, all possess the
further property that, as will be afterwards described, when
introduced into the bodies of susceptible animals they stimulate
the production of substances called antitoxins. The nature of
the antagonism between toxin and antitoxin will be discussed
later. Here, to explain what follows it may be stated (1) that the
molecule of toxin most probably forms a chemical combination
with the molecule of antitoxin, and (2) that it has been shown
that toxin molecules may lose much of their toxic power and
still be capable of uniting with exactly the same proportion of
antitoxin molecules. From these and other circumstances Ehrlich
has advanced the view that the toxin molecule has a very com-
plicated structure, and contains two atom groups. One of these,
the haptophorous (aTrmj/, to bind to), is that by which com-
THE THEORY OF TOXIC ACTION 171
bination takes place with the antitoxin molecule, and also with
presumably corresponding molecules naturally existing in the
tissues. The other atom group he calls the toxophorous, and it
is to this that the toxic effects are due. This atom group is
bound to the cell elements, e.g. the nerve cells in tetanus, by the
haptophorous group. Ehrlich explains the loss of toxicity which
with time occurs in, say, diphtheria toxin, on the theory that the
toxophorous group undergoes disintegration. And if we suppose
that the haptophorous group remains unaffected we can then
understand how a toxin may have its toxicity diminished and
still require the same proportion of antitoxin molecules for its
neutralisation. To the bodies whose toxophorous atom groups
have become degenerated, Ehrlich gives the name toxoids. The
theory may afford an explanation of what has been suspected,
namely, that in some instances toxins derived from different
sources may be related to one another. For example, Ehrlich
has pointed out that ricin produces in a susceptible animal body
an antitoxin which corresponds almost completely with that
produced by another vegetable poison, robin (vide supra),
though ricin and robin are certainly different. This may be ex-
plained according to the view that robin is a toxoid of ricin, i.e.
their haptophorous groups correspond, while their toxophorous
differ. The evidence on which Ehrlich's deductions are based
is of a very weighty character, and will be again referred to
in the chapter on Immunity.
With regard to the intracellular toxins we shall see -it is
difficult to determine whether or not they share with the extra-
cellular poisons the property of stimulating antitoxin formation,
— if they do not, then they may belong to an entirely different
class of substances. It is certain that a tolerance against such
poisons is difficult to establish and is not of a lasting character.
We thus cannot say what the mechanism is by which these
poisons act. It may be said that Macfadyen by grinding up
typhoid bacilli frozen by liquid air claimed that on thawing he
obtained the intracellular toxins in liquid form, and he further
stated that by using this fluid he could immunise animals not
only against the toxins but also against the living bacteria.
We have already pointed out that those who claim for the
aggressins a special character hold that the activity of these
bodies has as its effect the interference with the phagocytic
functions of the leucocytes. They .also hold that a special type
of immunity can be developed against the aggressive bodies.
CHAPTER VI.
INFLAMMATORY AND SUPPURATIVE CONDITIONS.
THIS subject is an exceedingly wide one, and embraces a great
many pathological conditions which in their general characters
and results are widely different. Thus in addition to suppuration,
various inflammations, ulcerative endocarditis, septicaemia and
pyaemia, will come up for consideration. With regard to these
the two following general statements, established by bacteriological
research, may be made in introducing the subject. In the first
place, there is no one specific organism for any one of these
conditions ; various organisms may produce them, and not in-
frequently more than one organism may be present together.
In the second place, the same organism may produce widely
varying results under different circumstances, — at one time a
local inflammation or abscess, at another multiple suppurations
or a general septicaemia. The principles on which this diversity
in results depends have already been explained (p. 151).
Furthermore, there are conditions like acute pneumonia, epidemic
meningitis, acute rheumatism, etc., which have practically the
character of specific diseases and yet which as regards their
essential pathology belong to the same class. The arrangement
followed is to a certain extent one of convenience.
It may be well to emphasise some of the chief points in- the
pathology of these conditions. In. suppuration the two main
phenomena are — (a) a. prn^rpsaive immigration of leucocytes,
chiefly of the polymorpho-nuclear (neutrophile) variety, and (6)
a liquefaction oT digestion pt 1M JJUppoTlllUr eleineut§ of the
tissue along with necrosis of the cells 01 tne part. The result"
is that the tissue aflectecT Le< -nines replaced by the cream-like
fluid called pus. A suppurative inflammation is thus to be
distinguished on the one hand from an inflammation without
destruction of tissue, and on the other from necrosis or death
172
NATURE OF SUPPURATION 173
en masse, where the tissue is not liquefied, and leucocyte
accumulation may be slight. When, however, suppuration is
taking place in a very dense fibrous tissue, liquefaction may be
incomplete, and a portion of dead tissue or slough may remain
in the centre, as is the case in boils. In the case of suppuration
in a serous cavity the two chief factors are the progressive
leucocytic accumulation and the disappearance of any fibrin
which may be present.
Many experiments have been performed to determine whether
suppuration can be produced in the absence of micro-organisms
by various chemical substances, such as croton oil, nitrate of
silver, turpentine, etc. — care, of course, being taken to ensure
the absence of bacteria. The general result obtained by in-
dependent observers is that as a rule suppuration does not follow,
but that in certain animals and with certain substances it may,
the pus being free from bacteria. It is still, however, questioned
by some whether the pus thus produced really corresponds
histologically and chemically with that due to bacterial action.
Buchner showed that suppuration may be produced by the
injection of dead bacteria, e.g. sterilised cultures of bacillus
pyocyaneus, etc. The subject has now more a scientific than a
practical interest, and the general statement may be made that
practically all cases of true suppuration met with clinically are
due to the action of living micro-organisms.
The term septicaemia is applied to conditions in which the
^organisms multiply within the blood and give rise to symptoms
j)f genera, i poisoning ragout, however, producing abscesses in the
.organs.^ In all cases of septicaemia the organisms are more
numerous in the capillaries of internal organs than in the
peripheral circulation, and, in the case of the human subject, it
may be impossible to detect any in the blood during life, though
they may be seen in large numbers in the capillaries of the
kidneys, liver, etc., post mortem. The essential fact in_pycemia, on
the Other hand, is the op.r-nrrenfp. of nmltiplft a.bsp.ftsaftsin internal
organs and other parts of the body. In most of the cases of
"typical pyaemia, common in pre-antiseptic days, the starting-point
of the disease was a septic wound with bacterial invasion of a
vein leading to thrombosis and secondary embolism. Multiple
foci of suppuration may be produced, however, in other ways, as
will be described below (p. 186). If the term "pyaemia" be
used to embrace all such conditions, their method of production
should always be distinguished.
174 INFLAMMATION AND SUPPURATION
BACTEKIA AS CAUSES OF INFLAMMATION AND SUPPURATION.
A considerable number of species of bacteria have been
found in acute inflammatory and suppurative conditions, and of
these many have been proved to be causally related, whilst of
some others the exact action has not yet been fully determined.
Ogston, who was one of the first to study this question (in
1881), found that the organisms most frequently present were
micrococci, of which some were arranged irregularly in clusters
(staphylococci), whilst others formed chains (streptococci). He
found that the former were more common in circumscribed acute
abscesses, the latter in spreading suppurative conditions. Rosen-
bach shortly afterwards (1884), by means of cultures, differentiated
several varieties of micrococci, to which he gave the following
special names : stayhylococcus pyogenes aureus.
pyogenes albus, streptococcus pi/o<jen< >•, micrococcus pyogenes ti-imls.
^ OllM' oTgaliislns are met with in suppuration, such as staphylo-
coccus pyogenes citreus. stavhvlococcus cereus albus. staphylococcus
cereus flavus neumococcus, pneumobacillus '(ITriedl&nder ba^ly*
pvodenes foetidus (assetj&aci^f/.s- c<Ji cummunis, laciUus I
bacillus cerogenes encapsitiatus^ bacillus pyoca
'
micrococcus tetraaenus\rmewnACQccu&^ pneumobacillus' dwlococcus
intraceUularis meningitidis, and others.
In secondary inflammations and suppurations following acute
diseases the corresponding organisms have been found in some
cases, such as gonococcus, typhoid bacillus, influenza bacillus, etc.
Suppuration is also produced by the actinomyces and the
glanders bacillus, and sometimes chronic tubercular lesions have
a suppurative character.
Staphylococcus Pyogenes Aureus. — Microscopical Characters.
— This organism is a spherical coccus about *9 ^ in diameter,
which grows irregularly in clusters or masses (Fig. 52). It stains
readily with all the basic aniline dyes, and retains the colour in
Gram's method.
Cultivation. — It grows readily in all the ordinary media at
the room temperature, though much more rapidly at the
temperature of the body. In stab cultures in peptone gelatin
a streak of growth is visible on the day after inoculation, and
on the second or third day liquefaction commences at the top.
As liquefaction proceeds, the growth falls to the bottom as a
flocculent deposit, which soon assumes a bright yellow colour,
while a yellowish film may form on the surface, the fluid portion
still remaining turbid. Ultimately liquefaction extends out to
STAPHYLOCOCCUS PYOGENES AUREUS 175
the wall of the tube (Fig. 53). In gelatin plates colonies may
be seen with the low power of the microscope in twenty-four
hours, as little balls somewhat granular on the surface and of
brownish colour. On the second day they are visible to the
FIG. 52. — Staphylococcus pyogenes aureus,
young culture on agar, showing clumps
of cocci.
Stained with weak carbol-fuchsin. x 1000.
j i
9
FIG.
of
naked eye as whitish yellow points,
which afterwards become more dis-
tinctly yellow. Liquefaction occurs
around these, and little cups are
formed, at the bottom of which the
colonies form little yellowish masses.
On agar, a stroke culture forms a
line of abundant yellowish growth,
with smooth, shining surface, well
formed after twenty-four hours at
37° C. Later it becomes bright
orange in colour, and resembles a
streak of oil paint. Single colonies on the surface of agar are
circular discs of similar appearance, which may reach 2 mm. or more
in diameter. On potatoes it grows well at ordinary temperature,
forming a somewhat abundant layer of orange colour. In bouillon
it produces a uniform turbidity, which afterwards settles to the
bottom as an abundant layer, which assumes a brownish yellow tint.
In the various media it renders the reaction acid, and it coagulates
53. — Two stab cultures
staphylococcus pyogenes
aureus in gelatin, (a) 10 days
old, (b) 3-weeks old, showing
liquefaction of the medium
and characters of growth.
Natural size.
176
INFLAMMATION AND SUPPURATION
milk, in which it readily grows. The cultures have a somewhat
sour odour.
It has considerable tenacity of life outside the body, cultures
in gelatin often being alive after having been kept for several
months. It also requires a rather higher temperature to kill
it than most spore-free bacteria, viz. 80° C. for half an hour
(Liibbert).
The staphylococcus pyogenes albus is similar in character,
with the exception that its growth on all the media is white.
The colour of the staphylococcus aureus may become less dis-
tinctly yellow after being kept for some time in culture, but it
never assumes the white colour of the staphylococcus albus, and
it has not been found possible to transform the one organism
into the other. A micrococcus called by Welch staphylococcus
epidermidis albus is practically always present in the skin
epithelium ; it is distinguished by its relatively non-pathogenic
properties and by liquefying gelatin somewhat slowly. It is
probably an attenuated variety of the staphylococcus albus.
The staphylococcus pyogenes citreus, which is less frequently
met with, differs in the colour of the cultures being a lemon
yellow, and is less virulent
than the other two.
The staphylococcus
cereus albus and staphy-
lococcus cereus flavus are
of much less importance.
They produce a wax-like
growth on gelatin without
liquefaction ; hence their
name.
Streptococcus pyo-
genes. — This organism
is a coccus of slightly
larger size than the
staphylococcus aureus
about 1 /x in diameter,
and forms chns which
FIG. 54.-Streptococcus pyogenes, young cul-
ture on agar, showing chains of cocci. may contain a large num-
Stained with weak carbol-fuchsin. x 1000. ber of members, especi-
ally when it is growing
in fluids (Fig. 54). The chains vary somewhat in length in
different specimens, and on this ground varieties have been dis-
tinguished, e.g. the streptococcus brevis and streptococcus longus
(vide infra). As division may take place in many of the cocci
STKEPTOCOCCUS PYOGENES
177
in a chain at the same time, the appearance of a chain of
diplococci is often met with. In young cultures the cocci are
fairly uniform in size, but after a time they present con-
siderable variations, many swelling up to twice their normal
diameter. These are to be regarded as involution forms. In
its staining reactions the streptococcus resembles the staphylo-
cocci described, being readily coloured by Gram's method.
Cultivation. — In cultures outside the body the streptococcus
pyogenes grows much more slowly than the staphylococci and also
V
T
v *
FIG/ 55. — Culture of the
streptococcus pyogenes on
an agar plate, showing
numerous colonies — three
successive strokes. Twenty-
four hours' growth. Natu-
ral size.
FIG. 56. — Bacillus pyocyaneus ; young
culture on agar.
Stained with weak carbol-fuchsin. x 1000.
dies out more readily, being in every respect a more delicate
organism.
In peptone gelatin a stab culture shows, about the second day,
a thin line, which in its subsequent growth is seen to be formed of
a row of minute rounded colonies of whitish colour, which may be
separate at the lower part of the puncture. They do not usually
exceed the size of a small pin's head, this size being reached about
the fifth or sixth day. The growth does not spread on the surface,
and no liquefaction of the medium occurs. The colonies in gelatin
plates have a corresponding appearance, being minute spherical
points of whitish colour. A somewhat warm temperature is
12
178 INFLAMMATION AND SUPPURATION
necessary for growth ; even at 20° C. some varieties do not
grow. On the agar media growth takes place along the stroke
as a collection of small circular discs of semi -translucent
appearance, which show a great tendency to remain separate
(Fig. 55). The separate colonies remain small, rarely exceeding
1 mm. in diameter. Cultures on agar kept at the body
temperature may often be found to be dead after ten days. On
potato, as a rule, no visible growth takes place. In milk it
produces a strongly acid reaction but no clotting of the medium.
It ferments lactose, saccharose, and salicin (Andrewes and
Horder) ; it produces no fermentation of inulin in Hiss's serum-
water-medium, in this respect differing from the pneumococcus.
It has a strong haBmoly tic action, as can be demonstrated by
growing it in blood-agar plates (p. 38). In bouillon, growth
forms numerous minute granules which afterwards fall to
the bottom, the deposit, which is usually not very abundant,
having a sandy appearance. The appearance in broth, however,
presents variations which have been used as an aid to distinguish
different species of streptococci. It has been found that those
which form the longest chains grow most distinctly in the form of
spherical granules, those forming short chains giving rise to a
finer deposit. To a variety which forms distinct spherules of
minute size the term streptococcus conglomeratus has been given.
Varieties of Streptococci. — Formerly the streptococcus pyogenes
and the streptococcus erysipelatis were regarded as two distinct
species, and various points of difference between them were
given. Further study, and especially the results obtained by
modifying the virulence (p. 182), have shown that these dis-
tinctions cannot be maintained, and now practically all authorities
are agreed that the two organisms are one and the same,
erysipelas being produced when the streptococcus pyogenes of a
certain standard of virulence gains entrance to the lymphatics Of
the skin. Petruschky, moreover, showed conclusively by inocu-
lation that a streptococcus cultivated from pus could cause
erysipelas in the human subject.
Streptococci have also been classified according to the length
of the chains. Thus there have been distinguished (a) strepto-
coccus longusy which occurs in long chains and is pathogenic to
rabbits and mice ; (l>) streptococcus brevis, which is common in
the mouth in normal conditions, and is usually non-pathogenic ;
and (c) streptococcus conglomeratus, so called from its forming in
bouillon minute granules composed of very long chains. It may
be stated that pathogenic streptococci obtained from the human
subject usually form fairly long chains on agar, whilst the short
STREPTOCOCCUS PYOGENES 179
streptococci obtained from the mouth and intestine are usually
devoid of virulence. But to these statements exceptions occur,
as short streptococci may be associated with grave lesions ; it
has also been found that the length of the chains is not a
constant feature. As in the case of other organisms attempts
have also been made to differentiate streptococci by means of
their fermentative properties. Mervyn Gordon introduced for
this purpose nine tests, namely : — (1) The clotting of milk,
(2) the reduction of neutral red, (3-9) the fermentation with
acid production of saccharose, lactose, raffinose, inulin, salicin,
coniferin, and mannite. Andrewes and Horder by means of
these have differentiated six varieties, of which five occur in the
human subject. These are : — (a) A short-chained form called
streptococcus mitis, which occurs chiefly in the saliva and faeces
as a saprophyte. (6) The streptococcus pyogenes, which is the
most important pathogenic variety, and has the characters
described above, (c) The streptococcus salivarius, which corre-
sponds to the streptococcus brevis of the mouth, and which, as
regards fermentative action, seems to bear the same relation to
the next variety as the streptococcus mitis does to the strepto-
coccus pyogenes. It has more active fermentative properties
and clots milk, (d) The streptococcus anginosus, which corre-
sponds with the so-called streptococcus scarlatinae and the strepto-
coccus conglomeratus. It usually clots milk and does not grow
on gelatin at 20° C. (e) The streptococcus fcecalis, a short-
chained form, which abounds in the intestine and which has
great fermentative activity. It forms sulphuretted hydrogen,
and is devoid of haemolytic action. (/) The sixth variety is the
streptococcus equinus, which is common in the air and dust of
towns, and appears to be derived from horse dung.1
Schottmuller has employed the appearance of the colonies of
^streptococci on blood agar as a means of separating varieties,
the medium used consisting of two parts human blood and five
parts melted agar. He distinguishes the streptococcus lon<jus or
erysipelcitis- which forms __grey colonies and^ _ ^
action i a, streptococcus mitior or mW^^o *
organism, which produces small green colonies and very little
-haemolysis-. and a streptococcus mucosus encapsulates, which, as
its name indicates, snows well-marked capsules, and produces
colonies which have a slimy consistence/ It should be noted
^thaton blood agar the ~pneumococcus forms green colonies andj
produces no hcemolysyf.
1 For further details reference niust be made to the original papers, Lancet,
September 1906, ii, 708, etc.
I
niust be mad
/VU>VA
180 INFLAMMATION AND SUPPURATION
It will be thus seen from this account that the streptococcus
pyogenes as described above is the organism most frequently
associated with the pathogenic processes, and that short-chained
forms are common saprophytes in the human body, although
they may be associated with conditions of disease ; these may
be subdivided according to their fermentative activity as
detailed. And lastly, there is the streptococcus conglomeratus
(anginosus), which is specially abundant in the throat in scarlet
fever, though it also occurs in other acute catarrhal states. No
definite statement can yet be made as to the etiological relation
of streptococci to scarlet fever ; we can only say that streptococci
are almost invariably present in the fauces, and that to them
many of the complications of the disease are due.
Bacillus coli communis. — The microscopic and cultural characters are
described in the chapter on typhoid fever. The bacillus lactis cerogenes
and the bacillus pyogenes f&tidus closely resemble it ; they are either
varieties or closely related species. The former is distinguished by
producing more abundant gas formation, and by its growth on gelatin,
etc., being thicker and whiter than that of the bacillus coli.
Bacillus aerogenes encapsulatus sometimes invades the tissues before
death, and is characterised by the formation of bubbles of gas in the
infected parts. Its characters are described in Chapter XVI.
Bacillus pyocyaneus. — This organism occurs in the form of minute
rods 1'5 to 3 fji. in length and less than '5 At in thickness (Fig. 56).
Occasionally two or three are found attached end to end. They are
actively motile, and do not form spores. They stain readily with the
ordinary basic stains, but are decolorised by Gram's method.
\Cultivation. — It grows readily on all the ordinary media at the room"
temperature, the cultures being distinguished by the formation of a
greenish pigment. In puncture cultures in peptone-gelatin a greyish
line appears in twenty- four hours, and at its upper part a small cup of
liquefaction forms within forty-eight hours. At this time a slightly
greenish tint is seen in the superficial part of the gelatin. The
liquefaction extends pretty rapidly; the fluid portion being turbid and
showing masses of growth at its lower part. The green colour
becomes more and more marked and diffuses through the gelatin.
Ultimately liquefaction reaches the wall of the tube. In plate cultures
the colonies appear as minute whitish points, those on the surface being
the larger. Under a low power of the microscope they have a brownish-
yellow colour and show a nodulated surface, the superficial colonies being
thinner and larger. Liquefaction soon occurs, the colonies on the surface
forming shallow cups with small irregular masses of growth at the
bottom, the deep colonies small spheres of liquefaction. Around the
colonies a greenish tint appears. On agarthe growth forms an abundant
slimy greyish layer which afterwards becomes greenish, and a bright
green colour diffuses through the whole substance of the medium. On
potatoes the growth is an abundant reddish-brown layer resembling that
of the glanders bacillus, and the potato sometimes shows a greenish
discoloration.
From the cultures there can be extracted by chloroform a coloured
body pyocyanin, which belongs to the aromatic series, and crystallises
EXPERIMENTAL INOCULATION
181
in the form of long, delicate bluish-green needles. On the addition of
a weak acid its colour changes to a red.
This organism has distinct pathogenic action in certain animals.
Subcutaneous injection of small doses in rabbits may produce a local
suppuration, but if the dose be large, spreading haemorrhagic oedema
results, which may be attended by septicaemia. Intravenous injection
may produce, according to the dose, rapid septicaemia with nephritis, or
sometimes a more chronic condition of wasting attended by albuminuria.
Micrococcus tetragenus.
— This organism, first de-
scribed by Gaffky, is char-
acterised by the fact that it
divides in two planes at right
angles to one another (Fig.
57), and is thus generally
found in the tissues in groups
of four or tetrads, which are
often seen to be surrounded
by a capsule. The cocci
measure 1 /* in diameter.
They stain readily with all
the ordinary stains, and also
retain the stain in Gram's
method.
It grows readily on all
the media at the room tem-
perature. In a puncture cul-
ture on peptone - gelatin a
F^alo'fthet^of "hee *»• 57 -Micrococcus tet.ag^s ; young
SS&SS& ^Jtewsteart -co.
of whitish colour. The gela-
tin is not liquefied. On the surface of agar and of potato the growth
is an abundant moist layer of the same colour. The growth on all
the media has a peculiar viscid or tenacious character, owing to the
gelatinous character of the sheaths of the cocci.
White mice are exceedingly susceptible to this organism. Subcutaneous
injection is followed by a general septicaemia, the organism being found
in large numbers in the blood throughout the body. Guinea-pigs are
less susceptible ; sometimes only a local arbscess with a good deal of
necrotic change results ; sometimes there is also septicaemia.
Experimental Inoculation. — We shall consider chiefly the
staphylococcus pyogenes aureus and the streptococcus pyogenes,
as these have been most fully studied.
It may be stated at the outset that the occurrence of suppura-
tion depends upon the number of organisms introduced into the
tissues, the number necessary varying not only in different
animals, but also in different parts of the same animal, — a smaller
number producing suppuration in the anterior chamber of the
eye, for example, than in the peritoneum. The virulence of the
182 IMFLAMMATION AND SUPPURATION
organism also may vary, and corresponding results may be pro-
duced. Especially is this so in the case of the streptococcus
pyogenes.
The staphylococcus aureus, when injected subcutaneously in
suitable numbers, produces an acute local inflammation, which
is followed by suppuration, in the manner described above.
The spread of the suppuration goes pari passu with the growth
of the cocci. If a large dose is injected the cocci may enter the
blood stream in sufficient numbers to cause secondary suppurative
foci in internal organs (cf. intravenous injection).
Intravenous injection in rabbits, for example, produces interest-
ing results which vary according to the quantity used. If a con-
siderable quantity be injected, the animal may die in twenty-four
hours of a general septicaemia, numerous cocci being present in
the capillaries of the various organs, often forming plugs. If a
smaller quantity be used, the cocci gradually disappear from the
circulating blood ; some become destroyed, while others settle in
the capillary walls in various parts and produce minute abscesses.
These are most common in the kidneys, where they occur both
in the cortex and medulla as minute yellowish areas surrounded
by a zone of intense congestion and haemorrhage. Similar small
abscesses may be produced in the heart wrall, in the liver, under
the periosteum, and in- the interior of bones, and occasionally in
the striped muscles. Very rarely indeed, in experimental
injection, do the cocci settle on the healthy valves of the heart.
If, however, when the organisms are injected into the blood,
there be any traumatism of a valve, or of any other part of the
body, they show a special tendency to settle at these weakened
points.
Experiments on the human subject have also proved the
pyogenic properties of those organisms. Garre inoculated
scratches near the root of his finger-nail with a pure culture, a
small cutaneous pustule resulting ; and by rubbing a culture over
the skin of the forearm he caused a carbuncular condition which
healed only after some weeks. Confirmatory experiments of
this nature have been made by Bockhart, Bumm, and others.
When tested experimentally the staphylococcus pyogenes albus
has practically the same pathogenic effects as the staphylococcus
aureus.
The streptococcus pyogenes is an organism the virulence of
which varies much according to the diseased condition from which
it has been obtained, and also one which loses its virulence
rapidly in cultures. Even highly virulent cultures, if grown
under ordinary conditions, in the course of time lose practically all
BACILLUS COLI COMMUNIS 183
pathogenic power. By passage from animal to animal, however,
the virulence may be much increased, andparipassu the effects of
inoculation are correspondingly varied. Marmorek, for example,
found that the virulence of a streptococcus can be enormously
increased by growing it alternately (a) in a mixture of human
blood serum and bouillon (vide page 41), and (6) in the body of
a rabbit ; ultimately, after several passages it possesses a super-
virulent character, so that even an extremely minute dose intro-
duced into the tissues of a rabbit produces rapid septicaemia, with
death in a few hours. It has been proved by Marmorek's experi-
ments, and those of others, that the same species of streptococcus
may produce at one time merely a passing local redness, at
another a local suppuration, at another a spreading erysi-
pelatous condition, or again a general septicaemic infection,
according as its virulence is artificially increased. Such experi-
ments are of extreme importance as explaining to some extent the
great diversity of lesions in the human subject with which strep-
tococci are associated.
Bacillus Coli Communis. — The virulence of this organism also
varies much and can be increased by passage from animal to
animal. Injection into the serous cavities of rabbits produces a
fibrinous inflammation which becomes purulent if the animal
lives sufficiently long. If, however, the virulence of the organism
be of a high order, death takes place before suppuration is
established, and there is a septica3mic condition, the organisms
occurring in large numbers in the blood. Intravenous injection
of a few drops of a virulent bouillon culture usually produces a
rapid septica3mia with scattered hemorrhages in various organs.
Other Effects.— It has been found by independent observers that in
cases where rabbits recover after intravenous injection of bacillus coli
communis, a certain proportion suffer from paralysis and sometimes from
atrophy of muscles, especially of the posterior limbs, these symptoms
being due to lesions of the cells in the anterior cornua of the spinal cord.
Somewhat similar results have been obtained by others after inoculations
with staphylococci and streptococci, a certain proportion only of the
animals showing paralytic symptoms and corresponding changes in the
spinal cord. The lesions are believed to be due chiefly to the action of
the products of the organisms on the highly organised nervous elements.
Much further research requires to be done before the importance of these
results can be properly estimated, but it is not improbable that they
will throw light on the causation of nervous lesions which occur in the
human subject, and the etiology of which at present is quite obscure.
Some observers, chiefly of the French school, consider that paralysis
associated with cystitis, in which the bacillus coli communis is often
present, may have such a causation, and that paralytic conditions
following acute infective fevers may be produced by the products of
pyogenic cocci, which frequently occur in these conditions.
1 - r CsTLAMMATKHT AKD SUPPUEATHW
in rnanT of tfe CMK off
LESIONS IX THE HUMAN SUBJECT 1-5
In pyaemia they are frequently present, though in most cases
associated with other pyogenic organisms. Some cases of
enteritis in infants — streptococcic enteritis — are also apparently
due to a streptococcus, which, however, presents in cultures
certain points of difference from the streptococcus pyogenes.
The bacillus coli communis is found in a great many inflam-
matory and suppurative conditions in connection with the ali-
mentary tract — for example, in suppuration in the peritoneum,
or in the extraperitoneal tissue with or without perforation of the
bowel, in the peritonitis following strangulation of the bowel, in
appendicitis and the lesions following it, in suppuration around
the bile-ducts, etc. It may also occur in lesions in other parts
of the body, — endocarditis, pleurisy, etc., which in some cases
are associated with lesions of the intestine, though in others such
cannot be found. It is also frequently present in inflammation
of the urinary passages, cystitis, pyelitis, abscesses in the kidneys,
etc., these lesions being in fact most frequently caused by this
or closely allied organisms.
In certain cases of enteritis it is probably the causal agent,
though this is difficult of proof, as it is much increased in
numbers in practically all abnormal conditions of the intestine.
We may remark that it has been repeatedly proved that the
bacillus coli cultivated from various lesions is more virulent than
that in the intestine,' its virulence having been heightened by
growth in the tissues.
The micrococcus tetragenus is often found in suppurations in
the region of the mouth or in the neck, and also occurs in
various lesions of the respiratory tract, in phthisical cavities,
abscesses in the lungs, etc. Sometimes it is present alone, and
probably has a pyogenic action in the human subject under
certain conditions. In other cases it is associated with other
organisms. Recently one or two cases of pyaemia have been
described in which this organism was found in a state of purity
in the pus in various situations. In this latter condition the pus
has been described as possessing an oily, viscous character, and
as being often blood-stained.
The bacillus pyocyaneus is rarely found alone in pus, though
it is not infrequent along with other organisms. We have met
with it twice in cases of multiple abscesses, in association with
the staphylococcus pyogenes aureus. Lately some diseases in
children have been described in which the bacillus pyocyaneus
has been found throughout the body ; in these cases the chief
symptoms have been fever, gastro-intestinal irritation, pustular
or petechial eruptions on the skin, and general marasmus.
186 INFLAMMATION AND SUPPURATION
Suppurative and inflammatory conditions, associated with the
organisms of special diseases, will be described in the respective
chapters.
Mode of Entrance and Spread. — Many of the organisms of
suppuration have a wide distribution in nature, and many also
are present on the skin and mucous membranes of healthy
individuals. Staphylococci are commonly present on the skin,
FIG. 59. — Minute focus of commencing suppuration in brain — case of acute
ulcerative endocarditis. In the centre a small haemorrhage ; to right
side dark masses of staphylococci ; zone of leucocytes at periphery.
Alum carmine and Gram's method, x 50.
and also occur in the throat and other parts, and streptococci
can often be cultivated from the secretions of the mouth in
normal conditions. The pneumococcus of Fraenkel and the
pneumobacillus of Friedlander have also been found in the
mouth and in the nasal cavity, whilst the bacillus coli communis
is a normal inhabitant of the intestinal tract. The entrance of
these organisms into the deeper tissues when a surface lesion
occurs can be readily understood. Their action will, of course,
be favoured by any condition of depressed vitality. Though in
normal conditions the blood is bacterium-free, we must suppose
ENTRANCE AND SPREAD OF BACTERIA 187
that from time to time a certain number of such organisms gain
entrance to it from trifling lesions of the skin or mucous surfaces,
the possibilities of entrance from the latter being especially
numerous. In most cases they are killed by the action of the
"healthy serum or cells of the body, and no harm results. If,
however, there be a local weakness, they may settle in that part
FIG. 60. — Secondary infection of a glomerulus of kidney by the staphylo-
coccus aureus, in a case of ulcerative endocarditis. The cocci (stained
darkly) are seen plugging the capillaries and also lying free. The
glomerulus is much swollen, infiltrated by leucocytes, and partly necrosed.
Paraffin section ; stained by Gram's method and Bismarck-brown. x 300.
and produce suppuration, and from this other parts of the body
may be infected. Such a supposition as this is necessary to
explain many inflammatory and suppurative conditions met with
clinically. In some cases of multiple suppurations due to
staphylococcus infection, which we have had the opportunity to
examine, only an apparently unimportant surface lesion was
present ; whilst in others no lesion could be found to explain
the origin of the infection. The term cryptogenetic has been
applied by some writers to such cases in which the original
188 INFLAMMATION AND SUPPURATION
point of infection cannot be found, but its use is scarcely
necessary.
The paths of secondary infection may be conveniently sum-
marised thus : First, by lymphatics. In this way the lymphatic
glands may be infected, and also serous sacs in relation to the
organs where the primary lesion exists. Second, by natural
channels, such as the ureters and the bile-ducts, the spread
being generally associated with an inflammatory condition of the
lining epithelium. In this way the kidneys and liver respectively
may be infected. Third, by the blood vessels : (a) by a few
organisms gaining entrance to the blood from a local lesion, and
settling in a favourable nidus or a damaged tissue, the original
path of infection often being obscure ; (b) by a septic phlebitis
with suppurative softening of the thrombus and resulting em-
bolism ; and we may add (c) by a direct extension along a vein,
producing a spreading thrombosis and suppuration within the
vein. In this way suppuration may spread along the portal vein
to the liver from a lesion in the alimentary canal, the condition
being known as pylephlebitis suppurativa.
Although many of the lesions produced by the bacteria
under consideration have already been mentioned, certain con-
ditions may be selected for further consideration on account of
their clinical importance or bacteriological interest.
Endocarditis. — There is now strong presumptive evidence
that all cases of endocarditis are due to bacterial infection. In
the simple or vegetative form, so often the result of acute
rheumatism, the micrococcus rheumaticus (p. 193) has been
cultivated from the valves in a certain number of cases, and is
probably the causal agent in most instances.
Endocarditis of the nlcerative
various organisms, chiefly pyogenic._ Of these the staphylococci
*anct streptococci are most frequently found. In some cases ol
ulcerative endocarditis following pneumonia, t.hp. pnftnmor;op.p.ns-
(Fraenkel's) is present ; in others pyogenic cocci, especially
streptococci. Other organisms have been cultivated from
'different cases of the disease, and some of these have received
special names ; for example, the diplococcus endocarditis encap-
sulatus, bacillus endocarditidis griseus (Weichselbaum), and
others. In some cases the bacillus coli cominunishas_beenjojiiid,
and occasionally in endocarcTillij following^ typhoid the typhoid
bacillus has been described as the organism present, but further
observations on this point are desirable. The^onococcr[s also
has been shown to affect the heart valves (p. 22o]7tnough this is
a very rare occurrence. TnT-raj-nlp. no^jplpa on the
ENDOCARDITIS
189
have been found in a few cases of acute tuberculosis, though no
vegetative or ulcerative condition is produced.
In some cases, though we believe not often, the organisms
may attack healthy valves, producing a primary ulcerative endo-
carditis, but more frequently the valves have been the seat of
previous endocarditis, secondary ulcerative endocarditis being
FIG. 61. — Section of a vegetation jn ulcerative endocarditis showing numerous
staphylococci lying in the spaces. The lower portion is a fragment
in process of separation.
Stained by Gram's method and Bismarck -brown. x 600.
thus produced. In some cases, especially when the valves have
been previously diseased, the source of the infection is quite
obscure. It is evident that as the vegetations are composed for
the most part of unorganised material, they do not offer the
same resistance to the growth of bacteria, when a few reach them,
as a healthy cellular tissue does. On microscopic examination
of the diseased valves the organisms are usually to be found in
enormous numbers, sometimes forming an almost continuous layer
on the surface, or occurring in large masses or clusters in spaces
in the vegetation (Fig. 61). By their action a certain amount
190 INFLAMMATION AND SUPPURATION
of softening or breaking down of the vegetations occurs, and the
emboli thus produced act as the carriers of infection to other
organs, and give rise to secondary suppurations.
Experimental. — Occasionally ulcerative endocarditis is produced by the
simple intravenous injection of staphylococci and streptococci into the
circulation, but this is a very rare occurrence. It often follows, however,
when the valves have been previously injured. Orth and Wyssokowitsch
at a comparatively early date produced the condition by damaging the
aortic cusps by a glass rod introduced through the carotid, and after-
wards injecting staphylococci into the circulation. Similar experiments
have since been repeated with streptococci, pneumococci, and other
organisms, with like result. Ribbert found that if a potato culture of
the staphylococcus aureus were rubbed down in salt solution so as to
form an emulsion, and then injected into the circulation, some minute
fragments became arrested at the attachment of the chordae tendinese and
produced an ulcerative endocarditis.
Acute Suppurative Periostitis and Osteomyelitis. — Special
mention is made of this condition on account of its comparative
frequency and gravity. The great majority of cases are caused
by the ^vogenic cocci, of which one or two varieties may be
present, the staphylococcus aureus, however, occurring most
frequently. Pneumococci have been found alone in some cases,
and in a few cases following typhoid fever, apparently well
authenticated, the typhoid bacillus has been found alone. In
others again the bacillus coli communis is present.
The affection of the periosteum or interior of the bones by
these organisms, which is especially common in young subjects,
may take place in the course of other affections produced by
the same organisms or in the course of infective fevers, but in a
great many cases the path of entrance cannot be determined.
In the course of this disease serious secondary infections are
always very liable to follow, such as small abscesses in the
kidneys, heart- wall, lungs, liver, etc., suppurations in serous
cavities, and ulcerative endocarditis ; in fact, some cases present
the most typical examples of extreme general staphylococcus
infection. The entrance of the organisms into the blood stream
from the lesion of the bone is especially favoured by the arrange-
ment of the veins in the bone and marrow.
Experimental. — Multiple abscesses in the bones and under the peri-
osteum may occur in simple intravenous injection of the pyogenic
cocci into the blood, and are especially liable to be formed when young
animals are used. These abscesses are of small size, and do not spread
in the. same way as in the natural disease in the human subject.
In experiments on healthy animals, however, the conditions are not
analogous to those of the natural disease. We must presume that in the
latter there is some local weakness or susceptibility which enables the
CONJUNCTIVITIS 191
few organisms which have reached the part by the blood to settle and
multiply. Moreover, if a bone be experimentally injured, e.g. by actual
fracture or by stripping off the periosteum, before the organisms are
injected, then a much more extensive suppuration occurs at the injured
part.
Erysipelas. — A spreading inflammatory condition of the
skin may be produced by a variety of organisms, but the disease
in the human subject in its characteristic form is almost in-
variably due to a streptococcus, as was shown by Fehleisen in
1884. He obtained pure cultures of the organism, and gave it
the name of streptococcus erysipelatis ; and, further, by inocu-
lations on the human subject as a therapeutic measure in
malignant disease, he was able to reproduce erysipelas. As
stated above, however, one after another of the supposed points
of difference between the streptococcus of erysipelas and that of
suppuration has broken down, and it is now generally held that
erysipelas is produced by the streptococcus pyogenes of a certain
degree of virulence. It must be noted, however, that erysipelas
passes from patient to patient as erysipelas, and purulent con-
ditions due to streptococci do not appear liable to be followed
by erysipelas. On the other hand, the connection between
erysipelas and puerperal septicaemia is well established clinically.
In a case of erysipelas the streptococci are found in large
numbers in the lymphatics of the cutis and underlying tissues,
just beyond the swollen margin of the inflammatory area. As
the inflammation advances they gradually die out, and after a
time their extension at the periphery comes to an end. The
streptococci may extend to serous and synovial cavities and set
up inflammatory or suppurative change, — peritonitis, meningitis,
and synovitis may thus be produced.
Conjunctivitis. — A considerable number of organisms are
concerned in the production of conjunctivitis and its associated
lesions. Of these a number appear to be specially associated
with this region. Thus a small organism, generally known as
the Koch- Weeks bacillus, is the most common- cause of acute
contagious conjunctivitis, especially prevalent in Egypt, but
also common in this country. This organism is very minute,
being little more than 1 //, in length, and morphologically
resembles the influenza bacillus : its conditions of growth are
jsven more restricted, as it rarely ^rows on blood agar, the best
"medium being serum ap^ar. un tnls medium it produces minute
transparent colonies like drops of dew. The obtaining of • pure
cultures is a matter of considerable difficulty, and it is nearly
always accompanied by the xerosis bacillus. It can readily be
192
INFLAMMATION- AND SUPPURATION
found in the muco-purulent secretion by staining films with
^^^^^^_ weak (1 : 10) carbol-
fuchsin, and is often to
be seen in the interior
of leucocytes (Fig. 62).
Another organism ex-
ceedingly like the prev-
ious, apparently differing
from it only in the rather
wider conditions of
growth, is Muller's bacil-
lus. It has been culti-
vated by^him in a con-
siderable proportion of
cases of trachoma, but
its relation to this con-
FIG. 62. -Film preparation from a case of dition is sti11 matter of
acute conjunctivitis, showing Koch-Weeks dispute. Another bacil-
bacilli, chiefly contained within a leucocyte, lus which is now well
(From a preparation by Dr. Inglis Pollock.) recognised is the diplo-
bacillus of conjunctivitis
first described by Morax. It is especially common in the more
subacute cases of conjunctivitis. Eyre found it in 2 '5 per cent
of all cases of conjunc-
tivitis. Its cultural char-
acters are given below.
The xerosis bacillus,
which is a small diph-
theroid organism (Fig.
123), has been found in r
xerosis of the conjunc-
tiva, in follicular con-
junctivitis, and in other
conditions ; it appears
to occur sometimes also
in the normal conjunc-
tiva. It is doubtful
whether it has any
pathogenic action of im-
portance. Acute con-
junctivitis is also pro
duced by the pneu mo-
coccus, epidemics of the
disease being sometimes due to this organism, and also by
FIG. 63. — Film preparation of conjunctival
secretion showing the Morax diplo-bacillus
of conjunctivitis. x 1000.
ACUTE RHEUMATISM 193
streptococci and staphylococci. True diphtheria of the con-
junctiva caused by the Klebs-LorHer bacillus also occurs,
whilst in gonorrha'al conjunctivitis, often of an acute purulent
type, the gonococcus is present (p. 225).
Diplo - bacillus of Conjunctivitis. — This organism, discovered by
Morax, is a small plump bacillus, measuring 1 x 2 /*, and usually occur-
ring in pairs, or in short chains of pairs (Fig. 63). It is non-motile,
does not form spores, and is decolorised by Gram's method. It does not
grow on the ordinary gelatin and agar media, the addition of blood or
serum being necessary. On serum it forms small rounded colonies
which produce small pits of liquefaction ; hence it has been called the
bacillus lacunatus. In cultures it is distinctly pleomorphous, and
involution forms also occur. It is non-pathogenic to the lower animals.
Acute Rheumatism. — There are many facts which point to
the infective nature of this disease, and investigations from this
point of view have yielded important results. Of the organism
isolated, the one which appears to have strongest claims is a
small coccus observed by Triboulet, and by Westphal and
Wassermann, the characters and action of which were first
investigated in this country by Poynton and Paine. It is now
usually spoken of as iho^nicrococcus rheumaticus. The organism
is sometimes spoken of as a diplococcus, but it is best described
as a streptococcus growing in short chains ; in the tissues, how-
ever, it usually occurs in pairs. It is rather smaller than the
streptococcus pyogenes, and although it can be stained by Gram's
method, it loses the colour more readily than the streptococcus.
In the various media it produces a large amount of acid, and
usually clots milk after incubation for two days ; on blood agar
it alters the haemoglobin to a brownish colour. Its growth on
media generally is more luxuriant than that of the streptococcus,
and it grows well on gelatin at 20° C. Intravenous injection
of pure cultures in rabbits often produces polyarthritis and
synovitis, valvulitis and pericarditis, without any suppurative
change — lesions which it is stated are not produced by the
ordinary streptococci (Beattie). In one or two instances
choreiform movements have been observed after injection. The
organism is most easily obtained from the substance of inflamed
synovial membrane where it is invading the tissues ; a part
where there is special congestion should be selected as being
most likely to give positive results. It is only occasionally to
be obtained from the fluid in joints. It has also been cultivated
from the blood in rheumatic fever, from the vegetations on the
heart valves, and from other acute lesions ; in many cases, how-
ever, cultures from the blood give negative results. Poynton
13
194 INFLAMMATION AND SUPPURATION
and Paine cultivated it from the cerebro- spinal fluid in three
cases where chorea was present, and also detected it in the
membranes of the brain. They consider that this disease is
probably of the nature of a slight meningo-myelitis produced by
this organism. The facts already accumulated speak strongly
in favour of this organism being causally related to rheumatic
fever, though this cannot be considered completely proved.
Andrewes finds that the organism has the same cultural characters
and fermentative effects as the streptococcus fsecalis, a common
inhabitant of the intestine. Even, however, if the two organisms
were the same, it might well be possible that rheumatic fever is
due to an infection of the tissues by this variety of streptococcus.
The clinical data, in fact, rather point to rheumatic fever being
due to an infection by some organism frequently present in the
body, brought about by some state of predisposition or acquired
susceptibility.
Vaccination Treatment of Infections by the Pyogenic Cocci.
— From his study of the part played by phagocytosis in the
successful combat of the pyogenic bacteria by the body, Wright
was led to advocate the treatment of such infections by the
origination during their course of an active immunisation by
dead cultures of the infecting agent. The treatment is applicable
when the infection is practically local as in acne pustules, in boils,
etc. (For the theoretical questions raised see Immunity.) It
is best to attempt to isolate the causal organism from the lesion
and to test the opsoriic index of the patient against it. To
prepare the vaccine an agar slope culture is taken and the
growth washed off with normal saline. The organism is then
killed by steaming for an appropriate time, and the efficacy of
the sterilisation tested by inoculating fresh agar tubes. The
strength of the emulsion is estimated by the method of counting
dead bacteria described on p. 67. The number of bacteria used
for an injection is from 250,000,000 to 500,000,000, and in the
details of the measurement of this quantity and in its injection
every aseptic precaution must, of course, be adopted. If
repeated injections are necessary Wright recommends that the
opsonic index should be observed every few days and the
injections only practised during a positive phase. If it is not
practicable to use the infecting strain for the preparation of the
vaccine, then laboratory cultures must be used, and in such
cases it is well to use a mixture of strains ; in skin infections
a mixture of staphylococcus aureus and albus may be employed.
Such means have been extensively used in the treatment of
acne, boils, sycosis, infections of the genito-urinary tract by the
METHODS -OF EXAMINATION 195
b. coli, infections of joints by the gonococcus, and in many cases
considerable success has followed the treatment.
Methods of Examination in Inflammatory and Suppurative
Conditions. — These are usually of a comparatively simple nature,
and include (1) microscopic examination, (2) the making of
cultures.
(1) The pus or other fluids should be examined microscopi-
cally, first of all by means of film preparations in order to
determine the characters of the organisms present. The films
should be stained (a) by one of the ordinary solutions, such
as carbol-thionin-blue (p. 98), or a saturated watery solution of
methylene-blue ; and (b) by Gram's method. The use of the
latter is of course of high importance as an aid in the recognition.
(2) As most of the pyogenic organisms grow readily on the
gelatin media at ordinary temperatures, pure cultures can be
readily obtained by the ordinary plate methods. But in many
cases the separation can be effected much more rapidly by the
method of successive streaks on agar tubes, which are then
incubated at 37° 0. When the presence of pneumococci is
suspected this method ought always to be used, and it is also to
be preferred in the case of streptococci. Inoculation experiments
may be carried out as occasion arises.
In cases of suspected blood infection the examination of the
blood is to be carried out by the methods already described
(p. 68).
CHAPTER VII.
INFLAMMATOKY AND SUPPURATIVE CONDITIONS,
CONTINUED: THE ACUTE PNEUMONIAS, EPI-
DEMIC CEREBRO-SPINAL MENINGITIS.
Introductory. — The term Pneumonia is applied to several con-
ditions which present differences in pathological anatomy and in
origin. All of these, however, must be looked on as varieties of
inflammation in which the process is modified in different ways
depending on the special structure of the lung or of the parts
which compose it. There is, first of all, and, in adults, the com-
monest type, the^acute croupous or lobar pneumonia, in which
an inflammatory process attended by abundant fibrjnous exuda-
J^ion affects, by continuity~jtlie entire tissue of a jobe or' of a^
large portion of the lun^'iT^Teparts trom ike course of an"
ordinary inflammation in that the reaction of the connective
tissue of the lung is relatively slight, and there is usually no
tendency for organisation of the inflammatory exudation to take
place. Secondly, there is the acute catarrhal or lobular
pneumonia, where a catarrhal inflammatory process spreads from
the capillary bronchi to the air vesicles, and in these a change,
consisting largely of proliferation of the endothelium of the
alveoli, takes place which leads to consolidation of patches of
the lung tissue. Up till 1889 acute catarrhal pneumonia was
comparatively rare except in children. In adults it was chiefly
found as a secondary complication to some condition such as
diphtheria, typhoid fever, etc. Since, however, influenza in an
epidemic form has become frequent, catarrhal pneumonia has
been of much more frequent occurrence in adults, has assumed
a very fatal tendency, and has presented the formerly quite
unusual feature of being sometimes the precursor of gangrene
of the lung. Besides these two definite types other forms also
occur. Thus instead of a fibrinous material the exudation may
196
TYPES OF PNEUMONIA 197
be of a serous, haemorrhagic, or purulent character. Cases
of mixed fibrinous and catarrhal pneumonia also occur, and
in the catarrhal there may be great leucocytic emigration.
Hsemorrhages also may occur here.
Besides the two chief types of pneumonia there is another
group of cases which are somewhat loosely denominated septic
pneumonias, and which may arise in two ways : (1) by the
entrance into the trachea and bronchi of discharges, blood, etc.,
which form a nidus for the growth of septic organisms ; these
often set up a purulent capillary bronchitis and lead to infection
of the air cells and also of the interstitial tissue of the lung ; (2)
from secondary pyogenic infection by means of the blood stream
from suppurative foci in other parts of the body. (See chapter
on Suppuration, etc.) In these septic pneumonias various
changes, resembling those found in the other types, are often
seen round the septic foci.
In pneumonias, therefore, there may be present a great variety
of types of inflammatory reaction. We shall see that with all of
them bacteria have been found associated. Special importance
is attached to acute croupous pneumonia on account of its course
and characters, but reference will also be made to the other
forms.
Historical. — Acute lobar pneumonia for long was supposed to be an
effect of exposure to cold ; but many observers were dissatisfied with
this view of its etiology. Not only did cases occur where no such
exposure could be traced, but it had been observed that the disease
sometimes occurred epidemically, and was occasionally contracted by
hospital patients lying in beds adjacent to those occupied by pneumonia
cases. Further, the sudden onset and definite course of the disease con-
formed to the type of an acute infective fever ; it was thus suspected by
some to be due to a specific infection. This view of its etiology was
promulgated in 1882-83 by Friedlander, whose results were briefly as
follows. In pneumonic lungs there were cocci, adherent usually in
pairs, and possessed of a definitely contoured capsule. These cocci
could be isolated and grown on gelatin, and on inoculation in mice they
produced a kind of septicaemia with inflammation of the serous membranes.
The blood and the exudation in serous cavities contained numerous
capsulated diplococci. There is little doubt that many of the organisms
seen by Friedlander were really Fraenkel's pneumococcus, to be presently
described.
By many observers it had been found that the sputum of healthy
men, when injected into animals, sometimes caused death, with the
same symptoms as in the case of the injection of Friedlander's coccus ;
and in the blood and serous exudations of such animals capsulated
diplococci were found. A. Fraenkel found that the sputum of pneumonic
patients was much more fatal and more constant in its effects than that
of healthy individuals. The cocci which were found in animals dead of
this "sputum septicsemia," as it was called, differed from Friedlander's
cocci in several respects, to be presently studied. Fraenkel further
198 THE ACUTE PNEUMONIAS
investigated a few cases of pneumonia, and isolated from them cocci
identical in microscopic appearances, cultures, and pathogenic effects,
with those isolated in sputum septicaemia. The most extensive investi-
gations on the whole question were those of Weichselbaum, published
in 1886. This author examined 129 cases of the disease, including
cases not only of acute croupous pneumonia, but of lobular and septic
pneumonia. From them he isolated four groups of organisms. (1)
Diplococcus pneumonice. This he described as an oval or lancet- formed
coccus, corresponding in appearance and growth characters to Fraenkel's
coccus. (2) Streptococcus pneumonice. This on the whole presented
similar characters to the last, but it was more vigorous in its growth,
and could grow below 20° C., though it preferred a temperature of 37° C.
(3) Staphylococcus pyogenes aureus. (4) Bacillus pneumonice. This was
a rod-shaped organism, and was identical . with Friedlander's pneumo-
coccus. Of these organisms the diplococcus pneumonias was by far the
most frequent. It also occurred in all forms of pneumonia. Next in
frequency was the streptococcus pneumonias, and lastly the bacillus
pneumoniae. Inoculation experiments were also performed by Weichsel-
baum with each of the three characteristic cocci he isolated. The
diplococcus pneumonias and the streptococcus pneumonias both gave
pathogenic effects of a similar kind in certain animals.
The general result of these earlier observations was to establish
the occurrence in connection with pneumonia of two species of
organisms, each having its distinctive characters, viz : —
1. Fraenkel's nneumncnc,r.u.^ whir».h is recognised to be identical
with the coccus of "sputum septicaemia," with Weichselbaum 's
diplococcus pneumoniae, and with his streptococcus pneumonise.
2. Friedldnder's ^^(^^^^,e (-n^™ known as Friedlander's
pneumobacillus), which is almost certainly the bacillus pneu-
monias of Weichselbaum.
We shall use the terms " Fraenkel's pneuinococcus " and
".Friedlander's pTiftiimobar'.illns." as these are now usually applied
to the two organisms.
Microscopic Characters of the Bacteria of Pneumonia.—
Methods. — The organisms present in acute pneumonia can best
be examined in film preparations made from pneumonic lung
(preferably from a part in a stage of acute congestion or early
hepatisation), or from the gelatino*us parts of pneumonic sputum
(here again preferably when such sputum is either rusty or
occurs early in the disease), or in sections of pneumonic lung.
Such preparations may be stained by any of the ordinary weak
stains, such as a watery solution of methylene-blue, but Gram's
method is to be preferred, with Bismarck-brown or Ziehl-Neelsen
carbol-fuchsin (one part to ten of water) as a contrast stain ;
with the latter it is best either to stain for only a few seconds,
or to overstain and then decolorise with alcohol till the ground
of the preparation is just tinted. The capsules can also be
BACTERIA IN PNEUMONIA
199
stained by the methods already described (p. 102). In such
preparations as the above, and even in specimens taken from
the lungs immediately after death (as may be quite well done
by means of a hypodermic syringe), putrefactive and other
bacteria may be present, but those to be looked for are capsulated
organisms, which may be of either or both of the varieties
mentioned.
(1) FraenkeVs Pneumococcus. — -This organism occurs in the form
of a small oval coccus, about 1 p in longest diameter, arranged
generally in pairs (diplo-
cocci), but also in chains /
of four to ten (Fig. 64). t ,/; *
The free ends are often >•;_
pointed like a lancet, hence * » <"'* ^
the term diplococcus lance-
Ltlajtus has also been ap-
plied to it. These cocci
have round them a capsule,
which, in films stained by
ordinary methods, usually
appears as an unstained
halo, but is sometimes
stained more deeply than
the ground of the pre-
paration. This difference
in Staining depends, in
nartfltlpfl^t on thpamount
partat least, on tneamount
of decolorisation to which
the preparation has been
subjected. The capsule
is rather broader than the body of the coccus, and has a sharply
defined external margin. This organism takes up the basic
aniline stains with great readiness, and also retains the stain in
(Ira/ml*. m.?thnd_ It is the organism of by far the most frequent
occurrence in true croupous pneumonia, and in fact may be said
to be rarely absent.
(2) Friedldnder's Pneumobacillus. — As seen in the sputum
and tissues, this organism, both in its appearance and arrange-
ment, as also in the presence of a capsule, somewhat resembles
Fraenkel's pneumococcus, and it was at first described as the
" pneumococcus." The form, however, is more of a short rod-
shape, and it has blunt rounded ends ; it is also rather
broader than Fraenkel's pneumococcus. It is now classed
amongst the bacilli, especially in view of the fact that elongated
FIG. 64.— Film preparation of pneumonic
sputum, showing numerous pneumococci
(Praenkel.s) with unstamed capsules;
some are arranged in short chains.
Stained with carbol-fuchsiu. x 1000.
200
THE ACUTE PNEUMONIAS
rod forms may occur (Fig. 65).
The capsule has the same
general characters as
that of FraenkePs organ-
ism. JFriedlanderV
pneumobacillus stains
readily with the basic
aniline stains, but loses
the stain in Gram's
method, and is accord-
ingly coloured with the
contrast stain, — fuchsin
or Bismarck -brown, as
above recommended. A
valuable means is thus
afforded of distinguish-
ing it from Fraenkel's
pneumococcus in inicro-
Fio. 65. — Friedlander's pneumobacillus, showing scopic preparations,
the variations in length, also capsules. Film
preparation from exudate in a case of pneu-
monia, x 1000.
sometimes it
Friedlander's organ-
ism is much less fre-
quently present in pneu-
is associated with the
inonia than Fraenkel's;
latter; very rarely it
occurs alone.
In sputum prepara-
tions the capsule of both
pneumococci may not be
recognisable, and the
same is sometimes true
of lung preparations.
This is probably due to
changes which occur in
the capsule as the result
of changes in the vitality
of the organisms. Some-
times in preparations
stained by ordinary
methods the difficulty of
recognising the capsule
when it is present, is
due to the refractive
index of the fluid in
which the specimen is
mounted being almost identical with that of the capsule.
FIG. 66. — Fraenkel's pneumococcus in serous
exudation at site of inoculation in a rabbit,
showing capsules stained.
Stained by Rd. Muir's method. x 1000.
This
CULTIVATION OF PNEUMOCOCCUS
201
difficulty can always be overcome by having the groundwork
of the preparation tinted.
The Cultivation of Fraenkel's Pneumococcus. — It is usually
difficult, and sometimes impossible, to isolate this coccus directly
from pneumonic sputum. On culture media it has not a vigorous
growth, and when mixed with other bacteria it is apt to be
overgrown by the latter. To get a pure culture it is best to
insert a small piece of the sputum beneath the skin of a rabbit
or a mouse. In about forty-eight hours the animal will die,
with numerous capsulated pneumococci
throughout its blood. From the heart-
blood cultures can be easily obtained.
Cultures can also be got post mortem from
the lungs of pneumonic patients by
streaking a number of agar or blood-
agar tubes with a scraping taken from
the area of acute congestion or commenc-
ing red hepatisation, and incubating them
at 37° C. The colonies of the pneumo-
coccus appear as almost transparent small
discs which have been compared to drops
of dew (Fig. 67). This method is also
sometimes successful in the case of
sputum.
The appearances presented in cultures FK} 67>_stroke cnlture of
by different varieties of the pneumococcus
vary somewhat. It always grows best
on blood serum or on Pfeiffer's blood
agar. It usually grows well on ordinary
agar or in bouillon, but not so well on
glycerin agar. In a stroke culture on
blood serum growth appears as an almost transparent pellicle
along the track, with isolated colonies at the margin. On
agar media it is more manifest, but otherwise has similar
characters. The appearances are similar to those of a culture
of streptococcus pyogenes, but the growth is less vigorous,
and is more delicate in appearance. A similar statement also
applies to cultures in gelatin at 22° C., growth in a stab culture
appearing as a row of minute points which remain of small
size ; there is, of course, no liquefaction of the medium. On
agar plates colonies are almost invisible to the naked eye,
but under a low power of the microscope appear to have a
compact finely granular centre and a pale transparent periphery.
In bouillon, growth forms a slight turbidity, which settles to the
Fraenkel's pneumococcus
on blood agar. The
colonies are large and un-
usually distinct. Twenty-
four hours' growth at
37° C. Natural size.
202 THE ACUTE PNEUMONIAS
bottom of the vessel as a slight dust-like deposit. On potatoes^
as a rule, no growth appears. Cultures on such media may be
maintained for one or two months, if fresh sub-cultures are made
every four or five days, but they tend ultimately to die out.
They also rapidly lose their virulence, so that four or five days
after isolation from an animal's body their pathogenic action
is already diminished. Eyre and Washbourn, however, have
succeeded in maintaining cultures in a condition of constant
virulence for at least three months by growing the organisms on
agar smeared with rab-
bits' blood. The agar
\ t must be prepared with
X ' p% "'• A s Witte's peptone, must
\ \ % n°t be neated over 100°
* i \ C., and after neutralisa-
tion (rosolic acid being
s** V | > used as the indicator)
i,,, \ '^jj must have '5 per cent of
^ ^ normal sodium hydrate
added. The tubes when
. inoculated are to be kept
^ ^ \ at 37' 5° C. and sealed to
prevent evaporation. In
%«^. none of the ordinary arti-
^ ficial media do pneumo-
cocci develop a capsule.
FIG. 68. — P raeukel s pneumococcus from a pure m, -,,
culture on blood agar of twenty-four hours' Thev Dually appear as
growth, some iu pairs, some in short chains, diplococci, but in pre-
Stained with weak carbol-fuchsin. x 1000. parations made from the
surface of agar or from
bouillon, shorter or longer chains may be observed (Fig. 68).
After a few days' growth they lose their regular shape and size,
and involution forms appear. Usually the pneumococcus does
not grow below 22° C., but forms in which the virulence has
disappeared often grow well at 20° C. Its optimum temperature
is 37° C., its maximum 42° C. It is preferably an aerobe, but
can exist without oxygen. It prefers a slightly alkaline medium
to a neutral, and does not grow on an acid medium. These
facts show that when growing outside the body on artificial
media, the pneumococcus is a comparatively delicate organism.
There has been described by Eyre and Washbourn a non-
pathogenic type of the pneumococcus which may be found in
the healthy mouth, and which may also be produced during the
saprophytic growth of the virulent form. From the latter it
CULTIVATION OF PNEUMOBACILLUS 203
differs generally in its more vigorous growth, in producing a
uniform cloud in bouillon, in slowly liquefying gelatin, and in
growing on potato.
The Cultivation of Friedlander's Pneumobacillus. — This
organism, when present in sputum or in a pneumonic lung, can
be readily separated by making ordinary
gelatin plate cultures, or a series of successive
strokes on agar tubes. The surface colonies
always appear as white discs which become
raised from the surface so as to appear like
little knobs of ivory. From these, pure
cultures can be readily obtained. The ap-
pearance of a stab culture in gelatin growth
is very characteristic. At the site of the
puncture, there is on the surface a white
growth heaped up, it may be fully one-eighth
I of an inch above the level of the gelatin ;
UHfeM along the needle track there is a white
^5
FIG. 69.— Stab culture
of Friedlander's
pneumobacillus in
peptone gelatin,
showing the nail-
like appearance ;
ten days' growth.
Natural size.
•«.•>*
.<«&.
••X.
^
&'*
FIG. 70. — Friedlander's pneumobacillus,1
from a young culture on agar, showing
some rod-shaped forms.
Stained with thionin-blue. x 1000.
granular appearance, so that the whole resembles a white round-
headed nail driven into the gelatin (Fig. 69). Hence the name
1 The apparent size of this organism, on account of the nature of its sheath,
varies much according to the stain used. If stained with a strong stain, e.g.
carbol-fuchsin, its thickness appears nearly twice as great as is shown in the
figure.
204 THE ACUTE PNEUMONIAS
"nail-like" which has been applied. Occasionally bubbles of
gas develop along the line of growth. There is no liquefaction
of the medium. On sloped agar it forms a very white growth
with a shiny lustre, which, when touched with a platinum needle,
is found to be of a viscous consistence. In cultures much longer
rods are formed than in the tissues of the body (Fig. 70). On
the surface of potatoes it forms an abundant moist white layer.
Friedlander's bacillus has active fermenting powers on sugars,
though varieties isolated by different observers vary in the degree
in which such powers are possessed. It always seems capable
of acting on dextrose, lactose, maltose, dextrin, and mannite, and
sometimes also on glycerin. The substances produced by the
fermentation vary with the sugar fermented, but include ethylic
alcohol, acetic acid, laevolactic acid, succinic acid, along with
hydrogen and carbonic acid gas. The amount of acid produced
from lactose seems only exceptionally sufficient to cause coagula-
tion of milk. It is said by some that this bacillus is identical
with an organism common in sour milk, and also a normal
inhabitant of the human intestine, viz. the bacterium lactis
aerogenes of Escherich.
The Occurrence of the Pneumobacteria in Pneumonia and
other Conditions. — Capsulated organisms have been found in
every variety of the disease — in acute croupous pneumonia, in
broncho-pneumonia, in septic pneumonia. In the great majority
of these it is Fraenkel's pneumococcus which both microscopic-
ally and culturally has been found to be present. Friedlander's
pneumobacillus occurs in only about 5 per cent of the cases. It
may be present alone or associated with Fraenkel's organism. In
a case of croupous pneumonia the pneumococci are found all
through the affected area in the lung, especially in the exudation
in the air-cells. They also occur in the pleural exudation and
effusion, and in the lymphatics of the lung. The greatest number
are found in the parts where the inflammatory process is most
recent, e.g. in an area of acute congestion in a case of croupous
pneumonia, and therefore such parts are preferably to be selected
for microscopic examination, and as the source of cultures. Some-
times there occur in pneumonic consolidation areas of suppura-
tive softening, which may spread diffusely. In such areas the
pneumococci occur with or without ordinary pyogenic organisms,
streptococci being the commonest concomitants. In other cases,
especially when the condition is secondary to influenza, gangrene
may supervene and lead to destruction of large portions of the
lung. In these a great variety of bacteria, both aerobes and
anaerobes, are to be found.
DISTRIBUTION OF PNEUMOBACTERIA 205
In ordinary broncho -pneumonias also Fraenkel's pneumo-
coccus is usually present, sometimes along with pyogenic cocci ;
in the broncho-pneumonias secondary to diphtheria it may be
accompanied by the diphtheria bacillus, and also by pyogenic
cocci ; in typhoid pneumonias the typhoid bacillus or the b. coli
may be alone present or be accompanied by the pneumococcus,
and in influenza pneumonias the influenza bacillus may occur.
In septic pneumonias the pyogenic cocci in many cases are the
only organisms discoverable, but the pneumococcus may also be
present. Especially important, as we shall see, from the point
of view of the etiology of the disease, is the occurrence in other
parts of the body of pathological conditions associated with the
presence of the pneumococcus. By direct extension to neigh-
bouring parts empyema, pericarditis, and lymphatic enlargements
in the mediastinum and neck may take place ; in the first the
pneumococcus may occur either alone or with pyogenic cocci.
But distant parts may be affected, and the pneumococcus may be
found in suppurations and inflammations in various parts of the
body (subcutaneous tissue, peritoneum, joints, kidneys, liver, etc.),
in ptitis media, ulcerative endocarditis (p. 188), and meningitis.
Tnese conditions may take place either as complications of
pneumonia, or they may constitute the primary disease. The
occurrence of meningitis is of special importance, for next to the
lungs the meninges appear to be the parts most liable to attack
by the pneumococcus. A large number of cases have been
investigated by Netter, who gives the following tables of the
relative frequency of the primary infections by the pneumococcus
in man : —
(1) In adults-
Pneumonia ..... 65 '95 per cent
Broncho-pneumonia 1 IK QK
Capillary bronchitis/ • • •
Meningitis . 13 '00
Empyema
Otitis
Endocarditis
Liver abscess
8-53
2-44
1-22
1-22
(2) In children 46 cases were investigated. In 29 the primary affection
was otitis media, in 12 broncho-pneumonia, in 2 meningitis, in 1
pneumonia, in 1 pleurisy, in 1 pericarditis.
Thus in children the primary source of infection is in a great
many cases an otitis media, and Netter concludes that infection
takes place in such conditions from the nasal cavities.
As bearing on the occurrence of pneumococcal infections
206 THE ACUTE PNEUMONIAS
secondary to such a local lesion as pneumonia, it is important to
note that in a large proportion of cases of the latter disease the
pneumococcus can be isolated from the blood.
Experimental Inoculation. — The pneumococcus of Fraenkel is
pathogenic to various animals, though the effects vary somewhat
with the virulence of the race used. The susceptibility of
different species, as Gamaleia has shown, varies to a considerable
FIG. 71. — Capsulated pneumococci in blood taken from the heart of a
rabbit, dead after inoculation with pneumonic sputum.
Dried film, fixed with corrosive sublimate. Stained with carbol-fuchsin and
partly decolorised. x 1000.
extent. The rabbit, and especially the mouse, are very sus-
ceptible ; the guinea-pig, the rat, the dog, and the sheep
occupy an intermediate position ; the pigeon is immune. In
the more susceptible animals the general type of the disease
produced is not pneumonia, but a general septiccemia. Thus, if
a rabbit or a mouse be injected subcutaneously with pneumonic
sputum, or with a scraping from a pneumonic lung, death
occurs in from twenty-four to forty-eight hours. There is some
fibrinous infiltration at the point of inoculation, the spleen is
EXPERIMENTAL INOCULATION 207
often enlarged and firm, and the blood contains capsulated
pneumococci in large numbers (Fig. 71). If the seat of inocula-
tion be in the lung, there generally results pleuritic effusion on
both sides, and in the lung there may be a process somewhat
resembling the early stage of acute croupous pneumonia in man.
There are often also pericarditis and enlargement of spleen.
We have already stated that cultures of the pneumococci on
artificial media in a few days begin to lose their virulence.
Now, if such a partly attenuated culture be injected sub-
cutaneously into a rabbit, there is greater local reaction ;
pneumonia, with exudation of lymph on the surface of the
pleura, and a similar condition in the peritoneum, may occur.
In sheep greater immunity is marked by the occurrence, after
subcutaneous inoculation, of an enormous local sero-fibrinous
exudation, and by the fact that few pneumococci are found in
the blood stream. Intra- pulmonary injection in sheep is
followed by a typical pneumonia, which is generally fatal. The
dog is still more immune ; in it also intra-pulmonary injection is
followed by a fibrinous pneumonia, which is only sometimes
fatal. Inoculation by inhalation appears only to have been
performed in the susceptible mouse and rabbit ; here also
septicaemia resulted.
The general conclusion to be drawn from these experiments
thus is that in highly susceptible animals virulent pneumococci
produce a general septicaemia ; whereas in more immune species
there is an acute local reaction at the point of inoculation, and
if the latter be in the lung, then there may result pneumonia,
which, of course, is merely a local acute inflammation occurring
in a special tissue, but identical in essential pathology with an
inflammatory reaction in any other part of the body. When a
dose of pneumococci sufficient to kill a rabbit is injected sub-
cutaneously in the human subject, it gives rise to a local inflam-
matory swelling with redness and slight rise of temperature, all
of which pass off in a few days. It is therefore justifiable to
suppose that man occupies an intermediate place in the scale of
susceptibility, probably between the dog and the sheep, and
that when the pneumococcus gains an entrance to his lungs, the
local reaction in the form of pneumonia occurs. In this con-
nection the occurrence of manifestations of general infection
associated with pneumonia in man is of the highest import-
ance. We have seen that meningitis and other inflammations
are not very rare complications of the disease, and such cases
form a link connecting the local disease in the human subject
with the general septicsemic processes which may be produced
208 THE ACUTE PNEUMONIAS
artificially in the more susceptible representatives of the lower
animals.
A fact which at first appeared rather to militate against the
pneumococcus being the cause of pneumonia was the discovery
of this organism in the saliva of healthy men. This fact was
early pointed out by Pasteur, and also by Fraenkel, and the
observation has been confirmed by many other observers. It
can certainly be isolated from the mouths of a considerable
proportion of normal men, from their nasal cavities, etc., being
probably in any particular individual more numerous at some
times than at others, and sometimes being entirely absent.
This can be proved, of course, by inoculation of susceptible
animals. Such a fact, however, only indicates the importance
of predisposing causes in the etiology of the disease, and it is
further to be observed that we have corresponding facts in the
case of the diseases caused by pyogenic staphylococci, streptococci,
the bacillus coli, etc. It is probable that by various causes the
vitality and power of resistance of the lung are diminished, and
that then the pneumococcus gains an entrance. In relation to
this possibility we have the very striking facts that in the
irregular forms of pneumonia, secondary to such conditions as
typhoid and diphtheria, the pneumococcus is very frequently
present, alone or with other organisms. Apparently the effects
produced by such bacteria as the b. typhosus and the b.
diphtherise can devitalise the lung to such an extent that
secondary infection by the pneumococcus is more likely to occur
and set up pneumonia. We can therefore understand how
much less definite devitalising agents such as cold, alcoholic
excess, etc., can play an important part in the causation of
pneumonia. In this way also other abnormal conditions of the
respiratory tract, a slight bronchitis, etc., may play a similar
part.
It is more difficult to explain why sometimes the pneumo-
coccus is associated with a spreading inflammation, as in croupous
pneumonia, whilst at other times it is localised to the catarrhal
patches in broncho-pneumonia. It is quite likely that in the
former condition the organism is possessed of a different order
of virulence, though of this we have no direct proof. We have,
however, a closely analogous fact in the case of erysipelas ; this
disease, we have stated reasons for believing, is produced by a
streptococcus which, when less virulent, causes only local
inflammatory and suppurative conditions.
Summary. — We may accordingly summarise the facts re-
garding the relation of Fraenkel's pneumococcus to the disease
PNEUMOCOCCUS INFECTION 209
by saying that it can be isolated from nearly all cases of acute
croupous pneumonia, and also from a considerable proportion
of other forms of pneumonia. When injected into the lungs of
moderately insusceptible animals it gives rise to pneumonia. If,
in default of the crucial experiment of intra-pulmonary injection
in the human subject, we take into account the facts we have
discussed, we are justified in holding that it is the chief factor in
causing croupous pneumonia, and also plays an important part
in other forms. Pneumonia, in the widest sense of the term, is,
however, not a specific affection, and various inflammatory con-
ditions in the lungs can be set up by the different pyogenic
organisms, by the bacilli of diphtheria, of influenza, etc.
The possibility of Friedlander's pneumobacillus having an
etiological relationship to pneumonia has been much disputed.
Its discoverer found that it was pathogenic towards mice and
guinea-pigs, and to a less extent towards dogs. Rabbits appeared
to be immune. The type of the disease was of the nature of a
septicaemia. No extended experiments, such as those performed
by Gamaleia with Fraenkel's coccus, have been done, and there-
fore we cannot say whether any similar pneumonic effects are
produced by it in partly susceptible animals. The organism
appears to be present alone in a small number of cases of
pneumonia, and the fact that it also appears to have been the
only organism present in certain septicaemic complications of
pneumonia, such as empyema and meningitis, render it possible
that it may be the causal agent in a few cases of the disease.
In the septic pneumonias the different pyogenic organisms
already described are found, and sometimes in ordinary
pneumonias, especially the catarrhal forms, other organisms,
such as the b. coli or its allies, may be the causal agents.
The Pathology of Pneumococcus Infection. — The effects of
the action of the pneumococcus, at any rate in a relatively
insusceptible animal such as man, seem to indicate that toxins
must play an important part. Pneumonia is a disease which
presents in many respects the characters of an acute poisoning.
In very few cases does death take place from the functions of
the lungs being interfered with to such an extent as to cause
asphyxia. It is from cardiac failure, from grave interference
with the heat-regulating mechanism, and from general nervous
depression that death usually results. These considerations,
taken in connection with the fact that in man the organisms are
found in the greatest numbers in the lung, suggest that a toxic
action is at work. Various attempts have been made to isolate
toxins from cultures of the pneumococcus, e.g. by precipitating
14
210 THE ACUTE PNEUMONIAS
bouillon cultures with alcohol or ammonium sulphate, and
poisonous effects have been produced by certain substances thus
derived ; but the effects produced are, as in so many other
similar cases, of a non-specific character and to be classed as
interferences with general metabolism. The general conclusion
has been that the toxins at work in pneumonia are intracellular ;
but no special light has been thrown on the common effects of
the members of this group of bacterial poisons. •
Immunisation against the Pneumococcus. — Animals can be
immunised against the pneumococcus by inoculation with
cultures which have become attenuated by growth on artificial
media, or with the naturally attenuated cocci which occur in the
sputum after the crisis of the disease. Netter effected immun-
isation by injecting an emulsion of the dried spleen of an animal
dead of pneumococcus septicaemia. Virulent cultures killed by
heating at 62° C., rusty sputum kept at 60° C. for one to two
hours and then filtered, and filtered or unfiltered bouillon
cultures similarly treated have also been used. In all cases one
or two injections, at intervals of several days, are sufficient for
immunisation, but the immunity has often been observed to
be of a fleeting character and may not last more than a few
weeks. The serum of such immunised animals protects rabbits
against subsequent inoculation with pneumococci, and if injected
within twenty-four hours after inoculation, prevents death. A
protective serum was obtained by Washbourn, who employed
pneumococcus cultures of constant virulence. This observer
immunised a pony by using successively (1) broth cultures killed
by one hour's exposure to 60° C. ; (2) living agar cultures ; (3)
living broth cultures. From this animal there was obtained a
serum which protected ' susceptible animals against many times
an otherwise fatal dose, and which also had a limited curative
action. It is stated that the serum of patients who have
recovered from pneumonia has in a certain proportion of cases
a protective effect against the pneumococcus in rabbits, similar
to that exhibited by the serum of immune animals.
The Klemperers treated a certain number of cases of human
pneumonia by serum derived from immune animals, and appar-
ently with a certain measure of success, and sera prepared by
Washbourn and by others have also been used. The results
obtained by different observers have, however, been rather con-
tradictory. The use of these sera apparently causes the tempera-
ture in some cases to fall, and even may hasten a crisis, but
further experience is necessary before their value in therapeutics
can be properly estimated.
PNEUMOCOCCUS INFECTION 211
There has been considerable difference of opinion as to the
explanations to be given of the facts observed regarding
immunisation against the pneumococcus and especially regarding
the properties of immune sera. At first these sera were supposed
to possess antitoxic qualities — largely on the ground that no
bactericidal effect was produced by them on the bacterium in
vitro. As no specific toxin has been proved to be concerned in
the action of the organism the development of an antitoxin
during immunisation must, in the present state of knowledge,
be looked on as not yet proved. To explain the action of a
serum in preventing and curing pneumococcal infections, it
has been thought to have the complex character seen in anti-
typhoid sera in which two substances — immune body and com-
plement (see Immunity) — are concerned, and the variability in
the therapeutic results obtained has been accounted for on
the view that there might be a deficiency of complement, such
as occurs in other similar cases. The absence of bactericidal
effect, however, raises several difficult points. It is stated that
no such effect is observable either in immune sera, or in the
serum of patients who have successfully come through an attack
of the natural disease. Some effect of the kind would be ex-
pected to be present if the anti-pneumonic serum were quite
comparable to the antityphoid serum. Within recent times
many have turned to the opsonic property of sera to account
for the facts observed. In this connection Mennes observed
that normal leucocytes only become phagocytic towards pneumo-
cocci when they are lying in the serum of an animal immunised
against this bacterium. Wright had in his early papers looked to
the phagocytosis of sensitised bacteria to explain their destruction
in the absence of bactericidal qualities in the serum alone, and
Neufeld and Rimpau have described the occurrence of an opsonic
effect in the action of an anti-pneumococcic serum. Further
work may show that along these lines lies the explanation of the
facts observed.
In studying further the relationship of the opsonic effect to
pneumococcal infection, inquiry has been directed to the opsonic
qualities of the blood of pneumonic patients, especially with a
view to throwing light on the nature of the febrile crisis.
According to some results the opsonic index as compared with
that of a healthy person is not above normal, but if the possible
phagocytic capacities of the whole blood of the sick person be
taken into account these will probably be much above normal in
consequence of the leucocytosis which usually accompanies a
successful resistance to this infection. It has been observed,
212 THE ACUTE PNEUMONIAS
however, that as the crisis approaches in a case which is to
recover the opsonic index rises, and after defervescence gradually
falls to normal. And further, as bearing on the factors in-
volved in the successful resistance of the organism to the
pneumococcus, it has been noted that avirulent pneumococci are
more readily opsonised than more virulent strains. Further
observations along such lines are to be looked for with interest,
and it may be said that Wright's vaccination methods have been
applied to the treatment of pneumonia cases, and in certain
instances are said to have been followed by favourable result.
It may be noted here, in conclusion, that in man it is probable
that immunity against pneumonia may be short-lived, as in a
good many cases of pneumonia a history of a previous attack is
elicited.
Agglutination of the Pneumococcus. — If a small amount of a
culture of Fraenkel's pneumococcus be placed in an anti-pneumo-
coccic serum, an aggregation of the bacteria into clumps occurs.
Such an agglutination, as it is called, is frequently observed
under similar circumstances with other bacteria. The pheno-
menon is not invariably associated with the presence of protective
bodies in a serum, but it has been used for diagnostic purposes
in the differentiation of sore throats due to pneumococcus
infection from those due to other bacteria. Whether the method
is reliable has still to be proved.
Methods of Examination. — These have been already
described, but may be summarised thus : (1) Microscopic.
Stain films from the densest part of the sputum or from
the area of spreading inflammation in the lung by Gram's
method and by carbol-fuchsin, etc. (pp. 99, 101), in the latter
case without decolorising the groundwork of the preparation.
(2) By cultures, (a) Fraenkel's pneumococcus. With similar
material make successive strokes on agar, blood agar, or blood
serum. The most certain method, however, is to inject some
of the material containing the suspected cocci into a rabbit. If
the pneumococcus be present the animal will die, usually within
forty-eight hours, with numerous capsulated pneumococci in its
heart blood. With the latter inoculate tubes of the above media
and observe the growth. In some cases of severe pneumococcic
infection the organism may be cultivated from the blood obtained
by venesection (p. 68). (b) Friedldnder's pneumobacillus can
be readily isolated either by ordinary gelatin plates or by
successive strokes on agar media.
EPIDEMIC CEREBRO-SPINAL MENINGITIS 213
EPIDEMIC CEKEBE.O-SPINAL MENINGITIS.
As the result of observations on this disease in different parts
of the world, it has been now established that the causal agent
is the dijdococcns intracdlidaris meningitidis first described by
WeicTiselbaum. This organism is a small coccus measuring
about 1 /x in diameter and usually occurs in pairs, the adjacent
sides being somewhat flattened against each other. In most
cases the cocci are chiefly contained within polymorphonuclear
leucocytes in the exudation (Fig. 72) ; in some cases, however,
the majority may be
lying free. It stains
readily with basic aniline
dyes, but loses the stain
Gram's metJtmcL tne
readiness with which the
organism decolorises
varying with different
strains. Both in appear-
ance and in its staining
reactions it is superfici-
ally similar to the gono-
coccus (vide infra). The
organism can readily be
cultivated outside the
body, but the conditions
of growth are somewhat
restricted — agar with an
admixture of serum or
blood (preferably human)
is most suitable.1 Strains
separated in different epidemics appear to present slight in-
dividual variations, but the following description may be taken
as summing up the common characters. Growth takes place
best at the temperature of the body, and practically ceases
at 25° C. On serum agar the colonies are circular discs of
almost transparent appearance and possessing a smooth shining
surface ; they have little tendency to become confluent. When
examined under a low magnification the colour is seen to be
somewhat yellowish and the margins usually are smooth and
1 A very good medium is one composed of 1 part of ascitic fluid and 6
parts of 1 per cent glucose agar ; the serum obtained aseptically^is added to
the agar in the melted state at 45° C. and the tubes are tested as regards
sterility by incxibation.
Fia. 72. — Film preparation of exudation from
a case of meningitis, showing the diplococci
within leucocytes.
Stained with carbol-thionin-blue. x 1000.
214 THE ACUTE PNEUMONIAS
regular, though on some media slight crenation may appear. The
colonies may be of considerable size, reaching sometimes a
diameter of 2 to 3 mm. on the third day. On plain agar the
colonies are very much smaller, and sometimes no growth
occurs ; sub-cultures especially often fail to give any growth
on this medium. In serum bouillon the organism produces a
general turbidity with formation of some deposit after a day or
two. It ferments maltose, galactose, and dextrose with acid
production, a property which distinguishes it from the micro-
coccus catarrhalis (vide infra}. Buchanan has pointed out that
this may be demonstrated by making up lots of Loifler's medium
(p. 40) in Petri dishes
. • with each of these sugars
* • •• * added. In all cases
/ *• *** ., * 5 „ . growth occurs best when
»*' *.%*** /V the medium has a neutral
v •» *"* ? • V * %^\ or very slightly alkaline
+ ^* -*, „, **; **** '-/ %*t*»* reaction. In cultures the
», .** •*%• "* '*.!V*.' *„ *•' « organism presents the
. *%**** •• V * same appearance as in the
**• - ** . • . , - . **, body and often shows
•' *«•%«*, * % * tetrad formation. There
. "' •* ** ***** T' is also a great tendency
i • » t^, ^ t to the production of in-
*-.+ .'• • f . * volution forms (Fig. 73),
,%«X,» \+* many of the cocci be-
* coming much swollen,
FIG. 73,-Pure culture of diplococcus intra- staining badly. and after'
cellularis, showing involution forms. wards undergoing disin-
tegration. This change,
according to Flexner's observations, would appear to be due to
the production of an autolytic enzyne, and he has also found that
this substance has the property of producing dissolution of the
bodies of other bacteria. The life of the organism in cultures is
a comparatively short one ; after a few days cultures will often
be found to be dead, but, by making sub-cultures every three or
four days strains can be maintained alive for considerable periods.
The organism is readily killed by heat at 60° C., and it is also
very sensitive to weak antiseptics ; drying for a period of a day
has been found to be fatal to it. The facts established accord-
ingly show it to be a somewhat delicate parasite.
As stated above, the organism occurs in the exudate in the
meninges and in the cerebro-spinal fluid, and it can usually be
obtained by lumbar puncture. In acute cases, especially in the
EPIDEMIC CEREBRO-SPINAL MENINGITIS 215
earlier stages, it is usually abundant ; but in the later stages of
cases of more sub-acute character, its detection may be a matter
of difficulty, and only a few examples may be found after a
prolonged search ; in extremely acute cases also the organism
may be difficult to demonstrate. In most cases the disease is
practically restricted to the nervous system, but occasionally
complications occur, and in these the organism may sometimes
be found. It has been observed, for example, in arthritis, peri-
carditis, pneumonic patches in the lung, and in other inflam-
matory conditions associated with the disease. In a small
proportion of cases it has been obtained from the blood during
life, but cultures in most instances give negative results.
Experimental inoculation shows that the ordinary laboratory
animals are relatively insusceptible to this organism. An
inflammatory condition may be produced in guinea-pigs by
intra-peritoneal injection, but large quantities of cultures must
be used, and none of the characteristic lesions found in the
human subject are reproduced. The intra-peritoneal injection
of the cerebro-spinal fluid or of cultures in mice is frequently
followed by death, the cocci being found in the exudate and even
in the blood. Flexner has shown that cerebro-spinal meningitis
may be produced in monkeys by injections of the organism into
the spinal canal. In such experiments the organism extends
upwards to the brain, and produces meningitis within a very
short time. The resulting lesions, both as regards their dis-
tribution and general characters, and also as regards the
histological changes, resemble the disease in the human subject.
Even these animals, however, are, in comparison with man,
relatively insusceptible, as a considerable amount of culture has
to be injected.
Many questions of great importance Math regard to the
spread of the disease still require further investigation. The
organism has been obtained by culture from the throat and
nasal cavities of those suffering from the disease in a consider-
able number of instances. It has also been obtained from the
same positions in healthy individuals, during an epidemic of
the disease. In some epidemics also a pharyngitis has been
found to occur, and the organism has been obtained from
the affected fauces. The general opinion is that the organism
spreads by means of the lymphatics from the pharynx or
nose to the base of the brain, but a spread by means of the
blood stream cannot be excluded, and infection by the alimentary
canal has also been suggested. Flexner in his experiments
found that when the organism was injected into the spinal
216 THE ACUTE PNEUMONIAS
canal marked congestion and inflammatory change in the
nasal mucous membrane followed, and in this position he was
able to find a Gram-negative diplococcus ; he was, however,
unable to recover the diplococcus intracellularis in culture from
this situation. These results would seem to indicate that the
organism might spread from the brain to the nasal cavity, but if
this be so, it also follows that an extension may take place in
the reverse direction. On the whole the evidence at present
tends to show that the entrance of the organism into the body
is by the naso-pharynx, and that this usually results by
inhalation of the organism distributed in fine particles of
expectoration, etc. In fact, as regards the mode and conditions
of infection, an analogy would appear to hold between this
disease and influenza.
Apart from the epidemic form of the disease, cases of sporadic
nature also occur, in which the lesions are of the same nature,
and in which the diplococcus intracellularis is present. The
facts stated would indicate that the origin and spread of the
disease in the epidemic form depend on certain conditions which
produce an increased virulence of the organism. We are, however,
as yet entirely ignorant as to what these conditions may be. In
simple posterior basal meningitis in children, a diplococcus is
present, as described by Still, which has the same microscopic
and cultural characters as the diplococcus intracellularis ; it has
been regarded as probably an attenuated variety of the latter.
Recently, however, Houston and Rankin have found that the
serum of a patient suffering from epidemic meningitis does not
exert the same opsonic and agglutinative effects on the diplococcus
of basal meningitis as on the diplococcus intracellularis; and
this result points to the two organisms being distinct, though
closely allied, species.
An agglutination reaction towards the diplococcus intracellu-
laris is given by the serum of patients suffering from the disease,
where life is prolonged for a sufficient length of time, but the
degree of the reaction does not possess much clinical significance.
It usually appears about the fourth day, when the serum may
give a positive reaction in a dilution of 1 : 50 ; at a later stage
it has been observed in so great a dilution as 1 : 1000. There is
thus no doubt that anti-substances are produced in epidemic
meningitis as in other diseases, and this is also found to be the
case on inoculation of animals with pure cultures. Attempts
had been made to obtain an anti-serum, and a certain measure of
success has been obtained so far as experimental results are
concerned. Flexner obtained such a serum from a goat, and
EPIDEMIC CEREBRO-SPINAL MENINGITIS 217
found that it had a certain protective effect in guinea-pigs and
monkeys against infection by the organism, but, on the whole,
better results were obtained with the serum of inoculated
monkeys. As yet no important applications towards the
treatment of the disease have been effected.
In the nasal cavity there occur other diplococci which have a
close resemblance to the diplococcus intracellularis. These occur
in the healthy state but are especially abundant in catarrhal
conditions ; of these the diplococcus catarrhalis has the closest
resemblance to the diplococcus p intracellularis. In addition to
occurring in health this organism has also been found in
"numbers in epidemic catarrh. Its microscopic appearances are
"practically similar to those described above, and it also occurs
within leucocytes. _Its colonies on pfiir™ QFQr Qro ™p™> ripQqnf__
than those of the diplococcus intracellularis, and they have a
tnan tnose 01 tne diplococcus intracellularis, ana tney u
tough consistence^so that they afe1 JjOInetmiejLJcejjQOvedLgft
bv the platinum needle. The organism grows on gela
The organism grows on gelatin at
iv C. without liquefying the medium, and it has none of the
. fermentative properties described itfjnvft s\^ Kplrmgmg- t^ the
Ljdiplococcus intracellularis. _.. Other species of Gram - negative
micrococci have also been isolated, and a Gram-positive diplo-
coccus called the diplococcus crassus is of common occurrence ;
this organism is rather larger than the diplococcus intracellularis,
and especially in sub-cultures may tend to assume staphylo-
coccal forms. It is thus evident that the nasal cavity is the
common habitat for a number of closely allied diplococci, and
that the identification of any suspected organism as the diplo-
coccus intracellularis can only be effected by cultivation tests.
Apart from the epidemic form of the disease, meningitis may
be produced by almost any of the organisms described in the
previous chapter, as associated with inflammatory conditions.
A considerable number of cases, especially in children, are due
to the pneumococcus. In many instances where no other lesions
are present the extension is by the Eustachian tube to the middle
ear. In other cases the path of infection is from some other
lesion by means of the blood stream. This organism also infects
the meninges not infrequently in lobar pneumonia, and in some
cases with head symptoms we have found it present where there
was merely a condition of congestion. The pneumobacillus also
has been found in a few cases. Meningitis is not infrequently
produced by streptococci, especially when middle ear disease is
present, less frequently by one of the staphylococci ; occasionally
more than one organism may be concerned. In meningitis
following influenza the influenza bacillus has been found in a
218 THE ACUTE PNEUMONIAS
few instances, but sometimes the pneumococcus is the causal
agent ; and in tubercular meningitis the tubercle bacillus of
course is present, especially in the nodules along the sheaths of
the vessels. An invasion of the meninges by the anthrax
bacillus occurs, but is a rare condition ; it is attended by diffuse
haemorrhage in the subarachnoid space.
In conclusion here it may be stated that mixed infections
may occur in meningitis. Thus the pneumococcus has been
found associated with the tubercle bacillus and also with the
diplococcus intracellularis. Further, in infection with the latter,
Gram-negative bacilli of a diphtheroid appearance have also been
observed ; the significance of these is unknown.
CHAPTER VIII.
GONORRHCEA, SOFT SORE, SYPHILIS.
GONORRHOEA.
Introductory. — The micrococcus now known to be the cause of
gonorrhoea, and now called the gonococcus, was first described
by Neisser, who in 1879 gave an account of its microscopical
characters as seen in the pus of gonorrhoeal affections, both of
the urethra and of the conjunctiva. He considered that this
organism was peculiar to the disease, and that its .characters
were distinctive. Later it was successfully isolated and cultivated
on solidified blood serum by Bumm and others. Its characters
have since been minutely studied, and by inoculations of cultures
on the human subject its causal relationship to the disease has
been conclusively established.
The Gonococcus. — Microscopical Characters. — The organism
of gonorrhoea is a small micrococcus which usually is seen in the
diplococcus form, the adjacent margins of the two cocci being
flattened, or even slightly concave, so that between them there is
a small oval interval which does not stain. An appearance is
thus presented which has been compared to that of two beans
placed side by side (vide Fig. 74). When division takes place in
the two members of a diplococcus, a tetrad is formed, which,
however, soon separates into two sets of diplococci — that is to
say, arrangement as diplococci is much commoner than as tetrads.
Cocci in process of degeneration are seen as spherical elements
of varying size, some being considerably swollen.
These organisms are found in large numbers in the pus of
acute gonorrhoea, both in the male and female, and for the most
part are contained within the leucocytes. In the earliest stage,
when the secretion is glairy, a considerable number are lying
free, or are adhering to the surface of desquamated epithelial
219
220 GONORRHOEA, SOFT SORE, SYPHILIS
\
cells, but when it becomes purulent the large proportion within
leucocytes is a very striking feature. In the leucocytes they lie
within the protoplasm, especially superficially, and are often so
numerous that the leuco-
5B|^^ cytes appear to be filled
with them, and their nuclei
are obscured. As the dis-
ease becomes more chronic,
the gonococci gradually
become diminished in
number, though even in
long-standing cases they
may still be found in con-
siderable numbers. They
are also present in the
purulent secretion of gon-
orrhoeal conjunctivitis, also
in various parts of the
female genital organs when
FIG. 74.— Portion of film of gonorrhceal pus, tkege partg are t^e geat Qf
showing the characteristic arrangement , i i • £ ,•
of the gonococci within leucocytes true gonorrhoeal infection,
Stained with fuchsin. x 1000. and they have been found
in some cases in the second-
ary infections of the joints in the disease, as will be described
below.
Staining. — The gonococcus stains readily and deeply with a
watery solution of any of the basic aniline dyes — methylene-blue,
fuchsin, etc. It is, however, easily decolorised, and it completely
loses the stain by Gram's method — an important point in the
microscopical examination.
Cultivation of the Gonococcus. — This is attended with some
difficulty, as the suitable media and conditions of growth are
somewhat restricted. The most suitable media are solidified
blood serum (especially human serum and rabbit's serum),
"blood agar," and Wertheim's medium, which consists of one
part of fluid serum added to two parts of liquefied agar at a
temperature of 40° C. and then allowed to solidify by cooling.
The serum may be obtained from the blood of the human
placenta ; pleuritic or other effusion may also be used.
Growth takes place best at the temperature of the body, and
ceases altogether at 25° C. Cultures are obtained by taking
some pus on the loop of the platinum needle and inoculating
one of the media mentioned by leaving minute quantities here
and there on the surface. The medium may be used either as
CULTIVATION OF GONOCOCCUS
221
ordinary "sloped tubes" or as a thin layer in a Petri's capsule.
The young colonies are visible within forty-eight hours, and often
within twenty-four hours. They appear around the points of
inoculation as small semi-transparent discs of irregularly rounded
shape, the margin being undulated and sometimes showing small
processes. The colonies vary somewhat in size and tend to
remain more or less separate. • They generally reach their
maximum size on the fourth or fifth day, and are usu'ally found
to be dead on the ninth day, sometimes earlier. On the
medium of Wertheim the period of active growth and the
duration of life are some-
what longer. Even if
impurities are present, pure
sub-cultures can generally
be obtained by the above
method from colonies of
the gonococcus which may
be lying separate. In the
early stage of the disease
the organism is present in
the male urethra in prac-
tically pure condition, and
if the meatus of the urethra
be sterilised by washing
with weak solution of cor-
rosive sublimate and then
with absolute alcohol, and
the material for inoculation
be expressed from the
deeper part of the urethra,
cultures may often be ob-
tained which are pure
from the first. By successive sub-cultures at short intervals,
growth may be maintained indefinitely, and the organism
gradually nourishes more luxuriantly. In culture the organisms
have similar microscopic characters to those described (Fig. 75),
but show a remarkable tendency to undergo degeneration,
becoming swollen and of various sizes, and staining very
irregularly. Degenerated forms are seen even on the second
day, whilst in a culture four or five days old comparatively few
normal cocci may be found. The less suitable the medium the
more rapidly does degeneration take place.
On ordinary agar and on glycerin agar growth does not take
place, or is so slight that these media are quite unsuitable for
FIG. 75. — Gonococci, from a pure culture
on blood agar of twenty - four hours'
growth. Some already are beginning to
show the swollen appearance common in
older cultures.
Stained with carbol-thionin-blue. x 1000.
222 GONORRHOEA, SOFT SORE, SYPHILIS
purposes of culture. The organism does not grow on gelatin,1
potato, etc.
Plate-Cultures. — The following ingenious method of plate-culture was
introduced by Wertheim for the culture of the gonococcus. The medium
of culture is a mixture of human blood serum and of ordinary agar (2
per cent) in equal parts. The serum, in a fluid and sterile condition, is
put in suitable quantities into two or three test tubes and brought to a
temperatuft of 40° C. These are then successively inoculated with the
pus or other material in the same manner as gelatin tubes for ordinary
plates (vide p. 52). To each tube is added an equal part of ordinary
agar which has been thoroughly liquefied by heating and allowed to
cool also to 40° C. The mixture is then thoroughly shaken up and
quickly poured out on a plate or Petri's dish and allowed to solidify,
the plates being then incubated at a temperature of 37° C. The colonies
of the gonococcus are just visible in twenty-four hours, and are seen
both in the substance of the medium a"nd on the surface. The deep
colonies when examined with a lens are minute and slightly nodulated
spheres, sometimes showing little processes, whilst those on the surface
are thin discs of larger diameter with wavy margin and rather darker
centre. In this way the gonococcus may be separated from fluids which
are contaminated with a considerable number of other organisms.
Relations to the Disease. — The gonococcus is invariably
present in the urethral discharge in gonorrlwea, and also in
other parts of the genital tract when these are the seat of true
gonorrhoeal infection. Its presence in these different positions
has been demonstrated not only by microscopic examination
but also by culture. From the description of the conditions of
growth in culture, it will be seen that a life outside the body
in natural conditions is practically impossible — a statement
which corresponds with the clinical fact that the disease is
always transmitted directly by contagion. Inoculations of pure
cultures on the urethra ' of lower animals, and even of apes, is
followed by no effect, but a similar statement can be made with
regard to inoculations of gonorrhoeal pus itself. In fact,
hitherto it has been found impossible to reproduce the disease by
any means in the lower animals. On a considerable number of
occasions inoculations of pure cultures have been made on the
human urethra, both in the male and female, and the disease,
with all its characteristic symptoms, has resulted. (Such
experiments have been performed independently by Bumm,
Steinschneider, Wertheim, and others.) The causal relationship
of the organism to the disease has therefore been completely
1 Turro has announced that he has cultivated the gonococcus on acid
gelatin, i.e. ordinary peptone gelatin which has not been neutralised. We
have failed to obtain any growth of the gonococcus on this medium, even
when inoculation was made from a vigorous growth on blood agar.
DISTRIBUTION OF GONOCOCCUS 223
established, and it is interesting to note how the conditions of
growth and the pathogenic effects of the organism agree with
the characters of the natural disease.
Intraperitoneal injections of pure cultures of the gonococcus in white
mice produce a localised peritonitis with a small amount of suppuration,
the organisms being found in large numbers in the leucocytes (Wertheim).
They also penetrate the peritoneal lining and are found in the sub-
endothelial connective tissue, but they appear to have little power of
proliferation, they soon disappear, and the inflammatory condition does
not spread. Injection of pure cultures into the joints of rabbits, dogs,
and guinea-pigs causes an acute inflammation, which, however, soon
subsides, whilst the gonococci rapidly die out ; a practically similar
result is obtained when dead cultures are used. These experiments show
that while the organism, when present in" large numbers, can produce a
certain amount of inflammatory change in these animals, it has little or
no power of multiplying and spreading in their tissues.
Toxin of the Gonococcus.— De Christmas cultivated the gonococcus in
a mixture of one part of ascitic fluid and three parts of bouillon, and
has found that the fluid after twelve days' growth has toxic properties.
At this period all the organisms are dead ; such a fluid constitutes the
"toxin." The toxic substances are precipitated along with the proteids
by alcohol, and the precipitate after being desiccated possesses the toxic
action. In young rabbits injection of the toxin produces suppuration ;
this is well seen in the anterior chamber of the eye, where hypopyon
results. The most interesting point, however, is with regard to its
action on mucous surfaces ; for, while in the case of animals it produces
no effect, its introduction into the human urethra causes acute catarrh,
attended with purulent discharge. He found that no tolerance to the
toxin resulted after five successive injections at intervals. In a more
recent publication he points out that the toxin on intracerebral injec-
tion has marked effects ; he also claims to have produced an antitoxin.
He states that the toxin diffuses out in the culture medium, and does
not merely result from disintegration of the organisms. This has, how-
ever, been called in question by other investigators.
Distribution in the Tissues. — The gonococcus having been
thus shown to be the direct cause of the disease, some additional
facts may be given regarding its presence both in the primary
and secondary lesions. In the human urethra the gonococci
penetrate the mucous membrane, passing chiefly between the
epithelial cells, causing a loosening and desquamation of many
of the latter and inflammatory reaction in the tissues below,
attended with great increase of secretion. There occurs also
a gradually increasing emigration of leucocytes, which take up a
large number of the organisms. The organisms also penetrate
the subjacent connective tissue, and are especially found, along
with extensive leucocytic emigration around the lacunae. Here
also many are contained within leucocytes. Even, however,
when the gonococci have disappeared from the urethral dis-
charge, they may still be present in the deeper part of the
224 GONORRHCEA, SOFT SORE, SYPHILIS
mucous membrane of .the urethra, possibly also in the prostate,
and may thus be capable of producing infection. The prostatic
secretion may sometimes be examinecLJby making pressure on
the prostate from the rectum when the patient has almost emptied
his bladder, the secretion being afterwards discharged along with
the remaining urine. (Foulerton.) In acute gonorrhoea there
is often a considerable degree of inflammatory affection of the
prostate and vesiculae seminales, but whether these conditions are
always due to the presence of gonococci in the affected parts
we have not at present the data for determining. A similar
statement also applies to the occurrence of orchitis and also of
cystitis in the early stage of gonorrhoea. Gonococci have,
however, been obtained in pure culture from peri-urethral abscess
and from epididymitis : it is likely that the latter condition,
when occurring in gonorrhoea, is usually due to the actual
presence of gonococci. During the more chronic stages other
organisms may appear in the urethra, aid in maintaining the
irritation, and may produce some of the secondary results.
The bacillus coli, the pyogenic cocci, etc., are often present, and
may extend along the urethra to the bladder and set up cystitis,
though in this they may be aided by the passage of a catheter.
It may be mentioned here that Wertheim cultivated the
gonococcus from a case of chronic gonorrhoea of two years'
standing, and by inoculation on the human subject proved it to
be still virulent.
In the disease in the female, gonococci are almost invariably
present in the urethra, the situation affected next in frequency
being the cervix uteri. They do not appear to infect the lining
epithelium of the vagina of the adult unless some other abnormal
condition be present •, but they do so in the gonorrhoeal vulvo-
vaginitis of young subjects. They have also been found in
suppurations in connection with Bartholini's glands, and some-
times produce an inflammatory condition of the mucous membrane
of the body of the uterus. They may also pass along the
Fallopian tubes and produce inflammation of the mucous mem-
brane there. From the pus in cases of pyosalpinx they have
been cultivated in a considerable number of cases. According
to the results of various observers they are present in one out
of four or five cases of this condition, usually unassociated with
other organisms. Further, in a large proportion of the cases in
which the gonococcus has not been found no organisms of any
kind have been obtained from the pus, and in these cases the
gonococci may have been once present and have subsequently
died out. Lastly, they may pass to the peritoneum and produce
DISTRIBUTION OF GONOCOCCUS 225
peritonitis, which is usually of a local character. It is chiefly to
the methods of culture supplied by Wertheim that we owe our
extended knowledge of such conditions.
In gonorrhceal conjunctivitis the mode in which the gonococci
spread through the epithelium to the subjacent connective
tissue is closely analogous to what obtains in the case of the
urethra. Their relation to the leucocytes in the purulent
secretion is also the same. Microscopic examination of the
secretion alone in acute cases often gives positive evidence, and
pure cultures may be readily obtained on blood-agar. As the
condition becomes more chronic the gonococci are less numerous
and a greater proportion of other organisms may be present.
Relations to Joint Affections, etc. — The relations of the gono-
coccus to the sequelae of gonorrhoea form a subject of great
interest and importance, and the application of recent methods of
examination shows that the organism is much more frequently
present in such conditions than the earlier results indicated.
The following statements may be made with regard to them.
First, in a considerable number of cases of arthritis following
gonorrhoea the gonococcus has been found microscopically, and
pure cultures have been obtained, e.g. by Neisser, Lang,
Bordoni-Uffreduzzi, and many others. A similar statement
applies to inflammation of the sheaths of tendons following
gonorrhoea. Secondly, in a large proportion of cases no organ-
isms have been found. It is, however, possible that in a number
of these the gonococci may have been present in the synovial
membrane, as it has been observed that they may be much more
numerous in that situation than in the fluid. Thirdly, in some
cases, especially in those associated with extensive suppuration,
occasionally of a pyaemic nature, various pyogenic cocci have
been found to be present. In the instances in which the gono-
coccus has been found in the joints, the fluid present has usually
been described as being of a whitish-yellow tint, somewhat
turbid, and containing shreds of fibrin-like material, sometimes
purulent in appearance. In one case Bordoni-Uffreduzzi culti-
vated the gonococcus from a joint-affection, and afterwards
produced gonorrhoea in the human subject by inoculating with
the cultures obtained. In another case in which pleurisy was
present along with arthritis the gonococcus was cultivated from
the fluid in the pleural cavity. The existence of a gonorrhoeal
endocarditis has been established by recent observations. Cases
apparently of this nature occurring in the course of gonorrhoea
had been previously described, but the complete bacteriological
test has now been satisfied in several instances. In one case
15
226 GONORRHCEA, SOFT SOEE, SYPHILIS
Lenhartz produced gonorrhoea in the human subject by
inoculation with the organisms obtained from the vegetations.
That a true gonorrhoeal septicaemia may also occur has also been
established, cultures of the gonococcus having been obtained
from the blood during life on more than one occasion (Thayer
and Blumer, Thayer and Lazear, Ahmann).
Methods of Diagnosis. — For microscopical examination dried
films of the suspected pus, etc., may be stained by any of the
simple solutions of the basic aniline stains. We prefer methy-
lene- or thionin-blue, as they do not overstain, and the films do
not need to be decolorised. Staining for one minute is sufficient.
It is also advisable to stain by Gram's method, and it is a good
plan to put at one margin of the cover-glass a small quantity of
culture of staphylococcus aureus if available, in order to have a
standard by which to be certain that the supposed gonococci are
really decolorised. Regarding the value of microscopic examina-
tion alone, we may say that the presence of a large number of
micrococci in a urethral discharge having the characters, position,
and staining reactions described above, is practically conclusive
that the case is one of gonorrhoea. There is no other condition
in which the sum total of the microscopical characters is present.
We consider that it is sufficient for purposes of clinical diagnosis,
and therefore of great value ; in the acute stage a diagnosis can
thus be made earlier than by any other method. The mistake
of confusing gonorrhoea with such conditions as a urethral chancre
with urethritis, will also be avoided. Even in chronic cases the
typical picture is often well maintained, and microscopic examina-
tion alone may give a definite positive result. When other organ-
isms are present, and especially when the gonococci are few in
number, it is difficult, and in some cases impossible, to give a
definite opinion, as a few gonococci mixed with other organisms
cannot be recognised with certainty. This is often the condition
in chronic gonorrhoea in the female. Microscopic examination,
therefore, though often giving positive results, will sometimes be
inconclusive. As regards lesions in other parts of the body
microscopic examination alone is quite insufficient ; it is practi-
cally impossible, for example, to distinguish by this means the
gonococcus from the diplococcus intracellularis of meningitis.
Cultures alone supply the absolute test, and when the organism
is present in an apparent condition of purity, Wertheim's
medium or blood -agar should be used. If other organisms
are present, we are practically restricted to Wertheim's plate
method.
SOFT SORE
227
SOFT SORE.
The bacillus of soft sore was first described by Ducrey
in 1889, who found it in the purulent discharge from the
ulcerated surface; and later, in 1892, Unna described its
appearance and distribution as seen in sections through the
sores. The statements of these observers regarding the presence
and characters of this organism have been fully confirmed by
other observers.
Microscopical Characters. — The organism occurs in the form
of minute oval rods measuring about 1 '5 /x in length, and '5 //,
in thickness (Fig. 76). It is found mixed with other organisms
in the purulent discharge
from the surface, and is
chiefly arranged in small
groups or in short chains.
When studied in sections
through the ulcer it is
found in the superficial
part of the floor, but more
deeply situated than other
organisms, and may be
present in a state of pur-
ity amongst the leucocytic
infiltration. In this posi-
tion it is usually arranged
in chains which may be
of considerable length,
and which are often seen FIG. 76. — Film preparation of pus from soft
lying; in parallel rows be- chancre, showing Ducrey's bacillus, chiefly
,, mi arranged in pairs ; stained with carbol-
tween the cells. Ine fuchsin and slightly decolorised, x 1500.
bacilli chiefly occur in
the free condition, but occasionally a few may be contained
within leucocytes.
There is no doubt that in many cases the organism is
present in the buboes in a state of purity ; it has been found
there by microscopic examination, and cultures have also been
obtained from this source. The negative results of some
observers are probably due to the organism having died off.
On the whole the evidence goes to show that the ordinary
bubo associated with soft sore is to be regarded as another lesion
produced by Ducrey's bacillus. Sometimes the ordinary pyogenic
organisms become superadded.
This bacillus takes up the basic aniline stains fairly readily,
228 GONORRHCEA, SOFT SORE, SYPHILIS
-'£»
h*->&v
but loses the colour very rapidly when a decolorising agent is
applied. Accordingly, in film preparations when dehydration
is not required, it can be readily stained by most of the
ordinary combinations, though Loffler's or Kiihne's methylene-
blue solutions are preferable, as they do not overstain. In
sections, however, great care must be taken in the process of
dehydration, and the aniline-oil method (vide p. 93) should be
used for this purpose, as alcohol decolorises the organism very
readily. A little of the methylene-blue or other stain may be
with advantage added to the aniline- oil used for dehydrating.
Cultivation. — Although for a long period of time attempts
to obtain cultures were unsuccessful, success has been attained
within recent years.
Benzangon, Griffon, and
Le Sourd obtained pure
cultures in four cases,
the medium used being
a mixture of rabbit's
blood and agar, in the
proportion of one part
of the former to two of
the latter. The blood
is added to the agar in
the melted condition at
45° C., and the tubes are
then sloped. Davis con-
firms these results, and
finds that another good
medium is freshly-drawn
human blood distributed
in small tubes; this
method is specially suitable, as the blood inhibits the growth
of various extraneous organisms. On the solid medium (blood-
agar) the growth appears in the form of small round globules,
wjiich attain their complete development in forty-eight hours,
having then a diameter of 1 to 2 mm.; the colonies do not
become confluent. Microscopic examination of these colonies,
which are dissociated with some difficulty, shows similar appear-
ances to those observed when the organism is in the tissues (Fig.
77), but occasionally long undivided filaments are observed which
Davis regards as degenerative forms. Within a comparatively
short period cultures undergo marked degenerative changes, and
great irregularities of form and shape are to be founct It
We are indebted to Dr. Davis for the use of Figs. 76 and 77.
FIG. 77. — Ducrey's bacillus from a 24-hour
culture in blood-bouillon. x 1500.1
SYPHILIS 229
would appear that a comparatively large amount of blood is
necessary for the growth of this organism, and even sub-cultures
on the ordinary media, including blood -serum media, give
negative results. Inoculation of the ordinary laboratory animals
is not attended by any result, but it has been found that some
monkeys are susceptible, small ulcerations being produced by
superficial inoculation, and in these the organism can be demon-
strated. Tomasczewski cultivated the organism for several
generations, and reproduced the disease by inoculation of the
human subject. The causal relationship of this bacillus must
therefore be considered as completely established, and the con-
ditions under which it grows show it to be a strict parasite — a
fact which is in conformity with the known facts as to the
transmission of the disease.
SYPHILIS.
Up till quite recent times practically nothing of a definite
nature was known regarding the etiology of syphilis. Most
interest for a long time centred around the observations of
Lustgarten, who in 1884 described a characteristic bacillus,
both in the primary sore and in the lesions in internal organs.
This organism occurred in the form of slender rods, straight, or
slightly bent, 3 to 4 /x in length, often forming little clusters
either within cells or lying free in the lymphatic spaces ; it took
up basic aniline dyes with some difficulty, but was much more
easily decolorised by acids than the tubercle bacillus.
Lustgarten stained the tissues for twenty-four to forty-eight hours
in aniline-water solution of gentian violet ; and then, after washing
them in alcohol, placed them for ten seconds in a 1'5 per cent solution
of permanganate of potassium. They were then treated with sulphurous
acid, which removes the brown precipitate formed, and decolorises the
sections. They were then washed in water, dehydrated, and mounted.
Much controversy arose regarding the significance of this
bacillus. Some considered it to be the tubercle bacillus, whilst
others supposed that it was the smegma bacillus which had
invaded the tissues. The etiological relationship of the organism
to the disease was, however, not generally accepted, and in view
of the recent work on syphilis, the organism cannot be regarded
as having any pathological importance.
Spirochsete pallida. — An entirely new light has been thrown
on the etiology of the disease by the work of Schaudinn and
Hoffmann which appeared in 1905. Since their first publication
a great amount of work has been undertaken in order to test
230 GONORRHOEA, SOFT SORE, SYPHILIS
their conclusions, and the general result may be said to be of a
confirmatory nature. These observers found in certain cases an
organism to which they gave the name spirochcete pallida ; it
now also goes by the name spironema pallidum. As described
by them, it is a minute spiral-shaped organism, showing usually
from six to eight curves, though longer forms are met with ; the
curves are small, comparatively sharp, and regular (Figs. 78, 79).
It may be said to measure 4-14 fj. in length, while it is extremely
thin, its thickness being only '25 p. In a fresh specimen, say a
scraping from a chancre suspended in a little salt solution, the
organism shows active movements, which are of three kinds —
rotation about the long axis, gliding movements to and fro,
FIGS. 78 and 79. — Film preparations from juice of hard chancre showing
spirochaete pallida,— Giemsa's stain, x 1000. (From preparations by
Dr. A. MacLennan.)
and movements of flexion of the whole body. The ends are
pointed and tapering. Its detection is comparatively difficult,
as the organism is feebly refractile, and more difficult to see than
most other organisms; the movement of small particles in the
vicinity, however, is of assistance in finding it.
In ulcerated syphilitic lesions other organisms are, of course,
present, and not infrequently another spiral organism, to which
the name spirochcete refringens has been given. This organism
is usually somewhat longer, and is distinctly thicker than the
spirochaete pallida. As the name implies, it is more highly
refractile, and is much more easily detected than the latter
organism ; its curves also are opener and much less regular,
and they vary in their appearance during the movements. In
stained films (see p. 107) the differences between the organisms
come out more distinctly, as can be gathered from the accom-
panying photograph (Fig. 81). The spirochaete pallida by the
SYPHILIS 231
Giemsa stain is coloured somewhat faintly, and of reddish tint,
whilst the regular spiral twistings are preserved ; the spirochsete
refringens shows natter, wave-like bends, and, like other organ-
isms, is stained of a bluish tint.
By using Loffler's stain for the
flagella of bacteria, Schaudinn was
able to demonstrate a single deli-
cate flagellum at each pole of the
spirochsete pallida, while no undu-
lating membrane could be detected ;
on the other hand, several other
species, including the spirochaete
refringens, showed a distinct un-
dulating membrane. Two flagella
at one pole of the spirochaete
pallida were also seen, an appear- FIG. 80. — Section of spleen from
ance which Schaudinn thought a case of congenital syphilis,
might represent the commencement showi^ .8ev*£l, examples of
PI ., T i ^ - spirochsete pallida ; Levaditi s
of longitudinal fission. mJetho(L x 1000.
The number of publications with
regard to the distribution of the spirochsete pallida is already
very large, and a summary of the results may be given. In the
primary sore and in the related lymphatic glands, the juice of
which can be conveniently obtained by means of a hypodermic
syringe, the organism has been
found in a very large majority of
cases. It has been also obtained
in the papular and roseolar erup-
tions, in condylomata and mucous
patches — in fact, one may say
generally, in all the primary and
secondary lesions. It has been ob-
tained from the spleen during life,
and on a few occasions, e.g. by
Schaudinn. also from the blood
during life in secondary syphilis.
In the congenital form of the disease
FIG. 81. — Spirochsete refringens ,, , , •
in film preparation from a case the organism may be present in
of balanitis. x 1000. large numbers, as was first shown
by Buschke and Fischer, and by
Levaditi. In the pemphigoid bullse, in the blood, in the in-
ternal organs, the liver, lungs, spleen, supra-renals, and even in
the heart its detection may be comparatively easy, owing to the
large numbers present (Fig. 80). It can readily be demonstrated
232 GONORRHCEA, SOFT SORE, SYPHILIS
in sections of the organs by the method described on p. 104. In
such preparations large numbers of spirochaetes, chiefly extra-
vascular in position, can be seen, and many may occur in the
interior of the more highly specialised cells, for example, liver-
cells ; in many cases examination has been made within so short
a period after the death of the child as to practically exclude the
possibility of contamination from without. It also abounds
sometimes on mucous surfaces, e.g. of the bladder and intestine
in cases of congenital syphilis. Shortly after the discovery of
the organism, Metchnikoff was able to detect it in the lesions
produced in monkeys by inoculation with material derived from
syphilitic sores, and his observations have since been confirmed.
Although various organisms may be associated with it in the
lesions of the skin or mucous membranes, there is a comparative
agreement amongst observers that this organism occurs alone in
syphilitic lesions where the entrance of bacteria, etc., from outside
is excluded. The high percentage of cases in which it is found
would, in view of the difficulty in detecting it, almost point to
its invariable presence, and, as a matter of fact, Schaudinn in
his last series of cases, numbering over seventy, found it in all.
Jn gummata and other tertiary lesions, however, the spirochaete
Vhas rarely, if ever, been detected, and it is probable, as Schaudinn
/suggests, that it has passed into some resting condition which
'has not yet been found. Another question of considerable
importance is, as to whether this organism has. been found in
other conditions. Observations show that in various conditions,
such as ulcerated carcinomata, balanitis, etc., spirochaetes are of
comparatively common occurrence. There is no doubt what-
ever that the great majority of these are readily distinguishable
by their appearance from the spirochaete pallida, but others
resemble it closely. Hoffmann, however, who has seen many of
these spirochaetes from other sources, considers that even by
their microscopic appearance they are capable of being dis-
tinguished, though with considerable difficulty. It must, of
course, be borne in mind that the finding of an organism in
non-syphilitic lesions with exactly the same microscopical char-
acters does not show that it is the same organism as the spiro-
chaete pallida. It cannot be claimed that the pathological
relation of this organism to the disease is absolutely demon-
strated ; but the facts stated are sufficient to form very strong
presumptive evidence that in the spirochaete pallida we have
the true cause of syphilis.
Transmission of the Disease to Animals. — Although
various experiments had previously been from time to time
SYPHILIS 233
made by different observers, in some cases with reported
successful result, it is to the papers of Metchnikoff and Roux
(1903-5) that we owe most of our knowledge. These observers
have carried on a large series of observations, and have shown
that the disease can be transmitted to various species of monkeys.
Of these the anthropoid apes are most susceptible, the chimpanzee
being the most suitable for experimental purposes. Their
results have been confirmed by Lassar, Neisser, Kraus, and
others. Inoculations made by scarification resulted in the
production of typical primary lesions in all of more than twenty
animals used. The primary lesion is in the form of an indurated
papule or of papules, in every respect resembling the human
lesion. Along with this there is a marked enlargement and
induration of the corresponding lymphatic glands. The primary
lesion appeared on an average about thirty days after inocula-
tion, and secondary symptoms appeared in rather more than
half of the cases after a further period of rather longer duration.
These were of the nature of squamous papules on the skin,
mucous patches in the mouth, and sometimes palmar psoriasis.
As a rule, the secondary manifestations were of a somewhat
mild degree, and in no instance up to the present has any
tertiary lesion been observed. By inoculation from the secondary
lesions, the primary manifestations with their typical characters
have been reproduced. The orang-outang has been found to be
less susceptible, whilst Roux's experiments on the gorilla have
been too few to admit of any conclusion. The disease may also
be produced in baboons and macaques (macacus sinicus is one of
the most susceptible), but these animals are less susceptible.
In the case of many of them no result follows, and when a lesion
is produced it is only of the nature of a primary papule,
secondary manifestations never appearing. There is thus no
doubt that the disease may be produced in apes, and, to speak
generally, the severity of the affection increases according to the
nearness of the relationship of the animal to the human
subject.
The production of the disease, experimentally, has supplied
us with some further facts regarding the nature of the virus.
It has been shown repeatedly that the passage of fluid con-
taining the virus through a Berkefeld filter deprives it completely
of its infectivity. In other words, the virus does not belong to
the ultra-microscopic group of organisms. The virus is also
readily destroyed by heat, a temperature of 51° C. being
fatal. With regard to the production of immunity, very little
of a satisfactory nature has so far been established. It has been
234 GONORRHCEA, SOFT SORE, SYPHILIS
found that the virus from a macaque monkey produces a less
severe disease in the chimpanzee than the virus from the human
subject, inasmuch as secondary lesions do not follow ; the virus
would thus appear to have undergone a certain amount of
attenuation in the tissues of that monkey. Recently corneal
ulcers in rabbits have been produced by Bertarelli and by Hoff-
mann by inoculation with syphilitic material ; they appear after
a long period of incubation, and the spirochaBte can be demon-
strated in the lesions. The effects of injecting emulsions of
tertiary lesions or of serum from syphilitic patients, at the time
of inoculation with the virus, appear to be practically nil ;
so also the employment of the virus rendered inactive by heating
has apparently no influence in acting as a vaccine. There is
some evidence that the serum from a patient suffering from the
disease when mixed with the virus before inoculation modifies
the disease to a certain extent, but further evidence on this
point is necessary. As mentioned above, the spirochsete pallida
has been found in the lesions in monkeys, Metchnikoff and
Roux obtaining positive results in more than 75 per cent of the
cases, and it is to be noted that here also the organism has been
found deep in the substance of the papules, unaccompanied by
any other organisms. Hoffmann failed to find any spirochsetes
in monkeys which had not been inoculated with syphilitic
material. This observer produced a lesion on the upper eyelid
of a macacus by inoculation with the blood of a man who had
suffered from the disease for six months, and a papule appeared
which contained spirochaetes. This result is in conformity with
that given by microscopic examination, and shows that the
organism is sometimes present in the circulating blood in severe
cases of the disease, and that the blood is accordingly infective.
Castellani has described in yaws or frambcesia the occurrence
of a spirochsete closely resembling the spirochsete pallida in
appearance, and to this organism he has given the name spiro-
chcete pertenuis. He has found it not only in the skin lesions
but also in the spleen and lymphatic glands of patients suffering
from the disease. He has produced the disease in monkeys by
direct inoculation and has found the spirochsete in the resulting
lesions. He finds that the immunity reactions of the two organ-
isms— spirochaete pallida and spirochsete pertenuis — are quite
distinct ; hence we have probably to deal with two distinct species.
CHAPTER IX.
TUBERCULOSIS.
THE cause of tubercle was proved by Koch in 1882 to be the
organism now universally known as the tubercle bacillus.
Probably no other single discovery has had a more important
effect on medical science and pathology than this. It has not
only shown what is the real cause of the disease, but has also
supplied infallible methods for determining what are tubercular
lesions and what are not, and has also given the means of
studying the modes and paths of infection. A definite answer
has in this way been supplied to many questions which were
previously the subject of endless discussion.
Historical. — By the work of Armanni and of Cohnheim and Salomonsen
(1870-80) it had been demonstrated that tubercle was an infective disease.
The latter observers found on inoculation of the anterior chamber of the
eye of rabbits with tubercular material that in many cases the results of
irritation soon disappeared, but that after a period of incubation, usually
about twenty-five days, small tubercular nodules appeared in the iris ;
afterwards the disease gradually spread, leading to a tubercular disorgan-
isation of the globe of the eye. Later still, the lymphatic glands became
involved, and finally the animal died of acute tuberculosis. The question
remained as to the nature of the virus, the specific character of which
was thus established, and this question was answered by the work
of Koch.
The announcement of the discovery of the tubercle bacillus was made
by Koch in March 1882, and a full account of his researches appeared in
1884 (Mitth. a. d. K. Gsndhtsamte., Berlin). Koch's work on this subject
will remain as a classical masterpiece of bacteriological research, both on
account of the great difficulties which he successfully overcame and the
completeness with which he demonstrated the relations of the organism
to the disease. The two chief difficulties were, first, the demonstration
of the bacilli in the tissues, and, secondly, the cultivation of the organism
outside the body. For, with regard to the first, the tubercle bacillus
cannot be demonstrated by a simple watery solution of a basic aniline
dye, and it was only after prolonged staining for twenty-four hours, with
a solution of methylene-blue with caustic potash added, that he was
able to reveal the presence of the organism. Then, in the second place,
235
236 TUBERCULOSIS
all attempts to cultivate it on the ordinary media failed, and he only
succeeded in obtaining growth on solidified blood serum, the method of
preparing which he himself devised, inoculations being made on this
medium from the organs of animals artificially rendered tubercular.
The fact that growth did not appear till the tenth day at the earliest,
might easily have led to the hasty conclusion that no growth took place.
All difficulties were, however, successfully overcome. He cultivated the
organism by the above method from a great variety of sources, and by
a large series of inoculation experiments on various animals, performed
by different methods, he conclusively proved that bacilli from these
different sources produced the same tubercular lesions and were really of
the same species. His work was the means of showing conclusively that
such conditions as lupus, "white swelling" of joints, scrofulous disease
of glands, etc. , are really tubercular in nature.
Tuberculosis in Animals. — Tuberculosis is not only the most
widely spread of all diseases affecting the human subject, and
produces a mortality greater than any other, but there is probably
no other disease which affects the domestic animals so widely.
We need not here describe in detail the various tubercular lesions
in the human subject, but some facts regarding the disease in
the lower animals may be given, as this subject is of great im-
portance in relation to the infection of the human subject.
Amongst the domestic animals the disease is commonest in cattle
(bovine tuberculosis), in which animals the lesions are very various, both
in character and distribution. In most cases the lungs are affected, and
contain numerous rounded nodules, many being of considerable size ;
these may be softened in the centre, but are usually of pretty firm
consistence and may be calcified. There may be in addition caseous
pneumonia, and also small tubercular granulations. Along with these
changes in the lungs, the pleurae are also often affected, and show
numerous nodules, some of which may be of large size, firm and pedun-
culated, the condition being known in Germany as Perlsucht, in France
as pommeliere. Lesions similar to the last may be chiefly confined to
the peritoneum and pleurae. In other cases, again, the abdominal organs
are principally involved. The udder becomes affected in a certain pro-
portion of cases of tuberculosis in cows — in 3 per cent according to Bang
— but primary affection of this gland is very rare. Tuberculosis is also
a comparatively common disease in pigs, in which animals it in many
cases affects the abdominal organs, in other cases produces a sort of
caseous pneumonia, and sometimes is met with as a chronic disease of
the lymphatic glands, the so-called "scrofula" of pigs. Tubercular
lesions in the muscles are less rare in pigs than in most other animals.
In the horse the abdominal organs are usually the primary seat of the
disease, the spleen being often enormously enlarged and crowded with
nodules of various shapes and sizes ; sometimes, however, the primary
lesions are pulmonary. In sheep and goats tuberculosis is of rare
occurrence, especially in the former animals. It may occur spontaneously
in dogs, cats, and in the large carnivora. It is also sometimes met with
in monkeys in confinement, and leads to a very rapid and widespread
affection in these animals, the nodules having a special tendency to
soften and break down into a pus-like fluid.
THE TUBERCLE BACILLUS 237
Tuberculosis in fowls (avian tuberculosis) is a common and very
infectious disease, nearly all the birds in a poultry-yard being sometimes
affected.
From these statements it will be seen that the disease in
animals presents great variations in character, and may differ in
many respects from that met with in the human subject. The
relation of the different forms of tuberculosis is discussed below.
Tubercle Bacillus — Microscopical Characters. — Tubercle
bacilli are minute rods which usually measure 2 '5 to 3 -5 ^ in
length, and '3 /A in thickness, i.e. in proportion to their length
they are comparatively thin organisms (Figs. 82 and 83). Some-
times, however, longer
forms, up to 5 /A or more
in length, are met with,
both in cultures and in the
tissues. They are straight *
or slightly curved, and are
of uniform thickness, or
may show slight swelling
at their extremities. When
stained they appear uni-
formly coloured, or may
present small uncoloured
spots along their course,
with darkly -stained parts
between. In such a min-
ute organism it is ex-
tremely difficult to deter^ FJG 82._Tubercle bacilli, from a pure
mine the exact natures. of culture on glycerin agar.
the unstained points. Ac- Stained with carbol-fuchsin. x 1000.
cordingly, we find that
some observers consider these to be spores, while others find that
it is impossible to stain them by any means whatever, and con-
sider that they are really of the nature of vacuoles. Against their
being spores is also the fact that many occur in one bacillus.
Others again hold that some of the condensed and highly-stained
particles are spores. It is impossible to speak definitely on the
question at present. We can only say that the younger bacilli
stain uniformly, and that in the older forms inequality in staining
is met with ; this latter condition is, however, not associated with
greater powers of resistance.
The bacilli in the tissues occur scattered irregularly or in
little masses. They are usually single, or two are attached end
to end and often form in such a case an obtuse angle. True
238 TUBERCULOSIS
chains are not formed, but occasionally short filaments are met
with. In cultures the bacilli form masses in which the rods are
closely applied to one another and arranged in a more or less
parallel manner. Tubercle bacilli are quite devoid of motility.
Aberrant Forms. — Though such are the characters of the
organism as usually met with, other appearances are sometimes
found. In old cultures, for example, very much larger elements
V
FIG. 83. — Tubercle bacilli in phthisical sputum ; they are longer than
is often the case.
Film preparation, stained with carbol-fuchsin and rnethylene-blue. x 1000.
may occur. These may be in the form of long filaments, some-
times swollen or clubbed at their extremities, may be irregularly
beaded, and may even show the appearance of branching. Such
forms have been studied by Metclmikoff, Maffucci, Klein, and
others. Their significance has been variously interpreted, for
while some look upon them as degenerated or involution forms,
others regard them as indicating a special phase in the life
history of the organism, allying it with the higher bacteria.
Recent observations, however, go to establish the latter view,
and this is now generally accepted by authorities. It has also
CULTIVATION OF TUBERCLE BACILLUS 239
been found that under certain circumstances tubercle bacilli in
the tissues produce a radiating structure closely similar to that
of the actinomyces. This was found by Babes and also by
Lubarsch to be the case when the bacilli were injected under
the dura mater and directly into certain solid organs, such as
the kidneys in the rabbit. Club-like structures may be present
at the periphery ; these are usually not acid-fast, but they retain
the stain in the Weigert-Gram method. Similar results obtained
with other acid-fast bacilli will be mentioned below, and these
organisms would appear to form a group closely allied to the
streptothriceae, the bacillary parasitic form being one stage of
the life history of the organism. This group is often spoken of
as the mycobacteria.
Staining Reactions. — The tubercle bacillus takes up the
ordinary stains very slowly and faintly, and for successful staining
one of the most powerful solutions ought to be employed, e.g.
gentian- violet or fuchsin, along with aniline-oil water or solution
of carbolic acid. Further, such staining solutions require to be
applied for a long time, or the staining must be accelerated by
heat, the solution being warmed till steam arises and the
specimen allowed to remain in the hot stain for two or three
minutes. One of the best and most convenient methods is the
Ziehl-Neelsen method (see p. 100). The bacilli present this
further peculiarity, however, that after staining has taken place
they resist decolorising by solutions which readily remove the
colour from the tissues and from other organisms which may be
present. Such decolorising agents are sulphuric or nitric acid
in 20 per cent solution. Preparations can thus be obtained in
which the tubercle bacilli alone are coloured by the stain first
used, and the tissues can then be coloured by a contrast stain.
Within recent years certain other bacilli have been discovered
which present the same staining reactions as tubercle bacilli ;
they are therefore called " acid-fast " (vide infra). The spores
of many bacilli become decolorised more readily than tubercle
bacilli, though some retain the colour with equal tenacity.
Bulloch and Macleod, by treating tubercle bacilli with hot alcohol
and ether, extracted a wax which gave the characteristic staining
reactions of the bacilli themselves. The remains of the bacilli, further,
when extracted witli caustic potash, yielded a body which was probably
a chitin, and which was acid-fast when stained for twenty-four hours
with carbol- fuchsin.
Cultivation. — The medium first used by Koch was inspissated
blood serum (vide p. 39). If inoculations are made on this
medium with tubercular material free from other organisms,
240
TUBERCULOSIS
there appear in from ten to fourteen days minute points of
growth of dull whitish colour, rather irregular, and slightly raised
above the surface (it is advisable to plant on the medium an
actual piece of the tubercular tissue and to fix it in a wound of
the surface of the serum). Koch compared the appearance of these
to that of small dry scales. In such cultures the growths usually
reach only a compara-
tively small size and re-
main separate, becoming
confluent only when many
occur close together. In
sub - cultures, however,
growth is more luxuriant
and may come to form a
dull wrinkled film of
whitish colour, which
may cover the greater part
of the surf ace of the serum
and at the bottom of the
tube may grow over the
surface of the condensa-
tion water on to the glass
(Fig. 84, A). The growth
is always of a dull ap-
pearance and has a con-
siderable degree of con-
sistence, so that it is diffi-
cult to dissociate a portion
thoroughly in a drop of
water. In older cultures
th° %™^ may acquire
a slightly brownish or buff
colour. When the Small
colonies are examined
•, ,. , ,
Under a lOW power OI the
microscope they are seen
to be extending at the periphery in the form of wavy or sinuous
streaks which radiate outward and which have been compared
to the flourishes of a pen. The central part shows similar
markings closely interwoven. These streaks are composed of
masses of the bacilli arranged in a more or less parallel manner.
On glycerin agar, which was first introduced by Nocard and
Roux as a medium for the culture of the tubercle bacillus,
growth takes place in sub-cultures at an earlier date and pro-
FIG. 84.-Cultures of tubercle bacilli on
glycerin agar.
A and B. Mammalian tubercle bacilli ; A is an
old culture, B one of a few weeks' growth.
C. Avian tubercle bacilli. The growth is whiter
and smoother on the surface than the others.
POWERS OF RESISTANCE 241
gresses more rapidly than on serum, but this medium is not
suitable for obtaining cultures from the tissues, inoculations
with tubercular material usually yielding a negative result. The
growth has practically the same characters as on serum, but is
more luxuriant. In glycerin broth, especially when the layer is
not deep, tubercle bacilli grow readily in the form of little white
masses which fall to the bottom and form a powdery layer. If,
however, the growth be started on the surface it spreads super-
ficially as a dull whitish, wrinkled pellicle which may reach the
walls of the flask; this mode of growth is specially suitable for
the production of tuberculin (vide infra}. The culture has a
peculiar fruity and not unpleasant odour. On ordinary agar
and on gelatin media no growth takes place.
It Avas at one time believed that the tubercle bacillus would only grow
on media containing animal fluids, but of late years it has been found that
growth takes place also on a purely vegetable medium, as was first shown
by Pawlowsky in the case of potatoes. Sander has shown that the
bacillus grows readily on potato, carrot, macaroni, and on infusion of
these substances, especially when glycerin is added. He also found
that cultures from tubercular lesions could be obtained on glycerin potato
(p. 46).
The optimum temperature for growth is 37° to 38° C.
Growth ceases above 42° and usually below 28°, but on long-
continued cultivation outside the body and in special circum-
stances, growth may take place at a lower temperature, e.g.
Sander found that growth took place in glycerin-potato broth
even at 22° to 23° C.
Powers of Resistance. — Tubercle bacilli have considerable
powers of resistance to external influences, and can retain their
vitality for a long time outside the body in various conditions ;
in fact, in this respect they may be said to occupy an inter-
mediate position between spores and spore-free bacilli. Dried
phthisical sputum has been found to contain still virulent bacilli
(or their spores) after two months, and similar results are obtained
when the bacilli are kept in distilled water for several weeks.
So also they resist for a long time the action of putrefaction,
which is rapidly fatal to many pathogenic organisms. Sputum
has been found to contain living tubercle bacilli even after being
allowed to putrefy for several weeks (Fraenkel, Baumgarten), and
the bacilli have been found to be alive in tubercular organs which
have been buried in the ground for a similar period. They are
not killed by being exposed to the action of the gastric juice for
six hours, or to a temperature of - 3° C. for three hours, even
when this is repeated several times. It has been found that
16
242 TUBERCULOSIS
when completely dried they can resist a temperature of 100° C.
for an hour, but, on the other hand, exposure in the moist
condition to 70° C. for the same time is usually fatal. It may
be stated that raising the temperature to 100° C. kills the bacilli
in fluids and in tissues, but in the case of large masses of tissue
care must be taken that this temperature is reached throughout.
They are killed in less than a minute by exposure to 5 per cent
carbolic acid, and both Koch and Straus found that they are
rapidly killed by being exposed to the action of direct sunlight.
Action on the Tissues. — The local lesion produced by the
tubercle bacillus is the well-known tubercle nodule, the
structure of which varies in different situations and according to
the intensity of the action of the bacilli. After the bacilli gain
entrance to a connective tissue such as that of the iris, their
first action appears to be on the connective-tissue cells, which
become somewhat swollen and undergo mitotic division, the
resulting cells being distinguishable by their large size and pale
nuclei — the so-called epithelioid cells. These proliferative changes
may be well seen on the fifth day after inoculation or even
earlier. A small focus of proliferated cells is thus formed in the
neighbourhood of the bacilli and about the same time numbers
of leucocytes — chiefly lymphocytes — begin to appear at the
periphery and gradually become more numerous. Soon, however,
the action of the bacilli as cell-poisons comes into prominence.
The epithelioid cells become swollen and somewhat hyaline, their
outlines become indistinct, whilst their nucleus stains faintly,
and ultimately loses the power of staining. The cells in the
centre, thus altered, gradually become fused into a homogeneous
substance and this afterwards becomes somewhat granular in
appearance. If the central necrosis does not take place quickly,
then giant-cell formation may occur in the centre of the follicle,
this constituting one of the characteristic features of the tuber-
cular lesion ; or after the occurrence of caseation giant-cells may
be formed in the cellular tissue around. The centre of a giant-
cell often shows signs of degeneration, such as hyaline change
and vacuolation, or it may be more granular than the rest of the
cell.
Though there has been a considerable amount of discussion
as to the mode of origin of the giant-cells, we think there can
be little doubt that in most cases they result from enlargement
of single epithelioid cells, the nucleus of which undergoes pro-
liferation without the protoplasm dividing. These epithelioid
cells may sometimes be the lining cells of capillaries. Some con-
sider that the giant-cells result from a fusion of the epithelioid
ACTION ON THE TISSUES 243
cells ; but, though there are occasionally appearances which
indicate such a mode of formation, it cannot be regarded as of
common occurrence. In some cases of acute tuberculosis, when
the bacilli become lodged in a capillary the endothelial cells of
its wall may proliferate, and thus a ring of nuclei may be seen
round a small central thrombus. Such an occurrence gives rise
to an appearance closely resembling a typical giant -cell.
According to the view here stated, both the epithelioid and the
giant-cells are of connective tissue origin ; and we can see no
sufficient evidence for the view held by some observers, chiefly
of the French school, that they are formed from leucocytes
which have emigrated from the capillaries.
There can be no doubt that the cell necrosis and subsequent
caseation depend upon the products of the bacilli, and are not due
to the fact that the tubercle nodule is non-vascular. This non-
vascularity itself is to be explained by the circumstance that
young capillaries cannot grow into a part where tubercle bacilli
are active, and that the already existing capillaries become
thrombosed, owing to the action of the bacillary products on
their walls, and ultimately disappear. At the periphery of
tubercular lesions there may be considerable vascularity and new
formation of capillaries.
The general symptoms of tuberculosis — pyrexia, perspiration,
wasting, etc., are to be ascribed to the absorption and distribution
throughout the system of the toxic products of the bacilli ; in
the case of phthisical cavities and like conditions where other
bacteria are present, the toxins of the latter also play an im-
portant part. The occurrence of waxy change in the organs is
believed by some to be chiefly due to the products of other,
especially pyogenic, organisms, secondarily present in the tuber-
cular lesions. This matter, however, requires further elucidation.
Presence and Distribution of the Bacilli, — A few facts may be
stated regarding the presence of bacilli, and the numbers in
which they are likely to be found in tubercular lesions. On the
one hand, they may be very few in number and difficult to find,
and on the other hand, they may be present in very large
numbers, sometimes forming masses which are easily visible under
the low power of the microscope.
They are usually very few in number in chronic lesions,
whether these are tubercle nodules with much connective tissue
formation or old caseous collections. In caseous material one
can sometimes see a few bacilli faintly stained, along with very
minute unequally stained granular points, some of which may
possibly be spores of the bacilli. Whether they are spores or
244
TUBERCULOSIS
not, the important fact has been established that tubercular
material in which no bacilli can be found microscopically, may
be proved, on experimental inoculation into animals, to be still
virulent. In such cases the bacilli may be present in numbers so
small as to escape observation, or it may be that their spores only
are present. In subacute lesions, with well-formed tubercle
follicles and little caseation, the bacilli are generally scanty.
FIG. 85. — Tubercle bacilli in section of human lung in acute phthisis. The
bacilli are seen lying singly, and also in large masses to left of field. The
pale background is formed by caseous material.
Stained with carbol-fuchsin and Bismarck -brown, x 1000.
They are most numerous in acute lesions, especially where
caseation is rapidly spreading, for example, in such conditions as
caseous catarrhal pneumonia (Fig. 85), acute tuberculosis of the
spleen in children, which is often attended with a good deal of
rapid caseous change, etc. In acute miliary tuberculosis a few
bacilli can generally be found in the centre of the follicles ; but
here they are often much more scanty than one would expect.
The tubercle bacillus is one which not only has comparatively
slow growth, but retains its form and staining power for a much
ACTION ON THE TISSUES
245
longer period than most organisms. As a rule the bacilli are
extra-cellular in position. Occasionally they occur within the
giant-cells, in which they may be arranged in a somewhat radiate
manner at the periphery, occasionally also in epithelioid cells and
in leucocytes.
The above statements, however, apply only to tuberculosis
in the human subject, and even in this case there are exceptions.
» FIG. 86. — Tubercle bacilli in giant-cells, showing the radiate arrangement
at the periphery of the cells. Section of tubercular udder of cow.
Stained with carbol-fuchsin and Bismarck-brown. x 1000.
In the ox, on the other hand, the presence of tubercle bacilli
within giant-cells is a very common occurrence ; and it is
also common to find them in considerable numbers scattered
irregularly throughout the cellular connective tissue of the lesions,
even when there is little or no caseation present (Fig. 86).
In tuberculosis in the horse and in avian tuberculosis the
numbers of bacilli may be enormous, even in lesions which are
not specially acute ; and considerable variation both in their
number and in their site is met with in tuberculosis of other
animals.
246 TUBERCULOSIS
In discharges from tubercular lesions which are breaking
down, tubercle bacilli are usually to be found. In the sputum of
phthisical patients their presence can be demonstrated almost
invariably at some period, and sometimes their numbers are very
large (for method of staining see p. 101). Several examinations
may, however, require to be made ; this should always be done
before any conclusion as to the non-tubercular nature of a case is
come to. In cases of genito-
urinary tuberculosis they
are usually present in the
&& ^ urine; but as they are
|^ much diluted it is difficult
to find them unless a de-
posit is obtained by means
of the centrifuge. This
deposit is examined in the
same way as the sputum.
The bacilli often occur in
little clumps, as shown in
Fig. 87. In tubercular
ulceration of the intestine
their presence in the faeces
may be demonstrated^ as
FIG. 87. — Tubercle bacilli in urine ; showing was first shown by Koch '}
one of the characteristic clumps, in which ^ut jn t^g cage fa^ <jis-
they often occur. . ni ,. -,. ,,-,
Stained with carbol-fuchsin and methylene- p°verv 1S ^ally of little
blue, x 1000. importance, as the intes-
tinal lesions, as a rule,
occur only in advanced stages when diagnosis is no longer a
matter of doubt.
Experimental Inoculation. — Tuberculosis can be artificially
produced in animals by infection in a great many different ways
— by injection of the bacilli into the subcutaneous tissue, into
the peritoneum, into the anterior chamber of the eye, into the
veins ; by feeding the animals with the bacilli ; and, lastly, by
making them inhale the bacilli suspended in the air.
The exact result, of course, varies in different animals and
according to the method of inoculation, but we may state
generally that when introduced into the tissues of a susceptible
animal, the bacilli produce locally the lesions above described,
terminating in caseation ; that there occurs a tubercular affection
of the neighbouring lymphatic glands, and that lastly there
may be a rapid extension of the bacilli to other organs by the
blood stream and the production of general tuberculosis. Of
EXPERIMENTAL INOCULATION 247
the animals generally used for the purpose, the guinea-pig is
most susceptible.
When a guinea-pig is inoculated subcutaneously with tubercle
bacilli from a culture, or with material containing them, such as
phthisical sputum, a local swelling gradually forms which is
usually well marked about the tenth day. This swelling becomes
softened and caseous, and may break down, leading to the
formation of an irregularly ulcerated area with caseous lining.
The lymphatic glands in relation to the parts can generally be
found to be enlarged and of somewhat firm consistence, about
the end of the second or third week. Later, in them also caseous
change occurs, and a similar condition may spread to other
groups of glands in turn, passing also to those on the other side
of the body. During the occurrence of these changes, the animal
loses weight, gradually becomes cachectic, and ultimately dies,
sometimes within six weeks, sometimes not for two or three
months. Post mortem, in addition to the local and glandular
changes, an acute tuberculosis is usually present, the spleen
being specially affected. This organ is swollen, and is studded
throughout by numerous tubercle nodules, which may be minute
and grey, or larger and of a yellowish tint. If death has been
long delayed, calcification may have occurred in some of the
nodules. Tubercle nodules, though rather less numerous, are
also present in the liver and in the lungs, the nodules in the
latter organs being usually of smaller size though occasionally in
large numbers. The extent of the general infection varies;
sometimes the chronic glandular changes constitute the out-
standing feature.
Intraperitoneal injection of pure cultures produces a local lesion in the
form of an extensive tubercular infiltration and thickening of the
omentum, sometimes attended with acute tubercles all over the
peritoneum, There is a caseous enlargement of the retroperitoneal and
other lymphatic glands, and later there may be a general tuberculosis.
Intravenous injection produces a typical acute tuberculosis, the nodules
being usually more numerous and of smaller size, while death follows
more rapidly, the larger the numbers of bacilli injected. Guinea-pigs,
when fed with tubercle bacilli, or with sputum or portions of tissue
containing them, readily contract an intestinal form of tuberculosis,
lesions being present in the lymphoid tissue of the intestines, in the
mesenteric glands, and later in the internal organs.
Rabbits are less susceptible than guinea-pigs, and in them the effects
of subcutaneous inoculation are very variable ; sometimes the lesions
remain local, sometimes a general tuberculosis is set up. Otherwise the
reactions are much of the same nature. Dogs are much more highly
resistant, but tuberculosis can be produced in them by intraperitoneal
injection of pure cultures (Koch), or by intravenous injection (Maffucci).
In the latter case there results an extensive eruption of minute miliary
248 TUBERCULOSIS
tubercles. Tuberculosis can also be easily produced in susceptible
animals by making them inhale the bacilli.
Varieties of Tuberculosis. 1 . Human and Bovine Tuberculosis.
—Up till recent years it was generally accepted that all
mammalian tuberculosis was due to the same organism, and
in particular that tuberculosis could be transmitted from the
ox to the human subject. The matter became one of special
interest owing to Koch's address at the Tuberculosis Congress
in 1901, in which he stated his conclusion that human and
bovine tuberculosis are practically distinct, and that if a
susceptibility of the human subject to the latter really exists,
infection is of very rare occurrence, — so rare that it is not
necessary to take any measures against it. Previously to this,
Theobald Smith had pointed out differences between mammalian
and bovine tubercle bacilli, the most striking being that the latter
possess a much higher virulence to the guinea-pig, rabbit, and other
animals, and in particular that human tubercle bacilli, on
inoculation into oxen, produce either no disease or only local
lesions without any dissemination. Koch's conclusions were based
chiefly on the result of his inoculations • of the bovine species
with human tubercle bacilli, the result being confirmatory of
Smith's, and, secondly, on the supposition that infection of the
human subject through the intestine is of very rare occurrence.
Since the time of Koch's communication an enormous amount
of work has been done on this subject, and Commissions of
inquiry have been appointed in various countries. We may
summarise the chief facts which have been established.
Practically all observers are agreed that there are two chief
types of tubercle bacilli which differ both in their cultural
characters and in their virulence — a bovine type and a human
type. The bacilli of the bovine type when cultivated are shorter
and thicker and more regular in size ; whilst their growtETon
various culture media is scantier than that of the human type.
From the latter character the British Royal Commission have
applied the term dysgonic to the bovine and eugonic to the
human type. As already stated there is also a great difference
in virulence towards the lower animals, the bacillus from the ox
having a much higher virulence. This organism when injected
in suitable quantities into the ox produces a local tubercular
lesion, which is usually followed by a generalised and fatal
tuberculosis ; whereas injection of human tubercle bacilli pro-
duces no more than a local lesion, which undergoes retrogression.
(In certain experiments, e.g. those of Delepine, Hamilton and
VARIETIES OF TUBERCULOSIS 249
Young, general tuberculosis has been produced by tubercle
bacilli from the human subject, but these results are exceptional).
Corresponding differences come out in the case of the rabbit ; in
fact, intravenous injection of suitable quantities in this animal is
the readiest method of distinguishing the two types — an acute
tuberculosis resulting with the bovine, but not with the human
type. In guinea-pigs and monkeys a generalised tuberculosis may
result from subcutaneous injection of bacilli of the human type,
but in this case also the difference in favour of the greater viru-
lence of the bovine type is made out. With regard to the dis-
tribution of the two types of organisms, it may be stated that so
far as we know the bacillus obtained from bovine tuberculosis is
always of the bovine type, and the same may be said to be true
of tuberculosis in pigs ; in fact this seems to be the prevalent
organism in animal tuberculosis. In human tuberculosis the
bacilli in a large majority of the cases are of the human type ;
but on the other hand, in a certain proportion bacilli of the
bovine type are present, the bacilli when cultivated being
indistinguishable by any means at our disposal from those
obtained from bovine tuberculosis. The Royal Commission
found the bovine type in 14 out of 60 cases of human
tuberculosis — a somewhat higher proportion than has been
obtained by most other investigators — and in all of these,
with one exception, the bacilli were obtained either from caseous
cervical glands, or from the lesions of primary abdominal tubercu-
losis, that is from cases where there was evidence of infection by
alimentation. It is also to be noted that almost all the tuber-
cular lesions from which the bovine type has been obtained have
been in children. The general result accordingly is that bovine
tubercle bacilli are present in a certain proportion of cases of tuber-
culosis in young subjects, and that these are especially cases where
infection by the alimentary canal has occurred. It must thus
be held as established that tuberculosis is transmissible from
the ox to man, and that the milk of tubercular cows is a common
vehicle of transmission.
Although most of the bacilli which have been cultivated
correspond to one of the two types, as above described, it is
also to be noted that intermediate varieties are met with. It
has also been found that the type characters of the bacillus are
not constant. Various observers have found it possible to
modify bacilli of the human type by passing them through the
bodies of certain animals, e.g. guinea-pigs, sheep, and goats, so
that they acquire the characters of bovine bacilli. In view
of these facts it is probable that bovine bacilli will undergo
250 TUBERCULOSIS
corresponding modifications in the tissues of the human subject
— what period of time is necessary for such a change we cannot
say. It is thus possible that the cases from which the bovine
type has been obtained do not represent the full number where
infection from the ox has occurred. It is quite likely that
although the bovine bacilli are more virulent to the lower
animals than the human bacilli are, this does not also hold in the
case of the human subject. In fact the comparative chronicity
of the primary abdominal lesions in children in the first instance
would point rather to a low order of virulence towards the
human subject. We may also add that there are cases, notably
those of Ravenel, in which accidental inoculation of the human
subject with bovine tubercle has resulted in the production of
tuberculosis.
2. Avian Tuberculosis. — In the tubercular lesions in birds there
are found bacilli which correspond in their staining reactions
and in their morphological characters with those in mammals,
but differences are observed in cultures, and also on experimental
inoculation. These differences were first described by Maffucci
and by Rivolta, but special attention was drawn to the subject
by a paper read by Koch at the International Medical Congress in
1890. Koch stated that he had failed to change the one variety
of tubercle bacillus into the other, though he did not conclude
therefrom that they were quite distinct species. The following
points of difference may be noted : —
On glycerin agar and on serum, the growth of ..tubercle bacilli from
birds is more luxuriant, has a moister appearance (Fig. 84, C), and,
moreover, takes place at a higher temperature, 43 '5° C., than is the
case with ordinary tubercle bacilli. Experimental inoculation brings
out even more distinct differences. Tubercle bacilli derived from the
human subject, for example, when injected into birds, usually fail to
produce tuberculosis, whilst those of avian origin very readily do so.
Birds are also very susceptible to the disease when fed with portions
of the organs of birds containing tubercle bacilli, but they can consume
enormous quantities of phthisical sputum without becoming tubercular
(Straus, Wurtz, Nocard). No doubt, on the other hand, there are cases
on record in which the source of infection of a poultry-yard has ap-
parently been the sputum of phthisical patients. Again, tubercle
bacilli cultivated from birds have not the same effect on inoculation
of mammals as ordinary tubercle bacilli have. When guinea-pigs are
inoculated subcutaneously they usually resist infection, though occa-
sionally a fatal result follows. In the latter case, usually no tubercles
visible to the naked eye are found, but numerous bacilli may be present
in internal organs, especially in the spleen, which is much swollen.
Further, intravenous injection even of large quantities of avian tubercle
bacilli, in the case of dogs, leads to no effect, whereas ordinary tubercle
bacilli produce acute tuberculosis. [The rabbit, on the other hand, is
comparatively susceptible to avian tuberculosis (Nocard).]
VARIETIES OF TUBERCULOSIS 251
There is, therefore, abundant evidence that the bacilli derived
from the two classes of animals show important differences, and,
reasoning from analogy, we might infer that probably the human
subject also would be little susceptible to infection from avian
tuberculosis. The question remains, are these differences of a
permanent character ? The matter seems conclusively settled by
the experiments of Nocard, in which mammalian tubercle bacilli
have been made to acquire all the characters of those of avian
origin. The method adopted was to place bacilli from human
tuberculosis in small collodion sacs (v. p. 123) containing bouillon,
and then to insert each sac in the peritoneal cavity of a fowl.
The sacs were left in situ for periods of from four to eight months.
They were then removed, cultures were made from their con-
tents, fresh sacs were inoculated from these cultures and intro-
duced into other fowls. In such conditions the bacilli are
subjected only to the tissue juices, the wall of the sac being
impervious both to bacilli and to leucocytes, etc. After one
sojourn of this kind, and still more so after two, the bacilli are
found to have acquired some of the characters of avian tubercle
bacilli, but are still non- virulent to fowls. After the third
sojourn, however, they have acquired this property, and produce
in fowls the same lesion as bacilli derived from avian tuber-
culosis. It therefore appears that the bacilli of avian tuberculosis
are not a distinct and permanent species, but a variety which
has been modified by growth in the tissues of the bird. Evidently
also there are degrees of this modification according to the
period of time during which the bacilli have passed from bird to
bird, as in some cases inoculation with tubercle bacilli of avian
origin has produced ordinary tubercle nodules in guinea-pigs
(Courmont and Dor). It is also interesting to note that
Rabinowitch has cultivated tubercle bacilli of the mammalian
type from some cases of tuberculosis in parrots kept in con-
finement.
3. Tuberculosis in the Fish. — Bataillon, Dubard, and Terre
cultivated from a tubercle-like disease in a carp, a bacillus which,
in staining reaction and microscopic characters^ closely agrees
with the tubercle bacillus. The lesion with which it was
associated was an abundant growth of granulation tissue in
which numerous giant-cells were present. It forms, however,
luxuriant growth at the room temperature, the growth being
thick and moist like that of avian tubercle bacilli (Fig. 89, c).
Growth does not occur at the body temperature, though by
gradual acclimatisation a small amount of growth has been
obtained up to 36° C. Furthermore, the organism appears to
252 TUBERCULOSIS
undergo no multiplication when injected into the tissues of
mammals, and attempts to modify this characteristic have so
far been unsuccessful. Weber and Taute have cultivated this
organism from mud, and also from organs of healthy frogs. It
is thus probably to be regarded as a saprophyte which is only
occasionally associated with disease in the fish. According to
the results of different experimenters it is possible to modify
human tubercle bacilli by allowing them to sojourn in the tissues
of cold-blooded animals, e.g. the frog, blind- worm, etc., so that
they nourish at lower temperatures. These results have, how-
ever, been recently called in question, as it has been stated the
organisms obtained were not modified tubercle bacilli but other
acid-fast bacilli which may be found in the tissues of normal
cold-blooded animals. This question must accordingly be
considered still an open one.
All the above facts taken together indicate that tubercle
bacilli may become modified in relative virulence and in con-
ditions of growth by sojourn in the tissues of various animals.
This modification appears slight, though of definite character in
the case of bovine tuberculosis, more distinct in the case of
avian tuberculosis, and much more marked, if not permanent, in
the case of fish tuberculosis, that is, of course, in their relations
to the bacilli from the human subject.
Other Acid-fast Bacilli. — Within recent years a number of
bacilli presenting the same staining reaction as the tubercle
bacilli have been discovered. Such bacilli have a comparatively
wide distribution in nature, as they have been obtained from
various species of grass, from butter and milk, from manure, and
from the surfaces of animal bodies. Microscopically, they agree
more or less closely with 'tubercle bacilli, though most of them
are shorter and plumper ; many of them show filamentous and
branching forms under certain conditions of culture. Moreover,
on injection, they produce granulation-tissue nodules which may
closely resemble tubercles, although on the whole there is a
greater tendency to softening and suppuration, and usually the
lesions are localised to the site of inoculation. The most im-
portant point of distinction is the fact that their multiplication
on artificial media is much more rapid, growth usually being
visible within forty-eight hours and often within twenty-four
hours at 37° C. Furthermore, in most instances, growth occurs
at the room temperature. The general character of the cultures
in this group is a somewhat irregular layer, often with wrinkled
surface, dry or moist in appearance, and varying in tint from
white to yellow or reddish brown. The number of such
OTHER ACID-FAST BACILLI 253
organisms is constantly being added to, but the following may
be mentioned as examples : —
Afoeller's Grass Bacilli, I. and II.— The former was found in infusions
of Timothy-grass (PMeum pratense}. It is extremely acid-fast, morpho-
logically resembles the tubercle bacillus, and in cultures may show club
formation and branching. The lesions produced closely resemble
tubercles. The colonies, visible in thirty-six hours, are scale-like and
of greyish-white colour (Fig. 89, a). Moeller's bacillus II. was obtained
from the dust of a hay-loft. The colonies at first are moist and some-
what tenacious, but afterwards run together, and are of a dull yellowish
colour. The general results of inoculation resemble those of grass
a 0 G
FIG. 88.— Moeller's Timothy-grass bacillus. FlG< 89.— Cultures of acid-fast bacilli
From a culture on agar. grown at room temperature.
Stained with carbol-fuchsin, and treated with Moeller>s Timotll s bacillus L
20 per cent sulphuric acid. x 1000. J6)' The Petri-Rabinowitch butter bacillus.
(c) Bacillus of fish tuberculosis.
bacillus I. but are less marked. Moeller also obtained a similar organism
from milk. He also discovered a third acid-fast bacillus which he
obtained from manure and therefore called the " Mistbacillus " (dung
bacillus). This organism has analogous characters, though presenting
minor differences. It also has pathogenic effects.
Petri and Rabinowitch independently cultivated an acid- fast bacillus
from butter ("butter bacillus") in which it occurs with comparative
frequency. The organism resembles the tubercle bacillus, although it is
on the whole shorter and thicker. Its lesions closely resemble tuber-
culosis, especially when injection of the organism is made into the
peritoneal cavity of guinea-pigs, along with butter, — the method usually
adopted in searching for tubercle bacilli in butter. This organism
produces pretty rapidly a wrinkled growth (Fig. 89, b) not unlike that
of Moeller's grass bacillus II. Korn has also obtained other two
bacilli from butter which he holds to be distinct from one another and
254
TUBERCULOSIS
\
t
V
from Rabinowitch's bacillus. The points of distinction are of a minor
character. Other more or less similar bacilli have been cultivated by
Tobler, Coggi, and others.1
Another bacillus of considerable interest is Johne's bacillus or the
bacillus of "chronic bovine pseudo-tuberculous enteritis," the lesions
produced by it being corrugated thickenings of the mucous membrane,
especially of the small intestine. The disease has now been observed in
various countries, and several cases in Britain have been recorded by
M'Fadyean. The bacilli occur in large numbers in the lesions, and can
readily be found in scrapings from the surface. They resemble the
tubercle bacillus in appear-
ance, but on the whole are
rather shorter ; they are
equally acid-fast. The organ-
ism has not yet been culti-
vated outside the body.
Smegma Bacillus. —This
organism is of importance,
as in form and staining re-
action it somewhat resembles
the tubercle bacillus and may
be mistaken for it. It occurs
often in large numbers in
the smegma prseputiale and
in the region of the external
genitals, especially where
there is an accumulation of
fatty matter from the secre-
tions. Morphologically it is
a slender slightly curved
organism, like the tubercle
bacillus but usually dis-
tinctly shorter (Fig. 90).
Like the tubercle bacillus it
stains with some difficulty
and resists decolorisation with strong mineral acids. Most observers
ascribe the latter fact to the fatty matter with which it is surrounded,
and find that if the specimen is treated with alcohol the organism is
easily decolorised. Czaplewski, however, who claims to have cultivated
it on various media, finds that in culture it shows resistance to decolor-
isation both with alcohol and with acids, and considers, therefore, that
the reaction is not due to the surrounding fatty medium. We have
found that in smegma it can be readily decolorised by a minute's exposure
to alcohol after the usual treatment with sulphuric acid, and thus can
be readily distinguished from the tubercle bacillus. We, moreover,
believe that minor points of difference in the microscopic appearances of
the two organisms are quite sufficient to make the experienced observer
suspicious if he should meet with the smegma bacillus in urine, and lead
him to apply the decolorising test. Difficulty will only occur when a few
scattered bacilli retaining the fuchsin occur.
Its cultivation, which is attended with some difficulty, was first
FIG. 90. — Smegma bacilli. Film preparation
of smegma.
Ziehl-Neelsen stain. x 1000.
1 For further details on this subject, vide Potet, Etudes sur les bacilles dites
acidophiles. Paris, 1902.
ACTION OF DEAD TUBERCLE BACILLI 255
effected by Czaplewski. On serum it grows in the form of yellowish-
grey irregularly rounded colonies about 1 mm. in diameter, sometimes
becoming confluent to form a comparatively thick layer. He found that
it also grew on glycerin agar and in bouillon. It is non-pathogenic to
various animals which have been tested.
Cowie has recently found that acid-fast bacilli are of common occur-
rence in the secretions of the external genitals, mammse, etc., in certain
of the lower animals, and that these organisms vary in appearance. He
considers that the term "smegma bacillus" probably represents a
number of allied species.
The question may be asked — do these results modify the
validity of the staining reaction of tubercle bacilli as a means of
diagnosis'? The source of any acid-fast bacilli in question is
manifestly of importance, and it may be stated that when these
have been obtained from some source outside the body, or where
contamination from without has been possible, their recognition
as tubercle bacilli cannot be established by microscopic examina-
tion alone. In the case of material coming from the interior of
the body, however, — sputum, etc., — the condition must be looked
on as different, and although an acid-fast bacillus (not tubercle)
has been found by Rabinowitch in a case of pulmonary gangrene
we have no sufficient data for saying that acid-fast bacilli other
than the tubercle bacillus nourish within the tissues of the human
body except in such rare instances as to be practically negligible.
(To this statement the case of the leprosy bacillus is of course
an exception.) Accordingly, up till now, the microscopic ex-
amination of sputum, etc., cannot be said to have its validity
shaken, and we have the results of enormous clinical experience
that such examination is practically of unvarying value. Never-
theless the facts established with regard to other acid-fast bacilli
must be kept carefully in view, and great care must be exercised
when only one or two bacilli are found, especially if they deviate
in their morphological characters from the tubercle bacillus.
Action of dead Tubercle Bacilli. — The remarkable fact has
been established by independent investigators that tubercle
bacilli in the dead condition, when introduced into the tissues
in sufficient numbers, can produce tubercle-like nodules. Prudden
and Hodenpyl, by intravenous injection in rabbits of cultures
sterilised by heat, produced in the lungs small nodules in which
giant -cells, but no caseation, were occasionally present, and
which were characterised by more growth of fibrous tissue than
in ordinary tubercle. The subject was very fully investigated
with confirmatory results by Straus and Gamaleia, who found
that, if the number of bacilli introduced into the circulation were
large, there resulted very numerous tubercle nodules with well-
256 TUBERCULOSIS
formed giant -cells, and occasionally traces of caseation. The
bacilli can be well recognised in the nodules by the ordinary
staining method. In these experiments the bacilli were killed
by exposure to a temperature of 115° C. for ten minutes before
being injected. Similar nodules can be produced by intra-
peritoneal injection. Subcutaneous injection, on the other
hand, produces a local abscess, but in this case no secondary
tubercles are found in the internal organs. Further, in many
of the animals inoculated by the various methods a condition of
marasmus sets in and gradually leads to a fatal result, there
being great emaciation before death. These experiments, which
have been confirmed by other observers, show that even after
the bacilli are dead they preserve their staining reactions in
the tissues for a long time, and also that there are apparently
contained in the bodies of the dead bacilli certain substances
which act locally, producing proliferative and, to a less extent,
degenerative changes, and which also markedly affect the general
nutrition. S. Stockman has found that an animal inoculated
with large numbers of dead tubercle bacilli afterwards gives the
tuberculin reaction.
Practical Conclusions. — From the facts above stated with
regard to the conditions of growth of the tubercle bacilli, their
powers of resistance, and the paths by which they can enter the
body and produce disease (as shown by experiment), the manner
by which tuberculosis is naturally transmitted can be readily
understood. Though the experiments of Sander show that
tubercle bacilli can multiply on vegetable media to a certain
extent at warm summer temperature, it is doubtful whether all
the conditions necessary for growth are provided to any extent
in nature. At any rate, the great multiplying ground of tubercle
bacilli is the animal body, and tubercular tissues and secretions
containing the bacilli are the chief, if not the only, means by
which the disease is spread. The tubercle bacilli leave the body
in large numbers in the sputum of phthisical patients, and when
the sputum becomes dried and pulverised they are set free in
the air. Their powers of resistance in this condition have already
been stated. As examples of the extent to which this takes
place, it may be said that their presence in the air of rooms
containing phthisical patients has been repeatedly demonstrated.
Williams placed glass plates covered with glycerine in the
ventilating shaft of the Brompton Hospital, and after five days
found, by microscopic examination, tubercle bacilli on the surface,
whilst Klein found that guinea-pigs kept in the ventilating shaft
became tubercular. Cornet produced tuberculosis in rabbits by
ACTION OF DEAD TUBERCLE BACILLI 257
inoculating them with dust collected from the walls of a con-
sumptive ward. Tubercle bacilli are also discharged in consider-
able quantities in the urine in tubercular disease of the urinary
tract, and also by the bowel when there is tubercular ulceration ;
but, so far as the human subject is concerned, the great means
of disseminating the bacilli in the outer world is dried phthisical
sputum, and the source of danger from this means can scarcely
be over-estimated. Every phthisical patient ought to be looked
upon as a fruitful source of infection to those around, and should
only expectorate on to pieces of rag which are afterwards to be
burnt, or into special receptacles which are to be then sterilised
either by boiling or by the addition of a 5 per cent solution of
carbolic acid.
Another great source of infection is in all probability the
milk of cows affected with tuberculosis of the udder. In such
cases the presence of tubercle bacilli in the milk can usually be
readily detected by centrifugalising it, and then examining the
deposit microscopically, or by inoculating an animal with it. As
pointed out by Woodhead and others, the milk from cows thus
affected is probably the great source of tabes mesenterica, which
is so common in young subjects. In these cases there may be
tubercular ulceration of the intestine, or it may be absent.
Woodhead found that out of 127 cases of tuberculosis in children,
the mesenteric glands showed tubercular affection in 100, and
that there was ulceration of the intestine in 43. It is especially
in children that this mode of infection occurs, as in the adult
ulceration of the intestine is rare as a primary infection, though
it is common in phthisical patients as the result of infection by
the bacilli in the sputum which has been swallowed. There is
less risk of infection by means of the flesh of tubercular animals,
for, in the first place, tuberculosis of the muscles of oxen being
very rare, there is little chance of the bacilli being present in the
flesh unless the surface has been smeared with the juice of the
tubercular organs, as in the process of cutting up the parts ; and
in the second place, even when present they will be destroyed if
the meat is thoroughly cooked.
We may state, therefore, that the two great modes of infection'
are by inhalation, and by ingestion, of tubercle bacilli. By the
former method the tubercle bacilli will in most cases be derived
from the human subject ; in the latter, probably from tubercular
cows, though inhaled tubercle bacilli may also be swallowed and
contamination of food by tubercular material from the human
subject may occur. Alike when inhaled and when ingested,
tubercle bacilli may lodge about the pharynx and thus come to
17
258 TUBERCULOSIS
infect the pharyngeal lymphoid tissue, tonsils, etc., tubercular
lesions of these parts being much more frequent than was
formerly supposed. Thence the cervical lymphatic glands may
become infected, and afterwards other groups of glands, bones,
or joints, and internal organs.
The Toxins of the Tubercle Bacillus. — Two outstanding
features of the action of the tubercle bacillus are the occurrence
of necrosis in the cells of tubercle nodules and the production
of general disturbances of metabolism accompanied by fever.
It is natural to refer these phenomena to the effects of toxins
formed by the organism. The study of such toxins centres
round the substance known as tuberculin which Koch brought
forward in 1890-1 as a curative agent for tubercular affections.
Koch's Tuberculin. — Koch stated that if in a guinea-pig
suffering from the effects of a subcutaneous inoculation with
tubercle bacilli, a second subcutaneous inoculation of tubercle
bacilli was practised in another part of the body, superficial
ulceration occurred in the primary tubercular nodule, the wound
healed, and the animal did not succumb to tuberculosis. This
reaction was further studied by means of tuberculin, which
consisted of a concentrated glycerin bouillon culture of tubercle
in which the bacilli had been killed by heat. Its essential
components probably were the dead and often macerated bacilli
and the substances indestructible by boiling which existed in
these bacilli, or which were formed during their growth. The
injection of '25 c.c. of tuberculin into a healthy man causes, in
from three to four hours, malaise, tendency to cough, laboured
breathing, and moderate pyrexia ; all of which pass off in
twenty-four hours. The injection (the site of the injection being
quite unimportant), however, of '01 c.c. into a tubercular person
gives rise to similar symptoms, but in a much more aggravated
form, and in addition there occurs around any tubercular focus
great inflammatory reaction, resulting in necrosis and a casting
off of the tubercular mass, when this is possible, as for instance
in the case of lupus. The bacilli are, it was shown, not killed in
the process.
Koch's theory of the action of the substance was that the tubercle
bacillus ordinarily secretes a body having a necrotic action on the tissues.
When this is injected into a tubercular patient, the proportion present
round a tubercular focus is suddenly increased, inflammatory reaction
takes place around, and necrosis of the spreading margin occurs very
rapidly, the material containing the living or dead bacilli being thrown
off en masse instead of being disintegrated piecemeal. It appears,
however, that this explanation may not be the true one ; for, on the one
hand, other substances besides products of the tubercle bacillus may
TOXINS OF THE TUBERCLE BACILLUS 259
give rise to similar effects in tubercular animals, and, on the other, a
similar reaction can take place in other diseases where there is locally in
the body a deposit of new tissue. Matthes has, for instance, found that
albumoses and peptones isolated from the ordinary peptic digestion of
various albumins give the same reaction in tubercular guinea-pigs. The
injection of milk, lactic acid, ricin, all give a similar result. Before the
discovery of tuberculin, Gamaleia had found that tubercular animals
were very susceptible to the toxins of the vibrio Metchnikovi ; and later
Metchnikoff found that a similar susceptibility existed towards the
toxins of the bacillus of fowl cholera. How complicated the tuberculin
reaction is is shown by the fact that a similar reaction has taken place
when tuberculin has been injected into persons suffering from diseases
other than tubercle, e.g. cancer, sarcoma, syphilis.
The hopes which the introduction of tuberculin raised, that a
curative agent against tuberculosis had been discovered, were soon
found not to be justified. It was very difficult to see how the
necrosed material which was produced and which contained the
still living bacilli, could be got rid of either naturally, as would
be necessary in the case of a small tubercular deposit in a lung
or a lymphatic gland, or artificially, as in a complicated joint-
cavity where surgical interference could be undertaken. Not
only so, but the ulceration which might be the sequel of the
necrosis appeared to open a path for fresh infection. Soon facts
were reported which justified these criticisms. Cases where
rapid acute tubercular conditions ensued on the use of tuberculin
were reported, arid in a few months the treatment was practically
abandoned.
The Use of Tuberculin in the Diagnosis of Tuberculosis in Cattle. —
This is now the chief use to which tuberculin is put. In cattle,
tuberculosis may be present without giving rise to apparent
symptoms. It is thus important from the point of view of human
infection that an early diagnosis should be made. The method is
applied as follows : — Tiie animals are kept twenty-four hours in their
stalls, and the temperature is taken every three hours, from four hours
before the injection till twenty-four after. The average tempera-
ture in cattle is 102 '2° F. ; 30 to 40 centigrammes of tuberculin are
injected, and if the animal be tubercular the temperature rises 2° or 3° F.
in eight to twelve hours and continues elevated for ten to twelve hours.
Bang, who has worked most at the subject, lays down the principle that
the more nearly the temperature approaches 104° F. the more reason for
suspicion is there. He gives a record of 280 cases where the value of
the method was tested by subsequent post-mortem examination. He
found that with proper precautions the error was only 3 "3 per cent.
The method has been largely practised in all parts of the world, and is of
great value.
While it is undoubted that tuberculin contains toxic products
formed by the bacilli, we know nothing of the nature of the
toxins present. From the fact that filtered cultures cause little
260 TUBERCULOSIS
toxic effect, and that trituration of the bacilli increases the
poisonous content of a culture, it is inferred that we have to deal
with endotoxins, but beyond this statement we cannot go.
Hitherto no success has attended attempts to gain a closer
knowledge of the nature of such substances. It has been stated
that albumoses of a special kind are present in tuberculin, but
nothing definite has emerged from the investigation of these
bodies.
Active Immunisation against the Tubercle Bacillus. —
Koch's Tuber culin-R. Our knowledge here centres round the
substance introduced by Koch in 1897 under the name of
" Tuberculin-R," or the new tuberculin. Koch's new researches
consisted (1) of an attempt to immunise animals against the
tubercle bacillus by employing its intracellular toxins ; (2) of
trying to utilise such an immunisation to aid the tissues of an
animal already attacked with tubercle the better to combat the
effects of the bacilli. The method of obtaining the intracellular
toxins was as follows. Bacilli from young virulent cultures were
dried in vacuo, and disintegrated in an agate mill, treated with
distilled water and centrifugalised. The clear fluid was decanted,,
and is called by Koch " Tuberculin-O." The remaining deposit
was again dried, ground, treated with w^ater and centrifugalised,
the clear fluid being again decanted, and this process was
repeated with successive residues till no residue remained.
These fluids put together constitute the " Tuberculin-R."
From the fact that tuberculin-O gave no cloudiness when
glycerin was added, Koch concluded that it contained the sub-
stances present in the glycerin-bouillon extracts originally used
by him, and he held this was borne out by the readiness with
which a tuberculin reaction could be caused by it. Similarly, as
tuberculin-R gave a cloudiness with glycerin and did not readily
originate a reaction, he considered that it contained different
products of the bacillus. When injected into animals in
repeated and increasing doses, TJy- mgrm. being the initial dose,
tuberculin-R is said to produce immunity against the original
extract, against tuberculin-O, and against living and virulent
tubercle bacilli. Another preparation has also been introduced
known as " Koch's new tuberculin " (Baiillenemulsion). This is
an emulsion of ground tubercle bacilli in water containing 50 per
cent of glycerin ; it thus really contains both tuberculin-O and
tuberculin-R. Both, especially tuberculin-R, have been used for
the treatment of tuberculosis in man, especially for early localised
lesions. In the case of both substances commencing with from
to yj^ mgrm, gradually increasing doses were given every
OPSONINS IN TUBERCULOSIS 261
second day, and the rule originally laid down for the regulation
of the dosage was that no amount should be given which raised
the temperature more than *5° F. Very various opinions have
been expressed as to the efficacy of such treatment. There is
little doubt that in certain cases of lojcal conditions, such as lupus,
tubercular joints, glands and genito-urinary tuberculosis, improve-
ment has followed its application ; but where febrile conditions
indicate that general disturbances are in existence, there is little
or no justification for its being applied, and even in many local
conditions the absence of benefit is so marked that by many
physicians the method has been abandoned.
Active Immunisation associated with Opsonic Observations. —
Within recent years attention has been directed to the possibility
of controlling the use of tuberculin-R by observations of its
effect on the opsonic qualities of the serum. Wright, early in
his work, showed that tubercle bacilli when sensitised by an
appropriate serum, were readily phagocyted by the polymorpho-
nucleate leucocytes, and the relative sensitising capacities of
serum from tubercular and non-tubercular cases has been widely
studied. According to Wright, in strictly localised tuberculosis
the opsonic index is persistently low, varying from '1 to '9,
while in tubercle with general disturbances it fluctuates greatly
from day to day, being sometimes below, sometimes above unity.
To take the former and simpler case, he holds that if the treat-
ment with injections of tuberculin-R be controlled by noting the
effect produced on the opsonic index, great improvement in the
patient's condition may result. Wright's interpretation of what
occurs is bound up with his views on the nature of the effects
produced. These views are briefly as follows. For reasons
unknown the opsonic qualities of the body fluids may become
abnormally low, and the tubercle bacilli, if they gain admission to
the body, can multiply locally. This multiplication is associated
with a still further local diminution of the opsonins. By the
introduction of such a substance as Koch's tuberculin-R the
bodily mechanism, whatever it is, which produces the opsonins
is stimulated, and a rise in the general opsonic index occurs.
Naturally this is accompanied by a passing to the site of infec-
tion of fluids more rich in opsonins than previously, the activity
of the phagocytes comes into play and the tubercle bacilli are
destroyed. But any such vaccination process must be controlled
by constant observations of the opsonic index, and it is only by
this means that not only good results can be obtained, but that
the production of harmful effects can be prevented. The reason
of this is that in a great many cases the injection of a bacterial
262 TUBEECULOSIS
vaccine is followed by a decrease in the opsonic qualities of the
serum, — the occurrence of a negative phase. J)uring such a
period of depression there is probably an increased susceptibility
to the action of the bacilli. Now, in order to get permanent
benefit from the vaccination process, repeated injections of the
tuberculin must be practised, and if an injection be given during
a negative phase, actual harm may be done. The course of a
successful vaccination is that, after the passing off of the negative
phase, the opsonic index should rise to above its original level,
— the occurrence of a positive phase. It is when this positive
phase is fully developed that a fresh inoculation can be practised
with success. The new negative phase which will now occur
may not cause a drop to below the level of the original state of
the serum, and the hope is that its succeeding positive phase
will carry the opsonic index still higher and ensure a still greater
resistance to the bacterium. The importance of the observations
of the opsonic index lies in this that in antibacterial vaccina-
tions the degree of active immunisation which can be attained is
always much less than is the case with immunisation against
such a substance as the diphtheria toxin, although in the latter
there also occur negative and positive phases of a precisely
similar character. If an injection be practised during a negative
phase, then a still further drop in the opsonic content of the
serum will occur and a fresh growth of the invading bacilli is
likely. There are very great variations in the capacities shown
by tubercular patients to react to a vaccination process. In certain
cases good positive phases are readily and quickly produced, while
in others after an inoculation the negative phase is long con-
tinued and may even show no tendency to pass into a positive
phase. The irregularities in the opsonic index in cases where
there is a general disturbance of metabolism Wright explains by
supposing that they result from very irregular auto-infections of
the patient's body by tubercular products from the local lesions,
— positive and negative phases being produced without the pur-
posive quality which ought to characterise a successful therapeutic
vaccination. Such auto-infections may come about in various
ways, and Wright is of opinion that exercise, for instance, may
disseminate both tubercular products and tubercle bacilli, — he
having noticed in tubercle patients a fall in the opsonic index
after muscular exertion.
With regard to the details of the immunisation, Wright's
chief point is that the repeated, uncontrolled injections of tuber-
culin such as were originally given may very likely have a harmful
result, and that when an injection is practised it is not necessary
OPSONINS IN TUBERCULOSIS 263
for constitutional effects to occur in order that a beneficial result
may follow. Hence much smaller doses of tuberculin than
hitherto are given by him. For ordinary cases with low opsonic
index and no evidence of constitutional disturbance, an amount
of tuberculin corresponding to from one-thousandth to a six-
hundredth of a millegramme of tubercle powder is a sufficient
dose, and if any dose seems to produce a pronounced negative
phase then a smaller dose ought to be tried at the next inocula-
tion. For cases clinically tubercular where the index is about
normal, then smaller doses, say, the equivalent of a two-
thousandth of a millegramme or less ought to be used, — the
effect on the index being carefully watched. In any case, the
dose which is found to give the highest positive phase is the
optimum dose and one which need not necessarily be increased.
Cases where there is constitutional disturbance should be as a
rule left untreated.
The general position of Wright and his school is, that it is
only by the observation of the opsonic index that the application
of the tuberculin treatment can be effectually controlled, —
deductions based on clinical data, such as absence of interference
with pulse rate, temperature, etc., or increase of body weight
after an inoculation being unreliable, and further evidence of the
unreliability of such tests is brought forward in the fact that, in
cases of apparent benefit from sanatorium treatment, the opsonic
index may still be very low. With regard to the results
obtained, many cases have been brought forward by Wright and
others where benefit has followed the putting into practice of the
principles enunciated, and there is little doubt that the work
done has given a fresh start to the active immunisation method
in the treatment of tuberculosis. An outstanding event of
Wright's work in this field has been his insistance on the good
effects produced by extremely small doses of tuberculin (down to
the four-thousandth of a millegramme) given at fairly long inter-
vals (say 10 days or more). With regard to the efficacy of the
opsonic method as affording an index to the progress of a case
it must be recognised that the method is still on its trial, and
it is doubtful if even in the work of the most careful observers
the limits of the experimental error of the opsonic method have
been sufficiently denned.
The whole question of immunisation against the tubercle
bacillus presents many difficulties, and it is the merit of Wright's
work that it has shed fresh light on some of these. One great
difficulty arises from the great chronicity of the results of the
infection in the majority of human cases. It is probably
264 TUBERCULOSIS
true not only of man but of many species of animals used in
experimental inquiries, that many individuals are on the border-
line between immunity and susceptibility. From the wide
spread of the bacilli in centres of human population, it is certain
that the opportunity for infection arises in a very large propor-
tion of the race ; in many cases no results follow infection, and
in many others small lesions occur which do not develop further ;
this has actually been shown by morbid anatomists to be the
case. The disease being thus so often characterised by transient
local effects without constitutional disturbance, the course of an
immunisation may be expected to be 'rather different from that
obtaining in an ordinary acute affection, though the underlying
processes may be of the same nature. It is difficult, for instance,
on account of the slowness of tubercular processes, to define
recovery from an attack of the disease, or to speak of an animal
recovering from the effect of an inoculation during an immunisa-
tion. It follows that little is known regarding an attenuation
of the tubercle bacillus analogous to what is an important
feature in immunisations against other organisms. It has
been thought by some that the tubercle bacilli from so-called
scrofulous glands are less virulent than those, say, from phthisis,
but apparently here sufficient attention has not been paid to the
difference of the numbers of bacilli injected in each case, and
this appears to be a very important point. Experiments have
also been brought forward which appear to show that the injec-
tion of bacilli from avian tuberculosis could protect the dog
against bacilli derived from man. But these are not yet conclusive.
Agglutinative Phenomena. — The serum of tubercular patients
has been found to exert an agglutinating action on the tubercle
bacillus. A convenient 'method is to add different amounts
of serum, commencing with, say, '1 c.c., to quantities of a
dilution of the new tuberculin (Bazillenemulsion) equivalent to
1 part of the bacterial bodies to 10,000 of diluent, and leave the
mixture for 24 hours before observing. As with other agglutina-
tive observations, it is difficult to correlate the degree of agglu-
tinating power of the serum with the degree of immunisation
possessed by the individual from which it was derived.
Antitubercular Sera. — Several attempts have been made to
treat tuberculosis with the serum of animals immunised by the
tubercle bacillus or its products. The most successful is perhaps
that of Maragliano. This author distinguishes between the
toxic materials contained in the bodies of the bacilli (which
withstand, unchanged, a temperature of 100° C.) and those
secreted into the culture fluid (which are destroyed by heat).
METHODS OF EXAMINATION 265
The substance used by him for immunising his animals consists
of three parts of the former and one of the latter. The animals
employed are the dog, the ass, the horse. The serum obtained
from these is capable of protecting healthy animals against an
otherwise fatal dose of tuberculin, but very little importance can
be attached to this result. Maragliano does not appear to have
studied the effects of this serum on tubercular animals, but it
has been tried in a great number of cases of human tuberculosis,
2 c.c. being injected subcutaneously every two days. Improve-
ment is said to have taken place in a certain proportion, especi-
ally of mild non-febrile cases.
An antitubercular serum has also been introduced by
Marmorek. This observer considers that the tubercle bacillus
cannot produce in ordinary media the toxins which it originates
when exposed to the antagonism of the bodily cells. He tries
to make good this defect by first growing it in a serum antago-
nistic to some of the phagocytic cells of the body ; for this a
leucotoxic serum is used. When the bacillus has grown in this
presumably favourable soil it is transferred to a medium con-
taining a substance which may be unfavourable : and for this
there is employed a medium containing liver extract, the
liver being an organ in which in man tubercular lesions are
comparatively rare. The bacilli being thus accustomed to an
unfavourable surrounding are used for immunising animals, the
serum of which is now suitable for the treatment of human tuber-
culosis. It is too soon to speak of th« effects of this line of
treatment.
Methods of Examination. — (1) Microscopic Examination.
Tuberculosis is one of the comparatively few diseases in which a
diagnosis can usually be definitely made by microscopic examina-
tion alone. In the case of sputum, one of the yellowish
fragments which are often present ought to be selected ; dried
films are then prepared in the usual way and stained by the
Ziehl-Neelsen method (p. 101). In the case of urine or other
fluids a deposit should first be obtained by centrifugalising a
quantity in a test-tube, or by allowing the fluids to stand in a
tall glass vessel (an ordinary burette is very convenient). Film
preparations are then made with the deposit and treated as
before. If a negative result is obtained in a suspected case,
repeated examination should be undertaken. To avoid risk of
contamination with the smegma bacillus the meatus of the
urethra should be cleansed and the urine first passed should be
rejected, or the urine may be drawn off with a sterile catheter.
As stated above it is only exceptionally that difficulty will arise
266 TUBERCULOSIS
to the experienced observer from this cause. (For points to be
attended to, vide p. 255.)
(2) Inoculation. — The guinea-pig is the most suitable
animal. If the material to be tested is a fluid it is injected
subcutaneously or into the peritoneum ; if solid or semi-solid it
is placed in a small pocket in the skin, or it may be thoroughly
broken up in sterile water or other fluid and the emulsion
injected. By this method, material in which no tubercle bacilli
can be found microscopically may sometimes be shown to be
tubercular.
(3) Cultivation. — Owing to the difficulties this is usually
quite impracticable as a means of diagnosis, and it is also un-
necessary. The best method to obtain pure cultures is to
produce tuberculosis in a guinea-pig by inoculation with tubercular
material, and then, killing the animal after four or five weeks,
to inoculate tubes of solidified blood serum, under strict aseptic
precautions, with portions of a tubercular organ, e.g. the spleen.
The portions of tissue should be fairly large, and should be well
rubbed into the broken surface of the medium.
CHAPTER X.
LEPROSY.
LEPROSY is a disease of great interest, alike in its clinical and
pathological aspects ; whilst from the bacteriological point of
view also, it presents some striking peculiarities. The invariable
association of large numbers of characteristic bacilli with all
leprous lesions is a well-established fact, and yet, so far, attempts
to cultivate the bacilli outside the body, or to produce the disease
experimentally in animals, have been attended with failure.
Leprosy, so far as is known, is a disease which is confined to the
human subject, but it has a very wide geographical distribution.
It occurs in certain parts of Europe — Norway, Russia, Greece,
etc., but is commonest in Asia, occurring in Syria, Persia, etc.
It is prevalent in Africa, being especially found along the coast,
in the Pacific Islands, in the warmer parts of North and South
America, and also to a small extent in the northern part of North
America. In all these various regions the disease presents the
same general features, and the study of its pathological and
bacteriological characters, wherever such has been carried on, has
yielded similar results.
Pathological Changes. — Leprosy is characteristically a chronic
disease, in which there is a great amount of tissue change, with
comparatively little necessary impairment of the general health.
In other w^ords, the local effects of the bacilli are well marked,
often extreme, whilst the toxic phenomena are proportionately at
a minimum.
There are two chief forms of leprosy. The one, usually called
the tubercular form — lepra tuberosa or tuberculosa — is character-
ised by the growth of granulation tissue in a nodular form or as a
diffuse infiltration in the skin, in mucous membranes, etc., great
disfigurement often resulting. In the other form, the anaesthetic,
— maculo- anaesthetic of Hansen and Looft — the outstanding
267
268
LEPROSY
changes are in the nerves, with consequent anaesthesia, paralysis
of muscles, and trophic disturbances.
In the tubercular form the disease usually starts with the
appearance of erythematous patches attended by a small amount
of fever, and these are followed by the development of small
nodular thickenings in the skin, especially of the face, of the
backs of hands and feet, and of the extensor aspects of arms and
££
m
FIG. 91. — Sections through leprous skin, showing the masses of cellular
granulation tissue in the cutis ; the dark points are clumps of bacilli deeply
stained.
Paraffin section ; Ziehl-Neelsen stain, x 80.
legs. These nodules enlarge and produce great distortion of the
surface, so that, in the case of the face, an appearance is produced
which has been described as " leonine." The thickenings occur
chiefly in the cutis (Fig. 91), to a less extent in the subcutaneous
tissue. The epithelium often becomes stretched over them,
and an oozing surface becomes developed, or actual ulceration
may occur. The cornea and other parts of the eye, the mucous
membrane of the mouth, larynx, and pharynx, may be the seat
of similar nodular growths. Internal organs, especially the
spleen, liver, and testicles, may become secondarily affected. In
BACILLUS OF LEPROSY 269
all situations the change is of the same nature, — a chronic
inflammatory condition attended by abundant formation of
granulation tissue, nodular or diffuse in its arrangement. In this
tissue a large proportion of the cells are of rounded or oval shape,
like hyaline leucocytes ; a number of these may be of compara-
tively large size, and may show vacuolation of their protoplasm
and a vesicular type of nucleus. These are often known as
" lepra-cells." Amongst the cellular elements there is a varying
amount of stroma, which in the earlier lesions is scanty and
delicate, but in the older lesions may be very dense. Periarteritis
is a common change, and very frequently the superficial nerves
become involved in the nodules and undergo atrophy. The
tissue in the leprous lesions is comparatively vascular, at least
when young, and, unlike tubercular lesions, never shows caseation.
Some of the lepra cells may contain several nuclei, but we do
not meet with cells resembling in their appearance tubercle
giant-cells, nor does an arrangement like that in tubercle follicles
occur.
In the ancesthetic form the lesion of the nerves is the out-
standing feature. These are the seat of diffuse infiltrations
which lead to the destruction of the nerve fibres. In the earlier
stages, in which the chief symptoms are pains along the nerves,
there occur patches on the skin, often of considerable size, the
margins of which show a somewhat livid congestion. Later,
these patches become pale in the central parts, and the periphery
becomes pigmented. There then follow remarkable series of
trophic disturbances in which the skin, muscles, and bones are
especially involved. The skin often becomes atrophied, parch-
ment-like, and anaesthetic ; frequently pemphigoid bullee or other
skin eruptions occur. Partly owing to injury to which the feet
and arms are liable from their anaesthetic condition, and partly
owing to trophic disturbances, necrosis and separation of parts
are liable to occur. In this way great distortion results. The
lesions in the nerves are of the same nature as those described
above, that is, they are the result of a chronic inflammatory
process, but the granulation tissue is scantier, and has a greater
tendency to undergo cicatricial contraction. This is to be
associated with the fact that the bacilli are present in fewer
numbers.
Bacillus of Leprosy. — This bacillus was first observed in
leprous tissues by Hansen in 1871, and was the subject of several
communications by him in 1874 and later. Further researches,
first by Neisser in 1879, and afterwards by observers -in
various parts of the world, agreed in their main results, and
270 LEPROSY
confirmed the accuracy of Hansen's observations. The bacilli as
seen in scrapings of ulcerated leprous nodules, or in sections,
have the following characters. They are thin rods of practically
the same size as tubercle bacilli, which they also resemble both
in appearance and in staining reaction. They are straight or
slightly curved, and usually occur singly, or two may be attached
end to end ; but they do not form chains. When stained they
fe-
*
•
***••**
* ^
4 -.
FIG. 92. — Superficial part of leprous skin ; the cells of the granulation tissue
appear as dark patches, owing to the deeply-stained bacilli in their interior.
In the upper part a process of epithelium is seen.
Paraffin section ; stained with carbol-fuchsin and Bismarck-brown, x 500.
may have a uniform appearance, or the protoplasm may be
fragmented, so that they appear like short rows of cocci. They
often appear tapered at one or both extremities ; occasionally
there is slight club-like swelling. Degenerated and partially
broken down forms are also seen. They take up the basic
aniline stains more readily than tubercle bacilli, but in order
to stain them deeply a powerful stain, such as carbol-fuchsin,
is necessary. When stained, they strongly resist decolorising,
though they are more easily decolorised than tubercle bacilli.
The best method is to stain with carbol-fuchsin as for tubercle
POSITION OF THE BACILLI
271
bacilli, but to use a weaker solution of sulphuric acid, say 5 per
cent, in decolorising ; in the case of films and thin sections,
decolorising with such a solution for fifteen seconds is usually
sufficient. Thereafter the tissues are coloured by a contrast
stain, such as a watery solution of methylene-blue (vide p. 101).
The bacilli are also readily stained by Gram's method. Regarding
the presence of spores practically nothing is known, though some
FIG. 93. — High -power view of portion of leprous nodule showing the
arrangement of the bacilli within the cells of the granulation tissue.
Paraffin section ; stained with carbol-fuchsin and methylene-blue. x 1100.
of the unstained or stained points may be of this nature. We
have, however, no means of testing their powers of resistance.
Leprosy bacilli are non-motile.
Position of the Bacilli. — They occur in enormous numbers
in the leprous lesions, especially in the tubercular form. In fact,
so numerous are they that the granulation tissue in sections,
properly stained as above, presents quite a red colour under a low
power of the microscope. The bacilli occur for the most part
within the protoplasm of the round cells of the granulation tissue,
and are often so numerous that the structure of the cells is quite
272 LEPROSY
obscured (Fig. 92). They are often arranged in bundles which
contain several bacilli lying parallel to one another, though the
bundles lie in various directions (Fig. 93). The appearance thus
presented by the cells filled with bacilli is very characteristic.
Bacilli are also found free in the lymphatic spaces, but the greater
number are undoubtedly contained within the cells. They are
also found in spindle-shaped connective-tissue cells, in endothelial
cells, and in the walls of blood vessels. They are for the most
part confined to the connective tissue, but a few may be
seen in the hair follicles and glands of the skin. Occasionally
one or two may be found in the surface epithelium, where they
probably have been carried by leucocytes, but this position is, on
the whole, exceptional. They also occur in large numbers in the
lymphatic glands associated with the affected parts. In the
internal organs — liver, spleen, etc., when leprous lesions are
present, the bacilli are also found though in relatively smaller
numbers. In the nerves in the anaesthetic form they are com-
paratively few, and in the sclerosed parts it may be impossible to
find any. There are few also in the skin patches referred to
above as occurring in this form of the disease.
Their spread is chiefly by the lymphatics, though distribution
by the blood stream also occurs. They have been said to be
found in the blood during the presence of fever and the eruption
of fresh nodules, and they have also been observed in the blood
vessels post mortem, chiefly contained within leucocytes. Recent
observations (e.g. those of Doutrelepont and Wolters) show that
the bacilli may be more widely spread throughout the body than
was formerly supposed. A few may be detected in some cases
in various organs which show no structural change, especially in
their capillaries. The brain arid spinal cord are almost exempt,
but in some cases bacilli have been found • even within nerve
cells.
Relations to the Disease. — Attempts to cultivate the leprosy
bacilli outside the body have so far been unsuccessful. From
time to time announcements of successful cultivations have been
made, but one after another has proved to be erroneous. A
similar statement may be made with regard to experiments on
animals. If a piece of leprous tissue be introduced subcutaneously
in an animal, such as the rabbit, a certain amount of induration
may take place around it, and the bacilli may be found unchanged
in appearance weeks or even months afterwards, but no multi-
plication of the organisms occurs. The only exception to this
statement is afforded by the experiments of Melcher andOrthmann,
who inoculated the anterior chamber of the eye of rabbits with
RELATIONS TO THE DISEASE 273
leprous material, the inoculation being followed by an extensive
growth of nodules in the lungs and internal organs, which they
affirmed contained leprosy bacilli. It has been questioned,
however, by several authorities whether the organisms in the
nodules were really leprosy bacilli, and up to the present wre
cannot say that there is any satisfactory proof that the disease can
be transmitted to any of the lower animals. Diphtheroid bacilli
of more than one variety have been cultivated from the blood
and tissues of leprous patients by Babes and others. Their
presence would appear to be by no means infrequent, but it is
not possible to say at present what their significance is.
It is interesting to note that a disease occurs under natural
conditions in rats which presents many points of close similarity
to leprosy. It has been observed in Russia, Germany, and
England, and an excellent description has recently been given by
Dean. In this affection there are lesions in the skin which
resemble those in leprosy, and the cells contain enormous
numbers of an acid -fast bacillus. The disease can be trans-
mitted to rats by inoculation with the tissue juices containing
the bacilli, but riot to animals of other species. All attempts to
cultivate the characteristic organism outside the body have
failed, but Dean has obtained a diphtheroid bacillus— a result of
interest in relation to what has been found in leprosy. Whether
this disease has any relation to leprosy in the human subject is
very doubtful, but the facts which have been ascertained may
prove of high importance in connection with the pathology of
the latter disease.
It would also appear that the disease is not readily inoculable
in the human subject. In a well-known case described by Arning,
a criminal in the Sandwich Islands was inoculated in several parts
of the body with leprosy tissue. Two or three years later, well-
marked tubercular leprosy appeared and led to a fatal result.
This experiment, however, is open to the objection that the
individual before inoculation had been exposed to infection in a
natural way, having been frequently in contact with lepers. In
other cases, inoculation experiments on healthy subjects and
inoculations in other parts of leprous individuals have given
negative results. It has been supposed by some that the failure
to obtain cultures and to reproduce the disease experimentally
may be partly due to the bacilli in the tissues being dead. That
many of the leprous bacilli are in a dead condition is quite
possible, in view of the long period during which dead tubercle
bacilli introduced into the tissues of animals retain their form and
staining reaction. There is also the fact that from time to time
18
274 LEPROSY
in leprous subjects there occur febrile attacks, which are followed
by a fresh outbreak of nodules, and it would appear that
especially at these times multiplication of the bacilli takes place
more actively.
The facts stated with regard to cultivation and inoculation
experiments go to distinguish the leprosy bacillus all the more
strongly from other organisms. Some have supposed that
leprosy is a form of tubercle, or tubercle modified in some way,
but for this there appears to us to be no evidence. Both from
the pathological and from the bacteriological point of view the
diseases are distinct. It should also be mentioned that tubercle
is a not uncommon complication in leprous subjects, in which
case it presents the ordinary characters.
The mode by which leprosy is transmitted has been the
subject of great controversy, and is one on which authorities still
hold opposite opinions. Some consider that it is a hereditary
disease, or at least that it is transmitted from a parent to the
offspring ; others again that it is transmitted by direct contact.
There appears to be no doubt, however, that on the one hand
leprous subjects may bear children free from leprosy, and that on
the other hand, healthy individuals entering a leprous district may
contract the disease, though this rarely occurs. Of the latter
occurrence there is the well-known instance of Father Damien,
who contracted leprosy after going to the Sandwich Islands. In
view of all the facts there can be little doubt that leprosy in
certain conditions may be transmitted by direct contact, though
its contagiousness is not of a high order.
Methods of Diagnosis. — Film preparations should be made
with the discharge from any ulcerated nodule which may be
present, or from the scraping of a portion of excised tissue, and
should be stained as above described. The presence of large
numbers of bacilli situated within the cells and giving the staining
reaction of leprosy bacilli, is conclusive. It is more satisfactory,
however, to make microscopic sections through a portion of the
excised tissue, when the structure of the nodule and the arrange-
ment of the bacilli can be readily studied. The points of
difference between leprosy and tubercle have already been stated,
and in most cases there is really no difficulty in distinguishing
the two conditions.
CHAPTER XL
GLANDERS AND EHINOSCLEROMA.
GLANDERS.
THE bacillus of glanders (bacillus mallei ; Fr., bacille de la
morve ; Ger., Rotzbacillus] was discovered by Loffler and Schutz,
the announcement of this discovery being made towards the end
of 1882. They not only obtained pure cultures of this organism
from the tissues in the disease, but by experiments on horses
and other animals conclusively established its causal relationship.
These have been fully confirmed. The same organism has also
been cultivated from the disease in the human subject, first by
Weichselbaum in 1885, who obtained it from the pustules in a
case of acute glanders in a woman, and by inoculation of animals
obtained results similar to those of Loffler and Schutz.
Within recent years a substance, mallein, has been obtained
from the cultures of the glanders bacillus by a method similar
to that by which tuberculin was prepared, and has been found
to produce corresponding effects in animals suffering from
glanders to those produced by tuberculin in tuberculous animals.
The Natural Disease. — Glanders chiefly affects the equine
species — horses, mules, and asses. Horned cattle, on the other
hand, are quite immune, whilst goats and sheep occupy an inter-
mediate position, the former being rather more susceptible and
occasionally suffering from the natural disease. It also occurs
in some of the carnivora — cats, lions, and tigers in menageries,
which animals are infected from the carcases of animals affected
with the disease. Many of the small rodents are highly
susceptible to inoculation (vide infra).
Glanders is also found in man as the result of direct inocula-
tion on some wound of the skin or other part by means of the
discharges or diseased tissues of an animal affected, and hence is
commonest amongst grooms and others whose work brings them
275
276 GLANDERS
into contact with horses ; even amongst them it is a comparatively
rare disease.
In horses the lesions are of two types, to which the names "glanders "
proper and "farcy" have been given, though both may exist together.
In glanders proper the septum nasi and adjacent parts are chiefly affected,
there occurring in the mucous membrane nodules which are at first firm
and of somewhat translucent grey appearance. The growth of these is
attended usually by inflammatory swelling and profuse catarrhal dis-
charge. Afterwards the nodules soften in the centre, break down, and
give rise to irregular ulcerations. Similar lesions, though in less degree,
may be found in the respiratory passages. Associated with these lesions
there is usually implication of the lymphatic glands in the neck, media-
stinum, etc. ; and there may be in the lungs, spleen, liver, etc., nodules
of the size of a pea or larger, of greyish or yellow tint, firm or somewhat
softened in the centre, and often surrounded by a congested zone. The
term "farcy" is applied to the affection of the superficial lymphatic
vessels and glands, which is specially seen where infection takes place
through an abrasion of the skin, such as is often produced by the rubbing
of the harness. The lymphatic vessels become irregularly thickened, so
as to appear like knotted cords, and the associated lymphatic glands
become enlarged and firm, though suppurative softening usually follows,
and there may be ulceration. These thickenings are often spoken of as
" farcy buds " and " farcy pipes." In farcy, also, secondary nodules may
occur in internal organs and the nasal mucous membrane. The disease
is often present in a "latent form," and its presence can only be detected
by the mallein test (vide infra). In the ass the disease runs a more
acute course than in the horse.
In man the disease is met with in two forms, an acute and a
chronic ; though intermediate forms also occur, and chronic cases
may take on the characters of the acute disease. The site of
inoculation is usually on the hand or arm, by means of some
scratch or abrasion, or possibly along a hair follicle, sometimes on
the face, and occasionally on the mucous membrane of the mouth,
nose, or eye. In the acute form there appears at the site of
inoculation inflammatory swelling, attended usually with spread-
ing redness, and the lymphatics in relation to the part also become
inflamed, the appearances being those of a " poisoned wound."
These local changes are soon followed by marked constitutional
disturbance, and by an eruption on the surface of the body, at
first papular and afterwards pustular, and later there may form in
the subcutaneous tissue and muscles larger masses which soften
and suppurate, the pus being often mixed with blood ; suppuration
may occur also in the joints. In some cases the nasal mucous
membrane may be secondarily infected, and thence inflammatory
swelling may spread to the tissues of the face ; in others it
remains free. The patient usually dies in two or three weeks,
sometimes sooner, with the symptoms of rapid pyaemia. In
THE GLANDERS BACILLUS
277
addition to the lesions mentioned there may be foci, usually
suppurative, in the lungs (attended of ten. with pneumonic con-
solidation),, in the spleen, liver, bone-marrow, salivary glands, etc.
In the chronic form the local lesion results in the formation of an
irregular ulcer with thickened margins and sanious, often foul,
discharge. The ulceration spreads deeply as well as superficially,
and the thickened lymphatics also have a great tendency to
ulcerate, though the lymphatic system is not so prominently
affected as in the horse. Deposits may form in the subcutaneous
tissue and muscles, and the mucous membrane may become
affected. The disease may
run a very chronic course,
lasting for months, and
recovery may occur,
though, on the other hand,
the disease may take on
a more acute character
and rapidly become fatal.
The Glanders Bacil-
lus. — Microscopical
Characters. — The glan-
ders bacilli are minute
rods, straight or slightly
curved, with rounded
ends, and about the same
length as tubercle bacilli,
but distinctly thicker. Fia 94._Glanders bacim among8t broken-
(Flg. 94 ). They show, down cells. Film preparation from a
however, considerable glanders nodule in a guinea-pig,
variations in size and in staiued with weak carbol-fuchsin. x 1000.
appearance, and their pro-
toplasm is often broken up into a number of deeply-stained
portions with unstained intervals between. These characters are
seen both in the tissues and in cultures, but, as in the case of
many organisms, irregularities in form and size are more pro-
nounced in cultures (Fig 95) ; short filamentous forms 8 to 12 ju in
length are sometimes met with, but these are on the whole rare.
The organism is non-motile.
In the tissues the bacilli usually occur irregularly scattered
amongst the cellular elements ; a few may be contained within
leucocytes and connective-tissue corpuscles, but the position of
most is extracellular. They are most abundant in the acute lesions,
in which they may be found in considerable numbers ; but in the
chronic nodules, especially when softening has taken place, they
278
GLANDERS
are few in number, and it may be impossible to find any in
sections. They have less powers of persistence, and disappear in
the tissues much more quickly than tubercle bacilli.
There has been dispute as to whether or not they contain
spores. Some consider certain of the unstained portions to be
of that nature, and it has
been claimed that these
can be stained by the
method for staining spores
(Rosen thai). But it is
very doubtful that such is
the case ; the appearances
correspond rather with
mere breaks in the proto-
plasm, such as are met
with in many other bacilli
which do not contain
spores, and the compara-
tively low powers of resist-
ance of glanders bacilli
containing these so-called
from 0 a spores, is strongly against
FIG. 95.— Glanders bacilli,
culture on glycerin agar.
carbol-fuchsinand partially decolorised to
show segmentation of protoplasm, x 1000.
Stained with tneir being of that nature.
The power of resistance is
after all the important
practical point.
Staining. — The glanders bacillus differs widely froin the
tubercle bacillus in its staining reactions. It stains with simple
watery solutions of the basic stains, but somewhat faintly (better
when an alkali or a mordant, such as carbolic acid, is added),
and even when deeply stained it readily loses the colour when a
decolorising agent such as alcohol is applied. We have obtained
the best results by carbol-thionin-blue (p. 98), and we prefer to
dehydrate by the aniline-oil method. In film preparations of
fresh glanders nodules the bacilli can be readily found by staining
with any of the ordinary combinations, e.g. carbol-thionin-blue
or weak carbol-fuchsin. By using a stain of suitable strength
no decolorising agent is necessary, the film being simply washed
in water, dried and mounted. M'Fadyean recommends that
after sections have been stained in Loffler's methylene blue and
slightly decolorised in weak acetic acid, they should be treated
for fifteen minutes with a saturated solution of tannic acid ;
thereafter they are washed thoroughly in water, and as a contrast
stain a 1 per cent solution of acid fuchsin may be applied for
CULTIVATION OF GLANDERS BACILLUS 279
half a minute ; they are then dehydrated, cleared, and mounted.
Gram's method is quite inapplicable, the glanders bacilli rapidly
losing the stain in the process.
Cultivation. — (For the methods of separation vide infra.)
The glanders bacillus grows readily on most of the ordinary
media, but a somewhat high temperature is necessary, growth
taking place most rapidly at 35° to 37° C. Though a certain
amount of growth occurs down to 21° C., a temperature above
25° C. is always desirable.
On agar and glycerin agar in stroke cultures growth appears
along the line as a uniform streak of greyish-white colour and
somewhat transparent appearance, with moist-looking surface,
and when touched with a needle is found to be of rather slimy
consistence. Later it spreads laterally for some distance, and
the layer becomes of slightly brownish tint. On serum the
growth is somewhat similar but more transparent, the separate
colonies being in the form of round and almost clear drops. In
sub-cultures on these media at the body temperature growth is
visible within twenty-four hours, but when fresh cultures are
made from the tissues it may not be visible till the second day.
Serum or potato, however, is much more suitable for cultivating
from the tissues than the agar media ; on the latter it is some-
times difficult to obtain growth.
In broth, growth forms at first a uniform turbidity, but soon
settles to the bottom, and after a few days forms a pretty thick
fiocculent deposit of slimy and somewhat tenacious consistence.
On potato at 30° to 37° C. the glanders bacillus flourishes well
and produces a characteristic appearance ; incubation at a high
temperature, however, being necessary. Growth proceeds rapidly,
and on the third day has usually formed a transparent layer of
slightly yellowish tint, like clear honey in appearance. On sub-
sequent days, the growth still extends and becomes darker in colour
and more opaque, till about the eighth day it has a reddish-brown
or chocolate tint, while the potato at the margin of the growth
often shows a greenish-yellow staining. The characters of the
growth on potato along with the microscopical appearances are
quite sufficient to distinguish the glanders bacillus from every
other known organism (sometimes the cholera organism and the
b. pyocyaneus produce a somewhat similar appearance, but they
can be readily distinguished by their other characters). Potato
is also a suitable medium for starting cultures from the tissues ;
in this case minute transparent colonies become visible on the
third day and afterwards present the appearances just described.
Powers of Resistance. — The glanders bacillus is not killed at
280 GLANDERS
once by drying, but usually loses its vitality after fourteen days
in the dry condition, though sometimes it lives longer. It is not
quickly destroyed by putrefaction, having been found to be still
active after remaining two or three weeks in putrefying fluids.
In cultures the bacilli retain their vitality for three or four
months, if, after growth has taken place, they are kept at the
temperature of the room; on the other hand, they are often
found to be dead at the end of two weeks when kept constantly
at the body temperature. They have comparatively feeble
resistance to heat and antiseptics. Loftier found that they were
killed in ten minutes in a fluid kept at 55° C., and in from two
to three minutes by a 5 per cent solution of carbolic acid.
Boiling water and the ordinarily used antiseptics are very
rapid and efficient disinfectants.
We may summarise the characters of the glanders bacillus by
saying that in its morphological characters it resembles some-
what the tubercle bacillus, but is thicker, and differs widely
from it in its staining reactions. For its cultivation the higher
temperatures are necessary, and the growth on potato presents
most characteristic features.
Experimental Inoculation. — In horses subcutaneous injection
of the glanders bacillus in pure culture reproduces all the
important features of the disease. This fact was established at
a comparatively early date by Lofner and Schutz, who, after one
doubtful experiment, successfully inoculated two horses in this
way, the cultures used having been grown for several generations
outside the body. In a few days swellings formed at the sites
of inoculation, and later broke down into unhealthy -looking
ulcers. One of the animals died ; after a few weeks the other,
showing symptoms of caohexia, was killed. In both animals, in
addition to ulcerations on the surface with involvement of the
lymphatics, there were found, post mortem, nodules in the lungs,
softened deposits in the muscles, and also affection of the nasal
mucous membrane, — nodules, and irregular ulcerations. The
ass is even more susceptible than the horse, the disease in the
former running a more rapid course, but with similar lesions.
The ass can be readily infected by simple scarification and
inoculation with glanders secretion, etc. (Nocard).
Of small animals, field-mice and guinea-pigs are the most
susceptible. Strangely enough, house -mice and white mice
enjoy an almost complete immunity. In field-mice subcutaneous
inoculation is followed by a very rapid disease, usually leading
to death within eight days, the organisms becoming generalised
and producing numerous minute nodules, especially in the spleen,
ACTION ON THE TISSUES 281
lungs, and liver. In the guinea-pig the disease is less acute,
though secondary nodules in internal organs are usually present
in considerable numbers. At the site of inoculation an inflam-
matory swelling forms, which soon softens and breaks down,
leading to the formation of an irregular crateriform ulcer with
indurated margins. The lymphatic vessels become infiltrated,
and the corresponding lymphatic glands become enlarged to the
size of peas or small nuts, softened, and semi-purulent. The
animal sometimes dies in two or three weeks, sometimes not for
a longer period. Secondary nodules, in varying numbers in
different cases, may be present in the spleen, lungs, bones, nasal
mucous membrane, testicles, ovaries, etc. ; in some cases a few
nodules are found in the spleen alone. Intraperitoneal injection
in the male guinea-pig is followed, as pointed out by Straus, by
a very rapid and semi-purulent affection of the tunica vaginalis,
shown during life by great swelling and redness of the testicles,
which changes may be noticeable in two or three days. By this
method there occur also numerous small nodules on the surface
of the peritoneum. Eabbits are less susceptible than guinea-
pigs, and the effect of subcutaneous inoculation is somewhat
uncertain. Accidental inoculation of the human subject with
pure cultures of the bacillus has in more than one instance been
followed by the acute form of the disease and a fatal result.
Mayer has found that when the glanders bacillus is injected along
with melted butter into the peritoneum of a guinea-pig, it shows
filamentous, branching, and club-shaped forms; in other words, it
presents the characters of a streptothrix. Lubarsch, on the other hand,
in a comparative study of the results of inoculation with acid-fast and
other bacilli, found none of the above characters in the case of the
glanders bacillus (cf. Tubercle).
Action on the Tissues. — From the above facts it will be seen
tl;at in many respects glanders presents an analogy to tubercle as
regards the general characters of the lesions and the mode of
spread. When the tissue changes in the two diseases are
compared, certain differences are found. The glanders bacillus
causes a more rapid and more marked inflammatory reaction.
There is more leucocytic infiltration and less proliferative change
which might lead to the formation of epithelioid cells. Thus
the centre of an early glanders nodule shows a dense aggregation
of leucocytes, most of which are polymorpho-nuclear, whilst in
the central parts many show fragmentation of nuclei with the
formation of a deeply staining granular detritus. And further,
the inflammatory change may be followed by suppurative
softening of the tissue, especially in certain situations, such as
282 GLANDERS
the subcutaneous tissue and lymphatic glands. The nodules,
therefore, in glanders, as Baumgarten puts it, occupy an
intermediate position between miliary abscesses and tubercles.
The diffuse coagulative necrosis and caseation which are so
common in tubercle do not occur to the same degree in glanders,
and typical giant cells are not formed. The nodules in the lungs
show leucocytic infiltration and thickening of the alveolar walls,
whilst the vesicles are filled with catarrhal cells ; there may also
be fibrinous exudation, whilst at the periphery of the nodules con-
nective-tissue growth is present in proportion to their age. The
tendency to spread by the lymphatics is always a well-marked
feature, and when the bacilli gain entrance to the blood-stream,
they soon settle in the various tissues and organs. Accordingly,
even in acute cases it is usually quite impossible to detect the
bacilli in the circulating blood, though sometimes they have been
found. It is an interesting fact, shown by observations of the
disease both in the human subject and in the horse, as well as
by experiments on guinea-pigs, that the mucous membrane of the
nose may become infected by means of the blood-stream — another
example of the tendency of organisms to settle in special sites.
Mode of Spread. — Glanders usually spreads from a diseased
animal by direct contagion with the discharge from the nose or
from the sores, etc. So far as infection of the human subject
goes, no other mode is known. There is no evidence that the
disease is produced in man by inhalation of the bacilli in the
dried condition. Some authorities consider that pulmonary
glanders may be produced in this way in the horse, whilst others
maintain that in all cases there is first a lesion of the nasal
mucous membrane or of the skin surface, and that the lung is
affected secondarily. Babes, however, found that the disease
could be readily produced in susceptible animals by exposing
them to an atmosphere in which cultures of the bacillus had
been pulverised. He also found that inunction of the skin
with vaseline containing the bacilli might produce the disease,
the bacilli in this case entering along the hair follicles.
Agglutination of Glanders Bacilli.— Shortly after the discovery of
agglutination in typhoid fever, M'Fadyean showed that the serum of
glandered horses possessed the power of agglutinating glanders bacilli.
His later observations show that in the great majority of cases of glanders
a 1 in 50 dilution of the serum produces marked agglutination in a few
minutes, whilst in the great majority of non-glandered animals no effect
is produced under these conditions. The test performed in the ordinary
way is, however, not absolutely reliable, as exceptions occasionally
occur iu both directions, i.e. negative results by glandered anmials and
positive results by non-glandered animals. He finds that a more delicate
METHODS OF EXAMINATION 283
and reliable method is to grow the bacillus in bouillon containing a small
proportion of the serum to be tested. In this way he has obtained a
distinct sedimenting reaction with a serum which did not agglutinate at
all distinctly in the ordinary method. Further observations are still
required to determine the precise value of the test.
Mallein and its Preparation. — Mallein is obtained from cultures of the
glanders bacillus grown for a suitable length of time, and, like tuber-
culin, is really a mixture comprising (1) substances in the bodies of the
bacilli and (2) their soluble products, not destroyed by heat, along with
substances derived from the medium of growth. It was at first obtained
from cultures on solid media by extracting with glycerin or water, but is
now usually prepared from cultures in glycerin bouillon. Such a culture,
after being allowed to grow for three or four weeks, is sterilised by heat
either in the autoclave at 115° C. or by steaming at 100° C. on successive
days. It is then filtered through a Chamberland filter. The filtrate
constitutes fluid mallein. Usually a little carbolic acid ( '5 per cent) is
added to prevent it from decomposing. Of such mallein 1 c.c. is usually
the dose for a horse (M'Fadyean). Foth has prepared a dry form of
mallein by throwing the filtrate of a broth culture, evaporated to one-
tenth of its bulk, into twenty-five or thirty times its volume of alcohol.
A white precipitate is formed, which is dried over calcium chloride and
then under an air-pump. A dose of this dry mallein is '05 to '07 grm.
The use of Mallein as a means of Diagnosis. — In using mallein for the
diagnosis of glanders, the temperature of the animal ought to be observed
for some hours beforehand, and, after subcutaneous injection of a suitable
dose, it is taken at definite intervals, — according to M'Fadyean at the
sixth, tenth, fourteenth, and eighteenth hours afterwards, and on the
next day. Here both the local reaction and the temperature are of
importance. In a glandered animal, at the site of inoculation there is a
somewhat painful local swelling, which reaches a diameter of five inches
at least, the maximum size not being attained until twenty-four hours
afterwards. The temperature rises 1 '5° to 2° C. , or more, the maximum
generally occurring in from eight to sixteen hours. If the temperature
never rises as much as 1 '5°, the reaction is considered doubtful. In the
negative reaction given by an animal free from glanders, the rise of
temperature does not usually exceed 1°, the local swelling reaches the
diameter of three inches at most, and has much diminished at the end
of twenty-four hours. In the case of dry mallein, local reaction is less
marked. Veterinary authorities are practically unanimous as to the
great value of the mallein test as a means of diagnosis.
Methods of Examination. — Microscopic examination in a
case of suspected glanders will at most reveal the presence of
bacilli corresponding in their characters to the glanders bacillus.
An absolute diagnosis cannot be made by this method. Cultures
may be obtained by making successive strokes on blood serum or
on glycerin agar (preferably the former), and incubating at 37° C.
The colonies of the glanders bacillus do not appear till two days
after. This method often fails unless a considerable number of
the glanders bacilli are present. Another method is to dilute the
secretion or pus with sterile water, to varying degrees, and then
to smear the surface of potato with the mixture, the potatoes
284 GLANDERS
being incubated at the above temperature. The colonies on
potatoes may not appear till the third day. The most certain
method, however, is by inoculation of a guinea-pig, either by
subcutaneous or intraperitoneal injection. By the latter method,
as above described, lesions are much more rapidly produced, and
are more characteristic. If, however, there have been other
organisms present, the animal may die of a septic peritonitis,
though even in such a case the glanders bacilli will be found to
be more numerous in the tunica vaginalis, and may be cultivated
from this situation. It is extremely doubtful whether the
application of mallein to diagnosis of the disease in the human
subject is justifiable. There is a certain risk, that it may lead
to the lesions assuming a more acute character ; moreover,
culture and inoculation tests are generally available. In the
case of horses, etc., a diagnosis will, however, be much more
easily and rapidly effected by means of mallein.
RHINOSCLEROMA.
This disease is considered here as, from the anatomical
changes, it also belongs to the group of infective granulomata.
It is characterised by the occurrence of chronic nodular
thickenings in the skin or mucous membrane of the nose, or
in the mucous membrane of the pharynx, larynx, or upper part
of the trachea. The nodules are of considerable size, sometimes
as large as a pea ; in the earlier stages they are comparatively
smooth on the surface, but later they become shrunken and the
centre is often retracted. - The disease is scarcely ever met with
in this country, but is of not very uncommon occurrence on the
Continent, especially in Austria and Poland. In the granulation
tissue of the nodules there are to be found numerous round and
rather large cells, which have peculiar characters and are often
known as the cells of Mikulicz. Their protoplasm contains a
collection of somewhat gelatinous material which may fill the
cell and push the nucleus to the side. Within these cells there
is present a characteristic bacillus, occurring in little clumps or
masses chiefly in the gelatinous material. A few bacilli also lie
free in the lymphatic spaces around. This organism was first
observed by Frisch, and is now known as the bacillus of
rhinoscleroma. The bacilli have the form of short oval rods,
which, when lying separately, can be seen to possess a distinct
capsule, and which in all their microscopical characters correspond
closely with Friedlander's pneumobacillus. They are usually
present in the lesions in a state of purity. It was at first stated
RHINOSCLEROMA 285
that they could be stained by Gram's method, but more recent
observations show that like Friedlander's organism they lose the
stain.
From the affected tissues this bacillus can be easily cultivated
by the ordinary methods. In the characters of its growth in
the various culture media it presents a close similarity to that
of the pneumobacillus, as it also does in its fermentative action
in milk and sugar-containing fluids. The nail-like appearance
of the growth or gelatin is said to be less distinct, and the
growth on potatoes is more transparent and may show small
bubbles of gas ; but it is doubtful whether any distinct line of
difference can be drawn between the two organisms so far as
their microscopical and cultural characters are concerned.
The evidence that the organisms described are the cause of
this disease consists in their constant presence and their special
relation to the affected tissues, as already described. From
these facts alone it would appear probable that they are the
active agents in the production of the lesions. Experimental
inoculation has thrown little light on the subject, though one
observer has described the production of nodules on the
conjunctivas of guinea-pigs. The relation of the rhinoscleroma
organism to that of Friedlander is, however, still a matter of
doubt, and the matter has been further complicated by the fact
that a bacillus possessing closely similar characters has been
found to be very frequently present in ozcena, and is often
known as the bacillus ozwnce. The last-mentioned organism is
said to have more active fermentative powers. From what has
been stated it will be seen that a number of organisms closely
allied in their morphological characters, have been found in the
nasal cavity in healthy or diseased conditions. There is no
doubt that rhinoscleroma is a specific disease with well-marked
characters and it is quite possible that one member of this group
of organisms may be the causal agent, though indistinguishable
from others by culture tests. There is, however, a tendency on
the part of recent investigators to consider the "bacillus of
rhinoscleroma " to be identical with the pneumobacillus and its
presence in the affected tissues to represent merely a secondary
invasion. The subject is one on which more light is still
required.
CHAPTER XII.
ACTINOMYCOSIS AND ALLIED DISEASES.
ACTINOMYCOSIS is the most important example of a group of
diseases produced by streptothrix organisms. It is related, by
the characters of the pathological changes, to the diseases which
have been described. The disease affects man in common with
certain of the domestic animals, though it is more frequent in
the latter, especially in oxen, swine, and horses. The parasite
was first discovered in the ox by Bol linger, and described by
him in 1877, the name actinomyces or ray fungus being from its
appearance applied to it by the botanist Harz. In 1878 Israel
described the parasite in the human subject, and in the following
year Ponfick identified it as being the same as that found in the
ox. Since that time a large number of cases have been observed
in the human subject, the result of investigation being to show
that it affects man much more frequently than was formerly
supposed.
It is, however, to be noted that the term " actinomyces," as
originally used, does not' represent one parasite but a number of
closely allied species, as cultures obtained from various sources
have presented considerable differences ; and, further, it is noted
that other distinct species of streptothrix have been cultivated
from isolated cases of disease in the human subject where the
lesions resembled more or less closely those of actinomycosis.
In one or two instances the organism has been found to be
"acid-fast," and there is no doubt that the actinomyces group is
closely related through intermediate forms with the tubercle
group (vide p. 239).
Naked-eye Characters of the Parasite. — The actinomyces
grows in the tissues in the form of little round masses or colonies,
which, when fully developed, are easily visible to the naked eye,
the largest being about the size of a small pin's head, whilst all
sizes below this may be found. When suppuration is present,
286
CHARACTERS OF THE ACTINOMYCES 287
they lie free in the pus ; when there is no suppuration, they are
embedded in the granulation tissue, but are usually surrounded
by a zone of softer issue. They may be transparent or jelly-
like, or they may be opaque and of various colours — white,
yellow, greenish, or almost black. The appearance depends
upon their age and also upon their structure, the younger colonies
being more or less transparent, the older ones being generally
opaque. Their colour is modified by the presence of pigment
and by degenerative change, which is usually accompanied by a
yellowish coloration. They are generally of soft, sometimes
tallow-like, consistence, though sometimes in the ox they are
gritty, owing to the presence of calcareous deposit. They may
be readily found in the pus by spreading it out in a thin layer
on a glass slide and holding it up to the light, They are some-
times described as being always of a distinctly yellow colour,
but this is only occasionally the case ; in fact, in the human
subject they occur much more frequently as small specks of
semi-translucent appearance, and of greenish-grey tint.
Microscopical Characters. — The parasite, which is now
generally regarded as belonging to the streptothrix group of the
higher bacteria (p. 14), presents pleomorphous characters. In
the colonies, as they grow in the tissues, three morphological
elements may be described, namely, filaments, coccus-like bodies,
and clubs.
1. The filaments are comparatively thin, measuring about
•6 IJL in diameter, but they are often of great length. They are
composed of a central protoplasm enclosed by a sheath. The
latter, which is most easily made out in the older filaments with
granular protoplasm, occasionally contains granules of dark
pigment. In the centre of the colony the filaments interlace
with one another, and form an irregular network which may be
loose or dense ; at the periphery they are often arranged in a
somewhat radiating manner, and run outwards in a wavy or even
spiral course. They also show true branching, a character
which at once distinguishes them from the ordinary bacteria.
Between the filaments there is a finely granular or homogeneous
ground substance. Most of the colonies at an early stage are
chiefly constituted by filaments loosely arranged ; but later, part
of the growth may become so dense that its structure cannot be
made out. This dense part, starting excentrically, may grow
round the colony to form a hollow sphere, from the outer
surface of which filaments radiate for a short distance (Fig. 96).
The filaments usually stain uniformly in the younger colonies,
but some, especially in the older colonies, may be segmented so
288 ACTINOMYCOSIS AND ALLIED DISEASES
as to give the appearance of a chain of bacilli or of cocci, though
the sheath enclosing them may generally be distinguished.
Rod-shaped and spherical forms may also be seen lying free.
2. Spores or Gonidia. — Like other species of streptothrix the
actinomyces when growing on a culture medium shows on its
surface filaments growing upwards in the air, the protoplasm of
which becomes segmented into rounded spores or gonidia. In
natural conditions outside the body these gonidia become free
FIG. 96. — Actinomycosis of human liver, showing a colony of the parasite
composed of a felted mass of filaments surrounded by pus.
Paraffin section ; stained by Gram's method and with safranin. x 500. •
and act as new centres by growing out into filaments. They
have somewhat higher powers of resistance than the filaments,
though less than the spores of most of the lower bacteria. An
exposure to 75° C. for half an hour is sufficient to kill most
streptothrices or their spores ; cultures containing spores can
resist a temperature from five to ten degrees higher than spore-
free cultures (Foulerton). It is probable that some of the
spherical bodies formed within filaments when growing in the
tissues have the same significance, i.e. are gonidia, whilst others
may be merely the result of degenerative change. Both the
CHARACTERS OF THE ACTINOMYCES 289
filaments and the spherical bodies are readily stained by
Gram's method.
3. Clubs. — These are elongated pear-shaped bodies which are
seen at the periphery of the colony, and are formed by a sort of
hyaline swelling of the sheath around the free extremity of a
filament (Figs. 97, 98). They are usually homogeneous and
structureless in appearance. In the human subject the clubs are
FIG. 97. — Actinomyces in human kidney, showing clubs radially arranged
and surrounded by pus. The filaments had practically disappeared.
Paraffin section ; stained with hsematoxylin and rubin. x 500.
often comparatively fragile structures, which are easily broken
down, and may sometimes be dissolved in water. Sometimes
they are well seen when examined in the fresh condition, but in
hardened specimens are no longer distinguishable. In specimens
stained by Gram's method they are usually not coloured by the
violet, but take readily a contrast stain, such as picric acid,
rubin, etc ; sometimes a darkly-stained filament can be seen
running for a distance in the centre, and may have a knob-like
extremity. In many of the colonies in the human subject the
clubs are absent. In the ox, on the other hand, where there are,
19
290 ACTINOMYCOSIS AND ALLIED DISEASES
much older colonies, the clubs constitute the most prominent
feature, whilst in most colonies the filaments are more or less
degenerated, and it may sometimes be impossible to find any.
They often form a dense fringe around the colony, and when
stained by Gram's method retain the violet stain. They have,
in fact, undergone some further chemical change which produces
the altered staining reaction. Occasionally in very chronic
FIG. 98. — Colonies of actinomyces, showing general structural arrangement
and clubs at periphery. From pus in human subject.
Stained Gram and safranin. x 60.
lesions in the human subject the clubs stain with Gram's
method. Clubs showing intermediate staining reaction have
been described in the ox by M'Fadyean. The club-formation
probably represents a means of defence on the part of the
parasite against the phagocytes of the tissue : the view, formerly
held, that the clubs are organs of fructification has now been
generally abandoned.
Tissue Lesions. — In the human subject the parasite pro-
duces by its growth a chronic inflammatory change, usually
ending in a suppuration which slowly spreads. In some cases
TISSUE LESIONS 291
there is a comparatively large production of granulation tissue,
with only a little softening in the centre, so that the mass feels
solid. This condition is sometimes found in the subcutaneous
tissue, especially when the disease has not advanced far, and
also in dense fibrous tissue. In most cases, however, and
especially in internal organs, suppuration is the outstanding
feature ; this is associated with abundant growth of the parasite
in the filamentous form presenting a honeycomb appearance.
In an organ such as the liver, multiple foci of suppuration are
seen at the spreading margin of the disease, whilst the colonies
of the parasite may be seen in the pus with the naked eye. In
the older parts the abscesses have become confluent, and formed
large areas of suppuration. The pus is usually of greenish-
yellow colour, and of somewhat slimy character.
In cattle the tissue reaction is more of a formative type,
there being abundant growth of granulation tissue, which may
result in large tumour-like masses, usually of more or less
nodulated character, and often consisting of well-developed
fibrous tissue containing areas of younger formation in which
irregular abscess formation is usually present. The cells immedi-
ately around the colonies are usually irregularly rounded, or may
even be somewhat columnar in shape, whilst farther out they
become spindle-shaped and concentrically arranged. It is not
uncommon to find leucocytes or granulation tissue invading the
substance of the colonies, and portions of the parasite, etc., may
be contained within leucocytes or within small giant-cells which
are sometimes present. A similar invasion of old colonies by
leucocytes is sometimes seen in human actinomycosis.
Origin and Distribution of Lesions. — The lesions in the
human subject may occur in almost any part of the body, the
paths of entrance being very various. In many cases the
entrance takes place in the region of the mouth — probably
around a decayed tooth — by the crypts of the tonsil, or by some
abrasion. Swelling and suppuration may then follow in the
vicinity and may spread in various directions. The periosteum
of the jaw or the vertebra may thus become affected, caries or
necrosis resulting, or the pus may spread deeply in the tissues of
the neck, and may even pass into the mediastinum. Occasionally
the parasite may enter the tissues from the oesophagus, and in a
considerable number of cases the primary lesion is in some part
of the intestine, generally of the large intestine. The parasite
penetrates the wall of the bowel, and may be found deeply
between the coats, surrounded by purulent material. Thence it
may spread to the peritoneum or to the extraperitoneal tissue,
292 ACTINOMYCOSIS AND ALLIED DISEASES
the retrocaecal connective tissue and that around the rectum
being not uncommonly seats of suppuration produced in this
way. A peculiar affection of the intestine has been described,
in which slightly raised plaques are found both in the large and
small intestines, these plaques being composed almost exclusively
of masses of the actinomyces along with epithelial cells. This,
however, is a rare condition. The path of entrance may also be
by the respiratory passages, the primary lesion being pulmonary
or peribronchial ; extensive suppuration in the lungs may result.
Infection may also occur by the skin surface, and lastly, by the
female genital tract, as in a case recorded by Grainger Stewart
and Muir, in which both ovaries and both Fallopian tubes were
affected.
When the parasite has invaded the tissues by any of these
channels, secondary or " metastatic " abscesses may occur in
internal organs. The liver is the organ most frequently affected,
though abscesses may occur in the lungs, brain, kidneys, etc.
In such cases the spread takes place by the blood stream, and it
is possible that leucocytes may be the .carriers of the infection,
as it is not uncommon to find leucocytes in the neighbourhood
of a colony containing small portions of the filaments in their
interior.
In the ox, on the other hand, the disease usually remains
quite local, or spreads by continuity. It may produce tumour-
like masses in the region of the jaw or neck, or it may specially
affect the palate or tongue, in the latter producing enlargement
and induration, with nodular thickening on the surface — the
condition known as " woody tongue."
Source of the Parasite. — There is a considerable amount of
evidence to show that outside the body the parasite grows on
grain, especially on barley. Both in the ox and in the pig the
parasite has been found growing around fragments of grain em-
bedded in the tissues. There are besides, in the case of the
human subject, a certain number of cases in which there was a
history of penetration of a mucous surface by a portion of grain,
and in a considerable proportion of cases the patient has been
exposed to infection from this source. The position of the
lesions in cattle is also in favour of such a view.
Cultivation (for methods of isolation see later). — The de-
scriptions of the cultures obtained by various investigators differ
in essential particulars, and there is no doubt that the organisms
described are different. The following is the account of the
organism as cultivated by Bostrom : —
On agar or glycerin agar at 37° C., growth is generally
CULTIVATION OF ACTINOMYCES
293
visible on the third or fourth day in the form of little transparent
drops which gradually enlarge and form rounded projections of
a reddish-yellow tint and somewhat transparent appearance, like
drops of amber. The growths tend to remain separate, and even
when they become confluent, the nodular character is maintained.
They have a tough con-
sistence, being with diffi-
culty broken up, and ad-
here firmly to the surface
of theagar. Older growths
often show on the surface
a sort of corrugated aspect,
and may sometimes pre-
sent the appearance of
having been dusted with
a brownish-yellow powder
(Fig. 99). "
In the cultures at an
early stage the growth is
composed of branching
filaments, which stain
uniformly (Fig. 100), but
later some of the super-
ficial filaments may show
segmentation into gonidia.
Slight bulbous thicken-
ings may be seen at the
end of some of the fila-
ments, but true clubs
have not been observed.
On gelatin the
FIG. 99. — Cultures of the actinomyces on
glycerin agar, of about three weeks' growth,
showing the appearances which occur. The
growth in A is at places somewhat corru-
gated on the surface. Natural size.
same
tendency to grow in little
spherical masses is seen,
and the medium becomes
very slowly liquefied.
When this occurs the liquefied portion has a brownish colour
and somewhat syrupy consistence, and the growths may be seen
at the bottom, as little balls, from the surface of which filaments
radiate.
The organism obtained in culture by Wolff and Israel (vide
infra} is probably the same as the one which has been recently
described in detail by J. H. Wright, who obtained it in pure con-
dition from fifteen different cases of the disease. It differs markedly
from Bostrom's organism in being almost a strict anaerobe and in
294 ACTINOMYCOSIS AND ALLIED DISEASES
ceasing to grow at a temperature a little below that of the body.
Under ordinary aerobic conditions either no growth occurs or
it is of a very slight character. On the surface of agar under
anaerobic conditions the organism produces dense rounded
colonies of greyish -white colour, which sometimes assume a
rosette form. A somewhat curious feature of growth is described
by Wright, namely, that in a shake culture in glucose agar the
colonies are most numerous, and form a dense zone about half
an inch from the surface of the medium, that is, at a level
where there is presum-
ably a mere trace of
oxygen obtainable (Fig.
101). In bouillon, growth
takes place at the bottom
of the medium in rounded
masses which afterwards
undergo disintegration.
Wright found when the
organism was grown in
the presence of serum or
other animal fluids that
the formation of true
clubs occurred at the ex-
tremity of some of the fila-
ments (Fig. 102). From
the conditions under
FIG 100.- Actinomyces, from a culture on which th QC he
glycerin agar, showing the branching of . . S ,
the filaments. 1S inclined to regard
Stained with fuchsin. . x 1000. it as a true parasite,
and doubts whether it
can have a saprophytic existence outside the body, e.g. on grain.
He is also of opinion that all cases of true actinomycosis, i.e. cases
where colonies visible to the naked eye are present, are probably
produced by one species, and that the aerobic organisms obtained
by Bostrom and others are probably accidental contaminations.
It is quite evident that further investigations are required in
the light of the results detailed. Certainly the parasite in
many cases of actinomycosis in the human subject does not
grow on ordinary media under aerobic conditions as Bostrom's
organism does.
Varieties of Actinomyces and Allied Forms. — It is probable that in
the cases of the disease described in the human subject there is more
than one variety or species of parasite belonging to the same group.
Gasperini has described several varieties of actinomyces bovis according
VARIETIES OF ACTINOMYCES
295
to the colour of the growths,
and a similar condition may
obtain in the case of the
human subject. Further-
more a considerable number
of streptothrices have been
found in cases of disease in
the human subject, the as-
sociated lesions varying in
character from tubercle-like
nodules on the one hand to
suppurative processes on the
other. The organisms culti-
vated from such sources
differ according to their
microscopic characters (for
example, some form "clubs"
whilst others do not) ac-
cording to their conditions
of growth, staining reac-
tions, etc. Of these only a
few examples may here be
mentioned, but it may be
noted that the importance
of the streptothrices as
causes of disease is con-
stantly being extended.
Wolff and Israel cultivated
from two cases of ' ' actino-
mycosis" in man a strep-
tothrix which differs in so
many important points from
the actinomyces of Bostrom
FIG. 102. — Section of a colony of actinomyces
from a culture in blood serum, showing the
formation of clubs at the periphery. x 1500.
FIG. 10 1.1 — Shake cultures of actinomyces in
glucose agar, showing the maximum
growth at some distance from the sur-
face of the medium.
that it is now regarded
as a distinct species. An-
other species was culti-
vated by Eppinger from a
brain abscess, and called
by him "cladothrixaster-
oides," from the appear-
ance of its colonies on
culture media. A case
of general streptothrix
infection in the human
subject described by
MacDonald was probably
due to the same organism
as Eppinger's. In the
tissues it grows in a
somewhat diffuse manner
and does not form clubs ;
1 For Figs. 101 and 102
we are indebted to Dr. J. Homer Wright of Boston, U.S.A.
296 ACTINOMYCOSIS AttD ALLIED DISEASES
in rabbits and guinea-pigs it produces tubercle-like lesions. Flexner
observed a streptothrix in the lungs associated witb lesions somewhat like
a rapid phthisis, and applied the name "pseudo-tuberculosis hominis
streptothricea " ; an apparently similar condition has been described by
Buchholz. Berestnew cultivated two species of streptothrix from
suppurative lesions, one of which is acid -fast and grows only in
anaerobic conditions. Birt and Leishman have described another acid-
fast streptothrix obtained from cirrhotic nodules in the lungs of a man.
This organism grows readily on ordinary media, forming a white
powdery growth which afterwards assumes a pinkish colour ; it is
pathogenic for guinea-pigs, in which it causes caseous lesions. There
is, further, the streptothrix madurse described below.
In diseases of the lower animals several other forms have been found.
For example, a streptothrix has been shown by Nocard to be the cause
of a disease of the ox, — "farcin du boeuf," — a disease in which also
there occur tumour -like masses of granulation tissue. Dean has
cultivated from a nodule in a horse another streptothrix, which produces
tubercle-like nodules in the rabbit with club-formation ; it has close
resemblances to the organism of Israel and Wolff. The so-called
diphtheria of calves and "bacillary necrosis" in the ox are probably
both produced by another streptothrix or leptothrix, which grows
diffusely in the tissues in the form of fine felted filaments. Further
investigation may show that some of these or other species may occur
in the human subject in conditions which are not yet differentiated.
Experimental Inoculation. — Inoculation of smaller animals,
such as rabbits and guinea-pigs, has usually failed to give positive
results. This was the case, for example, in the important series
of experiments by Bostrom, and it may be assumed that these
animals are little susceptible to the actinomyces. The disease
has, however, been experimentally produced in the bovine species
both by cultures from the ox and from the human subject.
Inoculation with the organism of Israel and Wolff produces
nodular lesions both in rabbits and in guinea-pigs, while Wright
found that characteristic colonies and lesions resulted although
the parasite did not grow to any great extent. Several of the
other species of streptothrix have been found to possess active
pathogenic properties.
Methods of Examination and Diagnosis. — As actinomycosis
cannot be diagnosed with certainty apart from the discovery of
the parasite, a careful examination of the pus in obscure cases
of suppuration should always be undertaken. As already stated,
the colonies can be recognised with the naked eye, especially
when some of the pus is spread out on a piece of glass. If one
of these is washed in salt solution and examined unstained, the
clubs, if present, are at once seen on microscopic examination.
Or the colony may be stained with a simple reagent such as
picrocarmine, and mounted in glycerin or Farrant's solution.
To study the filaments, a colony should be broken down on a
MADURA DISEASE 297
cover-glass, dried, and stained with a simple solution of any of
the basic aniline dyes, such as gentian- violet, though better
results are obtained by carbol-thionin-blue, or by carbol-fuchsin
diluted with five parts of water. If the specimen be over-stained,
it may be decolorised by weak acetic acid. Cover-glass prepara-
tions of this kind, and also of cultures, are readily stained by
these methods, but in the case of sections of the tissues Gram's
method, or a modification of it, should be used to show 'the
filaments, etc., a watery solution of acid fuchsin being afterwards
used to stain the clubs. By this method, very striking prepara-
tions may be obtained.
Cultures should be made both under aerobic and anaerobic
conditions. Tubes of agar or glycerin agar should be inoculated
and incubated at 37° C. ; deep tubes of melted glucose agar
should also be used, the inoculated material being diffused
through the medium, separate colonies may thus be obtained.
Owing to the slow growth of the actinomyces, however, the
obtaining of pure cultures is difficult, unless the 'pus is free from
contamination with other organisms.
MADUKA DISEASE.
Madura disease or mycetoma resembles actinomycosis both
as regards the general characters of the lesions and the occurrence
of the parasite in the form of colonies. or "granules." There is
no doubt, however, that the two conditions are distinct, and it
also appears established that the two varieties of Madura disease
(vide infra) are produced by different organisms. This disease
is comparatively common in India and in various other parts of
the tropics : it has also been met with in Algiers and in America.
Madura disease differs from actinomyces not only in its geo-
graphical distribution but also in its clinical characters. Its
course, for example, is of an extremely chronic nature, and
though the local disease is incurable except by operation, the
parasite never produces secondary lesions in internal organs.
Vincent also found that iodide of potassium, which has a high
value as a therapeutic agent in many cases of actinomycosis, had
no effect in the case of Madura disease studied by him. It most
frequently affects the foot ; hence the disease is often spoken of
as " Madura foot." The hand is rarely affected. In the parts
affected there is a slow growth of granulation tissue which has an
irregularly nodular character, and in the centre of the nodules
there occurs purulent softening which is often followed by the
298 ACTINOMYCOSIS AND ALLIED DISEASES
formation of fistulous openings and ulcers. There are great en-
largement and distortion of the part and frequently caries and
necrosis of the bones. Within the softened cavities and also in
the spaces between the fibrous tissue, small rounded bodies or
granules, bearing a certain resemblance to the actinomyces, are
present. These may have a yellowish or pinkish colour, com-
pared from their appearance to fish roe, or they may be black
like grains of gunpowder, and may by their conglomeration
form nodules of considerable size. Hence a pale variety and
a black variety of the disease have been distinguished ; in both
varieties the granules mentioned reach a rather larger size
than in actinomycosis.
These two conditions will
be considered separately.
Pale Variety. — When
the roe-like granules are
examined microscopic-
ally, they are found, like
the actinomyces, to show
in their interior an abun-
dant mass of branching
filaments with mycelial
arrangement. There may
also be present at the
periphery club-like struc-
tures, as in actinomyces ;
sometimes they are ab-
sent. These structures
FIG. 103. — Streptothrix Maclurse, showing £, i i . j
branching filaments. From a culture of °ften have «* elongated
agar. Stained with carbol-thionin-blue. wedge-shape, tormmg an
x 1000. outer zone to the colony,
and in some cases the
filaments can be found to be connected with them. Vincent
obtained cultures of the parasite from a case in Algiers, and found
it to be a distinct species : it is now known as the streptothrix
Maduroe. Morphologically it closely resembles the actinomyces,
but it presents certain differences in cultural characters. In
gelatin it forms raised colonies of a yellowish colour, with
umbilication of the centre, and there is no liquefaction of the
medium. On agar the growth assumes a reddish colour; the
organism flourishes well in various vegetable infusions in which
the actinomyces does not grow. On all the media growth only
takes place in aerobic conditions. Experimental inoculation of
various animals has failed to reproduce the disease. There is
MADUKA DISEASE 299
therefore no doubt that the streptothrix madurae and the
actinomyces are distinct species.
Black Variety. — The observations of J. H. Wright, who
obtained pure cultures of a hyphomycete, show that this variety is
a distinct affection from the pale variety. The pigment may be
dissolved by soaking the granules for a few minutes in hypochlorite
of sodium solution, and the granules may then be crushed out
beneath a cover-glass and examined microscopically. The black
granules are composed of a somewhat homogeneous ground-
substance impregnated with pigment, and in this there is a
mycelium of thick filaments or hyphse, many of the segments of
which are swollen ; at the periphery the hyphae form a zone
with radiate arrangement. In many of the older granules the
parasite is largely degenerated and presents an amorphous appear-
ance. Wright planted over sixty of the black granules in various
culture media, and obtained cultures of a hyphomycete from
about a third of these. The organism grows well on agar,
bouillon, potato, etc. ; on agar it forms a felted mass of greyish
colour, and in old cultures black granules appear amongst the
mycelium. Microscopically the parasite appears as a mycelium
of thick branching filaments with delicate transverse septa ; in
the older threads the segments become swollen, so that strings
of oval-shaped bodies result. No signs of spore-formation were
noted. Inoculation of animals with cultures gave negative
results, as did also direct inoculation with the black granules
from the tissues.
CHAPTER XIII
ANTHRAX.1
OTHER NAMES. — SPLENIC FEVER, MALIGNANT PUSTULE, WOOL-
SORTER'S DISEASE. GERMAN, MILZBRAND; FRENCH, CHARBON.2
Introductory. — Anthrax is a disease occurring epidemically
among the herbivora, especially sheep and oxen, in which
animals it has the characters of a rapidly fatal form of
septicaemia with splenic enlargement, attended by an extensive
multiplication of characteristic bacilli throughout the blood.
The disease does not occur as a natural affection in man, but may
be communicated to him directly or indirectly from animals, and
it may then appear in certainly two and possibly three forms. In
the first there is infection through the skin, in which a local lesion,
the " malignant pustule," occurs. In the second form infection
takes place through the respiratory tract. Here very aggravated
symptoms centred in the thorax, with rapidly fatal termination,
follow. Thirdly, an infection may probably take place through
the intestinal tract, which is now the first part to give rise to
symptoms. In all these forms of the affection in the human
subject, the bacilli are in their distribution much more re-
stricted to the local lesions than is the case in the T>X, their
growth and spread being attended by inflammatory oedema
and often by haemorrhages.
Historical Summary. — Historical researches leave little doubt that
from the earliest times anthrax has occurred among cattle. For a long
time its pathology was not understood, and it went by many names.
1 In even recent works on surgery the term "anthrax" may be found
applied to any form of carbuncle. Before its true pathology was known the
local variety of the disease which occurs in man and which is now called
"malignant pustule" was known as "malignant carbuncle."
a This must be distinguished from "charbon symptomatique, " which is
quite a different disease.
300
BACILLUS ANTHRACIS 301
During the early part of last century much attention was paid to
it, and, with a view to finding out its nature and means of spread,
various conditions attaching to its occurrence, such as those of soil and
weather, were exhaustively studied. Pollender in 1849 pointed out that
the blood of anthrax animals contained numerous rod-shaped bodies
which he conjectured had some causal connection with the disease. In 1863
Davaine announced that they were bacteria, and originated the name
bacillus anthracis. He stated that unless blood used in inoculation
experiments on animals contained them, death did not ensue. Though
this conclusion was disputed, still' by the work of Davaine and others
the causal relationship of the bacilli to the disease had been nearly
established when the work of Koch appeared in 1876. This constituted
that observer's first contribution to bacteriology, and did much to clear up
the whole subject. Koch confirmed Davaine's view that the bodies were
bacteria. He observed in the blood of anthrax animals the appearance
of division, and from this deduced that multiplication took place in the
tissues. He observed them under the microscope dividing outside the
body, and noticed spore formation taking place. He also isolated the
bacilli in pure culture outside the body, and, by inoculating animals with
them, produced the disease artificially. In his earlier experiments he
failed to produce death by feeding susceptible animals both with bacilli
and spores, and as the intestinal tract was, in his view, the natural path
of infection, he considered as incomplete the proof of this method of the
spontaneous occurrence of anthrax in herds of animals. Koch's observa-
tions were, shortly afterwards, confirmed in the main by Pasteur,
though controversy arose between them on certain minor points.
Moreover, further research showed that the disease could be produced
in animals by feeding them with spores, and thus the way in which the
disease might spread naturally was explained.
The Bacillus Anthracis. — Anthrax as a disease in man is of
comparative rarity. Not only, however, is the bacillus anthracis
easy of growth and recognition, but in its growth it illustrates
many of the general morphological characters of the whole group
of bacilli, and is therefore of the greatest use to the student.
Further, its behaviour when inoculated in animals illustrates
many of the points raised in connection with such difficult
questions as the general pathogenic effects of bacteria, immunity,
etc. Hence an enormous amount of work has been done in
investigating it in all its aspects.
If a drop of blood is taken immediately after death from an
auricular vein of a cow, for example, which has died from
arrEhfax, and examined microscopically, it will be found to
contain a great number of large non-motile bacilli. On making
a cover-glass preparation from the same source, and staining with
watery methylene-blue, the characters of the bacilli can be better
made out. They are about 1*2 ^ thick or a little thicker, and 6
to 8 p long, though both shorter and longer forms also occur.
The ends are sharply cut across, or may be slightly dimpled so
as to resemble somewhat the proximal end of a phalanx. Their
302
ANTHRAX
protoplasm is very finely granular, and sometimes appears sur-
rounded by a thin unstained capsule. When several bacilli lie
end to end in a thread, the capsule seems common to the whole
thread (Fig. 108). They stain well with all the basic aniline dyes
and are not decolorised by Gram's method.
Plate Cultures. — From a source such as that indicated, it is
easy to isolate the bacilli by making gelatin or agar plates. If,
after twelve hours' incubation at 37° C., the latter be examined
under a low objective, colonies will be observed. They are to
be recognised by beautiful wavy wreaths like locks of hair,
radiating from the centre and apparently terminating in a point
which, however, on ex-
amination with a higher
power is observed to be a
filament which turns upon
itself (Fig. 104). The whole
colony is, in fact, probably
one long thread. Such
colonies are very suitable
for making impression pre-
parations (vide p. 118)
which preserve perman-
ently the appearances de-
scribed. On examining
such with a high power,
the wreaths are seen to be
made up of bundles of
FIG. 104.— Surface colony of the anthrax l°ng filaments lying par-
bacillus on an agar plate, showing the allel with one another, each
characteristic appearances, x 30. filament consisting of a
chain of bacilli lying end
to end, and similar to those observed in the blood (Fig. 105).
On gelatin plates, after from twenty-four to thirty-six hours
at 20° C., the same appearances manifest themselves, and later
they are accompanied by liquefaction of the gelatin. In gelatin
plates, however, instead of the characteristically wreathed appear-
ance at the margin, the colonies sometimes give off radiating
spikelets irregularly nodulated, which produce a star-like form.
These spikelets are composed of spirally twisted threads.
From such plates the bacilli can be easily isolated, and the
appearances of pure cultures on various media studied.
In bouillon, after twenty-four hours' incubation at 37° C.,
there is usually the appearance of irregularly spiral threads
suspended in the liquid. These, on being examined, are seen
BACILLUS ANTHRACIS
303
to be made up of bundles of parallel chains of bacilli. Later,
growth is more abundant, and forms a flocculent mass at the
bottom of the fluid.
In gelatin stab cultures, the characteristic appearance can be
best observed when a low proportion, say 7J per cent, of gelatin
FIG. 105. — Anthrax bacilli, arranged in chains,
from a twenty-four hours' culture on agar
at 37° C.
Stained with fuchsin. x 1000.
is present, and when the tube is directly
inoculated from anthrax blood. In
about two days there radiate out into
the medium from the needle track
numberless very fine s pikelets which
enable the cultures to be easily recog- Plo.106._stab culture of
msed. Inese spikelets are longest at
the upper part of the needle track
(Fig. 106). Not much spread takes
place on the surface of the gelatin,
but here liquefaction commences, and
gradually spreads down the stab and
out into the medium, till the whole
of the gelatin may be liquefied. Gelatin slope cultures exhibit
a thick felted growth, the edges of which show the wreathed
appearance seen in plate cultures. Liquefaction here soon
ploughs a trough in the surface of the medium. Sometimes
" spiking " does not take place in gelatin stab cultures, only
the anthrax bacillus in
peptone-gelatin ; seven
days' growth. It shows
the "spiking" and also,
at the surface, com-
mencing liquefaction.
Natural size.
304 ANTHRAX
little round particles of growth occurring down the needle track,
followed by liquefaction. As has been shown by Richard Muir,
this property of spiking can be restored by growing the bacillus
for twenty-four hours on blood agar at 37° C. Agar sloped
cultures have the appearance of similar cultures in gelatin,
though; of course, no liquefaction takes place.
Blood serum sloped cultures present the same appearances as
those on agar. The margin of the surface growth on any of the
solid media shows the characteristic wreathing seen in plate
colonies.
On potatoes there occurs a thick felted white mass of bacilli
showing no special characters. Such a growth, however, is useful
for studying sporulation.
The anthrax bacillus will thus grow readily on any of the
ordinary media. It can usually be sufficiently recognised by its
microscopic appearance, by its growth on agar or gelatin plates,
and by its growth in gelatin stab cultures. The growth on
plates is specially characteristic, and is simulated by no other
pathogenic organism.
The Biology of the B. Anthracis. — Koch found that the
bacillus anthracis grows best at a temperature of 35° C. Growth,
i.e. multiplication, does not take place below 12° C. or above
45° C. In the spore-free condition the bacilli have comparatively
low powers of resistance. They do not stand long exposure to
60° C., and if kept at ordinary temperature in the dry condition
they are usually found to be dead after a few days. The action
of the gastric juice is rapidly fatal to them, and they are accord-
ingly destroyed in the stomachs of healthy animals. They are
also soon killed in the process of putrefaction. They can, how-
ever, be cooled below the freezing-point without dying. The
bacillus can grow without oxygen, but some of its vital functions
are best carried on in the presence of this gas. Thus in anthrax
cultures the liquefaction of gelatin always commences at the
surface and spreads downwards. Growth is more rapid in the
presence of oxygen, and spore formation does not occur in its
absence. The organism may be classed as a facultative anaerobe.
Sporulation. — Under certain circumstances sporulation occurs
in anthrax bacilli. The morphological appearances are of the
ordinary kind. A little highly refractile speck appears in the
protoplasm about the centre of the bacillus; this gradually
increases in size until it forms an oval body about the same
thickness as the bacillus lying in the bacillary protoplasm (Fig.
107). The latter gradually loses its staining capacities and
finally disappears. The spore thus lies free as*1 an oval highly
BIOLOGY OF THE B. ANTHRACIS
305
refractile body which does not stain by ordinary methods, but
which can be easily stained by the special methods described
for such a purpose (p. 102). When the spore is 'again about to
assume the bacillary form the capsule is apparently absorbed,
and the protoplasm within grows out, taking on the ordinary rod-
shaped form.
According to most observers sporulation never occurs within
the body of an animal suffering from anthrax. Koch attributes
this, probably rightly, to the absence of free oxygen. The latter
gas he found necessary to the occurrence of spores in cultures
outside the body. Many, however, are inclined to assign as the
cause of sporulation the
absence of the optimum
pabulum, which in the case
of anthrax is afforded by
the animal tissues. Besides
these conditions there is
another factor necessary
to sporulation, viz. a suit-
able temperature. The
optimum temperature for
spore production is 30° C.
Koch found that spore
formation did not occur
below 18° C. Above 42°
C. not only does sporula-
tion cease, but Pasteur
found that if bacilli were
kept at this temperature
FIG. 107. — Anthrax bacilli containing spores
(the darkly coloured bodies) ; from a
.. . , , three days' culture on agar at 37° C.
tor eight days they did not stained with carbol-fuchsin and methylene-
regain the capacity when blue, x 1000.
again grown at a lower
temperature. In order to make them again capable of sporing
it is necessary to adopt special measures, such as passage through
the bodies of a series of susceptible animals.
Anthrax spores have extremely high powers of resistance.
In a dry condition they will remain viable for a year or more.
Koch found they resisted boiling for five minutes ; and dry heat
at 140° C. must be applied for several hours to kill them with
certainty. Unlike the bacilli, they can resist the action of the
gastric juice for a long period of time. They are often used
as test objects by which the action of germicides is judged. For
this purpose an emulsion is made by scraping off a surface
culture and rubbing it up in a little sterile water. Into this
20
306 ANTHRAX
sterile silk threads are dipped, which, after being dried over
strong sulphuric acid in a desiccator, can be kept for long
periods of time in an unchanged condition. For use they are
placed in the germicidal solution for the desired time, then
washed with water to remove the last traces of the reagent and
laid on the surface of agar or placed in bouillon, in order that if
death has not occurred growth may be observed (see Chap. IV.).
Anthrax in Animals. — Anthrax occurs from time to time
epidemically in sheep, cattle, and, more rarely, in horses and
deer. These epidemics are found in various parts of the world,
although they are naturally most far-reaching where legal pre-
cautions to prevent the spread of infection are non-existent.
All the countries of Europe are from time to time visited by the
disease, but in some it is much more common than in others.
In Britain the death-rate is small, but in France the annual
mortality among sheep was probably 10 per cent of the total
number in the country, and among cattle 5 per cent. These
figures, however, have been largely modified by the system of
preventive treatment which will be presently described. In sheep
and cattle the disease is specially virulent. An animal may
suddenly drop down, with symptoms of collapse, quickening of
pulse and respiration, and dyspnoea, and death may occur in
a few minutes. In less acute cases the animal is apparently out
of sorts, and does not feed j its pulse and respiration are
quickened ; rigors occur, succeeded by high temperature ; there
is a sanguineous discharge^ from the bowels, and bloody mucus
may be observed about the mouth and nose. There may be
convulsive movements, there is progressive weakness, with cyanosis,
death occurring in from twelve to forty-eight hours. In the
more prolonged cases widespread oedema and extensive enlarge-
ment of lymphatic glands are marked features ; and in the glands,
especially about the neck, actual necrosis with ulceration may
occur, constituting the so-called anthrax carbuncles. Such
subacute conditions are especially found among horses, which
are by nature not so susceptible to the disease as cattle and
On post mortem examination of an ox dead of anthrax, the
most noticeable feature — one which has given the name " splenic
fever " to the disease — is the enlargement of the spleen, which
may be two or three times its natural size. It is of dark-red
colour, and on section the pulp is very soft and friable, sometimes
almost diffluent. A cover-glass preparation may be made from
the spleen and stained with watery methylene-blue. On examina-
tion it will be found to contain enormous numbers of bacilli
ANTHEAX IN ANIMALS 307
mixed with red corpuscles and leucocytes, chiefly lymphocytes
and the large mononucleated variety (Fig. 108). Pieces of the
organ may be hardened in absolute alcohol, and sections cut in
paraffin. These are best stained by Gram's method. Micro-
scopic examination of such shows that the structure of the pulp
is considerably disintegrated, whilst the bacilli swarm throughout
the organ, lying irregularly amongst the cellular elements. The
FIG. 108. — Scraping from spleen of guinea-pig dead of anthrax, showing the
bacilli mixed with leucocytes, etc. (Same appearance as in the ox.)
"Corrosive film" stained with carbol-thionin-blue. x 1000.
liver is enlarged and congested, and may be in a state of acute
cloudy swelling. The bacilli are present in the capillaries
throughout the organ, but are not so numerous as in the spleen.
The kidney is in a similar condition, and here the bacilli are
chiefly found in the capillaries of the glomeruli, which often
appear as if injected with them. The lungs are congested and
may show catarrh, whilst bacilli are present in large numbers
throughout the capillaries, and may also be found in the air cells,
probably as the result of rupture of the capillaries. The blood
throughout the body is usually fluid and of dark colour.
308 ANTHRAX
The lymphatic system generally is much affected. The
glands, especially the mediastinal, mesenteric, and cervical
glands, are enlarged and surrounded by oedematous tissue, the
lymphatic vessels are swollen, and both glands and vessels may
contain numberless bacilli. The heart may be in a state of cloudy
swelling, and the blood in its cavities contains bacilli, though in
smaller numbers than that in the capillaries. The intestines are
enormously congested, the epithelium more or less desquamated,
and the lumen filled with a bloody fluid. From all the organs
the bacilli can be easily isolated by stroke cultures on agar.
It is important to note the existence of great differences in
susceptibility to anthrax in different species of animals. Thus
the ox, sheep (except those of Algeria, which only succumb to
enormous doses of the bacilli), guinea-pig, and mouse are all very
susceptible, the rabbit slightly less so. The last three are of
course most used for experimental inoculation. We have no
data to determine whether the disease occurs among these in the
wild state. Less susceptible than this group are the horse, deer,
goat, in which the disease occurs from time to time in nature.
Anthrax also occurs epidemically in the pig, often from the
ingestion of the organs of other animals dead of the disease. It
is, however, doubtful if all cases of disease in the pig described
on clinical grounds as anthrax are really such, and a careful
bacteriological examination is always advisable. The human
subject may be said to occupy a medium position between the
highly susceptible and the relatively immune animals. The
white rat is highly immune to the disease, while the brown rat
is susceptible. Adult carnivora are also very immune, and the
birds and amphibia are in the same position.
With these differences in susceptibility there are also great
variations in the pathological effects produced in the natural or
artificial disease. This is especially the case when we consider
the distribution of the bacilli in the bodies of the less susceptible
animals. Instead of the widespread occurrence described above,
they may be confined to the point where they first gained access
to the body and the lymphatic system in relation to it, or may
be only very sparsely scattered in organs such as the spleen
(which is often not enlarged), the lungs, or kidneys. Neverthe-
less the cellular structure of the organs even in such a case may
show changes, a fact which is important when we consider the
essential pathology of the disease.
Experimental Inoculation. — Of the animals commonly used
in laboratory work, white mice and guinea-pigs are the most
susceptible to anthrax, and are generally used for test inocula-
ANTHRAX IN MAN 309
tions. If a small quantity of anthrax bacilli be injected into the
subcutaneous tissue of a guinea-pig a fatal result follows, usually
within two days. Post mortem around the site of inoculation the
tissues, owing to intense inflammatory oedema, are swollen and
gelatinous in appearance, small haemorrhages are often present,
and on microscopic examination numerous bacilli are seen.
The internal organs show congestion and cloudy swelling, with
FIG. 109. — Portion of kidney of a guinea-pig dead of anthrax, showing the
bacilli in the capillaries, especially of the glomerulus.
Paraffin section ; stained by Gram's method and Bismarck-brown. x 300.
sometimes small haemorrhages, and their capillaries contain
enormous numbers of bacilli, as has already been described in
the case of the ox (Fig. 109) ; the spleen also shows a corre-
sponding condition. Highly susceptible animals may be infected
by being made to inhale the bacilli or their spores, and also by
being fed with spores, a general infection rapidly occurring by
both methods.
Anthrax in the Human Subject. — As we have noted, man
occupies a middle position in the scale of susceptibility to
anthrax. It is always communicated to him from animals, and
310 ANTHRAX
usually is seen among those whose trade leads them to handle
the carcases or skins of animals which have died of the disease.
It occurs in two principal forms, the main difference between
which is due to the site of entrance of the organism into the
body. In one, the path of entrance is through cuts or abrasions
in the skin, or through the hair follicles. A local condition
called a "malignant pustule" develops, which may lead to a
general infection. This variety occurs chiefly among butchers
and those who work among hides (foreign ones especially). In
Britain the workers of the latter class chiefly liable are the hide-
porters and hide- workers in South-Eastern London. In the other
variety of the disease the site of infection is the trachea and
bronchi, and here a fatal result almost always follows. The
cause is the inhalation of dust or threads from wool, hair, or
bristles, which have been taken from animals dead of the disease,
and which have been contaminated with blood or secretions con-
taining the bacilli, these having afterwards formed spores. This
variety is often referred to as " woolsorter's disease," from its
occurring in the centres of the woolstapling trade (in England,
chiefly in Yorkshire), but it also is found in places where there
are hair and brush factories.
(1) Malignant Pustule. — This usually occurs on the exposed
surfaces — the face, hands, fore-arms, and back, the last being a
common site among hide-porters. One to three days after
inoculation a small red painful pimple appears, soon becoming a
vesicle, which may contain clear or blood-stained fluid, and is
rapidly surrounded by an area of intense congestion. Central
necrosis occurs and leads to the malignant pustule proper, which
in its typical form appears as a black eschar often surrounded
by an irregular ring of vesicles, these in turn being surrounded
by a congested area. From this pustule as a centre subcutaneous
oedema spreads, especially in the direction of the lymphatics ;
the neighbouring glands are enlarged. There is fever with
general malaise. On microscopic section of the typical pustule,
the central eschar is noticed to be composed of necrosed tissue
and degenerating blood cells ; the vesicles are formed by the
raising of the stratum corneum from the rete Malpighi. Beneath
them and in their neighbourhood the cells of the latter are
swollen and cedematous, the papillae being enlarged and flattened
out and infiltrated with inflammatory exudation, which also
extends beneath the centre of the pustule. In the tissue next
the eschar necrosis is commencing. The subcutaneous tissue is
also cedematous, and often infiltrated with leucocytes. The
bacilli exist in the periphery of the eschar and in the neigh-
ANTHRAX IN MAN 311
bouring lymphatics, and, to a certain extent, in the vesicles. It
is very important to note that widespread oedema of a limb,
enlargement of neighbouring glands, and fever may occur while
the bacilli are still confined to the immediate neighbourhood of
the pustule. Sometimes the pathological process goes no further,
the eschar becomes a scab, the inflammation subsides, and
recovery takes place. In the majority of cases, however, if the
pustule be not excised, the oedema spreads, invasion of the blood
stream may occur, and the patient dies with, in a modified
degree, the pathological changes detailed with regard to the
acute disease in cattle. In man the spleen is usually not much
enlarged, and the organs generally contain few bacilli. The
actual cause of death is therefore the absorption of toxins. It
may here be said that early excision of an anthrax pustule,
especially when it is situated in the extremities, is followed, in a
large proportion of cases, by recovery.
(2) Woolsorter's Disease. — The pathology of this affection
was worked out in this country especially by Greenfield. The
local lesion is usually situated in the lower part of the trachea or
in the large bronchi, and is in the form of swollen patches in
the mucous membrane often with haemorrhage into them, The
tissues are cedematous, and the cellular elements are separated,
but there is usually little or no necrosis. There is enormous
enlargement of the mediastinal and bronchial glands, and
hsemorrhagic infiltration of the cellular tissue in the region.
There are pleural and pericardial effusions, and hsemorrhagic
spots occur beneath the serous membranes. The lungs show
collapse and oedema. There may be cutaneous oedema over the
chest and neck, with enlargement of glands, and the patient
rapidly dies with symptoms of pulmonary embarrassment, and
with a varying degree of pyrexia. It is to be noted that in such
cases, though numerous bacilli are present in the bronchial
lesions, in the lymphatic glands and affected tissues in the
thorax, comparatively few may be present in the various organs,
such as the kidney, spleen, etc., and sometimes it may be im-
possible to find any.
(3) Infection occasionally takes place through the intestine,
probably by ingestion of spores as in the case of animals ;
but this condition is rare. In such cases there is a local lesion
in the intestinal mucous membrane, of similar nature to that in
the bronchial form, the central parts of the hsemorrhagic areas
being, however, sometimes necrotic and yellowish, and there is a
corresponding affection of the mesenteric glands. In a case of
this kind, recently recorded by Teacher hsemorrhagic meningitis,
312 ANTHRAX
associated with the presence of the bacilli in large numbers,
occurred as a complication.
The Toxins of the Bacillus Anthracis. — Various theories
were formerly held as to the mode in which the anthrax bacillus
produces its effects. One of the earliest was the mechanical,
according to which it was supposed that the serious results were
produced by extensive blocking of the capillaries in the various
organs by the bacilli. According to another, it was supposed
that the bacilli used up the oxygen of the blood, thus leading to
starvation of the tissues. The discovery of definite toxins which
accounted for the pathogenic effects of certain bacteria led to
such bodies being sought for in connection with the anthrax
bacillus. Among other workers, Sidney Martin investigated this
subject. This observer used alkali-albumin on which to grow
the bacillus, this medium approaching most closely to the
environment of the latter when growing in the animal body.
From cultures in this medium, concentrated by evaporation
either at 100° C. or in vacuo at 35° to 45° C., there were
isolated proto-albumose, deutero-albumose, and traces of peptone.
The albumoses differed from those which occur in ordinary
digestion, in being strongly alkaline in their reaction. This
alkalinity, Martin held, was due to traces of an alkaloidal body
of which the albumoses were the precursors, and which were
formed when the process of digestion of the alkali-albumin by
the bacillus was allowed to go on further. By the albumoses
and the alkaloid, pathogenic effects were produced in animals,
closely similar to those produced by the bacilli themselves.
Martin, to account for the symptoms of the disease, considered
that the fever was mostly due to the albumoses, while the
oedema and congestion were due to the alkaloid which acted as a
local irritant. He showed that prolonged boiling destroyed the
activity of the albumoses, but not that of the alkaloid. Further,
from the body fluids of animals dead of anthrax he isolated
poisonous bodies similar to those produced by the bacilli growing
in this artificial medium. Hankin and Wesbrook arrived at the
conclusion that the bacillus anthracis produces a ferment which,
diffusing out into the culture fluid, elaborates albumoses from
the proteids present in it. The bacilli also produce albumoses
directly without the intervention of a ferment. Marmier, after
cultivating the b. anthracis in peptone solution containing
certain salts, removed all the albumoses from the resultant
liquid, and from them, either by dialysis or extraction with
glycerin, isolated a body which gave no reactions of albuminoid
matter, peptone, propeptone, or alkaloid. This he considered the
SPREAD OF THE DISEASE IN NATURE 313
toxin. It killed animals susceptible to anthrax by a sort of
cachexia, and in suitably small doses could be used to immunise
them against subsequent inoculation with virulent bacilli. It
was chiefly retained within the bacilli when these were growing
in the most favourable conditions.. Unlike the toxins of
tetanus and diphtheria, and unlike ferments, it was not
destroyed by heating to 110° C. The toxin produced by the
b. anthracis growing in a fluid medium remains intimately
associated with the bacterial protoplasm, as such cultures when
filtered are relatively non-toxic.
It cannot be said that great light has been thrown on the
pathology of the disease by these researches. The effects of
infection by the b. anthracis are those shared by all other
organisms producing inflammation, the tendency to oedema
production of an unwonted degree being the chief special
feature and one with reference to which Martin's work may be
important. That toxic effects do occur in anthrax is undoubted,
for frequently, while the bacilli are still locally confined, there
may occur pyrexia and oedema spreading widely beyond the
pustule, but we have no definite information as to how these
effects are produced. In this disease we are probably dealing
with another example of the action of intracellular toxins,
regarding which, as in other cases, little is known.
The Spread of the Disease in Nature. — We have seen that
the b. anthracis rarely, if ever, forms spores in the body, and if
the bacilli could be confined to the blood and tissues of carcases
of animals dying of the disease, it is certain that anthrax in an
epidemic form would rarely occur. For it has been shown by
many observers that in the course of the putrefaction of such a
carcase the anthrax bacilli rapidly die out, and that after ten
days or a fortnight very few remain. But it must be remembered
that while still alive, an animal is shedding into the air by the
bloody excretions from the mouth, nose, and bowel, myriads of
bacilli which may rapidly spore, and thus arrive at a very re-
sistant stage. These lie on the surface of the ground and are
washed off by surface water. At certain seasons of the year the
temperature is, however, sufficiently high to permit of their
germination, and also of their multiplication, as they can un-
doubtedly grow on the organic matter which occurs in nature.
They can again form spores. It is in the condition of spores
that they are dangerous to susceptible animals. In the bacillary
stage, if swallowed, they will be killed by the acid gastric con-
tents ; but as spores they can pass uninjured through the
stomach, and gaining an entrance into the intestine, infect its
314 ANTHRAX
wall, and ultimately reach, and multiply in the blood. It is
known that in the great majority of cases of the disease in sheep
and oxen, infection takes place thus from the intestine. It was
thought by Pasteur that worms were active agents in the natural
spread of the disease by bringing to the surface anthrax spores.
Koch made direct experiments on this point, and could get no
evidence that such was the case. He thinks it much more
probable that the recrudescence of epidemics in fields where
anthrax carcases have been buried is due to persistence of
spores on the surface which has been infected by the cattle when
alive.
The Disposal of the Carcases of Animals dead of Anthrax.— It is ex-
tremely important that anthrax carcases should be disposed of in such a
way as to prevent their becoming future sources of infection. If anthrax
be suspected as the cause of death no post mortem examination should be
made, but only a small quantity of blood removed from an auricular
vein for bacteriological investigation. If such a carcase be now buried
in a deep pit surrounded by quicklime, little danger of infection will be
run. The bacilli being confined within the body will not spore, and
will die during the process of putrefaction. The danger of sporulation
taking place is, of course, much greater when an animal has died of an
unknown disease which on post mortem examination has proved to be
anthrax, but similar measures for burial must be here adopted. In some
countries anthrax carcases are burned, and this, if practicable, is of
course the best means of treating them. The chief source of danger to
cattle subsequently, however, proceeds from the infection of fields, yards,
and byres with the offal and the discharge from the mouths of anthrax
animals. All material that can be recognised as such should be burned
along with the straw in which the animals have lain. The stalls or
buildings in which the anthrax cases have been must be limewashed.
Needless to say, the greatest care must be taken in the case of men who
handle the animal or its carcase that they have no wounds on their
persons, and that they thoroughly disinfect themselves by washing their
hands, etc., in 1 to 1000 solution of corrosive sublimate, and that all
clothes soiled with blood, etc., from anthrax animals be thoroughly
boiled or steamed for half an hour before being washed.
The Immunising of Animals against Anthrax.— Having
ascertained that there was ground for believing that in cattle
one attack of anthrax protected against a second, Pasteur (in
the years 1880-82) elaborated a method by which a mild form
of the disease could be given to animals, which rendered
harmless a subsequent inoculation with virulent bacilli. He
found that the continued growth of anthrax bacilli at 42° to
43° C. caused them to lose their capacity of producing spores,
and also gradually to lose their virulence, so that after twenty-
four days they could no longer kill either guinea-pigs, rabbits,
or sheep. Such cultures constituted his premier vaccin, and
IMMUNISATION AGAINST ANTHRAX 315
protected against the subsequent inoculation with bacilli which
had been grown for twelve days at the same temperature, and
the attenuation of which had therefore not been carried so far.
The latter constituted the deuxieme vaccin. It was further
found that sheep thus twice vaccinated now resisted inoculation
with a culture which usually would be fatal. The method was
to inoculate a sheep on the inner side of the thigh by the
subcutaneous injection, from a hypodermic syringe, of about
five drops of the premier vaccin:, twelve days later to again
inoculate with the deuxieme vaccin • fourteen days later an
ordinary virulent culture was injected without any ill result.
This method was applicable also to cattle and horses, about
double the dose of each vaccine being here necessary. Extended
experiments in France generally confirmed earlier results, and
the method was, before long, used to mitigate the disease, which
in many departments was endemic and a very great scourge.
Since that time the method has been regularly in use. It is
difficult to arrive at a certain conclusion as to its merits.
Undoubtedly a certain number of animals die of anthrax either
after the first or second vaccination, or during the year following
vaccination. At the end of a year the immunity is lost in
about 40 per cent of the animals vaccinated ; and thus to be
permanently efficacious the process would have to be repeated
every year. Further, the immunity is much higher in degree
if, after the first and second vaccinations, an inoculation with
virulent anthrax is performed. Everything being taken into
account, however, there is no doubt that the mortality from
natural anthrax is much diminished by this system.
During the twelve years 1882-93 3,296,815 sheep were vaccinated,
with a mortality, either after the first or second vaccination, or during
the subsequent twelve months, of '94 per cent, as contrasted with the
ordinary mortality in all the flocks of the districts of 10 per cent.
During the same time 438,824 cattle were vaccinated, with a mortality
of '34 per cent, as contrasted with a probable mortality of 5 per cent if
they had been unprotected.
The immunisation of animals against anthrax Jhas always
been found to be a difficult proceeding. The most usual
technique has been to commence with Pasteur's vaccines, and to
follow these by careful dosage with virulent cultures. Marchoux
in this way produced immunity, and found that the serum of
immune animals had a certain degree of protective and curative
action. The most successful attempts in this direction have
been those of Sclavo and of Sobernheim. The former observer,
after trying various animals, came to the conclusion that the
316 ANTHKAX
ass was the most suitable. He first employed a method similar
to that of Marchoux; later, however, after noting the effects
of the serum of an animal so immunised, he commenced the
immunisation by injecting 5 to 15 c.c. of this serum along with
a slightly attentuated culture of the bacilli. A few days later
this was followed up with injections of virulent cultures which
could now be periodically introduced for many months, and a
high degree of immunity resulted. What was even more
important, the serum of such an animal had strongly protective
and curative properties. It has been extensively used in the
treatment of anthrax in man. In a case of malignant pustule
30 to 40 c.c. are injected in quantities of 10 c.c. into the
abdominal wall, and if necessary the injection is repeated on the
following day. In cases treated by Sclavo himself the serum is
alone employed, and its action is not aided by the excision
of the pustule usually practised. The results obtained have been
very good, — Sclavo, out of 164 cases, had only ten deaths or
about a fourth of the ordinary mortality in Italy. Sobernheim
independently elaborated an almost identical method of com-
bining passive with active immunisation for the obtaining of a
powerful anti-serum, and he has used this for the protective
inoculation of cattle. The technique is to inject the serum into
one side of the neck or into one thigh and the culture (Pasteur's
second vaccine) into the other side; the doses given are for
cattle or horses 5 c.c. of serum and '5 c.c. -culture, and for sheep
4 c.c. of serum and '25 c.c. culture. The method has been widely
used in Germany and in Brazil, and its originator claims as its
advantages simplification of application, in that one operation
instead of two is sufficient, less risk of death following the
immunisation procedure, and higher degree and more lasting
character of the immunity resulting. Whether this method is
really more efficient than that of Pasteur future experience will
show, but it might be preferable for developing protection in
herds at a time when an epidemic was raging. During the
development of active immunity it is likely in every case (see
Immunity) that there is a period of increased susceptibility to the
disease. Such a period would be more likely to occur with the
Pasteur method than with the Sobernheim procedure, where ,the
presence in the animal's body of the protective serum might tide
it over the stage when the action of the vaccine was lowering
its resistance.
The effects of the b. anthracis have been much studied with
a view to the shedding of light on the processes obtaining in
resistance and the development of immunity. Many puzzling
METHODS OF EXAMINATION 317
facts have long been known; for example, in the dog, which
shows great natural resistance, the serum has little if any
bactericidal action, while in the susceptible rabbit there is
present a serum capable of killing the organism. Such observa-
tions have hitherto been without explanation. Again the
properties of the serum of immune animals have been much
discussed. Sobernheim and others have been unable to detect
in it any trace of special bactericidal action. Sclavo found that
the serum when heated to 55° C. did not lose its protective
properties ; as the serum might have been complemented (see
Immunity) by the serum of the animal into which it was injected,
he simultaneously introduced an anti-complementary serum and
found that the heated serum was still effectual. From this he
deduces that in the action of the serum substances of the nature
of immune body and complement are not concerned. Many
have thought that the serum had a stimulating effect on the
leucocytes, but Cler has brought forward ground for supposing
that its effect is a sensitising one on the bacteria, and that thus
the effects are to be traced to opsonic action. With regard to
the formation of the protective substances, it is stated that the
spleen and bone-marrow are richer in these than the blood fluids.
In this connection an interesting fact may be mentioned, namely,
that Roger and Gamier found evidence of the liver and spleen
having special capacities for killing anthrax bacilli ; an otherwise
fatal dose could be introduced into the portal vein or the splenic
artery without causing death.
Methods of Examination. — These include (a) microscopic
examination ; (b) the making of cultures ; and (c) test in-
oculations.
(a) Microscopic Examination. — In a case of suspected
malignant pustule, film preparations should be made from the
fluid in the vesicles or from a scraping of the incised or excised
pustule, and stained with a watery solution of methylene-blue
and also by Gram's method. By this method practically con-
clusive evidence may be obtained ; but sometimes the result is
doubtful, as the bacilli may be very few in number. In all
cases confirmatory evidence should be obtained by culture.
Occasionally bacilli are so scanty that both film preparations
made from different parts and even cultures may give negative
results, and yet a few bacilli may be found when a section of
the pustule is examined. It should be noted that the greatest
care ought to be taken in manipulating a pustule before excision,
as the diffusion of. the bacilli into the surrounding tissues may
be aided and the condition greatly aggravated. The examination
318 ANTHRAX
of the blood in cases of anthrax in man usually gives negative
results, with the exception of very severe cases, when a few
bacilli may be found in the blood shortly before death, though
even then they may be absent.
(b) Cultivation. — A small quantity of the material used for
microscopic examination should be taken on a platinum needle,
and successive strokes made on agar tubes, which are then
incubated at 37° C. At the end of twenty-four hours anthrax
colonies will appear, and can be readily recognised from their
wavy margins by means of a hand lens. They should also be
examined microscopically by means of film preparations.
(c) Test Inoculations. — A little of the suspected material
should be mixed with some sterile bouillon or water, and
injected subcutaneously into a guinea-pig or mouse, or it may
be introduced into the subcutaneous tissue by means of a seton.
If anthrax bacilli are present, the animal usually dies within
two days, with the changes in internal organs already described.
CHAPTER XIV.
TYPHOID FEVER— BACILLI ALLIED TO THE
TYPHOID BACILLUS.
OTHER NAMES. ENTEEIC FEVER : GASTRIC FEVER. GERMAN,
TYPHUS ABDOMINALIS : ABDOMINALTYPHUS : UNTERLEIBS-
TYPHUS. FRENCH, LA FIEVRE TYPHOIDE.
Introductory. — The organism now known as the bacillus
typhosus was first described in 1880-1 by Eberth, who observed
its microscopic appearances in the intestinal ulcers and in the
spleen in cases of typhoid fever. It was first isolated (from
the spleen) in 1884 by Gaffky, and its cultural characters were
then investigated. In 1885 Escherich observed a bacillus, now
known as the bacillus coli communis, which occurs in the normal
intestine and which both microscopically and culturally closely
resembles the typhoid bacillus. Ordinarily the b. coli is no
doubt a harmless saprophyte, but under experimental conditions
in animals and also naturally in man it may manifest pathogenic
properties. Investigation has shown that these two bacilli
belong to a widespread group of organisms isolated from various
disease conditions, which all bear close resemblances to one
another and whose differentiation is often a matter of consider-
able difficulty. Other members, of this group are the para-
typhoid bacillus, the organism of bacillary dysentery, the b.
enteritidis of Gaertner, the psittacosis bacillus, and the bacillus
of hog cholera.
Bacillus Typhosus. — Microscopic Appearances. — It is some-
times difficult to find the typhoid bacilli in the organs of a
typhoid patient. Numerous sections of different parts of a
spleen, for example, may be examined before a characteristic
group is found. The best tissues for examination are a Peyer's
patch where ulceration has not yet commenced or where it is
319
320
TYPHOID FEVER
just commencing, the spleen, the liver, or a mesenteric gland.
The spleen and liver are better than the other tissues named, as
in the latter the presence of the b. coli is more frequent. From
scrapings of such solid organs dried films may be prepared and
stained for a few minutes in the cold by any of the strong
staining solutions, e.g. with carbol-thionin-blue, or with Ziehl-
Neelsen carbol - fuehsin
diluted with five parts
of distilled water. As a
^ rule decolorising is not
necessary. For the proper
V\ observation of the ar-
*A rangement of the bacilli
in the tissues, paraffin
sections should be pre-
pared and stained in
carbol-thionin-blue for a
fewminutes, orinLofner's
methylene-blue for one
or two hours. The bacilli
take up the stain some-
what slowly, and as they
FIG. 110.-A large clump of typhoid bacilli are also easily decolorised
in a spleen. The individual bacilli are the aniline-Oil method ot
only seen at the periphery of the mass, dehydration may be used
(In this spleen enormous numbers of with advantage (vide p.
typhoid bacilli were shown by cultures to g3)> Jn 8uch prepara.
be present in a practically pure condition. ) . > r. .
Paraffin section ; stained with carbol-thionin- tions the characteristic
blue, x 500. appearance to be looked
for is the occurrence of
groups of bacilli lying between the cells of the tissue (Fig. 110).
The individual bacilli are 2 ^ to 4 //, long, with somewhat oval
ends, and -5 //. in thickness. Sometimes filaments 8 ^ to 10 ^
long may be observed, though they are less common than in
cultures. It is evident that one of the short oval forms may
frequently in a section be viewed endwise, in which case the
appearance will be circular. This appearance accounts for some,
at least, of the coccus-like forms which have been described.
The bacilli are decolorised by Gram's method.
Isolation and Appearances of Cultures. — To grow the
organism artificially it is best to isolate it from the spleen, as it
exists there in greater numbers than in the other solid organs,
and may be the sole organism present even some time after
death. The spleen is removed whole, and a portion of its
ISOLATION AND APPEARANCES OF CULTURES 321
capsule is seared with a cautery to destroy all superficial con-
taminating organisms. A small incision is made into the organ
with a sterile knife, a little of the pulp removed by a platinum
needle, and agar or gelatin plates are prepared, or successive
strokes made on agar tubes. On the agar media the growths
are visible after twenty-four hours' incubation at 37° C. On
agar plates the superficial colonies are thin and film-like, circular
or slightly irregular at the margins, dull white by reflected light,
bluish-grey by transmitted light. Colonies in the substance of
the agar are small, and appear as minute round points. When
viewed under a low ob-
jective, the surface
colonies are found to be
very transparent (requir-
ing a small diaphragm
for their definition), finely
granular in appearance,
and with a very coarsely
crenated and well-defined
margin. The deep colonies
are usually spherical,
sometimes lenticular in
shape, and are smooth or
finely granular on the
surface, and more opaque
than the superficial
colonies. On making
rovpr ffW nrprarfltinn? Fm< UL— Typhoid bacilli, from a young
.over-glass preparations, culture Qn agai. showi some fiiamentous
the bacilli are tound to forms.
present the same micro- Stained with weak carbol-fuchsin. x 1000.
scopic appearances as are ~~
observed in preparations from solid organs, except that there
may be a greater number of the longer forms which may
almost be called filaments (Fig. 111). (The same is true of films
made from young gelatin colonies) Sometimes the diversity in
the length of the bacilli is such as to throw doubt on the purity
of the culture. Its purity, of course, can be readily tested by
preparing plates from it in the usual way. As a general rule in
a young (twenty-four to forty-eight hours old) colony, grown at
a uniform temperature, the bacilli are plump, and the protoplasm
stains uniformly. In old cultures, or in cultures which have
been exposed to changes of temperature, the protoplasm stains
only in parts ; there may be an appearance of irregular vacuola-
tion either at the centre or at the ends of the bacilli. There
21
322
TYPHOID FEVER
is no evidence that spore -formation occurs in the typhoid
bacillus.
Mobility. — In hanging-drop preparations the bacilli are found
to be actively motile. The smaller forms have a darting or
rolling motion, passing quickly across the field, whilst some show
rapid rotatory motion. The filamentous forms have an un-
dulating or serpentine motion, and move more slowly. Hanging-
drop preparations ought to be made from agar or broth cultures
Fm. 1.12. — Typhoid bacilli, from a young culture on agar, showing flagella.
Stained by Van Ermengem's method. x 1000.
not more than twenty-four hours old. In older cultures the
movements are less active.
Flagella. — On being stained by the appropriate methods (vide
p. 103) the bacilli are seen to possess many long wavy flagella
which are attached all along the sides and to the ends (Fig. 112).
They are more numerous, longer, and more wavy than those of
the b. coli.
Characters of Cultures. — Stab cultures in peptone gelatin give
a somewhat characteristic appearance. On the surface of the
medium growth spreads outwards from the puncture as a thin
APPEARANCES OF CULTURES
323
film or pellicle, with irregularly wavy margin (Fig. 113, A). It
is semi-transparent and of bluish-white colour. Ultimately this
surface growth may reach the wall of the tube. Not infrequently,
however, the surface growth is not well marked. Along the
stab there is an opaque whitish line of growth, of finely nodose
appearance. There is no liquefaction of the medium, and no
formation of gas. In stroke cultures there is a thin bluish-white
A. Stab culture of the typhoid bacillus in gelatin, five days' growth.
B. Stroke culture of the typhoid bacillus on gelatin, six days' growth.
C. Stab culture of the bacillus coli in gelatin, nine days' growth ; the gelatin is split
in its lower part owing to the formation of gas.
film, but it does not spread to such an extent as in the case of
the surface growth of a stab culture (Fig. 113, B). In gelatin
plates also the superficial and deep colonies present correspond-
ing differences. The former are delicate semi-transparent films,
with wavy margin, and are much larger than the colonies in the
substance, which appear as small round points (Fig. 114). These
appearances, which are well seen on the third or fourth day,
resemble those seen in agar plates, as already described in the
method of isolation ; but on gelatin the surface colonies are
324 TYPHOID FEVER
rather more transparent than those on agar. Their characters,
as seen under a low power of the microscope, also correspond.
In stroke cultures on agar there is a bluish-grey film of
growth, with fairly regular margins, but without any character-
istic features. This film is loosely attached to the surface, and
can be easily scraped off.
The growth on potatoes is important. For several days (at
incubation temperature) after inoculation there is apparently
no growth. If looked at obliquely, the surface appears wet,
and if it is scraped with the platinum loop, a glistening track
is left ; a cover-glass pre-
paration shows numerous
bacilli. Laterfhowever,
a slight pellicle with a
dull, somewhat velvety
surface may appear, and
this may even assume
a brown appearance)
These characteristic ap-
pearances are only seen
when a fresh potato with
an acid reaction has been
used.
In bouillon incubated
at 37° C. for twenty-four
hours there is simply a
FIG. 114. — Colonies of the typhoid bacillus uniform turbidity
(one superficial and three deep) in a gelatin Cover-glass preparations
plate. Three days growth at room tern- , ^
perature x 15. made from such some-
times show filamentous
forms of considerable length without apparent segmentation.
Conditions of Growth, etc. — The optimum temperature of the
typhoid bacillus is about 37° C., though it also nourishes well at
the room temperature. It will not grow below 9° C. or above
42° C. Growth takes place in anaerobic as well as in aerobic
conditions. Its powers of resistance correspond with those of
most non-sporing bacteria. It is killed by exposure for half an
hour at 60° C., or for two or three minutes at 100° C. Typhoid
bacilli kept in distilled or in ordinary tap water have usually
been found to be dead after three weeks (Frankland).
Bacillus coli communis. — This bacillus is the chief organism
present in the small intestine in normal conditions, and, with
many other bacteria, it also inhabits the large intestine. During
typhoid fever, and other pathological conditions affecting the
REACTIONS OF B. TYPHOSUS AND B. COLI 325
intestines, it is relatively and absolutely enormously increased
in the latter situation, where it may sometimes be almost the
only bacillus present. Its relations to various suppurative and
inflammatory conditions are described in the chapter on Suppura-
tion (p. 185). Microscopically it has the same appearances and
staining reaction as the typhoid bacillus, and like the latter also
presents variations in size, though it is usually somewhat shorter
(Fig. 115). It is motile, and possesses lateral flagella, which,
however, are fewer in number and somewhat shorter than those
of the typhoid bacillus. It is easily isolated from the stools
of men and animals by
any of the ordinary
methods. After twenty-
four hours' incubation at
37° C. on agar, there are
large superficial colonies
and small deep colonies
in the plates ; to the
naked eye they are
denser and more glisten-
ing than those of typhoid
when viewed by trans-
mitted light, and rather of
a brownish-white colour.
Under a low objective
the colonies again appear
denser than those of the
typhoid bacillus and more
granular. On ordinary
gelatin and agar media
the appearances are similar to those of the typhoid bacillus,
but the growth is whiter, thicker, and more opaque, and gives
the impression of having greater vigour. In the case of gelatin
stab cultures a few gas bubbles sometimes develop in the
medium (Fig. 113, C). On potatoes in forty-eight hours there
is a distinct film of growth of brownish tint and moist-looking
surface, which rapidly spreads and becomes thicker. This con-
trasts very markedly with the colourless film of the b. typhosus.
The Comparative Culture Reactions of the B. typhosus
and the B. coli. — The importance of the relationships between
the b. typhosus and the b. coli has caused great attention to be
paid to their biological characters, in order to facilitate the
distinction of the one from the other. Some of these we have
already noted. Of the cultural characters the growth on
FIG. 115. — Bacillus coli com munis. Film
preparation from a young culture on agar.
Stained with weak carbol-fuchsin. x 1000.
326 TYPHOID FEVER
potatoes is the most important. As lias been pointed out by
Wathelet, and also by Klein, differences exist in the growth of
the two bacilli in melted gelatin. A gelatin tube is inoculated,
and instead of being kept at the room temperature, is placed in
the incubator at 37° C., at which temperature it is of course
fluid. In such cultures, in the case of the b. typhosus, there is
a general turbidity of the gelatin, while with the b. coli there
are large flocculi developed which float on the surface. It is,
however, to physiological differences between the bacilli, rather
than to morphological, that importance is to be attached. In
detailing the following reactions we must note that all that can
be said is that under certain conditions certain effects are obtained.
We cannot profess to know the principles which underlie the
occurrence of these effects, and it may be that in several
apparently diverse reactions the same biological processes are
really at work.
(1) The Fermentation of Sugars. — Of these one of the most
important is the effect on lactose as first pointed out by
Chantemesse and Widal. This is usually demonstrated by
using a 1 per cent solution of the sugar in peptone-salt solution
placed in Durham's tubes (p. 76). If such a medium be
coloured with litmus the production of acid and gas by the
b. coli can easily be demonstrated. Similar changes caused by
this organism can also be observed in litmus milk and in
Petruschky's litmus whey.
Chantemesse and Widal first showed that the b. typhosus
does not act on lactose in bouillon though decolorisation of the
litmus may occur. It may be stated that under most conditions
of making the test an acid reaction does not result and there is
never any formation of gas. This organism is said, however, to
break up lactose in litmus milk and in litmus whey with some
acid formation. Much would thus seem to depend upon what
other constituents are present in the medium, and also, it may be
said, on its initial reaction.
The lactose fermenting power of the b. coli is of the greatest
importance ; and if MacConkey's bile-salt lactose fluid medium be
used, this organism and its congeners can be distinguished from
the b. typhosus, b. para typhosus, and from the dysentery bacilli
(v. infra), none of whose colonies are crimson on this medium.
The effects of the b. coli and the b. typhosus on other sugars
is also of great importance. As media to which the sugars may
be added, either peptone-salt solution or MacConkey's bile-salt
media are used (q.v.). To sum up the general results it may be
said that b. coli produces acid and gas in bile-salt glucose,
REACTIONS OF B. TYPHOSUS AND B. COLI 327
peptone-salt glucose, lactose and mannite, but not in cane sugar,1
while the b. typhosus produces acid without gas in bile-salt
glucose, peptone-salt glucose, and mannite, but not in lactose or
cane sugar. It can also cause similar changes in arabinose,
galactose, and fructose.
Gas production by the b. coli can also be demonstrated
by means of shake cultures. As ordinary bouillon contains
traces of glucose it is best to use peptone-salt solution to which
an appropriate sugar has been added and which has been
converted into a solid medium with 10 per cent gelatine. If
such a medium be inoculated in the fluid condition, shaken and
set aside till growth occurs, small bubbles of gas will form all
throughout it. In ordinary media inoculated with the b. coli
bubbles of gas are often developed along the needle track.
In the case of acid production by the b. coli or b. typhosus in
ordinary media the acid probably comes, as has been said, from
the glucose developed from the muscle sugar, but there may also
be a subsidiary acid formation from the breaking up of the
proteid elements.
In certain members of the coli-typhoid group it has been
observed that in such media as litmus milk or litmus whey an
acid reaction may be first produced, and this may be followed
after a few days by the formation of alkali, and in certain cases
this phenomenon may be helpful in differentiating the species.
Curdling of Milk by the B. coli. — This probably depends on
the fermentation of the lactose of the milk and the throwing
down of the casein by the resulting lactic acid ; but the reaction
may be a more complicated one, as milk can be curdled by
organisms which do not possess acid-forming properties. In any
case the observation of the reaction is important. The typhoid
bacillus produces no visible change in milk.
(2) Action on Media containing Neutral-Red. — While, as will
have been already gathered, neutral-red is used as an indicator,
there is some evidence that an actual breaking up of the
substance can take place by the action of the coli-typhoid group ;
the evidence for this lies in the fact that when the effects of
acid formation are observed the tint of the medium cannot be
brought back by the addition of alkali. The medium used here is
bouillon containing an appropriate sugar and *5 per cent of a 1
per cent watery solution of Grubler's neutral-red. In the case of
the typhoid bacillus no change occurs, but in the case of the b.
coli there is developed a beautiful canary yellow with a greenish
1 A variety of the organism which does ferment 'cane sugar has been
described under the name of the b. coli commuuior.
328 TYPHOID FEVER
fluorescence. Fitz- Gerald and Dreyer have shown that an
important factor here is the reaction of the medium, and that the
effects of the bacteria may be one of degree, — under certain
circumstances the effects described as characteristic of the two
organisms may be reversed.
(3) Formation of Indol. — Among the bacteria capable of
forming indol is to be classed the b. coli. Indol can be
recognised in bouillon cultures of the b. coli three to four days
•old by the usual tests (vide p. 77). As there is no evidence
that it can produce nitrites a small quantity of the latter must
be added. The typhoid bacillus never gives this reaction when
growing in ordinary conditions, but on the other hand, it
appears that some varieties of the b. coli fail to produce it also.
Peckham, however, has found that if the typhoid bacillus be
grown in peptone solution, after a few generations of three
days each it may acquire the property of producing indol. The
formation of indol by an organism after the first transference to
peptone solution from one of the ordinary media may, however,
be accepted as evidence in favour of the organism not being the
typhoid bacillus. It is to be noted here that the presence of
sugar in a medium retards the production of indol by the b.
coli. The indol reaction thus ought to be sought for in a sugar-
free medium.
(4) The Media of Capaldi and Proskauer. — The first of these
("No. 1 ") is a medium free of albumin, in which b. coli grows
well and freely produces acid, while the typhoid bacillus hardly
grows at all, and certainly will produce no change in the
reaction. Its composition is as follows : asparagin *2 parts,
mannite *2, sodium chloride '02, magnesium sulphate '01,
calcium chloride '02, potassium monophosphate '2, distilled
water to 100 parts. The second medium ("No. 2") contains
albumin, and is such that the b. coli produces no acid, while
the typhoid bacillus grows well and produces an acid reaction.
It consists of Witte's peptone 2 parts, mannite '1, distilled water
to 100 parts. After the constituents of each medium are mixed
and dissolved, it is steamed for one and a half hours and then
made neutral to litmus — the first medium, being usually naturally
acid, by sodium hydrate, the second, being usually alkaline, by
citric acid. The medium is then filtered, filled into tubes con-
taining 5 c.c., and these are sterilised. After inoculation for
twenty hours the reaction of the medium is tested by adding
litmus.
(5) The Application of the Agglutination Test in distinguish-
ing B. typhosus from B. coli. — The scope of the application of
PATHOLOGICAL CHANGES^ 329
this test will be discussed later (see Immunity). Here we may
say that a negative result obtained with a suspected b.
typhosus culture is of greater value than a positive result
obtained with a suspected b. coli culture. The test is to be
taken in conjunction with the other means of differentiating the
two organisms (cf. p. 340).
It will thus be seen that the diagnosis between the b.
typhosus and the b. coli is a matter of no small difficulty.
There is no evidence that the one organism ever passes into the
other. Great difficulties sometimes arise in consequence of a
bacillus being found which, while giving a number of the
characteristics of either one or the other, fails to give some of
the characteristic tests, or only gives them very slowly. This is
especially true of organisms related to the b. coli. It has
consequently become common to speak of the typhoid group
and the coli group in order that such varieties may be included.
Pathological Changes in Typhoid Fever. — Here we confine
our attention solely to the bacteriological aspects of the disease.
The inflammation and ulceration in the Peyer's patches and
solitary glands of the intestine are the central features. In the
early stage there is produced an acute inflammatory condition,
attended with extensive leucocytic emigration and sometimes
with small haemorrhages. At this period the typhoid bacilli are
most numerous in the patches, groups being easily found between
the cells. The subsequent necrosis is evidently in chief part the
result of the action of the toxic products of the bacilli, which
gradually disappear from their former positions, though they
may still be found in the deeper tissues and at the spreading
margin of the necrosed area. They also occur in the lymphatic
spaces of the muscular coat. It is to be remarked that
the number of the ulcers arising in the course of a case bears no
relation to its severity. Small ulcers may occur in the lymphoid
follicles of the large intestine.
The mesenteric glands corresponding to the affected part of
the intestine are usually enlarged, sometimes to a very great
extent, the whole mesentery being filled with glandular masses.
In such glands there may be acute inflammation, and occasionally
necrosis in patches occurs. Sometimes on section the glands
are of a pale-yellowish colour, the contents being diffluent and
consisting largely of leucocytes. Typhoid bacilli may be isolated
both from the glands and the lymphatics connected with them,
but the b. coli is in addition often present.
The spleen is enlarged, — on section usually of a fairly firm
consistence, of a reddish-pink colour, and in a state of congestion.
330 TYPHOID FEVER
Of all the solid organs it usually contains the bacilli in greatest
numbers. They can be seen in sections, occurring in clumps
between the cells, there being no evidence of local reaction
round them (Fig. 110). Similar clumps may occur in the liver
in any situation, and without any local reaction. In this organ,
however, there are often small foci of leucocytic infiltration, in
which, so far as our experience goes, bacilli cannot be demon-
strated. The bacillus is found, often in large numbers, in the
gall-bladder, where it may persist for years. Clumps of bacilli
may also occur in the kidney.
In addition to these local changes in the solid organs there are also
widespread cellular degenerations in the solid organs which suggest the
circulation of soluble poisons in the blood.
In the lungs there may be bronchitis, patches of congestion and of
acute broncho-pneumonia. In these, typhoid bacilli may sometimes be
observed, but evidence of a toxic action depressing the powers of resist-
ance of the lung tissue is found in the fact that the pneumococcus
frequently occurs in such complications of typhoid fever.
The nervous system shows little change, though meningitis associated
either with the typhoid bacillus, with the b. coli, or with the streptococcus
pyogenes has been observed.
The typhoid bacilli probably travel by the blood stream, and they can
be isolated from the blood much more readily than was formerly supposed.
Considerable quantities of blood (say, 4 c.c.) must of course be taken
(v. p. 68). They have been found in the roseolar spots which occur in
typhoid fever, but it cannot be yet stated that such spots are always due
to the presence of the bacilli. The fact that the typhoid bacilli are usually
confined to certain organs and tissues shows that they probably have a
selective action on certain tissues.
To sum up the pathology of typhoid fever, we have in it a
disease the centre of which lies in the lymphoid tissue in and
connected with the intestine. In this situation we must have
an irritant, against which the inflammatory reaction is set up,
and which in the intestine is sufficiently powerful to cause
necrosis. The affections of the other organs of the body suggest
the circulation in the blood of poisonous substances capable of
depressing cellular vitality, and producing histological changes.
The occurrence of bacilli in the blood and organs links typhoid
fever with septicaemic processes.
Suppurations occurring in connection with Typhoid Fever.—
With regard to the relation of the typhoid bacillus to such
conditions, statements as to its isolation from pus, etc., can be
accepted only when all the points available for the diagnosis of
the organism have been attended to. On this understanding
the following summary may be given : — In a small proportion
of the cases examined the typhoid bacillus has been the only
PATHOGENIC EFFECTS OF B. TYPHOSUS 331
organism found. This lias been the case in subcutaneous
abscesses, in suppurative periostitis, suppuration in the parotid,
abscesses in the kidneys, etc., and probably also in one or two
cases of ulcerative endocarditis. But in the majority of cases
other organisms, especially the b. coli and the pyogenic
micrococci, have been obtained, the typhoid bacillus having been
searched for in vain. It has, moreover, been experimentally
shown, notably by Dmochowski and Janowski, that suppuration
can be experimentally produced by injection in animals, especially
in rabbits, of pure cultures of the typhoid bacillus, the occurrence
of suppuration being favoured by conditions of depressed vitality,
etc. These observers also found that when typhoid bacilli were
injected along with pyogenic staphylococci, the former died out
in the pusjnore quickly than the latter. Accordingly, in clinical
cases where the typhoid bacillus is present alone, it is improbable
that other organisms were present at an earlier date.
Pathogenic Effects produced in Animals by the Typhoid
Bacillus. — There is no disease known to veterinary science
which can be said to be identical with typhoid, nor is there any
evidence of the occurrence of the typhoid bacillus under ordinary
pathological conditions in the bodies of animals. Attempts
to communicate the disease to animals by feeding them on
typhoid dejecta have been unsuccessful, and though pathogenic
effects have been produced by introducing pure cultures in
food, the disease has usually borne no resemblance to human
typhoid. The most successful experiments have been those of
Remlinger, who, by continuously feeding rabbits on vegetables
soaked in water containing typhoid bacilli, produced in certain
cases symptoms resembling those of typhoid fever (diarrhoea,
remittent pyrexia, etc.). An agglutinating action was observed
in the serum, and post mortem there was congestion of the
Peyerian patches, and typhoid bacilli were isolated from
the spleen.
While feeding experiments are thus rather unsatisfactory, the
same may be said of the results of subcutaneous or intraperitoneal
infection. Here, again, pathogenic effects can easily be produced
by the typhoid bacillus, but these effects are of the nature of a
short acute illness characterised by pyrexia, rapid loss of weight,
inability to take food, and frequently ending fatally in from
twenty-four to forty-eight hours. The type of disease is thus very
different from what occurs naturally in man. In such injection
experiments the results vary considerably, sometimes scarcely
any effect being produced by a large dose of a culture. This
is no doubt due to the fact that different strains of the bacillus
332 TYPHOID FEVER
vary much, in virulence. Ordinary laboratory cultures are often
almost non-pathogenic. They can, however, be made virulent
in various ways. Sanarelli used the method of injecting
sterilised cultures of the b. coli intraperitoneally at the same
time as the typhoid bacillus was introduced subcutaneously.
After this procedure had been repeated through a series of
animals a culture of typhoid was obtained of exalted virulence.
Sidney Martin has obtained virulent cultures by passing bacilli,
derived directly from the spleen of a person dead of typhoid
fever, through the peritoneal cavities of a series of guinea-pigs.
Sanarelli, studying the effects of the intraperitoneal injection
of a few drops of a culture of highly exalted virulence, found
that the Peyer's patches and solitary glands of the intestine
were enormously infiltrated, sometimes almost purulent, and
that they contained typhoid bacilli, as also did the mesenteric
lymphatics and glands, and the spleen. These results are
interesting, but have not been confirmed.
The Toxic Products of the Typhoid Bacillus. — Here very
little light has been thrown on the pathology of the disease, but
the general results may be outlined. We may state that there
exist in the bodies of typhoid bacilli toxic substances, that in
artificial cultures these do not pass to any great degree out into
the surrounding medium, and that though they produce effects on
the intestine, there is evidence that such effects are not character-
istic and not peculiar to the toxins of the b. typhosus. Sidney
Martin found that the bodies of bacteria killed by chloro-
form vapour were very toxic, — more so than filtered cultures.
Diarrhoea was a constant symptom after injection, but no change
in the Peyerian patches was observed. Martin found that
virulent cultures of the b. coli gave similar results when
similarly treated. Allan Macfadyen, by grinding up typhoid
bacilli frozen solid by liquid air, produced a fluid whose toxic
effect he attributed to the presence of the intracellular poisons.
The Immunisation of Animals against the Typhoid
Bacillus. — Earlier observers had been successful in accustoming
mice to the typhoid bacillus by the successive injections of small
and gradually increasing doses of living cultures of the bacillus.
Later, Brieger, Kitasato, and Wassermann found that the bacillus
when modified by being grown in a bouillon made from an
extract of the thymus gland no longer killed mice and guinea-
pigs. These animals after injection were moreover immune, and
it was also found that the serum of a guinea-pig thus immunised
could, if transferred to another guinea-pig, protect the latter
from the subsequent injection of a dose of typhoid bacilli to
PATHOGENICITY OF B. COLI 333
which it would naturally succumb. Chantemesse and Widal,
Sanarelli, and also Pfeiffer, succeeded in immunising guinea-pigs
against the subsequent intraperitoneal injection of virulent
living typhoid bacilli, by repeated and gradually increasing
intraperitoneal or subcutaneous doses of dead typhoid cultures
in bouillon. Experiments performed with serum derived from
.typhoid patients and convalescents indicate that similar effects
occur in those who have successfully resisted the natural disease.
The serum of such patients has antibacterial powers, but
there is no evidence that it contains any antitoxic bodies (see
chapter on Immunity). Pfeiffer, for example, found on adding
serum from typhoid convalescents to the bodies of typhoid
bacilli killed by heat, and injecting the mixture into guinea-
pigs, that death took place as in control animals which had
received these toxic agents alone. Pfeiffer also found that by
using the serum of immunised goats, he could, to a certain
extent, protect other animals against the subsequent injection
of virulent living typhoid bacilli. On trying to use the agent
in a curative way, i.e. injecting it only after the bacilli had
begun to produce their effects, he got little or no result.
The Pathogenicity of the B. coli and its Relation to that of the
Typhoid Bacillus. — We have already seen that the b. coli is probably
responsible for the occurrence of some of the abscesses which follow
typhoid fever. It is also apparently the cause of some cases of summer
diarrhoea (cholera nostras), of infantile diarrhoea, and of some food
poisonings. Its numbers in the intestine are greatly increased during
typhoid fever, and also during any pathological condition affecting the
intestine. Intraperitoneal injection in guinea-pigs is often fatal. Sub-
cutaneous injection may result in local abscesses, and sometimes in death
from cachexia. Sanarelli found that the b. coli isolated from typhoid
stools was much more virulent than when isolated from the stools of
healthy persons. He holds that the increase in virulence is due to the
effect of typhoid toxins. This increased virulence of the b. coli in the
typhoid intestine makes it possible that some of the pathological changes
in typhoid may be due, not to the typhoid bacillus, but to the b. coli.
Some of the general symptoms may be intensified by the absorption of
toxic products formed by it and by other organisms. It is to be noted
that lesions produced in guinea-pigs are very similar to those of the b.
typhosus. Differences of behaviour of the two bacilli in connection with
their pathological effects have been brought forward as confirmatory of
the fact of their being distinct species. Thus Sanarelli accustomed the
intestinal mucous membrane of guinea-pigs to toxins derived from an old
culture of the b. coli, by introducing day by day small quantities of the
latter into the stomach. When a relatively large dose could be tolerated,
it was found that the introduction in the same way of a small quantity
of typhoid toxin was still followed by fatal result. Pfeiffer also found
that while the serum of convalescents from typhoid paralysed the
typhoid bacilli, it had no more effect on similar numbers of b. coli than
the serum of healthy men.
334 TYPHOID FEVER
General View of the Relationship of the B. typhosus to
Typhoid Fever. — 1. We have in typhoid fever a disease having
its centre in and about the intestine, and acting secondarily on
many other parts of the body. In the parts most affected there
is always a bacillus present, microscopically resembling other
bacilli, especially the b. coli which is a normal inhabitant of
the animal intestine. This bacillus can be isolated from the
characteristic lesions of the disease and from other parts of the
body as described, and further, it is found by culture and serum
reactions to differ from other organisms. Here the important
point is that a bacillus giving all the reactions of the typhoid
bacillus has never been isolated except from cases of typhoid
fever, or under circumstances that make it possible for the
bacillus in question to have been derived from a case of typhoid
fever.
2. A difficulty in the way of accepting the etiological relation-
ship of the b. typhosus lies in the comparative failure of attempts
to cause the disease in animals. We have noted, however, that
in nature animals do not suffer from typhoid fever.
3. The observations of Pfeiffer and others on the protective
power against typhoid bacilli shown, on testing in animals, to
belong to the serum of typhoid patients and convalescents, and
the peculiar action of such serum in immobilising and causing
clumping of the bacilli (vide infra) are also of great importance
as indicating an etiological relationship between the bacillus and
the disease. Additional important evidence is found in the fact
that vaccination by means of the dead bacilli (vide infra) has a
marked effect in preventing the disease from arising in a popula-
tion exposed to infection, and also in lowering the mortality
when the fever attacks those who have been inoculated. These
facts may thus be accepted as indirect but practically conclusive
evidence of the pathogenic relationships of the typhoid bacillus
to the disease.
According to our present results we must thus hold that the
b. typhosus constitutes a distinct species of bacterium, and
that it is the cause of typhoid fever. Evidence of an important
nature confirmatory of this view is, we think, found in the fact
that cases have occurred where bacteriologists have accidentally
infected themselves by the mouth with pure cultures of the
typhoid bacillus, and after the usual incubation period has
developed typhoid fever. Several cases of this kind have been
brought to our notice and are not, we think, vitiated by the fact
that other similar instances have occurred without the subsequent
development of illness. These latter would be accounted for by
PARATYPHOID BACILLUS 335
a low degree of susceptibility on the part of the individual or to
a want of pathogenicity in the cultures.
The Paratyphoid Bacillus. — This organism, which was when
first described often denominated the paracolon bacillus, was
primarily isolated from abscesses occurring in apparently non-
typhoid cases. Widal noted its resemblances to b. typhosus and
b. coli, from the latter of which it differed in not producing
indol and in not fermenting lactose. Gwynn first isolated it
from the blood of a case presenting typhoid symptoms, and since
then it has been recognised as being the probable cause of the
disease effects in about 3 per cent of cases which clinically are to
be described as typhoid fever. The case-mortality in paratyphoid
fever is low, being only from 1 to 2 per cent. The organism
has been isolated from the blood, the roseolar spots, and from
the stools, — plates of MacCorikey's bile -salt neutral -red agar
with 1 per cent lactose added being recommended for use here.
Several strains showing slight differences in culture reactions
have been obtained. Generally speaking, the cultural reactions
resemble those of b. coli, though the growth on potato some-
times presents typhoid characteristics. It produces no indol,
or at least (with some strains) merely a trace, and its action
on sugars also differs. With regard to the latter, different
results have been obtained by different observers, but there is
general agreement that, like the b. coli, it produces acid and gas
in glucose, laevulose, sorbite, mannite, dextrin, maltose, dulcite,
galactose, and arabinose; but in lactose, like typhoid, it either
originates no change or only slight acid production without any
gas formation. It also causes no change in cane sugar, erythrite,
salicin, inulin, and raffinose. Probably the most important
reactions which will in any case aid in the recognition of the
paratyphoid bacillus are the agglutinating reactions. The
serum of a paratyphoid patient will agglutinate the bacillus in
high dilutions. Observations on the behaviour of such sera
towards the b. typhosus have in different cases yielded some
discordant results, but to speak generally, it may be said that
usually a very much stronger concentration is necessary to give
clumping, and often a paratyphoid serum will not clump the
typhoid bacillus except in such concentrations as might give
similar effects when normal sera are under observation. When
any serum clumps both the paratyphoid and the typhoid bacilli
the more closely the maximal clumping dilutions correspond, the
more likely is the case to be typhoid fever ; on the other hand,
if a high dilution will clump the paratyphoid bacillus, while
a low dilution is necessary for the typhoid bacillus, then
336 TYPHOID FEVER
the case is likely to be paratyphoid fever. With regard to the
effects of other sera on the paratyphoid bacillus, it may be said
that usually a typhoid serum will require to be used in greater
concentrations to clump this bacillus than are necessary to obtain
an effect with the typhoid bacillus itself. Similar effects are
observed when the sera of animals immunised against Gaertner's
bacillus or the bacillus of psittacosis are used. In all serum
tests the essential point is that deductions should alone be based
on comparative observations of the highest dilutions in which
a clumping effect is produced with any series of organisms
compared.
As has been indicated, a disease resembling typhoid fever is
not the only condition originated by the paratyphoid bacillus.
The organism has been isolated from cases of bone abscess, from
orchitis, and in WidaPs case from a thyroid abscess, and in such
cases the history of a previous typhoid-like illness may not be
elicited. It has also been found in ordinary faeces. In animal
experiments it produces in rabbits and guinea-pigs a fatal ill-
ness of a septicaemic type with serous inflammations.
Bacillus Enteritidis (Gaertner).— In 1888 Gaertner, in
investigating a number of cases of illness resulting from eating
the flesh of a diseased cow, isolated, from the meat and from the
spleen of a man who died, a bacillus closely resembling the
typhoid bacillus. Since then a great number of outbreaks of
gastro-enteritis due to eating diseased meat have been inquired
into, and very frequently similar bacilli have been found both in
the stools and in the organs. These bacilli closely resemble the
paratyphoid organism, — indol is not produced, and generally
speaking the fermentations of sugars also correspond. With
regard to the latter it may, however, be said that, according to
some, lactose is fermented, while other observers have found this
not to be the case. No doubt different strains differ somewhat
from one another. Here again much information may be
obtained from the agglutinating properties of the serum and also
from the effects on suspicious bacilli of the sera of animals
immunised against other strains and other members of the coli
group. It has also been found that the serum of persons suffer-
ing from meat poisoning sometimes clumps the typhoid bacillus,
though a higher concentration is required than in the case of
Gaertner's bacillus. The Gaertner group of organisms is very
pathogenic for laboratory animals. Often, whatever the channel
of infection, there is intense haemorrhagic enteritis, and very
usually there is a septicaemia with the occurrence of serous inflam-
mations ; the bacilli are recoverable from the solid organs and
PSITTACOSIS BACILLUS 337
often from the blood. In man, as the name of the bacillus
indicates, the symptoms are centred in the intestine, where there
is usually marked inflammation of the mucous membrane, some-
times attended with haemorrhage into it ; evidence of a septicaemic
condition may also exist. Infection may take place by the
bacillus itself where meat has been insufficiently cooked or
merely pickled, and here the illness usually appears within
twenty-four hours of the food being partaken of, but symptoms
may appear almost at once, in which case they are no doubt due
to the action of toxins ; here it is important to note that the
poisons formed by this group of organisms are relatively heat-
resisting, so that boiling for a time does not destroy the toxicity.
In cases of Gaertner bacillus poisoning, the animal whose
carcase is suspected has usually been found to have been itself
suffering from the action of the bacillus, but cases of meat
poisoning also occur where the meat of a healthy animal becomes
infected subsequently to slaughter with organisms pathogenic to
man. In such cases these organisms are often varieties of the
b. coli group, and indeed the b. coli itself may be the cause of
meat poisoning.
The Psittacosis Bacillus. — When parrots are imported from the
tropics in large numbers many die of a septicaemia condition in which an
enteritis, it may be hsemorrhagic, is a marked feature. There is intense
congestion of all the organs and peritoneal ecchymoses. From the
spleen, bone marrow, and blood there has been isolated a short,
actively motile bacillus with rounded ends which does not stain by
Gram's method. It, grows on all ordinary media, and on potato resembles
b. coli. It does not liquefy gelatin, does not ferment lactose, does not
curdle milk, and gives no indol reaction. Culturally the organism is
practically indistinguishable from the two bacilli last described. The
parrot is most susceptible to its action, but it also causes a fatal
hsemorrhagic septicaemia in guinea-pigs, rabbits, mice, pigeons, and
fowls, the bacilli after death being chieny in the solid organs. From
affected parrots the disease appears to be readily communicable to man,
chiefly, it is probable, from the feathers being soiled by infective
excrement. Several small epidemics have been recognised and in-
vestigated in Paris. After about ten days' incubation, headache,
fever, and anorexia occur, followed by great restlessness, delirium, vomit-
ing, often diarrhoea, and albuminuria. Frequently broncho-pneumonia
supervenes, and a fatal result has followed in about a third of the cases
observed. The organism has been isolated from the blood of the heart.
The psittacosis bacillus is evidently one of the typhoid group, a fact
which is further borne out by the observation that it is clumped by a
typhoid serum — 1 : 10 (normal serum having no result). The clumping
is, however, said to be incomplete, as the bacilli between the clumps may
retain their motility. It differs from the typhoid bacillus in its growth
on potatoes and in its pathogenicity.
The Serum Diagnosis of Typhoid Fever. — This method of
22
338 TYPHOID FEVER
diagnosis is based on the fact that living and actively motile
typhoid bacilli, if placed in the diluted serum of a patient suffer-
ing from typhoid fever, within a very short time lose their
motility and become aggregated into clumps. We shall find
(see Immunity) that in many diseases the serum has this property
of causing agglutination of cultures of the causal bacterium.
The principles on which the possession of the faculty depends and
also its significance, are obscure, and in the case of the typhoid
bacillus we do not know the true interpretation of some of the
facts which have been observed.
The methods by which the test can be applied have already
been described (p. 109).
(1) It will be there seen that the loss of motility and clumping
may be observed microscopically. If a preparation be made by
the method detailed (typhoid serum in a dilution of, say, 1 : 30
having been employed), and examined at once under the micro-
scope, the bacilli will usually be found actively motile, darting
about in all directions. In a short time, however, these move-
ments gradually become slower, the bacilli begin to adhere to one
another, and ultimately become completely immobile and form
clumps by their aggregation, so that no longer are any free
bacilli noticeable in the preparation. When this occurs the
reaction is said to be complete. If the clumps be watched still
longer a swelling up of the bacilli will be observed, with a
granulation of the protoplasm, so that their forms can with
difficulty be recognised. In a preparation similarly made with
non-typhoid serum the individual bacilli can be observed separate
and actively motile for many hours.
(2) A corresponding reaction visible to the naked eye is
obtained by the "sedimentation test," the method of applying
which has also been described (p. 111). Here at the end of
twenty-four hours the bacilli form a mass like a precipitate at
the bottom of the mixture of bacterial emulsion and diluted
typhoid serum, while the upper part remains clear. A similar
preparation made with normal serum shows a diffuse turbidity
at the end of twenty-four hours. The test in this form has the
disadvantage of taking longer time than the microscopic method,
but it is useful as a control ; in nature it is similar.
Such is what occurs in the case of a typical reaction. The
value of the method as a means of diagnosis largely depends on
attention to several details. The race of typhoid bacillus
employed is important. All races do not give uniformly the
same results, though it is not known on what this difference of
susceptibility depends. A race must therefore be selected
SERUM DIAGNOSIS 339
which gives the best result in the greatest number of undoubted
cases of typhoid fever, and which gives as little reaction as
possible with normal sera or sera derived from other diseases.
This latter point is important, as some races react very readily to
non-typhoid sera. Again, care must be taken as to the state of
the culture used. The suitability of a culture may be impaired
by varying the conditions of its growth. Continued growth of a
race at 37° C. makes it less suitable for use in the test, as the
bacilli tend naturally to adhere in clumps, which may be
mistaken for those produced by the reaction. Wyatt Johnson
recommended that the stock culture should be kept growing on
agar at room temperature and maintained by agar sub-cultures
made once a month. For use in applying the test, bouillon
sub-cultures are made and incubated for twenty-four hours at
37° C. The relation of the dilution of the serum to the
occurrence of clumping is most important. It has been found
that if the degree of dilution be too small a non-typhoid serum
may cause clumping. If possible, observations should always be
made with dilutions of 1 : 10, 1 : 30, 1 : 60, 1 : 100. To speak
generally, the more dilute the serum the longer time is necessary
for a complete reaction. Some typhoid sera have, however,
very powerful agglutinating properties, and may in a compara-
tively short time produce a reaction when diluted many hundreds
of times. With a too dilute serum not only may the reaction
be delayed, but it may be incomplete, — the clumps formed being
small and many bacilli being left free. These latter may either
have been rendered motionless or they may still be motile. No
diagnosis is conclusive which is founded on the occurrence of
such an incomplete clumping alone. Seeing that low dilutions
sometimes give a reaction with non-typhoid sera, it is important
to know what is the highest dilution at which complete
clumping indicates a positive reaction. The general consensus
of opinion, with which our own experience agrees, is (that when
a serum in a dilution of 1 : 30 causes complete clumping in half
an hour, it may safely be said that it has been derived from a
case of typhoid feverj Suspicion should be entertained as to
the diagnosis if a lower dilution is required, or if a longer time
is required.
The reaction given by the serum in typhoid fever usually
begins to be observed about the seventh day of the disease,
though occasionally it has been found as early as the fifth day,
and sometimes it does not appear till the third week or later.
Usually it becomes gradually more marked as the disease
advances, and it is still given by the blood of convalescents from
340 TYPHOID FEVER
typhoid, but cases occur in which it may permanently disappear
before convalescence sets in. How long it lasts after the end of
the disease has not yet been fully determined, but in many cases
it has been found after several months or longer. As a rule, up
to a certain point, the reaction is more marked where the fever
is of a pronounced character, whilst in the milder cases it is less
pronounced. In certain grave cases, however, the reaction has
been found to be feeble or almost absent, and accordingly some
hold that (a feeble reaction when the disease is manifestly severe
is of bad omenj In some cases which from the clinical symptoms
were almost certainly typhoid, the reaction has apparently been
found to be absent. Such cases should always be investigated
from the point of view of their possibly being paratyphoid fever.
It has been found that the reaction is not only obtained with
living bacilli, but in certain circumstances also with bacilli
that have been killed by heating at 60° C. for an hour, — if
a higher temperature be used, sensitiveness to agglutination is
impaired. The capacity is also still retained if a germicide be
employed. Here Widal recommends the addition of one drop of
formalin to 150 drops of culture. The reaction, however, tends
to be less complete.
Besides the blood serum it has been found that the reaction
is given in cases of typhoid fever by pericardial and pleural
effusions, by the bile and by the milk, and also to a slight
degree by the urine. The blood of a foetus may have little
agglutinating effect though that of its mother may have given
a well-marked reaction ; sometimes, however, the foetal blood
gives a well-marked reaction. It may here also be mentioned
that a serum will stand exposure for an hour at 58° C. without
having its agglutinating power much diminished. Higher tem-
peratures, however, cause the property to be lost.
The Agglutination of Organisms other than the B. Typhosus
by Typhoid Serum. — It was at first thought that' the reaction in
typhoid fever would afford a reliable method of distinguishing
the typhoid bacillus from the b. coli. Though many races of
the latter give no reaction with a typhoid serum, there are others
which react positively. Usually, however, a lower dilution and
a longer time are required for a result to be obtained, and the
reaction is often incomplete.' It has also been found that other
organisms belonging to the typhoid group (v. p. 335) react in a
similar way. The reaction as a method of distinguishing between
these forms is thus not absolutely reliable, but in certain cases it
is of great value in giving confirmation to other tests. The im-
portant point here is the determination of the highest dilution
SEBUM DIAGNOSIS 341
with which clumping is obtained. There is a point in this
connection regarding which further light is required. Many
races of b. coli in use have been isolated from typhoid cases,
and we as yet do not know what effect a sojourn in such circum-
stances may have on its subsequent sensitiveness to agglutination
by typhoid serum. Again, Christophers has pointed out that a
large proportion of sera from normal persons or from those
suffering from diseases other than typhoid will clump the b. coli
in dilutions of from 1 : 20 to 1 : 200, and no doubt many of the
reactions shown by typhoid sera towards b. coli are due to the
pre-existence in the individuals of an agglutinative property
towards the latter bacillus.
With regard to the value of the serum reaction there is little
doubt. In nearly 95 per cent of cases of typhoid it can be
obtained in such a form that no difficulty is experienced if the
precautions detailed above are observed. The causes of possible
error may be summarised as follows : the serum of the person
may naturally have the capacity of clumping typhoid bacilli ;
there may have been an attack of typhoid fever previously with
persistence of agglutinative capacity ; the case may be one of
disease caused by an allied bacillus ; the disease may have a
quite different cause, and yet the serum may react with typhoid
bacilli ; the disease may be typhoid fever and yet no reaction
may occur. The most important of these sources of error is
that with which diseases caused by allied organisms are
concerned, as it is probable that all the forms which these take
in men have not been recognised. The very wide application
of the reaction has elicited the fact that it is given in many
cases of slight, transient, and ill -denned febriculae, which occur
especially when typhoid fever is prevalent. Some of these may
be aborted typhoid, some may be paratyphoid. There is no doubt
that, if all the facts are taken into account, the cases where the
reaction gives undoubtedly correct information so far outnumber
those in which an error may be made that it must be looked on
as a most valuable aid to diagnosis. In conclusion here we may
say that the fact of a typhoid serum clumping allied bacilli in
no way, so far as our present knowledge goes, justifies doubt
being cast on the specific relation of the typhoid bacillus to
typhoid fever.
In connection with the phenomenon that a serum either from
a normal person or a typhoid patient may clump several varieties
of bacteria, some points arise. The theoretical consideration of
agglutination is reserved for the chapter on Immunity, but here
it may be said that agglutinating properties may be present
342 TYPHOID FEVER
normally in a serum or they may be originated by an animal
being infected with a particular bacterium. As the result of
injecting a bacterium not only may agglutinins capable of
acting on that bacterium appear in the serum, but the serum may
become capable of agglutinating other, and especially kindred,
bacteria ; further, any normal agglutinins for the infecting
bacterium present in the serum may be increased in amount.
The agglutinin acting on the infecting organism has been called
the primary or homologous agglutinin, while the others have been
called the secondary or heterologous agglutinins. But besides
what we know to be a fact, that infection by a single bacillary
species can originate agglutinins acting both on itself and on allied
species, we must consider the possibility of infections by more
than one species occurring in an animal, e.g. b. typhosus with b.
coli or with b. paratyphosus. In such a case each organism
may originate its primary agglutinin so that the presence of
multiple agglutinins in a serum may really be an indication of
a mixed infection. Some attention has been directed to the
diagnosis and differentiation of these conditions. Castellani
has introduced a method for their investigation. This depends
on the capacity manifested by bacteria of absorbing the ag-
glutinins from a serum. A small quantity of the agglutinating
serum, say '5 c.c., is taken either pure or diluted with bouillon,
there are added 4 to 8 loops of an agar culture of the germ which
originated it, the mixture is well shaken and set at 37° C. for 12
hours. Clumping of course occurs, and the clumps fall to the
bottom of the tube. The supernatant fluid is pipetted off and
is available for further tests. Castellani studied the primary and
secondary agglutinins produced in infections in rabbits ; he found
that when an animal had been infected with b. typhosus this
organism would absorb from its serum not only the primary
typhoid agglutinins but also such secondary agglutinins as those
acting on the b. coli. If, however, an animal had undergone
infection with, say, both the b. typhosus and the b. coli, then the
b. typhosus could not absorb from its serum the b. coli (primary)
agglutinin. Castellani thus put forward the view that by this
means primary could be differentiated from secondary agglutinins,
and therefore pure could be differentiated from mixed infections.
There is little doubt that this view possesses considerable validity,
though it is probably not of universal applicability. Safe
deductions can only be drawn when any serum is tested with
several species of fairly closely related organisms, such as those
of the coli group. Especially is it necessary that the highest
dilutions in which agglutination occurs should be compared. If
VACCINATION AGAINST TYPHOID 343
such precautions be adopted the absorption method can be util-
ised for the differentiation of the typhoid and paratyphoid organ-
isms and their infections and for similar investigations.
Vaccination against Typhoid. — The principles of the im-
munisation of animals against typhoid bacilli have been applied
by Wright and Semple to man in the following way. ^Typhoid
bacilli are obtained of such virulence that a quarter of a twenty-,
four hours' old sloped agar culture when administered hypo-
dermically will kill a guinea-pig of from 350 to 400 grammes)
Vaccination can be accomplished by such a culture emulsified
in bouillon, and killed by heating for five minutes at 60° C.
For use, from one-twentieth to one-fourth of the dead culture is
injected hypodermically, usually in the flank. The vaccine now
used, however, actually consists of a portion of a bouillon
culture similarly treated. The effects of the injection are some
tenderness locally and in the adjacent lymphatic glands, and it
may be local swelling, all of which come on in a few hours, and
may be accompanied by a general feeling of restlessness and a
rise of temperature, but the illness is over in twenty-four hours.
During the next ten days the blood of the individual begins to
manifest, when tested, an agglutination reaction, and further,
Wright has found that' usually after the injection there is a
marked increase in the capacity of the blood serum to kill the
typhoid bacillus in vitro. There is little doubt that these observa-
tions indicate that the vaccinated person possesses a degree of
immunity against the bacillus, and this conclusion is borne out
by the results obtained in the use of the vaccine as a prophy-
lactic against typhoid fever. Extensive observations have been
made in the British army in India, and in the South African
War the efficacy of the treatment was put to test. Though
in isolated cases not much difference was observed among
those treated as compared with those untreated, yet the broad
general result may be said to leave little doubt that on the
one hand protective inoculation diminishes the tendency for the
individual to contract typhoid fever, and on the other, if the
disease be contracted, the likelihood of its having a fatal result
is diminished. Thus in India of 4502 soldiers inoculated, '98
per cent contracted typhoid, while of 25,851 soldiers in the
same stations who were not inoculated, 2 '54 per cent took the
disease. In Ladysmith during the siege there were 1705
soldiers inoculated, among whom 2 per cent of cases occurred,
and 10,529 uninoculated, among whom 14 per cent suffered
from typhoid. Wright has collected statistics dealing in all
with 49,600 individuals, of whom 8600 were inoculated, and
344 TYPHOID FEVER
showed a case incidence of 2'25 per cent, with a case mortality
of 12 per cent; in the remaining 41,000 uninoculated the case
incidence was 5 '75 per cent and the case mortality 21 per cent.
The best results seemed to be obtained when ten days after the
first inoculation a second similar inoculation is practised.
Wright has found that in certain cases immediately after
inoculation there is a fall in the bactericidal power of the blood
(negative phase), and he is of opinion that this indicates a
temporary increased susceptibility to the disease. He therefore
recommends that when possible the vaccination should be carried
out some time previous to the exposure to infection. There can
be very little doubt that in this method an important prophy-
lactic measure has been discovered.
Antityphoid Serum. Chantemesse lias immunised animals with dead
cultures of the typhoid bacillus, and having found that their sera had
protective and curative effects in other animals, has used such sera
in human cases of typhoid with apparent good result. In the hands
of others, however, such a line of treatment has not been equally
successful.
Methods of Examination. — The methods of microscopic
examination, and of isolation of typhoid bacilli from the spleen
post mortem, have already been described. They may be isolated
from the Peyer's patches, lymphatic glands, etc., by a similar
method.
During life, typhoid bacilli may be obtained in culture in the
following ways : —
(a) From the Spleen. — This is the most certain method of
obtaining the typhoid bacillus during the continuance of a case.
The skin over the spleen is purified and, a sterile hypodermic
syringe being plunged into the organ, there is withdrawn from
the splenic pulp a droplet of fluid, from which plates are made.
In a large proportion of cases of typhoid the bacillus may be
thus obtained, failure only occurring when the needle does not
happen to touch a bacillus. Numerous observations have shown
that, provided the needle be not too large, the procedure is quite
safe. Its use, however, is scarcely called for.
(b) From the Urine. — Typhoid bacilli are present in the urine
in at least twenty -five per cent of cases, especially late in the
disease, probably chiefly when there are groups in the kidney
substance. For methods of examining suspected urine, see
p. 69.
(c) From the Stools. — During the first ten days of a case of
typhoid fever, the bacilli can be isolated from the stools by the
ordinary plate methods — preferably in MacConkey's lactose bile
METHODS OF EXAMINATION 345
salt neutral-red agar, or in the medium of Drigalski and Conradi
(v. p. 42). After that period, though the continued infectiveness
of the disease indicates that they are still present, their isolation is
very difficult. We have seen that after ulceration is fairly estab-
lished by the sloughing of the necrosed tissue, the numbers present
in the patches are much diminished, and therefore there are fewer
cast off into the intestinal lumen, and that in addition there is a
correspondingly great increase of the b. coli, which thus causes
any typhoid bacilli in a plate to be quite outgrown. From the
fact that the ulcers in a case of typhoid may be very few in
number, it is evident that there may be at no time very many
typhoid bacilli in the intestine. The microscopic examination
of the stools is of course useless as a means of diagnosing the
presence of the typhoid bacillus.
Isolation from Water Supplies. — A great deal of work has been done
on this subject. It is evident that if it is difficult to isolate the bacilli
from the stools it must a fortiori be much more difficult to do so when
the latter are enormously diluted by water. The b. typhosus has, how-
ever, been isolated from water during epidemics. This was done by Klein
in the outbreaks in recent years at Worthing and Rotherham. The b.
coli is, as might be expected, the organism most commonly isolated in
such circumstances. In the case of both bacteria, the whole series of
culture reactions must be gone through before any particular organism
isolated is identified as the one or the other ; probably there are saprophytes
existing in nature which only differ from them in one or two reactions.
In the examination of water, the addition of '2 per cent carbolic acid to
the medium inhibits to a certain extent the growth of other bacteria,
while the b. typhosus and the b. coli are unaffected. In examining
waters, the ordinary plate methods are generally used, but the Conradi-
Drigalski or MacConkey media may be employed with advantage.
Klein niters a large quantity through a Berkefeld filter and, brushing
off the bacteria retained on the porcelain, makes cultures. A much
greater concentration of the bacteria is thus obtained. From time to time
various substances havebeen used with the object of inhibiting the growth
of the b. coli without interfering with that of the b. typhosus. Most
of these have not stood the test of experience. Lately caffeine has been
used for this end. For use in examining waters the following is the
method employed. To 900 c.c. of the suspected water there are added
10 grammes nutrose dissolved in 80 c.c. of sterile water, and 5 grammes
of caffeine dissolved in sterile distilled water heated to 80° C. and cooled
to 55° C. before addition. After mixing the ingredients there is added
10 c.c. of '1 per cent crystal violet. The flask is incubated at 37° C. for
12 hours and then plates of Conradi-Drigalski medium are inoculated from
it. For investigation of faeces, a medium made up as above but with
ordinary sterile water may be infected and a similar procedure followed.
On the whole there is little to be gained from this attempt to isolate the
typhoid bacillus from water in any particular case, and it is much more
useful for the bacteriologist to bend his energies towards the obtaining
of the indirect evidence of contamination of water by sewage, to the
nature of which attention has been called in Chap. IV.
346 TYPHOID FEVER
BACTERIA IN DYSENTERY.
Dysentery has for long been recognised as including a number
of different pathological conditions, and within more recent times
amoebic and non-amoebic forms have been distinguished. Of the
latter bacteria have been believed to be the causal agents, and an
organism described by Shiga in 1898 has almost certainly been
established as the cause of a large proportion of cases. Shiga's
observations were made in Japan, and confirmatory results have
been obtained by Kruse in Germany, by Flexner and by Strong
and Harvie in the Philippine Islands, and more recently by Vedder
and Duval in the United States. It is now further recognised
that the epidemics of dysentery which from time to time occur
in lunatic asylums are usually due to bacilli of this type, and in
America the organism has been demonstrated in the summer
diarrhoea of children. The evidence for the relationship of the
organism to the disease consists chiefly in the apparently con-
stant presence of the organism in the dejecta in this form of
dysentery, and the agglutination of the organism by the serum
of patients suffering from the disease, but confirmatory evidence
has also come from animal experimentation. From different
epidemics a great many different strains of the dysentery bacillus
have been obtained, but these all possess common characters and
are undoubtedly closely related to one another. The various
strains resolve themselves into two chief groups, whose differences
lie in their behaviour towards certain sugars, in their capacities
of originating indol and in their agglutinating reactions. The
relation of amcebaB to dysentery will be discussed in the
Appendix.
Bacillus Dysenteriae (Shiga}. — Morphological Characters. —
This bacillus morphologically closely resembles the typhoid
bacillus, but is on the whole somewhat plumper, and filamentous
forms are comparatively rare. Involution forms sometimes occur,
especially in glucose agar. Most observers have found no trace
of motility, whilst others say that it is slightly motile. Vedder
and Duval have, however, by a modification of Van Ermengen's
process, demonstrated in the case of one strain the presence of
numerous lateral flagella, which are of great fineness, but of
considerable length. No spore formation occurs ; the organism
is stained readily by the ordinary dyes, but is decolorised by
Gram's method.
Cultural Characters. — In these also considerable resemblance
is presented to the typhoid bacillus. In gelatin a whitish line of
growth occurs along the puncture, but the superficial film-like
BACILLUS DYSENTERIC 347
growth is usually absent, or at least poorly marked. In plate
cultures also the superficial growths are smaller and have less of
the film-like character, than those of the typhoid organism. On
agar, growth occurs as a smooth film with regular margins, but
after two or three days, especially if the surface be moist, Vedder
and Duval describe an outgrowth of lateral offshoots on the
surface of the medium. On agar plates the colonies resemble
those of the typhoid organism, being of smaller size and less
opaque than those of the bacillus coli. 4^ peptone bouillon a
uniform haziness is produced. As has been indicated, different
strains of the bacillus behave differently towards different sugars,
and the results of all observers do not agree, so that only general
statements can be made. Without going into the question of
the particular strains to be placed in the two groups we may say
that, roughly, these may be classified into the Shiga-Kruse group
and the Flexner group. All produce acid in peptone-glucose and
in taurocholate peptone-glucose, none produce change in lactose or
cane-sugar. The Shiga group do not produce acid in maltose or
mannite, while the Flexner group do, and, generally speaking, the
former do not produce indol while the latter do. Forms inter-
mediate between the two groups occur. There is never any
evolution of gas observed in sugar media. In litmus milk there
is developed at first a slight degree of acidity, which is followed
by a phase of increased alkalinity ; no coagulation of the milk
ever occurs. On potato the organism forms a transparent or
whitish layer, which, however, in the course of a few days assumes
a brownish-red or dirty grey colour, with some discoloration of
the potato at the margin of the growth.
Relation to the Disease. — The organism has been found in
large numbers in the dejecta, especially in the acute cases, where
it may be present in almost pure culture. In the thirty-six cases
examined, Shiga obtained it in thirty-four from the dejecta, and in
the two others post mortem from the intestinal mucous membrane.
The organism does not appear to spread deeply or to invade
the general circulation. In the more chronic cases it is difficult
to obtain on account of the large number of the bacillus coli and
other bacteria present. Vedder and Duval found agar plates to
be the best method of culture, these being incubated at the
blood temperature. They also found that if the colonies which
appeared at twelve hours were marked with a pencil, there was
a greater probability of obtaining the bacillus of dysentery from
those which appeared later, most of those appearing early being
colonies of the bacillus coli. MacConkey's agar medium with
lactose added may be used for isolation from stools. A little of
348 TYPHOID FEVER
the faeces is rubbed up in broth and some of the mixture stroked
on the medium. The formation of acid by the coli colonies enables
them to be excluded, and, therefore, as the b. dysenteriae is not
a lactose f ermenter, the colourless colonies which develop after
twenty-four hours are picked out for further investigation.
As already stated, both acute and chronic cases are marked
by the presence of this organism. In the former, where death
may occur in from one to six days, the chief changes, according
to Flexner, are a marked swelling and corrugation of the mucous
membrane, with haemorrhage and pseudo-membrane at places.
There is extensive coagulation-necrosis with fibrinous exuda-
tion and abundance of polymorpho-nuclear leucocytes, and the
structure of the mucous membrane, as well as that of the
muscularis mucosae, is often lost in the exudation. There is
also great thickening of the sub-mucosa, with great infiltration of
leucocytes, these being chiefly of the character of " plasma cells."
In the more chronic forms the changes correspond, but are
more of a proliferative character. The mucous membrane is
granular, and superficial areas are devoid of epithelium, whilst
ulceration and pseudo-membrane are present in varying degree.
A feature of bacillary dysentery is the fact that abscess of the
liver does not occur as a complication.
Agglutination. — All the above-mentioned observers agree re-
garding the agglutination of this bacillus by the serum — that is,
in the cases of dysentery from which the organism can be cul-
tivated. The reaction may appear on the second day, and is
most marked after from six to seven days in the acute cases ; it
is usually given in a dilution of from one in twenty to one in
fifty within an hour, though sometimes much higher dilutions
give a positive result. In the more chronic cases the reaction
is less marked, and here the sedimentation method is to be
preferred. It is difficult to make any general statements with
regard to the effects of dysenteric sera on the different strains
of the bacilli, but it may be said that generally a serum
agglutinates the strain which produced it and the other strains
of the same group in higher dilutions than it does the strains of
the other group. Many observers have found that the serum
from a case associated with strains of the Shiga-Kruse group
have not agglutinated strains of the Flexner group, and
corresponding observations have been made in cases associated
with the Flexner group. Often the sera of animals immunised
with bacilli have been used for such tests, but apparently great
care must be exercised in basing diagnoses on such observations,
for some species of animals when treated with a particular strain
BACILLUS DYSENTERIC 349
will yield a serum which is active against many more strains
than other species will when immunised with that strain.
Agglutination of the organism has not been obtained with serum
from cases other than those of dysentery, nor has a similar bacillus
been cultivated from other such sources. The reaction is also
absent in those cases of dysentery which are manifestly of amoebic
nature.
Pathogenic Properties. — The organism is pathogenic in guinea-
pigs and other laboratory animals, but, in these, characteristic
changes in the intestine are often awanting. Shiga, however,
obtained such effects by introducing the organism into the
stomach of young cats and dogs, and confirmatory results were
obtained by Flexner. Such attempts have been specially
successful when the virulence of the organism has been previously
exalted by intraperitoneal passage. In two cases, apparently
well authenticated, a dysenteric condition has followed in the
human subject from ingestion of pure cultures of the organism.
It is probable that in the action of the bacillus a toxin is
concerned. It has been found that the nitrate from three weeks'
old cultures in alkaline bouillon is very toxic to animals, especially
rabbits, and that, however introduced into the body, it causes
a haBmorrhagic enteritis with a diphtheritic-like exudate on
the surface of the mucous membrane. According to some obser-
vers the toxin is more readily obtainable from the Shiga-Kruse
strains than from the Flexner strains. The toxin is fairly resist-
ant to heat, standing temperatures up to 70° C. without being
injured. From the fact, that by the maceration of cultures
whose filtrates are relatively non-toxic a stronger poison can be
obtained, the dysentery toxin has been thought to be an
endotoxin, but on this point no definite opinion can be expressed.
Immunisation Experiments. — Both large and small animals
have been immunised against the bacillus and also against its
toxic filtrates. In the former case the immunisation has been
commenced either with non-lethal doses of living cultures or with
cultures killed by heat. The nature of the immunisation is
probably complex. When cultures have been used, a bactericidal
serum, in which immune bodies and complements (vide Immunity)
are concerned, is developed. When the toxin is used for immun-
isation a serum protecting against the toxin is produced. Ac-
cording to some results animals immunised with cultures are
immune against the toxin, and vice versa. All races of animal
do not lend themselves to immunisation. Large animals (horses,
goats) have been immunised with the toxin with a view of ob-
taining sera for use in human dysentery, and in certain cases,
350 TYPHOID FEVER
notably in the work of Rosenthal, a distinct therapeutic effect
has been produced by the subcutaneous administration of the
serum, especially in early cases of the disease.
It will be seen that the evidence furnished is practically
conclusive as to the causal relationship between this bacillus and
one form of dysentery, a form, moreover, which is both wide-
spread and embraces a large proportion of cases of the disease ;
and especially of importance is the fact that observations made
independently in different countries have yielded practically
identical results on this point.
Bacillus Dysenteric (Ogata). — Ogata obtained this bacillus in an
extensive epidejnic in Japan in which no amoebae were present. He
found in sections of the affected tissues enormous numbers of small
bacilli of about the same thickness as the tubercle bacillus, but very
much shorter. These bacilli were sometimes found in a practically pure
condition. They were actively motile and could be stained by Gram's
method. He also obtained pure cultures from various cases and tested
their pathogenic effects. They grew well on gelatin, at the ordinary
temperature producing liquefaction, the growth somewhat resembling
that of the cholera spirillum. By injection into cats and guinea-pigs, as
well as by feeding them, this organism was found to have distinct
pathogenic effects ; these were chiefly confined to the large intestine,
hsemorrhagic inflammation and ulceration being produced. It still
remains to be determined whether this organism has a causal relation-
ship to one variety of dysentery.
BACILLUS ENTERITIDIS SPOKOGENES.
This organism was first isolated by Klein from the evacuations in an
outbreak of diarrhoea following the ingestion of milk which contained
the microbe, and it was subsequently found by him in certain cases of
infantile diarrhoea and of summer diarrhoea, in certain instances in milk,
and as a constant inhabitant of sewage (see Chap. IV.). In films made
from the stools in diarrhoea cases where it is present it can be micro-
scopically recognised as a bacillus 1 "6 /a to 4 '8 ^ in length and '8 n in
breadth, staining by ordinary stains and retaining the dye in Gram's
method. It often contains a spore near one of the ends, or sometimes
nearer the centre. It is slightly motile, and in cultures can be shown to
possess a small number of terminal flagella. It grows well under
anaerobic conditions in ordinary media, especially on those containing
reducing agents. On agar the colonies are circular, grey, and translucent,
and under a low power are seen to have a granular appearance. On this
medium spore formation does not occur, but is easily obtained if the
organism is grown on solidified blood serum, which, further, is liquefied
during growth. On gelatin plates liquefaction commences after twenty-
four hours at 20° C. It produces acid and gas in bile-salt glucose media,
and in peptone salt solution containing glucose or mannite. Spore
formation can be seen to take place in 2 per cent dextrose gelatin, but
the degree seems to be in inverse ratio to the amount of gas formation.
Very typical is the growth on milk, and it is by this medium that
isolation can be best effected. A small quantity of the material
SUMMER DIARRHCEA 351
suspected to contain the bacillus is placed in 15 to 20 c.c. of sterile
milk, which is then heated for ten minutes at 80° C. to destroy all
vegetative bacteria ; the tube is cooled, placed under anaerobic con-
ditions, and incubated at 37° C. for from twenty-four to thirty-six hours.
If the bacillus be present there is abundant gas formation, and almost
complete separation of the curd from the whey takes place. The former
adheres to the sides of the tube in shreds, and large masses gather with
the cream on the top of the fluid, all being torn by the gas evolved.
The whey is only slightly turbid and contains numerous bacilli. The
growth has an odour of butyric acid. If a small quantity (say 1 c.c.) of
the whey be injected into a guinea-pig, the animal becomes ill in a few
hours and dies in twenty-four hours. At the point of inoculation the
skin and subcutaneous tissues, and sometimes even the subjacent muscles,
are green and gangrenous and evil-smelling, there is considerable oedema,
and there may also be gas formation. The exudation is crowded with
bacilli, which, however, are not generally distributed in any numbers
throughout the body. These pathogenic properties of the bacillus
enteritidis sporogenes are important in its recognition, for its culture
reactions taken alone are very similar to those of the bacillus butyricus
of Botkin.
SUMMER DIARRHCEA.
As has been already stated, both the bacillus of dysentery, b. coli
and the b. enteritidis sporogenes have been found associated with
epidemics of this disease. This indicates that the condition may
be originated by a variety of organisms, and it is further probable
that the clinical features in different epidemics vary. The
multiple origin of the disease has been illustrated by the work
of Morgan, who, in a careful investigation of the disease in
Britain, has been unable to find evidence of the dysentery
bacillus being present. He has, however, very constantly found
in the stools and intestine a bacillus (" Morgan's No. 1 bacillus")
which is a motile Gram-negative organism producing acid and
slight gas formation in glucose, Isevulose, and galactose, and no
change in mannite, dulcite, maltose, dextrin, cane sugar, lactose,
inulin, amygdalin, salicin, arabinose, raffinose, sorbite, or ery-
thrite ; it further causes indol formation, and in litmus milk
slowly originates an alkaline reaction. It produces diarrhoea and
death in young rabbits, rats, and monkeys when these animals
are fed on cultures. It is thus possible that in this bacillus
we have still another cause of the disease.
CHAPTER
DIPHTHERIA.
THERE is no better example of the valuable contributions of
bacteriology to scientific medicine than that afforded in the case
of diphtheria. Not only has research supplied, as in the case of
tubercle, a means of distinguishing true diphtheria from condi-
tions which resemble, it, but the study of the toxins of the
bacillus has explainer! the manner by which the pathological
changes and characteristic symptoms of the disease are brought
about, and has led to ttie discovery of the most efficient means
of treatment, namely, the an ti- diphtheritic serum.
Historical. — The first account of the bacillus now known to be the
cause of diphtheria was given by Klebs in 1883, who described its
characters in the false membrane, but made no cultivations. It was
first cultivated by Loffler from a number of cases of diphtheria, his
observations being published in 1884, and to him we owe the first
account of its characters in cultures and of some of its pathogenic effects
on animals. The organism is for these reasons known as the Klebs-
Loffler bacillus, or simply, as Loffler's bacillus. By experimental in-
oculation with the cultures obtained, Lb'rfler was able to produce false
membrane on damaged mucous surfaces, but he hesitated to conclude
definitely that this organism was the cause of the disease, for he did
not find it in all the cases of diphtheria examined, he was not able to
produce paralytic phenomena in animals by its injection, and, further,
he obtained the same organism from the throat of a healthy child. This
organism became the subject of much inquiry, but its relationship to
the disease may be said to have been definitely established by the
brilliant researches of Roux and Yersin which showed that the most
important features of the disease could be produced by means of the
separated toxins of the organism. Their experiments were published in
1888-90. Further light has been thrown on the subject by the work of
Sidney Martin, who has found that there can be separated from the
organs in cases of diphtheria substances which act as nerve poisons, and
also produce other phenomena met with in diphtheria.
General Facts. — Without giving a description of the patho-
logical changes in diphtheria, it will be well to mention the out-
352
BACILLUS DIPHTHERIA 353
standing features which ought to be considered in connection
with its bacteriology. In addition to the formation of false
membrane, which may prove fatal by mechanical effects, the
chief clinical phenomena are the symptoms of general poisoning,
great muscular weakness, tendency to syncope, and albuminuria ;
also the striking paralyses which occur later in the disease,
and which may affect the muscles of the pharynx, larynx, and
eye, or less frequently the lower limbs (being sometimes of
paraplegic type), all these being grouped together under the
term " post-diphtheritic paralyses." It may be stated here that all
these conditions have been experimentally reproduced by the
action of the bacillus of diphtheria, or by its toxins. Other
bacteria are, however, concerned in producing various secondary
inflammatory complications in the region of the throat, such as
ulceration, gangrenous change, and suppuration, which may be
accompanied by symptoms of general septic poisoning. The detec-
tion of the bacillus of Loffler in the false membrane or secretions
of the mouth is to be regarded as supplying the only certain /
means of diagnosis of diphtheria. *
Bacillus Diphtherias. — Microscopical Characters. — If a film
preparation be made from a piece of diphtheria membrane (in
the manner described below) and stained with methylene-blue,
the bacilli are found to have the following characters. They are
slender rods, straight or slightly curved, and usually about 3 //.
in length, their thickness being a little greater than that of
the tubercle bacillus. The size, however, varies somewhat in
different cases, and for this reason varieties have been distin-
guished as small and large, and even of intermediate size. It is
sufficient to mention here that in some cases most are about 3 //,
in length, whilst in others they may measure fully 5 p. Corre-
sponding differences in size are found in cultures. They stain
deeply with the blue, sometimes being uniformly coloured, but
often showing, in their substance, little granules more darkly
stained, so that a dotted or beaded appearance is presented.
Sometimes the ends are swollen and more darkly stained than
the rest ; often, however, they are rather tapered off (Fig. 116).
In some cases the terminal swelling is very marked, so as to
amount to clubbing, and with some specimens of methylene-blue
these swellings and granules stain of a violet tint. Distinct
clubbing, however, is less frequent than in cultures. There is a
want of uniformity in the appearance of the bacilli when com-
pared side by side. They usually lie irregularly scattered or in
clusters, the individual bacilli being disposed in all directions.
Some may be contained within leucocytes. They do not form
23
DIPHTHERIA
chains, but occasionally forms longer than those mentioned may
be found, and these specially occur in the spaces between the
fibrin as seen in sections.
Distribution of the Bacillus. — The diphtheria bacillus may
be found in the membrane wherever it is formed, and may also
occur in the secretions of the pharynx and larynx in the disease.
<
W, i^^^ff-^m.
*H
f •*
* ik
Fro. 116.— Film preparation from diphtheria membrane ; showing
diphtheria bacilli. One or two degenerated forms are seen near the
centre of the field. (Cultures made from the same piece of membrane
showed the organism to be present in practically pure condition.)
Stained with nwthylene-blne. x 1000.
It may be mentioned that distinctions formerly drawn between
true diphtheria and non-diphtheritic conditions from the appear-
ance and site of the membrane, have no scientific value, the only
terue criterion being the presence of the diphtheria bacillus. The
occurrence of a membranous formation produced by streptococci
has already been mentioned (p. 184).
In diphtheria the membrane has a somewhat different
structure according as it is formed on the surface covered with
stratified squamous epithelium as in the pharynx, or on a surface
DISTRIBUTION OF THE BACILLUS 355
covered by ciliated epithelium as in the trachea. In the former
situation necrosis of the epithelium occurs either uniformly or in
patches, and along with this there is marked inflammatory
reaction in the connective tissue beneath, attended by abundant
fibriuous exudation. The necrosed epithelium becomes raised
up by the fibrin, and its interstices are also filled by it. The
FIG. 117. — Sectiou through a diphtheritic membrane in trachea, showing diph-
theria bacilli (stained darkly) in clumps, and also scattered amongst the
fibrin- Some streptococci are also shown, towards the surface on the
left side.
Stained by Gram's method and Bismarck-brown, x 1000.
fibrinous exudation also occurs around the vessels in the tissue
beneath, and in this way the membrane is firmly adherent. In
the trachea, on the other hand, the epithelial cells rapidly
become shed, and the membrane is found to consist almost
exclusively of fibrin with leucocytes, the former arranged in a
reticulated or somewhat laminated manner, and varying in
density in different parts. The membrane lies upon the base-
ment membrane, and is less firmly adherent than in the case of
the pharynx
356 DIPHTHERIA
The position of the diphtheria bacilli varies somewhat in
different cases, but they are most frequently found lying in oval
or irregular clumps in the spaces between the fibrin, towards the
superficial, that is, usually, the oldest part of the false membrane
(Fig. 117). There they may be in a practically pure condition,
though streptococci and occasionally some other organisms may
be present along with them. They may occur also deeper, but
are rarely found in the fibrin around the blood vessels. On the
surface of the membrane they may be also seen lying in large
numbers, but are there accompanied by numerous other
organisms. Occasionally a few bacilli have been detected in the
lymphatic glands. As Lo'fner first described, they may be
found after death in pneumonic patches in the lung, these
being due to a secondary extension by the air passages. They
have also been occasionally found in the spleen, liver, and
other organs after death. This occurrence is probably to
be explained by an entrance into the blood stream shortly
before death, similar to what occurs in the case of other
organisms, e.g. the bacillus coli communis. The diphtheria
bacillus may also infect other mucous membranes. It is found
in true diphtheria of the conjunctiva, and may also occur
in similar affections of the vulva and vagina : some of these
cases have been treated successfully with diphtheria antitoxin.
The pseudo-diphtheria bacillus, however, may also occur in these
situations.
Association with other Organisms. — The diphtheria organism is
sometimes present alone in the membrane, but more frequently
is associated with some of the pyogenic organisms, the strepto-
coccus pyogenes being .the commonest. The staphylococci, and
occasionally the pneumococcus or the bacillus coli, may be
present in some cases. Streptococci are often found lying side
by side with the diphtheria bacilli in the membrane, and also
penetrating more deeply into the tissues. In some cases of
tracheal diphtheria, we have found streptococci alone at a lower
level in the trachea than the diphtheria bacilli, where the
membrane was thinner and softer, the appearance in these cases
being as if the streptococci acted as exciters of inflammation and
prepared the way for the bacilli. It is still a matter of dispute
as to whether the association of the diphtheria bacillus with the
pyogenic organisms is a favourable sign or the contrary, though
on experimental grounds the latter is the more probable. We
know, however, that some of the complications of diphtheria
may be due to the action of pyogenic organisms. The extensive
swelling of the tissues of the neck, sometimes attended by
CULTIVATION OF THE BACILLUS
357"
FIG. 118. — Cultures of the
diphtheria bacillus on an
agar plate ; twenty - six
hours' growth.
(a) Two successive strokes ; (6)
isolated colonies from
plate.
suppuration in the glands, and also various hsemorrhagic con-
ditions, have been found to be as-
sociated with their presence ; in fact,
in some cases the diphtheritic lesion
enables them to get a foothold in
the tissues, where they exert their
usual action and may lead to exten-
sive suppurative change, to septic
poisoning or to septicaemia. In cases
where a gangrenous process is super-
added, a great variety of organisms
may be present, some of them being
anaerobic. Against such complica-
tions produced by other organisms
anti-diphtheritic serum produce no
favourable effect.
Cultivation. — The diphtheria
bacillus grows best in cultures at the
temperature of the body; growth
still takes place at 22° C., but ceases
about 20° C. The best media are the
following : Loffler's original medium
(p. 40), solidified blood serum, alkaline blood serum (Lorrain
Smith), blood agar, and
^^iJHlJ^^fe^ the ordinary agar media.
If inoculations be made
on the surface of blood
serum with a piece of
diphtheria membranes,
colonies of the bacillus
*^\ 5^ may appear in twelve
\ 5 hours and are well formed
«<* .^ *** < I within twenty-four hours
IgjNiq^j^ often before any other
^J^jSK growths are visible^ The
T§\» Jv?A» colonies are small circular
discs of opaque whitish
colour, their centre being
thicker and of darker
greyish appearance when
viewed by transmitted
light than the periphery.
On the second or third
mm. in size, but when numerous they
FIG. 119.— Diphtheria bacilli from a twenty-
four hours' culture on agar.
Stained with methylene-blue. x 1000.
day they may reach 3
358
DIPHTHERIA
remain smaller.
•••n
Fig. 120. — Diphtheria bacilli of larger size
than in previous figure, showing also ir-
regular staining of protoplasm. From a
three days' agar -culture.
Stained with weak carbol-fuchsin. x 1000.
On the agar media the colonies have much
the same appearance (Fig.
117) but grow less quickly,
and sometimes they may
be comparatively minute,
so as rather to resemble
those of the streptococcus
pyogenes. In stroke cul-
tures the growth forms a
continuous layer of the
same dull whitish colour,
the margins of which often
show single colonies partly
or completely separated.
On gelatin at 22° C. a
puncture culture shows a
line of dots along the
needle track, whilst at the
surface a small disc forms,
rather thicker in the
middle. In none of the
media does any liquefac-
the organism produces a turbidity
tion occur. In bouillon
which soon settles to the
bottom and forms a pow-
dery layer on the wall of
the vessel. By starting
the growth on the surface
and keeping the flasks at
rest a distinct scum forms,
and this is especially suit-
able for the development
of toxin. Ordinary bouillon
becomes acid during the
first two or three days,
and several days later
again acquires an alkaline
reaction. If, however, the
bouillon is glucose -free
(p. 75) the acid reaction Fig. 121.— Involution forms of the diphtheria
does not occur.
In these media the bacilli
show the same characters
as in the membrane, but the irregularity in staining is more marked
bacillus ; from an agar culture of seven
days' growth.
Stained with carbol-thionin-blue. x 1000.
POWERS OF RESISTANCE OF BACILLUS 359
(Figs. 119, 120). They are at first fairly uniform in size and
shape, but later involution forms are present. Many are swollen
at their ends into club-shaped masses which stain deeply, and the
protoplasm becomes broken up into globules with unstained
parts between (Fig. 121). Some become thicker throughout,
and segmented so as to appear like large cocci, and others show
globules at their ends, the rest of the rod appearing as a faintly-
stained line. Occasionally branched forms are met with. The
bacilli are non-motile, and do not form spores.
Staining. — They take up the basic aniline dyes, e.g. methy-
lene-blue in watery solution, with great readiness, and stain
deeply, the granules often giving the metachromatic reaction as
described. They also retain the colour in Gram's method,
though they are more easily decolorised than the pyogenic cocci.
By Neisser's stain (p. 108) the granules are stained almost black,
the rest of the bacillary substance yellowish brown.
Powers of Resistance, etc. — In cultures the bacilli possess
long duration of life ; at the room temperature they may survive
for two months or longer. In the moist condition, whether in
cultures or in membrane, they have a low power of resistance,
being killed at 60° C. in a few minutes. On the other hand, in
the dry condition they have great powers of endurance. In
membrane which is perfectly dry, for example, they can resist a
temperature of 98° C. for an hour. Dried diphtheria membrane,
kept in the absence of light and at the room temperature, has
been proved to contain diphtheria bacilli still living and virulent
at the end of several months. The presence of light, moisture,
or a higher temperature, causes them to die out more rapidly.
Corresponding results have been obtained with bacilli obtained
from cultures and kept on dried threads. These facts, especially
with regard to drying, are of great importance, as they show that
the contagium of diphtheria may be preserved for a long time
in the dried membrane.
Effects of Inoculation. — In considering the effects produced
in animals by experimental inoculations of pure cultures, we
have to keep in view the local changes which occur in diphtheria,
and also the symptoms of general poisoning.
As Loffler stated in his original paper inoculation of the
healthy mucous membranes of various animals with pure cultures
causes no lesion, but the formation of false membrane may
result when the surface is injured by scarification or otherwise.
A similar result may be obtained when the trachea is inoculated
after tracheotomy has been performed. In this case the
surrounding tissues may become the seat of a blood-stained
360 DIPHTHERIA
oedema, and the lymphatic glands become enlarged, the general
picture resembling pretty closely that of lar.yngeal diphtheria.
The membrane produced by such experiments is usually less
firm than in human diphtheria, and the bacilli in the membrane
are less numerous. Rabbits inoculated after tracheotomy often
die, and Roux and Yersin were the first to observe that in some
cases paralysis may appear before death.
Subcutaneous injection in guinea-pigs, of diphtheria bacilli in
a suitable dose, produces death within thirty-six hours. At the
site of inoculation there is usually a small patch of greyish
membrane, whilst in the tissues around there is extensive
inflammatory oedema, often associated with haemorrhages, and
there is also some swelling of the corresponding lymphatic glands.
The internal organs show general congestion, the suprarenal
capsules being especially reddened and often showing haemorrhage,
The renal epithelium may show cloudy swelling, and there is
often effusion into the pleural cavities. After injection the
bacilli increase in number for a few hours, but multiplication
soon ceases, and at the time of death they may be less numerous
than when injected. The bacilli remain practically local,
cultures made from the blood and internal organs giving usually
negative results, though sometimes a few colonies may be
obtained. If a non-fatal dose of a culture be injected, a local
necrosis of the skin and subcutaneous tissue may follow at the
site of inoculation.
In rabbits, after subcutaneous inoculation, results of the same
nature follow, but these animals are less susceptible than guinea-
pigs, and the dose requires to be proportionately larger. Roux
and Yersin found that -after intravenous injection the bacilli
rapidly disappeared from the blood, and when 1 c.c. of a broth
culture had been injected no trace of the organisms could be
detected by culture after twenty-four hours : nevertheless the
animals died with symptoms of general toxaemia, nephritis also
being often present (cf. Cholera, p. 408). The dog and sheep
are also susceptible to inoculation with virulent bacilli, but the
mouse and rat enjoy a high degree of immunity.
Klein found that cats also were susceptible to inoculation. The
animals usually die after a few days, and post mortem there is well-marked
nephritis. He also found that after subcutaneous injection in cows, a
vesicular eruption appeared on the teats of the udder, the fluid in which
contained diphtheria bacilli. At the time of death the diphtheria bacilli
were still alive and virulent at the site of injection. The most striking
result of these experiments is that the diphtheria bacilli passed into the
circulation and were present in the eruption on the udder. He considers
that this may throw light on certain epidemics of diphtheria in which
THE TOXINS OF DIPHTHERIA 361
the contagion was apparently carried by the milk. Other observers
have, however, failed to obtain similar results. Dean and Todd, in
investigating an outbreak of diphtheria traceable to milk supplied,
found a vesicular eruption on the teats of the udder in which diphtheria
bacilli were present. They, however, came to the conclusion that these
bacilli were not the cause of the eruption, but were the result of a
secondary contamination, probably from the saliva of the milkers. The
occurrence of a true infection with the diphtheria bacillus in the horse
has been described by Cobbett.
The Toxins of Diphtheria. — As in the above experiments
the symptoms of poisoning and ultimately a fatal result occur
when the bacilli are diminishing in number, or even after they
have practically disappeared, Roux and Yersin inferred that the
chief effects were produced by toxins, and this supposition they
proved to be correct. They showed that broth cultures of three
or four weeks' growth freed from bacilli by filtration were highly
toxic. The nitrate when injected into guinea-pigs and other
animals produces practically the same effects as the living bacilli ;
locally there is fibrinous exudation but a considerable amount
of inflammatory cedema, and, if the animal survive long enough,
necrosis in varying degree of the superficial tissues may follow.
The toxicity may be so great that "01 c.c. or even less may be
fatal to a guinea-pig in twenty-four hours.
After injection either of the toxin or of the living bacilli,
when the animals survive long enough, paralytic phenomena
may occur. The hind limbs are usually affected first, the
paralysis afterwards extending to other parts, though sometimes
the fore-limbs and neck first show the condition. Sometimes
symptoms of paralysis do not appear till two or three weeks
after inoculation. After paralysis has appeared, a fatal result
usually follows in the smaller animals, but in dogs recovery may
take place. There is evidence that these paralytic phenomena
are produced by toxone (p. 171), as they may occur when there
is injected along with the toxin sufficient antitoxin to neutralise
the more rapidly acting toxin proper. This toxone is supposed
by Ehrlich to have a different toxic action, i.e. a different
toxophorous group, from that of the toxin, and to have a weaker
affinity for antitoxin ; much of it may thus be left unneutralised.
It is to be noted in this connection that paralytic symptoms are
of not uncommon occurrence in the human subject after treatment
with antitoxin, the explanation of which occurrence is probably
the same as that just given. One point of much interest is the
high degree of resistance to the toxin possessed by mice and
rats. Roux and Yersin, for example, found that 2 c.c. of toxin,
which was sufficient to kill a rabbit in sixty hours, had no effect
362 DIPHTHERIA
on a mouse, whilst of this toxin even T^ c.c. produced extensive
necrosis of the skin of the guinea-pig.
Preparation of the Toxin. — The obtaining of a very active
toxin in large quantities is an essential in the preparation of anti-
diphtheritic serum. Certain conditions favour the development
of a high degree of toxicity, viz., a free supply of oxygen, the
presence of a large proportion of peptone or albumin in the
medium, and the absence of substances which produce an acid
reaction. In the earlier work a current of sterile air was made
to pass over the surface of the medium, as it was found that by
this means the period of acid reaction was shortened and the
toxin formation favoured. This expedient is now considered
unnecessary if an alkaline medium free from glucose is used, as
in this no acid reaction is developed : it is then sufficient to
grow the cultures in shallow flasks. The absence of glucose — an
all-important point — may be attained by the method described
above (p. 75), or by using for the preparation of the meat extract
flesh which is just commencing to putrefy (Spronck). L. Martin
uses a medium composed of equal parts of freshly prepared
peptone (by digesting pigs' stomachs with HC1 at 35° C.), and
glucose-free veal bouillon. By this medium he has obtained a
toxin of which y^ c.c. is the fatal dose to a guinea-pig of 500
grms. He finds that glucose, glycerin, saccharose, and galactose
lead to the production of an acid reaction, whilst glycogen does
not. The latter fact explains how some observers have found
that bouillon prepared from quite fresh flesh is suitable for toxin
formation. There is in all cases a period at which the toxicity
reaches a maximum, usually in 2-3 weeks, but earlier if the toxin
is rapidly formed ; later the toxicity diminishes. Martin found
that in his medium the maximum was reached on the 8th- 10th
day. It may be added that the power of toxin formation
varies much in different races of the diphtheria bacillus, and
that many may require to be tested ere one suitable is obtained.
Properties and Nature of the Toxin. — The toxic substance in
filtered cultures is a relatively unstable body. When kept in
sealed tubes in the absence of light, it may preserve its powers
little altered for several months, but on the other hand, it
gradually loses them when exposed to the action of light and
air. As will be shown later (p. 472), the toxin probably does not
become destroyed, but its toxophorous group suffers a sort of
deterioration so that a toxoid is formed which has still the
power of combining with antitoxins. Heating at 58° C. for
two hours destroys the toxic properties in great part, but not
altogether. When, however, the toxin is evaporated to dry ness, it
NATURE OF THE TOXIN 363
has much greater resistance to heat. One striking fact, discovered
by Roux and Yersin, is that after an organic acid, such as tartaric
acid, is added to the toxin the toxic property disappears, but that
it can be in great part restored by again making the fluid alkaline.
Guinochet showed that toxin was formed by the bacilli when
grown in urine with no proteid bodies present. After growth
had taken place he could not detect proteid bodies in the fluid,
but on account of the very minute amount of toxin present,
their absence could not be excluded. Uschinsky also found that
toxic bodies were produced by diphtheria bacilli when grown in
a proteid-free medium.1 It follows from this that if the toxin is
a proteid, it may be formed by synthesis within the bodies of the
bacilli. Brieger and Boer have separated from diphtheria cultures
a toxic body which gives no proteid reaction (vide p. 166).
Toxic bodies have also been obtained from the tissues of those
who have died from diphtheria. Roux and Yersin, by using
a filtered watery extract from the spleen from very virulent cases
of diphtheria, produced in animals death after wasting and
paralysis, and also obtained similar results by employing the
urine. The subject of toxic bodies in the tissues has, however,
been specially worked out by Sidney Martin. He has separated
from the tissues, and especially from the spleen, of patients who
have died from diphtheria, by precipitation with alcohol, chemical
substances of two kinds, namely, albumoses (proto- and deutero-,
but especially the latter), and an organic acid. The albumoses
when injected into rabbits, especially in repeated doses, produce
fever, diarrhoea, paresis, and loss of weight, with ultimately a
fatal result. As in the experiments with the toxin from cultures,
the posterior limbs are first affected ; afterwards the respiratory
muscles, and finally the heart, are implicated. He further found
that this paresis is due to well-marked changes in the nerves.
The medullary sheaths first become affected, breaking up into
globules ; ultimately the axis cylinders are involved, and may
break across, so that degeneration occurs in the peripheral
portion of the nerve fibres. Such changes occur irregularly in
patches, both sensory and motor fibres being affected. Fatty
change takes place in the associated muscle fibres. There may
also be a similar condition in the cardiac muscle. The organic
acid has a similar but weaker action. Substances obtained from
diphtheria membrane have an action like that of the bodies
1 Uschinsky's medium has the following composition : water, 1000 parts ;
glycerin, 30-40 ; sodium chloride, 5-7 ; calcium chloride, '1 ; magnesium
sulphate, '2- '4 ; di-potassium phosphate, '2- '25; ammonium lactate, 6-7;
sodium asparaginate, 3-4.
364 DIPHTHERIA
obtained from the spleen, but in higher degree. Martin con-
siders that this is due to the presence in the membrane of an
enzyme which has a proteolytic action within the body, resulting
in the formation of poisonous albumoses. According to this
view the actually toxic bodies are not the direct product of the
bacillus, but are formed by the enzyme which is produced by it
locally in the membrane. Cartwright Wood has also found that
when diphtheria cultures in an albumin-containing medium are
filtered germ-free and exposed to 65° C. for an hour (the supposed
ferments being thus destroyed), there still remain albumoses
which produce febrile reaction and are active in developing
immunity. In the present state of knowledge we are not in a
position to give an interpretation of such experiments, and we
cannot even say whether the proteids obtained by precipitation
from cultures and from the tissues are in themselves toxic, or
whether the true toxic bodies are carried down along with
them.
Immunity. — This is described in the general chapter on
Immunity. It is sufficient to state here that a high degree of
immunity, against both the bacilli and their toxins, can be
produced in various animals by gradually increasing doses either
of the bacilli or of their filtered toxins (vide Chap. XIX.).
Variations in the Virulence of the Diphtheria Bacillus. — In
cultures on serum the diphtheria bacilli retain their virulence
fairly well, but they lose it much more quickly on less suitable
media, such as glycerin agar. Roux and Yersin found that,
when the bacilli were grown at an abnormally high temperature,
namely, 39'5° C., and in a current of air, the virulence diminished
so much that they became practically innocuous. When the
virulence was much diminished, these observers found that it
could be restored if the bacilli were inoculated into animals along
with streptococci, inoculation of the bacilli alone not being
successful for this purpose. If, however, the virulence had fallen
very low, even the presence of the streptococci was insufficient
to restore it. As a rule, the cultures most virulent to guinea-
pigs are obtained from the gravest cases of diphtheria, though to
this rule there are frequent exceptions. Perhaps the majority of
observers have found that the bacilli of the larger form are
usually more virulent than those of the shorter form ; but this is
not invariably the case, as sometimes short forms are obtained
which possess an extremely virulent character. It has been
abundantly established that after the cure of the disease, the
bacilli may persist in the mouth for weeks, though they often
quickly disappear. Roux and Yersin found, by making cultures
BACILLI ALLIED TO DIPHTHERIA BACILLUS 365
at various stages after the termination of the disease, that these
bacilli in the mouth gradually become attenuated.
L. Martin, moreover, has shown that some races of diphtheria
bacillus are so attenuated that 1 c.c. of a 24 hours' growth
in bouillon does not cause death in a guinea-pig, yet their true
nature is shown not only by their microscopical characters, etc.,
but also by the fact that on more prolonged growth they form
small quantities of toxin, which is neutralised by diphtheria
antitoxin. The persistence of these non-virulent bacilli in the
throats of those who have suffered from the disease, and their
occasional presence in quite healthy individuals, may manifestly
be of importance in relation to the continuance of the infection
and the reappearance of epidemics of the disease.
Bacilli allied to the Diphtheria Bacillus.
Bacteriological examinations carried on within recent times
have shown that the diphtheria bacillus is merely a member of a
group of organisms with closely allied characters which are of
common occurrence and have a wide distribution. The terms
"pseudo-diphtheria bacilli "and "diphtheroid bacilli" have been
applied in a loose way to organisms which resemble the diphtheria
bacillus microscopically, especially as regards the beaded ap-
pearance. Such bacilli have been obtained from the mouth,
nose, skin, genital organs, and even from the blood in certain
diseases. They are to be met with sometimes in conditions of
health, and they have been obtained from many diverse morbid
conditions — from skin diseases, from coryza, from leprosy, and
even from general paralysis of the insane. As has been found
with other groups the differentiation is a matter of considerable
difficulty. Some are practically identical with the diphtheria
bacillus both morphologically and culturally, and a few even give
the characteristic reaction with ISTeisser's stain ; others again
differ in essential particulars. The fermentative action on
sugars T has also been called into requisition as a means of distin-
guishing them, but the results obtained cannot be said to be of a
definite character, and further work is necessary. It may be
stated, however, that most observers have found the diphtheria
bacillus of all the members of the group to be the most active
acid -producer, though here the difference seems to be one of
degree rather than of kind. The absence of the power of
fermenting certain sugars, notably glucose, may, however, be
accepted in any particular case as sufficient to exclude the
1 Vide a paper by Graham- Smith, Journal of Hygiene, vi. 286.
366 DIPHTHERIA
organism from being the diphtheria bacillus. From these facts,
and from what has been stated with regard to attenuated
diphtheria bacilli, it will be seen that an absolute decision as to
the nature of a suspected organism may in some cases be a
practical impossibility. It may be that some of the " diphtheroid "
organisms cultivated have really been non-virulent diphtheria
bacilli. The bearing of this on the practical means of diagnosis
will be discussed below.
The term " pseudo-diphtheria bacillus " is often restricted by
present writers to an organism frequently met with in the throat.
This organism, which is also known as Hofmann's bacillus, merits
a separate description.
Hofmann's Bacillus — Pseudo- Diphtheria Bacillus. — This
organism, described by Hofmann in 1888, is probably the
same as one observed by
Loffler in the previous
year, and regarded by him
as being a distinct species
from the diphtheria bacil-
lus. The organism is a
shorter bacillus than the
, diphtheria bacillus, with
usually a single unstained
septum running across it,
though sometimes there
may be more than one
(Fig. 122). -The typical
beaded appearance is rarely
^^^ seen, and the reaction with
Neisser's stain is not given.
FIG. 122.— Pseudo-diphtheria bacillus It grows readily on the
(Hofmann's). Young agar culture. same media as the diph-
Stained with thionin-blue. x 1000. theria baci]luS) but the
colonies are whiter and
more opaque. It does not form acid from glucose or other
sugars, and is non-pathogenic to the guinea-pig. Involution
forms may sometimes be produced by it. It is usually a com-
paratively easy matter to distinguish this organism from the
diphtheria bacillus.
Hofmann's bacillus is of comparatively common occurrence in
the throat in normal as well as diseased conditions, including
diphtheria ; it seems to be specially frequent in poorly nourished
children of the lower classes. Cobbett found it 157 times in an
examination of 692 persons examined, of whom 650 were not
SUMMAEY 367
suffering from diphtheria. Boycott's statistics show that the
time of its maximum seasonal prevalence precedes that of the
diphtheria bacillus. To what extent, if any, it is responsible for
pathological changes in the throat, must be considered a question
which is not yet settled. Hewlett and Knight have found
evidence that a true diphtheria bacillus may assume the characters
of Hofmann's bacillus, but attempts to effect the transformation
have met with negative results in the hands of other observers.
The general opinion is that the two organisms are distinct species
which are comparatively easily distinguished characters.
Xerosis Bacillus. — This term has been given to an organism first
observed by Kuschbert and Neisser in xerosis of the conjunctiva, and
which has been since found in many other affections of the conjunctiva
and also in normal conditions. Morphologically it is practically similar
to the diphtheria bacillus, and even in cultures presents very minor
differences. It is, however, non-virulent to animals, and does not pro-
duce an acid reaction in sugar-containing bouillon, or does so to only
a slight extent ; in this way it can be distinguished from the diphtheria
bacillus. Its morphological characters are shown in Fig. 123.
Action of the Diphtheria Bacillus — Summary. — From a
study of the morbid changes in diphtheria and of the results
produced experimentally
by the bacillus and its
toxins, the following sum-
mary may be given of its
action in the body. Locally,
the bacillus produces in-
flammatory change with
nbrinous exudation, but
at the same time cellular
necrosis is also an out-
standing feature. Though
false membranes have not
been produced by the
toxins, a necrotic action
may result when these are
injected subcutaneously. Fm> 123._xerosis bacillus from a young
The toxins also act upon agar culture, x 1000.
the blood - vessels, and
hence cedema and tendency to haemorrhage are produced ;
this action on the vessels is also exemplified by the general
congestion of organs. The hyaline change in the walls of
arterioles and capillaries so often met with in diphtheria is
another example of the action of the toxin. The toxins have
368 DIPHTHEKIA
also a pernicious action on highly-developed cells and on nerve
fibres. Thus in the kidney, cloudy swelling occurs, which may
be followed by actual necrosis of the secreting cells, and along
with these changes albuminuria is present. The action is also
well seen in the case of the muscle fibres of the heart, which
may undergo a sort of hyaline change, followed by granular dis-
integration or by an actual fatty degeneration. These changes
are of great importance in relation to heart failure in the disease.
Changes of a somewhat similar nature have been recently
observed in the nerve cells of the central nervous system, those
lying near the capillaries, it is said, being affected first. There
is also the striking change in the peripheral nerves, which is
shown first by the disintegration of the medullary sheaths as
already described. It is, however, still a matter of dispute to
what extent these nerve lesions are of primary nature or
secondary to changes in the nerve cells.
Methods of Diagnosis. — The bacteriological diagnosis of
diphtheria depends on the discovery of the bacillus. As the
bacillus occurs in largest numbers in the membrane, a portion of
this should be obtained whenever it is possible, and transferred
to a sterile test-tube. (The tube can be readily sterilised by
boiling some water in it.) If, however, membrane cannot be
obtained, a scraping of the surface with a platinum loop may be
sufficient. Where the membrane is confined to the trachea the
bacilli are often present in the secretions of the pharynx, and
may be obtained from that situation by swabbing it with cotton-
wool (non-antiseptic), the swab being put into a sterile tube or
bottle for transport. A convenient method is to twist a piece of
cotton-wool round the roughened end of a piece of very stout
iron wire, six inches long, and pass the other end of the latter
through a cotton plug inserted in the mouth of a test-tube
(compare Fig. 48, the wire taking the place of the pipette), and
sterilise. In use the wire and plug are extracted in one piece,
and after swabbing are replaced in the tube for transit. A
scraping may be made off the swab for microscopic examination,
and the swab may be smeared over the surface of a serum tube
to obtain a culture. This method of taking and treating swabs
is that usually employed in routine public health work. The
results obtained ordinarily suffice for the diagnosis of cases
suspected to be diphtheritic in nature.
The means for identifying the bacillus are (a) By micro-
scopical examination. — For microscopical examination it is
sufficient to tease out a piece of the membrane with forceps and
rub it on a cover-glass ; if it be somewhat dry a small drop of
METHODS OF DIAGNOSIS 369
normal saline should be added. The films are then dried in the
usual way and stained with any ordinary basic stain, though
methylene-blue is on the whole to be preferred, used either as
a saturated watery solution or in the form of Loffler's solution.
After staining for two or three 'minutes the films are washed in
water, dried, and mounted. As a rule no decolorising is
necessary, as the blue does not overstain. Neisser's stain (p. 108)
may also be used with advantage. Any secretion from the
pharynx or other part is to be treated in the same way. The
value of microscopical examination alone depends much upon
the experience of the observer. In some cases the bacilli are
present in characteristic form in such numbers as to leave no
doubt in the matter. In other cases a few only may be found,
mixed with large quantities of other organisms, and sometimes
their characters are not sufficiently distinct to render a definite
opinion possible. We have frequently obtained the bacillus by
means of cultures, when the result of microscopical examination
of the same piece of membrane was non-conclusive. As already
said, however, microscopical examination alone is more reliable
after the observer has had experience in examining cases of
diphtheria and making cultures from them.
(b) By making Cultures. — For this purpose a piece of the
membrane should be separated by forceps from the pharynx or
other part when that is possible. It should be then washed
well in a tube containing sterile water, most of the surface im-
purities being removed in this way. A fragment is then fixed in
a platinum loop by 'means of sterile forceps, and a series of
stroke cultures are made on the surface of any of the media
mentioned (p. 357), the same portion of the membrane being
always brought into contact with the surface. The tubes are
then placed in the incubator at 37° C., and, in the case
of the serum media and blood-agar, the circular colonies
of the diphtheria bacillus are well formed within twenty-four
hours. A small portion of a colony is then removed by means
of a platinum needle, stained, and examined in the usual way,
Neisser's stain being also applied. When the material has been
taken from the throat, an organism with all the morphological
and cultural characters of the diphtheria bacillus may for all
practical purposes be accepted as the diphtheria bacillus.
In cases where a suspicion arises that the organism found
is a pseudo-diphtheria bacillus, bouillon containing a trace
of glucose should be inoculated and incubated at 37° C.
The reaction should be tested after one and after two days'
growth. If it remains alkaline, the diphtheria bacillus may be
24
370 DIPHTHERIA
excluded. If an acid reaction results, then all the microscopical
and cultural characters must be carefully observed, and the
virulence of the bacillus may be ascertained by inoculating a
guinea-pig, say with 1 c.c. of a broth culture of two days' growth.
(See also pp. 359, 365.) A fatal result with characteristic
appearances may be taken as positive evidence, but if the animal
survive there is still theoretically the possibility that the
organism is an attenuated diphtheria bacillus (p. 364).
CHAPTER XVI.
TETANUS.1
SYNONYMS. LOCKJAW. GERMAN, WUNDSTARRKRAMPF.
FRENCH, TETANOS.
Introductory. — Tetanus is a disease which in natural conditions
affects chiefly: man and theJiorse. Clinically it is characterised
by the gradual onset of general stiffness and spasms of the volun-
tary muscles, commencing in those of the jaw and the back of the
neck, and extending to all the muscles of the body. These spasms
are of a tonic nature, and, as the disease advances, succeed each
other with only a slight intermission of time. There are often,
towards the end of a case, fever and rise of respiration and pulse
rate. The disease is usually associated with a wound received from
four to fourteen days previously, and which has been denied by
earth or dung. Such a wound may be very small. The disease
Is, in the majority of cases, fatal. Post mortem there is little to
be observed on naked -eye examination. The most marked
feature is the occurrence of patches of congestion in thejspinal
cord, and especially the medulla.
Historical. — The general association of the development of tetanus with
the presence of wounds, though these might be very small, suggested that
some infection took place through the latter, but for long nothing was
known as to the nature of this infection. Carle and Rattone in 1884
announced that they had produced the disease in a number of animals by
inoculation with material from a wound in tetanus. They thus demon-
strated the transmissibility of the disease. Nocolaier (1885) infected mice
and rabbits with garden earth, and found that many of them developed
tetanus. Suppuration occurred in the neighbourhood of the point of
1 This disease is not to be confused with the " tetany " of infants, which in
its essential pathology probably differs from tetanus (vide Frankl-Hochwart,
" Die Tetanie der Erwachsenen," Vienna, 1907). This remark of course does
not exclude the possibility of the occurrence of true tetanus in very young
subjects.
371
372 TETANUS
inoculation, and in this pus, besides other organisms, there was always
present, when tetanus had occurred, a bacillus having certain constant
microscopic characters. Inoculation of fresh animals with such pus
reproduced the disease. Nicolaier's attempts at its isolation by the
ordinary gelatin plate-culture method were, however, unsuccessful. He
succeeded in getting it to grow in liquid blood serum, but always in
mixture with other organisms. Infection of animals with such a culture
produced the disease. These results were confirmed by Rosenbach, who,
though failing to obtain a pure culture, cultivated the other organisms
present, and inoculated them, but with negative results. He further
pointed out, as characteristic of the bacillus, its development of terminal
spores. In 1889, Kitasato succeeded in isolating from the local suppura-
tion of mice inoculated from a human case, several bacilli, only one of
which, when injected in pure culture into animals, caused the disease,
and which was now named the b. tetani. This organism is the same as
that observed by Nicolaier and Rosenbach. Kitasato found that the
cause of earlier culture failures was the fact that it could only grow in the
absence of oxygen. The pathology of the disease was further elucidated
by Faber, who, having^ isolated bacterium-free poisons from cultures,
reproduced the symptoms of the disease.
Bacillus Tetani. — If in a case of tetanus naturally arising in
man, there be a definite wound with pus formation or necrotic
change, the bacillus tetani may be recognised in film preparations
from the pus, if the characteristic spore formation has occurred
(Fig. 124). If, however, the tetanus bacilli have not formed
spores, they appear as somewhat slender rods, without present-
ing any characteristic features. There is usually present in such
pus a great variety of other organisms — cocci and bacilli. The
characters of the bacillus are, therefore, best studied in cultures.
It is then seen to be a slender organism, usually about 4 /z to 5 /A
in length and '4 //, in thickness, with somewhat rounded ends.
Besides occurring as short rods it also develops filamentous
forms, the latter being more common in fluid media. It stains
readily by any of the usual stains and also by Gram's method.
A feature in it is the uniformity with which the protoplasm stains.
It is very slightly motile, and its motility can be best studied in
an anaerobic hanging- drop preparation (p. 64). When stained
by the special methods already described, it is found to possess
numerous delicate flagella attached both at the sides and at the
ends (Fig. 125). These flagella, though they maybe of consider-
able length, are usually curled up close to the body of the bacillus.
The formation of flagella can be best studied in preparations
made from surface anaerobic cultures (p. 62). As is the case
with many other anaerobic flagellated bacteria the flagella, on
becoming detached, often become massed together in the form
of spirals of striking appearance (Fig. 126). At incubation
temperature b. tetani readily forms spores, and then presents a
BACILLUS TETANI
373
very characteristic appearance. The spores are round, and in
diameter may be three or four times the thickness of the bacilli.
They are developed at one end of a bacillus, which thus assumes
what is usually described as the drumstick form (Figs. 124, 127).
In a specimen stained with a watery solution of gentian- violet or
methylene-blue, the spores are uncoloured except at the periphery,
FIG. 124. — Film preparation of discharge from wound in a case of tetanus,
showing several tetanus bacilli of "drumstick" form. (The thicker
bacillus with oval and not quite terminal spore, in the upper part of the field
towards the right side, is not a tetanus bacillus but a putrefactive
anaerobe which was obtained in pure culture from the wound.)
Stained with gentian- violet. x 1000.
so that the appearance of a small ring is produced ; if a powerful
stain such as carbol-fuchsin be applied for some time, the spores
become deeply coloured like the bacilli. Further, especially
if the preparation be heated, many spores may become free from
the bacilli in which they were formed.
Isolation. — The isolation of the tetanus bacillus is somewhat
difficult. By inoculation experiments in animals, its natural
habitat has been proved to be garden soil, and especially the
374 TETANUS
contents of dung -heaps, where it probably leads a saprophytic
existence, though its function as a saprophyte is unknown.
From such sources and from the pus of wounds in tetanus,
occurring naturally or experimentally produced, it has been
isolated by means of the methods appropriate for anaerobic
bacteria. The best methods for dealing with such pus are as
follows : —
(1) The principle is to take advantage of the resistance of
FIG. 125. — Tetanus bacilli, showing flagella.
Stained by Rd. Muir's method. x 1000.
the spores of the bacillus to heat. A sloped tube of inspissated
serum or a deep tube of glucose agar is inoculated with the
pus and incubated at 37° C. for forty-eight hours, at the end of
which time numerous spore-bearing bacilli can often be observed
microscopically. The culture is then kept at 80° C. for from
three-quarters to one hour, with the view of killing all organisms
except those which have spored. A loopful is then added to
glucose gelatin, and roll-tube cultures are made in the usual
way and kept in an atmosphere of hydrogen at 22° C. ; after
five days the plates are ready for examination. Kitasato com-
ISOLATION OF THE BACILLUS
375
pares the colonies in gelatin plates to those of the b. subtilis.
They consist of a thick
centre with shoots radi-
ating out on all sides.
They liquefy the gelatin
more slowly than the b.
subtilis. This method
of isolation is not always
successful, partly because
along with the tetanus
bacilli, both in its
natural habitats outside
the body and in the pus
of wounds, other spore-
forming obligatory and
facultative anaerobes oc-
cur, which grow faster
than the tetanus bacillus,
and 'thus overgrow it.
(2) If in any dis-
charge the spore-bearing
tetanus bacilli be seen
1
JfP
.
FIG. 126. — Spiral composed of numerous
twisted flagella of the tetanus bacillus.
Stained by Kd. Muir's method. x 1000.
\
^
on microscopic examination, then a
method of isolation
based on the same prin-
ciple as the last may
be adopted. Inocula-
tions with the suspected
material are made in
half a dozen deep tubes
of glucose agar, previ-
ously melteTand kept at
a temperature of 100°
C. After inoculation
they are again placed in
boiling water and kept
for varying times, say
for half a minute, for
one, three, four, five,
and six minutes respect-
ively. They are then
plunged in cold water
till cool, and thereafter
placed in the incubator at 37° C., in the hope that in one
or other of the tubes all the organisms present will have been
FIG. 127. — Tetanus bacilli ; some of which
possess spores. From a culture in glucose
agar, incubated for three days at 37° C.
Stained with carbol-fuchsin. x 1000.
376
TETANUS
killed, except the tetanus spores which can develop in pure
culture.
(3) Some method of anaerobically making plates, such as
that of Bulloch, may be employed. The isolation of the tetanus
bacillus is in many cases a difficult matter,
and various expedients require to be tried.
Characters of Cultures. — Pure cultures
having been obtained, sub-cultures can be
made in deep upright glucose gelatin or
agar tubes. On glucose gelatin in such a
tube there commences, an inch or so below
the surface, a growth consisting of fine
straight threads, rather longer in the lower
than in the upper parts of the tube, radiating
out from the needle track (Fig. 128). Slow
liquefaction of the gelatin takes place, with
slight gas formation. In agar the growth is
somewhat similar, consisting of small nodules
along the needle track, with irregular short
offshoots passing out into the medium (Fig.
131, A). There is slight formation of gas,
but, of course, no liquefaction. Growth also
occurs in blood serum and also in glucose
bouillon under anaerobic conditions. The
latter is the medium usually employed for
obtaining the soluble products of the or-
ganism. There is in it at first a slight
turbidity, and later a thin layer of a
powdery deposit on the walls of the vessel.
FIG. 128 -Stab cul- A]1 ^e cultures give out a peculiar burnt
ture of the tetanus odour of rather unpleasant character,
bacillus in glucose Conditions of Growth, etc. — The b.
gelatin, showing tetani grows best at 37° C. The minimum
the lateral shoots growth temperature is about 14° C., and
(Kitasato). .Natural , -, ^,^0 ^ ^1^1 i i i
gize below 22 C. growth takes place very slowly.
Growth takes place only in the absence of
oxygen, the organism being a strict anaerobe. Sporulation may
commence at the end of twenty-four hours in cultures grown
at 37° C., — much later at lower temperatures. Like other
spores, those of tetanus are extremely resistant. They can
usually withstand boiling for five minutes, and cAn be kept in
a dry condition for many months without being killed or losing
their virulence. They have also high powers of resistance to
antiseptics.
PATHOGENIC EFFECTS 377
Pathogenic Effects. — The proof that the b. tetani is the cause
of tetanus is complete. It can be isolated in pure culture, and
when re-injected in pure culture it reproduces the disease. It
may be impossible to isolate it from some cases of the disease,
but the cause of this very probably is the small numbers in
which it sometimes occurs.
(a) The Disease as arising Naturally, — The disease occurs
naturally, chiefly in horses and in man. Other animals may,
however, be affected. There is usually some wound, often of a
ragged character, which has either been made by an object
soiled with earth or dung, or which has become contaminated
with these substances. There is often purulent or foetid dis-
charge, though this may be absent. Microscopic examination
of sections may show at the edges of the wound necrosed tissue
in which the tetanus bacilli may be very numerous. If a
scraping from the wound be examined microscopically, bacilli
resembling the tetanus bacillus may be recognised. f[f these
have spored, there can be practically no doubt as to their
identity, as the drumstick appearance which the terminal spore
gives to the bacillus is not common among other bacilli.] Care
must be taken, however, to distinguish it from other Ahicker
bacilli with oval spores placed at a short distance from their ex-
tremities, such forms being common in earth, etc., and also met
with in contaminated wounds (Fig. 124). It is important to note
that the wound through which infection has taken place may be
very small, in fact, may consist of a mere abrasion. In some
cases, especially in the tropics, it may be merely the bite of an
insect. The absence of a definite channel of infection has given
rise to the term "idiopathic" tetanus. There is, however,
practically no doubt that all such cases are true cases of tetanus,
and that in all of them the cause is the b. tetani. The latter
has also been found in the bronchial mucous membrane in some
cases of the so-called rheumatic tetanus, the cause of which is
usually said to be cold.
The pathological changes found post mortem are not striking,
trhere may be haemorrhages in the muscles which have been the
subject of the spasms.i These are probably due to mechanical
causes. Naturally it is in the nervous system that we look for
the most important lesions. Here there is ordinarily a general
redness of the grey matter, and the most striking feature is the
occurrence of fa-regular patches of slight congestion/vhich are not
limited particularly to grey or white matter, or to any tract of
the latter. These patches are usually best marked in the grey
matter of the medulla and pons. Microscopically there is little
378 TETANUS
of a definite nature to be found. There is congestion, and there
may be minute haemorrhages in the areas noted by the naked
eye. (The ganglion cells may show appearances which have
been regarded as degenerative in nature,) and similar changes
have been described in the white matter. The only marked
feature is thus a vascular disturbance in the central nervous
system, with a possible tendency to degeneration in its specialised
cells. Both of these conditions are probably due to the action
of the toxins of the bacillus. In the case of the cellular degenera-
tions the cells have been observed to return to the normal under
the curative influence of the antitoxins (vide infra}. In the
other organs of the body there are no constant changes.
We have said that the general distribution of pathogenic
bacteria throughout the body is probably a relative phenomenon,
and that bacteria usually found locally may occur generally and
vice versa. With regard to the tetanus bacillus it is, however,
probably the case that very rarely, if ever, are the organisms
found anywhere except in the local lesion.
(b) The Artificially -produced Disease. — The disease can be com-
municated to animals by any of the usual methods of inoculation,
but does not arise in animals fed with bacilli, whether these
contain spores or not. Kitasato found that pure cultures,
injected subcutaneously or intravenously, caused death in mice,
rats, guinea-pigs, and rabbits. In mice, symptoms appear in a
day, and death occurs in two or three days, after inoculation
with, a loopful of a bouillon culture. The other animals
mentioned require larger doses, and death does not occur so
rapidly. (Usually in animals injected subcutaneously the spasms
begin in the limb nearest the point of inoculation. In intra-
venous inoculation the spasms begin in the extensor muscles of
the trunk, as is the case in the natural disease in man.J After
death there is found slight hypersemia without pus formation, at
the seat of inoculation. The bacilli diminish in number, and
may be absent at the time of death. The organs generally show
little change.
Kitasato states that in his earlier experiments the quantity of
culture medium injected along with the bacilli already contained
enough of the poisonous bodies formed by the bacilli to cause
death. The symptoms came on sooner than by the improved
method mentioned below, and were, therefore, due to the toxins
already present. In his subsequent work, therefore, he employed
splinters of wood soaked in cultures in which spores were present,
and subsequently subjected for one hour to a temperature of
80° C. The latter treatment not only killed all the bacilli, but,
TOXINS OF THE TETANUS BACILLUS 379
as we shall see, was sufficient to destroy the activity of the toxins.
When such splinters are introduced subcutaneously, death results
by the development of the spores which they carry, fin this way
he completed the proof that the bacilli by themselves can form
toxins in the body and produce the disease.) Further, if a small
quantity of garden earth be placed under the skin of a mouse,
death from tetanus takes place in a great many cases. [Some-
times, however, in such circumstances death occurs without
tetanic symptoms, and is not due to the tetanus bacillus but to
the bacillus of malignant oedema, which also is of common
occurrence in the soil (vide infra).] By such experiments, supple-
mented by the culture experiments mentioned, the natural
habitats of the b. tetani, as given above, have become known.
The Toxins of the Tetanus Bacillus. — The tetanus bacillus
being thus accepted as the cause of the disease, we have to
consider how it produces its pathogenic effects.
Almost contemporaneously with the work on diphtheria was the
attempt made with regard to tetanus to explain the general symptoms
by supposing that the bacillus could excrete soluble poisons. The
earlier results in which certain bases, (tetanin and tetanotoxinj were
said to have been isolated, have only a historic interest, as they were
obtained by faulty methods. In 1890 Brieger and Fraenkel announced
that they had isolated &(toxalbumin) from tetanus cultures, and this body
was independently discovered by Faber in the same year. Brieger and
Fraenkel's body consisted practically of an alcoholic precipitate from
filtered cultures in bouillon, and was undoubtedly toxic. Within recent
years such attempts to isolate tetanus toxins in a pure condition have
practically been abandoned, and attention has been turned to the
investigation of the physiological effects either of the crude toxin
present in filtered bouillon cultures, or of the precipitate produced from
the same by ammonium sulphate (cf. p. 167).
The toxic properties of bacterium-free nitrates of pure cultures
of the b. tetani were investigated in 1891 by Kitasato. This
observer found that when the filtrate, in certain doses, was
injected subcutaneously or intravenously into mice, tetanic spasms
developed, first in muscles contiguous to the site of inoculation,
and later all over the body. Death resulted, file found that
guinea-pigs were more susceptible than mice, and rabbits less sol
In order that a strongly toxic bouillon be produced, it must
originally have been either neutral or slightly alkaline.) fKitasato
further found that the toxin was easily injured by heat.) Exposure
for a few minutes at 65° C. destroyed it. It was also destroyed
by twenty minutes' exposure at 60° C., and by one and a half
hours' at 55° C. ^Drying had no effect. It was, however,
destroyed by various chemicals such as (pyrogallol (and also by
380 TETANUS
sunlight. Behring has more recently pointed out that after the
nitration of cultures containing toxin, (the latter may very rapidly
lose its power, and in a few days may only possess y^th of its
original toxicity. This he attributes to such factors as temperature
and light, and especially to the action of oxygen.] The effect
of these agents on the crude toxin is undoubtedly to cause a
degeneration of the true toxin into a series of toxoids similar to
those produced in the case of diphtheria toxin, and it is also
true here that the toxoids while losing their toxicity may still
retain their power of producing immunity against the potent
toxin. Further, altogether apart from the occurrence side by
side in the crude toxin of strong and weak poisons, it has been
shown that such crude toxin contains toxic substances of probably
quite a different nature. (jEhrlich has shown that besides the pre-
dominant spasm-producing toxin (called by him tetanospasmin),
there exists in crude toxin a poison capable of producing the
solution of certain red blood corpuscles. This haemolytic agent
he calls tetanolysin) It does not occur in all samples of crude
tetanus toxin, nor is it found when a bouillon culture of the
bacillus is filtered through porcelain. To obtain it the fresh
culture must be treated by ammonium sulphate, as described in
the method of obtaining concentrated toxins (p. 167). This
substance also has the power of originating an antitoxin so. that
certain antitetanic sera can protect red blood corpuscles against
its action. Madsen, studying the interactions of this anti-
tetanolysin with the tetanolysin, has shown that the phenomena
can be demonstrated similar to those noted by Ehrlich as
occurring with diphtheria toxin, and which he interpreted as
indicating the presence .of degenerated toxins (toxoids) in the
crude poison. With tetanus as with diphtheria toxin the action
of an acid is to cause an apparent disappearance of toxicity, but
if before a certain time has elapsed the acid be neutralised by
alkali, then a degree of the toxicity returns.
As with other members of the group, nothing is known of the
nature of tetanus toxin. Uschinsky has found that the tetanus
bacillus can produce its toxin when growing in a fluid containing
no proteid matter. The toxin may thus be formed independently
of the breaking up of the proteids on which the bacillus may be
living, though the latter no doubt has a digestive action on
these. The liquefaction (i.e. probable peptonisation) of gelatin
cultures advances pari passu with the development of toxins,
and filtered bacterium-free cultures will still liquefy gelatin. /It
is probable that there is an independent peptic ferment which
will, of course, also pass through a filter^ For if equal portions
if
TOXINS OF THE TETANUS BACILLUS 381
of the filtered culture be left in contact with equal portions of
gelatin for various lengths of time, there is no increase of toxicity
in those kept longest. There is thus no fresh development of
toxin during the advancing liquefaction of the gelatin. Thus
peptic digestion and toxin formation are apparently due to
different vital processes on the part of the tetanus bacillus.
Whatever the nature of the toxin is, it is undoubtedly one
of the most powerful poisons known. Even with a probably
impure toxalbumin Brieger found that the fatal dose for a
mouse was '0005 of a milligramme. If the susceptibility of
man be the same as that of a mouse, the fatal dose for an average
adult would have been '23 of a milligramme, or about -^ ^ ^-ths
of a grain. Animals differ very much in their susceptibilities
to the action of tetanus toxin. According to v. Lingelsheim, if
the minimal lethal dose per gramme weight for a horse be taken
as unity, that for the guinea-pig would be 6 times the amount,
the mouse 12, the goat 24, the dog about 500, the rabbit 1800,
the cat 6000, the goose 12,000, the pigeon 48,000, and the
hen 360,000.
("A striking feature of the action of tetanus toxin is the
occurrence of a definite incubation period between the introduc-
tion of the toxin into an animal's body and the appearance of
symptoms.J (The incubation period varies according to the species
of animal employed, and the path of infection. In the guinea-
pig it is from thirteen to eighteen hours, in the horse five days,
and the incubation is shorter when the poison is introduced into
a vein than when injected subcutaneously. In man the period
between the receiving of an injury and the appearance of tetanic
symptoms is from two to fourteen daysl
With regard to the action of the toxin, it has been shown to
have no effect on the sensory or motor endings of the nerves.
(it acts solely as an exciter of the reflex excitability of the motor
cells in the spinal cord./ The motor cells in the pons and
medulla are also affected, and to a much greater degree than
those in the cerebral cortex. When injected subcutaneously
the toxin is absorbed into the nerves, and thence finds its way
to that part of the spinal cord from which these nerves spring.
This explains the fact that in some animals the tetanic spasms
appear first in the muscles of the part in which the inoculation
has taken place. This is not the case with man, in whom usually
the first symptoms appear in the neck. In artificial injection of
toxin part finds its way into the blood stream, and if infected
animals be killed during the incubation period there is often
evidence of toxin in the blood and solid organs. In the guinea-
382 TETANUS
pig there is little doubt that tetanus toxin has an affinity solely
for the nervous system. In other animals, such as the rabbit,
an affinity may exist in other organs, and the fixation of the
poison in such situations may give rise to no recognisable
symptoms. In such an animal as the alligator, it is possible
that while some of its organs have an affinity for tetanus toxin
its nervous system has none. These facts are of great scientific
interest, and a possible explanation of them will be discussed in
the chapter on Immunity. If tetanus toxin be introduced into
the stomach or intestine, it is not absorbed, but to a large extent
passes through the intestine unchanged. Evidence that any
destruction takes place is wanting.
Within recent years some important light has been shed on
the mode of action of tetanus toxin. Marie and Morax studied
the path of absorption when the toxin was injected into the
muscles of the hind limb. The sciatic nerve in a rabbit was cut
near the spinal cord and toxin introduced into the muscles of the
same side ; after some hours the nerve was excised and introduced
into a mouse — the animal died of tetanus. But if the nerve were
cut near the muscles and the same procedure adopted, the mouse
did not contract the disease, though no doubt the cut nerve had
been surrounded by lymph containing toxin. If the same
experiment were performed and an excess of toxin injected into
the other limb, still only the nerve which was left in connection
with the muscle showed evidence of containing toxin. (From
this it was deduced that the toxin was absorbed by the end-
plates in the muscle and not from the lymphatics surrounding
the nerve.) It was further shown that a nerve in the process of
degeneration following section did not absorb toxin after the
manner of a normal nerve. By a similar method it was shown
that the absorption by the nerve was fairly rapid, as one hour
after injection the toxin was present in it, and from other
experiments the view was put forth that the toxin was centripetal
in its flow and did not pass centrifugally in a nerve to which it
artificially gained access. Further observations have been made
on this subject by Meyer and Ransom. These observers found
evidence that toxin is only absorbed by the motor filaments of a
nerve, for while tetanus could .be produced by injection into a
mixed nerve like the sciatic the introduction of a lethal dose into
such a sensory nerve as the infra-orbital was not followed by
disease symptoms. If a small dose of toxin be injected into the
sciatic nerve it reaches the corresponding motor cells of the cord,
and a local tetanus of the muscles supplied by the nerve results.
With a larger dose the poison passes across the commissure to
TOXINS OF THE TETANUS BACILLUS 383
the corresponding cells of the other side, and if still further
excess is present it passes up the cord to higher centres. The
affection of such higher centres can be prevented by section of
the cord. Meyer and Ransom £hold that when toxin is injected
subcutaneously or intravenously, it only acts by being absorbed
by the end-plates in muscles and thence passes to the cord, and
they consider that the incubation period is to be explained by the
time taken for this extended passage to occur.) In this connection
they point out that it is in the larger animals, where the nerve
path is longest, that the incubation period is also long. Like
Marie and Morax, they believe that absorption of toxin by its
bathing the lateral aspects of uninjured nervous structures does
not occur, and in support of this they bring forward the
observation that when intravenous injection is practised, the
occurrence of tetanus in a part of the body can be precipitated by
such a slight injury as may be caused by the injection of a drop
of normal saline into the corresponding part of the cord. With
regard to the action of tetanus toxin, Meyer and Ransom believe
that there is a double effect on the nerve cells — ferst, an exaggera-
tion of the normal tonus, which accounts for the continuous
stiffness of the muscles, and secondly, an increase in reflex
irritability, which is a prominent factor in the recurring spasms'^
While no absorption of toxin takes place by sensory filaments,
they have found evidence of sensibility of the sensory apparatus
in the occurrence of what they call {tetanus dolorosus) This is a
great hypersesthesia and a paroxysmal hyperalgesia which can be
caused by injecting toxin into the spinal cord or into a sensory
root on the spinal side of the posterior root ganglion. These
symptoms are unaccompanied by motor spasms, but the animal
may die from exhaustion. The same observers have also made
interesting observations on the action of antitoxin. They found
that the injection of this substance into the course of a mixed
nerve could prevent toxin from passing up to the cord, but that if
antitoxin were injected even in great excess intravenously, and a
short time thereafter toxin were introduced into a nerve, the
death of the animal was not prevented. This they attribute
to the fact that antitoxin can only neutralise the toxin which is
still circulating in the blood. This is a very far-reaching
conclusion, as it throws doubt on what has been held to be a
possibility, namely, (that toxin can be actually detached from
cells in which it is already anchored^ But a still more
significant observation was made, for in one case of an animal
actively immunised against tetanus, and which contained in its
serum a considerable quantity of antitoxin, the injection of toxin
384 TETANUS
into the sciatic nerve was followed by tetanus. This would
appear to militate against Ehrlich's position that antitoxin is
manufactured in the cells which are sensitive to the toxin (see
Immunity).
Reference may here be made to the effects of injecting tetanus
toxin into the brain itself, as investigated by Roux and Borrel.
ft was found that the ordinary type of the disease was not
produced, but what these observers called "cerebral tetanus. J
This consisted of general unrest, symptoms of a psychic
character (apparent hallucinations, fear, etc.), and epileptiform con-
vulsions. Death occurred in from twelve to twenty hours without
any true tetanic spasms. In this manifestation of tetanus the
incubation period was much shorter than with subcutaneous
injection, and (the fatal dose was one twenty-fifth of the minimal
subcutaneous dosel Further, the injection of antitoxin forty-eight
to ninety-six hours previously did not prevent an animal from suc-
cumbing to the intracerebral inoculation. (En the light of what has
been already said these results would seem to indicate a special
effect of the toxin when brought into direct contact with the
protoplasm of the brain cells)
We have seen that unless suitable precautions are adopted, in
experimental tetanus in animals death results not from inocula-
tion but from an intoxication with toxin previously existent in
the fluid in which the bacilli have been growing. According to
Vaillard, if spores rendered toxin-free, by being kept for a
sufficient time at 80° C., are injected into an animal, death does
not take place. It was found, however, that such spores can be
rendered pathogenic by injecting along with them such chemicals
as lactic acid, by injuring the seat of inoculation so as to cause
effusion of blood, by fracturing an adjacent bone, by introducing
a mechanical irritant such as soil or a splinter of wood (as in
Kitasato's experiments), or by the simultaneous injection of
other bacteria such as the staphylococcus pyogenes aureus. These
facts, especially the last, throw great light on the disease as it
occurs naturally, for tetanus results especially from wounds
which have been accidentally subjected to conditions such as
those enumerated, fcitasato now holds that in the natural
infection in man, along with tetanus spores, the presence of
foreign material or of other bacteria is necessaryJ Spores alone
or tetanus bacilli without spores die in the tissues, and tetanus
does not result.
Immunity against Tetanus. — Antitetanic Serum. — The arti-
ficial immunisation of animals against tetanus has received much
attention. The most complete study of the question is found
IMMUNITY AGAINST TETANUS 385
in the work of Behring and Kitasato in Germany, and of Tizzoni
and Cattani in Italy. The former observers found that such an
immunity could be conferred "by the injection of very small and
progressively increasing doses of the tetanus toxin. The degree
of immunity attained, however, was not high. Subsequent
work has shown that the less rich a crude toxin is in modifica-
tions of the true toxin the less useful it is for immunisation
procedures. In fact it is doubtful if small animals can be
immunised at all by fresh filtrates. In some cases it has been
found that the injection of non-lethal doses instead of commenc-
ing an immunity actually increases the susceptibility of the
animal. More successful are the methods of accompanying the
early injections of crude toxin with the subcutaneous introduction
of small doses of iodine terchloride, or of using toxin which has been
acted on with iodine terchloride or with iodine itself. Tizzoni
and Cattani have also used the method of administering pro-
gressively increasing doses of living cultures attenuated in
various ways, e.g. by heat. By any of these methods susceptible
animals can be made to acquire great immunity, not only
against many times the fatal dose of tetanic toxin, but also
against injections of the living bacilli. The degree of immunisa-
tion acquired by an animal remains in existence for a very long
time. Not only so, but when a high degree of immunity has
been produced by prolonged treatment, it is found that the
serum of immune animals possesses the capacity, when injected
into animals susceptible to the disease, of protecting them
against a subsequent infection with a fatal dose of tetanus
bacilli or toxin. Further, if injected subsequently to such
infection, the serum can in certain cases prevent a fatal result,
even when symptoms have begun to appear. The degree of
success attained depends, however, on the shortness of the time
which has elapsed between the infection with the bacilli or toxin
and the injection of the serum. In animals where symptoms
have fully manifested themselves only a small proportion of
cases can be saved. As with other antitoxins there is no
evidence that the antitetanic serum has any detrimental effect
on the bacilli. It only neutralises the Effects of the toxin.
The standardisation of the antitetanic serum is of the highest
importance. Behring recommends that for protecting animals
a serum should be obtained of which one gramme will protect
1,000,000 grammes weight of mice against the minimum fatal
dose of the bacillus or toxin. A mouse weighing twenty
grammes would thus require '00002 gramme of the serum to
protect it against the minimum lethal dose. In the injection
25
386 TETANUS
of such a serum subsequent to infection, if symptoms, have
begun to appear, 1000 times this dose would be necessary ; a
few hours later 10,000 times, and so on.
As the result of his experiments, Behring aimed at obtaining
a curative effect in the natural disease occurring in man. For
this purpose, as for his later laboratory experiments, he obtained
serum by the immunisation of such large animals as the horse,
the sheep, and the goat, by the injection of toxin accompanied
at first with the injection of iodine terchloride. It was found
that the greater the degree of the natural susceptibility of an
animal to tetanus, the easier was it to obtain a serum of a high
antitetanic potency. The horse was, therefore, the most
suitable animal. If now we take for granted that the relative
susceptibility of man and the mouse towards tetanus are nearly
equal, a man weighing 100 kilogrm. would require *1 grm. of
the serum mentioned above to protect him from inoculation
with the minimum lethal dose of bacilli or toxin. If symptoms
had begun to appear, 100 c.c. at once would be necessary, and
as the injection of such a quantity might be inconvenient,
Behring recommended that for man a more powerful serum
should be obtained, viz., a serum of which one gramme would
protect 100,000,000 grammes weight of mice.1 The potency is
maintained for several months if precautions are taken to
avoid putrefaction, exposure to bright light, etc. To this end
•5 per cent carbolic acid is usually added, and the serum is
kept in the dark. In a case of tetanus in man, 100 c.c. of
such a serum should be injected within twenty-four hours in
five doses, each at a different part of the body, and this
followed up by further injections if no improvement takes
place. Intravenous injection of the antitoxin has also been
practised, and, in cases which we have seen treated in this way,
has seemed to give better results than those obtained by the
subcutaneous method. The serum is warmed to the body
temperature and slowly introduced into a vein in the arm, the
pulse and respiration being carefully watched during the
proceeding. Ten to twenty c.c. can be injected every few hours,
and in all 100 c.c. should be given in as short a time as possible.
Henderson Smith has shown that when antitoxins to toxins of
the tetanus group are injected intravenously a high concentration
in the body fluid is maintained for some time and the op-
portunity for neutralisation of toxin is thus great. He suggests
1 The antitetanic serum sent out by the Pasteur Institute in Paris has a
strength of 1 : 1,000,000,000. Of this it is recommended that 50 to 100 c.c.
should be injected in one or two doses.
IMMUNITY AGAINST TETANUS 387
that vboth intravenous and subcutaneous injections should be
simultaneously practised. The former gives the quickly attained
concentration which is desirable, and when the antitoxin injected
intravenously is beginning to be eliminated that introduced
hypodermically comes into the circulation and the concentration
is maintained. The antitoxin has also been introduced intra-
cerebrally, very slow injection into the brain substance being
practised, but no better results have been obtained than by the
subcutaneous method.
Many cases of human tetanus have been thus treated, but
the improvement in the death-rate has not been nearly so
marked as that which has occurred in diphtheria under similar
circumstances. As in the case of diphtheria, however, the
results would probably be better if more attention were paid
to the dosage of the serum. The great difficulty is that, as a
matter of fact, we have not the opportunity of recognising the
presence of the tetanus bacilli till they have begun to manifest
their gravest effects. In diphtheria we have a well-marked
clinical feature which draws attention to the probable presence
of the bacilli — a presence which can be readily proved, — and
the curative agent can thus be early applied. In tetanus the
wound in which the bacilli exist may be, as we have seen, of
the most trifling character, and even when a well-marked
wound exists, the search for the bacilli may be a matter of
difficulty. Still it might be well, when practicable, that every
ragged, unhealthy-looking wound, especially when contaminated
with soil, should, as a matter of routine, be examined
bacteriologically. In such cases, undoubtedly, from time to
time cases of tetanus would be detected early, and their treat-
ment could be undertaken with more hope of success than at
present. However, in the existing state of matters, whenever
the first symptoms of tetanus appear, large doses, such as those
above indicated, of a serum whose strength is known, should be
at once administered. In giving a prognosis as to the probable
result, the two clinical observations on which, according to
Behring, chief reliance ought to be placed, are the presence or
absence of interference with respiration, and the rapidity with
which the groups of muscles usually affected are attacked. If
dyspnoea or irregularity in respiration comes on soon, and if group
after group of muscles is quickly involved, then the outlook is
extremely grave. In addition to these points the duration of the
incubation period is of high importance in forming a prognosis.
The shorter the time between the infliction of a wound and the
appearance of symptoms the graver is the outlook.
388 TETANUS
The theory as to the nature of antitoxic action will be
discussed later in the chapter on Immunity.
Methods of Examination in a case of Tetanus. — The
routine bacteriological procedure in a case presenting the clinical
features of tetanus ought to be as follows : —
(a) Microscopic. — Though tetanus is not a disease in which
the discovery of the bacilli is easy, still microscopic examination
should be undertaken in every case. From every wound or
abrasion from which sufficient discharge can .be obtained, film
preparations ought to be made and stained with any of the
ordinary combinations, e.g. carbol-fuchsin diluted with five parts
of water. Drumstick-shaped spore-bearing bacilli are to be-
looked for. The presence of such, having characters corre-
sponding to those of the tetanus bacilli, though not absolutely
conclusive proof of identification, is yet sufficient for all practical
purposes. If only bacilli without spores resembling the tetanus
bacilli are seen, then the identification can only be provisional.
The microscopic examination of wounds contaminated by soil,
etc., may, as we have said, in some cases lead to the anticipation
that tetanus will probably result.
(b) Cultivation. — The methods to be employed in isolating
the tetanus bacilli have already been described (p. 373). It
may be added, however, that if the characteristic forms are
not seen on microscopic examination of the material from the
wound, they may often be found by inoculating a deep tube of
one of the glucose media with such material, and incubating for
forty-eight hours at 37° C. At the end of this period, spore-
bearing tetanus bacilli may be detected microscopically, though
of course mixed with other organisms.
(c) Inoculation. — Mice and guinea-pigs are the most suitable
animals. Inoculation with the material from a wound should
be made subcutaneously. A loopful of the discharge introduced
at the root of the tail in a mouse will soon give rise to the
characteristic symptoms, if tetanus bacilli are present.
MALIGNANT (EDEMA (Septicemie de Pasteur).
The organism now usually known as the bacillus of malignant
cedema is the same as that first discovered by Pasteur and
named vibrion septique. He described its characters, distin-
guishing it from the anthrax bacillus which it somewhat
resembles morphologically, and also the lesions produced by it.
He found that it grew only in anaerobic conditions, but was
able to cultivate it merely in an impure state. It was more
MALIGNANT (EDEMA
389
fully studied by Koch, who called it the bacillus of malignant
oedema, and pointed out that the disease produced by it is not
really of the nature of a septicaemia, as immediately after death
the blood is practically free from the bacilli.
"Malignant oedema" in the human subject is usually
described as a spreading inflammatory oedema attended with
emphysema, and ultimately followed by gangrene of the skin
FIG. 129. — Film preparation from the affected tissues in a case of malignant
oedema in the human subject, showing the spore-bearing bacilli.
Gentian- violet, x 1000.
and subjacent parts. In many cases of this nature the bacillus
of malignant oedema is present, associated with other organisms
which aid its spread, whilst in others it may be absent. One of
us has, however, observed a case in which the bacillus was
present in pure condition. Here there occurred intense oadema
with swelling and induration of the tissues, and the formation
of vesicles on the skin. Those changes were attended with a
reddish discoloration, afterwards becoming livid. Emphysema
was not recognisable until the limb was incised, when it was
detected, though in small degree. Further, the tissues had a
390
MALIGNANT (EDEMA
peculiar heavy, but not putrid, odour. The bacillus, which was
obtained in pure culture, was present in enormous numbers in
the affected tissues, attended by cellular necrosis, serous
exudation, and at places much leucocytic emigration. The
picture, in short, corresponded with that seen on inoculating a
guinea-pig with a pure culture. The term " malignant oedema "
should be limited in its application to cases in which the
bacillus in question is present. In most of these there is a
mixed infection ; in some this bacillus may be present alone.
This bacillus has a very widespread distribution in nature,
being present in garden
soil, dung, and various
putrefying animal fluids ;
and it is by contamination
of lacerated wounds by such
substances that the disease
is usually set up in the
human subject. Malignant
oedema can be readily pro-
duced by inoculating sus-
ceptible animals, such as
guinea - pigs, wi th garden
soil. The bacillus is also
of ten present in the intestine
of man and animals, and has
been described as being
present in some gangrenous
conditions originating in
connection with the in-
testine in the human sub-
ject.
Microscopical Characters. — -The bacillus of malignant oedema
is a comparatively large organism, being slightly less than 1 //,
in thickness, that is, thinner than the anthrax bacillus. It
occurs in the form of single rods 3 //, to 10 /x in length, but both
in the tissues and in cultures in fluids it frequently grows out
into long filaments, which may be uniform throughout or
segmented at irregular intervals. In cultures on solid media it
chiefly occurs in the form of shorter rods with somewhat rounded
ends. The rods are motile, possessing several laterally placed
flagella, but in a given specimen, as a rule, only a few bacilli
show active movement. Under suitable conditions they form
spores which are usually near the centre of the rods and have an
oval shape, their thickness somewhat exceeding that of the
FIG. 130. — Bacillus of malignant oedema,
showing spores. From a culture iu
glucose agar, incubated for three days
at 37° C.
Stained with weak carbol-fuchsin. x 1000.
CHARACTER OF CULTURES
391
bacillus (Figs. 129, 130). The bacillus can be readily stained
by any of the basic aniline stains, but loses the colour in Gram's
method, in this way differing from the anthrax bacillus.
Characters of Cultures. — This organism grows readily at
ordinary temperature, but only under anaerobic conditions. In
a puncture culture in a deep tube of glucose gelatin, the growth
A B c
FIG. 131.— Stab cultures in agar, five days' growth at 37° C.
Natural size.
A. Tetanus bacillus. B. Bacillus of malignant oedema. C. Bacillus of
quarter-evil (Rauschbrand).
appears as a whitish line giving off minute short processes, the
growth, of course, not reaching the surface of the medium.
Soon liquefaction occurs, and a long fluid funnel is formed, with
turbid contents and nocculent masses of growth at the bottom.
At the same time bubbles of gas are given off, which may split
up the gelatin. The colonies in gelatin plates under anaerobic
conditions appear first as small whitish points which under the
microscope show a radiating appearance at the periphery,
resembling the colonies of the bacillus subtilis. Soon, however,
392 MALIGNANT (EDEMA
liquefaction occurs around the colonies, and spheres with turbid
contents result ; gas is developed around the colonies.
In deep tubes of glucose agar at 37° C., growth is extremely
rapid. Along the line of puncture, growth appears as a some-
what broad white line with short lateral projections here and
there (Fig. 131, B). Here also gas may be formed, but this is
most marked in a shake culture, in which the medium becomes
cracked in various directions, and may be pushed upwards so
high as to displace the cotton-wool plug. The cultures possess
a peculiar heavy, though not putrid, odour.
Spore formation occurs above 20° C., and is usually well
seen within forty-eight hours at 37° C. The spores have the
usual high powers of resistance, and may be kept for months in
the dried condition without being killed.
Experimental Inoculation. — A considerable number of animals
— the guinea-pig, rabbit, sheep, and goat, for example — are
susceptible to inoculation with this organism. The ox is said to
be quite immune to experimental inoculation, though it can,
under certain conditions, contract the disease by natural channels.
The guinea-pig is the animal most convenient for experimental
inoculation. When the disease is set up in the guinea-pig by
subcutaneous inoculation with garden soil, death usually occurs
in about twenty-four to forty-eight hours. There is an intense
inflammatory oedema around the site of inoculation, which
extends over the wall of the abdomen and thorax. The skin
and subcutaneous tissue are infiltrated with a reddish brown fluid
and softened ; they contain bubbles of gas and are at places
gangrenous. The superficial muscles are also involved. These
parts have a very putrid odour. The internal organs are con-
gested, the spleen soft but not much enlarged. In such con-
ditions the bacillus of malignant oedema, both in short and long
forms, will be found in the affected tissues along with various
other organisms. Spores may be present, especially when the
examination is made some time after the death of the animal.
If the animal is examined immediately after death, a few of the
bacilli may be present in the peritoneum and pleurae, usually in
the form of long motile filaments, but they are almost invariably
absent from the blood. A short time after death, however, they
spread directly into the blood and various organs, and may then
be found in considerable numbers.
Subcutaneous inoculation with pure cultures of the bacillus of
malignant oedema produces chiefly a spreading bloody oedema,
the muscles being softened and partly necrosed ; but there is
little formation of gas, and the putrid odour is almost absent.
BACILLUS BOTULINUS 393
When the bacilli are injected into mice, however, they enter
and multiply in the blood stream, and they are found in con-
siderable numbers in the various organs, so that a condition not
unlike that of anthrax is found. The spleen also is much
swollen.
The virulence of the bacillus of malignant oedema varies con-
siderably in different cases, and it always becomes diminished in
cultures grown for some time. To produce a fatal disease, a
relatively large number of the organisms is necessary, and these
must be introduced deeply into the tissues, inoculation by scari-
fication being followed by no result. A smaller dose produces a
fatal result when injected along with various other organisms
(bacillus prodigiosus, etc.).
Immunity. — Malignant oedema was one of the first diseases
against which immunity was produced by injections of toxins.
The filtered cultures of the bacillus in sufficient doses produce
death with the same symptoms as those caused by the living
organisms, but a relatively large quantity is necessary. Chamber-
land and Roux (1887) found that if guinea-pigs were injected
with several non-fatal doses of cultures sterilised by heat or freed
from the bacilli by filtration, immunity against the living organism
could be developed in a comparatively short time. They found
that the filtered serum of animals dead of the disease is more
highly toxic, and also gives immunity when injected in small
doses. These experiments have been confirmed by Sanfelice.
Methods of Diagnosis. — In any case of supposed malignant
oedema, the fluid from the affected tissues ought first to be
examined microscopically, to ascertain the characters of the
organisms present. Though it is not possible to identify ab-
solutely the bacillus of malignant oedema without cultivating it,
the presence of spore-bearing bacilli with the characters described
above is highly suspicious (Fig. 1 29). In such a case the fluid
containing the bacilli should be first exposed to a temperature
of 80° C. for half an hour, and then a deep glucose agar tube
should be inoculated. In this way the spore-free organisms are
killed off. Pure cultures may be thus obtained, or this procedure
may require to be followed by the roll -tube method under
anaerobic conditions. An inoculation experiment, if available,
may also be made on a guinea-pig.
BACILLUS BOTULINUS.
The term " meat-poisoning " embraces a number of conditions
produced by different agents, and the relation of the bacillus of
394 BACILLUS BOTULINUS
Gaertner to one class of case has already been discussed. Another
group was shown by van Ermengem in 1896 to be caused by an
anaerobic bacillus to which he gave the name bacillus lotulinus.
He cultivated the organism from a sample of ham, the ingestion
of which in the raw condition had produced a number of cases
of poisoning, some of them followed by fatal result. The
symptoms in these cases closely corresponded with those occur-
ring in the so-called " sausage poisoning " met with from time to
time in Germany and other countries where sausages and ham
are eaten in an imperfectly cooked condition. Such cases form
a fairly well-defined group, the symptoms in which are chiefly
referable to an action on the medulla, and, as will be detailed
below, similar symptoms have been experimentally produced
by means of the bacillus mentioned or its toxins. The chief
symptoms of this variety of botulismus, as detailed by van
Ermengem, are disordered secretion in the mouth and nose, more
or less marked ophthalmoplegia, externa and interim (dilated
pupil, ptosis, etc.), dysphagia, and sometimes aphagia with
aphonia, marked constipation and retention of urine, and in
fatal cases interference with the cardiac and respiratory centres.
Along with these there is practically no fever and no interference
with the intellectual faculties. The symptoms commence at
earliest twelve to twenty-four hours after ingestion of the poison.
From the ham in question, which was not decomposed in the
ordinary sense, van Ermengem obtained numerous colonies of this
bacillus, the leading characters of which are given below. It may
be added that Homer obtained practically the same results as van
Ermengem in a similar condition, and that the bacillus botulinus
has been cultivated by Kempner from the intestine of the pig.
Microscopical and Cultural Characters. — The organism is a
bacillus of considerable size, measuring 4 to 9 //, in length and
•9 to 1'2 /* in thickness; it has somewhat rounded ends and
sometimes is seen in a spindle form. It is often arranged in
pairs, sometimes in short threads. Under certain conditions it
forms spores which are oval in shape, usually terminal in position,
and a little thicker than the bacilli. It is a motile organism
and has 4 to 8 lateral flagella of wavy form. It stains readily
with the ordinary dyes, and also retains the colour in Gram's
method, though care must be employed in decolorising.
The organism can be readily cultivated on the ordinary
media, but only under strictly anaerobic conditions. In glucose
gelatin a whitish line of growth forms with lateral offshoots,
but liquefaction with abundant gas formation soon occurs. In
gelatin plates the colonies after four to six days are somewhat
MICROSCOPICAL AND CULTURAL CHARACTERS 395
characteristic ; they appear to the naked eye as small semi-
transparent spheres, and these on examination under a low
power of the microscope have a yellowish-brown colour and are
seen to be composed of granules which show a streaming move-
ment, especially at the periphery. Cultures in glucose agar
resemble those of certain other anaerobes ; there is abundant
development of gas, and the medium is split up in various
directions. The cultures have a rancid, though not foul, odour,
due chiefly to the development of butyric acid. The optimum
temperature is below that of the body, viz., between 20° and
30° C. ; at the body temperature growth is slower and less
abundant and spore formation does not occur.
Pathogenic Effects. — Like the tetanus bacillus the bacillus
botulinus has little power of nourishing in the tissues, whereas it
produces a very powerful toxin. Van Ermengem found that the
characteristic symptoms could be produced in certain animals
by administering watery extracts of the infected ham or cultures
either by the alimentary canal or by subcutaneous injection.
Here also there is a period of incubation of not less than six to
twelve hours before the symptoms appear, and when the dose is
small a somewhat chronic condition may result in which local
paralyses form a striking feature. The characteristic effects
can also be produced by means of the filtered toxin by either of
the methods mentioned, though in the case of administration by
the alimentary canal the dose requires to be larger. Here also,
as in the case of the tetanus poison, the potency of the toxin is
remarkable, the fatal dose for a guinea-pig of 250 grm. weight
being in some instances "0005 c.c. of the filtered toxin. In cases
of poisoning in the human subject the effects would accordingly
appear to be produced by absorption of the toxin from the
alimentary canal ; it is only after or immediately before death
that a few bacilli may enter the tissues. Van Ermengem
obtained a few colonies from the spleen of a patient who had
died from ham-poisoning. The properties of the botulinus toxin
have been investigated and have been found to correspond
closely, as regards relative instability, conditions of precipitation,
etc., with the toxins of diphtheria and tetanus. An antitoxin
has also been prepared by Kempner by the .usual methods, and
has been shown not only to have the neutralising property but
to have considerable therapeutical value when administered
some hours after the toxin. The direct combining affinity of
the toxin for the central nervous has been demonstrated by
Kempner and Schepilewsky by the same methods as Wassermann
and Takaki employed in the case of the tetanus toxin. The
396 QUARTER-EVIL
condition of the nerve cells in experimental poisoning with the
botulinus toxin has been investigated independently by Marinesco
and by Kempner and Pollack, and these observers agree as to
the occurrence of marked degenerative changes, especially in the
motor cells in the spinal cord and medulla. Marinesco also
observed hypertrophy and proliferation of the neuroglia cells
around them.
These observations, therefore, show that in one variety of
meat-poisoning the symptoms are produced by the absorption of
the toxins of the bacillus botulinus from the alimentary canal,
and, as van Ermengem points out, it is of special importance to
note that the meat may be extensively contaminated with this
bacillus and may contain relatively large quantities of its toxins
without the ordinary signs of decomposition being present.
The production of an extracellular toxin by this organism, with
extremely potent action on the nervous system, is a fact of great
scientific interest and has a bearing on the etiology of other
obscure nervous affections.
QUARTER-EVIL (GERMAN, RAUSCHBRAND ; FRENCH, CHARBON
SYMPTOMATIQUE).
The characters of the bacillus need be only briefly described, as, so
far as is known, it never infects the human subject. The natural disease,
which specially occurs in certain localities, affects chiefly sheep, cattle,
and goats. Infection takes place by some wound of the surface, and
there spreads in the region around, inflammatory swelling attended by
bloody oedema and emphysema of the tissues. The part becomes greatly
swollen, and of a dark, almost black, colour. Hence the name " black-
leg " by which the disease is sometimes known. The bacillus which
produces this condition is present in large numbers in the affected tissues,
associated with other organisms, and also occurs in small numbers in
the blood of internal organs.
The bacillus morphologically closely resembles that of malignant
oedema. Like the latter, also, it is a strict anaerobe, and its conditions
of growth as regards temperature are also similar. It is, however, some-
what thicker, and does not usually form such long filaments. Moreover
the spores, which are of oval shape and broader than the bacillus, are
almost invariably situated close to one extremity, though not actually
terminal (Fig. 132). The characters of the cultures, also, resemble
those of the bacillus of malignant oedema, but in a stab culture in
glucose agar there are more numerous and longer lateral offshoots, the
growth being also more luxuriant (Fig. 131, c). This bacillus is actively
motile, and possesses numerous lateral flagella.
The disease can be readily produced in various animals, e.g. guinea-
pigs, by inoculation with the affected tissues of diseased animals, and
also by means of pure cultures, though an intramuscular injection of a
considerable amount of the latter is sometimes necessary. The condition
produced in this way closely resembles that in malignant oedema, though
BACILLUS ^EROGENES CAPSULATUS
397
there is said to be more formation of gas in the tissues. Rabbits are
very immune against this disease, whilst they are comparatively suscep-
tible to malignant oedema. As in the case of tetanus, inoculation with
living spores which have been deprived of adherent toxin by heat does
not produce the disease. A toxin can be separated by nitration from
bouillon cultures. It is fairly resistant to heat, withstanding two hours
at 70-75° C. without being
destroyed, and it is also
very rapid in its action, being
capable in appropriate dose
of killing a horse in five
minutes.
The disease is one against
which immunity can be
readily produced in various
ways, and methods of pre-
ventive inoculation have been
adopted in the case of animals
liable to suffer from it. This
subject was specially worked
out by Arloing, Cornevin, and
Thomas, and later by others.
Immunity may be produced
by injection with a non-fatal
dose of the virus (i.e. the
oedematous fluid found in the
FIG. 132. — Bacillus of quarter- evil, showing
spores. From a culture in glucose agar,
incubated for three days at 37° C.
Stained with weak carbol-fuchsin. x 1000.
tissues of affected animals and
which contains the bacilli),
or by injection with larger
quantities of the virus at-
tenuated by heat, drying, etc.
It can be produced also by
cultures attenuated by heat and by the products of the bacilli obtained
by nitration of cultures. An antitoxin has been produced against the
toxins of the bacillus, and a method of protection in which the action
of this antitoxin is combined with that of the. virus has been used.
The antitoxin is said to increase the chemotactic properties of the
leucocytes.
BACILLUS ^ROGENES CAPSULATUS.
This bacillus, though sometimes aiding in the production of patho-
logical changes, is chiefly of interest on account of the extensive gaseous
development which it gives rise to in the tissues post mortem. It was
described by Welch and Nuttall in 1892 ; it is now recognised as being
identical with an organism found in gaseous phlegmon by E. Fraenkel,
and called by him the bacillus phlegmones emphyscmatosce. The organism
is a comparatively large one, measuring 3 to 6 //, in length and having a
thickness about the same as that of the anthrax bacillus ; its ends are
square or slightly rounded (Fig. 133). It often occurs in pairs, sometimes in
chains ; occasionally filamentous forms are met with. It usually shows
a well-marked capsule, hence the name ; it is non-motile and does not
form spores. It stains readily with the basic aniline dyes and retains
the stain in Gram's method. It grows readily on the ordinary media,
398
BACILLUS ^RROGENES CAPSULATUS
but only under anaerobic conditions ; the optimum temperature is that
of the body, growth at the room temperature being comparatively slow.
In a puncture culture in agar there is an abundant whitish line of growth
with somewhat indented margin ; the individual colonies are white and
of rounded or oval form. There is practically no liquefaction of gelatin,
though this medium becomes somewhat softened around the growth.
In all cases there is a tendency to abundant evolution of gas in the
cultures, and this is especi-
ally marked when ferment-
able sugars are present.
The organism appears to
be the most frequent cause
of rapid gaseous develop-
ment in the blood and
organs post mortem, this
depending upon an invasion
#HH of the blood immediately
before death. In such cases,
even within twenty - four
hours under ordinary con-
ditions, large bubbles of
gas may be present in the
veins, and the organs may
be beset with gas-contain-
ing spheres of various sizes ;
the liver is usually the
organ most affected, and its
appearance has been com-
Fio. 133. — Bacillus aerogeues capsulatu.s ; * £ed to that of Grilyere
film preparation from bone- marrow in a cheese. The invasion by
case where gas-cavities were present in the this organjsm is met with
organs, x 1000. from time to time in puer.
peral cases, and also in
connection with ulcerative or gangrenous conditions of the intestine ;
the bacillus is also found not infrequently in the peritoneum in
cases of perforation. . Although the striking changes in the organs
are due to a post-mortem development of the bacillus, there is no
doubt that its entrance into the blood stream often hastens death,
and may in some instances be the cause of it. As already stated, the
organism is also met with in some cases of spreading oedema' with
emphysema as a leading feature.
When tested experimentally the bacillus by itself is found to have
little pathogenic action. Injection of pure cultures in rabbits and
guinea-pigs may be followed by little result, but sometimes in the latter
animals "gaseous phlegmon" is produced, without suppuration unless
other organisms are present. If a small quantity of culture be injected
intravenously, e.g. in a rabbit, and then the animal be killed, bubbles
of gas are rapidly produced in the blood and organs, the picture corre-
sponding with that in the human cases.
CHAPTER XVII.
CHOLERA.
Introductory. — It is no exaggeration of the facts to say that
previously to 1883 practically nothing of value was known regard-
ing the nature of the virus of cholera. In that year Koch was
sent to Egypt, where the disease had broken out, in charge of a
Commission for the purpose of investigating its nature. In the
course of his researches he discovered the organism now generally
known as the " comma bacillus " or the " cholera spirillum."
He also obtained pure cultures of the organism from a large
number of cases of cholera, and described their characters. The
results of his researches were given at the first Cholera Conference
at Berlin in 1884.
Since Koch's discovery, and especially during the epidemic in
Europe in 1892-93, spirilla have been cultivated from cases of
cholera in a great many different localities, and though this
extensive investigation has revealed the invariable presence in
true cholera of organisms resembling more or less closely Koch's
spirillum, certain difficulties have arisen. For it has been found
that the cultures obtained from different places have shown con-
siderable variations in their characters, and, further, spirilla
which closely resemble Koch's cholera spirillum have been
cultivated from sources other than cases of true cholera. There
has therefore been much controversy, on the one hand, as to the
signification of these variations, — whether they are to be regarded
as indicating distinct species or merely varieties of the same
species, — and on the other hand, as to the means of distinguishing
the cholera spirillum from other species which resemble it. These
questions will be discussed below.
In considering the bacteriology of cholera it is to be borne in
mind that in this disease, in addition to the evidence of great
intestinal irritation, accompanied by profuse watery discharge, and
399
400 CHOLERA
.
often by vomiting, there are also symptoms of general systemic
disturbance which cannot be accounted for merely by the with-
drawal of water and certain substances from the system. Such
symptoms include the profound general prostration, cramps in
the muscles, extreme cardiac depression, the cold and clammy
condition of the surface, the subnormal temperature, suppression
of urine, etc. These taken in their entirety are indications of a
general poisoning in which the circulatory and thermo-regulatory
mechanisms are specially involved. In some, though rare, cases
known as cholera sicca, general collapse occurs with remarkable
suddenness, and is rapidly followed by a fatal result, whilst there
is little or no evacuation from the bowel, though post mortem the
intestine is distended
.^^••1^^^ with fluid contents. As
VJT* xi^ *?i > the characteristic organ-
<y *J *\ 2~~ *sms *n cn°lera are found
^^ ^ £*& i <f^ t c only in tne intestine, the
* f/ SQ <*?• general disturbances are
to be regarded as the
result of toxic substances
absorbed from the bowel.
is also to be noted
that cholera is a disease
of which the onset and
course are much more
rapid than is the case in
most infective diseases,
such as typhoid and
FIG. 134.-Cholera spirilla,- from a culture on Diphtheria : and that
agar of twenty-four hours' growth. recovery also, when it
Stained with weak carbol-fuchsin. x 1000. takes place, does SO more
quickly. The two factors
to be correlated to these facts are (a) a rapid multiplication of
organisms, (6) the production of rapidly acting toxins.
The Cholera Spirillum. Microscopical Characters. — The
cholera spirilla as found in the intestines in cholera are small
organisms measuring about 1'5 to 2 ju in length, and rather less
than *5 in thickness. They are distinctly curved in one direction,
hence the appearance of a comma (Fig. 134) ; most occur singly,
but some are attached in pairs and curved in opposite directions,
so that an S-shape results. Longer forms are rarely seen in the
intestine, but in cultures in fluids, as is especially well seen in
hanging-drop preparations, they may grow into longer spiral
filaments, showing a large number of turns. In film preparations
^ f
OJ- a
1 *<*,, J& >\vJV^ It
~j ''J •*% f^r !S th
~' '
THE CHOLERA SPIRILLUM
401
made from the intestinal contents in typical cases, these organ-
isms are present in enor-
mous numbers in almost
pure culture, most of the
spirilla lying with their
long axes in the same
direction, so as to give the
appearance which Koch
compared to a number of
fish in a stream.
They possess very active
motility, which is most
marked in the single forms.
When stained by the suit-
able methods they are seen
to be flagellated. Usually
a single terminal flagellum
is present at one end
only (Fig. 135). It is
very delicate, and measures
four or five times the length of the organism. In some varieties,
however, there may be a flagellum at both ends, or more than
one may be present ; cul-
tures obtained at different
places have shown con-
siderable variations in this
respect. Cholera spirilla
do not form spores. In
old cultures the organisms
may present great variety
in size and shape. Some
are irregularly twisted fila-
ments, sometimes globose,
sometimes clubbed at their
extremities, and also show-
ing irregular swellings along
their course. Others are
short and thick, and may
FIG. 135. — Cholera spirilla stained to show
the terminal flagella. x 1000.
have the appearance of large
cocci, often staining faintly.
All these changes in appear-
ance are to be classed to-
gether as involution forms.
Staining. — -'Cholera spirilla stain readily with the usual basic
26
FIG. 136. — Cholera spirilla from an old agar
culture, showing irregularities in size and
shape, with numerous faintly - stained
coccoid bodies — involution forms.
Stained with fuchsin. x 1000.
402 CHOLERA
aniline stains, though Lofner's methylene-blue or weak carbol-
fuchsin is specially suitable. They lose the stain in Gram's
method.
Distribution within the Body. — The chief fact in this con-
nection is that the spirilla are confined to the intestine, and -are
not present in the blood or internal organs. This was determined
by Koch in his earlier work, and his statement has been amply
confirmed. In cases in which there is the characteristic " rice-
water " fluid in the intestines, they occur in enormous numbers
— almost in pure culture. The lower half of the small intestine
is the part most affected. Its surface epithelium becomes shed
in great part, and the flakes floating in the fluid consist chiefly
of masses of epithelial cells and mucus, amongst which are
numerous spirilla. The spirilla also penetrate the follicles of
Lieberkiihn, and may be seen lying between the basement
membrane and the epithelial lining, which becomes loosened by
their action. They are, however, rarely found in the connective
tissue beneath, and never penetrate deeply. Along with these
changes there is congestion of the mucosa, especially around the
Peyer's patches and solitary glands, which are somewhat swollen
and prominent. In some very acute cases the mucosa may show
general acute congestion with a rosy pink colour but very little
desquamation of epithelium, the intestinal contents being a com-
paratively clear fluid containing the spirilla in large numbers.
In other cases of a more chronic type, the intestine may show
more extensive necrosis of the mucosa and a considerable
amount of haemorrhage into its substance, along with formation
of false membrane at places. The intestinal contents in such
cases are blood-stained and foul-smelling, there being a great
proportion of other organisms present besides the cholera spirilla
(Koch).
Cultivation. — (For Methods, see p. 413.)
The cholera spirillum grows readily on all the ordinary media,
and with the exception of that on potato, growth takes place at
the ordinary room temperature. The most suitable temperature,
however, is that of the body, and growth usually stops about
16° C., though in some cases it has been obtained at a lower
temperature.
Peptone Gelatin. — On this medium the organism grows well
and produces liquefaction. In puncture cultivations at 22° C. a
whitish line appears along the needle track, at the upper part of
which liquefaction commences, and as evaporation quickly occurs,
a small bell-shaped depression forms, which gives the appearance
of an air-bubble. On the fourth or fifth day we get the following
CULTIVATION
403
appearance : there is at the surface the bubble -shaped de-
pression ; below this there is a funnel-shaped area of liquefaction,
the fluid being only slightly turbid, but showing at its lower end
thick masses of growth of a more or less spiral shape (Fig. 137).
The liquefied portion gradually tapers off downwards towards the
needle track. (This appearance is, however, in some varieties not
produced till much later, especially when
the gelatin is very stiff, and, in other
varieties which liquefy very slowly, may not'
be met with at all.) At a later stage, lique-
faction spreads and may reach the side of
the, tube.
In gelatin plates the colonies are some-
what characteristic. They appear as minute
whitish points, visible in twenty-four to forty-
eight hours, the surface of which, under a low
power of the microscope, is irregularly granular
or furrowed (Fig. 138, A), and later has an
appearance which has been compared to
fragments of broken glass. Liquefaction
occurs, and the colony sinks into the small
cup formed, the plate then showing small
sharply - marked rings around the colonies.
Under the microscope the outer margin of
the cup is circular and sharply marked.
Within the cup the liquefied portion forms
a ring which has a more or less granular
appearance, whilst the mass of growth in the
centre is irregular and often broken up at its
margins (Fig. 138, B). The growth of the
colonies in gelatin plates constitutes one of
the most important means of distinguishing
the cholera spirillum from other organisms.
On the surface of the agar media a thin
almost transparent layer forms, which pre-
sents no special characters. On solidified
blood serum the growth has at first the same
appearance, but afterwards liquefaction of the medium occurs.
On agar plates the superficial colonies under a low power are
circular discs of brownish-yellow colour, and more transparent
than those of most other organisms. On potato at the ordinary
temperature, growth does not take place, but on incubation at a
temperature of from 30° to 37° C., a moist layer appears, which
assumes a dirty brown colour somewhat like that of the glanders
FIG. 137. — Puncture
culture of the cholera
spirillum in peptone
gelatin — six days'
growth. Natural size.
404 CHOLERA
bacillus ; the appearance, however, varies somewhat in different
varieties, and also on different sorts of potatoes.
In bouillon with alkaline reaction the organism grows very
readily, there occurring in twelve hours at 37° C. a general
turbidity, while the surface shows a thin pellicle composed of
spirilla in a very actively motile condition. Growth takes place
under the same conditions equally rapidly in peptone solution
(1 per cent with %5 per cent sodium chloride added).
In milk also the organism grows well and produces no coagu-
lation nor any change in its appearance, at least for several days.
On all the media the growth of the cholera spirillum is a
relatively rapid one, and especially is this the case in peptone
FIG. 138. — Colonies of the cholera spirillum in a gelatin plate ; three days'
growth. A shows the granular surface, liquefaction just commencing ; in B
liquefaction is well marked.
solution and in bouillon, a circumstance of importance in relation
to its separation in cases of cholera (vide p. 413).
The cholera organism is one which grows much more rapidly
in the presence of oxygen than in anaerobic conditions ; in the
complete exclusion of oxygen very little growth occurs.
Cholera-red reaction. — This is one of the most important tests
in the diagnosis of the cholera organism. It is always given by
a true cholera spirillum, and though the reaction is not peculiar
to it, the number of organisms which give the reaction under
the conditions mentioned are comparatively few. The test is
made by adding a few drops of pure sulphuric acid to a culture
in bouillon or in peptone solution (1 per cent) which has been
incubated for twenty-four hours at 37° C. ; in the case of the
cholera spirillum a reddish-pink colour is produced. This is due
to the fact that both indol and a nitrite are formed by the
spirillum in the medium. The addition of sulphuric acid causes
a nitroso-indol body to be produced from these, and this gives
POWERS OF RESISTANCE 405
the red colour. Here, as in testing for the production of indol
by other bacteria, it is found that not every specimen of peptone
is suitable, and it is advisable to select a peptone which gives
the characteristic reaction with a known cholera organism, and
to use it for further tests. It is also essential that the sulphuric
acid should be pure, for if traces of nitrites are present the
reaction might be given by an organism which had not the
power of forming nitrites.
Hcemolytic Test. — This method introduced by Kraus is
performed by means of agar plates, a small quantity of sterile
defibrinated blood being added to the agar and thoroughly
diffused ; if any organism has hsemolytic properties a clear zone
or areola forms around each colony by the diffusion of haemo-
globin. In no instance has an undoubted cholera organism been
found to produce haemolysis, whereas many species of spirilla
closely resembling it possess marked haemolytic action. This
test may accordingly be applied along with the others in
determining the identity of a supposed cholera organism.
Powers of Resistance. — In their resistance against heat,
cholera spirilla correspond with most spore-free organisms, and are
killed in an hour by a temperature of 55° C., and much more rapidly
at higher temperatures. They have comparatively high powers
of resistance against great cold, and have been found alive after
being exposed for several hours to the temperature of - 10° C.
They are, however, killed by being kept in ice for a few days.
Against the ordinary antiseptics they have comparatively low
powers of resistance, and Pfuhl found that the addition of lime,
in the proportion of 1 per cent, to water containing the cholera
organisms was sufficient to kill them in the course of an hour.
As regards the powers of resistance in ordinary conditions,
the following facts may be stated. In cholera stools kept at the
ordinary room temperature, the cholera organisms are rapidly
outgrown by putrefactive bacteria, but in exceptional cases they
have been found alive even after two or three months. In most
experiments, however, attempts to cultivate them even after a
much shorter time have failed. The general conclusion may be
drawn from the work of various observers that the spirilla do not
multiply freely in ordinary sewage water, although they may
remain alive for a considerable period of time. On moist linen,
as Koch showed, they can nourish very rapidly. When the
cholera organisms are grown along with other organisms
in fluids at a warm temperature, it is found that at first they
may multiply more rapidly than the others, but that after a
certain time they are outgrown by some of the organisms present,
406 CHOLERA
gradually dimmish in number, and ultimately disappear. It
must not, however, be inferred from such experiments that a
similar result will necessarily follow in nature, as any particular
saprophytic organism requires a special habitat — that is, certain
suitable conditions for its growth in competition with other
organisms. Though we can state generally that the conditions
favourable for the growth of the cholera spirillum are a warm
temperature, moisture, a good supply of oxygen, and a consider-
able proportion of organic material, we do not know the exact
circumstances under which it can nourish for an indefinite period
of time as a saprophyte. The fact that the area in which
cholera is an endemic disease is so restricted tends to show that
the conditions for a prolonged growth of the spirillum outside
the body are not usually supplied. Yet, on the other hand,
there is no doubt that in ordinary conditions it can live a
sufficient time outside the body and multiply to a sufficient
extent, to explain all the facts known with regard to the per-
sistence and spread of cholera epidemics.
Numerous experiments show that the cholera organisms are,
as a rule, rapidly killed by drying, usually in two or three
minutes when the drying has been thorough, and it is inferred
from this that they cannot be carried in the living condition for
any great distance through the air, a conclusion which is well
supported by observations on the spread, of the disease. Cholera
is practically always transmitted by means of water or food
contaminated by the organism, and there is no doubt that con-
tamination of the water supply by choleraic discharges is the
chief means by which areas of population are rapidly infected.
It has been shown that if flies are fed on material containing
cholera organisms, the organisms may be found alive within their
bodies twenty -four hours afterwards. And further, Haffkine
found that sterilised milk might become contaminated with
cholera organisms, if kept in open jars to which flies had free
access, in a locality infected by cholera. It is quite possible
that infection may be carried by this agency in some cases.
Experimental Inoculation. — In considering the effects of
inoculation with the cholera organism, we are met with the
difficulty that none of the lower animals, so far as is known,
suffer from the disease under natural conditions. Even in places
where cholera is endemic, no corresponding affection has been
observed in any animals. And further, before the discovery of
the cholera organism, various efforts had been made to induce
the disease in animals by feeding them with cholera dejecta, but
without success. It is therefore not surprising that the earlier
EXPERIMENTAL INOCULATION 407
experiments on animals by feeding them with pure cultures were
attended with negative results. As the organisms are confined
to the alimentary tract in the natural disease, attempts to induce
their multiplication within the intestine of animals by artificially
arranging favouring conditions, have occupied a prominent place
in the experimental work. We shall give a short account of
such experiments.
Nikati and Rietsch were the first to inject the organisms directly into
the duodenum of dogs and rabbits, and they succeeded in producing, in
a considerable proportion of the animals, a choleraic condition of the
intestine ; in their earlier experiments the common bile duct was
ligatured, but the later were performed without this operation. These
experiments were confirmed by other observers, including Koch. Think-
ing that probably the spirillum, when introduced by the mouth, is
destroyed by the action of the hydrochloric acid of the gastric secretion,
Koch first neutralised this acidity by administering to guinea-pigs 5 c.c.
of a 5 per cent solution of carbonate of soda, and sometime afterwards
introduced a pure culture into the stomach by means of a tube. As this
method failed to give positive results he tried the effect of artificially
interfering with the intestinal peristalsis by injecting tincture of opium
into the peritoneum (1 c.c. per 200 grm. weight), in addition to neutral-
ising as before with the carbonate of sodium solution. The result was
remarkable, as thirty out of thirty -five animals treated died. The
animals infected by this method show signs of general prostration, their
posterior extremities being especially weakened ; the abdomen becomes
tumid, respiration slow, heart's action weak, and the surface cold.
Death occurs after a few hours. Post mortem the small intestine is
distended, its mucous membrane congested, and it contains a coiourless
fluid with small fiocculi and the cholera organisms in practically pure
cultures. These experiments, which have been repeatedly confirmed,
therefore demonstrated that the cholera organisms could, under certain
conditions, set up in animals a condition in some respects resembling
cholera. Koch, however, found that when the spirilla of Finkler and
Prior, of Deneke, and of Miller (vide infra), were employed by the same
method, a certain, though much smaller, proportion of the animals died
from an intestinal infection. Though the changes in these cases were
not so characteristic, they were sufficient to prevent the results obtained
with the cholera organism from being used as a demonstration of the
specific relation of the latter to the disease.
Within later years some additional facts of high interest have been
established with regard to choleraic infection of animals. For example,
Sabolotny found that in the marmot an intestinal infection readily takes
place by simple feeding with the organism, there resulting the usual
intestinal changes, sometimes with hsemorrhagic peritonitis, the organ-
isms, however, being present also in the blood. It was found by Issaeff
and Kolle that young rabbits could be infected by merely neutralising
the gastric secretion with sodium carbonate, the use of opium being
unnecessary ; but of special interest is the fact, discovered by Metchnikoff,
that in the case of young rabbits shortly after birth a large proportion
die of choleraic infection when the organisms are simply introduced
along with the milk, as may be done by infecting the teats of the
mother. Further, from these animals thus infected the disease may be
408 CHOLERA
transmitted to others by a natural mode of infection. In this affection
of young rabbits many of the symptoms of cholera are present. The
organisms occur in large numbers in the intestine, and in some cases a
few may be found in the blood, and especially in the gall bladder. Many
of these experiments were performed with the vibrio of Massowah, which
is now admitted not to be a true cholera organism, others with a
cholera vibrio obtained from the water of the Seine.
It will be seen from the above account that the evidence
obtained from experiments on intestinal infection of animals,
though by no means sufficient to establish the specific relation-
ship of the cholera organism, is on the whole favourable to this
view, especially when it is borne in mind that animals do not in
natural conditions suffer from the disease.
Experiments performed by direct inoculation also supply
interesting facts. Intraperitoneal injection in guinea-pigs is
followed by general symptoms of illness, the most prominent
being distension of the abdomen, subnormal temperature, and,
ultimately, profound collapse. There is peritoneal effusion,
which may be comparatively clear, or may be somewhat turbid
and contain flakes of lymph, according to the stage at which
death takes place. If the dose is large, organisms are found
in considerable numbers in the blood and also in the small
intestine, but with smaller doses they are practically confined to
the peritoneum. Kolle found that when the minimum lethal
dose was used in guinea-pigs, the peritoneum might be free from
organisms at the time of death, the fatal result having taken
place from an intoxication (cf. diphtheria, p. 360). These and
other experiments show that though the organisms undergo a
certain amount of multiplication when introduced by the
channels mentioned, still the tendency to invade the tissues is
not a marked one. On the other hand the symptoms of general
intoxication are always pronounced. Hence arise questions as
to the nature and mode of action of toxic bodies produced by
the cholera organism.
Toxins. — Though there is no doubt that there are formed by
Koch's spirillum toxic bodies which produce many of the
symptoms of cholera, there is at present very little satisfactory
knowledge regarding their chemical nature. The following
summary may be given.
It has been shown, especially by R. Pfeiffer,1 that toxic
phenomena can be produced by injection of the dead spirilla
into animals. A certain quantity of a young culture on agar,
1 Pfeiffer obtained his earlier results with a vibrio from Massowah, which is
now known (as mentioned above) not to be a true cholera organism. This fact
shows that the effects described are not specific to the latter.
TOXINS OF KOCH'S SPIRILLUM 409
killed by exposure to the vapour of chloroform, when injected
intraperitoneally into a guinea-pig, may cause death in from
eight to twelve hours. There is extreme collapse, sometimes
clonic spasms occur, and the temperature may fall below 30° 0.
before death. Pfeiffer considers that the toxic substances are
contained in the bodies of the organisms — that is, they are in- '
tracellular, — and that they are only set free by the disintegration
of the latter. This opinion is grounded chiefly on the fact that
when bouillon cultures were filtered, he found that the filtrate
possessed very feeble toxic properties. He showed also that
when an animal is inoculated intraperitoneally with the cholera
organism, and then some time later anti-cholera serum which
produces bacteriolysis is injected, rapid collapse with a fatal
result may ensue, apparently due to the liberation of the intra-
cellular toxins. The dead cultures administered by the mouth
produce no effect unless the intestinal epithelium is injured, in
which case poisoning may result. He considers that the
desquamation of the epithelium is an essential factor in the
production of the phenomena of the disease in the human
subject. Pfeiffer found that the toxic bodies were to a great
extent destroyed at 60° C., but even after heating at 100° C. a
small proportion of toxin remained, which had the same physio-
logical action. Recently A. Macfadyen found that the product
obtained by grinding up the spirilla frozen by means of liquid air
had a very high degree of toxicity when injected intravenously.
Like Pfeiffer he found that the " endotoxin " was in great part
destroyed at 60° C.
On the other hand, other observers (Petri, Ransom, Klein,
and others) have obtained toxic bodies from filtered cultures.
Metchnikoff, E. Roux, and Taurelli-Salimbeni have demon-
strated the formation of such diffusible toxic bodies in fluid
media. By means of cultures placed in collodion sacs in the
peritoneum of animals, they found that the living organisms
produce toxic bodies which diffuse through the wall of the sac
and produce toxic symptoms. By greatly increasing the
virulence of the organism, then growing it in bouillon and
filtering the cultures on the third and fourth day, they obtained
a fluid which was highly toxic to guinea-pigs (the fatal dose
usually being i c.c. per 100 grm. weight). If the dose of the
toxin is very large, death follows in an hour or even less. The
symptoms closely resemble those obtained by Pfeiffer, the rapid
fall of temperature being a striking feature. They found that
the toxicity of the filtrate was not altered by boiling; appar-
ently the toxic substance is different from Pfeiffer's endotoxin.
410 CHOLERA
Attempts to investigate the chemical nature of the toxic bodies
have not led to definite results.
Experiments on the Human Subject. — Experiments have also
been performed in the case of the human subject, both intention-
ally and accidentally. In the course of Koch's earlier work, one
of the workers in his laboratory shortly after leaving was seized
with severe choleraic symptoms. The stools were found to
contain cholera spirilla in enormous numbers. Recovery, how-
ever, took place. In this case there was no other possible
source of infection than the cultures with which the man had
been working, as no cholera was present in Germany at the time.
Within recent years a considerable number of experiments have
been performed on the human subject, which certainly show that
in some cases more or less severe choleraic symptoms may follow
ingestion of pure cultures, whilst in others no effects may result.
The former was the case, for example, with Emmerich and
Pettenkofer, who made experiments on themselves, the former
especially becoming seriously ill. In the case of both, diarrhoea
was well marked, and numerous cholera spirilla were present in
the stools, though toxic symptoms were proportionately little
pronounced. Metchnikoff also, by experiments on himself and
others, obtained results which convinced him of the specific
relation of the cholera spirillum to the disease. Lastly, we may
mention the case of Dr. Orgel in Hamburg, who contracted the
disease in the course of experiments with the cholera and other
spirilla, and died in spite of treatment. It is believed that in
sucking up some peritoneal fluid containing cholera spirilla, a
little entered his mouth and thus infection was produced. This
took place in September 1894 at a time when there was no
cholera in Germany. On the other hand, in many cases the
experimental ingestion of cholera spirilla by the human subject
has given negative results. Still, as the result of observation of
what takes place in a cholera epidemic, it is the general opinion
of authorities that only a certain proportion of people are
susceptible to cholera, and the facts mentioned above are, in our
opinion, of the greatest importance in establishing the relation
of the organism to the disease.
Immunity.— As this subject is discussed later, only a few
facts will be here stated, chiefly for the purpose of making clear
what follows with regard to the means of distinguishing the
cholera spirillum from other organisms. The guinea-pig or any
other animal may be easily immunised against the cholera
organism by repeated injections (conveniently made into the
peritoneum) of non-fatal doses of the spirilla. It is better to
IMMUNITY 411
commence the process with non-fatal doses of cultures killed by
the vapour of chloroform or by heat, the doses being gradually
increased, and afterwards to proceed with increasing doses of the
living organism. In this way a high degree of immunity against
the organism is developed, and further, the blood serum of an
animal thus immunised (anti-cholera serum) has markedly pro-
tective power when injected, even in a small quantity, into a
guinea-pig along with five or ten times the fatal dose of
the living organism. Under these circumstances the spirilla
undergo a granular transformation and, ultimately, solution ; this
phenomenon is generally known as Pfeiffer's reaction, and was
applied by him to distinguish the cholera spirillum from organisms
resembling it. The following are the details : —
Pfeiffers Reaction. — A loopf'ul (2 uigrm.) of recent agar culture of the
organism to be tested is added to 1 c.c. of ordinary bouillon containing
'001 c.c. of anti-cholera serum. The mixture is then injected into the
peritoneal cavity of a young guinea-pig (about 200 grm. in weight), and
the peritoneal fluid of this animal (conveniently obtained by means of
capillary glass tubes inserted into the peritoneum) is examined micro-
scopically after a few minutes. If the spirilla injected have been cholera
spirilla, it will be found that they become motionless, swell up into
globules, and ultimately break down and disappear — positive result. If
they are found active and motile, then the possibility of their being
true cholera spirilla may be excluded — negative result. In the former
case (positive result) there is, however, still the possibility that the
organism is devoid of pathogenic properties and has been destroyed by
the normal peritoneal fluid. A control experiment should accordingly
be made with '001 c.c. of normal serum in place of the anti-cholera serum.
If no alteration of the organism occurs with its use, then it is to be
concluded that a true reaction has been given.
The serum of an animal immunised by the above method has
also marked agglutinative action against the cholera spirillum,
and this property closely corresponds with Pfeiffer's reaction as
regards specificity. Such a serum has, however, little protective
effect against the toxic action of the dead spirilla. On the other
hand, Macfadyen by injecting the endotoxin derived from the
spirilla by grinding obtained a serum which had antitoxic as well
as agglutinative and bacteriolytic properties (vide Immunity).
Metchnikoff and others have also obtained antitoxic sera which
act on the extra-cellular toxins obtained by filtration.
The serum of cholera convalescents has been found to possess
properties similar to those of immunised animals ; that is, it
affords protection against the cholera spirillum and may also
give Pfeiffer's reaction. These properties of the serum may be
present eight or ten days after the attack of the disease, but
are most marked four weeks after ; they then gradually become
412 CHOLERA
weaker and disappear in two or three months (Pfeiffer and
Issaeff).
Specific agglutinative properties have, however, been detected
in the serum of cholera patients at a much earlier date, in some
cases even on the first day of the disease, though usually a day
or two later. The dilutions used were 1 : 15 to 1 : 120, and these
had no appreciable effect on organisms other than the cholera
spirillum (Achard and Bensande). Needless to say, such facts
supply strong additional evidence of the relation of Koch's
spirillum to cholera.
Anti-Cholera Inoculation. — Haffkine's method for inoculation
against cholera exemplifies the above principles. It depends
upon (a) attenuation of the virus — that is, the cholera organism,
and (6) exaltation of the virus. The virulence of the organism
is diminished by passing a current of sterile air over the surface
of the cultures, or by various other methods. The virulence is
exalted by the method of passage — that is, by growing the
organism in the peritoneum in a series of guinea-pigs. By the
latter method the virulence after a time is increased twenty-fold
— that is, the fatal dose has been reduced to a twentieth of the
original. Cultures treated in this way constitute the virus exalte.
Subcutaneous injection of the virus exalte produces a local
necrosis, and may be followed Try the death of the animal, but if
the animal be treated first with the attenuated virus, the sub-
sequent injection of the virus exalte produces only a local oedema.
After inoculation first by attenuated and afterwards by exalted
virus, the guinea-pig has acquired a high degree of immunity, and
Haffkine believed that this immunity was effective in the case
of every method of inoculation — that is, by the mouth as well as
by injection into the tissues. After trying his method on the
human subject and finding it free from risk, he extended it in
practice on a large scale in India in 1894, and these experiments
are still going on. So far the results are, on the whole, distinctly
encouraging. In the human subject two or sometimes three in-
oculations were formerly made with attenuated virus before the
virus exalte was used ; now, however, a single injection of the
latter is usually practised. Wassermann and Pfeiffer, and also
Klein, have found that guinea-pigs immunised by Haffkine's
method are not immunised against intestinal infection when the
animal is treated by Koch's method (vide p. 407). Notwith-
standing this fact Haffkine's method may still have a beneficial
effect, though it may not be preventive in all cases.
Methods of Diagnosis. — In the first place, the stools ought
to be examined microscopically. Dried film preparations should
METHODS OF DIAGNOSIS 413
be made and stained by any ordinary stains, though carbol-fuchsin
diluted four times with water is specially to be recommended.
Hanging-drop preparations, with or without the addition of a
weak watery solution of gentian- violet or other stain, should also
be made, by which method the motility of the organism can be
readily seen. By microscopic examination the presence of spirilla
will be ascertained, and an idea as to their number obtained.
In some cases the cholera spirilla are so numerous in the stools
that a picture is presented which is obtained in no other con-
dition, and a microscopic examination may be sufficient for
practical purposes. According to Koch, a diagnosis was made
in 50 per cent of the cases during the Hamburg epidemic by
microscopic examination alone. In the case of the first appear-
ance of a cholera-like disease, however, all the other tests
should be applied before a definite diagnosis of cholera is made.
Dunbar has recently introduced a method for rapid diagnosis
which depends on the properties of an anti-cholera serum. Two
hang-drop preparations are made, each consisting of a small
portion of mucus from the suspected stool broken up in peptone
solution. To one a drop of a 50-fold dilution of normal serum
is added, to the other a drop of a 500-fold dilution of an active
cholera serum. If the spirilla present are cholera organisms
they retain their motility in the first preparation, while they lose
it and then become agglutinated in the second. By this method
a diagnosis may sometimes be given in a few minutes.
If the organisms are very numerous, gelatin or agar plates
may be made at once and pure cultures obtained.
If the spirilla occur in comparatively small numbers, the best
method is to inoculate peptone solution (1 per cent) and incubate
for from eight to twelve hours. At the end of that time the spirilla
will be found on microscopic examination in enormous numbers
at the surface, and thereafter -plate cultures can readily be made.
If the spirilla are very few in number, or if a suspected water is
to be examined for cholera organisms, the peptone solution
which has been inoculated should be examined at short intervals
till the spirilla are found microscopically. A second flask of
peptone solution should then be inoculated, and possibly again
a third from the second. By this method, properly carried out,
a culture may be obtained which, though impure, contains a
large proportion of the spirilla, and then plate cultures may be
made.
When a spirillum has been obtained in pure condition by
these methods, the appearance of the colonies in plates should
be specially noted, the test for the cholera-red reaction should
414 CHOLERA
be applied, and in many cases it is advisable to test the effects
of intraperitoneal injection of a portion of a recent agar culture
in a guinea-pig, the amount sufficient to cause death being also
ascertained. The agglutinating or sedimenting properties of the
serum of the patient should be tested against a known cholera
organism, and against the spirillum cultivated from the case.
The action of an anti-cholera serum, i.e. the serum of animal
immunised against the cholera spirillum, should be tested in a
similar manner.
Up till recent times there had been cultivated from sources
other than cholera cases, no organism which gave all the cultural
and biological tests (agglutination and Pfeiffer's reaction) of the
cholera spirillum. In 1905, however, Gotschlich obtained six
different strains of a spirillum which conformed in all these
respects. The organisms were obtained at El Tor from the
intestines of pilgrims who had died with dysenteric symptoms,
and there were no cases of cholera in the vicinity. The organisms
in question, however, differ from the cholera organism in having
marked hsemolytic action, and also in producing a rapidly acting
extra -cellular toxin. There has been diversity of opinion
with regard to the nature of these organisms, for while some
consider that they are a different species from the cholera
organism, others regard them as true cholera spirilla which had
been carried by the patients, although no symptoms of cholera
resulted from their presence. If they are not to be regarded as
cholera organisms, .we have the striking fact that they correspond
in the immunity reactions. This instance exemplifies well the
great difficulty which may surround the identification of a
particular organism obtained from non- cholera cases, and one
can hardly doubt that if cholera -like symptoms had been
present in the El Tor cases, the spirilla would have been accepted
as varieties of the cholera organism, though differing in their
haemolytic action. None of the facts ascertained, however, really
affect the question as to the causal relationship of Koch's spirillum
to cholera, although they indicate the difficulties which may
attend the bacteriological diagnosis in isolated cases of the
disease.
General Summary. — We may briefly summarise as follows
the facts in favour of Koch's spirillum being the cause of cholera.
First, there is the constant presence of spirilla in true cases of
cholera, which on the whole conform closely with Koch's
description, though variations undoubtedly occur. Moreover,
the facts known with regard to their conditions of growth, etc.,
are in conformity with the origin and spread of cholera epidemics.
SPIRILLA RESEMBLING CHOLERA SPIRILLUM 415
Secondly r, the experiments on animals with Koch's spirillum or
its toxins give as definite results as one can reasonably look for
in view of the fact that animals do not suffer naturally from the
disease. Thirdly, the experiments on the human subject and
the results of accidental infection by means of pure cultures are
also strongly in favour of this view. Fourthly, the agglutinative
and protective properties of the serum of cholera patients and
convalescents constitute another point in its favour. Fifthly,
bacteriological methods, which proceed on the assumption that
Koch's spirillum is the cause of the disease, have been of the
greatest value in the diagnosis of the disease. And lastly, the
results of Haffkine's method of preventive inoculation in the
human subject, which are on the whole favourable, also supply
additional evidence. If all these facts are taken together, we
consider the conclusion must be arrived at that the growth of
Koch's spirillum in the intestine is the immediate cause of the
disease. This does not exclude the probability of an important
part being played by conditions of weather and locality, though
such are very imperfectly understood. Pettenkofer, for example,
recognised two main factors in the causation of epidemics, which
he designated x and y, and considered that these two must be
present together in order that cholera may spread. The x is the
direct cause of the disease — an organism which he admitted
to be Koch's spirillum ; the y includes climatic and local con-
ditions, e.g. state of ground-water, etc.
Other Spirilla resembling the Cholera Organism. — These
have been chiefly obtained either from water contaminated by
sewage or from the intestinal discharge in cases with choleraic
symptoms. Some of them differ so widely in their cultural and
other characters (some, for example, are phosphorescent) that no
one would hesitate to classify them as distinct species. Others,
however, closely resemble the cholera organism.
The vibrio berolinensis, cultivated by Neisser from Berlin sewage
water, differs from the cholera organism only in the appearance of its
colonies in gelatin plates, its weak pathogenic action, and its giving a
negative result with Pfeiffer's test. It, however, gives the cholera-red
reaction. The vibrio Danubicus, cultivated by Heider from canal water,
also differs in the appearance of its colonies in plates, and also reacts
negatively to Pfeiffer's test ; in most respects it closely resembles the
cholera organism. Another spirillum (v. Ivanoff] was cultivated by
Ivanoff from the stools of a typhoid patient after these had been diluted
with water. The organism differed somewhat in the appearance of its
colonies and in its great tendency to grow out in the form of long
threads, but Pfeiffer found that it reacted to his test in the same way as
the cholera organism, and he considered that it was really a variety of
the cholera organism. No spirilla could be found microscopically in the
416 CHOLERA
stools in this case, and Pfeiffer is of the opinion that the organism
gained entrance accidentally. These examples will show how differences
of opinion, even amongst experts, might arise as to whether a certain
spirillum were really the cholera organism or a distinct species resem-
bling it.
A few examples may also be given of organisms cultivated
from cases in which cholera-like symptoms were present.
The vibrio of Massowah was cultivated by Pasquale from a case during
a small epidemic of cholera. The organism so closely resembles Koch's
spirillum that it was accepted by several authorities as the true cholera
organism, and, as already stated, Metchnikotf produced by it cholera
symptoms in the human subject, and also the cholera-like disease in
young rabbits. It possesses four flagella, has a high degree of virulence,
producing septicaemia both in guinea-pigs and pigeons, and its colonies
in plates differ somewhat from the cholera organism. Moreover, it
reacts negatively to Pfeiffer's test. Another organism, the v. Gindha,
was cultivated by Pasquale from a well, and was at first accepted by
Pfeiffer as the cholera organism, but afterwards rejected, chiefly because
it failed to give the specific immunity reaction. It also differs some-
what from the cholera organism in its pathogenic effects, and it fails to
give the cholera-red reaction or gives it very faintly.
Pestana and Bettencourt also cultivated a species of spirillum from a
number of cases during an epidemic in Lisbon — an epidemic in wrhich
there were symptoms of gastro-enteritis, although only in a few instances
did the disease resemble cholera. They also cultivated the same
organism from the drinking water. It differs from the cholera organism
in the appearance of its colonies and of puncture cultures in gelatin. It
has very feeble pathogenic effects, and gives a very faint, or no, cholera-
red reaction. To Pfeiffer's test it also reacts negatively. Another
spirillum (v. Romanus) was obtained by Celli and Santori from twelve
out of forty-four cases where there were the symptoms of mild cholera.
This organism does not give the cholera-red reaction, nor is it pathogenic
for animals. They look upon it as a "transitory variety " of the cholera
organism, though sufficient evidence for this view is not adduced.
We have mentioned these examples in order to show some of
the difficulties which exist in connection with this subject. It is
important to note that, on the one hand, spirilla which have
been judged to be of different species from the cholera organism,
have been cultivated from cases in which cholera -like symptoms
were present, and, on the other hand, in cases of apparently true
cholera considerable variations in the characters of the cholera
organisms have been found. Such variations have especially
been recorded by Colonel Cunningham in India. It is there-
fore quite an open question whether some of the organisms
in the former class may not be cholera spirilla which have under-
gone variations as a result of the conditions of their growth.
That such variations may occur we have a considerable amount
of evidence. The great bulk of evidence, however, goes to show
METCHNIKOFF'S SPIRILLUM 417
that Asiatic cholera always spreads as an epidemic from places
in India where the disease is endemic, and that its direct cause
is Koch's spirillum. It is sufficient to bear in mind that choleraic
symptoms may be produced by other causes, and that in some
of such cases spirilla which have some resemblance to Koch's
organism may be present in the intestinal discharges, though
rarely in large numbers.
A number of other spirilla have been cultivated, which are of
interest on account of their points of resemblance to the cholera
organism, though probably they produce no pathological condi-
tions in the human subject.
Metchnikoff's Spirillum (vibrio Metchnikovi). — This organism was ob-
tained by Gamaleia from an epidemic disease of fowls in Odessa, and is of
special interest on account of its close resemblance to the cholera organism.
In the natural disease, which
especially affects young _ ^^ ^ ^
fowls, the animals suffer . ***%%* *
from diarrhoea, pass into a
sort of stupor, sitting with Vt4T* *"«>
their feathers ruffled, and . &
usually die within forty- M
eight hours. The intestines ^
contain a greyish - yellow
fluid, sometimes slightly
blood-stained, in which the
spirilla are found. A few
of these spirilla may also be
found in the blood in the
younger fowls, though
generally absent from the
blood in the older.
Morphologically the or-
ganism is practically identi-
cal with Koch's spirillum
(Fig. 139). It is actively FIG. 139.— Metchnikoflfs spirillum, both in
motile, and has the same curved and straight forms ; from an agar
staining reactions. Its culture of twenty-four hours' growth,
growth in peptone gelatin Stained with weak carbol-fuchsin. x 1000.
also closely resembles that
of the cholera organism, though it produces liquefaction more rapidly
(Fig. 140, A). In gelatin plates the young colonies are, however,
smoother and more circular. After liquefaction occurs, some of the
colonies are almost identical in appearance with those of the cholera
vibrio, whilst others show more uniformly turbid contents. In puncture
cultures the growth takes place more rapidly, but in appearance
closely resembles that of the cholera organism a few days older. Its
growth in peptone solution too is closely similar, and it also gives the
cholera-red reaction.
This organism can, however, be readily distinguished from the
cholera organism by the effects of inoculation on animals, especially on
pigeons and guinea-pigs. Subcutaneous inoculation of small quantities,
27
418
CHOLERA
of pure culture in pigeons is followed by septicaemia, which produces a
fatal result usually within twenty-four hours. Inoculation with the same
quantity of cholera culture produces practically no result ; even with
large quantities death is rarely produced. The vibrio Metchnikovi
produces somewhat similar effects in the guinea-pig to those in the
pigeon, subcutaneous inoculation
being followed by extensive haemor-
rhagic redema and a rapidly fatal
septicaemia. Young fowls can be
infected by feeding with virulent
cultures. We have evidence from
the work of Gamaleia that the toxins
of this organism have somewhat the
same action as those of the cholera
organism.
The organism is therefore one
which very closely resembles the
cholera organism, the results on in-
oculating the pigeon offering the most
ready means of distinction. It gives
a negative reaction to Pfeiffer's test
— that is, the properties of an anti-
cholera serum are not exerted against
it. It may also be mentioned that
an organism which is apparently the
same as the vibrio Metchnikovi was
cultivated by Pfuhl from water, and
named v. Nordhafen.
Finkler and Prior's Spirillum. —
These observers, shortly after Koch's
discovery of the cholera organism,
separated a spirillum, in a case of
cholera nostras, from the stools after
they had been allowed to decompose
for several days. There is, however,
no evidence that the spirillum has
any causal relationship to this or any
other disease in the human subject.
Morphologically it closely resembles
Koch's spirillum, and cannot be dis-
tinguished from it by its micro-
scopical characters, although, on the
whole, it tends to be rather thicker
in the centre and more pointed at the ends (Fig. 141). In cultures,
however, it presents marked differences. In puncture cultures on
gelatin it grows much more quickly, and liquefaction is generally
visible within twenty-four hours. The liquefaction spreads rapidly,
and usually in forty -eight hours it has produced a funnel-shaped
tube with turbid contents, denser below (Fig. 140, B). In plate
cultures the growth of the colonies is proportionately rapid. Before
they have produced liquefaction around them, they appear, unlike those
of the cholera organism, as minute spheres with smooth margins. When
liquefaction occurs, they appear as little spheres with turbid contents,
which rapidly increase in size ; ultimately general liquefaction occurs.
On potatoes this organism grows well at the ordinary temperature, and
A B
FIG. 140. — Puncture cultures in
peptone-gelatin.
A. Metchnikoffs spirillum. Five
days' growth.
B. Finkler and Prior's spirillum.
Four days' growth. Natural size.
DENEKE'S SPIRILLUM
419
X 4 .t
in two or three days has formed a slimy layer of greyish-yellow colour,
which rapidly spreads over the potato. On all the media the growth
has a distinctly fcetid odour. A growth in peptone solution fails to give
the cholera-red reaction at the end of twenty-four hours, though later a
faint reaction may ap -
pear. As stated above, Koch
succeeded in producing,
by this organism, an intes-
tinal affection inguinea-pigs
after neutralising the stom-
ach contents and paralysing
the intestine with opium.
This occurs in a small pro-
portion of the animals ex-
perimented on, and the con- ^rv
tents of the intestine, unlike
what was found in the case
of the cholera organism, ^j
were turbid in appearance,
and had a markedly foetid
odour. When tested by in-
traperitoneal injection, its
effects are somewhat of the
same nature as those of the
'V
;^L I
\
FIG. 141. — Finkler and Prior's spirillum ; from
an agar culture of twenty - four hours'
growth.
Stained with carbol-fuchsin. x 1000.
cholera organism, but its
virulence is of a much lower
order.
An organism cultivated by
Miller (" Miller's Spirillum")
from the cavity of a decayed
tooth in a human subject is almost certainly the same organism as
Finkler and Prior's spirillum.
Deneke's Spirillum. — This organism was obtained from old cheese, and
is also known as the spirillum tyrogenum. It closely resembles Koch's
spirillum in microscopic appearances, though it is rather thinner and
smaller. Its growth in gelatin is also somewhat similar, but liquefaction
proceeds more rapidly, and the bell-shaped depression on the surface is
larger and shallower, whilst the growth has a more distinctly yellowish
tint. The colonies in plates also show points of resemblance, though the
youngest colonies are rather smoother and more regular on the surface, and
liquefaction occurs more rapidly than in the case of the cholera organism.
The colonies have, on naked-eye examination, a distinctly yellowish colour.
The organism does not give the cholera-red reaction, and on potato it
forms a thin yellowish layer when incubated above 30° C. When tested
by intraperitoneal injection and by other methods it is found to possess
very feeble, or almost no, pathogenic properties. Koch found that this
organism, when administered through the stomach in the same way as
the cholera organism, produced a fatal result in three cases out of
fifteen. Deneke's spirillum is usually regarded as a comparatively
harmless saprophyte.
CHAPTER XVIII.
INFLUENZA, PLAGUE, RELAPSING FEVER,
MALTA FEVER, YELLOW FEVER.
INFLUENZA.
THE first accounts of the organism now known as the influenza
bacillus were published simultaneously by Pfeiffer, Kitasato, and
Canon, in January 1892. The two first-mentioned observers
found it in the bronchial
- \r*J ' '*?. sputum, and obtained pure
. */ ;J *. i cultures, and Canon ob-
j* . % ' Kv f- vv» f^ served it in the blood in a
*'A x few cases of the disease.
fc * tf, ' fcf&s* " % t It is, however, to Pfeiffer 's
>;t ' , , work that we owe most of
» i jfv «£**/{' * 1 *..^> our knowledge regarding
- « ' s?v'^» | its characters and action.
*~" /,*?' S-is results have been
' c* % / amply confirmed by those
r of others in various epi-
fri? ' f '^\*. .'. V / demies of the disease, and
V ^ , « , this organism has been
. generally accepted as the
cause of the disease, al-
FIG. 142. -Influenza bacilli from a culture ^ugh absolute proof is
on blood agar. j.-n
Stained with carbol-fuchsin. x 1000. stl11 wanting.
Microscopical Char-
acters.— The influenza bacilli as seen in the sputum are very
minute rods not exceeding 1 '5 /A in length and '3 p in thickness.
They are straight, with rounded ends, and sometimes stain more
deeply at the extremities (Fig. 142). The bacilli occur singly
or form clumps by their aggregation, but do not grow into chains.
They show no capsule. They take up the basic aniline stains
420
CULTIVATION OF BACILLI 421
somewhat feebly, and are best stained by a weak solution (1 : 10)
of carbol-fuchsin applied for 5 to 10 minutes. They lose the
stain in Gram's method. They are non-motile, and do not form
spores.
In many cases of the disease, especially in the early stages of
the more acute, influenza bacilli are present in large numbers
and may be easily found. On the other hand, it is often
difficult or impossible to find them, even when the symptoms
are severe ; this may be due to the restriction of the organisms
to some part not readily accessible, or it may be that they
actually die out in great part while the effects of their toxins
persist. It has also been observed in recent epidemics, in which
the disease has been less widespread and on the whole less
severe, that the period during which the bacilli have been readily
demonstrable in the secretions has been on the average shorter
than in the previous epidemics.
Cultivation. — The best medium for the growth of the
influenza bacillus is blood agar (see page 38), which was intro-
duced by Pfeiffer for this purpose. He obtained growths of the
bacilli on agar which had been smeared with influenza sputum,
but he failed to get any subcultures on the agar media or on
serum. The growth in the first cultures he considered to be
probably due to the presence of certain organic substances in
the sputum, and accordingly he tried the expedient of smearing
the agar with drops of blood before making the inoculations.
In this way he completely succeeded in attaining his object.
The blood of the lower animals is suitable, as well as human
blood ; and the favouring influences of the blood would appear
to be due to the haemoglobin, as a solution of this substance is
equally effective. The colonies of the influenza bacilli on blood
agar, incubated at 37° C., appear within twenty-four hours, in
the form of minute circular dots almost transparent, like drops
of dew. When numerous, the colonies are scarcely visible to
the naked eye, but when sparsely arranged they may reach the
size of a small pin's head. This size is generally reached on the
second day. The bacilli die out somewhat quickly in cultures,
and in order to keep them alive sub-cultures should be made
every four or five days. By this method the cultures may be
maintained for an indefinite period. Growth on the ordinary
agar media is slight and somewhat uncertain ; there is, however,
evidence that growth is more marked when other organisms are
present, that is, is favoured by symbiosis. Neisser, for example,
was able to cultivate the influenza bacfllus on plain agar
through several generations by growing the xerosis bacillus
422 INFLUENZA
along with it ; dead cultures of the latter had not the sanu-
favouring effect. A very small amount of growth takes place
in bouillon, but it is more marked when a little fresh blood is
added. The growth forms a thin whitish deposit at the bottom
of the flask. The limits of growth are from 25° to 42° C., the
optimum temperature being that of the body. The infliu'ii/a
bacillus is a strictly aerobic organism.
The powers of resistance of this organism are of a low order.
Pfeiffer found that dried cultures kept at the ordinary tempera-
ture were usually dead in twenty hours, and that if sputum \\viv
kept in a dry condition for two days, all the influenza bacilli
were dead, or rather, cultures could be no longer obtained.
Their duration of life in ordinary water is also short, the bacilli
usually being dead within two days. From these experiments
Pfeiffer concludes that outside the body in ordinary conditions
they cannot multiply, and can remain alive only for a short time.
The mode of infection in the disease he accordingly considers to
be chiefly by means of fine particles of disseminated sputum, etc.
Distribution in the Body. — The bacilli are found chiefly in
the respiratory passages in influenza. They may be present in
large numbers in the nasal secretion, generally mixed with a
considerable number of other organisms, but it is in the small
masses of greenish-yellow sputum from the bronchi that they
occur in largest numbers, and in many cases almost in a state
of purity. They occur in clumps which may contain as many
as 100 bacilli, and in the early stages of the disease are chiefly
lying free. As the disease advances, they may be found in
considerable numbers within the leucocytes, and towards the
end of the disease a large proportion have this position. It is
a matter of considerable importance, however, that they may
persist for weeks after symptoms of the disease have disappeared*
and may still be detected in the sputum. Especially is this the
case when there is any chronic pulmonary disease. They also
occur in large numbers in the capillary bronchitis and catarrhal
pneumonia of influenza, as Pfeiffer showed by means of sections
of the affected parts. In these sections he found the bacilli
lying amongst the leucocytes which filled the minute bronchi,
and also penetrating between the epithelial cells and into the
superficial parts of the mucous membrane. Other organisms
also, especially Fraenkel's pneumococcus, may be concerned in
the pneumonic conditions following influenza. In some cases
influenza occurs in tubercular subjects, or is followed by tubercular
affection, in which cases both influenza and tubercle bacilli may
be found in the sputum. In such a condition the prognosis is
DISTRIBUTION OF MClLLl 423
very grave. Regarding the presence of influenza bacilli in the
other pulmonary complications following influenza, much in-
formation is still required. Occasionally in the foci of sup-
purative softening in the lung the influenza bacilli have been
found in a practically pure condition. In cases of empyema the
organisms present would appear to be chiefly streptococci and
pneumococci; whilst in the gangrenous conditions, which
sometimes occur, a great variety of organisms has been found.
Pfeiffer's observations on a large series of cases convinced
him that the organism was very rarely present in the blood —
that in fact its occurrence there must be looked upon as
exceptional. The conclusions of other observers have, on the
whole, confirmed this statement, and it is probable that the
chief symptoms in the disease are due to toxins absorbed from
the respiratory tract (vide infra). The bacillus may be present
in some of the lesions complicating influenza. Pfeiffer found
it in inflammation of the middle ear, but in a case of meningitis
following influenza FraenkePs diplococcus was present. In a
few cases of meningitis, however, the influenza bacillus has been
found, sometimes alone, sometimes along with pyogenic cocci
( Pf uhl and Walter, Cornil and Durante) ; Pfuhl considers that
in these the path of infection is usually a direct one through
the roof of the nasal cavity. This observer also found post
mortem, in a rapidly fatal case with profound general symptoms,
influenza bacilli in various organs, both within and outside of
the vessels. In a few cases also the bacilli have been found in
the brain and its membranes with little tissue change in the
parts around.
Extensive observations on the bacteriology of the respiratory
system show that influenza-like bacilli may be present in a great
variety of conditions ; we have, in fact, once more to do with a
group of organisms with closely allied characters, of which
Pfeiffer's influenza bacillus was the first recognised example.
These "pseudo-influenza" bacilli have been obtained from the
fauces, bronchi, and lungs in inflammatory conditions, and also in
various specific fevers. To this group belongs the bacillus which
has been cultivated from cases of whooping-cough by Spengler,
Jochmann, Davis, and others, and which is present in considerable
numbers in a large proportion of cases of this disease. Wollstein
has obtained a marked agglutinative reaction on this organism
by the serum of whooping-cough patients, all the sera examined
giving a positive reaction in a dilution of at least 1 : 100 on all
the strains of the organism isolated ; on the other hand, clumping
was never obtained with a normal serum in a greater dilution
424 INFLUENZA
than 1:10. Davis, by inoculation of the fauces of a healthy man
with this organism, produced inflammatory change with febrile
reaction, but not the characteristic symptoms of whooping-cough.
There is presumptive evidence in favour of the bacillus being
etiologically related to the disease, though the matter cannot yet
be considered settled. Miiller's "trachoma bacillus" (p. 192) is
a member of the same group. All these organisms are very
restricted in their growth, and require the addition of blood or
haemoglobin to the ordinary culture media ; hence they are some-
times spoken of as hsemophilic bacteria. Some of the examples
are a little larger than the influenza-bacillus, and tend to form
short filaments, but others are quite indistinguishable. All of
them also seem to have very feeble pathogenic properties towards
the lower animals. At present it can scarcely be claimed as
possible to identify PfeifTer's bacillus by its microscopic and
cultural characters.
Experimental Inoculation. — There is no satisfactory evidence
that any of the lower animals suffer from influenza in natural
conditions, and accordingly we cannot look for very definite
results from experimental inoculation. Pfeiffer, by injecting
living cultures of the organism into the lungs of monkeys, in
three cases produced a condition of fever of a remittent type.
There wras, however, little evidence that the bacilli had under-
gone multiplication, the symptoms being apparently produced
by their toxins. In the case of rabbits, intravenous injection of
living cultures produces dyspnoea, muscular weakness, and
slight rise of temperature, but the bacilli rapidly disappear in
the body, and exactly similar symptoms are produced by
injection of cultures .killed by the vapour of chloroform.
Pfeiffer, therefore, came to the conclusion that the influenza
bacilli contain toxic substances which can produce in animals
some of the substances of the disease, but that animals are not
liable to infection, the bacilli not having power of multiplying
to any extent in their tissues.
Cantani succeeded in producing infection to some extent in
rabbits, by injecting the bacilli directly into the anterior
portion of the brain. In these experiments the organisms
spread to the ventricles, and then through the spinal cord by
means of the central canal, afterwards infecting the substance
of the cord. An acute encephalitis was thus produced, and
sometimes a purulent condition in the lateral ventricles. The
bacilli were, however, never found in the blood or in other organs.
Similar symptoms were also produced by injection of dead
cultures, though in this case the dose required to be five or six
METHODS OF EXAMINATION 425
times larger. Cantani therefore concludes that the brain
substance is the most suitable nidus for their growth, but
agrees with Pfeiffer in believing that the chief symptoms are
produced by toxins resident in the bodies of the bacilli. He
made control experiments by injecting other organisms, and also
by injecting inert substances into the cerebral tissue.
The evidence, accordingly, that the influenza bacillus is the
cause of the disease rests chiefly on the well-established fact that
it is always present in the secretions of the respiratory tract in
true cases of influenza, and often in very large numbers. The
observed relationships of the organism to lesions in the lungs
and elsewhere leave no room for doubt that it is possessed of
pathogenic properties, but we cannot yet maintain that its causal
relationship to epidemic influenza is absolutely established. A
certain amount of confirmatory evidence has been supplied by
the results of experiment.
Methods of Examination — (a) Microscopic. — A portion of the
greenish-yellow purulent material which often occurs in little
round masses in the sputum should be selected, and film prepara-
tions should be made in the usual way. Films are best stained
by Ziehl-Neelsen carbol-f uchsin diluted with ten parts of water,
the films being stained for ten minutes at least. In sections of
the tissues, such as the lungs, the bacilli are best brought out,
according to Pfeiffer, by staining with the same solution as above
for half an hour. The sections are then placed in alcohol
containing a few drops of acetic acid, in which they are
dehydrated and slightly decolorised at the same time. They
should be allowed to remain till they have a moderately light
colour, the time varying according to their appearance. They
are then washed in pure alcohol, cleared in xylol, and afterwards
mounted in balsam.
(b) Cultures. — A suitable portion of the greenish -yellow
material having been selected from the sputum, it should be
washed well in several changes of sterilised water. A portion
should then be taken on a platinum needle, and successive
strokes made on the surface of blood-agar tubes. The tubes
should then be incubated at 37° C., when the transparent
colonies of the influenza bacillus will appear, usually within
twenty-four hours. These should give a negative result on
inoculation on ordinary agar media.
PLAGUE.
The bacillus of oriental plague or bubonic pest was discovered
independently by Kitasato and Yersin during the epidemic at
426
PLAGUE
Hong Kong in 1894. The results of their investigations, which
were published almost at the same time, agree in most of the
important points. They cultivated the same organism from a
large number of cases of plague, and reproduced the disease in
susceptible animals by inoculation of pure cultures. It is to
be noted that during an epidemic of plague, sometimes even
preceding it, a high mortality has been observed amongst certain
FIG. 143. — Film preparation from a plague bubo showing enormous numbers
of bacilli, most of which show well-marked bipolar staining.
Stained with weak gentian- violet, x 1000.
animals, especially rats and mice, and that from the bodies of
these animals found dead in the plague-stricken district, the
same bacillus was obtained by Kitasato and also by Yersin.
Bacillus of Plague — Microscopical Characters. — As seen in
the affected glands or buboes in this disease, the bacilli are
small oval rods, somewhat shorter than the typhoid bacillus,
and about the same thickness (Fig. 143), though considerable
variations in size occur. They have rounded ends, and in
stained preparations a portion in the middle of the bacillus is
BACILLUS OF PLAGUE
427
often left uncoloured, giving the so-called " polar staining." In
films from the tissues they
are found scattered ~ *•'
amongst the cells, for the
most part lying singly,
though pairs are also seen.
On the other hand, in cul-
tures in fluids, e.g. bouillon,
they grow chiefly in chains,
sometimes of considerable
length, the form known
as a streptobacillus result-
ing (Fig. 145). In young
agar cultures the bacilli
show greater variation in
size, and polar staining is
less marked than in the
tissues: sometimes forms Fm- ^^"^{J^LfaTr6 from a young
of considerable length are stained witn wTak'cLbolTchsiu. x 1000.
present. After a time in-
volution forms appear, especially when the surface of the agar
is dry ; but the formation
of these is much more
rapid and more marked
when 2-5 per cent of
• \ sodium chloride is added
\ to the medium, constitut-
ing the so-called " salt-
agar " (Hankin and Leu-
mann). On this medium,
especially with the higher
percentage, the in volution
forms assume a great size
and a striking variety of
shapes, large globular,
oval, or pyriform bodies
resulting (Fig. 146); with
FIG. 145.— Bacillus of plague in chains show- about 2 per cent sodium
iugboumronaiUiUg' Fr°ma youngculture chloride,aftertwenty-four
Stained with thionin-blue. x 1000. hours' incubation, the
most striking feature is a
general enlargement of all the bacilli. Sometimes in the tissues
they are seen to be surrounded by an unstained capsule, though
this appearance is by no means common. They do not form
428
PLAGUE
9
*
'
spores. Gordon, who has found that they possess flagella
which, however, stain with difficulty, states that they are
motile. Most observers, however, and with these we agree,
have failed to find evidence of true motility. They stain readily
with the basic aniline stains, but are decolorised by Gram's
method.
Cultivation. — From the affected glands, etc., the bacillus can
readily be cultivated on the ordinary media. It grows best at
the temperature of the body, though growth occurs as low as
18° C. On agar and on blood serum the colonies are whitish
circular discs of somewhat
'^ „ „ transparent appearance
* y and smooth, shining sur-
face. When examined
with a lens, their borders
*\ appear slightly wavy. In
£ ;^ stroke cultures on agar
there forms a continuous
line of growth with the
same appearance, showing
partly separated colonies
at its margins. When
agar cultures are kept at
the room temperature,
some of the colonies may
show a more luxuriant
growth with more opaque
FIG. 146. — Culture of the bacillus of plague &
on 4 per cent salt agar, showing involution appearance than the rest
forms of great variety of -size and shape. of the growth, the appear-
Stained with carbol-thionin-blue. x 1000. ance in fact being often
such as to suggest the
presence of impurities in the cultures. In stab cultures in
peptone gelatin, growth takes place along the needle track as
a white line, composed of small spherical colonies. On the
surface of the gelatin a thin, semi-transparent layer may be
formed, which is usually restricted to the region of puncture,
though sometimes it may spread to the wall of the tube ; some-
times, however, there is practically no surface growth. There is
no liquefaction of the medium. In gelatin plates the superficial
colonies develop first and form slightly raised semi-transparent
discs with somewhat crenated margins \ the deeper colonies are
smaller and of spherical shape with smooth outline. In bouillon
the growth usually forms a slightly granular or powdery deposit
at the foot and sides of the flask, somewhat resembling that of
DISTRIBUTION OF BACILLI 429
a streptococcus. If oil or melted butter is added to the bouillon
so that drops float on the surface, then a striking mode of growth
may result, to which the term " stalactite " has been applied.
This consists in the growth starting from the under surface of
the fat globules and extending downwards in the form of
pendulous, string-like masses. These masses are exceedingly
delicate, and readily break off on the slightest shaking of the
flask ; accordingly during their formation the culture must be
kept absolutely at rest. This manner of growth constitutes an
important but not absolutely specific character of the organism ;
unfortunately it is not supplied by all races of the organism, and
varies from time to time with the same race. The organism
flourishes best in an abundant supply of oxygen ; in strictly
anaerobic conditions almost no growth takes place.
The organism in its powers of resistance corresponds with
other spore-free bacilli, and is readily killed by heat, an exposure
for an hour at 58° C. being fatal. On the other hand it has
remarkable powers of resistance against cold ; it has been exposed
to a temperature several degrees below freezing-point without
being killed. Experiments on the effects of drying have given
somewhat diverse results, but as a rule the organism has been
found to be dead after being dried for from six to eight days,
though sometimes it has survived the process for a longer period ;
exposure to direct sunlight for three or four hours kills it. When
cultivated outside the body the organism often loses its virulence,
but some races remain virulent in cultures for a long period of
time.
Anatomical Changes and Distribution of Bacilli. — The
disease occurs in several forms, the bubonic and the pulmonary
being the best recognised ; to these may be added the septiccemic.
The most striking feature in the bubonic form is the affection of
the lymphatic glands, which undergo intense inflammatory
swelling, attended with haemorrhage, and generally ending in
a greater or less degree of necrotic softening if the patient lives
long enough. The connective tissue around the glands is
similarly affected. The bubo is thus usually formed by a
collection of enlarged glands fused by the inflammatory swelling.
True suppuration is rare. Usually one group of glands is
affected first, constituting the primary bubo — in the great
majority the inguinal or the axillary glands — and afterwards
other groups may become involved, though to a much less
extent. Along with these changes there is great swelling of
the spleen, and often intense cloudy swelling of the cells of the
kidneys, liver, and other organs. There may also occur secondary
430
PLAGUE
areas of haemorrhage and necrosis, chiefly in the lungs, liver,
and spleen. The bacilli occur in enormous numbers in the
swollen glands, being often so numerous that a film preparation
made from a scraping almost resembles a pure culture (Fig.
143). In sections of the glands in the earlier stages the bacilli
are found to form dense masses in the lymph paths and sinuses
FIG. 147. — Section of a human lymphatic gland in plague, showing the
injection of the lymph paths and sinuses with masses of plague bacilli — seen
as black areas.
Stained with carbol-thionin-blue. x 50.
(Fig. 147), often forming an injection of them; they may also
be seen growing as a fine reticulum between the cells of the
lymphoid tissue. At a later period, when disorganisation of
the gland has occurred, they become irregularly mixed with the
cellular elements. Later still they gradually disappear, and
when necrosis is well advanced it may be impossible to find any
— a point of importance in connection with diagnosis. In the
spleen they may be very numerous or they may be scanty,
according to the amount of blood infection which has occurred ;
EXPERIMENTAL INOCULATION 431
in the secondary lesions mentioned they are often abundant.
In the pulmonary form the lesion is the well-recognised " plague
pneumonia." This is of broncho-pneumonic type, though large
areas may be formed by confluence of the consolidated patches,
and the inflammatory process is attended usually by much
haemorrhage ; the bronchial glands show inflammatory swelling.
Clinically there is usually a fairly abundant frothy sputum often
tinted with blood, and in it the bacilli may be found in large
numbers. Sometimes, however, cough and expectoration may
be absent. The disease in this form is said to be invariably
fatal. In the septiccemic form proper there is no primary bubo
discoverable, though there is almost always general enlargement
of lymphatic glands ; here also the disease is of specially grave
character. A bubonic case may, however, terminate with septi-
caemia ; in fact all intermediate forms occur. An intestinal form
with extensive affection of the mesenteric glands has been
described, but it is exceedingly rare — so much so that many
observers with extensive experience have doubted its occurrence.
In the various forms of the disease the bacilli occur also in the
blood, in which they may be found during life by microscopic
examination, chiefly, however, just before death in very severe
and rapidly fatal cases. The examination of the blood by means
of cultivation experiments is, however, a much more reliable
procedure. For this purpose about 1 c.c. of blood may be with-
drawn from a vein and distributed in flasks of bouillon (p. 68).
It may be said from the results of different investigators that
the bacillus may be obtained by culture in fully 50 per cent of
the cases, though the number will necessarily vary in different
epidemics. The Advisory Committee, recently appointed, found
that in some septica3mic cases the bacilli may be present in the
blood in large numbers two, or even three, days before death,
though this is exceptional.
The above types of the disease are usually classified together
under the heading pestis major, but there also occur mild forms
to which the term pestis minor is applied. In these latter there
may be a moderate degree of swelling of a group of glands,
attended with some pyrexia and general malaise, or there may
be little more than slight discomfort. Between such and the
graver types, cases of all degrees of severity are met with.
Experimental Inoculation. — Mice, guinea-pigs, rats, and
rabbits are susceptible to inoculation, the two former being on
the whole most suitable for experimental purposes. After sub-
cutaneous injection there occurs a local inflammatory oedema,
which is followed by inflammatory swelling of the corresponding
432
PLAGUE
lymphatic glands, and thereafter by a general infection. The
lesions in the lymphatic glands correspond in their main
characters with those in the human subject, although usually
at the time of death they have not reached a stage so advanced.
By this method of inoculation mice usually die in 1-3 days,
guinea-pigs and rats in 2-5 days, and rabbits in 4-7 days.
Post mortem the chief changes, in addition to the glandular
enlargement, are congestion of internal organs, sometimes with
haemorrhages, and enlargement of the spleen ; the bacilli are
numerous in the lymphatic glands and usually in the spleen
(Fig. 148), and also,
though in somewhat less
^f -f* if* **2t *f\. degree, throughout the
mf ^ * fcP^v blood. Infection can
also be produced by
flf++ * 1 1 *J£I0>\ smearjng the material
* '•: 4K, t _ .*•* "* • «• &< ,r • ,-
on the conjunctiva or
mucous membrane of
the nose, and this method
of inoculation has been
successfully applied in
cases where the plague
bacilli are present along
with other virulent
organisms, e.g. in sputum
along with pneumococci.
FIG. 148. — Film preparation of spleen of rat Rats and mice can also
after inoculation with the bacillus of plague, be infected by feeding
!^!Yi^^e^1T,bacilli' m°st °f wMch either with pure cultures
x 1000.
are somewhat plump.
Stained with carbol-thionin-blue.
or with pieces of organs
from cases of the disease,
though in this case infection probably takes place through the
mucous membrane of the mouth and adjacent parts, and only
to a limited extent, if at all, by the alimentary canal. Monkeys
also are highly susceptible to infection, and it has been shown
in the case of these animals that when inoculation is made on
the skin surface, for example, by means of a spine charged with
the bacillus, the glands in relation to the part may show the
characteristic lesion and a fatal result may follow without there
being any noticeable lesion at the primary seat. This fact
throws important light on infection by the skin in the -human
subject. The disease may also extensively affect monkeys by
natural means during an epidemic.
Paths and Mode of Infection. — Plague bacilli may enter
PATHS AND MODE OF INFECTION 433
the system by the skin surface through small wounds, cracks,
abrasions, etc., and in such cases there is usually no reaction
at the site of entrance. This last fact is in accordance with
what has been stated above with regard to experiments on
monkeys. The path of infection is shown by the primary
buboes, which are usually in the glands through which the
skin is drained, those in the groin being the commonest site.
Absolute proof of the possibility of infection by the skin is
supplied by several cases in which the disease has been acquired
at post-mortem examinations, the lesions of the skin surface
being in the majority of these of trifling nature ; in only two
was there local reaction at the site of inoculation. In most of
these cases the period of incubation has been from two to three
days ; under natural conditions of infection the average period
is within five days. While infection may occur by accidental
inoculation through small wounds of the skin surface, it appears
in the majority of cases to take place by means of the bites
of fleas. For some time it had been known that plague bacilli
might be found for some time afterwards in the stomach of fleas
allowed to feed on animals suffering from plague, and some
observers, for example Simond, had succeeded in transmitting
the disease to other animals by means of the infected insects.
Most observers, however, had obtained negative results, and it
was only by the work of the Advisory Committee appointed by
the Secretary of State for India in 1 905 l that the importance
of this means of infection was established. By carefully planned
experiments the Committee showed that the disease could be
transmitted from a plague rat to a healthy rat kept in adjacent
cages when fleas were present ; whereas this did not occur when
means were taken to prevent the access of fleas, though the
facilities for aerial infection were the same. The disease can
also be produced by fleas removed from plague rats and trans-
ferred directly to healthy animals, success having been obtained
in fully 50 per cent of experiments of this kind. When plague-
infected guinea-pigs are placed amongst healthy guinea-pigs
comparatively few of the latter acquire the disease when fleas
are absent or scanty ; whereas all of them may die of plague
when fleas are numerous. This result demonstrates the com-
paratively small part played by direct contact, even when of
a close character. Important results were also obtained with
regard to the mode of infection in houses where there had been
cases of plague. It was found possible to produce the disease
in susceptible animals by means of fleas taken from rats in
1 See Journal of Hygiene, vi. 421 ; vii. 323.
28 *
434 PLAGUE
plague houses. When animals were placed in plague houses
and efficiently protected from fleas they remained healthy;
whereas they acquired the disease when the cages were free
to the access of fleas in the neighbourhood.
The following are some of the experiments which were conducted. A
series of six huts were built which only differed in the structure of their
roofs. In two the roofs were made of ordinary native tiles in which rats
freely lodge ; in two others flat tiles were used in which rats live, but in
which they have not such facilities for movement as in the first set, and in
the third pair the roof was formed of corrugated iron. Under the roof in
each case was placed a wire diaphragm which prevented rats or "their
droppings having access to the hut, but which would not prevent fleas
falling down on to the floor of the hut. The huts were left a sufficient
time to become infected with rats, and then on the floor in each case
healthy guinea-pigs mixed with guinea-pigs artificially infected with
plague were allowed to run about together. In the first two sets of huts
to which fleas had access the healthy guinea-pigs contracted plague,
while in the third set they remained unaffected, though they were freely
liable to contamination by contact with the Bodies and excreta of the
diseased animals. In the third set of huts no infection took place as
long as fleas were excluded, but when accidentally these insects obtained
admission, then infection of the uninoculated animals commenced.
Other experiments were also performed. In one case healthy guinea-pigs
were suspended in a cage two inches above a floor on which infected and
flea-infested animals were running about. Infection occurred in the cage,
but if the latter were suspended at a distance above the floor higher than
a flea could jump, then no infection took place. Again, in a hut in which
guinea-pigs had died of plague, and which contained infected fleas, two
cages were placed, each containing a monkey. One cage was surrounded
by a zone of sticky material broader than the jump of a flea. The
monkey in this cage remained unaffected, but the other monkey con-
tracted plague.
Other experiments showed that when plague bacilli were
placed on the floors of houses, they died off in a comparatively
short period of time. After forty-eight hours it was not found
possible to reproduce plague by inoculation with material from
floors which had been grossly contaminated with cultures of the
bacillus. Afterwards, however, animals placed in such a house
might become infected by means of fleas. In all these experi-
ments the common rat-flea of India — pulex cheojris (Rothschild)
— was used, but it has been shown that this flea, when a rat is
not available, will bite a man. These results are manifestly of
great practical importance. They show that direct infection by
dust and other material through small lesions of the skin plays,
probably, a comparatively small part in the spread of the disease,
fleas apparently being in the majority of cases the carriers of in-
fection. They also point to important preventive measures,
which will no doubt be put to a practical test before long.
TOXINS, IMMUNITY, ETC. 435
In primary plague pneumonia, from a consideration of the
anatomical changes and the clinical facts, the disease may be
said to be produced by the direct passage of the bacilli into the
respiratory passages. Nevertheless there must be certain factors,
still imperfectly understood, which determine the incidence of
this form ; as in some epidemics of the highest virulence plague
pneumonia has been practically absent, though opportunities for
infection by inhalation must have been present. On the other
hand, a case of plague pneumonia is of great infectivity in
producing other cases of plague pneumonia. If we except
infection through the respiratory passages in such cases, it may
be said that direct infection from patient to patient is relatively
uncommon. This is in accordance with the fact that in bubonic
plague the bacilli are not discharged from the unbroken surface
of the body, and are only present in the secretions in severe cases.
The occurrence of the disease in rats was early recognised, and
there is no doubt that it plays a very important part in the
spread of epidemics. The disease in these animals has, in fact,
been the means of rapidly distributing infection over wide areas
of a town or district. This has been abundantly proved in
the case of Bombay, where observations have shown that the
migration of plague-infected rats to quarters comparatively free
from the disease, has been followed by extensive outbreaks in
these places. The facts stated above show how the disease is
spread among these animals by fleas, and how it is conveyed
by them to the human subject.
Toxins, Immunity, etc. — As is the case with most organisms
which extensively invade the tissues, the toxins in plague cultures
are chiefly contained in the bodies of the bacteria. Injection of
dead cultures in animals produces distinctly toxic effects ; post
mortem haemorrhage in the mucous membrane of the stomach,
areas of necrosis in the liver and at the site of inoculation, may
be present. The toxic substances are comparatively resistant to
heat, being unaffected by an exposure to 65° C. for an hour. By
the injection of dead cultures in suitable doses a certain degree
of immunity against the living virulent bacilli is obtained, arid,
as first shown by Yersin, Calmette, and Borrel, the serum of
such immunised animals confers a degree of protection on
small animals such as mice. On these facts the principles of
preventive inoculation and serum treatment, presently to be
described, depend. It may also be mentioned that the filtrate
of a plague culture possesses a very slight toxic action, and the
Indian Plague Commission found that such a filtrate has practi-
cally no effect in the direction of conferring immunity.
436 PLAGUE
1. Preventive Inoculation — Hq/kine's Method. — To prepare
the preventive fluid, cultures are made in flasks of bouillon with
drops of oil on the surface (in India Haffkine employed a
medium prepared by digesting goats' flesh with hydrochloric
acid at 140° C. and afterwards neutralising with caustic soda).
In such cultures stalactite growths (vide supra) form, and the
flasks are shaken every few days so as to break up the stalactites
and induce fresh crops. The flasks are kept at a temperature
of about 25° C., and growth is allowed to proceed for about
six weeks. At the end of this time sterilisation is effected by
exposing the contents of the flasks to 65° C. for an hour;
thereafter carbolic acid is added in the proportion of '5 per cent.
The contents are well shaken to diffuse thoroughly the sediment
in the fluid, and are then distributed in small sterilised bottles for
use. The preventive fluid thus contains both the dead bodies
of the bacilli and any toxins which may be in solution. It is
administered by subcutaneous injection, the dose, which varies
according to the "strength," being on an average about 7*5 c.c.
Usually only one injection is made, sometimes two ; though the
latter procedure does not appear to have any advantage. The
method has been systematically tested by inoculating a certain
proportion of the inhabitants of districts exposed to infection,
leaving others uninoculated, and then observing the proportion
of cases of disease and the mortality amongst the two classes.
The results of inoculation, as attested by the first Indian
Commission, have been distinctly satisfactory. For although
absolute protection is not afforded by inoculation, both the
proportion of cases of plague and the percentage mortality
amongst these cases have been considerably smaller in the
inoculated, as compared with the uninoculated. Protection
is not established till some days after inoculation, and lasts for
a considerable number of weeks, possibly for several months
(Bannerman). In the Punjab during the season 1902-3 the case
incidence among the inoculated was 1*8 per cent, among the
uninoculated 7*7 per cent, while the case mortality was 23*9 and
60 '1 per cent respectively in the two classes, the statistics being
taken from villages where 10 per cent of the population and
upwards had been inoculated.
2. Anti-plague Sera. — Of these, two have been used as therapeutic
agents, viz. that of Yersin and that of Lustig. Yersin's serum is
prepared by injections of increasing doses of plague bacilli into the
horse. In the early stages of immunisation dead bacilli are injected
subcutaneously, thereafter into the veins, and, finally, living bacilli are
injected intravenously. After a suitable time blood is drawn off and
the serum is preserved in the usual way. Of this serum 10-20 c.c.
METHODS OF DIAGNOSIS 437
are used, and injections are usually repeated on subsequent days.
Lustig's serum is prepared by injecting a horse with repeated and
increasing doses of a substance derived from the bodies of plague bacilli,
probably in great part nucleo-proteid. Masses of growth are obtained
from the surface of agar cultures, and are broken up and dissolved in a
1 per cent solution of caustic potash. The solution is then made slightly
acid by hydrochloric acid, when a bulky precipitate forms ; this is
collected on a filter and dried. For use a weighed amount is dissolved
in a weak solution of carbonate of soda and then injected. The serum
is obtained from the animal in the usual way. Extensive observations
with both of these sera show that neither of them can be considered
a powerful remedy in cases of plague, though in certain instances
distinctly favourable results have been recorded. The Indian Com-
mission, however, came to the conclusion " that, on the whole, a certain
amount of advantage accrued to the patients both in case of those
injected with Yersin's serum and of those injected with Lustig's serum."
It may also be mentioned that the Commission found, as the result of
experiments, that Yersin's serum modified favourably the course of the
disease in animals, whereas Lustig's serum had no such effect
3. Serum Diagnosis. — Specific agglutinins may appear in the blood of
patients suffering from plague, as also they do in the case of animals
immunised against the plague bacillus. It is to be noted, however, that
in clinical cases the reaction is not invariably present, the potency of
the serum is not of high order, and the carrying out of the test is com-
plicated by the natural tendency of the bacilli to cohere in clumps. For
the last reason the macroscopic (sedimentation) method is be preferred
to the microscopic (p. 111). A suspension of plague bacilli is made by
breaking up a young agar culture in 75 per cent sodium chloride
solution ; the larger flocculi of growth are allowed to settle, and the
fine, supernatant emulsion is employed in the usual way. According to
the results of the German Plague Commission and the observations of
Cairns, made during the Glasgow epidemic, it may be said that the
reaction is best obtained with dilutions of the serum of from 1 : 10 to
1 : 50. Cairns found that the date of its appearance is about a week
after the onset of illness, and that it usually increases till about the end
of the sixth week, thereafter fading off. It is most marked in severe
cases characterised by an early and favourable crisis, less marked in
severe cases ultimately proving fatal, whilst in very mild cases it is
feeble or may be absent. The method, if carefully applied, may be of
service under certain conditions ; but it will be seen that its use as a
means of diagnosis is somewhat restricted.
Methods of Diagnosis. — Where a bubo is present a little of
the juice may be obtained by plunging a sterile hypodermic
needle into the swelling. The fluid is then to be examined micro-
scopically, and cultures on agar or blood serum should be made
by the successive stroke method. The cultural and morpho-
logical characters are then to be investigated, the most important
being the involution forms on salt agar and the stalactite
growth in bouillon, though the latter may not always be obtained
with the plague bacillus : the pathogenic properties should also
be studied, the guinea-pig being on the whole most suitable for
438 RELAPSING FEVER AND AFRICAN TICK FEVER
subcutaneous inoculation. In many cases a diagnosis may be
made by microscopic examination alone, as in no other known
condition than plague do bacilli with the morphological char-
acters of the plague bacillus occur in large numbers in the lym-
phatic glands. The organism may be obtained in culture from
the blood in a considerable proportion of cases by withdrawing
a few cubic centimetres and proceeding in the usual manner.
On the occurrence of the first suspected case, every care to
exclude possibility of doubt should be used before a positive
opinion is given.
In a case of suspected plague pneumonia, in addition to
microscopic examination of the sputum, the above cultural
methods along with animal inoculation with the sputum should
be carried out ; subcutaneous injection in the guinea-pig and
smearing the nasal mucous membrane of the rat may be recom-
mended. Here a positive diagnosis should not be attempted by
microscopic examination alone, especially in a plague-free dis-
trict, as bacilli morphologically resembling the plague organism
may occur in the sputum in other conditions.
RELAPSING FEVER AND AFRICAN TICK FEVER.
At a comparatively early date, namely in 1873, when practi-
cally nothing was known with regard to the production of disease
by bacteria, a highly characteristic organism was discovered in
the blood of patients suffering from relapsing fever. This
discovery was made by Obermeier, and the organism is usually
known as the spirillum or spirochcete Obermeieri, or the spirillum
of relapsing fever. He described its microscopical characters,
and found that its presence in the blood had a definite relation
to the time of the fever, as the organism rapidly disappeared
about the time of the crisis, and reappeared when a relapse
occurred. He failed to find such an organism in any other
disease. His observations were fully confirmed, and his views
as to its causal relationship to the disease were generally accepted.
Later, the disease was produced in the human subject by inocula-
tions with blood containing the organisms, and a similar con-
dition has been produced in apes.
Recently it has been shown that the so-called "tick fever"
prevalent in Africa is also due to a spirochsete of similar
character, and results of the highest importance have been
established with regard to the part played by ticks in the trans-
mission of the disease. Doubt still obtains as to the relationship
of the organisms of the two diseases, but all are agreed that they
CHARACTERS OF THE SPIRILLUM 439
are closely similar, if not identical. As a matter of convenience,
and in accordance with the history of the investigations, we shall
give the chief facts regarding these diseases separately. It has
also been shown that spirillar diseases or " spirilloses," as they
are called, are widespread in the animal kingdom. Such
infections have been described in geese by Sacharoff, in fowls by
Marchoux and Salimbeni, in oxen and sheep by Theiler, and in
bats by Nlcolle and Comte, and it is interesting to note that in
the case of the spirilloses of oxen and fowls the infection is
transmissible by means of ticks.
The work of Schaudinn(v. p. 548) has led to these spirochsete
being regarded by various authorities as members of the protozoal
group. As, however,
longitudinal division has
not been satisfactorily
observed and no cycle of
development has been /£
determined amongst them, JB
we are not justified at fl|
present in removing them
from the class of bacteria.
Characters of the
Spirillum. — The organ-
£\
isms as seen in the blood
J.OI11O CtO OV/V1J- AA-l UAJLV; KS±\S\S\A.
during the fever are deli-
cate spiral filaments which
have a length of from two
to six times the diameter
of a red blood corpuscle. FlG 149.— Spirilla of relapsing fever in
They are, however, exceed- human blood. Film preparation. (After
ingly thin, their thickness Koch.) x about 1000.
being much less than that
of the cholera spirillum. They show several regular sharp curves
or windings, of number varying according to the length of the
spirilla, and their extremities are finely pointed (Fig. 149).
There are often to be seen in the spirals, portions which are
thinner and less deeply stained than the rest, and which suggest
the occurrence of transverse division. They are actively motile,
and may be seen moving quickly across the microscopic field
with a peculiar movement which is partly twisting and partly
undulatory, and disturbing the blood corpuscles in their course.
Novy and Knapp have found that there is a single flagellum at
one end of the organism.
They stain with watery solutions of the basic aniline dyes,
440 RELAPSING FEVER
though somewhat faintly, and are best coloured by the
llomanowsky method or one of its modifications. When thus
stained they usually have a uniform appearance throughout, or
may be slightly granular at places, but they show no division
into short segments. They lose the stain in Gram's method.
In blood outside the body the organisms have a considerable
degree of vitality, and when kept in sealed tubes they have been
found alive and active after many days. They are readily
killed at a temperature of 60° C., but may be exposed to 0° C.
without being killed. There is no evidence that they form spores.
Relations to the Disease. — In relapsing fever, after a period
of incubation there occurs a rapid rise of temperature which
lasts for about five to seven days. At the end of this time a
crisis occurs, the temperature falling quickly to normal. In the
course of about other seven days a sharp rise of temperature
again takes place, but on this occasion the fever lasts a shorter
time, again suddenly disappearing. A second or even third
relapse may occur after a similar interval. The spirilla begin to
appear in the blood shortly before the onset of the pyrexia, aud
during the rise of temperature rapidly increase in number.
They are very numerous during the fever, a large number being
often present in every field of the microscope when the blood is
examined at this stage. They begin to disappear shortly before
the crisis : after the crisis they are entirely absent from the
circulating blood. A similar relation between the presence of
the spirilla in the blood and the fever is found in the case of the
relapses, whilst between these they are entirely absent. Munch
in 1876 produced the disease in the human subject by injecting
blood containing the - spirilla, and this experiment has been
several times repeated with the same result. Additional proof,
that the organism is the cause of the disease has been afforded by
experiments on animals. Carter in 1879 was the first to show that
the disease could be readily produced in monkeys, and his experi-
ments were confirmed by Koch. In such experiments the blood
taken from patients and containing the spirilla was injected sub-
cutaneously. In the disease thus produced there is an incubation
period which usually lasts about three days. At the end of that
time the spirilla rapidly appear in the blood, and shortly after-
wards the temperature quickly rises. The period of pyrexia
usually lasts for two or three days, and is followed by a marked
crisis. As a rule there is no relapse, but occasionally one of
short duration occurs.1 White mice and rats are also susceptible
1 Norris, Pappenheimer, and Flournoy, in their experiments on monkeys
in America, found that several relapses occurred.
IMMUNITY 441
to infection. In the former animals the disease is characterised
by several relapses, in the latter there is, however, no relapse.
Numerous attempts to cultivate this organism outside the
body have all been attended with failure, and it has been
abundantly shown that it does not grow on any of the media
ordinarily in use. Koch found that on blood serum the
filaments of the spirilla increased somewhat in length, and
formed a sort of felted mass, but that no multiplication took
FIG. 150. — Spirillum Obermeieri in blood of infected mouse. x 1000.
place. Recently Norris, Pappenheimer, and Flournoy have
found that a considerable amount of multiplication may take
place in the citrated blood of man and the rat.
Immunity. — Metchnikoff found that during the fever the
spirilla were practically never taken up by the leucocytes in the
circulating blood, but that at the time of the crisis, on dis-
appearing from the blood, they accumulated in the spleen and
were ingested in large numbers by the microphages or poly-
morphonuclear leucocytes. Within these they rapidly under-
went degeneration and disappeared. It is to be noted in this
connection that swelling of the spleen is a very marked feature
in relapsing fever. These observations were entirely confirmed
442 RELAPSING FEVER
by Soudake witch, who also produced the disease in two monkeys
(cercocebus fuliginosus) from which the spleen had been previously
removed, the animals having been allowed to recover completely
from the operation, and found that in these cases the spirilla
did not disappear from the blood at the usual time, but rather
increased in number, and a fatal result followed on the eighth
and ninth days respectively. Recent observations, however, in-
dicate that, as in the case of so many other diseases, the all-
important factor in the destruction of the organisms is the
development of antagonistic substances in the blood. Lamb
found in the case of the monkey (macacm radiatus) that the
removal of the spleen of an animal rendered immune by an
attack of the disease did not render it susceptible to fresh
inoculation, and attributed the immunity to the presence of
bactericidal bodies in the serum. He found, for example, that
in vitro the serum of an immune animal brought the movements
of the spirilla to an end, clumped them, and caused their dis-
integration ; and further, that when the spirilla and the immune
serum were injected in one case into a fresh monkey no disease
developed. In opposition to Soudakewitch, Lamb found that
with a monkey from which the spleen had been removed death
did not occur after it was inoculated with the spirilla. Sawt-
schenko and Milkich found that there are developed during the
disease an immune body and an agglutinin, while Novy and
Knapp in their recent important work distinguish germicidal,
immunising, and agglutinating substances. They found that
the blood of the rat has no germicidal properties during the
onset of the disease, but that these appear and become well
marked during the decline. They produced a " hyper-immunity "
in rats by repeated injections of blood containing the spirilla,
and found that the serum of such animals had a markedly cura-
tive effect, and could cut short the disease in rats, mice, and
monkeys.
In the case of the human subject it has been found that a
second attack of the disease can follow the first after a com-
paratively short period of time, and it is often said that one
attack does not confer immunity. It is probably rather the case
that the immunity conferred is of very short duration. The
course of events in the disease might be explained by supposing
that immunity of short duration is produced during the first
period of pyrexia, but that it does not last until all the spirilla
have been destroyed, some still surviving in internal organs
or in tissues where they escape the bactericidal action of the
serum. With the disappearance of the immunity the organ-
AFRICAN TICK FEVER 443
isms reappear in the blood, the relapse being, however, of
shorter duration and less severe than the first attack. This is
repeated till the immunity lasts long enough to allow all the
organisms to be killed. The production of anti-substances
during the febrile attack is an established fact, and the experi-
mental results above detailed show that the disease as met with
in the human subject will probably be eminently amenable to
serum therapeutics.
The fact that other like spirillar diseases may be conveyed
by the bites of insects makes it extremely probable that relaps-
ing fever may also be transmitted in this way, and a number of
facts point to the bed-bug as the means of transmission. The
presence of the spirilla within the bodies of bugs has been
demonstrated, and it has also been shown that they may be
present for a considerable time after the insects have sucked the
blood, — according to Karlinski for forty days. Tictin, by inject-
ing the blood removed from a number of bugs which had been
allowed to bite infected monkeys, produced the disease in other
healthy monkeys, but so far as we know the crucial experiment
of infecting man by means of the bites of these insects has not
yet been successfully carried out.
African Tick Fever.
The disease long known by this name as prevalent in Africa
has also been shown to be caused by a spirillum or spirochaete.
Organisms of this nature had been seen in the blood of patients
in Uganda by Greig and Nabarro in 1903, and Milne and Ross
in the end of 1904 recorded a series of observations which led
them to the conclusion that tick fever was due to a spirochsete.
It is, however, chiefly owing to the work of Button and Todd in
the Congo Free State, on the one hand, and of Koch in German
East Africa, on the other; that our knowledge of this disease has
been thoroughly established. The former gave a full account of
the organism, and by means of experiments showed that the
disease could be transferred by means of ticks to healthy
animals. The latter published interesting observations on the
infection of the ticks and the transmission of the organisms to
the young, and also important facts with regard to the extent to
which ticks were infected in certain districts.
The following are the chief facts regarding this fever.
Clinically the fever closely resembles relapsing fever, but the
periods of fever are somewhat shorter, rarely lasting for more
than two or three days. It is rarely attended with a fatal result
444
AFRICAN TICK FEVER
unless in patients debilitated by other causes. The spirilla are
considerably fewer in the blood than in the European relapsing
fever, and sometimes a careful search may be necessary before they
are found. Morphologically they are said to be practically
identical, although Koch thought that the organisms in tick
fever tended on the whole to be slightly longer ; the average
length may be said to be 15 to 35 /x. Dutton and Todd showed
that it was possible to transmit the disease to certain monkeys
.• /*
FIG. 151. — Film of human blood containing spirillum of tick fever, x 1000. *
(cercopitheci) by means of ticks which had been allowed to bite
patients suffering from the disease, the symptoms in these
animals appearing about five days after inoculation. The disease
thus produced is characterised by several relapses, and often
leads to a fatal result. In one case they produced the disease
by means of young ticks hatched from the eggs of ticks which
had been allowed to suck the blood of fever patients, and they
came to the conclusion that the spirilla were not simply carried
mechanically by the ticks, but probably underwent some cycle of
1 We are indebted to Colonel Leishman, R.A.M.C., for the preparations
from which Figs. 150-152 were taken.
AFRICAN TICK FEVER 445
development in the tissues of the latter. The species of tick
concerned is the ornithodorus moubata. These results were con-
firmed and extended by Koch. He found that after the ticks
had been allowed to suck the blood containing the organisms,
these could be found for a day or two in the stomachs of the
insects. After this time they gradually disappeared from the
stomach, but were detected in large numbers in the ovaries of
the female ticks, where they sometimes formed felted masses.
FIG. 152. — Spirillum of human tick fever (Spirillum Duttoni) in blood of
infected mouse. x 1000.
He also traced the presence of the spirilla in the eggs laid by the
infected ticks, and in the young embryos hatched from them.
He was thus able to demonstrate how the infection might be
continued within the tissues of ticks from generation to
generation ; in the process of transmission, however, the spirillar
form was always observed, and there was no evidence that the
organism went through a cycle of change. Koch also made
extensive observations on the ticks in German East Africa, and
found that of over six hundred examined 11 ,per cent of these
insects along the main caravan routes contained spirilla, and
in some localities almost half of the ticks were infected. In
446 MALTA FEVER
places removed from the main lines of commerce he still found
them, though in smaller number. It has also been demonstrated
that in some places the ticks are found to be infected with the
spirilla although the inhabitants do not suffer from tick fever, a
circumstance which is probably due to an acquired immunity
against the disease.
Although our knowledge regarding the relationships of these
to other spirilla is far from complete, certain differences between
the organisms of European relapsing fever and of African tick
fever have been established. Zettnow, for example, has found
that the organism of tick fever possesses numerous lateral
flagella, whereas, as already stated, the sp. Obermeieri has a
single terminal flagellum. This observation, however, has not
yet been confirmed. Differences are also brought out by animal
inoculation. In addition to the more severe illness produced by
the spirillum of tick fever in monkeys, it has been found by
Breinl and Kinghorn that a considerable number of animals
are susceptible to the African spirillum, including rabbits and
guinea-pigs, which appear to be refractory to the organism of
relapsing fever. Breinl also compared the immunity conferred
by the sp. Obermeieri and by the tick fever spirillum, and found
that each conferred a relative active immunity against itself, but
not against the other. It is thus highly probable that they
represent two distinct species. Spirillar fever has also been
found in India, but its relations to the European and African
fevers have not yet been fully worked out.
MALTA FEVER.
Synonyms — Mediterranean Fever : Rock Fever of Gibraltar :
Neapolitan Fever, etc.
This disease is of common occurrence along the shores of the
Mediterranean and in its islands. Since its bacteriology has
been worked out, it has been found to occur also in India, China,
and in some parts of North and South America, its distribution
being much wider than was formerly supposed. Although from
its symptomatology and pathological anatomy it had been re-
cognised as a distinct affection, and was known under various
names, its precise etiology was unknown till the publication
of the researches of Colonel Bruce in 1887. From the spleen
of patients dead of the disease he cultivated a characteristic
organism, now known as the micrococcus melitensis, and by
MICROCOCCUS MELITENSIS 447
means of inoculation experiments established its causal relation-
ship to the disease. Wright and Semple applied the agglutina-
tion test to the diagnosis of the disease, while within recent
years the mode of spread of the disease has been fully studied
by a Commission, and it has been demonstrated that goat's milk
is the chief means of infection.
The duration of the disease is usually long— often two or
three months, though shorter and much longer periods are met
with. Its course is very variable, the fever being of the con-
tinued type with irregular remissions. In addition to the usual
symptoms of pyrexia, there occur profuse perspirations, pains
and sometimes swellings in the joints, occasionally orchitis,
whilst constipation is usually a marked feature. The mortality
is low — about 2 per cent (Bruce).
In fatal cases the most striking post-mortem change is in the
spleen. This organ is enlarged, often weighing slightly over a
pound, and in a condition of acute congestion ; the pulp is soft
and may be diffluent, and the Malpighian bodies are swollen and
indistinct. In the other organs the chief change is cloudy
swelling ; in the kidneys there may be in addition glomerular
nephritis. The lymphoid tissue of the intestines shows none of
the changes characteristic of typhoid fever.
Micrococcus Melitensis. — This is a small, rounded, or slightly
oval organism about '4 /A in diameter, which is specially abundant
in the spleen. It usually occurs singly or in pairs, but in
cultures short chains are also met with (Fig. 153). (Durham
has shown that in old cultures kept at the room temperature
bacillary forms appear, and we have noticed indications of such
in comparatively young cultures ; the usual form is, however,
that of a coccus.) It stains fairly readily with the ordinary
basic aniline stains, but loses the stain in Gram's method. It is
generally said to be a non-motile organism. Gordon, however,
is of a contrary opinion, and has recently demonstrated that it
possesses from one to four flagella, which, however, are difficult
to stain. In the spleen of a patient dead of the disease it occurs
irregularly scattered through the congested pulp ; it may also be
found in small numbers post mortem in the capillaries of various
organs. It may be cultivated from the blood during life in a
considerable proportion of cases; for this purpose 5-10 c.c. of
blood should be withdrawn from a vein and distributed in small
flasks of bouillon. The micrococcus was found by the members
of the Commission in the urine of Malta fever patients in 10 per
cent of the cases examined ; it was sometimes scanty, but some-
times present in large numbers.
448
MALTA FEVER
Cultivation. — This can usually readily be effected by making
stroke cultures on agar tubes from the spleen pulp and incub-
ating at 37° C. The colonies, which are usually not visible
before the third or fourth day, appear as small round discs,
slightly raised and of somewhat transparent appearance. The
maximum size — 2-3 mm. in diameter — is reached about the
ninth day ; at this period by reflected light they appear pearly
white, while by transmitted light they have a yellowish tint in
the centre, bluish white at the periphery. A stroke culture
shows a layer of growth of similar appearance with somewhat
serrated margins. Old
Hfefe^ cultures assume a buff tint.
The optimum temperature
is 37° C., but growth still
occurs down to about 20°
C. On gelatin at summer
temperature growth is ex-
tremely slow — after two or
three weeks, in a puncture
culture, there is a delicate
line of growth along the
needle track and a small
flat expansion of growth
„ * on the surface. There is
no liquefaction of the
rfrn medium. In bouillon there
FIG. 153. — Micrococcus rnehtensis, from a . . ,.,
two days' culture on agar at 37° C. occurs a general turbidity
Stained with fuchsin. x 1000. with flocculent deposit at
the bottom ; on the surface
there is no formation of a pellicle. The reaction of the media
ought to be very faintly alkaline, as marked alkalinity interferes
with the growth. On potatoes no visible growth takes place
even at the body temperature, though the organism multiplies
to a certain extent. Outside the body the organism has
considerable powers of vitality, as it has been found to sur-
vive in a dry condition in dust and clothing for a period of two
months.
Relations to the Disease. — There is in the first place ample
evidence, from examination of the spleen, both post mortem and
during life, that this organism is always present in the disease.
The experiments of Bruce and Hughes first showed that by
inoculation with even comparatively small doses of pure cultures
the disease could be produced in monkeys, sometimes with a
fatal result. And it has now been fully established that inocula-
MODE OF SPREAD OF THE DISEASE 449
tion with the minutest amount of culture, even by scarification,
leads to infection both in monkeys and in the human subject.
Rabbits, guinea-pigs, and mice are insusceptible to inocula-
tion by the ordinary method. Durham, by using the intra-
cerebral method of inoculation, has, however, succeeded in
raising the virulence so that the organism is capable of produc-
ing in guinea-pigs on intra-peritoneal injection illness with some-
times a fatal result many weeks afterwards. An interesting
point brought out by these experiments is that, in the case of
animals which survive, the micrococcus may be cultivated from
the urine several months after inoculation.
Mode of Spread of the Disease. — The work of the recent
Commission has resulted in establishing facts of the highest
importance with regard to the spread of the disease. In the
course of investigations Zammitt found that the blood of many
of the goats agglutinated the micrococcus melitensis, and
Horrocks obtained cultures of the organism from the milk.
Further observations showed that agglutination was given in
the case of 50 per cent of the goats in Malta, whilst the organism
was present in the milk in 10 per cent. Sometimes the organism
was present in enormous numbers, and in these cases the animal
usually appeared poorly nourished, whilst the milk had a some-
what serous character. In other cases, however, the organism
was found when the animals appeared healthy and there was
no physical or chemical change in the milk. It was also
determined that the organism might be excreted for a period
of two to three months before any notable change occurred in
the milk. Agglutination is usually given by the milk of infected
animals, and this property was always present when the micro-
coccus was found in the milk. It was, moreover, found that mon-
keys and goats - could be readily infected by feeding them with
milk containing the micrococcus, the disease being contracted
by fully 80 per cent of the monkeys used. It was therefore
rendered practically certain that the human subject was infected
by means of such milk, and the result of preventive measures
by which milk was excluded as an article of dietary amongst
the troops in Malta has fully borne out this view. After such
measures were instituted, the number of cases in the second
half of 1906 fell to 11 per thousand, as contrasted with 47
per thousand in the corresponding part of the preceding year.
The various facts with regard to the epidemiology of the disease
have thus been cleared up. For example, it is more prevalent
in the summer months, when more milk is consumed, and there
is a larger proportion of cases amongst those in goad social
29
450 MALTA FEVER
position, the officers, for example, suffering more in proportion
than the privates. Another interesting fact, pointed out by
Horrocks, is that the disease has practically disappeared from
Gibraltar since the practice of importing goats from Malta has
stopped.
The work of the Commission, so far as it has gone, has
been to exclude other modes of infection as being of practical
importance, by dust, by the bites of mosquitoes, etc., and if it
is conveyed by contact at all, this is only when the contact is
of an intimate character, and even then it is probably of rare
occurrence. Although numerous patients suffering from the
disease come to England, there is no known case of fresh
infection arising under natural conditions.
Agglutinative Action of Serum. — The blood serum of patients
suffering from Malta fever possesses the power of agglutinating
the micrococcus melitensis in a manner analogous to what has
been described in the case of typhoid fever. The reaction
appears comparatively early, often about the fifth day, and may
be present for a considerable time after recovery — sometimes
for more than a year. Distinct agglutination with a 1 : 20
dilution of the serum in half an hour may be taken as a positive
reaction, sufficient for diagnosis. The reaction is, however,
usually given by much higher dilutions, e.g. 1 : 500, and even
higher. It is to be noted that normal serum diluted 1 : 2 may
produce some agglutination. As regards relation to prognosis,
the observations of Birt and Lamb and of Bassett-Smith have
given results analogous to those obtained in typhoid (p. 340).
The Commission has recently found that vaccination with
dead cultures of the micrococcus confers a certain degree of
protection amongst those exposed to the disease. As a rule two
injections were made, 200-300 million cocci being the dose of
the first injection, and about 400 million of the second. The
use of vaccines has also been carried out in the treatment of
the disease, but the observations are not sufficiently numerous
to allow a definite statement to be made as to its value.
Methods of Diagnosis. — During life the readiest means of
diagnosis is supplied by the agglutinative test just described
(for technique, vide p. 109).
Cultures are most easily obtained from the spleen either
during life or j>o*t mortem. Inoculate a number of agar tubes
by successive strokes and incubate at 37° C. Film preparations
should also be made from the spleen pulp and stained with
carbol-thionin-blue or diluted carbol-fuchsin (1 : 10).
YELLOW FEVER 451
YELLOW FEVER.
Yellow fever is an infectious disease which is endemic in the
West Indies, in Brazil, in Sierra Leone and the adjacent parts
of West Africa, though it is probable that it was from the
first-named region that the others were originally infected.
From time to time serious outbreaks occur, during which
neighbouring countries also suffer, and the disease may be
carried to other parts of the world. In this way epidemics
have occurred in the United States, in Spain, and even in
England, infection usually being carried by cases occurring
among the crews of ships. In the parts where it is endemic,
though usually a few cases may occur from time to time, there
is some evidence that occasionally the disease may remain in
abeyance for many years and then originate de novo. There is,
therefore, reason to suspect that the infective agent can exist
for considerable periods outside the human body. It is possible,
however, that continuity may be maintained by the occurrence
of a mild type of the disease which may be grouped with the
" bilious fevers " prevalent in yellow-fever regions. This would
explain the degree of immunity which is shown during a serious
epidemic by the older immigrants.
Great variations are observed in the clinical types under
which the disease presents itself. Usually after from two to
six days' incubation a sudden onset in the form of a rigor
occurs. The temperature rises to 104°- 105° F. The person is
livid, with outstanding bloodshot eyes. There are present great
prostration, pain in the back, and vomiting, at first of mucus,
later of bile. -The urine is diminished and contains albumin.
About the fifth day an apparent improvement takes place, and
this may lead on to recovery. Frequently, however, the remission,
which may last from a few hours to two days, is followed
by an aggravation of all the symptoms. The temperature rises,
jaundice is observed, hemorrhages occur from all the mucous
surfaces, causing, in the case of the stomach, the " black vomit "
— one of the clinical signs of the disease in its worst form.
Anuria, coma, and cardiac collapse usher in a fatal issue. The
mortality varies in different epidemics from about 35 to 99
per cent of those attacked. Both white and black races are
susceptible, but those who have resided long in a country are
less susceptible than new immigrants. An attack of the disease
usually confers complete immunity against subsequent infection.
Post mortem the stomach is found in a state of acute gastritis,
and contains much altered blood derived from hemorrhages
452 YELLOW FEVER
which have occurred in the mucous and submucous coats. The
intestine may be normal, but is often congested and may be
ulcerated ; the mesenteric glands are enlarged. The liver is in
a state of fatty degeneration of greater or less degree, but often
resembling the condition found in phosphorus poisoning. The
kidneys are in a state of intense glomerulo-nephritis, with fatty
degeneration of the epithelium. There is congestion of the
meninges, especially in the lumbar region, and haemorrhages
may occur. The other organs do not show much change,
though small haemorrhages under the skin and into all the
tissues of the body are not infrequent. In the blood a feature
is the excess of urea present, amounting, it may be, to nearly
4 per cent.
Etiology of Yellow Fever. — Although a large amount of
bacteriological work has been done on yellow fever, this has
now chiefly a historical interest, as it is now known that the
causal agent is not one of the ordinary bacteria, but belongs to
the group of ultra-microscopic organisms.1 A mosquito acts as
the intermediate host, and the facts detailed below point to the
organism passing through some cycle of development in the
body of the insect. The analogy of malaria makes it extremely
probable that the organism is a protozoon, but as this has not
yet been completely proved we have not felt justified in altering
the position of the disease and placing it amongst the protozoal
infections. As bacteriological work led up to the establishment
of our knowledge regarding the nature of the disease, some
reference must be made to it.
A very full research into the bacteriology of yellow fever
was that of Sternberg,- the result of which was that of the
varied organisms isolated, one which he called the bacillus x
appeared possibly to have some relationship to the disease.
Sanarelli in 1897 obtained cultures of an organism which he
called bacillus icteroides, and which he considered to be the
cause of yellow fever ; it is probably identical with the bacillus
x of Sternberg. Subsequent observations made by others
gave conflicting results, some finding this bacillus, others
failing to do so. The bacillus icteroides, as described by
Sanarelli, belongs to the paratyphoid group, possessing lateral
flagella, growing on gelatin without liquefaction, and fermenting
glucose but not lactose. Reed and Carroll found that it was
practically identical with the bacillus of swine cholera. It
1 In several diseases the existence of such causal factors is suspected.
Other examples are foot and mouth disease, South African horse-sickness, and
the contagious pleuro-pneumonia of cattle.
ETIOLOGY OF YELLOW FEVER 453
must now be considered merely as an organism which may
occur in the organs and tissues in yellow fever as a secondary
infection, but without any etiological significance.
The facts of importance which have been established
regarding the etiology of the disease are due to the labours of
the United States Army Commission, which began its work in
1900. The members of the Commission first directed their
inquiries towards determining whether the bacillus icteroides
was present in the blood during life, and a series of cases were
investigated bacteriologically, with entirely negative results in
each instance. They then resolved to test the hypothesis of
Dr. Carlos Finlay of Havana, promulgated several years pre-
viously, that the disease was carried by mosquitoes. Selecting
mosquitoes which they reared from eggs, they allowed them to
bite yellow fever patients and then to bite healthy men. Of
several experiments of this nature two were successful in the
first instance, the first individual to be infected in this way
being Dr. James Carroll, a member of the Commission, who
passed through a severe attack of typical yellow fever. Experi-
ments were then performed on a larger scale, with completely
confirmatory results, as to the conveyance of the disease by
mosquitoes. Of twelve non-immunes living under circumstances
which excluded natural means of infection, ten contracted
yellow fever after having been bitten by mosquitoes which had
previously bitten yellow fever patients; happily all of these
recovered. Two of the men who were thus infected had been
previously exposed to contact with fomites from yellow fever
patients without results. These results were confirmed by
Guiteras, whose investigations were carried out along similar
lines; of seventeen individuals bitten by infected mosquitoes,
eight took yellow fever, and three of these died.
The species of mosquito used by the American Commission
was the Stegomyia fasciata, and up to the present time no other
species has been found capable of carrying the infection. It has
also been determined that a certain period must elapse after the
insect has bitten a yellow fever patient before it becomes infec-
tive to another subject. In summer weather this period is about
twelve days ; at a lower temperature somewhat longer. This
probably means that, as in the case of malaria, the parasite must
pass through certain stages of development before it reaches the
salivary gland and is thus in a position to be transferred to a fresh
subject. Infected mosquitoes, however, retain the power of
infection for a considerable time afterwards, probably as long as
sixty days. It has also been shown that mosquitoes may become
454 YELLOW FEVER
infective after biting a patient on the first, second, or third day
of the disease, but at a later period the results are usually
negative, apparently because the virus is no longer present in
the blood.
Interesting results were also obtained with regard to the
communication of the disease directly from patient to patient,
the conclusion arrived at, after careful experiments, being that
the disease cannot be transferred in this way, even when the
contact is of a close character. In a specially constructed house
seven men were exposed to the most intimate contact with the
fomites of yellow fever patients for a period of twenty days each,
the soiled garments worn by the patients being in some cases
actually slept in by these men ; the result was that not one of
them thus exposed contracted the disease. The conclusions on
this point have been subsequently confirmed by other workers.
The American Commission also found it possible to transmit
yellow fever to a healthy man by injecting small quantities of
blood or of serum taken from a yellow fever patient at any
period up till the third day of the disease. The period of
incubation in this case is somewhat shorter than when the disease
is conveyed by the bite of mosquitoes, the average duration in
the former case being about three days, and in the latter about
four days, though these times may be considerably exceeded.
It is also interesting to know that in these experimental injec-
tions the blood or serum used was found to be free from bacteria.
Up till the present time, we know of only these two methods of
infection, namely, indirectly by the bite of a mosquito infected
with the yellow fever germ, or directly by the injection of some
of the blood from a yellow fever patient. In these respects
there is a striking similarity to what has been established in the
case of malarial fever.
Experiments with regard to the nature of the yellow fever
organism were carried out by Reed and Carroll, and interest-
ing results were obtained. They found that the organism of
the disease was very easily killed by heat, as blood from a
yellow fever patient lost its infective power on being heated
to 55° C. for ten minutes. On the other hand, blood or serum
was found to be still infective after having been passed through
a Berkefeld filter. This has been confirmed by the French
Commission, with the additional result that the virus passes
through a Chamberland F filter, but not through a Chamber-
land B. These facts would show that the parasite is of
extremely minute size, and apparently belongs to the group of
ultra - microscopic organisms. Up till the present time all
ETIOLOGY OF YELLOW FEVER 455
attempts to find by microscopic examination the yellow fever
parasite, either in the blood of patients suffering from the
disease, or in the tissues of infective mosquitoes, have been
attended with negative results.
Though nothing has been determined regarding the actual
nature of the virus, yet the results already obtained have
supplied the basis for preventive measures against the disease,
these being directed towards the destruction of mosquitoes and
the protection of those suffering from yellow fever, and also the
healthy, against the bites of these insects. Already a striking
degree of success has been obtained in Havana. Such measures
came into force in February 1901, and in ninety days the town
was free of yellow fever and f9r fifty-four days later no new
cases occurred; and although subsequently the disease was
reintroduced into the town, no difficulty was experienced in
stamping it out by the same measures. In recent years the
results have also been highly gratifying, and the disease may
be said to be practically eradicated from Havana. In other
places also successful results have been obtained, and epidemics
would appear to be now under control if the proper measures
are taken.
CHAPTER XIX.
IMMUNITY.
Introductory. — By immunity is meant non-susceptibility to a
given disease or to a given organism, either under natural
conditions or under conditions experimentally produced. The
term is also used in relation to the toxins of an organism.
Immunity may be possessed by an animal naturally, and is then
usually called natural immunity, or it may be acquired by an
animal either by passing through an attack of the disease, or by
artificial means of inoculation. It is to be noted that man and
the lower animals may be exempt from certain diseases under
natural conditions, and yet the causal organisms of these diseases
may produce pathogenic effects when injected in sufficient
quantity. Immunity is, in fact, of very varying degrees, and
accordingly the use of the term has a correspondingly relative
significance. This is not only true of infection by bacteria, but
of toxins also : — when the resistance of an animal to these is of
high degree, the resistance may in certain cases be overcome by
a very large dose of the toxic agent. For example, the common
fowl may be able to resist as much as 20 c.c. of powerful tetanus
toxin, but on this amount being exceeded may be affected by
tetanic spasms (Klemperer). On the other hand, in cases where
the natural powers of resistance are very high, these can be still
further exalted by artificial means, that is, the natural immunity
may be artificially intensified.
Acquired Immunity in the Human Subject. — The following
facts are supplied by a study of the natural diseases which affect
the human subject. First, in the case of certain diseases, one
attack protects against another for many years, sometimes
practically for a lifetime, e.g. smallpox, typhoid, scarlet fever,
etc. Secondly, in the case of other diseases, e.g. erysipelas,
diphtheria, influenza, and pneumonia, a patient may suffer from
several attacks. In the case of the diseases of the second group,
456
ARTIFICIAL IMMUNITY 457
however, experimental research has shown that in many of them
a certain degree of immunity does follow ; and, though we
cannot definitely state it as a universal law, it must be con-
sidered highly probable that the passing through an attack of an
acute disease produced by an organism, confers immunity for a
longer or shorter period. The immunity is not, however, to be
regarded as the result of the disease per se, but of the bacterial
products introduced into the system ; as will be shown below,
by suitable gradation of the doses of such products, or by the
use of weakened toxins, a high degree of immunity may be
attained without the occurrence of any symptoms whatever.
The facts known regarding vaccination and smallpox
exemplify another principle. We may take it as practically
proved that vaccinia is variola or smallpox in the cow, and that
when vaccination is performed, the patient is inoculated with a
modified variola (vide Smallpox, in Appendix). Vaccination
produces certain pathogenic effects which are of trifling degree
as compared with those of smallpox, and we find that the degree
of protection is less complete and lasts a shorter time than that
produced by the natural disease. Again, inoculation with lymph
from a smallpox pustule produces a form of smallpox less
severe than the natural disease but a much more severe con-
dition than that produced by vaccination, and it is found that
the degree of protection or immunity resulting occupies an inter-
mediate position.
Immunity and Recovery from Disease. — Recovery from an
acute infective disease shows that in natural conditions the vims
may be exhausted after a time, the period of time varying in
different diseases. How this is accomplished we do not yet
fully know, but it has been found in the case of diphtheria,
typhoid, cholera, pneumonia, etc., that in the course of the
disease certain substances (called by German writers Antikb'rper)
appear in the blood, which are antagonistic either to the toxin
or to the vital activity of the organism. In such cases a process
of immunisation would appear to be going on during the pro-
gress of the disease, and when this immunisation has reached a
certain height, the disease naturally comes to an end. It can-
not, however, be said as yet that such antagonistic substances
are developed in all cases ; though the results already obtained
make this probable.
ARTIFICIAL IMMUNITY.
Varieties. — According to the means by which it is produced,
immunity may be said to be of two kinds, to which the terms
458 IMMUNITY
active and passive are generally applied, or we may speak of
immunity directly, or indirectly, produced. We shall first give
an account of the established facts, and afterwards discuss some
of the theories which have been brought forward in explanation
of these facts.
Active immunity is obtained by (a) injections of the organisms
either in an attenuated condition or in sub-lethal doses, or (b)
by sub-lethal doses of their products, i.e. of their " toxins," the
word being used in the widest sense. By repeated injections
at suitable intervals the dose of organisms or of the products
can be gradually increased ; or, what practically amounts to the
same, an organism of greater virulence or a toxin of greater
strength may be used. A proportionate degree of resistance or
immunity can thus be developed, which degree in course of time
may reach a very high level. Such methods constitute the
means of preventive inoculation or vaccination. Immunity of
this kind is comparatively slowly produced and lasts a consider-
able time, the duration varying in different cases.
Passive immunity depends upon the fact that if an animal
be immunised to a very high degree by the previous method, its
serum may have distinctly antagonistic or neutralising effects
when injected into another animal along with the organisms, or
with their products, as the case may be. Such a serum, generally
known as an anti-serum, may exert its effects if introduced into
an animal at the same time as infection occurs or even a short
time afterwards ; it can, therefore, be employed as a curative
agent. The serum is also preventive, i.e. protects an animal
from subsequent infection, but the immunity thus conferred
lasts a comparatively short time. These facts form the basis of
serum therapeutics. When such a serum has the power of
neutralising a toxin it is called antitoxic ; when, with little or
no antitoxic power, it protects against the living bacterium in a
virulent condition, it is called antimicrobic or antibacterial (vide
infra).
In the accompanying table a sketch of the chief methods by
which an immunity may be artificially produced is given. It
has been arranged merely for purposes of convenience and to
aid subsequent description ; the principles underlying all the
methods are the same.
ARTIFICIAL IMMUNITY.
A. Active Immunity — i.e. produced in an animal by an in-
jection, or by a series of injections, of non-lethal doses of
an organism or its toxins.
ARTIFICIAL IMMUNITY 459
1 . By injection of the living organisms.
(a) Attenuated in various ways. Examples : —
(1) By growing in the presence of oxygen, or in a
current of air.
(2) By passing through the tissues of one species
of animal (becomes attenuated for another
species).
(3) By growing at abnormal temperatures, etc.
(4) By growing in the presence of weak antiseptics, or
by injecting the latter along with the organism,
etc.
(b) In a virulent condition, in non-lethal doses.
2. By injection of the dead organisms.
3. By injection of filtered bacterial cultures, i.e. toxins ; or of
chemical substances derived from such filtrates.
These methods may also be combined in various ways.
B. Passive Immunity, i.e. produced in one animal by injection
of the serum of another animal highly immunised by the
methods of A.
1. By antitoxic serum, i.e. the serum of an animal highly
immunised against a particular toxin.
2. By antibacterial serum, i.e. the serum of an animal highly
immunised against a particular bacterium in the living
and virulent condition./
A. Active Immunity.
1. By Living Cultures. — (a) Attenuated. — In the earlier
work on immunity in the case of anthrax, chicken cholera, swine
plague, etc., the investigators had to deal with organisms of
high virulence, which had accordingly to be reduced before the
organisms could be injected in the living state. It is now found
most convenient as a rule to start the process of active immunisa-
tion with the injection of dead cultures. The principle is the
same as that of vaccination, and both attenuated cultures and
also the dead cultures used for injection are often spoken of as
vaccines. The virulence of an organism may be diminished in
various ways, of which the following examples may be given.
(1) In the first place, practically every organism when culti-
vated for some time outside the body, loses its virulence, and
in the case of some this is very marked indeed, e.g. the pneumo-
460 IMMUNITY
coccus. Pasteur found in the case of chicken cholera, that
when cultures were kept for a time in ordinary conditions, they
gradually lost their virulence, and that when sub-cultures were
made the diminished virulence persisted. Such attenuated
cultures could be used for protective inoculation. He considered
the loss of virulence to be due to the action of the oxygen of
the air, as he found that in tubes sealed in the absence of oxygen
the virulence was not lost. Haffkine attenuated cultures of the
cholera spirillum by growing them in a current of air (p. 412).
(2) The virulence of an organism for a particular animal may
be lessened by passing the organism through the body of another
animal. Duguid and Burdon Sanderson found that the virulence
of the anthrax bacillus for bovine animals was lessened by its being
passed through guinea-pigs, the disease produced in the ox by
inoculation from the guinea-pig being a non-fatal one. This dis-
covery was confirmed by Greenfield, who showed that the bacilli
cultivated from guinea-pigs preserved their property in cultures,
and could therefore be used for protective inoculation of cattle.
A similar principle was applied in the case of swine plague by
Pasteur, who found that if the organism producing this disease
was inoculated from rabbit to rabbit, its virulence was increased
for rabbits but was diminished for pigs. The method of vaccina-
tion against smallpox depends upon the same principle. There
is also evidence to show that the virulence of the tubercle
bacillus becomes modified according to its host, being often
diminished for other animals.
(3) Many organisms become diminished in virulence when
grown at an abnormally high temperature. The method of
Pasteur, already described (p. 314), for producing immunity in
sheep against anthrax bacilli, depends upon this fact. A virulent
organism may also be attenuated by being exposed to an elevated
temperature which is insufficient to kill it, as was found by
Toussaint in the case of anthrax.
(4) Still another method may be mentioned, namely, the
attenuation of the virulence by growing the organism in the
presence of weak antiseptics. Chamberland and Koux, for
example, succeeded in attenuating the anthrax bacillus by
growing it in a medium containing carbolic acid in the
proportion of 1 : 600.
These examples will serve to show the principles underlying
attenuation of the virulence of an organism. There are, how-
ever, still other methods, most of which consist in growing the
organism in conditions somewhat unfavourable to its growth, e.g.
under compressed air, etc.
BY LIVING CULTURES 461
(b) Immunity by living Virulent Cultures in Non-lethal
Doses. — Immunity may also be produced by employing virulent
cultures in small, that is non-lethal, doses. In subsequent
inoculations the doses may be increased in amount. For example,
immunity may thus be obtained in rabbits against the bacillus
pyocyaneus. Such a method, however, has had a limited
application in the case of virulent organisms, as it has been
found more convenient to commence the process by attenuated
cultures, and then to continue with living cultures.
Exaltation of the Virulence. — The converse process to
attenuation, i.e. the exaltation of the virulence, is obtained
chiefly by the method of cultivating the organism from animal
to animal — the method of passage discovered by Pasteur (first,
we believe, in the case of an organism obtained from the saliva
in hydrophobia, though having no causal relationship to that
disease). This is most conveniently done by intraperitoneal
injections, as there is less risk of contamination. The organisms
in the peritoneal fluid may be used for the subsequent injection,
or a culture may be made between each inoculation. The
virulence of a great number of organisms can be increased in
this way, the animals most frequently used being rabbits and
guinea-pigs. This method can be applied to the organisms of
typhoid, cholera, pneumonia, to streptococci and staphylococci,
and in fact to those organisms generally which invade the
tissues.
The virulence of an organism, especially when in a relatively
attenuated condition, can also be raised by injecting along with
it a quantity of a culture of another organism either in the living
or dead condition. A few examples may be mentioned. An
attenuated diphtheria culture may have its virulence raised by
being injected into an animal along with the streptococcus
pyogenes ; an attenuated culture of the bacillus of malignant
oedema by being injected with the bacillus prodigiosus ; an
attenuated streptococcus by being injected with the bacillus coli,
etc. A culture of the typhoid bacillus may be increased in
virulence, as already stated, by being injected along with a dead
culture of the bacillus coli. In such cases the accompanying in-
jection enables the attenuated- organism to gain a foothold in the
tissues, and it may be stated as a general rule that the virulence
of an organism for a particular animal is raised by its growing
in the tissues of that animal.
Combination of Methods. — The above methods may be com-
bined in various ways. By repeated injections of cultures at
first in the dead condition, then living and attenuated and
462 IMMUNITY
afterwards more virulent, and by increasing the doses, a high
degree of immunity may be obtained.
2. Immunity by Dead Cultures of Bacteria.— In some cases
a high degree of immunity against infection by a given microbe
may be developed by repeated and gradually increasing doses
of the dead cultures, the cultures being killed sometimes by
heat, sometimes by exposure to the vapour of chloroform. In
this method the so-called endotoxins will be injected along with
the other substances in the bacterial protoplasm, but the resulting
immunity is chiefly directed against the vital activity of the
organisms — is antibacterial rather than antitoxic (vide infra).
The cultures when dead produce, of course, less effect than when
living, and this method may be conveniently used in the initial
stages of active immunisation, to be afterwards followed by
injections of the living cultures. The method is extensively
used for experimental purposes, and is that adopted in anti-
plague and anti-typhoid inoculations.
3. Immunity by the Separated Bacterial Products or
Toxins. — The organisms in a virulent condition are grown in
a fluid medium for a certain time, and the fluid is then filtered
through a Chamberland or other porcelain filter. The filtrate
contains the toxins, and it may be used unaltered, or may be
reduced in bulk by evaporation, or may be evaporated to
dryness. The process of immunisation by the toxin is started
by small non-lethal doses of the strong toxin, or by larger doses
of toxin the power of which has been weakened by various
methods (vide infra). Afterwards the doses are gradually
increased. This method was carried out with a great degree
of success in the case, of diphtheria, tetanus, malignant oedema,
etc. It appears capable of general application in the case of
organisms where it is possible to get an active toxin from the
filtered cultures. It has also been applied in the case of snake
poisons by Calmette and by Fraser, and a high degree of im-
munity has been produced.
Immunity may also be obtained by means of certain chemical
substances separated from filtered bacterial cultures, though
these substances are generally in a more or less impure
condition. Hankin was the first to obtain this result by means
of an albumose separated from anthrax cultures.
The following may be mentioned as some of the most
important examples of the practical application of the principles
of active immunity, i.e. of protective inoculation : — (1) Inocula-
tion of sheep and oxen against anthrax (Pasteur) (p. 314); (2)
Jennerian vaccination against smallpox (p. 503) ; (3) Anti-
BY BACTERIAL PRODUCTS OR TOXINS 463
cholera inoculation (Haffkine) (p. 412); (4) Anti-plague
inoculation (Haffkine) (p. 434) ; (5) Anti-typhoid inoculation
(Wright and Seinple) (p. 343) ; (6) Pasteur's method of inocu-
lation against hydrophobia, which involves essentially the same
principles (p. 516).
Vaccines as a Method of Treatment. — Up till recently the
principles of active immunity had not been directly applied in
the treatment of an existing disease except in the case of
tuberculosis. The work of Wright, however, shows that active
immunisation in such circumstances is not only possible, but is
also probably capable of wide application. From his study of
the part played by phagocytosis in the successful combat of
bacteria by the body, he was led to advocate the treatment of
bacterial infections by carrying on an active immunisation
against the causal agents by the injection of dead cultures of
the latter. The justification for such a procedure lies in his
contention that in many cases infections are to be looked on as
practically entirely localised, e.g. the cases of an acne pustule,
or a boil. The reason for the local growth of bacteria in the
part of the body affected is that there is for unknown causes a
deficiency of the opsonic power (vide p. 483) of the body fluids,
which is essential for the phagocytosis of the invading bacteria.
Still more marked in such cases is the deficiency in the opsonic
qualities of the fluids in the actual site of infection. Any
procedure which will raise the opsonic power of the body fluids
as a whole, and therefore of the fluids in the focus of infection,
will aid the destruction of the bacteria by sensitising them to
phagocytic action. Such a procedure is found in the active
immunisation which results from the injection of a vaccine
consisting of a dead culture of the causal bacteria. The appli-
cation of a vaccine of this kind must, however, be controlled by
constant observation of the opsonic index of the patient's serum
during the treatment. When a local infection occurs the general
opsonic index is usually found to be below unity. If dead
bacteria be injected into the individual there may occur during
the following few days a further fall in the opsonic index, —
what Wright calls the occurrence of a negative phase. In a case
where the treatment is successful, this negative phase is succeeded
by a rise in the opsonic index above its original level, — ocurrence
of positive phase, — and with this reaction there is an improve-
ment in the local condition. Usually in such cases repeated
injections are required to effect a cure, and the important point,
according to Wright, is to avoid giving an injection when a
negative phase is in progress. If this point is not attended to
464 IMMUNITY
only an aggravation of symptoms is to be looked for (vide
pp. 194, 261).
With regard to the details of the preparation of the vaccines,
an agar culture is taken, the growth removed into normal saline
and killed by steaming for a sufficient time — say 1J hours.
The efficiency of the sterilisation is tested by inoculating tubes
of appropriate media. The strength of the emulsion is then
estimated by the method of counting dead bacteria described on
p. 67. The number of bacteria employed for a vaccination is
usually from 250,000,000 to 500,000,000, and in the details of
the measurement of this quantity and in its injection, every
aseptic precaution must, of course, be adopted. Such vaccines
have been used extensively in the treatment of acne, boils,
sycosis, infections of the genito-urinary tract by the b. coli,
infections of joints by the gonococcus, and in many cases con-
siderable success has followed the treatment.
Active Immunity by Feeding. — Ehrlich found that mice
could be gradually immunised against ricin and abrin by feeding
them with increasing quantities of these substances (vide p. 169).
In the course of some weeks' treatment in this way the resulting
immunity was of so high a degree that the animals could tolerate
on subcutaneous inoculation 400 times the dose originally fatal.
Fraser also found in the case of snake venom that rabbits could
be immunised, by feeding with the poison, against several times
the lethal dose of venom injected into the tissues.
By feeding animals with dead cultures of bacteria or with
their separated toxins, a degree of immunity may in some cases
be gradually developed. But this method is so much less certain
in results, and so much more tedious than the others, that it has
obtained no practical applications.
Active immunity of high degree developed by the methods
described may be regarded as specific, that is, is exerted only
towards the organism or toxin by means of which it has been
produced. A certain degree of immunity, or rather of increased
general resistance of parts of the body (for example the peri-
toneum), can, however, be produced by the injection of various
substances — bouillon, blood serum, solution of nuclein, etc.
(Issaeff). Also increased resistance to one organism can be thus
produced by injections of another organism. Immunity of this
kind, however, never reaches a high degree.
B. Passive Immunity.
Action of the Serum of Highly-Immunised Animals. — 1.
The serum of an animal A, treated by repeated and gradually
PASSIVE IMMUNITY 465
increased doses of a toxin of a particular microbe, may protect
an animal B against a certain amount of the same toxin when
injected along with the latter, or a short time before it. As
would be expected, it has less effect when injected some time
afterwards, but even then within certain limits it has a degree
of curative or palliative power. Seeing that the serum of animal
A appears to neutralise the toxin, the term antitoxic has been
applied to it.
2. The serum of an animal A, highly immunised against a
bacterium by repeated and gradually increasing doses of the
organism, may protect an animal B against an infection by the
living organism when injected under conditions similar to the
above. This serum is therefore antimicrobic, or antibacterial,
i.e. preventive against invasion by a particular organism. (In
addition to the preventive or protective action in vivo, such a
serum may exert certain recognisable effects on the corresponding
organism in vitro. Thus (a) it may lead to the death or solution
of the organism — bactericidal or lysogenic action', (6) it may
produce an increased susceptibility to ingestion by phagocytes —
opsonic action ; (c) it may lead to the clumping of the organism
— agglutinative action.)
These two kinds of anti-sera — antitoxic and antibacterial —
exert their effect when injected along with the toxin or organism
respectively or some time previously ; as would be expected,
they have less effect when injected some time afterwards, though
even then they may have a certain degree of curative or palliative
power. The two properties, antitoxic and antibacterial, are essen-
tially different in kind, the former leading to a neutralisation of
the toxin, the latter to some alteration in the vital activity of
the bacterium ; in other words, the point of attack in the case
of the two sera is different. A serum may, however, possess
both properties in varying degree. The fundamental fact in
passive immunity, viz. that immunity can be transferred to
another animal, shows that the serum in question differs from
the serum of a normal animal in containing antagonistic sub-
stances to the toxin or bacterium as the case may be, — these
being generally spoken of as anti- substances. It will accordingly .
be convenient to speak of anti-substances in general.
The development of anti-substances, first observed in the case
of the injection of toxins, is found to occur when a great many
different substances are introduced into the tissues of the living-
body. We can, in fact, divide organic molecules into two classes
— those which give rise to the production of anti-substances, and
those which have not this property. Amongst the former are
30
466 IMMUNITY
various toxins, ferments, molecules of tissue cells, bacteria, red
corpuscles, etc. They are all probably of proteid nature, though
their true constitution is not known, and none of them have
been obtained in a pure condition. Amongst the latter may
be placed the various poisons of known constitution, glucosides,
alkaloids, etc. We may .also state at present that the anti-
substance forms a chemical or physical union with the particular
substance which has led to its development, and shall discuss
the evidence for this later. Furthermore, the anti-substance has
apparently a specific combining group which fits, as it were, a
group in the corresponding substance, the two groups having
been compared to a lock and key. It is, however, to be noted
that this specificity is a chemical one rather than a biological
one. An anti-serum, for example, developed by the injection of
bacterium A may also have some effect on bacterium B, and
thus appear not to be specific. We have, however, evidence to
show that some of the preponderating molecules in bacterium A
are also present in bacterium B, and thus the theory of chemical
specificity is not invalidated. The number of different anti-
substances, as judged by their combining properties, would
appear to be almost unlimited, a fact which throws new light
on the complexity of the structure of living matter. When anti-
substances are studied as regards their action on the substances
with which they combine, they may be conveniently arranged
in three classes corresponding to Ehrlich's three classes of
receptors (vide p. 491). In the first place, the anti-substance
may simply combine with the substance without, so far as we
know, producing any change in it, and to this group the anti-
toxins and anti-ferments belong. In the second place, the
anti-substance, in addition to combining, may produce some
recognisable physical alteration. In other words, it possesses
an active or zymotoxic group as well as a combining group.
The agglutiniris may be mentioned as examples of this group.
In the third place, the anti-substance after combination leads to
the combination of another body normally present in serum
called complement or alexine, and this latter, which has a con-
stitution very similar to that of a toxin, may lead to physical
change, for example, death or solution of a cell. Anti-substances
of this class are known as immune -bodies or amboceptors
(Ehrlich) or as sensitising substances — substances sensibilisatrices
of French writers. Anti-substances of the second and third
groups are met with especially, though not exclusively, when
.f formed elements such as bacteria, red corpuscles, or tissue cells,
etc., are injected, the anti-serum developed possessing agglu-
ANTITOXIC SERUM 467
tinating, solvent, or other properties towards the particular
substance.
After this preliminary statement in explanation we shall
consider the actual properties of the two classes of serum, and
later we shall resume the theoretical consideration.
Antitoxic Serum. — The best examples are the antitoxic
sera of diphtheria,. ^nd^tatanus, though similar principles and
methods are involved in the case of the anti-sera to ricin and
abrin, and to snake poison. We shall here speak of diphtheria
and tetanus. The steps in the process of preparation may be
said to be the following : First, the preparation of a powerful
toxin. Second, the estimation of the power of the toxin. Third,
the development of antitoxin in the blood of a suitable animal
by gradually increasing doses of the toxin. Fourth, the estima-
tion from time to time of the antitoxic power of the serum of
the animal thus treated.
1. Preparation of the Toxin. — The mode of preparation and
the conditions affecting the development of diphtheria toxin
have already been described (p. 362). In the case of tetanus the
growth takes place in glucose bouillon under an atmosphere of
hydrogen (vide p. 60). In either case the culture is filtered
through a Chamberland filter when the maximum degree of
toxicity has been reached. The term " toxin " is usually applied
for convenience to the filtered (i.e. bacterium-free) culture.
2. Estimation of the Toxin. — The power of the toxin is
estimated by the subcutaneous injection of varying amounts in
a number of guinea-pigs, and the minimum dose which will
produce death is thus obtained. This, of course, varies in
proportion to the weight of the animal, and is expressed accord-
ingly. In the case of diphtheria, in Ehrlich's standard, the
minimum lethal dose — known as M.L.D. — is the smallest amount
which will certainly cause death in a guinea-pig of 250 grms.
within four days. Behring uses the term " normal diphtheria
toxin of simple strength" (DTN1), as indicating a toxin of
which '01 c.c. is the minimum lethal dose under these conditions.
A toxin of which the minimum lethal dose is '02 will be of half
normal strength (DTN>5); and so on. The testing of a toxin
directly is a tedious process, and in actual practice, where many
toxins have to be dealt with, it is found more convenient to test
them by finding how much will be neutralised by a certain
amount of a standard antitoxic serum, viz. an "immunity unit"
(p. 468).
3. Development of Antitoxin. — The earlier experiments on
tetanus and diphtheria were performed on small animals, such
468 IMMUNITY
as guinea-pigs, but afterwards the sheep and the goat were
used, and finally horses. In the case of the small animals it was
found advisable to use in the first stages of the process either a
weak toxin or a powerful toxin modified by certain methods.
Such methods are the addition to the toxin of terchloride of
iodine (Behring and Kitasato), the addition of Gram's iodine
solution in the proportion of one to three (Roux and Vaillard),
and the plan, adopted by Vaillard in the case of tetanus, of
using a series of toxins weakened to varying degrees by being
exposed to different temperatures, viz. 60°, and 55°, and 50° C.
In the case of large animals immunisation is sometimes started
with small doses of unaltered toxin ; and the doses are gradu-
ally increased. The toxin is at first injected into the sub-
cutaneous tissues, later into a vein. Ultimately 300 c.c., or
more, of active diphtheria toxin thus injected may be borne by
a horse, such a degree of resistance being developed after the
treatment has been carried out for two or three months. In all
cases of immunising the general health of the animal ought not
to suffer. If the process is pushed too rapidly the antitoxic
power of the serum may diminish instead of increasing, and a
condition of marasmus may set in and may even lead to the
death of the animal (v. p. 494). (In immunisation of small
animals an indication of their general condition may be obtained
by weighing them from time to time.)
4. Bttwn&ting the Antitoxic Power of, or " standardising," the
Serum. — This is done by testing the effect of various quantities
of the serum of the immunised animal against a certain amount
of toxin. Various standards have been used, of which the two
chief are that of Ehrlich and that of Roux. Ehrlich has
adopted as the immunity unit the amount of antitoxic serum
which will neutralise 100 times the minimum lethal dose of
toxin, serum and toxin being mixed together, diluted up to
4 c.c. and injected subcutaneously into a guinea-pig of 250
grms. weight, the prevention of the death of the animal within
four days being taken as the indication of neutralisation, As a
standard in testing, Ehrlich employs quantities of serum of
known antitoxic power in a dry condition, preserved in a vacuum
in a cool place, and in the absence of light. A thoroughly dry
condition is ensured by having the glass bulb containing the
dried serum connected with another bulb containing anhydrous
phosphoric acid. With such a standard test-serum any newly
prepared serum can readily be compared. A " normal " antitoxic
serum is one of which 1 c.c. contains an immunity unit. 1 c.c.
of a serum, of which '02 c.c. will protect from a hundred times
USE OF ANTITOXIC SERA 469
the lethal dose, will possess 50 immunity units, and 20 c.c. of
this serum 1000 immunity units. Sera have been prepared of
which 1 c.c. has the value of 800 units or even more.
Roux adopts a standard which represents the animal Aveight in
grammes protected by 1 c.c. of serum against the dose of virulent bacilli
lethal to a control guinea-pig in thirty hours, the serum being injected
twelve hours previously. Thus, if "01 c.c. of a serum will protect a
guinea-pig of 500 grins, against the lethal dose, 1 c.c. (1 grm.) will protect
50,000 grms. of guinea-pig, and the value of the serum will be 50,000.
During the process of development of antitoxin a small
quantity of the blood of the animal is withdrawn from time to
time, and the antitoxic power tested in the manner described
above. After a sufficiently high degree of antitoxic power has
been reached the animal is bled under aseptic precautions, and
the serum is allowed to separate in the usual manner. It is then
ready for use, but some weak antiseptic, such as *5 per cent
carbolic acid, is usually added to prevent its decomposing. Other
antitoxic sera are prepared in a corresponding manner. Some
further facts about antitetanic serum are given on p. 384.
Use of Antitoxic Sera. — In all cases the antitoxic serum ought
to be injected as early in the disease as possible, and in large
doses. In the case of diphtheria 1500 immunity units of anti-
toxic serum was the amount first recommended for the treatment
of a bad case, but the advisability of using larger doses has
gradually become more and more evident. Sidney Martin
recommends that as much as 4000 units should be administered
at once, and that if necessary this quantity should be repeated.
A strong serum prepared by Behring contains 3000 units in
5-6 c.c., but even stronger sera may be obtained. Even very
large doses of antitoxic serum are without any harmful effects
beyond the occasional production of urticarial and erythematous
rashes. Where large quantities of serum require to be ad-
ministered, as is always the case with antitetanic serum, injections
must be made at different parts of the body; preferably not
more than 20 c.c. should be injected at one place. The immunity
conferred by injection of antitoxic serum lasts a comparatively
short time, usually a few weeks at longest.
Sera of Animals immunised against Vegetable and Animal
Poisons. — It was found by Ehrlich in the case of the vegetable
toxins, ricin and abrin, and also by Calmette and Fraser in the
case of the snake poisons, that the serum of animals immunised
against these respective substances had a protective effect when
injected- along with them into other animals. Ehrlich found,
for example, that the serum of a mouse which had been highly
470 IMMUNITY
immunised against ricin by feeding as described above, could
protect another mouse against forty times the fatal dose of that
substance. He considered that in the case of the two poisons,
antagonistic substances — " anti-ricin " and " anti-abrin " — were
developed in the blood of the highly-immunised animals. A
corresponding antagonistic body, to which Fraser has given the
name "antivenin," appears in the blood of animals in the process
of immunisation against snake poison.
Theso investigations are specially instructive, as such vegetable
and animal poisons, both as regards their local action and the
general toxic phenomena produced by them, present, as we have
seen, an analogy to various toxins of bacteria.
Nature of Antitoxic Action.— This subject is only part of the
general question with regard to the relation of anti-substances
to their corresponding substances, but it is with regard to anti-
toxic action that most of the work has been done. We have to
consider here two points, viz. (a) the relation of antitoxin to
toxin, and (b) the source of the antitoxin. With regard to the
former subject there has been much diversity of opinion, but
the evidence now available goes to show that the antagonism
between toxin and antitoxin is not a physiological one but that
the two bodies unite in vitro to form a compound inert towards
the living tissues, there being in the toxin molecule an atom
group which has a specific affinity for the antitoxin molecule or
part of it. We shall consider the facts in favour of this view,
and in doing so we must also take into account the anti-sera of
the vegetable toxins, of snake poisons, etc.
When toxin and antitoxin are brought together in vitro it
can be proved that their behaviour towards each other resembles
what is observed in chemical union. Thus it has been found
that a definite period of time elapses before the neutralisation
of the toxin is complete, that neutralisation takes place more
rapidly in strong solutions than in weak, and that it is hastened
by warmth and delayed by cold. C. J. Martin and Cherry, and
also Brodie, have shown, that in the case of diphtheria toxin and
in that of 'an Australian snake poison the toxin molecules will
pass through a colloid membrane (p. 166), whilst those of the
corresponding antitoxin will not. Now if a mixture of
equivalent parts of toxin and antitoxin is freshly prepared and
at once filtered, a certain amount of toxin will pass through, but
the longer such a mixture is allowed to stand before nitration
the less toxin passes, till a time is reached when no toxin is
found in the filtrate. Further, if the portion of fluid which at
this stage has not passed through the filter be injected into an
NATURE OF ANTITOXIC ACTION 471
animal no symptoms take place ; this shows that after a time
neutralisation is complete. Again, in cases when the toxin has
some definite physical effect demonstrable in vitro, e.g. lysis,
agglutination, coagulation, or the prevention of coagulation, its
action can be annulled by the antitoxin ; in such circumstances
manifestly no physiological action of antitoxin through the
medium of the cells of the body can come into play. These
facts are practically conclusive in favour of antitoxin action
depending upon a direct union of the two substances concerned.
The evidence usually brought forward against the direct union of
toxin and antitoxin rests chiefly on certain observations of Calmette,
who found that the antitoxin to a snake venom was more easily destroyed
by heat than the toxin, and stated that when a neutral mixture of the
two was heated at a temperature sufficient to destroy free antivenin, the
toxic properties in part returned. Hence he concluded that the two
bodies existed in an uncombined condition in the mixture. Martin and
Cherry, however, on repeating these experiments, found that the above
result was not obtained if sufficient time for complete combination was
allowed ; but if this precaution was not taken, then the presence of the
free toxin was revealed when the antitoxin was destroyed by heat.
Even, however, if Calmette's results were quite correct, they cannot be
considered to constitute a proof that chemical union does not occur :
they would only prove that the toxin has not been destroyed. If
two complicated chemical compounds of unequal stability are in loose
chemical union, it is quite conceivable that the less stable may be
destroyed (e.g. by heat), whilst the more stable escapes.
Although practically all authorities are now agreed as to the
direct combination of toxin and antitoxin there is still much uncer-
tainty as to the exact nature of this union. Controversy on this
subject may be said to date from the important work of Ehrlich
on the neutralisation of diphtheria toxin. Using an immunity
unit of antitoxin (the equivalent of 100 doses of toxin) he deter-
mined with any example of crude toxin the largest amount of
toxin which could be neutralised completely, so that no symptoms
resulted from an injection of the mixture. This amount he
called the limes null dose, expressed as L0. He then investigated
the effects of adding larger amounts of toxin to the immunity
unit and observed the quantity which was first sufficient to
produce a fatal result, that is, which contained one M.L.D. of
free toxin ; this amount he called the limes todtlich, fatal limit,
expressed as Lt. Now if, as he supposed, the union of toxin and
antitoxin resembled that of a strong acid and base, Lt - L0 ought
to be the equivalent of a minimum lethal dose of the toxin alone.
This, however, was never found to be the case, the difference
being always considerably more than one M.L.D. For example,
in the case of one toxin, M.L.D. = '0165 gr., Lt = 1*26 gr., L0 =
472 IMMUNITY
•9 gr. ; difference = -36 gr., i.e. 21 '9 M.L.D. This, in brief, is
what is known as the "Ehrlich phenomenon," and it has been
explained by him as the result of the presence of toxoids (vide
p. 171), i.e. toxin molecules in which the toxophorous group has
become degenerated. He distinguishes three possible varieties
of such bodies according to the affinity of the haptophorous
group, namely prototoxoid with more powerful affinity than the
toxin molecule, epitoxoid with less powerful affinity, and syntoxoid
with equal affinity. The presence of epitoxoids would manifestly
explain the above phenomenon. The L0 dose would represent
toxin + epitoxoid molecules all united to antitoxin molecules and
the addition of another M.L.D. of toxin would not result in
there being a free fatal dose, but in the added toxin taking the
place of epitoxoid. Several lethal doses would need to be added
before the mixture was sufficient to produce a fatal result ; that is,
Lt - L0 would equal several M.L.D.s. Ehrlich observed another
fact strongly in favour of the existence of toxoids, namely that
in the course of time the toxin might become much weakened,
so that in one case observed the M.L.D. was three times the
original fatal dose, and still the amount of antitoxin necessary to
neutralise it completely was the same as before. Ehrlich also
investigated the effects of partial neutralisation of the L0 amount
of toxin, that is, he added to this amount different fractions of an
immunity unit and estimated the toxicity of the mixture. He
found by this method that the neutralisation of the toxin did
not take place gradually, but as if there were distinct bodies
present with different combining affinities — the graphic repre-
sentation of the mixture not being a curve but a step-stair line.
Thus he distinguished proto-, deutero-, and trito-toxins (with
corresponding toxoids). It will thus be seen that Ehrlich regards
the combination toxin-antitoxin to be a firm one, and that the
neutralisation phenomena are to be explained by the complicated
constitution of the crude toxin.
The chief criticism of Ehrlich's views has come from the
important work of Madsen and Arrhenius. Their main con-
tention is that the toxin-antitoxin combination is not a firm one
but a reversible one, and is governed by the laws of physical
chemistry. For example, in the case of a mixture of ammonia
and boracic acid in solution, there is a constant relation between
the amounts of each of the substances in the free condition and
the amounts in combination, — the combination is reversible, so
that if some of the free ammonia were removed a certain amount
of the combined ammonia would become dissociated to take its
place ; further, if to the mixture, in a state of equilibrium,
MODE OF PRODUCTION OF ANTITOXINS 473
more ammonia or more boracic acid were added, part would
remain free while part would combine. Accordingly, if toxin
and antitoxin behaved in a similar manner an explanation of the
Ehrlich phenomenon would be afforded. Madsen and Arrhenius
have worked out the question in the case of a great many toxins
and find that the graphic representation of neutralisation is in
every case a curve which can be represented by a formula. It
should be noted in connection with this controversy that there
are two questions which may be independent of each other, viz.
(1) does the "toxin" in any particular case represent a single
substance or several ? (2) What is the nature of the combination
of any one constituent substance and its anti-substance — is it
reversible or is it not ? It may be said that it is practically
impossible to explain the facts with regard to diphtheria toxin
on the hypothesis of a single substance even if this should have
its combining and toxic actions equally weakened ; " toxoids " in
Ehrlich's sense must in our opinion be supposed. Then there is
an important fact established by Danysz and by v. Dungern,
namely that the amount of toxin neutralisable by a given amount
of antitoxin is different according as the toxin is added in several
moieties or all at once — in the latter case the amount of toxin
neutralisable is greater. There seems no explanation of this
according to the view of Madsen and Arrhenius as the same state
of equilibrium ought to be reached in the two cases, that is, the
amounts of toxin neutralised should be the same. On the other
hand we have instances of the combination of a substance and its
anti-substance being reversible — the example of a haemolytic
immune-body may be cited (p. 481) — and there is no doubt that
there are varying degrees of firmness of the union. It is quite
evident that if there should be several toxic bodies in a "toxin,"
and that if the union of some of these with antitoxin should be
reversible, the problem, becomes one of extreme complexity.
There has recently been a tendency on the part of some
authorities to consider that the union of toxin-antitoxin does not
correspond to what takes place in ordinary chemical union, but
is a physical interaction of bodies in a colloidal state, the action
being one of the so-called absorption phenomena. The smaller
toxin molecule becomes entangled, as it were, in the larger
antitoxin one, very much as a dye becomes attached to the
structure of a thread. Bordet has long maintained a theory of
this nature and gives reasons for believing that there is no
definite quantitative relationship in the combination of the
molecules of the two substances, different amounts of antitoxin
affecting in varying degree all the molecules of a given amount
474 IMMUNITY
of toxin. A statement on the general question is at present
impossible ; we can only say that combination of the two bodies
does occur ; that sometimes, probably often, the " toxin " con-
tains different toxic bodies with varying affinity ; and that in a
few instances the combination has been proved to be reversible,
but to what extent this is the case remains still to be determined.
The next question to be considered is the source of antitoxin.
The following three possibilities present themselves : (a) antitoxin
may be formed from the toxin, i.e. may be a " modified toxin " ;
(6) antitoxin may be the result of an increased formation of
molecules normally present in the tissues ; (c) antitoxin may be
an entirely new product of the cells of the body. It can now be
stated that antitoxin is not a modified toxin. It has been shown,
for example, that the amount of antitoxin produced by an animal
may be many times greater than the equivalent of toxin injected ;
and further, that when an animal is bled the total amount of
antitoxin in the blood may some time afterwards be greater
than it was immediately after the bleeding, even although no
additional toxin is introduced. This latter circumstance
shows that antitoxin is formed by the cells of the body. If
antitoxin is a product of the cells of the body, we are almost com-
pelled, on theoretical grounds, to conclude that it is not a newly-
manufactured substance, but a normal constituent of the living
cells which is produced in increased quantity. We have, however,
direct evidence of the presence of antitoxin under normal con-
ditions,— the presence of such being sho\vn by its uniting with
toxin and rendering it inert. Normal horse serum, to mention
an example, may have a varying amount of antitoxic action to
the diphtheria poison, ox-bile has a similar action to snake poison,
whilst in the case of other anti-substances — such as agglutinins,
bacteriolysins, hasmolysins, etc. — whose production is governed
by the same laws, numerous examples might be given. It is,
however, rather to the protoplasm of living cells than to the serum
that we must look for the source of antitoxins. In the first place,
we have evidence that in the living body bacterial toxins enter
into combination with, or, as it is often expressed, are fixed by
the tissues — presumably by means of certain combining affinities.
This has been shown by the experiments of Donitz and of
Heymans with tetanus toxin. We have, in such cases, however,
no evidence as to where the toxin is fixed beyond that supplied
by the occurrence of symptoms. Another line of research which
has been followed is to bring emulsions of various organs into
contact with a given toxin and observe whether any of the
toxicity is removed. This was first carried out by Wassermann
CHEMICAL NATURE OF ANTITOXINS 475
and Takaki, who investigated the action of emulsions of the
central nervous system of the susceptible guinea-pig on tetanus
toxin. They found in this way that the nervous system con-
tained bodies which had a neutralising effect on the toxin.
For example, it was shown that 1 c.c. of emulsion of brain and
spinal cord was capable of protecting a mouse against ten times
the fatal dose of toxin. These observations have been confirmed,
though their significance has been variously interpreted. It
would, however, be out of place to discuss at length the opposing
views, and we accordingly simply state the facts ascertained.
We may note, however, that it is not a serious objection that in
certain animals other tissues than that of the central nervous
system can combine with tetanus toxin — this might take place
with or without resulting symptoms ; the important fact is that
in the nervous system certain molecules have an affinity for the
toxin.
It will be seen from what has been stated with regard to the
relation of toxin and antitoxin, that the fixation of toxin by the
tissues leads up theoretically to the possible production of anti-
toxin. In other words, the substance which, when forming part
of the cells, fixes the toxin and thus serves as the means of
poisoning, may act as an antitoxin when free in the blood.
This will be discussed below in connection with Ehrlich's theory
of passive immunity. We may conclude by saying that anti-
toxin is probably represented by molecules normally present in
the cells or (more rarely) in the fluids of the body.
Of the chemical nature of antitoxins we know little. From
their experiments C. J. Martin and Cherry deduce that while
toxins are probably of the nature of albumoses, the antitoxins
probably have a molecule of greater size, and may be allied to
the globulins. Hiss and Atkinson have also come to the con-
clusion that antitoxin belongs to the globulins. They found
that the precipitate with magnesium sulphate from anti-
diphtheria serum contained practically all the antitoxin, and that
any substance obtained which had an antitoxic value gave all
the reactions of a globulin. They also found that the per-
centage amount of globulin precipitated from the serum of the
horse increased after it was treated in the usual way for the
production of antitoxin. Such a supposed difference in the
sizes of the molecules might explain the fact, observed by
Fraser and also by C. J. Martin, that antitoxin is much more
slowly absorbed when introduced subcutaneously than is the case
with toxin.
Antitoxin, when present in the serum, leaves the body by
476 IMMUNITY
the various secretions, and in these it has been found, though
in much less concentration than in the blood. It is present in
the milk, and a certain degree of immunity can be conferred on
animals by feeding them with such milk, as has been shown by
Ehrlich, Klemperer, and others. Klemperer also found traces of
antitoxin in the yolk of eggs of hens whose serum contained
antitoxin. Bulloch also found in the case of hremolytic sera
(vide infra) that the anti-substance (" immune -body ") is trans-
mitted from the mother to the offspring.
Antibacterial Serum. — The stages in the preparation of
antibacterial sera correspond to those in the case of antitoxic
sera, but living, or, in the early stages, dead cultures are used
instead of toxin separated by nitration, and in order to
obtain a serum of high antibacterial power a very virulent
culture in large doses must be ultimately tolerated by the
animal. For this purpose a fairly virulent culture is obtained
fresh from a case of the particular disease, and its virulence
may be further increased by the method of passage. This
method of obtaining a high degree of immunity against the
microbe is specially applicable in the case of those organisms
which invade the tissues and multiply to a great extent within
the body, and of which the toxic effects, though always existent,
are proportionately small in relation to the number of organisms
present. The method has been applied in the case of the
typhoid and cholera organisms, the bacillus of bubonic plague,
the bacillus coli communis, the pneumococcus, streptococcus
(Marmorek), and many others. In fact, it seems capable of very
general application.
The important result obtained by such experiments is, that if
an animal be highly immunised by the method mentioned, the
development of the immunity is accompanied by the appearance
in the blood of protective substances, which can be transferred to
another animal. The law enunciated by Behring regarding
immunity against toxins thus holds good in the case of the
living organisms, as was first shown by Pfeiffer. The latter
found, for example, that in the case of the cholera organism, so
high a degree of immunity could be produced in the guinea-pig,
that *002 c.c. of its serum would protect another guinea-pig
against ten times the lethal dose of the organisms, when in-
jected along with them. Here again is presented the remark-
able potency of the antagonising substances in the serum,
which in this case lead to the destruction of the corresponding
microbe.
The anti-streptococcic serum of Marraorek may be briefly described, as
PROPERTIES OF ANTIBACTERIAL SERUM 477
it has come into extensive practical use. This observer found that he
could intensify the virulence of a streptococcus by growing it alternately
in the peritoneal cavity of a guinea-pig and in a mixture of human blood
serum and bouillon (vide p. 41). The virulence became so enormously
increased by this method, that when only one or two organisms were
introduced into the tissues of a rabbit a rapidly fatal septicaemia was
produced. Streptococci of this high degree of virulence were used first
by subcutaneous, afterwards by intravenous injection, to develop a high
degree of resistance in the horse. Injections were continued over a con-
siderable period of time, and the protective power of the serum was
tested by mixing it with a certain dose of the virulent organisms, and
then injecting into a rabbit. The serum of a horse highly immunised in
this way constitutes the anti-streptococcic serum which has been
extensively used with success in many cases of streptococcic invasion in
the human subject. Marmorek, however, found that this serum had
little antitoxic power, that is, could only protect from a comparatively
small dose of toxin obtained by filtration of cultures.
Anti- typhoid, anti-cholera,1 anti-pneumococcic, anti-plague,
and other sera are all prepared in an analogous manner.
Properties of Antibacterial Serum. — We have here to
consider the three main actions mentioned above, viz. (a)
bactericidal and lysogenic action, (6) opsonic action, and (c)
agglutinative action.
(a) Bactericidal and Lysogenic Action. — Pfeiffer found that
if certain organisms, e.g. the cholera spirillum, were injected
into the peritoneal cavity of a guinea-pig highly immunised
against these organisms they lost their motility almost immedi-
ately, gradually became granular, swollen, and then disappeared
in the fluid — these changes constitute " Pfeiffer's phenomenon."
Further, he found that the same phenomenon was witnessed if
a minute quantity of the anti-serum was added to a certain
quantity of the corresponding organisms, and the mixture
injected into the peritoneal cavity of a non-treated animal.
Pfeiffer found that the serum of convalescent cholera patients
gave the same reaction as that of immunised animals. He
obtained the same reaction also in the case of the typhoid
bacillus and other organisms. From his observations he con-
cluded that the reaction was specific, and could be used as a
means of distinguishing organisms wilich resemble one another.
He accordingly considered that a specific substance was developed
in the process of immunisation and that this was rendered
actively bactericidal by the aid of the living cells of the body.
It was subsequently shown, however, by Metchnikoff and by
Bordet that lysogenesis might occur outside the body by the
1 A true antitoxic cholera serum has been prepared by Metchnikoff, E.
Roux, and Taurelli-Salimbeni.
478 IMMUNITY
addition of fresh peritoneal fluid or normal serum to the heated
immune-serum. PfeifFer also found that an anti-serum heated to
70° C. for an hour produced the reaction when injected with the
corresponding organisms into the peritoneum of a fresh animal.
The outcome of these and subsequent researches is to show that
when an animal is immunised against a bacterium a substance
appears in its serum with combining affinity for that particular
organism. This substance which is generally known as the
immune-body, amboceptor (Ehrlich), or substance sensibilisatrice
(Bordet) is comparatively stable, resisting usually a temperature
of 70° C for an hour. It cannot produce the destructive effect
alone but requires the addition of a substance normally present
in the serum, which is spoken of under various names — com-
plement (Ehrlich), alexine or cytase (French writers). The com-
plement is relatively unstable, being rapidly destroyed by a
temperature of 60° C., and it is not increased in amount during
the process of immunisation. Though ferment-like in its in-
stability, it differs from a ferment in being fixed or used up in
definite quantities.
The phenomenon of lysogenesis is, however, only seen in the
case of certain organisms when an animal is highly immunised
against them ; the typhoid and cholera group are outstanding
examples. It is also to be noted that it sometimes is seen in the
case of a normal serum (vide Natural Immunity). In other oases
the bactericidal effect of a serum may occur without the rapicLdis-
solution characteristic of lysogenesis though other structural
changes may be produced. In still other cases a bactericidal
effect may be wanting ; nevertheless it may be shown that an
immune-body is developed by the process of immunisation. This
may be done by observing the increased amount of complement
which is fixed through the medium of the anti-serum (immune-
body), sensitised red corpuscles being used as the test for the
presence of free complement. The following scheme will show
the mode of experiment, which is carried out in a series of
small test-tubes : —
(1) Bacteria + immune-body (anti-serum heated at 55° C.) + complement.
(The same amount of bacteria and immune-body in each tube, varying
amounts of complement in different tubes.)
(2) Incubate at 37° C. for one and a half hours.
(3) Add to each tube red corpuscles treated with the corresponding
immune-body, and incubate for another hour.
The " immune-body " is in each case the anti-serum deprived
of complement (by heating at 55° C.), obtained from animals
injected with the bacteria and red corpuscles respectively. The
ILEMOLYTIC AND OTHER SERA 479
control is got by substituting in another experiment the same
amount of heated normal serum for the anti-serum. If there is
free complement left there will be corresponding lysis of the red
corpuscles ; if the complement has all been fixed there will be no
lysis. To take an example from Muir's experiments,— it was found
that an emulsion of the bacteria alone took up "03 c.c. of guinea-
pig's complement, whilst the same amount of bacteria treated
with immune-body took up '13 c.c. The all-important action of
the immune-body is thus to bring an increased amount of com-
plement into union with bacteria ; whether death of the bacteria
will result or not will depend ultimately on their sensitiveness
to the action of the particular complement.
It is to be noted that with a bactericidal serum there is
an optimum amount of immune-body which gives the greatest
bactericidal effect. If this amount be exceeded the bactericidal
action becomes diminished and may be practically annulled.
This result, which is generally known as the Neisser-Wechsberg
phenomenon, has been the subject of much controversy, and
cannot yet be said to be satisfactorily explained. It would
accordingly be out of place to discuss here the different views
with regard to it. (Regarding some theoretical considerations
as to the therapeutic applications of antibacterial sera, vide
p. 489.)
The laws of lysogenesis are, however, not peculiar to the
case of solution of bacteria by the fluids of the body, but, as has
been shown within the last few years, hold also in the case of
other organised substances, red corpuscles, leucocytes, etc., when
these are introduced into the tissues of an animal as in a process
of immunisation. Of such sera the hsemolytic have been most
fully studied, and owing to the delicacy of the reaction and the
ease with which it can be observed, have been the means of
throwing much light on the process of lysogenesis, and thus on
one part of the subject of immunity. A short account of their
properties may now be given.
Hcemolytic and other Sera. — It has been known for some time
that in some instances the blood serum of one animal has, in
certain degree, the power of dissolving the red corpuscles of
another animal of different species ; in other instances, how-
ever, this property cannot be detected. Bordet showed that
if one animal were treated with repeated injections of the
corpuscles of another of different species, the serum of the former
acquired a marked haemolytic property towards the corpuscles of
the latter, the property being demonstrated when the serum is
added to the corpuscles. Bordet also found that the hsemolytic
480 IMMUNITY
property disappeared when the haeraolytic serum was heated at
55° C., but, as in the case of a bacteriolytic serum, was regained
on the subsequent addition of some serum from a fresh (i.e. non-
treated) animal. These observations have been fully confirmed
and greatly extended. Ehrlich and Morgenroth analysed the
phenomena in question, and showed that the specially developed
and heat-resisting substance, "immune-body," entered into com-
bination with the red corpuscles at a comparatively low
temperature, namely at 0° C. ; whereas complement does not
combine at this temperature. In this way a method is supplied
by which the immune-body can be removed from a hsemolytic
serum while the complement is left. They came to the con-
clusion that immune - body combined with the complement
though the combination was less firm and only occurred
at a higher temperature — best about 37° C. They therefore
consider that the immune-body acts as a sort of connecting link
between the red corpuscle and the complement, hence the term
" amboceptor " which Ehrlich afterwards applied. It may be
stated, however, that the direct union of complement and
immune-body has not been conclusively demonstrated. Bordet,
on the other hand, holds that the immune-body acts merely as
a sensitising agent — hence the term substance sensibilisatrice —
and allows the ferment-like complement to act. It is quite
evident from his writings, however, that he does not mean, as
is often assumed, that the immune-body causes some lesion in
the corpuscle which allows the complement to act, but simply
that it produces in the molecules (receptors) of the red corpuscles
an avidity for complement. All that we can say definitely at
present is that the combination of receptor + immune-body takes
up complement in firm union while neither does so alone ;
whether the immune-body acts as a link between the two or not
must be left an open question. Even after the corpuscles are
laked with water the receptors are not destroyed: Muir and
Ferguson have shown that they can still take up immune-
body and, through its medium, complement, just as the intact
corpuscles do. Ehrlich and Morgenroth showed that in some
cases the red corpuscles can take up much more immune-body
than is necessary for their lysis, and Muir found in one case
studied, that each further dose of immune-body led to the fixation
of more complement, so that as many as ten times the haemolytic
dose of complement might thus be used up. It is a matter of
considerable importance that the union of immune-body and red
corpuscles can be shown to be a reversible action. If, as was
found by Morgenroth and Muir independently, corpuscles treated
ELEMOLYTIC AND OTHER SERA 481
with several doses of immune-body and then repeatedly washed
in salt solution be mixed with untreated corpuscles and allowed
to remain for an hour, then sufficient immune-body will pass
from the former to the latter, so that all become lysed on
the addition of sufficient complement. The combination of
complement, on the other hand, is usually of very firm nature.
It has been a disputed point whether there are several distinct
complements in a normal serum with different relations to
different immune-bodies, for which Ehrlich and his co-workers
have brought forward a large amount of evidence, or whether,
as Bordet holds, there is a single complement which may, how-
ever, show slight variations in behaviour towards different
immune-bodies. There is at least no doubt that all the com-
plement molecules in a serum are not the same. Workers of
the French school also hold that complement does not exist
in the free condition in the blood, but is liberated from the
leucocytes when the blood is shed ; though this cannot be held
as proved, there is evidence that the amount of free complement
increases after the blood is shed and some time later gradually
diminishes.
The hsemolytic action of a normal serum can be shown in
many cases to be of the same nature as that of an immune-
serum, that is, complement and the homologue of an immune-
body can be distinguished. For example, the guinea-pig's serum
is hsemolytic to the ox's corpuscles ; if a portion of serum be
heated at 55° C. the complement will be destroyed ; if another
portion be treated with ox's corpuscles at 0° C., the natural
immune-body will be removed and only complement will be left.
Neither portion is in itself hsemolytic, but this property becomes
manifest again when the two portions are mixed. Haemolytic
sera are of great service in the study of the question of specificity.
Each is specific in the sense already explained (p. 466), but the
serum developed against the corpuscles of an animal may have
some action on those of an allied species, that is, some receptors
are common to the two species. This fact can be readily shown
by the usual absorption tests, for example, in the case of an
anti-ox serum tested on sheep's corpuscles. A close analogy
holds to what has been established in the case of agglutinins.
It is further of great interest to note that by the injection of red
corpuscles into an animal its serum not only becomes haemolytic,
but in many cases when heated at 55° C. possesses also agglu-
tinating and opsonic properties towards the red corpuscles used.
And further, it would appear that in some cases at least the
immune-body, haBmagglutinin, and hsemopsonin are distinct
31
482 IMMUNITY
substances. These facts abundantly show how close an analogy
obtains between anti-bacterial and hsemolytic sera, and how
important a bearing haemolytic studies have on the questions of
immunity in general.
In addition to hsemolytic sera, anti-sera have been obtained
by the injection of leucocytes, spermatozoa, ciliated epithelium,
liver cells, nervous tissue, etc. The laws governing the pro-
duction and properties of these are identical, that is, each serum
exhibits a specifk-4)roperty towards the body used in its produc-
tion— i.e. dissolves leucocytes, immobilises spermatozoa, etc.
The specificity is, however, not so marked as in the case of
sera produced against red blood corpuscles ; thus a serum pro-
duced against tissue cells is often hsemolytic ; this is probably
due to various cells of the body having the same receptors.
Here again when the anti-serum produces -no destructive effect
on the corresponding cells, the presence of an immune-body may
be demonstrated by the increased amount of complement which
is taken up through its medium. It may also be mentioned
that each anti-serum usually exhibits toxic properties towards
the animal whose cells have been used in the injections, e.g. a
hsemolytic serum may produce a fatal result, with signs of
extensive blood destruction, haemoglobinuria, etc., i.e. it is
haemotoxic for the particular animal ; a serum prepared by
injection of liver cells has been found to produce on injection
necrotic changes in the liver in the species of animal whose liver
cells were used. These are mentioned as examples of a very
large group of specific activities.
With regard to the sites of origin of immune-bodies our
information is still very deficient. Pfeiffer and Marx brought
forward evidence in the case of typhoid, and Wassermann in the
case of cholera, that the immune-bodies are chiefly formed in
the spleen, lymphatic glands, and bone-marrow. According to
certain workers of the French school, the chief source of anti-
substances acting on cells such as red blood corpuscles is the large
mononucleated leucocytes, whilst those acting on bacteria are
chiefly derived from the polymorpho-nuclear leucocytes (vide
p. 495). Another view is that immune-bodies are chiefly formed
by the large mononucleated leucocytes, whilst complements are
products of the polymorphs. That these cells are concerned in
the production of antagonistic and protective substances is
almost certain, though another possible source of wide extent,
viz. the endothelium of the vascular system, has been largely
overlooked. As yet, definite statements cannot be made on this
point.
OPSONIC ACTION 483
Methods of Hsemolytic Tests. — A hsemolytic serum is usually pre-
pared by injecting the red corpuscles of an animal into the peritoneum
of an animal of different species — the corpuscles of the ox are most
frequently used, and the rabbit is the most suitable animal for injection.
The corpuscles ought to be completely freed of serum by repeatedly
washing them in sterile salt solution and centrifugalising. An injection
of the corpuscles of 5 c.c. of ox's blood followed by two injections, each
of 10 c.c., at intervals of ten days, will usually give an active serum.
The animal should be killed by bleeding it, aseptically as far as possible,
seven to ten days after the last injection ; the serum which separates
may be collected in suitable lengths of quill glass-tubing drawn out at
the ends, which are afterwards sealed in the flame. To ensure sterility
when the serum is to be kept some time, it is advisable to heat it for an
hour at 55° C. on three successive days ; we have always found that
serum treated in this way remains sterile. It is of course devoid of
complement. The test amount of corpuscles is usually 1 c.c. of a 5 per
cent suspension of corpuscles in '8 per cent sodium chloride solution :
that is, the corpuscles of 5 c.c. blood are completely freed of serum by
repeatedly washing in salt solution, and then salt solution is added to
make up 100 c.c. In any investigation it is necessary to obtain the
minimum haemolytic dose (M.H.D.) of the immune-body and of the
complement to be used. (It is to be noted that as complement does not
increase during immunisation, the hsemolytic dose of the fresh serum
will come far short of representing the amount of immune-body present.)
In testing the dose of immune-body, the fresh serum to be used as com-
plement must be devoid of hsemolytic action (in the present instance
rabbit's serum will be found suitable) and more than sufficient to produce
lysis with immune-body is added to each of a series of tubes. Varying
amounts of immune-body are added to the tubes, the contents are
shaken, made up to 1*5 c.c. and incubated for two hours. The amount
of lysis is then noted and the tubes are placed in a cool chamber till
next morning, when a final reading is takeri. The smallest amount of
immune-body which gives complete lysis is of course the M.H.D. :
sometimes this may be as low as '001 c.c. for the test amount of
corpuscles. When further observations are to be continued on the same
day, the reading after incubation must be taken as the working
standard. To estimate the M.H.D. of complement, proceed in a
corresponding manner ; to each of a series of tubes add several doses of
immune-body, and then to the several tubes different amounts of com-
plement. The activity of a serum as complement varies considerably,
and each sample must be separately tested. The above will serve as an
indication of the fundamental methods ; for further details special
papers on the subject must be consulted.
(b) Opsonic Action. — The presence of a substance in an
immune-serum which makes the corresponding organism sensitive
to phagocytosis was first demonstrated by Denys and Leclef in
1895, in the case of an anti-streptococcal serum. They also
showed that the serum produced this effect by acting on the
organism, not on the leucocytes. It is, however, chiefly to the
researches of Wright and his co-workers that this subject has
come into special prominence. Wright and Douglas in their
first paper showed that the phagocytosis of staphylococci by
484 IMMUNITY
leucocytes depended on a body in the normal serum which
became fixed to the cocci and made them a prey to the
phagocytes. To this they gave the name of " opsonin " (vide
pp. 194, 261). There is no phagocytosis of cocci by leucocytes
washed in salt solution ; normal serum heated to 55° C. is also
without effect in inducing this phenomenon. They could not
demonstrate any effect of the opsonin on the leucocytes. On the
other hand, if bacteria be exposed to the fresh serum, and they
be freed from the excess of serum and then exposed to phago-
cytes also washed free from serum, they will be readily taken
up by the cells. It has been abundantly shown that the opsonic
action of the serum is increased by the process of immumsa/EToh
against an organism, and the opsonic index represents the degree
of immunity in one of its aspects as already explained (p. 111).
The matter has, however, become complicated by the circum-
stance that in an immune-serum an opsonin may still be present
after the serum is heated at 55° C., as has been shown by Dean
and others. Some observers consider that this opsonin is simply
an immune-body, but the results brought forward by others would
point to their being different substances, at least in certain cases,
notably in haemolytic sera. We are, however, probably safe in
saying that the thermostable opsonin of an immune-serum is a
true anti-substance\ possessing the specific characters of anti-
substances in general and comparable in this respect and in its
mode of production with an agglutinin. Muir and Martin have,
however, found that the thermolabile opsonin of a normal serum
has different characters. For example, when a normal serum is
tested on a particular bacterium, the opsonic effect on that
bacterium may be removed by treating the serum with other
bacteria ; in other words, the thermolabile opsonin of normal
serum does not possess the specific character of the opsonin
developed in the process of immunisation./ They have also
found that various substances or combinations of substances
which act as "complement -absorbers" also remove the opsonic
property from a normal serum, while they have no effect on an
immune-opsonin. According to this view the opsonic effect of
the unheated serum of an actively immunised animal or person
would represent the sum of the effects of the two kinds of
opsonin.
Further study will be necessary before the exact relationships
of these substances are fully understood, and other questions with
regard to them have as yet scarcely been touched upon.
Increased phagocytic action had long been known by the work
of Metchnikoff to be associated with the development of active
AGGLUTINATION 485
immunity and the theory of stimulation of leucocytes was
supported by many. The work on opsonins has caused a swing
of the pendulum in the other direction, and points to the
development of anti-substances in the serum as the all-important
factor. It remains to be determined to what extent the opsonic
and directly bactericidal properties taken together will explain
the phenomena of natural and acquired immunity.
(c) Agglutination. — Charrin and Koger in 1889 observed
that when the bacillus pyocyaneus was grown in the serum of
an animal immunised against this organism, the growth formed
a deposit at the foot of the vessel ; whereas a growth in normal
serum produced a uniform turbidity. Gruber and Durham, in
investigating Pfeiffer's reaction, found that when a small quantity
of an anti-serum is added to an emulsion of the corresponding
bacterium, the organisms become agglutinated into clumps,
this phenomenon depending upon the presence of definite bodies
in the serum called agglutinins.
It had been already found that the serum of convalescents
from typhoid fever could protect animals to a certain extent
against typhoid fever, and, in view of the facts experimentally
established, it appeared a natural proceeding to enquire whether
such serum possessed an agglutinative action and at what stage
of the disease it appeared. The result, obtained independ-
ently by Griinbaum and Widal, but first published by the latter,
was to show that the serum possessed this specific action shortly
after infection had taken place; in other words, the develop-
ment of this variety of anti- substance can be demonstrated at
an early stage of the disease. Agglutination is also observed in
the case of cholera, Malta fever, bacterial dysentery, glanders,
plague, infection by Gartner's bacillus, b. coli, etc. Furthermore
the phenomenon is not peculiar to bacteria ; it is seen, for
example, when an animal is injected with the red corpuscles of
another species, haemagglutinins appearing in the serum, which
have a corresponding specificity.
The physical changes on which agglutination depends cannot
as yet be said to be fully understood. Gruber and Durham
considered that the agglutinin produced a change in the envelope
of the bacterium, causing it to swell up and become viscous, and
the facts first established appeared to be in favour of this view.
On the other hand, this is not the full explanation, as it has
been shown by Nicolle and by Kruse that if an old bacterial
culture be filtered through porcelain, the addition of some of
the corresponding anti-serum produces a sort of granular
precipitate in it, and that when, as in the agglutination of bacteria,
486 IMMUNITY
minute inorganic particles are added to the mixture they become
aggregated into clumps. The phenomenon would thus appear
to be the result of the interaction of the agglutinin and some
substance in the bacterial cell which is known as the agglutin-
able substance or as the agglutinogen, seeing that it is probably
the element in the bacterial structure which in the tissues of the
animal or person leads to the development of the agglutinin.
Joos has found in the case of the typhoid bacillus that
there are two agglutinable substances which differ in their
resistance to heat — a- and ^-agglutinogen, and that they give rise
to corresponding agglutinins. Further, as the result of a com-
parative study of the agglutinins of a motile and a non-motile
variety of the hog cholera bacillus Theobald Smith has come
to the conclusion that there is an agglutinin which is produced
by and acts on the flagella and another which is similarly related
to the bacterial bodies. The former acts in very much higher
dilutions than the latter, and this is regarded as an explanation
of the fact that in the case of non-motile organisms the
agglutinating serum acts only in proportionately high concentra-
tion as compared with the case of most motile forms. Another
factor necessary for the phenomenon of agglutination is a proper
salt content. Bordet showed that if the clumps of agglutinated
bacteria are freed from salt by washing in distilled water they
become resolved, and that on the addition of some sodium
chloride they are formed again, and Joos has also brought
forward striking confirmatory evidence as to the necessity for
the presence of salts. It is thus probable that in the pheno-
menon of agglutination as ordinarily understood more than one
factor is concerned, and it is possible that in part it may depend
on some altered molecular relationship of the bacteria to the
surrounding fluid analogous to altered surface tension. t? }
As stated above, the agglutinins are usually placed in the
second order of anti-substances, and are regarded as possessing
a combining group and an active or agglutinating group. The
constitution would thus be analogous to that of a toxin, and in
conformity with this view Eisenberg and Volk consider that the
agglutinating group may be destroyed while the combining
group remains, the result being an agglutinoid. The evidence
for this lies in the fact that when an agglutinating serum is
heated to a certain temperature, not only does it lose its
agglutinating action but when the bacteria are treated with
such a serum their agglutination by active serum is interfered
with, a sort of plugging up of the combining molecules having
apparently taken place. Other facts have, however, been
AGGLUTINATION 487
brought forward in opposition to this view, and the existence of
agglutinoids cannot be said to be completely proved. Like
immune-bodies agglutinins are not destroyed at 55° C. (a
temperature sufficient to annul bactericidal action), and the
question arises as to the relation of the two bodies ; discussion
has also taken place as to how far agglutination is an indication
of immunity. It may be said that in the case of certain sera
investigated it has been shown that the immune-body and the
agglutinin are separate substances, and that the agglutinative
power does not vary pari passu with the degree of immunity —
a serum may be strongly agglutinative and feebly bactericidal
and vice versa. But while probably as a rule the two substances
are distinct, it would not be justifiable to say this is always the
case — that is, that an immune-body never has an agglutinating
action. And while the agglutinative power cannot in itself be
taken as the measure of the degree of immunity, agglutinins and
immune-bodies are the products of corresponding reactive pro-
cesses, and their formation is governed by corresponding laws.
Agglutinins become fixed in definite proportion by the receptors
of the bacteria ; that is, the agglutinin becomes used up in the
process of agglutination, and it has been shown that bacteria
may take up many times the amount necessary to their
agglutination — a corresponding fact to what has been established
with regard to immune -bodies of hasmolytic sera. The
agglutinins are specific in the sense which has been explained
above (p. 466). It can be shown by the method of absorption
that in an agglutinating serum there may be several agglutinins
with different combining groups, some of which may be taken up
by organisms allied to that which has given rise to the anti-
serum. Whether or not the combination of an agglutinin with
the bacterial receptors is a reversible action must be left an open
question.
Besides those stated above, other phenomena have been
observed in the interaction of anti-sera and the corresponding
bacteria. For example, it has been shown that when certain
bacteria — e.g. the typhoid bacillus, b. coli, and b. proteus — are
grown in bouillon containing a small proportion of the homo-
logous serum, their morphological characters may be altered,
growth taking place in the form of threads or chains which are
not observed in ordinary conditions. In other instances a serum
may inhibit some of the vital functions of the corresponding
bacterium.
Precipitins. — This subject does not strictly belong to bacteriology,
but the general phenomena are so closely allied to those just described,
488 IMMUNITY
especially to agglutination, that some reference may be made to it.
When the serum of an animal is injected in repeated doses into another
animal of different species, after the type of an immunisation, there
appears in the serum of the animal treatea a substance called precipitin,
which causes a cloudiness or precipitate when added to the serum used.
This precipitate results from the union of the precipitin in the anti-serum
with a body in the homologous serum, the latter being known as the
precipitinogen. (In the case of rabbits doses of 3 to 4 c.c. of the serum
may be injected intraperitoneally at intervals of four to five days, a
precipitin usually appearing at the end of about three weeks.) The
reaction, which is a very delicate one, is conveniently observed by adding
a given amount of the anti-serum, say '05 c.c., to varying amounts of
the homologous serum '1, *01, etc. c.c., in a series of small test tubes,
the volume being then made up with salt solution to 1 c.c. In this way
a definite reaction may be observed with '001 c.c. of the serum or even
less. The reaction is specific in the sense explained above. It is always
most marked towards the serum of the species used in the immunisation ;
but while this is so, there may also be a slight reaction .towards animals of
allied species. An anti-human serum, for example, gives the maximum
reaction with human serum, but also a slight reaction with the serum
of monkeys, especially of anthropoid apes ; it, however, gives no reaction
with the serum of other animals. The precipitin test has thus come to
be employed as a means of differentiating human from other bloods.
Another interesting phenomenon is what is known as the "deviation of
complement," which is produced by the combination of the two sub-
stances in the serum and anti-serum respectively. If mixtures be made
according to the above method, and then a small quantity of complement,
say fresh guinea-pig serum, be added, it will be found that the comple-
ment becomes absorbed, as may be shown by subsequently adding a test
amount of sensitised red blood corpuscles. This deviation phenomenon
is even a more delicate reaction than the precipitin test, it being often
possible to demonstrate by its use from a tenth to a hundredth of the
smallest amount of serum which will give a perceptible precipitate ; it
also is specific within the same limits.1
Therapeutic Effects of Anti-Sera. — As will have been
gathered, the chief human diseases treated by anti-sera are
diphtheria, tetanus, streptococcus infection, pneumonia, plague,
and snake bite. Of the results of such treatment most is known
in the case of diphtheria. Here a very great diminution in the
mortality has resulted. The diphtheria antitoxin came into
general use about October 1894, and the statistics published by
Behring towards the end of 1895 indicated results which have
since been confirmed. In the Berlin Hospitals the average
mortality for the years 1891-93 was 36'1 per cent, in 1894 it
was 21*1 per cent, and in January- July 1895, 14*9 per cent. The
objection that in some epidemics a very mild type of disease
prevails is met by the fact that similar diminutions of mortality
1 For an account of precipitins, vide Nuttall, "Blood Immunity and
Relationships," Cambridge 1904 ; and of complement deviation, Muir and
Martin, Journ. of Hyg. vi., 1906, p. 265.
THERAPEUTIC EFFECTS OF ANTI-SERA 489
have occurred all over the world. Loddo collected the results
of 7000 cases in Europe, America, Australia, and Japan, in
which the mortality was 20 per cent as compared with a former
mortality in the same hospitals of 44 per cent. It has also been
observed that if during an epidemic the supply of serum fails,
the mortality at once rises ; and in two instances recorded it
was doubled. It must here be remembered that from the
spread of bacteriological knowledge the diagnosis of diphtheria
is now much more accurate than formerly. Another effect of
the antitoxic treatment has been that when tracheotomy is
necessary the percentage of recoveries is now much higher, being
73 per cent instead of 27 per cent in a group of cases collected
by the American Pediatric Society. In the London fever
hospitals since 1894 the recoveries after tracheotomy have been
5 6 '4 as compared with 32'1 per cent previous to the intro-
duction of antitoxin. One of the most striking results obtained
in the same hospitals is a reduction of the death-rate in post-
scarlatinal diphtheria from 50 per cent to between 4 per cent
and 5 per cent. As the disease here occurs while the patient is
under observation the treatment is nearly always begun on the
first day. It is a matter of prime importance that the treat-
ment should be commenced whenever the disease is recognised.
Behring showed that in cases treated on the first and second
days of the disease the mortality was only 7*3 per cent, and this
has been generally confirmed, whilst after the fifth day it was of
little service to apply the treatment. In order to obtain such
results it cannot be too strongly insisted on that attention
should be given to tho dosage. When bad results are obtained
it may be strongly suspected that this precaution has not been
observed. In the treatment of acute^ tetanus by the antitoxin
the improvement in results has no? been marked, but some
chronic cases have been benefited, and as already stated (p. 386)
better results are obtained in acute cases if intravenous in-
jection be practised. In the case of Yersin's anti-plague serum,
though benefit has appeared to follow its use, experience
with its effects has been too limited to enable a judgment
to be formed. The same may be said to be true of the anti-
streptococcic and anti-pneumonic sera, though in the case of the
first mentioned numerous cases of apparently successful result
have been recorded. With regard to anti-venin, Lamb has shown
that, if a cobra with full glands bites a man, many times the
minimal lethal dose are probably injected. In cases of slight
bite, however, benefit may accrue from the use of the anti-
serum.
490 IMMUNITY
As has been shown above, antibacterial sera require for their
complete action a sufficiency of complements, and as these
diminish in amount when a serum is kept, the unsatisfactory
results with this class of sera may be due to a deficiency of
complement. Or it may be as Ehrlich has suggested, that the
complement naturally existing in human serum does not suit
the immune-body in the anti-serum — that is, is not taken up
through the medium of the latter and brought into combination
with the bacterium. And there is still the further possibility
that even though the complement should be taken up, the
zymotoxic group of the latter is not sufficiently active towards
the bacterium to effect its death. In both cases it will appear
that an extracellular bactericidal action cannot be produced by
the particular immune-body in association with the complement
of the animal in question. There is no doubt that this question
of complements is one of importance, and that both combining
affinity and toxic action of complements must be considered in
each case.
Theories as to Acquired Immunity.
The advances made within recent years in our knowledge
regarding artificial immunity, and the methods by which it may
be produced, have demonstrated the insufficiency of various
theories which had been propounded. Only a short reference
need be made to these. The theory of exhaustion, with which
Pasteur's name is associated, supposed that in the body of the
living animal there are substances necessary for the existence of
a particular organism, which become used up during the sojourn
of that organism in the tissues ; this pabulum being exhausted
the organisms die out. Such a supposition is, of course, quite
disproved by the facts of passive immunity. According to the
theory of retention the bacteria within the body were considered
to produce substances which are inimical to their growth, so that
they die out, just as they do in a test-tube culture before the
medium is really exhausted. Such a theory only survives now
in the view that antitoxins are modified toxins, the evidence
against which has already been discussed (p. 470). There then
came the humoral theory and the theory of phagocytosis, but
neither of these is tenable in its pure form, and the distinction
between them need not be maintained. For, on the one hand,
any substance with specific property in the serum must be the
product of cellular activity, and on the other hand, the facts
with regard to passive immunity go far beyond the ingestive
and digestive properties of phagocytes, though these cells may
EHRLICH'S SIDE-CHAIN THEORY 491
be in part the source of important bodies in the serum. At the
present time interest centres around two theories, viz. Ehrlich's
side-chain theory and Metchnikoff 's phagocytic theory as further
developed. These will now be discussed, and it may be noted
that the ground covered by each is not coextensive. For the
former deals chiefly with the production of anti-substances and
its biological significance, the latter deals with the defensive
properties of cells, either directly by their phagocytic activity
or indirectly by substances produced by them after the manner
of digestive ferments. It will be seen, however, that each has
a normal process as its basis, viz. that of nutrition.
1. Ehrlich's Side-Chain Theory. — This may be said to be an
application of his views regarding the nourishment of proto-
plasm. A molecule of protoplasm (in the general sense) may be
regarded as composed of a central atom group or executive
centre (Leistungskern) with a large number of side- chains
(Seitenketten), i.e. atom groups with combining affinity for
food-stuffs. It is by means of these latter that the living
molecule is increased in the process of nutrition, and hence
the name receptors given by Ehrlich is on the whole preferable.
These receptors are of three chief kinds corresponding to the
classes of anti-substances described (p. 466) ; the first has a
single unsatisfied combining group and fixes molecules of
simpler constitution — receptor of the first order : the second
has a combining group for the food molecule, and another
active or zymotoxic group, which leads to some physical change
in it — receptor of the second order ; the third has two com-
bining groups, one for the food molecule and another which
fixes a ferment in the fluid medium around — receptor of the
third order or amboceptor. These latter receptors come into
action in the case of larger food molecules which require to be
broken up by ferment -action for the purposes of the cell
economy. In considering the application of this idea to the
facts of passive immunity, it must be kept in view that all the
substances to which anti-substances have been obtained are, like
proteids, of unknown but undoubtedly of very complex chemical
constitution, and that in apparently every case the anti-substance
enters into combination with its corresponding substance. The
dual constitution of toxins and kindred substances, as already
described (p. 170), is also of importance in this connection.
Now, to take the case of toxins, when these are introduced into
the system they are fixed, like food-stuffs, by their haptophorous
groups to the receptors of the cell protoplasm, but are unsuitable
for assimilation. If they are in sufficiently large amount the
492 IMMUNITY
toxophorous part of the toxin molecule produces that disturb-
ance of the protoplasm which is shown by symptoms of
poisoning. If, however, they are in smaller dose, as in the
early stages of immunisation, fixation to the protoplasm occurs
in the same way ; and as the combination of receptors with
toxin is supposed to be of firm nature, the receptors are lost
for the purposes of the cell, and the combination R.-T. (receptor
+ toxin) is shed off into the blood. The receptors thus lost
become replaced by new ones, and when additional toxin
molecules are introduced, these new receptors are used up in
the same manner as before. As a result of this repeated loss
the regeneration of the receptors becomes an over-regeneration,
and the receptors formed in excess appear in the free condition
in the blood stream and then constitute antiiojdn_jiiojejcules.
There are thus three factors in the process, namely, (1) fixation
of toxin, (2) over-production of receptors, (3) setting free of
receptors produced in excess. Accordingly these receptors
which, when forming part of the cell protoplasm, anchor the
toxin to the cell, and thus are essential to the occurrence of
toxic phenomena, in the free condition unite with the toxin, and
thus prevent the toxin from combining with the cells and exert-
ing a pathogenic action. The three orders of receptors, when
separated from the cells, thus give the three kinds of anti-
substances. Ehrlich does not state what cells are specially
concerned in the production of anti-substances, but from what
has been stated it is manifest that any cell which fixes a toxin
molecule, for example, is potentially a source of antitoxin.
Cells, to whose disturbance, resulting from the fixation of toxin,
characteristic symptoms of poisoning are due, will thus be
sources of antitoxin, e.g. cells of the nervous system in the case
of tetanus, though the cells not so seriously affected by toxin
fixation may act in the same way. The experimental investiga-
tion of the source of antitoxins has, however, yielded little result,
and no definite statement can be made on the subject.
When we come to consider how far Ehrlich's theory is in
harmony with known facts, we find that there is much in its
favour. In the first place, it explains the difference between
active and passive immunity, e.g. difference in duration, etc. ; in
the former the cells have acquired the habit of discharging anti-
substances, in the latter the anti-substances are simply present
as the result of direct transference. It is also in harmony with
the action of antitoxins, etc., as detailed above, and especially it
affords an explanation of the multiplicity of anti-substances.
For, if we take the case of antitoxins, we see that this depends
EHELICH'S SIDE-CHAIN THEORY 493
uppn the combining affinity of the toxin for certain of the cells
of the body, and this again is referred back to the complicated
constitution of living protoplasm. Furthermore, the biological
principle involved is no new one, being simply that of over-
regeneration after loss. It would appear likely that the integrity
of the executive centres of the protoplasm molecules would be
essential to the satisfactory production of side- chains, and this
would appear in accordance with the fact that antitoxin
formation occurs most satisfactorily when there is no marked
disturbance of the health of the animal.
It is to be noted, however, that it does not explain active
immunity apart from the presence of anti-substances in the
serum. For example, an animal may be able to withstand a
much larger amount of toxin than could be neutralised by the
total amount of antitoxin in its serum. This might theoretically
be explained by supposing a special looseness of the cell re-
ceptors so that the toxin-receptor combination became readily cast
off. The question, however, arises whether there may not be really
an increased resistance of the cells to the toxophorous affinities.
An observation recently made by Meyer and Ransom (v. p. 383)
is also difficult of explanation according to the view that antitoxin
is formed by the cells with which the toxin combines and on
which it acts. They found that in an animal actively immun-J
ised against tetanus and with antitoxin beginning to appear in
its blood, the injection of a single M.L.D. of tetanus toxin into a
peripheral nerve brought about tetanus with a fatal result. On
the other hand, the injection of antitoxin into the sciatic nerve
above the point of injection of toxin prevented the latter from
reaching the cells of the cord. One can scarcely imagine an
explanation of these facts if antitoxin molecules were in process
of being shed off by the cells of the nervous system. Further,
when the serum of an animal contains a large amount of anti-
toxin, how does the additional toxin injected reach the cells
in order to influence them as we know it does? This also is
difficult to understand unless the toxin has a greater affinity for
the receptors in the cells than for the free receptors (antitoxin)
in the serum. A supersensitiveness of the nerve-cells of an
animal to tetanus toxin, sometimes observed even when there is
a large amount of antitoxin in the serum, has been often brought
forward as an objection. But this also may perhaps be explained
by there having occurred a partial damage of the cell protoplasm
by the toxophorous action in the process of immunisation — an
explanation which, of course, demands that in some way the
freshly introduced toxin may reach the cells in spite of the anti-
494 IMMUNITY
toxin in the blood. Further investigation alone will settle these
and various other disputed points, and may remove many of the
apparent objections. At present we may say, however, that
Ehrlich's theory is the only one which even attempts to explain
the cardinal facts of this aspect of immunity.
In connection with the condition of supersensitiveness referred to
above, an interesting phenomenon has recently been -described by
Theobald Smith, and is now generally known as "serum anaphylaxis,"
or "Theobald Smith's phenomenon." It is briefly the following : — If a
guinea-pig be injected with a quantity, say 5 c.c., of horse serum, no
disturbance follows ; if, however, the animal be previously treated,
say fourteen davs before, with a very small quantity of horse serum,
'001 c.c. (even less is sufficient), and then the 5 c.c. of serum be injected,
the animal usually dies within an hour with characteristic symptoms.
The general lesions are of hsemorrhagic nature, as pointed out by Gay and
Southard,1 and occur especially in the stomach. The condition of super-
sensitiveness to the horse serum lasts fora long period of time. Accord-
ing to Gay and Southard the phenomenon depends upon a substance in
the horse serum which they call anaphylactin, and which persists for a
long period of time in the blood of the guinea-pig. This body they con-
sider to act as a slight irritant to the cells of the guinea-pig, and to
produce an increased affinity for the molecules in the horse serum. Ac-
cordingly when the second injection is made the rapid combination of these
substances with the cells result in the disturbances referred to. What-
ever may be the explanation, the phenomenon is of extreme importance
as showing the profound alterations in metabolism which may be induced
by a minute quantity of serum of a normal animal.
The facts relating to hypersensitiveness raise the question of
whether in any immunisation procedure an injury may not be
constantly done to the cells forming the anti-substances. We
have already drawn attention to the occurrence of what Wright
has called the negative phase in the course of the increase of the
opsonic power of the serum aimed at in a bacterial vaccination.
There is evidence that such negative phases are common in all
immunisations. They have been also noted in the formation of
antitoxins, of immune-bodies, and of agglutinins. Thus in the case
of the first, Salamonsen and Madsen showed that the fall in the
content of an animal's serum in antitoxin after a fresh toxin injec-
tion was greater than could be accounted for by the neutralisa-
tion of the free antitoxin in the blood by the toxin introduced,
Iand they attributed the occurrence to an injury to the producing 1
cells temporarily diminishing the productive activity. The4
normal course of every immunisation may be said to consist in
a succession of positive and negative phases, and an effective
immunisation is one where each succeeding positive phase brings
1 Vide Gay and Southard, Journ. Med. Research, xvi., 1907, 143.
THE THEORY OF PHAGOCYTOSIS 495
the serum to a higher content in anti-body. Again, in no case
is the capacity of producing anti-bodies unlimited. In certain
reactions the limit of possible increase is less than in others.
Thus it is not possible to raise the opsonic power of a serum
higher than a not very great multiple of its original opsonic
content. On the other hand, when we are dealing with the
reaction against bacterial toxins we find that the mechanism
producing antitoxin can react in an extraordinary degree, and
a serum many thousand times stronger than that produced
during the early days of immunisation may ultimately be
attained. The animal body also exhibits great power of forming
agglutinins, and the capacity of forming immune bodies seems
to occupy an intermediate position between the opsonic reaction
and the antitoxin reaction. But even in the antitoxin reaction
a time comes in a high immunisation when evidence of exhaus-
tion of the producing mechanism is manifest, so that the injection
of fresh toxin is no longer efficient, and the negative phase is
not followed by a positive phase. From the practical stand-
point it is the aim of the immuniser to select the time just
preceding such an event for the bleeding of an animal. If the
cells of the latter be given a few months rest then the capacity
for producing antitoxin usually reappears. But such facts
emphasise what we have said as to the possibility of every
immunisation entailing the infliction of an injury on some bodily
mechanism.
2. The Theory of Phagocytosis.— This theory, brought
forward by Metchnikoff to explain the facts of natural and
acquired immunity, has been of enormous influence in stimu-
lating research on the subject. Looking at the subject from the
standpoint of the comparative anatomist, he saw that it was a
very general property possessed by certain cells throughout
•the animal kingdom, that they should take up foreign bodies
into their interior and in many cases digest and destroy them.
On extending his observations to what occurred in disease, he
came to the conclusion that the successful resistance of an
animal against bacteria depended on the activity of certain cells
called phagocytes. In the human subject he distinguished two
chief varieties, namely, (a) the microphages, which are the
" polyinorpho-nuclear " finely granular leucocytes of the blood,
and (b) the macrophages, which include the larger hyaline
leucocytes, endothelial cells, connective tissue corpuscles, and, in
short, any of the larger cells which have the power of ingesting
bacteria. Insusceptibility to a given disease is indicated by a
rapid activity on the part of the phagocytes, different varieties
496 IMMUNITY
being concerned in different cases, — an activity which may
rapidly destroy the bacteria and prevent even local damage. If
the organisms are introduced into the tissues of a moderately
susceptible animal, there occurs an inflammatory reaction with
local leucocytosis, which results in the intracellular destruction
of the invading organisms. Phagocytosis is regarded by
Metchnikoff as the essence of inflammation. He also showed
that the bacteria may be in a living and active state when they
are ingested by leucocytes. On the other hand, he found that
in a susceptible animal phagocytosis did not occur or was only
imperfect. He also showed that when a naturally susceptible
animal was immunised, the process was accompanied by the
appearance of an active phagocytosis. The ingestion of bacteria
by phagocytes is undoubtedly a phenomenon of the greatest
importance in the defence of the organism. It is known that
amoebae and allied organisms have digestive properties which
are specially active towards bacteria, and from what can be
directly observed, as well as indirectly inferred, there can be no
doubt that such a faculty is also possessed by the phagocytes of
the body. Thus bacteria within these cells are in a position
favourable to their destruction and do in many instances
become destroyed. In fact, observations on phagocytosis in
vitro show that such destruction may in the case of some
organisms occur so rapidly that the actual number observable in
the leucocytes is no indication of the activity of the process.
In other instances, e.g. in gonorrhoea, the ingested organisms
would appear to survive a considerable time without undergoing
change. Undoubtedly phagocytosis is of the highest importance
in active immunity, as. by its means organisms which would not
undergo an extra-cellular death may be killed off. In the process
of immunisation of a susceptible animal we see a negative or
neutral chemiotaxis becoming replaced by positive chemiotaxis.
This has been explained by Metchnikoff as due to an education
or stimulation of the phagocytes. The recent work on opsonins
shows, however, that this is not the case, as leucocytes from an
immunised animal are not more active in this direction than
those of a normal animal, the all-important factor being the
development of an opsonin in the immune animal. Thus this
phase of immunity comes to be merely a part of the subject of
anti-substances in general.
The digestive ferments of phagocytes or cytases are, according
to Metchnikoff, retained within the cells under normal conditions,
but are set free when these cells are injured, for example, when
the blood is shed. They then become free in the serum by
THE THEORY OF PHAGOCYTOSIS 497
the breaking up of the cells— the process known as phagolysis —
and they then constitute the alexines, or complements of Ehrlich.
Of these, as has already been said, Metchnikoff thinks there
are probably two kinds — one called macrocytase, contained in
the macrophages, which is specially active toward the formed
elements of the animal body, protozoa, etc. ; and the other,
microcytase, contained within the poly morpho-nucl ear leucocytes,
which has a special digestive action on bacteria. It is the
microcytase which gives blood serum its bactericidal properties.
It appears to us, however, that Metchnikoff has gone too far in
distinguishing the activities of the two classes of cells so much
as he has done.
When the properties of antibacterial sera, as above described,
are considered in relation to phagocytosis, Metchnikoff gives the
following explanation. He admits that the immune -body is
fixed by the bacteria (or red corpuscles, as the case may be),
though he does not state that a chemical combination takes
place ; hence he calls it a fixative (fixateur). The immune-bodies
are to be regarded as auxiliary ferments (ferments adjuvants)
which aid the action of the alexine. Unlike the latter, however,
they are formed in excess during immunisation and set free in
the serum. He compares their action to that of enterokinase, a
ferment which is produced in the intestine and which aids the
action of trypsin. Thus, when the bacteria have fixed the immune-
body their digestion is facilitated either within the phagocytes, or
outside of them when the alexine has been set free by phagolysis.
He, however, maintains that extracellular digestion or lysogenesis
does not take place without the occurrence of phagolysis. The
source of immune-bodies is, in all probability, also the leucocytes,
as these substances are specially abundant in organs rich in
such cells — spleen, lymphatic glands, etc. ; here again the mono-
nuclear leucocytes are probably the source of the immune-bodies
concerned in haemolysis, the polymorpho-nuclear leucocytes the
source of those concerned in bacteriolysis. Although the im-
mune-bodies are usually set free in the serum, this is not always the
case ; sometimes they are contained in the cells, and this probably
occurs when there is a high degree of active immunity against
bacteria without a serum having an antibacterial action, the
powers of intracellular digestion being in such cases increased.
In this way the facts of immunity can be explained so far as
these concern the destruction of bacteria.
Metchnikoff 's work has less direct bearing on the production
of antitoxins. He admits the fixation of the toxin by the anti-
toxin to form a neutral compound, and he apparently considers
32
498 IMMUNITY
that leucocytes may also be concerned in the production of
antitoxins. Apart, however, from antitoxin formation, he con-
siders the acquired resistance of the cells themselves of high
importance in toxin immunity.
When we consider MetchnikofFs theory as thus extended to
cover recently established facts, it must be admitted that it affords
a rational explanation of a considerable part of the subject,
though the elucidation of the chemiotactic phenomena during
immunisation as explained above detracts from the importance
which he attached to the leucocyte. It, however, does not afford
explanation of the multiplicity and specificity of antitoxins as
Ehrlich's does ; on the other hand, it is more concerned with the
cells of the body as destroyers or digesters of bacteria. As
regards the subject of antibacterial sera, the results of these two
workers may be said to be in harmony in some of the funda-
mental conceptions. And it is of interest to note that Metchni-
koff, starting with the phenomena of intracellular digestion, has
arrived at the giving off of specific ferments by phagocytes ; whilst
Ehrlich, from his first investigations on the constitution of toxins,
has arrived at an explanation of antitoxins and immune-bodies
also with a theory of cell-nutrition as its basis. Within the last
few years marked progress has thus been made towards the
establishment of the fundamental laws of immunity. -;
NATURAL IMMUNITY.
We have placed the consideration of this subject after that of
acquired immunity, as the latter supplies facts which indicate in
what direction an explanation of the former may be looked for.
There may be said to be two main facts with regard to natural
immunity. The first is, that there is a large number of bacteria
— the so-called non-pathogenic organisms — which are practically
incapable, unless perhaps in very large doses, of producing patho-
genic effects in any animal ; when these are introduced into the
body they rapidly die out. This fact, accordingly, shows that
the animal tissues generally have a remarkable power of destroy-
ing living bacteria. The second fact is, that there are other
bacteria which are very virulent to some species of animals,
whilst they are almost harmless to other species ; the anthrax
bacillus may be taken as an example. Now it is manifest that
natural immunity against such an organism might be due to a
special power possessed by an animal of destroying the organisms
when introduced into its tissues. It might also possibly be due
to an insusceptibility to, or power of neutralising, the toxins of
NATURAL BACTERICIDAL POWERS 499
the organism. For the study of the various diseases shows that
the toxins (in the widest sense) are the weapons by which morbid
changes are produced, and that toxin-formation is a property
common to all pathogenic bacteria. There is, moreover, no
such thing known as a bacterium multiplying in the living tissues
without producing local or general changes, though, theoretically,
there might be. As a matter of fact, however, natural immunity
is in most cases one against infection, i.e. consists in a power
possessed by the animal body of destroying the living bacteria
when introduced into its tissues : such a power may exist though
the animal is still susceptible to the separated toxins. We shall
now look at these two factors separately.
1. Variations in Natural Bactericidal Poivers. — The funda-
mental fact here is that a given bacterium may be rapidly
destroyed in one animal, whereas in another it may rapidly
multiply and produce morbid effects. The special powers of
destroying organisms in natural immunity have been ascribed to
(a) phagocytosis, and (b) the action of the serum.
(a) The chief factors with regard to phagocytosis have been
given above. The bacteria in a naturally immune animal, for
example, the anthrax bacillus in the tissues of the white rat, are
undoubtedly taken up in large numbers and destroyed by the
phagocytes, whereas in a susceptible animal this only occurs to
a small extent ; and Metchnikoff has shown that they are taken
up in a living condition, and are still virulent when tested in a
susceptible animal. Variations in phagocytic activity are found
to correspond more* or less closely with the degree of immunity
present, but are probably in themselves capable of explanation.
The fundamental observations of Wright and Douglas show that
in many cases at least, leucocytes do not ingest organisms in a
neutral saline solution, and that this is not due to the medium
in which they are, is readily shown by subjecting the organisms
to the action of fresh serum and then washing them ; thereafter,
they are rapidly taken up by the leucocytes in salt solution.
In most cases this result is due to the labile opsonin of normal
serum which has combining affinities for a great many organisms
as already stated. In other cases more specific substances may
be concerned. But the all-important fact is that whether
phagocytosis occurs or not, appears to depend upon certain bodies
in the serum. As yet we cannot say whether the phagocytosis
in a given serum, observed according to the opsonic technique,
always runs parallel with phagocytosis in the tissues of the
animal from which the serum has been taken. This is a subject
on which extended observations are necessary. But whether or
500 IMMUNITY
not phagocytosis in vivo corresponds with that in vitro it is
probably to be explained in the same way ; that is, it probably
depends upon the content of the serum. The composition of the
latter, no doubt, is the result of cellular activity, and in this
the leucocytes themselves are in all probability concerned, but
the movements and phagocytic activity of these cells seem to
be chiefly if not entirely controlled by their environments.
Ingestion is, however, only the first stage in the process ; intra-
cellular destruction is the second, and is of equal importance.
What may be called intracellular bactericidal action probably
varies in the case of leucocytes of different animals, but regarding
this our knowledge is deficient, and, further, bacteria may some-
times survive the cells which have ingested them.
(b) When it had been shown that normal serum possessed
bactericidal powers against different organisms, the question
naturally arose as to whether this bactericidal power varied in
different animals in proportion to the natural immunity enjoyed
by them. The earlier experiments of Behring appeared to give
grounds for the belief that this was the case. He found, for
example, that the serum of the white rat, which has a remark-
able immunity to anthrax, had greater bactericidal powers than
that of other animals investigated. Further investigation, how-
ever, has shown that this is not an example of a general law,
and that the bactericidal action of the serum does not vary pari
passu with the degree of immunity. In many cases, however,
non-pathogenic and also attenuated pathogenic bacteria can be
seen to undergo rapid solution and disappear when placed in a
drop of normal serum. The bactericidal action of the serum
was specially studied by Nuttall, and later by Buchner and
Hankin, who believe that the serum owes its power to certain
substances in it derived from the spleen, lymphatic glands,
thymus, and other tissues rich in leucocytes. To these
substances Buchner gave the name of alexines] as already
explained, they correspond with MetchnikofFs cytases and
Ehrlich's complements described above. They can be pre-
cipitated by alcohol and by ammonium sulphate, and in this
respect and in their relative lability correspond with enzymes or
unorganised ferments. Variations in bactericidal power of the
serum as tested in vitro, however, do not explain the presence
or absence of natural immunity against a living bacterium. In
some cases, for example, it has been found to be considerable,
while the organisms flourish in the body, and the animal has no
immunity. In such a case Metchnikoff says that there occurs in
the living body no liberation of alexines by the phagocytes, and
NATURAL SUSCEPTIBILITY TO TOXINS 501
hence no bactericidal action such as occurs when the blood is shed.
In the case of the haemolytic action of a normal serum, it has
been shown in many instances that in addition to complement a
natural immune-body is also concerned (p. 481), and this would
appear to be the rule ; the process being analogous to what is
seen in the case of an artificially developed hsemolytic serum.
In certain instances an analogous condition appears to obtain in
a normal bactericidal serum. For example the dog's serum heated
at 58° C. contains a natural immune-body to anthrax which can
be activated by the addition of normal guinea-pig's serum so as to
produce a bactericidal action, though the latter is by itself with-
out any such effect. At present, however, the possibility of
bactericidal action by complement alone cannot be excluded, as
it appears to combine with many bacteria without any inter-
mediary. Further work is necessary to determine whether all
the facts regarding natural immunity are explainable by the
opsonic and bactericidal properties of the serum.
2. Variations in Natural Susceptibility to Toxins. — We must
here start with the fundamental fact, incapable of explanation,
that toxicity is a relative thing, or in other words, that different
animals have different degrees of resistance or non-suscepti-
bility to toxic bodies. In every case a certain dose must be
reached before effects can be observed, and up to that point the
animal has resistance. This natural resistance is found to
present very remarkable degrees of variation in different animals.
The great resistance of the common fowl to the toxin of the tetanus
bacillus may be here mentioned (v. p. 381), and large amounts of
this poison can be injected into the scorpion without producing
any effects whatever; the high resistance of the pigeon to
morphia is a striking example in the case of vegetable poisons.
This variation in resistance to toxins applies also to those which
produce local effects, as well as to those which cause symptoms
of general poisoning. Instances of this are furnished, for
example, by the vegetable poisons ricin and abrin, by the snake
poisons, and by bacterial toxins such as that of diphtheria. We
must take this natural resistance for granted, though it is
possible that ere long it will be explained.
According to Ehrlich's view of the constitution of toxins, it
might be due to the want of combining affinity between the
tissue cells and the haptophorous group of the toxin ; or, on the
other hand, supposing this affinity to exist, it might be due to
an innate non-susceptibility to the action of the toxophorous
group. Certain investigations have been made in order to
determine the combining affinity of the nervous system of the
502 IMMUNITY
fowl with tetanus toxin, as compared with that obtaining in a
susceptible animal, but the results have been somewhat contra-
dictory. Accordingly, a general statement on this point cannot
at present be made, though in all probability variations in the
susceptibility to the toxophorous group will be found to play a
very important part. It has been shown by Muir and Brown-
ing by means of hsemolytic tests that the toxic activity of com-
plement, after it has been fixed to the corpuscles, varies very
much; in some instances an amount of complement, which would
rapidly produce complete lysis of one kind of corpuscle, may
have practically no effect on another, even though it enters into
combination. These results are of importance in demonstrating
how the corresponding molecules of different animals may vary
in sensitiveness to toxic action.
APPENDIX A.
SMALLPOX AND VACCINATION.
SMALLPOX is a disease to which much study has been devoted,
owing, on the one hand, to the havoc which it formerly wrought
among the nations of Europe — a havoc which at the present
day it is difficult to realise, — and on the other hand, to the
controversies which have arisen in connection with the active
immunisation against it introduced by Jenner. Though there is
little doubt that a contagium vivum is concerned in its occurrence,
the etiological relationship of any particular organism to smallpox
has still to be proved ; and with regard to Jennerian vaccination,
it is only the advance of bacteriological knowledge which is now
enabling us to understand the principles which underlie the
treatment, and which is furnishing methods whereby, in the near
future, the vexed questions concerned will probably be satis-
factorily settled. We cannot here do more than touch on some
of the results of investigation with regard to the disease.
Jennerian Vaccination. — Up to Jenner's time the only means
adopted to mitigate the disease had been by inoculation (by
scarification) of virus taken from a smallpox pustule, especially
from a mild case. By this means it was shown that in the great
majority of cases a mild form of the disease was originated. It
had previously been known that one attack of the disease
protected against future infection, and that the mild attack
produced by inoculation also had this effect. This inoculation
method had long been practised in various parts of the world,
and had considerable popularity all over Europe during the
eighteenth century. Its disadvantage was that the resulting
disease, though mild, was still infectious, and thus might be the
starting-point of a virulent form among unprotected persons.
Jenner's discovery was published when inoculation was still
considerably practised. It was founded on the popular belief
that those who had contracted cowpox from an affected animal
503
504 SMALLPOX AND VACCINATION
were insusceptible to subsequent infection from smallpox. In the
horse there occurs a disease known as horsepox, especially tend-
ing to arise in wet cold springs, which consists in an inflammatory
condition about the hocks, giving rise to ulceration. Jenner
believed that the matter from these ulcers, when transferred by
the hands of men who dressed the sores to the teats of cows
subsequently milked by them, gave rise to cowpox in the latter.
This disease was thus identical with horsepox in epidemics of
which it had its origin. Jenner was, however, probably in error
in confounding horsepox with another disease of horses, namely,
grease. Cowpox manifests itself as a papular eruption on the
teats; the papules become pustules; their contents dry up to
form scabs, or more or less deep ulcers are formed at their sites.
From such a lesion the hands of the milkers may become infected
through abrasions, and a similar local eruption occurs, with
general symptoms in the form of slight fever, malaise, and loss of
appetite. It is this illness which, according to Jenner, gives rise
to immunity from smallpox infection. He showed experimentally
that persons who had suffered from such attacks did not react
to inoculation with smallpox, and further, that persons to whom
he communicated cowpox artificially, were similarly immune.
The results of Jenner's observations and experiments were
published in 1798 under the title .4 ft Inquiry into the Causes and
Effects of the Variola Vaccince. Though from the first Jennerian
vaccination had many opponents, it gradually gained the con-
fidence of the unprejudiced, and became extensively practised all
over the world, as it is at the present day.
The evidence in favour of vaccination is very strong. There
is no doubt that inoculation with lymph properly taken from a
case of cowpox, can be maintained with very little variation in
strength for a long time by passage from calf to calf, and such
calves are now the usual source of the lymph used for human
vaccination. When lymph derived from them is used for the
latter purpose, immunity against smallpox is conferred on the
vaccinated individual. It has been objected that some of the
lymph which has been used has been derived from calves
inoculated, not with cowpox, but with human smallpox. It is
possible that this may have occurred in some of the strains of
lymph in use shortly after the publication of Jenner's discovery,
but most of the strains at present in use have probably been
derived originally from cowpox. The most striking evidence in
favour of vaccination is derived from its effects among the staffs
of smallpox hospitals, for hefe, in numerous instances, it is only
the unvaccinated individuals who have contracted the disease.
RELATIONS OF SMALLPOX TO COWPOX 505
While vaccination is undoubtedly efficacious in protecting
against smallpox, Jenner was wrong in supposing that a vaccina-
tion in infancy afforded protection for more than a certain
number of years thereafter. It has been noted in smallpox
epidemics which have occurred since the introduction of vaccina-
tion, that whereas young unprotected subjects readily contract
the disease, those vaccinated as infants escape more or less till
after the thirteenth to the fifteenth years. It has become,
therefore, more and more evident that revaccination is necessary
if immunity is to continue; and where this is done in any
population, smallpox becomes a rare disease, as has happened in
the German army, where the mortality is practically nil. The
whole question of the efficacy of vaccination was investigated in
this country in 1896 by a Royal Commission, whose general
conclusions were as follows. Vaccination diminishes the liability
to attack by smallpox, and when the latter does occur, the
disease is milder and less fatal. Protection against attack is
greatest during nine or ten years after vaccination. It is still
efficacious for a further period of five years, and possibly never
wholly ceases. The power of vaccination to modify an attack
outlasts its power wholly to ward it off. Revaccination restores
protection, but this operation must be from time to time repeated.
Vaccination is beneficial according to the thoroughness with
which it is performed.
The Relationship of Smallpox (Variola) to Cowpox
(Vaccinia). — This is the question regarding which, since the
introduction of vaccination, the greatest controversy has taken
place ; a subsidiary point has been the inter-relationships within
the group of animal diseases which includes cowpox, horsepox,
sheep-pox, and cattle-plague.. With reference to smallpox and
cowpox the problem has been, Are they identical or not ? There
is no doubt that cowpox can be communicated to man, in whom
it produces the eruption limited to the point of inoculation, and
the slight general symptoms which vaccination with calf lymph
has made familiar. Apparently against the view that cowpox is
a modified smallpox are the facts that it never reproduces in
man a general eruption, and that the local eruption is only
infectious when matter from it is introduced into an abrasion.
The loss of infectiveness by transmission through the body of a
relatively insusceptible animal is a condition of which we have
already seen many instances in other diseases, and the uniformity
of the type of the affection resulting from vaccination with calf
lymph finds a parallel in such a disease as hydrophobia, where,
after passage through a series of monkeys, a virus of attenuated
506 SMALLPOX AND VACCINATION
but constant virulence can be obtained. We have seen that
there are good grounds for believing that the virus of calf lymph
confers immunity against human smallpox. In considering the
relationships of cowpox and smallpox, this is an important
though subsidiary point; for at present it is questionable
whether there are any well -authenticated instances of one
disease having the capacity of conferring immunity against
another. The most difficult question in this connection is what
happens when inoculations of smallpox matter are made on
cattle. Chauveau denies that in such circumstances cowpox is
obtained. He, however, only experimented on adult cows. The
transformation has been accomplished by many observers,
including, in this country, Simpson, Klein, Hime, and Copeman.
The general result of these experiments has been that if a series
of calves is inoculated with variolous matter, in the first there
may not be much local reaction, though redness and swelling
appear at the point of inoculation, and some general symptoms
manifest themselves. On squeezing some of the lymph from
such reaction as occurs, and using it to continue the passages
through other calves, after a very few transfers a local reaction
indistinguishable from that caused by cowpox lymph generally
takes place, and the animals are now found to be immune
against the latter. Not only so, but on using for human
vaccination the lymph from such variolated calves, results
indistinguishable from those produced by vaccine lymph are
obtained, and the transitory illness which follows, unlike that
produced in man by inoculation with smallpox lymph, is no
longer infectious. In fact, many of the strains of lymph in use
in Germany at present -have been derived thus from the variola-
tion of calves. The criticism of these experiments which has
been offered, namely, that since many of them were performed
in vaccine establishments, the calves were probably at the same
time infected with vaccinia, is not of great weight, as in all the
recent cases at least, very elaborate precautions have been
adopted against such a contingency. And at any rate it would
be rather extraordinary that this accident should happen to
occur in every case. We can, therefore, say that at present
there is the very strongest ground for holding not only that
vaccinia confers immunity against variola, but that variola
confers immunity against vaccinia. The experimentum crucis
for establishing the identity of the two diseases would of course
be the isolation of the same micro-organism from both, and the
obtaining of all the results just detailed by means of pure
cultures or the products of such. In the absence of this
BACTERIA IN SMALLPOX 507
evidence we are at present justified in considering that there is
strong reason for believing that vaccinia and variola are the same
disease, and that the differences between them result from the
relative susceptibilities of the two species of animals in which
they naturally occur.
With regard to the relation of cowpox to horsepox, it is
extremely probable that they are the same disease. Some
epidemics of the former have originated from the horse, but in
other cases such a source has not been traced. Cattle-plague
from the clinical standpoint, and also from that of pathological
anatomy, resembles very closely human smallpox. Though
each of the two diseases is extremely infectious to its appropriate
animal, there is no record of cattle-plague giving rise to small-
pox in man or vice versa. When matter from a cattle-plague
pustule is inoculated in man, a pustule resembling a vaccine
pustule occurs, and further, the individual is asserted to be now
immune to vaccination ; but vaccination of cattle with cowpox
lymph offers no protection against cattle-plague, though some
have looked on the latter as merely a malignant cowpox. Sheep-
pox also has many clinical and pathological analogies with
human smallpox, and facts as to its relation to cowpox vaccina-
tion similar to those observed in cattle -plague have been
reported. Smallpox, cowpox, cattle-plague, horsepox, and sheep-
pox, in short, constitute an interesting group of analogous
diseases, of the true relationships of which to one another we
are, however, still ignorant.
Micro-organisms associated with Smallpox. — Burdon Sander-
son was among the first to show that in vaccine lymph there
were certain bodies which he recognised as bacteria. Since
then numerous observations have been made as to the occurrence
of such in matter derived from variolous and vaccine pustules.
In especially the later stages of the latter, many of the pyogenic
organisms are always present, e.g. staphylococcus aureus and
staphylococcus cereus flavus, and many of the ordinary skin
saprophytes also are often present, but no organism has ever
been isolated which on transference to animals has been shown
to have any specific relationship to the disease. Streptococci
have also been described as agglutinable by the sera of smallpox
patients and of vaccinated persons ; such sera it may be said
had no effect on other strains of streptococci. Calmette and Guerin
have described very minute granules in the lymph which could
not be cultivated but which persisted after all the bacteria had
been removed. (The method by which the latter was accom-
plished was by exciting a leucocytosis in a rabbit's peritoneum and
508 SMALLPOX AND VACCINATION
then introducing the vaccinal lymph ; the leucocytes phagocyted
the bacteria so that the lymph no longer gave cultures on ordinary
media. It was, however, still potent to produce vaccinia.)
Klein and also, independently, Copeman, have observed an organism
in lymph taken from a vaccine pustule in a calf on the fifth and sixth
days, in human vaccine lymph on the eighth day, and in lymph from
a smallpox pustule on the fourth day. To demonstrate the bacilli,
cover-glass films are dried and placed for five minutes in acetic acid (1
in 2), washed in distilled water, dried, and placed in alcoholic gentian-
violet for from twenty-four to forty-eight hours, after which they are
washed in water and mounted. Copeman and Kent also found the
bacilli in sections of vaccine pustules stained by Lbffler's methylene-blue,
or by Gram's method. The organisms are "4 to '8 jj. in length, and
one-third to a half of this in thickness. They are generally thinner and
stain better at the ends than at the middle. They occur in groups of
from three to ten in both the lymph and the tissues. In the centre of
their protoplasm there is often a clear globule, which is looked on as a
spore. They have hitherto resisted the ordinary isolation methods, a
fact which is rather in favour of their non-saprophytic nature. By
inoculating fresh eggs with the crusts of smallpox pustules Copeman has,
however, obtained a growth of a bacillus resembling that found by him
in the tissues. Though subcultures on ordinary media have been
obtained, the pathogenic effects of these have not been fully investigated,
and thus the identity of this bacillus with that seen in the tissues is
not proved.
Various observers have described appearances in the epithelial
cells in the neighbourhood of the smallpox or vaccine pustules,
which they have interpreted as being protozoa. Thus Ruffer
and Plimmer describe as occurring in clear vacuoles in the cells
of the rete Malpighii at the edge of the pustule, in paraffin
sections of vaccine and smallpox pustules carefully hardened in
alcohol, and stained by the Ehrlich-Biondi mixture, small round
bodies of about four times the size of a staphylococcus pyogenes,
coloured red by the acid fuchsin, sometimes with a central part
stained by the methyl-green. These are described as multiplying
by simple division, and in the living condition exhibiting
amoeboid movement. Similar bodies have been described by
Reed in the blood of smallpox patients and of vaccinated
children and calves.
These are probably the bodies described by Guarnieri and to
which considerable attention has been paid. They are from
1-8 /A in diameter, are round, oval, or sickle -shaped, and stain
by ordinary nuclear dyes. They lie in the cells in spaces
often near the nucleus, and are readily demonstrable in vaccine
pustules and also in the experimental lesions which can be
produced in the rabbit's cornea, the larger bodies being defined
in the cells towards the centre of the lesion. These bodies
NATURE OF VACCINATION 509
have been looked on by many as protozoa, and Guarnieri himself
stated that multiplication could be seen occurring in them in
fresh lymph, but Ewing and also Prowazek have brought forward
strong evidence for the appearances being due to nuclear changes,
Still the question of the specificity of these changes to variolous
lesions remains, and here it may be said Wasielewski has shown
that they persist through 46 transfers on the cornea of the
rabbit and further no similar appearances have been found in
other skin lesions. Prowazek examined material fixed in a hot
mixture of two- thirds saturated perchloride of mercury and
one -third 90 per cent, alcohol, washed in 40 per cent, iodine
alcohol and stained in Grenacher's haematoxylin, and found
bodies in the epithelial cells 1-4 fj. in size, sharply contoured
and having ragged edges as if made up of massed chromosomes.
These were often broader at one end than at the other, and
appearances have been seen which suggest longitudinal division.
Prowazek has also seen these " lymph-bodies " as he has called
them in the lymph, and he inclines to the idea that they may be
protozoa. Bonhoff and also Carini have described spirochaetes
as occurring in variolous lesions, but this has not been confirmed.
Future investigations must show what significance is to be
attached to these various observations.
The causal organism of smallpox is probably very small as,
though there has been some difference in opinion on this point,
there is little doubt that it will pass through the coarser porcelain
filters.
The Nature of Vaccination. — As we are ignorant of the cause
of smallpox, we can only conjecture what the nature of vaccina-
tion is. From what we know of other like processes, however,
we have some ground for believing that it consists in an active
immunisation by means of an attenuated form of the causal
organism. As to how immunity is maintained after vaccination,
we do not know much. Some, including Beclere, Chambon,
and Menard (who jointly investigated the subject), maintain that
in the blood of vaccinated animals substances exist which, when
transferred to other animals, can confer a certain degree of
passive immunity against vaccination, and which have also a
degree of curative action in animals already vaccinated. Beumer
and Peiper, on the other hand, could not find evidence of the
existence of such bodies.
APPENDIX B.
HYDROPHOBIA.
SYNONYMS. RABIES : FRENCH, LA RAGE : GERMAN, LYSSA,
DIE HUNDSWUTH, DIE TOLLWUTH.
Introductory. — Hydrophobia is an infectious disease which in
nature occurs epidemically chiefly among the carnivora, especi-
ally in the dog and the wolf. Infection is carried by the bite
of a rabid animal or by a wound or abrasion being licked by
such. The disease can be transferred to other species, and
when once started can be spread from individual to individual
by the same paths of infection. Thus it occurs epidemically
from time to time in cattle, sheep, horses, and deer, and can be
communicated to man. It is questionable whether infection
can take place from man to man as the saliva of a person
suffering from hydrophobia appears not to contain the virus.
It is to be noted that the virus is apparently extremely potent,
as cases of infection taking place through an unabraded mucous
membrane by the licking of a rabic animal are on record and the
experimental applications of the virus to such surfaces as the
mucous membrane of the nose or the conjunctiva is often followed
by infection.
In Western Europe the disease is most frequently observed
in the dog ; but in Eastern Europe, especially in Russia,
epidemics among wolves constitute a serious danger both to
other animals and to man. All the manifestations of the disease
point to a serious affection of the nervous system ; but inasmuch
as symptoms of excitement or of depression may predominate,
it is customary to describe clinically two varieties of rabies, (1)
rabies proper, or furious rabies (la rage vraie, la rage furieuse :
die rasende Wuth) ; and (2) dumb madness or paralytic rabies (la
rage, mue : die stille Wuth). The disease, however, is essentially
the same in both cases. In the dog the furious form is the
510
PATHOLOGY OF HYDROPHOBIA 511
more common. After a period of incubation of from three to
six weeks, the first symptom noticed is a change in the animal's
aspect ; it becomes restless, it snaps at anything which it touches,
and tears up and swallows unwonted objects ; it has a peculiar
high-toned bark. Spasms of the throat muscles come on,
especially in swallowing, and there is abundant secretion of
saliva ; its supposed special fear of water is, however, a myth,
— it fears to swallow at all. Gradually convulsions, paralysis,
and coma come on ; and death supervenes. In the paralytic
form, the early symptoms are the same, but paralysis appears
sooner. The lower jaw of the animal drops, from implication
of the elevator muscles, all the muscles of the body become
more or less weakened, and death ensues without any very
marked irritative symptoms.
In man the incubation period after infection varies from
fifteen days to seven or eight months, or even longer, but is
usually about forty days. When symptoms of rabies are about
to appear, certain prodromata, such as pains in the wound and
along the nerves of the limb in which the wound has been
received, may be observed. To this succeeds a stage of nervous
irritability, during which all the reflexes are augmented — the
victim starting at the slightest sound, for example. There are
spasms, especially of the muscles of deglutition and respiration,
and cortical excitement evidenced by delirium may occur. On
this follows a period in which all the reflexes are diminished,
weakness and paralysis are observed, convulsions occur, and
finally coma and death supervene. The duration of the acute
illness is usually from four to eight days, and death invariably
results. The existence of paralytic rabies in man has been
denied by some, but it undoubtedly occurs. This is usually
manifested by paralysis of the limb in which the infection has
been received, and of the neighbouring parts ; but while in such
cases this is often the first symptom observed, during the whole
of the illness the occurrence of widespread and progressive
paralysis is the outstanding feature. In man then also occur
cases where the cerebellum and also the sympathetic system seem
to be specially affected.
The Pathology of Hydrophobia.— In hydrophobia as in
tetanus, to which it bears more than a superficial resemblance,
the appearances presented in the nervous system, to which all
symptoms are naturally referred, are comparatively unimportant.
On naked-eye examination, congestions, and, it may be, minute
haemorrhages in the central nervous system, are the only features
noticeable. Microscopically, leucocytic exudation into the peri-
512 HYDROPHOBIA
vascular lymphatic spaces in the nerve centres has been observed,
and in the cells of the anterior cornua of the grey matter in the
spinal cord, and also in the nuclei of the cranial nerves, various
degenerations have been described. Round the nerve cells in
the grey matter of the cord and medulla Babes described
accumulations of newly-formed cells, and Van Gehuchten observed
a phagocytosis of the cells in the posterior root ganglia and also
in the sympathetic ganglia. Both of these conditions were at one
time thought to be specific of rabies, but this has been found not
to be the case. In the white matter, especially in the posterior
columns, swelling of the axis cylinders and breaking up of the
myeline sheaths have been noted, and similar changes occur also
in the spinal nerves, especially of the part of the body through
which infection has come. In the nervous system also some
have seen minute bodies which they have considered to be cocci,
but there is no evidence that they are really of this nature. The
changes in the other parts of the body are unimportant.
Experimental pathology confirms the view that the nervous
system is the centre of the disease by finding in it a special
concentration of what, from want of a more exact term, we must
call the hydrophobic virus. Earlier inoculation experiments
made by subcutaneous injection of material from various parts
of animals dead of rabies had not given uniform results, as,
whatever was the source of the material, the disease was not
invariably produced. Pasteur's first contribution to the subject
was to show that the most certain method of infection was by
inserting the infective matter beneath the dura mater. He
found that in the case of any animal or man dead of the disease,
injection, by this method, of emulsions of any part of the central
nervous system, of the cerebro-spinal fluid, or of the saliva,
invariably gave rise to rabies, and also that the natural period of
incubation was shortened. Further, the "identity of the furious
and paralytic forms was proved, as sometimes the one, sometimes
the other, was produced, whatever form had been present in the
original case. Inoculation into the anterior chamber of the eye
is nearly as efficacious as subdural infection. Infection with the
blood of rabic animals does not reproduce the disease. There is
evidence, however, that the poison also exists in such glands as
the pancreas and mamma. Subcutaneous infection with part of
the nervous system of an animal dead of rabies usually gives
rise to the disease.
In consequence of the introduction of such reliable inoculation
methods, further information has been acquired regarding the
spread and distribution of the virus in the body. Gaining
THE VIRUS OF HYDROPHOBIA 513
entrance by the infected wound, it early manifests its affinity for
the nervous tissues. It reaches the central nervous system
chiefly by spreading up the peripheral nerves. This can be
shown by inoculating an animal jsubcutaneously in one of its
limbs, with virulent material, ^tf now the animal be killed
before symptoms have manifested themselves, rabies can be
produced by subdural inoculation from the r^erves of the limb
which was infected. Further, 'rabies can often^be produced from
such a case by subdural infection with the part of the spinal
cord into which these nerves pass, while the other parts of the
animal's nervous system do not give rise to the disease. This
explains how the initial symptoms of the disease (pains along
nerves, paralysis, etc.) so often appear in the infected part of the
body, and it probably also explains the fact that bites in such
richly nervous parts as the face and head are much more likely
to be followed by hydrophobia than bites in other parts of the
body. Again, injection into a peripheral nerve, such as the
sciatic, is almost as certain a method of infection as injection
into the subdural space, and gives rise to the, same type of
symptoms as injection into the corresponding limb. Intravenous
injection of the virus, on the other hand, differs from the other
modes of infection in that it more frequently gives rise to
paralytic rabies. This fact Pasteur explained by supposing
that the whole of the nervous system in such a^ case becomes
simultaneously affected. In certain animals the: virus seems to
have an elective affinity for the salivary glands, - as well as for
the nervous system. Roux and Nocard found that the saliva of
the dog became virulent three days before the first appearance
of symptoms of the disease.
The Virus of Hydrophobia. — While a source of infection
undoubtedly occurs in all cases of hydrophobia, and can usually
be traced, all attempts to determine the actual morbific cause
have been unsatisfactory. In this connection various organisms
have been described as being associated with the disease.
Thus Memmo has isolated an organism which resembles a yeast, but
which he places amongst the blastomycetes, and with which he states
he has produced both types of rabies in rabbits and dogs. Bruschettini
also, by using media containing brain substance, has grown a bacillus
having some resemblances to the members of the diphtheria group, and
with which he claims to have produced paralytic rabies in rabbits. In
the case of the work of neither of these observers has there been con-
firmation from independent sources, and in neither case is there evidence
of the crucial test having been applied, namely, that of immunising
animals against the ordinary hydrophobia virus by means of pure
cultures of the alleged causal organism. With regard to other possible
33
514 HYDROPHOBIA
causal agents, Grigorjew thinks such may be found in a protozoon which
he has constantly observed after inoculation in the cornea.
In 1903 Negri described certain bodies as occurring in the
nervous system in animals dying of rabies to which considerable
attention has since been devoted, and regarding the significance
of which opinion is still divided. It may be said that Negri's
observations have been generally confirmed, and as it is probable,
whatever the final opinion as to the nature of the bodies
may be, that their occurrence is specific to the disease and hence
may be used for diagnosis, we shall describe the methods for
their demonstration. In doing so we shall chiefly follow the
work of the American observers, Williams and Lowden, who,
more than any others who have confirmed Negri, have used
methods widely employed in the investigation of similar
appearances.
Their chief method is to take a piece of the brain tissue, to squeeze it
between a slide and cover-glass, and, sliding off the latter, to make a
smear which is then fixed in methyl alcohol for five minutes and stained
by Giemsa's stain (p. 107) for half an hour to three hours ; the prepara-
tion is then washed in tap water for 2-3 min. and dried. For rapid work,
after fixation, equal parts of distilled water and stain are used instead of
the more dilute mixture.
For sections the tissues are left in Zenker's fluid l for 3-4 hours, then
placed in tap water for five minutes, 80 per cent alcohol with enough
iodine added to give it a port wine colour for 24 hours ; 95 per cent
alcohol and iodine, 24 hours ; absolute alcohol, 4-6 hours ; cleared with
cedar oil and embedded in paraffin of melting point 52° C. ; sections should
be 3-6 /u, thick. For staining, Mallory's methylene - blue eosin is
recommended ; the steps are as follows : xylol ; absolute alcohol ; 95
per cent alcohol and iodine, £ hour ; 95 per cent alcohol, ^ hour ;
absolute alcohol, £ hour ; eosin solution (5-10 per cent aqueous solution),
20 minutes ; rinse in tap water ; Unna's polychrome raethylene-blue
solution diluted 1-4 with distilled water, 15 minutes ; differentiation in
95 per cent alcohol for 1-5 minutes (the preparation being kept in
motion and its progress watched with a low power) ; rapid and careful
dehydration and clearing.
The bodies vary much in size, measuring from -5 //- to 25 p.
They are round, oval, or angular in outline. They are found in
the protoplasm of the nerve cells and of their processes. They
have a hyaline appearance with a sharply -defined outline, and in
their substance they contain granular material. Taking for granted
their cellular structure we may say that with the Giemsa mixture
1 Zenker's fluid is of the following composition : potassium bichromate
2'5 gr., sodium sulphate 1 gr., perchloride of mercury 5 gr., glacial acetic
acid 5 c.c., water to 100 c.c. Dissolve the perchloride of mercury and the
bichromate of potassium in the water with the aid of heat and add the
acetic acid.
THE VIRUS OF HYDROPHOBIA 515
their cytoplasm stains blue and the granules a blue-red, — by
Mallory's stain the cytoplasm is magenta and the granules a
deep blue. The cytoplasm is homogeneous, and in it is a
nucleus -like body whose chromatin particles in the larger
individuals are arranged round the periphery, there being a
clear centre containing a nucleolus ; in the smaller forms the
nucleus is a mere chromatin spot. Round the central definite
nuclear body are some chromatoid particles which are irregular
in outline and size, are sometimes elongated, and do not take
on such a pure chromatin stain as the nucleus. There is
evidence of division of the nucleus, and sometimes there may
apparently be three or four nuclei in one body without division
of the protoplasm having occurred. Sometimes the chromatin
appears to fragment and break up into a large number of small
particles, and in such bodies active budding of the protoplasm
may be seen. Sometimes the bodies seem to go on dividing
again and again, with the result that some very small forms may
be produced, these sometimes appearing in mulberry masses.
The Negri bodies have been found in nearly all cases of
street-rabies examined by many observers, and have never been
found in other conditions of brain disease. They occur in all
parts of the central nervous system, but are said to be most
abundant in the cells of the cornu Ammonis. They are
apparently not so readily found, at least in their larger forms,
in animals dying from the inoculation of virus fixe. What the
significance of these bodies is, it is at present, impossible to say ;
but whatever may be their nature, there is now considerable
evidence that their presence is specific of rabies, and that thus
in their recognition a much quicker means of diagnosis is possible
than by the longer method of awaiting symptoms in an in-
oculated rabbit. Many have looked on these bodies as protozoa,
and their appearance is not inconsistent with such a view. . The
objection which has been raised, that if they were protozoa
they could not pass through a porcelain filter (vide infra) as
the virus does, is met by the fact of the occurrence of minute
forms, and by the fact that similar small forms probably exist
in certain trypanosomes (see Appendix E). The occurrence of
minute forms would also account for the non-recognition of the
parasite in the more acute forms of the disease where there had
been an active vegetative condition, and thus no time for the
larger forms to develop.
There is no doubt that between rabies and the bacterial
diseases we have studied there are at every point analogies, the
most striking being the protective inoculation methods which
516 HYDROPHOBIA
constitute the great work of Pasteur ; and everything points to
a micro-organism being the cause. The organism, whatever it is,
is, in its infective form, probably very small, as it can pass
through the coarser Berkefeld niters, and also occasionally
through the coarser Chamberland candles. Evidence that it is
the organism itself which passes through, is found in the fact
that when an animal dies from infection with the nitrate, a
small portion of its central nervous system will originate the
disease in a fresh animal. Judging from our knowledge of
similar diseases we would strongly suspect that it is actually
present in a living condition in the central nervous system, the
saliva, etc., which yield what we have called the hydrophobic
virus, for by no mere toxin could the -disease be transmitted
through a series of animals, as we shall presently see can be
done. A toxin may, however, be concerned in the production
of the pathogenic effects. Remlinger found that death with
paralytic symptoms sometimes followed the injection of filtered
virus, but that the nervous system of the dead animals did not
reproduce rabies. He explains this occurrence by supposing that
the filtrate contained a toxin but not the actual infective agent.
The resistance of the virus to external agents varies. Thus a
nervous system containing it is virulent till destroyed by putre-
faction ; it can resist the prolonged application of a temperature
of from - 10° to - 20° C., but, on the other hand, it is rendered
non-virulent by one hour's exposure at 50° C. Again, its
potency probably varies in nature according to the source.
Thus, while the death-rate among persons bitten by mad dogs is
about 16 per cent, the corresponding death-rate after the bites
of wolves is 80 per cent. Here, however, it must be kept in
view that, as the wolf is naturally the more savage animal, the
number and extent of the bites, i.e. the number of channels of
entrance of the virus into the body, and the total dose, are
greater than in the case of persons bitten by dogs. As we shall
see, alterations in the potency of the virus can certainly be
effected by artificial means.
The Prophylactic Treatment of Hydrophobia. — Until the
publication of Pasteur's researches in 1885, the only means
adopted to prevent the development of hydrophobia in a person
bitten by a rabid animal had consisted in the cauterisation of
the wound. Such a procedure was undoubtedly not without
effect. It has been shown that cauterisation within five minutes
of the infliction of a rabic wound prevents the disease from
developing, and that if done within half an hour it saves a
proportion of the cases. After this time, cauterisation only
PROPHYLACTIC TREATMENT OF HYDROPHOBIA 517
lengthens the period of incubation ; but, as we shall see
presently, this is an extremely important effect.
The work of Pasteur has, however, revolutionised the whole
treatment of wounds inflicted by hydrophobic animals. Pasteur
started with the idea that, since the period of incubation in the
case of animals infected subdurally from the nervous systems of
mad dogs is constant in the dog, the virus has been from time
immemorial of constant strength. Such a virus, of what might
be called natural strength, is usually referred to in his works as
the virus of la rage des rues, in the writings of German authors
as the virus of die Strasswuth. Pasteur found on inoculating a
monkey subdurally with such a virus, and then inoculating
a second monkey from the first, and so on with a series of
monkeys, that it gradually lost its virulence, as evidenced by
lengthened periods of incubation on subdural inoculation of
dogs, until it wholly lost the power of producing rabies in dogs,
when introduced subcutaneously. When this point had been
attained, its virulence was not diminished by further passage
through the monkey. On the other hand, if the virus of la
rage des rues were similarly passed through a series of rabbits
or guinea-pigs, its virulence was increased till a constant strength
(the virus fixe) was attained. Pasteur had thus at command
three varieties of vims — that of natural strength, that which had
been attenuated, and that which had been exalted. He further
found that, commencing with the 'subcutaneous injection of a
weak virus and following this up writh the injection of the
stronger varieties, he could ultimately, in a very short time,
immunise dogs against subdural infection with a virus which,
under ordinary conditions, would certainly have caused a fatal
result. He also elucidated the fact that the exalted virus con-
tained in the spinal cords of rabbits such as those referred to,
could be attenuated so as no longer to produce rabies in dogs by
subcutaneous injection. This was done by drying the cords in
air over caustic potash (to absorb the moisture), the diminution
of virulence being proportional to the length of time during
which the cords were kept. Accordingly, by taking a series of
such spinal cords kept for various periods of time, he was
supplied with a series of vaccines of different strengths. Pasteur
at once applied himself to find whether the comparatively long
period of incubation in man could not be taken advantage of to
"vaccinate" him against the disease before its gravest manifesta-
tion took place. The following is the record of the first case
thus treated. The technique was to rub up in a little sterile
bouillon a small piece of the cord used, and inject it under the
518
HYDROPHOBIA
skin by means of a hypodermic syringe. The first injection was
made with a very attenuated virus, i.e. a cord fourteen days old.
In subsequent injections the strength of the virus was gradually
increased, as shown in the table : —
July 7, 1885, 9 A.M., cord of June 23, i.e. 14 days old.
, 7
6 P.M. „ 25
12
8
9 A.M. ,, 27
11
8
6 P.M. ,, 29
9
9
11 A.M., cord of July 1
8
10
3
7
11
5
6
12
7
5
13
9
4
14
11
3
15
13
2
16
15
1 day'
old.
The patient never manifested the slightest symptom of hydro-
phobia. Other similarly favourable results followed ; and this
prophylactic treatment of the disease quickly gained the con-
fidence of the scientific world, which it still maintains. (The
principal is, of course, the same as in artificially developing a
high degree of active immunity against a bacterial infection.)
The only modification which the method has undergone lias been in
the treatment of serious cases, such as multiple bites from wolves,
extensive bites about the head, especially in children, cases which come
under treatment at a late period of the incubation stage, and cases where
the wounds have not cicatrised. In such cases the stages of the treat-
ment are condensed. Thus on the first day, say at 11 A.M. and 4 P.M.
and 9 P.M., cords of 12, 10, and 8 days respectively are used ; on the
second day, cords of 6, 4, and 2 days ; on the third day, cords of 1 day ;
on the fourth day, cords of 8, 6, and 4 days ; on the fifth, cords of 3 and
2 days ; on the sixth, cords of 1 day ; and so on for ten days. In each
case the average dose is about 2 c.c. of the emulsion.
The success of the treatment has been very marked. The statistics
of the cases treated in Paris are published quarterly in the Annales de
I'Institut Pasteur, and general summaries of the results of each year
are also prepared. As we have said, the ordinary mortality formerly
was 16 per cent of all persons bitten. During the ten years 1886-95,
17,337 cases were treated, with' a mortality of '48 per cent. It has been
alleged that many people are treated who have been bitten by dogs that
were not mad. This, however, is not more true of the cases treated by
Pasteur's method than it was of those on which the ordinary mortality
of 16 per cent was based, and care is taken in making up the statistics
to distinguish the cases into three classes. Class A includes only persons
bitten by dogs proved to have had rabies, by inoculation in healthy
animals of parts of the central nervous system of the diseased animal.
Class B includes those bitten by dogs that a competent veterinary surgeon
has pronounced to be mad. Class C includes all other cases. During
1895, 122 cases belonging to Class A were treated, with no deaths ; 940
belonging to class B, with two deaths ; and 449 belonging to Class C,
METHODS 519
with no deaths. Besides the Institute in Paris, similar institutions exist
in other parts of France, in Italy, and especially in Russia, as well as
in other parts of the world ; and in these similar success has been
experienced. It may be now taken as established, that a very grave
responsibility rests on those concerned, if a person bitten by a mad
animal is not subjected to the Pasteur treatment. Sometimes during or
after treatment there appear slight paralytic symptoms with obstinate
constipation and it may be retention of urine, but these pass off within
a few weeks and leave behind no ill etfects.
Antirabic Serum. — In the early part of the nineteenth century
an Italian physician, Valli, showed that immunity against rabies
could be conferred by administering through the stomach pro-
gressively increasing doses of hydrophobic virus. Following up
this observation, Tizzoni and Centanni have attenuated rabic
virus by submitting it to peptic digestion, and have immunised
animals by injecting gradually increasing strengths of such virus.
This method is usually referred to as the Italian method of
immunisation. The latter workers showed from this that the
serum of animals thus immunised could give rise to passive
immunity in other animals ; and further, that if injected into
animals from seven to fourteen days after infection with the
virus, it prevented the latter from producing its fatal effects,
even when symptoms had begun to manifest themselves. They
further succeeded in producing in the sheep and the dog an
immunity equal to from 1-25,000 to 1-50,000 (vide p. 454), and
they recommended the use, in severe cases, of the serum of such
animals in addition to the treatment of the patient by the
Pasteur method. A like serum has been obtained from animals
treated by the ordinary Pasteur method.
Methods, (a) Diagnosis. — When a person is bitten by an
animal suspected to be rabid, the latter must under no circum-
stances be killed. Much more can be learned by watching it
while alive than by post-mortem examination. In the latter case
only such things as the occurrence of broken teeth, marked
congestion of the fauces, or the presence of unwonted material
in the stomach throw any light on the condition ; nothing of a
positive nature can be learned from examining the nervous
system. On the other hand, in the living animal the develop-
ment of the characteristic symptoms can be watched, and death
will occur in not more than five days. If the suspected animal
has been killed, then a small piece of its medulla or cord must
be taken, with all aseptic precautions, rubbed up in a little
sterile '75 per cent sodium chloride solution, and injected by
means of a syringe beneath the dura mater of a rabbit, the latter
having been trephined over the cerebrum by means of the small
520 HYDROPHOBIA
trephine which is made for the purpose. Symptoms usually
occur in from ten to twenty-three days and death in fifteen to
twenty-five days. When such inoculation has to be practised it
is evident that the diagnosis is delayed. When the material for
inoculation has to be sent any distance this is best effected by
packing the head of the animal in ice. The virulence of organs
is not lost, however, if they are simply placed in sterile water or
glycerin in well-stoppered bottles. When the brain of the
suspected dog is available either through its death or its being
killed, the Negri bodies should be sought for especially in the
cornu Ammonis by the methods described above.
(b) Treatment. — Every wound inflicted by a rabid animal
ought to be cauterised with the actual cautery as soon as possible.
By such treatment the incubation period will at any rate be
lengthened, and therefore there will be better opportunity for
the Pasteur inoculation method being efficacious. The person
ought then to be sent to the nearest Pasteur Institute for treat-
ment. It is of great importance that in such a case the nervous
system of the animal should also be sent, in order that the
diagnosis may be certainly verified.
APPENDIX C.
MALARIAL FEVER.
IT has now been conclusively proved that the cause of malarial
fever is a protozoon of which there are several species. They
belong to the hsemosporidia (a sub-class of the sporozoa) which
are blood parasites, infecting the red corpuscles of mammals,
reptiles, and birds. The parasite was formerly known as the
hcematozoon or plasmodium malarias, although the use of the
latter term is incorrect ; the term hcemamoeba is, however, now
generally employed. The parasite was first observed by Laveran
in 1880, and his discovery received confirmation from the in-
dependent researches of Marchiafava and Celli, and later from
the researches of many others in various parts of the world.
Golgi supplied valuable additional information, especially in
relation to the sporulation of the organism and the varieties in
different types of malarial fever. In this country valuable work
on the subject was done by Manson, and to him specially belongs
the credit of regarding the exflagellation of the organism as
a preparation for an extra-corporeal phase of existence. By in-
duction he arrived at the belief that the cycle of existence outside
the human body probably took place in the mosquito. It was
specially in order to discover, if possible, the parasite in the
mosquito, that Ross commenced his long series of observations,
which were ultimately crowned with success. After patient and
persistent search, he found rounded pigmented bodies in. the
wall of the stomach of a dapple-winged mosquito (a species of
Anopheles) which had been fed on the blood of a malarial
patient. The pigment in these bodies was exactly similar to
that in the malarial parasite, and he excluded the possibility of
their representing anything else than a stage in the life cycle of
the organism. He confirmed this discovery and obtained cor-
responding results in the case of the proteosoma infection of
birds, where the parasite is closely related to that of malaria.
521
522 MALARIAL FEVER
In birds affected with this organism, he was able to trace all
stages of its development, from the time it entered the stomach
along with the blood, till the time when it settled in a special
form in the salivary glands of the insect. Ross's results were
published in 1898. Exactly corresponding stages were after-
wards found in the case of the different species of the human
parasite, by Grassi, Bignami, and Bastianelli ; and these with
other Italian observers also supplied important information
regarding the transmission of the disease by infected mosquitoes.
Abundant additional observations, with confirmatory results,
were supplied by Koch, Daniels, Christophers, Stephens, and
others. Wherever malaria has been studied the result has been
the same. Lastly, we may mention the striking experiment
carried out by Manson by means of mosquitoes fed on the blood
of patients in Italy suffering from mild tertian fever. The
insects, after being thus fed, were taken to London and allowed
to bite the human subject, Hanson's son, Dr. P. Thurburn
Manson, offering himself for the purpose. The result was that
infection occurred ; the parasites appeared in the blood, and
were associated with an attack of tertian fever. Ross's discovery
has not only been a means of elucidating the mode of infection,
but, as will be shown below, has also supplied the means of
successfully combating the disease.
From the zoological point of view the mosquito is regarded
as the definitive host of the parasite, the human subject as the
intermediate host. But in describing the life history, it will be
convenient to consider, first, the cycle in the human body, and,
secondly, that in the mosquito. Various terms have been
applied to the various stages, but we shall give those now
generally used.
The Cycle in the Human Subject. — With regard to this
cycle, it may be stated that the parasite is conveyed by the bite
of the mosquito in the form of a small filamentous cell —
sporozoite or exotospore, which penetrates a red corpuscle and
becomes a small amoeboid organism or amoebula. There is then
a regularly repeated asexual cycle of the parasite in the blood,
the length of which cycle determines the type of the fever.
During this cycle there is a growth of the amcebulae or
trophozoites within the red corpuscles up to their complete
development ; sporulation or schizogony then occurs. The onset
of the febrile attack corresponds with the stage of sporulation
and the setting free of the spores (enhsemospores or merozoites),
i.e. with the production of a fresh brood of parasites. These
spores soon become attached to, and penetrate into the interior
FORMS OF THE MALARIAL PARASITE 523
of the red corpuscles, becoming intra-corpuscular amoebulse ; the
cycle is thus completed. The parasites are most numerous in
the blood during the development of the pyrexia, and, further,
they are also much more abundant in the internal organs than in
the peripheral blood ; in the malignant type, for example, the
process of sporulation is practically confined to the former.
In addition to these forms which are part of the ordinary
asexual cycle, there are derived from the amoebulse other forms,
which are called gametocytes, or sexual cells. These remain
unaltered during successive attacks of pyrexia, and undergo no
further change until the blood is removed from the human body.
In the simple tertian and quartan fevers (vide infra] the gameto-
cytes resemble somewhat in appearance the fully developed
amcebulas before sporulation, whereas in the malignant type they
have a characteristic crescent-like or sausage-shaped form ; hence
they are often spoken of as " crescentic bodies."
The various forms of the parasite seen in the human blood
may now be described more in detail.
1. The Enhoemospores (Lankester) or Merozoites are the
youngest and smallest forms resulting from the segmentation of
the adult amoebula — sporocyte or schizont. They are of round
or oval shape and of small size, usually not exceeding 2 //. in
diameter; the size, however, varies somewhat in the different
types of fever. A nucleus and peripheral protoplasm can be
distinguished (Fig. 159). The former appears as a small
rounded body which usually remains unstained, but contains a
minute mass of chromatin which stains a deep red with the
Romano wsky method ; the peripheral protoplasm is coloured
fairly deeply with methylene-blue. The spores show little or
no amoeboid movement ; at first free on the plasma, they soon
attack the red corpuscles, where they become the intra-corpuscular
amcebulre. If the blood, say in a mild tertian case, be examined
in the early stages of pyrexia, one often finds at the same time
sporulating forms, free spores, and the young amcebulas within
the red corpuscles.
2. Intra-corpuscular Amoebulce or Trophozoites. — These include
the parasites which have attacked the red corpuscles ; they are at
first situated on the surface of the latter but afterwards penetrate
their substance. They usually occur singly in the red corpuscles,
but sometimes two or more may be present together. The
youngest or smallest forms appear as minute colourless specks,
of about the same size as the spores. As seen in fresh blood,
they exhibit more or less active amoeboid movement, showing
marked variations in shape. The amount and character of the
524 MALARIAL FEVEK
amoeboid movement varies somewhat in different types of fever.
As they increase in size, pigment appears in their interior as
minute dark brown or black specks, and gradually becomes
more abundant (Figs. 155, 156). The pigment may be
scattered through their substance, or concentrated at one or
more points, and often shows vibratory or oscillating movements.
This pigment is elaborated from the haemoglobin of the
red corpuscles, the parasite growing at the expense of the latter.
The red corpuscles thus invaded may remain unaltered in
appearance (quartan fever), may become swollen and pale (tertian
fever), or somewhat shrivelled and of darker tint (malignant
fever). In stained specimens a nucleus may be seen in the
parasite as a pale spot containing chromatin which may be
arranged as a single concentrated mass or as several separated
granules, the chromatin being coloured a deep red by the
Romanowsky method. The protoplasm of the parasite, which
is coloured of varying depth of tint with methylene-blue, shows
great variation in configuration (Fig. 156). The young parasites
not unfrequently present a "ring-form," a portion of the red
corpuscle being thus enclosed by the parasite. These ring-forms
are met with in all the varieties of the parasite, but they are
especially common in the case of the malignant parasite, where
they are of smaller size and of more symmetrical form than in
the others (Fig. 160); the pigment is usually collected in a
small clump at one side.
Within the red corpuscles the parasites gradually increase
in size till the full adult form is reached (Fig. 157). In this
stage the parasite loses its amoeboid movement more or less
completely, has a somewhat rounded form, and contains a
considerable amount of pigment. In the malignant form it only
occupies a fraction of the red corpuscle. The adult parasites
may then undergo sporulation, but not all of them do so ; some
become degenerated and ultimately break down.
3. Sporocytes or Schizonts. — In the process of schizogony
the chromatin becomes divided into a number of daughter
nuclei which are scattered through the protoplasm ; the latter
then undergoes corresponding segmentation and the small
merozoites or enhsemospores result. The pigment during the
process becomes aggregated in the centre and is surrounded by
a small quantity of residuary protoplasm. Schaudinn has found
in the case of the tertian parasite that schizogony begins by a sort
of primitive mitosis, which is then followed by simple multiple
fission. The spores or merozoites are of rounded or oval shape,
as above described, and are set free by the rupture of the
FIG. 154.
FIG. 155.
FIG. 156.
FIG. 157.
,
t*
FIG. 158. FIG. 159.
FIGS. 154-159. — Various phases of the benign tertian parasite.
Fig. 154. Several young ring-shaped amcebulae within the red corpuscles, one of the
latter enlarged and showing a dotted appearance. Fig. 155. A larger amcebula con-
taining pigment granules. Fig. 156. Two large amoebulse, exemplifying the great
variation in form. Fig. 157. Large amcebula assuming the spherical form and showing
isolated fragments of chromatin — preparatory to sporulation. Fig. 153. Sporocyte
or schi/ont, which has produced eighteen spores, each of which contains a small
collection of chromatin. Fig. 159. A number of spores which have just been set
free in the plasma, x 1000.
FIG. 160.
FIG. 161.
FIG. 162.
FIG. 163.
•
FIG. 164. FIG. 165.
FIGS. 160-165. — Exemplifying phases of the malignant parasite.
Fig. 160. Two small ring-shaped amcebulse within the red corpuscles. Fig. 161. A
"crescent" or gamete showing the envelope of the red corpuscles; also an amoebula.
Figs. 162-165 illustrate the changes in form undergone by the crescents outside the
body. In the interior of the spherical form in Fig. 164 evidence of the flagella can be
seen. Fig. 165. A male gametocyte which has undergone exflagellation, showing the
thread-like microgametes or spermatozoa attached at the periphery. xlOOO. (The
figures in this plate are from preparations kindly lent by Sir Patrick Hanson.)
FORMS OF THE MALARIAL PARASITE 527
envelope of the red corpuscle. The pigment also becomes free
and may be taken up by leucocytes. The number and arrange-
ment of the spores within the sporocyte vary in the different
types. In the quartan there are 6-12, and the segmentation is
in a radiate manner, giving rise to the characteristic daisy-head
appearance; in the tertian they number 15-20 or more, and
have a somewhat rosette-like arrangement (Fig. 158) ; in the
malignant there are usually 6-12 spores of small size and
somewhat irregularly arranged.
Gametocytes. — As stated above, these are sexual cells which
are formed from certain of the amcebulae, and which undergo
no further development in the human subject. In the mild
tertian and quartan fevers they are rounded and resemble some-
what the largest amcebulae. The female cells, macrogametocytes,
are of large size, measuring up to 1 6 ft in diameter ; they con-
tain coarse grains of pigment, and the protoplasm stains somewhat
deeply with methylene-blue. The male cells, microgametocytes,
are smaller, and the protoplasm stains faintly ; the nucleus,
generally in the centre, is rich in chromatin. In the malignant
fevers the gametocytes have the special crescentic form mentioned
above. They measure 8-9 ^ in length, and occasionally a fine
curved line is seen joining the extremities on the concave aspect,
which represents the envelope of the red corpuscle (Fig. 161).
They are colourless and transparent, and are enclosed by a
distinct membrane ; in the central part there is a collection of
pigment and granules of chromatin. It is stated that the male
crescents can be distinguished from the female by their appear-
ance. In the former the pigment is less dark and more scattered
through the cell, and there are several granules of chromatin ;
in the latter the pigment is dark and concentrated, often in a
small ring, and there are one or two masses of chromatin in the
centre of the crescent. According to the Italian observers the
early forms of the crescents are somewhat fusiform in shape and
are produced in the bone-marrow. The fully developed crescents
do not appear in the blood till several days after the onset of
the fever, and they may be found a considerable time after the
disappearance of the pyrexial attacks. They are also little, if at
all, influenced by the administration of quinine.
It is well known that after a patient has apparently recovered
from malarial fever a relapse may take place without fresh
infection occurring, and Schaudinn has published interesting
observations bearing on this point. He has found that the
macrogametocyte of tertian fever may by a process of partheno-
genesis give rise to merozoites, which in their turn infect the red
528 MALARIAL FEVER
corpuscles and start the cycle again. As described and figured
by him, the chromatin of the macrogametocyte divides first into
two portions, one of which is smaller and stains more deeply
than the other. This more deeply-staining portion then divides,
and the protoplasm becomes segmented as in ordinary schizogony,
and a young brood of parasites results. The more faintly-staining
chromatin along with part of the protoplasm breaks up and
disappears.
The Cycle in the Mosquito. — As already explained, this
starts from the gametocytes. After the blood is shed, or after
it is swallowed by the mosquito, two important phenomena
occur, viz. (a) the full development of the sexual cells or
gametocytes, and (b) the impregnation of the female. If the
blood from a case of malignant infection be examined in a moist
chamber, preferably on a warm stage, under the microscope,
both male and female gametocytes may be seen to become oval
and afterwards rounded in shape (Figs. 162-164). Thereafter,
in the case of the male cell, a vibratile or dancing movement of
the pigment granules can be seen in the interior, and soon
several flagella-like structures shoot out from the periphery
(Fig. 165). They are of considerable length but of great fineness,
and often show a somewhat bulbous extremity. By the
Romanowsky method they have been found to contain a delicate
core of chromatin, which is covered by protoplasm. They
represent the male cells proper, that is, they are sperm-cells or
spermatozoa ; they are also known as microgametes. They
become detached from the sphere and move away in the
surrounding fluid. The female cell also assumes the rounded
form, and maturation- takes place by the giving off of part of
the nuclear chromatin. Impregnation occurs by the entrance of
a microgamete, the chromatin of the two cells afterwards
becoming fused. Impregnation was first observed by MacCallum
in the case of halteridium, and he found that the female cell
afterwards acquired the power of independent movement or
became a "travelling vermicule." He also observed the
impregnation of the malignant parasite. The fertilised female
cell is now generally spoken of as a zygote or ookinete.
It has been established that the phenomena just described
occur within the stomach of the mosquito, and that the fertilised
cell or zygote penetrates the stomach wall and settles between
the muscle fibres ; on the second day after the mosquito has
ingested the infected blood small rounded cells about 6-8 // in
diameter and containing clumps of pigment may be found in
this position. (It was in fact the character of the pigment
VARIETIES OF THE MALARIAL PARASITE 529
which led Ross to believe that he had before him a stage in the
development of the malarial parasite.) A distinct membrane
called a sporocyst forms around the zygote, and on subsequent
days a great increase in size takes place, and the cysts come to
project from the surface of the stomach into the body cavity.
The zygote divides into a number of cells called blastophores or
sporoblasts, and these again divide and form a large number of
filiform cells which have a radiate arrangement; these were
called by Ross "germinal rods," but are now usually known as
sporozoites or exotospores (in contradistinction to the enhaemospores
of the human cycle). The full development within the sporocyst
occupies, in the case of proteosoma, about seven days, in the
case of the malarial parasites a little longer. When fully
developed the cyst measures about 60 /* in diameter, and appears
packed with sporozoites. It then bursts, and the latter are set
free in the body cavity. A large number settle within the
large veneno-salivary gland of the insect, and are thus in a
position to be injected along with its secretion into the human
subject. The sporozoites enter red corpuscles and become
amcebulse as above described. Daniels found that in the case
of the malignant parasite an interval of twelve days at least
intervened between the time of feeding the mosquito and the
appearance of the sporozoites in the gland.
It will thus be seen that in the human subject the parasite
passes through an indefinite number of regularly recurring asexual
cycles, with the giving off of collateral sexual cells, and that in
the mosquito there is one cycle which may be said to start with
the impregnation of the female gamete.
Varieties of the Malarial Parasite. — The view propounded
by Laveran was that there is only one species of malarial parasite,
which is polymorphous, and presents slight differences in
structural character in the different types of fever. It may,
however, now be accepted as proved that there are at least three
distinct species which infect the human subject. Practically all
are agreed as to a division into two groups, one of which
embraces the parasites of the milder fevers — " winter-spring "
fevers of Italian writers — there being in this group two distinct
species, for the quartan and tertian types respectively ; whilst
the other includes the parasites of the severer forms — " sestivo-
autumnal " fevers, malignant or pernicious fevers of the tropics,
or irregularly remittent fevers. There is still doubt as to
whether there are more than one species in this latter group.
Formerly Italian writers distinguished (1) a quotidian ; (2) a
non-pi gmented quotidian ; and (3) a malignant tertian parasite,
34
530 MALARIAL FEVER
though the morphological differences described were slight.
Further observations have, however, thrown doubt on this dis-
tinction, and the evidence rather goes to show that there is a
single species. Opinion also varies as to the cjcle of this
parasite ; according to some observers it is twenty-four hours,
according to others forty-eight hours, though it is generally
admitted that variations occur. The fever is often of an
irregular type and multiple infection is probably common.
Although the question cannot be considered as finally settled, we
shall speak of three species of human parasites. The zoological
position may be shown by the following scheme, generally
followed by English writers, the terminology being chiefly that
of Grassi and Feletti : —
Family : H^MAMCEBID^ (Wasielewski).
Genus I. Hsemamoeba. The mature gametes resemble in form the
schizonts before segmentation lias occurred.
Species 1. Ncemamceba Danilewski or halteridium.
Parasite of pigeons, crows, etc.
Species 2. Hcemamceba relida or proteosoma.
Parasite of sparrows, larks, etc.
Species 3. fTcemamceba malarice.
Parasite of quartan fever of man.
Species 4. ffcemamceba vivax.
Parasite of tertian fever of man.
Genus II. Haemomenas. The ganietocytes have a special crescentic
form.
Species : Hcemomenas prcecox.
Parasite of malignant orsestivo-autumnal fever of man.
In addition there are other species belonging to the same
family of blood parasites, which infect frogs, lizards, bats, etc.,
especially in malarial regions.
We shall now give the chief distinctive characters of the
three human parasites.
1. Parasite of Quartan Fever, — The cycle of development in
man is seventy-two hours, and produces pyrexia every third clay ;
double or triple infection may, however, occur. In fresh speci-
mens of blood the outline is more distinct than that of the tertian
parasite, and amceboid movement is less marked. Only the
smaller forms show movement, and this is not of active character.
The infected red corpuscles do not become altered in size or
appearance, and the pigment within the parasite is in the form
of coarse granules, of dark brown or almost black colour. The
VARIETIES OF THE MALARIAL PARASITE 531
fully developed schizont has a "daisy-head" appearance,
dividing by regular radial segmentation into six to twelve
merozoites which, on becoming free, are rounded in form.
2. The Parasite of Mild Tertian Fever. — The cycle of develop-
ment is completed in forty-eight hours, though a quotidian type
of fever may be produced by double infection. The amoebulae
have a less refractile margin than in the quartan type, and are
thus less easily distinguished in the fresh blood ; the amoeboid
movements are, however, much more active, while longer and
more slender processes are given off. The infected corpuscles
become swollen and pale, and may show deeply stained points
by the Romahowsky method — " Schiiffner's dots." The pig-
ment within the parasite is fine and of yellowish-brown tint.
The mature schizont is rather larger than in the quartan, has
a rosette appearance, and gives rise to fifteen to twenty merozoites,
though sometimes even more occur ; these have a somewhat oval
shape.
In both the quartan and tertian fevers all the stages of
development can be readily observed in the peripheral blood.
3. The Parasite of Malignant or ^Estivo-autumnal Fever, or
Tropical Malaria. — The cycle in the human subject probably
occupies forty-eight hours, though this cannot be definitely stated
to be always the case (vide supra). The amoebulae in the red
corpuscles are of small size, and their amoeboid movements are
very active ; they often, however, pass into the quiescent ring
form (Fig. 160). The pigment granules, even in the larger
forms, are few in number and very fine ; the infected red
corpuscles have a tendency to shrivel and assume a deeper or
coppery tint. The fully developed schizont occupies less than
half the red corpuscle, and gives rise to usually from six to
twelve merozoites, somewhat irregularly arranged and of minute
size. Schizogony takes place almost exclusively in the internal
organs, spleen, etc., so that, as a rule, no sporocytes can be
found in the blood taken in the usual way. The proportion
of red corpuscles infected by the amcebula3 is also much larger
in the internal organs. The gametes have the crescentic form,
as already described.
Cases of infection with the malignant parasite sometimes
assume a pernicious character, and then the number of organisms
in the interior of the body may be enormous. In certain fatal
cases with coma the cerebral capillaries appear to be almost
filled with them, many parasites being in process of sporulation ;
and in so-called algid cases, characterised by great collapse, a
similar condition has been found in the capillaries of the
532 MALARIAL FEVER
omentum and intestines. The process of blood destruction,
present in all malarial fevers, reaches its maximum in the
malignant class, and the brown or black pigment elaborated by
the parasites— in part after being taken up by leucocytes, chiefly
of the mononuclear class — becomes deposited in various organs,
spleen, liver, brain, etc., especially in the endothelium of
vessels and the peri vascular lymphatics. In the severer forms
also brownish-yellow pigment is apparently derived from liberated
haemoglobin, and accumulates in various parts, especially in the
liver cells ; most of this latter gives the reaction of haemosiderin.
General Considerations. — The development of the malarial
parasites in the mosquito and infection of the human subject
through the bites of this insect, have, by the work of Ross and
others, as detailed above, become established scientific facts.
These facts, moreover, point to certain definite methods of pre-
vention of infection, which have to a certain extent already been
practically tested. The extensive observations recently carried
out go to show that all the mosquitoes which act as hosts of the
parasite belong to the genus anopheles] of these there are a
large number of species, and in at least eight or nine the
parasite has been found. Some of these anopheles occur in
England, especially in regions where malaria formerly prevailed.
The opportunity for infection from cases of malaria returning
from the tropics to this country thus exists, and such infection
has occurred. The breeding-places of the insects are chiefly in
stagnant pools and other collections of standing water, and
accordingly the removal, where practicable, by drainage of
such collections in the vicinity of centres of population, and
the killing of the larvae by petroleum sprinkled on the water,
have constituted one of the most important measures. This
procedure has been carried out in various places with marked
success. Another measure is the protection against mosquito
bites by netting, it being fortunately the habit of the anopheles
to rarely become active before sundown. The experiments of
Sambon and Low in the Campagna proved that individuals
using these means of protection may live in a highly malarial
district without becoming infected. The administration of
quinine to persons living in highly malarial regions, in order to
prevent infection, has also been recommended and carried out.
In the tropics the natives in large proportion suffer from malarial
infection, and one would accordingly expect that infection of the
mosquitoes in the neighbourhood of native settlements will be
common. This has been found to be actually the case, and it
has accordingly been suggested that the dwellings of whites
THE PATHOLOGY OF MALARIA 533
should as far as possible be at some distance from the native
centres of population.
So far as is known none of the lower animals have been found
to take the place of man as intermediate host to the parasite of
malaria, but the possibility of such being the case cannot be as
yet definitely excluded. On the death of infected mosquitoes
the exotospores or sporozoites will become set free, and therefore
theoretically there is a possibility that they may enter the
human subject by inhalation or by some other means. We have
no facts, however, to show that this really occurs, and the
evidence already obtained establishes the bites of mosquitoes as
the most important if not the only mode of infection.
It may also be mentioned as a scientific fact of some interest,
though not bearing on the natural modes of infection, that the
disease can also be communicated from one person to another by
injecting the blood containing the pa.rasites. Several experi-
ments of this kind have been performed (usually about J to 1 c.c.
of blood has been used), and the result is more certain in
intravenous than in subcutaneous injection. In such cases there
is an incubation period, usually of from seven to fourteen days,
after which the fever occurs; the same type of fever is re-
produced as was present in the patient from whom the blood
was taken.
The Pathology of Malaria. — While much work has been
done on the malarial parasite, relatively less attention has been
directed to the processes by which it produces its pathogenic
effects. It may be said that the organisms are not always
equally prevalent in the circulating blood, and probably at
certain stages tend to be confined in the solid organs ; thus they
may be scanty at the height of the paroxysm. Some of the
pathogenic effects are probably associated with particular stages
in the life cycle. Thus the pyrexia occurs when the stage of
sporulation is actively in progress. No opinion can be stated,
however, as to the cause of the fever, — whether it is due to a
toxic process or to general disturbance of metabolism. We can
better explain the anaemia which is so pronounced in cases where
the disease is of long standing, and which is due to the actual
destruction of red blood corpuscles. The parasite in its sojourn
in these cells absorbs their pigment and thus destroys their
function ; this is further evidenced by the activity displayed by
the red marrow in its attempts to make good the loss sustained
by the blood. One of the most interesting events in malaria,
and one that links it with bacterial infections, is the reaction of
the colourless cells of the blood. It has been shown that during
534 MALARIAL FEVER
the apyrexial stages the total number of leucocytes may be
diminished but that there is always an increase of the mono-
nuclear cells, these frequently numbering 20 per cent of the
whole, and sometimes even outnumbering the polymorphs.
This is such an important feature that in cases where the
parasites themselves cannot be demonstrated in the blood, the
mononuclear reaction, along with the presence of pigment in the
mononuclear cells (due to phagocytosis of pigmented parasites),
has been taken as evidence that the case is really one of malaria.
The mononuclear reaction is specially interesting from the fact
that in other protozoal diseases an activity of the same elements
has been observed.
The question of the possibility of immunity to malaria being
developed naturally arises, and this is specially interesting in
the light of the leucocytic reaction which we have seen must be
looked on as an element in immunity against bacterial infection.
With regard to Europeans developing immunity it is difficult to
speak. In such a malaria-stricken region as the West Coast of
Africa the death-rate in residents of more than four years'
standing is less than in the previous years, but this may be due
to the survival of the more resistant immigrants. But there can
be little doubt that malaria in the negro is a much less serious
condition than in the European. Koch from his observations in
New Guinea attributes this to the infection of the native children
leading to the development of immunity in the adult community.
He found, what has been independently noted by Stephens and
Christophers in West Africa, that the greater number of the
children harboured malarial parasites in their blood. The wide-
spread presence of parasites in children might appear to preclude
the immunity of the adult being due to survival of the most
resistant, but the infant mortality in these regions may be very
high, and such a survival may be the real explanation. On the
other hand, Koch states that while an immunity appears to exist
in native adults in malarial districts, this is only true of those
born in the locality ; natives coming from neighbouring non-
malarial districts into the malarial region being liable to contract
the disease. At present it must be held that the facts available
do not enable us to determine the relative parts played by the
development of artificial immunity on the one hand, and the
existence of a natural immunity on the other, in apparent un-
susceptibility to malaria.
Our knowledge on the relationship of blackwater fever to
malaria is also in an unsatisfactory condition. Blackwater fever
is a condition often occurring, especially in Europeans, in tropical
METHODS OF EXAMINATION 535
countries. It is characterised by pyrexia, darkly-coloured urine,
— the colour being due to altered haemoglobin pigment, — delirium
and collapse, frequently ending in coma and death. By some
the condition has been looked on as a separate disease, by others
as the terminal stage of a severe malaria. With regard to the
former view no special parasite has yet been demonstrated.
Stephens sums up the evidence for the second view by saying
that malaria, apart from the occurrence of blackwater fever, is a
relatively non-fatal disease, that in the great majority of cases
there is direct or indirect evidence of the subject of the condition
having suffered from repeated attacks of malaria, that while in
all cases there must be an agent at work causing hadinolysis,
there is evidence that in many cases there is the possibility of
that agent being quinine. This last point is of great interest.
It has been shown that in certain individuals the taking of this
drug is sometimes followed by hsemoglobinuria. The conditions
under which this occurs are unknown, and in the case of black-
water patients, neither is the serum haemolytic for normal
corpuscles, nor do the red corpuscles seem to be specially
sensitive to haemolysis by quinine, in fact, the latter do not
appreciably differ from ordinary red cells. The whole subject
of the pathology of the condition is thus very obscure.
Methods of Examination. — The parasites may be studied by
examining the blood in the fresh condition, or by permanent
preparations. In the former case, a slide and cover-glass having
been thoroughly cleaned, a small drop of blood from the finger
or lobe of the ear is caught by the cover-glass, and allowed to
spread out between it and the slide. It ought to be of such a
size that only a thin layer is formed. A ring of vaseline is
placed round the edge of the cover-glass to prevent evaporation.
For satisfactory examination an immersion lens is to be preferred.
The amoaboid movements are visible at the ordinary room
temperature, though they are more active on a warm stage.
With an Abbe condenser a small aperture of the diaphragm
should be used.
Permanent preparations are best made by means of dried
films. A small drop of blood is allowed to spread itself out
between two cover-glasses, which are separated by sliding the
one on the other. The films are then allowed to dry. A very
good method is that of Manson, who catches the drop of blood
on a piece of gutta-percha tissue (a piece of cigarette-paper also
does well), and then makes a film on a clean slide by drawing
the blood over the surface. The dried films are then fixed by
one of the methods already given (p. 87), or by placing in
536 MALARIAL FEVER
absolute alcohol for five minutes (Manson). The films thus
prepared and fixed may be stained for two or three minutes in a
saturated watery solution of methylene blue or in carbol-thionin-
blue (p. 98) ; the solutions must be carefully filtered (especially
the latter), and the films must be washed well after staining.
They are then dried and mounted in balsam. In the case of
thionin-blue, sharper results are obtained by dehydrating in
alcohol and clearing in xylol before mounting. The best results
are, however, obtained by one of the Romanowsky methods as
described on p. 106.
The fact that in many cases the parasites may be few in
number led Ross to devise a method for making their recognition
more easy by using blood films of unusual thickness. Here
about as much blood as is used in a haemoglobin determination
(20 c.mm.) is taken on a slide, and, being spread out only so
much as to occupy the area of an ordinary cover-glass, is allowed
to dry. There is then dropped on it by means of a glass rod a
little of the watery eosin used in making up the Romanowsky
dye (vide p. 106). This is allowed to act for about a quarter
of an hour, and then very gently washed off with distilled water.
The Romanowsky methylene-blue solution is then applied for a
few seconds and also carefully washed off, and the preparation
dried and mounted. The haemoglobin of the red corpuscles is
washed out by the eosin solution, and the smaller forms of the
malarial parasite stand out as round circles containing the char-
acteristic chromatin dots ; and in consequence of the greater
number present in an area of unit size as compared with an
ordinary preparation their recognition is very easy. For the
large forms of the parasite Ross has found it useful to make
such a film and, haemolysing the red cells with distilled water,
to examine it unstained. The presence of pigment in the para-
sites enables these to be readily seen.
APPENDIX D.
AMCEBIC DYSENTERY.
IN a previous chapter it has been pointed out that the term
"dysentery" has been applied to a number of conditions of
different etiology, and the relations of bacteria as causal agents
have been there discussed (vide p. 346). We shall here consider
that variety of tropical dysentery which is believed to be due to
an amoeba, and hence often known as amoebic dysentery.
Amongst the early researches on the relation of organisms to
dysentery probably the most important are those of Losch, who
noted the presence and described the characters of amoebae in
the stools of a person suffering from the disease, and considered
that they were probably the causal agents. Further observations
on a more extended scale were made by Kartulis with confirma-
tory results, this observer finding the same organisms also in
liver abscesses associated with dysentery. Councilman and
Lafleur, working in Baltimore, showed that this variety of
dysentery can be distinguished from other forms, not only by
the presence of amoebae but also by its pathological anatomy.
The intestinal lesions, to which reference is made below, are of a
grave character, mortality is relatively high, and recovery, when
it occurs, is protracted on account of the extensive tissue
changes. The subject was, however, complicated by the fact
that a similar organism — the amoeba coli — had been previously
found in the intestine in normal conditions and in other
diseases than dysentery (by Cunningham and Lewis and others),
and additional research confirmed these results. It may now be
regarded as established that the amoeba of dysentery and the
common amoeba of the colon are two distinct species. This has
especially been shown by the researches of Schaudinn, who has
given the terms entamoeba histolytica and entamoeba coli to the
two organisms.
Entamoeba histolytica as seen in the dysenteric stools occurs
537
538 AMCEBIC DYSENTERY
in the form of rounded, oval, or pear-shaped cells, measuring
12-30 /x. in diameter. When at rest a somewhat clear, highly
refractile ectoplasm and a granular endoplasm can be distin-
guished, a feature which differentiates the organism from the
eritamoeba coli. The nucleus is rounded, or oval, and is seen
with difficulty ; its position is usually eccentric arid is sometimes
quite at the margin of the ectoplasm. In the fresh condition,
and especially when examined on a warm stage, the organism
shows very active amoeboid movements. The pseudopodia, which
are quickly protruded and retracted, are blunt and appear to be
of tough consistence (Fig. 166), a property which Schaudinn
considers of importance as enabling the organism to penetrate
the mucous membrane, etc. The amoebic movements are often
m
Flo. 166. — Anioebse of dysentery.
a and b, amoeb;e as seen in the fresh stools, showing blunt amoeboid processes of
ectoplasm. The endoplasm of a shows a nucleus, three red corpuscles and numerous
vacuoles ; that of b, numerous red corpuscles and a few vacuoles.
c, an amoeba as seen in a fixed film preparation, showing a small rounded nucleus
(Kruse and Pasquale). x 600.
of an active kind and locomotion may be fairly rapid ; not
infrequently red corpuscles, bacteria, cells, etc. may be seen in
the interior. The organism usually dies and undergoes disinte-
gration in a comparatively short time after being removed from
the body ; the stools ought therefore to be examined in as fresh
a state as possible. Multiplication takes place by simple amitotic
division and also by budding. The entamceba coli is an organism
of about the same size. When at rest it shows no differentiation
into ectoplasm and endoplasm, and the nucleus, usually situated
in the centre, shows a highly refractile membrane with chromatiri
masses scattered in the interior. During amoeboid movement
some delicate processes of ectoplasm come into view.
DISTRIBUTION OF THE AMCEB^E 539
Both organisms have now been shown to pass into a resting
stage with formation of cysts, the character and mode of forma-
tion of which are markedly different in the two cases. The cyst
formation of the entamoeba histolytica is specially seen when the
disease is in process of cure and the stools are beginning to have
a less fluid character. In the earliest stage of the change the
nuclear membrane becomes broader and fades into the proto-
plasm, whilst the chromatin becomes dispersed through the
entoplasm in the form of small chromidia. Buds then form
on the surface and into these some of the chromatin passes.
Around these buds concentric striation can be seen, and then
a hyaline cyst wall is formed, which is highly refractile in
character. The cyst then becomes separated from the rest of the
cell. Several cysts which measure 2-7 /x in diameter may be
formed from the same amoeba, and the remnant of the cell under-
goes disintegration. These cysts, as will be shown below, repre-
sent a resting stage with high powers of resistance to external
agencies, and are concerned in producing infection of another
subject. The cellular changes in the encysting of the entamceba
coli have also been worked out by Schaudinn. They are of
a somewhat complicated character, involving the formation
of reduction bodies and copulation of nuclei, but the ultimate
result is the formation of a fairly large cyst, which contains
eight small cells. The process of cyst formation accordingly in
the two organisms is of a widely different character.
Cultivation. — Various attempts have been made to cultivate
the amoeba of dysentery, and Kartulis considered that he obtained
growth in straw infusions. Recently Lesage has announced that
he has obtained cultures on plates of agar which had been
washed in wTater for eight days. Both the vegetative and the
cystic forms were used for inoculation. In some cases a growth
of a colon bacillus was made on the agar and afterwards removed,
this procedure interfering with the development of such bacilli
present in the material used for inoculation. The plates were
kept in the sloped position, and the inoculations were made in
the lower part ; the amoeba? moved to the upper part, where they
were got in pure .condition. He succeeded in obtaining cultures
in seven out of thirty cases, and in some instances cultivated the
organisms for more than sixty generations. The amoebae multi-
plied by simple amitotic division, and in certain cases produced
small cysts. These cysts, as described and figured by him,
correspond in all important respects with the changes observed
by Schaudinn in dysenteric stools.
Distribution of the Amoebae. — As already stated, they are
540 AMCEBIC DYSENTERY
usually found in large numbers in the contents of the large
intestine, in tropical amoebic dysentery. They also, however,
penetrate into the tissues, where they appear to exert a well-
marked action. In this disease the lesions are chiefly in the large
intestine, especially in the rectum and at the flexures, though
they may also be present in the lower part of the ileum. At
first there are seen local swellings on the mucous surface, chiefly
clue to a sort of inflammatory gelatinous oedema with little
leucocytic infiltration; soon, however, the mucous membrane
becomes partially ulcerated, more or less extensive necrosis of
the subjacent tissues oc-
curs, and gangrenous
sloughs result. The ulcers
thus come to have irregular
and overhanging margins,
and the excavation below
is often of wider extent
than the aperture in the
mucous membrane. The
amoebae are found in the
mucous membrane when
ulcers are being formed,
but their most character-
istic site is beyond the
ulcerated area, where they
may be seen penetrating
FIG. 167. — Section of wall of liver abscess, deeply into the submucous,
showing an amoeba of spherical form with and even into the muscular
vacuolated protoplasm. From a case T .-, ...
published by Surgeon-Major D. G. C0ats' In these Positions
Marshall, x 1000. they may be unattended
by any other organisms,
and the tissues around them show oedematous swelling and more
or less necrotic change without much accompanying cellular
reaction, beyond a certain amount of swelling and proliferation
of the connective-tissue cells. This action of the amoebae on
the tissues explains the character of the ulcers as just de-
scribed. These lesions are considered to be characteristic of
amoebic dysentery.
As a complication of this form of dysentery, liver abscesses
are of comparatively common occurrence. They are usually
single and of large size, sometimes there are more than one, and
occasionally numerous small ones may be present. The contents
are usually a thick pinkish fluid of somewhat slimy consistence
and are largely constituted by necrosed and liquefied tissue with
EXPERIMENTAL INOCULATION 541
admixture of blood in varying amount. Microscopic examination
shows chiefly necrosed and granular cells and debris resulting
from their disintegration, whereas ordinary pus corpuscles are
scanty or may be practically absent. In such abscesses associated
with dysentery the amoebae are usually to be found, and not
infrequently are the only organisms present, no cultures of
bacteria being obtainable by the ordinary methods (Fig. 167).
They are most numerous at the spreading margin, and this
probably explains a fact pointed out by Manson, that examination
of the contents first removed may give a negative result, while
they may be detected in the discharge a day or two later. The
action here on the tissues is of an analogous nature, namely, a
necrosis with softening and partial liquefaction, attended by
little or no suppurative change. The amoebae have also been
found in the sputum when a liver abscess has ruptured into the
lung, as not very infrequently happens. Kartulis records two
cases of brain abscess occurring secondarily to dysentery in
which numerous amoebae were present.
Experimental Inoculation. — The anatomical changes in
dysentery, as above described, gives strong presumptive evidence
as to the causal relationship of the amoebae, and practically con-
clusive evidence is afforded by animal experiments. Dysentery
occurs occasionally in animals, but it is of rare occurrence. The
disease sometimes results in the dog by experimental inoculation
with dysenteric material. Kartulis, for example^ records two
cases, in one of which liver abscess was present. Cats are,
however, found to be more susceptible, especially young animals.
Dysenteric changes have been produced in this animal by
Kartulis, Kruse and Pasquale, and others. The method generally
adopted is the introduction of a small quantity of mucus from
a dysenteric case into the rectum. The resulting disease is of
an acute character, and sometimes leads to a fatal result. The
changes in the large intestine resemble those found in the
human disease, and microscopic examination shows the amoebae
penetrating the wall of the bowel in the characteristic manner.
Kruse and Pasquale obtained corresponding results when the
material from a liver abscess, containing amoebae without any
other organisms, was injected. Quincke and Koos obtained no
effects when the amoebae were administered by the mouth, but
they obtained a fatal result in two out of four cases when the
cyst-like forms were given. They also found that the cysts,
unlike the amoebae, were still present even after the material
had been kept for two or three weeks. Extremely important
confirmatory evidence with regard to infection by the cysts has
542 AMCEBIC DYSENTERY
been supplied by experiments of Schaudinn. Dysenteric material
was obtained from China, and portions of it which were found
to contain the cysts were thoroughly dried. Some of this
material was given with food to cats by the mouth, and typical
dysentery resulted, the amoebae being found in the stools. No
results follow when the material ingested merely contains the
vegetative form of the organism, as it is readily destroyed in the
contents of the stomach.
Investigations with regard to entamoeba coli show that it is
a harmless organism and that it is frequently present in the
intestines of healthy individuals. Schaudinn found that in East
Prussia as many as 50 per cent of the population were infected
with it. The administration of the amoebae, or of the cysts by
the methods mentioned above, produces no result in animals.
It has, however, been shown that when the eight-celled cysts
are swallowed by persons who are free from the parasite, the
entamceba coli appears in the large intestine in a comparatively
short period of time. It accordingly appears that in the case of
both organisms it is the cysts alone which give rise to infection.
From the above facts, all of which have received ample
confirmation, there can be no doubt that the amoeba described
is the cause of the form of dysentery with which it is associated.
We are still ignorant whether the organism has any life history
outside the body, but it has been shown that the cysts have
high powers of endurance and almost certainly form the means
of infection when they are swallowed in drinking-water or in
food. It is also of importance to note that the serum of patients
suffering from amoebic dysentery gives no agglutinating reaction
with Shiga's bacillus of dysentery (vide p. 348).
Methods of Examination. — The faeces in a case of suspected
dysentery ought to be examined microscopically as soon as
possible after being passed, as the amoebae disappear rapidly,
especially when the reaction becomes acid. A drop is placed
on a slide without the addition of any reagent, a cover-glass is
placed over it but not pressed down, and the preparation is
examined in the ordinary way or on a hot stage, preferably by
the latter method, as the movements of the amoebae become more
active, and it is difficult to recognise them when they are at rest.
Hanging-drop preparations may also be made by the methods
described. Dried films are not suitable, as in the preparation of
these the amoebae become broken down ; but wet films may be
fixed with corrosive sublimate or other fixative (vide p. 88). In
sections of tissue the amoebae may be stained by methylene-blue,
by safranin, by haematoxylin and eosin, etc. Benda's method of
METHODS OF EXAMINATION 543
staining with safranin and light-green is also a very suitable one.
Sections are stained for several hours in a saturated solution
of safranin in aniline oil water (p. 98), they are then washed
in water and decolorised in a \ per cent solution of light-green
in alcohol till most of the safranin is discharged, the nuclei,
however, remaining deeply stained. In this method the nuclei
of the amoebae are coloured red (like these of the tissue cells),
the protoplasm being of a purplish tint.
APPENDIX E.
TRYPANOSOMIASIS— KALA-AZAR—PIROPLASMOSIS.
THE PATHOGENIC TRYPANOSOMES.
THE trypanosomata are protozoal organisms belonging to the
sub-class Flagellata, and during the last decade several members
of the genus have come to be recognised as living in the blood
and tissues in various animals and as causing important disease
conditions. As long ago as 1878 the Trypanosoma Leivisi was
observed infesting the blood of rats, and it has been found to be
sometimes capable of causing death. Other diseases in which
similar organisms have been found are Surra, which occurs in
cattle, horses, and camels in India, and which is associated with
the Tr. Evansi ; Dourine, a condition affecting horses in especially
the Mediterranean littoral (Tr. equiperdum or Rougeti) ; Mai de
Caderas, a disease of South American horses (Tr. equinum or
Elmassiani) ; Tse-tse Fly Disease or Nagana, affecting horses
and herbivora in South Africa (Tr. Brucei) ; trypanosomiasis of
African cattle (Tr. Theileri) ; and — most important from the
human standpoint — the trypanosomiasis and sleeping sickness
of West and Central Africa associated with the Tr. gambiense
and Tr. ugandense, which are now believed to be the same
organism. These diseases present many general resemblances to
one another. They tend to be characterised by wasting, cachexia,
anaemia, fever often of an intermittent type and irregular oedemas,
and often lead to a fatal result. In many cases the infective
agent is conveyed from a diseased to a healthy animal by the
agency of blood-sucking insects.
General Morphology of the Trypanosomata. — If a drop of
blood containing trypanosomes be examined, the organism will
be seen to be a fusiform mass of protoplasm which at one end
passes into a pointed flagellum. In the living condition the
trypanosome is usually actively motile by an undulatory move-
544
THE PATHOGENIC TRYPANOSOMES 545
ment of its protoplasm and a lashing of the flagellum. The size
varies, but those mentioned above are about 30 //. long and about
1 '5 to 3 //, broad. From the fact that in progression the flagellum
is in front, the flagellated end is denominated the anterior end
of the organism. It is stated that the method of examining
the fresh blood merely allowed to spread itself out in a
fairly large drop beneath a cover-glass is more likely to reveal
the presence of trypanosomes, if these are present in small
numbers, than is the examination of stained specimens; but the
minuter structure of the organisms can best be studied in dried
preparations stained by Romanowsky dyes such as those of
Leishman or Giemsa.
For staining trypanosomata (or the Leishman-Donovan bodies) in
sections so as to bring out the chromatin structures, Leishman recom-
mends the following method: — Sections of 5 //. thickness are made and
carefully fixed on slides. The paraffin is very thoroughly removed by
melting it before applying the first xylol, and then washing with
alternate baths of alcohol and xylol three or four times. The last
alcohol is thoroughly washed off by distilled water, and the excess of
water is removed with cigarette paper. A drop of fresh blood serum is
then placed on the preparation and allowed to soak in for five minutes.
The excess is removed by blotting, and the remainder is allowed to
dry on the section, which is now treated with a mixture of two parts
of Leishman's stain and three of distilled water, and placed in a Petri
dish for 1 to 1^ hours. The preparation is very deeply stained, the
nuclei being almost black and decolorisation and differentiation are
effected by alternately applying the acetic acid and caustic soda solutions
(commencing with the acid) used in the application of the stain to
ordinary histological sections (v. p. 106), the effects being carefully
watched with a low power. The essential part of the method is the
application of the blood serum, though what effect this has is not
known ; Leishman suggests that it restores the normal alkalinity of the
tissue.
In preparations stained by the above methods the protoplasm
of trypanosomata stains blue, and in some species some parts are
more intensely coloured than others. Sometimes it contains
violet-coloured granules (chromatin granules), and occasionally
there appears in it slight longitudinal striation. Two bodies are
always present in the protoplasm. Usually near the middle there
is an oval granular body staining purple, — the nucleus or macro-
nucleus, — and towards the posterior end is a minute intensely
stained purple granule known as the micronucleus or centrosome
(that this body represents the centrosome is strongly held by
Laveran from the analogy of appearances in certain spermatozoa
which closely resemble trypanosomes in structure). This micro-
nucleus is often surrounded by an unstained halo, and in its
neighbourhood, in certain species, a vacuole has been described
35
546 TRYPANOSOMIASIS
as existing ; this has been considered by some to be analogous
to the contractile vacuole present in many protozoa, and its
shape and position have been made the basis of specific dis-
tinctions ; Laveran, however, thinks it is an artefact. From
the micronucleus or from its neighbourhood there arises an
important structure in the trypanosome, — the undulatory
membrane. This is of varying breadth, has a sharp undulating
free margin, and surmounts the protoplasm of the organism
like a cock's comb; it narrows towards the anterior end
where it passes into the flagellum. Motion is chiefly effected by
the undulations of this membrane and of the flagellum. The
latter is continuous with the protoplasm of the body of the
organism ; it stains uniformly like it, except the free edge which
has the reddish hue of the chromatin. In different species
of trypanosomes variations occur in shape, in length, in breadth,
in the position of the micronucleus (and therefore in the
length of the undulating membrane), in the breadth ,of the
membrane, in the length of the free part of the flagellum, in the
shape of the posterior end, which is sometimes blunt, sometimes
sharp, and in the presence or absence of free chromatin granules
in the protoplasm.
Multiplication in the body fluids ordinarily occurs by
longitudinal, amitotic division (see Fig. 168). First of all the
micronucleus divides, sometimes transversely, sometimes longitud-
inally, then the nucleus and undulating membrane, and lastly
the protoplasm. In some species the root of the flagellum only
divides so that in the young trypanosomes the flagellum is
short and subsequently increases in length (Tr. Lewisi) ; usually
the whole flagellum takes part in the general splitting of the
organism.
In the cases of several of the trypanosomata it has been
found possible to cultivate them outside the body, the chief
work here having been done by Novy and MacNeal, who have
succeeded with the Tr. Lewisi, Tr. Evansi, and Tr. Brucei.
The most suitable medium is made as follows : —
125 grammes rabbit or ox flesh is treated with 1000 c.c. distilled
water, as in making ordinary bouillon, and there are added to the
meat extract 20 gr. Witte's peptone, 5 gr. sodium chloride, 20 gr.
agar, and 10 c.c. normal sodium carbonate. The medium is placed in
tubes and sterilised in the autoclave at 110° C. for thirty minutes. It is
cooled to 50° C. , and there is added to the medium in each tube twice its
volume of defibrinated rabbit blood, which has been prepared with all
aseptic precautions ; the tubes are allowed to set in the inclined position.
In inoculating such tubes they are placed in an upright position for a
few minutes and then the infective material is introduced.
THE PATHOGENIC TRYPANOSOMES 547
Multiplication goes on readily on such, a medium, the
organisms dividing longitudinally as in the tissues. Sometimes
very small forms result, and often these are found in rosettes
which are formed by a number of individuals arranging them-
selves in a circle with the flagella directed towards the centre
of the agglomeration. By repeated sub-cultures several of the
trypanosomata named have been kept alive for more than a year,
and when re-introduced into appropriate hosts have been found
not to have lost their infective properties.
Within recent years considerable attention has been directed
to the question of whether in the trypanosomes a sexual
cycle occurs. It cannot be said that the existence of such has
been definitely established, and we shall merely give a short
account of certain views which have been advanced. The
starting point lies in the slight differences in form which have
been observed in the organisms in the body fluids of the
vertebrate hosts. Such differences have been described in
Tr. Lewisi and Tr. Brucei by Prowazek, and in Tr. ugandense
by Minchin, and have been made the basis of a classification into
three types which are looked on as representing male, female, and
indifferent individuals. The male type is rather slender both in
body and in nucleus, the free part of the flagellum is longer than
the body and the protoplasm is free from granules ; the female
is broader, its nucleus is larger and rounder, the undulating
membrane narrower, the free part of the flagellum is shorter
than the body, and the protoplasm contains many chromatin
granules, which are looked upon as reserve food material. The
indifferent individuals present intermediate characters. All
multiply by fission as described, and the indifferent individuals
can on occasion become differentiated into male or female
forms. The females are the most hardy, and next come the
indifferent individuals. If all but the females die out, these
can undergo parthenogenesis, and representatives of all three
types can be again reproduced. The sexual cycle is represented
as occurring in the invertebrate host. In Tr. Lewisi, according
to Prowazek, this is found in the rat louse, hcematopinus
spinulosus. When this insect sucks the blood of an infected
rat, copulation occurs by the male trypanosome entering the
female near the micronucleus and the various parts of the two
individuals becoming fused. A non-flagellated ookinete results,
which, passing through a spindle-shaped gregarine-like stage,
can develop into a trypanosome in the stomach of the louse.
A resting-stage in an immature trypanosome -like form is
described as occurring between or attached to the intestinal
548 TRYPANOSOMIASIS
epithelium, and the parasite is supposed to reach the body
cavity, and ultimately the pharynx of the insect, and thus to
find the opportunity for passing into the body of a fresh host.
A still further development of the views held as to the life-history of
the trypanosomes is found in the work of Schaudinn, who investigated .
the trypanusoma noctuce found in the owl (athenc noctua), and which is
carried from bird to bird by the common mosquito (culex pipiens). In
the blood of the owl is a halteridium haemamoeba showing pigmented
male and female forms, closely corresponding to those observed by
Macallum in the crow. These, according to Schaudinn, on reaching the
mosquito's stomach undergo ordinary changes — the microgametocyte
develops microgametes, one of which fertilises the macrogamete. An
oval motile ookinete results, and in the formation of its nucleus from
the male and female,' elements a reduction of chromosomes takes place,
while the superfluous nuclear structures along with the pigment are cast
out of the cell. In these ookinetes a differentiation into male, female,
and indifferent forms can be recognised, but the important new departure
is that each can go on to develop into a trypanosome. In the indifferent
ookinete a portion of the new nucleus breaks off by a sort of hcteropolar
division, and the broken-otf part becomes the micronucleus or blepharo-
plast of the trypanosome and gives origin to the undulatory membrane.
Longitudinal division of these forms in the mosquito's intestine may
occur, and here it may be said that Schaudinn differs from other observers
in holding that on division the membrane does not split, but that one of
the individuals gets its membrane by this being laid down along the root
of that already in existence. Further, a resting-stage of the trypanosome
may occur, in which it becomes attached to the intestinal epithelium,
and losing more or less its flagellate form, may resemble a gregarine.
The female ookinete is plumper and contains more chromatin granules
in its protoplasm than the male and indifferent forms, gives rise to a
trypanosome after a more complicated division of its nucleus, is less
motile, does not reproduce itself by longitudinal fission, soon attaches
itself to the intestinal epithelium, is in virtue of the reserve material
in its protoplasm much more resistant than the male or indifferent
forms ; when these die out, as they do when, for instance, the insect is
starved, it reproduces all three forms by a process of parthenogenesis.
In the female ookinete the smaller nucleus which goes to form the
blepharoplast of the indifferent trypanosome divides into eight small
nuclei, all of which perish, and the blepharoplast and membrane are
formed by a fresh division in the large nucleus remaining. In the male
ookinete, which differs from the female in the clearness of its protoplasm,
a similar heteropolar mitosis takes place, and again eight small nuclei
are produced. These evidently represent the essentially male element,
for they persist, and each, appropriating to itself a portion of the
cellular protoplasm, detaches itself so that eight small trypanosomes are
budded off from the ookinete. This male ookinete Schaudinn holds to
be homologous with the microgametocyte occurring in the blood of the
.owl, and the small trypanosomes are similarly homologous with the
microgametes formed when the blood of the host reaches the stomach
of the mosquito. These small trypanosomes and the male trypano-
somes readily die, probably because, by a reduction process in their
genesis, the assimilative powers of the larger nucleus have been
diminished. In degenerating they often are found in the intestinal
THE PATHOGENIC TRYPANOSOMES 549
tract of the mosquito arranged in rosettes, with sometimes the anterior
ends, sometimes the posterior ends, directed towards the centre of the
rosette. The next stage of development takes place when the parasites
by the mosquito's bite reach the blood of the vertebrate host, and it is
in connection with this stage that Schaudinn's observations are very
far-reaching. The indifferent forms by this time, by repeated longi-
tudinal division, have become small. In the owl they attach them-
selves to red blood corpuscles, which they penetrate, and, assuming a
halteridium form, grow in size for twenty-four hours ; they then leave
the cells, elongate, again assume the flagellate form, move freely in the
blood till the next night, when they again enter fresh cells. This cycle
is repeated for six nigiits, the organism gaining in size with each sojourn
in a corpuscle. When the full size is thus attained, repeated longi-
tudinal division occurs, and when the smallest forms are again reached
the intra- cellular/ cycle recommences. The largest female forms prob-
ably cannot pass through the mosquito's proboscis, but when small
forms reach the owl they enter the red corpuscles. They assimilate
food material, and appear not to migrate so frequently as the indifferent
forms. They lose their capacity of assuming the free trypanosoma
form, and ultimately their capacity of migration, so that they are
found lying surrounded by the remains of the last cell they entered.
The male forms when they reach the blood of the host rapidly die, and
the microgarnetocytes are always recruited from the indifferent forms,
or from parthenogenesis of the female forms.
It may be said that according to Schaudinn the trypanosomes gain
access' to the tissues of the mosquito by perforating the intestinal wall,
and, passing through the body till they reach the wall of the pharynx,
they burst through this, and are in a position to be ejected when next
the insect bites.
Such are the views put forward by this observer on the cycle
of life-history of the Tr. noctuse. It will be recognised that
the essential point is the occurrence, both in the vertebrate and
invertebrate host, of halteridium stages alternating with those
in which the trypanosome form is assumed. It is evident that
if this were substantiated important effects would follow in our
views as to the morphology not only of the trypanosome group,
but also of that to which the malarial parasites belong.
Certain criticisms of these results have been made, especially by
Novy and MacNeal, who sought confirmatory evidence by means of their
culture method. These observers are of opinion that the appearances
described by Schaudinn were due to his dealing with a mixed infection
of the owls by trypanosomes on the one hand and haemamoebae of the
halteridium type on the other. They examined a very large number of
different species of birds, and established the fact that infection with
halteridium parasites on the one hand and with trypanosomes on the
other is extremely common, and further, that in the blood of the same
bird both halteridium and trypanosome infection could be observed.
The bird trypanosomes could be readily cultivated, and it was observed
that no cultures were obtainable from birds in which halteridia were
alone found, and further, that when a trypanosome isolated from a case
where both forms of parasite had been seen was injected into a fresh
550 TRYPANOSOMIASIS
bird, only trypanosome forms were found to develop in its body. These
results are, of course, not quite conclusive, as the halteridium stage
might only follow on the sexual portion of the cycle. In this connection,
however, the fact may be mentioned that both with Tr. Brucei and Tr.
ugandense attempts to produce disease by means of the contents of the
stomachs of infected insects have failed. Bruce was of opinion that
infection took place in nagana by means of the insect carrying the
trypanosomes in the tube of its proboscis, where he observed them to be
freely motile for forty-eight hour_s after the insect had fed, and with
regard to the sleeping sickness organism Minchin held a similar opinion,
and showed that if a glossina bit an infected animal, and then in succes-
sion two healthy animals, only the first of the latter would contract the
disease — the proboscis being apparently cleaned by the biting process.
Reference may here be made to the views put forward by
Schaudinn regarding the relationship of certain spirilla to the
trypanosomes. In the athene noctua, besides the Tr. noctuae
already referred to, there is a protozoal parasite infesting the
leucocytes known as spirillum Ziemanni or leucocytozoon
Ziemanni, whose invertebrate host is also the culex pipiens.
Ziemann had described the male and female forms in the owl,
and microgametes had been observed forming from the micro-
gametocytes. Schaudinn observed the formation of an ookinete
in the mosquito ; in certain cases this ookinete elongates, and
the vermicule rolls itself up into a ball with great proliferation
of the nucleus. Each little nucleus attaches to itself a portion
of the protoplasm, and becoming a miniature trypanosome,
swarms off and becomes free. These minute trypanosomes
elongate and develop into typical spirilla by rolling their ribbon-
shaped bodies spirally along their longitudinal axes, the
individuals possessing male, female, or indifferent characters,
just as in Tr. noctuae. - These 'spirilla multiply by longitudinal
division, and often after fission the two individuals remain
attached to each other by their posterior ends, and in this way
there is made possible what is often seen in spirilla, namely, a
capacity to move in either direction. The spirilla often divide
so frequently that ultimately the individuals become invisible by
means of the microscope and can only be seen when lying in
clumps. In this stage Schaudinn thinks the organism would be
able to pass through a Chamberland filter, and this may be a
very important observation, as throwing light on the etiology of
certain diseases, such as yellow fever, in which no visible parasites
have been found.
Schaudinn's views on the trypanosomal characteristics of Sp. Ziemanni
have raised important questions regarding the morphology of other
similar forms which have been long familiar, such as Sp. Obermeieri,
and also of the Spirochsete pallida which Schaudinn himself .discovered.
TRYPANOSOMA LEWISI 551
It is as yet too soon to express any opinion on the ultimate effect of
these views. According to Schaudinn a trypanosomal spirillum consists
of a central thread which represents the posterior nucleus of the
trypanosome ; round this thread the undulating membrane is spirally
wound, and the principal nucleus is represented by minute chromatin
dots sometimes seen in the course of the spiral. Whether all spirilla
have this structure must be left for future investigation to determine.
Difficulties arise with regard to the significance of an undulatory
membrane as a spirillary characteristic and also with regard to the
terminal flagellum which Schaudinn himself found in Spirochaete pallida,
and which he had previously thought did not occur in protozoal spirilla.
It is possible that two groups of organisms have hitherto been classed
together under the name spirillum, and that one of these may still
have to be placed with the bacteria.
Trypanosoma Lewisi. — In 1878 Lewis described in rats in
India the occurrence of the parasite which now goes by his
name, and since that time this trypanosome has been found to
be very common in the blood of rats all over the world, though
the percentage of animals affected varies in different localities.
The condition is of great interest, as, though the infection runs
a very definite course, it is very rarely fatal ; in fact, many
observers have been unable to' produce death by infecting even
large series of animals. There is, however, little doubt that a
fatal issue does occur sometimes in young individuals, especially
when these are infected with strains of the organism imported
from other localities. The trypanosome, which is actively
motile, is of the usual length but is somewhat narrow, and its
protoplasm does not contain any granules. It multiplies by
fission, of which Laveran describes two varieties. In one, the
organism splits longitudinally and gives rise to smaller individuals
than the parent. In the other, the trypanosome loses its
ordinary shape and becomes more oval; nuclear division,
which is often multiple, then takes place, and on subsequent
division of the protoplasm a number of small flagellate organisms
result ; these last may attain the full form and size before
dividing again, or they may divide when still small. When a
rat is infected by injection into the peritoneum active multi-
plication goes on in the cavity for a few days and then comes to
an end. Very soon after infection the organisms begin to appear
in the blood and there rapid multiplication occurs, the extent
of which is sometimes so great that the trypanosomes may seem
to equal the red blood corpuscles in number. The animal
usually shows no symptoms of illness. The infection goes on
for about two months, and then the organisms gradually dis-
appear from the blood. In the great majority of cases the rat
is now immune against fresh infection. If trypanosomes be
552 TRYPANOSOMIASIS
introduced into its peritoneum they are, according to Laveran,
taken up by mononucleate phagocytes and destroyed. The
serum of a rat which has been infected shows agglutinating
capacities towards the trypanosomes, causing them to agglomer-
ate in rosettes in which the flagella are directed outwards, and
the serum of immune rats has a certain degree of protective
action if injected along with the organism into a susceptible
animal. As has already been noted, this trypanosome has been
cultivated on artificial media, on which it multiplies freely,
large numbers of small forms being often produced. These
when injected into rats give rise to the usual infection, but not
so rapidly as when blood from an infected animal is used.
The organism multiplies at the body temperature, but a lower
temperature is preferable, and at 20° C. Novy and MacNeal
succeeded in carrying a growth through many sub-cultures. This
trypanosome is very resistant to cooling, and has been exposed
for fifteen minutes to the temperature of liquid air ( - 191° C.)
without being killed. The means by which the rat becomes
infected naturally is not known, but probably this comes about
by the bite of a flea or louse.
Nagana or Tse-tse Fly Disease. — This is a disease affecting
under natural conditions chiefly horses, cattle, and dogs ; it is
prevalent especially in certain regions of South Africa, though
it probably may occur elsewhere. In the horse the chief
symptoms are the following : — The animal is observed to be
out of condition, its coat stares, it has a watery discharge
from the eyes and nose, and the temperature is elevated ;
swellings appear on the under surface of the abdomen and in
the legs, it gradually becomes extremely emaciated and
anaemic, and dies after an illness of from two or three weeks to
two or three months. In other animals the symptoms are of
the same order, though the duration of the disease varies much ;
thus in the dog the illness does not last more than one or two
weeks, while in cattle it may continue for six months. It is
doubtful whether a domestic animal attacked by the disease
ever recovers. The popular idea regarding the etiology of the
disease was that it was contracted by animals passing through
certain rather restricted and sharply defined areas or belts
characterised by heat and damp, usually lying beside rivers,
and always infested by the tse-tse fly (glossina morsitans), to the
bite of which the disease was attributed ; in this connection it
is important to note that though man is frequently bitten by
the tse-tse fly he does not contract nagana. Modern know-
ledge on the subject dates from the discovery made by Bruce
NAGANA OR TSE-TSE FLY DISEASE 553
in 1894 that the blood of animals suffering from nagana
swarmed with a trypanosome now known as the Tr. Brucei,
and in 1895 he was instructed by the Governor of Natal to
undertake the investigation which led him to work out the true
etiology of the disease. It may be said that this research
forms the starting-point of the important work done during
the last decade with regard to infections by trypanosomes.
In his earlier work Bruce found that the parasite was present
in the blood of every animal suffering from nagana and absent
from the blood of healthy animals in the affected districts;
further, that the fever which marks the onset of the disease was
accompanied by the appearance of the trypanosome in the
blood; and finally, that the transference of the smallest
quantity of blood from an affected to a healthy animal origin-
ated the disease. He then proceeded to investigate the part
played by the tse-tse fly in the condition. He found that if
flies taken from the fly belt were transported to a place where
nagana did not occur, kept for a few days, and then allowed to
bite susceptible animals, the latter did not contract the disease —
this result showing that it was not, as had been supposed by
some, a poison natural to the insect which was the pathogenic
agent. But if such a fly was allowed to bite a dog suffering
from the disease and then to bite a healthy dog, the latter
contracted the malady and abundant trypanosomes were found
in its blood. Again, threads dipped in the blood of an infected
animal and allowed to dry caused the disease in healthy animals
up to but rarely beyond 24 hours after being dried ; if, however,
the blood were kept moist, then it retained its infectiveness up
to between 4 and 7 days; up to 46 hours living trypano-
somes could be seen in the tube of the fly's proboscis.
This corresponds roughly with what was found regarding the
limits of the infectiveness of a fly, in that 24 hours after it has
been fed on an infected animal its bite is usually innocuous.
Further, Bruce showed that infection did not occur by any food
or water partaken of by an animal while going through a fly
belt, for he took horses through such a region without allowing
them to eat or drink, and found that they still contracted the
infection, if during their few hours' journey through the belt
they had been bitten by the tse-tse fly. Finally, he showed
that if flies were taken from an infected area to a healthy one
a few miles off and allowed at once to bite infected animals, the
latter contracted nagana.
By those experiments it was thus determined that nagana
could be transmitted by the blood of the infected animal, that
554
TRYPANOSOMIASIS
is, without the agency of the fly ; that the latter had no inherent
power to produce the disease ; that it could, however, by
successively biting infected and healthy animals transmit the
disease to the latter ; and that specimens of the insect caught in
infected areas harboured the parasite and were thus infective.
The question remained as to how the flies might become infected
in nature. It had been observed that in districts where the
FIG. 168. — Trypanosoraa Brucei from blood of infected rat. Note hi two
of the organisms commencing division of micronucleus and undulating
membrane, x 1000.
tse-tse fly lived the prevalence of the disease in imported animals
was related to the presence in the locality of wild herbivora.
Bruce now found that, if considerable amounts of the blood
of the latter were taken to another locality and' injected into
dogs, these in a proportion of cases contracted nagana, and from
this he deduced that the wild animals harboured the parasites
in small numbers in their blood and thus kept up the
possibility of infection. A further and as yet unexplained
fact was that other blood -sucking flies besides the tse-tse
NAGANA OR TSE-TSE FLY DISEASE 555
appeared incapable of acting as carriers of infection. Brace's
work as a whole pointed to the trypanosome as the cause of
nagana, and this has since been finally established by the
origination of the disease by artificial cultures of the organism.
The Tr. Brucei (Fig. 168), according to Laveran, measures
in the horse from 28-33 /x long and from 1*5 to 2*5 /x broad ; in
the rat and dog it is somewhat shorter. It is motile, but its
activity is less than that of Tr. Lewisi. When stained it
presents the usual appearances ; its posterior end is usually
blunt, and the body often contains granules in the anterior
portion of its protoplasm. It divides longitudinally, and
according to Bradford and Plimmer a form of longitudinal
conjugation occurs in the blood. According to the same
observers, it can be kept alive for 5-6 days in blood outside
the body. It is less resistant to the action of cold than Tr.
Lewisi, perishing in a few days at 5-7° C., but like the other
organism it can withstand short exposures to temperatures down
to -191° C. ; it is quickly killed at 44-45° C. Novy and
MacNeal succeeded in cultivating this trypanosome also,
though here it was very difficult to obtain a first growth from
the blood on their blood-agar medium ; once started, however,
it was kept alive through many sub-cultures, the optimum
temperature of growth being 25° C., and it was from these
sub-cultures that the infection was obtained which definitely
proved the organism to be the cause of the disease. In cultures,
as with Tr. Lewisi, short forms occur, and there is sometimes a
rosette formation with the flagella directed outwards ; agglutina-
tion phenomena are also observable in defibrinated blood.
Under unfavourable conditions involution forms occur, the
organism dividing frequently to form round flagellated
individuals.
Nearly all laboratory animals are susceptible to infection,
and the duration of the illness corresponds to what has been
observed in the natural infection of these animals. The rat
has been largely used for experiment and usually succumbs in
about ten days, there being very few symptoms up till a few
hours before death. A very important fact has been observed
with regard to this animal, namely, that individuals which have
gone through infection with Tr. Lewisi and which are immune
are still susceptible to the Tr. Brucei; from this it has been
deduced that the two organisms are to be looked on as -distinct
species.
Trypanosoma of Sleeping Sickness. — Since the year 1800
the disease called sleeping sickness, sleeping dropsy, or negro
556 TRYPANOSOMTASIS
lethargy has been recognised as prevailing on the West Coast of
Africa from the Senegal to Lagos and in the parts lying behind
the coast between these regions. It has also been found to be
rife from Cameroon to Angola and in the Congo valley, and to a
less extent up the Niger and its tributaries. In 1901 it began
to appear in the Uganda Protectorate, and it is in that region
that the investigations have been carried on which have led
to a knowledge of its cause ; here it has wrought very serious
havoc amongst the native population. It is characterised
in the early stages by a change in disposition leading to
moroseness, apathy, disinclination for work or exertion, and
slowness of speech and gait. There may be headache, indefinite
pains about the body, the evening temperature may be elevated
several degrees, the pulse tends to be soft and rapid, and in a
very large number of cases the superficial glands of the body are
enlarged. In a rapid case the lethargy becomes more pronounced ;
fine tremors, especially of the tongue and arms, develop; pro-
gressive emaciation occurs ; blood changes appear, consisting of a
progressive diminution of the red cells and of the haemoglobin,
and of a lymphocytosis in which the percentage of both the
large and small mononuclear cells is increased, so that the former
may constitute from 20 to 30 and the latter from 30 to 40 per
cent of all the white cells present. As the disease progresses
the drowsiness increases till it deepens into a coma from which
the individual cannot be roused. Often during the disease there
occur irregular cedematous patches on the skin and sometimes
erythematous eruptions, and effusions into the serous cavities.
Not every case runs a progressively advancing course. Some-
times along with enlargement of glands the chief early feature
is the occurrence from time to time of attacks of fever which
may be mistaken for malaria, and from these apparently com-
plete recovery may take place ; recurrence, however, follows
as a rule, and ultimately the typical terminal phenomena
may commence. Such cases may go on for years, and it is
probable that many patients die of pneumonia without exhibiting
typical manifestations of the malady from which they really
suffer. The disease is an extremely fatal condition, and probably
no case where the actual lethargy is developed ever recovers.
On considering the disease from the standpoint of patho-
logical anatomy there is little to be said. As Mott described,
the most striking feature is the presence of a chronic meningo-
encephalitis and meningo-myelitis. The pia-arachnoid is some-
times opaque and slightly thickened and may be adherent
to the brain, and its vessels usually show some congestion.
TKYPANOSOMA OF SLEEPING SICKNESS 557
The sub-arachnoid fluid is sometimes in excess and occasionally
may even be purulent. The membranes of the spinal cord
show similar changes. The chief other feature is the presence
of enlarged lymphatic glands in the body, but otherwise there is
nothing special to note. With regard to the microscopic
changes the chief feature, according to Mott, is a proliferation
and overgrowth of the neuroglia cells, especially of those which
FIG. 169. — Trypanosoma gambiense from blood of guinea-pig. x 1000.
are related to the sub-arachnoid space and the perivascular lymph
spaces, with accumulation and probably proliferation of lympho-
cytes in the meshwork. He further points out that the changes
in the lymph glands are of similar nature and resemble the in-
filtration of the perivascular lymphatics of the central nervous
system. These changes are specially significant in view of the
lymph ocytosis present in the blood, which has already been
noted, and which so often occurs in protozoal infections. In the
nervous structures there is comparatively little change, there
being merely, according to Mott, some atrophy of the dendrons
558 TRYPANOSOMIASIS
of the nerve cells, a diminution of Nissl's granules, and an
excentricity of the nucleus.
Trypanosoma gambiense. — Before going further we must
refer to the observation of a trypanosome in the blood of persons
not evidently suffering from sleeping sickness. The first case of
this was recorded by Button in 1901, the patient being a
European then living at Bathurst on the Gambia. The progress
of the disease was here very slow, and was characterised by
general wasting and weakness, irregular rises of temperature,
local oedemas, congested areas of the skin, enlargement of spleen,
and increased frequency of pulse and respiration ; death occurred
a year after the case came under observation after an access of
fever, and a striking fact was the absence of any gross causal
lesion. During the time the patient was under observation
trypanosomes were repeatedly demonstrated in the peripheral
blood, and they also developed in the bodies of monkeys
and white rats inoculated with the blood. Pursuing further in-
quiries, Dutton and Todd demonstrated similar parasites in other
Europeans and in several natives in the Gambia region, whilst
about the same time Manson reported a case of the same kind in
the wife of a missionary on the Congo. It thus came to be
recognised that in man there occurred a disease having characters
somewhat resembling nagana and in which trypanosomes could
be demonstrated in the blood, and this was usually referred to as
human trypanosomiasis, or trypanosoma fever, — the trypanosome
being named the Tr. gambiense.
Relation of Trypanosomes to Sleeping Sichiess. — Several
views as to the etiology of this disease had been advanced. A
Portuguese Commission in 1902 described a diplococcus tending
to grow in chains which they isolated from the cerebro-spinal
fluid taken from cases during life, and to which they were
inclined from the constancy of its occurrence to attribute a
causal role. The seriousness of the epidemic in Uganda had led
the Royal Society of London in 1902, at the instigation of the
Foreign Office, to despatch a Commission to investigate the
condition on the spot. Soon after commencing work, Dr.
Castellani found in some cases in the cerebro-spinal fluid,
especially when this was centrifugalised, living trypanosomes
resembling the Tr. gambiense ; he also found in 80 per cent of
the cases post mortem a coccus resembling if not identical with
that observed by the Portuguese Commissioners. At first
Castellani was inclined to look on the presence of the protozoan
as accidental, but Colonel Bruce on going out with Nabarro
and Greig in 1903 to pursue the work of the Commission
TRYPANOSOMA OF SLEEPING SICKNESS 559
realised the significance of the observation, urged Castellani to
further inquiries, which he himself continued after the departure
of the latter, with the result that in a series of examinations
carried out in several infected localities, the trypanosome was
demonstrated in every case of the disease. This work formed
the starting-point for inquiries, the results of which make it
practically certain that the parasite is the causal agent of the
condition. The organisms were not demonstrable in the cerebro-
spinal fluid of patients dying of other diseases in the sleeping sick-
ness area. On the other hand, it was found that if cerebro-spinal
fluid withdrawn from cases of the disease was injected into monkeys
(especially macacus rhesus) trypanosomes appeared in the blood,
and in many cases in three or four months the animals died of an
illness indistinguishable from sleeping sickness and with the para-
sites in the central nervous system. It was further found that in
the parts round the north end of Victoria Nyanza where sleeping
sickness was rife, the distribution of the disease exactly corre-
sponded with the distribution of a blood-sucking insect, the
glossina palpalis, a species closely allied to the glossina morsitans
of nagana. It was found that, when one of these flies was fed on
a sleeping sickness patient and then allowed to bite a monkey,
frequently trypanosomes appeared in the animal's blood, and
that when fresh flies caught in the sleeping sickness area were
placed on a monkey a similar occurrence took place.
The trypanosome of sleeping sickness is 17-28 /A long and
1*4-2 //. broad (Fig. 169) (when about to divide it is both longer
and broader). According to Laveran the free part of the
flagellum often equals a fourth of the whole length, but occasion-
ally the body protoplasm extends quite to the end of the
organism. The undulating membrane is narrow, and the
posterior end may be either sharp or blunt. The trypanosome
contains the macro- and micronucleus characteristic of the group,
and the protoplasm often shows chromatin granules. Castellani
attached great importance to a vacuole often seen in the neigh-
bourhood of the micronucleus, but, as stated above, Laveran
holds this to be an artefact. The organism divides longitudin-
ally in the usual manner, and often two can be seen to approach
each other and lie more or less side by side, but whether this
indicates conjugation or not is not known. The organism does
not usually long survive removal from the body, but it has been
found to be motile for nineteen days when kept on rabbit-blood
agar at 22° C. As we have said, when Tr. ugandense is in-
oculated into monkeys they often contract an illness which
ultimately presents the features of typical sleeping sickness.
560 TRYPANOSOMIASIS
Inoculation of other species of animals is not usually so
successful, though in nearly every case, e.g. in the guinea-pig, a
proliferation of the parasite, as indicated by its appearing in the
blood, takes place ; but often either no disease occurs or this runs
a very chronic course. The relative insusceptibility of animals,
especially of the dog, to the Tr. ugandense is taken as evidence
that this organism is essentially different from Tr. Brucei.
By means of microscopic examination the organisms may be
found in the cerebro-spinal fluid, the blood, or the juice of
glands. In the case of the first about 10 c.c. of the fluid is to
be centrifuged for fifteen minutes and the deposit placed under
a cover-glass for examination ; it is better to make a little cell
on a slide by painting a ring of ordinary embedding paraffin,
to place the droplet of fluid in its centre, and to support the cover-
glass on the paraffin ; in this way injury to the delicate structure
of the organism is avoided. In fresh cerebro-spinal fluid the
trypanosomes can be seen to be actively motile ; the number in
which they occur varies very much, and the same is true to a
greater degree of the blood, in which they are, however, usually
very scanty. With regard to the examination of the blood
Bruce and Nabarro state that it is difficult by ordinary centri-
fugalisation to concentrate the organisms, as they are not readily
precipitated. They accordingly recommend that the blood be
mixed with citrate of sodium solution (equal parts of blood and of
a one per cent citrate solution) and centrif ugalised for ten minutes,
that the plasma be removed and centrifugalised afresh for
the same time, and that this be repeated three times, the deposit
from each centrifugalisation after the first being carefully
examined. Greig and Gray have insisted that the examination
of the glands in a suspected case forms the most ready means of
arriving at a diagnosis, and this opinion has found strong
support from the work of Button and Todd. The method is to
push a hypodermic needle into the gland, suck up a little of the
juice, and blow it out on to a slide. In all cases where films of
any kind are to be prepared the staining methods of Leishman
or Giemsa are to be recommended. Often in cerebro-spinal fluid
and gland juice the staining of the chromatin is difficult, but
good preparations are obtained by the procedure recommended
by Leishman for studying the parasite in sections (p. 545).
Greig and Gray have studied the trypanosome in the body
of the glossina. They found evidence of its multiplying in the
stomach of the insect, and it also was seen to undergo changes
not elsewhere observed. These consisted in alterations in the
position of the micronucleus, which often became anterior to the
TRYPANOSOMA OF SLEEPING SICKNESS 561
macronucleus ; there also occurred rosettes, consisting of from
four to twenty individuals attached by their posterior extremities.
Oval forms were also observed. It was found that monkeys
could not be inoculated with the trypanosomes from the stomach
of the fly, and this observation corresponds with what Bruce
found to be true of the trypanosome of nagana in glossina
morsitans.
Early in the Uganda investigations the question arose as to
whether the trypanosoma of sleeping sickness was different from
Tr. gambiense. This was forced on the inquirers by the fact
that a very large proportion of the natives in the sleeping
sickness area were found to harbour trypanosomes in their
blood, although not apparently suffering from the disease.
Several cases were carefully examined in which trypanosomes
were constantly present in the blood, but in which the patients
from time to time suffered from fever, and during these pyrexial
periods trypanosomes were found in the cerebro- spinal fluid.
It was suggested that these cases were on the way to develop
sleeping sickness. A very important observation was that
while in sleeping sickness areas a large proportion of the native
population harboured trypanosomes, this was not the case
where sleeping sickness did not occur. Further, it was found
that trypanosomes from the cerebro -spinal fluid of sleeping
sickness cases and from the blood of persons harbouring try-
panosomes, but not suffering from disease symptoms, gave rise
in monkeys to the same group of chronic effects which resembled
the last stages of the disease in man. These facts led the
Commissioners to incline to the idea that trypanosoma fever
and sleeping sickness are due to the same cause, and represent
different stages of the same disease. It has already been
pointed out that a fatal termination can occur in trypanosoma
fever by an acute febrile attack or from intercurrent disease,
and thus the terminal lethargic stage may only develop in a
certain proportion of cases. The view of the identity of the
two conditions has continued to gain ground. The best
authorities are agreed that morphologically no difference
between the two organisms can be recognised, and the con-
tinued observation of prolonged cases of trypanosoma fever,
both in Uganda by Greig and Gray and in this country
by Manson, has shown that sometimes the termination of a
case is by the onset of typical sleeping sickness.
The prevalence of trypanosomes in the blood of apparently
healthy natives has raised the question of the possibility of
tolerance existing and of immunity being established. It is
36
562 TRYPANOSOMIASIS
possible that both phenomena occur, that not every infection
results in multiplication of the parasite in the body of the
victim, and that in certain cases where multiplication does
occur a resistance is developed which enables the body to kill
the parasites. The occurrence of the mononuclear reaction is
here significant ; it has been suggested that, when this resist-
ance is weak, the organism gains entrance to the spinal canal,
and that then sleeping sickness results.
The whole of the recent work on the disease is of the highest
interest and importance. Short of the prolonged subculture of
the parasite and the reproduction of the disease by such cultures,
the strongest evidence may be said to exist that the Tr.
ugandense is the cause of sleeping sickness.
Other Pathogenic Trypanosomata. — It is beyond the scope
of this work to deal at length with the other diseases of animals
caused by trypanosomes. The chief of these have been
mentioned in the opening paragraph, but it may be said that
many others have been described in various species of mammals,
birds, and fishes, and that these are spread either by flies or by
leeches. The most interesting of those mentioned is Dourine,
a condition resembling in many ways nagana. It, however,
presents this peculiarity, that infection does not take place by
an intermediate host, but apparently directly through coitus, as
it occurs only in stallions and in mares covered by these.
In several of the trypanosomal infections of animals it
appears as if, as in the case of Tr. Lewisi, the animal suffers
little inconvenience from the presence of the parasite in its
blood, and the view has even been put forward that with all
pathogenic trypanosomes there exists a host which carries the
organism without being affected by its presence more, for
example, than is the rat by Tr. Lewisi. Though no opinion
can be expressed on this point, it is necessary to bear the fact
in mind that either natural or acquired immunity can exist
against such protozoa. Not only is this important from the
point of view of the investigation of the conditions under which
such tolerance arises, but also from the bearing which the
existence of this tolerance may have on the spread in nature of
the parasites to a susceptible species from immune animals which
still harbour trypanosomes in their blood. We are, however,
as yet quite ignorant of many of the processes at work in the
body during a trypanosomal infection, and of the causes of the
symptoms and other morbid effects.
KALA-AZAR 563
Ki.LA-AZAK.
(Synonyms : Cachectic Fever, Dum-Dum Fever, Non-malarial
Remittent Fever.)
Leishman noticed in several soldiers invalided from India for
remittent fever and cachexia that the most careful examination
of the blood failed to reveal the presence of the malarial
parasite. From the fact that such patients had almost invari-
ably been quartered during their service at Dum-Dum, an
unhealthy cantonment near Calcutta, he suspected he had
to deal with an undescribed disease. In 1900 he noticed in
the spleen of such a case peculiar bodies which, from comparison
with certain appearances found in degenerating forms of Tr.
Brucei, he suggested might be trypanosomes, and on publishing
his observations in 1903 he put forward the view that
trypanosomiasis might prevail in India and account for the
aberrant cases of cachexial fever met with there. Soon after
Leishman's paper appeared, his observations were confirmed in
India by Donovan, and the bodies associated with the disease
are usually called the " Leishman " or the " Leishman-Donovan "
bodies. They were found by Bentley, and later by Rogers, in
the disease known in Assam as Kala-azar, the pathology of
which had long puzzled those who had worked at it, from
the fact that, while it resembled malaria in many ways, no
parasite could be demonstrated to occur in those suffering
from it.
Kala-azar (or "black disease," — so called from the hue
assumed by chocolate-coloured patients suffering from it) has
been known since 1869 as a serious epidemic disease in Assam,
where it has spread from village to village up the Brahmaputra
valley. The disease is now known to occur in various sub-
tropical centres south of the forty-ninth parallel — cases where
the Leishman bodies have been found having been met with in
many parts of India, China, North Africa, and Arabia. The
disease is characterised by fever of a very irregular type,
by progressive cachexia, and by anaemia associated with
enlargement of the spleen and liver, and often with ulcers of
the skin and dropsical swellings. Rogers has pointed out that
there occurs a leucopenia which differs from that of malaria in
that it is almost always more marked, — the leucocytes usually
numbering less than 2000, and further in that they are always
reduced in greater ratio than the red corpuscles, which condition,
again, does not occur in malaria. The disease is chronic,
564 KALA-AZAR
often going on for several years, and is extremely fatal, — in
fact, it is difficult to say if recovery can ever take place.
Post mortem, there is little to note beyond the enlargement of
the liver and spleen, but in the intestine, especially in the
colon, there are often large or small ulcers, and there is evidence
of proliferation in the bone marrow, the red marrow encroaching
on the yellow.
In a film made from the spleen and stained by Leishman's
Fia. 170. — Leishman- Donovan bodies from spleen smear. x 1000.
stain, the characteristic bodies can be readily demonstrated
(Fig. 170). They are round, oval, or, as Christophers has
pointed out, cockle-shell shaped, and usually 2*5 to 3 -5 //, in
diameter, though smaller forms occur. The protoplasm stains
pink, or sometimes slightly bluish, and contains two bodies
taking on the bright red colour of nuclear matter when stained
by the Romanowsky combination. The larger stains less
intensely than the smaller, is round, oval, heart-shaped, or
bilobed, and lies rather towards the periphery of the body — in
the region of the " hinge " in the cockle-shaped individuals.
KALA-AZAR 565
The other chromatin body is usually rod-shaped, and is set
perpendicularly or at a tangent to the larger mass, with which
only exceptionally it appears to be connected. Usually the
protoplasm contains one or two vacuoles. Though in spleen
smears many free bodies are seen, the study of sections shows
that ordinarily their position is intra-cellular, — the cells con-
taining them being of a large mononuclear type (Fig. 171). The
view held is that on their entering the circulation they are
taken up by the mononuclear leucocytes and by such cells as
the endothelial lining of the splenic sinuses or by those lining
capillaries or lymphatics, that in these cells multiplication takes
place — it may be to such
an extent as to rupture ^
the cell, — and that if
thus the bodies become
free, they are taken up
by other cells and the
process is repeated. The
clusters of bodies some-
times seen in smears are
probably held together
by the remains of rup-
tured phagocytes. In
capillaries the endothelial
cells after phagocyting
the bodies probably be-
come detached from the
capillary wall, as they
f/ , T £ FlG. 171. — Leishmau-Donovan bodies within
are often observed free endothelial cell in spleen, x 1000.
in the lumen of the
vessel, — this being well seen in the hepatic capillaries.
In the body generally the parasites are found in greatest
abundance in the spleen, liver, and bone marrow, and also in
mesenteric glands, especially in those draining one of the
intestinal ulcers • less frequently they occur in the skin ulcers,
and in other parts of the body. Whether they can be demon-
strated microscopically in the blood is disputed. Donovan
described them as occurring in the blood, and also as being
present within red blood corpuscles, but though Laveran agreed
with Donovan's description, the observation has not been
confirmed by other observers.
In the body the parasite multiplies by simple fission, both
nuclei dividing amitotically, and two new individuals being
formed ; but sometimes a multiple division takes place, each
566 KlLA-AZAR
nucleus dividing several times within the protoplasm and a
corresponding number of new parasites resulting.
In view of Leishman's original opinion an extremely important
discovery was made by Rogers and later confirmed by Leishman
himself, to the effect that in cultures a flagellate organism
developed from the Leishman-Donovan body. Cultivation was
effected by taking spleen juice containing the parasite, placing it
in 10 per cent sodium citrate solution and keeping at 17-24° C.
Under such conditions there occurs an enlargement of the
organism, but especially of the larger nucleus. This is followed
by the appearance of a pink-staining vacuole in the neighbourhood
of the smaller nucleus. Along with these changes, in from 24 to
48 hours the parasite becomes elongated and the smaller nucleus
and its vacuole move to one end ; from the vacuole there then
appears to develop a red-staining flagellum, which when fully
formed seems to take its origin from the neighbourhood of the
small nucleus. The body of the parasite is now from 20-22 //,
long and 3-4 p broad, with the flagellum about 22 /x long. The
whole development occupies about 96 hours. The formation of
an undulating membrane was not observed, and, although the
flagellated organism moved flagellum first, like a trypanosome,
it is evident that here the relationship of the micronucleus is
different as this structure lies anterior to the macronucleus.
In his cultures, which kept alive for four weeks, Leishman made
a still further important observation. In certain of the flagellate
forms he saw chromatin granules develop in the protoplasm often
in couples, a larger and a smaller. There then occurred a very
unequal longitudinal division of the protoplasm, and a hair-like
undulating individual containing one of the pairs of chromatin
granules would be split off. At first these would be non-
flagellate, but later a red-staining flagellum would appear at one
end. The analogies between these observations and those of
Schaudinn (v. p. 550) on the relations of spirochaetes to
trypanosomes will be at orice apparent; the further develop-
ment of these spirillary forms in Leishman's organism could
not, however, be traced.
The facts just detailed have caused considerable discussion as
to the classification of the organism, which now usually goes by
the name Leishmania donovani, originally given to it by Ross.
According to one view, it is to be looked on as a trypanosome,
and although, as we have noted, its flagellated form differs from
the typical trypanosoma form, it bears considerable resemblance
to the members of this group, and as Leishman has pointed out,
his cultures may not represent the full development of the
KALA-AZAR 567
organism in the trypanosoma direction. Others have looked
on it as a piroplasma, but Minchin suggests that in the present
incomplete state of knowledge it may be well to place it in a
provisional genus, Leishmania, of the flagellata. In this genus
there would be at present two species, the Leishmania donovani,
and the organism seen in Dehli sore, Leishmania tropica,
presently to be alluded to.
The question arises, given that the Leishmania donovani is
the cause of kala-azar, how is infection spread1? On this we
have as yet no certain information. The fact that in some
centres of the disease natives who are supplied with good water
are less liable than those who rely on the ordinary polluted
native cisterns, has led to the opinion that water may be the
carrier of infection. On the other hand, the possible relation-
ship of the organism to the trypanosomata naturally suggests
the idea of an insect as an intermediary, and Rogers has brought
forward some evidence that the bed-bug is the extra-human host,
but the organism has not as yet been demonstrated in the body
of this insect. It has been objected to the theory of an insect
carrier that the organism probably does not occur in the blood,
but it has been pointed out that invisible spirillary forms may
be the instruments of infection, and that such may exist in the
blood. It may be said here that all attempts to communicate
the disease to animals have been hitherto unsuccessful.
With regard to kala-azar as a whole, we may say that we are
dealing with a disease fairly widespread in various sub-tropical
regions, which is, there is every reason to believe, a separate
entity. All attempts to include it among the malarial cachexias,
which clinically it so much resembles, have failed. In this atypical
cachexial fever there is always present a parasite of very special
characters belonging or closely allied to a group which contains
many varieties capable of giving rise to similar diseases.
Beyond this we cannot go, but at present we must admit that
there is strong presumptive evidence of the parasite described
being the cause of the disease.
Methods of Examination. — The Leishmania donovani can
be readily seen in films or sections of the organs in which we
have mentioned its occurrence. These should be stained by the
Romanowsky stains. Fluid taken from the enlarged spleen during
life may be examined, but it is probable that in this disease
puncture of the spleen may not be a very safe operation, as
death from haemorrhage from this organ is a not uncommon
natural terminal event. During life the main points on which
a pathological diagnosis may be based are the absence of
568 KALA-AZAR
malarial parasites from the blood, and the features of the
leucopenia which have been alluded to.
Delhi Sore. — In various tropical and sub-tropical regions
there is widely prevalent a variety of very intractable chronic
ulceration which goes by various names in different parts of
the world, Delhi sore, tropical ulcer, Aleppo boil, etc. Various
views have been held as to the pathology of the condition, but
the work of J. H. Wright, which has been confirmed by other
observers, makes it extremely probable that a protozoal parasite
is concerned in its etiology. In the discharge from the ulcer
and in sections of a portion of tissue excised from a case coming
from Armenia, Wright observed great numbers of round or oval,
sharply defined bodies, 2-4 ya in diameter. When stained by a
Romanowsky combination there was found to be a peripheral
portion coloured a pale blue and a central portion tending to be
unstained; there were also two chromatin bodies, one larger,
occupying a fourth or a third of the whole and situated in the
periphery, another smaller, round or rod-shaped, and of a deeper
colour than the larger mass. It was found that the bodies
were usually intra-cellular in position in the lesion, as many as
twenty being in one cell, and that the type of cell containing
them was, as in kala-azar, that derivable from endothelial tissues.
There can be little doubt that these bodies are of the same
type as those occurring in kala-azar, and the question of the
identity of the two parasites has been raised. At present the
tendency is to regard them as distinct. As we have seen,
although skin ulcers are common in kala-azar, it is difficult to
find the parasite in this lesion of the disease, while, on the
other hand, in Wright's case at least the number of organisms
present in the ulcer was enormous. Provisionally Minchin
calls this parasite the Leishmania tropica and includes it as the
second species of the genus Leishmania.
PlROPLASMOSIS.
Up to the present no human disease has been proved to be associated
with the presence of piroplasmata. The observations of Donovan,
which seemed to indicate that the parasite of kala-azar might be found
within the red blood corpuscles, and which led Laveran to denominate
the Leishmania donovani the piroplasma donovani, have, as already
indicated, not been confirmed ; the same is true of the association of
piroplasms with the occurrence of the Kocky Mountain spotted fever
sometimes prevalent in Montana. But several important diseases of the
lower animals are almost certainly caused by protozoan parasites of this
group, and a short account of the organisms may be given.
The piroplasmata are pear-shaped unicellular organisms about 1-1 '5 /*
PIROPLASMOSIS 569
longhand varying in breadth. The peripheral part is denser than the
central, which often appears as if vacuolated, and at the broad end there
is a well-staining chromatin mass. Sometimes irregular and ring-, rod-,
or oval-shaped individuals occur. The organisms are found within the
red blood corpuscles of the infected animal and also free in the blood.
In the former situation there is sometimes only one within a cell, but
the numbers vary under different circumstances and in different species.
Multiplication takes place by fission, and the new individuals, remaining
for longer or shorter times in apposition, account for some of the
appearances seen in cells. Especially in the forms free in the blood
pseudopodial prolongations of the protoplasm, usually from the pointed
end, are developed, and it may be by means of such pseudopodia that
entrance to the red cells is obtained. Infection is usually carried from
infected animals by means of ticks. In one case Koch has described the
development in the organism, in the stomach of the tick, of spiked proto-
plasmic processes sprouting out from the broad end of the piroplasm,
and the occurrence of conjugation of two such individuals by their
narrow ends to form a zygote. Further observations, however, here are
necessary, and nothing is known of the further history of the parasite
within the insect except that the eggs in the ovary may become infected,
so that insects developed from these can carry infection to animals.
Frequently when an animal has passed through an attack of a piro-
plasmosis it is immune to the disease, and with regard to this immunity
in certain cases very interesting facts have been observed. For instance,
the condition may not be associated with the disappearance of the
parasite from the blood of the immune animal, and the latter may thus
be a source of danger to other non-immune animals with which ticks
harboured by it may come in contact.
The following are the chief piroplasmata causing disease in animals : —
(1) Piroplasma bigeminum. This was first described by Theobald Smith
and is the cause of Texas or red-water fever, a febrile condition associated
with hsemoglobinuria, which occurs in the Southern States of America,
the Argentine, South and Central Africa, Algeria, various parts of
Northern Europe, and in Australia. The organism gets its name of
bigeminum from the fact that it is often present in the red cells in pairs,
which may be attached to one another by a fine thread of protoplasm ;
this probably results from the complete separation of two individuals
being delayed after division has occurred. Infection is here spread by
the tick boophilus boms, and some of the characteristics of the disease
epidemiologically are explained by the fact that this insect goes through
all its moultings on the same individual host. (2) Piroplasma parvum.
This organism was discovered by Theiler in the blood of cattle suffering
from African East Coast fever, a disease closely resembling Texas fever,
which prevails endemically in a narrow strip along a long extent of the east
coast, and which occurs epidemically inland. As its designation implies,
the organism is small, and it is also attenuated. Its insect host is the tick
rhipicephalus appetidiculatus, and it may be noted that this tick drops
off the animal on wThich it may be feeding when it is about to go through
one of its several moultings. It can thus carry an infection much more
quickly and widely through a herd than can the carrier of ordinary red-
water fever. It may be said that in England there occurs a red-water
fever also associated with the presence of a piroplasm in the blood, but
the, relationship of this organism to the other varieties has not yet been
fully worked out. (3) Piroplasma equi. This organism gives rise to
biliary fever in horses, another South African disease, and it is carried
570 PIROPLASMOSIS
by the tick rhipicephalus evertsii. In this disease Theiler made the
interesting observation that when the blood of a donkey which had
recovered from the disease was injected into a horse the latter suffered a
slight illness only, although the organisms were present in the blood
injected. Such a fact is of importance, as attenuation of virulence in
pathogenic protozoa seems, so far as our present knowledge goes, a not
very common event. (4) Piroplasma canis. This causes a piroplasmosis
occurring in dogs.
With regard to the pathology of infection by piroplasmata we know
nothing. The diseases are often extremely fatal, carrying off nearly
every individual attacked, but we do not know the nature of the
changes originated.
BIBLIOGRAPHY.
GENERAL TEXT- BOOKS.— In English the student may consult the follow-
ing : "Micro-organisms and Disease," E. Klein, 3rd ed., London, 1896.
" Bacteriology and Infective Diseases," Edgar M. Crookshank, London,
1898. "A Manual of Bacteriology," George M. Sternberg, New York,
1st ed. 1893, 2nd ed. 1896 (this book contains a full bibliography). "Text-
Book upon the Pathogenic Bacteria," Joseph M'Farland, London, 5th ed.,
1906. "Practical Bacteriology," A. A. Kanthack and J. H. Drysdale,
London, 1895. " Bacteria and their Products," G. S. Woodhead, London,
1891. "Bacteriological Technique," Eyre, London, 1902. The articles
on bacteriological subjects in Clifford Allbutt's "System of Medicine,"
London, are of the highest excellence, and have full bibliographies
appended.' For the hygienic aspects of bacteriology see "System of
Hygiene," Stevenson and Murphy, London, 1892-94.
For non-pathogenic bacteria occurring in connection with pathological
work consult Heim, op. cit. infra. For fungi see De Bary, "Comparative
Morphology and Biology of the Fungi, Mycetozoa and Bacteria," transl.
by Garnsey and Balfour, Oxford, 1887 ; Sachs, "Text-Book of Botany,"
ii., transl. by Garnsey and Balfour, Oxford, 1887.
In German: "Die Mikroorganismen," by Dr. C. Fliigge, 3rd ed.,
Leipzig, 1896. " Lehrbuch der pathologischen Mykologie," by Baum-
garten, Braunschweig, 1890. " Handbuch der pathogenen Mikro-
organismen," Kolle and Wassermann, Fischer, Jena, 1904.
In French : Roger, " Les maladies infectieuses," Paris, 1902.
PERIODICALS.— For references to current work see (1) Centralbl. f.
Bakteriol. u. Parasitenk., Jena. This publication commenced in 1887.
Two volumes are issued yearly. In 1895 it was divided into two parts.
Abtheilung I. deals with Medizinisch-hygienische Bakteriologie und
thierische Parasitenkunde. The volumes of this part are numbered con-
secutively with those of the former series, the first issued thus being vol.
xvii. Commencing in 1902 with volume xxxi., each volume of Ab-
theilung I. was further divided into two parts, one consisting of Originate
the other of Referate. Abtheilung II. deals with Allgeineine landwirt-
schaftlich-technologische Bakteriologie, Garungs-physiologie und Pflanzen-
pathologie. The first volume is entitled Zweite Abtheilung, Bd. I. It
contains original articles, JRcferate, etc. (2) Bull, de I'lnsi. Pasteur,
Paris, Masson. Besides bacteriological abstracts this journal contains
many valuable reviews and analyses relating to protozoology. (3) "Ergeb-
nisse der allgemeinen Pathologic," Lubarsch and Ostertag, Wiesbaden,
Bergmann. This from time to time contains valuable critical reviews.
The most complete account of the work of the year is found in the
Jahresb. u. d. Fortschr. . . . d. path. Mikroorganismen, conducted by
571
572 BIBLIOGRAPHY
Baumgarten, and published in Braunschweig. This publication com-
menced in 1887. Its disadvantage is that the volume for any year does
not usually appear till two years later.
Bacteriology is also dealt with in the Index Medicus. For valuable
lists of papers by particular authors see Royal Society Catalogue of
Scientific Papers and Internat. Cat. Sc. Lit. (Section, Bacteriology).
The chief bacteriological periodicals are the Journ. Path, and Bacterial.,
Cambridge, edited by G. Sims Woodhead ; the Ztschr. f. Hyg. u. Infec-
tionskrankh., Leipzig, edited by Koch and Fliigge, and the Ann. de I'Inst.
Pasteur, Paris, edited by Duclaux ; Journ. Exper. Med. , New York,
edited by Flexner ; Journ. Hyg., Cambridge, edited by Nuttall ; Journ.
Med. Research, Boston, edited by Ernst ; Journ. Infect. Diseases, Chicago,
edited by Hektoen ; Journ. of Royal Army Medical Corps, edited by
Bruce.
Valuable papers also from time to time appear in the Lancet, Brit.
Med. Journ., Deutsche med. Wchnschr., Berl. klin. Wchnschr., Semaine
med., Arch. f. Hyg., Arch. f. exper. Path. u. Pharmakol. Besides these
periodicals the student may have to consult the Reports of the Med. Off.
of the Local Government Board, which contain the reports of the medical
officers, also the Proc. Roy. Soc. London, the Compt. rend. Acad. d. sc.
Paris, the Compt. rend. Soc. de biol. Paris, and the Arb. a. d. k. Gsndht-
samte. (the first two volumes of the last were denominated Mitthcilungen}.
CHAPTER I. — GENERAL MORPHOLOGY AND BIOLOGY.
Consult here especially Fliigge, "Die Mikroorganismen." De Bary,
"Bacteria," translated by Garnsey and Bayley Balfour, Oxford, 1887.
Zopf, "Zur Morphologic der Spaltpflanzen," Leipzig, 1882; "Beitr. z.
Physiologic uud Morphologic niederer Organismen," 5th ed., Leipzig,
1895. Cohn, Beitr. z. Biol. d. Pftanz., Bresl. (1876), ii. v. Nageli,
"Die niecleren Pilze," Munich, 1877; " Untersuchungen iiber niedere
Pilze," Munich, 1882. Migula, "System der Bakterien," Jena, 1897.
Duclaux, "Traite de microbiologie," Paris, 1898-99. For general
morphological relations see Marshall Ward, art. "Schizomycetes,"
Ency. Brit., 9th ed. xxi.' 398 ; xxvi. 51. Engler and Prantl, "Die
natiirlichen Pflanzenfamilien," Lieferung, 129. " Schizophy ta " (by W.
Migula). STRUCTURE or BACTERIAL CELL. — Biitschli, "Uber den Bau
der Bakterien," Leipzig, 1890; "Weitere Ausfuhrungen iiber den Bau
der Cyanophyceen und Bakterien," Leipzig, 1896. Fischer, op. cit. in
text. Buchner, Longard and Riedlin, Centralbl. f. Bakteriol. u. Para-
sitenk. ii. 1. Ernst, Ztschr. f. Hyg. v. 428 ; Babes, ibid. v. 173.
Neisser, ibid. iv. 165. MOTILITY. — Klein, Biitschli, Fischer, Cohn,
loc. cit. Loftier, Centralbl. f. Bakteriol. u. Parasitenk. vi. 209 ; vii. 625.
PIGMENTS. — Zopf, loc. cit. ; Galeotti, Ref. in Centralbl. f. Bakteriol. u.
Parasitenk. xiv. 696. Babes, Ztschr. f. Hyg. xx. 3. SPORULATION. —
Prazmowski, Biol. Centralbl. viii. 301. A. Koch, Botan. Ztg. (1888),
Nos. 18-22. Buchner, Sitzungsb. d. math.-phys. Cl. d. k. layer. Akad.
d. Wissensch. zu Munchen, 7th Feb. 1880. R. Koch, Mitth. a. d. k.
Gsndhtsamte. i. 65. CHEMICAL STRUCTURE OF BACTERIA. — Nencki,
Ber. d. deutsch. chem. Gesellsch. (1884), xvii. 2605. Cramer, Arch. f.
Hyg. xvi. 154. Buchner, Berl. klin. Wchnschr. (1890), 673, 1084 ; vide
Fliigge, op. cit. CLASSIFICATION OF BACTERIA. —For general review
see Marshall Ward, Ann. of Botany, vi. 103 ; Migula, loc. cit. supra.
BIBLIOGRAPHY 573
FOOD OF BACTERIA. — Nageli, Cohn, op. cit. Pasteur, "Etudes sur la
biere," 1876. Hueppe, Mitth. a. d. k. Gsndhtsamte. ii. 309. RELATIONS
TO OXYGEN. — Pasteur, Compt. rend. Acad. d. sc. Hi. 344, 1142 ; Kitasato
and Weyl, Ztschr. f. Hyg. viii. 41, 404 ; ix. 97. TEMPERATURE. — Vide
Fliigge, op. cit. ; for thermophilic bacteria, Rabinowitsch, Ztschr. f.
Hyg. xx. 154 ; Macfadyen and Blaxall, Journ. Path, and Bacterial, iii.
87. ACTION OF BACTERIAL FERMENTS. — Salkowski, Ztschr. f. Biol.,
N.F., vii. 92 ; Pasteur and Joubert, Compt. rend. Acad. d. sc. Ixxxiii. 5 ;
Sheridan Lea, Journ. Physiol. vi. 136 ; Beijerinck, Centralbl. f. Bak-
teriol. u. Parasitenk., Abth. II. i. 221 ; E. Fischer, Ber. d. deutsch. chem.
Gcsellsch. xxviii. 1430 ; Liborius, Ztschr. f. Hyg. i. 115 ; see also
Pasteur, "Royal Society Catalogue of Scientific Papers." VARIABILITY.
— Cohn, Nageli, Fliigge, op. cit. Winogradski, " Beitr. z. Morph. u.
Physiol. d. Bakt.," Leipzig, 1888 ; Ray Lankester, Quart. Journ. Micr.
Sc., N.S. (1873), xiii. 408 ; (1876), xvi. 27, 278. NITRIFYING ORGANISMS.
—Winogradski, Ann. de Vlnst. Pasteur, iv. 213, 257, 760 ; v. 92, 577.
Maze, ibid. xi. 44 ; xii. 1, 263.
CHAPTER II. — METHODS OF CULTIVATION OF BACTERIA.
For GENERAL PRINCIPLES. — Pasteur, Compt. rend. Acad. d. sc. 1.
303 ; li. 348, 675 ; Ann. de chem. Ixiii. 5 ; Tyndall, "Floating Matter
of the Air in relation to Putrefaction and Infection," London, 1881 ;
H. C. Bastian, "The Beginnings of Life," London, 1872. METHODS OF
STERILISATION. — R. Koch, Gatt'ky, and Lbffler, Mitth. a. d. k. Gsndht-
samte. i. 322 ; Koch and Wolffhiigel, ibid. i. 301. CULTURE MEDIA.
— See text -books, especially Kanthack and Drysdale, Eyre ; Pasteur,
"Etudes sur la Mere," Paris, 1876; R. Koch, Mitth. a." d. k. Gsndht-
samte. i. 1 ; Roux et Nocard, Ann. de Vlnst. Pasteur, i. 1 ; Roux, ibid.
ii. 28 ; Marmorek, ibid. ix. 593 ; Kitasato and Weyl, op. cit. supra • P.
and Mrs. Percy Frankland, "Micro-organisms in Water," London, 1894.
Fuller, Rep. Amer. Pub. Health Ass. xx. 381. Theobald Smith,
Centralbl. f. Bakteriol. u. Parasitenk. vii. 502 ; xiv. 864. Durham,
Brit. Med. Journ. (1898), i. 1387. "Report of American Committee on
Bacteriological Methods," Concord, 1898. MacConkey, Thompson-
Yates and Johnston Lab. Rep. vol. iii. pt. iii. 151 ; vol. iv. pt. i. p. 151 ;
Journ. Hyg. v. 333. Drigalski and Conradi, Ztschr. f. Hyg. xxxix.
283.
CHAPTER III. — MICROSCOPIC METHODS, ETC.
Consult text-books, especially Klein, Kanthack and Drysdale, Hueppe,
Gunther, Heim ; also Bolles Lee, "The Microtomist's Vademecum, "
London, 1905 (this is the most complete treatise on the subject).
Rawitz, op. cit. in text ; Koch, Mitth. a. d. k. Gsndhtsamte. i. 1 ;
Ehrlich, Ztschr. f. klin. Med. i. 553 ; ii. 710. Gram, Fortschr. d.
Med. (1884), ii. No. 6 ; Nicholle, Ann. de Vlnst. Pasteur, ix. 666 ;
Kiihne, " Praktische Anleituug zum mikroscopischen Nachweis der
Bakterien im tierischen Gewebe," Leipzig, 1888 ; van Ermengem, ref.
Centralbl. f. Bakteriol. u. Parasitenk. xv. 969 ; Richard Muir, Journ.
Path, and Bacterial, v. 374 ; Mann, " Physiological Histology,"
Oxford, 1902. For Romanowsky methods see Jenner, Lancet (1899),
574 BIBLIOGRAPHY
i. 370 ; Leishman, Brit. Med. Journ. (1901), i. 635 ; (1902), ii. 757 ;
Journ. of Roy. Army Med. Corps (1904), ii. 669 ; Geirasa, Deutsche
Med. Wchnschr. (1905), 1026 ; Ann. de I'Inst. Pasteur, xix. 346 ;
MacNeal, Journ. Inf. Dis. iii. 412; Wright, J. H., Journ. Med. Res.
vii. 138.
AGGLUTINATION.— Delepine, Brit. Med. Journ. (1897), ii. 529, 967.
Widal and Sicard, Ann. de I'Inst. Pasteur, xi. 353. Wright, Brit. Med.
Journ. (1897), i. 139 ; (1898), i. 355.
CHAPTER IV. — BACTERIA OF AIR, SOIL, WATER. ANTISEPTICS.
AIR, SOIL, AND WATER. — Petri, Ztschr. f. Hyg. iii. 1; vi. 233.
Fliigge, ibid. xxv. 179. Sticher, ibid. xxx. 163. Weyl, " Handbuch
der Hygiene," Jena, 1896, et seq. Houston, Rep. Med. Off. Local Gov.
Bd. xxvii. (1897-98) 251 ; xxviii. (1898-99) 413, 439, 467 ; xxix. (1899-
1900) 458, 489. Sidney Martin, ibid. xxvi. (1896-97) 231 ; xxvii.
(1897-98) 308; xxviii. (1898-99) 382. Horrocks, "Bacteriological
Examination of Water," London, 1901. Percy and G. C. Frankland,
"Micro-organisms in Water," London, 1894. Dibdin, " Purification of
Sewage and Water," London, 1897. Ann. Rep. Bd. Health, Mass.,
Boston, 1890, ^ seq. Savage, " The Bacteriol. Exam, of Water Supplies,"
London, 1906. Lewis, Rideal, and Walker, Journ. Roy. San. Inst.
(1903), xxiv. 424.
ANTISEPTICS. — R. Koch, Mitth. a. d. k. Gsndhtsamte. i. 234. Behring,
Ztschr. f. Hyg. ix. 395. Ritchie, Trans. Path. Soc. London, 1. 256.
Rideal, "Disinfection and Disinfectants," London, 1898.
CHAPTER V. — RELATIONS OF BACTERIA TO DISEASE, ETC.
As the observations on which this chapter is based are scattered
through the rest of the book, the references to them will be found under
the different diseases.
CHAPTER VI. — INFLAMMATORY AND SUPPTJRATIVE CONDITIONS.
Ogston, Brit. Med. Journ. (1881), i. 369. Rosenbach, " Mikro-
organismen bei den Wundinfectionskrankheiten des Menschen," Wies-
baden, 1884. Passet, Fortschr. d. Med. (1885), Nos. 2 and 3. W.
Watson Cheyne, "Suppuration and Septic Diseases," Edinburgh, 1889.
Grawitz, Virchow's Archiv, cxvi. 116 ; Deutsche med. Wchnschr. (1889),
No. 23. Steinhaus, Ztschr. f. Hyg. v. 518 (micrococcus tetragenus) ;
'•'Die Aetiologie der acuten Eiterung," Leipzig, 1889. Christmas-
Dirckinck-Holmfeld, " Recherches experimentales sur la suppuration,"
Paris, 1888. Muir, Journ. Path, and Bacteriol. vii. 161 ; Trans. Path.
Soc. London, 1902. Garre, Fortschr. d. Med. (1885), No. 6. Marmorek,
Ann. de I'Inst. Pasteur, ix. 593. Petruschky, Ztschr. f. Hyg. xvii. 59 ;
xviii. 413; xxiii. 142; (with Koch, xxiii. 477). Liibbert. " Biologische
Spaltpilzuntersuchung," Wiirzburg, 1886. Krause, Fortschr. d. Med.
(1884), Nos. 7 and 8. Ribbert, Fortschr. d. Med. (1886), No. 1. Widal
and Besaii9on, Ann. de I'Inst. Pasteur, ix. 104. .v. Lingelsheim, Ztschr.
BIBLIOGRAPHY 575
/. Hyg. x. 331 ; xii. 308. Behring, Centralbl. f. BaTcteriol. u. Parasitenk.
xii. 192. Thoinot et Masselin, Rev. de med. (1894), 449. Orth and
Wyssokowitsch, Centralbl. f. d. med. Wissensch. (1885), 577. Netter,
Arch, de physiol. norm, et path. (1886), 106. Weichselbauni, Wien. med.
Wchnschr. (1885), No. 41 ; (1888), Nos. 28-32 ; Centralbl. f. Bakteriol. u.
Parasitenk. ii. 209 ; Beitr. z. path. Anat. u. z. allg. Path. iv. 127.
Becker, Deutsche med. Wchnschr. (1883), No. 46. Lannelongue et
Achard, Ann. de I'lnst. Pasteur, v. 209. Fehleisen, "Die Aetiologie
des Erysipels," Berlin, 1883. Welch, Am. Med. Journ. Sc. (1891), 439.
Lemoine, Ann. de I'lnst. Pasteur, ix. 877. Kurth, Arb. a. d. Tc.
Gsndhtsamte. vii. 389. Knorr, Ztschr. f. Hyg. xiii. 427. Bulloch,
Lancet (1896), i. 982, 1216. Bordet, Ann. de I'lnst. Pasteur, xi. 177.
Booker (streptococcus enteritis), Johns Hopkins Hosp. Rep. vi. 159.
Hirsch, Centralbl. f. Bakteriol. u. Parasitenk. xxii. 369. Libman, ibid.
xxii. 376. Wright and Douglas, Proc. Roy. Soc. Lond. Ixxiv. 147.
Wright, Clinical Journal (1906), May 16.
Streptococci. — Hiss, Journ. Exper. Med. vi. 317. Schottmiiller,
Munchen. med. Wchnschr. (1903), 849. Gordon, Reports Med. Officer
Local Gov. Board (1905), 388 ; Lancet (1905), ii. 1400. Andrewes and
Horder, Lancet (1906), ii. Ruediger, Journ. Infect. Dis. iii. 755.
Besredka, Bull, de I'lnst. Pasteur, iii.
Conjunctivitis.— Morax, Ann. de I'lnst. Pasteur (1896), x. 337. Eyre,
Journ. Path. and. Bacteriol. vi. 1. Miiller, Wien. med. Wchnschr.
1897 ; Inglis Pollock, Trans. Ophthalm. Soc. 1905 ; Axenfeld, in Lubarsch
and Ostertag, "Ergebnisse der allgem. Pathol. u. Path. Anat.," 1901 ;
"Die Bakteriologie in der Augeuheilkunde," 1907 (full references).
Acute Rheumatism. — Triboulet and Cay on, Bull. Soc. med. d. h6p. de
Paris (1898), 93. Westphal, Wassermann, and Malkoff, Berl. klin.
Wchnschr. (1899), 638. Poynton and Paine, Lancet (1900), ii. 861, 932
(full references). Trans. Path. Soc. Lond. (1902), liii. 221 ; Lancet,
December 1905. Beaton and Walker, Brit. Med. Journ. (1903), i. 237.
Shaw, Journ. Path, and Bacteriol. (1903), ix. 158. Beattie, ibid. ix. 272 ;
Journ. Med. Research, xiv. 399 ; Journ. Exper. Med. ix. 186. Cole,
Journ. Infect. Dis. i. 714.
CHAPTER VII. — INFLAMMATORY AND SUPPURATIVE CONDITIONS,
CONTINUED : ACUTE PNEUMONIAS, EPIDEMIC CEREBRO - SPINAL
MENINGITIS.
Friedlander, Fortschr. d. Med. i. No. 22 ; ii. 287 ; Tirchow's Archiv,
Ixxxvii. 319. A. Fraenkel, Ztschr. f. klin. Med. (1886), 401. Salvioli
and Zaslein, Centralbl. f. d. med. Wissensch. (1883), 721. Ziehl, ibid.
(1883), 433 ; (1884), 97. Klein, ibid. (1884), 529. Jiirgensen, Berl. klin.
Wchnschr. (1884), 270. Seibert, ibid. (1884), 272, 292. Senger, Arch,
f. exper. Path. u. Pharmakol. (1886), 389. Weiohselbaum, Wien. med.
Wchnschr. xxxvi. 1301, 1339, 1367 ; Monatschr. f. Ohrenh. (1888), Nos.
8 and 9 ; Centralbl. f. Bakteriol. u. Parasitenk. v. 33. Gamaleia, Ann.
de I'lnst. Pasteur, ii. 440. Guarnieri, Atti d. r. Accad. med. di Roma
(1888), ser. ii. iv. Kruse and Pansini, Ztschr. f. Hyg. xi. 279. E.
Fraenkel and Reiche, Ztschr. f. klin. Med. xxv. 230. Sanarelli, Centralbl.
f. Bakteriol. u. Parasitenk. x. 817. Lannelongue, Gaz. d. Mp. (1891),
379. Netter, Bull, et mim. Soc. med. d. hdp. de Paris (1889) ; Cpmpt.
rend. Acad. d. sc. (1890) ; Compt. rend. Soc. de. biol. Ixxxvii. 34.
576 BIBLIOGRAPHY
G. and F. Klemperer, Berl. klin. Wchnschr. (1891), 893, 869. Fok and
Bordoni-Uffreduzzi, Deutsche med. Wchnschr. (1886), No. 33. Emmerich,
Miinchen. med. Wchnschr. (1891), No. 32. Issaeff, Ann. de I'lnst.
Pasteur, vii. 260. Grimbert, Ann. de I'lnst. Pasteur, xi. 840. Wash-
bourn, Brit. Med. Journ. (1897), i. 510 ; (1897), ii. 1849. Eyre and
Washbourn, Journ. Path, and Bacterial, iv. 394 ; v. 13. See also
Brit. Med. Journ. (1901), ii. 760; Neufeld and Rimpau, Ztschr. f.
Hyg. Ii. 283.
Meningitis. — Weichselbaum, Fortschr. d. Med. (1887), v. 573, 620.
Jaeger, Ztschr. f. Hyg. xix. 351. Councilman, Mallory, and Wright,
"Epidemic Cerebro-spinal Meningitis," Rep. Bd. Health Mass., Boston,
1898 (full references). Gwyn, Johns Hopkins Hosp. Bull. (1899), 109.
v. Lingelsheim, Klin. Jahrb. xv. 373. Kolle and Wassermann, ibid.
p. 507. Kutscher, Deutsche med. Wchnschr. (1906), 1071. Bettencourt
and Franca, Ztschr. f. Hyg. xlvi. 463. Durham, Journ. Infect. Dis.
Suppl. No. 2, p. 10. Goodwin and von Sholly, ibid. p. 21. Arkwriglit,
Journ. of Hyg. vii. 145. Flexner, Journ. Exper. Med. ix. 105. Van-
steenberghe et Grysez, Ann. de I'lnst. Pasteur, xx. 69.
CHAPTER VIII.— GONORRHOEA, SOFT SORE, SYPHILIS.
GONORRHOEA. — Neisser, Centralbl. f. d. med. Wissensch. (1879), 497 ;
Deutsche med. Wchnschr. (1882), 279 ; (1894), 335. Bumm, " Der Mikro-
organismus der gonorrhoischen Schleimhauterkrankungen," Wiesbaden,
1885, 2nd ed. 1887 ; Miinchen. med. Wchnschr. (1886), No. 27 ; (1891),
Nos. 50 and 51 ; Centralbl. f. Gynak. (1891), No. 22 ; Wien. med. Presse
(1891), No. 24. Bockhart, Monatsh. f. praTct. Dermat. (1886), v. No. 4 ;
(1887), vi. No. 19. Steinschneider, Berl. Tclin. Wchnschr. (1890), No.
24 ; (1893), 'No. 29 ; Verhandl. d. deutsche dermat. Gesellsch. I. Congress,
Wien (1889), 159. Wertheim, Wien. Tclin. Wchnschr. (1890), 25 ;
Deutsche med. Wchnschr. (1891), No. 50 ; Arch. f. Gynak. xli. Heft 1 ;
Centralbl. f. Gynak. (1891), No. 24 ; (1892), No. 20 ; Wien. klin.
Wchnschr. (1894), 441. Gerhardt, Charity-Ann. (1889), 241. Leyden,
Ztschr. f. klin. Med. xxi. 607 ; Deutsche med. Wchnschr. (1893), 909.
Bordoni-Uffreduzzi, ibid: (1894), 484. Councilman, Am. Journ. Med.
Sc. cvi. 277. Finger, Ghon, and Schlagenhaufer, Arch. f. Dermat. it.
Syph. xxviii. 3, 276. Lang, ibid. (1892), 1007 ; Wien. med. Wchnschr.
(1891), No. 7; "Der Venerische Katarrh, dessen Pathologic und
Therapie," Wiesbaden, 1893. Klein, Monatschr. f. Geburtsh. u. Gynaek.
(1895), 33. Michaelis, Ztschr. f. klin. Med. xxix. 556. Heiman, New
York Med. Eec. (1895), 769 ; (1896), Dec. 19. Foulerton, Trans. Brit.
Inst. Preven. Med. i. 40. De Christmas, Ann. de I'lnst. Pasteur, xi.
609. Nicolaysen, Centralbl. f. Bakteriol. u. Parasitenk. xxii. 305.
Rendu, Berl. klin. Wchnschr. (1898), 431. Wassermann, Ztschr. f.
Hyg. xxvii. 298 ; Miinchen. med. Wchnschr. (1901), No. 8. Lenhartz,
Berl. klin. Wchnschr. (1897), 1138. Thayer and Lazear, Journ. Exper.
Med. iv. 81. Kb'nig, Berl. klin. Wchnschr. (1900), No. 47. De Christmas,
Ann. de I'lnst. Pasteur\(19QQ), xiv. 331. Raskai, Deutsche med. Wchnschr.
(1901), No. 1. Jundell, Centralbl. f. Bakteriol. u. Parasitenk. xxix.
224. Colombini, ibid. xxiv. 955. Bressel, Miinchen. med. Wchnschr.
(1903), No. 13. Holler, Arch. f. Dermat. u. Syph. (1904), Ixxi. 269.
Wynn, Lancet (1905), i. 352. Prochaska, Arch. f. klin. Med. Ixxxiii.
Heft 1-2. Strong, Journ. Am. Med. As., May 1904.
BIBLIOGRAPHY 577
SOFT SORE. — Ducrey, Monatsh. f. prakt. Dermat. ix. 221. Krefting,
Arch.f. Dermat. u. Syph. (1892), 263. Jullien, Journ. d. mal. cutan. et
syph. (1892), 330. Unna, Monatsh. f. prakt. Dermat. (1892), 475 ; (1895),
61. Quinquand, Semaine mtd. (1892), 278. Petersen, Centralbl. f.
Bakteriol. u. Parasitenk. xiii. 743 ; Arch. f. Dermat. u. Syph. (1894),
419. Audrey, Monatsh. f. prakt. Dermat. (1895), 267. Colombini,
Centralbl. f. Bakteriol. u. Parasitenk. xxv. 254. Nicolle, Presse medicale
(1900), 304. Bezan9on, Griffon, and Le Sourd, Ann. de dermat. et de
syphilolog. (1901); tome ii. 1. Lenglet, ibid. (1901), tome ii. 209. Simon,
Compt. rend. Soe. Biol. (1902), 547. Tomasczewski, Ztschr. f. Hyg.
(1903), Bd. 43, p. 327. Davis, Journ. of Med. Research (1903), ix. 401.
SYPHILIS. — Lustgarten, Wien. med. Wchnschr. (1884), No. 47.
Doutrelepont and Schiitz, Deutsche med. Wchnschr. (1885), No. 19.
Gottstein, Fortschr. d. Med. (1885), No. 16. De Michele and Radice,
Gior. internaz. di sc. med. (1892), 535. Sabouraud, Ann. de I'Inst.
Pasteur, vi. 184. Golasz, Journ. d. mal. cutan. et syph. (1894), 170.
Markuse, Vrtljschr. f. Dermat. u. Syph. (1883), No. 3. Van Niessen,
Centralbl. f. Bakteriol. u. Parasitenk. xxiii. 49. Metchnikoff and Roux,
Ann. de I Inst. Pasteur, xvii.-xix. Lassar, Berl. klin. Wchnschr. (1903),
1189. Neisser, Deutsche med. Wchnschr. (1904), 1369, 1431. Schaudinn
and Hoffmann, Arb. a. d. kaiserl. Gesundheitsamte (1905), Bd. 22 ;
Deutsche med. Wchnschr. (1905), No. 18 ; Berl. klin. Wchnschr. (1905);
Nos. 22, 23. Schaudinn, Deutsche med. Wchnschr. (1905), No. 22.
Hoffmann, Berl. klin. Wchnschr. (1905), No. 46; "Selected Essays on
Syphilis and Smallpox," New Sydenham Society, 1906. Metchnikoff,
La Semaine med. (1905), 234. Levaditi, ibid. (1905), 247. Siegel,
Miinchen. med. Wchnschr. (1905). 1321, 1384. Herxheimer, ibid. (1905),
1857. Shennan, Lancet (1906), i. 663, 746. Maclennan, Brit. Med. Journ.
(1906), i. 1090. Levaditi, Ann. de I'Inst. Pasteur (1906), 41.
CHAPTER IX.— TUBERCULOSIS.
Klencke, " Untersuchungen und Erfnhrungen im Gebiet der Anatomic,
etc.," Leipzig, 1843. Villemin, " De la virulence et de la specificite de
la tuberculose, " Paris, 1868. Cohnheim and Fraenkel, " Experimented
Untersuchungen iiber der tlbertragbarkeit der Tuberculose auf Thiere."
Cohnheim, "Die Tuberculose vom Standpunkt der Infectionslehre,"
1879. Various Authors, "Discussion sur la tuberculose," Bull. Acad.
de med. (1867), xxxii., xxxiii. Armanni, "Novimento med.-chir.,"
Naples, 1872. Baumgarten, "Lehrb. d. path. Myk.," 1890. Straus,
"La • tuberculose et son bacille," Paris, 1895. Koch, Berl. klin.
Wchnschr. (1882), 221 ; Mitth. a. d. k. Gsndhtsamte., 1884 ; Deutsche
med. Wchnschr. (1890), No. 46a. ; (1891), Nos. 3 and 43 ; (1897), No. 14.
Bulloch, Lancet (1901), ii. 243. Nocard, "The Animal Tuberculoses,"
(trans.), London, 1895. Cornet, Ztschr. f. Hyg. v. 191. Nocard and
Roux, Ann. de I'Inst. Pasteur, i. 19. Pawlowsky, ibid. ii. 303.
Sander, Arch. f. Hyg. xvi. 238. Coppen Jones, Centralbl. f. Bakteriol.
u. Parasitenk. xvii. 1. Prudden and Hodenpyl, New York Med. Eec.
(1891), 636. Vissman, Virchoufs Archiv, cxxix. 163. Straus and
Gamaleia, Arch, de med. expe"r. et d'anat. path. iii. No. 4. Courmont,
Semaine med. (1893), 53 ; Revue de med. (1891), No. 10. Hericourt and
Richet, Bull. mtd. (1892), 741, 966. Williams, Lancet (1883), i. 312.
Pawlowsky, A nn. de I'Inst. Pasteur, vi. 116. Maffucci, "Sull' azione
37
578 BIBLIOGRAPHY
tossica del prodotti del bacillo della tuberculosi " ; CentralbL f. allg.
Path. u. path. Anat. i. 404. Kruse, Beitr. z. path. Anat. u. z. allg.
Path. xii. 221. Bellinger, Munchen. med. Wchnschr. (1889), No. 37 ;
Verhandl. d. Gesellsch. deutsch. Naturf. u. Aertze (1890), ii. 187.
Hofmann, Wien. med. Wchnschr. (1894), No. 38. Straus and Wiirtz,
Cong. p. V etude de la tuberculose, Paris, July 1888. Gilbert and Roger,
Mem. Soc. de Uol. (1891). Diem, Monatsh. f. prakt. Thierh. iii. 481.
Weyl, Deutsche med. Wchnschr. (1891), 256. Buchner, Centralbl. f.
Bakteriol. u. Parasitenk. xi. 488. Courmont and Dor, Province rntd.
(1890), No. 50. Tizzoni and Centanni, Centralbl. f. Bakteriol. u.
Parasitenk. xi. 82. Eibbert, Deutsche med. Wchnschr. (1892), 353.
Virchow, ibid. (1891), 131. Hunter, Brit. Med. Journ. (1891), July
25. Kiihne, Ztschr. f. Biol. xxix. 1 ; xxx. 221. Krehl, Arch. f. exper.
Path. u. Pharmakol. xxxv. 222. Krehl and Matthes, Hid. xxxvi. 437.
Bang, "La lutte contre la tuberculose en Danemark," Geneva, 1895.
Maragliano, " Le serum antituberculeux et son antitoxin," Paris, 1896 ;
Berl. klin. Wchnschr. (1896), 409, 437, 773. Nocard, Ann. de I'lnst.
Pasteur, xii. 561. Stockman, Brit. Med. Journ. (1898), ii. 681. Mara-
gliano, ref. Brit. Med. Journ., Epitome (1896), i. 63. Baumgarten and
Walz, CentralbL f. Bakteriol. u. Parasitenk. xxiii. 587. Smith, T.,
Journ. Exper. Med. iii. 451. Koch. 'Brit. Med. Journ. (1901), ii. 189 ;
Trans. Internat. Congr. of Tuberc., London, 1901. Delepine, Brit.
Med. Journ. (1901), ii. 1224. Bataillon, Dubard, and Terre (fish
tuberculosis), Compt. rend. Soc. de biol. 1897, 446. Dubard, Rev. de la
tubercul. (1898), 13, 129. Ravenel, Med. Bull. Univ. Pennsylvania, May
1902. Koch, Deutsche med. Wchnschr. (1902), No. 48. Koch, Schutz,,
Neufeld, and Miessner, Ztschr. f. Hyg. 51, 300. De Jong, Centralbl. f.'
Bakteriol. u. Parasitenk. 38 (Orig.), 146. Ravenel, Univ. of Pennsylvania
Med. Bulletin, 1902. Kossel, Weber, and Heuss, Tuber kulosearbeiten
aits d. Kaiserl. Gsndhtsmte., Berlin, 1904-1905. Weber and Tante, ibid.
1905. Salmon and Smith, "Tuberculosis," U.S. Department of Agri-
culture, Washington, 1904. Wolbach and Ernst, Journ. Med. Research,
x. 313. Interim Reports of the Royal Commission on Tuberculosis,
London, 1904, 1907. Wright and Douglas, Proc. Roy. Soc. Lond., Ixxiv.
159. Wright, Clinical Journal, Nov. 9, 1904; ibid., May 15, 1906;
Med. Chir. Trans. Ixxxix. (1905). Wright and Reid, Proc. Roy. Soc.
Lond. Ixxvii. 194, 211.
Other acid-fast bacilli. — Moeller, Deutsche med. Wchnschr. (1898), 376.
Centralbl. /. Bakteriol. u. Parasitenk. xxv. 369 ; ibid. xxx. 513. Petri,
Arb. a. d. k. Gsndhtsamte. (1898), 1. Rabinowitch, Deutsche med.
Wchnschr. (1897), No. 26 ; (1900), No. 16 ; Ztschr. f. Hyg. xxvi. 90.
Korn, Arch. f. Hyg. xxxvi. 57 ; Centralbl. f. Bakteriol. u. Parasitenk.
xxvii. 481. Schulze, Ztschr. f. Hyg. xxxi. 153. M. Tobler, ibid, xxxvi.
120. Lubarsch, ibid. xxxi. 187. Holscher, Centralbl. f. Bakteriol. u.
Parasitenk. xxix. 425. Potet, " Etude sur les bacilles dites ' acido-
philes,'" Paris, 1902. Abbott and Gildersleeve, Pennsylv. Med. Bullet.,
June 1902. Johne and Frothingham, Deutsche Ztschr. f. Thiermed. (1895),
438. M'Fadyean, Journ. Compar. Path. xx. (1907), 48.
CHAPTER X.— LEPROSY.
Hansen, Norsk. Mag. f. Lcegevidensk., 1874 ; Virchow 's Archiv, Ixxix.
32 ; xc. 542 ; ciii. 388 ; Firchotv's Festschr. (1892), iii. See paperstby
BIBLIOGRAPHY 579
Neisser and Cornil and Suchard in " Microparasites in Disease" (New
Sydenham Soc., 1886). Hansen and Looft, "Leprosy," Bristol, 1895.
Doutrelepont and Wolters, Arch. f. Dermat. u. Syph. (1892), 55.
Thoma, Sitzungsb. d. Dorpater Naturforsch. , 1889. Unna, Dermat.
Stud. Hamburg (1887), iv. Bordoni-Uffreduzzi, Ztschr. f. Hyg. iii.
178 ; Berl. klin. Wchnschr. (1885), No. 11. Arning and Nonne,
Virchow's Archiv, cxxxiv. 319. Gairdner, Brit. Med. Journ. (1887),
i. 1269. Hutchinson, Arch. Surg. (1889), i. V. Torok, "Summary of
Discussion on Leprosy at the 1st Internat. Congr. for Dermatol. and
Syph." v. Monatsh. f. praTct. Dermat. ix. 238. Profeta, Gior. internaz.
d. sc. med. 1889. See Journal of the Leprosy Investigation Committee,
1890-91. Philippson, Virchow's Archiv, cxxxii. 529. Danielssen,
Monatsh. f. prakt. Dermat. (1891), 85, 142. Wesener, CentralU. f.
Bakteriol. u. Parasitenk. ii. 450 ; Miinchen. med. Wchnschr. (1887),
No. 18. Uhlenhuth and Westphal, CentralU. f. Bakteriol. u. Parasitenk.
xxix. 233. Dean, Journ. of Hyg. v. 99. Babes in " Erganzungsband "
of Kolle and Wassermann's Handbuch der Pathogenen Mikroorganismen.
CHAPTER XI. —GLANDERS— RHINOSCLEROM A.
Loffler and Schultz, Deutsche med. Wchnschr. (1882), No. 52. Loffler,
Mitth. a. d. . k. Gsndhtsamte. i. 134. "Weichselbaum, Wien. med.
Wchnschr. (1885), Nos. 21-24. Preusse, Berl. thierarztl. Wchnschr.
(1889), Nos. 3, 5, 11 ; ibid. (1894), Nos. 39, 51. Gamaleia, Ann. de
I'Inst. Pasteur, iv. 103. A. Babes, Arch, de med. exper. et d'anat. path.
(1892), 450. Straus, Compt. rend. Acad. d. sc. cviii. 530. M'Fadyean
and Woodhead, Rep. National Vet. Assoc. 1888. Baumgarten, CentralU.
f. Bakteriol. u. Parasitenk. iii. 379. Silviera, Semaine m^d. (1891),
No. 31. Bonome, Deutsche med. Wchnschr. (1894), 703, 725, 744.
Kalning, Arch. /. Veterindrwissensch. (St. Petersburg), i. Apr. May.
Foth, Centralbl. f. Bakteriol. u. Parasitenk. xvi. 508,550. M'Fadyean,
Journ. Comp. Path, and Therap., 1892, 1893, 1894. Leclainche and
Montane, Ann. de I'Inst. Pasteur, vii. 481. Leo, Ztschr. f. Hyg. vii.
505. Marx, Centralbl. f. Bakteriol. u. Parasitenk. xxv. 275. Mayer,
ibid, xviii. 673. Bonome, Centralbl. f. Bakteriol. u. Parasitenk. Refer,
xxxviii. 97. Anderson, Chalmers, and Buchanan, Glasgow Med. Journ.,
Oct. 1905. Nicolle, Ann. de I'Inst Pasteur, xx. 623, 698, 801.
RHINOSCLEROMA. — Frisch, Wien. med. Wchnschr. (1882), No. 32.
Cornil and Alvarez, Arch, de physiol. norm, et path. (1895), 3rd series,
vi. 11. Paltauf and Eiselsberg, Fortschr. d. Med. (1S86), Nos. 19, 20.
Wolkowitsch, Centralbl. f. d. med. Wissensch., 1886. Dittrich, Ztschr.
f. Heilk. viii. 251. Babes, Centralbl. /. Bakteriol. u. Parasitenk. ii.
617. Pawlowski, ibid. ix. 742; "Sur 1'etiologie et la pathologic du
rhinosclerome," Berlin, 1891. Paltauf, Wien. med. Wchnschr. (1891),
Nos. 52, 53 ; (1892), Nos. 1, 2. Wilde, Semaine mid. (1896), 336.
Klemperer and Scheier, Ztschr. f. klin. Med. xlv. Heft 1-2. Lanzi,
Centralbl. f. Bakteriol. u. Parasitenk. Refer, xxxiv. 627. Schablowski,
ibid, xxxviii. 714.
CHAPTER XII.— ACTINOMYCOSIS, ETC.
Bollinger, Centralbl. f. d. med. Wissensch., 1877. J. Israel, Virchow's
Archiv, Ixxiv. 15 ; Ixxviii. 421. Ponfick, Breslau. aertzl. Ztschr., 1879 ;
580 BIBLIOGRAPHY
"Die Aktinomykose des Menschen," 1882. 0. Israel, Firchow's Archiv
xcvi. 175. Chiari, Prag. med. Wchnschr., 1884. Langhans, Cor.-BL f.
schweiz. Aerzte (1888), xviii. Liining and Hanau, ibid. (1889), xix.
Shattock, Trans. Path. Soc. London, 1885. Acland, ibid., 1886. Delepine,
ibid., 1889. Harley, Med.-Chir. Trans., London, 1886. Crookshank, ibid.,
1889; "Manual of Bacteriology," London, 1896. Ransome, Med.-Chir.
Trans., London, 1891. M'Fadyean, Journ. Comp. Path, and Therap.,
1889. Bostrom, Beitr. 2. path. Anat. u. z. allg. Path., 1890. Wolff and
Israel, Virchow's Archiv, cxxvi. 11. Illich, " Beitrage zur Klinik der
Aktinomykose," Wien, 1892. Grainger Stewart and Muir, Edin. Hosp.
Rep., 1893. Leith, ibid., 1894. Gasperini, Centralbl. f. Bakteriol. u.
Parasitenk. xv. 684. Hummel, Beitr. z. klin. Chir. xiii. No. 3. Paw-
lowsky and Maksutoff, Ann. de I'lnst. Pasteur, vii. 544. Neukirch,
Ueber Strahlenpilze, Strassburg, 1902. Doepke, Munehen. med. Wchnschr.,
1902. Sillurschmidt, Ztschr. f. Hyg. xxxvii. 345. J. Homer Wright,
Publications of the Massachusetts General Hospital, Boston, May 1905.
Allied Streptothrices. — Nocard, Ann. de I'lnst. Pasteur (1888), ii. 293.
Eppinger, Beitr. z. path. Anat. u. z. allg. Path. ix. 287 ; in Lubarsch and
Ostertag, "Ergebnisse der allgem. Path." iii. 328. Buchholz, Ztschr. f.
Hyg. xxiv. 470. Berestnew, ibid. xxix. 94. Cozzolino, ibid, xxxiii. 36.
Flexner, Journ. Exper. Med. iii. 435. Dean, Trans. Path. Soc. London
(1900), 26. Birt and Leishman, Journ. of Hyg. ii. 120. Mertens,
Centralbl. f. Bakteriol. u. Parasitenk. xxix. 694. Foulerton, Trans.
Path. Soc. London (1902), 56. M 'Donald, Trans. Med.-Chir. Soc. Edin.
xxiii. 131. Norris and Larkins, Journ. Exper. Med. v. 155. Butter-
field, Journ. Infect. Diseases, vi. 421.
MADURA DISEASE. — Carter "On Mycetoma or the Fungus Disease of
India," London. Bassini, Ref. in Centralbl. f. Bakteriol. u. Parasitenk.
iv. 652. Lewis and Cunningham, \\th Ann. Rep. San. Com. India.
Kobner, Fortschr. d. Med. (1886), No. 17. Kanthack, Journ. Path, and
Bacterial, i. 140. Boyce and Surveyor, Proc. Roy. Soc. London, 1893.
Vandyke Carter, Trans. Path. Soc. London, 1886. Vincent, Ann. de
I'lnst. Pasteur, viii. 129. Wright, J. H., Journ. Exper. Med. iii. 421.
Oppenheim, Arch. f. Dermat. u. Syph. Ixxi. 209.
CHAPTER XIII.— ANTHRAX.
Bellinger in Ziemssen's "Cyclopaedia of Medicine." Greenfield,
"Malignant Pustule" in Quain's "Dictionary of Medicine," London,
1894. Pollender, Vrtljschr. f. gerichtl. Med. viii. ; Davaine, Compt.
rend. Acad. d. sc. Ivii. 220, 351, 386 ; lix. 393. Koch, Cohn's Beitr. z.
Biol. d. Pftanz. (1876), ii. Heft 2. Mitth. a. d. k. Gsndhtsamte. i. 49.
Pasteur, Compt. rend. Acad. d. sc. xci. 86, 455, 531, 697 ; xcii. 209.
Buchner, Vir 'chow's Archiv, xci. Chamberland, Ann de I'lnst. Pasteur,
viii. 161. Chauveau, Compt. rend. Acad. d. sc. xci. 33, 648, 880 ; xcvi.
553. Czaplewski, Beitr. z. path. Anat. u. z. allg. Path. vii. 47.
Gamaleia, Ann. de. I'lnst. Pasteur, ii. 517. Marshall Ward, Proc. Roy.
Soc. London, Feb. 1893. Petruschky, Beitr. z. path. Anat. u. z. allg.
Path. iii. 357. Weyl, Ztschr. f. Hyg. xi. 381. Behring, ibid. vi. 117 ;
vii. 171 ; Osborne, Arch. f. Hyg. xi. 51. Roux, Ann. de I'lnst. Pasteur,
iv. 25. Hankin, Brit. Med. Journ. (1889), ii. 810 ; (1890), ii. 65.
Hankin and Wesbrook, Ann. de I'lnst. Pasteur, vi. 633. Sidney Martin,
Rep. Med. Off. Loc. Govt. Board (1890-91), 255. Marmier, Ann. del'Inst.
BIBLIOGRAPHY 581
Pasteur, ix. 533. Rd. Muir, Journ. Path, and Bacteriol. v. 374. Sclavo,
Rivista d' Igiene e Sannita pubblica, vii. Nos. 18, 19 ; Sulla stato
presente della Sierotherapia anticarbonchiosa. Turin, Pozzo, 1903 (see
Legge, Lancet (1905), i. 689, 765, 841). Sobernheim in Kolle and Wasser-
mann's Handbuch, iv. 793. Cler, Centralbl. f. Bakteriol. und Para-
sitenk. (Orig.) xl. 241. Bail, ibid, xxxiii. 343, 610. Sanfelice, ibid.
xxxiii. 61. Roger and Gamier, Compt. rend. Soc. BioL Iviii. 863.
Teacher, Lancet (1906), i. 1306.
CHAPTER XIV.— TYPHOID FEVER, ETC.
Eberth, Virchoufs Archiv, Ixxxi: 58 ; Ixxxiii. 486. Koch, Mitth. a.
d. k. Gsndhtsamte. i. 46. Gaffky, ibid. ii. 80. Klebs, Arch. f. exper.
Path. u. Pharmakol. xii. 231 ; xiii. 381. Escherich, Fortschr. d. Med.
(1885), Nos. 16, 17. Emmerich, Arch. f. Hyg. iii. 291. Rodet and
Roux, Arch, de med. exper. et d'anat. path. iv. 317. Weisser, Ztschr. f.
Hyg. i. 315. Klein, " Micro-organisms and Disease," London, 1896 ;
Rep. Med. Off. Loc. Govt. Board (1892-93), 345 ; (1893-94), 457 ; (1894-
95), 399, 407, 411. Babes, Ztschr. f. Hyg. ix. 323. Vincent, Compt.
rend. Soc. de biol. ser. ix. ii. 62. Birch- Hirschfeld, Arch. f. Hyg. vii.
341. Buchner, Centralbl. f. Bakteriol. u. Parasitenk. iv. 353. Pfuhl,
ibid. iv. 769. Petruschky, ibid. vi. 660. Hunter, Lancet (1901), i. 613.
Kitasato, Ztschr. f. Hyg. vii. 515. Chantemesse and Widal, Bull. med.
(1891), No. 82 ; Ann. de I'lnst. Pasteur, vi. 755 ; vii. 141. Pere, Ann.
de I'lnst. Pasteur, vi. 512. Neisser, Ztschr. f. klin. Med. xxiii. 93.
Nicholle, Ann. de I'lnst. Pasteur, viii. 853. Quincke and Stiihlen,
Berl. klin. Wchnschr. (1894), 351. A. Fraenkel, Centralbl. /. klin. Med.
(1886), No. 10. E. Fraenkel and Simmonds, ibid. (1886), No. 39.
Achalme, Semaine mtd. (1890), No. 27. Grawitz, Charite-Ann. xvii.
228. Beumer and Peiper, Centralbl. f. klin. Med. (1887), No. 4 ; Ztschr.
f. Hyg. i. 489 ; ii. 110, 382. Sirotinin, ibid. i. 465. R. Pfeiffer and
Kolle, Ztschr. f. Hyg. xxi. 203. R. Pfeiffer, Deutsche med. Wchnschr.
(1894), 898. Sanarelli, Ann. de I'lnst. Pasteur, vi. 721 ; viii. 193, 353.
Brieger and Fraenkel, Berl. klin. Wchnschr. (1890), 241, 268. Brieger,
Kitasato, and Wassermann, Ztschr. f. Hyg. xii. 137. Widal, Semaine
med. (1896), 295, 303. Achard, ibid. 295, 303. Grunbaum, Lancet,
Sept. 1896. Delepine, Brit. Med. Journ. (1897), i. 529, 967 ; Lancet,
Dec. 1896. Remlinger and Schneider, Ann. de I'lnst. Pasteur, xi. 55,
829. Widal and Sicard, ibid. xi. 353. Peckham, Journ. Exper. Med.
ii. 549. Richardson, ibid. iii. 329. Wright and Semple, Brit. Med.
Journ. (1897), i. 256. Wright and Lamb, Lancet (1899), ii. 1727.
Wright, ibid. (1900), i. 150 ; ii. 1556 ; ibid. (1901), i. 609, 858, 1272,
1532 ; ii. 715, 1107 ; ibid. (1902), ii. 651 ; Brit. Med. Journ. (1900), ii.
113 ; ibid. (1901), i. 645, 771. Wright and Leishman, ibid. (1900), i.
622. See also discussion at the Clin. Soc. London, Brit. Med. Journ.
(1901), ii. 1342. Sidney Martin, ibid. (1898), i. 1569, 1644 ; ii. 11, 73.
Bokenham, Trans. Path. Soc. London (1898), xlix. 373. Macfadyen,
Proc. Roy. Soc. London, B. Ixxvii. 548. Macfadyen and Rowland,
Centralbl. f. Bakteriol. u. Parasitenk. (Orig.) xxxiv. 618, 765.
Chantemesse and Widal, Ann. de I'lnst. Pasteur, vi. 755. Christophers,
Brit. Med. Journ. (1898), i. 71. Remy, Ann. de I'lnst. Pasteur, xiv.
555, 705. Wyatt Johnson, Brit. Med. Journ. (1897), i. 231 ; Lancet
(1897), ii. 1746. Durham, Lancet (1898), i. 154 ; ibid. ii. 446.
582 BIBLIOGRAPHY
Lorrain Smith and Tennant, Brit. Med. Journ. (1899), i. 193. Gordon,
Journ. Path, and Baderiol. iv. 438. Castellani, Ztschrft. f. Hyg. u.
Infectionskrankh. xl. i. (B. paratyphosus), Boycott, Journ. Hyg. vi.
33. (Bacillus Enteritidis, Gaertner), refs. vide Bauragarteu's Jahres-
bericht, iv. 249 ; vii. 297 ; xii. 508. Van Ermengem, in Kolle and
Wassermann, Handbuch, vol. ii. (Psittacosis), Baumgarten's Jahres-
bericht, xii. 496. (Bacillus Enteritidis Sporogenes), Klein, Rep. Med.
Off. Local Govt. Board, xxv. 171 ; xxvii. 210.
' BACTERIAL DYSENTERY. — Shiga, Centralbl.f. Bakteriol. u. Parasitenk.
xxiii. 599 ; xxiv. 817, 870, 913. Kruse, Deutsche med. Wchnschr.
(1900), 637. Flexner, Bull. Johns Hopkins Hosp. (1900), xi. 39, 231 ;
Brit. Med. Journ. (1900), ii. 917. Strong and Musgrave, Journ. Amer.
Med. Assoc. (1900), xxxv. 498. Vedder and Duval, Journ. Exper. Med.
(1902), vi. 181. Ogata, Centralbl. f. Bakteriol. u. Parasitenk. xi. 264.
See various authors in Studies from the Rockefeller Institute for Medical
Research (1904), vol. ii. Park, Collins, and Goodwin, Journ. Med.
Research (1904), xi. 553. Hiss, ibid. (1905), xiii. 1. Torrey, Journ.
Exper. Med. (1905), vii. 365. Weaver, Tunniclitfe, Heinemann, and
Michael, Journ. Inf. Dis. ii. 70. Doerr, Das Dysenterietoxin,
Jena, 1907.
SUMMER DIARRHCEA. — Morgan, Brit. Med. Journ. (1906), i. 908;
(1907), ii. 16.
CHAPTER XV.— DIPHTHERIA.
' Klebs, Verhandl. d. Cong. f. innere Med. (1883), ii. Loffler, Mitth.
a. d. k. Gsndhtsamte. (1884), 421. Roux and Yersin, Ann. de I'Inst.
Pasteur, ii. 629 ; iii. 273 ; iv. 385. Brieger and Fraenkel, Berl. klin.
Wchnschr. (1890), 241, 268. Spronck, Centralbl. f. allg. Path. u. path.
Anat. i. No. 25 ; iii. No. 1. Welch and Abbott, Johns Hopkins Hosp.
Bull., 1891. Behring and Wernicke, Ztschr. f. Hyg. xii. 10. Loffler,
Centralbl. f. Bakteriol. u. Parasitenk. ii. 105. v. Hofmann, Wien.
med. Wchnschr. (1888), Nos. 3 and 4. Cobbett and Phillips, Journ.
Path, and Baderiol. iv. 193. Peters, ibid. iv. 181. Wright, Boston
Med. and S. Journ. (1894), 329, 357. Kanthack and Stephens, Journ.
Path, and Bacteriol. iv. '45. Klein, Brit. Med: Journ. (1894), ii. 1393 ;
(1895), i. 100. Rep. Med. Off. LOG. Govt. Board (1890-91), 219 ; (1891-
92), 125. Guinochet, Compt. rend. Soc. de biol. (1892), 480. Roux and
Martin, Ann. de I'Inst. Pasteur, viii. 609. Cartwright Wood, Lancet
(1896), i. 980, 1076; ii. 1145. Sidney Martin, a Goulstonian Lectures,"
Brit. Med. Journ. (1892), i. 641, 696, 755 ; Rep. Med. Off. LOG. Govt.
Board (1891-92), 147 ; (1892-93), 427. Escherich, Wien. med. Wchnschr.
(1893), Nos. 47-50 ; Wien. klin. Wchnschr. (1893), Nos. 7-10 ; (1894),
No. 22; Berl. klin. Wchnschr. (1893), Nos. 21, 22, 23. Behring, "Die
Wassermann, Deutsche med. Wchnschr. (1894), 353. Funck, Ztschr. f.
Hyg. xvii. 401. Prochaska, ibid. xxiv. 373. Madsen, ibid. xxiv. 425.
Neisser, ibid. xxiv. 443. L. Martin, Ann. de I'Inst. Pasteur, xii. 26.
Salomonsen and Madsen, ibid. xii. 763. Woodhead, Brit. Med< Journ.
(1898), ii. 893 ; Rep. Metrop. Asyl. Bd., London, 1901. Metin, Ann. de
I'Inst. Pasteur, xii. 596. Madsen, ibid. xiii. 568, 801. Dean and Todd,
BIBLIOGRAPHY 583
Journ. of Hyg. ii. 194. Cobbett, ibid. i. 485. Graham-Smith, ibid. iv.
258 ; vi. 286. Petrie, ibid. v. 134. Hist, Compt. rend. Soc. de biol.
(1903), No. 25. Neisser, Berl. klin. Wchnschr. (1904), No. 11. Knapp,
Journ. Med. Research (1904), 475. Morgenroth, Ztschr. f. Hycj. xlviii.
177. Bolton, Lancet (1905), i. 1117. Theobald Smith, Journ. Med.
Research, (1905), xiii. 341. Boycott, Journ. of Hyg. v. 223.
CHAPTER XVI.— TETANUS, ETC.
Nicolaier, " Beitrage zur Aetiologie des Wundstarrkrampfes," Inaug.
Diss. Gottingen, 1885. Rosenbach, Arch. f. klin. Chir. xxxiv. 306.
Carle and Rattone, Gior. d. r. Accad. di med. di Torino, 1884. Kitasato,
Ztschr. f. Hycj. vii. 225 ; x. 267 ; xii. 256. Kitasato and Weyl, ibid.
viii. 41, 404. Vaillard, Ann. de I'lnst. Pasteur, vi. 224, 676. Vaillard
and Rouget, ibid. vi. 385. Behring, " Abhaiidlungen z. atiol. Therap.
v. anst. Krankh.," Leipzig, 1893; Ztschr. f. Hyg. xii. 1, 45; " Blut-
serumtherapie, " Leipzig, 1892 ; "Das Tetanusheilserum," Leipzig, 1892.
Brieger and Fraenkel, Berl. klin. Wchnschr. (1890), 241, 268. Sidney
Martin, Rep. Med. Off. LOG. Govt. Board (1893-94), 497 ; (1894-95), 505.
Uschinsky, Centralbl. f. Bakteriol. u. Parasitenk. xiv. 316. Tizzoni
and Cattani, Arch. f. exper. Path. u. Pharmakol. xxvii. 432 ; Centralbl.
f. Bakteriol. u. Parasitenk. ix. 189, 685 ; x. 33, 576 (Ref.) ; xi. 325 ;
Berl. klin. Wchnschr. (1894), 732. Madsen, Ztschr. f. Hyg. xxxii. 214.
Ritchie, Journ. of Hyg. i. 125. Danysz, Ann. de I'lnst. Pasteur,
xiii. 155. Marie and Morax, Ann. de I'lnst. Pasteur, Paris, xvi. 818.
Meyer and Ransom, Proc. Roy. Soc. London, Ixxii. 26 ; Arch. f. exper.
Path. u. Pharmakl., Leipzig, xlix. 269. Roux and Borrel, Ann. de
I'lnst. Pasteur, Paris, xii. 225 ; Henderson Smith, Journ. Hyg. vii. 205.
Kitt, see ref. in Centralbl. f. Bakteriol. u. Parasitenk. Jena, Referate,
xxxii. 359.
MALIGNANT OEDEMA. — Pasteur, Bull. Acad. de med., 1881, 1887.
Koch, Mitth. a. d. k. Gsndhtsamte. i. 54. Kitt, Jahresb. d. k. Centr.-
Thierarznei-Schule in Munchen, 1883-84. W. R. Hesse, Deutsche med.
Wchnschr. (1885), No. 14. Chauveau and Arloing, Arch. vtt. (1884),
366, 817. Liborius, Ztschr. f. Hyg. i. 115. Roux and Chamberland,
Ann. de I'lnst. Pasteur, i. 562. Charrin and Roger, Compt. rend. Soc.
de biol. (1877), ser. viii. vol. iv. p. 408. Kerry and S. Fraenkel, Ztschr.
f. Hyg. xii. 204. Sanfelice, ibid. xiv. 339. Leclainche and Velle, Ann.
de I'lnst. Pasteur, xiv. 202, 590.
BACILLUS BOTULINUS. — v. Ermengem, Centralbl. f. Bakteriol. u.
Parasitenk. xix. 443 ; Ztschr. f. Hyg. xxvi. 1. Kempner, ibid,, xxvi.
481. Kempner and Schepilewsky, ibid, xxvii. 213. Kempner and
Pollack, Deutsche med. Wchnschr. (1897), No. 32. Brieger and Kempner,
ibid. (1897), No. 33. Marinesco, Compt. rend. Soc. de biol. (1896), No. 31.
Schneidemiihl, Centralbl. f. Bakteriol. u. Parasitenk. xxiv. 577, 619.
Romer, ibid, xxvii. 857.
QUARTER-EVIL. — See Nocard and Leclainche, "Les maladies micro-
biennes des animaux," Paris, 1896. Arloing, Cornevin, et Thomas,
" Le charbon symptomatique du bceuf," Paris, 1887. Nocard and Roux,
Ann. de I'lnst. Pasteur, i. 256. Roux, ibid. ii. 49. See also Journ.
Comp. Path, and Therap. iii. 253, 346 ; viii. 166, 233.
BACILLUS ^ERG-GENES CAPSULATUS. — Welch and Nuttall, Bull. Johns
Hopkins Hosp. (1892), 81. Welch and Flexner, Journ. Exper. Med. i. 5.
584 BIBLIOGRAPHY
E. Fraenkel, Centralbl. f. Bakteriol. u. Parasitenk. xiii. 13. Durham,
Bull. Johns Hopkins Hosp. (1897), 68. Norris, Am. Journ. Med. Sc.
cxvii. 172.
CHAPTER XVII.— CHOLERA.
Koch, Rep. of 1st Cholera Conference, 1884 (v. " Microparasites in
Disease," New Sydenham Soc., 1886). Nikati and Rietsch, Compt. rqnd.
Acad. d. sc. xcix. 928, 1145. Bosk, Ann. de I'Inst Pasteur, ix. 507.
Pettenkofer, Munchen. med. Wchnschr. (1892), No. 46 ; (1894), No. 10.
Sawtschenko, Centralbl. f. Bakteriol. u. Parasitenk. xii. .893. Pfeiffer,
Ztschr. f. Hyg. xi. 393. Kolle, ibid. xvi. 329. Issaeff and Kolle, ibid.
xviii. 17. Wassermann, ibid. xiv. 35. Sobernheim, ibid. xiv. 485.
Metchnikoff, Ann. de I'Inst. Pasteur, vii. 403, 562 ; viii. 257, 529.
Fraenkel and Sobernheim, Hyg. Rundschau, iv. 97. Duribar, Arb. a. d. k.
Gsndhtsamte. ix. 379. Pfeiffer and Wassermann, Ztschr. f. Hyg. xiv. 46.
Wesbrook, Ann. de V Inst. Pasteur, viii. 318. Scholl, Berl. klin. Wchnschr.
(1890), No. 41. Griiber and Wiener, Arch.f. Hyg. xv. 241. Cunningham,
Sclent. Mem. Med. Off. India, 1890 and 1894. Hueppe, Deutsche med.
Wchnschr. (1889), No. 33. Klemperer, ibid. (1894), 435; Berl. klin.
Wchnschr. (1892), 969. Lazarus, ibid. (1892), 1071. Reincke, Deutsche
med. Wchnschr. (1894), 795. Koch, Ztschr. f. Hyg. xiv. 319. Voges,
Centralbl. f. Bakteriol. u. Parasitenk. xv. 453. Pastana and Bettencourt,
Centralbl. f. Bakteriol. u. Parasitenk. xvi. 401. Dieudonne, ibid. xiv.
323. Celli and Santori, ibid. xv. 289. Neisser, ibid. xiv. 666. Sanarelli,
Ann. de I'Inst. Pasteur, vii. 693. Ivanoff, Ztschr. f. Hyg. xv. 485.
Issaeff, ibid. xvi. 286. Pfuhl, ibid. x. 510. Rumpel, Deutsche med.
Wchnschr. (1893), 160. Klein, Rep. Med. Off. Loc. Govt. Board, 1893 ;
"Micro-organisms and Disease," London, 1896. Haffkine, Brit. Med.
Journ. (1895), ii. 1541 ; Indian Med. Gaz. (1895), No. 1 ; "Anti-cholera
Inoculation," Rep. San. Com. India, Calcutta, 1895. Pfeiffer in
Fliigge, "Die Mikroorganismen," 3rd ed. 1896; Gamaleia, Ann. de
I'Inst. Pasteur, ii. 482, 552. Achard and Bensande, Semaine med.
(1897), 151. Rumpf, "Die Cholera Asiatica und Nostras," Jena, 1898.
Kraus and Pribram, Centralbl. f. Bakteriol. xli. (Orig.), 15, 155. Kraus
and Prantschoff, ibid. 377, 480. A. Macfadyen, ibid. xlii. (Orig.),
365. Gotschlich, Scientific Reps. Sanitary, Maritime, and Quarantine
Council of Egypt, Alexandria, 1905, 1906. For discussion, vide Supple-
ment to Centralbl. f. 'Bakteriol. Referate, xxxviii. 84. Dunbar, Berlin,
klin. Wchnschr. (1902), No. 39.
CHAPTER XVIII.— INFLUENZA, ETC.
INFLUENZA. — Pfeiffer, Kitasato, and Canon, Deutsche med. Wchnschr.
xviii. 28, and Brit. Med. Journ. (1892), i. 128. Babes, Deutsche med.
Wchnschr. xviii. 113. Pfeiffer and Beck, ibid. (1892), 465. Pfuhl,
Centralbl. f. Bakteriol. u. Parasitenk. xi. 397. Klein, Rep. Med. Off.
Loc. Govt. Board (1893), 85. Pfeiffer, Ztschr. f. Hyg. xiii. 357. Huber,
Ztschr. f. Hyg. xv. 454. Kruse, Deutsche med. Wchnschr. (1894), 513.
Pelicke, Berl. klin. Wchnschr. (1894), 524. Pfuhl and Walter, Deutsche
med. Wchnschr. (1896), 82, 105. Cantani, Ztschr. f. Hyg. xxiii. 265.
Pfuhl, Ztschr. f. Hyg. xxvi. 112. Wassermaim, Deutsche med. Wchnschr.
(1900), No. 28. Clemens, Munchen. med. Wchnschr. (1900), No. 27.
BIBLIOGRAPHY 585
Wynecoop, Journ. Med. Ass., February 1903. Neisser, Deutsche med.
Wchnschr. (1903), No. 26. Auerbach, Ztschr. f. Hyg. (1904), xlviii. 259.
WHOOPING-COUGH. — Jochmann, Arch. f. klin. Med. Ixxxiv. 470.
Spengler, Deutsche med. Wchnschr. (1897), 830. Davis, Journ. Infect.
Dis. iii. 1. Bordet and Gengou, Ann. de I'lnst. Pasteur, xx. 731.
PLAGUE. — Kitasato, Lancet (1894), ii. 428. Yersin, Ann. de I'lnst.
Pasteur, viii. 662. Lowson, Lancet (1895), ii. 199. Yersin, Calmette,
and Borrel, Ann. de I'lnst. Pasteur, ix. 589. Aoyama, Centralbl. f.
Bakteriol. u. Parasitenk. xix. 481. Zettnow, Ztschr. f. Hyg. xxi. 164.
Yersin, Ann. de I'lnst. Pasteur, xi. 81. Gordon, Lancet (1899), i. 688.
Simond, Ann. de I'lnst. Pasteur, xii. 625. Haffkine, Brit. Med. Journ.
(1897), i. 424. Wyssokowitz and Zabolotny, Ann. de I'lnst. Pasteur,
xi. 663. Ogata, Centralbl. f. Bakteriol. u. Parasitenk. xxi. 769. Childe,
Brit. Med. Journ. (1898), ii. 858. See also Brit. Med. Journ. and
Lancet, 1897-99. Lustig and Galeotti, Deutsche med. Wchnschr. (1897),
No. 15. Markl, Centralbl. f. Bakteriol. u. Parasitenk. xxiv. 641, 728 ;
xxix. 810. Cairns, Lancet (1901), i. 1746. Montenegro, " Bubonic
Plague," London, 1900. Netter, "La peste et son bacille," Paris, 1900.
Mitth. der Deutschen Pest-Kommission, Deutsche med. Wchnschr. (1897),
Nos. 17, 19, 31, 32. "Report of the Indian Plague Commission (1898-
99)," London, 1900-1901. Also numerous papers in the Lancet and Brit.
Med. Journ., 1897-1901. Regarding Glasgow epidemic see ibid. (1900),
ii. "Reports on Plague Investigations in India," Journ. Hyg. (1906),
vi. 422 ; (1907), vii. 323.
RELAPSING FEVER.— Obermeier, Centralbl. f. d. med. Wissensch.
(1873), 145 ; and Berl. klin. Wchnschr. (1873), No. 35. Miinch,
Centralbl. f. d. med. Wissensch., 1876. Koch, Deutsche med. Wchnschr.
(1879), 327. Moczutkowsky, Deutsches Arch. f. klin. Med. xxiv. 192.
Vandyke Carter, Med.-Chir. Trans., London (1880), 78. Lubinoff,
Virchow's Archiv, xcviii. 160. Metchnikoff, ibid. cix. 176. Soudake-
witch, Ann. de I'lnst. Pasteur, v. 545. Lamb, Scient. Mem. Med. Off.
India (1901), pt. xii. 77. Sawtschenko and Melkich, Ann. de I'lnst.
Pasteur, xv. 497. Tictin, Centralbl. f. Bakteriol. xxi. 179. Karlinski,
Centralbl. f. Bakteriol. (1902), Orig. xxxi. 566. Gabritschewsky, Ztschr.
f. klin. Med. (1905), Bd. 56. Norris, Pappenheimer, Flournoy, Journ.
Infect. Dis. iii. 266. Novy and Knapp, ibid. 291. Zettnow, Ztschr. f.
Hyg. (1906), Iii. 485; Deutsche med. Wchnschr., 1906.
AFRICAN TICK FEVER.— Ross and Milne, Brit. Med. Journ. (1904),
ii. 1453. Button and Todd, Thompson- Yates Laboratory Rep. (1905), vi.
pt. ii. Koch, Deutsche med. Wchnschr., 1905 ; Berl. klin. Wchnschr., 1906.
Hodges and Ross, Brit. Med. Journ. (1905), i. 713. Breuil and Kinghorn,
ibid. i. 668. Breuil, Lancet (1906), i. 1806. Levaditi, Compt. rend. Soc.
biol., 1906.
MALTA FEVER.— Bruce, Practitioner, xxxix. 160 ; xl. 241 ; Ann. de
I'lnst. Pasteur, vii. 291. Bruce, Hughes, and Westcott, Brit. Med.
Journ. (1887), ii. 58. Hughes, Ann. de I'lnst. Pasteur, vii. 628 ; Lancet
(1892), ii. 1265. Wright and Semple, Brit. Med. Journ. (1897), i. 1214.
Wright and Smith, ibid. (1897), i. 911 ; Lancet (1897), i. 656. Welch,
ibid. (1897), i. 1512. Gordon, ibid. (1899), i. 688. Durham, Journ.
Path, and Bacterial, v. 377. Bruce in Davidson's "Hygiene and
Diseases of Warm Climates," Edinburgh and London, 1893. Birt and
Lamb, Lancet (1899), ii. 701. Brunner, Wien. klin. Wchnschr. (1900),
No. 7. Bruce, Journ. Roy. Army Med. Corps (1904), ii. 487, 731 ; (1907),
viii. 225. Horrocks, Proc. Roy. Soc. London, Series B (1905), Ixxvi.
586 BIBLIOGRAPHY
510. "Reports of the Commission on Mediterranean Fever," 1904-1907
(reprinted in Journ. Roy. Army Med. Corps). Eyre in Kolle and
Wassermann's Handbuch d. Pathoq. Mikro-organismen, Erganzungsband,
1906.
YELLOW FEVER. — Sternberg, Rep. Am. Pub. Health Ass. xv. 170.
Sanarelli, Ann. de I'Inst. Pasteur, xi. 433, 673, 753 ; xii. 348. David-
son, art. in Clifford Allbutt's "System of Medicine," vol. ii., London,
1897. Sternberg, Centralbl. f. Bakteriol. u. Parasitenk. xxii. 145 ; xxiii.
769. Sanarelli, ibid. xxii. 668. Reed and Carroll, Medical News, April
1899. Reed, Journ. of Hyg. ii. 101 (with full references). Durham,
Thompson- Yates Laboratory Rep. (1902), iv. pt. ii. 485. Gorgas, Lancet,
1902, Sept. 9 ; 1903, March 28. Marchoux, Salimbeni, and Simond,
Ann. de I'Inst. Pasteur, xvii. 665 ; xx. 16, 104, 161. Bandi, Ztschr. f.
Hyg. (1904), xlvi. 81. Otto and Neumann, Ztschr. f. Hyg. (1905), Ii.
Heft 3. Reed, Carroll, Agramonte, Lazear, Proc. Amer. Health Ass.,
1900; Journ. Amer. Med. Ass., Feb. 1901. Carroll, New York Med.
Journ., Feb. 1904 ; Amer. Medicine (.1906), xi. 383.
CHAPTER XIX.— IMMUNITY.
For oarly inoculation methods (e.g. against anthrax, chicken cholera,
etc.), see " Microparasites in Disease," New Syd. Soc. 1886. Duguid
and Sanderson, Journ. Roy. Agric. Soc. (1880), 267. Greenfield, ibid.
(1880), 273 ; Proc. Roy. Soc. London, June 1880. Toussaint, Compt.
rend. Acad. d. sc. xci. 135. Haffkine, Brit. Med. Journ. (1891), ii.
1278. Klein, ibid. (1893), i. 632, 639, 651. Klemperer, Arch. f. exper.
Path. u. Pharmakol. xxxi. 356. Buchner, Milnchen. med. Wchnschr.
(1893), 449, .480. Ehrlich, Deutsche med. Wchnschr. (1891), 976, 1218.
R. Pfeiffer, Ztschr. f. Hyg. xviii. 1 ; xx. 198. Pfeiffer and Kolle, ibid.
xxi. 203. Bordet, Ann. de I'Inst. Pasteur, ix. 462 ; xi. 106. Metchni-
koff, Firchow's Archiv, xcvi. 177 ; xcvii. 502 ; cvii. 209 ; cix. 176 ;
Ann. de I'Inst. Pasteur, iii. 289 ; iv. 65 ; iv. 193 ; iv. 493 ; v. 465 ; vi.
289 ; vii. 402 ; vii. 562 ; viii. 257 ; viii. 529 ; ix. 433. Calmette, Ann.
de I'Inst. Pasteur, viii. 275 ; xi. 95. Fraser, Proc. Roy. Soc. Edin. xx.
448. Marmorek, Ann. de I'Inst. Pasteur, ix. 593. Metchnikoff, Roux,
and Taurelli-Salimbeni, ibid. x. 257. Charrin and Roger, Compt. rend.
Soc. de biol. (1887), 667. Griiber and Durham, Miinchen. med. Wchnschr.
(1896), March. Durham, Journ. Path, and Bacteriol. iv. 13. Cart-
wright Wood, Lancet (1896), i. 980 ; ii. 1145. Sidney Martin, "Serum
Treatment of Diphtheria," Lancet (1896), ii. 1059. Ransome, "On
Immunity to Disease," London, 1896. Burdon Sanderson, " Croonian
Lectures," Brit. Med. Journ. (1891), ii. 983, 1033, 1083, 1135. Discus-
sion on Immunity, Path. Soc. London, Brit. Med. Journ. (1892), i. 373.
Fodor, Deutsche med. Wchnschr. (1887), No. 34. Hueppe, Berl. klin.
Wchnschr. (1892), No. 17. Nicholle, Ann. de I'Inst. Pasteur, xii. 161.
Salomonsen and Madsen, ibid. xi. 315 ; xii. 763. Roux and Borrell, ibid.
xii. 225. Salimbeni, ibid. xi. 277. Wassermann and Takaki, Berl.
klin. Wchnschr. (1898), xxxv. 4. Blumenthal, Deutsche med. Wchnschr.
xxiv. 185. Ransom, ibid. xxiv. 117. Meade Bolton, Journ. Exper.
Med. i. 543. Fraser, T. R., Brit. Med. Journ. (1895), i. 1309 ; ii. 415,
416 ; (1896), i. 957 ; (1896), ii. 910 ; (1897), ii. 125, 595. Calmette,
Ann. de I'Inst. Pasteur, vi. 160, 604 ; viii. 275 ; ix. 225 ; x. 675 ; xi.
214 ; xii. 343. C. J. Martin, Journ. Physiol. xx. 364 ; Proc. Roy. Soc.
BIBLIOGRAPHY 587
London, Ixiv. 88. C. J. Martin and Cherry, ibid. Ixiii. 420. Gautier,
"Les Toxines microbiennes et animales," Paris, 1896. Wassermann,
Berl. klin. Wchnschr. (1898), 1209. Pfeiffer and Marx, Ztschr. f. Hyg.
xxvii. 272. Bordet, Ann. de I'Inst. Pasteur, xii. 688. Ehrlich, Deutsche
med. Wchnschr. (1898), xxiv. 597. "Die Wertbemessung des Diph-
therieheilserums," Jena, 1897 ; Croonian Lecture, Proc. Roy. Soc.
London, Ixvi. 424 ; Deutsche med. Wchnschr. xxvii. (1901), 866, 888,
913; Nothnagel's " Specielle Pathologie und Therapie," Bd. viii.
Schlussbetrachtungen. Ehrlich and Morgenroth, Berl. klin. Wchnschr.
(1899), xxxvi. 6, 481 ; (1900), xxxvii. 453, 681 ; (1901), xxxviii. 251,
569, 598. Weigert, in Lubarsch and Ostertag, " Ergebnisse der
Allgemeinen Pathologie " (1897), iv. Jahrg. (Wiesbaden, 1899). Morgen-
roth, Centralbl. f. Bakteriol. u. Parasitenk. xxvi. 349. Bulloch, Trans.
Jenner Inst. 2nd ser. p. 46. Dbnitz, Deutsche med. Wchnschr. (1897),
xxiii. 428. Bordet, Ann. de rinst. Pasteur, xii. 688 ; xiii. 225, 273 ;
xiv. 257 ; xv. 303 ; xvii. 161 ; xviii. 593 ; Metchnikoff, ibid. xiii. 737 ;
xiv. 369 ; xv. 865. Gengou, ibid. xv. 232. Sawtschenko, ibid. xvi. 106.
Ritchie, Journ. of Hyg. ii. 215, 251, 452 (with full references) ;
"General Pathology of Infection," in Clifford Allbutt's "System of
Medicine," 2nd ed. 1906, vol. ii. pt. i. p. 1. Metchnikoff, " L'imnmnite
dans les maladies infectieuses," Paris, 1901. Neisser and Wechsberg,
Munchen. med. Wchnschr. 1901, No. 18. Von Dungern, ibid. (1899),
1288 ; (1900), 677, 973. Ehrlich, Collected Studies on Immunity
(English trans.), 1906, Bordet. Joos, Ztschr. f. Hyg. xxxvi. 422; xl.
203 ; Centralbl. f. Bakteriol. (Orig. ) xxxiii. 762. Eisenberg and Volk,
Ztschr. f. Hyg. xl. 155. Dreyer and Jex-Blake, Journ. Path, and Bacterial.
xi. 1. (Precipitius) Nuttall, Blood Immunity and Blood Relationship,
Cambridge, 1904.
OrsoNiNS. — Denys and Leclef, La cellule, 1895, 177. Sawtschenko,
Ann. de I'Inst. Pasteur, 1902, 106. Wright and Douglas, Proc. Hoy.
Soc. London, Ixxii. 357 ; Ixxiii. 128 ; Ixxiv. 147. Wright and Reid, ibid.
Ixxvii. 211. Bulloch and Atkin, ibid. Ixxiv. 379. Bulloch and Western,
ibid. Ixxvii. 531. Neufeld and Rimpau, Deutsche med. Wchnschr. (1904),
1458. Hektoen and Ruediger, Journ. Infect. Diseases, 1905, 128.
Leishman, Trans. Path. Soc. Lond. 1905. Muir and Martin, Brit. Med.
Journ. 1906, ii. ; Proc. Eoy. Soc. London, Ixxix. 187.
APPENDIX A. — SMALLPOX.
Jenner, "An Inquiry into the Causes and Effects of the Variola
Vaccinte," London, 1798. Creighton, art. "Vaccination" i\\Ency. Brit.,
9th ed. Crookshank, " Bacteriology and Infective Diseases." M'Vail,
"Vaccination Vindicated." Chauveau, Viennois et Mairet, "Vaccine
et variole, nouvelle etude experimentale sur la question de 1'identite de
ces deux affections," Paris, 1865. Klein, Hep. Med. Off. Loc. Oovt. Board
(1892-93), 391 ; (1893-94), 493. Copeman, Brit. Med. Journ. (1894), ii.
631 ; Journ. Path, and Baderiol. ii. 407 ; art. in Clifford Allbutt's
"System of Medicine," vol. ii. L. Pfeiffer, "Die Protozoen als Krank-
heitserreger," Jena, 1891. Ruffer, Brit. Med. Journ. (1894), June 30.
Beclere, Chambon, and Menard, Ann. de I'Inst. Pasteur, x. 1 ; xii. 837.
Copeman, "Vaccination," London, 1899. Calmette and Guerin, Ann.
de I'Inst. Pasteur, xv. 161. Guarnieri, Centralbl. f. Bakteriol. u. Para-
sitenk. xvi. 299. Ewing, Journ. Med. Research, xiii. 233. Prowazek,
588 BIBLIOGRAPHY
Arb. a. d. kaiserl. Gesundheitsamte, xxii. 535 ; xxiii. 525. Wasielewski,
Ztschrft. f. Hyg. xxxviii. 212. Bonhoff', Berl. klin. Wchnschrft. 1905,
p. 1142. Carini, Centralbl. f. BakterioL u. Parasitenk. (Orig.) xxxix. 685.
APPENDIX B. — HYDROPHOBIA.
Pasteur, Compt. rend. Acad. d. sc. xcii. 1259 ; xcv. 1187 ; xcviii. 457.
1229 ; ci. 765 ; cii. 459, 835 ; ciii. 777. Schaffer, Ann. de I'lnst. Pasteur,
iii. 644. Fleming, Trans. 7th Internal. Cong. Hyg. and Demog. iii. 16.
Helman, Ann. de I'lnst. Pasteur, ii. 274 ; iii. 15. Babes and Lepp, ibid.
iii. 384. Nocard and Roux, ibid. ii. 341. Roux, ibid. i. 87 ; ii. 479.
Bruschettini, Centralbl. f. BakterioL u. Parasitenk. xx. 214 ; xxi. 203.
Memmo, ibid. xx. 209 ; xxi. 657. Frantzius, ibid, xxiii. 782 ; xxiv.
971. Remlinger, Ann. de I'lnst. Pasteur, xvii. 834 ; xviii. 150 ; xix.
625. Negri, Ztschrft. f. Hyg. u. Infectionskrankh. xliii. 507 ; xliv. 519.
Williams and Lowden, Journ. Inf. Dis. iii. 452.
APPENDIX C. — MALARIAL FEVER.
^ Laveran, Bull. Acad. de med. (1880) ser. ii. vol. ix. 1346 ; "Traitedes
fievres palustres," Paris, 1884 ; "Du paludisme et de son hernatozoaire,"
Paris, 1891. Marchiafava and Celli, Fortschr. d. Med., 1883 and 1885 ;
also in Vircliows Festschrift. Golgi, Arch, per le sc. med., 1886 and 1889 ;
Fortschr. d. Med. (1889), No. 3 ; Ztschr. f. Hyg. x. 136 ; Deutsche med.
Wchnschr. (1892), 663, 685, 707, 729 ; Sternberg, New York Med. Rec.
xxix. No. 18. James, ibid, xxxiii. No 10. Councilman, Fortschr. d. Med.
(1888), Nos. 12, 13. Osier, Trans. Path. Koc. Philadelphia, xii. xiii.
Grass! and Feletti, Kiforma med. (1890), ii. No. 50. Canalis, Fortschr. d.
Med. (1890), Nos. 8, 9. Danilewsky, Ann. de I'lnst. Pasteur, xi. 758.
"Parasites of Malarial Fevers," New. Syd. Soc., 1894 (Monographs by
Marchiafava and Bignami, and by Mannaberg, with Bibliography).
Manson, Brit. Med. Journ. (1894) i. 1252, 1307 ; Lancet (1895), ii. 302 ;
Brit. Med. Journ. (1898), ii. 849 ; Koch, Berl. klin. Wchnschr. (1899),
69. Ross, Indian Med. Gaz. xxxiii. 14, 133, 401, 448. Nuttall,
Centralbl. f. BakterioL u. Parasitenk. xxv. 877, 903 ; xxvi. 140 ; xxvii.
193, 218, 260, 328 (with full literature). Manson, Lancet (1900),
i. 1417; (1900) ii. 151. Gray, Brit. Med. Journ. (1902), i. 1121.
Leishman, ibid. (1901), i. 635 ; ii. 757. Daniel, ibid. (1901), i. 193.
Celli, ibid. (1901), i. 1030. Nuttall and Shipley, Journ. of Hyg. i. 45,
269, 451 (with literature). Ross, Nature, Ixi. 522 ; "Mosquito Brigades
and how to organise them," London, 1902. Celli, "Malaria," trans, by
Eyre, London, 1900. Lankester, Brit. Med. Journ. (1902), i. 652.
Ewing, Journ. Exper. Med. v. 429; vi. 119. Schaudinn, Arbeit, ans d.
kaiserl. Gesundheitsamte, xix. ; Argutinsky Archiv mikroskop. Anat.
lix. 315 ; Ixi. 331. Ruge in Kolle and Wassermanns Handbuch d.
pathogen. Mikroorganismen, Erganzungsband, 1907 (full literature).
Ross, Lancet (1903), i. 86. Minchin, "The Sporozoa," London, 1903.
Stephens, art. " Blackwater Fever," in Allbutt's "System of Medicine,"
vol. ii. pt. ii., London, 1907.
BIBLIOGRAPHY 589
APPENDIX D. — DYSENTERY.
Losch, Virchow's Archiv, Ixv. 196. Cunningham, Quart. Journ.
Micr. Sc., N.S. xxi. 234. Kartulis, Virchow's Archiv, cv. 118 ; Centralbl.
f. BakterioL u. Parasitenk. ii. 745 ; ix. 365. Koch, Arb. a. d. k.
Gsndhtsamte. iii. 65. Councilman and Lafleur, Johns Hopkins Hosp.
Rep. (1891), ii. 395. Maggiora, Centralbl. f. BakterioL u. Parasitenk.
xi. 173. Ogata, ibid. xi. 264. Schuberg, ibid. xiii. 598, 701. Quincke
and Roos, Berl. klin. Wchnschr. (1893), 1089. Kruse and Pasquale,
Ztschr. f. Hyg. xvi. i. Ciechanowski and Nowak, Centralbl. f. BakterioL
u. Parasitenk. xxiii. 445. Howard and Hoover, Am. Journ. Med. Sc.
(1897), cxiv. 150, 263. Harris, Vir chow's Archiv, clxvi. 67. Schaudinn,
Arbeit, ans d. kaiserl. Gsndhtsamte. (1903), xix. 547. Lesage, Ann.
de VInst. Pasteur (1905), xix. 9. Kartulis in Kolle and Wassermann's
Handbuch d. pathoy. Mikroorganismen, Erganzungsband, 1906 ; Cen-
tralbl. f. BakterioL (Originate) 1904, xxxvii. 527.
APPENDIX E. — TRYPANOSOMIASIS, ETC.
GENERAL. — Laveran and Mesnil, Trypanosomes et trypanosomiasis,
Paris, Masson, 1904. Minchin, in Clifford Allbutt's "System of Medicine,"
2nd ed. vol. ii. pt. ii. p. 9, London, Macmillan, 1907. Schaudinn,
Arbeit, a. d. kaiserl. Gesundheitsamte, xx. 387. Mense, Handbuch der
Tropenkrankheiten, Leipzig, 1906, Barth. Novy and MacNeal, Journ.
Inf. Dis. ii. 256. Leishman, Journ. Hyg. iv. 434.
SLEEPING SICKNESS. — Mott, Reports of the Sleeping Sickness Com-
mission of the Royal Society, pt. vii. No. 15, London, Bale, Sons and
Danielsson, 1906. Dutton and Todd, Brit. Med. Journ. (1903), i. 304.
Button and Todd, Thompson- Yates Lab. Rep. v. pt. ii. i. ; v. pt. ii. 97.
Dutton, Todd, and Christy, ibid. vi. pt. i. p. 1. Manson and Daniels, ibid.
(1903), i. 1249. Idem, ibid. (1903), ii. 1461. Low and Mott, ibid. (1904), i.
1000. Bettencourt, Kopke, Resende, and Mendes, ibid. (1903), i. 908.
Castellani, Reports of the Sleeping Sickness Commission of the Royal
Society, No. 1, i. 1, London, Harrison and Sons, 1903. Bruce and
Nabarro, ibid. (1903), No. 1, ii. 11. Bruce, Nabarro, and Greig
ibid. (1903), No. 4, viii. 3. Greig and Gray, ibid. (1905), No. 6, ii.
3. Leishman, Journ. Hyg. iv. 434. Minchin, Gray, and Tulloch,
Reports of the Sleeping Sickness Commission of the Royal Society, No.
8, xxi. 122, London, H.M. Stationery Office, 1907. Manson, Brit.
Med. Journ. (1903), ii. 1249, 1461. See Discussions at British Medical
Association, Brit. Med. Journ. (1903), ii. 637 ; (1904), ii. 365. Thomas,
Thompson- Yates Lab. Rep. vi. pt. ii. 1.
KALA-AZAR. — Leishman, Brit. Med. Journ. (1903), i. 1252. Idem,
Clifford Allbutt, "System of Medicine," 2nd ed. vol. ii. pt. ii. 226, London,
Macmillan, 1907. Idem, Mense, Handbuch der Tropenkrankheiten, iii.
591, Leipzig, Barth., 1906. Leishman and Statham, Journ. of Roy.
Army Med. Corps, iv. 321. Donovan, Brit. Med. Journ. (1903), ii. 79.
Rogers, Quart. Journ. Micr. Soc. xlviii. 367. Idem, Brit. Med. Journ.
(1904), i. 1249 ; ii. 645. Idem, Proc. Roy. Soc. Ixxvii. 284. Bentley
590 BIBLIOGRAPHY
Brit. Med. Journ. (1904), ii. 653 ; ibid. (1905), i. 705. Christophers,
Scicntif. Mem. by Off. of the Med. and San. Dept. of the Govt. of India,
Nos. 8, 11, 15. Ross, Brit. Med. Journ. (1903), ii. 1401. See discussion
at Brit. Med. Assoc., Brit. Med. Journ. (1904), ii. 642.
DELHI SORE. — Wright, J. H., Journ. Med. Research, x. 472.
PIROPLASMOSIS. — See Minchin, loc. cit. supra. Koch, Deutsche, med.
Wchnschrft. (1905), No. 47 ; Ztschrft. f. Hyg. u. InfektionsTcrankh. liv. i.
Nuttall, Journ. Hyg. iv. 219. Nuttall and Graham -Smith, ibid. v.
237 ; vi. 586.
INDEX
INDEX
Abrin, 169
immunity against, 464, 469
Abscesses (v. also Suppuration)
bacteria in, 174
in dysentery, 540
Absolute alcohol, fixing by, 89
Acid-fast bacilli, 239, 252
stain for, 100
Acid formation, observation of, 44, 77
Acquired immunity in man, 456
theories of, 490
Actinomyces, 15
characters of, 287
cultivation of, 292
inoculation with, 296
varieties of, 294
Actinomycosis, 286
anaerobic streptothrices in, 293
diagnosis of, 296
lesions in, 290
origin of, 292
Active immunity, 458, 459
Aerobes, 17
culture of, 57
separation of, 51
^Estivo-autumnal fevers, 529
Agar media (v. also Culture media), 35
separation by, 55
Agglutinable substance, 486
Agglutination by sera, 485
in relapsing fever, 442
methods, 109
of b. mallei, 282
of b. typhosus, etc., 337
of cholera vibrio, 412
of m. melitensis, 450
of plague bacillus, 437
of red blood corpuscles, 481, 485
theories regarding, 485
38 593
Agglutinins, primary (homologous),
342
secondary (heterologous), 342
Agglutinogen, 486
Agglutinoids, 486
Aggressins, 164
Air, bacteria in, 126
examination of, for bacteria, 126
Albumose of anthrax, immunity by,
312
Albumoses, 165
in diphtheria, 363
Alcohols, higher, fermentation of, 75
Aleppo boil, 568
Alexines, 478, 500
Amboceptors, 480, 491
Amoebic dysentery, 537
Amcebulae of malaria, 523
Anaerobes, 17
cultures of, 60
separation of, 57
toxins of, 60
Anaerobic Esmarch's tubes, 60
Anaerobic fermentation tubes, 60
Anaerobic plate cultures, Bulloch'.s
apparatus for, 58
Anaesthetic leprosy, 269
Aniline oil, dehydrating by, 93
water, 98
Aniline stains, list of, 94
Animals, autopsies on, 123
inoculation of, 120
Anthrax, 300
anti-serum, 315
bacillus, 301
biology of, 304
cultivation of, 302
inoculation with, 308
toxins of, 312
594
INDEX
Anthrax, diagnosis of, 317
in animals, 306
in man, 309
protective inoculation, 314
spread of, 313
Anti-abrin, 470
Anti-anthrax serum, 315
Anti-bacterial sera, 476
properties of, 477
Anti-cholera vaccination, 412
Anti-diphtheritic serum, 467
Antikorper, 457
Anti-plague inoculation, 436
Anti-plague sera, 436
Antipneumococcic serum, 210
Antirabic serum, 519
Anti-ricin, 470
Antiseptics, 141
actions of, 144
standardisation of, 143
testing of, 142
Antisera, therapeutic action of,
488
Antistreptococcic serum, 489
Antitetanic serum, 384
preparation of, 467 et seq.
Antitoxic action, nature of, 470
bodies in normal tissues, 474
sera, use of, 469
serum, 467
cholera, 411
standardisation of, 468
Antitoxins, chemical nature of, 470
origin of, 474
Antitubercular serum, 264
Antityphoid serum, 344
Appendicitus, 185
Arthrospores, question of occurrence
of, 7
Artificial immunity, varieties of,
457 et seq.
Attenuation of virulence, 457
Autoclave, 29
Autolysis of bacteria, 162
Autopsies on animals, 123
Avian tuberculosis, 250
Bacilli, acid-fast, 239, 252
stain for, 100
characters of, 12
Bacillus acidi lactici, 20
aerogenes capsulatus, 188, 397
anthracis, 301
botulinus, 393
Bacillus coli communis, lesions caused
by, 183 et seq.
characters of, 324
comparison with b. typhosus, 325
coli in soil, 133, 134
pathogenicity of, 333
diphtherias, 353
dysenteric Shiga-Flexner, 346
enteritidis (Gaertner), 336
enteritidis sporogenes, 350
in soil, 133, 134
of glanders, 277
icteroides, 452
of influenza, 420
Koch- Weeks, 191
lactis aerogenes, 180
lacunatus, 193
of leprosy, 269
of malignant oedema, 390
Miiller's, 192
mycoides in soil, 132
ozoenae, 285
paratyphosus, 335
of plague, 426
pneumonice, 199
pseudo-diphthericus, 366
of psittacosis, 337
pyocyaneus, 180
agglutination of, 485
occurrence of, 185
pyogenes fcetidus, 174
of quarter-evil, 396
of rhinoscleroma, 284
of smegma, 254-
of soft sore, 227
subtilis, 57
of syphilis, 229
tetani, 372
of Timothy grass, 253
of tubercle, 237
of typhoid, 319
differentiation from b. coli, 325
of xerosis, 367
Bacteria, action of dead, 154
aerobic (v. Aerobes), 17
anaerobic (v. Anaerobes), 17
biology of, 16
capsulated, 3
chemical action of, 21
composition of, 9
classification of, 11
cultivation of, 25
death of, 141
effects of light on, 19
INDEX
595
Bacteria, food supply of, 16
higher, 14
lower, 11
microscopic examination of, 85
morphological relations of, 2
motility of, 7
movements of, 19
multiplication of, 4
nitrifying, 23
parasitic, 21
pathogenic, action of, 149
effects of, 155
saprophytic, 21
separation of, 51
species of, 23
spore formation in (v. also Spores),
5, 56
structure of, 3
sulphur- containing, 9
temperature of growth of, 18
toxins of, 161
variability among, 23
virulence of, 150, 461
Bacterial ferments, 22, 168
pigments, 10
protoplasm, structure of, 8
treatment of sewage, 139
Bactericidal powers of serum, 477
substances, 477
Bacteriological diagnosis, 118
examination of discharges, 116
Beer wort agar, 44
Beggiatoa, 15
Behring ondmmunity, 385, 468
Bile-salt media, 43
Bismarck-brown, 95
Blackleg, 396
Blackwater fever, 534
Blastophores (malaria), 529
Blood-agar (v. also Culture media),
00
DO
Blood, examination of, 68, 88
in malarial fever, 521
in relapsing fever, 438
serum, coagulated, as medium, 40
Bone-marrow in leucocytosis, 156
Bordet's phenomenon, 477
Botulism, bacillus of, 393
Bouillon (v. also Culture media), 32
Bovine tuberculosis, 248
Bread paste, 47
Brieger and Boer, 166
Fraenkel, 162
Buboes, 227
Bubonic pest, 429
Buchner on alexines, 500
Bulloch's apparatus for anaerobic
culture, 58
Biitschli on bacterial structure, 9
Butter bacilli, acid fast, 253
Calmette, 435, 462, 469, 471
Canon on influenza, 420
Cantani on influenza, 424
Capaldi and Proskauer, media of, 328
Capsules, staining of, 102
Carbol-fuchsin, 99
-methylene-blue, 98
-thionin-blne, 98
Carbolic acid as antiseptic, 147
Carroll's method of making anaerobic
cultures, 60
Carter on relapsing fever, 440
Cattle plague, 507
Cerebro- spinal fluid, examination by
lumbar puncture, 68
Chamberland and Eoux, attenuation
of b. anthracis, 460
Chamber-land's filter, 70
Chemiotaxis, 20, 495
Chlorine as antiseptic, 144
Cholera, 399
immunity against, 410
inoculation of man with, 410
methods of diagnosis of, 412
preventive inoculation against, 412
-red reaction, 404
Cholera spirillum, 400
distribution of, 402
inoculation with, 406
powers of resistance of, 405
relations to disease, 414
toxins of, 408
Cladothrices in soil, 132
Cladothrix, 15
asteroides, 295
Clubs in actinomyces, 289
Cocci, characters of, 11
Collodion capsules, preparation of,
123
Colonies, counting of, 65
Comma bacillus, 399
Commission on tuberculosis, 248
vaccination, 505
Complement, 478
deviation of, 488
Conjunctivitis, 191
Conradi-Drigalski medium, 42
596
INDEX
Copeman on smallpox, 508
Cornet's forceps, 87
Corrosive films of blood, etc., 89
Corrosive sublimate, as antiseptic, 145
fixing by, 90
Councilman and Lafleur on dysentery,
537
Coimting of colonies, 65
dead bacteria in a culture, 67
living bacteria in a culture, 66
Cover-glasses, cleaning of, 87
Cowpox, relation to smallpox, 505
Cresceutic bodies in malaria, 523
Cultivation of anaerobes, 57
Culture media, preparation of : agar,
36
alkaline blood serum, 41
blood agar, 38
serum, 39
bouillon, 32
bread paste, 47,
glucose agar, 37
broth, 32
gelatin, 35
glycerin agar, 37
broth, 35
litmus whey, 44
Loffler's serum medium, 40
Marmorek's serum media, 41
meat extract, 31
peptone gelatin, 35
solution, 38
potatoes, 44
serum agar, 38
Cultures, destruction ol, 83
filtration of, 69
from organs, 117, 124
hanging-drop, aerobic, 63
anaerobic, 64
incubation of, 79
microscopic examination of, 86
permanent preservation of, 82
plate, 52j
pure, 48
"shake," 77
Cutting of sections, 92
Cystitis, 185, 224
Cytases, 478, 496
Cytolytic sera, 482
De Bary, definition of species, 23
Decolorising agents, 97
Deep cultures, 60
Delhi sore, 568
Dehydration of sections, 93
Delepine, 110
Deneke's spirillum, 419
Dextrose-free bouillon, 75
Diagnosis, bacteriological, 115, 118
Diphtheria, 352
diagnosis of, 368
immunity against, 467
origin and spread of, 365
paralysis in, 353, 361
results of treatment, 488
Diphtheria bacillus, action of, 358
bacilli allied to, 365
characters of, 353
distribution of, 354
inoculation with, 359
isolation of, 368
powers of resistance of, 359
staining of, 108, 359
toxins of, 163, 361
variations in virulence of, 364
Diplo-bacillus of conjunctivitis, 193
Diplococcus, 12
catarrhalis, 217
crassus, 217
endocarditidis encapsulatus, 188
intracellularis meningitidis, 213
pneumonise, 199
Disturbances of metabolism by
bacteria, 159
Drigalski-Conradi medium, 42
Drying of sera, etc., in vacuo, 78
Ducrey's bacillus, 227
cultivation of, 228
Dum-Dum fever, 563
Durham's fermentation tubes, 76
Dysentery, amoebic, 537
bacteria in, 346
characters of amoeba of, 537
Dysentery, methods of examination
in, 347, 542
East coast fever in cattle, 569
Eberth's bacillus, 319
Ehrlich on ricin and abrin, 464, 469
on toxins, 170
side-chain theory of antitoxin for-
mation, 491
Eisner's medium, 46
Embedding in paraffin, 91
Empyema, 205, 423
Endocarditis, bacteria in, 188
Enhsemosphores (malaria), 523
Entamceba coli, 537
INDEX
597
Entamoeba histolytica, 537
cultivation of, 539
Enteric fever, 319
Enteritis, dysenteric, 347, 540
Epidemic cerebrospinal meningitis,
213
Eppinger's streptothrix, 295
Ermengem on botulism, 394
stain for flagella, 104
Erysipelas, 191
Escherich's bacillus, 319
Esmarch's roll-tubes, 55, 59
anaerobic, 60
Exaltation of virulence, 461
Examination of water, 135
Exhaust-pump, 70
Exotospores (malaria), 522
False membrane, 184, 353
Farcy, 276
Feeding, immunity by, 464
Fermentation by pneurno- bacillus,
204
by bacillus coli, 326
methods of observing, 74
of sugars by bacteria, 74
test of bacterial action, 74
tubes, 76
anaerobic, 60
Ferments formed by bacteria, 22,
168
in diphtheria, 364
in tetanus, 380
Fever, 159
Film preparations, dry, 86
wet, 88
staining of, 95
Filter, porcelain, gelatin ed, 166
Filtration of cultures, 69
Finkler and Prior's spirillum, 418
Fish, tuberculosis in, 251
Fixateurs, 497
Fixation of tissues, 89
Flagella, nature of, 8
staining of, 103
Flagellated organisms in malaria, 528
Fliigge, 14
Forceps for cover-glasses, 87
Formalin as antiseptic, 146
Foth's dry mallein, 283
Fraenkel's pneumococcus, 192, 198,
199
stain for tubercle, 101
Framboesia, spirochsetes in, 234
Frankland, on water bacteria, 137
Fraser, T. E., 462, 469, 475
Friedlander's pneumobacillus, 198,
203
Frisch on rhinoscleroma, 284
Fuchsin, carbol-, 99
Gamaleia on pneumonia, 206
Gaiuetocytes (malaria), 527
Gangrenous emphysema, 389, 392
pneumonia, 423
Gas formation, observation of, 44, 76
Gas -regulator, 80
Geissler's exhaust-pump, 70
Gelatin media, 35
phenolated, 345
separation by, 51
Gelatined porcelain filter, 166
Gentian-violet, 98
Germicides, 141
Geryk pump, 78
Giemsa's stain, 107
stain for spirochsetes in films, 107
Glanders, 275
diagnosis of, 283
in horses, 276
in man, 276
lesions in, 281
Glanders bacillus, 277
agglutination of, 282
inoculation with, 280
Glossina morsitans, 552
palpalis, 559
Glucose media, 35 et seq.
Glucosides, fermentation of, 75
Glycerin media, 35 et seq.
potato as culture medium, 46
Golgi on malaria, 521
Gonidia, 15
Gonococcus, characters of, 219
inoculation with, 222
toxin of, 223
Gonorrhoea, 219
Gonorrhoeal conjunctivitis, 225
endocarditis, 225
septicaemia, 226
Gram's method, 99^
Weigert's modification of, 100
Grease, 504
Greenfield on anthrax, 311, 446, 460
Griiber and Durham's phenomenon,
485
Guarnieri bodies in smallpox, 508
598
INDEX
Gulland (methods), 89, 92
Haemamceba Danilewski, 530
malarise, 530
prsecox, 530
relicta, 530 *
vivax, 530
Hsematozoon malaria?, 520
Hsemolytic sera, 479
Hsemolytic tests, methods of, 483
Haff kine on anti - cholera inocula-
tion, 412
Haffkine's inoculation method against
plague, 436
Halteridium, 528, 530
Hanging-drop cultures, 63
examination of, 8,5
Hankiu, 312
Hansen, leprosy bacilli, 269
Hesse's tube, 127
Hiss's serum water media, 41
Hofmann's bacillus, 366
Horsepox, 504
Houston on bacteriology of soil, 1 31
Hueppe, 7, 14
Hydrogen, supply of, 58
Hydrophobia, 510
diagnosis of, 519
Negri bodies in, 514
prophylactic treatment of, 516
the virus of, 513
Hypodermic syringes, 121
Immune-bodies, 478
origin of, 482
Immunity (v. also Special Diseases),
456
acquired, theories of, 490
active, 458, 459
artificial, 457
by feeding, 464
by toxins, 462
methods, 459
natural, 498
passive, 458, 464
unit of, 468
Impression preparations, 118
Incubators, 79
Indol, formation of, 77
Infection, conditions modifying, 149
nature of, 153
Inflammatory conditions due to
bacteria, 157
Influenza, 420
bacilli, pseudo-, 423
Influenza, bacillus, cultivation of, 421
bacillus, inoculation, 424
lesions in, 422
sputum in, 422
Inoculation, methods of, 120
of animals, 120
separation by, 56
protective, 462 et seq.
Intestinal changes in cholera, 402
amoebic dysentery, 539
bacterial dysentery, 347
typhoid fever, 329
Intestinal infection in cholera (ex-
perimental), 407
Involution forms in bacteria, 4
Iodine solution, Gram's, 99
terchloride, 468
as antiseptic, 145
lodoform as antiseptic, 148
Issaeff, 464
Ivauoff s vibrio, 415
Japanese dysentery, 350
Jenner on vaccination, 503
Jeuner's stain, 106
Johne's bacillus, 254
Joints, gouococci in, 225
Kala-azar, 563
Kipp's apparatus, 58
Kitasato on bacillus of influenza, 420
of plague, 425
of tetanus, 372 et seq.
Klebs-Loffler bacillus, 352
Klein, 345, 508
Klemperer on pneumonia, 210
Koch on avian tuberculosis, 250
bacillus of malignant oedema, 388
bovine tuberculosis, 248
cholera spirillum, 399
cultivation of b. anthracis, 301
leveller for plates, 53
tubercle bacillus, 235
tuberculin, 258
"tuberculin 0," and "R," 260
Koch-Weeks bacillus, 191
Korn's acid-fast bacillus, 253
Kruse and Pasquale on dysentery, 541
Kubel-Tiemann litmus solution, 42
Kiihne's methylene-blue, 98
modification of Gram's method, 100
Lamb on relapsing fever, 442
Laveran's malarial parasite, 521
Leishmau- Donovan bodies, 563
INDEX
599
Leisbman-Donovan bodies, cultivation
of, 566
Irishman's opsonic technique, 111
serum method for staining try-
panosomes, 545
stain, 106
Leishmauia donovaui, 567
tropica, 567, 568
Lenses, 85
Lepra cells, 269
Leprosy, 267
bacillus, 269
distribution of, 271
staining, 100, 270
diagnosis of, 274
etiology of, 272
Leprosy-like disease in rats, 273
Leptothrix, 15
Lesions produced by bacteria, 155
Leucocidin, 165
Leucocytosis, 156, 495
Leucomaines, 161
Levaditi's method for staining spiro-
chsetes, 104
Litmus solution, Kubel-Tiemann's, 42
whey, 44
Liver abscess in dysentery, 540
Lockjaw, 371
Loffler's bacillus, 352
methylene-blue, 98
serum medium, 40
and Schutz' glanders bacillus, 275
Losch, amceba of, 537
Lumbar puncture, 68
Lustgarten's bacillus, 229
Lustig's anti-plague serum, 436
Lymph, vaccine, 506
Lymphangitis, 184
Lysogenic action of serum, 477
towards blood corpuscles, 479
MacConkey's bile-salt media, 43
medium, use of in dysentery, 347
in examining water, 136
in paratyphoid fever, 335
M'Fadyean on glanders, 282
Macrocytase, 497
Macrophages, 495
Madura disease, 297
Malaria, cycle in man, 522
in mosquito, 528
pathology of, 533
prevention of, 532
question of immunity against, 534
Malarial fever, examination of blood
in, 535
malignant, 523, 531
mosquitoes in, 532
Malarial parasite, 52-1
inoculation of, 522
staining of, Leishman's method,
106
Romanowsky methods, 106
varieties of, 529
Malignant cedema, bacillus of, 388
diagnosis of, 393
immunity against, 393
Malignant pustule, 310
Mallein, 283
Malta fever, 446
methods of diagnosis, 450
spread of disease, 449
Mann's method of fixing sections, 92
Manson, 521
Maragliano's anti - tubercular serum
264
Marchiafava and Celli on malaria
521
Marmorek, on streptococci, 183
antistreptococcic serum, 476
Marmorek's serum media, 41
antitubercular serum, 265
Martin, Sidney, on albumoses, etc., 165
on anthrax, 312
on diphtheria, 363
Martin, C. J., on toxins, 166
on antitoxins, 475
Massowah vibrio, 416
Measuring bacteria, 119
Meat extract, 31
Meat-poisoning by bacillus botulinus,
393
by Gaertner's bacillus, 336
Mediterranean fever, 446
Meningitis, bacteria in, 217
epidemic cerebro-spinal, 174, 213
in influenza, 423
pneumococci in, 205
posterior basal, 216
Mercury perchloride as antiseptic, 145
Metabolism, disturbances of, by
bacteria, 159
Metachromatic granules, 8
Metchnikoff on cholera in rabbits, 407
relapsing fever, 441
Metchnikoff's phagocytosis theory, 495
spirillum, 417
Methylene-blue, 95, 98
600
INDEX
Methyl-violet, 94
Meyer and Ransom on tetanus toxin,
382
Micrococci of suppuration, 174
Micrococcus, 12
of gonorrhoea, 219
melitensis, 447
pyogenes tennis, 174
tetragenus, 181
lesions caused by, 185
urese, 20 """
Microcytase, 497
Microphages, 495
Microscope, use of, 85
Microtomes, 90
Migula, 12
Mikulicz, cells of, 284
Milk as culture medium, 46
Moller's stain for spores, 102
Moeller's Timothy-grass bacillus, 253
Morax, bacillus of, 192, 193 '
Mordants, 97 •
Morgan's bacillus, No. 1, 351
Mosquitoes, in malaria, 528, 532
role in yellow fever, 453
Moulds, media for growing, 44
Muencke's filter, 72
Miiller's bacillus, 192
Mycetoma, 297
Myelocytes, neutrophile, 156
Nagana, 552
Natural immunity, 498
Neelsen's stain for tubercle, 101
Negative phase in immunisation, 262,
494
Negri bodies in rabies, 514
Neisser's gonococcus, 219
stain for b. diphtherise, 108
Neucki, 10
Neutral-red as indicator for media, 43
use of, 38
with b. typhosus, 327
Neutrophile leucocytes, 156
myelocytes, 156
Nicolaier, tetanus bacillus, 371
Nicolle's modification of Gram's
method, 100
Nikati and Rietsch on cholera, 407
Nitrifying bacteria, 23
Nitroso-indol body, 78
Nordhafen vibrio, 418
Novy and MacNeal, medium for
culture of trypanosomes, 546
Obermeier's spirillum, 438
(Edema, malignant, 388
Ogata's dysentery bacillus, 350
Ogston, 174
Oil, aniline, for dehydrating, etc., 93
Oil immersion lens, 85
Ookinete, 528, 547, 548
Opsonic action, nature of, 483
technique, 111
Opscnins, 112
absorption of, 484
in tuberculosis, 261
thermolabile, 484
thermostable, 484
Organisms lower than bacteria, 2, 452
Oriental plague, 425
Osteomyelitis, 190
Otitis, 205, 423
Oxygen, nascent, as antiseptic, 145
Ozoena bacillus, 285
Para-colon bacillus, 335
Paraffin embedding, 91
Paratyphoid bacillus, 335
Passage, 461
Passive immunity, 458, 464
Pasteur on exaltation of virulence of
bacteria, 461
on hydrophobia, 516
on vaccination against anthrax, 314
septicemie de, 388
Pathogenicity of bacteria, 149
Peptone gelatin (v. Culture media), 35
solution, 38, 404
Periostitis, acute suppurative, 190
Peritonitis, 184, 224
Perlsucht, 236
Pestis major, 431
minor, 431
Petri's acid-fast bacillus, 253
capsules, 52
sand-filter for examining air, 128
Petruschky's litmus whey, 44
Pettenkofer on cholera, 410, 415
Pfeffer, 20
Pfeiffer on anti-serum, 477
cholera, 411
influenza, 420
typhoid, 333
Pfeiffer's phenomenon, 411, 477
Phagocytes, 156
Phagocytosis theory of Metchnikoff,
495
Phenol broth, 347
INDEX
601
Phenol-phthalein as indicator, 33
Phenomenon of Bordet, 477
Gru'ber and Durham, 485
Pfeiffer, 411, 477
Pigments, bacterial, 10
Pipettes, 66, 108, 110, 116
Piroplasmata as causes of disease, 569
Piroplasmosis, 568
Pitfield's flagella stain, 103
Plague, bacillus of, 426 et seq,
Haffkiue's inoculation against, 436
immunity against, 435
infection in, 432
involution forms, 427
part played by rat fleas in the
spread of, 433
preventive inoculation against, 436
serum diagnosis, 437
stalactite growths of, 429
varieties of, 431
Plasmolysis, 9
Plate cultures, agar, 55
gelatin, 51
gonococcus, 222
Platinum needles, 49
Piieumobacillus ( Fried! ander's), 199,
203 et seq.
Pneumococcus (Fraenkel's), 199,
201 et seq.
immunity against, 210
in endocarditis, 188
lesions caused by, 204 .
toxins of, 209
Pneumonia, bacteria in, 197
gangrenous, 423
in influenza, 422
methods of examination of, 212
septic, 197
varieties of, 196
Polar granules, 8
Positive phase in immunisation, 262,
494
Potassium permanganate as antiseptic,
147
Potatoes as culture material, 44
Poynton and Payne on acute
rheumatism, 193
Precipitius, 487
Preparations, impression, 118
Protective inoculation, 462 et seq.
Proteosoma, 530
Protozoa described in hydrophobia,
513
smallpox, 508
Protozoou inalarise, 521
Pseudo- diphtheria bacillus, 365
-tuberculosis streptothricea, 296
Psittacosis bacillus, 337
Ptomaines, 161
Puerperal septicaemia, 184
Pus, examination of, 87, 195
Pustule, malignant, 310
Pyaemia, 184 et seq.
nature of, 173
Quartan fever, 530
Quarter-evil, bacillus of, 396
Quotidian fever, 529
Rabies, 510
Rabinowitch's acid-fast bacillus, 253
Rauschbrand bacillus, 396
Ray-fungus (actinomyces), 286
Reaction of media, standardising of,
oq
OQ
Receptors, 491
Recovery from disease, 457
Red stains, 95
Red- water fever in cattle, 569
Reichert's gas regulator, 80
Relapsing fever, agglutination of
spirillum, 442
bactericidal serum in, 442
spirillum of, etc., 439
Reversibility of toxin-antitoxin reac-
tion, 472
Rheumatism, acute, 193
Rhinoscleroma, bacillus of, 284
Ricin, 169
immunity against, 464, 469
Rivers, bacteria in, 137
Robin, 169
Rock fever, 446
Roll-tubes, Esmarch's, 55, 59
Romanowsky stains, 105
Roseubach (bacteria in suppuration),
174
Ross, on malaria, 521
thick film method for malarial
parasite, 536
Roux on antitoxic sera, 469
and Yersin (diphtheria), 361 etseq.
Sabouraud's medium, 44
Safraniu, 95
Salt-agar as medium for b. pestis, 427
Sanarelli (typhoid fever), 332
Sanderson, Burden, 460, 507
602
INDEX
Saprophytes, 149
Sarciiia, 12
Sausage poisoning, bacillus botulinus
in, 394
Schaudinn 011 biology of trypauosomes,
548
on amoebae of dysentery, 537
on morphology of spirilla, 550
on spirochsete pallida, 229
on spirillum Ziemanni, 550
Schizomycetes, 3
Schizophyceae, 3
Schizophyta, 3
Schiiffuer's dots, 106, 531
Sclavo's anti-anthrax serum, 315
Scorpion poison, 169
Section-cutting, 90
Sections, dehydration of, 93
Sedimentation methods, 109
test for typhoid, 338
Seiteuketten, 491
Septicaemia, nature of, 173
puerperal, 184
sputum, 197
Septicemie de Pasteur, 388
Septic pneumonia, 197
Sera, haemolytic, 479
Serum agar. 38
Serum, agglutinative action of, 485
anaphylaxis, 494
antibacterial, 476
anti- cholera, 411
antidiphtheritic, 467
anti-plague, 436
antipneumococcic, 210
antirabic, 519
antistreptococcic, 476
antitetanic, 384
antitoxic, preparation of, 467 et seq.
antitubercular, 264
antityphoid, 344
bactericidal action of, 477
blood (v. Culture media), 39
diagnosis, 485
methods, 109
of typhoid, 337
inspissator, 39
lysogenic action of, 477
towards blood corpuscles, 479
Serum media, 39
Serum- water media, 41
Sewage, bacterial treatment of, 139
contamination of water by, 136
Shake cultures, 77
Sheep-pox, 507
Shiga's bacillus, 346
Side-chain theory, Ehrlich's, 491
Sleeping sickness, 555
Slides for hanging-drops, 63, 64
Sloped cultures, aerobic, 48
anaerobic, 62
Smallpox, 503
bacteria in, 507
Guarnieri bodies in, 508
Siuegma bacillus, 254
Smith's, Lorrain, serum medium, 41
Smith, Theobald, phenomenon of,
494
Snake poisons, 169
activating of, by serum, 170
constituents of, 169
immunity against, 462
Sobernheim's anti-anthrax serum, 316
Soft sore, 227
Soil, examination of, for bacteria, 131
Soudakewitch on relapsing fever, 442
Spinal cord, lesions by pyogenic
organisms, 183
Spirilla, characters of (v. also Vibrio),
14, 550
like cholera spirillum, 417
Spirillosis in animals, 439
Spirillum Metchuikovi, 417
of cholera, 400
Deneke, 419
Fiukler and Prior, 418
Miller, 419
relapsing fever, inoculation with,
etc., 438
Spirochsete, 14, 229, 550
pallida, 229
staining of, 104, 107
pertenuis, 234
refringeus, 230
Spirochsetes in syphilis, 229
in yaws, 234
staining of, in films, 107
staining of, in sections, 104
Spironema pallidum, 229
Splenic fever, 300
Spore formation, arthrosporous, 7
endogenous, 5
in b. anthracis, 304
Spores, staining of, 102
Sporoblasts, 529
Sporocyst (malaria), 529
Sporocytes, in malaria, 524
Sporozoites, 529
INDEX
603
Sporulation of malarial parasite, 522
Sputum, amoebae in, 541
influenza, 422, 425
in plague, 238
in pneumonia, 200
phthisical, 241, 255, 265
septicaemia, 197
Staining methods, 94 et seq.
of capsules, Welch's method, 102
Richard Muir's method, 102
of flagella, 103
of leprosy bacilli, 270
of spores, 102
of tubercle bacilli, 100
principles, 94
Stains, basic aniline, 94
Standard of immunity, 468
Standardising reaction of media, 33
Staphylococci, lesions caused by, 184
Staphylococcus, 12
cereus albus, 176
flavus, 176
pyogenes albus, 176 — '
aureus, characters of, 174
inoculation with, 182
citreus, 174
Steam steriliser, Koch's, 27
Stegomyia fasciata, 453
Sterilisation by heat, 26 et seq.
at low temperatures, 29
by steam at high pressure, 29
Streptococci in diphtheria, 356
in false membrane, 184
lesions caused by, 184
varieties of, 178
Streptococcus, 12
anginosus, 179
brevis, 178
conglomerate, 178, 179
equinus, 179
erysipelatis, 191 -
faecalis, 179
longus, 178
mitis, 179
pneumoniae, 198
pyogenes, characters of, 176
inoculation with, 182
in air, 130
in soil, 133
salivarius, 179
Streptothrices allied to actinomyces,
294
Streptothrix, 15
actinomyces, 287
Streptothrix, anaerobic in actino-
mycosis, 293
madurae, 297
Subcultures, 49
Sugars, classification of, 74
fermentation of, 74
Sulphurous acid as antiseptic, 147
Summer diarrhoea, bacteria in, 351
Suppuration, bacteria of, 174
gonococci in, 223
methods of examination of, 195
nature of, 172
origin of, 186
pneumococci in, 205
typhoid bacillus in, 330
Symptoms caused by bacteria, 161
Syphilis, bacillus of, 229
spirochaete pallida in, 229
transmission to animals, 233
Syringes for inoculation, 120, 121
Tabes mesenterica, 257
Taurocholate media, 43
Tertian fever, 530, 531
Test-tubes for cultures, 47
Tetanolysin, 380
Tetanospasmin, 380
Tetanus, 371
anti-serum of, 384, 467 et seq.
intravenous injection of, 386
cerebral, 384
dolorosus, 383
immunity against, 384
methods of examination in, 388
treatment of, 386, 489
Tetanus bacillus, 372
inoculation with, 378
isolation of, 373
spores of, 373
toxins of, 163, 379
Tetrads, 12
Texas fever, 569
Theory of exhaustion, 490
of phagocytosis, 495
of retention, 490
humoral, 490
Thermophilic bacteria, 18
Thermostable opsonius, 484
Thionin-blue, 95, 98
Thiothrix, 15
Tick fever, African, 443
Timothy-grass bacillus, 153
Tissues, action of bacteria on, 155
fixation of, 89
604
INDEX
Tizzoni and Cattani on tetanus, 385
Toxalbumius, 162
Toxic action, theory of, 170
Toxicity, estimation of, 467
Toxins, concentrated, method of ob-
taining, 167
constitution of, 491
early work on, 161
effects of, 158
immunisation by, 467
intra- and extra- cellular, 162
nature of, 165
non-proteid, 166
of anthrax, cholera, etc. (vide
Special Diseases)
production, 154
susceptibility to, 491
vegetable, 169
Toxoids, 171, 472
Toxones, 171
Trachoma, bacteria in, 192, 424
Trichophyta, media for growing, 44
Tropical ulcer, 568
Trypanosoma gambiense, 558
Lewisi, 544, 551
noctuse, 548
of sleeping sickness, 555
• ugandense, 544, 558
ugandense, relation to Tr. Gam-
biense, 561
Trypanosomata associated with
various diseases, 544
culture of, 546
morphology of, 544
sexual cycle in, 548
Trypanosomiasis, 544
Tse-tse fly disease, 552
Tubercle bacillus, 237
action of dead, 255
avian, 250
cultivation of, 239
distribution of, 243
immunity against, 260
inoculation with, 246
powers of resistance of, 241
in sputum, etc., 255, 265
toxins of, 258
' stains for, 100, 239
giant cells, 242
methods of examination of, 265
Tubercles, structure of, 242
Tubercular leprosy, 268
Tuberculin, 258
" Bazillenemulsion/' 260
Tuberculin, "0" and "R," 260
Tuberculosis, 235
aviau, 250
bovine, 248
its relation to human, 248
diagnosis by tuberculin, 259
Tuberculosis, in animals, 236
in fish, 251
modes of infection, 256
precautions in diagnosis of, 255
Tubes, cultures in, 47
Typhoid bacillus, 319
comparison with b. coli, 325
examination for, 344
immunity against, 332
inoculation with, 331
isolation from water supplies, 345
toxins of, 332
serum diagnosis, 337
suppurations in, 330
vaccination against, 343
Typhoid fever, 319
pathological changes in, 329
Ulcerative endocarditis, 188
experimental, 190
gouococci in, 225
Unit of immunity, 468
Urine, examination of, 69
staining of bacteria in, 88
tubercle bacilli in, 246, 265
typhoid bacilli in, 344
Vaccination against smallpox, 502
against hydrophobia, 516
against typhoid, 343
for infection by pyogenic bacteria,
194
nature of, 509
Variola, 505 et seq.
Venins, 169
Vibrio (see also Spirillum), 14
berolinensis, 415
of cholera, 400
Danubicus, 415
Deneke's, 419
Finkler and Prior's, 418
Gindha, 416
Ivanoff, 415
Massowah, 408, 416
Metchnikovi, 417
Nordhafen, 418
of Pestana and Bettencourt, 416
Romanus, 416
Vibrion septique, 388
INDEX
605
Virulence, attenuation of, 459
exaltation of, 461
of bacteria, 150
Water, bacteria in, 135
contamination of by sewage, 138
examination of, 135
supplies, typhoid bacilli in, 345
Weichselbaum on pneumonia, 198
Weigert's method of dehydration, 93
modification of Gram's method, 100
Wertheim's medium, 220, 226
Whooping cough, bacteria in, 423
Widal on serum diagnosis, 485
Widal's reaction, synonym for agglu-
tination of b. typhosus, q.v.,
109, 337
Winogradski, 23
Winter-spring fevers, 529
Wolff and Israel's streptothrix, 295
Woodhead on tuberculosis, 257
Woody tongue, 292
Woolsorter's disease, 311
Wright's, A. E., calibrated pipette,
108
diluting pipette, 66
method of counting dead
bacteria, 67
Wright's, A. E., opsonic technique,
112
vaccination against tuberculosis,
261
vaccination treatment of pyo-
genic infections, 194
Wright, J. H., on anaerobic strepto-
thrices, 293
Romanowsky stain, 107
Xerosis bacillus, 367
Xylol, 93
Yaws, spirochaetes in, 234
Yellow fever, 451
bacteria in, 452
etiology of, 452
mosquitoes in relation to, 453
Yersin (v. also Roux), on plague,
435 et seq.
Yersin's anti-plague serum, 436
Ziemanni, spirillum, 550
Ziehl-Neelsen stain, 101
Zoogloea, 3
Zygote (malaria), 328
6
YOUNG J. PENTLAND'S
PUBLICATIONS
CONTRIBUTIONS to CLINICAL MEDICINE. By Sir
THOMAS M'CALL ANDERSON, M.D., Professor of Medicine in
the University of Glasgow ; Physician, Glasgow Western
Infirmary. 8vo, Cloth, pp. xii., 416, with 28 Illustrations.
Price 10s. 6d. nett.
TEXT-BOOK of GENERAL BOTANY. By Dr. W. J.
BEHRKNS. Translation from the Second German Edition. Re-
vised by PATRICK GEDDES, F.R.S.E., Professor of Botany in the
University of Dundee. 8vo, Cloth, pp. viii., 374, with 408 Illus-
trations, finely engraved on Wood, and 4 Analytical Tables. New
Edition. Price 5s.
THE ELEMENTS of OPHTHALMOSCOPIC DIAG-
NOSIS. FOR THE USE OF STUDENTS ATTENDING OPHTHALMIC
PRACTICE. By GEORGE A. BERRY, M.B., F.R.C.S.Ed., Ophthalmic
Surgeon, Edinburgh Royal Infirmary ; Lecturer on Ophthalmology
in the University of Edinburgh. Crown 8vo, Cloth, pp. xii., 83
Price 3s. 6d.
DISEASES of the EYE. A PRACTICAL TREATISE FOR STUDENTS
OP OPHTHALMOLOGY. By GEORGE A. BERRY, M.B., F.R.C.S.Ed.,
Ophthalmic Surgeon, Edinburgh Royal Infirmary ; Lecturer on
Ophthalmology in the University of Edinburgh. Second Edition,
revised and enlarged. 8vo, Cloth, gilt top, pp. xxiv., 728, with
197 Illustrations, including many Coloured Figures. Price 25s.
(Pentland's Medical Series, Volume Second.
MANUAL of PRACTICAL OPHTHALMOLOGY. By
GEORGE A. BERRY, M.B., F.R.C.S.Ed., Senior Ophthalmic Sur-
geon, Edinburgh Royal Infirmary ; Surgeon, Edinburgh Eye
Dispensary ; Ophthalmic Surgeon, Royal Hospital for Sick Chil-
dren ; Lecturer on Ophthalmology, University of Edinburgh.
Crown 8vo, Cloth, pp. xx., 570, with 223 Illustrations, mostly
from Original Drawings. Price 10s. 6d. nett.
(Pentland's Students' Manuals.
2 MEDICAL PUBLICATIONS ISSUED BY
THE NATIONAL MEDICAL DICTIONARY. INCLUDING
ENGLISH, FRENCH, GERMAN, ITALIAN, AND LATIN TECHNICAL
TERMS USED IN MEDICINE AND THE COLLATERAL SCIENCES, AND
A SERIES OF TABLES OF USEFUL DATA. By JOHN S. BILLINGS,
A.M., M.D., LL.D. Harv. and Edin., D.C.L. Oxon., Member
of the National Academy of Sciences, Surgeon U.S.A., etc. In
Two Volumes, Imperial 8vo, pp. xlviii., 1530. Price 50s. nett.
DISEASES of the HEART and THORACIC AORTA. By
BYROM BRAMWELL, M.D., F.RC.P.Ed., Lecturer on the Principles
and Practice of Medicine, and on Practical Medicine and Medical
Diagnosis, in the Extra- Academical School of Medicine, Edin-
burgh ; Physician, Edinburgh Royal Infirmary. Large 8vo,
Cloth, pp. xvi., 783. Illustrated with 226 Wood Engravings,
and 68 pages of Lithograph Plates, exhibiting 91 Figures, —
317 Illustrations in all. Price 25s.
INTRACRANIAL TUMOURS. By BYROM BRAMWELL, M.D.,
F.R.C.P.Ed., Lecturer on the Principles and Practice of Medicine
in the Extra - Academical School of Medicine, Edinburgh ;
Physician, Edinburgh Royal Infirmary. 8vo, Cloth, pp. xvi., 270,
with 116 Illustrations. Price 14s.
ILLUSTRATIONS of the NERVE TRACTS in the MID
AND HIND BRAIN, AND THE CRANIAL NERVES ARISING THERE-
FROM. By ALEXANDER BRUCE, M.D., F.R.C.P.Ed., Lecturer on
Medicine in the School of Medicine ; Physician, Royal
Infirmary, Edinburgh. Illustrated with a series of 27 Coloured
Plates from Original Drawings, with Descriptive Letterpress,
and 27 Engravings throughout the Text. Oblong 4to, Cloth.
Price 50s. nett.
THE PRINCIPLES of TREATMENT, and their APPLICA-
TIONS IN PRACTICAL MEDICINE. By J. MITCHELL BRUCE, M.A.,
LL.D., M.D., F.R.C.P., Consulting Physician to the Charing
Cross Hospital and to the Hospital for Consumption, Brompton.
Third Impression. 8vo, Cloth, gilt top, pp. xviii., 614. Price 16s.
(Pentland's Medical Series, Volume Seventh.
DISEASE in CHILDREN: A MANUAL FOR STUDENTS AND
PRACTITIONERS. By JAMES CARMICHAEL, M.D., F.R. C.P.Ed.,
Physician, Royal Hospital for Sick Children ; University Lecturer
YOUNG J. PENTLAND.
on Disease in Children, Edinburgh. Crown 8vo, Cloth, pp. xviii.,
591. Illustrated with Charts. Price 10s. 6d.
(Pentland's Students' Manuals.
TUBERCULOUS DISEASE of the BONES and JOINTS.
By W. WATSON CHEYNE, F.R.S., F.R.C.S., Professor of
Surgery, King's College, London. 8vo, Cloth, pp. xvi., 374.
Illustrated with numerous Wood Engravings throughout the
Text. Price 14s. nett.
SUPPURATION and SEPTIC DISEASES. THREE LEC-
TURES DELIVERED AT THE ROYAL COLLEGE OP SURGEONS OP
ENGLAND. By W. WATSON CHEYNE, F.R.S., F.R.C.S., Professor
of Surgery, King's College, London. 8vo, Cloth, pp. xii., 102,
with 4 Illustrations. Price 5s.
THE TREATMENT of WOUNDS, ULCERS, and
ABSCESSES. By W. WATSON CHEYNE, F.R.S., F.R.C.S.,
Professor of Surgery, King's College, London. Second Edition.
Crown 8vo, Cloth, pp. xvi., 197. Price 3s. 6d.
THE DIAGNOSIS and TREATMENT of INTUSSUS-
CEPTION. By CHARLES P. B. CLUBBE, M.R.C.S. Eng., Hon.
Surgeon to the Royal Prince Alfred Hospital, and to the Royal
Alexandra Hospital for Children, Sydney ; Joint Lecturer in
Clinical Surgery at the Sydney University. 8vo, Cloth, pp. xii.,
92. Price 3s. 6d. nett.
ATLAS of the DISEASES of the SKIN. By H. RADCLIPPE
CROCKER, M.D., F.R.C.P., Physician to the Department for
Diseases of the Skin, University College Hospital, London.
In Two large handsome Folio Volumes, Half Morocco, gilt tops,
containing 96 plates, exhibiting 238 Figures in colours from
Original Drawings specially prepared for the work, with Descrip-
tive Letterpress. Price £18, 18s. nett.
MANUAL of PRACTICAL ANATOMY. By D. J. CUN-
NINGHAM, M.D., F.R.S., Professor of Anatomy, University of
Edinburgh. Fourth Edition, thoroughly revised. In Two
Vols., Crown 8vo, Cloth. Volume First — Upper and Lower
Limb ; Abdomen. Volume Second — Thorax ; Head and Neck.
Illustrated with 573 Engravings, many in colours. Price
10s. 6d. each nett. (Pentland's Students' Manuals.
4 MEDICAL PUBLICATIONS ISSUED BY
TEXT-BOOK of ANATOMY. Edited by D. J. CUNNINGHAM,
M.D., F.R.S., Professor of Anatomy, University of Edinburgh.
Second and thoroughly Revised Edition. In One Volume, Royal
8vo, Cloth, pp. xxxvi., 1388. With 936 Wood Engravings from
Original Drawings, many printed in colours. Price 31s. 6d.
nett ; or in half leather binding, price 35s. nett.
ALSO IN FOUR SECTIONS.
Sections I. to III. — First Edition.
Section IV.— Second Edition.
Section I. — General Embryology ; Osteology ; Articulations or
Joints ; The Muscular System. Pp. xxxii., 438. Illustrated with
307 Wood Engravings, many printed in colours, in extra cloth
binding. Price 12s. 6d. nett.
Section II. — The Nervous System ; Organs of Sense and the
Integument. Pp. vi., 344. Illustrated with 234 Wood Engrav-
ings, some in colours, in extra cloth binding. Price 10s. 6d.
nett.
Section III. — Vascular System ; Respiratory System ; Diges-
tive System. Pp. viii., 364. Illustrated with 184 Wood Engrav-
ings, many printed in colours, in extra cloth binding. Price
10s. 6d. nett.
Section IV. — Urogenital System; Ductless Glands; Surface
and Surgical Anatomy. Pp. viii., 259. Illustrated with 139 Wood
Engravings, some printed in colours. Second Edition. In extra
cloth binding. Price 7s. 6d. nett.
%* The list of Contributors is as follows :— Professor A. H.
YOUNG (Manchester), Dr. ARTHUR ROBINSON (Birmingham),
Professor ARTHUR THOMSON (Oxford), Professor D. H. HEPBURN
(Cardiff), Dr. HAROLD STILES (Edinburgh), Professor A. M.
PATERSON (Liverpool), Professor R. HOWDEN (Newcastle), The
late Professor A. BIRMINGHAM (Dublin), Professor A. F. DIXON
(Dublin), and the EDITOR.
HYGIENE and DISEASES of WARM CLIMATES,
IN A SERIES OP ARTICLES BY EMINENT AUTHORITIES. Edited by
ANDREW DAVIDSON, M.D., F.R.C.P.Ed., late Visiting and Super-
intending Surgeon, Civil Hospital, and Professor of Chemistry.
Royal College, Mauritius ; Author of Geographical Pathology.
Royal 8vo, Cloth, pp. xx., 1016. Illustrated with 7 full-page Plates
and 97 Engravings throughout the Text. Price 31s. 6d.
YOUNG J. PENTLAND.
GEOGRAPHICAL PATHOLOGY. AN INQUIRY INTO THE
GEOGRAPHICAL DISTRIBUTION OF INFECTIVE AND CLIMATIC DIS-
EASES. By ANDREW DAVIDSON, M.D., F.R.C.P.Ed., late Visiting
and Superintending Surgeon, Civil Hospital, and Professor of
Chemistry, Royal College, Mauritius. Two Vols., Large 8vox
Cloth, pp. xvi., 1008. Illustrated with Maps and Charts. Price
31s. 6d.
TEXT-BOOK of NERVOUS DISEASES. BY AMERICAN
AUTHORS. Edited by FRANCIS X. DERCUM, M.D., Ph.D., Clinical
Professor of Nervous Diseases in the Jefferson Medical College
of Philadelphia. Royal 8vo, Cloth, pp. 1056, with 341
Engravings in the Text, and 7 Coloured Plates. Price 25s.
nett.
THE TOXIC AMBLYOPIAS : THEIR CLASSIFICATION, HIS-
TORY, SYMPTOMS, PATHOLOGY, AND TREATMENT. By G. E. de
SCHWEINITZ, A.M., M.D., Professor of Ophthalmology in the
Philadelphia Polyclinic. Large 8vo, Cloth, pp. 238, with 46
Illustrations and 9 Plates. Price 16s. nett.
THE EDINBURGH MEDICAL JOURNAL. Edited by
ALEXIS THOMSON, M.D., F.R. C.S.Ed., and HARVEY LITTLEJOHN,
F.R. C.S.Ed. Issued monthly. Price 2s. each part; Subscrip-
tion 21s. per annum (in advance), post free. For the Colonies
and Abroad, 24s. per annum (in advance), post free.
THE ERASMUS WILSON LECTURES on the PATH-
OLOGY AND DISEASES OF THE THYROID GLAND. DE-
LIVERED BEFORE THE ROYAL COLLEGE OF SURGEONS OF ENGLAND
ON FEBRUARY 18TH, 20ra, AND 22ND, 1901. By WALTER
EDMUNDS, M.A., M.C. (Cantab.), F.R.C.S. (Eng.), Surgeon to
the Tottenham Hospital ; Surgeon to Out-Patients Evelina
Hospital for Sick Children. 8vo, Cloth, pp. 47, with 24 Illus-
trations. Price 2s. 6d. nett.
EXAMINATION QUESTIONS, SET FOR THE PROFESSIONAL
EXAMINATIONS IN EDINBURGH UNIVERSITY DURING A PERIOD OF TEN
YEARS, SELECTED FROM THE CALENDARS. By W. RAMSAY SMITH,
M.B., C.M., B.Sc., Formerly Demonstrator of Anatomy, Edinburgh
6 MEDICAL PUBLICATIONS ISSUED BY
School of Medicine, Minto House ; late Senior Assistant to the
Professor of Natural History, University of Edinburgh.
NATURAL HISTORY, arranged and annotated. Price Is. nett.
BOTANY, arranged and annotated. Price Is. 6d. nett.
ANATOMY, answered and annotated. Price 2s. nett.
PHYSIOLOGY, answered and annotated. Price 2s. nett.
MATERIA MEDICA, answered and annotated. Price 2s. nett.
MIDWIFERY AND GYNECOLOGY, answered and annotated.
Price Is. 6d. nett.
SURGERY, answered and annotated. Price Is. 6d. nett.
PRACTICE OF PHYSIC, answered and annotated. Price Is. 6d.
nett.
MEDICAL JURISPRUDENCE AND PUBLIC HEALTH, answered and
annotated. Price 2s. nett.
PATHOLOGY, answered and annotated. Price Is. 6d. nett.
THE FUNDUS OCULI, with an OPHTHALMOSCOPIC
ATLAS, ILLUSTRATING ITS PHYSIOLOGICAL AND PATHOLOGICAL
CONDITIONS. By W. ADAMS FROST, F.R.C.S., Ophthalmic
Surgeon, St. George's Hospital ; Surgeon to the Royal West-
minster Ophthalmic Hospital, London. In one handsome 4to
Volume, extra Cloth, gilt top, with 47 Plates, exhibiting 107
beautifully Coloured Figures and numerous Engravings in the
Text. Price £3, 3s. nett.
DISEASES of the HEART and AORTA. By G. A. GIBSON,
M.D., D.Sc., F.R.C.P.Ed., Physician to the Royal Infirmary; Con-
sulting Physician to the Deaconess Hospital ; Lecturer on Medicine
at Minto House, and on Clinical Medicine at the Royal Infirmary,
Edinburgh. 8vo, Cloth, pp. xxiv., 932, with 210 Illustrations.
Price 24s. (Pcntland's Medical Series, Volume Sixth.
TEXT-BOOK of MEDICINE. By BRITISH TEACHERS. Edited
by G. A. GIBSON, M.D., D.Sc., F.R.C.P.Ed., Lecturer on the
Principles and Practice of Medicine in the Edinburgh Medical
School ; Physician, Edinburgh Royal Infirmary. Two Volumes,
Royal 8vo, Cloth, pp. xxxvi., 1734, with 122 Illustrations. Price
25s. nett.
%* The list of Contributors is as follows : — J. O. AFFLECK,
HENRY ASHBY, A. G. AULD, JOHN ROSE BRADFORD, ALEXANDER
BRUCE, J. MITCHELL BRUCE, Sir LAUDER BRUNTON, W. S. COLMAN,
G. A. GIBSON, A. LOCKHART GILLESPIE, Sir WILLIAM GOWERS,
V. D. HARRIS, HERBERT P. HAWKINS, W. ALLAN JAMIESON, the
YOUNG J. PENTLAND.
late A. A. KANTHACK, CLAUDE B. KER, T. W. P. LAWRENCE,
A. P. LUFF, HECTOR MACKENZIE, PATRICK MANSON, SIDNEY
MARTIN, Sir JOHN WILLIAM MOORE, F. W. MOTT, THOMAS OLIVER,
W. PASTEUR, R. W. PHILIP, S. RISIEN RUSSELL, WILLIAM RUSSELL,
RALPH STOCKMAN, FREDERICK TAYLOR, JAMES TAYLOR, W. ALDREN
TURNER, W. HALE WHITE, R. T. WILLIAMSON, G. E. CARTWRIGHT
WOOD, G. SIMS WOODHEAD.
THE NERVOUS AFFECTIONS of the HEART. BEING
THE MORISON LECTURES DELIVERED BEFORE THE ROYAL COLLEGE
OF PHYSICIANS OF EDINBURGH IN 1902 AND 1903. By GEORGE
ALEXANDER GIBSON, M.D., D.Sc., F.R.C.P.Ed., F.R.S.E., Hon-
orary Member of the Medico-Chirurgical Society of Norwich ;
Honorary Corresponding Member of the Therapeutical Society
of Paris ; Physician to the Royal Infirmary, and Lecturer on
Medicine and Clinical Medicine in the Medical School of the
Royal Colleges, Edinburgh ; Examiner in Medicine in the
University of Oxford. Second Edition. 8vo, Cloth, pp. x., 106,
with 35 Illustrations. Price 5s. nett.
PHYSICAL DIAGNOSIS : A GUIDE TO METHODS OF CLINICAL
INVESTIGATION. By G. A. GIBSON, M.D., D.Sc., F.R.C.P.Ed.,
Physician, Edinburgh Royal Infirmary ; Lecturer on the Principles
and Practice of Medicine in the Edinburgh Medical School ; anc
WILLIAM RUSSELL, M.D., F.R.C.P.Ed., Assistant Physician, Edin-
burgh Royal Infirmary ; Lecturer on Pathology and Morbid
Anatomy, School of Medicine, Edinburgh. Third Edition, revised
and enlarged. Edited by FRANCIS D. BOYD, C.M.G., M.D.,
F.R.C.P.Ed., Assistant Physician, Royal Infirmary, Edinburgh.
Crown 8vo, Cloth, pp. xx., 448, with 144 Illustrations. Price
9s. nett. (Pentland's Students' Manuals.
HYDATID DISEASE in its CLINICAL ASPECTS.
By JAMES GRAHAM, M.A., M.D., late Demonstrator of Anatomy,
Sydney University ; Medical Superintendent, Prince Alfred
Hospital, Sydney. 8vo, Cloth, gilt top, pp. xii., 204, with 34
full-page Coloured Plates. Price 16s.
HANDBOOK of OBSTETRIC NURSING. By F. W. N.
HAULTAIN, M.D., F.R.C.P.Ed., Lecturer on Midwifery and
Diseases of Women, School of Medicine ; Physician for Diseases
8 MEDICAL PUBLICATIONS ISSUED BY
of Women, Deaconess' Hospital ; Obstetric and Gynecological
Physician to the Royal Dispensary, Edinburgh ; and J. HAIG
FERGUSON, M.D., F.R.C.P.Ed., Lecturer on Midwifery and
Diseases of Women, School of Medicine and Jubilee Institute
for Nurses, Edinburgh. Fifth Edition, revised and enlarged.
Crown 8vo, Cloth, pp. xvi., 267. Illustrated with Frontispiece
and 37 Wood Engravings. Price 5s.
HUMAN MONSTROSITIES. By BARTON COOKE HIRST, M.D.
Professor of Obstetrics in the University of Pennsylvania ; and
GEORGE A. PIERSOL, M.D., Professor of Embryology and Histology
in the University of Pennsylvania. In 4 Fasciculi, folio size. Illus-
trated with Engravings and 39 full -page Photographic Plates from
Nature. Price 25s. nett. each Fasciculus. Sold only by Subscription.
DISEASES of the SKIN : A MANUAL FOR STUDENTS AND
PRACTITIONERS. By W. ALLAN JAMIESON, M.D., F.R.C.P.Ed.,
Extra Physician for Diseases of the Skin, Edinburgh Royal
Infirmary ; Lecturer on Diseases of the Skin in the University of
Edinburgh. Fourth Edition, revised and enlarged. 8vo, Cloth,
pp. xx., 660, with 9 Coloured Illustrations. Price 21s.
(Pentland's Medical Series, Volume First.
BOTANY : A CONCISE MANUAL FOR STUDENTS OF MEDICINE AND
SCIENCE. By ALEXANDER JOHNSTONE, F.G.S., Lecturer on Botany,
School of Medicine, Edinburgh. Crown 8vo, Cloth, pp. xvi., 260,
with 164 Illustrations and a series of Floral Diagrams. Price 6s.
(Pentland's Students' Manuals.
A NEW PRONOUNCING DICTIONARY of MEDICAL
TERMS. By JOHN M. KEATING, M.D., Fellow of the College
of Physicians of Philadelphia, late Visiting Obstetrician to the
Philadelphia Hospital, and Lecturer on Diseases of Women and
Children. New impression. Large 8vo, Cloth, pp. 818. Price
7s. 6d. nett.
CLINICAL GYNAECOLOGY, MEDICAL and SURGICAL.
By AMERICAN AUTHORS. Edited by JOHN M. KEATING, M.D.,
LL.D., and HENRY C. COE, M.D., M.R.C.S., Professor of Gynae-
cology, New York Poly clinic. Two Volumes, Royal 8vo, Cloth
extra, gilt tops, pp. xviii., 994. Illustrated with 34 full-page Plates
and 377 Engravings in the Text. Price 25s. nett.
YOUNG J. PENTLAND.
GYNAECOLOGICAL OPERATIONS, EXCLUSIVE OF THOSE
INTERFERING WITH THE PERITONEAL CAVITY. By SKENE KEITH,
F.R.C.S.Ed. 8vo, Cloth extra, pp. xii., 118. Illustrated with 44
Figures. Price 5s. nett.
TEXT-BOOK of ABDOMINAL SURGERY: A CLINICAL
MANUAL FOR PRACTITIONERS AND STUDENTS. By SKENE KEITH,
F.R.C.S.Ed.; assisted by GEORGE E. KEITH, M.B. 8vo, Cloth,
gilt top, pp. xx., 508. Price 16s.
(Pentland's Medical Series, Volume Fourth.
RENAL GROWTHS : THEIR PATHOLOGY, DIAGNOSIS, AND TREAT-
MENT. By T. N. KELYNACK, M.D. (Viet.), M.R.C.P. (Lond.).,
Pathologist, Manchester Royal Infirmary ; Demonstrator and
Assistant Lecturer in Pathology, Owens College, Manchester.
8vo, Cloth, pp. xvi., 269, with 96 Illustrations. Price 12s. 6d. nett.
THE PARASITES of MAN, AND THE DISEASES WHICH PROCEED
FROM THEM. A TEXT-BOOK FOR STUDENTS AND PRACTITIONERS.
By RUDOLF LEUCKART, late Professor of Zoology and Comparative
Anatomy in the University of Leipsic. Translated from the
German, with the Co-operation of the Author, by WILLIAM E.
HOYLE, M.A. (Oxon.), M.R.C.S., F.R.S.E., Curator of the
Museums, Owens College, Manchester. NATURAL HISTORY OF
PARASITES IN GENERAL — SYSTEMATIC ACCOUNT OF THE PARASITES
INFESTING MAN — PROTOZOA — CESTODA. Large 8vo, Cloth, pp.
xxviii., 772. Illustrated with 404 Engravings. Price 31s. 6d.
TRAUMATIC INFECTION. HUNTERIAN LECTURES DELIVERED
AT THE ROYAL COLLEGE OF SURGEONS OF ENGLAND. By CHARLES
BARRETT LOCKWOOD, F.R.C.S., Hunterian Professor, Royal College
of Surgeons of England ; Assistant Surgeon to St. Bartholomew's
Hospital ; Surgeon to the Great Northern Central Hospital,
London. Crown 8vo, Cloth, pp. xii., 138. Illustrated with 27
Wood Engravings in the Text. Price 3s.
ASEPTIC SURGERY. By CHARLES BARRETT LOCKWOOD,
F.R.C.S. Second Edition. Crown 8vo, Cloth, pp. xvi., 264.
Price 4s.
io MEDICAL PUBLICATIONS ISSUED BY
THE RADICAL CURE of HERNIA, HYDROCELE, and
VARICOCELE. By CHARLES BARRETT LOCKWOOD, F.R.C.S.
Crown 8vo, Cloth, pp. xii., 279, with 23 Illustrations. Price
4s. 6d.
DISEASES of the THROAT, NOSE, and EAR. A CLINICAL
MANUAL FOR STUDENTS AND PRACTITIONERS. By P. M'BRIDE,
M.D., F.R.C.P.Ed., Lecturer on the Diseases of the Ear and
Throat- in the University ; Aural Surgeon and Laryngologist,
Royal Infirmary, Edinburgh. Third Edition, thoroughly revised
and partly re- written. 8vo, Cloth, pp. xviii., 744, with 48 Coloured
Illustrations from Original Drawings. Price 25s.
(Pentland's Medical Series, Volume Third.
ATLAS of VENEREAL DISEASES. A SERIES OF ILLUSTRA-
TIONS FROM ORIGINAL PAINTINGS, WITH DESCRIPTIONS OF THE
VARIED LESIONS, THEIR DIFFERENTIAL DIAGNOSIS AND TREATMENT.
By P. H. MACLAREN,M.D., F.R.C.S.Ed., Surgeon, Edinburgh Royal
Infirmary ; Formerly Surgeon in Charge of the Lock Wards,
Edinburgh Royal Infirmary ; Examiner in the Royal College of
Surgeons, Edinburgh. In One handsome Royal 4to Volume, Extra
Cloth. Price 63s. nett.
FUNCTIONAL and ORGANIC DISEASES of the
STOMACH. By SIDNEY MARTIN, M.D., F.R.S., F.R.C.P.,
Physician, University College Hospital ; Professor of Pathology,
University College, London. 8vo, Cloth, pp. xx., 506. Illus-
trated with numerous Engravings throughout the Text. Price 16s.
(Pentland's Medical Series, Volume Fifth.
THE NERVOUS SYSTEM and its DISEASES. A PRAC-
TICAL TREATISE ON NEUROLOGY, FOR THE USE OF PHYSICIANS
AND STUDENTS. By CHARLES K. MILLS, M.D., Professor of
Mental Diseases and of Medical Jurisprudence in the University
of Pennsylvania ; Clinical Professor of Neurology in the Women's
Medical College, Penn. 8vo, Cloth, pp. xxx., 1056, with 459
Illustrations. Price 25s.
THE NURSING and CARE of the NERVOUS and the
INSANE. By CHARLES K. MILLS, M.D. Crown Svo, Cloth,
pp. 147, with 14 Illustrations. Price 4s. 6d.
YOUNG /. PENTLAND.
A SYSTEM of GENITO - URINARY DISEASES,
SYPHILOLOGY, AND DERMATOLOGY. Edited by PRINCE
A. MORROW, M.D. 6 Vols., Large 8vo, of about 550 pages each,
fully Illustrated. Price 14s. per Volume, carriage free.
MANUAL of BACTERIOLOGY. By ROBERT Mure, M.D.,
F.R.C.P.Ed., Professor of Pathology, University of Glasgow; and
JAMES RITCHIE, M.D., B.Sc., Superintendent, Research Labora-
tory, R.C.P., Edinburgh ; late Professor of Pathology, University
of Oxford. Fourth Edition, revised and enlarged. Crown 8vo,
with Illustrations in the Text. In Press.
(Pentland's Students' Manuals.
PRESCRIBING and TREATMENT IN THE DISEASES OF
INFANTS AND CHILDREN. By PHILIP E. MUSKETT, L.R.C.P. &
S.Ed., late Surgeon to the Sydney Hospital ; formerly Senior
Resident Medical Officer, Sydney Hospital. Third Edition, re-
vised, enlarged, and rearranged. 18mo, pp. xx., 334, in Flexible
Leather binding for the pocket. Price 6s. 6d.
LEAD POISONING, IN ITS ACUTE AND CHRONIC FORMS. BEING
THE GOULSTONIAN LECTURES DELIVERED AT THE ROYAL COLLEGE
OF PHYSICIANS. By THOMAS OLIVER, M.D., F.R.C.P., Physician
Royal Infirmary, Newcastle-on-Tyne ; Professor of Physiology,
University of Durham. 8vo, Cloth, pp. xii., 122, with Coloured
Illustrations. Price 10s. 6d.
THE SCIENCE and ART of OBSTETRICS. By THEO-
PHILUS PARVIN, M.D., LL.D., late Professor of Obstetrics and
Diseases of Women and Children in Jefferson Medical College,
Philadelphia, and one of the Obstetricians to the Philadelphia
Hospital. Third Edition, thoroughly revised. Large 8vo, Cloth,
pp. 685, with 269 Wood Engravings, and 2 Coloured Plates.
Price 18s.
COMPEND of HUMAN ANATOMY, INCLUDING THE ANATOMY
OF THE VISCERA. By SAMUEL O. L. POTTER, M.A., M.D., Cooper
Medical College, San Francisco. Seventh Edition, revised and
enlarged. Crown 8vo, Cloth, pp. 372, with 138 Engravings ; also
numerous Tables and 16 full-page Plates. Price 5s. nett.
12 MEDICAL PUBLICATIONS ISSUED BY
TEXT-BOOK of PHYSIOLOGY. By BRITISH PHYSIOLOGISTS.
Edited by E. A. SCHAFER, F.R.S., Professor of Physiology in
the University of Edinburgh. Two Volumes. Vol. I., Royal
8vo, Cloth, pp. xx., 1036, with 3 Plates and 93 Illustrations in
the Text. Price 31s. 6d. Vol. II., pp. xxiv., 1365, with 449
Illustrations in the Text. Price 42s.
%* The list of Contributors is as follows : — Professor Sir
BURDON SANDERSON (Oxford), Professor GAMGEE, Dr. GASKELL
(Cambridge), Professor GOTCH (Oxford), Professor SHERRINGTON
(Liverpool), Professor M'KENDRICK (Glasgow), Professor HALLI-
BURTON (King's College), Professor HAYCRAFT (Cardiff), Dr.
PEMBREY (Guy's), Professor STARLING (University College), W.
H. R. RIVERS (Cambridge), J. N. LANGLEY (Cambridge), Professor
WAYMOUTH REID (Dundee), B. MOORE (Charing Cross), F. G.
HOPKINS (Cambridge), LEONARD HILL (London Hospital),
J. S. EDKINS (St. Bartholomew's), A. A. GRAY (Glasgow), D. NOEL
PATON (Edinburgh), and the EDITOR.
CLASSIFICATION of the CELLS found in the BLOOD
IN HEALTH AND DISEASE. By the Hon. G. H. SCOTT,
M.A., M.D. (Cantab.). 8vo, sewed, pp. 24, with 3 Plates.
Price 2s. 6d. nett.
ILLUSTRATIONS of ZOOLOGY, INVERTEBRATES AND VERTE-
BRATES. By WILLIAM RAMSAY SMITH, M.B., B.Sc., and J. STEWART
NORWELL, B.Sc. New and Cheaper Edition. Crown 4to, extra
Cloth, gilt top, with 70 Plates, exhibiting over 400 Figures.
Price 7s. 6d.
PRACTICAL GUIDE to the EXAMINATION of the
EYE. FOB STUDENTS AND JUNIOR PRACTITIONERS. By SIMEON
SNELL, F.R.C.S.Ed., Ophthalmic Surgeon, Sheffield General
Infirmary. Crown 8vo, Cloth, pp. xvi., 177, with 88 Illustrations.
Price 5s.
YOUNG J. PENTLAND. 13
LECTURES on GIDDINESS and on HYSTERIA in the
MALE. By Sir THOMAS GRAINGER STEWART, M.D., LL.D.,
late Physician-in-Ordinary to Her Majesty the Queen for Scotland,
and Professor of the Practice of Physic and of Clinical Medicine in
the University of Edinburgh. Crown 8vo, Cloth, pp. x., 89,
with 13 Illustrations. Price 2s. 6d. nett.
EPIDEMIC OPHTHALMIA: ITS SYMPTOMS, DIAGNOSIS, AND
MANAGEMENT. By SYDNEY STEPHENSON, F.R. C.S.Ed., Ophthal-
mic Surgeon, Evelina Hospital and N.E. Hospital for Children,
London. 8vo, Cloth, pp. xvi., 278. Illustrated with a Coloured
Plate and 25 Figures in the Text. Price 9s. nett.
SURFACE and SURGICAL ANATOMY. By HAROLD J.
STILES, M.B., F.R.C.S. Ed., Surgeon Royal Edinburgh Hospital
for Sick Children ; Lecturer on Applied Anatomy, University,
Edinburgh. Second Edition thoroughly revised. Royal 8vo,
Cloth, pp. viii., 260, with 139 Wood Engravings, many printed
in Colours. Price 7s. 6d. nett. (Forming Section IV. of
Cunningham's Text-Book of Anatomy.)
APPENDICITIS and PERITYPHLITIS. By CHARLES TALA-
MON, Physician to the Tenon Hospital, Paris. Translated from
the French by RICHARD J. A. BERP,Y, M.B., C.M., late President
of the Royal Medical Society, Edinburgh. Crown 8vo, Cloth, pp.
viii., 239. Price 6s.
THE PATHOLOGY and TREATMENT of VENEREAL
DISEASES. By ROBERT W. TAYLOR, M.D., Clinical Professor
of Venereal Diseases at the College of Physicians and Surgeons
(Columbia College), New York ; Surgeon to Belle vue Hospital ;
and Consulting Surgeon to the City (Charity) Hospital, New
York. Royal 8vo, Cloth, pp. 1002. Illustrated with 230 Engrav-
ings in the Text, and 7 Coloured Plates. Price 22s. nett.
OUTLINES of ZOOLOGY. By J. ARTHUR THOMSON, M.A.,
Regius Professor of Natural History in the University of Aber-
deen. Fourth Edition, revised and enlarged. Crown 8vo, Cloth,
pp. xx., 856, with 378 Illustrations in the Text. Price 12s. 6d.
nett. (Pentland's Students' Manuals.
H MEDICAL PUBLICATIONS ISSUED BY
MANUAL of SURGERY. By ALEXIS THOMSON, M.D.,
F.R. C.S.Ed., Assistant Surgeon, Edinburgh Royal Infirmary;
Surgeon to the Deaconess' Hospital, Edinburgh ; and ALEXANDER
MILES, M.D., F.R.C.S.Ed., Assistant Surgeon, Edinburgh Royal
Infirmary ; Surgeon to Leith Hospital. Two Volumes. Crown
8vo, Cloth. Price 10s. 6d. each nett.
VOL. I. GENERAL SURGERY, Second Edition revised and enlarged,
pp. xxiv., 808, with 313 Illustrations in the Text.
VOL. II. REGIONAL SURGERY, Second Edition revised and enlarged,
pp. xvi., 816, with 203 Illustrations in the Text.
(Pentland's Students' Manuals.
PRACTICAL GUIDE to MEAT INSPECTION. By THOMAS
WALLEY, M.R.C.V.S., formerly Principal of the Edinburgh Royal
(Dick) Veterinary College ; Professor of Veterinary Medicine
and Surgery, etc. Fourth Edition, rewritten and enlarged by
STEWART STOCKMAN, M.R.C.V.S., Professor of Pathology;
Lecturer on Hygiene and Meat Inspection, Edinburgh Royal
(Dick's) Veterinary College. Post 8vo, Cloth, pp. xvi., 296, with
14 Coloured Plates and 79 Illustrations in the Text. Price
10s. 6d. nett.
DISEASES of the LIVER, GALL BLADDER, and
BILIARY SYSTEM : THEIR PATHOLOGY, DIAGNOSIS, AND SURGICAL
TREATMENT. By H. J.. WARING, M.S., B.Sc., F.R.C.S., Assistant
Surgeon and Lecturer on Surgical Anatomy at St. Bartholo-
mew's Hospital ; Surgeon to the Metropolitan Hospital, London.
8vo, Cloth, pp. xvi., 386, with 58 Illustrations. Price 12s. 6d.
nett.
MANUAL of OPERATIVE SURGERY. By H. J. WAR!NG,
M.S., B.Sc., F.R.C.S., Assistant Surgeon, Lecturer on Surgical
Anatomy, and Demonstrator of Practical Surgery, St. Bartholo-
mew's Hospital ; Consulting Surgeon to the Metropolitan Hospi-
tal, London ; Member of Board of Examiners, Royal College of
Surgeons, England. Second Edition. Crown 8vo, Cloth, pp. xxx.,
659, with 472 Illustrations in the Text. Price 10s. 6d. nett.
(Pentland's Students' Manuals.
YOUNG J. PENTLAND. 15
RESEARCHES in FEMALE PELVIC ANATOMY. By
J. CLAKENCE WEBSTEK, B.A., M.D., F.R.C.P.Ed., Professor of
Midwifery and Diseases of Women in the University of Chicago ;
formerly Assistant to the Professor of Midwifery and Diseases of
Women, University of Edinburgh. Demy 4to, Cloth. Illustrated
with 26 full-page Coloured Plates from Original Drawings.
Price 30s. nett.
TUBO-PERITONEAL ECTOPIC GESTATION. By J.
CLARENCE WEBSTER, B.A., M.D., F.R.C.P.Ed. Demy 4to,
Cloth. Illustrated with 11 full-page Plates, several Coloured,
exhibiting numerous Figures. Price 16s. nett.
TEXT -BOOK of PHARMACOLOGY and THERA-
PEUTICS. Edited by W. HALE WHITE, M.D., F.R.C.P.,
Physician and Lecturer on Medicine, Guy's Hospital, London.
Royal 8vo, pp. x., 1040. Price 21s. nett.
* x* The list of Contributors is as follows : — JOHN ROSE BRAD-
FORD, J. H. BRYANT, J. T. CASH, A. R. CDSHNY, WALTER DIXON,
MICHAEL G. FOSTER, A. E. GARROD, J. S. HALDANE, HOBART
AMORY HARE, LEONARD HILL, F. G. HOPKINS, D. J. LEECH,
EDWARD W. LUCAS, HECTOR MACKENZIE, C. R. MARSHALL,
SIDNEY MARTIN, THOMAS OLIVER, M. S. PEMBREY, G. V. POORE,
GEORGE ROVVELL, G. H. SAVAGE, J. V. SHOEMAKER, SAMUEL E.
SOLLY, WALTER GEORGE SMITH, RALPH STOCKMAN, NESTOR
TIRARD, J. W. WASHBOURN, W. HALE WHITE, R. B. WILD.
DIABETES MELLITUS: AND ITS TREATMENT. By RICHARD
T. WILLIAMSON, M.D., M.R.C.P., M.R.C.S., Medical Registrar,
Manchester Royal Infirmary ; Hon. Medical Officer, Pendleton
Dispensary ; Assistant to the Professor of Medicine, Owena
College, Manchester. 8vo, Cloth, pp. xvi., 417, with 18 Illus-
trations (two coloured). Price 12s. 6d. nett.
1 6 MEDJCAL PUBLICATIONS.
PRACTICAL PATHOLOGY. A MANUAL FOR STUDENTS AND
PRACTITIONERS. By G. SIMS WOODHEAD, M.D., F.R.C.P.Ed.,
Professor of Pathology in the University of Cambridge ; formerly
Director of the Laboratories of the Royal Colleges of Physicians
(London) and Surgeons (England). Fourth Edition, 8vo, with
Coloured Illustrations, mostly from Original Drawings. In
Press.
YOUN-G J. PENTLAND,
EDINBURGH : n TEVIOT PLACE.
LONDON:
21 WARWICK LANE, PATERNOSTER ROW, E.G.
I
UNIVERSITY OF TORONTO
LIBRARY
Do not
Acme Library Card Pocket
Under Pat. "Ref. Index File."
Made by LIBRARY BUREAU, Beaton
to |
is
fe
ft
Live Music Archive
Librivox Free Audio
Metropolitan Museum
Cleveland Museum of Art
Internet Arcade
Console Living Room
Books to Borrow
Open Library
TV News
Understanding 9/11