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GI? 


PY.P.H Person 





Pentland’s Students Manuals. 





MANUAL OF BACTERIOLOGY. 


Pentland’s Students’ Manuals. 


MANUAL OF BACTERIOLOGY. 





Pentland’s Students’ Manuals. 


MANUAL OF BACTERIOLOGY. 





MANUAL 


oF 


BACTERIOLOGY 


BY 


ROBERT MUIR, M.A., M.D., F.R.C.P.Ep. 
vnorel#on OF PATHOLOGY, UNIVERSITY OF GLASGOW 
AND 


JAMES RITCHIE, M.A., M.D., BSc. 


SUPERINTENDENT UF TAK ROYAL COLLEGE OF PHYSICIANS’ LABORATURY, EDIXBUROI 
‘YORMERLY PROFESGOR OF PATHOLOGY IX TRE UNIVERSITY OF OXYORD 


FOURTH EDITION 


WITH ONE HUNDRED & SEVENTY-ONE ILLUSTRATIONS 


NEW YORK 
THE MACMILLAN COMPANY 
EDINBURGH AND LONDON: YOUNG J. PENTLAND 


1907 |, 
x. 


EDINBURGH : PRINTED FOR YOUNG J. PENTLAND, 11 THVIOT PLACE 
AND 21 WARWICK LANE, FATERNOSTER ROW, LONDON, FCs 
BY R. AND R. CLARK, LIMITED 


All rights reserved 





195° 


PREFACE TO THE FOURTH EDITION. 


In the present edition the whole subject has been carefully 
revised. During the five years since the last edition was 
published, valuable additions to our knowledge have been 
made in practically every department, whilst in the case of 
several diseases there have been discoveries of the highest 
importance. Our object has been to incorporate this new 
matter and at the same time to maintain the primary object 
of the work as a text-book for students of medicine. Thus 
whilst we have dealt with all the facts having a direct bearing 
on clinical medicine we have also given considerable prominence 
to matters at present under discussion from the scientific point 
of view. In this way we have endeavoured to give a faithful 
representation of the subject as it at present stands both in 
its practical and theoretical aspects. In the case of several 
diseases which up till recent times have been investiguted by 
purely bacteriological methods there is now considerable evidence 





that the causal agent is of protozoal nature. Amongst such 
conditions the most important are those in which spirochates 
are concerned, syphilis and the relapsing fevers being outstand- 
ing examples, As, however, the exact biological relationships 


of these organisms are still matters of dispute we have keyt 


¥ S931 


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. 


October 1907. 


PREFACE 10 THE FIRST EDITION. 


Tue science of Tacteriology has, within recent years, become 
‘so extensive, that in treating the subject in a book of this size 
we ane necessarily restricted to some special departments, unless 
the deseription 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 buman subject have been considered. We have 
made it & chief endeavour to render the work of jsrvctical utility 
for beginners, and, in the account of the more important 
methods, have given elementary detaila which onr experience in 
the practical teaching of the subject has shown to be necessary. 

Tn the systematic description of the various bacteria, an 
Attempt has been made to bring into prominence the evidence of 
their faving an etiological relationship to the corresponding 
dikouses, to point out the general laws governing their action us 
producers of disease, and to consider the effects in particular 
jnstaneus of various modifying circumptances, Much research 
on certain subjects is 20 recent that conclusions on many points 
must nocessarily be of a tentative character. We have, therefore, 
im our statemont of results aimed at drawing a distinction 
between what ix proved and what is only probable, 

Than Appendix we have treated of four diseases; in two of 
these the ciusal organist is uot a bacterium, whilst in the other 
‘two its nature ie not yet dotermincd. Those ‘liseases have been 

vil 





fO THE FIRS’ 





EDITION 





“ACE 










= whieh they illustrate. 
te Professor Greenfield for his kind 
ertain parts of the work, We have 





connection with 





“at pleasure in nowledging our indebtedness to 
Dr. Patrick Manson, who kindly lent us the negatives or pre- 
ions from which Figs. 160-165 hav 
« convinced that to any one engaged in practical 
photographs and photomicrographs supply the most useful 
have used these almost exclusively in 
ic description. These have been | 
of the University of | 
and Muir, ‘The line drawings were 





been executed. 












act information, w 








illustration of the system 





ted in the Pathological Laborator 






inburgh by Mr. 
prepared for us by Mr. Alfred Robinson, of the University 
Musewn, Oxford. 





ppended w short Bibliography, which, 
no pretension to completeness, will, we hope, be of 
in putting those who desire further information on the track 











of the principal papers which have heen published on each of 


the subjects considered 





June 1897, 


CONTENTS. 


+ 
CHAPTER I. 


GeveRaL Morpuorocy anp Browoey. 


Ixrropucrony—Terminology—Structure of the bacterial cell— 
Reproduction of bacteria—Spore formation — Motility — 
Minuter structuro 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 naturo—Methods of bacterial action— 
Variability among bacteria. ¢ a 


CHAPTER II. 


Metsops or Cortivation oF Bacteria. 





Introductory—Methods of sterilisation—Preparation of culture 
media—Use of the culture media—Methous of the separation 
of aerobic organisms—Principles of the culture of anaerobic 
organisms — Miscellaneous methods — General laboratory 
rules. . . . . + . . 





CHAPTER III. 


Microscoric Metaops—Grngrat BacTEertoLocical. 
Dracyosis—Inocunation oF ANIMALS. 


The microscope—Examination of hanging-<drop cultures—Film pre- 
parations—Examination of bacteria in tissues—The cutting, 
6 ix 


x CONTENTS 


of sections—Staining principles—Morlants and decolorisers 
—Formule of stains—Gram's method and its modifications 
—Stain for tubercle and other acid-fast bacilli—Staining of 
spores and flagella—The Romanowsky stains—Obsorvation of 
agglutination and sedimentation—Method of measuring the 
phagocytic capacity of the leucocytes—Routine bacteriological 
examination—Methods of inoculation—Autopsies on animals 











CHAPTER IV. 
Bacteria tx Arr, Som, 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 . 


CHAPTER V. 


Re.ations or Bacrerta To Diszass—Tue Propuction 
or Toxins By Bacrerta. 


Introductory—Conditions modifying pathogenicity—Modes of 
bacterial action—Tissue changes produced by bacteria—Local 
lesions—General lesions—Disturbanco 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 é 


CHAPTER VI. 
IxFLamMatory aND Suppurative ConpITIoNs. 


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—Uleerative 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 





Paar, 


85 


126 


149 


172 


CONTENTS 


CHAPTER VII. 


xi 


InPLaumatony ap SuPPURATIVE CONDITIONS, CONTINUED : 


‘Tae Acute Prrvmowias, Eprpemic CergpRo-Srivat 
Menrnorms. 


Introductory — Histories] — Bactoria in pneumonia — Fraenkel’s 
pheumococcus—Friedlaender's pneumococcus—Distributionof 
pneumobacteria— Experimental inoculation— Pathology of 
Pneumococcus—Methods of examination, Epidemic cerebro- 
spinal meningitis 





CHAPTER VIII 


Gowonanaa, Sort Sork, SYPHILIS. 


‘The gonococcus — Microscopical characters — Cultivation — Rela- 
tions to the disease—Its toxin—Distribution—Gonococeus in 
joint affections—Methods of diagnosis—Soft sore—Syphilis— 
Spirocheete pallida—Transmission of the disease to animals . 


CHAPTER IX. 
TUBERCULOSIS. 


Historical—Tubereulosis in animals—Tubercle becillus—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 Tubereulin-R—Agglutinative phenomena—Methods 
of examination . . i 











CHAPTER X, 
Leprosy. 


Pathological changes—Bacillus of leprosy—Position of the bacilli 
—Relations to the disease—Methods of diagnosis 





PAGE 


196 


219 


235 


28t 


xii CONTENTS 


CHAPTER XL 
GLanpeRs anD RHINOSCLEROMA. 


Glanders: The natural diseaso—The glanders bacillu-—Cultiva- 
tion of glanders bacillus—Powers of resistance—Experimental 
inoculation—Action on the tissues—Mode of spread—Mallein 
and its preparation—Methods of examination, Rhinosoleroma 





CHAPTER XII. 
Activomycosis aND ALLIED Diskases, 


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 . 





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 . 





CHAPTER XIV. 


TypHorp Fever—Baciti aLuizp TO TRE TYPHOID 
Bactiivs. 


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 diarrhea 











275 


286 


300 


319 


Introductory—Historical—Bacillus tetani- 





CONTENTS 


CHAPTER XV. 


DiparHerta. 


istorical—General facts—Bacillus diphtherie— 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 : a 








CHAPTER XVI. 
TErANvs. 


Isolation of bacillus 
tetani—Characters of cultures—Conditions of growth—Patho- 
genic effects—Experimental inoculation—Tetanus toxins— 
Antitetanic serum — Methods of examination — Malignant 
cxdema—Characters of bacillus—Experimental inoculation— 
Methods of diagnosis—Bacillus setulae Guat evil— 
Bacillus erogenes capsulatus . 2 





CHAPTER XVII. 


CHOLERA. 


Introductory —The choleraspirillum—Distribution of thespirilla— 


Cultivation—Powers of resistance—Experimental inoculation 
Toxins of cholera spirillum—Inoculation of human subject— 
Immunity—Methods of diagnosie—Ceneral summary—Other 
spirilla resembling the cholera organism— Metchnikoif's 
apirillum—Finkler and Prior's spirillum—Deneke's spirillum 





CHAPTER XVIIL 


Inrivenza, Prague, Retrsixa Fever, Mavta FEVER, 
Yeuiow Fever 





Influenza bacillus— Microscopical characters—Cultivation—Dis- 





tribution—Experimental inoculation—Methods of examina- 


xiii 


PAGE 


352 


371 


399 


CONTENTS 


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 disgnosis— 
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 . 








CHAPTER XIX. 


Iuxonrry. 


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’ 
theory of the constitution of toxins—Antibacterial serum— 
Bactericidal and lysogenic action— Hemolytic and other 
sera— Methods of hemolytic tests—Opsonic action— Ag- 
glutination—Precipitins—Therapeutic effects of anti-sera— 
‘Theories as to acquired immunity—Ehrlich’s side-chain theory 
—Serum anaphylaxis— Theory of phagocytosis — Natural 
immunity—Natural bactericidal powors—Natural suscopti- 
bility to toxins . : 








APPENDIX A. 


Smaturox anp Vacernation. 


Jennerian vaccination—Relationship of smallpox to cowpox— 


Tntruisary Pathology Thiiras af hydropbobl 


Micro-organisms associated with smallpox—The nature of 
vaccination é » 


APPENDIX B. 


Hypropuosta. 





Prophylaxis 
—Antirabic serum—Methods 


420 


456 


508 


510 


CONTENTS 


APPENDIX C. 
Matantan FEVER 
‘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 a s 


APPENDIX D. 
Amapic Dyseyrery. 


Amabie dysentery—Characters of the ameba—Distribution of the 
‘smorbe—Experimental inoculation—Methods of examination 


APPENDIX E 
‘TryPaNnosoMrasis—K ALA-4ZAR—PIROPLASMOSIS. 


The pathogenic trypanosomes—General morphology of the trypauo- 
somata—Trypanosoma Lewisi—Nagana or tso-tso fly disease 
—Trypanosome of sleeping sickness—Try panosoma gambiense 
—Kala-dzar—Debli sore—Piroplasmosis 








BIBLIOGRAPHY . 


INDEX 


xv 


521 


544 


593 








LIST OF ILLUSTRATIONS. 


— 


|. Forms of bacteria 
. Hot-air steriliser 
. Koch's steam steriliser . 
. Autoclave 
. Steriliser for blood serum 
. Meat press z 
. Hot-water funnel 
Blood serum inspissator 
. Potato jar 
. Cylinder of potato eut obliquely 
. Ehrlich's tube containing piece of potato 
2. Apparatus for filling tubes A 
1. Tubes of media ‘ 
. Platinum wires in glass handles 
. Method of inoculating solid tubes 
. Rack for platinum needles 
. Petri’s eapwule . 
. Koch's levelling apparatus for tse in vrepning Plater 
. Koch's levelling apparatus 
). Esmarch’s tube for roll culture 
. Apparatus for supplying hydrogen for anaerobic cultures 
. Esmarch’s roll-tube adapted for culture containing anaerobes 
. Bulloch's apparatus for anaerobic plate cultures 
. Flask for anaerobes in liquid media. 
. Flask arranged for culture of anaerobes which develop gas 
. Tubes for anaerobic cultures on the surface of solid media 
. Slides for hanging-drop cultures 
. Graham Brown's chamber for anaerobic hanging-drops 
. Apparatus for counting colonies =. 
). Wright's 250 c.anm. pipette fitted with nipple 
|. Geissler’s vacuum pump for filtering cultures . 
. Chamberland’s candle and flask arranged for filtration 
xvii 











Paar 


62 


xviii LIST OF ILLUSTRATIONS 


33. 
34, 
35. 
36, 


37. 
38. 
39, 
40. 
al. 

42, 
43, 


44, 
45. 
46. 


47. 
48. 


49. 
50. 
51. 
52, 


53, 
ba. 

55, 
66, 
87. 
68. 
59. 

60. 


61. 
62, 


63, 


65. 


8s 


Chamberland’s bougie with lamp funnel 

Bougie inserted through rubber stopper. 

Muencke’s modification of Chamberland’s filter. 

Flak Sttod with porslain bougie for tering large auntie 
of fluid 





‘Tubes for demonstrating gas-formation by bacteria 

Goryk air-pump for drying én eacuo i 

Reichert's gas regulator 

Hearson's incubator for use at 87° C. 

Cornet’s forceps for holding cover-glasses 

‘Needle with square of paper on end for manipulating peratin 
sections . . 

Syphon wash-bottle for distilled water 

Wright's 5 omm. pipette. 

‘Tubes used in testing. sgslutnatingand sedimenting propetin 
ofserum 

Wright's blood-capsule 

Test-tube and pipette arranged for obtaining fluids containing 
bacteria : 

Hollow needle for intraperitoneal inoculations 

Hesse’s tube 

Petri's sand filter 

Staphylococcus pyogenes aureus, young culture om ager. 
1000 

‘Two stab cultures of staphylococcus pyogenes aureus in gelatin 

Streptococcus pyogenes, young culture on agar. x 1000 

Culture of the streptococcus pyogenes on an agar plate 

Bacillus pyocyaneus ; young culture on agar. x 1000 

Micrococous tetragenus. x 1000 4 : 

Streptococci in acute suppuration. 

Minute focus of commencing suppura ‘ 

Secondary infoction ofa glomeralu of kidney by th wtaphylo- 
coecus aureus. 300 ts 

Section of a vegetation in ulcerative endocarditis, x 600 

Film preparation from a case of acute conjunctivitis, showing 
the Koch-Weeks bacilli. x 1000 : 

Film preparation of conjunctival secretion showing the he dp 
bacillus of conjunctivitis, 1000 . 

Film preparation of pneumonic sputum, showing uumerous 
pneumococci (Fraenkel’s), 1000 . 

Friedlinder's pneumobacillus, from exudate in a case of 
Pneumonia, x 1000 

Fraenkel's pncumococeus in serous exudation. x 1000 

Stroke culture 6f Fraenkel’s pneumococens on blood agar 














LIST OF ILLUSTRATIONS 


ro. 
88. Praabale postmen from s pure culture on Bhat fete 
* Stab pea of! Friedhinder's poeumobsoil us + 
‘a proumobed{lius, from a young culture on ager, 
1000. 
71. Cepwulated posumeconsk in blood taken from the heart of & 
rabbit. %1000 . 
rs Filmpreparatimotxadetiontiom acamot meningitis, x «1000 
73, Pure culture of diplococeus intracellulatis ss 
T4. Portion of film of gonorrheeal pus. 1000 f 
75. Gonocose, from a pure culture on blood agar, 1000 - 
76. cree apy eae een shoring Door’ 
‘bacillus, =< 1500 
72, Duccey’s bacillus, 1000. | 
78 nod 79, Film preparations from juice of hard chanere showing 
apirechats pallida. 2000 
450, Seotion of splesn from * ase of congenital phi, showing 
1000 


api 

Hi, Bplicchede schingaas. 1000 z 

82, Tuberole bacilli, from a pare culture on glycerin agar, «1000 

Bi. Tubercle bacitli in phthisical sputum, 1000, : 

‘4. Cultures of tubersio bacilli on glycerin agar 

#5. Tubervle bavilli in ection of human les in aoute pit 
~1000 0. 

60; Tubercle bacilli io glantoella. x 1000. 

87, Tubercle bacilli in urine, x1000 . ~ 

85. Moollor’s Timothy-grass bacillus. «1000 

$9. Cultures of acid. 








Bi. Beotion through leprous skin, showing the masses of cellular 
granulation tinme in the cutis, «80 
#2, Superticlal part of leprous skin. 500 
a3, ah porter vi view of portion of leprons nodule showing the 
‘arrangemont of the bacilli within the cells of the rane 
tion tisens, 1100 : 
M4. Glanders bacilli amongst brokeusdown cell” x 1000 
95. Glusstors bacilli, x1000 
96. Aetinomycosis of human Liver. 500 
97. Astinomyons in human kidney, $00 
BB, Colouics of sctinemyers. » 60 F 
9% Cultures of the aotinomyees on glyverin agar 3 
100. Actinomyces, frum a cultare on glycerin agar. * 1000 
101. Shake enitures of sotinomyces in glucose agar 5 
102, Section of « colony of actinomyors (rom « culture in Mood 
serum. 1600 








$ ER BEEBE g ts fe 


xx LIST OF ILLUSTRATIONS 


103, Streptothrix Madune, 1000. 
104. Surtuce colony of the anthrax bacillus on an agar plate. 
30 
195, Anthrax becilli, arranged in cain from’ a twenty-four 
hours’ culture on agar at 87°C, x 1000 . A 
10s. stab culture ofthe wehrax baci in peptone- estatin je 
167, Anthrax bacilli containing spores. x 1000 . 2 
raping from wpleen of guinea-pig dead of anthrax. x 1000 
109, Portion of kidney of a guinea-pig dead of anthrax. x00 . 
110, A large clump of typhoid bacilli in a spleen. x 600 3 
111, Typhoid bacilli, from a young culture on agar, showing some 
filamentous forms, x 1000 
112. Typhoid bacilli, from a young culture on ager, showing 
flagella, x 1000. 
Culture of the typhoid bacillus and of the becillus coli. 
olonies of the typhoid bacillus in a gelatin plate, x 15 
. Bacillus coli communis. x 1000 
Film preparation from diphtheria membrane ; showing 
numerous diphtheria bacilli. x1000 ae: 
ttn hen ibrar Fecha, shi 
1g diphtheria bacilli, x 1000 5 
Cultures of the diphtheria bacillus on an agar plate 
. Diphtheria bacilli from a twenty-four hours’ culture on 
agar. 1000 ‘i : : 
iphtheria bacilli, from a three days’ agar culture. x 1000 
+ Involution forms of the diphtheria bacillus. x 1000 
22. Po ria bacillus (Hofmann’s). 1000. . 
. Xorosis Iucillus from a young agar culture. x 1000 : 
. Film preparation of discharge from wound in a case of 
ux, showing several tetanus bacilli of “ drumstick ” 
form, x1000 : : 
nus bacilli, showing Hagella. x 1000 | z 
Spiral composed of numerous twisted Angela ofthe tetanus 
bacillus. 1000, > 
illl, some af which fomeas spores. 1000 
of the tetanus bacillus in glucose gelatin. 
paration from the affected tissues in a case of 
malignant > 1000 : . x 
. Bacillus of malignant wdema, showing spores. 1000. 
1, Stab cultures in agar ctanus bacillus, bacillus of malignant 
vedema, and bacillus of quarter-evil z 5 
spores. 1000 






































































152, Bacillus of quarter-evil, sho 
15%, Baeillus erogenes capsulatus R i i 
121. Cholera spirilla, trom a culture on agar of twenty-four hours’ 





growth, «1000 


vag. 


298 
302 


308 
303 
305 
307 
309 
320 


921 


322 
323 
324 
325 


354 


355 
387 


357 
358 
358 
366 
367 


373 
art 
375 
375 
376 


389 
390 


391 
397 
398 


400 


LIST OF ILLUSTRATIONS 
no. 
135. Cholera spirilla stained to show the terminal flagella. 
x10 H ‘ : 
136. Cholera spirilla from an old agar culture, 1000 . 
137. Puncture culture of the cholera spirillum : 
188, Colonies of the cholera spirillum on a gelatin plate . fi 
139. Metchnikoff’s spirillum. 1000. . ‘ 
140. Puncture cultures in peptone-gelatin 
141. Finkler and Prior's spirillum. 1000. ; 
142. Influenza bacilli from a culture on blood agar. x1000  . 
143. Film preparation from a plague bubo. x 1000 2 : 
144. Bacillus of plague from # young culture on agar. 1000 . 
145. Bacillus of plague in chains. x 1000 if 
146. Culture of the bacillus of plague on 4 per cent salt agar. 
x1000 : 
147. Section of a human lymphatic gland in plague. x50. 
148. Film preparation of spleen of rat after inoculation with the 
bacillus of plague. x 1000 3 j R 
149. Spirilla of relapsing fever in human blood. x about 1000 . 
150. Spirillum Obermeieri in blood of infected mouse. 1000 . 
151. Film of human blood containing spirillum of tick fever. 





x 1000 
152, Spirillum of human tick fever (Spirilium Duttoni) in blood 
of infected mouse. x 1000 i X i 


153. Micrococcus melitensis. 1000. A 3 

154-159. Various phases of the benign tertian parasite. : 

160-165. Exemplifying phases of the malignant parasite . : 

166. Amcebee of dysentery . a 

167. Scotion of wal of liver abscess, showing au amebs of spheri 
form with vacuolated protoplasm. x 1000 

168. Trypanosoma Brueei from blood of infected rat. Note in two 
of the organisms commencing division of micronucleus and 
undulating membrane. 1000. é 7 

169. Trypanosoma gambiense from blood of guinea-pig. x 1000. 

170. Leishman-Donovan bodies from spleen smear. x 1000, 

171. Leishman-Donovan bodies within endothelial cell in spleen. 
x1000 








xxi 
PAGE 


401 
401 
403 
404 
417 
418 
419 





MANUAL QF BACTERIOLOGY 


MANUAL OF BACTERIOLOGY. 


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 /ower 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 
1p (xxbou inch), ‘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 Jacilli, (3) A 
group in which the shape is that of a curved or spiral rod. 
These are called spiritla. 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 





2 GENERAL MORPHOLOGY AND 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.—The 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 smnall, unicellular organisms, which may represent the most primitive 
forms of life before diferentiation into unimal and vegetable types had 
occurred. This would inelude the flagellata and infusoria, the myxomy- 
cetes, the lower alge, and the bacteria. To the lower alge the bacteria 
possess many similarities. “These alge are unicellular masses of proto- 
plasm, ig gencrally the same ahapen as the bacteria, and largely 
multiply by fission, Endogenous sporulation, however, does not occur, 
nor is ted with the possession of flagella, Also theit 

















protoplasm from that of the bacteria in containing chlorophyll and 
another 1 pigment ealled phycoe From the morpholozical 
resemblances, however, between these alge and the bacteria, and from 
the fact that’ fission plays a predominant part in the multiplication of 


STRUCTURE OF THE BACTERIAL CELL = 3 


Hoth ey Inve ben grouped fn one clase as the Sch 
te St) a tw itu 


rahe fe splitting alge am denominated. the 
3 ite Casters or aplitting fungi 


Mimaneirers |, Spaltpilzen). The bact 
oo Sap seen te Ce ‘ea ata wh 
im con ought probably 
abel ing, chlorop hy! 


‘Gexmntan Moxrnoney or re Bacrmnta. 


‘The Structure of the Bacterial Cell—On account of the 
minateness of bacteria the invest of their structure is 
attended te onpeeten eae tne the 
Microscope, in natural condition, eg. in water, they aj 

as colourless refractile bodies of the different ape 

Spore formation and motility, when these exist, can 

aleo be observed, but little ele bors be made ont, tena 
otal, epee ae ig ways taken of the fact of 
their affinities for various dyes, especially those which are usually 
See eee atin for the nuclei of animal cells, Certain 
points have thus been determined. The bacterial cell consists 
‘of a sharply con Rg trred susesot protoplasm which reacts to, 
especially basic, aniline dyos like the nucleus of an animal coll 
—thongh from this fact we cannot deduce that the two are 
identical in composition. A healthy bacterium when thus 


stmeture. The protoplasm is surrounded by an 

envelope which can in some cascs be demonstrated by over- 
a specimen with a strong aniline dye, when it will appear 

asa 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 
sem by ving the latter the appearance af being 
by a membrane. It is only, however, in somo of 

higher forma that 4 trac membrane occurs. Sometimes the 

ose i of the envelope is sharply defined, in which case 
ium appears to have a distinct capsule, and is known 

Pa tee kacterions (ride Fig. l. No. 3; and Fig, 64). 
sion of bacteria into masses depends largely on the 
character of the envelope, If the latter ix glutinous, then a 
large mass of the same species may occur, formed of individual 
embedded int what appears to be a mass of jelly. When 

Upeeurs, it fa known as 4 soogleea mass, On the other hand, 
‘envelope hak 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 ontirely free from one another. Many: 
PA the higher bacteria possess a shoath which has a much more 
definite structure than is found among the Jower forma Tt 
Fesiats external influonces, possesses elasticity, and serves to bined 
the clements of the erganiam together, 
lution among the Lower Bacteria.—When a bacterial 
cell iz placed in favourable surroundings it multiplies ; as has 
boon said, this, in the great majority of cases, takes place by 
simple fission. In the process a constriction appears in the 
middie and a transverse unstained line develops aeross the 
roteplagm 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 separute, scomding: to the 
character of the envelope, as already exphined, 
bacterin growth and multiplication go on with great npaiity 
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 us the tubercle bacillus, 
aa ly much more slowly. ‘Sometimes division prococda #0 
y that the young individualn do not reach the adult size 
Faire wultiplieation again occurs, ‘This may give rise to 
anomalous appearances, When bacteria are placed in unfavont- 
able conditions as regards food, cte., growth and multiplication 
take place with difficulty, In the great majority of cases this is 
evidenced hy changes in the appearance of the protoplasm, 
Instead of its maintaining the regularity of shape seen in healthy 
bacteria, various aberrant appearances are proacnted, 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 protopiagm 
ulso 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 a small 
proportion of the bacteria may stain at all, Sometimes, on the 
other hand, « degenensted bacterium contains intensely stained 
grapules or globules which may be of large size, Such aberrant 
and degenerate appearances are roferred 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 takee place, Man; 
individuals have undoubtedly died, and the remainder whi 

























SPORE FORMATION 5 


live and develop inte typical forms may sometimes have lost 
some of their propertios, 


a eteath. ‘The crgauiam is Freqcontly atteohed a8 cna end ta some 
‘or to another individual, It grows toa certain length and. then 


Dee ae oe 
in al oloment of the filament suck as 
in ehe growth of the latter, In some one, howe “livison takes 
Reet cect ue ae Sy peers taGeien 
certain timo before becoming attached, and in this stage are 
wwe motile. ‘Thoy ara usually rodslike in shape, somotines 
a ‘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 
pa sesult in the formation of bodies culled spores, to which 
the vital activities of the original bacteria are transferred, 
Spore formation occurs chiefly among the ie and in some 
spirilla, Its commencement in a bacterium is indicated by the 


in the i refructile 
CS ordinary mothods, This inereases in 
‘size, and axsumes 4 roand, oval, or short rod-shaped form, always 
shorter but often broader than the original bacterium. Tn the 
process of spore formation the rest of the bacterial protoplasm 
may remain unchanged in appearance and staining power for a 
considerable tine (ey, b, tetani), or, on the other hand, it may 
soon lose its neg of pabiog and ultimately disappear, leaving 
‘the spore ia the remains of the onvelope (eg. b, anthracis), 
‘This method ey spore a poe + aoe endogenous. ee! 
are nommotile. ‘The spore may appear in the 
aharieiatia ce tt rhe Pe kt ooh Ashes Gun 
ee from ono extromity (Fig. 1, No. 11). Inatiucture 
dalieehi a tonsa oh peotooiears surrounded by adense 
‘This can be domonatmted “by methods which will 
the underlying principle of which is the prolongest 

of @ powerful sain. The membrane is supposed to 
0m the spore its characteristic feature, namely, great 
of al_influcnces such as heat or 
och, for instance, in one serics of experi- 

ments, found that while the bacillus anthracis in the unspored 
| Killed by a two minutes’ exposure to 1 per cont catbolie 

Of the same organism resisted on exposure of from 













spore is placed in suitable surroundings for yrowtn 


—_— 





6 GENERAL MORPHOLOGY AND BIOLOGY 


it again assnmes the original bacillary or spiral form, The 
capsule dohivees either longitudinally, or torminally, or trans- 
versely. In the last case the dehiscence may be and the 
new individual may remain fora time attached by its ends to 
the hinged spore-ease, or the debisconee 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 apore formation 
is rarely, if ever, to be considered as a method of multiplication. 
Trat least the great majority of cases only one spore ik 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 bacterium, and is to be contrasted with the stage 
when active multiplication takes place. The latter ia 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 us representing the highest stage in the vital activity 
of e 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 ite greatost 
vitality, Such « rejuvenescence, as it wore, through sporulation, 
is known in many alge. snpport of this view there are 
cortain facts. Tn many eases, for instance, spore formation only 
occur at temperatures specially favourable for growth and 
multiplication, There is often a temperature below which, 
while vegetative growth still takes place, sporulation will not 
ooear ; and in tho case of b. anthracis, if the organiam be kept 
wt 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 
hactaria preferring the absence of axygen for vegetative growth, 
the presence of this gue favours sporulation, Most bacteriologists 
ure, however, of opinion that when a bacterium forms a spore, 
it only does so when its sarroundings, 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 anulogous 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 @ bacterium is constantly supplied with fresh food (the 




















SPORE FORMATION 7 
‘other conditions of life keing equal). ‘The p 





since not will the food suj around 
in be cae eres own 
inimical matters will be all the more rapid. 

We must note that the usually applied tests of a body 
developed within a baeteriam being a spore a (1) its staining 
reaction, namely, resistance to ordinary ateiping Snide but but 
capacity of bei Ste ty he el 

pt 3) rae he fnet as the iia 


‘Vegetative = ies inortat a 


bei 
the Se 
ee oe the smaller bactes 
beep is very difficult to say whether they spore or oot 
sr taal bad in such organistns small unstained spots the 
which it is very difficule to determine. 


‘The Question of Arthrosporons Bacteria.—It is stated by Huoppe that 
eertain oryanioms, ey, some atreytococci, certain individu may, 
sporulation, take on resting stage. Tliese become 

snollen, stain woll with ordinary staina, and they are stated to have 
-of resintanoe then tho other forme ; further, when vegetative 

aaa is from them thot multiplication is said to take rae 


Phenomena noted oan be explained we the undoul 

Baeeitear y growth there ie verg great variation ameng the 
Tepes in their powers of resistance to external conditions, 
“Motility.—As has been stated, many bacteria are motile. 
ean bo studied by moans of hanging drop preparations 

‘ The movernents are of a darting, rolling, or 
character, The degres of motility depends on the 
temperature, the age of the growth, and on the 
Which the bacteria are growing. Sometimes the 
# are most active just after the cell has multiylicd, 








8 GENERAL MORPHOLOGY AND BIOLOGY 


sometimes it goea on all through the life of the bacterium, 
sometimes it ceases when sporalation is about to occur. Motility 
is associated with the possession of fine wavy thread -like 
appendages called flagella, which for their demonstration require 
¢ application of special staining methods (ride Fig. 1, No, L2; 
and Fig. 112). They have heen shown to occur in mauy bacilli 
and spirilla, bat only in a few species of cove. ‘They vary in 
hi, but may be several times the length of the bacterium, 
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 ocours in many algie, they are not actual 
prolorgutions of the bucterial protoplasm, but merely appendages 
of the envelope, and have doubted whether they are really angus 
f locomotion. There is now, however, little doubt that they 
belong to the protoplasm. By appropriate means the central 
parts of tus latter can be made to shrink away from the peripheral 
(vitte infra, “plusmolysis"). In xnch 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, thoy have boon 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 tho special spiral forms, and in most 
of the highor bacteria, motility 18 probably duc to contractility 
of the protoplasm itself, 


‘The Minuter Structure of tho Bacterial Protoplasm.—Many attom| 
have been mile to obtain deeper information as to the structure of 
bucterisl coll, and especially as to its bebayiour in division. ‘Thuee 
have largely turned on tho interprotation to be put on certain appears 
‘pcos which havo been obse ‘These appearances aro of two kinds, 
First, under certain cireumstances irregular deoply-stained granules are 
‘observed in the protoplaam, often, when they ocour in a bacillus, giving 
the latter the appearance of a short chain of cocci. They are often 
called metachromatic granules (vide Pig. 1, No. 19) trom the fact that 
Uy aperopriate prooodice they can be stained with one dye, and the 
protoplasm in which thoy lio with another ; sometimes, when » single 

each ax methylene bluo, thoy sesumo a alightly dilferont 

protoplasm, 
For tho demonstration of the metachromatic granalex two mothoda 
have beem advanced, Ernst recommends that a few drops of Liftlor's 
methylene blue (vite p. 98) be placed on a cover-glass preparation ai 
the Inttor passed backwards and forwards over 4 Bunsen Name for half 
4 minute after steam bogius to ris. ‘The proparation i+ thea washod 
in water and counterstained for ono to two minutos in watery Bismaroke 
The granulos are here stained blue, the protoplasm brown, 
























STRUCTURE OF BACTERIAL PROTOPLASM 9 


‘proparstion fuchsin, waahow 
$i ein pr eo tai th Latier’s bine, 


apes, blue. = The general 
iis frie is mh brea pax the Girnt more 


which ean sometimes bo seem in specimens 
fu the oosirrence of  concenteation of the 


a ‘a m grown ander 

ee here ie food i ecmiyg xin ‘exhausted. Some 
pearances mig jue toa process alliod to 

ee penrae bit division, but of thix ther is ne 


Sinapts young bacteria, ces of 
fornsatton yas teouslation toay Be gehen rode at 
eotte im the oveurrence of what i Ky known as p! 


whew 
Berto: semen 


ig conpentrati 
of oxmncls thy water bei in the protoplasn passes 
bra the mean and, the protoplasi reteactiny fun 


Furthermore it is often most readi 
‘Stharwive enfoebled cultures, 
from « study of some largo sulphur-containing forms, con- 
cts. the grestas part of the bectarial ell may correspond to 8 
Byasd that this senrrounded ty a thin layer of protoplasm which 
notice, unl ho bacillé, it 


capes 
east ts at thy code ot ‘of tho cells, Fi ite mae be maid, looks 
‘BpRenrances econ in Blitsehli’s proparations os due to plasmolysis, 


‘The Chemical Composition of Bacteria.—In the bodies of 
bg many definite cubstances oeeur. Some bacteria have 
as containing chl ihyl!, but these are properly 
None ote with the schizophycem” Sulphur is found in some 
forms, and starch granles are also described aa 
Many species of bacteria, when growing in masses, 
Fischer, “*Untersuchnugen Uber Haxterien,” Berlin, 1896; 

den Han der Cyanophyceen und Bakterien,” Jena, L897. 








10° GENERAL MORPHOLOGY AND BIOLOGY 


ane brilliantly coloured, though few bacteria associated with the 
production of disease give rise to pigments, In some of the 
‘corganisins classed a bacteria a pigment named Leap hriaed ae 
hae been observed in the protoplasm, and similar intracellular 
pigments probably oceur 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 {ree prodyction 
of pigment abundant oxygen supply is necessary; but sometimes, 
as in the exe 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. pyocyanons be exposed to the temperature of 42° C. 
for a certain time, it loves ita 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 usture of bacterial pigments. 

pf, however, has found that many of them beloug to a group of 
colotting matters which coour widdy in the vogviablo end animal 
kingdoms, viz, the Lipochromes, These tiposhromes, which get their 
namo from the colouring matter of animal fat, inelude the eolourin, 
Sati bn Ube pars of anosealscen, the fellow rignients of erame ats 
ofthe solke ofexgs, and many bacterial pigments. ‘Tie Lipochromes ure 
characterised by their solubility in chloroform, aleobol, ether, and 
potoloun, and by their giving indigo-bloc cryxtals with strong sulphuric 
acid, and’a green volour with iodine dimsolved in potaminm iodide. 
‘Though orystalline compounds of these have been obtained, their 
chetnieal constitution is entirely unknown and even their percentage 
‘composition ix disputed. 





Some observations have been made on the chemical structure 
cof bagterlal protoplasm, Nencki isolated from the bodies of 
certain putrolactive bacteria proteid bodies which, according to 
Ruppel, appear to have beon allied to peptone, and which 
differed from nucleo-proteids in not containing phosphorus, 
but many of the proteids isolated by other chemists have 
boon 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 caseing, and he addoces evidence 
to show that it ix to these that the characteristic staining 
properties are due. Various observers have isolated similar 
phosphoruscontaining 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 ae 


varions mineral salts, espoelally those of sodium, potassium, and 
|. ‘The amonot of different constituents varies ac- 
to the eth culbare aid the toedinmn sed lob 


growth, and 2 Soe takes place in the com- 
es, 


Classification of Bactoria.—Thoro have been numerous 
schemes set forth for the classification of tucteria, the funda- 
mental principle running through all of which has been the 

os the two sub-groups and the type forme mentioned 
in 9 fumeraph above. In the attempts to still 
farther su! rite the group, eoohel Seth a 
as to the characters ou which sub-cl ure to be based, Our 


that in every epecios there should be 
studied the habitat, best food supply, condition us to gascous 
environment, range of growth, temperature, morphology, life 
‘special propertios and pathegenici 
We must thus be content with a provisional and incomplete 
classification. We have said that the division into lower and 
bacteria is recognised by all, though, as in every other 
classification, thors occur transitional forma Tn subdividing 





bacilli, and spirilla, though the higher aro more difficult to deal 
with, Sutsidiary, though important, points in still further stib- 
division are the planes in which fission takes place and the 
| eraeg or itetnes of sporea, The recognition of actual species 
‘often a matter of great difficulty. ‘The points to be observed 
will be discussed later (p. 115), 
‘L The Lower Bacteria.'—Thess, as we have seon, are 
fmitiate unicellular casces of protoplism surrounded by an 
envelope, the total vital capacities of a species being represented 
im Gvery cell. They prosent three distinct type forma, the 
eoceas, the bacillus, and the spirillum ; endogenous sporulation 
may oceur. They may also be motile. 

T. The Corei.—In this group tho cells rango in different 
from “5 p to 2 pindiameter, but most mensure about I yx, 
division they may increase in siz: in all directions. The 

ate ‘are usually classitied acconling to the method af division. 
Por 4 Mustrntion of this and the sacceeding aystewmtic parsgrepha, 


-_ 


cy 





12 GENERAL MORPHOLOGY AND BIOLOGY 


Af tho 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 ton bunch of grapes, and the species is often called 
w staphylococcus. Wivision may take place in two axes at right 
angles to one another, in which ease cocoi ndhorent to cach other 
Packets of four (called detrads) or sixtoen may be found, 
former number being the more frequent, To all these forms 
the word mierococous is aften geuenlly applied. The individuals 
in a growth of micrococei often show a tondency to remain 
carted wu tora: Sloss are spoken of as diplococer, but this is 
not a distinctive character, since every coceus as a result of 
division becomes a diplococcus, though in samo species the 
tondeney to romain in pairs ix well marked, The adhesion of 
cocci to one another depends on the character of the capsule. 
Often this has a woll-marknd outer limit (micrococeus tetragenus), 
somotimes it is of geoat extent, its diameter being many times 
that of the coccus (streptococcus mesenteriodes). It is especially 
among the streptococci and staphylococe’ that the pheaomenon 
of the formation of arthrospores ia said to occur, In none of 
tho cocci have endogenous spores been cortainly observed. ‘The 
number of species of the streptococe! and staphylococel probably 
exceeds 150, Usually included in this group are eoccuslike 
organisms which divide in three axe# at right angles to one 
another, These are usually referred to as sarc. If the cells 
are lying single they are round, but usually they are seen in 
cubes of cight with the sides which are in contact slightly 
flattened. Large numbers of such cubes may be lying together. 
The surcinw are, a¥ a rule, rather larger than the other members 
of the group. Most of the eocei are non-motile, but a few motile 
species possvesing flagella have been described. 

2, Bacilli.—These consist of long or short cylindrical cells, 
with rounded or sharply rectangular ends, usally not mons than 
1 broad, but varying very greatly in length. They may bo 
motile or non-motile. Where flagella occur, these may be 
distributed all round the organism, or only at one or both of 
the poles (preudomonas). Several species are provided with 
sharply-marked capsules (b. pncumoniac), In many species 
endogenous sporulation occurs, ‘The spores may be central or 
terminal, round, oval, or spindle-shaped, 









Grest confusion in tiomenolature has ariewn in this group in cone 
Aoquonce of the different artiticial meanings assigned to the essentially 
synoUymous terms bacteriuin and bacillus. Migula, for instance, applies 


THE LOWER BACTERIA 13 
6 
& 

i § & & e 2% % at, 
1 3 5 


Qu 






2b 


i{ y 


Wy 


2. Streptococcus, 3. Staphylococcus. 4. Capmulated diplococeus. 

spel coceus, 6, Tetras. 7. Sarci i 

Giger tle). 9, Nor 

fa) comima-shaped element : 

1000, 11. Types of spore fo 

Bacteria produiced by yrlaxmol we Fiselier). 14. with terminsl prot 
fa} Hacilius eonsposed of ve protoplasmic mneshien 















jer Low ovr 


ny ia 
‘containing cocctsike Uudiew ; GE) Mle 


14 GENERAL MORPHOLOGY AND BIOLOGY 


the former term to non-motile species, the latter to the motile. Hueppe, 
on tho other hand, calls those in which endogenous sporulation 

not occur, bacteria, and those where it does, bacilli. In the ordinary 
terainology of aystematio Baoterislogy the word bacterium. has beed 
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 cholera 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 serew-like contraction of the protoplasm. Most of 
the motile spirilla, however, possess flagella. Of the latter there 
miay 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, spirochete, 
vibrio, Migula makes “ vibrio” synonymous with ‘microspira,” which 
ho applies to members of the group which posses only. one or two polar 
fiagella; spirllum” he applies to similar epecies which have bunches 
of polar flagella, while “ spirochvete ” is reserved for the long unflagellated 
spiral cells, Hneppe applies the term “‘spirochete” to forms without 
exudospores,  sibrio” to those with endospores in which during sparala- 
tion the organism changes its form, and ‘spirillum’” to the latter 
when no change of form takes place’ in sporulation, Fingge, another 
systematist, applies “‘spirochwte” and ‘spirillum” indiveriminately to 
any wavy or corkscrew form, and ‘‘vibrio” to forms where the undula- 

+" not so well marked. It is thus necessary, in denominating such 
rium 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 nunseptate 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 mnst thus be left an open question. 

IL. 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 regalar intorvals are ent 
3 rod-shaped or curved clements, Such 
Jess interdependent on one another, and 

jods are often necessary to demonstrate the 


— of Seay for neon one 
frequently concerned merely in attachi 
pan other object, The greatest ayiate 
in tho setting apart among most of the A 
terminations of the filaments for the 
ae init described (jp. 2). There 
ich the nec of the higher bacterin 
Hnowlsdgn of, thom ia all scmesiat 
members baye not yet been artificially 
soap on consists “of free reinalig 
suction. 


Hi 


bacteria 


Be 
al 


= 
va 


EEE 


group Fe 
Tast in structure, and the ae also contains 
ules: but the filaments are attached at one ond, 
other form gonidia. ‘The keptothrix group resembles 
Sy the thiothrix group, but the protoplasm does not contain 
In the eladothriz group there is the appearance 

which, however, is of a falas kind. What happons 
terminal cell divides, and on dividing again, it pushes 
its first division to one side. There ary thus two 

colle tying side by sido, and as each goos on dividing, 
of branching is given. Here, again, there 
formation ; and while the parent organism is in some 
‘of its cloments motile, the gonidia move by means of flagella, 
‘The nee dovolopment is in the atreplothrix group, to which 
atreptothrix actinomyces, or the actinomyces bovis, and 

te important pathogenic agents. Here the organism 

of a felted mass of non-septate filaments, in which true 
branching occurs Under certain circumstances 

{grow out, and produce chains of coccus-like bodies from 

few individnals can be reproduced. Such bodies are eften 

‘tons apores, but thoy have not tho mmo staining reactions 
re powers of so “high w dogroe as ordinary bacuerva 






To 
Hg 


Hi 


A 





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 xtreptothrix 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. 


Generat BroLocy or THE Bacteria. 


There are five prime factors which must he considered in 
the growth of bacteria, namely, food supply, moisture, relation to 
gascous environment, temperature, and light. 

Food Supply.—The bacteria are chielly found living on the 
complicated organie substances which form the bodies of dead 
plants and animals, or which are excreted by the latter while 
they are yet alive. "Seeing that, asa general rule, many bacteria 
grow side hy 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 
‘k may therefore be poor substitutes 














under which we yrow 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 exeretions 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, ey. 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 aurround- 


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 bacterin require nitrogen to be present in 
some form, and ~Wany require-to derive their carbon from 
carbohydrates. Mineral salts, especially sulphates, chlorides, and 
phosphates, and aleo 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 docs 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, ey. the cholera 
spirillum, wor gow 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 diphtheriw still 
more. In the case of spores the periods are much longer. 
Anthrax spores will survive drying for several years, but here 
again moisture cnables 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 properti 

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 aerohes is given. Ornet 
bacteria will only grow when no oxygen is present. ‘These exe 





























18 GENERAL MORPHOLOGY AND BIOLOGY 


called obligatory anacrobes, In atill other bacteria tho presence 
eee gen is w matter of indifference. is group: 





might ‘thooretically ye divided into those which are preferably 
awrobea, can be anaerobes, and those which aro Pacer 
anaerobes, but can be aerobes, As a matter of fact 
differences are manifested to a slight degree, but all such 
or are usually ped fiacultatine anaerobes, ie. pre 
ferabl ey aerobic at teat, ing withoutoxygon. Examples 
of oblig werobes are b. proteus vulgaris, b. subtilis; of 
obligatory anaerobes, bs, tetani, b. oedematie maligni, while the 
reat majority of pathogenic bacteria are facultative anaerobes. 
litert rd to anaerobes, hydrogen and nitrogen are indifferent 
any anaerobes, however, do not flourish well in an 
cencintiaes ‘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 lowe ita power of sporing, but it seems to have 
no effect on its vitality or on that of the b. typhoms, » With 
the bacillus pyocyaneus, however, it is suid to destroy life. 
Temperature.—For every species of bacterium there is a 
terpemture at which it grows best. ‘Thisis called the “ optimum 
temperature,” ‘There is also in ench case a maximum tempera 
ture above which growth does not take plice, 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 ax the 
average optimum, while for organisms normally inhabiting animal 
tissues 35° to 39° C is a fair average, The lowest limit of 
erdinary growth is from 12° to 14° C., and the upper is from 
42° to 14°C, In exceptional eases 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 boon 
isolated from dung, the intestinal tract, ote. ‘Theso have boon 
called thermophilic bacteria, It is to be noted that while growth 
does not take place below or above a cortain limit it hy 
no means follows that death takoa placo outside auch limita, 
‘Organisms can resist cooling below their 
huyond their maximum without being kill 
is meroly paralysed, Especially is this trae of the offect of colt 
on bacteria, ‘Tho results of different observers vary; but if we 
fake ns an example the cholera vibrio, Ki 
the minimum temperature of growth was 16° C,, a cultare 


















































CONDITIONS AFFECTING BACTERIAL MOTILITY 19 


if grown above theic 0 ‘optimum tery and some 
Ruceotelntrae wat of =. eather lu trspern 
me lose their mpacity of producing pigment, ¢g. spiritium 


een Light.—Of recent years much attention has beon 
to this factor in the life of bacteria, Direct sunlight ts 
to have a very inimical effect. It has been that 
an of dry wnthnix spores for one and a half hours to 
onlight Kille then, When they are moist, a much longer 
‘exposure is necewury, Typhoid bacilli are killed in about 
one and a half hours, Pay? similar results have been obtained 
with many other organisms Tm snch experiments the thickness 
‘of the medium surrounding the growth is an important point, 
Death takes place more reilly if the medium is scanty or if the 
ae are suspended in water, Any fallacy which might 
from the effect of the heat rays of the sun has been ex- 
duded, light plus heot is more fatal than light alone. 
Th direct sunlight it is chiefly the green, violet, and, it may be, 
the ultm-violet rays which are fatal, Diffnse daylight has also a 
bad effect upon bacteria, thongh it takes a much longer exposure 
to do werious harm, A powerful electric light is as fatal as sun- 
Hore, a4 with other factors, the results vary.very much 
the species under observation, and a distinction must be 
ears Between a mers cessation of growth and the condition of 
actual death, Some hicteria especially oceurring ou the dead 
hodies of fresh fish are phosphoruscent, ) 
Conditions affecting tho Movoments of Bacteria, —In some 
cakes differences are observed in the behaviour of motile bacteria, 
contemporaneous with changes in their life history. Thus, in 
the ease of bacilius subtilix, movement ceases when sporulation 
He about to take place. On the other band, in the bacillus of 
anthrax, movement continues while sporulation is 
Under ordinary circumstances motile hwcteria 
‘net to be constantly moving but oeensionally to rest 
p every case the movements become more active if the 
be misod. Most interest, however, attaches to the 
fact that booilli may be attracted to certain substances and 
by Others. Schenk, for instance, observed that motile 


leap toa warm point in @ way which S\\ vs 





20 ©=GENERAL MORPHOLOGY AND BIOLOGY 


positive Rare negative chemictaris,  DPfctfor investigated 
subject in many lowly organisms, including bacterium terme 
and spirillam anda. The method was to fll with th agent 
pillary tube, closed at one end, to introduce this into a 
drop of Bald’ contataing the bacteria ‘under a cover-glass, and 
to witch the effect through the microscope, The general result 
‘was to indicate that motile bacteria may be either attmeted or 
by the fluid in the tube. The effect of a given fluid 
differs in different organisms, and fluid chemiotactic for one 
orgunism may not acton another. Degree of concentration ix 
ices ‘but the nature of the fluid is more eo. Of inorganic 
lies salts of potassium are the most powerfully attracting 
bodies, and in comparing organic bodies the important factor 
is the molecular constitation. These observations have beon 
confirmed by AliCohen, 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 thesn observations have a 
‘Most important bearing on the action of bacteria, though wo do 
not yet know their true significance, Corresponding chemio- 
tuetic phenomena are shown also by certain animal cells, eg. 
Jencocytes, to which reference is made below, 
‘Tho Parts played by Bacteria in Nature. 
the chief effect of bacterial action in natu 
ile tio ple® Molecules o 
sulistances which form the bodies of plants and animals, or 
which aro excreted by them. In some cases we know some of 
the stages of disintegration, but in most casea we know anly 
ral principles and sometimes only results In the case of 
milk, for instance, we know thot lactic acid ia produced from 
the lactose by the nection of the bacillus weidi lactici and of 
other bacteria, and that from ret ammoninm carbonate 
in produced by the micrococcus ureae. ‘That the very comp 
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 


‘tako place in the passing of the organic 
f tod Many. ross wt ral ere a 
putrefactive are also bacterial in their origin. The souring 
to, the becoming rancid of butter, the 

jing of cream and of el ‘aro all due to bacteria, 
‘cortain comparatively small number of bacteria have been 
causal agents in some diseasn procesans 
mala, and plants ‘This means that the 
living bodies are, under certain circum 
stances, a suitable pabulum for the bacteria Involved. The 
of the action of thee bacteria ure analogous to those 


i 


pissé 
Hl 
bet 


are 
the diseato-producing effects of bacteria form the tasis of 


eed eas and and tly und 
mi ntly ler no 
ee ee ceo 


as They are normally engyged in breaking up 
ye vogotable matter. Others normally live on 





‘or in tho bodies of plants and animals and produce dissas, 
‘These are known as Tarssitie bacterie Sometinies an attompt 
“Sod to draw a hard™ani lind between the asprap/ytes 


parasites, and obligntory “ophy or ites are 
Bie itrmaegrnste\ delinesoa, Same herders, 
‘normally saprophytes can produce pathogenie effects 
oedematis maligni), and it is consistent with our 
that the best-known parasites may have been derived 
saprophytes. On the other hand, the fact that moot 
associated with disease process, and proved to be 
of the latter, can be grown in artificial media, shows 
time at kast such parasites can be saprophytic. As 
far such a xprophytic existence of diseas-producing 
as ‘occtrs in nature, we ure in many instances still ignorant, 
of Bacterial Action.The processes which 

undergo in being split up by bacteria depend, first, on 
‘nature of the bodies involved and, secondly, on the 
the bacteria which are acting, The destruction of 
bodies which is mostly involved in the wide and 
| precest of putrefaction can be undertaken by whole 
“Of different varictios of bacterin, ‘Tho action of tho 


He 


if 


r 






Bisuch substances is analogous to what takes pincer whem 
fare subjjected to andinary gastric and Wntesinmh Agena. | 









22. GENERAL MORPHOLOGY AND BIOLOGY 


In these circumstances, therefore, the production of albumoses, 
peptones, ete., similar to those of ordinary digestion, can be 
recognised in putrefying solutions, rae the process of destrac- 
tion always goes further, and till simpler substances, ey. indol, 
and, it may be, crystalline bodies Birt, ulkaloidal 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, 
9. some pathogenic forms, though not concerned in ondary 
putrofactive processes, have a similar digestivo eepeaity, 
carbohydrates are being split up, then various alcohols, Pili 
and acids are produced, During bacterial growth there ix 
not infrequently the abundant production of such gases as 
sulphuretted hydrogen, carbon dioxide, methane, ete. For an 
exact knowledge of the destructive capacities of any particular 
bacterium there must be an accurate chemical examination of its 
éffects whon 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 
hy bucteria, 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 congulate cascin, which 
split np trea 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 p 
within, or in the immediate proximity of, the bacteria, sometimes 
wherovor the soluble fermonts reach the organie substances. 
And in certain cases the ferments diffused ont 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 cortain 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 
ucteria, shortly after the process bas begun, are killed or 
lysed by chloroform, then only  peptonisation of the 
occurs, without the further splitting up and gaseous pro- 









































VARIABILITY AMONG BACTERIA 23 


duction being observed, Se 

by has been shown to occar 

in the case of the micrococens ureas, which from urea forms 

Ree ee aeeary satoe water to the Desk mibiemnle 

action has commenced, the bacteria are filtored 
2 a carbonate takes 

jt has been dissolved out into che 

the bacteria be extracted with 

absolute alcohol or ether, which of course destroy their vitality, 

ure of a ferment, whieh, when 


‘a sabstance is obtained af the nat 
urine, 


‘In cousidering the effects of bacteria in nature it must be recognised 
fore specea are capable of building up somplex autetancea out of 
jla chemical compounds. Examples of these are found in thi 

fhinh in the soil make nitrogen more available for plant mutris 

tion by converting aimonis tnto nitrites and nitrates. Winogradski, by 

tising inedia containing non-nitrogenous salts of maguesiuim, potassiuin, 

asd smmoniam, snd free of ongauio matter, has demonstrated the 

if forms which eonvert, by oxi mn, ammonia into nitrites, 

other forms which convert these nitrites into nitrates. Both can 
‘earbon from alkaline carbonates. Other bactaris 


les do not dorelop, 
ants are poor and stunted, Bactorin 
‘sn important part in tho enrichment and fertilisation of the 


‘The Ooccrrance of Variability among Bacteria —Tiie yumtion af the 
division of the group of bacteria into definite species has given rise to 
tuueh discunsior io 


ni animal morphologists, and at 
|. Som 


14, even thought that the 
diseavo,—at another 
Pratioal_mosnimatiy: that 
the other lower plants and siimals, 
defining the eoncopt of a species fs an 
the latter. Still, we can ay that among the 
hited (to use tho words of Dy Bary) ‘the eamne 
ied course of development within certain empirically 
rte of variation” whieh justifies, among Mgher forma of 
ipwoire to be recognised. What’ at firat'raisod doubts ax to the 
of species among the bacteria was the observation ip certain 
foot of nhac pvertatons Dy thie to meatt tons ce 
ij aosuin ifferent Limes different forms, ¢ 
f am bacillus, or a leptothrix. Undoubtedly, m 
this wae to have been otuerred con 
the medern techaique for the olisining of pure calturea, 
the present day there are cases where evilence ayyente Vo exis 


24 GENERAL MORPHOLOGY AND BIOLOGY 


of the occurrence of pleomorphism. This is especially the case with 
certain bacilli, and it may lead to such forms being classed among the 
higher bacteria. Pleomorphism is, howover, a rare condition, and with 
regard to the bacteria as a whole we may say that each variety tends to 
conform to a definite typo 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 organisin 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 olaboration 
of certain ferments or of certain pigments may be impaired. Also the 
characters of the growths on various media inay 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 trom 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 obse 
vation within twenty-four hours, 























CHAPTER IL 
METHODS OF CULTIVATION OF BACTERIA. 


Introductory.—In onder to stully the characters of any species 
‘of bacterium it ix necessary to have it growing apart from ta eTEY 
other a In the great majority of casee wherv bacteria 

occur in nature, this condition is not fulfilled, Only in the 
Blood anil tisnes In some diseases do particular species occur 
singly and alone. We usually have, therefore, to remove 


artificial food medium. When we have succeeded in separating 
it, and have got it to grow on a medium which suits it, we are 
maid to have obtained n pure ewlture. Tho recoguition of 
different species of bacteria depends, in fact, far more on the 
ehameters prosonted by pure cultures and their behaviour in 
different food media, than on microscopic examination. The 
Inter in most cases only enables us to refer a given bacterium 
to its class Again, in inquiring aa to the possible possession 
of tae pee roperties by a bactorinm, the obtaining of pure 
aeetataly omential, 
obtain pure cultures, then, is the first requisite of 
ical research Now, as bacteria aro practical 
wo must first of all havo means of destroying all 
extraneous organiama which may be present in the food media 
to be weed in the vessel which the food media are contained, 
and on all instruments which are to come in contact with our 
€altures Tho technique of this destructive process is called 
sterileation. We must therefore study the methods of sterilisation, 
ied ‘of bacteria in other than their natural surroundings 
farther the preparation of sterile artificial food media, 
ual when we have such media prepared we bave still to look at 
the tochnique of the separation of microorgamiems fram mixtures 
‘and the maintaining of pure cultures when the otter 
been obtained. Wo shall here find that different methods 

















26 METHODS OF CULTIVATION OF BACTERIA 


are necessary according as we are dealing with aorobes oranacrotes. 
Each of these methods will be considered in turn. 


Tax Micrnops ov Strercasarion, 


‘To exclude extraneous organisms, all food materials, 
vessels containing them, wires used in transferring bacteria from 
one culture medium to another, instruments used in making 
antopsick, ote., mitet be sterilised. ‘These objects belay 50 
diferent, various methods ure necessary, but underlying these 
methods is the general principle that all’ bacteria are destroyed 
by heat, ‘The temperature nocessary varies with different 
bacterin, and the vehicle of heat is also of great importance. 
The two vehicles employed are hot air and hot water or steam, 
‘The former ix usually referred to ax “dry heat,” the latter ns 
“moist heat.” Ax showing the different offects 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° ©, to effect death. 
Both forms of heat may be applicd at different temperatures — 
in the case of moist heat above 100° C., o 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, 
atv so constantly in use, A dull heat is used for eanteries, the 
points of forceps, und may be used for the incidental sterilisation 
of small glass objects (coverslip, slides, occasionally when 
necessary oven test-tubes), care of course being taken not to 
melt the glass, ‘The heat is obtained by an ordinary Bunsen 
burne 









A. (2) Storilisation by Dry Hoat in a Hot-Air Chambor, — 
‘The per (Fig. 2) cousisia of an outer aud inner case of 
sheet iron Tn the bottom of the outer ther @ 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 ehamber, half- 
way up which ite bulb should be situated. It is found os a 
matter of experisnos, that an exposure in such a chamber for 


STERILISATION BY MOIST HEAT 27 
toa temperature tate ©., is snflicient to kill all the 


‘which asnally 

a aetna 
boratory, though cireum- 
where thin 
insufficient. This 


Hi 


| 


Hi 
= 
i 


i 
tad 3 


z 
ig 
E 


the temperature falls, Sud- 

aatise te cooling is apt 
fo canan glass to crack. ‘The 
mothed is manifestly unsuitable Fin, 2.—Blot alr sterilisar, 





Ki. Steritieation by Moist Heat, 


B. (1) By Boiling.—The boiling of n liquid for five minutes 
is sufficient to kill ordinary germs if no 
spores be present, and this method is usefal 
for sterilising distilled or tap water which 
may be required in various manipalations, 
It is host to sterilise knives and instruments 
used in antopsios by boiling in water to 
Which o little sodium carbonate has been 
added to prevent rusting, Twenty minutes’ 
boiling will here be sufticiont, The boiling 
of any fluid at 100° C. for one and a half 
hours will ensure sterilisation ander almost 
any clreumstances, 

B. (2) By Steam at 100° C.. 
far tho moet useful 








potato steamer placed on a kitchen pot. 
Ihe apparatus ordinarily used is “ Koch's 
steam sterilisor” (Fig. 3). ‘This consists 
of a tall metal eylinder on legs, provided 
with » lid, and covered exterwily \y 





a 


28 METHODS OF CULTIVATION OF BACTERIA 


some bed 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 atmoxphore 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 chainber. With such a “Koch” in the laboratory 
a hot-water filter is not needed. As has becn 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, us with long boiling, gelatin tends to lose ite physical 
property of solidification, ‘The method adopted in this case 
is to weam for a quarter of an hour on each of three succeeding 
days. 


This is a modification of what is known as ‘Tyndall's intormittent 
sterilisation.” The fundamental principle of this method is that all 
bacteria in @ nou-spored form are killed by tho temperature of boiling 
water, while if im  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 romaining alive. During the twenty-four hours which 
intervene before the next heating, these spores, bing in a favourable 
medium, are likely to assume the non-spored form. ‘The next heating 
kills these. In caso 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 mediuin 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 


B. (3) Sterilisation by Steam at High Pressure. —This is 
the most rapid and effective means of sterilisation. It is effected 
in an autoclave (Fi ‘This is a gun-metal cylinder supported 
in a cylindrical sheet-iron case; its top is fastened down with 
serews 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 
allow the apparatus to cool well below 100° 
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 ont 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 xteam 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 iot 
the sterilisation of blood serum, which will coaguletell expoweA ou 











Fig, 4.— Autoclave, 


Safety-valve, 
1 Blow-of pipe. 
©. Gauge, 











30 METHODS OF CULTIVATION OF BACTERIA 


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. Theapparatusshown 
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 

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- 
|—Steriliver for blood Wards be performed at a higher 

serum, temperature. 








Tue Preparation or CottuRE Mepra, 


The gencral principle to be observed in the artificial culture 
of bacteria ix 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 
ble the juices of the body. The serum of the blood 
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, ete., constitute 
differences which are valuable in the identification of 
bacteria, ‘The most commonly used media have as their basis 
-y extract of meat, Most bacteria in growing in such an 

y a grey turbidit eat advance resulted 

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 thero- 
fore another ‘inous substance which does not 
melt below 98° C., was substivated. Ser =e Let cape 
extmet, gelatin, ond media, and the modifications 
these, constitute the hie materials in which bacteria are 
grown. 


Preparation of Meat Extract. 


The tlesh 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 dimdvantage for certain purposes of containing a larger 

ion of fermentable sugar. The 


FF 
E 
E 
i 
u 
5 


ly 
a shallow dish. Set aside in a cool 
twenty-four hours. Skim off 
Present, removing the last traces 
the surface of the fluid with 
paper. Placo a clean Linon 
the mouth of a large filter 
strain the fluid through it 
Pour the minced meat into 
the cloth, and gathering up the cdges 
of thes latter in the left hand, squoeze ric, «—Moat prose 
ont the Juice still held tack in the con- 
tained teat, Finish this oxpression by putting tho cloth and its 
fnto @ meat preas (Fig. 6), similar to that used by 
iin prepering extracts; thus squeeze out the last drops. 
Sees fluid contains the soluble albumins of 
ible talte, extractives, and colouring matter, 
hamoglobis. It is now boiled thoroughly for two hours, 
[process the alburina coagulable by heat are coagulated 
through a clean eloth, boil for another half hour, and 
through white Swedish filtcr paper (best, C. Schleicher u 
al h distilled water. 
resulting Muid ought to be quite transparent, of a yellowish 
any red tint, If there is any redness, the fluid 
led and filtered till this colour dimppears, ather 
tn the later stages it will become opalescent. A large 
éf the extract nay bo made at a time, and what wor 


Bab ee 
sy 





nfl 


i 


F 








Ht 
it 


Pa 





82 METHODS OF CULTIVATION OF BACTERIA 


immediately required is put into « lange 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 chiofly 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 ix of avid 
reaction. We have now to see how, by the addition of proteid 
and other matter, it may be transformed into proper eulture 
modiy 


ia. 
1, Bouillon Modia.— These consist of meat extract with the 
addition of certain substances to render them suitable for the 
of bacteria. 





(1) (a). Poptone Broth or Bonillon.—This bas tho com- 
position <— 

Meat extract J = 6 1000-05, 

Sodium chlor 6 4 y 5 grms. 


Peptone alimmin. . =... 10 


Boil till the ingredients are quite dissolved, and neutralise 
with o saturated solution of sodium hydrate, Add tho 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. Neutrulisation must be 
practised with great care, a4 under certain circumstances, 
depending on the relative proportions of the different phosphutes 
of sodium and potassium, what ie known as the amphoteric 
reaction is obtained, ie. red litmus is turned blue, and blac red, 
by the same solution. The sodium hydrate must be added till 
rod litmms is turned slightly but distinetly blue, and blue litmus 
ig not at all tinted red. After alkalinisation, allow the fluid to 
become cold, filter through Swedish filter paper into flasks, male 
up to original volume with distilled water, plug the flasks with 
cotton wool, and storilixe by mothods B (2) ov (3), (pp. 27, 29). 
This method of neutralisation is to be rec vended for all 
ordinary work. 















In this medium the place of the original albumin of the meat is 
taken by peptone, » soluble proteld not congulated by heat. Here it 
tay he remarked that the commereial poptoue albumin is not pure 
peptone, but a mixture of alburnoscs (vee footnote, p. 185) with variable 
Amount of pare peptone, ‘The addition of tho sodium chloride ix 
necessitated hy the fact that alkalinisation precipitates some of the 
phosphates and carbonates present. Experience haa shown that sodinm 
chloride can quite well bo substituted, The svason for the alkalinisation 
is thas ie found that most bactori: m & medium stighthe 
alkaline to litmos Some, ¢.9. tho cholera vibrio, will not grow at all 
on even «slightly acid medium. 


















STANDARDISING THR REACTION OF MEDIA 33 


ion of Reaction of Media. While the above 

fa ailing with tha reaction of ‘a medium is sufficient 
es hs been thonght advisable to have a more 
exact method for making media to be used in growing organisms, 
Pee tse cand rae for 
a hens Such a method should also be used in 

sens in reaction produced in a medium by the 
‘bacteria. vine Tt however, involves considerable dificalty, 

and sald wt te be undertaken by tho beginner, It entails the 
os sdb tla rege which may be used 

itienicing i original reaction of tho medium, and for 
tely it of a definite degree of alkalinity, Normal™ 
decinormal solutions of sodium hydrate and hydrochloric 


Pa gocereety Bolutions.—The first requisites hore are 
lution of acid and alkali, |The latter fe propared as followe 
‘of pore sodit dull redness 

platinum ves sarees clineed oe eeu tia us exsiecator 

just aver 54 of sodium carbonate should now be prosent. Any 
excess fs qt removed, und the rest being dissolved fa oue litre of 
distifled normal solution is obtained. A measured quantity is 


water, 
t's porcelain dish, ood a fow drops of a "5 per cont solution of 
Pawn yee fn gauteal methylated apicit fs adel to ct aaldicater, 
lial ‘the at 


eh 








apy Mt adhleralball (ahaa the daotsormal ealaion 

A montioned below) may be derived, 
As Byre has suggested, the reaction of a medium may be 
Stine hy the sign + or to indicate acid or 


atts & number to indicate the number of 

"normal acid or alkaline solution necessary 

paged @ litre of the medium neutral to phenol-phthaleine. 
eee gag “reaction = — 15,” will mean that the medium 

is requires 15 co. of normal HC] to make a litro 
4 AYnormal” solition of any aalt is preparnd by dissolving su “equivalent” 
Tal that salt (um litre of distilled water, If the molal of 
ionotaleat, ie. if it Re replaceable in a compound by one maton 

f feg. sodium), an equivalent in the molecular weight in 
pmmien. In the case of Nol, it would be 685 grammes (atomic weight of 
pel Cle 355). If the metal be bivalent, Le. reqairing two atoms of Ht 
it in & componnd (eg. calctum) an equivalent ix the 


m fu grammes divided by two. Thus kn the ome of Cals am 
E would be 55-5 grammes (atomic weight of Caw A0, oh Cy= Nis 


wal _ 





fA 





34 METHODS OF CULTIVATION OF BACTERIA 


neutral. Tt has been found that when a medium such as bouillon 
reacts neutral to litmus, ite ee to phenol-phthaleing, 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 avidently difficult to eay exactly what precise degroe of 
alkalinity is the optimum for bacterial growth. It is probably 
safe to say, however, that when a medium has been rendered 
neutral to phenolphthaleine by the addition of NaOH, the 
optimum degree is generally attained by the addition of from 
10 to 15 ee. of normal HCI per lit « the optimum reaction 
ie +10 to +19, In other rei the optinmm reaction for 
bacterial growth lies, as Fuller has pointed out, about midway 

between the neutral point indicated by phenol-phthaleine te 
the neatral point indicated by litmus, 

‘The only objection to the ue of phenol-phthaleing is that its 
ction is somewhat ritiated if free CO, be present. This can 
by completely obviated ws follows Before testing, any medium 
it is boiled in the porcelain dish into which titration takee place, 

ate soda solutions are hest stored in bottles such as thot shown 

4 on the air inlet u little bottle filled with soda 
tiki with tubes fitted as in the large one. The CO, 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 and put in 
a porcelain dish, add 25 ¢.c. distilled water, and 1 ¢.c. phenol- 
phthaleine solution. Run in decinormal soda till neutral point 
is reached, indicated by the finst trace of pink colour, the mixture 
being kept hot.! Repeat process thrice, and take the mean; 
this divided by 10 will give the amount (x) of normal soda 
required to neutralise 25 cc. of medium; then 40 « = amount 
necessary to neutralise a litre; and 40 «-10 = amount of 
normal soda necessary to give o 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 way find considerable difficulty in recognising the firmt 
tint of pink in the yellow bouillon. A good way of getting over thie ix to 
take two eumples of the medium, adding the indicator to one only ; then to 
rin the sox into thess from separate bnrettos ; for nach fow drops run into 
the medium containing the indicator the same amount 18 ran into the other, 
has the recognition of tho fimt permanent chxuge in tint will be at ouoe 
recogulsed Ly comparing the two lote of solution, 































GELATIN MEDIA 35 


ws cetera times normal which 
is out of a 1 ao. pipette divided into hi ths ; this 
to. Inrge extent, the error introduced by inersesing 

of the medium on the addition of the neutralising 


solut 
VO. Glucose Broth.—To the other constituents of 1 (a) 
added 1 or 2 per cent of grape sugar, ‘The steps in 
tf supe en pace aoa Heat rr ct 
10 oxygen can exist in a medium containing it, and 
pe ‘broth is used as a culture fluid for anaerobic 


a vew: (c}. Vega Broth.—The initial steps are the same as in 

1 (o) ba -jileration 6 to 8 per cant tof ghyerin (gras 125) 
is added, Uehie medium is es used for growing the 
tubsrele bacillus when the aolubl = s Mee ipgctet the 
latter arc required. 

2. Gelatin Media.—These aro simply the above broths, with 
gelatin added a3 a polatifying' body. 


2 (a) Peptone 
Meat extract = 1000 ec. 
Sodium chloride Se 5 gems, 
Feptone albumin. =. . . 10 y 
Gelttin 2. 100-150 


th a label" gelatin of Coignet ct Cie, Paris, is the best). 

fi cat into small pieces, and added with the other 
constituents to the extract ; they are 
‘then thorot melted ona mnd bath, 
or in the “ ” The fluid medium 


in 1 (a), and filtered through filter 
paper. As the medium must not be 
‘allowed to solidify «luring the process, 
it must be kept warm. ‘This is effected 
ty pting the fas and fone into a 
tall Koch's steviliser, in which case 
the funnel must be supported on a 
tripod or diaphmgm, os thore is great 
‘of the neck of the flask breaking 
if it bas to support the funnel and 
its contents The filtration may also F 
‘be carried out in a funnel with water- 
haces which is heated, as shown in Fig. 7. Whichever instrument 
ased, before filtoring shake up the melted mediom, ws We aye 











Hot-wnter funnel, 


_— — 





36 METHODS OF CULTIVATION OF BACTERIA 


while melting to bave settled into layers of different density. 
Sometimes what first comes through ix 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 
onght to be quite transparent. Tf, however, it is partially opaque, 
add the white of an ogg, shake up well, and boil thoroughly over 
the sand bath. The consequent coagulation of the allumin 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, Tho flask containing it is then plugged with cotton 
woo! and sterilised, best by method B (2), p. 27. Tf the 
antoclave 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, ax has been said, causes a gelatin medinm to 
Jose ite property of solidification, ‘The exact percentage of 
gelatin used in its preparation depends on the temperature at 
which growth ix to take place. Its firmness is its most valuable 
characteristic, and to maintain this in summer weather, 15 parte 
per 100 are necessary. A limit is placed on higher percentages 
hy the fuct 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 ane the same as 
2 (@), with the addition of 1 to 2 per cont of grape sugar. The 
mothod of preparstion is identical, This medium is used for 
growing arwerobic organisms at the ordinary temperatures, 

5, Media (French, ‘‘gélose ")—The disadvantage of 
gelatin ia that at the blood temperature (38° ©,), at which most 
pathogenic orgapisms grow best, it is liquid. To get « medium 
which will he solid at this temperature, agar is used as the 
utiffoning agent instead af gelatin, Unlike the latter, which is 
a proteid, agar isa carbohydrate. It is derived from the stems of 
various sea-weeds growing in the Chinese seas, popularly classed 
together as Ceylon Moxs,” For bacteriological purposes the dried 
stems of the seaweed may be used, but there is in the market @ 
purified product in the form of a powder; this is preferable, 

3 (a). “Ordinary” Agar.—This has the following composi- 
tion :— 



























Meat extract ‘ 1000 exes 
Sodium chloride 5 germs 
Poptone albumin . 10, 


Receidies 5 16 jy 


AGAR MEDIA 37 





from the second flask by a bent glass tube passing from just 
‘Deneath the cork to beneath the surface of the medium (K\ 


3 
ss 
: 
z 
E 
i 
a 
r 
a 
g 
e 
Zz 
= 
z 
: 


‘over the filter funnel to prevent condensation water 
roping off the roof of the steriliaor into the medium. If 
of turbidity may bo tolcmted, it is aufficient to 
a felt bag or jelly stmince, Flug the flask con- 
trate, and storilise either in autoclave for fifteen 
in Koch's steriliser for one and a half houra. v 
100° ©, on cooling solidifies about 39° C. 
Glycerin Agar.—To 3 (a) after filtration add 6 to 8 
pir cont of glycerin and steriliso as above, ‘This is used 
especially for growing the tubercle bacillus, 
$ (ch. Glucose Agar —Prepure us in 3 (a), but add 1 to 2 
red cent of grape angar along with agar. This medium is used 
the culture of anaerobic organisms at temperatures above the 
melting-point of gelatin. It is also a superior culture medium 
for nome avrobes, e.g. the b. diphthoriv. 
‘These bouillon, gelatin, and agar prepamtions constitute 
the mest frequently wed media, Growtha in bouillon do not 
show any characteristic appearances which facilitate 
tion, but such a medium is of great uso in investigating 
soluble toxic products of bacteria, The most characteristic 
of organisms take place on the gelatin media, 
Thesn have, however, tho disadvantage of not being available 
When growth is to take placo at any tamporature above 24” C, 
Por higher tempermtures agar must be ean 
ever, never so trinsparcnt, n 
saliping fing it always becomes slightly opaque. g 
upon it are never so characteristic as those on gelatin. It is, 


for instance, never liquefied, whereas some organisms, by their 
th, liquefy gelatin and others do not—a fact of prime 


iY 





i 





f 





et 
a 
Hi 





nus Media —To any of the above modi \trus Grew, 


—_— 





38 METHODS OF CULTIVATION OF BACTERIA 


tournesol) may be added to a ipa nie reaction during 
bacterial growth. The litmus is added, boforo sterilisation, ax 
a strong watery solution the Kubel-Tiemann solution; vide 
# 42) in sufficient quantity to give the medium a distinctly 
bluish tint, During the development of an acid reaction the 
colour changes toa pink and may subsoquontly 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, expecially in the examination of water. ‘The media found 
most suitable are agar or bonillon containing “5 per cent of 
glucose, to which °5 per cent of a one per cent watery solution 
of neutral red ig added, The use of these media and their 
probable value are described below {vide Typhoid Fover). 

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, casily grown on 
the ordinary media, ey. the gonococcus and the pneumococcus, 
Human blood or the blood of animaly may be used. “Sloped 
tubes " (vide p. 48) of agar wre omployed (glycerin agur is not so 
suitable) Purify a finger first with 1-1000 corrosive sublimate, 
dry, and then wash with absolute aleohol to rmmove the sublimate. 
Allow the aleohol to evaporate. Prick with a needle sterilised 
by heat, and, entching a drop of blood in the loop of a sterile 
platinum wire (vide p. 49), smear it on the surface of the agar. 

s excess of the blood runs dewn and Jeaves a film on the 
surface, Cover the tubes with india-rubber cape, and incubate 
them for ane or two days at 38° C. before use, to make certain 
that they are sterile, Agar poured out in a thin layer ina 
Potri 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° 
has been used for the cultivation of the bacillus of eoft sore. 

Serum agar ix 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 


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 -d per cent NaCl, is dissolved 
in distilled water by heating. ‘The fluid is then filtered, placed 
im tubes and sterilised. Tho reaction is usually distinotly 
alkaline, which condition is suitable for most purposes, For 








BLOOD SERUM 39 


special purposes the reaction may be standardised. In such « 
solution the cholera vibrio grows with remarkable rapidity. Tt 
is also mach used for testing tho formation of indol by a 
yarticular bacterium ; and by tho addition of ono of the sugars 
to it the fermentative powers of an orginism may be tested 
{p. 75). Litmus may be added to show any change in reaction. 


* Blood Seren, 


Koch introduced this medium, and it wed ns follows : 
Plog tho mouth of » tall cylindrical glass (say of 1000 ec. 
eaqucity) with cotton wool, and sterilise by steaming iv in a 

Koch's steriliser for one and a half honrs. Tuke 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, ote., to 
Cee fill the voasol with the blood snbsequontly shed. Carry 

lly back to the laboratory without shaking, and place for 
twenty-four hours in a cool place, preferably an ice chest, The 
eer sernm will separate from the clotted blood. If a centrifuge 
is available, # large yield of veram may be obtained by centri 
the freshly drawn blood. Tf coagulation has occurred, 

the clot must first be thoroughly broken up, With a sterile 10 
‘ne. pipette, transfor thie quantity of eerum to each of a series 
of tést-tubes which must previously have been sterilised by dry 
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 ix therefore kept for one hour at thie 
on eich of eight succemive days, It is always 

te incubate it for a day at 37° C. before use, to see that 

the result is successful. After sterilisstion it ix “inspissated,” 
vag hich process a clear solid medium is obtained.“ Inspissa 
* is probably an initial stage of coagulation, and is effccted 

the serum at 65°C, till it stiffens. ‘This temperature 

ig just below the coagulation point of the serum. The more 
‘slowly the operntion is performed the clearer will be the serum. 
‘The apparatus used for the purpose is one of the various forms 
of sernm steriliser (eg. Fig 8), generally a chamber with water- 
Gackot Beated with a Bunsen below. The temperature is con- 
‘trolled ly a gas rogulator, and such an apparatus can, by altering 
be used either for sterilisation or inspixsation, 


temperature, 
‘As is ovident, the preparation of this medium is tedions, wor Ws 

















(i 








with atrict aseptic precan- 
tiona, then sterilisation of 
the serum is unnecessary. 
To this ond the mouth of 


inserted in it 
which wo bent glass tubes 
pass, The outer ond 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 elaughtor-house the 
cap is removed and the 
tube is inserted into the 
blood-vessel as a cannula, 
‘Tho cylinder is thus onsily 
filled, Another method is 
to conduct the blood to 
the cylinder by means of 
a sterilised cannula and 
F1G, §.—Blood seram inepissutor. india-rubber tube, the 
former being inserted in 
the blood-vessel. In every case the scrum must bo incubated 
before use, to make sure that it is sterile, 

Ooagulated Blood Serum.—If fresh serum be placed in 
sterile tubes and be steamed in the sloped position for an hour 
it couguiates, and there is thus obtained « solid medium yery 
useful for the growth of the diphtheria bacillus for diagnostic 


ae psier's Blood Sorum.—Thie is the best medium for the 

















BLOOD SERUM 41 


b. diphtheria and may be uscd for other 
organisms. It bas the following composition, Three parts of 
calfs or lnmb’s blood serum are mixed with one part ordinary 
nentral bouillon made from veal with 1 per cent of grape 
sugar to it, ‘Though this is the original fornimla it can 
from ox or sheep serum and beef bouillon without its 

markedly impaired, Storiliso by mothod B (4) 


Ne 
Blood Serum (Lorrain Smith's Method) —To each 
the serum obtained as before, add 1 to Ini ee, of 
‘cent solution of sodium hydrate and shake gontly, Put 
of the mixture into each of a series of test-tubes, and 
ther on thelr sides, sterilise by method B (2). If the 
of storilisation be carried ont too quickly, bubbles of gas 
to form before the serum is solid, and theso interfere with 
fulness of the medium. Dr, Lorrain Smith informs us 


ie 


Fa 
rs 


‘organisms for which Koch's scrum is used, and especially for the 
growth of the b.diphtheria. Its great advantage ix that aseptic 

ons in obtaining blood from the animal are not necessary, 
and it is easily sterilised. 





1, Haman eerum 2 parts, bouillon 1 part. 

2. Pleuritic or ascitic serum 1 part, bouillon 2 parts, 
B. Asses’ or mules’ serum 2 parts, bouillon 1 part. 
4, Horse serum 2 parts, bouillon I part. 


Human serum can be obtained from the Wleod shed in 
vunosetion, the usual aseptic precautions being taken. In the 
east of these media, sterilisation is effected by method B (4), and 

are usext fluid. 

i Serum Water Media.—These are composed of one 

art of ox’s scrum and threo parts of distilled water with 1 
: fitmus; various sugurs in a pure condition are added in 
he proportion of 1 per cent. The develoyment of acid by 

is shown by the alteration of the eolowt amd Wy 


Aermentaion 
(ali 








42 METHODS OF CULTIVATION OF BACTERIA 


‘ion of the medium, Thea media do not coagulate at 
100° C. and thus can be sterilised in the steam steriliser, 
Haid ae been extensively used by American workers in 

iyi 


ing the fermentative properties of the b, dysenterim, 
b, coli, ote. 


Drigalaki and Conradi's Medium.—This ix one of the media used for 
the study of intestinal bacteria and especially for the isolation of the 





tzphoid group of organs (a) Taree of meat are treated with 
tive litres of water ovornight ; the fluid is separuted as usual, Loiled 
for an hour, filtered, and there are addod 90 grammes Witte’s peptons, 


2 grammes outron! 10 gramunes sodium chloride; the mixture Js thes 
boiled for gn hour, GO grammos finest agar ary added, anc \t ls placea tn 
the autoclave till melted (usually one hour) : it ix then rendered slight! 
alkaline to litmes, filtered, and boiled forhalfan hour, (é) 260 c.0. Kubel 
‘Tiomann litmus? solution is boiled for ten minutes, 90 ames mille 
lly pure) are added, and the mixture is boiled for fifteen 
(a) and (b) are thon mixed hot, well shaken, and, if necessary, 
‘the slightly alkaline reaction restored, ' There are thn added 4 ec. of 
#10 por cent sterile solution of warer-ftoe sodium hydrate and 20 oc, of 
eitrouhty) Drepated solution wade by dissolving gtarnme cerystal-violet. 
B, Hocobiat, im 100 c.c, hot aterile distilled water. is the finished 
modiam, and groat euro must be taken not to overhoat it or to hoat it too 
Jong, ax changyx in the Iastose may be originated. It is convenient to 
distribute the medium in 80 ce. fhisks. 

‘The principle of tho medium ix that while there is # food eupply vory 
favourable to the b. typhionus aud the b. ooli the antiveptic action of the 
srystal-violet ten¢ rit the growth of other bacteria likely to 
ocour in material which has boon subjected to intestinal contamination. 
In oxamining ficor a little i rubbed wp in from ton to twonty timen ite 
volume of storile normal salt solution ; in the easo of urine or water thn 
fluid ix centrifiigalised and the deposit or lower portion is used for the 
inoculation procedures. 

For oe the medium is distribated in Petri cayeules in » rather thicker 
Jayor thon is customary in an ordinary plate, The sheet of medium 
rons be transparent, but must not be leve than 2 mm. in thickness 
in fact, ought to be about 4mm. After being poured, the cajaules aro 
Iaft with the covers off for an hour or so, to allow the xnperfioial 
lnyers of the modinm to become sot hard. The effect of this is 
Unt during incubation no water of condensativn forms on the lid 
of the capsule, and thus the danger of this fluid dropping ou to the 
developing colonies is avoided. The antisoptio nature of tho orystal- 
violot is sufficient to prevent the growth of any acrial organisms falling 
































1 Nutrose is an alkaline preparation of casein. 

* The Litmus solution i made as fellows Solid commercial litmus ia 
Aigosted with pure epirit at 90°C. till on adding fresh alcohol the latter 
deoomes only of light violet colour. A saturated solution of the rosie tt 
‘thon sve in distilled water and Hitered. When this is diluted with a Iittle 
isto water it ix of a violet colour, which further dilution turox te a pare 
Due To sucha tue wlation very weak wulphuric noid (made by adding 
two drops of dilute sulphuric acid to 200 cc. water) is added til} the blue 
colour is turned tomwino-rod, Thon the saturated solution of the dye ia added. 
4 the blue colour returns 





BILE-SALT MEDIA 43 





to the air. The platee are usually 
ala made by bending three inches of & 
to the reat of the tod. This partis 
and smeared in all directions over the 
succemively without any interroning 

a poms sin exposed to the air after inovalation 
Hien toutieted Be Conty-lnw heer At Us eal 

h a period b. eoli colonios ar 2 to € min. in diameter, stained 
i wnsparont. Colonies of the h typhosne are 

they are Line or bluish-violet in colour, are 
have a single contour. Sometimes 
ners appear, and colonies of these 


stents ia luo colour. Theis xrowth ox howover, moro exuberant 
{hat ofthe tzid sli bing ofan heap the one 
out, 











jsenteriie, eto. 

“uy The 

‘Mock solution is the following :—Commorcial sodium taurceholate, O° 
tte’ peptone, 0 


i 

Fern tuquld mediuls there ts 
“1 per cont solution of neutral rol” and the sugar,—when 
is used 05 por otbor sugary 


solution in the 
af. Neutral red and 
“hea 

8 
are. 


q original medium was «per cont hilesalt lactose agar 
swith mo todicator, aud was used for the decection of intestinal bacteria 
in Sueh * medium is unfavourable to all the eonmoan spore 
found in water, and by incubating at 42° C. tubes, in 

Ghleh Mace te probably a iced lnfetion from sual « source, the growth 
Fauoat ether wator bacteria in inhibited. B, coli and b. typbesus, on 
hand, grow readily. With the former the surfaoe colonies aro 
Arreguisr, and tat, of opaque colour, and with w «mall spot of 
Fellow or orange in the centre, a the colony is surrounded by a hare; 
he ‘solonies are Ious-shaped, of orange colour, and are likewise 
mrgendel byahhare, With tho typhoid oncaninm st she end of forty-eight 
‘eolonies are small, round, raised, and seinistransparent, 


Philo the deep colonics are lens-shaped, have no surrounding 


eet = Didoep erliason with ackle and a yellow-rvd with 












a 





44 METHODS OF CULTIVATION OF BACTERIA 


Jae, The haze in the eave of bea is due tothe ready production of 
‘acid from the lactose causing a procipilate of the tauroc! ‘Ang othor 
organinm capable of producing aohl from lactose will give a simélur 
robotion, and the hiss can be rosdily cleared up by floating & drop of 
ammonia on the surface of the mediim. MacConkey also ased a 
slunilar object a 6 per cent glucose bile-xalt bouillow tinted with seutral 


Fitmnus ns in Drigalaki's ; tis 
With reference to MacCoakey's fluid media, organisms are divided into 
{2} shove which prodnes both ald and gua (2) thoes podulng oad eat 
those growing but not produtny sitter acid or gue; (4) thon 
incapable of growing. 18 soli belongs to the first gronp and b, typhoxtia 
to the and to these groupe also belong mint onlinaey organise 
rowing in feces, practically none of whieh aro found in the third and 
th claws, ‘Thus if any growth takes place on this modiua when 
inooulated with, my, wator, the probability is that the bwsteria have bean 
denied from foeees, bat of course their identification might presemt some 
difficulty. With the noutral-red solid :nedia the eo{onies of any organism 
giving ris to acid will bo of a beautiful ros rod eolow 

Potruschky's Litmus Whey.—The proparation of this medium, which 
is somewhat diflicult, is as follows:—Freat milk is slightly warmed, 
‘ond auificient vory dilute hydrochloric acid is added to eauve yroolpita- 
tion of the casoin, whioh ie now filerod off, Dilute sodium hydrate 
solution is added dp to, but not beyond, the point of neutralisation, and 
the finid steamed for one to two hours, by which procedure any cascin 
which lias been converted into acid albumin by ie hydrochlorie acid 
is precipitated. ‘This is filtered off, and « clear, colourless, porfuotly 
neutral fluid should result. Its chief constituent, of course, will be 
lnotove. ‘To this sullicient Kubel-Ticmann solution of litmus ts added, 
the medium ix pat into tubes and then sterilised, After growth has 
taken placa, the amount of asd formed van be estimated by dropping. 
Jn standardised soda solation til the tint of an uninoculated tube 
reached, 

Media for growing Trichophyta, Moulds, eto.—1. Beer Wort dgare 
Tube Dorr work un btalaable Gove the tronery on diluie elle hese 
fg. of 1100, Add 15 por cent of ‘powdered ugar ani heat in tha Kooh 
till it ie dissolved (usually about two houre are twceaary). Filter 
rapidly and fill into tutes. " Sterilise in the Koch for twenty minutes on 
three successive days, If the medium {8 heated too long it loses the 
capacity of rolidifying. 

D, Sabouraud's medium (motised), Take 40 grammes maltoos and 
10 grammes Witto's poptone and dissolve thevo in ono litre of water, then 
13 grammes of powdered agar. Heat in the Koch till the ager is 
jolved, filter and fil into tubes, aterilise in the autoclave for twanty. 
minntes at 12¢ a 

To use those for isolating, say, the Tinea tonsurans, piok out am 
{afooted hair, yaah in abvoluts alschol for a few socoude, then waa ti 
‘ilo water and atab the lisir into the aurface of the medium 
‘of places; inoubate at 24° C, Usually it is sufficient to 
stab the hair as itis picked from the skin into the wiedium, 






















































Potatoes aa Culture Material, 


(a) In Potato Jars,—The jar consiste of a round, shallow, 
glass vesec! 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 
r, moistened with the same, is lnid in its bottom. On this 
Pare placed four sterile watch 
glasses, ‘Two firm, healthy, small, 
round potatoes, ax free from eyes 
wo 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 jn 
the Koch's steriliser for thirty 
misutes or longer, or in the auto Fre, 9,—Potato jar, 
clave fora quarterof an hour. When 
cold, each is grasped between tho left thumb and forefinger 
(whieh have been sterilised with sub- 
Timate) and eut through the middle with 
a sterile ki It is best to Lave the 
cover of the jar mised by an assistant, 
and to perform the eutting beneath it, 
Fu.t0—Cylinderof potato ach half is put in one of tho wateh 
‘aa auliqoay.” glasses, the cut surfaces, 
which ‘are then ready for 
fnoenlation with a bacterial growth, being upper- 
most. Smaller jars, cach of which holds half of « 
aecdeld ‘also used in the same way and are very 








(b) By Slices in Tubes,—This method, intro- 
duced by Ehrlich, is the best moans of utilising 
potatoes asa medinm, A large, long potato is well 
washed and scrubbed, and peeled with a clean kaife, 

Mise then, bored. from ito fateror with aa 
‘corer or a lurge cork borer, and is cut obliquely, 

asin Fig. 10, Two wedges are thus obtained, each 
2 which is placed bread und downward in a test-tabo 
form (seo Fig, 11, In the wide part at 

ee tom of this tube ix placed u piece of cotton 
wool, which catches any condensation water which 
may form. The wedge rests on the constriction Pr. 11. 








above this bulbous portion. ‘The tubes, washed, [BMrtch's 
tind with cotton wool in the bottom and in the thy" Niece of 


fre Atcrilined before the slices of potato are potato, 
introduced. After the latter are inserted, the tubes 
fire Atorilised in the Koch steam steriliser for one hour, or in 
the autcelave for fifteen minutes, at 115°C. An ordinary texte 








46 METHODS OF CULTIVATION OF BACTERIA 


tube may be used with « piece of sterile absorbent wool in its 
Soe ESL fas Sy Peaks Per, 

lyeerin potato, suitable for growth tubercle 
bacillus, may ala aie covering the elices in the tubes 
with 6 per cent solution of glycerin in water and steaming for 
halfan hour. The fuid ix then poured off and the sterilisation 
continued for another half hour. 

Potatoes ought not to be prepared long before being used, wx 
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 ucenrately extimated 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 ncidity 
or alkalinity is obtained by adding the necessary quantity of 
HCI 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 bo incubated at 37° C, for a night, 
to moke sure that their sterilisation has been successful, 

Elmer's Mecium.—This is one of the medla introduced in the stud 
of the comparative reactions of the typhoid bacillus amd the B, coll. 
‘The preparation is na follows: 500 grammes potato up ina 
ito of water, allowed to stand over night, then strained, and added to 
fan. equal quantity of ordi t peptone golatin which has not 
eon neutral Normal sodium hydrate solution is added tll the 
reaction fs feobly acid tolitmus, the whole boiled together, Gltered, and 
sterilised. Just bofore use potassium iodide is aided 90 as to constitute 
‘one por cent of the medium, Moore has used a similaragar preparation, 
Hore 500 grammos potato are seraped up in on liteo of water, allowed to 
stand for three hours, strained, nnd put wide over night ‘The clear 
fluid ix poured aif, made ap to one litre, rendered slightly alkaline, 20 
grammos agar are udded, and the whole is treated as in making ordinat 

1 is letrfbuted in taststubes—10 c-0. to exch—eual 
6, of a goluti f 10 grammes: 























Milk ax a Culture Medium. 


‘This is a convenient medium for observing the effecta of 
bacterial growth in changing the reaction, in coagulating the 
soluble albumin, and in fermenting the lactose. It ix prepared 
as follows: fresh milk ix taken, preferably after having had the 
cream “ separated” by contrifugalimtion, as is practised in the 
best dairies, and ix steamed for fifteen minutes in the Koch, it 
ix 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 

ieee eno capes Se ae eee 

great accuracy: legree of reaction 

ta otal ye a we Te is then placed in 
sterilised by methods B (2) or B (3). 


Bread Paste, 


This is useful for growing torule, moulds, ete. Some 
peares into slices, and then dried in i 

it is so dry that it can be pounded to a fine powder in 
or mbbed down with the fingers and passed throngh a sieve. 
Some 100 cc. finsks 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, tho flasks are atcrilised in the 
Koch's steriliser by method B (2), The reaction is slightly acid, 





Tae Use or rae Covrunk Mepis. 


oa ae bacteria is usually carricd on in test-tubes 
in Thean ought to be very thoroughly 
ve ore tones, and thoir mouths plugged with plain 
cotton wool. They are then sterilised for one hour at 170" C. 
Tf the tates 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 medinm thus 
protected will remain sterile for yeors, Whenever a protectin 
plog is removed for even a short time, the sterility of the 
contents may be endangered. It is well to place the bouillon, 
‘agar media in the test-tubes directly after filtration. 

‘modia can then be sterilieed in the test-tubes, 

Tn filling tubes, care must be taken to run tho liquid down 
the centro, 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 
conixet, otherwise the latter will subsequently stick to 
the and its removal will be difflenlt. Tn the casn of 

media, tost-tubes are filled about one-third full. With 

‘solid media the amount varies, Tn the caso of gelatin 
media, tubea filled one-third fall and allowed to solidify 
While standing upright, aro those commonly uscd. With 


48 METHODS OF CULTIVATION OF BACTERIA 


organisms needing an abundant supply of oxygen the best 
growth takes place on the wurface of the medium, and for 
practical purposes the surface ought thns to be as lange as 
possible, ‘To this end “sloped” agar and gelatin tubes are 
hued. 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 





Fro. 1, Tubes of media, 


nary upright taba. Sloped tube, 
jeep tube for cultures of annervbes. 








Pro. 12.—Apparatue which 
may bo used for fling tuts. 
‘The apparatus explains Iteelf. 
‘Tho {ulla-ruller stopper with 
its tubes ought to be sterilised 
Defore use, 
to 4 inches up, giring 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 whieh blood 
serum is Inspisated, Tubes, especially thoee of the loss 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 beon inoculated with a bacterium, and on which 

wh has taken place, is called a “culture.” A “pure culture” 
ix one in which only one organism is present. ‘The methods of 

















USE OF CULTURE MEDIA 49 


obtaining cultures will presently be deserihed, When a 
frosh tobe of media ix ingulated from an alady exiting 
culture, the resulting growth issaid to be a “sub-culture” of ist 
first. All manipulations involving the transference of small 
aps ‘of growth either from one medium to another, as in tho 
ingeulation of tubes, or, ax will be soon later, to cover-glaases for 
microscopic examination, are effected by pivces of platinum wire 
(Nos, 24 or 2F Birmingham wim gange—the former being the 
thickor) fixed in gliss rods8 inchoa long.) Evory worker should 
petite wach wires, Two are 2) inches long, one of these 
ne we a), and the other having a loop turned 
‘The latter is referred to as the platinum 
or ati “oyolot,” and is used for many purposes. 


Fro. 14.—Platinnm wirns in glave handles. 


1% Straight ote for onary pune wowalabons, Pit 
amg newts for toreniatleg * sop ae 


“Taking a Joopfal” is a phrase constantly usod. ‘The third wire 
(Pig L, 14, ) ought to be 43 inches long and straight. It in used 
making anaerobic culties. Tt is also very useful to have 
Athanda platinum-iridium spud, ‘This consists of x pioce of 
platinarm iridium about 1} inches long, 2 mm, broad, and of 
sufficient thickness to give it a firm consistence ; ita distal end is 
‘expanded into a dlaxnond shape, and its proximal Is screwed 
into an aluminium rod, It is very uscfal for making sorapings 
froan organs aud for disintegrating felted bacterial cultures ; in 
such timnipulations the ordinary platinum wire is awkward to 
work with az it bends so easily. Cultures on a solid medium 
are referred to (1) as “yruncture” or “stab” caltures 
Stichkultur), or (2)as “stroke" or “slant” cultures ( 
‘secording as they are made (1) on tubes solidified in tho upright 

oroinid ‘or (2) on sloped tubes. 
+ Altaniniatn reds are made whieh are ver 
Beife, the plationm wiro ls insert 

on it In & view. 




















event. The end Is split 
wadl fined by plnshing the 





‘4 









50 METHODS OF CULTIVATION OF BACTERIA 


To inoculate, sy, ove ordinary upright gelatin tube from 
Apothog the tee Subse ore held in ba, Lxvertodpoalion, bobween 
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 
eure they are not 
ing to the glasx The 
ehort, straight platinum 
wire is then heated to 
redness from point to 
N insortion, and 2 to 3 
a inches of the glass rod 
- are also passed two or 
three times through the 
Bunsen flame. Itisheld 
between the right fore 
and middle fingers, with 
Hes mens projecting 
P10, 15 —Anothor method of inooulat backwards, 1.6, Sway 
es trom the right palin 
Remove plug from eul- 
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, roplace plug. In tho 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 thenimmediatelysterilised. 
A variation in dotail of thia [~~ 
method ia to hold the plug } 4 | 
of the tabe next the thumb “~~ 
betweon the fore and middlo 
fingers, and the plag of tho "4. 16.—Rack for platinum needion 
other between the middle and 
ring fingers, then to make the inoculation (Fig. 15). 
contain a liquid medium, it must be held ina sl position 
between the same fingers, as above, For a strc Jtare the 
platinum loop is used, and a little of the culture ix smeared in 
Dlinealong the surface of the medium from below upwards, In 
inoculating tubes, it is always well, on removing the plugs, to 











Tf a tube 





SEPARATION OF BACTERIA 51 


make sure that no strands of cotton fibre are adhering to the inalde 

of the necks, Ax thew might be touched with the charged needle 

and the thus be contaminated, they must be removed by heat- 

lating needle red-hot and scorching them aif with it. 

‘the platinom wires are not in use they may be laid in « 

mck made by bending up the Parte Secale 

16. To prevent contamination of cultures by bacteria 

the plugs while these are exposed to the air during 

i manipulations, some bacteriologists singe the plugs 

in the flame hefore 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. 





‘Tue Mernops or Tue Seraration or Axnonic Onoastams, 
Poare Cucrones. 


ple underlying the methods of 6 
is as Miura the bacteria in one of the selis Sa 
by heat and the dilution of the mixture eo that the 
re by the individual bacteria—called colonics— 
be suitably apart. In order to render the colonies easily 
accessible, the medium is made to solidify in as thin a layer as 
pcs pl Rata being poured out on glass plates—honce the torm 
As the optimum temperature varies with different bacteria, 
it ig necewmry to uso both gelatin and agar media. Many 
pathogenic organisms, ¢.g. pneumococcus, b. diphtheris, etc, 
‘too slowly on gelatin to allow its ready use, On the other 
many organisms, ¢g. some occurring in water, do not 
on agar incubated at 37° ©, 
ae by Gelatin Media. —As the nakedaye aul micro 
pearances of colonies are often very characteristic, 
Hata alture, borides veo in separ 
are often taken advantage of 
description of individual 
The plate -cultare 
method can also be used to test 
Eee tube culture is or is not 
‘The suspected colture is 
Fitea {three plates 1 pre. 
Fare, SP ber Géecizel). It via a—Peete capuala, 
egal are the same, then (Cover shown partially mised.) 


a held to be pure. 
cong plates of glasa 4 inches by 3 inches are wow, 


La _— | 











52 METHODS OF CULTIVATION OF BACTERIA 





‘or, what are more convenient, circular gliss cella with similar 

corer ‘The Jatter are known as Petrie dishes or 

(Fig. 17) They are usually 3 inches in diameter aud 

fan inch deep. The advantage of these is that they do aut 

require to be kept level by a special apparatus while the medium 

ix solidifying, and can be readily handlod 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 sheetiron 
boxes made for the purpose sec (Fig, 18), 

1, Glass Capauler.—While in certain circumstances, as when 
the number of colonics has to be counted, it is beat to use plates 
‘of glass ; in tho usual laboratory routine Petri’s capsules aro to 
be preferred for the above reasons, 

‘The contents of three golatin tubes, marked a, J, 6) are 
Tiquefied by placing in a beaker of water at any temporature 
between 25° OG. and 38° C. Tnoculate a with the bacterial 
mixture, The amount of the latter to be taken varies, and can 
ovly bo regulated by experience, If the microscope shows 
enormous numbers of differont kinds of bacteria prosont, just as 
much as adheres to the point of a straight platinum needle is 
sufficient. If the number of bucilli ix «mall, one to three loops 
of the mixture may be transferred to the medium. Shake @ 
well, but not so as to cause many fine airbubbles to form, 
‘Trunsfer two loops of gelatin froma to b. Shuke Gund transfer 
five loops to «The plugs of the tubes are in exch ease replaced 
and the tubes returned to the beaker. The contents of the 
three tubes ure then poured out into three capsules. In doing 
0 the plug of each tube is removed and the month 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 ary labelled and 
sot aside till yrowth takes place, 

For accurate -work it will be found convenient to carry out 
the dilutions in definite proportions. ‘The following is the pro- 
eodury which wo have found very serviceable, Tn a number of 
small sterile test-tubca ‘05 cc. sterile water is put, To the first 
tube we add ‘05 ce. of the bir 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 cc. of fluid is transferred from the first tube to the second. 
By a similar procedure ‘05 ce. is transferred from the second to 















4 Yor tarking gluse wesnls it ix convenient to use the red, blue, or yellow 
tics made for the purposs by Faber. 


KOCH'S METHOD OF PLATE CULTURES 53 


third and #0 on. Thero ia thus effected a twenty-fold 
in ewch successive tube. After these steps have been 
amount, say, ‘00 oc. ia transferred from 

pais ‘boing aftor- 
growth occurs, 
mbes paraty will vary according to the 
bacteria in the original mixture, but usually four or 
sufficient, It is quite evident that this method not 
Vs to sepurate bucteria, but if necessary gives us a 
exactly the number in the mixture, 
colonies: us minute rounded points, whitish or 


aad 
BE 
it 
ee 
aa 
Ue 





ue 


of the gelatin, otc., the colonics can be classified into eee 
Further aid in the grouping of the varieties in obtained by 
making film preparations and examining them microscopically, 
Gelatin or agar tubes may then be inoculated from a colony of 
each variuty, and the growths obtained are then examined both 
as to their purity and as to their special characters, with @ view 
to their identification (p. 115). 


2, Glass Plates (Koch),—When plates of ginst aro to bo used, an 

‘on which they may be ear bare Jevol while the medium ts solids. 

a An spparatin dovised by Koch fx 

Figs 18, 1h. This comsiata ofa circuler plate of glass (with the 

d, the lower polished) on which the plate used for 

“ine Plpak nay median Ane The = protected from the air 

solidification by a bell jar. ylate and bell jar rowt 

aga Bein fof a-effeular gins trough, which tw filial quita fall with 

ice and water, to fuollitate the lowering of the temperature 

seiner iiplaned beneath the ball jar 'Tho plam tzough bests oa 

eerks on the bottom of a large circular trough, which catches any water 

be spilled, ‘This trough in turn reste on a wooden triangle 

vis het at eh corner, the aaigat of whioh can be sdjusted, and 
which thus constitutes the levellin, 














ain A spirit level i placed 





where th to go, and feral ‘of the ground glass plate thus 
aware. is abo prop a “damp chamber, in whieh the 
Plates are to bo stored after being made. This consists of a circular 






Tah with» similar cover, “It is storilisod by boing washod out 
jad inside with perchloride of mercury 1-1000, and a circle of filt 


‘Moiatened with the eamo ix laid on its bottom. Glass bench: 
lates may be laidl are similarly porifiod. 

(separate organisms by this method three tubes, a, 8, ¢, are Inocu- 
ie alo Petr capsules (p. The bands’ b tern 


sy 
fide of moroury 1-1000 and dried, the plate box is 
ae & plate lifted Ly its opposite odges and teanaterad to Yom 

















a 





54 METHODS OF CULTIVATION OF BACTERIA 


Jovelled ground glass (ax in Figs. 1, 19), ‘Tho bell jar of the Jeveller 
being now lifted s little, the gelatin in tube a is poured out on the 
surfice of the sterile plate, aud while still Quid, is spread by stroking 





Fio, 18.—Koch's levelling apparatus for swe in preparing plates 
ands shown in first position for transferring sterile plate from {row 
box to beneath bell jar, where it subsequently hea the medium poured 
owt upon it. 





After the medium solidisies, the plate 
miber as rapidly as possible, so os to voi 





with the rim of the tul 
transforred lo the moist © 





is shown in second 
round glass «untae 





1a. 19.—Koch's levelling apparutus 1 
position jtist aa the plate in lowered on to th 





iy executing the transference of the plate from the box in this way, 
the surfaco which was andermost tn the latter is upperront iti the 
Tevoller, aud thus never meots a current of air which might con: 


taminate (t. 


In doing this, it is advisable to have ah 


atmospheric contamination, 
Tubes > and ¢ aro similarly treated, 


assistant to raise tho glass covers. 


SEPARATION BY AGAR MEDIA 55 


ofa, The chamber 
ihbshed ant ia Sa ae fo a ite clets open ot 


Rorapea ther provedure ie of the ame wature es with 


caine rare the bacterial mixture as 
in moking cultures, but inatend of being 
poured a is rolled in a nearly horizontal 





a constriction a short distance below the plug of 
cotton wool (Fig. 20), ‘The great disdvantage of 
‘the mothod 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 
Uncle era ‘of the two media. Ayar, 

said, melts at 98" C., and becomes again 
mat f little under 40° C. As itis dangerous Fyn, 20, 
fo expose organisms to a temperature much Eamnnrch’s tabe 
ubore 42° C., it is necessary in preparing tubes for roll culture 
to be used in plato cultures to firet 
agar, by boiling m a vessel of water for a few minutes, 
© 
fore 





cool them to about 42° C. before inoculating, ‘The 
must be rapidly carried out, us the margin of 
solidification occurs, is narrow; otherwise the 
details are the same as for gelatin, Esmarch’s tubes are not 
for use here, as the agar docs not adhere well to the 
Tf to the agar 2 per cont of a strong watery solution of 
Ma gum arabic i added, Esmarch's tubes may, however, be 


Sh Sarin ly Stroking Mixture om Surface of Agar 


ure, instead of being mixod in the 
medium, ee spread ont on its surface, The method may be used 
‘beth when the the bacteria to be separated are ina fluid, and when 


‘contained in a fairly eolid tisuc or substance, auch 48 & Wars, 











eile 
56 METHODS OF CULTIVATION OF BACTERIA 


pe Sie In the case of a tissue, for example, 
small portion entangled in the loop of a platinum needle is 
feo in successive parallel longitudinal strokes on sloped 
same aspect being brought in contct with the agar in 
cline three strokes may be made on each tube, and 
three tubes are usually soflicient. In this process the o1 
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 lee numerous than in the earlier, and 
metres fs apart to enable parts of them to be picked aff 
without the needle touching nny 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 provionly 
sterilised (nice Diphtheria). Iu the case of liquids, the loop is 
charged and similarly stroked, Tubes thus inoculated must be 
pe in the incubator in the upright position and must be 
dled carefully 40 that the condensation water, which always 
is present in incubated agar tubes, may not run over the surface, 
Agar, poured ont in a Petri’s capsule and allowod to atand till 
firm, may be used instead of successive tubes. Hore a sufticiont 
number of strokes can be sade in one capsule. Sloped blood- 
sernm tubes may be used instead of agar, ‘The method is rapid 
and eagy, and gives good results, 

Separation of Pathogonic Bacteria by Inoculation of 
Animals,—It is found difficult and often impossible to sepwrate 
by ordinary y plate mothods certain pathogenic organisms, seh 
‘as b, tuberculosis, b, mallel, and the pnoumococeus, when such 
occur in conjunction with other Lacteria. These grow best on 
Special media, and the first two (especially the tabercls bacillns) 
grow so slowly that the other organisms present outgrow them, 
cover the whole plates, and make separation impossible. The 
method adopted in such cases ix to inoculate an aniwal with 
the mixture of bacilli, wait until the particular disease develops, 
kill the animal, and with all aseptic precautions (vide p. 128) 
inoculate tubes of suitable media from charncteristic lesions 
situated away from the seat of inoculation, eg. from spleen in 
‘the cave of b, tuberculosis, spleen or liver in the case of b, 
mallei, and heart blood in the case of pneumococeus. 

Separation by killing Non-spored Forms by Heat.—This ix 
fa 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 
‘Fogetutive forms of this and other bacteria, then if the mixture 








' ‘SEPARATION OF ANAEROBES oT 


‘This nu be casily tested in the case of 


i 


‘Tax Peexctrnes oF THe COLTORE or ANAEROBIC 
OnGantsms, 


All ordinary media, after preparation, may contain traces of 
free oxygen, and will absorb more from the air on standing. @ 
For naa aromth of nurobes this exygen may be expelled by 

ing of an inert gas, such a& hydrogen, through 
the medium (lave Rah ie assiap} hin ea esbank eat 
be kept in an atmosphere of the same gas, while growth is going 
on, Media for anaerobes may be kept in contact with the 
air, if they contain a reducing agent which docs not interfere 
with bacterial growth, Such an agent takes up any oxygen 
which may ulready be in the modium, and prevents further 
absorption. The reducing body used is generally glucose, though 
formate of sodiwm may be similarly employed, -'The preparation 
of such media has already been described (pp. 36, 3), In this 
caso the mectinm ought to be of considerable thickness, 


The Supply of Hydrogen for Anaerobic Cultures 
in a large Kipp’s apparatus from pure salyhuric 
is through three vash-bottles, ax in Tn the first ix 
Placed a solution of lead acetate (2 in 10 of water) to remove auy traces 
of ited hydrogen. In tho second is placed a 1 in 10 solution of 
iiver nitrate to remove any arsenictted hydrogen which may be present 
4€ the wine ix not quite pure. In the third ls a 10 per cont slation 
of lie acid in caustic potash solution (1; 10) to remove any 
Hates of exrgon. The tubo leuding. from the last bottle ta the rome) 

medium ought to be sterilised by pasaing through » 
Bunsen flame, and should have s small plag of cotton wool tn it to filter 
the hydrogen germ-frov. 


Separation of Anaerobic Organisma.—(«) Ay Holl-tubea— 

A Tj inch test-tubo bas a» much gelatin put inte it as would be 
im the Esmarch roll-tube method. Tt is corked with an 
stopper having two tubes passing through it, as in 

Fig. 22. The ends of the tubes are partly drawn out as shown, 






pure zine. It 








ral 





58 METHODS OF CULTIVATION OF BACTERIA 


and covered with plugs of cotton wool. Three such test-tnbes: 
tare propared, and they'are storilised in the steam storiliser (p. 27). 
Aftor sterilisation the gelatin is melted and one tube inoculated 
with the mixture containing the anaerobes; the second is inocu- 
Jated from the first, and the third from tho second, as in making 
ordinary golatin plates, After inoculation the golatin is kept 
liquid by the lower ends of the tubes being placed in water at 
lout 30° C., and irogen is passed in throogh tube = for 
hice minutes, ‘The gas-eupply tubes are then poe 
off at x and F, and each test-tube is rolled as in Esrnat rch 
method till the gelatin solidifies aa a thin layer on the internal 

















a o . @ 
Fra, 21.—Appaeatus for supplying hydrogen for annerobie cultures, 
- Kiny's spyaratas for manoachure of hydrouyn. |. Wash bottle containing 
140 cof Toad agntate, e- Wash-tottle containing 1-10 solution of sliver 
hitintes sk Wasli-botie eontalalug 1-10 solution of pyteqallle ais (yf 0 


aro Intentionally drawn to layger seals thin a to abow detail.) 


surface, A little hard paraffin may be ran betweon the rim of the 
test tube and the stopper, and round the perforations for the gas- 
supply tubes, to ensure that the ican 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 mothod is 
admirably suited for all anwerobes which grow at the ordinary 
temperature. 

(8) Bulloch's Apparatus for Anaerobic Culture. ‘Thie can 
be recommended for plating out mixtures containing anaerobes, 
and for obtaining growths (especii 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 ungucntum resinas, In 














ae u 
He 


serie 


acid 
Culture 


rt 
fe 
28 


ik 


dt 


if 


by passing 





& 


hase-plate a shallow 
less diameter than that 
ving a little heap 
from two to four grammes of dry 
in it towands one 


bell jar, an 


al 
the edges of the dish, 
éning culture tnbes ean 
in it, ‘The bell 
tion so that the longer glass 
ix situated over that part of the “"sine adapted for culture 
‘of the challow dish farthest away Containing anaerobes, 
the pyrogallic acid, and the bottom 
are lated. The air in the bell jar is now expelled 
current of hydrogen through the short glaas-tobe, 


in posit 


CULTURE OF ANAEROBES 59 


the bell jar aro two apertures furnished with 
and throught each of the latter 0 glass tube 


One tube, bont ly just after 


stopper, extends 


apparatus 


ia thon 





Fig. 22.—Ksmarch’s roll- 


‘and both stoppers are closed. A 
partial vacuum is then effected in 
the jar by connecting ap the short 
tubé with an airpump, opening 
the tap, and giving a few strokes 
of the latter. A solution of 109 
grms. solid canstic potash dinsolved 
in 145 e.c. water is made, and 
into the vessel containing it a 
rubber tube connected with the 
Jong glass tube is made to dip, 
and the stopper of the latter 
being opened, the fluid is forced 
into tho chamber and spreads over 
the bottom of the shallow dish ; 
potassiam pyrogallate is thus 


YASS Pallets aypersive gor fred, which alworba any’ free 


imunerobic plate cultures. 


oxygen still present, Bofore the 
whole of tho fluid is foreed in, 


the rabber tube is placed ina little boiled water, and this, passing 
throngh the glass tubes, washes out the potash and prgwonta 





60 METHODS OF CULTIVATION OF BACTERIA 


erosion of the glass. ‘The whole apparatus may be placed in the 
ineubator 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 ineubating for 24 or 48 hours, to apply an 
anacrobie 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 boon 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 muat be filled toa depth of 4 
inches, They are storilised as usual and are called “deep” 
tubes. The Tong straight platinum wire ia 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, bat in the lower part of the needle track growth 
occurs. From such “deep” cultures growths may be maintained 
indotinitely by successive sub-cultares in similar tubes, Even 
ordinary ae and agar can be used in the same way if the 
medinm is heated to boiling-point before use to expel any 
absorbed oxygen. 

Carroll Wethet 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 tabea. 
‘hore 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 Ustube are smeared with vascline and a rubber tube 
slipped over each ; the end of the culture tube being similarly 
tronted, 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. Tn thedry tube one or two grammes of pyrogallic 
faciil are placed and the powder is packed down with a layer of 
filter paper. ‘Ten or twenty cubic centimetres of a ten per cont_ 
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 
aodium pyrogallate and the conditions for anocrobic growth 
are fulfilled, 

‘Cultures of Anaerobes in Liquid Media. —It is nocessary to 
employ such in order to obtain the toxic products of the growth 


















CULTURE OF ANAEROBES IN LIQUID MEDIA 61 


of anacrobis Glucose broth ia most convenient. Tt is placed 
either (1) ino conical flask with a Jaterul opening and a perfor: 
ated indiwrubber stopper, through which a bent gluss tube passes, 
ns in. Hig 24, 24, a, by which hydrogen may be delivered, or (2) in 
a conical fask with a rubber stopper furnished with two 

as in Fig. 24, 4, through a tube in one of which hydrogen is 
delivered, while through the tubs. in the other the gas escapes. 
Tho inner end of the gas dolivery tube must in cithor 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 must of coure be above the surface of the liquid, 
Tho single tube in the one caso and the two tubes in the other 





Pra, 24, 


He Hlak for anaerobem In Ngult nia Lateral nore and ecpper fitted for 
epregen supply. f. A stopper arranpod for a flask without lateral noszis. 


ought tobe eyertaly drawn out ina flame to facilitate subsequent 
eomplete sealing. ‘The ends of tho tubes through which the gas 
is to pass are proviously protocted by pieces of cotton wool ted 
‘on them, It is well previously to place in the tube, through 
pie Ge | drogen is to be delivered, a litsle plug of cotton 
kk boing thus prepared, it ia sterilised by methods 

B HoH or sy 3), On cooling it is ready for inoculation, In the 
the with the lateral nozzle, the cotton-wool covering 

having been momentarily removed, a wire charged with the 
ia parend down to the bouillon, In the other kind of 
Husk the stopper must be removed for an instant to admit the 
wire. ‘The flask ix then connected with the hydrogen apparatus 
Dy means of a short piece of sterile india-rabber tubing, and 
Akpdrogen ix passed through for half an hour. In the cane of 








62 METHODS OF CULTIVATION OF BACTERIA 


flask (1), the Interal nozale is 
eet with alternate layera 








lugged with molten 
cotton wool and paraffin, the 


whole being tightly bound on with string. The entrance tube 


is now completely drawn off in the flame bofore bei 





Fro, 25.—Flaxk arranged for culture of 
anaerobes which develop gna. 


bin & trough of yonreury lato whigh exit 
tubo dips. 


connected from the 
apparatus Ta the case of 
flask (2), first the exit tube 
and then tho entrance tube 
are ecaled off in the flame 
before the flask ix discon- 
neoted from the bydrogen 
apparatus, It ia woll in the 
caso 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 
will occur, provision must be 
made for’ its escape. This 
ix conveniently done by lead- 
ing down the exit tubs, and 


letting the end just dip into a trough of mercury (Fig. 25), 
or into mercury in a little bottle tied on to tho end of the 
exit tube, ‘The proasuro of gas within causes an oseape at the 


mercury contact, which at the 
valve. 


same time acts as an efficient 
The thethod of culture in fluid media is used to obtain 


the soluble products of such anaerobes as the tetanus bacillus 





é 


Fin. 26.—Tudes for unserobie cultures on the surface of sotid 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, 


@and f, may bo 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 bacterin free from admixture of 
medium, as in the case of staining flagella. 


Misexttaxnous Mrntons. 


Cultures.—It is often necessary to observe 

a alive, either to watch the method and rate of 

their est or to investigate whether or not they are 
motile This is offectoed by making hanging-drop cultures, ~The 





900022 LALA AAI OO DALAL P LL SI PLEA ILD. 








os ®@ 


Fre. 27. 5 
A. Motlew-grovist site for tanging-crop cultures xhown In plan and seetion, 
B. Another form of slide for siinjlar euloaros. 


method in the form to be described is only suitable for aerobes, 
For this special slides are necessary, ‘Two forma are in use and 
ro 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 Bexplains itself. ‘The slide to be used and a coversglass are 
sterilised by hot air in a Petri’s dish, or simply by being heated 
im a Bunsen ond laid in a sterile Petri to cool. In the case of 
4 ‘one or other of two manipulation methods may be employed. 
ye the organism be growing in a liquid cultare, a loop of the 
lid in placed on the middle of the under surface of the sterile 
cover-glaas, which is held in forceps, the points of which have 
ie sterilised ina Bunsen flame. If the organism be growing 
i & 40h medium, @ loopiul of sterile bouillon is placed on tho 


od in the same position, and a very small quantity of 
the culture (picked up with a platinum needle) is cubbed wy im 











(ai ee 





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 ede of the coverylass is covered: with 
vaseline, and the preparation is then complete and may be 
placed under the microscope. If necessary, it may bo first in- 
cubated and then examined on a warm stage. (2) The sterile 
cover-gliss is placed on a sterile plate (an ordinary glass piste 
used for plate cultures ix convenient), ‘The drop ia then placed 
on its upper suiface, the details being the tame as in the last 
case. ‘The edge of the cell in the slide ix then painted with 
vaseline, and the slide, held with the hollow surface downwards, 
is lowered on to the cover-glass, to the rim of whieh it of course 
adheres. The slide with the cover attached is then quickly 
turned right side up, and the preparation is complete. 

In the caso of B the drop of fluid is placed on the contre of 
the table «. The drop must be thick enough to come in contact 
with the coverglasa 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 coverglass. The method of 
microscopic examination i8 described on page 85, 





Anaervbie Hangingatrop Cultures, The growth and examination of 
dacterix in hanging-drops under anaorobie conditions involve consider 





¥10, 28,—Grubom Brown's chamber for anaerobic hanging-dropa, 


(A portion of one adge of uppar plate te shown ent away.) 


able diticulty, but may be carried out in an apparatus devieed by 





Graham Brown (Fig. 25). Tt cousiats of two brass plates (a und a) 
which oan be approximated by scrows, and which have rounded 
Apertures in their middle ¥ iu. in diameter. ‘These support two rubber 
tings, an npper thinner ono (h) and a lower thick ono (d), the inner 





inwnovers being the samo us that of 





pertures inthe plates Betwoon 






COUNTING OF COLONIES 6 
‘enitalile sizo (e) ; d is separated 

eg era 
i Mothod of we: Bis 4p 


egainet 


ft st the 
‘the under surface of 


as co oa 
the dro) par turgagh one’ef the 
nieceiwe nae 

ned from time tortime. 
‘The Counting of Colonies.—An approximate estimate of the 
sumber of bacteria present ina given amount of a fluid (say, 
ip tain be arrived at 











4 
Hf 
i 





the counting, Fw. 20.—Apparstus for counting colontes, 

Qn apparatus such ax 
shown in Fig. 29 is employed. This consiste of a shoot of glass 
‘ruled into squares as indicated, and supported by its corners on 
woolen blocks The table to which these blocks are attached 
Fc surface. The plate-enlture eontaining the colonies 
the top of the ruled glasa The numbers of colonies 
twenty of the smaller squares, are then counted, and an 
The total number of squares covered by the 
ig then taken, and by a simple calculation the total 
of jies present can bs obtained. Plate-cultares in 
are sometimes employed for purposes of count- 

‘The bottoms of such dishes are, however, never flat, and 
‘of the medium thus varice in different parte, 
are to be used, a circle of the same size as the 
wn with Chinese white on a black card, the 
into equal arcs, and mdi drawn. The 


eer 
H me 

















66 METHODS OF CULTIVATION OF BACTERIA 


dish is then laid on the card, the sumber of colonies in a 

few of the sectors counted, and an average struck aa before, 

In sounting colonics it is always beat to aid the eye with a small 
-lens. 

‘Method of counting Living Bacteria in a Culture,—This 
ix accomplished by putting into practice a dilution method 
such a8 that described on p. 52. 
Measured amounts of high dilutions 
aro plated, and the wumbers of 
colonies whieh eubsequently develop 
are counted. In applying such a 
method it is necessary to have pipettes eapable 
of measuring small quantities of fluid. ‘Those 
250 SM discharging “06 and *1 0.6. will be found con- 
zs = venient, and such pipettes can have sub- 

divisions which enable them to be used for 
menguring still smaller fractions of « cubic 
centimetre. Pipettes of this kind can be 








ae: Pas obtained at the instrament makers, Wright 
hos described a method by which a pipette 
2 4s (Fig. 30) for measuring small quantities of 
fluid can be made from ordinary quill tubing. 
16 + The method is as follows:—A picco of quill 
tubing about 15 cm. long is drawn out to a 
oO capillary stem, A standard 5 ¢.mm, pipette 


(such as that of the Gower's hemocytometer), 
or the pipette described later on p, 108, is 
fillod with inereury and the motal transferred 
Caan to the capillary stem and run down to near 


is then displaced up the tube 
viously distal ond is at the proximal 
f the two marke, and a third mark ia 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 tubo beyond the lowest mark ix broken off, ‘Thus on the 
capillary part of the pipette we have five divisions, each cap- 
able of folding Scanm. of fluid, ‘The rest of the pipette is now 
calibrated 80. # that part capable of containing. 
225 mm. and This is done by placing a rubber 
nipple on the wide end of the pipette and sucking up some 
water tinted with, say, methyleno-blue till the 25 c.mm. mark 
is reached; a small air-bubble is then allowed to enter the 





ile 








METHOD OF COUNTING BACTERIA 67 


pipette, then other 25 c.mm. of fluid, then another bubble, and 
till nine volumes each of 25 ¢.mm. have been sucked up. 
‘is then made on the tube at the upper level of this 
amount, other 25 o.mm. are sucked up, and another mark made. 
The fluid ia expelled, the tube dried, and that part containing 
the nd 250 marks is drawn ont 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 
acenrate calforation for these amounts ix attained. Tn order to 
form a safety chamber a second bulb is formed by drawing out 
the tube a little higher up aa in the figure, and finally the upper 
inch or two ure bent at right angles to the calibrated limb, ma 
doing this a loop may Le thrown on the plastic melted ea 
oxactly in the way in which « similar loop may be t rsd 
‘on @ piece of cord. With such a pipette any required dilution 
of'a cultnre can be Janda 9 on the principles already described 
Method of counting the Bacteria in Dead 
Cultures.—In the making of vaccines for usc in Wright's pro- 
cedures it is necessary to know the total sumber of bacterial 
cella, whether dead or living, prosont in a cultnro, for the dead 
as well as tho living contain the toxins which may stimulate 
the therapeutic capacities of the body. The method consists 
in making a mixture of blood (whoan content in red blood 
epmie is known) with tho bacterial cultars and comparing 
the sumber of bacteria with the number of corpuscles. The 
observer firet estimates the red cells in his Mood ; a enpillary 
pipette with a rubber nipplo and with a mark near ite capillary 
extremity ix then taken, blood is sucked up to the mark, then 
an airbubble, then (according to the empirical estimate the 
observer forms of the strength of his hacterial emulsion) either 
one volume of culture and three volumes of diluting fluid 
(eg. 85 per cent sodium chloride) or two of cultare and two 
of fail, and so on; the five volumes are thoroughly mixed by 
boing drawn backwards and forwards in the wide part of the 
Pegi ‘a drop is then blown out on to a slide, and a blood 
is spread teres may be stained by Leishman's method, 
‘The bacteria and blood-corpuseles are now separately enumorated 
in & rerics of fields in different parts of the preparation, If 
& dilution bas been taken in which a large number of bacteria 
aff present, an artificial field may be used, made by drawing 
with the oil pencil a swall square on a cirenlar cover’ glasw and 


the latter on to the diaphmgm of the microscope 
Suppose, now, that the observer's blood contained 
900 





i 


red cells per cmm., 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. Tt is evident that in the undiluted cultnre for 
500 red colls there would have been 2400 bacteria, Now 
500 : 2400 :; 5,000,000: 24,000,000, which last figure is the 
pole of bacteria per emm, of the emulsion, 

"The Bacteriological Examination of the Blood.—(a) ‘This 
may be done by taking « amall drop ne the ekin surface, aq. the 
lobe of the car, The part should be thoroughly washed with 
1-1000 corrosive sublimate and dried with sterile eotton 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 hood is 
canght with sterile platinuz loop and smeared on the surface 
of agar or blood serum, Film preparations for inicroscopic 
exainination may be made at the sume time. tis rare to obtain 
growths from the blood of the human snbject by this method 
(vide special chaptors), and if colonies appoar 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 eatisfactory 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 storilised, 
the voin is made turgid by pressure, and tho needle of a syringe 
of 10-15 c.c. capacity, carefully sterilised, is thon plunged 
obliquely through the skin into the lamen of the vessel. Several 
cubic centimetres of blood can thus be withdrawn into the 
Some of the blood (¢.g. 1 ce.) should be added to small 

ini of bouillon; the rest may be used for 
smearing the surface of agar tubes or may be added to melted 
agar at 42° ©,, 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, ete, 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 tho skin 
‘Gyar 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, vive p. 87.) 

Bacteriological Examination of the Cerebro-spinal Fluid — 
Lumbar Pancture.—This dingnostie procedure, which is some- 


times called for in cases of meningitis, can be carried ont with 
























| RXAMINATION OF CEREBROSPINAL FLUID 69 
a sterilised “antitoxin needle" as follows, ‘The patient ahould 
Hoon te right side, with Ines comewhat drawn up and left 
shoulder tilted somewhat forward, so that the is fully 
exposad. The skin over the lumbar region is then 

asabove desoribed, and the hands of the operator should 
timnilnrly treeted. The spines of the lumbar rertebro having 
counted, the left thumb or forefinger ix pressed into the 
ace between the Srd and 4th spines in the middle line; the 
is then inserted about half an inch to the right of the 
Tine at this level and pushed through the tissues, its 
being directed thet and upwards, till it enters 
n 


5 


Tf 


iz 


PETE 
i 
ge 
Li 
= 
A 
2 
i 
2 


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 “pate length of the needle mast be cia 
accordingly, In making the puncture it is convenient to have 
either a storile ageatsachsa, or to hawo tho thick end of 
the needle with a pad of sterile wool, which is of course 
gemoved at once when the fluid begins to flow. It is advisable 
to use the platinam needles which are specially made for the 
aa snidden movement of the patient may snap an 
Ueliary steel needle 
‘The Bacteriological Examination of Urine—In such an 
examination care must be taken to prevent the contamination 
‘of the utino by extrancous organisms. In tho malo it ia usually 


‘aa rogards external cleansing, the catheter must be 
teed. Tho lattor must bo boiled for half an hour, and anointed 
with olive oil sterilised by half au hour's exposure in a plugged 
flack to a temperature of 120°C. Hera, again, it is well to 

‘the uring first passed. It ix often advimable to allow the 

fo stand ina cool place for some hours, to then withdraw 
ower portion with a sterile pipette, to centeifugalise this, 
amd to use the urine in the lower parts of the centrifuge tubes 
for microscopic exainination or culture. 

Filtration of Cultures.—lor inany purposes it is necessary 


(ti La, 


F 


70 METHODS OF CULTIVATION OF BACTERIA 


to filtor all the organiama from fluids in which they may have 
been growing. This is done especially in obtaining the soluble 
toxie products of bac- 
teria. Tho only filter 
capable of keeping back 
such minute bodies as 
bacteria is that formed 
from a tube of unglazed 
porcelain as introduced 
by Chamberland. The 
elficioney of such a filter 
depends on the fineness 
of the grain of the clay 
from which it ia made ; 
tho finest ia the Kitasato 
filter and the Chamber- 
land “Te” pattern; the 
noxt finest is the Cham. 
berland “F" pattern, 
which is quite good 
enough for ordinary 
Fiv, 81,—Geielor’s vactum pup arranged with work, ‘There are several 
per otteiati ra age (te Seing igs 
feale thn the tasometer Dourd to show i detail, all possessing 
Getaile) the common principle 
Sometimes the fluid is 
forced through the porcelain tube, Tn ona form the filter 
consists practically of an ordinary tap screwed into the top 
ofa porealuin tube, Through the latter the fluid is forced and 
passes into a chamber formed by a 
metal cylinder which surrounds the 
poreelain tube. ‘The fluid exeapes 
y an aperture at the bottom, Such 
a filter is very suitable for domestic 
‘use, or for use in surgical operating 
theatres, As considerable prossure 
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, pyc, 92, -Chamberland’s candle 
they are forced through a porcelain ani flask arranged for Atration. 
eylinder by compressed carbonic 
acid gas. For ordioary bacteriological work, filters of various 
Kinds ore in the m (such as those of Klein and 























THE FILTRATION OF CULTURES 7 


‘the most convenient is that in which 
is sucked through porcelain by exhausting the 


F 
tl 






air in the receptacle into which it ix to flow. This is con- 
yenicntly done by moans of a Geistler’s water-exhaust pump 
(Fig. 31, 9), which must be fixed to a tap leading directly from 
GMaeiei Tie wisnection with the tap must be effected. by 
means of of thick-walled rubbertubing as short as 
possible, to tap and pump, nnd firmly Iahed externally 
‘with many turns of strong tape. Before lashing with the tape 
‘the tube may be hened by fixing round it with rubber 
solution strips of the rubborod canvas used for mending punctures 
< 





Pro, 88,—Chamberland’s boagie Pia, 34.—Bongie in. 


‘areanged with lorop faunel for sorted through 

Witering a amall quantity of rubber stopper 

laid, for same purpose 
os in Pig. 33, 


In the outer case of a bicycle tyre. A manometer tube (6) and 
& receptacle (¢) (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 
teard a, as in Fig. 31. Between the tubo fand the pump g, 
‘and betwoen the tube d and the filter, it is convenient to insert 
lengths of flexible lead-‘ubing connected up at each end with 

stout-walled rubber-tubing, 

ters are arranged in various ways (a) An apparatus is 








oy! “Into this a “candle” or “bougie” of 
Porcelain dips. From the upper end of the bougic a glass tube 
with thick rubber counections, as in Fig, 32, proceeds to flask 4 








reste by a 





pavers ion 009 of the two perforations with which the 


the ask i# farnished, 


Pe Ratna cae ope kts Sak This apparatus 
good, but not suitable for small quantities of — 


apparatus can be arran; 
«rel dougie. a 


(0) A ver 
funnel and 
two ways, fi) An 


-— 


with a lamp 
These may be fitted ap in 


indisrabber washer is placed round the 
bougie ¢ ab its glazed end (vide Fig, 33), 


Ga this the. nasrom 


ond of the funnel d, which tnt, of course, be of an appropriate 
size, rests. A broad band of shect rubber ix then wrapped 





Fig. 96.—Muvneke's modification of 
Chamberland’s filter, 


(2) This modification is shown in Fi 
of the funnel an 








diarubber bung 


round the lower end of th 
funnel, and the projecting 
yurt of the bougie. It ix 
firmly wired to the funnel 
above and to the 
below, The extreme paar 
cof the Jatter is loft exposed, 
and the whole apparatus, 
Deing supported on a stand, 
ia connectod by a glass tube 
with the lateral tube of the 
flask b; the tube @ is con- 
nected with the exhanst- 
pump. The fluid to. be 
filtered is placed between 
the funnel and the bougie 
in the space ¢, and ia sucked 
through into the flask 6, 
4, Into the narrow part 
fitted, with a perforation 


in it wufliciontly large to receive the candle, which it should grasp 


tightly. 


(o) Mnencks's modification of the Chamberland filter is 


ween in Big. 35, 
part conical, the upper 





Lt consists of a thick-walled Haak a, the lowor 
‘lindrical, with « strong flange on the 


lip. There ure two lateral tubes, one horizontal to connect with 


exlaust-pipe, and one sloping, by whieh the 
poured out, Passing into the upper cylindrical part of 
is a hollow porcelain cylinder 4, of less diamet 
Tt is closed below, 
jecting rim on the flange of the flask, an asbestos 
The fluid to be filtered is placed 


cylindrical pat of flak 2 





washer, c, interposed. 





the contents may be 
ask 
the 


and 







er thi 
above, 








THE FILTRATION OF CULTURES 3 


in the | ler, and the whole top covered, ax shown 
at fy an eap with & central perforation ; the 
tube d is connected with the 


=> und the tube ¢ plugged with « rubber stopper. 
CS— > — When a lange 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 aj tne 
being woshipped, but it is difficult 
to get the tap to fit so accurately ax not to 
allow air to pass into the vacwam chamber. 
For filtering small quantities of fuid the 
apparatus sown in Fig. 37 may be used. 
Tt consists of a small Cham! 
fitted by a rubber tube to a funnel, the stem 
of which has been passed 





angular flask with sido 
arm for connection with 
exhanst, | 
Before any ono of 
the above apparatus is 
used, it ought to be con- 
Fre 26,—Fissk fitted nocted up ax far as pos 
with porcelain sible and sterilised in 
filtering the Koch's steriliser, 
The ends of any im- 
portant unconnected 
Parts ought to have pieces of cotton wool 
tied over them After us» the bongie is 
to be storilisod in tho autoclave, and alter 
being driod is to be passed carefully throngh 
s Burisen flame, to burn off all organic 
matter. Tf the lator is allowed to accumn- 
Tate the pores become fillod up, 
‘The success of filtration must be tested 
ppaseciting tubes of media from the . 
tad obverving if grow takes plac, Fe 37.— Yi er 
aia enay.b the ttering «mall quanti 
candles suificiontly large to all lk eee 
to puss through. Filterod fluids keep for a long time if the 
Openings of the glass vessels in which they aro placed are kept 











4 





74 METHODS OF CULTIVATION OF BACTERIA 


thoronghly closed, and if these vessels be kept in a cool in 
the dark, A layer of sterile toluol about half an inch thick 
ought to be ran on to the top of the filtered fluid to protect the 
latter from the re 0, 

Tustead of being filtered GF the Dacteria may be killed by 
various antiseptics, chiefly volatile oily, such as oil of mustard 
(Roux). ‘These olle aro 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 thojr soluble products are to be studied, as many 
of the lattor are deetroyod by a lower temperature than ia required 
to kill the bacteria themselves. 

Bacterla can be almost entirely removed from (aid cultures 
by spinning the latter in a centrifuge of very high speed (eg. 
©. J, Martin's turbine centrifuge), and this mothod is sometimes 
adopted in practice. 

The Observation of Bacterial Fermentation of Sugars, ete, 
—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 
ase the dofinition 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 isthus -CHOH ~CO—. The sugars are 
divided into monosaccharides or monoses, disaccharides (dioses), 
and polysaccharides (polyoses). ‘Tho members of the Jast two 
groups may be looked on ag derived from the combination of two 
or more molecules of « monosaccharide with the elimination of 
water (¢g. 2C,F),0,=C,,H,,0,, + H,O). 

Monowaccharrid ese are classified according to the 
number of C atoms they contain, The pontoses ordinarily used 
are arabinose (obtained from gum arabic), rhamnose and xylose 
(from wood), Among the hexoses are glucose (dextrose) with 
dextro-rotatory properties, Glucoso is an aldehyde alcohol 
(aldose), In fruit there is also a ketone alcohol (ketose) called 
frnetose, which from its evo-rotatory properties ix also known 
as lavulose, Other hoxoses aro wannose (from the vegetable 
ivory nut) and galactose (a hydrolytic derivative of lactose), 

jisaccharides (CysHy.0,,).—The ordinary members of this 
ip are maltose (derived from starch), lactose, and cane sugar 
(sucrose, saccharove). 

Polysaccharides. —Examples are starch, raffinose, inulin (from 

dublin roots), dextrin, arabin, glycogen, cellulose, 











BACTERIAL FERMENTATION OF SUGARS 175 


Tf we consider sugars saxo from the point af view of 
the capacity of yeast to originate alcoholic fermentation in them, 
we may say that the simpler the constitution of the ir the 
more oasily ia it ‘ote een oe ee = ue 

more easily acted east tl or moe 
Canal an independent process resulting in the splitting of the 
Mighee into the Lower ia prelininary | to the aleoholio formentation, 

‘Yeast first inverts cane sugar into glucose and fructose and 
then acts on thee products. From what is known it ix protable 
that sisnilar facts hold with regard to the action of bacteria, 


broken down ty bacterial action, and these bodies have been 
used for  diperentiating ¢ ‘the properties of allied bacteria. Among 
thewes substances rake mentioned the trihydric aleohol glycerol 
), the tetrabydric erythritol and the hexahydric dulcitol 

ania mannitol (umnnite), and sorbitol (sorbite). 

Similarly certain glucosides, such as saliein, coniferin, ete., 
have boon used for testing the fermentative propertics of 
bacteria, Other substances allied to sugars (eg. inosite) have 
also been used, 

‘The end products of bacterial fermentations may be various, 
They differ wecording to the sugar employed and according to 
icapacter of taceriom under observation, and frequently a species 

will formont one sugar has no effect on another, "The sub- 
stances finally produced, speaking roughly, may be alcohols, acida, 
or gaseous bodies aoe carbon dioxide, hydrogen, and methane), 
For the estimation of the first groups complicated chemical 
procedure may bo necowary. ‘Tho tosts usually employed for 
the detection of ordinary fermentative processes depend on two 
Kinds of changes, namely (@) the evolution of gases and (6) the 
formation of acids, Gonorally speaking, we may say that such 
teste aro reliable and the methods to be puratied aro simple, 
fashion gach gnece ax) those named some orgahiama give rise 
to ealphurotted hydrogen by breaking up the proteid, The 
formation of this gue can be detected by tho blackening of lead 
acetate whon it is added to the gascontaining medium. 

Th testing the effect of a bacterium on « given sugar it ix 
Gesemtiol that this sugar alone be present; tho basis of the 
medium ought therefore to be either peptone solution (vm p. 38) 
Of & dextrose-free bouillon (v. infra). The sugar or other 
mubstance ix added in the proportion of from a half to one 
jper cent, and care is taken not to overheat during sterilisation, 

To obtain  ‘ doxtroao-f uillow it is usual to inoculate ordinary 
Houillon with some organism, such ax b. coli, whieh is known to Corum 














Ai — 





76 METHODS OF CULTIVATION OF BACTERIA 


dextrose, and allow the latter to sot for forty-eight hours. The bouillon 
fa then Sitared and ro-sterilived, A antnplo ia texted for another period of 
fight hours with bh, cols, to make certain that all the dh has 
been removed. If no fresh’ gax- formation ts absoryed, 
romuinder of the bouillon the sugar to be investigated may 
Its preferable that the addition should be mado in the form of sterile 
wolution, Ifthe sugar in solid form be placed in the bouillon and this 
then storilisod, thore ie danger that chemical changes may take place 
in the sugar, ‘in consquonce of its being heated in the prosonce of 
substances (anch ax the alkali) which may act deleteriously upon st; im 
asuy vage atorilisstion should not be at a temperature above 100° 


For the observation of gas.formation either of the following 
methods may be employed -— 

(1) Durham's Tubes (Fig. 38, b)—The plug of @ tube which 
contains about one-third more than weual of a gold medium is 














© 
Fra, 98,—Tubes for demonstrating gus-formation by tueterin, 


a, Lube with *ehake” culture 





& Ordinary form of fermentation tbe 


removed, and a small test-mbe is slipped into the Jatter mouth 
downwards, ‘Tho plug is replaced and the tube atorilised thrice 
for ten minutes at 100° C, he air remaining in the smaller 
tube is thereby expelled. The tabe is then inoculated with the 
hacterium to be tested. Any gas developed collects in the upper 
part of the inner tube. 

(2) The Fermentation Tube (Fig. 38, ¢).—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 gusforming ongunisinn, and when growth occurs the gus 

collects in the 1 Ries ce Sheena ica ho eas ining 
into 





organisms throughout the jelly. It is then allowed to solidify, 
and is set asideatawuitable temperature, If the bacterium used 
is a gasforming one, then, asgrowth occurs, little bubbles appear 
round the colonies 

Tn 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 bo added to the medium if the effects on this 
sugir are to be observed with certainty. The shake culture 
methed may be utiliwd {or observing formontation in other 
sugars by adding to peptone solution containing the sugar 
10-15 per cent of gelatin. 

The development ofan arid reaction is demonstrated by the 
widition of an indicator to the medium, Htmus belog generally 
weed. The dotails of composition of such media have already 
been given. In Hiss's serum water media the production of 
acid leads to coayulation of the mediom. Sometimes acid 
fx formed very slowly from sugars, 40 that it is well to keop the 
énltures under observation for several days. 

‘Add and gas-formation may be simultaneonsly tested for, by 
placing the fluid medium containing tho indicator in Durham's 


Tn all tests in which sugars are used a control uninoculated 
tube ought to be incubated with the bacterial cultures, ax changes 
in sometimes spontaneously oceur in media contaising 

sugars. 

‘The capacity of an organism to produce acid may be measured 
by taking a standard amount of a fluid modium and allowing 
growth to take place for a standard time, and then adding an 
amount of, say, decinormal soda solution sufficient to bring the 
fitmus back to the tint of the original medium. 

The Observation of Indolformation by Bacteria. —The 
formation of indol from albumin by « bacterium. sometimes con- 
‘stitutes an important specific characteristic. ‘To observe indol 





—- ae 





78 METHODS OF CULTIVATION OF BACTERIA 


production the bacterium is grown, preferatly at ineubation 
temperature, in a fiuid medinm containing ivan ‘The latter 

may either be ordinary bouillon or preferably peptone solution 
io p. 8). Indol production is recognised hy the fact that when 





aeted on by nitric acid in the nce of nitviter, a nitrosoindel 
compound juced, w! Sekt bass rosy red colour, Some 
Dacterin (ey. the cholera vibrio) produce nitrites as well as indol, 


but anally in making the test (eg. in the ense of b, coli) the 
nitrites must be added, This is effected by adding to an exdinary 
tube of medium 1 c.c. of a 02 per cent solution of potassium 
nitrite, und testing with pure nitric or sulphuric acid. In any 
couse only a drop of the acid need be added to, say, 10 eo. of 
medium, If no result bo obtained at once it is well to allow 
the tube to stand for an hour, as sometimes the reaction is very 
slonty: epe In many instances incubation at 37° €. for 

m may be neceesary before the presence of indol is 
cin. ‘The amount of indol produced by a bacterium 
seems to vary very much with certain unknown qualities of the 
poptone, It is well therefore to test a series of peptones with 
fn organism (such as the b. coli) known to produce indo), and 
noting the sample with which the best reaction is obtained, to 
reserve it for making media to be used for tho detection of thia 
product. 

‘The Drying of Substances in vacuo,.—As many substances, 
for example toxins and antitoxins, with which bacteriology ix 
eoncerned 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 tn 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 
sueh a vacuum there mnst always be water vapour present, An 
airpwmp ia therefore to be employed, Here we have found the 
Geryk pump most efficient, and iv has this farther advantage, 
that its internal parts are lubricated with an cil of very low 
vapour density vo that almost a perfect vacuum ix obtainable, 
‘The apparates ia shown in Fig, 89. ‘Tho vacuum chamber 
consists of a belljar set on brass plate. A perforation in the 
centre of the latter leads into the pipe a, which can be connected 
hy strong-walled rubbertubing with the ainpump, and which 
can be cut off from the latter by a sto ck. In using the 
apparatus the substance to be dried is poured ent in flat dishes 
(one-half of a Petri capsule does very well), anil these are stucked 
alternately with similar dishes of ‘strong sulphuric acid on m 











STORING AND INCUBATION OF CULTURES 79 


stand which rests on the brass plate. The edge of the bell-jar 
‘woll luted with unguentum resin and placed in position and 

exhausted. In a few hours, af, as is always advis- 
Modi didiave contained only, @ thin ayer ef Atif. 2ha 
Il be complete. The vacuum is then broken by 
air very slowly through a bye-pass ¢, and the bell-jar 
In such an apparatus it is always advisable, us ix 
in the figure, to have interposed between the pump and 
vacuum chamber a Wolff's bottle containing sulphuric acid. 
is protects the oil ef the pump from contamination with 


BEETEGEES 
He 





Fro, 30,—Geryk air pump for drying én eacwa. 


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 ia exhausted to covor 
the vacnum chamber and the Welff’s bottle with wire guards 
eoverad with strong cloth, in case, under the external pressure, 
tho glaan vessels give way. 

Storing and Incubation of Oultures.- Gelatin cultures: 
must be grown at a temperature below their melting-point, ae. 
far 16 por cent gelatin, below 22°C, Thy are usually kept in 

rooms, which vury, of course, in temperature at different 
it which have usually a range of from about 12° C. to 
18° 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 organiams grow best, usually 37° C. in the ense of 
pathogenic Organtams, For the purpose of maintaining 4 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 fluid (water or glycerin and 
wator) is placed, which, when raised to & 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 glam. Hat ix supplied from a: burner 

fixed below. These burners vary much in 
design. Sometimes w mechani devised in 
Koch's lnboratory is affixed, which auto. 
matically turns off the gas if the light be 
accidentally extinguished, Between the tap 
supplying the gas, and the burner, is inter- 
posed a gow regulator, Such regulators 
vary in design, but for ordinary chambers 
which require to be kept at a constant tem- 
perature, Roichortis is as good and simple 
any and is not expensive, It is shown 
in Fig. 40, 

Tr consists of a long tube felosedl at the lower 
eau, open at the upper, and farnished with two 
Tateral tubes, ‘The lower part is filled with 
merery up to a point above tho level of the lower 
lateral tube. ‘Tho end of the lator ix closed by « 
brass cap through which » screw d panes the 
{nner ond of which Lies free in the merenry. " The 
height of the latter in the perpendicular tube eam 
thus be varied by increasing or decreasing the 
capacity of the laieral tubo by turning the eorew 
few tums out of ur into it. Into the npspor 
_ opon end of the perpendicular tube fits accurately 
Fie, 40,—Relehert’y x heut tubo y, drawn out below to s comparativuly 

gas regulator, ginal open point c and having in it» side a 
little above the point a rminute needle-hole 

called the peophole or bye-pass «. To fix the apparatus the long 
tmereary Dufb is plased in the jacket of tho chamber to be controlled, 
tube @ ta connected to gas supply, tubs # with the burner, ‘The appar 
level of the mercury should bo sore distance below the lower open end 
of tube e The burner ix now lit. The gas passes in at « through ¢ 
and ¢ and ont at 6 to the buraer. Whoa the thermometer in the 
interior of the chamber indicates that the desired tomporavuro haa boen 
reaclicd, the screw d is turned till the meroury roaches tho end of the 
tubes ' Gas oan only now pass through the peophole ¢, and the Same 

4 down. ‘The contents of the jackot cool, the mercury contencts off 
fire end of tube ¢,and the flame ris ‘his alternation yong on, the 





























temperatutwof the charobers kept very nearly coustant.. Ifthe merea 
cuts off the ges supply before the desired temperature Ls reached, ani 


‘STORING AND INCUBATION OF CULTURES 81 


the setow dla far out as it will go, then somo of the moreury must be 
temored. Similerly, if when the temperature is reached and the 
stew of eax fear in ax it can go, the mercury does not mach ¢, vam more 
must bs introduced. If the amount of gas which Faames, throagh the 
Deephole is sutficient still to enise che cowpersture of the chamber when 
inched ly the rive of te mersary, then the pexpbols i too large 
¢-niast be unshipped and ¢ plastered over i 






these ean be obtained. In all cases y ought to be fixed tod with a turn 





Pui, 41.—Hearon"s incubator for we at 87° 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 unsatisf ‘They easily 
get ont of order and are difficult to repair. We havo 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 weing an 
imeubstor 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 evaportion from the surface of cultures 

Within incubators, otherwise they may quickly dry up. 
it is thus udvimble to raise the amount of water vapour in the 
interior by having in the bottom of the ineubator a flat dish Cult 

6 










—~ — 





82 METHODS OF CULTIVATION OF BACTERIA 


of water from which evaponition may take place, Tubes which 
will require to be long in the incubator should have their plugs 
covered either by indiarubber 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 
ina flame. “Cool” incubators are often used for incubating 

tin at 21° to 22°C. An incubator of this kind fitted with a 

temperature Hearson’s regulator is in the market, 

Method 6 mounting Cultures as Permanent 
Musenm Rates (Richurd Muir).—(a) Stab or stroke 
cultures in nutrient gelatin or agar media.— When the culture 
shows typical charueters, further growth is arrested by placing 
tube in a formol vapour chamber, or by saturating the cotten- 
wool plug with strong formalin, ‘Then leave for a day or two, 
Make up following :— 


(2) Thymet Water (saturated tn cold) - 1006.0. 
20 oc. 
‘Auttatn of Potash . oo) Bgrams 
Colguet's (gold Inbel) Gelatin — | 10 grams 


Render the mlstitre asld to litmas with soot acid ; clear with white 
of ogg and filtor, 

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 o position that a thin layer of the preserving medinm 
remains completely covering the growth und 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) Thymel Water (saturated in cold) 





Glycerin 5 
Acntat of Potash gram 
: Cover top of tube with a small piece of paper so ay to keep 


out dust, allow to stand for a day or two co that small air-bells 
may rise to the surface. 

To seal tube, pour melted paraffin gently on to the surface 
of finid to near the top of tube ; allow to aolidify, Cover paraffin 
with layer of alcoholic orange shellac cement ; allow this to sot 
nd repeat until the cement becomes level with top of test tube. 
When sot, a fow drops of black lacquer are put on and a circular 
cover-glass of about the same diameter as the mouth of tube is 
placed #0 as completely to seal it. 

















GENERAL LABORATORY RULES 83 


8) Th folloning meth’ in wefal for preserving plate cultures, 
Tostend of making the cultures in Petri's capsules, use ordinary 
a ‘The watehluss is sterilised in  Petri's capsule, 

and the inoculated medium is poured out into the wateh-glans, 
solidify in the usual way, and loft in the Petry’s 
san ea sclosien of rome tava developed: "The washe 
glass is now removed from capsule and a layer of the preserving 
tat teetiara Uh; to which lnvo been aed few dropa of 
strong formulin, is allowed to spread over the xurface of the cultars 
medium. When the layer is solidified the wateh-glass is filled up 
with the same, and a clean square or oblong piece of glans 
(hich of course should be of slightly larger diatneter than the 


clas 
ay be closely applied to the glass cover and left in position 
until the gelatin hn solidified. The superfluous gelatin is now 
removed, and the glasses sealed first with tho orange shellac 
eement, then with black lacquer. It is now finished off by 
wecireular mask of suitable size. 
various kinds of solid media used in the cultivation of 
bacteria, such as blood serum, potato, bread paste, ete, can bo 
in the same mauner with excellent results. 
General Laboratory Rules.—On the working bench of every 


hangin at rl etc, which have contained bactena and 
bas finished, ought to be at once plunged (in the 
ease of tabea the tube and plag should be put in sepanitely). 
‘On no account whatever are such infected articles to be left 
ying about the laboratory. The basin is to be repeatedly 
jeanod out. All tho glass is carefully washed in repeated 
‘changes of tap water to remove the last trace of perchloride of 
> & very minnte quantity of which is sufficient to inbibit 
Ih, Old coltures which hare been stored for a time and 
which fresh subcultures have been made ought to be 
steamed in the Koch’s steriliser for two or three hours, or in the 
aiitoelare for a shorter period, and the tubes thoroughly washed 
out. Besides a basin of mercuric chloride solution for infected 
apparatus, ete, there ought to be a sccond reserved for the 
worker's hands in case of any accidental contamirmtion, When, 
as in publichealth work, a lange number of tubes are being 
daily pat out of usc, thoy may bo placed in an enamelled slop: 
pail and this when full is placed in the steam steriliser. 
A white glozed tile on whieh a bell-jar can be et ix very 


—_ ao 


84 METHODS OF CULTIVATION OF BACTERIA 


convenient to have on a bench. Infective material in wateh 
glasses can be placed thas under cover while investigution is 
going on, and it anything ix 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 remuin wet with this 
solution. No food ought to be yurtaken of in the laboratory, 
and pipes, ete, are not to be Inid with their mouth-picces on 
the bench, No label is to be licked with the tongue, Defore 
Toaving 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 Huid containing bacteria 
being accidentally spilt on the bench or floor, 1-100 mereuric 
chloride is to be at once poured on tho spot. Tho air of the 
laboratory ought to be kept as quict as possible. 














CHAPTER IIL. 


MICROSCOPIC METHODS._GENERAL BACTERIO- 
LOGICAL DIAGNOSIS—INOCULATION OF ANIMALS, 


Tho Microscope. —Hor ordinary bucteriological work a good 
microscope is essential. Lt t to have a heary 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 
It is best to havo three 


diaphragm, and a triple nose-picce, 
objectives, either Zeiss A, D, and 4 
Tenses of other makers corresponding to those. The oil immer- 
aion Ions is emential, It is well to have two eye-picces, my 
Nos 2 and 4 of Zeiss or lenses of corresponding strengths. 
‘The student must be thoroughly familiar with the focussing of 
the light on tho lens by means of tho condenser, and aleo with 
the use of the immorsion lens It may here be remarked that 
when iv is desired to bring out in sharp relief the margins of 
tanetained objects, «g. living bactoria in a fluid, a narrow 
ayeare, of tho diaphragm should be ueed, whereas, in the ease 
stained bacteria, when a puce colour picture is desired, the 
diaphragm onght to be widely opened ‘The flat side of the 
mirror ought to bo nsed along with the 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 
fine. If tho oil has dried on the lens it may be 
moistened with xylol—never with alcohol, which will dissolve 
the material by which the Jens is fixed in its metal carrier. 
je Examination of Bacteria. 1. Hanging-drop 
Preparations. —Micro organisms may be examined; (1) alive or 
ead) in Huids; (2) in film preparations ; (3) in sections of 
fines In the two last cases advantage is always taken of the 
AMinity of bucteria for cortain stains. Whon they are to be 
@xamined in fluids u drop of the liquid may be pinced on a ave 
85 

















86 MICROSCOPIC METHODS 


and covered with a coverglasa! It is more wsnal, however, to 
employ hanging-drop prepartions. The technique of making 
these has alreacly been described (p. 63). In examining them 
microscopically, it is necessary to use @ vory «all diaphraga. 
Tt 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 centro of the field, to clamp the preparation in this 
position, A high-power Jens is then turned into position and 
lowered by the coarse adjustment to a short distance above its 
focal distance ; it is now carofully scrowed down by the tine 
adjustment, the eye being kept at the tubo 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 somo purposes tho oll immersion ik 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 nom 
motile form such as anthrax, In fluid preparations the natural 
appearanos of bacteria may bo studied, and their rate of growth 
determined, The great use of such preparations, however, is to 
find whether or not the hcteria are , ani for determining. 
this point it is advienble to use either broth or agar cultures 'niot 
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, saya small crystal of yentian violet to 100 cc, af 
houilion. Such a degree of dilution will not have any effect on 
tho vitality of the bacteria. Ordinarily, living bacterin will not 
take up a stain, but even though y do not, the contmet 
between the nnstained bacteria and the tinted fluid will enable 
the observer more easily to recognise them. 

2. Pilm Preparations, («) Dry Method. ‘This is tho moet 
extensively applicable method of microscopically examining 
bacteria, Flnids containing bacteria, such ax blood, pus, 
of organs, can be thus investignted, as also cultures 
in fluid and solid media, The first requimte is a perfectly clean 
cover-glass Many methods are recommended for obtaining 
auch, ‘The test of this being necomplished is that, when the 
drop of fluid containing the bacterin 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 Yo tmoteriologiosl work it ie exwatial that covwrglases of No. thick 
Tew (Ge. “14 ram, thick) should be sed, ws thone of greater thickness nre not 
wuitable for a \4-in. lens, 



































FILM PREPARATIONS 87 


The best method ia that rocommended by Van Ermengem, The 
eover-glasses are placed for some time in a mixture of con- 
centrated sulphuric acid 6 parta, potassium bichromate 6 parts, 
water 100 then washed thoroughly in water and stored in 
absolute alcohol, For use, a coverg! is either dried by 
‘ping with a clean duster or is simply allowed to dry. This 
method will amply repay the trouble, and really eaves time in 
the end. A clean cover having been obtained, the film pre 
can now be made. If a fluid is to be examined a 
may Lan ag on the cover-glass, and either spread 

eu 


out over tho with the needle, or another clean cover 
may bo on the top of the first, the drop thus spread 
coup between them and the two then drawn apart. When 


th 
4 culture on a solid medium is to be cxamined a loopfal of 
distilled water is placed on the 
seed pe ————__} 

rubbed up init, (> 
the gas. The Sy =e) 
aaa By bry Fra. 42.—Cornet's forceps for holding 

b coven glasses, 

point of the 
should just touch the surface of the culture, and 
is rabbed up in the droplet of water and the film dried, 
Td be an opaque cloud jast visible on the cover-glass, 
film has boon spread it must next be dried by being 
wards and forwards at arm’slength above a Bunson 
film must then be fixed on tho glass by being 
or four times slowly through the flame, In doing 

lan is to hold the coverzlass between the right 
and thumb; if the fingers just escape being burned 
‘will accrue to the bacteria in the film. 
making films of a thick fluid such as pws it in best to 


i 


2 
af 


He 
Ly 





<HALtiift 
ae 






spread it out on one cover with the needle. ‘The result will be 
# fills of irrogular thickness, but sufficiently thin at many parts 
for projer examination. Scrapings of organs may be smeared 
directly on the covor-glasses, 

Ths the case of Wood, a fairly large drop should be alloweel to 
spreed iteolf betwoen two clean covorglases, which are then to 

ipped apart, and boing held betw inger ond 
Ree steal bys rs met i te 
air, A film reel in this way me at one odge, 


Dab at the othor is beautifully thin. If it is desired to preserve 
blood corpuscles in such a film it nay be fixed by one 
following methods: by being placed (a) Ww a eetvake 


 . “ 











88 MICROSCOPIC METHODS: 


chamber at 120° C. for half an hour, (4) ina mixture of equal 
parts of aleohol and ether for half an hour, then washed and 
ed ( fa formolaleohol (Gulland) (formalin 1 part, absolute 
alcobol ports) 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. TL 
shows a film prepared by the last mothod.) In wang the 
Romanowsky stains no previous fixation is necessary (vide infra). 
Tn the ease of urine, the specimen must be allowed to stand, and 
films mado from any deposit which occurs; or, what is still 
better, the urine is centrifugulised, and films made from the 
deposit which forms, After dried films ure thus made from 
Senin advantage to place a drop of distilled water on the 
film and heat gently to dissolve the deposit of salts ; then wash 
in wator and dry, In this way a much clearer picture is 
obtained when the preparution ix stained. 

Within recent years it has become common to make blood 
films on ordinary microscopic slides inatead of upon cover- 
glasses. Here the slides inust be clean, ‘This can be effected by 
washing thoroughly first with weak alkali and then with water 
and storing in alcohol. For uso, w slide is taken from the 
alcohol aid the fluid adhering to it set on fire aud allowed to 
burn off, a dry clean slide being thus obtained. ‘To make a film 
on such, @ small drop of blood ia placed near one end, the ¢¢ 
of a second clean slide is lowered throngh the drop on to S 
surface of the glass on which the blood has been placed. This 
socond slide i held at an angle to the first, on which it 
its edge. ‘Tho droplet of blood by capillarity spreads i 
theangle between thetwoalides. ‘The edge of the second alide is 
then stroked slong the surface of the first: slide, and in this 
procedare the blood is spread out in a film whose thickness ean 
be regulated by the angle formed by the second slide. Large: 
sized films ean thus be obtained, und when these are stained they 
are often examined without at er-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 upplication of 

















Films dried and fixed by the above methods are now ready to 
be stained by the methods to be described below, 

(6) Wet Method.—If it is desired to examine the fine 

histological structure of the cella of a discharge aa well aa to 

rem to substitute 

ims for the “dried ” films, the preparation of which has 

Boon described. ‘Tho nuclear structure, mitotic figures, ete, are 








EXAMINATION OF BACTERIA IN TISSUES 89 





bly 
stages in the preparation of 
wet films ure the same as above, but instead of being dried in 
ee ee eek ise at film downwards in the 
fixative. The following ure some of the best fixing methods — 





A saturated solation of perchloride of mercury in °75 per cant 
ebloride; fie for five minntes Thon plaoe the films for talfan 
with ozcasioual king, in “75 per cout sodium chloride 





soll to wash ont the corrsive subliimate ; Chey are thereafter washed 
iu successive strengths of tr mpirit, After this treatment the 
dl treated thoy wore sections. >, 
Ferol-aleobol— formalin 1 absolute sloohol ® Fix filme 
for three minutes: then wash woll in’ mo:hylated spirit. This is an 
excellent and very rapid method. 

(e), Another excol}ent mothod of fixing has been devised by Gulland. 
‘The fixing solution has the composition—absolute aleohol 46 ,¢., pure 
other 25 c.2., sleoholic solution of correaive sublimato (2 gra. in 10 oe 
of alcohol) about 5 drops. Tho filme are placed in thin solution for five 
minutes or longer. ‘They are then washed well in water, and arn ready 
for staining. A contrast «tain can be applied at the same time as the 
fixing solution, by saturating the 25 c.c. of alcohol with eosin before 
mixing. Theecafter the bacteria, etc., may be atained with methylene- 
Mae or other stain, ax described bolow. ‘This method has the advantage 
over (o) that, as a small smount of corrosive sublimate is used, leas 
‘washing is necoseary to remove it from the preparation, and doposite are 
ows linhle to ooeur, 


5, Examination of Bacteria in Tiseues.—For the examina- 
tion of bacteria in the tines, the latter must be fixed and 
hardened, in propartion for being cut with a microtome, 
Fixation consists in eo treating a tissue that it shall permanently 
amaintain, 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 («9. Cutheart’s or one of the newer instrn= 
ments in which the freezing is aecowplihed by compressed 
carbonic acid gas), but far finer results can be obtained by 
embexiding the tissue in solid paraffin nnd cutting with some of 
the more delicate microtomex of which, for pathological purposes, 
the small Cambridge rceker is by far the best. Por bacterio- 
Rael purposes embedding in celloidin is not advisable, us the 

jidin takes on the aniline dyes which are used for staining 
bacteria, and ix apt thus to spoil the preparation, and besides 
thinner sections can be obtained by the paratin method. 

‘The Fixation and Hardening of Tissues.—The following 
fre Amongst the best mothods for bacteriological purposes »— 

(a) Absolace atcohot may be ased for the double purpose of fixing amd 
Wardening, If the pices of tissue is not wore than } inch im thiaksaas 4% 











oo 








90 MICROSCOPIC METHODS: 


is mufliciont to keop it in thie reagent for a fow hours. If the ji 
are thicker « longer exposure is necessary, aud in such easee it ie 
toeliange the aleshol at the end of the tirat twenty-four hours. The 
tinue must be tough without being hard, and the necexsry consistence, 
ap estimated by feeling with the fingers, oan only be judged of after 
come experience, If the tienes are not to be out at once, they may be 
preservod in 60 per cent spirit 

(0) Formol-aleohol—formalio 1, slaoluite alechol 9. Fix for not more 
than Beaty et boars ; then place in absolnte aleohol ifthe ticene is 
to be embedded at onee, Lu 50 per cent spirit if it is to be kept for sonu 
Aime. For sinall pisces of tisue fixation for twelve hours oF even leas is 
sufficient. The method in a rapid and very entiafuctory one, 

(e) Corrosive rubimnte is an exoollont Fixing ageut. It ix best used 
sa saturated solation in ‘7S per cont sodium chloride solution, Dix 
solve the sublimate in the salt sotntion by hoat ; the 
crystals on cooling sliows that the solution tx suturated, 
ices oftionte &tnch in thickness, twelve hour innuersion is wuflcint 

If the pivoes are larger, twenty-four hovas is neosssary. Thos should. 
then be tied up ina plese of gauze, and placed ino stream of eunning 
wator for from twelve to tweaty-fonr hours, according to the kizo of the 
ieee, to wash ont the exness of sublimate, They are then placed for 
‘twenty-four hours in each of the following strengths of methylated 
splrle (Ree from pth): 20 per cout, 00 per cent, aud! 90 per cent 
Finally they aro placed in absolute alcohol for twenty-four hours and 
‘ero then realy to be propared for outing, 

If the tisue ie very small, as in the case of minute pivcos removed 
for diagnosis, the stages may be all compressed into twenty-four hours. 
Tn fact after fixation in corrosive the tiesue may be transferred directly 
tw absolute alcohol, the perehioride of mercury being removed after the 
sections mre cut, as will be afterwards described. 

(d) Methylated Spiril.—B8mall piccos of timne may be placed in 
methylated spirit, whioh is to bp changed aftor the first day, In from 
six to sevon days thoy will be hardened. If tho picoss arw large, « 
longer time is nosessary. : 























The Cutting of Sections..1. By Means of the Freeing 
tome—Pieces of tissue hardened by any of the above 
methods must have ull the alcohol removed from thean by wale 
ing in running water for twenty-four hours They are then 
placed for from twolve to twonty four hours (according to their 
size) in a thick syrupy solution containing two parts of gam 
farabie and ono part of sugar. They are then cut om w freezing 
microtome and placed for a fow hours in a how! of water so that 
the ara and syrup may dissolve out, ‘They are then stained or 
they may be stored in methylated spirit 






















4m Beitain ordinary commercial methylated wpirit has wood naphtha 
ailded to it to diecsuragn ite being used asa beverage. ‘The naphtha being 
fusclable in water a uilky Guid results from the dilution of the spirit. By 
Jaw; chemists oun only sell $ ounces of pure spirit at a time, Mort patho- 
Laboratories are, lowever, licensed by the Esoise to buy ‘industrial 
spirit," which contains only ono-ninetoonth of wood noplitha, 





THE CUTTING OF SECTIONS a 


2. Kubedding and Cutting in Solid Pavagfin—This method 
gives by far the finest results, and should always be 
when prcticable. The principle ix the impregeation of the 
tissue with paraffin in the mclted state. This parafin when it 
solidifies gives support to all the tissue elements. The method 
involves after hardening, the tismo hall bo thoroughly 
dnd then thoroughly permeated by some solvent 
of paraffin which will oxpel the dehydmting Auid and prepare 
for the entrance of tho paraifis. ‘Tho aolvents most in uae are 
I seals xylol, and tarpentine ; of these chloroform 


i 





mest Matogical laboratories, We have usd for 
mixture of one part of parattin, melting at 48°, and. 
two parts of melting at 54° C. This mixture hos a 
melting-point between 52° and 53° C. i ordin 
well. An excel 


tot obtained by using paratin melting about DS” C., anch as ix 
in 
youre a 








‘1, Pieces of tissue, however hardened, are placed fn fresh absolute 
for twenty-four hours in orer to their complete dehydration, 
rnow tom mixtare of equal parts of absolute alcohol and 
for twenty-frar hours, 

to pure chloroform for twenty-four hours or Jonger. At 

time the tissnes slionid xink er float heavily 
& Transfer now to a mixture of equal parts of eliloroform and paraffin 
‘807 place on the top of the orwn for from twelve to twenty-four hours, 
Hthe temperature thero is not sutticient to keop the mixture melted 








in the oven for twenty-four houry, 
ig the ticenes, seuall tinh 
nooks will be found very suitable, Tin 
Ne exerax of paradin over the bulk of tissue presnt, otherwive 
Chloroforin will be present to witiate the final res) 

give the ately hard block obtained with pure puraifin. 


wx 
persistence of tho slightest tmoe of chloroform ean be 


by smoll 


i — 








92 MICROSCOPIC METHODS 
In the ense of ory stnall pices of tisane the time 





for cach stage 


may be much shortened, and where haste in Wosirable Now 2 and 4 may 
bo omitted. Otherwise it is otter to carry out the process ax di 









By Jayis 
trough ts formed 
fn a soparate diab, 


restangular 
parallin taken from a stock 
cn of tanto, whieh i 1ifted 
The direction in 
becomes opaque. 
trong have 
Linaro 





The Cuiting of Paraffin Sections,—Sectiona must be cut as 
thin as possible, the Cambridge rocking microtome being, on 
tho whole, most suitable. They should not exeeed 8 pin thick- 


———$—  _| =] 


Pid, 48, —Neeille with square of paper on 
soctions, 








1d for manipulating paraitin 


ness, and ought, if possible, to be about 44, For their mani- 
palation it is best to have two noodles on handles, two camel’s- 
hwir brushes on handles, aud a noodle with a rectangle of stiff 
writing paper fixed on it as in the diogram (Fig. 43). When 
ent, sections are floated on the surface of beaker of water kept 
At & temperature abont 10° C. below the melting-point of the 
parafin, On the surface of the warm water they become perfectly 
flat, 





Fination on Onlinary Slides. (a) Gulland’s Method,—A mpply of 
Widos well cteanod being at hand, one of thom is thrust obliquely into 
the water bolow the section, a corner of the scotion is fixed on it with a 
neodle and the slide withdrawn. The surplus of water being wiped off 
witha cloth, the slide is placed ona sapport, with the avetion down 
wards, und allowod to remain on the top of the porallin oven or in 
bacteriologion! incubator for from twelve to twenty-four hours. It will 
then be snificiently fixed on the slide to withstand all the manipulations 
necessary dicring staining and mounting. 

(0) Pisation by Mann's Mehod,—This bus tho advantage of bein 
more rapid than the provious one, A solution of albumin is propared 
hy mixing the white of a freah ox with teu parts of distilled 
filtering. Slides aro mado perfectly cloan with alcohol, 0: 
Ante the solution and its edge ix thon crawn over ane surface of another 
slide so.as to leave on it « thin film of altumin. ‘This ix repeated with 
the others, Ax cach is thus coated, it is leant, with the film down 

















DEHYDRATION AND CLEARING 93 


pont ge peor itl petal armen ain ill ed 

Hoating ont is performed ax before. 

Side of the aie i casily by the fact that if 

i breathed on, the ett dey not cone oh ‘The great 
advantage of this 


‘meth 
ates’ drying ot 97° CIF the ton Kardoned i 
De sbasbcecsl slate tad'emibedded sn pects, tora sen 
method of fixing the seotions on the slide muat be used. 


Soo Fat eaccaaiice alleen meter der 
ing, the paraffin must be removed from tho section, This ix 
extra die ag bor bottle, When the 

ved out, the superfluous pei is wiped off with 

a sin and a little absolute alcohol dropped on, When the 
xylol is removed the superfluous alcohol is wiped off and a 
little 50 per cont methylated spirit dropped on, During these 
lures sections must on no account be allowed to dry. 

‘sections are now ready to be stained, Deposits of crystala 
‘of corrosive xublimate often occur in sections which have been 
fixed by this reagent, These can be removed by placing the 

before staining, for a few minutes in equal parts of 
Grams iodine solution (p. 99) and water, and then washing ont 
the iodine with methylated spirit. 

‘To save rupetition we shall in treating of stains suppose that, 
with faraflin 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 n specimen is stained, 
Dry films after being stained aro washed in water, dried and 

xylol balsam ; wet lina and sections must be 
cleared, and then mounted in xylol balsam, 

uniration is most commonly effected with absolute aleohol. 
Alcohol, however, sometimes decolorises the stained organisms 
oe than is desirable, and therefore Weigert devised the 
method of dehydrating and clearing by aniline oil, 

tilich thowsh it may decolorise somewhat, does not do 20 tothe 
same extent ax alcohol. As much as possible of the water being 
fomoved, 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 i¥ 
iy bene and becomes clear. The process may be accelerated 
ing gently, ‘The preparation is then treated with a 
eettns of tro parts of aniline oil and one part of xylol, and 

then wi with xylol alone, after which it is mounted in xylol balsam, 
Baleam os ordinarily’ supplied has often an acid reaction, anc 





dehy 














4 MICROSCOPIC METHODS: 


preparations stained with aniline dyes are apt to fade when 
mounted init, It is accordingly a great advantage to uae the 
acid-free Inlsam sepplied by Gribler. Paraffin soctiona can 
usually be dehydrated and cloared by the mixture of aniline oil 
and xylol alone, 

Sections stained for bacteria should always be clorred, at 
least. finally, in xylol, for the sume reason that xylol balsam. is 
to be nsed for mounting filins, vis, that it dissolves out aniline 

less readily than such clearing reagonts as clove cil, etc. 
ylol, however, requires the previous dehydration to have been 
more complete ‘than clove oil, which “will clear a section readily 
whon the dohydmtion has beon only partially otfectod by, say, 
methylated spirit. If a little decelorisation of @ section is still 
required before mounting, clove oil may be used to commence 
the clearing, the process boing finished with xylol, With a little 
experience the progress, not only of these processes but also of 
staining, can be very uccurstely judged of by observing the 
Appearances under low objective, 


Tux Srarexc or Bactenta. 
Staining Prinelplos,—To spoak generally, the protoplasm of 


bacteria reacts to stains in a maguer similar to the nuclear 
chromatin, thongh sometimes more and sometimes lest actively. 
‘The bacterial stains par excellence are the basic aniline dyes, 
‘These dyes are mors or less complicated compounds derived 
from the coal-tar product aniline (CyH,.NH,). Many of them 
have the constitution of salts Such compounds are divided 
into two groups according as the staining action depends an the 

ic or the acid partion of the molecule. ‘Thus the acetate of 
rosaniline derives its staining action from the rosaniling. Tt is 
therefore called a basie aniline 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 o apecial atfinity for the nuclear chromatin, 
the acid for the protoplasm and various formed elements. Thus 
it is that the former—the basie aniline dyes—are especially the 
bacterial staing. 
























‘Tho wambor of basic aniline stains is vory large, ‘The following are 
the mast commonly sed :— 
Ges Biotan Methyl violet, WER (rynonyme: Hollnann's vile, 


f 
Gentian-violet (aynonyr#: bonzyl-violet, Pyoktanin). 





THE STAINING OF BACTERIA 95 


3 | violet. 
Bie oae "Meter div! (eynonym ; phonylene-blae), 


Pea paete=—Noslsfocbela (eyncny es :‘bane rabtn, wayhats), 
Safranis ( + fucbain, Girvile) 

Brews Stoin,—Biswarck-trown (synonyms; vesuriv, phenslene- 
brown) 

This of the importance that the stains used by the 
bueteriologist Id be good, and therefore it is advisable to 
obtain those prepared by Grubler of Leipzig. One is then 
perfectly ure 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 blacs, methylone-blue 
ae gives the best differentiation of structure, and it is 

fflcult. to overstain with it. Thionin-blue also gives good 

Sitwentintion and does not readily overstain. Its tone is deoper 

than that of methylene-blue and it approaches the violets in’tint, 

Bismarek-brown is n weak stain, Int is useful for some purposes, 

Formerly it was much used in photomicrographie work, as it 

was less actinic than the other stains. It is not, however, 
now, on account of the improved sensitivenees ‘of plates, 

Te ik most convenient to keep saturated alcoholic solutions 
of the #tains made up, and for ues to filter a little into about 

ten times its bulk of distilled water in a wateh-glans. A solution 
of wo | pay is thus obtained. Most Imcteria (except thos of 

‘a oe @ few others) will stain in a short time in 
auch w regan vt latery solutions may also be made up, eg. 
saturated watery solution of methylene-blue or a 1 per cent 
solution of gentian-violet. Stains must always be filtered before 
tite; atherwies there may be deposited on the preparati 
granules which it is impossible to wash off. ‘The violet stnins in 
solition in water have a great tendency to decompose. Only 
small quantities should therefore be prepared at a time. 

The Staining of Cover-glase Films.—Vilms arc made from 
cultures as described abore. The coverglass may be fleated on 
the surface of the stain in a watch.glass, or the coverloss held in 
foreepa with film side uppermost may have ne much stain poured 
Oh itasit will hold. When the preparation has been exposed for 

Tequisite time, usually a few minntes, it is well washed in 

Swater in a bowl, or with distilled water with «uch a simple 
Pontrivance aa that figured (Fig. 44), ‘The figure explains iteelf. 


TDDAE be to be distingnieded fom methyt-bine, which O& @ different com: 


























al —— 





96 MICROSCOPIC METHODS 


When the film has been washed the surplus of water is drawn 
off with a picce of filter-paper, the preparation ix carefully dried 
Ligh over a flame, a drop of xylol balsam is applicd, and the 
cover-gliss mounted on a slide, Tt is sometimes advantageous 
to examine filma in a drop of water in place of balaam. The 
films can be subsequently dried and mounted permanently, To 
the caae of tubercle, special stains are necessary (p, 100), but with 
this exception, practically all bacterial films mado from cultures 
‘can be stained in this way, Some bac- 
teria, eg. typhoid, glanders, take up the 
stains rathor slowly, and for these the 
more intensive stains, red or violet, are 
to be preferred. 

Filia of stwids from the body (Vlood, 
pus, ote.) can be gonerally atained 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 auch 
fluids, if the histological elements also 
claim attention it ix 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 cella. The Romanowsky 
stains (v. p. 105) are here most useful, as 
by these the preparations are fixed as 
Wellas stained. Fixation by heat which 
is apt toinjure delicate cellular structures 

is thus avoided. Tn tho case of films 
Rothe for dutilled wale! made from urine, whore thero is little 
nised in wushing prepara. i 
tions. or no albuminous mat’ present, the 
Ducteria may be imperfectly fixed on 
the slide, and are thus apt to be washed off, Tn such a case it 
is well to modify the staining mothod, 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, 
4 drop of water is placed at ono eido of the cover glaze and a 
little piece of filter-ymper at the other side, The result is that 
the stain is sucked out by the filte adding fresh 
drops of water and using fresh pieces of filter-paper, the epeci 
mien is washod without any violent application of water, and 
the bacteria are not displacec 





Pio. 44. 



































MORDANTS AND DECOLORISING AGENTS 97 


For the general staining of films a mturnted watery solution 
‘of methylene-blue will be found to be the best stain to com 
menco with, the Gram method (v. infre) is thon Lida 
subsequently ‘special staina which may ay wh le, 

The Use of its and Decolorising —In films of 
Blood and pas, and still snore so in sections of tissues, ff the above 
‘mothods are used, the tissue olements may be stained to euch an 
extent as to quite obscure the bacteria, Hence many methods 
rave been in which the general principle may be said to 
be (a) the use of substances which, while increasing the staining 
power, tond to fix tho stain in the bacteria, and (4) the subex 

treatment by substances which decolorise the overstained 
40 & greater or less extent, while they leave the tacteria 
coloured, ‘The staining capacity of a solution may be incressed— 

(a) the addition of substances auch ae earbolie acid, 
‘aniline oil, or metallic salts 

(8) By the addition of alkalies, such as caustic potash or 
Ammonium carbonate, in weak solution, 

the employment of heat, 
Jong duration of the staining process 


As decolorising agents wo use chicfly mineral ncids (hydro- 
chloric, nitric, sulphuric), vegetable acids (especially acetic acid), 
alcohol (either methylated spirit or absolute alcahol), or a com= 
bination of spirit and acid, ¢¢. methylated spirit with a drop or 
two of hydrochloric acid added, also various oils, ¢. fina, 
clove, ete. In most cases about thirty drops of avetic acid i 
howl of water will be sufficient to remove the excess of stain 
from overstained films and sections More of the acid may, of 
course, be if necessary. 

‘Hot water also decolorises to a certain extent ; over-stained 
films can be readily decolorised by placing a drop of wuter ou 
the film and heating gently over a flarue. 

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. 

he methods embracing the use of a stain with a mordant, 
and a deeoloriser, are very numerous, and we can only enumerate 
the best of them. 

Dilferent organisms take wp and retain the stains with 
Warlots degrees of intensity, and thus duration of staining and 

miust be modified accordingly. We sometimes have 


ag Tmecteria which show a special tendency to be 








This tendency can be obviated by adding » We 


a ale 





98 MICROSCOPIC METHODS 


of the stain to the alcohol, or aniline oil, employed in dehydray 
tion. In the latter ease a little of the stain ix rubbed down in 
tho 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 « 

When methylene-bltte, methyl-violet, or gentian-violot is used, 
tho stain can, after the proper degree of decolorisation has been 
reached, be fixed in the tissues by treating for a minute with 
ammonium molybdate (24 per cent in water), 

‘The Formule of some of the more commonly used Stain Combinations, 

1. Lifler's Methylene-bluc. 





Saturatod eolution of mothyleue-blue in wloohel ss 
Solution of potassium hydrate in distilled water (1-10,000) : 100 ,, 
(This dilute solation may be conveniently mado by adding 1 oc, of & 





1 per cont solution to 99 0,0. of wator.) 


Sections may be stained in this mixture for from a quarter of an hour 
tosoverul hours, They do uot readily overstain, ‘The tissue containiny 
the bacteria is thon deoolorived if necessary with 4-1 per cent acotic acid, 
till it is m palo bluc-groon. Tho ssotion in washed in water, rapidly 
dehydrated with aleobel or aniline oil, eleared in xylol, and mounted. 

‘Tho tissue may be contrast stained with eosin. If this ix doxird, 
after dooolorisation wash with water, place for afow seconds in 1 por cent 
solution of eosin in absolute alcohol, rapidly complete dehydration with 
Pure absolute alcohol, and proceed as before. 

ms way be stained with Laiiler’s blue by five minutes’ 

longer in the cold. Thoy usually do not roquite decolori 
tissue elements are not overstained. 


2 Kivhne's Methylenerdtwe. 


















posure oF 
tion, ax the 








Mothylone-tlan 3 + Wer 
Atwolute aleohol > 1 1006 
Carbolis acid solution (1-20) . 100 


Stain and decolorise as with Lattor's blue, or decolorise with vory 

weak hydrochloric acid (a fow drops in a bowl of water). 

4% Carbot- Phionin-bive,—Make up a stock solution conristing of 1 
rainme of thionin-blue dissolved in 100.0. carbollo aold solution (1-40), 
‘or tise, dilute 1 yoluine with 3 of water and filter. Stain sections for 

five minutes or upwards, Wash very thoroughly with water, otherwine 
i deposit of cryxtale may ooour in the subsoquent stages, Decolorise 
with very weak nostic sold, A fow drops of the acid added to m bowl 
of water are quite sufiieient, Wash ayain thoroughly with water. 
Debydrate with absolute alcohol, ‘Thiouin-luv stains more deoply 
than methylenc-blue, and gives equally good differentiation, It ix very 
mitable for staining typhoid and glandors bacilli in sections, Cover 
lass Proparations stained by this method do not usually require 
dosolorisation. Ax 4 contrast stain, 1 per cont watery solution of eowin 
tay he used before staining with the thionin, 



























A. Gentian-riotet in Ani Oi Water wo solutions have here to 
bbe mode up. (a) Antlino oll water, Add about. § ec. aniline oil to 
109 6.6 dintilled wator in a flank, ond shake violently till ax much ns 








Pombo of the oil lias dissolved. Filtor and keep in a covered bottle 


GRAM'S STAIN 99 


lst" When she sat ts" be wa tpt of 
¢ 

ite fo), and the mature ie te ata shoul 

irit. Sometimes: 


it @ little hydrochloric 
tution is ee ‘mach 






ob bit 
= 


A 
(eee p. 101).—Thix 


6, Cortol-Fucksin very powerfal stain, and, 
when toed in the tmcilnted condition, 41 minutes staining ie nwtally 
Saflclet., [fle batter, however, to dilute with from tivo to ten times ite 
Yolume of water and siain forx few minutes. Tn this form it has a very 
while application. Mothyisted syirit with or without a few ropa of 

in the most convenient decolorixing agent. Then dehydrate 
", clear, and mount, 


Gram's Method and its Modifications —In the methods 
maeiy, described the tissues, and more especially the wuelei, 
retain some stain when decolorisation haa reached the point to 
which it can safely go without the bacteria themselves being 
affected. Tn the method of Gram, now to be detuiled, this does 
not ocenir, for the stain can here be removed completely from 
the ordinary tresnes, and left only in the bacterin, All kinda of 
bacteria, however, do not retain the stain in this method, and 
therefore in the systematic description of any species it ix 
eittomary to state whether it is, or is not, stained by Grams 
‘method—by this is meant, as will be understood from what has 
been said, whether the particular organien retains the colour 
iafler the latter has been completely vemovedl from the tisaues It 
must, bowever, be remarked that somo tiasuc elements may retain 
stain as firmly ns any bacteria, ey. keratinised epithelium, 
ealeified ywrticles, the granules of mast cells, and sometimes 
‘altered red blood corpuscles, ete. 
Ti Gram's method the cascntial feature is the treating of the 
Hiss, after staining, with a solution of iodine. This solution is 
spoken of as Gram’s solution, and has the following composition :— 





F 


2 1 part 
Potamsium iodide 2 parts 
Distilled water r 300, 





‘The following is the method :— 


See eet be ne oil gontian-violot or in carbol-gentian-violet (ride 
‘about five minutes, nnd wash in water 
fhe stotion or film with Grams solution till its colour 
[a parplish blick—venera(ly about half a sninate of a minute is 
feaction to take place. 

















100 MICROSCOPIC METHODS 


3. Decolorise with absolute alcohol or methylated spirit. till the 
colocr has almont entirely disappeared, the fiat having only « faint 
vi tint, 

4. Dehydrste completaly, elear with xylol and mount. 
of ies poopasations, the «pectmon ix simply washed in wal 
mounted. 

Iu stage (0) the process of ‘decolorisation is amore sallafutorl 

formed by using clove oil after suificient dehydration with alcohol, 

he clove oil being aftcewanks removed by zylol. 

Aaa contrast stain for the tiesues oarm or lithia earmine is neod 
dofore staining with gentian-violet (1). As a contrast stain for other 
Dacteria which are decolorised by Gram’s method carbol-fuchein dilnted 
with tan volumes of water or a saturated watery solution of Biemarck+ 
Inown may bo weed before mtage (4), 


‘The following modifications of Gram’s method may be given:— 


1. Weigert’s Modification. —The contrast staining of tho tissues and 
stages (1) und (2) ars performed as above, 

(a) After using the icdine solution the preparation {s dried by 
Plotting and thon decolorisod by aniline-xylol (aniline-oil 2, xylol 1). 

(4) Wash wellin zylol and mount ia xylol balun. Film preparations 
after being washod in xylol may bo dried and thereafter dilute earbol- 
fuchsin may be used to stain buctoria which have bean decolarised. 

This modification probably gives the most auiformly successful results, 

2. Nicole's Modification —Carbol-gontian-violet is used as the stain. 
Treatment with iodino is carried out as above and decolorisation is 
effected with a mixture of acetone (1 part) and alcohol (2 parts). 

3. Kuhne's Modifieation.—(1) Stain for ve minutes in a solution 
made up of equal parts of saturated alooholio solution of crystal-violot 
(* Keystall-violet™) and 1 per cent solution of ammonium carbonate. 

(2) Wash in water. 

(3), Place for two to thro minutes ix Gran’s iodine solution, or in 
the following modlifieation by Kuhne -— 

TAMEE re ce 2 parts 

Potassium iodide 2 sf ky 

Dirtillod water . . . . » 0, 
For ure, dilute with water to make a shorry-coloured solution, 

(4) Wash in water, 

(8) Decoloriss in ® saturated alcoholic solution of fluoresvein (a 
saturated solution in methylated spirit doce equally well). 

(G) Debiydrate, clear and monn 

‘Thore in great variability in tho avidity with which organisme tained 
by Grom rotain the dye when washed with alcohol, and sometimes 
lmeutty 1 exporienced’in snying whether an organism does ar does not 
tain by thie method. 


Stain for Tubercle and other Acid-fast Bacilli—These 
Dacilli cannot he well stained with a simple watery solution of a 
Luisic anilino dyo. This fact can easily bo tostod tempting 
y of o tubercle culture with euch 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 


In the case 
dried and 







































TUBERCLE STAINS 101 
by a short of beat. Wher once stained, however, 





Bichl-Neeleen Carvol-Fuchein Stain, 


Basicfuchsn =... ST part 
Absolute alcohol 2 | 10 parts 
Solution of carbolic ucid (1:20) 100 ,, 


Plaee the speoimen in this fluid, and having heated it till steam 

iw if to remain thers for five minutos, or allow it to remain in 

atsin for from twelve to twontyfour houre. (Filins and pairallin 

‘sro timally staineci with hot stain, loose wsctions with cold ; in 

‘stain the latter shrink.) 

Decolorise with 20 par cent solution of strong sulphuric acid, uitrie 

or shioric acid, in water. In this the tissuos become La 

‘Wath well with water, ‘The tissues will regain a ie tint. 

‘is distinctly red, the decolorisat al 

must be rvturned to the acid, Ax a matter of practioo, it 
the yauparation from the anid every fow xeoonds a 

replacing the specimen in the acid and re-washing till 
pink tint is obiained. ‘Then wash in alcohol for half « 

water, 

stain with a aaturotod watery solution of methytene-blue 
half & minute, or with satursted watory Biamarck-brown for from 
te three minutox. 

5. Wah weil with water. Tn the case of films, dry and mount, Tn 
‘case of sections, debydrate, clear and mount, 


UG; 









ebe 





: 
i 





| 





f 


iN 
i 


i 


S. 


Fat f 
ie 


Braenkel’'s Metification of the Zishl-Necleen Stain, 


Hore the procoss fs shortened by using a mixture containing 
both the decolorising agont and the contrwst stain. 


The sections or films are stained with the carbol-fuchsin aw above 
Aosoribed, aud then placed in the following solution -— 


Distilled water oan. ~ 60 parts 
Absolute alcohol. . Pain! . DRY, 
Ritzis mid - »« Dy 





Mathylene-bloe in o 


faze trestod with this till the red colour Ine quite dissppeered and 
Inapdiced by bloc. ‘The subssqnont stages are the eame an in No. 
re 


ws —_— 


to saturation, 








102 MICROSCOPIC METHODS 


Leprosy bacilli are stained in the same way, but are rather 
more easily decolorised than tubercle bacilli, and it ix better to 
‘use only 5 per cont sulphuric acid in decolorising, 

Tn the case of specimens stained either by the original Ziehl- 
Neelson method, or by Fraenkel’a modification, the tabercle or 
loproay bacilli ought to be bright red, and tho tissue blue or 
brown, according to the contrast stain used. Other bacteria 
which may be present ate algo coloured with the contrast stain, 

‘Tho 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 protoplaam of the bacilli or they have a resisting 
enyelope 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 corer-ginxs films as for tuborols bacilli 

‘2. Decolorise with 1 per cont sulpharic acid in water or wich methyl» 
ated spirit. ‘This removes the stain from the bacilli. 

5. Wash in water. 

4. Stain wich saturated watery methylene-blue for half a minute, 

5. Wash i dry, and mount in baleam, 


Tho result ix that tho *pores aro stained red, the protoplaam of the 
banilli blue. 

‘The apores of some organisms lose the stain more readily thau those 
of other, and for some, methylated spirit is a sufficiently strong 
deoolorising agent for use. If sulphurio acid, stronger than 1 per cont 
is used the spores of many bacilli aco readily decolorised. 

AMolier's Methed.—Vho following mothod, recommended by Moller, ts 
rnuph more satisfictory than the provious,  Boforn being stained, the films 
are placed in chloroform for % minutes, and then in a6 per cant solution 
of chromic acid for -2 minutes, the preparation being well washod 
ater each reagent, ‘Thereafter they ace stained and docolorised as above. 


‘The Staining of Capsules.—The two following methods may 


be recommended in the cave of capsulated bacteria :— 

















a) Welch's Method.—'This depends on the fact that in many cases 
the capsules can be fixed with glacial acotie acid, 

Filme when still wot are placed in this it for a few seoonds. 

‘The superfluous avid is removed with tiiter-paper aud the preparation 
{is treated with gentiau-vielet In aviline vil water repeatedly til all the 
acetic acid in removed. 

Then wash with 1-2 por ont solution of sodinm chloride and examine 
in the same solution, 

Tho capsule appears as 4 pale violet halo around tho deeply stained 
pete = : ee 

@) Richard Muir's Method (ne recently modified), 

1, The film containing the bacteria must be vory thin. Iv is dried 
nd stained in tered carbel-fachsin for half «minute, the preparation 

ted. 











‘STAINING OF FLAGELLA 103 


ft and thon well 
miboertahneraguadssr t's Se tans 


solation of cormsive sublimate . 2 party 
ee: lution—20 percent =... 2g 
tion of potush alam == SS By 


4. Wash well in water. 
& ‘Treat with methylated spirit for about a minute. 


The Bas a pale redilish 

& Wash wel in wate. coee Ears 

nak Gommtertain ‘with watery solution of endinary mothylenc-blue for 
& Dehdeate in alcoho), clear in ayo, and mont tn balsam, 


srimxon and the expsulos of a blue tint. ‘The 
caren bs Tateria'tn oaiterensasy poraeticae be demonstrated by ihfo 





Bes Staining of Plagella.—The staining of the flagella of 
bacteria is the most diffienlt of all bacteriological procedures, 
‘and it requires considcrable practice to ensure that good results 
ball be obtained. Many methods have been introduced, of 
which the two Pesce ne are the most satisfactory. 

Preparation of Filmse,—In all the methods of staining 

tate or cultures on agar should be regia @ culture 
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 « little water in a watch-glass ; 
the amount should be euch ws to produce scarcely any vurbidity 
Jn the water. A film ix then made by placing a drop on a 
‘cloan cov and carofully spreading it out with the needle, 
Ap is all to dry in the air and is then passed twice or thrice 
nob to o The 


‘sulphuric acid and potassium bichromate described on page 87, 


1, Piifield’s Metiod nx ventifial by Richard Muir. 
Prepare the following wolutions ;— 
AL The Montane, 
Tanto acid, 10 per cout watery solation, filtered . 10 ¢6 
Corrosive sublimate, wturated watery solation 5 
Alum, saturated watery solution Sel: 
Carbol-fuchaln (wide p 101). rt 
Ge iborsughly. A precipitate forms, which must bs allowed to 
Majo ter oentrifugalising or simply by allowing 10 stand, 
Ramovo the clear fluid with = pipette and transfer to « clean bottle, 
oe Keeps woll for one or two weeks. 

















urated watery solution Wes, 
turated alcoholic solution 











104 MICROSCOPIC METHODS: 


‘The stain should not be more than two or three days old when used, 
Temay be substituted in the mordant in place of the carbol-fuchsin. 

‘The film having been prepared ax above deveribed, pour over it as 
muoh of the mordant na the cover-glass will hold. Heat gently over a 
(ame till steain beging to rivo, allow to steam for about « minute, and 
thon wash well in a stremn of rauning water for about two minutes 
Then dry carefully over the fame, 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 mountin a drop of xylol balsam. 

This mothod has yielded the best resulta ie our Landa, 


2, Fan Hemengem's Method for Staining Flagelia, 








‘Phe films are prepared as above described. ‘Tiree solutions wre hero 
ncoauary : 
Solution A. (Bain foeateur)— 
Osaile acid, 2 per cent solution 2: ae ba hese 
Tannin, 10-26 percent wolution , . . . parts 


Place the films tn this for one hour at room temperature, or heat over 
4 flaue till ston rises aud keop in the hot stain for five minutes, 
Wash with distilled water, then with absolute alcohol for tree to four 
minutes, and again in distilled water, and treat with 

Solution B. (ain sensibitisatewr)— 

S per ceut volution of nitrate of silver in distilled water, Allow 
films to be in thisa few eevonds. ‘Then without washing tranafer to 


Solution C. (Sain redueteur et reinforgatewr)— 





Gallic acid. c - Sgrm, 
‘Twnnin 3 = : ees oy 
Fused potassium acetate . . . - - = 10 
Distilled water =. |} ] st Bie 





‘Thon treat ayain with Solution B till 
gins to turn black, Wash, dry, and mount, 

Morvyn Gordon recommends, fo leave the specimen ia 
B for two minutos and then to transfer to C for our and « half to two 
minutes, and pot to trenelor again to B. Jt will also bo found an 
advantage to uses frush supply of © for vach preparation, a «mall 
quantity being snificient, Tho heginner will find the typhoid bacillns 
or the bacillas coli communis very suitable orgaulams to stain by this 
method. 

Although the rosalts obtained by this method aro sometimes oxcellont, 
thoy vary considerably. Frejuontly both tho orgeuisine and flagella 
sppear of abnormal thickness. ‘This is due to the Riot that the process 
on which the method depends {x a procipitation rather than a trne 
Maining Tho pictures om the wholy ary Jess fait than inthe frat 
method, 


Staining of Spirochete in Sections.—The following im. 


Prognation method, which is practically that of Ramon-y-Cajal 















THE ROMANOWSKY STAINS 105 


for nerve frill, has boon applied for this purpose by Levaditi 
and exeollent maulta. 

‘The tissnes, which ought to be in thin slices, about L mm. 

i are best fixed in 10 per cent formalin solution for 


iv hours, 
‘Thoy are washed for an hour in water and then brought 
into 96 per cont aloahol for twenty-four hours, 

3) They are than placed in Ih per cent solution of nitrate 
ina dark bottle, and are kept in an incubator at 37° ©, 
for threo days, 
(4) They are washed in water for about twenty minutes, and 

are Pik placed in the following mixture, namely — 


a 


Pyrogallic acid, 4 
Formalin, 5 parts, 
Distilled ‘water up to 100 parts. 


They are kept in this mixture in a dark bottle for forty-eight 
at 


‘hours ut room temperature, 
(5) They are then washed in water for a few minutes, taken 
through increasing strengths of uleohol, and embedded in 


Gor the etaining of spircohutes In films see p. 107.) 


‘The Bomanowsky Stains.— Within recent years the numerous 
tnodifications of the Romonowsly stain have been extensively 
The dye concerned is the compound which is formed 
When watery solutions of medicinal methylene-blue and water- 
solulile eosin are brought together. ‘This compound is insoluble 
TM water but soluble in alcohol—tho alcohol employed being 

aloohol, The stain was originally used by Romanowsky 

for the talarinl parnsite, and its spocial quality ix that it 
Tnparts to certain clomenta, such as the chromatin of this 
Onganiam, a reddish-purple hue. This was at first thought to be 
imply dine to the combination of the methylenc-blue and the 
Dut it ix now recognised that certain changes, such as 
Geour in mothylene-blue solutions with age, are necessary. In 
the wedern formule these changes are brought about by 
Hrattment with alkalies, especially alkaline carbonates, ax wux 
wed by Unna in tho prvparation of his polychrome 

Ine. It is not certainly known to what yartieuas 


Vm. — 


i 

















106 MICROSCOPIC METHODS 


new body the reddish hue is due, but it may be to methyl-violet 
or to methylazure, 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 buctcria in tissues or 
exudates, the malaria parasite, trypanosomes, tho pathogenic 
spirochetes (such as the spirochwte pallida), and protozoa 


The following are the chief formalin in use — 


1. Jenner's Stoin.—This is an excellent blood stain, butis not se good 
for ‘the study of ‘paraites as the others to bo mentioned. In ite 
Preparation no alkali is uvvd, It is made by mixing equal purty of (0) 
& 12 to 1-25 por cont solution of Gribler's water soluble eosin (yellow 
shade) in distilled water and (b) 1 per cont Gritbler’s medicinal mothy- 
fene-blue (also a watery solution), ‘The mixture is allowed to stand 
tweaty-foor hours, is filtered, and’ tho residue is dried ut 65" C.;. the 
powder {s shakon up in distilled water, filsorod, washod with distilled 
waterand dried. Of the powdor, 5 grr. aro dissolved in 100-0.c. Merck's 
methyl alcohol. For usi a few drops are placed on the dried unfixed 
film for ono to throo sninntes, the dye is poured off, and the preparation 
washed with distilled water till it presenta a pink colour; it is then 
dried botwoen filter-paper and mounted in xylol balsam. 

2. Leiehnan's Siain.—The following solatiens are propared 1 (a) to 
1 por cont solution of modioinal methylono-blue is added “b per cont 
sodium carbouate ; the mixture is kept at 65°C. for twolve hours and 
then for Wn days at room temperature ("25 yer cent formalin may be 
added as a preservative) ; (0) 1-100 solution of eosin, extra BoA, in 
distilled water. Equal volumes of the two solutions are anbxed ‘and 
allowed to ataud for six to twelve hours with occasional stirring, the 

ocipitate is collected, filtorod, washed with distilled water and dried. 
for uso ‘15 par ont is dissolved in Merck's mothy! alcohol (‘for 
analysis, agotone free”) as follows: the powder is placed in m clean 
toriar, a little of the’alcohol ix added and well rubbed up with « 
tle; the undiwolved powder ia allowed to suttle and the fluid 
lecanted into adry bottle; the process is repeated with froash fractions 
of the solvent till practically ail the stain in diasolved, and the bottle 
ia wall wtopperod ; the atain will keop for a long perlod. For the 
staining of filma & fow drops of the stain are placed on the unfixed 
Proparation for fifteen to thirey waconds so as to cover it with a 
shallow layer (the staln sa aveniently spread over the film 
with m glam rod) and the fila fs tilted to and fro s0 ax to prevent 
drying, This treatment efficiently fixes the film ky tho action of tho 
méithyl aloohol. About double the quantity of distilled water is now 
dropped on tho film, and the stain and diluent are anscnly mixed with 
the rod. Five minttes are now allowed for staining, and the stain ix 
thon gently wasliod off with distilled water. A little of the water is 
Kept on the Hlm for half » minute to intensify the colour eoutrasts in 
the various cols. For certain special structures such as Schiilfacr's dots 
or Maurvr's dots in the nulurial parasite u longer staining (up to one 
hour) may be nooewary, snd in any caso it is well to practise being able 
toxontrol the depth of tho staining effect by observation with a low 
power objective. If proparation ie to be stained for a long time it 




























































be and if in it ix formed 
Bical betec sal label of Yack td cxose ay 

cor ar 
sesored by wasting the tia with scetis acl, 11600, The fm 4 





between prot al raount 
Jor staining sections a little medéoation ix nocesary. A paral 
‘section is taken into distilled water as ayual, tho excess of water fe drained 
‘eff, aud & mixture of one of stain and two parts of distilled water 
ts placed on it. Thestain is allowed to act for five to ten minutes till the 
ewe a dev Oxford blue; it is thon decolurived with 31-1500 
‘scetic aa affect being watched under a low-power lous. ‘The bine 
to fous ou and tho to-go on till only the 
romain biue. The section is thet ith distilled water, 
rapidly dehydrated with alcohol, cleared and mounted. If, ax some: 
Aimes happens, the eosin tint be too well marked it can be lightened 


of 1.7000 solution of caustle soda, this being washed off 


desired colour has been attained, 
rights Stain.—In this ion 3 por cent methylene 
Bhrliob’s rectified) and } por cent sodium carbonate (both 
in water) are mixed and placed in a Kooh’s sterilisor for an hour. When 
ees ait. 1000 aulonaextra HA. saint aided ei he mixtare 
‘purpli a granular precipitate appesre in 
suspension (about £00 co. own to 100 o.c. methylene blue solution ate 
the precipitate (s filtered off and dried without being washed. 
A-maturated solution of this & made in tho pure methyl aloohol ; thie is 
Sitored asl dilated by adding to 20 o.r, of the saturated solution 90 €.0 
na application of the taln is almost the same at 
ropa are placed on the preparation for 
winate for fixation; water is thon dropped on till a green iridescent 
Hauin appears on the top of the fluid sad oaining goes on for about 
ws minutes ; the stain is then washed off with distilled water and a 
little & allowed to romain on the fiim till differentiation ix complete ; 
is oprefully dried with ste Peres end mounted, 
's Stain —Glemes belloves that the reddish-blue hue 
ie of the Ramanonsky atain fs dus to. the formation of 
Lacare, and he has preparod this by a method of his own under 
Aror 1." From this, by the addition 
aioinal methylene blue, he prepares what bo el 
from in by the addition of cosis he prepares 
‘The Batest formula for tho Gnixhed wtain is as follows 
bi Azar 11.8 gr.» Glycerin (Merck, chemoally par 
Sieotot 8 






En 
x 
= 

















Agar U..cosin % 
) 250 gr, Methyl 
extauaively mand 














(Kelibsum, 1.) 250 gr. This stain hax 
demonstrating the Spirochacte pallida, but it can be used for auy 
to which the Romanowaky stains aro applicable. Por the 





the following ary Giomnsa's direetion 
1) Fix films in absolute aleohol for fifteen to twenty minut 
ter. ) Dilute stain with distilled water—c 
stain to Loc. water (she mixture being well shaken). (Sometinos the 
water by the addition of one deop of 1 per vaut p 
















carbonate water). (3 ain for fifteou minutes. (4) ¥ 

Drink stresin of distilled water, (5) Drain with filter-paper, dry, and 
‘mount in Canada bales. 

With regard to the Jennor and Giemsa stains it {s best to obtain the 

from Grater ready for use; the powder for Leishman's stain 

inay be obtained from the mame source aad the solution wimle wy tg 





om 





108 MICROSCOPIC METHODS 


‘the worker himself, Cabot states that Wright's stain can be obtained 
from the Harvard Co-operative Society, Boslston Street, Boston, U.S.A. 
Nolsser’s Stain.—Neisscr introduced the following stain as an aid to 
the diagnosis of the diphtheria hactilux ‘Two solutions are used ne 
follaws: (a) 1 grim. wethylene-blue (Gribler) is dissolved in 20 cc. of 
96 per cent aloohol, aud to the solation are aided 960 co, of distilled 
water and 60 66, of glacial soctic oid ; (b} 2 gras, Bismarck-brown 
(vouuvin) dissolved du litre of distilled water, Films wro stained iu 
(a) for 1-3 seconds or a little Jonger, washed in water, stained for 2.5 
seconde in (4), dried, and mounted. "The protoplasm of the diphtheria 
ductus Is stained & faint brown colour, the granoles a blue colon. 
Noiasor cousiders that this reaction ix charactoristic of the organinin, 
fovided that cultures oa Léflar's werum aru used and examined 
24 hours incubation at $486" O, Satisfactory results are not alwayn 
obtained in the ease of tilme preparod from 
membrane, ote., but there i no doubt that heew 
also the method ix ono of considerable value, 


















Srectat BacrunorogicaL Mirwops. 


‘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 auch 
Pipettes are not always available, und by 

rights technique if a Gower's 5 e.mm. 
hasmocytometer pipette be ut 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 
4 till it is of the thickness of a hair bares be 

cokes 5 Still possessing a bore, Tf the point be 

ine pinata 4? broken off this hair and msroury be Tn 
oe ‘, inte the tube the metal will be caught 
where the tube narrows and will pass no 
further —in fact, though sir will pass, 

seer chpacity «Die mercury will n Into tho wide end of 

W, hair capillary, this tube 5 ¢ of mercury, measured 

from 2 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, 
wnd the tube is carefully cut through at the mark, A pices 
of ordinary quill tubing is drawn out and broken off just 
below whore its narrowing has begun, the capillary tube has 

















TESTING OF PROPERTIES OF SERUM 109 


some wax moulded round ite middle, the hair end ie al 
the broken-off end just mentioned, and the tube is 
in position #s shown In the figure. A ee pee placed on 
eel ee Bi Fees capes See as fees) 
the nipple the air be expelled from eget 
‘mercury, exactly 5 c.tam, will be taken up. ‘Thus 
ws ec on the ine is relaxed, other tubes ean be very: 
calibrated by the mercury being expelled into them and 
its limits marked on their bores, 
For measuring equal parts of different fluids the pipette 
in connection with agglutination ie very useful 
{ece Fig, 46 d). 


‘The Testing of Aggintinative and Sedimenting Properties 
of Serum. 


By agglutination is meant the aggregation into clumps of 
uniformly disposed bactorin in a fluid: by sedimentation tho 
formation of a spesi compeeod of euch elampa when the fluid 

ia allowed to stand, Sedimentation is thus the naked-eye evidence 
of agglutination, ‘Tho blond serum may acquire this ehunping 
power towards 4 particular organism undor certain conditions ; 
wg chiefly met with when the individual is suffering 


artificially y injections of the organism, Tho nature of this 
be discussed later. Here we shall only give the 
technique by which the presence or absance of the property may 
bbe tested, ‘Thorn aro two chief methods, a microsoopic and a 
naked eye, corresponding to the effects mentioned above. Tn 
Doth, the essential process is the bringing of the diluted serum 
nto contact with the bacteria uniformly disposed ina fuid. In 
the former this is done on a glass lide, and the result ix watched 
minder the microsoope; the occurrence of the phenomenon ix 
shown by the aggregation of the tacteria into chimps, 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- 
tion ia shown by the formation within a given time (say 
24 hours) of a somewhat floccalent layor at the bottom, the uid 
@hove being clear. ‘Two points should bo attended to: (a) 
controls should always be made with normal serum, and (6) the 
forum to be tested should never be brought in the undilated 












a em 


110 MICROSCOPIC METHODS: 
condition into contact with tho bacterin, ‘The stages of pro: 
codure are the following -— : 


1. Blood is conveniently obtained by wicking the Tobe of the nar, 
which should previously ave been washed with a mixture of aleohol 
‘aud ether and allowed to 
dry. | Tho blood ix drawa. 
up int a Wright's blood 
capsule (Fig. 47) of into 
the bulbous portion of a 
capillary pipotte, stich ax 
in Fig. 46,4. (Thevo pip- 
cettes can be readily mado 
by drawing out quill glass 
tubing ina flame. It in 
converiont always to have 
several realy for ase.) 
The pipette is kept iu the 
upright position, one end 
boing cloned, For purposes 
of transit, break off the 
balb at the constriction 
ud seal tha enda. “Alter 
the serum has separated 
from the eoagulum the 
bulb is broken through 
nwar ite upper end and tho 
sorum romeved by means 
of anager capillary Hip. 
tts. ‘The serum is then 

4 to bo diluted, 

2 The serum may be 
Ailuted (w) hy weane of a 
grad antod pipotte—oither 
& leneonytometer pipette 

a (Pig. 40, 6) or some corres 
1 sponding form, In this 

} m way successive dilations 
of 1510, 1690, 14 100, 

tte, eon ba rapidly mace. 

' This ts tho best: method. 

















(2) By means of capillary 
pipette mish neck on the 
tube, the serum is drown 
up to the mark and then 
blown out into a glen 
rapoule; oqual quantities 
of bouillounresuzcessively 
measured in the same way 
nd abled till the requisite dilution is obtained. [c) By meane of a 
platinum needlo with a loop at the ond (Delépine’s rmcthod). A loopful 
of seruns Ss placed on o slide and the desired number of similar loopfuls 
of bouillon Are aoparatoly placed around on the slide, ‘The drops are 
then mixed. 

Avery convenient and rapid method of combining the steps 1 an¢ 





Fin, 48, —‘Taikor used in tenting agglotinating and 


sedimenting properties of serum 




















THE OPSONIC TECHNIQUE mm 


Lien ea fe btn after Ute ul lok don 
in 


i! vad draw tho bouillon after { fillet. 
of-20 times ix thus obdéained. ‘Then blow the mixture into m 
ce Pg. ed eratsiogalie or Spy slow the ra 





corpuscles to separate taney 
sion eel ars than 1 pare ser sera we comida cleat 
Ganmst thavetare bat tance in ooxiparing Feruite! 


The preeenos of red 
cane of | ; mr ne eae puts ings 
corpuscles should bo 2 
thedld be takes from pice se, 

Bip not more than twenty-four hors old, inowbated at 37” C. 

te used either as» bouillon culture or as an emulsion made 

Ey aiding salt portlon of anager culture to bouilion. In tho latter 
c mcteri p shoul 











the fF the Bald ine. waveh-glom 

fuabidity is thus obtained, any romsining fregments shoald fat be 

etercsiand then ihe Organisms should be aniformly mixed with the 

mat of the flaid. The tncierial emulsion ongh 

distinct turbidity. (Whoa the exact 
is to be tested —o 


sn th ‘w have ‘2 faint but 

wer of a 
proses us (ke highest allution bs wise 
ete sedimentation within ‘recat; four hours—: dard 


Fredeore comp 
it) of bactoria must be te vets of 
Wein Tae fines rca tages = ONY 
+ 











“pe 
test microsopicatty, mix equal quantities (measured by « 
marked pais. 9! pette) of the di ted wera a the Taoteial 
teunlsiou ns glans aide cover with a corer-gins, and examine under 
Pes eriexontore, ‘The form of glass slide drop cultures 
(Fig. 27) will bo found very suitable. The ultimate dilution of the 
serum will of cote, be double the orginal dilution, 

To 6 seimicatation mix equal parte of ituted serum and of 
actorlal emulsion and plaos fn» thin las tube—a simple tube with 
loved end or o Uinte. Keep in upright position for twenty-four 
Hours, One of Wright's sedimentation tubes is ahown in Fig, 48, ay 
Diluted serum is drawn up to fill the space mm, 9 smal) quantity of ait 
Geauvked up after {t to voparste it from the bacterial emulsion, which 
Gothen dravn up in tho same quantity ; tho diluted sarum will thet 
‘oveupy the position a The Haida arw then drawn aqveral times up 
Anto the bulb and returned to the capillary tube 0 as to mix, and flual 

Mm carefully down close to the lower end, which is then sealal off. 
sediment collects at the lower extremity. 

Tt may be maid that it is often important to observe not only the 
Bisengyteencentration of» wera which will produce agglatination tat 

est. 


Method of measuring the Phagocytic Capacity of the 
Leucocytes—the Opsonic Technique.—'his was first done 
Leishman by a very simple method as followa: A piece 
quill tubing is drawn out to a capillary diameter eo ns 
fo make a pipette about six inches long. The point is 
roken off and « rubber nipple adjusted to the wide end, 

is made with an oil pencil about three-quarters of an 
above the orifice. Blood is drawa from tho finger up 
Moths mark, then an airbubble is allowed to asain. A shin 


i — 


2g 





m2 MICROSCOPIC METHODS 


emulsion of the bacterium to be tested having bean prepared, a 
quantity of this is also drawn up to the mark. The two flui 
are thon thoroughly mixed by tate first. blown out on to a 
sterile slide and then being drawn back into the pipette and 
expelled,—thix being repeated several times. A coverglass ix 
placed over the drop, ik the slide is placed in the incubator at 
37° ©. for fifteen minutes, The cover-gluss is then slipped off 
xous to make a film proparation which in the caso of ordinary 
bacteria may be stained by Leishman's method. The number 
of bacteria present in, say, 50 polymorphonuclear cells sneces- 
sively examined is determined and an average struck. Tho 
method was first used for showing that in casos of ataphylococeus 
infection the average number of ia taken up was leas than 
ina control fo which the eame bacterial emulsion waa exposed 
to tho blood of a healthy individual, In making euch 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 othor to 
the other, —the two films being then stained us one. 

According to Wright's view the process of phagocytosis in 
blood outside the body is not « simple one, and before a 
leucocyte takes up n bacterium the latter must be acted on 
in some way by substances present in the serum, which Wright 
calls opsonine (sce Immunity). The technique by which the 
netions of these opsonins ia studied has been elaborated by 
Wright and his co-workers in connection with his work on 
‘Dacterial vaccines, especially in relation to infection by the 
pyogonie cocei and tho tubercle bacillus ‘This technique 
involves (1) the proparation of the bacterial emulsion, (2) the 
Proparation of the leucocytes, (3) the preparation of samples of 
(a) seram from a normal person, (b) serum from the infected 

POR. 

(1) Preparation of bacteriol emulsion. Tn the caso of the 
pyogwnte cocel a little of a twenty-four hour living cultare off a 
#loped agar tube ix taken and rubbed up in a wateh-glass with 
“8D per cont ealine. ‘The mixture is placed in a tube and centri- 
fugulised so a8 to deposit any masses of bacteria which may be 
present, Only hy experience ean a knowledge be gained of the 
amount of culture to be wed in the first instance, but the 
resultant craulsion 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 ecm, If too atrong an 
‘emulsion be used the leucocytes may take up #o many organisms 
that these cannot be accurately enumerated. In the case of 











PREPARATION OF THE SERA 113 


the tubercle bacillus ax short a variety of tho organism ax 










l 


should bo selected, and a mass of growth off a solid 
is taken (bacilli in mass can be obtained in the market 
chemists) and is well washed with changes of 
dried on cepilns in a Petri dish and 
bbed up with a little 18 por cent saline in an 
#0 mx to disintegrate the bacterial masses and 
sion “ral pate as far as possible of individual bacilli, 
he cor by microscopic examination, A thick 
be obtained, and this should be sterilised by 
for half an hour on three successive days. Beforo 
ig convenient to eeal up the stock emulsion in 
in a number of pieces of quill tubing so that in 
ont procedures only amall portions of the emulsion 

fo aerial contamination at one time, For actual 

one of those tubes is opened, a little is withdrawn with a 

pil and a weak emulsion made in the same way as 
yylococeas except that 1:5 per cent saline is used. 
may be ecaled with wax and kept for use again, 

A fresh emulsion ought to be made up for each day’s work. 

9) Preparation of leucceytes. Wore tho observer uses his 
coll, A 1°5 por cent solution of sodium citrate in 
cent sodium chloride is prepared. This is placed in a 
tnbe thron inches long made by drawing out a piece of 
jing to a point, the tube being filled nearly to the 
handkerchief being bound round the finger, this is 
and the blood allowed to flow directly inte the 
bottom of which it sinks, The tube ought to be 
between the addition of every few drops of blood so ne 
Ulood in contact with the citrate and prevent 
The equivalent of about ten to twenty drops of 
should be obtained. ‘The diluted blood is then eentri- 
and when the corpuscles are separated the saper- 
fitid is removed, “86 per cent saline is substituted and 
‘gentrifugalieation repeated. The fluid is again removed, 
being taken not to disturb the layer of white celle lying on 
top of the red corpuscles, This layer is then pipetted off 
& Watch-ginss or tube and the le ytea required are thus 


iH 
i 





it 


zz 
u 


i 
i 


E 






z 







iH 


> 








i 


Hi 









if 








F 








3 


| Preparation of the sera. serum whose sensitising 
bon the bacteria it ix desir ih 

A“ blood capsule” is made hy drawing a prc 
ll tubing into the shape shown in Fig, 47, the part not 
inch in length. Tt is comsenen ve 





















ia 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 extremitics are broken off, the finger ine, and bloed 
allowed to pass into the capsule through the bent limb till 
the capsule is about half full, The air remaining in the capsule 
ia rarefied by passing the straight end through a flame and 
then scaling it off, “hy 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, Tt ia 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 instrament, 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 ther. This is done by taking 
4 plece of quill tubing and drawing it 
out to a capillary point so as to make 
& 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 
4 an oil pencil. From the wate 
ey aenaine mt: containing the soparated leucocytes u 
rane. portion is sucked up to the mark and 
then an airbabble js allowed to pass 
in, A similar portion of the serum ia drawn up, and then another 
air-bubble, and finally a similar portion of the bacterial emulsion, 
The three droplets are carefully blown on to » slide and are 
thoroughly mixed with one another by being alternately 
drawn up into the tubo and oxpellod everal times, The 
mixturo is thon drawn into the tube and the end sealed off in the 
ubber nipple is removed and the tube placed in the 
incubator at 37° for fiftecn minutes. A slide is now prepared 
by rubbing it once or twice with very fine emery paper 
(No. 000) and thoroughly wiping it. is ix a procedure 
adopted by Wright to caus» an evenly distributed film to be 
made. ‘The tube being romoved from the incubator and the 
end#broken off, its contents are again inixed by expelling 
and ‘drawing up into the tube, A minute droplet is placed 
‘on the prepared slide, and by means of tho 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 (g.v.) or with carbol-thionin bing, In the 
former case no fixation is necessary, in the latter it is usual to 
fix in saturated perchloride of mercury for 1} 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 
merenry, washed thoroughly, stained with carbolfuchsin as 
teaual, decolorised with 25 per cent sulphuric acid, cleared with 
4 per cent acetic acid, counterstained with watery solution of 
mothylene-blue, and dried, 

In applying the tochniqne two preparations are made, in both 
of the same emulsion and the same Ioucocytes 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 hormal porson—usually the observer 
Himself. Each of these in now examined microseopically with 
@ movable stage, the number of bacteria in the protoplasn of 
at Teast 50 perme fcenclianed leucocytes is counted and an 
average per yte struck ; the proportion which thia average 
in the case of the abnormal serum bears to the average in the 
preparation in which the healthy serum was used, constitutes the 
opsouce inder,—that of the healthy eeram 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. 





Geserat BrcrrrtotocicaL DiaGwosr. 


Under this heading we have to consider the general routine 
which is to be obsarved by the bacteriologist when any material 
i submitted to him for examination. ‘The object of euch 
examination may be to determine whether wny organisms aro 

5, and if 40, what organisms ; or the bacteriologist may 
simply be asked whether a particular organism is or ix not 
present. In any case his inquiry mnst consist (1) of a micro 
feopic examination of the material submitted ; (2) of an attempt 
to idolate the organisms present ; and (3) of the identification of 
the organisms isolated. Wo must, however, before considering 
thens points look at a matter often neglected by those who seek 
a tuctoriological opinion, viz. : the proper methods of obtaining 
ahd transferring to the bacteriologist the material which he ix te 
Be asked to examine. The gencral principles here are (1) that 
‘every precaution must be adopted to prevent the material tow 





6 BACTERIOLOGICAL DIAGNOSIS 
es with extraneous oe Pasaaivs ae 


the fy aad (ay fab whe lester Hatskgit ebtsle tha neietal 
‘ue possible after it has teen removed from its natural 





ecru 
The sources of materials to be examined, even in patho- 
logical tacteriology alone, are of course xo vuried that we can 
but mention a few examples. It ix, for instance, often necessary 
to examine the contents of an abscess. Here the akin must be 
carefully purified by the usual surgical methods; the knife used 
| for the incision is preferably to be sterilised by boiling, the first 
ut of the pus which eseapes allowed to flow away (as it might 
containing some of the antiseptics used in the 
parifleation) and a Tittle of what snbsequently escapes allowed 
to flow into a atorile toat-tube. Tr tert-tubes 


ordinary test-tube may be » quarter filled with 
water, which is then well boiled over a spirit 
lamp, Tho tubo is then emptied and pl 
mitha plug of cotton wool, the outside of w] 
has been xinged in w flame, Small stoppered 
bottles may be sterilieed and used in the mame 
way. A discharge to be examined nay be so 
small in quantity ax to make the procedure 
oseribod impracticable. Tt may be caught on 
n piece of storile plain gauze, or of plain ab- 
sorbent wool, which is then placed in w 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 
antisoptic impregnated in it, ax the Jatter may 
Xcill the bacteria presout and make the obtaining 
of cultures impossible, 
icaperibggted Fluids from the body cavities, urine, ete. 
anit pipette w- may be spoured with aterile pipettes, ‘To make 
Muafitidccomats, 016 of these, take nine inches of ordinary « _ 
ing bacteria, gliss-tubing, draw out one end to a capillar 
diameter, and place a little plag of cotton weal 
in the other end. Invert this tube through the eotton plug of an 
ordinary test-tube and sterilise by heat. To use it, remove 
test-tube plug with the quill tnbe in its centre, anck up some 
‘of tho fluid into the latter, and replace in ite former position 
the ipsitabe (Fig, 48). ‘Another method very convenient for 
tor make two constrictions on the glass tube at 


















ROUTINE EXAMINATION OF MATERIAL 117 


suitable distances, according to the amount of fluid to be taken. 
‘The Guid is drawn up into the part between the constrictions, 
but x0 as not to fill it completely. The tube is then broken 
through at both constrictions and the thin enda are scaled by 
‘ing in a flame. 

organs to be examined should, if possible, be obtained 
whole. They may be treated in one of two ways 1. The 
warface over ono part about an inch broad is seared with a 
eautory heated to dull red heat, All superficial organisms arc 
thus killed. An incision is made in this seared zone with a 
sterile: sealpel, and amall quantities of the juice are removed by 
4% platinum epud to make cover-glass preparations and plate or 
smear cultures. 2. An alternative method is as follows:—The 
surface is sterilised by vonking it well with 1 to 1000 corrosive 
eublimate for half on hour. It is then dried, and the capsule of 
the organ is cut through with « sterile knife, the incision being 
further deepened by tearing. In this way a perfectly uncou- 
taminated surface is obtained. Hints are often obtained from 
the elimical history of the case as to what the procedure onght 
to be in examination. Thua, as a matter of practice, cultures 
‘of tubercle and often of glanders bacilli ean be sasily obtained 
only by inoculation experiments, ‘Typhoid bacilli noed hardly 
be looked for in the fwces after the first ten days of the disease, 
and soon, 

Procedure im Bacteriological Examination of 
Material.—Ln the case of « discharge regarding which nothing ix 
kaown the following procedure should be adopted :—(1) Several 

lass proparations should be made, One onght to be 

with saturated watery mothylene-blue, one with a stain 
containing « miordant such as ZiehlNeelsen carbol-fuchsin, one 
by Gram’s method. (2) (@) Gelatin plates should be made and 
kept at room temperature, (4) a series of agar plates or successive 
on agar tubes (p. 55) should be made and incubated at 

87°C. Method (+) of course gives results more quickly. If 
microscopic investigation reveals the presence of hweteria, it ix 
‘well to keep the material ina cool place till next day when, if 
m0, has appenred in the incubated agur, some other culture 
(eg. blood serum or agar sineared with Mood) may be 

If growth hna taken place, my ih the agur plates, 

‘ome with about 200 or fewer colonies should be made the chief 
Paxin for research. In such a plate the first question to be 
elaired up is: Do all the colonics present conaist of the same 
Bacterinm! The shape of the colony, its size, the appearance of 
the margin, the graining of the substance, its colour, ete., axe Wh 






Wiese 





118 GENERAL BACTERIOLOGICAL DIAGNOSIS 


to be noted. One precaution is 1 viz, it must be noted 
whether the colony ia 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 w 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 fame. It ix 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 bacilins, The 
colonies on a plate having been classified, 2 microscopic examina. 
tion of cach group may be mado by means of coverglass 
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 udded for aay reference, 

‘The general lines along which observation is to be made in the 
ease of a particular bacterium may be indicated as follows :— 

1. Microscopic Appearances,—For ordinary deseriptive pur- 
poses young cultures, say of 24 hours’ growth, on agar should 
be used, though appearances in older cultures, such as involution 
forms, otc., 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 mothod of grouping, (5) the staining reactions. Has it 
a capsule? Does the bnetorium stain with simple watery 
solutions! Docs it require the use of stains containing 
mordanits? How does it behave towards Gram's method? Tt is 
important to investigate the first four points both when the 
‘organiam is in the fluids or tisauos of the body and when growing 
in artificial media, os slight variations occur. It must also 
be borne In mind thar slight variations are observed according 
to the Ikind and consistence of tho medium in which the organism 
is growing. (6) Ts it motile and has it flagella? Tf so, how are 
they arranged’? (7) Dous it form spores, and if so, under what 
conditions us to temperature, ete. t 

[2 Growth Characteristics—Horw tho most important points 
on which information is to be asked are, What are the 
of growth and what are the relations of growth (1) to 














GROWTH CHARACTERISTICS 19 


temperature, (2) to oxygen? These can be answered from some 
the following experiments -— 
A. Growth on gelatin. (1) Stab culture, Note (a) rate of 
growth ; (6) form of growth, (a) on surface, (8) in substance ; (¢) 
‘or absence of liquefaction : (d) colour; (e) .¢0 OF 
ice of gas formation and of characteristic «moll ; (7) rolation 
to reaction of medium. (2) Streak culture, (3) w culture. 
(t) Plate cultures Note appearances of colonies (@) superticial, 
O Ly ad Growth in fluid gelatin at 37°C, 
B on agus at 37°C. (1) Stab, (2) Streak, Also 
on glycerin agar, blood agar, etc. Appearances of colonies in 
C, Growth in bouillon, (a) charactor of growth, (4) smell, (c) 


a 


= 


£ 


D. Growth on special media. (1) Solidified blood sernm. 
(2) Potatoes, (3) Lactose and other a media. Does fermenta- 
tion occur and is gas formed? (4) Milk, Is it curdled or turned 
sour! (5) Litmus media. Note changes in colour. (6) Peptone 
solution. Is indol formed t 

E, What is the viability of organism on artificial media? 

3. Rerults of inoculation expertiments on animals. 

By attontion to such points as these w considerable knowledge 
ia attained regarding the bactcrium, which will lead to ite 
identification. In the case of many well-known organisms, how: 
ever, a few of the above points taken together will often be 
mificient for the recognition of the species, and experience 
teaches what are the cssential points an regards any individual 
organiam. In the course of the systematic description of the 
pathogenic organisms, it will be found that all the above points 
‘will be referred ta, though not in every case, 


‘The methods by which the morphological and biological characteristics 
of any growth may bo clocrved havo already boen fully deaoribed, It 
faeed only bo pointed cut lero tbat in Siving descriptions of bactoria the 
teat core must be taken to state every detail of investigation, Thus 

any description of microscopic appearances tha age of the growth from 
which the pooparation was wazle, tho sodium ormployed, the temperature 
























BE which deroloyment took place must bo noted, ith the stain 

‘was used ; and with regard to tho latter it is always preforablo to 
employ one of the well-known st mbinations, sush as Léftler's 
methylene-blue. special caro yin stating the site of a 





Bacterium. Tho apparant «zn often 
Bal the etuin used noel the growth conditi 
umasurements of bacteria can only be made by preparing microphotor 

of a definite mgnification and measuring the sises on the 
Begatives. From those the actual sizes can caaily bo caloulatol In 
aseribing bacteria! cultures it must bo Loruein mind that the apyearancan 


slight warlations dependent 
i the culture. Accurate 











10 INOCULATION OF ANIMALS 


often vary with the age. Tt in suggested that in the case of cultures 
frown af from 36° to 37° C, the appearances betwoon 24 and 48 hours 
should be made the basis of desoription, and in the case of onltures 
groan betwoon 18° and 27°C. the appearances botwoen 48 and 72 hours 
Shoald bo employed. Tho culture Hulda used must be made up and 
heuteaised Uy the precise methods already desried, | The investigator 
mast give orery dotail of the methoda he has employed in onder that his 
observations may bo capable of repetition. 


TxoouLarton ov Animazat 


‘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 ans-very easily killed. Dogs are, as a rule, rather inansceptible 
to microbie discase, and the larger animals are too expensive for 
erdinary 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 varicties, and between 
the white and brown varicties 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, ench individual should be kept in a sepamte 
cage, as they show great tendencies to cannibalism, Of all the 
ordinary animals the most. suaceptible to microbie disease ix thie 
guinea-pig, Practically all inoculations are porformod by moans 
of the hypodermic syrings. ‘The best variety is made on the 
ordinary mode! with metal mountings, asbestos washers, and 
preferably furnished with platinuro-iridinm needles. Before use 
‘the syringe and the needle are sterilised by boiling for five minutes, 
‘The materials used for inoculation are cultures, aulinal exudations, 
or the juice of organs, If the bacteria already exist in a fluid 
thore is no difficulty. ‘The ayringe is moat conveniently filled out 
‘of @ 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 organixms growing on the surface 
of @ solid medium, cithor a little ought to be scraped off and 
shaken up in sterile distilled water or “85 per cent sult solution 
to makean emulsion, ora little sterile fluid is poured on the growth 
wnd the latter scraped off into it, ‘This fluid is then filtered into 
the test-glass through a plug of sterile gluss wool. This is casily 
wlfected by taking picce of in. glass-tubing 3 in. long, drawing 

‘ nents on anlioals, of vourse, counot be performed in this country 
Without a leone granted by the Home Sccrctary. 
































INOCULATION OF ANIMALS 1 


oue cd out to a fairly narrow point, plugging the tube with 
Baie aera alinge Gan alot ware tba xaromtng commeaven sil 

by heat, By filtering an emulsion through such a 
pipette, i 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 im a sterile porcelain or metal erueiblo with a 
little sterile distilled water, by means of a sterile glass rod, and 
the emulsion filtered as in the last cxse. 

‘The methods of inoculation geuorally used are: (1) by 
searification of the skin; (2) by subcutancous injection ; (3) by 
jutmaperitoncal injection ; (4) by intravenous injection ; (6) by 
injections into special regions, such as the anterior eharnber af the 
eye, the substance of the lung, ote. Of these (2) and (3) are 
snost frequently used, When an anwstheticis to he administered, 
this ix conveniently done by placing the animal, along with a 

ieee of cotton-wool or sponge soaked in chloroform, ander 
jar or inverted glaas beaker of suitable size, 

1. Scurisicution,—A few parallel scratches ure nade in the 
skin of the abdomen proviously cleansed, just sufficiently deep 
to draw blood, and the infective material ie rubbed in with a 

inum eyelet. ‘The disadvantage of this method is that the 

tion is easily contaminated. The method ts only 
nahi A hargod 

2. Sutcutancous Injection—A hypodermic ayringe is char; 
with the fluid to be inoculated. ‘The hair is cut 
off the part to be inocalated, and the skin 
purified with 1 to 1000 corrosive sublimate, we 
The skin is thea pinched up and, the needle 








performed by means of a sj 
The needle is curved, and has its opening not at 

the point, but in the side in the middle of the 

arch (Pig. 49). Tho hair over the lower part 

‘of the aldomen is cut, and the skin purified with 

fn sntimptic. The whole thicknos of the ob- Fe 4 —Hollow 
dominal walls is then pinched up by an assistant, ueedie with 
Fetween the forefingers and thumbs of the two — taster! aperture 
Hands, The needle is then plunged through (02) tre 
tho fold thus formed, ‘The result is that the — Sonlatio 
fiole ip the side of the needle is within the 

Widominal cavity, and the inoculation can thos be made. 
Titeaperitoncal inoculation can also be practised with ow 











4 igject tort. 
one of the suriewlar voing 
ekin is purified, and tho vein made 
‘between the point of inoculation and the 


occurs in subeutaneots 
firat laid bars by anipping the skin over it, ‘The needle is then 
introduced. 

5. Inoculation into the Anterior Chamber of the Eye—Local 


anioathosin is established by applying a few drops of 2 per cent 
solution of hydrochlorate of Reais The a is fad by 
pinching up the orbital conjunctiva with a pait of fine forceps, 
‘und the edge of the cornea twing perforated by the hypodermic 
needle, the injection is easily accomplished, 

Sometimes inoculations are made by planting small pieces of 
pathological tisanes in the subcutaneous tissue. ‘Thisis especially 
done in the cage of glandors and tubercle, The skin over the 
back is parified, and the haircat, A small incision is made with 
asterile knife, and the skin being separated from the subjacent 
tissues by means of the ends of a blunt Peas of forceps, a little 
pocket is formed into which a piece of the suspected tissue ix 
inserted. ‘The wound is then closed with suture, and collodion 
is applied, In the case of gninea-piys, the abdominal wall i to 
be proferred as the «ite of inoculation, as the skin over tho back 
is extremely thick, 

Thjections are sometimes made into other parts af the body, 
«g. the plenne and tho cranium, It i# unnecossary to describe 
these, as the application of the general principles cinployed above, 

ther with those of modern aseptic surgery, will sufficiently 
‘guide the investigator ax to the techniques which is requisite. 
After inoculation, the animals ought to be kept in comfortable 
which must b pable’ of easy and thorough disinfection 
Hibesquantly. For this parpose galvanised iron wire cages are the 
‘best. ‘Thoy can oasily be sterilised by boiling them in the lange 
“fiskokettle which it is uscful to have in a bacteriological laboratory 
‘such a purpose, Tt is preferable to have the cages opening 





COLLODION CAPSULES 128 


from above. Otherwise material which may be infective may be 
soratched out of the cage by the animal The general condition 
bs aniatal sto be otourved, howe far Ut difoc fom the vormal, 
whether there is increased rapidity of breathing, ete. The 
temperature is usually to be taken, ‘This ix generally done yer 
rectum. ‘Tho thermometer (the ordinary 5 min, clinical variety) 

ae smeared with vaseliv, and the bulb inserted just within 
sphincter, whore it is allowed to remain for a minute; it is then 
| a ‘woll into the rectum for five minutes. If this precaution 
4 reflex contraction of the vessels may take place, 

which is likely to vitiate the result by giving too low a readi 
Collodion Capsules.—These have been used to allow ‘tho 
sojourn of bacteria within the animal body without their coming 
into contact with the cells of the tissues. Various substances 
in solution cin poss in eithor direction through the wall by 
diffusion, but the wall is impermeable alike to bacteria and 
‘The following method of preparing such capeules is 
that of M‘Rae modified by Harris. A gelatine capsules, such a 
iw used by veterinary rurgeons, is taken, and in one ond there 
in fixed a small piece of thin glass tubing by gently heating the 
glass and inserting it, The tubo becomes fixed when quite cold, 
and the junction is then painted round with eollodion, which 
ik allowed to dry Meet The bore of the tubing is cleared 
of any obstructing gelatine, and the whole capsule is dipped into 
a solation of collodion a0 as to coat it completely, ‘The callodien 
He allowed to dry and the coating ia repeated ; it is also advise 
able to strengthen the layer by further painting it at the 
extremity and at the junction. ‘The interior of the capsule is 
thon filled with water by a fine cay vist! pipette, and the capaulo 
is placed in hot water in order to Nauety ihe golatino, whiek can 
be removed from the interior by means of the fine pipette. ‘The 
‘40 is filled with bouillon and is placed ina tube of bouillon. Tr 
is then sterilised in tho autoclave. A small quantity of the 
bouillon is removed, and the contents are inoculated with the 
Inoterium to be studied or an emulsion of the bacterium 
is added, ‘The glass tubing is scized in sterile foreepe und i 
gealed off in a small flame a short distance above the junction. 
Whe closed sac ought then to be placed in a tube of sterile 
bouillon to test its impermeability. The result is satisfactory if 
0 growth cours in the surrounding medium, ‘The ee with ite 
eoutents can now be transferred to the peritoneal cavity of an 

unimal. 











Antopsies on Animals dead or killed after Inoculation. — 
These should be made as soon as possible after death. is 


ee 








1m INOCULATION OF ANTMALS 


necessary to have some shallow hs, constructed either of 
metal or of wood covered with metal, conveniently with sheet 
Jead, and having a perforation at cach corner to admit a tay 
strong cord. ‘The animal is tightly stretched out in the trough and 
tied in position. The size of the trough will therefore haye to vary 
with the sizeof theoutstretched body of the animal to be examined. 
In certain cases it is well to sonk 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 
dixinfects 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 necesary are 
scalpels (preferably with metal handles), dissecting force} 
seimors, ‘Thoy are to be sterilised by boiling for five mi 
‘This is conveniently done in one of the small portable st 
used hy surgeons. ‘Two xets at Ioast ought to be used in an 
autopsy, and they may be placed, after boiling, on a sterile glass 
plate covered hy a bell jar, It is also nccossary to have a medium- 
sized hatchet-shaped cautery, or other similar piece of metal, To 
is well to have prepared a few freshly-drawn-out capillary tubes 
stored in a sterile cylindrical glass vessel, and also some larger 
sterile ghiss pipettes The hair of the abdomen of the animal ix 
removed. If some of the peritoneal fluid ix wanted, a band 
should be eauterised down the lineaalba from the sternum to the 
pubes, and another at right angles to the upper end of this ; an 
ision should be inade in the middle of these hands, and the 
abdominal walle thrown to each side. One or more capillary 
tubes should then be filled with the Huid collected in the flanks, 
the fluid being allowed to run up the tube and the point sealed 
off; or a Jurger quantity, if desired, is taken ina sterile pipette. 
Jf peritoneal fluid be not wanted, then an incision may be made 
from the episteraum to the pubes, and the thorax and abdomen 
opened in the usual way. The organs ought to he removed with 
another set of instruments, and it ix convenient to place them 
pending examination in deep Petri’s capsules (sterile), It is 
gencmlly udyisable 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 
imiddle of the part seared, and remove some of the blood witha 
capillary tube for fature examination, or, introducing a platinum 
ae. inoculate tubes and make cover-glasa preparations at once, 
examine any organ, scar the surface with acantory, cut into it, 
und inoculate tubes aud make film preparations with a platinum 
Toop. 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. 


Ir 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. 


Ar. 


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 suificient to expose gelatin plates for 
defini :imes 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 @ gelatin 
plate. 











126 


METHODS OF EXAMINATION 127 


More complete results aro available when souje mothod is by 
‘which the bactoris ina giv ity of a 6 oldost 
thod employed, and one is atill ueed, ie that of Home. Tho 





mel 
Sey ix shown in Fig, 50. It consists of o cylindrical tube « about 

inches long and 2 inches in diameter. At ono and this ix closed by a 
rubber eork having « pivce of quill tubing, J, passing through it and 
projecting somo distance into the interior. For use the tube is sterilised 
ma tall “Koob,” and then « quantity of Peptowe gelatin, auliclent 10 
cover the whole interior to the thickness of an omlinary gelatin plate, i 





‘tubing into the lumen of 
the large tube. A plug 
ote op) is now 
‘in the outer en: 

ofthe quill tubing, Over 
‘the other end of tho large 

ix tind a shvet of 
rubber having a hole 
about a quarter of an inch 
in diameter in its cvntro, 
thie i 
eos of similar but une 
foratod sheet rubber. 













ja em 
ig rollod. after the manner 
an Ramaroh’s tube 
(ee.), till the geletio 
is set as a layer over 
its interior, a is then 
horizontally on 
tripod asshown. The 
other part of tho appar- 
Atug i an aspirator by 
means of which » known 
tity of air can be 
fin contact with Fra, 50.—Hame's tubo, mounted for nse. 

the gelatin, It consints 
Sf two conical glass flasks connected by means of 2 tube which pases 
Hinongh the cork af each down to the bottom of the flask. When this 
tube is filled with water, it, of course, can act axa syphon tube between 
volumes of water in the flasks, Such a syphon eystem being established, 
the levels of the water are marked on tho flasks, and to one @ litre of 
water In added, and by dopressing flask 4 tho whole litre can be get into 
Stand tho connesting tube cis thon clamyed. Tho two flasks are then 
Renneited by 2 ruber tote with the tube, tho clamp on « i opened, 
nd the passing of a litre of watar into @ will draw a litre of air throagh 
Hie golatin tule, when the outer rubber sheet is removed from the end 
BidthoclampAopencd, By disconnceting at g and reversing the ayphon 



























(snothor litre can be sucked through, and so any desired quantity 
fair can be brought in contact with the gelatin. The ought net 
Ho be more than one litro in two minutes, mpd im such a came YeMXwentig 





128 BACTERIA IN AIR 


all the organisms will be found to have fallen aut of the air on to the 
gelatin in the coures of their transit, ‘Thin fact oan bo toxtod 
interposing between the tube a and the aspirator a second tube 

in the samo way, which ought, of course, to show no growth. When 
forty-eight hours at 20° © or four days at lower temperature have 
clapredd, the colonies which develop in a may be counted. ‘The dit 
advantage of tho method is that if particles of dust carrying moro than 
‘one bacterium alight on tho gelatin, these bacteria develop {x one colony, 
and thus the enumoration nefulte may bo too low ; difficultios may ala 
arise from liquefying colonies developing in the tpper part of the tuba 
and running over the gelatin. 

Petri's Sand-Filter Metiod—A glass tube opou at both ends aud 
about $4} inches long and half an inch wido, ix taken, and ia itx coutra ix 

Placed a transverse diaphragm of very fino iron 

ganzo (Fig: 61, ¢); on each sido of this is placed 

Ff Some fine quarts sind which Tas boon well washed, 

dried, and burned to remove all impurities, and this 

4s kept in position by cotton plugs, The wholo ix 

sterilised by dry heat. Ono plug is removed oud a 

2 atorile rubber cork, ¢, fusorted, through which m tube, 

‘ di, passos to an exhausting apparatus. Tho tubo in 

‘then clamped in an pt position in the atmo~ 

sphere to be examined, with the romaining plug, 

appermost, Tho latter ie removed and the ait 

‘sucked through, Difficulty may be experienced 

from the resistance of the sand if quick filtration be 

4 attompted. The beat means to adopt is to use an 

sinpump—the amount of air drawn par stroke. of 

which is accurately known—and have a manometer 

(an in Wig. $1) nuerponed between the tuba and the 

pump. Between each two strokes of the air-pamp: 

the mercury is allowed to return to zero, After 

+-® the required amount of alr has passed, tho sand a 

is removed, and is distributed emong a number of 

@ —sturila gelatin tubex which aro well shaken ; plate 

cultnres are then made, and whon growth has oooirred 

vetn's the colontas are onumerated.; the sand 6 {s shmilatly 
» —Vetris tryated and acts ay a control. 

saad filter: When it is necossary to examine air for partioular 

organisms, spocial methods must often be adopted 

‘Thus in the ease of the mspectod presence of tubersle bacilli a given 

quantity of air ig drawn through a small quantity of water and then 

injected into a guinea-pig. 

Tt must be admitted that comparatively little information 
bearing on the harmlessness or harmfulnoss of the air ix obtain- 
able by the mere enumeration of the living organisms present, 
for under certain conditions the number may be increased by 
tho presence of many individuals of a purely non-pathogenic 
charactor, The organisms found in the air belong to two 
groups—firstly, a great varicty of bacterin; secondly, yeasts and 
the spores of moulds and of the lower fungi. With regard to 
tho spores, the organisms from which they are derived often 
































PETRI'S SAND-FILTER METHOD 129 
Sarees sis cea from which are thrust into the 


is 
substratum that the organisms become free. The entminee of 
bacteria into the air, therefore, is astociated with conditions 
which favour the presence of dust, minute droplets of fluid, ete. 
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 


together of a number of healthy human beings. On 

the other hand, there is no doubt nih ar germs can be 
dimeminated by means of the air. spore of this 
be heen shown experimentally by iin the mouth with the 
b, prodigiosus, which is easily reengnised by its brilliantly 
‘coloured colonies, and thon studying its subsequent distribation. 
Most important here is the infection of the air from sick persona, 
‘The actions of coughing, sncexing, speaking, and even of deep 
breathing, distribute, often to m considerble distance, minute 
‘of secretions from the mouth, throat, and nose, and theso 

may float in the air for a considerable time. Even five hours 
nat ‘an atmosphere has been thus infected evidence may be 
found of bacteria still floating free. Before this time, however, 

+ most of the bacteria have scttled upon various objects, where 
ters rapidly dry, and aro no longer displaceable by ordinary le 
diseases of known etiology where infection can 

anes Sel diphtheria, influenza, pneumonia, and phthisis ; 
and here also probably whooping-<ough, typhus fever, and measles 
‘are to be added, though the morbific agents are unknown, Tn 
the ease of phthisix, the alighting of tubercle bacilli has been 
een on cover-glasses hold before the mouths of patients 
While talking, and animals made to breathe directly in front of 
“mouths of such patients have become infected with 
jlosis. Apart from direct infection from individuals, 
however, pathogenic bacteria may be spread in some cases from 
ing of infected water, as from a sewage outfall 


‘Thie llity has to be recognised especially in tho casea of 
= chelom. Besides infection through fuid yartieveny a 





180 BACTERIA IN AIR 


infection can be caused in the air by dust coming, say, from 
infected skin or clothes, ete, Pli in dealing with this 


tho particles alight they cannot be displaced by currents of air 
except when these are moving at, at least, 5 metres per 

but the brushing, shaking, or beating of objecta may, of course, 
diatribute them. In the case of the finer dust the particles will 
romain for long suspended, and when they have settled can be 
more eaaily displaced, as by the waving of an art, breathing, 
ets, With mgand to infection by dust, a most important factor, 
however, is whether or not the infecting agent can preserve its 
vitality ina dry condition. In the case of a sporing onganisin 
sich as anthrax, vitality is preserved for long periods of time, 
and great resistance to drying ia also possessed by the tubercle 
and diphthoria ; but apart from such cases there is little 
doubt that infection is usually necessarily nasocinted with the 
transport of moist particles, and ix 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- 
reales of the latter diaewoc, which have apparently been sproad 
by dusty winds, although we have the fact that the typhoid 
hacilins 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 tho case of tho soll and of water, presently to be described, 
attompts have boon made to obtain indirect evislonce of the contamination 
of the alr by man. ‘Thux Gordon has shown that certain «treptococol 
‘are common in the mulira : these resemble the streptococeus pyogenes, tnt 
fare relatively non-pathogenic, grow well at 37" G. and under anaerebie 
conditions, cause clotting and acid-formation in litmus milk at. 97% and 
in deutral-red modia havo an action resembling that of b, coli. ‘These 
characters verve, acoording to Gordon, to dilforentinte organisms of 
human origin from ordinary streptococci eecurring in the air and which 
he states grow better at about 22° C., aro facultative anaorobes and 
do not produce the changes in milk and in neutral-red media, ‘Thos 
‘the finding of streptocoos! of the first group in plates exposed to air 
‘would indleste that « human source was jaobable, and, if tho obvervation 
‘werd made on air from tha neighbourhood of a sick person, that risk of 
the dissemination of disease germs was prevent. The value of this ae a 
‘Practionl mothod has yet to be determined. 

















BACTERIA IN SOIL 131 


fnvestigation of the bacteria which may be found in the 
‘undertaken tom vroa poat of view. Information 


desired as to the its composition undergoes by a 
action, the result ol wee eatied an pian i 


ues 
iF 
= 
fe 
if 
Hi 
ul 
a 


SEE 
Huy 
HIE 
iMtliG 
Free 
Fz 

i fe 
fetal 
Esty 
. a 
3 fas 
seer 


papers that the following account is largely taken, 


NS ee of rs examination LO oe 
far from surface, Houston recommends tin troughs 10 is, by 8 in, and 


ited at one enirralt te be wrapped in layers of atorilised 
earset ce aaweeaaas sal can be well 
ited psu sample securvd and placed in m sterile test-tabe for 
ag soon as convenient after collection. IF samples aro to 
utes ‘at some depth beneath the varios, tka 
0 variotieg have bona dev be wed. ‘The general 
these is that of = gigan' fet stoutly Ler lar 
rs the point of the fsttsent tho shalt has fa it's hollow chamber, 
ing lateral door in this can be opened and shut by * mechaniam 
salict gna andi, The chaser Deing ateriliend and olowsd, the 
{astrament is bored to the required depth, the door is slid back, and by 
i devices it is effected that the chamber is filled with earth ; the 

‘ia reclosed and the {nstrament withdrawn. 
nny, veil tho two important lines of inquiry ar fret as tothe tetal 
of onganiams (axoally reckoned pur gramme of th fresh sample), 
seonily as to the varieties of organi ‘The number of 

nt iu a soil is often, hor 

to submit only a fraction of a 
smuployed is to woigh the tabe containing the sell; shake out an 
of about the nize of a boun into a litre of dintiSled water, and re- 


istrumNnt 




















‘The amount placed im the water is distributed ay 
ly ax possible ig, and, if necessary, by rubbing down 
‘sterile ‘quantitins moasured from s gradaated 





for the investigation, For estimating the total umber of 
present ia the portion of soil used, small quantities, aay “I ec, 
Tine, of the fluid aro wdded to melted tubes of ordinary alkaline 





after boing shaken, the gelatio in gated, iumakatvel xt 





132 BACTERIA IN SOIL 


22° C,, ond the colonies aro counted as loto ox the liquefaction, which 
always osettra round wom of them, will allow. From those surabors the 

‘nimber of organiama preswnt in the ammount of sail originally present 
an be ealoalated, 


‘The numbers of bucteria in the soil vary ery much. Aceord- 
ing to Houston's results, fewest occur in unenltivaterl sandy soils, 
these containing on an average 100,000 per gramme. Ponty soils, 
though rich in organic matter, also give low results, it being 
possible that the ncidity of such soils inhibits free bacterial 
growth. Garden soils yield usually about 1,500,000 bacterin 
per grams, but the greatest numbers are found in soils which 

ave been polluted by sewage, when the figures may rise to 
115,000,000. In addition to the enumeration of the numbers 
of bacteria presont, it is a question whether something may not 
be gained from a knowledge of the number of spores prosent in 
a soil relative to the total number of bacteria. This isu point 
which demunds further inquiry, especially by the periodic investi- 
gation of examples of different classes of soils, The method is to 
take 1 .c. of such a soil emulsion as that just described, add it 
to 10 cc of gelatin, heat for ten minutes at 80° C. to destroy 
the non-spored bacteria, plato, incubate, and count as before. 

Besides the enumertion of the numbers of bacteria present in 
a sail, an important question in its bacteriological examination 
ios in inquiring what kinds of bacteria are present in any pars 
ticnlar caac. tically this resolves iteclf into studying the 
most common bacteria prevent, for the complete examination of 
tho bactorial flora of any one samplo would occupy far too mach 
time, Of these common bacteria the most important are those 
from whose presence indications can be gathered of the con- 
tamination of the aoil by sewage, for from the public health 
standpoint this ia by far the most important question on which 
Dactoriology can shed light. 















horacteristio appearanos. At fret under a low 
anes of laments throwing out irrogular shoots 
from tha centre, jr to the naked eye thew appear ta be in the 
form of thick threads like the growth of & mould, ‘They rapidly spread 
over the surface of the medium, and the whole resembles a picee of wet 
teased-out cotton wool. ‘The gelatin is iquelied. 

YAricet.—Of those several kinds are common in the soil, ‘The 
ordinary cladothriz dichotoma is among them. This crganiém appears 











BACTERIA IN SOIL 133 










1s colourless tlocouten' hwith an opaque centre, and can be seen 

‘under the microseope to tent ont into the medium ay iy branched 

sat Sete ter 

consist of ise rods may divide wt sny 

pins, and be pushed along the sheath. 
moe the sheath rt Ute extraion of thew 

dis 

Te 

forming lourton describes as oecurring in 





rove characters pps as bro 

ne iffusing a bismarck-brown pigment 

ote. 

made with an ordinary Geld soil will, howevor, 
tid bacteria: 








str ns Thi f the 

fetound i We chapter on Typhiold Fever ; of the third in Chap. VI. 

Bate detection of these bactoria Houston recommends the following 
‘The B. col group, A third of a gramme of soil is added to 10.0.0. 

Paenni bch (oes chapter 00 Typhold evar) und, focubatad a 37°C. 


‘rll grow, ao that if aflee tmunty-four hours a tarbidi , 
Strap of the latter may: be stspeeted to be preseut, In auch «cate 6 


ae disti if, and of 
‘one or two loopfiula are xpread bs ‘of & solid plate of 


a mnail 










rrogenes. "To searoh for this organism 
ly distributed in 100 c.c. sterile distilled 
d *O1c.c. is added to cach of three st oe 


A£97"C. for twenty-four hours. If thn elaraoteristic appear- 
iabtea seen In such cultures of the b. enteritidis (g.e,) are developed, 
peeeey nity safely be deduced thas it is this organism which has 

i, 








 Sereptococes. The method here is to pour ont a tube ef ager 
yeale, and when it has solidified to spmmad out *l ac. of 

at Meiahutan of veil over it and incabste at 37°O, for bwents-four hoare 
eieiieeparatare many of tho non-pathegents bacteria’ grow with 
f, and thus the number of coloules which develop is relatively 

sma fea having appearances resembling those ef thy atreptoceocas 
18 (g.0.) can thus be investigated. Much work has bees devoted 
Hothe queetion of these fecal atreptoeocei preventing wyerifia dhaxuAarn sey 





= 





134 BACTERIA IN SOIL 


which they could bs diforentisted from othor streplosoesl. No defikte 
Tevults hava ay yet Ween obtained, Houston gives as the gen 
obaraatrs of those engantams that the ‘ually grow ++ Saat 
Bat they produce uniform turbidity in broth, that they they ae cee Or 
ud clot 48 tition milk at 31" Cand that ‘they are non-pathogenie 
mice. ‘The important point Is to reoognise that streptaoocel of 
ordinary types exist in great numbers in human faeces, and. that wien 
nag seas Sena ose nahin pnnosted ohn Sedation oP 
stroptocooat atrengtheus the euspicion, 


We may now give in brief the results at which Houston has 
arrived by the application of thete methods. First of all, un- 
cultivated soils contain very few, if any, representatives of the 

6, mycoides, and thia is also true to a less extent of the 
cladothrices, Cultivated soils, on the other hand, do practically 
always contain these organiams, With regard to the 4. cold ite 
presence in a soil must be looked on as indicative of recent 
pollution with excremental matter. The presence of b. 
enteritidis ta aleo evidence of gneh pollution, but from the 
fact that it is a sporing organism this pollution may not have 
been recent, With, regard to the sreptococe’, on the other 
hand, the opinion ix 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 theso 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 
snbject 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 havo 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 theae 
organisms when they are present in such bacterial mixtures as 
naturally occur in the soil. Now there is evidence that bacteria 
when growing togethar often influence ench other's growth in ap 
unfavourablo way, so that it is only by studying the organiama in 
question when growing in unsterilised soils that information can 
be obtained as to what occurs in nature. For inataneo, it has 
boon found that tho b, typhosus, when grown in an organically 
oe soil which has been sterilised, can maintain ite vitality 

it fifteen weeks, but if the conditions occurring naturally be so 
Jar imitated by growing it in soil in the prosence of a pare culture 














Me 


BACTERIA IN WATER 135, 


ee ea area wl ee 1 Soce i o 
bacillus, sometimes the soil bacterium in the course of a 
Reinvents few days, disappears. Further, the char- 
acter of the soil exercises an important effect on what happens ; 
for instance, the typhoid bacillus soon dies ont in a virgin sandy 
aisiice: ahier't is tee cay mated peewee In experiments 
made by sowing cultures of cholera and diphtheria in plots in a 
fot ie wan fonnd thas aftr, at the longo, forty days they ware 
no longer , Further, it is a question whether 
ordinary disease organisms, even if they romain alive, can 


feeteesie the capacity for remaining ae escent condition of 
Potential pathogenicity is, Se much greater, The most 
important. ile to be deduced from these riments is that 
‘the ordinary conditions of soil rather tend to be unfavourable 
to the continued oxiatence of pathogenic bacteria, eo that by 
natural processes soi] tends to purify itself. Tt 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 examination of water thre lines of 
inquiry may have to be followed. Pirst, the number of bacteria 
ic centimetra may be estimated. Second, the kinds of 
invent may be investigated. Third, it may be necessary 
Gukis particular organism is present, and, if so, in what 
mmumber per e.c. it occurs 





Mothoda —In the two first came a small quantity (5-1 ec.) ix takon 
ean mate Piestbe ‘and added toa tube of golatin, wl iol it 
at the reom temperature. In the case of 
a the water should be allowed to run for several hours 
Before the samplo is taken, a» water standing in Fine in. house is under 
welt favourable conditions for ee of bacteria taki 























if thie precaut be not edopted an altogether errones 
bee frresent may bo obtained. In the case of the es 
fiver wate the platas caught to be prepared on the poe; st any 
ietyths time clsaping beiween the sowple bring taken and thw glates 


jepured inust be as short as posible, otuerwise the bacteria will 

ly, and again an crroncous ides of thelr number be obtained. 
When samples havo to be taken for transport to the laboratory, these 
fre best collected in four-ounos, widemouthed stoppered bottles, whiok 
mma to be atertlised Uy dry heat (tho stopper mut be sterilised separately 
from the bottle and not {nsertod in tho latter till both are cold, other cid 
FEwill bo vo tightly beld a» to make removal rery difficnit|.” [a ual 
‘Bich w bottle it is best to immerse it in the water ani Yawn oave en 











136 BACTERIA IN WATER : 


stopper with forcopa. Cary must be taken not to touch the water. 
ta the cogptatle oaller covering fk eoutaine aa anager 


‘The bottles ought to be packed i ice and tad plates trast be 
Te a retire rg ee 
view is to determine number of bacteria per cuble centimetre, it in 





important to note that water Lacteria grow at very varied rates, and 
therefore it is well that tho sume time should always elapse before the 
lly. allowed ts forky= 







ig! oni i amber 
of colonies which derolop.. A slightly greater degree of alkalinity’ than 
peptone gvlatin, ag ordinarily pry Yah youemnoraeatl an fretaeel 
leyree as that caused by the addition of “OL grim NaCO, to 10 cc 
Peptone gelesin—nill gives ureater yield of colenis that the ondisney 
‘Again, the natural temperature of the growth of water banteria 
fo temperate lituates is comparatively low, buing not often above 18°C. 
and, on aeconnt of this, gulatin suggests itself as tho most suitable 
medium. ‘This can be sven by comparing the growth on an agar plate 
inoculated with » given quantity of water, ancl incubated at 37° C., with 
fhe growsh on a prociely similar gelatin plate jnoubated at 20° Cy as 
will be fownd that many more colonies have developed on the latter. 
‘This fast aay be taken advantage of when pathogonic bacterin are being 
songht for in a watur, Tho latter axaally grow wel! at 37° O., and thas 
Af agur pintos be used the search may he fheilitated. Apart from the 
difference of incubation temperatures, however, fx such a caso. as that 
citol, iis probable that agar in loss suitable inediun than gulatin for 
the growth of water bacteria, for in plates incubated at the rame 
temperature the colonins which’ grow on the agar aro often fower than 
those on tin. Probably mo one aiodtom will support the growth. 
of all the organisms present in a given sample of water, and ander 
certain ciroumstances special media must therefore be used, Thas 
Mansen found that in testing waters to be used in brewing it was 
advisable to have in the medium employed same sterile wort of beer, 80 
that tho organisms ia tho test exporiments should be jrovided with tho 
food materials whioh would bo prosent in the commercial uso of the water, 
Manifastly this principle applies gonorally in the bastoriological examina 
tion of wanars to bo used for Industrial purposes. 

In ordinary public health work i may be taken that the most 
frequent and important inquiry is directed towards tho presence. oF 
abssuoe of the b. coli and its congeners. Many methcds ate hove used 
but we considor that in which MacConkey's bile-salt media aro omployed 
tho most conveniont. For small qasntitins of water,—up to 1 ec,,—the 
satnplo is simply added to a Durhatn’s tubo of bile-aalt ylvcowe neutrals 
red broth and inoubated for 48 hours, When it is necessary to oxamino 
larger sarmples it is conveulent, es Savage recommends, to have the bile. 
salt broth made of double, treble, or quadruple ith usual strength, 
‘The water to be ¢zamined tv used wx the diluent by which the meditm 
is bronght down to the ontinary concentration, If gas forms, somo of 
thn coll group ate nimost certainly prasent. The organisms may bo 
plated out by amoaring a little of the broth om bile-sait agar for further 
isolation wud examination, 


























With regard to the objects with which the bacteriological 


examination of water may be undertaken, though these may 
be of a eclentific character, ‘uoually aim at contribut- 
ing to the settlement of questions relating to the potability of 
waters, to their use in commence, and to the effi ef pro- 
cesses undertaken for the purification of waters whieh have 

pollution. ‘The last of these objects is often closely 
associated with the first two, as the question so often arises 


E 
2 


whether & purification process ia €0 efficient as to 
water again fit for use, 
colle ae from any natural source contains bacteria, 
asin case of some artesian wells and some apri 
the numbers may be very amall, eg. 4 to 100 per oc. Toca 
snow, and ice there are often great numbers, those in the first two 
ced from the air. Great attention bas been paid to 
the rial content of wells and rivers, With regard to the 
former, precautions are necessary in arriving at a judgment, 
If the water in a well us been standing for seme time, 
multiplication of bascati may ima ‘alin ee sont oe 
ity, if practical 18 wel it to be pum ry 
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. ‘in, if the sediment of the well bas been stirred 
upa high value is obtained. Ordinary wells of medium depth 
contain from 100 to 2000 perce. With regard to rivers very 
varied results ure obtained. Moorland streams are usually very 
In an ordinary river the numbers present vary at 
clade ct lis reer, lidat Abe. provera fempentoas 
the presence or absence of decaying vegetation, or of washings 
from land, ond 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 
pring water entering, and very little water as washings off land. 
Tu the caso of other rivers the bacterin have been found to be 
fewest in winter. A great many circumstances must therefore 
he taken into account in dealing with mere cnumorations of 
water bacteria, and such cnumerations are only useful when 
‘they are taken stimultaneously over a streteh of river, with 
fal reference to the sources of the water entering the river. 
it is usually found that immediately below a sewage 
efluont the bacterial content rises, though in a comparatively 
short distance the numbers may markedly decrease, and it may 
be that tho river as far as numbers are concerned may appear 
to return to its previous bacterial conteat. The sumbers of 


—_ 





138 BACTERIA IN WATER 


bacteria present in rivers: 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 SoS Such a method is thus of great 
use in ostimating the efficacy of the filter beds of a town water 
supply. Thove usually remove from 95 to 98 per cont of the 
bacteria present, and a town supply ws it issues from the filter 
beds should not contain more than 100 bacteria per oc. , 
ia it found that the stor of water diminishes the number of 
bacteria present, The hi, counts of bacteria per cc. are 
observed with sewage ; for example, in the London sewage the 
numbers range from six to twelve millions, 

Much more important than tho 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 ne 
Europe ie concerned, lies in the fact that typhoid fever is #0 
frequently water-borne, but cholera and certain other intestinal 
diseases have a similar source. The search in waters for the 
organistie concerned in these diseases is a matter of the greatest 
difficulty, for cach belongs to a group of organisms morpho- 
logically similar, very widespread in nature, and many of which 
have little or no pathogonie action. ‘The biological characters 
of thoso orgunisma will be given in the chapters devoted to 
the diseases in question, but here it may be said that from 
the pablic 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 
bacterin can exist for some time in water—at least this 
hax been found to be tho case when sterile water haa boon 
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 anfely suid that by no known method ean the proven 
‘of either be demonstrated in the complex mixtures which occur 
in natare, With regard to the typhoid bacillus, of late the 
tondency has boon to seek for the presenes of indirect bacterlo- 
logical ‘evidence which might point in the direction of the 
possibility of the presence of this organism. The methods 
series and the lines along which such investigations have 
ono havo already been alluded to in connection with soil. 
The whole question turns on the possibility of recognising 
Iucteriologically the contamination of water with sewage. 
Klein and Houston here insist on the fact that in crude sewage 
the b coli or the mombers of the coli group are practically 





BACTERIAL TREATMENT OF SEWAGE 139 


forms a proportion of the total 

number of organisms pi then there is grave reason for 

a aarp ip sewage pollution. Hoon hls a tho arr 2 

jority of the coli onganiema in a wator approach to the typical 

reactions of coli the more a SeenaS ecs ae 
present. The reactions by him am, 

producti gelatin abake ion of iosoh 


Ss 
& 
& 
: 
3 
if 
se 
= 
2 


etl As tho b. tol italy Gieeratte nature, Klein 
and Houston hold that valuable anyy porting evidence i 


A further it hero is that it is well, whorever practicable, that 
the i Salih erilenteax' w tha’ ality oP center ia 


mentioned. It eos fone thet br ace LOeAO yl 
With sewage containing them, they can be detected by 
methods in mixtures from ten to a hundred times more 
te than thoec in which the pollution can be detected by 
a chemical methods, 

Bacterial Treatment of Sewage.—Of late years the opinion 
has been growing that the most appropriate method of dealing 
With the disposal isposal of sewage is to imitate as far as possible the 
processes which occur in nature for the breaking up of orgunic 
material. Theso practically depend entirely on baeterial action. 
Honce the rationale of the most approved methods of 91 
Aispasal is to encourage the growth of bacteria which  F 
exist in sewage, and which are capable of braking up orpanic 

ainda and of converting the nitrogen into nitrates and 

‘The technique by which this iv accomplished is very 

waried and sometimes rather empirical, but probably the goueral 
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 backed 











140 BACTERIA IN WATER 


aro 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 ranning 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 
Ieaving tho bed to rest for xome hours before rechurging. ‘The 
final result is better if the effiuent be afterwards ran over another 
wimilar coke-bed. According to some authorities the sewage, ax 
it rns into the flvst bed, takes up from the air considerable free 
oxygen, which, however, soon disappears during the stationary 
period, #0 that on loaving the first bed the sewago contains little 
oxygen, In the latter part of its stay it has thus been submitted 
to annurobie conditions. Further, while by the passage of the 
offluont out of the first bed oxygen ix 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 tho filter anacrobie conditions prevail, 
under which the bacteria can act on the depeait left on the coke, 
On thia latter point there is difference of opinion, for, in examin 
ing London sowage, Clowes has found oxygen prosent 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 
boda. Probably the beat 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- 
iminary stage tho treatment of the swage in what ia called a 
septic lank, and the method has been adopted at Exeter, Sutton, 
and Yeovil in this country, and very fully worked at in America 
by the Stare Board of Health of Massachusetta In the explana. 
tion given of the mtionale of this process, sewage it looked on as 
existing in three stages. (1) Firat of all, fresh sewage—the newly 
mixed and very varied material as it enters the main sewera, 
(2) Secondly, stale sewage—tho ordinary contents of the main 
towers, Here there is abundant oxygen, and.as the sewage flows 
along there occurs by bacterial action a certain formation of 
carbon dioxide and atmonia whieh combine to form ammoninm 
carbonate. ‘Thisis the sewage as it reaches the purification works, 
Here a preliminary mechanical screening may be adopted, after 
which it is run into an airtight tank—the septic tank. (3) 
It remains thore for from twenty-four to thirty-six hours, and 
becomes a foulsmelling fluid—the septic sewage. The chemical 














ANTISEPTICS Ml 


changes which take place in the septic tank are of « inost complex 
nature. The entering it contains little free 





comparatively simple compounds of hydi with carbon, 
sulphur, and phosphorus. As a result thore is a great reduction 
in the amount of organio nitrogen, of albuminoid ammonia, and 
of carbonaceons miatter. The latter fact is important, as the 
clogging of ordinary filter beds is largely due to: the accumulation 
of such material, and of matters gonerally consisting of colluloas, 
One further important effect in that the size of the deposited 
matter is decreased, and therefore it is more easily up 
in the next stage of the process, This consista of running the 
eilluent from the septic tank on to filter beda, preferably of coke, 
where a further purification process takes place, By this method 
thore is first an anaerobie treatment succeeded by an aerohle ; 
in the latter the process of nitrification occurs by moans of the 
‘ial bacteria concerned. The results areof a satinfactory natare, 
being often a marked diminution in the sumber of coli 
organiems it, 

‘Often the offlacnt from a sewage purification ayster contains 
as many bacteria as the sewage entering, but, especially by means 
‘of the septic tank method, there is often a marked dimiwation. 
Tt is said by somo that pathogenic bacteria do not live in sewage, 
The typhoid bacillus has been found to die out when placed in 

but it certainly can live in this fluid for @ much longer 
period than that embraced by any purification method. ‘Thus 
‘the conatant presence of b. coli, b. enteritidis, and atroptococei 
which has been obsereed in sewage efllucnts must here atill be 
Jooked on as indicating’ a poasible infection with the typhoid 
bacilli, and it is only by great dilution aud Prolonged eure 
to the conditions present in ranning water that sue! si ement 
ean be again a part of a potable water. 


Aptisxrrics, 


‘The death of bacteria is judged of by the fact that when 
they are placed on a suitable food medium no development 
takos place. Microscopically it would be observed that division 
no longer ovcurred, and thnt in the ese of motile epecies move- 
ment would have ceased, but such an observation has only 
scientific interest. From the importance of being able to kill 

an enormous amount of work has been done in the way 
‘of investigating the means of doing to by chemical weana, 


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. 


Mothods.—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- 

istance, the metliod was unsatisfactory, and now an antiseptic is usually 
judged of by its effects on pure cultures of definite pathogenic microbes, 









lococcus 
id, choles 


antiseptic 
nd then test the condition of the bacteria, and if the inert 
bstances are fluid there iy no objection to this proceeding, but if in 
the process # precipitate results, then it is better not to have recourse 
to such method, as xometimes the bacteria are carried down with the 
precipitate and may escape the culture test. Tho 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 sil threads in an emulsion 
of anthrax spores aud dry them. These were then subjected to the 
action of the antiseptic, well washed in water, and laid on the surface of 
agar. It wns found, however, that with threads oxposed 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 ou 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 ‘ture fluid, hr be 
followed weep men wre of ee ioe in belog 
hos been paid to the curds ot racists, 

pee watery solution of cartel asid ts new generally talen te the 
standard with which other antinoptice aro compa Rideal and 
‘Walker point out oe 110 parts by weight of i carbolio acid i aoa) 
100 pons weight of phenol, and they reooumend the foll 

ofstandaniising. To bec. of a particular dilution of the disinfectant 
add 5 drops of a 24-hour old bonillow culturw of the o: usualy 
bh typhosns) whioh hag heen incubated at 47°C. Shake the mixture a 
make subcultures every 24 minutes to 1 minutes. Perform a parallel 
terles of exqarinenta: with cartollo ocld’ acd express the cumparstive 
result in multiples of the earbolic acid doing the same work. 


‘The Action of Antiseptics.—In inquiries into the actions of 
antiseptics attention to a great variety of factors ix necewmry, 
ly when the object is not to compare different antis an 
with one another, but when the absolute value of any 

being investigated. ‘Thus the medium in which the bacteria to 
‘be Killed are situated, is important ; the more albuminous the 
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 tho antiseptic be, or the shorter the 
exporure necomury for a given offect to take place. ‘he most 
important factor, however, to be considered is the chemical 
nature of the substances employed. Though nearly sry sub- 
stance which is not a food to the animal or vegetable body is 
‘more or lees harmful to bacterial lifc, yet certain bodies have 
@ more marked action than others Thus it may be said that 
the most important antiseptics ars the salts of the heavy metals, 

cortain acids, especially mineral acida, certain oxidising and 
pone agents, a great variety of substances belonging to the 
‘aromatic series, and volatile oils genorally. Tn 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 molecular; in other words, the action of a molecule of 








one must be compared with the action of « molecule of 
another body. This can be done by dissolving the molecular 
weight in grammes in say a litre of water (see p. When 





substances with high molecular ‘weight are moro pens 
eect low molecalar weight—thus butyric alcohol is more 
powerful than ethylic aleohol—and important differences ances, 








Mt ANTISEPTICS 


the aromatic bodies are associated with their chemical constitu- 
tion. ‘Thus among the cresols the ortho- and para-bodies re- 
somble each other in general chemical properties, and stand apart 
from metacresol ; they also are similar in antiseptic action, and 
ure much stronger than the meta-body. The same may be 
observed in the other groups of ortho, meta, and parwbodies, 
Appin, such a property as acidity is important in the action of a 
neg, 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 
increwsing bactericidal potency. Thus in the cnse of auch ne 
ducers as sulphurous acid and formie acid, the effect in apparently. 
chiefly due to the fact that these substances are acids. Formic 
acid is much more efficient than formate of sodium, Tn the case 
of permanganate of potassium, which is usually taken as the 
type of oxidising agenta in this eonncetion, it ean be shown: that 
greater amount of the oxidation which takes place when this 
agent is brought into contact with bacteria cecurs after the 
organiams are killed. Such an observation is, howover, not 
conclusive as to the non-clficiency of the oxidation process, for 
the death of the bacteria might be due to the oxidation of a 
vory 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 playod by auch 
factors is exemplified in the important fact that a strong salation 
acting for « short time will have the same effect as a weaker 
solution acting for a longer time. From what haa heen sald it 
will be realised that the real causes of  matorin! being an 
antiseptic are very obscure, and at present we ean only have a 
remote idea of the factors at work. 

The Actions of certain Antiseptics,—Hore we can only 
briefly indicate certain results obtained with the more common 
mombera of the group. 

CMorine.—M\ the balogens have been found to be powerful 
antisoptics, but from tho cheapness with which it ean be produced 
chlorine has been most used; not only is it the chief active 
agent in the somewhat complex action of bleaching powder, but 
itis alao the chief constituent of several propriotary substances, 
of which  Eloctroxone” ina good example. Thia last aubstance 
is made from electrolysing sea- water, when magnesia and 
chlorine being liberated, magnesium hypochlorite and magnesinm 
chloride are formed, In the action of this substance free hypo- 

















ACTIONS OF CERTAIN ANTISEPTICS M5 


chlorous acid is formed, and the effect produced is thus similar 
to that of bleaching powder, Nissen, investigating tho action of 
the latter, found Hat per cont ka spo bil in aes 
and Rideal found that | part to 400-500 disinfected sewage in 
fourteen minutes, and Delépine’s results show that 1 part to 50 
(equal to *66 per cont of chlorine) rapidly kills the tubercle 
bacillus, and | part to 10 (equal to 33 per cent) killed anthrax 
spores. Klein found that 05 pee cont of chlorine killed most 
‘bacterial spores in five minutes, 

Jodine Terchloride—This is a very unatable compound of 

Oe aileer though it has been much used as am 
that the substance only remains as ICL, in 

an son of chlorine gas, it is open to doubt whether the 

described aro not due to a very complicated action of 
ee acid, hydriodie acid, of oxyacids of chlorine 

Aa fotline prodaced by its decomposition, and also, in certain 
‘eases, of organic iodine compounds formed from its contact with 
pee material, Tt is stated that the action is very potent : 
cent solution is said instantly to kill even anthrax spores, 

at lb te sone be in bouillon, death oecars 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 "This ischiafly available in two ways—firstly, 
when in the breaking up of ozone the free third atom of the 
eaone molecule is seeking to unite with another similar atom ; 
secondly, when peroxide of hydrogen is broken up into water 
wod an oxygen atom is thereby liberated. In commerce the 
eerily of “Sanitas” compounds ix due to the formation of 

tao by th the slow oxidation of the resin, camphor, and thymol 


Thesiloride of Merowry.—Of all the salts of the heavy metals 
this hax been most widely employed, and must be regarded as 
‘one of the most powerful and useful of known antiseptica In 

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 sporecaso an albuminate of 
mereury wna formed which prevented the contained protoplasm 
from developing, while not depriving it of life. Tt has been 
found, however, that this sult ina strength of 1-100 will kill the 
spores in twonty minutes, although an hour's exposure to 1-1000 
has no effect. The best results are obtained by the addition to 
Who corrosive sublimate solution of “5 per cont of sulphuric acid 
67 hydrochloric acid ; tho spores will then be killed by a saxant- 

20 





146 ANTISEPTIOS 


minute exposure ton 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 140,000, and 
in water by 1-500,000, Plague kacilli are killed by one to two 
minutes’ exposure to 1-3000, Generally speaking, it may bo eaid 
that w 1-2000 solution must be used for the pructically instan+ 
taneous killing of vegetative organisms. 

Porchloride of mereury in one of the sibstances which have 
‘been used for disinfecting rooms hy distributing it from a 
producer, of which the Equifex may be taken as a type. 
guch o machine it in calculated that 1 oz, of porehloride of 
mercury used in a solution of 1-1000 will probably disinfect 3000 
square fect of surface. Such a procedure has been extensively 
used in the disinfection of plague houses, but the use of a stronger 
solution (1-500 acidulnted) ia probably proferable. 

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 disadvant however, to its use is that, when 
diluted and exposed to air, amongst other changes which it 
undergoes it may be traneformed, under little understood 
conditions, into trioxymethylene and paraformaldehyde, these 
being polymers of formaldehyde. The bactericidal values of these 
mixtores 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 m re a5 pus contal 
pceente organisins a 10 por cent solution acting for half an 

wis necessary, In the case of pure cultures, a 5 per cent 
solution will kill the choler organism in three minutes, anthrax 
bacilli in a quarter of am hour, and the spores in five hours, 
When such organiams 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 ease of anthrax 
8 , 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 atudied, ax ite 
née constitutes a cheap method of treating infected rooms, in 
which case some spray-producing machine is employed. Tt is 
stated that a mixture of 8 ce. of formalin with 48 ec. of water 
is sufficient when vapourised to disinfect one cubic metre, «0 far 
‘AS nom-sporing organisms are concerned. It is stated that 1 part 














ACTIONS OF CERTAIN ANTISEPTICS = 147 


puters beens in es hous the aaphyboooes brown 
as in hours, ‘pyogenes 
in six hours, and sathrax ‘spores in Yee Tu the case 
reiacane dry it is probable, however, 
that much i. xen o neceszary, but on this point wo 
have not informa 
spams gas has SSP limited application ; it has little 
on dry ‘organisms, and in the cass of wet vain, in 
ora be ltetiee; probably era nipe 80 a 
ive the moisture & proportion analogous to the strengths stat 
sieeaiwih ih regard ‘8 the wapour. 
Sulpherowe Acid.—This substance has long been in ube, 
largely from the cheapness with which it can be produced by 
sulphur in the air. An atmosphere containing Se 
I kill the pyogenie cocci in two minutes if 
bry in twenty minutes if they are dry ; and anthrax fed 
killed by thirty minates’ exposure, but to kill anthrax spores an 


chet ane of Nese pease of ‘ot no i 
even by an atmosphere containi 
of the sulphurous acid gas. The ssa pea apintbed ‘of this 
agont are fore limited, 

Potasrium Permanganate—The action of thix agent very 
teh depends on whether it enn obtain froe access to the 





to act for about a day; for most onganiams « aimilar solution 
acting for shorter periods has been found sutticient, and in the 
ease af the pyogunic coce a | per cent solution will kill in ten 
minutes, ee is Tittle doubt that such weaker solutions are of 
value in disinfecting the throat on account of thelr non-irritatin, 
properties, and rosnits in this connection have been obtained 
im cases of diphtheria, A solution of 1 in 10,000 has been 
found to Kill plague bacilli in five minutes, 
Cartelic Acil_—Of all tho aromatic series this is the most 
‘extensively omployed antieoptic. All ordinary bactoria in the 
eth le tng ‘and of these the staphy! OA PLOT 


148 ANTISEPTICS 


is the most msistant, are killed in lesa than five minutes by a, 
2-3 ‘and poise in heecta that the 5 sot solution 
ly employed in surgery leaves a mangin foty. But for 
the killing of such onganisins ax anthrax spon a very much 
longer expovure is necessary; thus Koch found it pes to 
sree these spores for four days to ensure disinfection. The 
f such spores being present in ordinary surgical procedure 
may be overlooked, but thera might be rikk of tetanus spores 
not being killed, as these will withstand fifteen hours’ exposure 
to : 5 per cent solution. 
sais podhcta ‘of the distillation of coal thers occur, besides 
eatboll acid, many bodies of « similar chemical constitution, and 
many mixtures of these are in the market—the chief being ereolin, 
iual, and lysol, all of which are agents of vulue. Of these Lysol 
is perhaps the most noticenble, as from its natuns it acté ax a soap 
and thus can remove fat and dirt from the hands. A one-third 
per cont solution is said to destroy the typhoid and cholera 
organistas in twenty minutes. A 1 per cent solution is sufficient 
for She surgical procedures, 
rodoform.—This is an agent regarding the efficacy of-which 
cas has been much dispute. There is little doubt that it owes 
its officiency to ita capacity for being broken up by bacterial 
action in such a way as to act free iodine, which acta as a powerful 
disinfectant, The substance is therefore of value in the treatment: 
of foul wounds, such as those of the mouth and rectum, where 
Sai bacteria are abundantly present. It acts more slightly 
nore ame are only pyogenic cocci, and it seems to have & 
san beneficial effect in tubercular affections. In certain 
casos ite action may apparently be aided by the presonee of the 
producta of tissuc degeneration. 

From the results which have been given it will easily be 
recognised that the eholce of an antiseptic and the precise 
manner in which it is to bo employed depond entirely on the 
environment of the bacteria which are to be killed. In amany 
cases it will be quite impossible, without original inquiry, to say 
what course is likely to be attended with most success, 











CHAPTER Y. 


RELATIONS OF BACTERIA TO DISEASE—THE 
FRODUCTION OF TOXINS BY BACTERIA. 


Tt has already been stated that a strict division 
of microorganisms into soprophytes and true parasites cannot be 
made. No doubt there are organisms such as the bacillus of 
leprosy which ax yet havo not boon cultivated outside the animal 
body, and others, such as the gonococcus, which are in natural 
conditions always parasites associated with disease. But thests 
latter can lead a saprophytic exiatonce in specially prepared 
conditions, and there are many of the disease-producing organisms, 
such as the organisms of typhoid and cholera, which can flourish 
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 etatemont applies to the terms pathogenic and 
saprophytic, and even to the terms pathogenic and non mic. 
By the term pathogenic is meaut the power which an organist 
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 orgnn- 
fxm which will in all cirenmstances produce disease in all 
animals, und, 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 
dimase, though in the scicnce of human pathol it i» often 
sumed for convenionce us implying that the organism produces 
disease i) man in natural conditions 

Modifying itions. —In studying the pathogenic efecdam, 

1d 








- 





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 aleo 
its exact condition at the time of infection. In other words, 
the resulting disease ix the product of the sum total of the 
charactors of the infecting agent, on the one hand, and of the 
subject of infection, on the other. We may, therefore, state somo 
of the chief cireumstances which modify each of these two factors 
involved and, consequently, the diseased condition produced. 

1. The Infeeting Agent,—In tho cave of a particular speci 
of bacterium its effect will depend chiefly upon (a) its ps iy 
and () the number introduced into the body. To these may 
‘be added (¢) the path of infection. 

The wirutence, (.e. the power of multiplying in the body and 
producing disease, varies greatly in different conditions, and the 
mothods by which it can be diminished or increased will be 
afterwards described (mde Chapter XIX,)- Ono important 
point is that when o bacterium has been enabled to invade 
and multiply in the tissues of an animal, its virulence for that 
spocica ig often increased. » This ix well xeon in the case of 
certain bacteria which are normally present on the skin or 
mncous 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 atill more increased by inogulating from 
rie erin) tn another in eerlec—the rahod ct passage: < WAG 
diffrent effects aro, of course, produced on tho virulenes being 
altered, For example, a streptococcus which produces inerely 
1 local inflammation or suppuration, may produce « rapidly fatal 
A SOR when ite virulence is raised, Virulence aleo 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 animala, 
‘but become less virulent for rabbits. (Knorr.) The theoretical 
consideration of virulence must be reserved for a Inter 
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 «certain number of pathogenic organisms 
of given virulence, and it is only is instances that one or 
two organisms introduced will produce a fatal disease, eg. the 
case of anthmax in white mice. The healthy peritoneum of a 
rabbit can resist and destroy a considerable number of pyogenic 
micrococei without any scrious result, but if a larger dose be 








CONDITIONS MODIFYING PATHOGENICITY 151 


introduced, « fatal peritonitis follow. Agnin, a certain 
quantity of pom rariiooler genta tejected subcut i 


‘only a local inflammatory change, but i regedie! 
in 
nie sn" i oy» te 


irae may alter the result, serious effects often 
following a direct entrance into the blood stream. Staphylococci 
injected subcutancously in a rabbit may produce only a local 
abscess, whilst on intravenous injection multiple see in 
certain organs may result and death may follow, Local inflam- 
matory reaction with subsequent dostruction of the organisma 
may be restricted to the site of infection or may occur also in 
the |, ic glands in relation, The latter therefore act as a 


are very rapidly destroyed in the blood stream, 
cnt lone bis mari in the Sie eubeutancous injection 
the pneamoooceus produces death more newdily than intra 


ous injection. 
bat The Subject of Infection— Amongst healthy individuals 
lity and, in inverse ratio, resistance to a yarticular 

may vary aecording to (a) species, (4) race and individual 
pocaliarities, (c) age. Different species of the lower animals 
show the widest Pin. pe in this respect, some being extremely 
susceptible, others highly resistant. ‘Then there are diseases, 
such ax leprosy, gonorrhcea, ete, which appear to be peculiar to 
‘the human faigont and) and have not yet been transmitted to animals. 
And farther, there are other, such as cholera and typhoid, which 
do not naturally affect animals, and the typical lesions of which 
ly reproduced in them, or appear aay, 
though pathogenic effects follow inoculation with 
the onganisins, In the case of the human subject, differences in 
lity to a certain disease are found amongst different 
‘maces aud alto amongst individuals of the sme tnce, ax is woll 
eeu in the case of tabercle and other diseases. Age also playx 
‘an important part, young subjects being more Hable to certain 
diseases, ey. to diphtheria, Further, at difforent periods of life 
certain parts of the body aro more susceptible, for example, in 
‘early life, tho bones and joints to tubercular and acute suppura- 

tive affections. 


He 





152 RELATIONS OF BACTERIA TO DISEASE 


In increasing the susceptibility of a given individual, condi- 
tions of focal or diminished vitality play the most 
important part. It has been experimentally proved that 
conditions such a& exposure to cold, fatigue, starvation, ete, 
all diminish the natural resietance to bacterial infection, Rats 
naturally immane can be rendered suacoptible to Mere by 
being fed with phloridain, which produces « sort of diabetes, « 
Jarge amount of sugar being exorated in the urine (Leo). 
Guinew-pige may resist subcutaneous injection of a certain does 
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 guneral infection, Also a local susceptibility 
any 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 scttle there and set up an ulcerative endocarditis ; 
or if a bone be injured, they may produce suppuration at the 
part, whereus in ordinary circumstances these lesions would not 
take place, ‘The action of one species of bacterium in also often 
aided by the simultaneous presence of other species, In this 
case the latter may act simply as additional irritante which 
osaon tho vitality of tho tissues, but in somo casos their presencp 
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), illusteate the important part which local or 

weral 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 
‘ond tissues of tho body, with the exception of the skin and 
certain of the mucous surfaces, are bacterium-free, and if a few 
organisms gain entranes, they are destroyed, But if the vitality 
becomes lowered their entrance bocomes caaier and the poseibility 
of their multiplying aud producing disease greatly increased. In 
this way the favouring part played by fatigne, cold, ete., in the 
production of disvases of which the direct caus is n bacterium, 
may be understood. It is important to keep in view in this con= 
nection that many of the inflazamation-producing and pyogenic 
onganiama are normally present on the ¢kin and varions mucous 
surfaces. ‘The action of a certain orgunism may dovitalise tho 
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 variots 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 


Fyrom pn re rows oes th ine a eo 

severe character. It is not uncommon to find in the bodics of 
Show who have dnd fo croc wang ee, elleios of 
micrococci or bacilli in the eapillaries of various organs, which 
have entered in the later hours of life; that is to say, the 
dacteriumfree condition of the blood has been lost in the period 


of death, 

Mba anathods by which tba natural redstance may be specie 
ally increased belong to the subject of immunity, and are 
a Seater oa habenty et 
by of Bacterial Action.—In the production of disease 

sme ther aro two iain factors involved, namely, 
sewer ‘of the living organisms after they have 
entered the body, and (4) the production ete 
which may joa ea pepe nla eee! 
onl ‘The former corresponds to infection, ise et 
nature of intoxication or poisoning. In different diseasss one 
of these is usually the more prominent feature, but both are 
Jess concerned. 
Lids and Distribution of the Bacteria in the Body.— 
= Sima bacteria have invaded the tissues, or in other 
vaftor bacterin has taken place, their further 
behaviour varies greatly in different cases In certain vat 
ih nea multiply in the blood streun, producing 
the lower unimal this multiplication of the 
antes tks bond throughout the body may be very exten 
sive (for example, the septicarmia produced by the pneumococcus 
in rabbits); bat in septicemia in man, it very seldom, if ever, 





‘cecurs to a0 great a di the organisms rarely romain in large 
aumbers in the cireulating blood, and their detection in it during 
life by jie examination is rare, and even culture methods 
may give results unless a large amount of blood ix 


mased. In such cases, however, the organisms may be found 
lying in large numbers within the oe ot 









may rerain local, 

Hitthe reaction aroand them, as in tetanus, oF a well: 
marked: as in diphtheria, pneumonia, etc. Or in the 
second place, they rosy pase by the lymph or blood stream to 


164 RELATIONS OF BACTERIA TO DISEASE 


other parts or organs in which they settle, multiply, and produce 
Tosions, as in tubercle, 
. Production of Chemical Poisons,—In all these cases the 
growth of tho organisms ix accompanied by the formation af 
chemical producto, Which act generally or locally in carving egroa 
as toxic substances, The toxic substances become diffused 
thronghont the system, and their effects are manifested chiefly 
wy symptoms such as the occurrence of fover, disturbances of 
© circulatory, respiratory, and nervous systema, cte, In some 
cases corresponding changes in the tissues are found, for example, 
the changes in the nervous system in diphthoria, to be after- 
wards doseribed. ‘Tho gensral toxic olfocts may bo #0 slight ax 
to be of no importance, as in the case of « local suppuration, 
or they may be very intense as in tetanus, or again, leas severe 
lh producing cachexia by their long continuance, as in tuber 
culosis, 

‘The occurrence ef local tissue changes or lesions produced in 
the neighbourhood of the bacteria, as alroady mentioned, ix one 
of tho moat striking results of bacterial action, but these aleo 
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 mors difficult to demonstrate the mode of 
action, for, in the tissues the chernical products are formed by 
the bacteria slowly, continuously, and in a certain degree of 
concentration, and these conditions cannot be exactly reproduced 
by he ea It is also to be noted that more than one poison 

may be produced by « given bacterium, eg. the tetanus 
bacillus (p. 380). Further, it is very doubtful whether all the 
chowical 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 u local toxic action of 
very intense chameter, evidenced often by extensive necrotic 
changs 

The injection of large quantities of many different pathogenic 
‘organiems in the dead conditions resulta in the production of a 
local inflammatory ehange which may be followed by suppura- 
tion, this effect being possibly brought about by ecrtain sub- 
atances 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 parte 
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 (wide Tuberculosis). 

Summary.—We may wy then that the action of bacteria as 
disease-prednours, as in fact their power to exist and multiply in- 
the living body, depends upon the chemical products formed 
direstly or indirectly by them. This action it kbown by tieswe 
changes produced in the vicinity of the bacteria or throughout 
the system, und by taxie eymptoms of great variety of degree and 


Wo shall first consider the effects of bacteria on the body 
generally, and afterwards the nature of the chetnical products. 


Evrrets or Bactrntat. Action. 


‘These may be for convenisnce arranged in a tabular form as 
iallowa — 


A, Tis Changes, 
(2) Local changes, se, changes produced in the neigh- 
bourhood of the bacteria, 
Poaltion (#) At primary losion. 
(8) At secondary foci. 
Character (a) Tiseue reactions | Acute or 
(2) Degeneration and necrosis f Chronic. 


(2) Produced at n distance from the bacteria, directly or 
indircetly, by the absorption of toxins 
(a) In opecial tissues: 
(a) as the result of damage, eg. nerve cells and 
fibres, eversting cells, vee! walls, or 
(B) changes of a reactive nature in the blood 
forming organs. 
(0) General anatomical changes, the effects of mal- 
nutrition or of inereasd waste. 


B. Changes in Metabolism. 


‘The occurrence of fever, of errors of assimilation and 
elimination, ete. 


A. Tissue Changes produced by Bacteria —The oifects of 
bacterial action are 90 various as to include almost all known 
logical changes. Howover varied in character, they may 
lassified undor two main headings —(«) those of a degencra- 
tive or necrotic nature, the direct result of damage, and (4) 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 oneere woes for the benefit of the In 
‘the means of defence both leweocytes and the fixed cells of the 
tissues are concerned, Both show phagocytic Se ie. 
have the power of taking up bacteria Sto ther toplaam. ‘The 
cells ure guided towards the focus of infection uss chemtaelsia 
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 neatrophile leuco- 
cytes of the blood are chiefly concerned. When the local lesion 
is of some extent there is usually an inerease of these cells in 
the blood—a neutrophile Icucocytosis, And further, recent 
observations have shown that associated with this there is in the 
bone-marrow an increased number of the mothor-cells of these 
leucocytes—the neutrophile myelocytes. The passage of the 
neutrophile leucocytes from the marrow into the Bloods with the 
resulting leucocytosis, is also wpparently due to the absorbed 
bacterial toxins acting chemiotactically on the marrow. ‘Those 
facts abundantly show that the means of defence is not a mere 
local mechanism, but that increased proliferative activity in 
Gistant tissuos is called into play, In addition to dirvct phagoeyt- 
cals by these leucocytes, there is now abundant evidence that 
an important function is the peed in the body of bactericidal 
and other antagonistic aubstancea In other cases the cells 
chieily involved aro 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, whore the epecifio bacillus appear to have little 
or no action on the neutrophile leucocytes. In other cases, 
wgain, the reaction is chiefly on the part of the connective 
cells, though their proliferation fs always associated with some 
variety of leucooytic infiltration and usually also with the form 
tion of new blood vessels. Such a connective tissue reaction 
oceurs especially in slow infections or in the later stages of an 
acute infection. The tisie changes resulting from cellular 
activity in the presence of bacterial invasion are naturally very 
vuried—exaruples of this will be found in subsequent chapbers— 
but they may be said to be manifestations of the two funda- 
mental processes of (a) increased functional activity movement, 
phagocytosis, secretion, etc.—and (2) increased formative activity 
—cell growth and division. The exudation from the blood 
vossols bas ben variously interproted. There is no doubt that 
the exudate has bactericidal propertics and also acts as a diluting 














LOCAL LESIONS: 17 


2 eens be held as uncertain whother the process 
ought to be regarded as primarily cones or aa 
the direct result of damage to the endothelium of the vessels, 
may algo be pointed out that the various changes referred to 
are Rechasct Shan poeuiarto bartacal invasion ; they are examples 
‘of the general laws of tissue change under abnormal conditions, 
and they can all be reproduced by Sr nbaleupiinstaieoatasian 
or in a particulate state, What "oasttienda their special feature 
ia “s Progressive or spreading nature, dus to the bacterial 
1 Eetons 


upon the point of eritrance, ¢g. malignant pustule and the 
conditions ee thers again, there is 
a special tendency for certain parts to be affected, as the upper 
Seen cee instars Tn some cases the site has a 
Seis wet a the blood frm 
‘organiéme gain an entrance to: 8 primary 
lesion, the organs specially liable to be affeeted vary greatly in 
different di Pyogenie cocci show a 9 tendency to 
settle in tories capillaries of the kidneys produce 
abscesses, whilst these Iesions rarely occur in ths spleen. On 
the other hand, the nodules in disseminated tubercle or glanders 


ieoner: int is that the ition of Ye disseminated lesions 
is not to mat bya Mparcerin dlspa such aa embolism, 
‘but depends upon a special relation between the organisms and 
the tissues, which may be spoken of either ax a selective power 
on the ie of the organisms ora special susceptibility of tissucs, 
possibly in part due to their affording to the organiams more 
suitable conditions of nutriment, Even in the case of the 
Jesions produced dead tubercle bacilli, a certain selective 


Mewle Local Lerion. Tho local inflaminatery reaction 
presents different characters in diflorent conditions. Tt may be 
sige oe 2 by abundant Gbrinous exudation, or by it 

© the case of an epithelial surface), or by hemorrhage, 

mm; it may be localised or spreading in character; it 

hs followed by suppurution, and may be accompanied or 
‘up to necrosis of the tissues of the part, a good example of 


event being found ina boil, Examples will be given 
it ehaptera The necrotic or degenerative changes 


ia —_ 





158 RELATIONS OF BACTERIA TO DISEASE 


affecting especially the more highly developed elements of tissues 
are chiefly produced by the dinset nection of the bacterial poi 

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 
oceupy a medium position in the scale of susceptibility, and that 
fn organism which cauecs a general infection in a cortain animal 
rid produce only a local inflammation when its virulence ix 


Chronic Local Lerions.—In a considerable numbor of diseases 
produced by bacteria the local tissue reaction is a more chronic 
teed than those described, In other words, the specifie 
irritant is lesa intense, so that there is leas vascular disturbance 
and a greater proponderance of the proliferative processes, 
Teading 10 new formation of connective tissue or a modified 
connective tissue. This formation may ocenr in foci hera and 
there, eo that nodules of tor or leas consistence result, or it 
may bo more diffuse. Such changes especially occur in the 
diseases often known as the infective granulomata, of which 
tubercle, leprosy, glanders, cotlnomyeosia, syphilis, ete, are 
examples, A herd and fast line, however, cannot be drawn 
between such conditions and those described above ws acute, 
Tn glanders, for example, especially in the hnman 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 ehange is the aame—vir a ne 
nection to an irritant of minor intensity—the exact structural 
charncters and arrangement vary in different diseases. In somo 
causes the disease may be identified by the histological changes 
alone, but on the other hand, this is often impossible. These 
changes often inelude the occurrence of degenerations or of 
actual necrosis in the newly formed tissue. In the granulomata, 
infection of other parts from the primary lesion takes place 
chiotly by the blood vessols and lymphaties, though sometimes 
along natural tubes such as the bronchi, intestine, etc. 

(2) Genenal Lesions produced by Toxins.—In the various in- 
fective conditions produced by bacteria, changes commonly 
oceur in certain organs unassocinted with the prosence of the 
bacterin; these are produced by the action of bacterial pro- 
ducts cirenlating in the blood. Many atch lesions ean be pro- 
duced experimentally. ‘Tho socroting colle 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 grannlar disintegration, is 











DISTURBANCES OF METABOLISM, ETC. 159 


commoa, Hyaline change in the walle of arterioles may occur, 
and in certain chronic conditions waxy change is rrcentaict 
ina similar manner. Tho latter has been produced in animals 
aay injection of tho staphylococcus surena, 


inte. in the blood plasma, ax evidenced ean te 
Binal hamorrhages may 
tp mae geen meas 
eg, rien and al eruptions 

real pr oe lan Shin er be gehid 
though in many of these diseases the causal organism has not 

yet been isolated. We have, however, the important fact that 

eerenoading skin eruptions may be produced by poisoning with 
onopheenen the nervous system degenerative changes 

havo been in diphi theta, botb fa) tie opioalicond sad ts 

Pal spree nerves, und have been reproduced experimentally 

Products of the diphtheria bacilli. There is also experi- 

met evidence that the Prcillus ‘coli communis and the strepto- 

pyogenes may, by means of their products, produce areas 

aa mfionkog it in the spinal cord, and this may furnish an explana- 

tion of some of the lesions found clinically. I is also possible 

that some serous inflammations may be produced in the same 


way. 

B Disturbances of Metabolism, ote,—It will oasily 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 
ocemrrence, and it ix a striking fact found by experimont that 
after injection of lacterial products, eg, of the diphtheria bacillos, 
a marked low of body weight often occnrs which may be pro- 

ive, leading to tho death of the animal. In bacterial 
realy assimilation is often imperfect, for the digestive glands 
are affected, it may be, by actual poisoning hy bacterial products, 
it may he by the occurrence of fever. The fatty degenerations 
which aro a0 common are indicative of a breaking down of the 
[piotekd molecules, and are amocinted with increased urea produc- 
‘tion, while the degeneration of the kidney epithelium renders 
the exeretion of waste products deficient or impossible, and this 
_ ‘not infroquently the immediate canso of death. But of all 
the changes in metabolism the most difficult to understand ix 
the ocenrrence of that interference with the heat-requlating 
mechanism which rewalta in fever. The degree and course of the 
Hatter vary, sometimes conforming to a more or less definite type, 


ali _ 


160 RELATIONS OF BACTERIA TO DISEASE 


where the bacilli are selective in their field of operation, as in 
croupous pnoumonia 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 pywmic affections, Th 
main point of interost regarding the development of fover is aa 
to whether it is a direct offect of the circulation of bacterial 
toxins, or if it Is to be looked on ax part af the reaction of the 
body against tho irritant. ‘This question has still to be sottled, 
and all that we can do is to adduce certain facts bearing on it, 
‘Thus in diphtheria and tetanus, where toxic action leuding to 
ons plays such an important part, fover may be a very 
subsidiary foature, except in the terminal stage of tho latter 
disease ; and in fact in diphtheria pean) toxic effects may 
be produced with little or no interference with heat regulation. 
‘On the other hand, in bacterial disease, whore defensive and re- 
parative processes predominate, fever ia rarely absent, and it is 
nearly always presont when att active leucooytosis 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 
oceurs; while the injection of a large amount of the same ix 
followed by aubnormal temperatures and rapidly fatal collapse. 
Tt might appear as if this indicated that the occurrence of fever 
had beneficial effect, but this ix one of the points at issue, 
Certainly such an effect ix not due to the bacteria being unable 
to multiply at the higher degroes 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, ctc., 
in very doubtful; a production of antagonistic substances may 
be efficted 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 agnin are in difficulties, It might appear 
as if the tissue destruction, indicated by the occurrence of fatty 
dogencration, would lead to heat development, but frequently 
excessive heat production with increased proteid metabolism 
oceurs without any discoverable changes in the tissues; and 
further, in phosphorus poisoning there is little fover with great 
tissuo doatruction, The increased work performed by the heart 
in most bacterial infections no doubt contributes to the rise of 
bodily temperature, But we rovst bear in mind that in fever 
there ia more than mere increase of heat production—there is 
‘also & diminished loss of heat from interference with the nervous 

jam of the sweat apparatus. The known facts would 














THE TOXINS PRODUCED BY BACTERIA 161 


indicate that in fever there is a fnetor involving the aervous 
systom to be taken into account. The whole subject is thus 
very obscure, 


Symptoms.—Many of the symptoms occurring in bacterial 
affections are the hiv jical changes mentioned, 
as can be understood ; whilat in the case of others, corre- 
ing changes have not yet been discovered, Of the latter 
associated with fever, with its disturbances of metabolisni 
and manifold affections of the various systems, are the most 
The nervons system is especially liable to be 
affected —convuisions, spasms, coma, parilysia, ete, being 
common, Tho symptoms due to disturbance or abolition of the 
functions of secretory glands also constitute an important group, 
forming, aa they do, a striking analogy to what is found in the 
action of various di 


of bacterial poiaone or have an analogy in the aztion of druge, 


Tae Toxtxs rropucey wy Bactxata, 


Early Work on Toxins —Wo know that bacteria are capable 
of giving rise to poisonous bodies within the animal body and 
also in artificial media, We know, however, eompuratively little 


duction of stich poisonous bodies was Brieger. ‘This observer 
from putrefying substances, and also from bacterial 


Similar bodies occurring in the ordinary metabolic processes of 
the body hud previously been desoribed ‘and called /eucomaines, 

fines izolated from pathogenic bacterin 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 wore faulty, and they have therefore 
only a historic interest, 
» 





vory 
tubercle bacilli are killed by heat and injected into the body 
tissues of a susceptible animal tubercular nodules are found 
to dovolop round tho sites whore thoy havo eae From 
this it is inferred that they must have contained teristic 
toxing, seeing that characteristic lesions result. The bodior 
of the cholora vibrio are likewize toxic, Such intracellular 
toxins, as they have been called, may appear in the fluids 
/ in which the bacteria are living (1) ‘by excretion in an un- 
altered or altered condition, (2) by vhe disintegration of the 
bodies of tho organiama which we know are always dying 
in any bacterial growth. The death of bucteria occurs alo 
in the body of an infected animal, and the disintegration of 
these dead bactoria constitutes an important means by which 
the poisons they contain are absorbed, There is same evidence 
that often bacteria produce during growth poisons which 
are hurtful to their own vitality, and alzo 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 hos been called, may have an important effect in 
liberating intmeellular toxins. We do not, however, under: 
stand at that takes place under such circumstances; for the 
dead bodies of many bacteria, such as those of anthrax and 
@iphthoria, 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 aro 

















‘ACTS REGARDING BACTERIAL TOXINS 163 


injected into animals, When effects oro by such 
Dee te Mack eae etek cae oo 
‘They are of the nature of general disturbances of 


Fok 
a 
wet 
ai 
ike 
Pe 
tH 
ip 


examples of bacteria thus 
the diphtheria and tetanus bacilli, 
when bouillon cultures are filtered bact 
a porcelain filter, toxic fuids are obtained, 


& 

z 

E 

5 

oe 
fe: 


E 
: 


ieee 

spl 

ee 
HL 
HL 
iar 
pete 
ill 


‘or whother they are prodnced 

‘on tho constituents of the media, We 
therefore cannot as yet draw a hard and fast line between 
fntra- and extracellular toxins, but the terms are convenient, 


i 


vibrio, where the polaons which dissolve out into the culture 
fluid are probably different in their nature from those which act 
‘whon the dead bacteria aro injected into an animal, ‘The extra- 


: 


toxins are the mors easily obtainable in large quantities, 
is their nature and effects which are beet known. No 
however, has been discovered of obtaining them in a 
and our knowlege of their properties is exclusively 
from the study of the toxic filtrates of bouillon cultures 
filtrates being usually reforred to simply as the toxins, 
fins differ in their effects from the intracellular poisons 
ific uetions on certain tissnes are often manifested, 
toxins of the diphtheria, the tetanue, and tho botu- 
all act on the nervous system; with some of the 
ou the other hand, poisons, probably of 
prodnce solution of red blood corpuscles (eis 


nn 


Tie 
i 


" 





164 THE TOXINS PRODUCED BY 





last may explain, in at least, the anemias so common in 
th amit dc) he acon of any of Shwe oxi 


has 

forward aaa ibility that some bacteria aro onty capable of 
producing toxins within the animal tissees, and or 
Yeon thought possible that bacteria, such as, for 03 
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 mueb studied, and the 
ham agyreassine hos been given to them. ‘The evidence adduced 
for tho existence of these agressins asa separate group of bacterial 
poivons is of the following kind. An animal és killed by a dose 
of the typhoid, dysentery, ebolera, ce tubercle bueilins, or by a 
staphylococens, the organiatn being introduced into one of the 


these cases is present, is removed, and centrifogalised to remore 
tho tmctorin #0 far as this can be done by sach w procedure ; 
tho bactoria which are left aro killed by sbaking the flaid up with 
toluol and leaving it to stand for some days. Tt is stated that 
such a fluid is of itself without pathogenic effect, but has the 
proparty of transforming a non-lethal dose of the bacteriam used 
nto ono having fatal effect, Further, the effects of the combined _ 
actions of the bacteria and aggressins are often of a much more 
acute charoter than can be obtained with toxic products 
dovelopad fn vitro, ‘Thus, in the case of the action of a nom 
lothal close of tho tubercle hacilius plas its aguressin, death may 
oeour in twenty hours, « result nevor obtained with artificial 
cultures of the organiam. ‘The results obtained are attributed 








Aggromin action have been observed with macerated cultmnes,— 
tho deduction boing that in the aggressins we aru merely doal- 
ing with concentmted intracellular toxins, Ou the other band, 








anaemia by ite luemolytic properties. 
bacterial disease may thus in reality be due to vory di 


oe tated by nee of mach 


peptone. Like 
conpalted 9 
Elise ceca ood wes by heat0l minmated waiphate cf'Cncala, Both 
‘are yecoiptiated by alcohol. ‘The first alliamoaes forme in digestion are 
‘xo hotero-altmmose, whieh differ In the insotobility of the 
‘tn hot and cold water (insolubitity and coagulability are quite difereat 
0 


‘They hare Yeon called the primary albuiwows, By farthor 
fame nto the ecwndary altumoey, deuteroalbamom, whieh 

‘hamoleal resctions from the parent bodies, ag. it oxnpot be 
from watery solutions by saturated scrltum chiara eben, 


ee ae 














ns 
‘¢ have seen that in cortain cases there ia diffiealty in under. 
es dupe elaine apn beara ee 


Noplasm does not seem very toxic. Yet we often see effects 
laced at a distance from the foens of infection, «g. in 


name aggressina has been given to them. ‘The evideneo adduced 
for the oxistonce af these agurussins as a scparnto group of bacterial 
poisons is of the following kind, An animal is killed by & dose 
of the typhoid, dysentery, cholera, or tuberele bacillus, or by a 
ataphylococcua, the organism boing introduced into one of the 
serous cavities, After death the serous exudation, which in all 
these: cases is present, is removed, and ceatrifugalised to remove 
the bacteria so far ax thie can he done ; 
tho buctoria which aro Joft are killed by shaking the fluid up with 
toluol and Jeaving it to stand for some a Tt is stated that 
such s fluid is of itself without pathogenic effect, but has the 
property of transforming « non-lothal lowe of the bacterium used 
into oné having fatal effect, Further, tho effects of the combined 
actions of the bacteria and aggressins are often of a much mone 
neute character than can be obtained with toxic ucts 
developed in witro, Thus, in the case of the action of a non- 
Jethal dose of the tubercle bacillus plus its aggressin, death may: 
ocenr in twenty hours, a result never obtained with artificial 
cultures of the organiém. The results obtained are attributed 
tow paralysing action which the aggrossin is supposed to have 
on the phagocytic functions of the leucocytes. The subject is 
fall of difficulties, and in the case of certain of the organisms 
employed, it is stated that results similar to those attributed to 

in 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, 











recogn) 
ooh en en ok lrelinoae a A wipro 
death of these colla in connection with pm aang efi 
Pe a a ea cian a oe 
1 in. “The ap rey im of such poisons must be of the 
Seria weet irtance in of the hate Arter 
in protection of the infection, and. 


ft is possible that toxins having a fuel otic ta strong cone 

centrations, may, when faired be responsible for the phenomena 

of attraction or repulsion of leucocytes which we know occur 
round w focus of bacterial growth in the body. 

It is to be noted that in the ease of any particular bacteriam 

several different toxins may be at work, and it is also possible 

tan toxin may have “lifferent effects on different tissues 





known 

cellular toxins. “the earlier Siriaas pon toxins suguested 
that analogies exist between the modes of bacterial action and what 

takes place in corey deca 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 albamoees,' but the natuml fluids of the body or definite 


* fm the digestion of albumins by the gastric and pancreatic juices the 
ihcspena}ane 6 proen of bodice formed prlininarly o the prodution of 
prptone, ¢ Latter they differ from tho albumins in thelr not being 
‘congulatert mt Dent, and to tng slighty dlalyabie, They difer from the 


awl heteco-albumese, which differ in the imolability of the 

fn het and cold water (iusclubility and coagulability aro quite differat 
aged have ber called the primary altmmoses Hy farther 
pass into the secondary albamose, doutero-altmnose, whieh 

sMlightly in ebemical sesetions from the parent bodies, eg, it cannot be 
from watery tolutious by saturated solum chiovsle anlwm % 








165 THE TOXINS PRODUCED BY BACTERIA 


tolutions of albumins, and, further, never subjected the results 
of the Lucterial growth to heat ubove 40° C., or to uny stronger 
agent than absolute alcohol. He found that albumoses and 
sometimes peptones were formed by the action of the patho- 

ic bacteria studied, and further, that the precipitate contain- 

these albumoses was toxic. In certain casos the of 
splitting up of the albumins went further than in peptic diges- 
tion, and organic bases or acids might be formed. According w 
Martin, the characteristic symptoms of the diseases could be 
explained by compound actions, in which tho albumoscs wero 
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 Kahne, 

Furthor evidence that bacterial toxins are cither albumoses 
or bodies having « still stnaller molecule is furnished by C. J. 
Martin. ‘This worker, by filling the pores of & Chamberland 
bougie with tin, has obtained what is practically a etrongl 
supported colloid membrane through which dialysis can be na 
to take place under great pressure, say, of compressed oxygen. 
He finds that in such an apparatus toxins—at lenst two kinds 
tried,—will pass through just as an albumose will. 

Brieger und Boer, working with bouillon cultures of diphtheria 
and tetanus, have, by precipitation with zine chloride, separated 
bodies which show characteristic toxic properties, but which have 
the reactions neither of peptone, albusose, nor albuminate, and 
‘the nature of which is unknown. It has also been found that 
tho bacteria of tubercle, tetanus, diphthoria, and cholera can 
produce toxins when growing in proveid-free fluids, In the case 
of diphtheria when the toxin is produced in such a fluid a proteid 
eaction appears, Of course this need not necessarily be caused 
by the toxin, Further investigution is here required, for 
Uschinsky, applying Brieger and Boer's method to a toxin so 

juced, states that the toxie body is not precipitated by zinc 
ealte, but remains free in the medium, Lf 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 those poisons (in 








mice of acetic acid be present.  Dyxalbumose te probably merely ® temporary 
modification of hetero-allminow, Further digestion of deutero-albumose 
rosults in the formation of peptone. 














real natars we know lyre, ae Tn « recent research Friedberger 


and Moreschi have shown the intravenous injection in tho 
human subject of a fraction of a loopful of a dead typhoid 
culture gives rise to toxie aymptoms, including marked febrile 
reaetion. Such injections are followed by tho appearance of 
agglatinating and bactoriolytic substances im the serum, These 
ane intracellular toxins may be comparable with 


toxins 30 far as concerns the iy small dose 
sufficient to produce toxic effects, 
the ies of the oxtracellular toxins are 


the following, am certainly all uncrystallisable; they 
are soluble im water and they are dialysablo; they are pro- 
cipitated along with peouaiaa he concontrated alcohol, and also 
by ammonium sulphate; if they are protoids they are either 
albumoses or allied to the albumoses ; they are often relatively un- 
stable, having their toxicity diminished or destroyed by host (the 
of heat which is destructive varies much in different cases), 

it, and by certain chemical agents. Their potency is often 

in the ipitations practised to obtain them in o pure 

or concentrated ition, bub among the precipitants arnmoninm 
sulphate has little if any harmful effect. Regarding the toxins 
are more intimately associated with the kacterial proto- 

we know much less, but it is probable that their nature is 
‘similar, h some of thern at least are not so easily injured by 
eg. those of the tubercle bacillus, already mentioned. In 
erageer ‘the fatal awe for an aes cpt 

species, body weight, age, previous conditions as to 
foed, temperature, ete. In estimating the minimal lethal dose 


is contains the 
goon, placed in watoh glanse, ax 
in the dark, also tn oneso, ot in an 





itakning strong sulphuric acid. For use the contents of one 
up ina little normal saline sotation, 


comparison of tho action of bacteria in the tissuos in 


‘of these toxins to what takes place in the gpeinve 
a a 















‘apart 
tetanus and dij in, u digestive wetion may occur, analogies have: 
been drawn Mepiatlanin soutien teak are 


ity 
activity of much on undoubted ferment as that of the gustric 
juice. 


sperature, 
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 mjection of the toxic 
bodies and the appearance of symptoms, This may be inter- 
roted ag showing that after the introduction of, say, a filtered 
illon cultare, further chornical substances aro 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 loast, the delay may be explained by the fact 
that the poison opparently has to travel up the nerve trunks 
befors the ral poisonous action is developed. Further, with 
some Peluons presently to le mentioned whieh are closely allied 
to the bacterial toxina an incubation period may not exist, 
Tt would not be prudent to dogmatise ws to whether the toxins 
do or do not belong to such an ilbdefined group of substances 
as the formenta. 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 addueed that toxins fulfil this condition, Another 
property of fermente ia that eo long as the producte 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 
oeeurrence has been found. A certain amount of a toxin is 
dlways oxsociated with a given amount of discase effect, though 
w process of elimination of waste products must be all the time 


VEGETABLE AND ANIMAL POISONS 169 


going on in the animal’ body. Again, too much importance 
must not bo attached to Joes of toxicity by toxins at relatively 
low temperatures. This is not true of all toxins, and further- 
more many protelds show a tendency to change at such 
temperatures ; for instance, if oes, albumin be kept vat} 

at 55° C. nearly the whole of it will be coagulat 

re maintain an open mind on this subject. 

‘Similar Vegetable and Animal Potsons.—Within reoent youre it 
Delong to a of toro bodies 





aac U ii * acts Se oie udacacta 

ius Gequirity) reametively. | From. the 

another I bel ring to the same group ls vblained. The 

Tesctions of rieka and abcin corsespoud to those of the bacterial 

fotina, They are soluble in water, they are pro:ipitable by aleobol, but 

ily dialymblo than the aJbumous they have been called 

ing. Thwir toxicity is seriously impaired by boiling, and they 

also gradually become less ‘toxic on being kept. Hoth are among tho 

most oairy fo sys ed ee the a peeectah xen ‘they: 

are injected #ul wourly & period of twenty-four hours usually el 

SE ahasason'Da ths dome--Lalens ayziptaess sonia. Buh toad te prekion 

great tion at the seat of inoculation, which in the ease of ricin 

edecd ‘in an acute necrowix ; in fatal caves hetnorrhagic enteritie and 

nephritix be found. Both act as irritants to roucons membranes, 
Shain eopenially Telng capable of stig up most acae conn 

Tt is also certain that Che poisons of scorpions and of poisonous snakes 

te tho same group, The poisous derived from the latter aro 

tallod venins, and a vory Tepresentative group of wach vonin 

derived from different species has beou utudiod. To speak generally 

there is derivable from tho natural secretions of the poison glands & 

series of venine which have all the reactions of the bodies previously 

Like ricin and abrin, they are not sp easily dinlysable ax 

ated toxins, and therefore have also been classed as toxalbuming, 











ties are also similar ; auany of them aro destroyed by heat 
but Segre necessary here also varies rauch, and some will «tan 
boiling. “There ix also evidence that in a crude venin there may be several 


poisons differently sensitive 10 heat. All the venins are very powerful 
poisons, but hers there is practically no period of incubation—the elfeats 
sre-sinost immediate, An outstanding feature of is 
com le secret an) 
Soa GS ators a tumenieg op the resulta’ of mu 
pointed ont that different venoms been found 
more of the following poisons: a neurotoxin soting on the reepiratory 
centre, a metrotasin acting on the nerve-endings in muscle, m toxin 
eatising hemolysis, toxins acting on other eg the endothelium of 
Dhood-vessnls (this from its edfects has bs ned hweruorthaghn), 
merve-sells 9 toxin causing thrombosis, « toxin having an 
(te effect and preventing co: tion, toxin neutralising the 


1 qualities of the bo: and thus favouring putrefaction, 
a toxin agglutination of the red blood coryaseles, a yroedkytve: 




























170 THE TOXINS PRODUCED BY BACTERIA 
formont, a toxin causing systolic standatill of the exoised heart, Ai 





artioular venom containe « mixture in varying proportions of xe 
toxins, und the different elfwots produced hy the bites of diiferent snakes 
lurgely depend on thin variability of composition. Tho neurotoxic, the 
thrombotic, aud the hemolytic toxins are very important constituents 
of any yonem. The toxicity of different venoms varies much, and 9 
genoral statomont can bo made with rogurd to the toxicity of ifferont 
Polsons towarde man. Lamb has oalculated that the fatal dove of rade 
sobra venom for man is probably about «015 of a gramme, and that if 
such snake bitex with full glands many times this dose would 
probably be injected, but, of course, the amount omitted depends largely 
Sn the peciod which bas clapeed sing th auisal Int emptied its glands 
‘When o dove of « yenom not sufiiciont to couse immediate death from 
gonoral ollects be given, very rapid and widespread neorosts often may 
Scaur in a few hours round the «ite of inoculation 

An extromely important faot was discovered by Flexner and Noguchi, 
namely, that the luemolytic toxin of cobra venom in certain eaxex bas 10 
action by itself, but produges rapid solution of cod corpuscles when sono 
norma} scrum ix added, the latter containing w Iabilo complement-like 
body, which activates the venom, In thia thoro ie a clovo analogy to 
what holds fn tho euso of « hamolytic serum deprived of complement by 
heat at 65°C. (p. 479). Kyou and Sacha further showed that in addition 
fo serum-complement a substance with definitely known constitution, 
namely lecithin, had the property of activuting the hiamolytic substances 
fn cobra venom, the two apparently uniting lo form an actively toxic 
mbatanco. Later still, Kyos suooseded in demonstrating tho union of 
the two wmbstanoos to form a cobra-lecithid, and in separating the 
latter as a practically puro compound, which is, unlike lecithin, 
insoluble in other, but solable fn chiornferm. So far no example of 
activating a bacterial toxin is known, bat the results meatloned point to 
the possibility of this ovcurring 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 
pines ricin, abrin, snake poisons, and scorpion poisons, 

ides 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 antitoxinu. The nature of 
the antagonism between toxin and antitoxin will be discussed 
later, Hore, to explain what follows it may be stated (1) that the 
tnolecule of toxin most probably forms a chemical combination 
with the molecule of antitoxin, and (2) that it hay been shown 
that toxin molecules may lose much of their toxic power and 
still be capable of uniting with exactly tho same proportion of 
antitoxin molecules From those and othor cireumstances Ehrlich 
has advanced the view that the toxin molecule has a very com- 
plicated structure, and contains two atom groups. One of these, 
the Aaptophorous (arrev, to bind to) is that by which com: 








THE THEORY OF TOXIC ACTION 171 


oes ore mol peer existing in Ge 
tissues, The other atom @ calls the foxephorous, it 
Jorto this Yat tho torle eiicta are due. This atom is 


bound to tho coll cloments, «g. the nerve cells in tetanus, by the 
eee Rh. Ehrlich explains the loss of toxicity which 
occurs in, say, diphtheria toxin, on the theory that the 
toxophorous group undergoes disintegration. And if we euppoee 
that the group remains unaffected we can then 
understand how in way have its toxicity diminished and 
atill require the «ame proportion of antitoxin molocules for its 
neutralisation, ‘To the bodies whose toxophorous atom grow 
Daa cise egenerated, (strat gives’ the sanicicerotsee The 
theory may afford an explanation of what hus been suspected, 
namely, that in some instances toxins derived from different 
sources may bo related to one another, For example, Bhrlich 
has pointed out that ricin produces in a susceptible animal hody 
fn antitoxin which corresponds almost completely with that 
produced by another vegetable poison, robin (vide supra), 
though ricin and robin are certainly different, This be ex- 
ore according to the view that oe ae ia of o ie 
2 pa groups corres while toxephorous 
differ, evidence on which Ehrlich’s deductions are based 
fe of @ very weighty character, and will be again referred to 
in the chapter on Lammnity, 

With regard to tho 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 
elass of substances It is certain that « tolerance against such 
ae is difficult to extablish and is not of a lasting character, 

thus cannot my what the mechanism is by whieh these 
poisons act. It may be sid that Macfadyen by grinding up 
bacilli freaen by liquid oir claimed that on thawing he 
the intracellular toxins in liquid form, and he further 
‘stated that by using this fluid he could immunise animals not 
the toxins but also agninst the living bacteria. 
fe have already pointed out that those who claim for the 
ae @ special charactor hold that the activity of these 
lies has as its effect the interference with the phagocytic 
functions of tho leucocytes, They also hold that a special type 
Of immunity can be developed against the aggressive bodies, 





— ————e | 


CHAPTER VL 
INFLAMMATORY AND SUPPURATIVE CONDITIONS. 


‘Tus subject is an exceedingly wide onc, and embraces a great 
many pathological conditions which in their general characters 
and results are widely different, ‘Thus in addition to anppuration, 
various inflammations, wlcorative endocarditis, septicwmia and 
pywinia, will come up for consideration, With regard to these 
the two following general statements, established by bacteriological 
research, may be made in introducing the aubject. In the first 
place, there is no one specific organism for any one of these 
conditions ; various organisms may produce them, and not ine 
frequently ‘more than one organism may be present togeth 
In the second place, the same organixm may produce widely 
varying results under different circumstances—at one time a 
local inflammation or abscess, at another multiple suppurations 
or & general sopticwmin, The principles on which this diversity 
in results depends have already been explained (p, 151). 
Farthermor, there are conditions like acute pneumonia, epidemic 
meningitis, acute rheumatism, ote. which have practically the 
character of specific diseases and yet which as regards their 
esxcutial pathology belong to the same class. ‘The arrangement 
followed is to a certain extent one of convenience, 

It may be woll to emphasise some of the chief points in the 
pathology of these conditions, In suppuration the two main 
Phenomena are—(a) a progressive immigration of leneocy 
ehietly of the polymorphonuclear (noutrophile) variety, and (4) 
a liquefaction or digestion of the supporting clements of the 
tissue along with necrosis of the cells of the part. ‘The result 
ia that the tissue affected becomes replaced by the cream-like 
fluid called pas A suppurative inflammation is thus to be 
distinguished on the one hand from an inflammation without 
destrnction of tissue, and on the other from necrosis or death 


17a 























NATURE OF SUPPURATION Ws 





et masse, whore the tissue is not liquefiod, and leucocyte 


accumulation may be slight, When, however, tion is 
# very dense fibrous ti liquefaction muy be 
im @ portion of dead tissue or may remain 


tie 
Which may be present, 
Many fener have been performed to Me ree whether 
suppuration can be produced in the absence isms 
Warious chemical substances, such as epoton il, nivate of 
Iver, turpentine, ete.—care, of course, being taken to ensure 
the absence of ‘bacteria. ‘The geueral result obtained by in- 
dependent observers is that asa rule suppuration does not follow, 
but that in cortain animals and with certain substances it may, 
bs pus ete trea we = Tris treacle! ‘questioned 
some wi a8 juced really 
eesgieslty saa chamically with aie to eae oe 
Buchner showed that suppurntion may be produced by the 
injection of dead lacteris, ¢g. sterilised cultures of bacillus 
ote. The subject has now more a scientific than a 
interest, and the general statement may be made that 
tet all cases of trae ee met with clinically are 
the action of living micro-organisms, 
“here spelled to sonditons 4 in which the 
ano within and give rise to symptoms 
of general poisoning, without, however, tring abscesses in the 
In 


may be Sexpeutbe t to detect any in the blood tartup Hie 
seen in large numbers in the capillaries of ong 
i liver, ete, post mortem, The essential fact in pycewia, on 
the hand, ix the occurrence of multiple abscesses in internal 
‘Organs and othor parts of the body. In most of the casos of 
typical pyremia, common in proantisoptic days, the starting-point 
of the diseas: was a septic wound with bacterial javasion of a 
leading to thrombosis and secondary einbolism, Multiple 
i of suppuration may be produced, however, in other WOys 8 
be described below (p. 186). If the term “ pywmia" be 
mised to embrace all such conditions, their method of production 

should always be distinruiahod. 


a 





Bacrensa a8 Cavars or 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, whilat of 
ne ee ey determined, 

‘ho was one of the first to study this Gina Go 
ee ‘nn ee the Ai most frequently 
1 ler 
(streptococei| le 


epi 
Fiona) Breyer sraneyre ead 
id hat the former were seen common in eee acute | 
abscesses, the latter in gy suppurative conditions, Rosen- 


bach shortly afterwards (1884), by moans of cultures, differentiated 
several varieties of mivrococei, to which he gaye’ the following 
special (ales see ronda) pgeckconlae aureus, Lies tcl 
pyogenes 
‘Other organisms Teese upton such ns spat 
eoceus pyogenes citreus, staphylococous cereus albus, staphylococeus 
cereus, f, pneumococeus, pneumobacrilus (Friodlunder bacillus 
eenet fte (Passct), bacidlus coli cNpiy veoh bgaewdin., lactis 
pono — ea 
‘microsoceus preumocoecur, pneumobacillua, 
yt Fntracelularis meningitidéa, and others, Vig 
)* — “Tarswoniary inflammations and suppurations following acute 
diseases the corresponding organisma have been found in some 
cases, such as gonococeus, typhoid bacillus, influenza bacillus, ete, 
Suppuration ia also produced by the ‘actinomyces and” the 
glunders bacilIns, and sometimes chronic tubercular lesions have 
suppurative charactor. 
phylococcus Pyogenes Aureus. —Microwopical Characters. 
—This orguniem is a spherical coccus about "9 x in diameter, 
which grows ele in clusters or masses (Fig. 52). Itstaing 
readily with all basic aniline dyes, and retains the colour in 
+ Grams met] 
rvation.—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 
fa streak of growth is visible on the day after inoculation, aud 
d on the sscond or third day iquefiction commences at the top. 
As liquefaction proceeds, the growth falls to the bottom as a 
floceulent deposit, which soon assumes a bright yellow colour, 
while a yellowish film may form on the surface, the fluid portion 
atill remaining turbid. Ultimately liquefaction extends out to 


























STAPHYLOCOCCUS PYOGENES AUREUS 178 





Fie. 52.—Staphylococons pyogenes aureus, 
youtg culture on agar, showing elnmps 


of ecent. 
Btained with weak carbol-fuchsin. » 1000. 


naked eye as whitish yellow points, 
which afterwards become more dis- 
tinetly yellow, Liquefaction occurs 
these, little cups are 
formed, at the bottam of which tha ” ‘ 
colonies form little yellowish masses, Fi, 5% —Two stab calters 
‘On ager, a atroke culture forms ef staphylococous pyogenss 
line of abundant yellowish growth, Svea In goat, (a) 10 days 
with smooth, shining surface, well  firtnesion of the mania 
formed after twenty-four hours at aud charscters of growth, 
37 CO. Later it noes bright Natural sine, 
in colonr, and resembles « 
of oil paint, Single colonies on the surface of agnr are 
etreular discs of similar appearance, whichmay reach 2 mara, or more 
in diameter. On potatoes it grows well at ordinary temperature, 
forming 4 somewhat abandant layer of orange colour. In dowillon 
it produces & uniform turbidity, which afterwards settles to the 
bottom asan abundant layer, which assumes a brownish yellow tint. 
Tn the various medin it renders the reaction acid, and it coagnlatos 

















ee 





176 INFLAMMATION AND SUPPURATION 
ns Popa et 2B Ec The cultures have a somewhat 


Say Maa considerable tonacity of life ae the Pita 
in gelatin often being alive after baie en ni kept 
months. Jt also requires & rather higher ee to rapt 
Hs MESES sporefree bacteria, viz, 30° C. for half an hour 
(Lal 

The staphylococcus pyogenes albus is similar in charsoter, 
with the exception that its growth on all the media is white. 
‘The colour of the staphylococcus aureus may become lees dis 
tinetly yellow after being kept for some time in cultura, but it 
never assumes the white colonr of the staphylococcus albus, and 
it has not been found possible to transform the one i 
into the other. A micrococcus called by Welch s weEe 

ails is practically always present in the skin 
opithelium ; it is distinguished by its relatively non-pal ee 
by liquofying golatin somewhat slowly. 
profubly an attenuated variety of the staphylococcus si fa 
The staphylococcus pyogenes citreus, which ix less frequently 
mot with, diffors in the colour of tho cultures being a lomon 
yellow, and is loss virulont: 
than the other two, 

The — staphylococeus 
cereus albus and staphy- 
fococcus cereus flavus are 
of amch loss irnportance, 
They produce a wax-like 
growth on gelatin without 
Hquefaction ; hance their 
name. 

Btroptococeus pyo- 
genes. —This organisin 
is  cocens of ‘ty 
larger size than 
staphylococcus aureus 
about 1 in diameter, 

—Streptovoccus pyogenes, young ov). and forma chains which 
ely a, seoateg aint rid may contain a large num- 
Stained with weak carbol-fuchsin, * 1000. ber of members, especi- 
ally when it is growing 
in fluids (Fig. 64), Tho chains vary somewhat in length in 
different specimens, and on this ground varieties have been dis- 
tin, d, e.g. the atreptorocens brevis and streptococcus 
(wide infra). As division may take place in many of the cocci 








STREPTOCOCCUS PYOGENES wt 


in a chain at the sme time, the appearance of a chain of 
diplococe’ is often met with. In young eultures the cocci are 
fairly uniform in siz, 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. Tu 
its staining reactions the streptococons resembles the staphylo- ~ 
soosi described, being readily coloured by Gram’s method. 
Cultiention.—In_ cultures outside the body the streptococcus 
pyogenes ercexunch mary slowly than the staphylococe!, and algo 


Poe 55.—Caltaw of the 





veges on 
au ngar peepee ‘showing Fro, $8,—Bacillus pyoeyaneur ; young 
mamervas colonics—three oulture on agar, 
smaccombrectrokes. Twenty- ‘Stained with weak carbol-fuchsin. x 1000, 
fear bowre’ growth, Neto 

is oat mare. rally, bring io every respect « mere delicate 


rape stone gelatin a stab culture shows, about the aecond day, 
thin fine, which In ite subsequent growth is seen to be formed of 
4 row ef tninute rounded colonies of whitish colour, which may be 
separate at the lower part of the puncture. They do not uwsinally 
‘exceed the sizeof « sinall pin's head, this sizs being reached about 
the fifth orsixth day. Tho growth. does not spread on the snrfi 
wad wo Hasuainction ‘of tho medium occura. The colonies in g 
plates have a corresponding appearauce, being minute spherical 
einlsot whitish colour. A somewhat warm temperature is 





We 


i 





178 INFLAMMATION AND SUPPURATION 


ec for growth ; even at 20° C. some varieties do not 


‘On the frowth tales piace slong the sirka 
as a collection rams 

Stine great tendency e remiin mane 

(He 55), ‘The soparate colonies remain ecall rarely exeoeding 

mm, in diameter, Cultures on kopt at the body 

iota ‘may often be found to be after ten days, On 


potato, ax a tule, no visible growth takes place. In milk it 
pram a strongly qcid reaction but no clotting of the medium, 
forments Inctoac, anccliirote, ond salicin (Andrewes and 
Horder) ; it produces no fermentation of inulin in Hiss's serum- « 
water-medinm, in this respect differing from the pneumococens, 
Tt has a tence tag action, as can bo Cencaneratea by 
owing it in blood-agar plates (p, 38), Tn bouillon, 
forms numerous minute granules which afterwards fall ms 
the bottom, the deposit, which is usually not very abundant, 
having a sandy appearance. ‘The appoarance in broth, however, 
presents variations which have been used as au aid to distinguish 
different #pecies 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 gi tise ton 
finer deposit. To a variety which forms distinct spherules of 
minute size the term streptococous eonglomeratus han been given, 

Faricties of Streptococci. —Yormerly the streptococcus pyogenes 
and the streptococcus erysipelatis were regarded ux two distinet 
wpecios, and various points of difference betwoon them wero 
given, Further study, and especially the results obtained by 
modifying the virulence (p. 182), have shown that thes: dis- 
tinctions cannot be maintained, and now practically all authorities 
‘aro ngrocd that the two organisms are one_an me, 
erysipelas being produced when the streptococcus pyogenes of a 
certain standard of virulence gains entmnee to the lymphatics of 
thy ekin. Potruschky, moreover, showod conclusively by inocu 
lation that a streptococcus cultivated from pus could canse 
eryaipelas in the human subject. 

Streptococci have also beon classified according to the length 
of the chaina ‘Thua there have beon clistinguished (c) strapton 
coccus Longs, which occurs in long chains and is pathogenic to 
rabbits and mice ; (+) reptocoe brevs.whlch ja common in 
the mouth in normal conditions, and Ta untally non pathogenic j 

a called fi 


its forming in 

















ject. Thess are :—(a) A’ short-chained form called 
mitis, which occurs chiefly in the saliva and farce 
G which is the 


more active fermentative ies 
atreplococens ani which corre- 





towns, and appears to be derived from horse dung.* 
Schottmillor has cmployed the appoarance of the colonies of 
‘streptococel on blood agar us a means of separating varicties, 
the medinm used consisting of two parts human blood and five 
meltod agar. He distinguishes the streptccoceus Longue or 
ich forms grey colonics and has « hemolytic 
} & atreplococeus mitior or viridans, ~ short-chained 
| which produces small groon colonies and very little 
is, and a atreptococeis mucomua encapeulatus, which, as 
tes, shows well-marked capsules and produces 
have a slimy consistence. It should be noted 
‘that oe blood agar the pneamococcus forms grown colonies and 

| prodiaoes io hiernolyais, 
reference mst be made to the original papers Laweet, 

as 








180 INFLAMMATION AND SUPPURATION 


Tt will bo thus seen from this acount that the streptococcus 
pyogenes as described above is the organism most frequently 
associated with the pathogenic processes, and that short-chained 
forme are common sapro; in the human body, although 
they may be associated with conditions of disease; these may 
be subdivided sccording to their fermentative activity ax 
detailed. And lastly, there is the streptococcus conglomeratus 
{enginoew), which i# specially abundant in the throat in scarlet 
‘ever, though it also occurs in other acute catarrhal states. No 
definite statement can yet be made us to the etiological relation 
of streptococci to scarlet fever ; we ean only say that stroptococe! 
are almost invariably present in the fauces, and that to them 
muny of the complications of the dixease are due, 


Bacillus coll communi —The microscopic and euleural charactors are 
oworibed in the chapter on typhoid fover. ‘The bacitiua tactis 

and the bucitlus pyogenes fielidus closely resemble it; they are either 
Yaristion or lowly related apocios. formor is distiaguished Ly 
producing more abundant gas formation, and by its growth on gvlatin, 
‘to., boing thicker and whiter than that of the bacillus voli. 

Bacillus mrogones encapsulatas sometimes invades the tisanes bofore 
death, and is ohatucteriend by the formation of bubblos of yas in the 
infooted parts, Its characters are desoribed in Chapter XVI, 

Bacillus pyocyaneus. This orgauism ocours in the furm of minuto 
rode 1°5 to 8 w in longth and lew than Sw in thickueas (Fig. 66). 
Occasionally two or three are found attached ond to end, 'Th 
aotively motile, and do not form spores. They stain row!ily w 
ordinary taxi sting, but are deooloriad ty Grams need. 

Cultivation: Te GROWS really ow MIT Thin ordinary wedTa wt the room 
temperature, the cultures being distinguished by tho formation of @ 
“pani pigment. In puncture cultures in poptone-golatin a greyish 

appears in twenty-four hours, and at ita upper part n amall cup of 
Viquefaction forma within forty-olght hours. Jit thie time a slightly 
ro tint is soon in the superficial part of the gnlatin, ‘The 
ffitirtiotion ‘extends pretty rapidly, the fluid portion belng turbid and 
showing moss of growth ut its lower part. The grecu colour 
becomes moro and more marked and diffuses through the gelatin. 
Ultimately liqnefistion reachos the wall of the tube, In plate oulturos 
the colonies appear ne minute whTthpoIbts, tose on the surfuce boing 
tho larger. Under a low power of the microscope they have a brownish. 
yolloweolour and show a nodulated surface, the supericial colonies being 
thinner sad larger, Liquefaction soon coours, the wolonies on the aurfnce 
forming shallow cups with small irregular masses of growth at the 
Bottoms the deep colonics sasall epheros of Liquefaction. Around. the 
colonies « groonieh tint appears. On agar the growth forma an abundant 
slimy groyish layer which afterwards hocomes greenish, and a bright 
green colour diffuses throngh the whole autstance of the medinm. On 
potatoos the growth is an abundant reddish-brown layer resonbling that 
Of the glander bacillus, and tho potato sometimes shows a greonish 
discoloration. 

From the cultures thoro ean be extractod by chloroform a coloured 

body pyouyanin, which belongs to the aromatic sories, and crystallisos 























EXPERIMENTAL INOCULATION 18! 


‘in tho form of 1 delioate binsh-gevon neodlen Ou the addition of 


@ weak acid its co! ae toa rod. 
‘This orga distinct Pathogenio in certain animals. 














fay, prodiine, nocd fife treet ny 
r Tisers vibes vena or ieee Sionted by atone 








Tt grows readily on all 
‘the media at the unter 
geratn” Ina puncte Sak 
ture on ina in 
pf iti Tine F% ST-—Mlrocoonns tetngrous young |b) =| 

{ 


uf ‘ultore on r abowi 
ieee eo se Stifued with weehtootbel fucka. > 1000, 
E 


Mia is wot as ‘On ete satus of agar and of potato the we erowsh 
a gin abundant moist layer of the same colour, Tre gre 
pevuliar viscid or tenacious character, ey Pay hs 
ol tous cart fo seat eee peer 
ite mice are ex susceptible to this eanieotts 
injestion is followed igs tee I eepteats, epi spacey th oy found 
ing urnbers in the blood throaghoat the badly. nea ge are 
etimes oly a foal atiwcese with i pots ayy 
pen sashes j somotimes there is also septionmia, 


Experimental Inoculation —We shal] consider ehietly the 
Maphylococcus pyogenes aureus and the streptococcus calhg 
as these have fees most fully stuitied. 

Te may be stated at the outset that the occurrence of xuppara- 
tion depends upon the number of organisms introduced into the 
tissues, the number necessary var; not only in different 
‘animals, but also in different parts of the same animal,—a smaller 

ing suppuration in the anterior chamber of the 


ssumnber prox 
‘€7¢, for example, than in the peritoncum. ‘The virulence of the 











li 








results be 
carrion 





‘blood stream in sufficient numbers to cvuse secondary suppurative 
foci in internal orguns (cf. intravenous injection), 
Entravenous injection in rabbits, for example, produces interest 
ing results which vary Aesistas he the quantity used. Ifa con- 
animal may ¢ oad in tea, oe 
rope ie septicamin, "vara Cisse as s, Paaeet 
the various organs, often form a 
amaller Tees he nsed, the cocei gradually disay at eas 
cireulating blood ; some become destroyed, while a pottlo a 
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 
zone of i a congestion and hwmorrhage. Similar small 
ea uy be produced in the heart wall, in the liver, under 
periosteum, and in the interior of Line, and ooeasionally in 


tho striped musclos. Very in experimental 
Facake Atle pare mela ce i kro valves of the heart 
If, however, when the organisms are injected into the blood, 
thore be any traumatiom of a valve, or of any other part of the 
vs they show a special tendency to sottle at these weakened 


amines on the human mbject have also proved the 
pyogenic propertion of those organisms, Garré inoculated 
scratches near the root of his fingernail with a pure culture, a 
smal] entancous pustale resulting ; and by rubbing a culture over 
‘the akin of the forearm he caused a carbuncular condition which 
healed only after some weeks. Confirmatory experiments of 
this nature have been made by Bockhart, Buram, and others. 

‘Whon tested experimentally the staphylococcus pyogenes albus 
has practically the same pathogenic effects as the staphylococcus 
murets. 

‘The streptococens pyogenes is un 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 
uader ordinary conditions, in the course af time logo practically all 























BACILLUS COLI COMMUNIS 183 


Breucneey By passage from animal to animal, however, 
acne ae nos cen ureenes, Ord eateries Cmte st 

ure correspondingly varied. epieigigieeen ante 
Meet sies unarvioce ct seeps on ‘be enormously 
increased by growing it alternately (a) in a mixture of human 
eee esta aaa meek noe (0) Sn aeee 


a rabbit; ultimately, after ast 
Siete rea nn pecan ncteslc nae eee 
duced into tho tissucs of a rabbit produces haar 


diversit; Tin the hu subject with which 
great ine ty ol in Tinney strep- 
Bacillus 


Injection i 

bene inflammation which becomes purulent if animal 
lives suificiently long. If, however, the virulence of the a 
2 ee eed death takes place before ela 

stabliabod, and thore is a soptiewmic condition, the orguniams 
‘cccurring in large numbers in the blood. Intravenous injection 
of a few drops mea virulent bouillon eulture usually produces a 
mpid septicwmia with seattered hamorrhages in various organs, 


‘Other Bffects.—It hax heen found ly independent oluervers that in 
‘eases whore rabbits recover after intravenous injection of bacillus colt 
Searle, vertaln Fovportion snl from pannleals a | aametinea from 

wusoles, eaposially of the F limbs, these 
ths coll in the aot cord, 


peta etree ‘the anterior cornus of the #| 


‘similar results hare been obtsined by others after inoculations 
bpocel and strepicovce, a cartan proportion only of the 
atalytio symojacms scat corresponding chengte fo the 


ae oeence neal el to be duc chiefly to the action of 


jams on tho hij fet organised nervous elenents. 
further research requires to be done fore the importance of these 
ly atimated, but it ix aot improbatlo that thuy 
Sar tae tg tehtor ue ‘eausation of nervous lesions whieh ooeur in the 
) subject, and ie of which at present 
aeeareacs he ench school, consider that pazalya 
hk the focillin coli commands ke o 
es hare ti iytio conditions 
seute infective fevers toay be produced by the products of 
‘oveed, which frequently occur in those conditi an. 


= | 





186 INFLAMMATION AND SUPPURATION 


Lesions in the Human Subject. —The following statement 
may be made with regard to the occurrence of the chief organisms 
mentioned, in the various suppurative and inflammatory con- 
ditions in the human aubject. ‘The account is, however, by no 
means exluustive, as clinical bacteriology has shown thit practi- 
cally every part of the body may be the site of a lesion produced 
by the pyogenic bacteria It may also be noted that acute 
catarrhal conditions of cavitics or tubes are in many cases aleo 
to be ascribed to their presence. 

"The staphylocoeet ure the most common cuusal agents in 
localised absovesos, in pustules on the kin, in carbuneles, boils, 

cto, in acute suppurntive 
periostitis, in catarrhs of 
mucous surfaces, in ulcers 
ative endocarditis, and in 
various pyemicconditions, 
‘They may also be present 
in septicumia, 

Streptococes are especi~ 
ally found in. sprewling 
inflammation with ar with- 
out suppuration ; in diffuse 
phlegmonous and. eryai- 
pelatons conditions, aup- 
purations in serous mer: 
branes and in joints (Fig. 
58). They also occur 
Fin, 68—Streptoooecl in acute suppuration, Ute suppurative perio- 

easy tlm; stained by Grams method stitis ond ulcerative en- 
and safrauin, 1000, docarditi. Becondary 
abscesses in lymphatic 

glands and lymphangitis are also, wo believe, more frequently 
caused by streptococe’ than staphylococci, ‘They also produce 
fibrinows exudation on the mucous surfaces, leading to the 
formation of false membrane in many of the cases of non- 
Aiphtheritic inflammation of the throat, which are met with in 
scarlatina ! and other conditions, and they are also the organisms 
mest frequently present in acute catarrhal inflammations in this 
situation. In puerperal peritonitis they are frequently found in 
& condition of purity, and they also appear to be the most 
frequent cause of puerperal septicemia, in which condition they 
may be found after death in the capillaries of various organs. 


2 True diphtheria may also occasionally be associated with this siseas, 
Usually as a sequel. 

















‘LESIONS IN THE HUMAN SUBJECT = 185 


medlstod wih lier Spegurh eeguians * Bown se ot 


streptococcus, which, 
certain points of diffarence from the 
‘The bacitlus colt communis is found in a growt 
matory and suppurative conditions in connection with the ali- 
Se tion in the peritoneum, 
ss or withou' perforation af tho 
Powel, in the peritonitis following stra ion of the bowel, in 
Cote eer fap ped cciagirs around 
hile-ducts, te, It may also occur in lesions in other parts 
ise bra peel gl ae toy pea 
‘aro aasociatod with lesions of the intestine, though in othors such 
Paeeng eel It is ulso frequently present in inflammation 
of the urinary passages, cystitis, pyelitis, abscesses in the kidneys, 
ate., these lesions being in fact most frequently caused by this 
aan gree 
Th cortain cases of enteritis it is probably the causal pare 
vem is difficult of proof, as it is much in in 
i ehaegratd pes beon, eraerpattes bry the 
it 
pesteetat caltietel from various lesions is more virulent than 
‘that in the intestine, its virulonce baving been heightened by 
growth in the tissues, 
The micrococcus tetraenus is often found in sappurations in 
os Beagle I ee ered geared ocenrs in 
various lesions of the respiratory tract, in phthisical cavities, 
pee ra tt Lats ee rg a a present alone, and 
probably has a pyogenic action in the human subject under 
certain conditions. In other cues it is associated with other 
organisms, Recently one or two cass of pywmin have been 
Gmecribed 3 in which rgunistn was found in a state of purity 
in the pus in various situations. In this latter condition the pus 
has beon described as possessing an oily, viscous chaructor, and 










infrequent ate with othor orgunisma We have met 
‘with it twico in cases of multiple abscesses, in sseociation with 
the staphylocovcus pyogenes aureus Lately some diseases in 
children have been deserited in which the bacillus pyocyancus 
“Thos bees found Sep mee the bedy; im those cases the chief 
‘symptoms have been fever, gastro-intestinal irritation, pustular 
f or petechial eruptions on the skin, and general marasmus 


222 





186 INFLAMMATION AND SUPPURATION 


Suppurative aud inflammatory conditions, associated with the 
onmnisms of special diseases, will be described in the respective 


bar Hr 

of Entrance and Spread.—Many of the organisms of 
suppuration have a wide distribution in nature, and many also 
are present on the ekin and mucous membranes of healthy 
individuals, Staphylococci are commonly proscnt on the skin, 





Fic. 50.—Minnte foous of commencing suppuration in brain—case of acute 
uleerstive endcearditis. In tho oantrs » «mall homorrhage ; to right 
wide dark maser of staphylosocel : rove of loucocytos at periphery, 

Alum earmine and Gratn's method. —% 50, 


and leo occur in the throat and other parts, and streptococci 
can often be cultivated from the seerctions of the mouth in 
normal conditions, The ynenmococens of Fraenkel and the 
pneumobacillus of Friedlinder have also boon found in the 
mouth and in the nasal cavity, whilst the bacillus coli communis 
ina normal inhabitant of the intestinal tract, The entrance of 
these organisms into the deoper tissues when a surface lesion 
occurs can be readily anderstood, ‘Their action will, of course, 
be favoured by any condition of depressed vital Though in 

conditions the blood is bacterinm-free, we must suppose 

















ENTRANCE AND SPREAD OF BACTERTA 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 tho latter being especially 
numerous In meat cases thoy are killed by the action of the 
healthy serum or cells of the body, and no harm results, If, 
howaver, there be « local weakness, they may settle in that part 





Pia, €0.—Secondary infection of » glomerulus of kidney by the staphylo+ 
occas survus, in & case of ulcerative endocarditie. The coeci (stained 
Aarkly) sre soon plugging the enpillaries ant nso lying fre. ‘The 
glomeraiax ch swollen, infiltrated ty leucocytes, and partly necrosed, 

Paraffin ection ; stained by Gram's method and Bisrmarck-brown, 300. 








and produce suppurstion, and from this other parts of the body 
‘be infected. Such a supposition as this is necesary to 
many inflammatory and suppurative conditions mot with 
In some cases of multiple suppurations due to 
Maphylococens infection, which we have had the opportunity to 
examine, only an apparently unimportant surface lesion was 
ty whilat in others no lesion could be found to explain 

ie sic of the infection. The term eryplogenetic has been 
spptied by some writers to stich otses in which the ovigieah 


wal _ re 














188 INFLAMMATION AND SUPPURATION 
pois = infection cannot be found, but ite nae is scarcely 


“Nhe sath 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 10 the the 
‘organs where the primary lesion exists. Second, by nat 
channels, such as the ureters und the bileducts, the pees) 
being generally associated with an inflammatory condition of the 
fining epithelium, In this way tho kidnoye 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 ofton being obscure ; (4) by a septic phicbiti 
with suppurative softening of the thrombus and resulting em- 
Kolism ; and we may add (c) by a diroct extension along a vein, 
producing a spreading thrombosis and suppuration within the 
vein. In this way suppuration may apread along the portal vein 
to the liver from a lesion in the alimentary canal, the condition 
boing known as pylephlobitis suppurativa, 

Although suny 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 stroug presumptive evidence 
that all cases of endocarditis are due to bueterial infection. In 
the simple or yogetative form, so often the result of acute 
rheumatism, the micrococcus rheumaticus (p. 193) has been 
cultivated from the valves in a certain number of eases, and is 
probably the causal agent in most instances, 

Endocarditis of the ulcerative type may be produced by 
various organisms, chiefly pyogenic. Of thexe the staphylococei 
and xtruptococet Ar tly found. In some cases of 
ulcerative endocarditis following pheumonia, the pneumococeus 
(Frucnkel’s) is present; in others pyogenic cocci, especially 
streptococci, Other organisms have heen cultivated from 
different cases of the discasc, and some of these have received 
apecinl names; for example, the diplococcus endocarditis encap- 
sulatus, bacillus endocarditidis griseus (Weichselbanm), and 
others. In some cases the bacillus coli communis has been found, 
and occasionally in endocarditis following typhoid the typhoid 
Dacillus hus been described aa the organism present, but further 
observations on this point aro desirable, ‘The gonococcus also 
has boon shown to affect the heart valves (p, 225), though this is 
& vory rare occurrence, Tubercle nodules on the heart valves 




















ENDOCARDITIS 189 


have been found in a few cues of acute tuberculosis, though no 
vegetative or ulcerative condition is prodiiced. 

In some cases, though we believe not often, the organisms 
muy attack healthy valves, producing a primary ulcerative endo- 
carditis, bat more frequently the valves have been the sent of 
previous endocarditis, secondary ulcotative endocarditia being 





Fa 61.—Seotion of  rryntation in alcorative endecarditie, showing numerous 
Hephyloooa tying io the spaces, The lowor portion ls » fragment 


in procem of separation, 
Btained by Gratn’s metho! and Bisroarck-brows. + 600. 


thas produced. Tn some cases, especially when the valves have 
been previously dissed, the source of the infection is quit 
obscure. It is erident 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, 
tx a healthy cellular tissue does On microscopic examination 
of the dissased valves the organisms are usually to be found in 
eormons numbers, sometimes forming an almost continuous layer 
‘on the surface, or occurring in largo musaes or clusters in spaces 
in the vogotation (Fig. 61). By their action a certain amoant 








+ 2 


Suey 


fntas rr 


lea Oar! 


lads, 





190 = INFLAMMATION AND SUPPURATION 


of softening or ing down of the v ions cecurs, and the 
embolus eae as the orien infection to other 
organs, and give rise to secondary euppurations. 


Peperimental,—Ocoasionally ulonrative endocarditis is produced by the 
simple intravenous injeotion of staphylocopel and streptocoel Into the 
elroulation, but this isa very rare occurrence. It often follows, howerer, 
‘when tho Yalvos havo boon yroviously injared. Orth and Wystokowitech 
at a comparatively early dato produced the condition by damaging tho 
nortic ousps by a ginea ‘vod introduced through the carotid, ond after 
wards injecting staphylococe! into the circalation, Similar oxperimenta 
have xinco heoa repented with xtreydocorei, pneumenoee, and other 
organisms, with lke result, Ribbert found that if a potato oulture of 

‘staphylococeas anzens were rubbed down ia salt solation #0 aa to 
form an omul and then injected into the circulation,"some minute 
fragmonts beoamp arrested at the attachment of the chordw tendinew and 


jnood an uloarative endocarditis. 
Acute 81 Perle is and Qsteomyslitis-—Spesial 
mention is made of thie condition on account of its comparative 
froquency and gravity, The great majority of cases are caused 
by the pyogenic cocci, of which one or two varietios may be 
mesent, the staphylococcus aureus, however, occurring most 
frequently. Phetmmororet fave been found alone in ome case, 
‘and in a few cases following typhoid fever, apparently well 
authenticated, the typhoid bacillus has been found alone, Tn 
others again the bacillus coli communis is present, 
‘The affection of the periosteum or interior of the bones by 








Arrathese organisms, which is expecially common in young subjects, 


may take place in the course of other affections produced by 
tho same organisms or in the course of infeetive fevers, but in a 
great many cues 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 nbscomes in the 
Kidneys, heart-wall, lungs, liver, ete, suppurations in, serous 
cavities, und ulcerative endocarditis; in fact, some cases present 
the most typical examples of extreme gencral staphylococcus 
infection, ‘The entrance of the organisms into the blood stream 
from the lesion of the boue is especially favoured by the arrange- 
ment of the veins in the bono and marrow. 


Beperimentat,—Multiple abscomios tu the bones and under the pari 
cateum may occur in siinple intravenous injection of the pyogenic 
‘coos! into the blood, end are especially linble to be formed when young 
animals aro used, ‘Those abscesses are of stall size, and do not aprea 
in the same way a4 in the natural disoaso in the human sutject. 

In oxperiments on healthy animals, however, the conditions are not 
analogous to those of the natural diseaso, Wo uiust presume thot in the 
Intter there ts some local weakness or susceptibility which eusbles the 





few whlch hare reached the part bythe Mood to mtle aud 
Taper cute tis pting ot tn pacliote bebe Pas acon 
iene, Ter emo ati injured 









Capes human sabject as a therepeutic measure in 
tt diseans, he was able to reproduce orywipelas, As 
pale asere ser one after another of the suppored points 


degree of virulence. Tt must be noted, however, that erysipelas 
i Sb patient to patient as erysipelas, and pmrnlent con- 
do not appear liable to be freer 

the connection 


comes to an en: 

Series may sent to he aa synovial cavitics 

Cin aopereatd or suppurative change,—peritonitis, met 

‘and synovitis may thus be luced. 

ivitis—A considerable number of organisms are 
leslie pe pesca of conjunctivitis and its usociated 
Tesions. Of number 9 to be spocially nssocinted 
with aed they ‘Thus a small organism, higeré known as 
the Koch-Weeks bacillus, is the most common cause of acute 
oe conjunctivitis, especially prevalent in Egypt, but 
‘also common in this country, This organism is very minute, 
Ss, Tittle more than 1 jx in length, and morphologically 
bles the influenza bacillus ; conditions of growth are 
more restricted, as it rarely Hoots ‘on blood agar, the best 
Deing serum agar. On this medium it produces minute 
Weolonies like drops of dew. The obtaining of pure 
fs & matter of considerable difficulty, and it is nearly 
yx accompanied by tho xeroeis bacillus, It can readily be 













192 INFLAMMATION AND SUPPURATION: 
found i the muco-purulent secretion by staining films with 


weak (1:10) carbol- 
fuchsin, and in often to 


BR Biv sae 





paar be ween in the interior 
eA Ayal of lencoostes (Fig 62). 

Another organi ox. 
ey Led coedingly like the prov 
pin Oe fous, apparently differing 
f a We from it only in the rather 


wider conditions of 
rowth, is Milller’s bacil- 
jus It has been eulti~ 
vated by him in a eon. 
sidemblo proportion of 
- J cases of trachoma, but 
Sat = its relation to this con 
#10, 62,—Film preparation from a esse of ‘ition is etill matter of 
acute conjnnetivitis, showing Koch-Weeks dispute. Another bacil- 
‘bacilli, chiefly contained withis leucocyte, Ina which is maw well 
(From « preparation by Dr. Inglis Pollock) recognised ix the diplo- 
ed bacillus of conjunctivitis 
first described by Morax, Tt ix especially common in the more 
subacute cases of conjunctivitis, Eyry fonnd it in 25 por cent 
of all casca of conjunc 
tivitis Its caltural char 
acters are given below. 
‘Tho xerosis bacillus, 
which is a small di 
theroid organism (Fi 
123), has boon found in 
xerosis of the co 
tiva, in follicular con- 
fonetivitia, and in other 
conditions ; it_ appears 
to occur sometimes also 
in the normal conjune- 
tiva. It ia doubtful 
whether it has any 
pathogenic action of im- 
portance, — Acnte [60M "pia 63, ram preparation of onnjanctival 
Junctivitis is also! pro“ Sscrotion showing the Morax diplo-tacil!an 
duced by the pneumo: — of onjumotivitix, x 1000. 
coccux, epidemics of the 
disoasc being sometimes due to this organism, and also by 

















ACUTE RHEUMATISM 193 


and staphylococci, ‘True diphtheria of tho con- 
junctiva caused by Klobs-Lafler us alo orenra, 
Whilst in gonorrheral conjunctivitis, often of an acute purulent 
typo, the gonococeus is present (p, 225), 





eet east at eT a 
Mori; p y Maausing 1 sa stay osu 
‘ing. In patre, or in short. chaite’of pairs (fig, fi 
Aes nat er ora erie iran hethed. 
tow the cna gelatin aad gar din the elton 
ing neceasacy. On serum ft fora small rounded colonies 
hic ch yon snl pis of Lgwction ; hemos thas boon called he 
(ine locunotus. In cultures it is distinetly pleomorphor and 
iepplation Tera elas Gotces ST fu sacryathnpeets take hace ealioahe 


Bhoumat ‘There are many fuets 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 coceus obwrved by Triboulet, and by Westphal and 
Wasermann, the characters and action of which were first Pee 
investiguted is this conatry by Poynton and Paine. It is now (™ 
usually spoken of us the microeceeus rhewnaticus, The organism (eco ¢ 
ia somotimes spoken of as a dip est described 
48 & streptococcus xrowing in short chains; in the tissues, how- 
‘over, it ustally occurs in pairs, It is rather smaller than the 
strepiecncean Pyogenes, and although it can be stained hy Gram's 

‘od, it loaes the colour more rendily than the streptococcus, 
Ts the various media it produces a large amount of acid, and 
‘asvally clots milk after incabstion for two days; on blood agar 
it alters tho bamoglobin co a brownish colour.” Ite growth on 
modia generally is moro Inxuriant than that of the streptococcus, 
aod it grows well on gelatin at 20°C. Tatravenous injection 
of pure cultures in rabbits often produers polyarthritis and 
ovitis, valvulitis and pericarditis, without any suppurative 
—lesions which it is stated are not ses by the 
ordinary streptococct (Beattie). In one or two instances 
choreiform moruments have boen observed after infection, The 
onganiam is most casily obtained from tho substance of inflamed 
synovial membrane where it is invading the tismes; a part 
Where chere is special congestion shoul be selected as being 
mest likoly to give positive results. It i only occasionally to 
be obtained from the Muid in joiata It has also been cultivated 
from the blood in rhenmatic fever, from the vegetations on the 
heart valves, and from other acute lesions; in many cases, how 
‘over, cultures from the blood give negative roslée Poynton 
13 




















194 INFLAMMATION AND SUPPURATION 


and Paine cultivated it from the cerebro-xpinal fluid in three 
cases where chorea was present, and also detected it in the 
mombrines of the brain, They consider that this discase is 
probably of the nature of a slight meningo-myelitix produced by 
this organism, ‘The facts already accumulated spenk strongly 
in favour of this organism being causally rolated to rhoumatie 
fever, though this cannot be considered completely proved, 
Andrewes finds that the organiam has the same enltaral chareters 
and formentative effects aa the streptococcus fmcalis, a common 
inhabitant of the intestine, Even, however, if the two organiama 
were the same, it might well be possible that rheumatic fever is 
due to.an infection of tho tissuna by this varinty of streptococeas, 
‘Tho clinical data, in fact, rather point to rhoumatic fover being 
due to an infection by some organism [requenty present in the 
body, bronght about by some state of prediaposition or acquired 
ansceptibility. 

Vaccination Treatment of Infoctions by the Pyogenic Cocci. 
—From his study of the part played by phagocytosis in the 
successful combat of the pyogenic bactoria by the body, Wright 
was lod to advocate the treatmept of auch infections by the 
origination during their course of an active immunisation by 
dead cultures of the iafecting agent. The treatment is applicable 
when the infection is practically local as in acne pustules, in boils, 
ete. (For the theoretical questions raised sce Immunity.) It 
is best to attempt to isolate the causal orgunism from the lesion 
and to test the opsonic index of the pationt 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 effiency of 
tho sterilisntion tested by inoctilating 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 useptic precaution must, of course, be adopted. If 
repeated injections are necessary Wright recommends that the 
copsonic index should be obsorved every few days and the 
injections only practised during a positive phase, If it is not 
practicable to use the infecting steain for the preparation of the 
vaceine, then laboratory cultures must be usod, and in such 
cases it is well to usc a mixture of strains; in skin infections 
a mixture of staphylococens 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 


1b, coli, infections of joints by the gonococcus, and in many eases 
rable ues followed the treatment. 


Oonditions ‘These are usually of a comparatively simple nature, 
and inelude (1) microscopic examination, (2) the making of 
cultures, 

(1) The pus or other fluids shonld be examined microscopi- 
cally, first of all by means of film preparations in order to 
determine the characters of the orgunisms present. The films 
should be stained (a) by one of the ordinary solutions, such 
‘as carbol-thionin-blue (jx 98), or a saturated watery solution of 
methylene-blue; and (5) 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 ean be 
readily obtained by the ordinary plate methods But in many 
cas the separation can be effected much more rapidly by the 
method of successive streaks on agar tubes, which are then 
incubated at 37°C. When the presotice of pnenmococe’ ix 
suspected this method ought always to be used, and it ie also to 
be preferred in the case of atreptococci. Tnoculation experiments 
may be carried ont as oceasion arises. 

Tn cases of suspected blood infection the examination of the 
paren to be carried out by the methods alroady described 
iP 





CHAPTER VIL 


INFLAMMATORY AND SUPPURATIVE CONDITIONS, 
CONTINUED: THE ACUTE PNEUMONIAS, EPT- 
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 pnoumonin, in which 
an inflammatory process attended by abundant fibrinous exmda- 
tion affects, by continuity, the entire tissue of a lobe or of a 
large portion of the lung. It departs from the course of an 
ordinary inflammation in that the reaction of the connective 
tissue of the Jung ix relatively slight, and there is usually no 
tondency for organisation of the inflammatory exudation to take 
place. ~ Secondly, thero is the acute catarrhal or lobular 
puewmonia, 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 endotholium of the 
alveoli, ta ce which leads to consolidation of patches of 
the Tung tissue, Up till 1889 acute cxtarrhal pneumonia was 
comparatively rare exeept in children. Tn adults it was chiefly 
found as a secondary complication to some condition such as 
diphtheria, typhoid fever, etc. Since, howe 
epidemic form has become frequent, eatarrhy 
been of much more frequent occurrence in adults, lias 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 tho exudation may 
106 




















TYPES OF PNEUMONIA 17 


be of a serous, biemorrhagic, or purulent character. Cases 
of falsed fibrinous and courial poeancat also. occur, and 
im the cotarrhal there may great loucoeytic emi; I 
Hamorrhages alu may occur here. Se 

Besides the two chief types of pnenmonin there ix another 
group of cases which are somewhat loosely denominated saptic 
pneumonias, and which may arise in two ways : ag the 
entrance inte the trachea and bronchi of discharges, te, 
which forma nidus for the growth of septic organisms ; these 
often set up a purulent capillary bronchitis and lead to infection 
of the air cells und also of the interstitial tisnue of the lang; (2) 
from secondary pyogenic infection by means of the blood stream 
from suppurative foci in other parts of the body. (See cha 
on Suppuration, ete.) Tn e septic paeumonias various 
changes, waembling thos fonnd in the other types, are often 
seen round the septic foci, 

Ta proumonias, 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 noute cronpous pneumonia on account of its course 
and characters, but reference will also be made to the other 


Historical. —Acute lobar jeer for long was powrd to be 
effect of exposure to cold; but many observers were tinted wit! 
this viow of its otic! Not only did casos ecour where no wnoh 

had beon observed that the disease 


specific 
FoR in 186235 by redlinder, whove roelte wary bristly as 
In pheumonic fungs there wore eocal, mihorent ustialiy in 
‘anu ‘of 4 definitely contoured capsale. These cooei 
could be isolated and grown on gelatin, and on inveuilation in tive they 
Jraincada kind of epelcamla with inflammation ofthe serous membrane, 
blood and the exulation in scrous cavities contained mumerous 
r |. There is little doubt that many of the organisina 


capwulated 4i 
seen bg rindlsaer wore relly Yraxa's peumocoonus to be preeently 


were found. A. Fraonkcel found that ¢ 
iemta was tngeh tore fatal and tore constant 
Of healthy individuals, The cocel which wero found {n animals dead of 
this “sputum septicemia,” as it was called, differed from Priedlinder’s 
eocel bm several respouls, to be presently studied, Wrweukal 








198 THE ACUTE PNEUMONTAS 


investigated « fow cusex of pnoumonia, and isolated from them cocel 
fdentiont in microscopic appearances, cultures, and pathogenic effects, 
with those isolated in spatim septicumia, The most extanaive investi 
gations on the whole question were thone of Weicheeltauin, publisied 
in 1886. This author examined 129 cases of the disease, including 
croupous pucumonia, but of lobular and soptic 
fi ys of organiame (1 

oval or lancet: 
th characters to Fraenkel's 

















Brewmonic. This was 
‘ried landers pneamo- 
conus. Of these onganismn the diplococens puetimoniie was by far the 
most frequent, [f also ooourred in all forms of pneumonia, Next in 
frequency was the streptovovens Jucumonie, and lastly the bacthlus 

umotie. Tnozalation experiments were also porformed by Weichsel- 

uni With oaeh of the three characteristic coool ho isolated. The 
Aiplococous puonmonie and the streptococcus ymeumonia both gave 
pathogente effects of a similar kind in certain animals. 





‘The general result of these earlice obwcrvations was to establish 
the occurrence in conection with pneumonia of two species of 
organiems, each having its distinctive characters, vi 

1. Fraenkel's prenmococcus, which is recognised to be identical 
with the coccus of “sputum septicunia,” with Weichselbaum's 
diplococcus pneumonia, and with hia streptococcus pnenmonisn, 

2. Friedldnder's pneumocceous (now known a8 Friedliinder’s 
pocumobacillus), which is almost certainly the bacillus pneu- 
moni of Weichselbauan. 

We shall use the terme “ Kyaenkel’s pnenmococeus” and 
“Friedlinder’s pneurnobacillus,” as these are now usually applied 
to the two organisms, 

Microscopic Characters of the Bacteria of Pneumonia.— 
Methods. —The organi#ms 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 
hopatisation), or from the gelatinous parts of pneumonic sputum 
{here again profembly when such sputum is cither rnsty or 
oceurs early in the disease), or in sections of pneumonic lung. 
Such propamtions may be stained by any of the ordinary weak 
stains, such ax a watery solution of methylene blue, but Gram's 
method is to be preforred, with Bismarck-brown or Ziehl-Neelsen 
carbol-fachain (one part to ten of water) as a contrast stains 
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 PNEUMONTA 199 


Agranaie se"u nyptiarmis erage oneal ote 
bacteria may be present, but those to be looked for are capsulated 
Creve abate ilo" ‘cor both of the varieties 


(L) Fraenhel’s Pacumococeus. —Thisorganiat occurs inthe form 
‘of a small oval coccus, about 1 in longest diaweter, arranged 
geremlly in pairs (sliplo- 

coeci), but also in chains 
of four to ten (Fig. 64). 
The free ends are often 





Uotjecolorisation te which (traetkel's) with ondaloed , epeclen 
the iiepege has been Stained with bt fuclala 1000, 


capeule 
is rather The iti Wed ol i Soca, tal Va sharply 
defined external margin. This organism takes up the basic 
aniline stains with great remdincss, and also vetming the stain in 
Gram's method. Tt is the organism of by far the most frequent 
oceurrones in true croapous pneumonia, and in faet may be mid 
to be mrely absent. 

(2) Pruediiinder's Pacumobariflus.—As ween in the sputurn 
and tieuos, this organiem, both in its appearance and armnge- 
mont, na also in the presence of a capsnle, somewhat resembles 

‘s Le eegir sears and it was at firat described o# ee 
 pretutnioceects form, however, is more of a short ro 
shape, and it bas blunt rounded ends; also rather 
bromder than Fracnkel’s pneumecocous, It is now elasend 
amongst the bacilli, macenkally 3 in view of the fact that clowgatesd 









200 THE ACUTE PNEUMONIAS 


rod forms may occur (Fig, 65). The eapsule has the same 
general charactors as 


ism. Fricdlindor’s 
pneumobacillus stains 





methel, and is 
ingly colonred with the 
contrast stain, —fachsin 
or Bismarck-brown, as 
above recommended. A 
valuable moans is thus 
aifonded of distinguish- 
ing it from Fra 
pnonmococens i 
Pia, 65,—Friedliinder’s poeumobscillus showing scopic preparations: 
the variations in Tongth, also capsules. Fin — Fyjedliinder’s orgun- 
Preparation from axaste i 8 en Of YU Gao iy such. lew. fe 
quontly presont in pucw 
monia than Frenkel's; sometimes it ix associated with the 
latter; very murely it 
‘occurs alone. 

In sputum prepara 
tions the capsule of both 
pheumococe! may not be 
recognisable, and the 
some is sometimes trac 
of lung preparations. 
This is probably duc to 
changes which occur in 
the eapsule as the result 
of changes in the vitality 
of the orguniams, Some- 
times in preparations 
stained by ordinary 
methods the difficulty of 
nssognsaing ‘the capsule Fiv, 66.—Fraenkel’s pnoumococens in serous 


when it 14 present, fs “™.cudation at site of inoculation in @ rabbit, 
due to the refructivo — jhowing capaules stained, 


index of the fluid in Stained by Led. Muir's method. x 1000, 
which the specimen 
monnted being almost identical with that of the capsule. ‘This 

















CULTIVATION OF PNEUMOCOCCUS: 201 


difficalty can alwuye be overcome by having the groundwork 
of the aye 

‘The Cultivation of Fraenkel's Pneumococens.—It is usually 
difficult, and sometimes imposible, to isolate this cocous directly 
from pooumonio sputum. On culture media it has not o vigorous 
growth, and when mixed with other buctoria it is apt to be 
overgrown by the latter, To get a pure culture it is best to 
insert a small picce of the «putum beneath the skin of a mbbit . 
or a niouse, In about forty-eight hours the animal will dic, 
with numerous capsulated pneumococei 
throughout its blood. From tho heart- 
blood cultures can be casily obtained, 
‘Cultures can also be got post mortem fro 
the lungs of pmeumonic 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 thern 
at 37° ©. The colonies of the pneumo- 
cocens appear as almost transparent anal 
discs which have beon compared to drops 
‘of dew (Fig. 67). This method is also 
sometimes sucossful in the case of 








spuitam, 
‘The appearances presented in cultures 
by different varietins of the pneuimoooccus ""y;Sayalt emomtaneas 
vary somewhat, It always grows beat on bond agar, The 
on blood serum or on Pfeiffer’s’ blood colonies are lange atl une 
agar, It usually grows well on ordinary prvelly dinioot. Dents 
tose of in bouillon, but not so well on $2"6.""Natonl sion, 
agar. In a stroke colture on 
trum growth spyrare as an almost transparent pellicle 
wlomg the track, with isolated colomics at the imargin, On 
it is more manifest, but otherwiee bas similar 
‘Tho appearances are similar to those of a culture 
‘of atroptococous pyogenes, but the growth is lass vigorous, 
and ie more delicate in appearance. A similar statement also 
applies to cultures in gelatén at 22° C., growth ina stab caltane 
as a row of minute points which remain of small 
+ there is, of courte, no liquefaction of the medium, On 
agar plates colonics aro almost invisible to the naked eye, 
but under a Jow power of the microscope appear to havo « 


i 








eonipauct fincly granular centre and a pale transparent periphery 


touilion, growth forms a slight turbidity, which settles to Vim 
—_— A 





F 





202 THE ACUTE PNEUMONIAS 


bottom of the i light dustlike deposit, On potatoes, 
replete adel Fort fe Serpette 


every four or five days, but they tend ultimately to die ont. 
‘They also rapidly lose their viraleneo, xo that four or five days 
after isolation from an animal's beet their pathogenic action 
is already diminished. Eyre and Washbourn, however, have 





fed in maintaining cultures in a condition of constant 
virulence for at least three months by growing the enganiams on 
‘smaenred with rab- 





lated are to be kept 
at 37° 5° ©. and sealed to: 
prevent evaporation, In 
none of the ordinary arti- 
; ficial media do — 
- cocci develop & €aj 
'S.—Fraankel's pnyumococous fron a pi 

ie titare onload fear af trentycour haus. They wanally appear 
growth, some in palrs, some in short chains, diplococci, but in yre- 
Stained with wenk carbo!-fuclisin, 1000, parations made from the 
surface of agar or from 

bouillon, shorter or longer chains may be ‘becrvol (Fi 68), 
After a few days’ growth they lose their regalar shape sail size, 
and involution forms appear. Usually the pneumococeus dos 
not grow below 22° C., but forma in which the virulence has 
disappeared often grow well at 20° C, Its optimum temperature 
is 37° G, its n 42°C. Tt is preferably an aerobe, but 
can exist without oxygen. It preferaa slightly alkaline medium 
to a nentral, and docs not grow on an acid medium. ‘These 
facts show that when growing outside the body on artificial 
modia, the pnoumococeus is a comparatively delicate organism, 
‘Thoro hus been doscribod by Eyre and Washbourn a none 
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, Krom tho latter it 














CULTIVATION OF PNEUMOBACILLUS — 203 


differs generally in ite mor vi wth, in producing « 
uniform clond in bonilton, in slowly higuetging gelatin, and in 


emo tivation 
‘The of Friedltinder's Pneumobacillus,— This 
organism, when present in spntam or in a poeumonie I can 
aly separated by maki inary 


on agar tubes. ‘The surface colonics 
always appear as white dises which become 
ralzed from the surface 80 aa to appear tike 
Tittle knobs of . From these, pure 
cultures can be readily obtained. The ap- 
pearance of a stab eulture in golatin 
i very characteristic. At site of the 
puucture, there is on the surface # white 
growth heaped up, it may be fully one-ebghth 
of an inch above the lavel of the gekitin ; 
along the needle track there is a white 













Vi4.69_—Stab eultere 
0 Friedlander’ 
poesia 
peptone ye thy 
showing the wail jetlinder's puenmobacillus,! 
Whe appearauce ; ealture «in agar, showing 
tan stays gromth. ypedl forms. 
Natural sire. Di thiomitiblue, x YOUR 


granelar ayyearance, 0 that the whole resembles a white roand- 
headed nail driven into the gelatin (Fig. 69), Hence the name 
1 cThe spparwnt size of thie crganian, on seomnt af th 


‘aceoeting to the stain uned. If vain] wit 
His thickness appears nearly twice ax great 


cof ite sheath 










204 THE ACUTE PNEUMONIAS 


“naillike” which has been applied. Occasionally bubbles of 
gas develop along the line of growth. There ix no liquefaetion 
of the medium On eloped agar it forms a very white growth 
with a shiny lustre, which, when touched with a platinum noedle, 
is found to be of a viscous consistence. In cultures much h c 
rods are formed than in the times of the body (Fig. 70). 

the surface of potatocs it forma an abundant moist white layer. 
Friedliinder’s bacillus has active fermenting powers on sugars, 
thongh varieties isolated by different observers vary in the degree 
in which such powers are possessed, It always seoms capable 
of acting on dextrose, lactose, maltose, dextrin, and mannite, and 
sometimes also on glycerin. The substances produced 
fermentation vary with the sugar fermented, but include ethylie 
alcahol, acetic weid, levolnetic acid, succinic acid, along with 
byiogen ‘and carbonic. acid gas. The amount of acid produced 
from lactose sem only exceptionally suilicient to cause: coagula~ 
tion’ of milk, It is said by somo that this bacillus is identical 
with an organism common in sour milk, and also a normal 
inbabitant of the Loman intestine, vi, the bacterium lactis 
wrogenes of Escherich, 

Occurrence of the Phoumobacteria in Pnoumonia and 
other Conditions —Capsulated organisins have been found in 
every variety of the disease—in acute croupons pneumonia, ft 
broncho pneumonia, in eptic pneumonia, In the groat majority 
of these it is Fracukel's pmeumecoccus which both microscopic- 
ally and culturally has been found to be present. Friedlinder's 
pheumobwoillus oceurs 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 Inng, especially in the exudation 
in the aircells, They also occur in the pleural exudation and 
effasion, and in the fyaplation of the lung. ‘The greatest number 
are found in the parts where the inflammatory process is most 
recent, eg. iN an area of acute congestion in a ease of croapous 
pneumonia, and therefore such parts are preferably to be selected 
for micruscopicexumination, and as the source of cultures, Some= 
times there occur in pneumonic consolidation areas of suppura- 
tive noftening, which may spread diffusely. In such areas the 
pneumoc cour with or without ordinary pyogenic organisms, 
streptocneci being the commonest concomitants. Tn other cases, 
espocially when tho condition is secondary to influenza, gangrone 
may supervene und lead to destruction of large portions of the 
lung. In these a great variety of bucteria, both aerobes and 
anaerobes, are to be found. 




















DISTRIBUTION OF PNEUMOBACTERIA — 205 


In ae en i tno Fracnkel’s pneumo: 
corens is nsnall nt, sometimes nlong with pyogenic cocci ; 
in the rag pal secondary to diphtheria it may be 
accompanied by the diphtheria tacillon and also by pyogenic 
coeci ; in typhoid pocumonias the typhoid bacillus or the b. eli 
may be alone present or be accampanied by the pmeumococens, 
and in influenza pnoumonias the influenza bacillus may occur, 
In septic pneumonias the pyogenic cceci in twany cases are the 
only organiams discoverable, but the pnenmacnecus may also be 
present, Especially important, as wo shall seo, from the point 
of view of the etiology of the disease, is the occurrence in other 
parts af the body of pathological conditions associated with the 
eos poe of the pneumococcus By ee ea to noigh- 

ig parts empyema, tia, and Ly! ic aoe 
[spel oleate aerate Prepchamerei 
fostmoceres may ooctr either alone or with pyogenic ceca 
ut distant parts may be affected, and the pneumococeus may be 
fonnd in suppurations at inflammations in various parte of the 
berly (subcutaneous tisane, peritoneum, joluts, kidneys, liver, ete.), 
in otitis media, ulcerative endocarditis (p. 188), and seeing 
‘These conditions may take place cither as complications of 
or they may constitute the primary disease. The 
oreurrence of meningitis is of special enpextance, for next to the 
Jungs the meninges appear to be the parte most liable to attack 
the pneumococeus. A large number of cases have been 
investigated by Netter, who gives the following tables of the 
relative frequency of the pritnary infections by the pneumococens 





GE-8G pr cont 
WAS ow 
bw, 


Beet Shale he 
+o ae 





om eas 46 cares wore investigated. In 20 the primary affection 
was otitis media, in 12 boncho-pmeumonia, in 2 meningitis, in 1 
pheumonin, in 1 plensiey, in 1 pericarditis 


‘Thus it children the primary source of infection ie in a great 
ually cases an otitis im 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 i it to 
note that ina large proportion of cases of the latter the 
opted esac ate ore peas ve ait 

ital Inoculation —The of Fraenkel is 


pathogenic to various animals, the effects wary somewhat 
with the virulence of the race used. The susceptibility of 
different species, as Gamaleia has shown, varies to a considerable 





Ft. 71,-—Copsolated poentnovoest in Mood taken from the heart of a 
rabbit, dewl after inoculation with puoumooufe apatum, 

‘Dried tim, Axed with corrosive eublimaw, Stuinéel with earbol-fuchsin and 
partly decotorised, x 1000, 


extent. The rabbit, and especially the mouse, are yery sus 
ceptible; the guinea-pig, the rat, the dog, and the sheep 
occupy an intermediate position ; the pigeon is immune. In 
the wore suaceptible animals the general type of the disease 
produced is not pnenmonia, but a general septicemia. Thus, if 
a rabbit orm mouse be injocued subcutaneously with pneumonie 
sputum, or with a scraping from o pneumonic lung, death 
occurs in from twenty-four to forty-eight hours, There ia some 
fibrinous infiltration at the point of inoculation, the spleen is 








EXPERIMENTAL INOCULATION 207 


and firm, and the blood contains 
pneumococci in large numbers (Fig. 71), IU the oat othnoetien 
tion be in the lung, there generally results pleuritie effusion on 
in 


phoumonia, with exudation of Lym 
(apes ond @ similar condition in peritoneum, may occur. 


‘exudation, and by the fact that few pacumococei are found in 
the blood stream, Tntra-pulmonary injection in sheep is 


dog is still more immune ; in it also intra ‘injection ix 

wed by a fibrinous pneumonia, which is only sometimes 

Taceulation by inhalation appears only to have been 

performed in the susceptible mouse and rabbit; hero also 
resulted. 

‘The general conclusion to be drawn from these experiments 
nade chat fo highly susceptible animals virulent pnewmococe! 
produce a general septicwmia ; whereas in more immune species 
‘there ix 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 « local acute inflammation occurring 
in a special tissue, but identical in essential pathology with an 
inflammatory renction in any other part of the body. When « 
dose of i sufficient to kill a rabbit is injected sub- 
cuthneously in the human subject, it gives rite 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 
mippose that man cecupics an intermediate place in the scale of 
wasceptibility, probably between the dog and the sheop, and 
that when the pncumococcus gains an entrance to his lungs, the 
local reaction in the form of pneumonia occurs. In this con- 
neetion the occurrence of manifestations of genoral infection 
amociated with pneumonia in man is of the highest import- 
ance. We have seen that meningitis and other inflammations 
‘aro not very rare complications of the disease, and such cates 
form a link connecting the local disease in the human subject 
‘with the general septicamic processes which may be produced 


walk _ 





208 THE ACUTE PNEUMONIAS 


artificlally in the more suacoptible representatives of the lower 
animale, 

A fact which at firat appeared rather to militate against the 
pneumococeus being the cause of pnenmonia was the di 
of this organism in the saliva of healthy men, ‘This fact was 
early pointed out by Pastour, and also by Froenkel, and the 
observation has been confirmed by many other observers. Tt 
can certainly be isolated from the mouths of a considerable 
proportion of normal mon, from their nazal cavities, ete,, being: 
probably in wny 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 
animale. 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 
caue of tho divsases caneed by pyogenic staphylocovci, streptococei, 
the hncillus coli, ete. It is probable that by various causes the 
vitality and power of resistance of the lung are diminished, and ~ 
‘that then the pnewmeccecus 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 pneumococeus is very frequently 
present, alone or with other organisms, Apparently the effects 
produced by such bacteria as the b. typhosus and the b, 
diphtheria can devitalice the lung to such an extent that 
secondary infection by the pneumococeus is more likely to occur 
and set up pneumonia, We can therefore understaid how 
much less definite devitalising agents such as cold, wleoholic 
excess, etc, can play an important part in the causation of 
pneamonia. In this way also other abnormal conditions of the 
respiratory tract, « slight bronchitis, ete, may play a similar 
part. 

It is more difficult to explain why semetimes the pneumo- 
coceus is associated with a spreading inflammation, a9 in e 
nonin, Whilst at other times it is localised to th 
yatches in broncho.pneamonia. Tt is quite lilo 
former condition tho orgnniam is posaoased of a 
of virulence, though of this we have no direct proof, We lave, 
however, a closely analogous fact in the case of erysipelas ; this 
disoase, we have stated roasons for belioving, ia produced by a 
streptococeas which, when less virulent, causes only local 
inflammatory and suppurative conditions, 

Summary.—We may accordingly summarise the facts re- 
garding the rolation of Fraonkel's proumococeas to tho disease 


























PNEUMOCOCCUS INFECTION 209 


by saying that it can be isolated from nearly all cases of acute 
eroupous pneumonia, and also from a. considerable 


Siting in tho Tangs can be act up by the diferent. pyeyenic 


The ibility of Friedlindor’s pneumobaciiiue bar au 
edaicpiesl TaidU ohn) Sal peanrctonla tan Has dipeted, 
tes found that it was pathogenic towards mice and 
guinea-pigs, and toa less extent towards dogs, Rabbits appeared 


septicnm 
pneumonia, such as erapyema and meningitis, render it possible 
tit may be the causal agent in a few cases of the disease, 
tho septic pnenmonins the different pyogenic organisms 
ry a found, and aioe ordinary 
ins, expecially the catarrhal forms, other organisms, 
the b, coli or its allies, may be the cansal ts, 
Pnoumococcus Infoction.The effects of 
of the paeumococeus, ab any rate in a relatively 
lo animal such as man, seem to indicate that toxins 
‘an important part, Pnousnonin is a dimaso which 
many respects the characters of an acute poisoning. 
cases does death take place from the functions of 
‘ing interfered with to such an extent as to cause 
Tt is from cardiac failure, from grave interference 
the heatrgulating mechanism, and from general nervous 
Lara that death usually resulta, These considerations, 
in connection with the fact that in man tho organieme aro 
founil in the greatest cumbers in the lung, sagyest that a toxic 
‘attion is at work. Varions attempts have been mado to isolate 
toxins from cultures of the pneumococens, e.g. by qreciyitaniog, 


 _ 


TG 
i 
i 


F 
une 


I 


Hl 


210 THE ACUTE PNEUMONIAS 


Douillon cultures with. alcohol or ammonium sulphate, aud 

igonona effects have been produced by certain substances this 
hee ived; but the offects produced are, as in so many other 
similar cases, of non-specific character and to be clawed aw 
interferences with guneral metabolism. ‘The general conelusion 
has been that the toxing at work in pneumonia are intracellular ; 
but no vise light has been thrown on the common effects of 
the members of this group of bacterial poisons, 

Jimunisation aguinst the Paeumococeus—Animals ean be 
immunised agninst the pnoumococcus by inoculation with 
cultures which have become attenuated by growth on artificial 
media, or with the naturally attenuated cocet which occnr in the 
sputum after the crisis of the disease. Netter effected immnan- 
isation by injecting an emulsion of the dried spleen of an animal 
dead of pneumococcus septicemia, Virulent cultures killed by 
heating at 62° C., rosty spatam 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 al! cases one 
‘or two injections, at intervals of several days, are sufficient for 
immunisation, but the immunity hus often been observed to 
be of a fleeting character aod may not last more than a few 
weeks. The sernm of snch immunised animals protects mbbita 
against subsoquent inoenlation with pneumococei, and if injected 
within twenty-four hours after inoculation, prevents death. A 
Protective scrum was obtained by Washbourn, who employed 
Pnenmococcus cultures of constant virulence. This observer 
immunised a pony by using successively (1) broth cultures killed 
by one hour’s expomre to 60° C.; (2) living agar cultures ; (3) 
living broth enltures, From this animal there was obtained a 
serum Wl protected susceptible animals against many times 
an otherwise Kat dose, and which alao had a limited curative 
action, It is stated that the serum of patients who have 
recovered from pnenmonia has in a certain proportion of cases 
a protective effect against the pneumococeus in rabbite, similar 
to that exhibited by the serum of immune animals 

‘The Klemperors 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 sora prepared by 
mused, ‘The results 
ned by different observers have, however, been rather eons 
‘Tho use of thess sora apparently canses the tempera- 
ture in some cases to fall, and even may hasten u crisix, but 
further experience is necessary before their value in therapenties 
ean be properly estimated, 



















Washbourn and by others have also bh 
obtai 








PNEUMOCOCCUS INFECTION 2 


‘There has been considerable difference of opinion as to the 
explanations to be given of the facts observed regarding 
immunisation against the pnenmococens and expecially reyandi 
‘the properties of immune sera, At first these vera mae Ep 

to powsoss antitoxie qualities—largely on the ground that no 
Iuctericidal eifect was produced by them on the bacterium in 
vitro. Ax 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, 


plement (s0e Immunity)—aro concerned, and the variability in 
the ic results obtained haa been accounted for on 
the view that there might be a defleicncy of complement, such 
as ocenrs in other similar cases, Tho absence of bactericidal 
effect, howovor, raises goveral difficult points, It is stated that 
no anch effect is observable cither in immune sera, or in the 
serum of patients who have suecessfally come throngh an attack 
of the natural disease, Somo effect of the kind would be ex. 
to be present if the anti-pneumonic serum were quite 
compurable to the antityphoid serum. Within recent 
many have turned to the opsonie property of sera to account 
for the facts obeerved. In this connection Mennes observed 
that normal leucocytes only become phagocytic towards pneumo- 
eoosi whon they are lying in the serum of an animal immunised 
sane ca tb pa this tk eryae cana Q an paaaeT 
epee tosis of sensitised bacteria to explain their destruction 
in absence of bactericidal qualities in the serum alone, and 
Neufeld and Rimpau have described the cecurrence of an opsonic 
‘effect in the action of an anti-pneumococcic serum, Further 
may show that along these lines lies the oxplanation of the 
observed. 

In studying furthor the relationship of tho opsonic offoct to 
Jheumecoccal infection, inquiry has been directed to the opeonic 
qualities of the blood of pneumonic patients, expecially with « 
view to throwing light on the nature of the febrile crisia, 

to nome results the opsonic index as compared with 
hat of person: is not above normal, but if the passible 
phagoertic capacities of the whole blood af the sick person be 
taken into account those will probably be much above normal in 
consequence of the leucocytosis which usually accompanies a 
succesful resistance to this infection, Tt hax been alwerved, 


— 





212 THE ACUTE PNEUMONIAS: 


however, that as the crisis approaches in a case which ix to 
recover the opsonie index rises, and after defervescence gradually 
falls to normal. And further, as bearing on the factors ine 
volved in the successful resistance of the organism to the 
pncumococcus, it has been noted that avirulent pusumocoeei are 
more readily opsonised than more virulent strains Further 
observations along such lines are to be looked for with interest, 
and it may be suid that Wright’s vaccination methods have been 
applied to the tratment of pneumonia cases, and in certain 
iustances are said to have been followed by favourble result, 
Tt may be noted here, in conclusion, that in man it is probable 
that immunity agninst pocumonia may be short-lived, ae in a 
ood many cases of pneumonia a history of a previous attack ix 
elicited, 

Agglutination of the Preumococcus—If small amount of @ 
culture of Fraenkel’s pneumococens be placed in an anti-pmeumo- 
covele serum, an aggregation of the bacteria into clumps occurs: 
Such an agglutination, aa it is called, is frequently observed 
Seiiseistllas elisinsteaciens retshs C4059 bears aes pheno: 
menon is not invariably associated with the presence of protective 
bodies in a verum, but it has been used for diagnostic purposes 
in the differentiation of sore thronte due to pneumococous 
infection from those due to other bacteria, Whether the method 
is relinble hms still to be proved. 

of Examination, These have been already 
described, bot may be summarised thus; (1) Microscopic. 
Stain films from the densest part of the sputum or from 
the area of spreading inflammation in the ling by Gram's 
mothod and by carbol-fuchsin, cte. (pp. 99, 101), im the Inttor 
case without decolorising the groundwork of the preparation, 

(2) By cultures. (a) Mreniel's pmenmacocens, With similar 
material make successive strokes on agar, blood agar, or blood 

The most certain method, h ja to inject: seme 
paterial containing the auspected encei into a rabbit, Tf 
the menmococcns be present the animal will die, usually within 
forty-cight hours, with numerous capanlated pneumococei in ite 
heart blood. With the latter inoculate tabes of the above media 
observe the growth, In some eases af severe pnenumncocele 
infeotion the organism may be cultivated from the blood obtained 
by venesection (p. 68). (0) Friadldnder’s pneumobacillus can 
be readily isolated either by ordinary gelatin plates or by 
successive strokes on agar media, 

































EPIDEMIC CEREBRO-SPINAL MENINGITIS 213 


Eprpemic CEREBRO-NPINAL 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 diploracrus intrardHlularis meningitidis first described by 
Weicliselanm. Tis ‘oceus measuring 
about 1 in diameter and usually occurs in pairs, the adjacent 
sides being somewhat flattened “against each other. In most: 

wes the cocel are chiefly contained within_polymorphonuclear 
leneveytes in the exudation (Fig. 72); in some eases, however, 
the majority may be 
lying free, It stains 
realily with basic anilin 
dyes, but loses thes 
in Graitsmethis 
ness with which the 
decolori 
with different 
Both in appear- 
ance and in its stainin 
reactions it is supertic 
ally similar to the you 
coceus (ride infra). The 
organism can ‘readily: be 
cultivated outside — the ’ 
but the conditions 
rowtht are xomiewhitt 4 pit pmmparation of ex 
restricted: ager Wit a A ot ments showing Uh 
admixture of serum or \ithin Tewocytes 
Mood (preferably human) — Stained with eatbol- 
is most * Strains 
separated in different epidemies appear to present slight in- 
dividual but the following description nay be taken. 
as suming up the common charac Growth takes place 
est at the temperature of the body, and. practically cenmes 
rum agar the colonies are circular dises of 
nd possessing a smooth shining 
little tendency to berome contluent. When 
mined nnider a low magnification the colour is seen to be 
somewhat yellowish and the margins usnally are smeuth and 











































jon from + 
diplacoeei 














blue, 1000. 





io 




























of 1 part of ascitic tuid and 6 
tal cally is aslded to 
“C.aud the tuber are tested as regards 


Ya very good mei 
parts of 1 per vent glu 
the azar in the meltel xtate at 
sterility by incubation, 











ou THE ACUTE PNEUMONIAS 


regular, though on some media slight crenation may appear. ‘The 
colonies may be of considerable siae, reaching sometimes a 
diameter of 2 to 3 mm. on the third day. On plais ager the 
colonies are very much smaller, and sometimes no growth 
ocenrs; subcultures expecially 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. Tt ferments maltose, galactose, and dextrose with seid 
production, a property which distinguishes it from the micros 
‘ecoccus catarrhalis (wird infra). Buchanan has pointed out that 
this may be demonstrated by making up lots of Léffter's medium 
(p. 40) in Petri dishes 
with each of these sugars 
added. In all cases 
growth occurs best when 
the medium has « neutral 
or very slightly alkaline 
reaction. In cultures the 
organiam = presents the 
samo appearance as in the 
body and aften shows 
totrad formation. ‘There 
is also a great tendency 
to the production of in- 
volution forms (Fig. 73), 
many of the cocci be- 
coming much swollen, 
Fre: 7&—Pare culture of diplococeus Istra. **#ining badly, and aftar- 
collateris, showing Involvtion forms, Wards undergoing disin- 
togration. ‘This change, 
According (9 Floxner's obsoryations, would appear to be due to 
the production of an autolytic enzyno, and ho has also found that 
this snlstance has the property of producing dissolution of the 
bodies of other bacteria, The life of the organiem in cultures is 
& comparatively short one; after a few days cultures will often 
We found to be dead, but, by making sub-cultures every three or 
four days strains can be maintained alive for considerable periods. 
‘The organiam 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, Tbe facts established necord- 
ingly show it to be a somewhat delicate parasite, 
As stated above, the organism occurs in the exudate in the 
meninges and in the cerebrospinal fluid, and it con usually be 
obtained by lumbar puncture. In acute casos, expecially in the 



































EPIDEMIC CEREBROSPINAL MENINGITIS 215 


earlier stages, it is usully abundant; but in the lator 
eases of more #ubaeute character, ite detection may be a matter 


be found. It has been observed, for oxample, in arthritis, 
carditis, pncumonic patches in the lung, and in other inflam- 
matory conditions associated with the disease. Tn a small 
proportion of cases it has been obtained from the blood during 
life, but cultures in most instances give nogative resulta, 

‘imental inoculation shows that the ordinary laboratory 
animals are relatively insuxcoptible to thie organism, An 
condition may be produced in guinoapigs by 
intra-peritoncal injection, but large quantities of cultures must 
he used, and none of the characteristic lesions found in the 
human ‘subject are juced. ‘The intra-peritoneal injection 
‘of the corvl al fluid or of cultures in mice ix froquently 
followed by death, the cocci being found in the exudate and even 
in te Wlood. Flexner has shown that cerebro-spinal meni 

produced in monkeys by injections of the iam 

lols tn mh ee nts the orgoni teats 
oes the irate scnoces ieningitis within & very 
time. The rout lesions, both as regards their dis 
tribution and general Sage and iin au ac 
aun nee 16 disease in the human subject. 

Even ey animals, however, are, in comparison with man, 
=e as a considerable amount of culture hax 


importance with regard to the 
|) require further investigation, The 
has been obtained by culture from the throat and 
aries of those suffering from the disease in « consider- 
instances. Tt has also been obtained from the 
in healthy individuals, during an epidemic of 
An some epidemics also a pharyngitis hes been 
and the cngunism has been obtained from 
‘The general opinion ix that the organism 
a ‘the lymphatics from the pharyex or 
the brain, but » spread by means of the 
be excluded, and infection by the alimontary 
8 suggested Floxner in his ex = 
hen the orgunism was injected into 


nie 


ni 
Hi 
ee 


ELE EFEE 
ie 
rte i 


F 





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 diplocoocus; he was, however, 
unable to recover the diplococcas intracellularis in culture from 
this situation. ‘These results would seem to indicate that the 
onganism might spread from the brain to the nasal cavity, but if 
this be #0, it also follows that an extension may take placo in 
the reverse direction. Ou the whole the evidence at present 
tends to show that the entrance of the organiam into the body: 
Led the nasopharynx, und thot this usually results by 
i tion of the organism distributed in fine particles of 
expectoration, ete. In faet, as regards the mode and conditions 
of infection, an analogy would appoar to hold between this 
disease and influenza, 

Apart from the epidemic form of the diswise, eases of sporadic 
nature also occur, in which the lesions are of the same rat 
and in which the diplococeus intmcellularis is provent. The 
facts stated would indiente that tho origin and spread of the 
disease in the epidemic form depend on certain conditions which 
produce an inereased virulence of the organisms, We are, however, 
as yet entirely ignorant as to what these conditions may be. In 
simple posterior basal meningitis in children, a diplococcus ix 
present, as described by Still, which has the samo microscopic 
and cultural characters as the diplococeus intracellalaria; it hax 
been regarded us probably an attcauated variety of the latter: 
Recently, however, Houston and Rankin have found that the 
serum of a patient suffering from epidemic meningiti docs aot 
exert the same opsonie and agglutinative effects on the diplococeus 
of basal meningitis as on the diplococens intracellnlariss and 















degree of the reaction does not possess much clinical significance. 
Tt usually appears about the fourth day, when the serum may 
give a positive reaction ina dilution of 1:50; ats later stage 
it has been obacrved in wo great « dilution as 1: 1000, ‘There is 
thus no doubt that snti-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 au anti-serum, anda certain measure of 
success has been obtained so far ax experimental results ane 
concerned. Flexner obtained such a scrum from a goat, aid 








EPIDEMIC CEREBROSPINAL MENINGITIS 217 


face agi hic bf oy in guinen-pigs and 
monkeys: infeetion by the organism, but, on the whole, 
better its were obtained with rerum of inoculated 
monkeys. As yet no Smoportant pt ta towards the 
treatment of the disease have been effected. 

‘Tn the nasal cavity there ocour other dij ‘i which have a 
close resemblance to the diplococeus in avis, These occur 
in the healthy state but an: especially abundant in eatarrhal 
conditions ; of these the diplococeus eutarvhalia tna tho closest 
resemblance veka eee wl doen raha a Ler leie to 
occurring in ro 
numbers in_ epidemic ‘bari ta rlerootopla oppearances are 

similar to those described see pepo aa, 
Tencocytes. Its colonies on serum agar are more opaque 
than hose of tho ighsaoeras Lakes slialicny aaelitaep are 
tough consistence, Ls that cerita sometimes removed ot maze 
organism grows on gelatin at 
20° 0. without rieereea the medium, and it has now of the 
fermentative properties described above as belonging to the 
diplococens intracellularis. Other species of Gram - erate ta 
micrococei have also been isolated, und a Gram-positive diplo- 
scons alisd the diploeocews erases Art coma ssmeyanet 
this organism is rather larger than the diplococous intracellularia, 
‘and especially in subeultares may tend to assume staphylo- 
coccal forms It is thus evident that the nasal cavity is the 
common habitat fora number of closely allied diplococci, and 
that the identification of any suspected organism as the diplo- 
‘eoccus intracellularis can only ke effected by cultivation tests. 

Apart from the epidemie form of the disease, meningitis may 

Je produced by almost any of the organisms described in the 

a4 associated with inflammatory conditions. 

number of cases, expecially in chitin, are due 

to the pnoumococcus. In many instances where no other lesions 

fare present the extension is by the Eustachian tube to the middle 

ear, In other cases the path of aie is from some other 

means of the blood stroam. This organism also infocts 

the not infrequently in lobar pneumonia, and in some 

cases with symptoms we have found it present where there 

was merely a condition of congestion. The pneumobacillus also 

has been in a fow ie Meningitis ia not infrequently 

prodiiced by streptococci, especially when middle ear disease is 
oot frequently by one of the staphylococci ; oe: 

nie organiam may bo concerned. In meningit 

influenza the influenza bacillus has been found ina 











218 THE ACUTE PNEUMONIAS 


few instances, but sometimes the pnenmoeoceus is the causal 
agent; and in tubercular meningitia the tubercle bacillus of 
course is present, especially in the nodales 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 
lwomorrhage in the subarachnoid space. 

Tn conclusion here it may be stated that mixed infections 
may occur in meningitis, Thus the pneumococcus has been 
found associated with the tubercle bacillus und aleo with the 
diplococeus intracellularis, Further, in infection with the latter, 
Gram-negative bacilli of a diphtheroid appearance have also been 
observed ; the signiticance of these is unknown. 








CHAPTER VIII. 


GONORRHGA, SOFT SORE, SYPHILIS. 


Goxonnuass, 


—————t 
Introductory, —The micrococcus now known to be the cause of 
gonorrhor, und now called the gonococcus, was first described 
by Noisser, who in 1879 wave an account of ita microseopical 
charactora a8 seon in the pus of gonorrhoeal affections, of 
the urethra and of the conjunctiva. He considered that this 
organism was peculiar to the dissws, and that its characters 
‘wore 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 buman subject its caus! ‘relationship to the disease has 


been conclusively established, 
‘The Microscopical Characters.—The organisin 
of is u small micrococeus whieh usually it seen in the 


form, tho adjacent margins of the two cocci being 
Mitrened, or even slightly concave, 20 tl tween them there is 
a sznall oval Joterral which om 1 zat Pa eae a appearance ix sf 
thos to that of two beans 
neaty ade (vide Fig, 74). 1 When division takes place in oF ¢ 
the two members of a diplococcus, a tetrad ix formed, which, ( 
however, soon saparntes into two sets af diplococei—thut is to ()_) 
fay, arrangement as diplococci is much commoner than as tetmda 
Goeeci in process of degeneration are seen as spherical clen a 
heres ses some being considerably swollen, gris | 
aro found in large numbers in tho pas of ft TA 
pice both in the male and female, and for the most) Yr» | 
‘Part are contained within the leacocytes Tn the enrliest stage, |¢ .f 
when the secretion ix glairy, a consideraklo number aro lying 
free, oF are adhering to the surface of desquamated epithelial 
29 


 - Se] 








220 GONORRHGA, SOFT SORE, SYPHILIS 


cells, but when it becomes purulent the large proportion within 
leucocytes ix a very striking feature. In the leucocytes they lie 
writhin_ghe protoplasm, especially superficially, and are often 0 








numerous that the leuco- 
cytes appear to be filled 
with them, and their nuclei 
are obscured, As the dis- 
ease becomes more chronic, 
the gonocoeci gradually 
become diminished — in 
number even in 
long-standing cases they 
may still be found in con- 
siderable numbers. They 
are also present in the 
purulent secretion of gon- 
orrhwal conjunctivitis, also 
in various parts of the 
female genital organs when 








—Vortion of film of youorrheral pus, these parts are the seat of 
‘showing the characteristic arrangement tone gonorthwal infecti 
of the gonocorei within leucocytes. gonorthwal infection, 
Stained with fuchsin, > 1000. and they have heen found 
in some c in the second- 
ary infections of the joints in the disease, as will be described 
below, 

Staining.--The yonococeus stains readily and deeply with a 
watery solution of any of the basic aniline dyes—methylene-blue, 
fuchsin, ete. 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,” aud 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 hy cooling. 
The serum may be obtained from the blood of the humau 
placenta; pleuritic or other effusion may also he used. 
Growth takes place best at the temperature of the body, and 
ceases altogether at 25° C. Cultures are obtained by taking 
some jus on the loop of the -platinum needle and inoculating 
ane of the media mentioned by leaving minute quantities here 
“(a hmd there on the surface. The medium may be used cither ag 


. est * TENG 





















CULTIVATION OF GONOCOCCUS 221 


ordinary “sloped tubes” or as a thin layer in a Petri eapsnle, 
The young colonies are visible within forty eight hours, and often 
within twenty-four hours, They aj around the points of 





with absolute alcohol, and Wis, 75,—Gonococel, from # pure cultare 
thematerial forinoenlation on blood agar of twenty-four bourse” 
be from. the "th, Bome already are Yegioning to 
deeper partof the urethra, Syjse Pao eee one, 
cultures may often be ob- staimest with earbol-thionin-blne, x 1000, 


from the first, By anccessive aub-cultures at short intervals, 
growth may be nisintained indefinitely, and the organism 
ic aeet aleg more luxutriantly, Tn culture the organisms 
ve itnilar microscopic characters to those described (Fig. 75), 
remarkable tendency to undergo degeneration, 
wollen and of various sites, and staining very 
rie Degenerated forms are seen even on the second 
lst in & culture four or five days old comparatively few 
may be found, ‘The less suitable the medium the 
does degeneration take place. 
On ord ‘agar and on glycerin agar growth does not take 
place, or ix so slight that these media are quite unsuitable for 


 s fil 











222. GONORRHEA, SOFT SORE, SYPHILIS 


purposes of culture, The organism docs not grow on gelatin,* 
potato, ete, 


Plate-Cultures,—The following ingentons method of plate-culture wax 
introduced by Wertheim for the oultare of the gonococeus, ‘The medium 
of culture ina mixture of haman blood serum and of ordinary agar 
per cont) in equal parts, ‘The serum, ina Haid and sterile condition, 
put in wuitablo quantities into two or three test tubes and brought toa 
Temperature of 40°C, ‘Those are then snocossively inocalated with the 
pus or other material in the same manner as gelatin tubes for ordinary 
plates (ride p, 62), To each tube is added an equal part of ordivary 
ayar which has been thoroughly liquefied by hoating and allowed to 
con) also to 40" C. The mixtare is then thoroughly sbaken up end 
quickly poured out on a plate or Botr's dish and allowed, to eolldity, 
Bhs plate bing then inoabated at a torperatire of 27°C, Thnealenies 
of the gonocorons are just visible in twenty-four hours, and ore seen 
both in the substance of the medium and on the surfase. ‘The dee 
colonies wlien examined with « Jens are minule and slightly nodulat 
apheros, sometimes abowing little procomes, whilat those on the aueface 
aro thin dinos of larger diameter with wavy margin and rather darker 
centro. In this way the gononoeons may be separated from Aaids which 
are contaminated with a considerable nimber of other organisms. 





























Relations to the Disease.—Tho gonococeus is invariably 
present in the urethral discharge in gonorrhom, and also in 
other parts of the genital tract when these are the seat of trae 
gonorrhea) infection. Its presence in these different positions 
has been demonstrated not ouly by microscopic examination 
but also by culture. From the description of the conditions of 
growth in cnlinre, it will he seen that a life ontside the body 
in natural conditions is pructically impossible—o statement 
which corresponds with the clinical fact that the disease ix 
always transmitted directly by contagion, Tnoculations of pare 
cultares on the urethra of lower animuls, and even of apes, is 
followed by no effect, but a similar statement can be made with 
ganl to inoculations of gonortheeal pus itself, Tn fact, 
erto it has heen found impossible to repmoduce the disease by 
means in the lower animals, On a considerable number of 
‘occasions inoculations of pure cultures have been. mad the 
human urethra, both in the male and female, and the disease, 
with all ita characteriatic symptoms, has resulted, (Such 
xporimenta have boen performed independently by Bumm, 
Stuinschovider, Wertheim, and others) The causal relationship 
of the organism to the disease has therefore been completely 
























* Porro hax announemt that te has culsiy gonococens om eld 
glatin, he onlinary peptowe gelatin which bas not been neutralised. We 
have failed to obtain any growth of the gonococcus on this meliam, even 
when inoonlation was made from ® vigorous growth on blood agar. 





t 














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 eharacters of the natural discuse, 


itcineal injections of pury cultures of the ganococcus in white 
— Gelet annie ate itis with a small amount of suppuration, 


Bel fond tala num bees in the ley verthel 
Le koe | 
Brite ‘conneotive tise, but thoy appear to have little of 
Eepeprent: dnjeser pees eafcnrs into the juntas rabbitn, Hoge, 
I. of cultures nts 
apatite: lets a aale iaBatitaatiea,” whl which, henry ae 0 
val similar 


that while the orsanist, when humbers, ean produce a 
eertain amount ol inflammatory chango hango in these animale, it has little or 


co f multi) and spreading in their thee 
1 Fon ofthe of the Gonboocsua De Chricmas cultivated the si 





Pes all the organisms are dead ; euch « Avid eee the 
{ctasin.” The toxin wabstances ar precipitated slong with the oat 

by alsohol, and the precipitate after being dest toxie 
setion, In young ‘abhi injection of the toxin 

this is well seen fn the anterior chamber of” 

rooted Tha roost tntercating point, pois 

sction cn neous surfaces; for, while in the case of animals it fark e 
no ‘its introduction into the human urethra cinees acnty oatarrh, 


i ‘He found that no tolerance to the 
rexull Injeations at Interval. In x mone 
Jpeg el polermepe he poreisisat out Men the toxin on it 

has marked effeots ; he also claims to have produced a 
‘He states that the toxin diffuses out in tho culture mi anid does 
not macy result fom disintegration of tho organisms. This has, how 
eter, hewn éalied in question hy other investigators, 


Distribution in the Tissuos.— ‘The gonoesceus havi sonal 








attended with great increase of sceretion, There ocenrs also 
— emigration of leucocytes, whieh take up a 
Jarge ‘of the organisms ‘The organisms also penetrate 
‘the subjacent connective tiene, and are expecially found, along 
cues Tencocytic emigration around the lacuna, Here 
‘are contained within leucocytes Even, however, 

be itn gonoceee! have diseppeared from the urethral dis- 
“they may still be present in tho deeper part of the 


ae 





324 GONORRHEA, 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 examined by making pressure on 
the prostate from the rectum when the patient has almost emptied 
his bladder, the seerction being afterwards discharged along with 
the remaining urine. (Foulerton.) In acute gonorrhas there 
is often a considerable degree of inflammatory affection of the 
prostate and vosicule seminales, but whethor 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 applica to the occurrence of orchitis and also of 
cystitis in the carly stage of gonorrhea,  Gonococci have, 
however, been obtained in pure culture from pori-urvthral abscess 
and from opididymiti: it is likely that the latter condition, 
when cecurring in gonorehoca, is usually duc to the actual 
presence of gonococei, During the more chronic stages other 
organisma may appear in tho urethra, aid in maintaining the 
irritation, and may produce some of tho secondary results, 
The bacillas coli, the pyogenic cocci, ete., are often present, and 
may extend along the urethra to the bladder and set mp cystitis, 
though in thia they may be aided by the «of a catheter, 
It may be mentioned here that Wertheim cultivated the 
gonocoveus from a cass of chronic youorrhan of two years 
standing, and by inoculation on the human subject proved it to 
be still virulent. 

Tn the disease in the femal mococci ure almost invariably 
present in the urethra, the situation affected next in frequeney 
being the corvix uteri. They do not appear to infoet the lini 
epithelium of the vagina of the adult unless some other abnorm 
condition be present, but they de so in the gonorthaal vulvo- 
vaginitis of young subjects. They have also been found in 
suppurntions 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 pase along the 
Fallopian tubes and produce inflammation of the mucous mem- 
brane there, From the pus in cases of pyosalpinx they have 
boon enltivated ins considerable number of cases, According 
to the results of various obsorvers they aro present in one out 
of four or five cases of thi« condition, wasnally unasaociated with 
other organisms, Further, ina large proportion of the cass in 
Which the gonococeus hus not been found no organisms of any 
kind have been obtained from the pus, and in these cases the 
gonococci may have been ones present and have subsequently 
died out, Lastly, they muy paws to the peritoneum and produce 














DISTRIBUTION OF GONOCOCCTS 226 


peritonitie, 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 gonorrhea! conjunctivitis the mode in which tho gonococci 
through the epithelium to the subjacent connective 
ati is closely analogous to what obtains in the case of the 
‘Their relation to the leucovytes in tho purulent 
pee in also the mmc, Microscopic examination of the 
secretion alone in acute cases often gives positive evidences, and 
Bae cultures may be readily obtained on blood-agur. As the 
condition becomes more ehronie the gonococei aro lee numerous 
and a greater proportion of other organisms may be present. 
Rdations to Joint Afections, ete—The relations of the gono- 
eocens to tho sequelm Zit reid form a subject of great 
interest and importance, the application of recent methods of 
examination shows that the organism is much more frequently 
nt. in such conditions than the earlier results indicated, 
following stotoments may be made with rogard to them. 
Firat, in a considerable Baal ee Van of arthritis ile i 
gonorrhom jococens has been found miecroseopically, 
ure cultures have been obtained, «eg. by Neiyser, Lang, 
Bondoni-Utireduzn, and Soa others. A similar statement 
we sheaths of tendons followlag 


E 
if 
ng 
@ 


hreds of fibrin-like material, sometimes 
in appearance. In one case Bordoni-Uffreduzzi culti- 
the gonocoecus froma a jointaffection, and afterwards 
gonorrhas in the human subject by inoculating with 
tures obtained. In another case in which pleurisy was 
with arthritis the as was cultivated from 
wavity. ‘he existence of a genorrhwal 

has teen established by recent observations, Cas 
vet this ema oceurring in the course of gonorrhas 
previously described, but the complete bacteriological 
now been satisfied in eeveral instances. In owe ene. 


Hl 


if 





i 


a 
rit 





226 ©GONORRHGA, SOFT SORE, SYPHILIS 


Lenhart produced gonorrhea in the human subject by 
inoculation with the organisms obtained from the vegetations, 
‘That a true gonorrheead septicemia may also occur has also been 
established, cultures of the gonoeocens having been obtained 
from the blood daring life on more than one occasion (‘Thayer 
and Blumer, Thayer ond Lazear, Abmann). 

Methods of Diagnosis.—For micreweopieal examination dried 
films of the suspected pus, ete., may be stained by any of the 
simple solutions of the basic aniline stains. We lor 
lene- or thionin-blus, as they do not overstain, the films 
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 coverglass a small quantity of 
culture of staphylococcus aureus if available, in order to have 
standard by whieh to be certain that the supposed gonococet are 
roally decolorised. Regarding the value of microscopic examina- 
tion alone, we may say that the presence of a large number of 
micrococei in a urethral discharge having the characters, position, 
and staining renctions described above, is practically conclusive 
that the case is one af gonorrhea, There is no other conditi 
in which the sum total of the microscopical characters is present. 
We consider that it is sufficiont for purposes of clinical diagnosis, 
and therefore of groat valne ; in the acute stage a dingnosia 
thus be made earlier than by any other method, The mistake 
of confusing gonorrhcea with such conditions asa urethral chanere: 
with urethritis, will also bo avoided, Even in chronic eases 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 gonococel ara few in 
number, it is difficult, and in some cases impoesible, 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 gonorrhaa in tho fomalo, Microscopic examination, 
therefore, though often giving positive resulta, will sometimes be 
inconclusive, As regards lesions in other parts of the 
microscopic examination alone is quite insufficient ; it is I= 
cally impossible, for example, to distinguish by this means the 
gonococens from the diplococcus intracellularis of meningitis, 
Cultures alone supply the absolute test, and when the organism 
is present in an apparent condition of purity, Werthoim’s 

lium or blood-agar should be used. tr other organisina 
are present, we are practically restricted to Wertheitn’s plate 
method, 


SOFT SORE 7 


Sorr Sore. 


‘The bacillus of soft sore was first described by Ducrey 
in 1839, who found it in the purulent rags tet the 
ulcerated surface; and later, in 1892, Unna ribod its 
appenrance and distribution as seen in sections through the 
sores. The statements of these observers regarding the presence 
tind characters of this orgunism have been fully confirmed by 
other observers. 

Microseapicat Characters —The organism occurs in the form 
of minute oval rods measuring about 15 p in length, and ° 
in thieknoss (Fig. 76). It is found mixed with other organisms 


through the ulcer it is 
found in the superficial 
part of the floor, but more 
ooply situated than other 
‘and may be 

resent in a state of pur- 
the leneoeytie 
infiltration. In this posi- 
tion it is usually arranged 
in chains whieh may be 





and which are often son Fra. 78, Ves preparation of pus Srontiate 

ii paral 7 chanore, showing Ducrey's haoillas, chiet 
lying a of ver feranget in pata; shuined wih carbo 
faci chief occur i, sbein aml slightly decolericl. «1500, 
the fre condition, but occasionally a few may be contained 
within leucocytes. 

‘There is no doubt that in many cass the organism te 
presont in the buboes in a state of purity; it has been found 
there by ey xa Se-cah and cultures have also been 
obtained from this souree. negative results of some 
obacrvers are probably duo to ibe organism having died off 
‘On the whole the evidence gocs to show that the o 

Tubo sieciiated with sift nore is to be regarded a another lesion 
produced by Ducruy's tmeillus. Sometimes tho ordinary pyogenic 


organiams become snperadded. 
GNM UAiiig takes up tha,basic aniline staina faicly veasity, 














228 GONORRH®A, shied irs 
eee 


binations, though Loffler’s or Kihne's methylene 
ee ee ee In 
tions, however, great care must be taken in the process of 
ridecoihad and the aniline-cil method (vide p. 93) should be 
used for this purpose, as alcohol decolorives tho organism very 
readily, A little of the methylene-blus or other stain may be 
with advantage added to the aniline-oil used for dehydrating. 
Cultivation. —Although for along 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 
mu mixture of 
blood and agar, in the 
Proportion of one part 
‘of the former to two of 
the lattor, The blood 
is added to the in 
the melted condit 
45°C, and the tubes are 
then sloped, Davis con= 
firms these results, and 
finds that anothor good 
2. 77-—Ducrey’s bacillus from w 24-hour medium is froshly-drawn 
Pitre biead-nlon, 4200. “una blood distebuted 
in small tubes; this 
method is specially suitable, as the blood inhibits the 
of various extraneous organisms, On the solid medium (blood: 
agar) the growth appears in the form of mid} round globules, 
which attain their complete development in forty-eight hours, 
having thon a diameter of | to 2 5 the colonies do not 
become confluent. Microscopic. examination of these colonies, 
which are dissociated with some difficulty, shows similar appenr- 
ances to those observed when tho organiam is in the tieses (Fig, 
77), bat occasionally long undivided filaments are observed ‘which 
Davis mgards as degenerative forms, Wi capitis 
short period cnltures undergo marked degenerative changes, and 
great irrogularitics of form and shape are to be found, It 


We aro indobted to Dr. Davis for the une of Pigs 76 and TT. 











SYPHILIS 229 


would aj ttre a iP amount of blood is 
necessary Nor the growth ce ‘even sub-cultures 


le pg 
generations and re serpiey ns disease by inoculation of the 
human subject. The causal relatiouship of this bacillus must 
therefore be considered as completely established, and the con- 
ditions under which it grows show it to be a strict 
fact which ix in conformity with the known facts as to the 
‘tranemission of the disease. 


Syrmms. 


‘Up till quite recent times practically naihiog of a definite 
nature was known regarding tho Reni Ranta 
interest for a long time centred arow 

who in 1884 described a characteristic bacillus, 

eee remy erent in the lesions in internal organs, 
‘This organism occurred in the form of slender rods, straight, or 
slightly bent, 3 to 4 4 in length, often forming little clusters 
either within colls or Ising fron in the lymphatie spaces ; it touk 
up basic aniline dyes with some difficulty, but was much more 


va weids than the tubercle bacillus. 





‘Much controversy arose regarding the sagt of this 
bacillas Some considered it to be the tubercle bacillus, whilst 
others sapposed that it was the smegma bacillus which had 
invaded thetissues The etiological relationship of the orgunism 
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. 

An entirely new light has been thrown 

‘Of tho dissaso by the work of Schaudinn and 
Hota wish perce in 1905, Since their first publication 
& grtat amount of work has been undertaken in order to veh 





> 


230 GONORRHGA, SOFT SORE, SYPHILIS 





also the i Widum, 3 described 
iy theay fa sinatospelahaped oranizn shoving usally 
from six to eight though longer forms are met with ; the 
curved ‘comparatively sharp, and regular (Figs. 78, 79). 





Fins. 78 and 79.—Film preparations from juices of hart chanere showing 
‘apirochate pallids,—Glonxn's stain, x 1000, (From preparations ty 
Dr, A. MacLennan.) 


and movements of flexion of the whole body. ‘The onde ane 
Pointed and tapering. Its detection is comparatively difficult, 

us the organism is feebly refractile, and more difficult tosew than 
most other organisms; the movement of small particles in the 
vicinity, however, is of azsistanoe in finding it, 

In ulcerated syphilitic lesions other organisms are, of course, 
present, und not infrequently another spiral organism, to whiglt 
the name spirochalgywfringens hos been given. ‘This organisa 
is usually somewhat longer, and is distinctly thicker than the 
spirochete_pallida. As the name implies, it ix more highly 
refractile, and is much more easily detected ‘than the latter 
organiam ; ite curves also axe opener and much loss regalar, 
and they vary in their appearance during the movements, In 
stuined filins (see p. 107) the differences between the organisms 
caine out moro distinctly, as ean bo gathered from the accom 
pacying photograph (Rig. $1). The spirochwte pallida by the 








yery lange, and a summary of the results may oy Tn the 
primary sore aud in the related lymphatic the Juice of 
which can be conveniently obtained by means of a hypodermic 

syringe, the organism 


patches—in fact, one may say 
generally, in all the primary and 
lesions. It has been ob- 
tainod from the spleen during life, 
and on a fow occasions, 4.9. 
Schaudinn, also from the blood 
ey life in secondary syphilis, 
._ Inthe congenital form of the disease 
2 the organist may be present in 
a the numbers, as was first shown 
by Busehke and Fischer, and by 
Lovaditi, In the aol bulls, in the blood, in the in 
tergal organs, the liver, lungs, spleen, supra-renals, and even in 
‘the heart its detection may be comparatively easy, owing to the 
Targe numbers present (Fig 80), It can readily be demonstrated 





by their appearance from the spirochwte  p 
rosomble it closely. Hoffmann, however, who has seen many of 
these spirochates 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 
noivayphilitic lesions with exactly the sme microscopical Chars — 
acters does not show that it is the sume orgunixm as the spiro-_ 
chaste pallida. Tt cannot bo claimed that the pathological 
relation of this organism to the discase ia absolutely demon- 
strated ; but the facts stated are to form very strong 
presumptive evidence that in the spirochmte pallida we havo 
the trap cause of ayphilis, 

Transmission of the Disease to Animals, — Although 


ions experiments bad previously been from time to tine 








wh 


observers 
& large series of observations, and have shown 
[rcpt ae roster pera to various a monkeys, 
these the ant id apes are most ible, i Zee 
Sgn gate pone gr 
results have been confirmed by Luxsar, Neisser, Kraud, and 
others. Stoner place Pithleste agers otralte the 

‘typical primary lesions in more twen! 
sates rst The pri lesion is in of fecmaset 
or in 


every Teseml 

there is § marked enlargement and 
indurntion of the corresponding lyu primary 
lesion on an average after inocula. 


mucous 58 oat, ol aerate Lo pu 
Asa the secondary manifestations were of a somewhat 
os Boe MI a a TM ened! 

ion boon observed. By inoculation from the secondary 


in ‘and macaques (macicws sinicus is one of 
the most suscoptible), but these animals are less ble. 
Th the ease of many of ther no result follows, and when a lealon 
is produced it is only of the nature of a pear 
secondary ifestations never appearing. There ix thus no 
doubt that the disease may be produosd in apes, and, to speak 
wenerally, the severity of the affection increases according to the 
nearness of the relator ship of the auimal to the human 

ite luction of the dissase, experimentally, has supplied 
us with some further facts regarding the naturo of the ices 
Te has been shown repeatedly that the passage of fluid con- 


i 
: 
: 
i 


rh a Berkefeld filter deprives it completely 
In other words, the virua docs not belong to 


the i ic group of organisias The virus is also 
ty a temperature of St Co being 
fatal. regard to the production of immunity, very little 


Of & satisfactory nature hus so far becu established. tt has ten 


= | 


& 
F 
5 
ES 





234 GONORRHGA, SOFT SORE, SYPHILIS 
found that the virus from a macaque monkey produces « less 


| 
2 
: 
i 


by 
mann by inoculation with enate material ; they oe after 
© long period of incubation, and the spivochsete 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 virua rendered inactive by beating 
has apparently no influence in acting as a vaccino. There ix 
vome evidence that the seram from a pationt 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 spirochate pallida 
hos been found in the lesions in monkeys, Metchnikoff and 
Roux obtaining positive results in more than 7 per eeut of the 
cases, and it ig to be noted that here also the organiam has been: 
found deep in the substance af the papules, unaccompanied by 
any other organisws. Hoffuann failed to find any spirochetes 
in monkeys which had not been inoculated with syphilitic 
material, This observer produced o lesion on the upper eyolid 
of a macacus by inoculation with the blood of a man who had 
suffered fron the disease for six months, and a papule appeared 
which contained spirochates, This result isin 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 infectivo, 
Castellani has desoribed in yaws or frambovsia the occurrence 
of a spirochwte closely resembling the spirochete pallida in 
appearance, and to this organism he has given the name spiro 
cheste pertenwis. He hus found it not only in the kin lesions 
but also in the spleen and lymphatic glands of patients enffering 
from the disewse. He hus produced the disease in monkeys by 
direct inoculation and his fund the spirochwte in the resulting 
lesions, He finds that the immunity reactions of the two organ 
ixms—spirochiete pallida and spirochwte pertenuis—are quite 
distinet; hence we haye probably to deal with two distinct species. 











CHAPTER IX. 


TUBERCULOSIS. 


‘Tue 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 & more important 
effect on medical science and pathology than this, It has not 
ouly shown what is the real cans: of the disease, but has also 
supplied infallible methods for determining what are tubercular 
Jesions. and what aro not, and has also given tho mcans of 
studying the wodes and paths of infection. A dofinite answer 
has in this way heen supplied to many questions which were 
proviouly the subject of endless discussion. 


‘Bistorioal.—By the work of Armanni and of Cohnbeim and Salomonsen 
(1870-80) it hiad been demonstrated that tubercle wasn infective disease. 
‘Tho latter observers found 
‘eye of mbbits with tuborcular material that in ma 
irritation soon disappeared, but that after period of incutation, usually 

tive ays, small tubercular nodules appeared in the iris; 

ienana gradually spread, ening tox tgbarelar dlsorgan 

ho eye. Later atill, the lymphatic glauda became. 

iually the auiial died.of acute tuberculosis, The question 

to the nature of the virus, the specific charsetor of which 

Mes thas sitablished, and this question was antwered by the work 
ol 

‘The announcement of the disovery of the tubercle baoilins was made 

rum 











‘Koch ii March 18&2, and a full account of his researches appearedt in 

USGE (AA, 0644. X, Gamthtoomée., Berlin). Koch's work on this subject 
will a4. n classios| masterpiceo of bacteriological research, both on 
aecount of the great difficulties which he scceasfully overoane and the 
tentas with which he demonstrated tho relations of the organism 

to the disease. ‘Tho two chinf ditheulties wvry, firt, the demonstration 
of the bacilti in the tissues, anc, secondly, the cultivation of the organism 
outside iy, Vor, with regard to the first, the tubercle bacillus 
cannot te demonstrated by a simplo watery solution of a basic aniline 
ye, and it was only aftor prolonged staining for twouty-four hours, with 
js solution Of wethylene-blue with caustic potash added, that he was 
able to reveal the presence of the organism. ‘Then, in the second place, 


25 











all attempts to onltivate it on the media failed, and be i 
‘thawing owe ied oad wean, the eliod gf 
preparing whioh he himsel 


os 
of glands, oto., are really tuberoular in nature. 


Tuberculosis in Antmals—Tuberculosis is not only the most 
widely aproad of all diseases affecting the human su i 
jucee a mortality geeater than any other, but there ie proba 
Beales cles shot ante he dame 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 animale may be given, as this subject is of great im 

portance in relation to the infection of the human subject, 
Amongst the domestic aulmals the discase is commonest it cattle 


(bovine tuboroulo: in which animals the lesions aro ¥¢ 
‘in character and distribution, In most casos the lungs: 










‘re allot 






in dogs, cata, 
in_monke: 





nl in the large carnivor 
confinement, and I 





5 








THE TUBERCLE BACILLUS 287 


‘Tuberculosis in fowls (avian tubernolovie) ie & common and very 
infectious disease, nearly all tho birds in. poultzy-yanl helag sometimes 


a pe aye pap abe ates pear - 
animals prosonte great variations in charncter, differ in 
fay ropa htt i a the nae ‘The 
of the different forma of tuberculosis is discussed below. 
Tubercle Bacillus Microscopical Characters. — Tubercle 


eogth, and ‘9 px in thickness, te. in ou ta then Ieoth 
they are comparatively thin organisms (Figs. $2 and 83). Snes 
times, 





tremely difficult to deter. pya, 52 —tTuberele bail, from a pure 
mine the exact nature of calture foal pci} agar, 
the unstained points. Ac- Stained with carbol fuchsin.» 1000, 
cordingly, we find that 
ome consider these to be spores, while others find that 
it ia it ible to stain them by any moans whatever, and cox. 
sider they are really of the nature of vacuoles, Against their 
heing 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 ia impossible to speak definitely on the 
qnestion at present. We can only say that the younger bacilli 
stain uniformly, and that in the older forma inequality in staining 
is mot with; this latter condition ig, however, not associated with 
(greater powers of resistance. 

‘The bacilli in the tismes occur scattered irregularly or in 
Tittle mason. ‘They are usnally single, or two are attached ond 
to end and often form in euch a caso an obtuse angle, Tras 





TUBERCULOSIS 


238 

chains are not formed, but occasional filaments are met 

‘with. ‘Ta cultures the bacilli mm ih are 
one 





\ - 
yp. 
Fin, 69.—Tuberole bacill in phthisical sputum ; they are Longer than 
{a often the amie, 
Film properation, stained with oarbal-fuchsia anil mothylene-blue. * 1000. 


may occur. These may be in the form of long filaments, some- 
times swollen or clubbed at their extremities, 1ay be irregularly 
beaded, and may even chow the appearance of branching. Such 
forms have been studied by Motchnikoff, Maffucci, 
others. ‘Their significance has been variously interpreted, for 
while seme Jock npon 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 Intter view, 
and this is now generally accopted by authorities, It has also 





=a! 


CULTIVATION OF TURERCLE BACILLUS 239 


been found that under certain circumstances tubcrele bacilli in 
a tiseans eros»: ridiating structure closely similar to that 
This was found by Babes and also by 
case when the bacilli were injected under 
directly into certain solid organs, euch as 
kidneys in the rabbit, Club-like structures may be presont 
the poriphery ; these are usually not acid-fast, but: rotain 
in the Weigert Gram method. Similar resulta ol 
acid-fast bacilli will be mentioned below, and these 
organisms would appear to form a group closely allied to the 


a 


FREGESE 
ri 
BE 
= 


fe 


g 
i 
i 
a 
E 
ef 
zs 
a 


2 
zF 


i 


solution being warmed till steam arises and the 
to rmain in the hot stain for two or three 


method (see p. 100) eee 
Jeeullarty, homewer, that after sainng as taken pace 
resist docolorixing by solutions which readily remove the 

from the tissues ond from other organisms which may be 
Such decolorising agents are sulphuric or nitric acid 
cunt solution. tions exn thus be obtained in 


REST ee ibe 

a 
erie id 
Here 
ia 
£ i 


the tissues can then be coloured by a cominast stain. 
ithin recent years certain other bacilli have been discovered 
present the same staining reactions a4 tubercle bwcilli ; 
fare therefore called "acid-fast ” (ride infra). ‘The spores 
‘of many bacilli become decolorisod more readily than tubercle 
bacilli, though some retain tho colour with equal tenacity. 
Balloch anil Movies, by treating tubercle becili with hot slochol 
ether, extracted a wax wie ens the characteristic staluing 
rections OF the bacilli thernsed remains of the bacilli, farther, 
“Een ea 
acon : 


EE 


ff 


HE 


‘Cultivation. The medium first used by Koch was inapiasated 
blood serum (wile p, 39). If inoculations are made on this 
medium with tubercolar material free from other otgawvnus, 


— il 








240 TUBERCULOSIS 


there appear in from ten to fourteen days minute points of 
growth of dull whitish colonr, rather frrogalar, and slightly raisod 
above the aurface (it is advisble to plant en the medium an 
actual picce of the tubercular tissue and to fix it in a wound of 
the surface of the serum). Koch comparvd the appearance of these 
to that of amalldry scales. In such cultures the growths usually 
reach only a compara. 
tively sinall size and re 
main separate, becoming 
confluent only when many 
occur close together, Tn 
sub-cultures, however, 
growth is moro luxuriant 
and may come to form a 
dull wrinkled film of 
whitish colour, whieh 
may cover the tar 
af in eurtoceel On wirte 
and at the bottom of the 
tube imay grow over the 
surface of the condensa- 
tion water on to the glass 
(Fig. 84,4), The growth 
is always of a dull ap. 
pearance and has a com: 
siderable degree of eon 
siatence, 4 that it ix diffi- 
cult to dissociate a portion 
thoroughly in a drop of 
water. Tn older nltures 
ay ailearaa ‘al ole bactiti on #8, Growth may aoquire 
ches peperiak lca slightly brownish or buff 
Aud 8 Maslin tubercle wets A ws an colour. Wher the small 
iM cot th, colonies are examined 
eran nora ‘hia the omer” under a low power of the 
microscope they are seen 
to be extending at the periphery in the form of wavy or sinuous 
atroals which radiate outward and which have beén eompared 
to the flourishes of a pen. The central part shows similar 
markings closely interwoven. ‘These streaks are composed of 
tusses 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 subcultures at an earlier date and pro- 





























POWERS OF RESISTANCE 241 


greases moro than on sorum, but this medium is not 
suitable for ol cultures from the tissues, inoculations 
with tubercular tee sn result, ‘The 


however, the growth be started on the Seat supers 
ficially asa dull whitish, wrinkled pellicle which may reach the 
walls of the flask ; this mode of growth is specially suitable for 


Tk wa» at onetime believed that the tubercle tncillus would only 
‘on mindls contalufag animal Oulds, Dut of late yours has been it 
rp cele. purely vegas lium, a ran rt 

the potatom. Sander bea shown 


carrot, Tana 
these FP wha glycerin ls wid 
that cultures from tubercular lesions conld bv obtained om glycerin potase 


‘The optimum temperature for growth is 87° to 38" 
Growth ceases above 42° and usually below 28°, but on long- 
‘continized ctiltivation outside the body and in special eireum- 

ae orth may tako place at a lowor temporaturo, #9. 

found ‘hat go took place in glycerin-potato broth 
pe 29° to 23) 

zene of Beastial: —Taberele bacilli: have considerable 





Wve bev found to be alive in tubereular orgnna which 
buried in the ground for a similar period. —'Thoy are 
ing expored to the action of the gnatric juice for 


th belli 
have beer: 
not Killed 
ax! eipesatire of — 3° C, for three hours, even 
when this i repeated several times Tr hax been found oat 














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 Jocul 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 43 


} bt, though there are occasionally a] whieh 
Soar eke elo et formation, it cannot be regarded aa of 
common pecarrence. Tn some cases of acute tuberculosis, when 
the bacilli hecome Todged in.a eapillary the endothelial cells of 
its wall may proliferate, and thus a ring of nucloi may be seen 


round a Cea nae ete ely ives rise 

to ans losely resemblis a 
pape here Berend, Yt he tol 

giant-ecolls are of connective fe bieeey ca wenie wtiee 





wast 
the of the 
the case of phthisioal cavities and conditions toes other 
bacteria are present, the toxins of the latter also play an im 
ipod ‘The occurrence of waxy change in the organs is 
i by some to be chietly due to the products of other, 
ally pyogenic, organisins, secondarily present in the tuber- 
cularlesions. This matter, however, requires farther elucidation. 
Pepe pom and Distribution of the Bacilli.—A few fuets may be 
the presence of bacilli, and the numbers in 
ry they-ars likely to be found in tubercular lesions. Ou the 
one hand, they may be very few in number and diffienlt to find, 
and om the other hand, they may be present in very largo 
numbers, sometimes forming masses which are easily visiblo under 
the low power of the microscope. 











ion 
can sometimes see n few bacilli faintly xtainod, along with very 
minute anequally stained granular poets some of which muy 
pomibly be spores of the bacilli, Whether they axe sores oy 





erty TUBERCULOSIS 


aot, the important fact has been established that tubercular 
Peer ae te hrm piece Tapp AEN | 
be proved, on. imental inoculation into animals, to 

hark saath a facilt ansy be proton in eumelenae 
small us to observation, or it may be that their spores only 
are it. In subaente lesions, with well-formed tubercle 
folli and little cascation, the bacilli are genorlly scanty, 





Fiv, 85,—Tuberole bacilli in section of human lung in acute phtbisle, ‘The 
bacilli aro seen lying singly, and also in large masses to left of fold. ‘The 
pale background fs formed by exseous material, 

Stained with earbol-fuchen and Bisarek-brown. 1000. 


‘They are most numerous in acute lesions, especially where 
caseution is rapidly spreading, for example, in such conditions ax 
caseous catarrhal pneumonia (Fig, 85), acute tuberculogia of the 
spleen in children, which is often attended with a good deal of 
rapid cascous change, Th acute miliary tuberculosia a few 
tncilli can generally be found in the centre of the follicles; but 
here they are often much more scanty thon one would ex] 

‘The tubercle bacillus is one which not only has comparatively 
slow growth, but: retains its form and staining powor for a much 








ACTION ON THE TISSUES 245 


Jonger period than most organisms Ax a rule the tncilli are 
eee Ge position. Occasionally they occur within tho 
xiant-cells, in which they may be arranged in'a somowhat radiate 
Munnner af te porphery, oeastonaly also in epithelioid calls and 
in 


Tho statemonta, however, apply only to tuberculosia 
in the human subject, and oven in this case there are exceptions. 





Hr 8h —Teberele bacilli in giatit-colls, showing the radiate arrangement 
‘ab the periphery of the celis. Section of tuberealar wider af cow. 
‘Stalsed with carbolfuchoin and Bismarck:Lrown. 1000, 


eta on the other band, the presence of tubercle bacilli 
ianbeells is a vory common occurrence; and it ix 
pester common to ‘ea them in considerable numbers scattered 
throug! t the cellular connective tissue of the lesions, 

‘even when there is little or no cessation present (Fig. 46), 
‘Tn tuberculosis in the horse and in avian tuberculosis the 
of bacilli may be enormous, even in lesion# which are 
‘not spreially acute; and considerable variation both in their 
und in their site is met with in tuberculosis of other 





— = | 





46 TUBERCULOSIS: 


a ech fe faba, lana a ee - 
illi are usually to be found, 19 thames 
sical pationts their presence can be domo almost 


to find them unless a de- 
posit igobtained by means 
of the centrifuge, This 
doposit is examined in the 
same as the spnrum. 
‘The bacilli often cour in 
live clumps, as shown in 
Fig. 87. In tubercular 
‘ulceration of the intestine 
their presence in the fees 
demonstrated, ns 
Fin, 87.—Tuberole taollll In urine ; showing was first shown by Koch > 
ue of the characteristic lume, im which ut im thin ease their dis: 
Stained with carbol-fuchuin and mothylene- COVerY is usually of little 
bluw «1000, importance, as the intes: 
tinal Jesions, 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 peritoneam, into the anterior chamber of the eye, into the 
voing ; by feeding the animale with the bacilli; and, Instly, 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 
#enerally that when introduced into the tiseucs of a susceptible 
animal, the bacilli produce locally the lesions above described, 
terminating in caseation ; that there occurs a tuberenlar affeetion 
of the neighbouring lymphatic glands, and that lastly ses 
may be » mpid extension of the bacilli to other organs By 
blood stream and the production of geueral tuberculosia OF 








EXPERIMENTAL [NOCULATION aT 
used for the purpose, the guinea-pig is 
inoculated subcutaneously with tubercle 
phthisieal oh pal a local swelling gradually forme seh 3 


marked about the tenth day. Thi swelling 
and cascous, and may break down, leading to the 





changes, ite eee usually ‘the spleen 
an neu n] u 0 
being ally affected, This organ ix a egpetigny oe 


injection of pure cultures preduces a local lesion in the 
fer of an extensive tuberetlar lotitration ud thickening of the 
omentum, sotuetimes attended with seuto tubercles all over the 
Feritonnsin, ‘There ix a camous eolargement of the retroperitoneal and 





mors the larger the numbers of bacilli injected. inn 
ned Jmsilll-ox with spatzn. cr portions of tlaews 
Sestalniag them, vesdily contriet an intectinal form of tubereulocis 
lesions Present in the lymphoid tiene of the intestines, in the 
tmesontaric ‘and later in tha internal organs. 
Rabbits are jess succeptible than guinea-pigs, and in them the etfects 
of sul Ingsulation are yory variable ; sometimes the lesious 
femal local, sometines a general tuberculosis is act up. Otherwise the 
Tesctiows are much of the samo nature. Doge aro much more highly 
Teslitant, But tubercatlosis can be produced in them hy intraperitoneal 
pore ealtures (Koeh), or hry intravenous injection (MatTueei), 
la ‘case Chere results an extensive eruption of minute miliary 





(li 





248 TUBERCULOSIS 


iberolés. Tubereulosis also be juced in siacaptible 
Rica bfuating tion lalate tbe eee 


Varieties of Tuberculosis, 1. Hunan cand Bovine Tuberculoria, 
—Up till recent years it was generally accepted that vt 
mammalian tuberculosis was dae to the same organism, and 
in particular that tubereulosia could be transmitted from the 
oe aes ae ‘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 lutter really exists, 
infection is of very rare cecurrence,—so rare that it is not 
nécessiry to take any measures ngainst it. Previously to this, 
‘Theobald Smith had pointed out differenets between mammalian 
and bovine tuberele bacilli, the most striking being that the latter 
passeas a much higher virulence to theguinea-pig, rabbit, and other 
animal, and in yortionlar that human tubercle bacilli, on 
inoculation into oxen, produce either no discase or cay Wed 
lesions without any dissemination. Koch's conclusions were based 
chiofly on the result of his inoculations of the bovine species 
with human tubercle bacilli, the result being confienat of 
Smith's, wud, secondly, on the supposition that infection 
human subject Sioa the intestine is of very rare occurrence, 

Since the time of Koch’s communication 4s enormous amount 
‘of work has been done on this subject, aud Commissions of 
inquiry haye been appointed in yarious countries We 
wummarise the chief facts which have been estal 
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 bucilli of the bovine typy when cultivated are #horter 
and thicker and more regular in size; whilst their growth on 
various culture media is seantier than that of the human type 
From the latter character the British Royal Commission have 
applied the term dyegonic to the bovine and cugonic to the 
human type. As already stated there is also a great difference 
in virulence towards the lower animals, the bacillus from the ox 
amuch higher virulence This organism when inj 
in suitable quantities into the ox prodaces a local tubercular 
loxion, which is usually followed by a generalised and fatal 
tuberculosis ; whereas injection of human tubercle bacilli pro- 
duces no more than a local Jesion, which undergoes retrogression. 
(Jo certain experiments, eg. those of Deldy Hamilton and 


al 

















VARIETIES OF TUBERCULOSIS 249 


Young, general general tuberculosis has been produced by tubercle 
from the human subject, but these results are exee} ). 
ing differences come out in the case of the rabbit; in 
eh irarbceen Sajosion of eatable quantities in this animal is 
the resiest method of distinguishing the two n acnte 
boar Palle the bovine, but not with the human 
type. and monkeys a generalised tuberculosis 
result from subcatancous injection of bacilli of sete ead 
‘bit in this case also the differwnce in favour of tho greater virn- 
lence of tho bovine type is made out. With regard to the dis 
tribution of the two types of organisms, it may be stated that so 
far ax we know the kacillus obtained from bovine tuberculosis is 
always of the bovine typo, and tho same may be said to be true 
of tuboreulosis in pigs; in fact this sesms to be the prevalent 
organism in animal tuberculosis, Th human tuberculosis the 
bacilli in a tajority of the cases are of the human type ; 
a pec ba, 10° cartel peoporln eel of ty 


present, the bacilli when cultivated being 
any Means at our diay from those 
from tuberculosis, ‘The Koyal Commission 


Iemeppie sets es whe theca Pea ce hans of tion by 
alimontation. It is also to be noted that almost all the tuber: 


the ox to man, nnd that the milk of tubercular cows is a common 
vehiele of transmission. 
most of the bacilli which have boen cultivated 
to one of the two types, as above described, it is 
also to be noted that intermediate varictics are met with, It 
has also heen 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 passin m through the 
bodies of certain ani nage eg. quince pigs, sheep, and goats, 20 
emer eck: the charactors of bovino kucilli, In tiew 
these facts it is probable that Lovine bacilli will undergo 





— : 





250 TUBERCULOSIS: 


corresponding inodifications in the tissues of eee 
oka pedi. of tice fa necessary for such a eh: 
me At is thus possible that the eases from whit ie ier 
been obtained do not represent the full umber where 
ehghipetanfenciec ths teseeaeallas quite likely that 
althoneh the bovine bacilli are more virulent to the lower 
thon the human bacilli are, this does not aleo hold in the 
case of the human subject. In fuct the comparative chronicity 
of the primary abdominal lesions in children in the first instance 
would point rather to a low order of virulence towanls the 
human subject. We may also add that there aro cases, 
those of Ravenel, in which accidental inoculation of the human 
subject with bovine tubervle has resulted in the production of 


tuberculosis. 

2. ail Shlomi the tubercular lesions in birds there 
are found bacilli which correspond in their staining reactions 
and in their morphological charactors with those in matmal 
but differences are observed in cultures, and also on experiment 
inoculation. These differences were first deseribed by 
and by Rivolta, but special attention was drawn to the pine 
by a paper read by Koch at the International Medical Congress in 
1890. Koch stated that he had failed to change the one oe 
‘of tubercle bacillus into the other, though be did not conclude 
therefrom that they were quite distinct specicy. The following 
points of difference may be noted -— 


n plyoetn spur and ‘on serum, the growth of taberae beclll Cam 
birda fe more luxtiriant, has a moister appearance (Big. 
morvaver, takes plo at x higher tomperature, 43:5" C., than Mhaths 
case with onlinary tubercle bacilli, xperimontal inoculation 
out even more distinet differences. ‘Tubercle bacilli derived from 
roman aubjech for sxampla, when injected Into Links usally fall ta 
rontuse tuberduloity whist thowe of avian orig mdi 
Hinds are very susceptible to the discus wh 
‘of the organs of bids containing tubercle bacilli, but they ean consmme 
enormous quantifes of phihisical sputuan without becomlug tubereular 
(Straus, Wurts, Nocard). No doubt, ou the other hand, there are eases 
‘on record in which the source of infection of » poultry-yant has 
parently bean the sputum of phthisial petients, Asin tuber 

Ili cultivated frum birds have not the same effect on mooulation 
‘of muammale ue ele bacilli have. When guinea-pigs are 
inoculated suber y thay usually resist infection, thongh oekn 
sionally a fatal result follows. In the latter case, usally no tuberoles 
to the nak re found, bnt numerous bacilli may be presen 
ly in the epleon, which is much swollen. 
Further, intravenous injection even of large quantities of avian tnbarele 
bacilli, In the enso of dogs. leads to no etfeot, whereas onlinary tubercle 
Yweillé produce acute tuberculosis. [The mbbit, on the other handy is 
comparatively susceptible to avian tuberculosis (Nocund).} 





















































made to acquire all The chaste of Se 6 AP 


© tuberculosis in small collodion mies (v. p, 128) containing bonillen, 
and then to ingert each me in the poritoncal coy ta fo 
Fpemaroneac Eh ist fx pesiela of froma Ee 
‘They were Diz seecranea enikaraat wari ia econ lal ee 
Tee Lon yer my Dea from these cultures and intro 
Beira bats In such conditions the bacilli am 
ssa yely to: tie ea eset tes ala the ili 
impervious to bacilli to leucocytes, ete, After one 
this kind, and still more so after two, the bacilli are 


8 
it 
3 


found to have acquired some of the characters of avian tubercle 
Wall tt se still non-virulent to fowls. After the third 
sojourn, they have acquired this property, aud produce 
iu fowls the same Iesion as Iucilli derived from avian tuber: 
enlosis, Tt armen syeeee stat the bacilli of avian tuberentosis 
are not a distinct permanent species, but a variety which 
haa been. Ey grout in the tinaea of the bind, Evidently 
also there are d of this modification according to the 

‘of time during which the bacilli have passed from bird to 


aie ee see ease : bacilli of avian 
linary tal nodules in guino] 

(Courmont and Dor), It is also interesting eae en 

itch has cultivated tubercle bacilli of the mammalian 

rea Seem some ciaea of tuberculosis in parrota kept in eon- 


3, Tuterewlosix in the Fish—Bataillon, Dubard, and Terre 
ieee sation ont nie disease i "er carp, a “soeres whieh, 
in mn and microscopic charters, closely a 
with the tubercle bacillus The leion with which ocvey 
ameeinted was an abundant growth of geunulation titsve in 
which numerous giant-colla were present, It forma, however, 

growth at the room temperature, the growth being 
thick and moist like that of avian tubercle bacilli (Fig. 8%, ¢). 
Growth dows not occur at the body temperature, though by 
gmdual mclimatisation « small amount of growth hus beet 
obtained upto 36° C. Furthermors, the organism appears to 


i 


959 TUBERCULOSIS 


undergo no multiplication when injected into the tissmes of 
pera, and ee ie ‘this chareteristic have eo. 


organism from marl, and alko from organs of att ee - 
iw thus probably to be regarded as a sa) Adee 
cooasicanlly associated with discane in the 5 "Lending ts 
the results of diffrent experimenters it ix posible to modify 
hutnan tubercle bacilli by allowing them to sojourn in the tissues 
of cold-blooded animals, ¢g. the frog, blind-worm, ete., #0 that 
‘they flourish at lower temperatures. These results have, how- 
ever, been recently called in question, as it has boon 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 tuborele 
bacilli may become modified in relative virulence and in con — 
ditions of growth by sojourn in the tisues of various animale — 
‘This modification appears slight, though of dofinito character in 
the case of bovine tuberculosis, more distinct in the case of 
avian tuberculosis, and much mors marked, if not Petite in 
the case of fish tuberculosis, that ie, of course, in their relations || 
to the bacilli from the human subject. 

Other Acid-fast Bacilli—Within recent years a number of 
‘ili prosonting the same “St a reaction ax the tubercle 
bacilli have been disecoverid, Such bucilli have a comparutively 
wide distribution in nature, as they have been obtained from 
virions species of grass, from butter and milk, from manure, and 
from the surfaces of animal bodies Mieroseopically, they bes 
more or less closely with tubercle bacilli, though most of 

ture shorter and plumper; many of them show filamentous and 
branching forms under certain conditions of enlture. 

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 ite 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° ©, Furthermore, in most instances, growth occurs 
at the room temperature. The general character of the cultures: 
in this group is n somewhat irregular layor, ofton with wrinkled 
surfaco, dry or moist in appearance, and varying in tint from 
white to yellow or reddish brows, The number of such 








tained with earbol-fuclin, and treated with 


OTHER ACID-PAST BACTLLT 253 


organiams i constantly being added to, but the following may 
bo mentiined a8 eciae pate 


Mostler's Gross Bact, I. ond 17.—Tho former Coiba, eae 
of Timothy.grace (Alen pratense). It is extremely acid-t morpho: 
MNtseis eoeeatls Gn teherole belli: ae fa cnlbares wary Shey ab 
forumtion aad Drovching. The lesions. produced closely  reveeble 
tubereles, ‘The colonion Fiaiblo in thirty-atx hours, aro soale-like and 
af greyish-whito colour (Fig. 89, a).  Moulier’ bucillue If. was obtained 
from dust of a» hay.to! ‘The colonies at first are moist and some- 
‘whet tennaiony but afterwards run together, and are of « dull yellowish 
colour, ‘The goueral cestilte of iuoculation resemble these of grasa 








~ 0 
Pid, 88—Moelice’s Timothy-gras bacillus. Pio. $9.—Cultares of acid-fast bacilli 
Prom a culturo ou apar. grown at room temperatire, 





fo) Moetlars Timothy gran tal, 
20 per cent snlpburie maid. «1000, fh) the Petre tuatuncirticn butter tacit 
{9 Beciine of huh tobereuonts 








bacillus 1. but are less marked, Moollor also obtained a similar organisa 
from milk. He also discovered a third qctd-fast bacillus which be 
obtained from manure and therefore called tha ‘ Misthaoillns” (dong 
Vacilles) ‘This organism has avalogons eharacters, though presenting 
minor differences. It also has pathogenic olfcets. 

Petri and Rablnowitch indepontently cultivated at acid-fast bacillus 
from batter (butter bacillux”) in which it occurs with comparative 
‘The organism rosembles tho tuberole bacil}ns, althongh it i 

on the whole oe thicker. Its leatons closely resemble tuber. 
Walon, especially when injection of tho organism t% made into the 
Peritoneal cavity of guinea-pigs, along with buttor,—the method uaxally 
ado} in a for rors bacilli in butter, This organism 
qretty rapidly a wrinkled growth (Fig. 0, 4) not unlike that 

EP Mesibers grat tecilius If. Korn has alio obtained other two 
bacilli from butter which ho holds to be distinct from one anodiwe awa 











4 








254 TUBERCULOSIS 


trom Kabinowiteh's baciline. Th pointe af distinetion are of w minor = 
Spear sgt gL ml sit Aacilli have benn cultivated hy 
es 


of considerable interost is Johne's bacillus or the 
tuberoaloay enteritin" the lesions 
mucous 





various es, 

M'Fadyean. ‘The bacilli gocur in large numbers in the lesions, aud can 

teodlly be found in scropings from tho antface. ‘They resemble the 
‘tubercle bacillus i 





i To 
action it somewbat’ rewato 
the tubercle bacllausandanay 
‘be miataken for it. Te ocaurs 
often in large numbers mts 
inthe region of ‘Ke external 





S a slender at ae 
ongpaniem, like the taberele 

Fre. 90S bec. Fila preparation bostitay "but cali eta 

aTuegTa, tinetl; ver 20) 

Ziehl-Nealwon stain. 1000. eg ee 

stains with some difficulty 

and resists décolorisation with strong mincrel cide, Moxt obvervars 
aseribo the latter fact to the fatty matter with whioh it is smzronnded, 
and find that if the xpecimen ix treated with alcohol the organism ts 
casily decolorived, Csaplowaki, howsver, who claims to have cultivated 
it on various media, finds thet in culture it shows tance to decolor 
sation both with aleoliol and with acids and considers therofre, that 
the reaction ta not due to the surrounding fatty medium. We hare 
found that in snegma it can be readily decolorised by a minuto axpoanre 
to alechol after the usual treatment ‘with sulpharic acid, and thus ean 
























be readily distinguished frum the tubercle bacillus moreover, 
Lietiove tliat minor points of difference ix the mictoacople appearanees of 
the to organisinn are quite sufficient to make the experienced observer 


snspiciour if he should moct with the «megma bacillas in urine, and ead 
him to aj py the deoolerising test. Diltioulty will only ooeur when a fue 
seatterod bucilit retaining the fuchsin oceae. 

Tts cultivation, wirich ix attended with some difficulty, was first 





dotailson this subject, vile Potet, Btvudes eur lew bacilies dates 
ri, 1002, 


1 For 


seikepsraten 








ACTION OF DEAD TUBERCLE BACILLI 255 


by Craplewskii On serum it grows in the form of yellowish 
irregularly rounded colonies about 1 mtn. in diameter, sometimes 
es pees ta for x cmmpantively thick bayer. Hie foul that 
i se in agar and in bouillon, Te 
aka glach bate bean ted etn. SY 


Cowie hus recently found that acid-fast bacilli are of common ecour. 
rence in thir snoretions of the external genitals, iiammm, ote., tn certain 
tha Jonge satin, and that thane erganiam vary tn appearance, He 

the term ‘‘ smegma at proba! represents 
umber of allied species. = = 


‘The question may be asked—do these resulls modify the 
validity ot the staining reaction of tubercle bacilli as a means of 
diagnosis? The source of any acid-fast bacilli in question ia 
manifestly of importance, and it may be stated that when these 
have been obtained from some sonree ontside the body, or where 
contamination from without has been ible, their recognition 
as tuberole bacilli cannot be established by microseopic examina- 
tion alone. En the case of material coming from the interior of 
the body, however,—sputum, ete.,—the condition mast be looked 
on ns diferent, and although an acid-fast bacillus (not tubercle) 
has been found by Rabinowiteh in a case of pulmonary gangrene 
we have no sufficient data for saying that acid fast bacilli other 
than the taberele bacillus flourish within the tismos of the Auman 
body in such rare instances as to be practically negligible, 
(Tothix statement the case of the lepmsy bucillus ix of course 
an exception.) Accordingly, up till now, the microscopic ex- 
amination of sputum, ete., cannot be sid to have its validity 
shaken, and we have the results of cnormous clinical experience 
that sueh examination is prctically 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 tacilli are found, especially if they deviate 
in their Hie oa pe ee charactors from the tubercle bacillus 

Tubercle Bacilli—The remarkable fact bas 
ioe established by independent investigators that tubercle 
bacilli in the dead condition, when introduced into the tisanes 
in sufficient numbers, can produce tuberele-like nodules, Prudden 
‘end Hexlenpyl, by intravenous injection in rabbits of cultures 
sterilised by heat, produced in the lungs «mall nodules in which 
#evs-cells, but no caseition, were occasionally present, and 
which were characterised by more growth of fibrous tissue than 
is ordinary tubercle, The snhject was very investigated 
with confirmatory results by Strane and , who found 
that, if the number of bacilli introduced into the eireulution were 
Tange, there resulted very numerous tubercle nodules with well 


















28 TUBERCULOSIS 


formed insta and oomaionally traces of caseation, The 
bacilli can be well recognised in the nodules by the ordinary 
staining method. Tn 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 
hand, produces local abscess, but in this case no secondary 
tubercles are found in the internal organs Farther, in many 
‘of the animals inoculated by the various methods a condition of 
inarasiius sets in and puaually aa a fatal result, there 
being great emaciation before death. ‘These experiments, which 
have been confirmed by other observers, show that even after 
the bucilli ars dead they preserve their staining renetions in 
‘the tissues for a long time, and ples that ie 8 
contained in the bodies of the dead bacilli certain sul 

which act locally, producing proliferative and, to & less extent, 
degenerative changes, and which also markedly affeet the general 
nutrition, 8, Stockman has found that an animal inoculated: 
with largo 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 tho tubercle bacilli, thoir 
powers of resistance, and the paths by which they ean enter the 
ody and produeo disease (as shown by experiment), the manner 
by which tuberculosis is naturally transmitted can be rendil 
understood. Though the experiments of Sander show that 
tubercle bacilli can ioultiply on vegetable media to m certain 
extent at warm summer temperature, it is doubtful whether all 
tho conditions necessary for growth are provided to any extent 
in natare. Atany rate, the great multiplying ground of tubercle 
bacilli fs the animal body, and tnbercular tissues and secretions 
the bacilli are the chiof, if not tho only, meana by 
disease is spread, The tubercle bacilli leave the body 
in large numbers in the apatarm of phthisical patients, and when: 
the sputum becomes dried and pulverisod they are sot froe 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 presance in the alr of roome 
containing phthisical patients has beon repeatedly domonstrated. 
Williams plicod glass plates covered with glycerine in the 
ventilating shaft of the Brompton Hospital, and after five days 
found, by microscopic examination, tabercle bacilli on the surface, 
whilst Klein found that guinea-pigs loept in the ventilating shaft 
decame tubercular. Cornet produced tuberculosis in rabbits by 








sputus, and the source of from this means can scarcely 

sited it ought to be looked 
‘upon as a fruitful souros of infection to thoe around, and should 
‘only expectorate on to pieces of which are afterwards to be 
‘burnt, of into special receptacles wl are to be then sterilised 
either by boiling or by the addition of 5 per cent solution of 


CA gE rl Al Dah 
milk of cows affected with tbercalosis of the adder, Tn such 
canes the presence of tubercle bacilli in the milk ean usually be 
eat aaa isi ioe mrp 
it, microscopically, i i ith i 

Polat out by Woodhead 

alfected is bly tho great source of tabs mesenterica, which 
ix so common in young subjects, Tn theso cases there may be 












is rare as a primary infection, though 
it is common in phthisical paticnts as the result of infection by 
bacilli in the sputum which has been swallowed. There is 


the 
hati of infection by means of the flesh of tuberenlar animals, 


ongans, as in the process of cutting up the parts; and 
fn the second place, even when present they will be destroyed if 
the meat t4 thoroughly cooked. 





from the human subject ; in the latter, probably from tubercular 
‘cows, though inhaled tubcrele tucilli may aleo be swallowed and 
contamination of food by tubercalar material from the human 
subject may cecur. Alike when inhaled and when ingosted, 
‘tuberele bacilli may lodge about the pharynx and thus come te 


i” - A 


208 TUBERCULOSIS 


infeet the pharyngeal lymphoid ces tonsils, ete, tubercular 
lesiona of thesn parte being rch more frequent than was 
formerly supposed, ‘Thonce the cervical lymphatic glands may 
become infected, and afterwards other groups of glands, bones, 
or Joints, and Internal organs. 

The Toxins of the Tubercle Baciilus.—T'wo outstanding 
features of the action of the tubercle bacillus aro the occurrence 
of necrosis in the cells of tubercle nodules and the production 
of general disturbances of metabolism accompanied by fovor. 
Ib is natural to refer theao phenomena to the offecta of toxins 
formed by the organism. 6 study of such toxins centres 
round the substance known 4s tuterculin which Koch brought 
forward in 1890-1 a8 a enrative agent for tubercular affections. 

Koch's Tuberculin.—Koch stated that if in a guinea 
suffering from the effects of a subcutaneous inoculation “te 
tubercle bacilli, a second subeutaneons inoculation of tubercle 
bacilli was praotied 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 tubereulin, which 
consisted of a concentrated glyccrin bouillon culture of tubercle 
in which the bacilli had been killed by heat. Tts 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 cc. of tuberculin into a healthy man causes, in 
from three to four hours, malaise, tendoncy to cough, laboured 
breathing, and moderate pyrexia; all of which pass off in 
twenty-four hours. ‘The injection (the sito of the injection being 
quite unimportant), however, of “Ol cc. into a tuboreular 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 necrosia and a casting 
off of the tubercular mass, when this is poesiblo, as for instance 
in the case of lupus, The bacilli are, it was shown, not killed in 
the process, 


= 














theory of the action of the substance was that the tubercle 

‘ly secretes w body having a wecrotie action on the tissues 
‘When thie Ss Sijevted into « tuboroulaz:patiant, She proportion, rissa 
round a tubercular foous is suddenly increased, inflammatory roaetion 
takos place around, and necrosis of tho sproading margin ocours vory 
Tapidy, the material containing the living or dead bueilli being thraven 

en masse tnatead of toing disintegrated piecemeal. Th appeaty 














howeyer, that this explanation may not be oue ¢ for, on the one 
Baud, oiber substances Veaides products of the tuberce baclllus snay 








TOXINS OF THE TUBERCLE BACILLUS 289 


He 
at 
it 
E 
e 
: 


in 
‘tismno,  Matthos has, for instanos, found that 
mes sclsted from the andiary of 


Hy 
i 
ae 
A 
i) 
2 





imals 
to the toxins of the vibrio Motolmikoyi ; and later 
found that a similar susceptibility e: tow: 

the bacillus of fowl cholera. How complicated the taborenlin 
is is shown by the fact that a similar reaction has taken place 
tuberenlin has been injected inte persous suffering frum diseases 
tian tuberelo, eg. canorr, sarcoma, ayphilia. 


Hi 


curative agent against tuberculosis had been discovered, were soon 
found not to be fied. Th was very difficult to see how the 
necrowed ial 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 « small tubercular deposit’ in a Tung 
ora tic gland, or artificially, as in « complicated joint- 
cavity whore surgical interference could be undertaken, Not 
only 9, but the ulceration which might be the sequel of the 
necrosis appeared to open 1 path for frosh infection. Soon facta 
were reported which justifiod those criticisms. Cases whore 
rapid acute tubercular conditions ensued on the use of tuberculin 
‘wore reported, and in a fow months the treatment was practically 
abandoned. 








The oe of Fiterculie én tha Diagnosis of Tubercuteais tw Cotte — 
‘This is now the rine use to wae tuberculin is put. In cattle, 
tubereulosia —niay present without givin tim to apparent 
wpmptome Te ih thus Important trom ‘the palnt of view of human 
infection that an eat! “ations should be mode, ‘The method ie 
lied a8 follows :—The animals are kopt twenty-four hours in their 
‘andl the temperature is taken every Unrve hours, Crom four hours 
‘the injection till twenty-four alter, ‘The average temper: 
in cattle is 102-2" F.; 30 to 40 centigrammes of tuberculin aro 
J, and if the animal be tubercular the tomperatare rises 2° or 8 F. 
to twelvo hours and continnes elevated for ten to twelve hours. 
has worked most at the subject, lays down the prineiplo that 

y the temperature apyroaches 108" F. the mor reason for 
He gives a record of 250 cases wherw the value of 
teatod by subsequent post-mortem examination. Ifo 
Proper precautions the error was only 8°3 per cent, 
been largely proctised in all parts of the world, and iv of 


e 





tH 


q 


SPSit 
He 
533 


is undoubted that tabereulin contains toxic products 
‘by the bacilli, we know nothing of the nature of the 
‘present. From the fact that filtered cultures canae Wthe 


F Hs 


a 


260 TUBERCULOSIS 


toxie 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 succom bus attended attempts to gain a cloeor 
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 Tuberculin-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 nx follows, Bacilli from young virulent cultures werg 
dried in wrewo, and disintegrated in an agnte mill, treated with 
distilled water and centri liaed. The clear fluid was decanted, 
and is called by Koch * Tuberculin-O,” ‘The remaining depoxi 
was asin dried, ground, treated with water and contttfoaltaal 
the clear fluid being again decanted, and this process wax 
rvpeated with successive residues till no residue remained. 
‘Those fluids pat together constitute the “ Tuberculin-R,” 

From the fact that tuber 
glycerin was added, Koch con 
stances present in the glycerin-bouillon extracts originally msed 
by him, and he held this was borne out by the readiness with 
which « tuberculin reaction could be caused by it. Similarly, as 
tubercnlin-R gave a cloudiness with glycerin and did not readily 
originate w reaction, he considered that it contained different 
products of the bacillus. When injected into animals in 
repeated and increasing doses, y4q mgrm., being the initial dose, 
tuberculin-R is said to produce immunity agninst the original 
extract, against tubereulin-O, and against living and virulent 
tubercle bacilli. Another preparation hax also been introduead 
Known as “Koch's new tuberculin” (Bavillenemulsion). This is 
an emulsion of ground tubercle bacilli in water containing 60 per 
cent of glycerin ; it thus really contains both tuberculin-O amd 
tuberculin-R. Both, especially tuberculin-R, have boon used for 
the treatmont of tuberculosis in man, especially for early localised 
lesions. To the case of both substances commencing with from 
aby 10 ghy murm, yrulually increasing doses were given every 

































a 
eram from tubercular and non-tubereular cases hms 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 gonoral disturbances it fluctuates greatly 
from day to day, being sometimes below, sometimes above unity, 
‘To take the former and simpler case, he holils that if the treat- 
ment with injections of tubercalin-R be controlled by noting the 
effect produced on the opaonic indox, great improvement in the 
patient's condition result. Wright's interpretation of what 
occurs is bound up with his views on the nature of the effects 
produced. ‘Thess views aro brietly as follows, For reasons 
unknown the opsonic qualities of the body fluids may become 
low, and the tubercle bacilli, if they gain aduission to 

the body, ean multiply locally, ‘This multiplication is associated 
with a still farther local diminution of the opeonins. By the 
introduction of such a substance as Koch's tuberculin-R the 
Lodily mechanisen, whatever it is, which produces the opsonins 
is stimulated, and a rise in the genoral opeonie index occurs. 
faturally this is accompanied by a passing to the site of infec- 
tion of finids more rich in opsonins thin previously, the activity 
of the phagocytes comes into play and the tubercle bacilli are 
But any such vaccination proces must be eontrolled 

constant observations of the opsonie index, and it is only by 
‘moans that not only good resnite can be obtained, bot that 
the ion of harmful effocts can bo prevented. The reason 
Of this is that in a great many eases the injection of a bacterial 


a _ an 


262 TUBERCULOSIS 


vaccine is followed hy a decrease in the opkonic qualities of the 
aoram,—the occurrence of a nogutive phase. During such a 
period of depression there is probably an increased susceptibility 
to the action of the bacilli, Now, in order to got permanent 
benefit from the vaccination process, repeated injections of the 
tuberculin must be practised, and if an injection be given during 
a negative phuss, 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 pears 
phase is fully developed that afresh inoculation can be practised 
with success. Tho now negative phaso which will now occur 
may not cana a drop to below the level of the original state of 
the serum, and the bope is that its succeeding positive Lies 
will carry tho opwonic index still highor and ensure a etiil 
resistance bo the bacterium. The importance of the heer aa 
of the opsonic index lies in this that in antibacterial yaccinu- 
tions the degree of active immunisation which can be attained is 
always much loss than is the caso with immunisation inst 
such w substance as the diphtheria toxin, although in the 

there also ecour negative and positive phases of a precisely 
similar character, Lf an injoction 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 renct ton 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 
that they result from very irregular auto-infections of 
ient's body by tubcreular products from the local lesions, 
—positive and negative phases being produced without the pur 
posive quality which ought to characterize a successful therapeutic 
vaccination, Such auto-infections may come about in various 
ways, and Wright is of opinion that execcise, 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, Wrights 
chicf point is that the repeated, uncontrolled injections of tuber» 
ulin such as wero originally given may very likely have a harmful 
result, and that when an injection is practised it is not necessary 





ing to from one-thousandth to a sia- 

‘of a mill of tubercle powder is a sufficient 

to produce a pronounced negative 

sht to be tried at the next inocula- 

For cases clinically tu! lar where the index is about 

does, my, the equivalent of a two- 

legramme or less ought to be used,—the 

being carefully watched. Tn any came, the 
found to give the highest positive is 

Ria end ech ticsentelly $a Soectatal 








dove and ono . 
is constitutional disturbance should be asa 


deductions based on clinical data, such as absence of interference 
with pulse rate, temperature, cte., or increase of body weight 
after an inoculation being unreliable, and further evidence of the 
unroliability of such tosts is brought forward in the fact that, in 
cases of apparent benefit from sanatorium treatsnent, the opsonic 
index may ‘be very low. With regard to the results 
opiataed me ‘eases have bon bronght forward by Wright and 
others whore bonefit has followed the putting into practice of the 
i enunciated, and there is little doubt that the work 
given a fresh start. to the active immunisation method 

in the treatment of tuberculosia. An outstanding vent of 
Wright's work in this field has beon his insistance on the good 
effects produced by extremely sual doses of tuberculin (down to 
ith of a millegrammo) given at fairly long inter- 

ith rogard to the efleaop of tho 


it must be recognised that the method is ull ‘on its trial, and 
heceraee “a yen in ae work of bert ‘most careful eee 

limits of imental error of the opsonic method have 
teen sufficiently detined. 

“Tho whole question of immunisation against the tubercle 
bacillas presents many difficulties, and it is tho morit of Wright's 
work that it has shed fresh light on some of thess, One great 
frises from the great chronicity of the results of the 
im the majority of human cases. It is probably 





264 TUBERCULOSIS 


trae not only of man but of many species of animals used in 
experimental inquiries, that many individuals are on the border- 
line betwoon immunity and susceptibility, From the wide 
spread of the bucilli 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 othore emall lesions occur which do not develop further ; 
this has actually been shown by morbid anatomiats to be the 
case. ‘The disease being thus so often characterined by trausient 
jocal effecta without constitutional disturbance, the course of ax 
immunization 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 instanoe, 
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 immunissne 
tion. It follows that little is known regurding an attenuation 
of the tubercle bavillus analogous to whet is an important 
feature in imumunimtions against other organisma It has 
been thonght by some that the tubercle bacilli from so-called 
serofulous glands arp less virulent than these, say, from phthisia, 
bat 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 ahow that the injec 
tion of bacilli from avian tuberenlosia could protect the dog 
against bacilli derived from man, But these are not yet conclusive, 
Agglutinative Phonomona,—Tho serum of tubercular pationte 
has been found to exert an agglatioating action on the tubercle 
meillua, A convenient method f to add different amounts 
of serum, commencing with, aay, “1 cc. to quantitie of a 
dilution of the now tuberculin (Basillenemulsion) equivalent te 
1 part of the bacterial bodies to 10,000 of diluent, and leave the 
mixtury for 24 hours before observing, Ax with other agglatina- 
tive observations, it ix difficult to correlate the degree of agelu 
tinating powor of the seram with the degree of umounisation 
possess by the individaal from whieh it was derived. 
Antitubercular Sera, — Several atternpts have been made te 
treat tuberculosis with the serum of animals immunised by the 
tubvrole bacillus or it products. The most successful is perlaps 
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 suid (whieh are destroyed by heat). 














































S OF EXAMINATION 265 


apatite. immunising is animals consists 
and ono of the latter. The animals 
The seram obtained 


yi eiecamnnly aoe eee wee Lae eee 
every two days. Improve- 

ment is te haw taken place in a certain proportion, eapeci- 
ally of mild non-febrile case, 
As axtituberenlar serum has also been introduced by 
Marmorek, This observer considers that the tubercle bacillus 


presumably favourable eoil it is transferred to a medium con- 
‘a substance which may be unfavourable: and for 
there is employed a medium containing liver extract, th 
fiver being an orgun in which in man tubercular lesions are 
coniparitively rare. Tho baoilli being thus accustomed to an. 
‘anfavourable surrounding are used for immunising animals, the 
serain of which is now suitable for the treatment of human tuber- 
culosis. It is too soon to epeak of the effects of this line of 


treatment. 
Methots of Examination. —(1) Microwopic Biemination. 
Tubervtilosis i one of the comparntively few disuases in which « 










in w test-tube, or by allowing tho fluids to stand in a 
glass vowel (an ordinary burette i4 very convenient). Film 
are then made with the deposit and treated as 

me. If a negative result ix obtained in a suspected case, 
¢xamination should be underteken, To avoid risk of 

ion with the smogma bacillus the meatus of the 
should be cleansed and the urine first passed should be 
oF the urine may be drawn off with a sterile catheter: 
above it is only exceptionally that difficulty will ariee 


















TUBERCULOSIS 


to the experienced observer from this cause, (For points to be 
attended to, vide p. Bae 
(2) Lnoculation. eas is the most suitable 
animal. If the material to be tested fa'a uid. Tt ty {nected 
sabcutancously or into the peritoneum ; if solid or semisolid it 
is placed in a small pocket in the skin, or it may be 
broken up in sterile water or other fluid and the emulsion 
injected. By this method, material in which no tubercle bacilli 
ean be found microscopically may sometimes be ahown to be 
tabereuls 
(3) Cultivation.Owing to the difficulties this is usually 
quite impracticable ns 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 scrum, under strict aseptic 
mecantions, with portions of a tubercular organ, «g. the spleen. 
The portions of tissue should be fairly large, and should be well 
rubbed into tho broken surface of the medium, 

















CHAPTER X. 
LEPROSY. 


Larxosy is u disease of great inturest, alike in its clinics! and 
pathological aspects ; whilet from the ee point of 
view alg, it its some striking invariable 
association o! large numbers of wteriatie tachi with all 
Jeprous lesions is a wellestablished 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 fur as ix known, is a disease whieh ix confined to the 
aman subject, but it has « tae wide geographical distribution. 
It occurs in certain purts of Europo—Norway, Russia, Groce, 
ete., but is commonest in Asia, occurring in Syria, Persia, ete, 
It is prevalent in Africa, being especially found along the coast, 
‘in the Pacifie Islands, in the warmer parts of North and South 
America, and also to o smallextent in the northern part of North 
America, Th all these various regions the disease presents the 
same general features, and the study of its pathological and 
Bacteriological charncters, wherever such has been carried on, has 
yielded similar results, 

08, —Loprogy is charactoristically a chronic 
disease, in which thore is a great amount of tissue change, with 
comparatively little necessary inpairment of the rissa Maa: 
Ta other words, the local effects of the bacilli are well marked. 
efter: extreme, whilst the toxic phenomena are proportionately at 
« 

‘There are two chief forms of leprosy. ‘The one, usually called 
the tokerenlar form—lepra. tuheross ot tuberculonr—is character- 
Seed by the growth of granulation tissue in a nodular form or aa a 
diffuse infiltration in ite skin, in mucous membranes, ete, great 

often resulting. In the other form, the anesthetic, 
—thuculo~anesthetic of Hansen and Looft—the outstanding 





268 LEPROSY 


changes are in the nerves, with consequent anesthesia, paralysia 
of muscles, and sree disturbances, 

Tn the tubercular ferm the disease usually starts with the 
appearance of erythematous patches seen a small amount 
of fever, and these are followed by the development of small 
nodular thickenings in the skin, especially of face, of the 
bucks of hands und feet, aud of the extensor aspects of arma and 





Fiw, 01.—Seotions through leprous skin, showing the nassox of ealalar 
ranolation tiseue tn the oatie; the dark pointe are clamps of tweilli deeply: 
Maliied, 

Paraffia section ; Zieb)-Neclsen stain, * 80. 


legs, ‘These nodules enlarge and produce great distortion of the 
surfaeo, 6 that, in the caso of the face, an appearance tx jmoduoed 
bas been described a8 “Jeonine.” ‘The thickenings occur 
chiefly ia the cutis (Fig, 91), tow less extent in the subentaneous 
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 Lnternal orguns, especially the 
spleen, liver, and testicles, may become secondarily affected. In 














BACILLUS OF LEPROSY 209 
all situations the change is of the same nature,—a chronic 


tively size, and - show vacuolation of their ponies 
SUR Res Ga arise Sheek am chs katenias 


of 

delicate, bat in aa Tesions moe vory dense, Periarteritis 
is & common and very frequently tho superficial nerves 
become involved in the nodules and undergo atrophy, The 
a in the ly lesions is comparatively vascular, at least 

when young, and, unlike tubercular lesions, never shows cascation, 
Some of tho lopra cells may contain soveral nuclei, but we do 
not meet with cells resembling in their appearmice tubercle 
giant-cella, nor does an arrangement like that is tubercle follicles 


ocour, 

Tn the anesthetic 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  oectar patches on the skin, often of considerable size, the 

of which show a somewhnt livid congestion. ter, 

these patehes become pale in the central parts, and the periphery 
becomes pigmented, There then follow remarkable series of 
fic disturbances in which the skin, muscles, and bones are 
expecially involved. The skin often becomes atrophied, pareh. 
ment-like, and anuesthetic ; frequently pemphigoid bullac or other 
akin eruptions occur. Partly owing to injury to which the feet 
‘and arms are liable from their anwsthetic condition, and partly 
owing to trophic disturbances, necrosis and separation of parts 
‘sro liable to occur. In this way great distortion results Th 
Tesions in the nerves aro of the sume nature as those described 
shore, that ix, they aro the result of a chronic inflammatory 
process, but the granulation tissue is ecantier, and has a greater 
tendency to undergo cicatricial contraction. This ix to be 
costes with the fact that the bacilli are present in fewer 











eatin of Leprosy.—This bacillus was first observed in 
eee by Hanoe in 1871, and was the subject of several 
communications by him in 1874 and later. Purtiwer researches, 
int by Neier in | and afterwards by observers im 
Various parts of the world, agreed in their main results, and 











bese a 








270 LEPROSY 


confirmed the accuracy of Hansen's observations, ‘The bacilli as 
soon in scrapings of ulcerated lepromx nodules, or in sections, 
have tho following charetera, They are thin of 
es same size as tubercle bacilli, which they also resemble 
Spree and in staining reaction. They are straight or 
ly eurved, and usually occur singly, or two may be attached 
to end; but they do not form chains, When stained they 





Fio, 92—Suprerficial part of leprous skin ; the cells of the yrunulation tissue 
appear as dark patches, owing to the deeply-atained bacilli in thelr interior, 
In the upper part a proses of epithelive ts ween. 

Poraffin section ; stained with carbol-fuchsin and Biamarck-brown, » 600, 


may have a uniform appearance, or the protoplasm may be 
fragmented, #0 that they appear like short rows of coce. ‘They 
often appear tapered at one or both extremities ; occusionally 
there is slight clublike swelling, Dexencrated and partially 
broken down forms are also scen, take up the basic 
aniline stains more readily than tubercle bacilli, but in order 
to stain them deeply a poworful stain, much ax 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-fuchain as for tubercle 





POSITION OF THE BACILLI 271 


bacilli, but to uze a weaker solution of briny cap a 
cont, in decolorising ; in the case of films and thin eectic 

decalorisinye with such a solution ike: titteen: secon fi anual 
sufficient, ‘Thereafter the tissues are coloured by a contrast 
stain, euch 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 





Fic. 98.—High-power riew of portion of leprous notule showing the 
seratgenent of thn bacilli within the cells of the granulation thse, 
Parafiln seetion ; stalued with carbol-fuchsin and methylene-blae, x 1100, 


‘of the unstained or stained points may be of this nature We 
have, however, no means of testing their powers of resistance. 
i are non-motile. 
of the Bacilli.—They occur in enormous numbers 
in the leprous lesions, experially in the tubercular form. In fact, 
80 numerous are they that the granulation tise in sections, 
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 grarmulation tissue, 
jend are offen so numerous that the structure ft th cella Ye quike: 


ae — 








272 LEPROSY 
sJascurod often bundles whieh 
ol (Pig. ot rs ace arminged in = 


contain several Parallel to one another, 
‘bundles lie in various iets (Pig. 93), ‘The appearance thas 
resented by the cells filled with bacilli is very characteristic. 
Pail aro lao found free in the lymphatic spaces, but the 
number aro undoubtedly contained within the cells. are 
im found in eplndle shaped sonpoctiva-tisene cells ia eaceetital 
colle, and in tho walle of blood vossels, ‘They are for the most 
part confined to the connective tissus, bat a few may be 
seon in the hair follicles and glands of the skin, Ovensionally 
one or two may be found in the surface epithelium, where they 
probably havo ae tartiod 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, ete, whon leprous lesions are 
present, the bacilli are also found though in relatively smaller 
numbers. In the nerves in the anesthetic form they are com- 
paratively few, and in the sclerosed parts it may be impossible to 
find any. ‘There few aléo in the skin patches referred to 
above as occurring in this form of the disense. 

Thoir spread ik chiofly 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 pone 
of fresh nodules, am ty have also been observed in the blood 
vessels post mortem, chiefly contained within leucocytes Recent 
observations (ag. those of Doutrelepont and Wolters) show thut 
the bacilli may be more widely spread throughout the body than 
was formerly supposed. A few may be detected in some caves 
in various organs whieh show no structural change, expecially in 
their capillaries, ‘The brain and spinal cord are almost exempt, 
but in Home cases tacilli have been found even within nerve 
colle. 

Relations to the Disease,—Attempts to cultivate the leprosy 
tucilli outside the body bave so far been unsuecessfal. From 
time to time announcements of successful cultivations have been 
made, but one after another haa proved to be erroneous, A 
similar statement may be made with regard to experiments on 
animals. Ifa piece of leproua tiene be introduced subentanconsly 
in an animal, such ax the rabbit, & certain amount of induration 
may take place around it, and the bacilli may be found unehay, 
in appearance weeks or even months afterwards, but no multi- 
plication of the organixms occurs, The only exception to this 
statement is afforded by theexperiments of Melcher and Orthmann, 
who inoculated the anterior chamber of the eye of mlbbits with 























RELATIONS TO THE DISEASE , 758 
material, the iuoculation being followed by an extensive 
‘of nodules in the Innga and internal organs, which 

tained leprmy bacilli, It has bean questi 
authorities whether the organistns in the 
really luproay bacilli, and ap to the present we 
thore is any satisfuatory proof that the dimstse ean 
any of the lower animals. Diphtheroid bacilli 
variety have been cultivated from the blood 
leprous Patients by Bahes and others. Their 
would appear to be by no means infrequent, but it is 
not possible to say at present what their significance is. 

Te is interesting to note that a diseases occurs under natural 
conditions in rate which prosents many points of close similarity 
to leprosy. It has been observed in Rassia, Germany, and 
England, and an excellent description has recently been given by 
Dean. In thie affection there are lesions in the skin which 
resemble these in leprosy, and the celle contain enormous 
numbers of an fast bacillus. The disease can be trans 


i 


rH 
i 


l 





this disease hos any relation to leprosy in the human subject is 
wery doubtful, but the facts which have been ascertained may 
prove of high’ importance in connection with the pathology of 
tho latter disease, 

Tr woald also appear thut the disease is not rendily inocalable 
in the human subject. In a well-known case described by Arning, 
‘@ criminal in the Sandwich Islands was inoculated in several parts 
of the body with loprosy tissuc. ‘T'wo or three years later, woll 
taatked tubercular leprosy nppeared and led to a fatal rewult. 
This oxporiment, however, is open to the objection that the 
fadividual before inoculation had been, oxposad to infection in a 
natural way, having been frequently in contact with lepers. In 
‘aber cases, inoculation experiments on healthy subjects and 
inoenlations in other parts of leprous individuals have given 

resulta, It has boen supposed by some that the failure 

fo obtain cultures end to reproduco the disease experimentally 

may be partly due to the bacilli in the tissmes being dend. That 

the loprous bacilli are in a dead condition is quite 

in view of the long period during which dead tubercle 

futroduced into the tissues of animals retain their form 

Haining maction, ‘There is also the fact that from thie t tinoe 
is 


—E_ =z. £ 














ort 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 
‘experimenta go to distinguish the Icproay bacillus all the more 
strongly from other organisms Some have supposed that 
loproay is a form of tubercle, or tubercle modified im 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 aro distinct. Tt should alao be mentioned that tubercle 
is not uncommon complication in loprows subjects, in which 
case it presents the ordinary characters, 

‘The mode by which leproxy 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 ia transmitted by direct cantact, 
‘There appears to be no doubt, however, that on the ono 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 e high order, 

Methods of Diagnosis.—Film preparations should be made 
with the discharge from any uleemtcd nodule which may be 
present, or from the scraping of « portion of excised tissue, and 
shonld he stained ax above described. Tho presence of largo 
numbers of hacilli situated within the cells and giving the staining 
reaction of leprosy bacilli, in conclusive, Ibis more satisfactory, 
however, to make microscopic sections through a portion of the 
excised tissue, when tho structure of the nodule and the arrange- 
ment of the bacilli can be readily studied. Tho points of 
difference between leprosy and tubercle have already been stated, 
and in most cases thero ls really no difficulty in distinguishing 
the two conditions. 











CHAPTER XI. 
GLANDERS AND RHINOSCLEROMA, 


GLANDERS. 
‘Tar bacillus of lara (bacillus mallei; Fr., Dacille de tx 
morve; Gor., illus) was discovered by Léffler and Schuta, 
the neement of this discovery being made towards the end 


‘ease of acute landers in a woman, and by inoculation of animals 
obtained results similar to those of Loftler and Schutz. 

Within recent years a substance, madlein, haa 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 prodnce corresponding ffects in animals suffering from 
landers to those produced by tuberculin in tuberculous animals. 

~The Natural Disease.—Glanders chiefly affects the equine 
speciee—horens, mules, and ascon. Horned eattlo, on the other 
hand, are quite immune, whilst goats and sheep occupy an inter- 
medinte position, the former being rather more susceptible and 
oceasionally suffering from the natural disease. It also occurs 
fn some of the carnivora—cata, lions, and tigers in menngerics, 
Which animals are infected from the carcases of animals affectod 
with the disease. Many of the small rodents are highly 


seas inoculation (vide infre). 
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 amonget grooms and others whose work briwes Yxewn 
- 275 Ei 


— 





eae none ene oe 


In horses the lesions are of two types, to which the names ‘ if 
ee aud * farey have been given, though both, may exist 
In gleera abner ts the septum nasi and adjacent parts 
‘zootie men bpane nection which are at Bax 





nan ie en se a 
attended usually 
eerie einare te Solar hin ai inion 
give rime to irregular ul nations, ions, tt tic Ly 

may be found in the respiratory jon, 
there jv umually yinpeaton of “ite lymph ign pandas Se ea 
atioum, ote. 5 are may be in the Longa, spléan, ees ata motiules 
Of to ste aaa oF Iyer; of greyish of Suilow tat, faker Sema 
ffm in Eecente, al en won, by w een zone, 


term “fary" is applied to the affection of tl phate 
vomels iS fy soe whore tifoction! tin a 


wails, which is plage 
as ‘sie often produced by the rabbing 
harnews. The lymphatio vosscle booome itreyulasly thickened, ee 
pear like Knotted cores, and the associated Lymphatio glans 
bosoms enlarged and firm, thon ippurative softening asnally follows, 
fund there ay be ulesration. “Tho thlckextags are efven speked/Bf ba 
© firoy buds” and *farey pipes.” Tn fatoy, also, secondary nodules may 
coour in futornal orgens ond. the nasal mucous membrane ‘The 
fs often prosont in a latent fort,” and its presonce can only be dstected 
the mailein text (cide infra). In the swe tho dita rune x MOFe 
ssonte sourse than in the hone 
























Tn_man the disease ia met with in two forms, an route 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 bait follicle, sometimes on 
the face, and occasionally on the mucous membrane of the mouth, 
nose, or eye. In the cute form there appenrs at the alte of 
i i inflammatory awelling, attended usually with apread- 
icas, and the lymphatics in relation to the part aleo became 
cal, the appearances being those of @ “poisoned wound,” 
‘These local changes are soon followed by marked constitutional 
disturbance, and by an eruption on the surface of the body, at 
firet papular and afterwards pustular, and later there may form in 
the subcotaneous tistue and muscles larger masaes whieh shes 
and suppurate, the pus being often mixed with blood ; ae 
may occur also in the joints. In some cases the m 
mernbrane may be secondarily infected, and thenee indiana oey 
swelling may spread to the tisuex of the face; in others it 
remains free, The patient asually dics in two or three weeks, 
sometimes sooner, with the symptoms of rapid pywmin In 















THE GLANDERS BACILLUS 277 


pane to ‘the lesions mentioned there may be foci, amally 
a os the Innge (attended often with pnoumonie con- 
spleen, liver, bone-marrow, salivary glands, ole 
in sere ey the Jocal Jesion results in the formation of a 
ulcer with thickened margins and sanious, often foul, 
‘The ulceration spreads deeply as well as euperticially, 
0) the thickened lymphatics also have a great tendency to 
nicerate, Hough the lymphatic system is not so prominently 
affected in the horse. Dvposite may form in the subcutaneous 
tissue and muscles, and the mucous membrane may beoome 
affectey!. The dlisens may: 


rum a very chronic course, 
lasting foe ‘months, and 


reovvery may occur, 
thongh, onthe other hand, 
the distazs may take on 
& more uente character 
andl rapidly becoine fatal. 

The Glanders Bactl 





lus. — Microscopical 
Characters. —The  glan- 
ders bacilli are minute 
rods, straight or slightly 
carved, rounded 
ends, and about the same 
length as tubercle bacilli, 
ut distinctly thicker Fic. Glandere bacilli amongst broken 
(ig 84). They show, aoen cata "Mim pesperaice from 6 
however, considerable __glaniers nodule in » guinea pig. 
Sariations in size aud in Stained wiv week cartol fuchaiu. <1000, 
appearance, wnd their pror 

toplasm is often broken up into a number of deeply-stained 
portions with unstained intervals between, ‘These characters are 
s6en both in the tiemes and in cultures, but, aa in the case of 











WARY Organisms, irregularities in form aud size aro more pro 
nounced in cultures (Fig hort filamentous forma 8 to 12 # in 
Teagth are sometimes met , but these are on the whole mun 


‘The organism is non-motile, 
Thi thus tixsxes the bacilli usually occur irregularly conttered 
Gusooget the cellular clements ; a few may be contained within 
Gnd connective-tinue ph mere but the position of 

moet isextracellular. ‘They are most abundant in tho acute lesions, 
fi which thoy may be found in considerable numbers ; but in the 
chromic nodules, especially when softening hax taken. yas, Shey 











al 








278 GLANDERS: 


are few in number, and it may be impossible to find any in 
sections. They have less powers of persistence, and 
the tissues much moro quickly than tubercle bicilli. 
‘Thore hax been dispute as to whether or not 
spores. Some consider certain of the unstained portions to be 
of thut mature, and it has 


pl 
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 
Fi, 96.—Glanders baci ree, is atronply aypaizat 
‘oulture ou glycerin agar Stained cir being of that nature, 
carbob fuchsin and partinily decolorised to The power of resistance is 
showsegmentation of protoplam. x 1000. after all the important 
practical point, 
Staining.—The glanders bacillus differs widely from the 
tmborele bacillus in its staining reactions It stains with si 
watery solutions of the basic stains, but somewhat faintly (better 
when an alkali or a mordant, such as carbolic acid, is added), 
and even whon deeply stained it readily loses the colour when « 
decoloriaing 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, Tn film preparations of 
fresh glanders nodules the bacilli ean be readily found by stai 
with any of tho ordinary combinations, ¢g. earbol-thionin-bue 
or weak carbohfachsin, By using a stain of suitable sti 
no decolorising agent ix necessary, tho film being simply 
in water, dried and mounted,” M'Fadyean recommends that 
after sections have been atained in Létiler'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 a6 a contrast 
stain a 1 por cent solution of weid fuchsin may be applied for 








from a pare 









CULTIVATION OF GLANDERS BACILLUS 279 
aires pater oes and mounted. 


eee ieaces tors to 21° ‘One a ealink ae 


25° ©. is always desirable. 
On ager ant giyerén azar in stroke cultures growth appears 
long the line 08 uniform streak of greyish-white colour and 


somewbat transparent appearance, with moist-looking surface, 
und when touched with a needle is found to be of mther slimy 
consistence. Later it spreads laterally for some distance, and 
the layer becomes of slightly brownish tint. On serum the 
growth ix somewhat similar but more transparent, the separate 
colonies being in the form of round and almost clear drops, In 
sub-caltures on these media at the body temperature growth is 
Visible within twenty-four hours, bat when fresh cultures are 
rade from the tissues it may not be visible till the second day. 
Serum or oy however, is much more suitable for cultivating 
from the than the agar media; on the latter it is some- 
times difficult to obtain growth, 

In broth, growth forma at first a uniform turbidity, but eoon 
snttles to the bottom, and after a few days forms a pretty thick 
floccufent deposit of slimy and somewhat tenacious consistence. 

On potato at 30° to the glanders bacillus fourishes well 
and products 1 characteristic appearance ; incubation at a high 
temperature, however, being necessary. Growth proceeds rapidly, 
ancl on the third day has usually formed a transparent layer of 
slightly tint, like clear honey in appearance. On sub: 

the growth still extends and becomes darker in colour 

more opaue, till akxwt the eighth day it has a reddish-brown 

¢ 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: 











b, pyoeyancus produce a somewhat similar appearance, but they 
ean bo readily distinguished by their other characters). Potato 
is also 4 suitable medium for starting cultures from the tiasues ; 
in this case minute transparent colonics become visible on the 
third day and afterwards present the appearances just described. 

Powors of co. —The glandors kacillus is not killed at 








a GLANDERS 


onos by drying, but tsuully 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 bs still 
active after remaining two or three weeks in putrefying fluids 
Tn cultures the bacilli retain their vitality for three or four 
months, if, after growth has taken place they are kept at the 
tempenture of ee rotay 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. Léffler found that they were 
killed in ten minutes in w fluid kept at 55° C., and in from two: 
to three minutes by a 5 per cent solution of carbolie neid, 
Boiling water and the ordinarily used antiveptics ure very 
rapid and efficient disinfectants. 

We may summarize the characters of the glanders bacillus by 
saying that in its morphological chareters it resembles. some 
what the tubercle bacillus, but is thicker, und diffors widely 
from it in its staining reactions. For its cultivation the higher 
temperatures ans necessiry, and the growth on potato presents 
tnogt chamctoristic features, 

imental Inoculation, —In horses subcutaneous injection 
of the glinders Iweillus in pure culture reprodnees all the 
important features of the disease. This fact was eatnbliahed at 
a comparatively carly date by Loifler and Schutz, who, after one 
doubtful experiment, successfully inoculated two horses: in thix 
way, the cultures used having been grown for several generations 
outside the bedy, In a few days swollings formed at the sites 
of inoculation, and later broke down into unbealthy-looking 
ulcers, One of the animals died ; after a fow weeks the other, 
showing symptoms of cachexia, was killed. Ln both animals, in 
addition to ulccrations on the surface with involvement of the 
lymphatics, there were found, post mortem, nodules in Uhe laugs, 
softened deposits in the muscles, and also affection of the nasal 
mucous mombrane,—nodules, and irregular ulcerations, ‘The 
ass is even more susceptible than the horse, the disease in. the 
former running a more course, bat with similar Tesions, 
The as cin bo readily infected by simple 
inoculation with glanders seeroti » (N 

Of sinall animals, field-mice avd guine 
euscoptib Strangely enough, honso-mice and white mice 
enjoy an almost complote immunity. In fiold-mice sabeutaneous 
inoculation is followed by a very rapid di usually lending 
to death within eight days, the organisms becoming generalised 
and produving numerous minute nodule, expecially in the spleen, 














































ACTION ON THE TISSUES 281 


Panes liver, he ie guinea-ply the disease is Jess acute, 
omah eoeondary jules in internal organt are uenall, 

fn considerable numbers, At tho site of inoculation rd inflam- 
matory swelling forms, which soon softens and breaks down, 


adi wo the of an irregular erateriform uloor with 
indurated margins, ‘The lyzaphutic vessels become infiltrated, 
thes inphutic glands beoone enlarged to the 


a longer period. Secondary nodules, in varying numbers in 
different cases, may be present in the spleen, lings, bones, tmsal 
MmMoons Membranc, testicles, ovaries, ete. ; in some canes w fow 
nidiakes are found in the spleen alone. Intraperitoneal injection 
in tlie uale yuinen-pig is followed, as rire oa it by Straus, by 
avery pu ‘and semi-purnlent affection of the tunica vaginalis, 
shorn during life by great «welling and redness of the testicles, 
whieh changes may be noticeable in two or three days, Hy this 
method there occur ale numerous small nodules on the surface 
of the peritoneum. Rabbits are les bes Hoe than guinea. 

ition is somewhat 
4 hject with 
pe cultures of the bucillus has in more than one instance been 

followed by the acute form of the disease and a fatal result, 











a comparative study of the results of Teaakbes with acid-fast and 
tae Rall, found nove of the abore characters in tho awe of the 
giamilers baci!tas (¢f. Tuberelo}. 


Action on tho Tissues.rom the above facts it will be seen 
that in many respects glanders presents an analogy to tubercle as 
the general characters of the lesions and the mode of 
spread. When the timue changes in the two diseases are 
compared, certain differences are found, The glanders bacillus 
faust a more mpid und more murked inflammatory reaction 
‘There is more loucorytic infiltention and less proliferstive change 
Which might lead to the formation of cpithclioid cells Thus 
Leal ‘of an carly ghnders nodule sh aggregation 
moat of which are polyme sar, whilst in 
te ‘eontral parts many show fmgmentation of nuclei with the 
formation of a deeply staining granular detritus, And further, 
the inflammatory change may be followed by suppurative 
sottening of the tissue, expecially in certain situations, muck om 


= 








282 GLANDERS: 


ie, Lemire Irese ane glands, The nodules, 
erefore, in as oo puts it, an 
intermediate position between mi} abscesses oul dabelee 
‘Tho diffuse coagulasive necrosis and caseation which aro 80 
common in tubercle do not occur to the same degree in glandera, 
and typical giant cells are not formed. ‘The nodales in the li 
show loucocytic infiltration and thick: of the alveolar 
whilst the vesicles are filled with cat cells; there may also 
‘be fibrinous exudation, whilst at the periphery of the nodules con- 
nective-tiasue growth is nt in proportion vo their The 
tendency to spread by the lymphatics is always a well-marked 
feature, and when the bycilli gain entrance to the blood-stream, 
they soon settle in the various tissues and organs. Acco! . 
oven in aouto cases it ia usually quite impossible to detect 
bacilli in the circulating blood, though sometimes they have been 
found. It is au interesting fact, shown by observations of the 
disease both in the human subject and in the hors, a8 well as 
by experiments on einen yi that the mucous membrane of the 
now may become infected by menus of the blood-stream: 
example of the tendency of organisms to settle in special sites 
Mode of Spread.—Glanders usually spreads from a diseasod 
animal by direct contagion with the discharge from the nose or 
from the sores, etc. So far ws infection of the human subject 
goes, no other mode is known. There is no evidence that the 
disease in 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 dispase 
could be readily produced in susceptible animals by exposin, 
them to an atmosphore in which cultures of the bacillus 
been pulverised, He also found that inunction of the skin 
with vaseline containing the bacilli might produce the disease, 
tho bacilli in this case entering along the hair follicles. 





Agglutination of Glanders Bacilli. Shortly after the discovery of 
agglitination ia typhoid fover, MFudyoan showed that the verum of 
glandered horsee possowsed tho power of agglatinating glaudore bacilli, 
is later observations show that in the grost wwajority of casos of glanders 
# Lin 60 dilution of the serum produons marked agglutination ina few 

wutes, whilst in the great majority of non-glandered animals no effect 
duced under these conditions. ‘The test performed in the ordinary 
way is, however, aot absolutely reliable, as exceptions occasionall 
coour iu both directions, ie. nogative reaults by glandered aumiala an 
Positiverasalts by non-glandered axizaals- He dots that b more délioate 

























METHODS OF EXAMINATION 283 


Pres bapmel bed i fa pres the bation tu bo Nlan poiatng a well 
of the serum to be tested. In this way he hax obtained « 
ting reaction with » serum which did not agglutinate at 
method. Further ns are still 


obtained trom oultures of 
h of ir oe pis ‘be 
tances in 





ee een metic bya ‘tractiog with gl alpen water fat 








from oul tures in glyearia be Such aculture, 

So grow for thres or four weal, by 
caeee atl ‘at ae . or by steam 1 On HuiCObeR Fe 
days Te ix thon filtered thn a Chamber! er The hee 


Serwttates full mallets, Ussatly 2 Untle carboll sad (6, per ent) ts 
Pyne adr a a ates 
& hone 0 2 
mallein by throwing the filtrate of a broth culture, Srp to one 
‘tumth of its bulk, into twon aves Hale ins Ie voli 28 saath 

‘A white precipitate i 
then underanair-pump, A dose of this dr is 0 
_ The use of Multein ax 0 means of Dic Ta using mallein for the 
disgnosis of glanders, the temperoture of tho animal ought to be observed 
ferns hours befordhand, end, fer eaboutansousinjetion of « suitable 
it ix taken at dofinite intervals,—according to M'Fadyean at the 
nachionth, ual eighteenth: hours, trerwards, nal 'ou: tb 
next day. ‘ers both the looal reaction and the temperature are of 
Ins glandored animal, at tho site of inoculation there is 
painful local swolling, whioh reaches a dianweter of five inches 
at — fe mazima an not being attained until twenty four hours 
temperature rises 10" tod" O., cr more, the masiauss 
os in fom eight toixtoon hours. If the temperature 
‘as minh as 15°, the reaction is considered doubtful, kn the 
auimal, free from glanders, ths rise of 
re ear: usually exceed 1°, the local swelling’ reaches the 
a moat, and has much diminished at the end 
of four hours, In the care of dry mallein, local reaction is lees 
warked. Veterinary authorities are practically unanimous as to the 
‘great valuo of the mallein teat as a means of diagnonls. 


Methods of Examination.—Microscopic examination in a 
ease of suspected glanders will at most roveal the presonce of 
ing in their characters to the glanders bacillus. 
An absolute diagnosis cannot be made by this method. Cultures 
may be Sa by making successive strokes on bloc serum or 
‘on glycerin agar (proferably tho former), and incubating at 37° C, 
The colonies of | es 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 
seuretion or pus with storilo water, to varying degrees, and then 
fo smear the surface of potato with the mixture, the potatoes: 


he 





a 





























284 GLANDERS: 


being incabated at the above temperatures The colonies on 
potatoes nay not appear till the third day. The meet certain 
method, however, is by inoculation of x pine, ether 
subcutancows or intraperitoneal injection. By the latter m 
asubove described, lesions are much more rapidly prodaced, and 
are more characteristic. If, however, there lave been other 
organisms present, the animal may die of a septic peritonitis, 
though even in mush a caoe the glanders bncill wil be founalte 
be more numerous in the tunica vaginalis, and may be cultivated 
from this situation. It ix extremely doubtful whether the 
application of mullein to diagnosis of the disease in the human 
subject is justifiable, There is a certain risk, that it may lead 
to the lesions wsuming a mors actte chunteter; moreover, 
culture and inoculation tests are erally available, In the 
ease of horses, ote, a diagnosis will, however, be much more 
eswily and rapidly effected by means af mullein, 


Rurtwosctrnoata. 


This discuss is considered here ax, from the anatomic 
changes, it also belongs to the group of infective granulomata, 
It iy 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, vormetimes: 
‘ay large as a pea; in the earlicr stages they arc comparatively 
smooth on the surface, but later they become shranken and the 
contr is often retructod. ‘The discase ix searcoly ever met with 
in ths country, but is of not very uncommon occurrence on the 
C especially in Austria and Poland. In the granulation 

he nodules there are to be found numerous round anil 
rathor large cells, which have peculiar characters and are often 
known as the cells of Mikulica, Their protoplasm contains 
collection of somewhat gelatinous material which may fill the 
coll and push the nucleus to the side, Within these cella there 
ia prosent a characteristic bacillus, occurring in little elumps or 
masses chiefly in the gelatinous material. A few hucilli also He 
free in the lymphatic spaces around, ‘This organiam was first 
observed by Frisch, and is now known aa the bacillus of 
thinoseleroma. The bacilli have the form of short oval rods, 
which, when lying sepantely, 
capsule, and whieh in all their microscopical characters correspond 
clowly with Friedlindor’s pnoumobucillus. ‘They are usually 
present in the lesions in a state of purity. It was at first stated 















































RHINOSCLEROMA. 285 


| Sees: by Gram’s method, but more recent 
Friedliinder’s organism they lose the 


F 


to the affected tissues, as already 

facts alone it would appear probable that thoy are Shiny 
in the production of the lesions, Experimental 
maicen Hitle Tight i the saint thongh ‘one 
reeoy the nodules on the 
Tete bi the rhinoseloroma, 
it wot Fe Friedlinder is, however, still a matter of 
matter has been further complicated by the fact 
possessing closely similar characters has been 

very frequently present in oaena, and i: 
aria own, ‘Tho last-montioned organism ix 
jor active formentative powers, From what has 
Ba fs peanut w atcalbes OF wrpnioan closely 
moepbologial characters, have been found in. the 
healthy or diseased conditions. There is no 
Pdikonclarerba is o specific disease with wellanarked 
ra anid it is quite possible that one member of this group 
may be the causal agont, though indistinguishable 
by culture teats, Thoro ia, however, @ tendency on 
ft rocent investigators to consider the “bacillus of 
* to be identical with the pneamotucilins and its 
the atfoctod tissues to represent morely a secondary 
The subject is one on which more light is etill 


i 


THIPAGE 
oe 


TEE 
7 


a 








CHAPTER XU. 
ACTINOMYCOSIS AND ALLIED DISEASES, 


Actisomycoats 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 uffects man in common with 
certain of the domestic animals, though it is more frequent in 
thé latter, expecially in oxen, swine, and horses, ‘The parasite 
was first discovered in the ox by Bollinger, and deseribed by 
him in 1877, the name aetinomyces ot 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 homan subject, the result of investigation being to show 
that it affocta man rauch more frequently than was formerly 
supposed, 

Tt is, however, to he noted that the term “actinomyces,” as 
originally used, docs not represent one parasite but a number of 
closely allied st species, as cultures obtained from various sources 
have presented considerable differences ; and, further, it is noted 
that other distinet species of streptothrix have boon cultivated 
from isolated casos of disease in the human subject whore tho 
lesions resembled more or less closely those of actinomycosis, 
In one or two instances the organisin has been found to be 
“acid-fast,” and there ia no doubt that the actinomyces group is 
closely related through intermediate forms with tho tubercle 
wroup (wide p, 239), 

Naked-eye Characters of the Parasite—The actinomyees 
grows in tho 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 amall pin’s head, whilst all 
sizes below thi4y may be found, When suppuration i# prosont, 

286 











j i 
subject they occur much eee serena small specks of 
lucent ap) and of greenish-grey tint. 

Oharacters.— The parasite, which is now 
regarded as belonging to the stroptothrix group of the 
ighee in (p. 14), presents pleomorphous characters. In 
colonies, as grow in the tissues, three morphological 

elements may be described, nomely, filaments, coccus-li i 


eluks, 

fs Waments are comparatively thin, measnring abont 

in diameter, but thoy are often of great longth. Thoy are 

a central protoplasm enclosed by a sheath. The 

latter, which is most easily cea in the older Benois rie 
granular protoplasm, occasionally contains granules jar 
e e contre of the nee the filaments interlace 

and form an irregular network which may be 
or dense; at the periphery they are often arranged in a 
‘somowhat radiat ting manner, and run outwards in a wavy or even 
al course, ey also show truc branching, a character 

at onee distinguishes them from the ordinary bacteria, 
the filaments thero i a finely granular or homogeneous 
wubstance, Most of the colonies at an carly stage are 


“made out. ‘This dense part, starting excentrically, may grow 
colony to form » hollow sphere, from the outer 

‘of which filaments radiate for a short distance (Fig. 96). 

usually stain uniformly in the younger colonies, 

capecially in the older colonies, may be segmented so 





288 ACTINOMYCOSIS AND ALLIED DIBEASES 


ax to give the appearance of a chain of tacilli or of cocel, though 
the ebeath ‘enclosing. them may generally be  datingalabed 
Rod-shapod and spherical forms may also be seen lying fre. 

2. Spores or Gonidia.—Like other specie of streptottrix the 
actinomyees when growing on a cultare medium shews on its 
surface filaments wrowing upwards in the air, the protoplasm of 
which becomes segmented into rounded spores or gonidia, In 
natural conditions outside the body these gonidia became free 





16. —Actinomycenis uf human fiver, showing a colony of the parasite 
d of a felted mass of flaments snrrounded by pur 
section ; stained by Grams method and with eafrenin, * $00, 









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 #pores; cultures containing spores can 
resist a temperature from five to ten degrees higher than spore 
free caltures (Foulerton). It is probable that some 
spherical bodies formed within filaments when grow 

tissues have the same significance, .« are gonidia, whilst others 
may be merely the result of degenerative change, Both the 

















CHARACTERS OF THE ACTINOMYCES 289 
Glaments and the eal bodies are readily stained by 
spherical ily v 


$, Clubs.—Theee are elongated poarshaped bodies which are 
seen ut the periphery of the colony, and are formed by a sort of 
hyaline swolling of the shoath around the froc extromity of a 
filament (Figs, 97, 98). They aro usually homogenoous and 
structureless in appearance. In the human subject the clubs are 





Fru, 97. —Actinomyces in human kidney, showing claby radially arrange! 
une surrounded by pus. The flaaouts had. practically disappeared. 
Paraffin section ; stained with hematoxylin and rubin, » 600. 


often tively fragile structures, which ure easily broken 
down, a may scitiasat as Claw ted instars. Brsakttaony 
they are well scen whon examined in tho freah condition, but in 

specimens are no longer distinguishable. Tn specimens 
stained by Gram's method thoy are usually not coloured by the 
Wiolst, but take rewlily a contrast stain, much aa picrie acid, 
tubin, ete; sometimes a darkly-stained filament can bo seen 
inning for u distance in the centre, and may have a knob-like 
@xtromity. In many of the colonies in the human subject tho 
Clubsare absent. In the ox, on tho other hand, where thers sem 

we 





Mise 


290 ACTINOMYOOSIS AND ALLIED DISEASES 


much older colonies, the clubs constitute the most prominent 
feature, whilst in most colonies the filaments are more or loss 
degenerated, and it may sometimes be impossible to find any. 
They often form a dense fringe around the colony, aud when 
stained by Grams mothod rotain the violet stain. ‘They have, 
in fact, undergone somo {urthor chemical change which produces 
the altered staining reaction. Occasionally in very chronic 





Fia, 98, Colonies of actinomyces, showing general structural arrangement 
and clubs at periphery. From pus in human subject, 
Stained Gram and safranin, —% 60, 


lesions in the human subject the clubs stain with Gram’s 
method. Clubs showing intermediate staining reaction have 
been desoribed in the ox by M‘Fadyean. The club-formation 
probably represents a means of defence on the part of the 
parasite againat the phagocytes of the tissue: the view, formerly 
eld, that the clubs are organs of fructificntion has now been 
generally abandoned. 
Tisane Lesions.—In the human subject the parnsite pro- 
duces by its growth a chronic inflammatory change, usually 
ending in a suppuration which slowly spreads. Tn some cases 





nana aes 


pee 


ip 


a von st the parasite 
heneyoomb sppearance, 
=n foci of pe eee are 


ef 
fe 


Be 
4 


whilst the colonies 


of somewhat slimy character. 
tine ion i 
abundant growth of 
result in tumour-like masses, usually 
noulated chameter, and often pean of well-developed 
containing areas of younger formation in which 
abscess formation is usually present, The cella immedi- 
pia the colonies are usually irregularly rounded, or may 
even Le somewhat columnar in shape, whilst farther out they 





| 


is sometimes seen in hnman aoclanpel 
and Distribution of Lesions.—Vho losions in the 
ur in almost any part of the body, the 
In many cases the 
— probably 
food by the crypts of the tonsil, or by some 
welling und suppuration may then follow in the 
spread in various diroctions. Tho periosteum 
or the vertcbre may thus become affected, caries or 


i 


ire 


le number of cases the primary lesion is in some part 
Racin fuioetine, generally of tho large The parnsite 
' wall of the bowel, and may be found doopl: 

Dee Gals accpuadhd by poruleak reterial oi Tbaceaies 


“apread to the peritonenm or to the extraperitoneal tisaue, 
i A 








292 ACTTINOMYCOSIS AND ALLIED DISRASES 


the retrocweal connective tissue and that around the rectum 
being not uncommonly seats of suppuration Pea in this 
way. A poculiar affection of the intestine has been dese: 

in which slightly raised plaquos are found both in the largo and 
‘small intestines, these plaques being composed almost exclusively 
of masses of the aetinomyens along with vepithelial cells. -Thikg 
however, ia a rare conditi ‘The path of entrance may aleo be 
by the respiratory passagos, the primary lesion boing 

or peribronchial ; extensive suppuration in See may result. 
Infection may also oceur by the skin surface, and lastly, by the 
foralo guuital tract, as in & caso recorded by Grainger Stewart 
oe a » in which both ovaries and both Fallopian tubes were 
a 

When the parasite has invaded the tissues by ang of these 
channels, eecondary or “ metastatic” abscovses may occur in 
internal organs. The liver is the organ most frequently affected, 
though abscesses may oecur in - lungs, brain, kidneys, ete. 
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 ie not uncommon to find leucocytes in the neighbourhood 
of a colony containing small portions of tho filaments in their 
interior, 

Tn the ox, on the other hand, the disease usually remaing 
quite local, or spreads by continuity. It may produce tumour- 
like masses in the region of the jaw or neck, or it may pecially 
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 
evidenos 0 show that outside the body the parasite grows on 
gmin, especially on barley. Both in the ox and in tho pig the 
parasite is boon found growing around fragmenta 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 algo in favour of such o view. 

Cultivation (for methods of isolation see later)—The de- 
scriptions of the cultures obtained by various investigators differ 
in essontial particulars, and there is no doubt that the organisms 
described are different. The following is the account of the 
organism as cultivated by Bostrim :— 

On eyar 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, 

which enlarge and form rounded projections of 
a tint and somewhat transparent appearance, like 
drops of amber. The growths tend to remain separate, and even 
when vad ‘hecome confluent, the nodular character is maintained. 


elng wil at 


appearance: 
having been dusted with 
1a brownish-yellow powder 
(Fig. 99). 

To the cultures at an 
carly the growth is 
comn| of branching 
filaments, which stain 
oniformly (Fig. 100), but 
later some of the sy 
ficial filaments may show 
segmentation intogonidia. 
Sight Bulbous thickon- 

‘may bo econ at tho 
an em some of the fila 
ments, but true clubs a, 
have not been obeerved. B 

On gelatin the samo Pio. 99—Cultures of the actinomyces on 
tendency to grow in Little slyeerin ssnof 

eal oases is geen, — “Wing thea > 
fand thé medium becomes Serna thn amrtade Nesoal ans 
very slowly liquefied, 
When this occurs the liquefied portion has a brownish colour 
aol somewhat syrupy conaletence, and the growths may be seen 
at the bottom, as little balls, from the surfuce of which filamonts 
radiate. 

‘The organism obtained in culturo by Wolff and Tsrnel (wide 
Safin) is probably the samo as the one which has been recently 
Geseribed in detail by J. H. Wright, who obtained it in pure con- 
dition from fiftecndifferent cases of the disease, It differs markedly 
from Bostriim’s organism in being almost a strict anacrabe and in 
















294 ACTINOMYCOSIS AND ALLIED DISEASES 


eat eae eee ee eee 
Under ordinary aerobic conditions either no growth occurs or 
aitdiartr tal pieabaciar On the surfaces of agar under 
anaerobic conditions the ba ae produces dense rounded 
ii ww, which sometimes at a 


cygen obtainable 

101), Inbouillon,, 

takes place at the bottom 
of the medium in rounded 
masses which afterwards 
undergo disintegration. 
Wright found when the 
corganiam was grown in 
the presence of serum or 
other animal Maida that. 
the formation of trae 
clubs occurred at the ex- 





= a 
Fic. 100.—Aetinomyees, from a eniture on 
ireaia war, atowiny tho tranching of i ponies 

Stained with fuchsin, 1000, it os a true 

and doubts ae rs 

can have a saprophytic existence outside the body, eg. on grain. 
He ik also of opinion that all cases of true netinomyecosia, Le. cases 
whore colonies visible to the naked eye are present, are probably 
produced by one apices and thatthe werobie organi obtained 
hy Bostrim and others are probably accidental contaminations 
It ix quite evident that farther investigations are required in 
the light of the results detailed. Certainly the parasite in 
many cases of actinomycosis in the Immun subject does not 
grow on ordinary media under aerobic conditions as Bostrom's 
organism docs. 


Varloties of Actinomyces and Allied Forms.—It is probable thet in 
the eases of the disense described in the hua subject: thero is more 
ian one variety or spocice of parasite bolonging to the same group. 

Gsxperini has described sovorsl varieties of actinomyces bore according 


=i 





VARIETIES OF ACTINOMYCES 295 









HUeEstt 
i 
fee 


it 
i 
i 





fn man a strep- Fi. 102.!—Shake cultures of uctinomyces in 

which differs in so glucone agar, rie the maximum 
‘many im portant points from growth at sowe distance from the mur- 
‘the actinomyors of Bostrom face of the medinta, 


that it is now regarded 
asa distinct species. An- 
other species was enltl- 
std by Hopinger from» 
brain afeoess, and called 
hy him “eladothrix aster- 
oldes,” from the appear- 
noe of ita. oolomies «5 
culture media. A case 
of general streptotheix 
infection in the human 
subject described ky 
MasDonald was probably 
dub tot conve panier 
in the 
eit. rome ta 
Fic. 102 —Section of a colony of actinomyow pannewhat ditfuee manner 
from & eultsre in bloc! serum, showing the gurl doos not farm ela 
formation of cluts at the periphery. x 1500. 











1 For Figs, 101 «nd 102 
We are Indlebyied to Dr. J. Homer Wright of Boston, U.S.A. 


— i 


296 ACTINOMYCOSIS AND ALLIED DISEASES 


ican tenet rire 9 diy ohio relma Flexner 
ings asaociated with lesions somewhat like 
ni ph Ch SEEN et rakereuloais hominis 
apparently similar condition haw pod doseribed. by 
Buchhols “ponte SuUsvatad tne Spaces. of) Aewyiaet cat's 
suppurative lesions, ons of wi rt only is 
anaerobic couditions. Birt und Lelahman have ocala 
fast atroptothris obtained from elrehotic nodules in the 
This organiam grows reulily on ordinary media, forniee whl 
weit which fterwards, aowctice « pinkish colour it fs 
[gone for guinoe-plga, in which {¢ causes canons lesions, 
‘further, the streptothris madara desorbed tl 
in disatana of the lower sulmials eoveral other forma hae been foand, 
For example, a streptothrix has been Gert by Nocard to be the cause 
ore dinbae, of ANE txt feede a Bo discase in which also 
there ccour tumour-like mae of a time Dean 
oultivated from a nodule in a horse another at tchrix, whieh produces 
taborole-like nodules in the rabbit with club-formation: it has close 
resemblances to the orgauiam of Israel and Wolff. ‘The 
diphtheria of calves and ‘by robably 
oduoed by another stray ix or Jeptothrix, which grows 
Situ ly in the tissues in the form of fine felted fnuents. Purther 
investigation may ahow that somo of these or other species may ooour 
in the haman subject in conditions which are not yet di fforentintea. 


Experimental Inoculation.—Inoculation of smaller Animals, 
sach aa rabbite and guinow-pign, has usually failod to give positive 
results ‘This was the case, for example, in the important seties 
caf 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 4 

both by cultures from the ox and from the human subject. 
Inoculation with the organism of Iaracl and Wolff produces 
oaila Teelons both so rebbiia audi guinea-pigs, while Wright 
found that characteristic colonies aud Jesions resulted although 
the parasite did not grow to any great extent. Several of the 
other species of streptothrix have been found to possews active 
juthogenic properties, 

Mathods of Examination and Diagnosis, —As actinomyoosix 
cannot be diagnosed with cortainty apart from the diseovery of 
the parusite, o caroful 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, expecially 
when some of the pun ie apread out on a picce of glass, If one 
of these is washed in salt sclation and examined unstained, the 
clubs, if present, are at once seen on microscopic examination, 
Or the colony may be stained with a simple reagent sueh aa 
picrocarmine, and mounted in glycerin or Farrant’s solution, 
To study the filaments, a colony should be broken down on @ 








































‘MADURA DISEASE 297 


, and stained with a simple solution of any of 

dyes, such aa gentian-violet, th better 
obtained by carbol-thionin-blue, or by carbolfuchain 
five parts of water. If the be over-stained, 
decolorised by acetic 


and also of cultures, are rf 
Send mua tise 


the 
of it, should be used to show the 
ation of acid fuchsin being afterwards 


ha 
i 


u 
g 


é 


aye 


colonies may thus be obtained. 
the aetinomyces, however, the 
ficult, unless the pus is free from 







i 






Mapura Drarase 


general 
pepe the form of colonies or * grant i 
however, that the two conditions are distinct, and 
‘established that the two varictics of Madura disease 
are produced by different organisms. ‘This disease 
‘comparatively common in India and in varions othor parta of 
‘the tropics; it has also Leon mot with in Algiers and in America, 
‘Madara disease differs from actinomyces not only in its geo- 
eal distribution but also in its clinical characters, Its 
, for example, is of an extremely chronie nature, and 
‘the local disease is incurable except by operation, the 
never produces secondary lesions in internal organs 
also found that iodide of potaesium, which has a high 
therapeutic agent in many cases of actinomycosia, hid 
in the cus of Madura disease studied by him. It maost 
affects the foot; hence the disease is often spoken of 
ma foot.” ‘The hond is rarely affected, In the parte 
¢ is a slow growth of granulation tissue which has an 
“nodular charucter, and in the centre of the nodules 
purulent eoftening which is often followed yy Une 






















298 ACTINOMYCOSIS AND ALLIED DISEASES 


formation of fistulous openings and uleors ‘There are great en- 
largement and distortion of the part and frequently caries and 
necrosis of the bones. Within the softened caviti 
Sees between the fibrous tissue, small rounded bodies or 
granules, bearing a cortain resemblance to the actinomyves, are 
present, ‘Theso may have a yellowish or pinkish colour, 
from their appearance to fish ro, or they may be 
like grains of gunpowder, and may by their wneration 
form nodules of considerable size. Hence a pale variety and 


dant mass of branehin 
filaments with rmyoolial 
arrangement, ‘hero may 
also be present at the 
periphery club-like struc: 
tures, as in actinomyoes 5 
sometimes they are ab- 
sent. These structures 
Fro, 103. —Strptothriz Madurm, showing 
‘ining Biante: From. 2 tulture we often havo an slongated 
Agae. Stained with carbolhionin-bing, Wedge-shape, forming an 
x 1000. outer zone to the colony, 
and in some cases the 
filamenta can be found to be connected with them. Vincent 
obtained cultures of the parasite from a case in Algiers, and found 
it to bo a distinct species: it is now known ax the streptothrin 
Maclure, Morphologically it clovely rosorables the actinomyces, 
but it presents certain differences in cultural charmeters. In 
gelatin it forma raised colonies of a yellowish colour, with 
umbilication of the centro, and there is no liquefaction of the 
medium. On agur the growth assumes « reddish colour; the 
organism flourishes well in various vegetable infasions 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 





MADURA DISEASE 299 


therefore no doubt that the streptothrix madare and the 
are distinet species, 
Black Varicty.—The obsorvations of J. H. Wright, who 
eres earecenl « byrhoaiyeas aur that this variety is 
affection from the pale variety, The pigment may be 
diol by soaking the gramiles fora few minutes in hypochlodte 
‘of sodium solution, and the granules may then be crushed ont 
‘beneath ere gan and examined microscopically. The black 
compared of a somewhat homogeneons ground- 
“prt with pigmont, and in this there is 
= of filaments or hyphoxy, me ‘of the segments of 
which are swollen; at the pe iphery the hyphw form a zone 
with radiate a many of the older niles the 
esiia alargely degscerated and presents an amorphous appear 
ght planted over sixty of the black granules in various 
cultures of a hyphomycete from 
‘The organism growe well on am 
bouillon, potato, ete. ; on agar it forms a felted mass of grey 
‘colour, and in old cultures: black gmunules appear amongst seo 
sr Microscopically tho ite appears aa a mycelium 
thick branching filaments with delicate transverse septa ; in 
ths older threads the segments. become swollen, so that strings 
bodies result, No signs of spore-formation were 
ria Inoculation of animals with cultures gave negative 
results, as did also direct inoculation with tho black granules 
from the tissues. 






CHAPTER XIII 
ANTHRAX." 


OTHKR NAMSS.—SYLENIC FEVER, MALIONANT PUSTULE, WOOL 
SORTER'S DISEASE. G#/MAN, MILZRKAND; YKNCH, CHARBON 


Introductory.—Anthrax ia a disease occurring epldeaielly 
‘among the herbivora, capecially sheep and oxen, in whi 
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 dows not occur as 4 nutunal affection in man, but may 
be communicated to him directly or indirectly from animals, and 
it may then uppenr in certainly two and possibly three forms. In 
the first there is infection through the #kin, in which a local lesion, 
the “malignant pustule,” occurs, In the second form infection 
takes place through the respiratory tract, Hero vory aggravated 
symptoms centred in the thorax, with rapidly fatal termination, 
follaw. Thirdly, an infection may probably take place throngh 
the intestinal tract, which is now tho first part to give rise to 
aymptoms, 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 ox, their 
growth and spread being attended by inflammatory codema 
and often by hatorrhages. 


Historical Summary.—Historiosl researches loave little doubt that 
from the earliest times anthrax has ocourred among enttle, For ® long 
time it pathology wax not understood, and it went by many namo 
* Im evon recent works on surgery the term “anthrax” may be found 
applied to ang form of carbuncle,  Bofore its true pathology wus known the 
local variety of the disease which occurs in tian and which Ly now called 
‘““gpaliguant puatule” was known as “‘apaliguant carbuncle,” 

4 This must be distinguished from “charbon symptomatique,” which ta 
quite ao different diseaes, 














800 


BACILLUS ANTHRACIS 301 


‘the early part of last contury much attention was paid 
with a view to fining ont {ta nature and iene fea 
‘variows conditions attaching to i ‘and 
poten, wece exhaustively stadied, Pol Pollen in ie fe TA late out that 
of contalned orias 
He atest dst ata conection wth th dense, In 868 


He ‘tated that anlese Lived used in inoon)ation 
on animals contatned them, death did notensue. Thongh 
conclusion was disputed, still by the work of Dayaine and others 
the eaussl relationship of the bacilli to tho, dincan 

when the Koah 








cestabliabed ‘work of Kooh ap} 

that obserrur's firs) contribution to logy, and did much to clear up 

the whole subject, Kock confirmed Davaine's view that the bodies wors 
le in the of anthrax animals tho a; J 

of avi ‘and from this deduced that multiplication took place in the 
atin yea thom under the mi ividing outside the 





boat and org formation taking ples, He lio alate the 
in pure aracaltaré ental th bod body, and, ty ioonlstig animale with 
produced the disease artificially. In experiments. he 
encniace ‘death by feeding snsceptible animals both with bacilli 

sn a8 thy nfeaitoal rack was, fa hs view, the netural fit 

of te comidered ns incomplete the proof ofthis method of the 
OCCUFFEDCS thras in Kooh’s obser 


a ee oan eee in tho main b; Pantcur, 
theage controversy sro between, them cn certain mluor polats 
Moreover, farther research showed that the disease could be produced 
in animale by feeding them with and thus the way in which tha 
See RD spond uatsrally wee txpllad: 

‘The Bacillus Anthracis,—-Anthrax aa 4 disease in man is of 
comparative rarity. Not ouly, however, is the bacillus anthracis 
easy of growth and recognition, but in its growth it illustmtes 
many of the general morphological chamcters of the whole group 
of bacilli, and is therefore of the greatest use to the student, 
Further, its behaviour when inoculated in animals illustrmtes 
many of the points raissxi in connection with such difficult 

« questions as the gencral pathogenic effects of bacteria, immunity, 
etc, Hence an cnormous amount of work has bees done in 
ing it in all ite aspects. 

Hf a drop of blood is taken immodiately after death from an 

vein of a cow, for example, which has died from 

anthrax, and examined microscopically, it will be found to 
hepa ‘A great number of large non-motile bacilli, On making 
Preparation from the same source, and staining with 
a: the characters of the bacilli can be better 
aro about 1°2 p thick or « litele thicker, and 6 

Pies, though both shorter and longer forms also ooenr. 

me ane sharply ext across, or may be alightly dimpled so 

Ae fo rewmble somewhat the proximal end of « phalanx. Theix 





Ua 





302 ANTHRAX: 


protoplasm is very finely granular, and sometimes sur- 
rounded by a thin unstained capsule. When noveral hac 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. Tf, 
after twelve hours’ incubation at $87" C., tho latter be examined 
under a low objective, colonics will be observed. They are to 
be recognised by beautifal wavy wreaths like locks of hair, 
radiating from the centre and. Gas He terminating in a point 

‘ich, howover, on ex- 
amination with a er 
awer Ia observed to 
lament which turn 1 
iteolf (Fig. 104). The whole 
ae 
ona long thread 8 
colonies are very suitable 
for making impression pre- 
parations (vide p. 118) 
which preserve perman- 
cay the Speen de- 
m1 examining 
mee with a ah 
a reste need 
made up of bundlos of 
Pia, 104,—Surface colony of the anthrax long filaments lying par- 

‘bacillns on an agur plate, showing the allel with one another, cach 

charseteritio nppearances,” 30. filament consisting of 

chain of bacilli Tying ond 
to end, and similar to those observed in the blood (Fig. 105), 

‘On gelatin plates, after from twonty-foar to thirty-six hours 
at 20°C, the seme appearances manifest themselves, and later 
they are accompaniod by liquefaction of the gelatin, In gelatin 
plates, howover, instead of the characteristically wreathod aj 
nance at the margin, the colonies sometimes give off radiating 
spikelets irregularly nodulated, which produce a starlike form. 
‘Those spikelots 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 umally the appearance of irregularly spiral threads 
suspended in the liquid. These, on being examined, are seen 










BACILLUS ANTHRACIS 303 


p be made up of bundles of parallel chains of bacilli. Later, 
| atl fae ey forms a flocculent mass at the 
‘of the fluid, 


ni gelatin stab cultures, the characteristic appearance can be 
best, whon a low proportion, say 7) per cont, af gelatin 





Fim, 105—Avthrax lmeilli, arranged i chains, 
from » twenty-four hours’ cultare on agar 


a 
‘Stained with fuchsin. x 1000. 


is present, and when the tubo is directly 
inocu from anthmx blood. — In 
about two days there ridinte out into 
the medium from the needle track 
aussberiess very fine spikeeta which 
enable the cultures to be easily recog: py 
ples iThaes azikelots nro, longest at. 420g, cab culumat 
the upper part of the needle tmck  peptone-celatin ; sven 
Gig. 106). Not much spread takes aye growth, Itshows 
fon the surface of the gelatin, the spiking "and al 
horo liquefaction commences, and — yieneing  Inquriuction, 

ly spreads down the stab ond Natural size. 

‘out into the medium, till the whole 

‘of tho golatin may be liquefied, Gelatin slope cultures exhibit 
fa thick felted growth, the edges of which show the wreathed 
jee soon in plate cultures. Liquefaction here soon 
a trough in the surface of the medium. Sometimes 
" does not take place in gelatin stab cultures, only 








(ie ax 





304 ANTHRAX 


Tittle 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 rastored by ‘tho bacillus 


for twenty-four hours on blood agar at 37° C, Agar sloped 
cultures have the aj of anlar ealicran tan pane 


though, of course, no liquefaction takes place, 

Blood serum sloped cultures int the same appearances 08 
thore 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 oocure 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 
mieroscopie appearance, by its growth on agar or gelatin pilates, 
and ita growth in gelatin stab cultures. Tho growth on 
sutants specially characteristic, and is simulated by no other 
pathogenic organism, 

‘The Biology of the B. Anthracis—Koch found that tho 
bacillus anthracis grows best at a temperature of 35°C. Growth, 
te. multiplication, does not take place below 12" C. or above 
45°C. Tn the spore-free condition the bacilli have comparatively 
low powers of reistance, They do not stand long exposure to 
60° CO, and if kept at ordinary temperature in the dry condition 
they are usually found to be dead after few days. ‘The action 
of the gastric juice ia rapidly fatal to them, and they are accord- 
ingly destroyed in the stomachs of healthy animals. or 
also soon killed in the process of putrefuction. They can, how» 
ever, be cooled below the freezing-point without dying. The 
bacillus can grow without oxygen, tut eame of its vital functions 
are best carried on in the presence of this gas. ‘Thus in anthrax 
eultures the liquefaction of gelatin always commences at the 
surface and spreads downwards. Growth ia more rapid in the 
presence of oxygen, and spore formation docs not occur im its 
absence, The organism may be classed ws a facultative anaerobe, 

‘Sporulation.—Under certain circumstances sporulation occurs 
in anthrax bacilli, Tho morphological appearmnoes are of 
ordinary kind, A little highly refractile speck appears in. the 
protoplaam about th contre of the bacillus; this gmdually 
increases in sive until it forms an oval body about tho same 
thickness as tho bacillus lying in the bacillary protoplasm (Fig. 
107), The latter gradually loses its staining capacities and 
fivally disappears, ‘The spore thna lies froe a8 an oval highly 








BIOLOGY OF THE B. ANTHRACIS 305 


mfractilo body which doea not atain by ordh methods, but 
= aie 
& purpose (ps ). en te is agai mat to 
assume the bacillary form the capsule arageetinly absorbed, 
‘within growe out, taking on the ordinary tod- 


a 
F 


to most obeervers sporulation never oceurs within 


the body of an animal suffering from anthrax. Koch attributes 
this, oy rightly, to the whence of free oxygen. The latter 
gas ho fe necessary to the occurrence of spores in cultures 
outside the body. Many, however, are inelined to assign as the 
can of sporulation the 
abeonce of the optimum 
pabulum, which in the case 
‘of anthrax is afforded by 

iden 


re production is 30° 
eeh found that spore 
formation 

below 18°C, Above 42° 
Q. not only does spornla- 
tion cease, but uur 
found that if bacilli were Pi, 107—Anthrax bacilli con 
kept at this temperature (the darkly coloured. bodies 
foreightdays they didnot yt ota 
regain the capacity when pine. 1000, 


again grown at a lowor 
tem In order to make them again capable of sporing 


it is neceeeary to adopt special measures, such as passage thi 
the bodies of a series of susceptible animals, 

Anthrax spores have extromely high powwrs of resistance. 
In adry condition they will remain viable for 9 year or more. 
Koch found they resisted boiling for five minutes ; and dry beat 
at 140° C. must be applied for several to kill them with 
‘certainty. Unlike the bacilli, they can rosist the action of the 
gastric juice for « long period of time. They aro often used 
it test objects by which the w of germicides ts judged, For. 
this purpose an emulsion is made by scraping off a surface 
faltare and rubbing it up in o little sterile water. nto thik 

» 














ic 


306 ANTHRAX 


sterilo silk threads are dipped, which, after being drind over 
sulphuric acid in a desiccator, ean be Kept for long 

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 Inet traces of the reagent and 
Jnid on the surface of agar or placed in bouillon, in if 
death has not occurred growth inay be observed (see Chap. TV.) 
Anthrax in Animals.—Anthrax ocenra from time to 
epidemically in sheep, cattle, and, more rarely, in horses 
deer. These epidemics are found in various parts of the world, 
although they are natnrally most farrenching where legal pre: 





‘among sheep was probably 10 per cont of the total 
number in the country, and among cattle 5 per cent, These 
figures, however, have been largely modified by the system of 
Pproventive treatmont 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 
palse and respiration, and dyspnam, and death may occur in 
a few mimutes. In leas acute cases the animal is apparently out 
of sorts, and does not feed; its pulve and respiration are 
quickened ; rigors ocenr, succeeded by high temperature: there 
isa sanguinous discharge from the bowels, and bloody mucus 
may be observed about the mouth and nose. There may be 
convulsive movements, thereis progressive weakness, witheyanosis, 
death occurring in from twelve to fortycight hours. In the 
moro prolonged cases widespread c:dema and extensive enlarge- 
ment of lymphatio glands are inarked features ; and in the glands, 
especially about the neck, actual necrosis with ulceration 
occur, constituting the so-called anthmx carbunclea 61 
subacute conditions are especially found among horses, which 
are by nature not so susceptible to the disease as cattle and 
hoop. 

On post mortem examination of an ox dead of anthrax, the 
moat noticeable feature—one which has given the name “splenic 
fover" to the diseaso—is the enlargement of the apleen, which 
may be two ot three times its natural size, Ib is of dark-rod 
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 methylen-bhie, On examina 
tion it will be found to contain enormous mumbers of bacilli 














ANTHRAX TN ANTMALS 307 


mixed with red corpuscles and len I 

and the large mononueleated variety Te 100) Pores at ihe 
‘organ may be hardened in absolute alcohol, and sections cut in 
paraitin. Thoso are best stained by Gram's method. Micro- 
examination of euch shows that the structure of the pulp 
considerably disintegrated, whilst the bacilli swarm 

tho organ, lying irrogularly amongst the cellular clomente. The 





Fie. 108. —Sormping from spleen of guinea-pig dead of anthrax, showing the 
Bacilif mized with loucoeytes, ete. (Sa ince ax in tho 0x.) 
“Corrosive film” stained with carte 





Tivor ia enlarged and congested, and may be in a state of acute 
elouidy swelling. Tho bacilli are proscnt in the capillaries 
throughont the organ, but are not so numerous as in the spleen. 
‘The kidney is in a similar condition, and here the bacilli are 
chiefly fonnd in the capillaries of the glomeruli, which often 
Appear us if injected with them, The lungs any congested and 
may show catarrh, whilst bacilli are present in large numbers 


t the capillaries, and may ales be found in the air cella, 
as the result of Js of the capillaries, ‘The blood 


it the body is wsnally fluid and of dark colour. 
ss a 





308 ANTHRAX 


The lymphatic system generally ix much affected. The 
glands, cxpecially the mediastinal, mesenteric, and cervical 
are enlarged and surrounded by o:dematous tissue, the 
lymphatic vessels are swollen, and both glands and vessels may 
contain numborloss bacilli. The heart may be in a state of cloudy 
awelling, and the blood in its cavities contains bacilli, though in 
smaller numbers than that in the capillaries, The intestines are 
enormonsly congested, the epithelium more or Inss desquamatad, 
and the lumen filled with a bloody fluid. From all the organs 
tho bacilli can be easily isolated by stroke cultures on agar. 
: Tris important to note the existence of great differences in 
susceptibility to apthrax in different apecies of animale, Thus 
the ox, aheop (excopt those of Algoria, which only snooamb to 
enormous doses of the bacilli), guinea-pig, and mouse are all very 
susoaptible, the rabbit slightly less so. ‘The last three aro 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 ure the honw, deer, 
goat, in which the disease occurs from time to time in nature. 
Anthrax also occnrs opidemically in the pig, often from the 
ingestion of the organs of other animals dead of the disease, Tt 
iz, however, doubtful if all cases of discaso in the pig described 
on clinical grounds as anthrax are really such, and a careful 
bacteriolovical examination is always advisable. ‘The human 
subject may be aaid to occapy 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 suscoptible. Adult carnivora are also very immune, and the 
birds and amphibia are in the same position. 

With these differences in susceptibility there are also 
variations in the pathological effects produced in the natural or 
artificial disoase. This is especially the case when we consider 
the distribution of the bacilli in the bodies of the less snsceptible 
animals, Instead of the widespread occurrence described abave, 
thay may be confined to the point where they finst gained accoss 
to the body and tho 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 Inngs, or kidneys, Neverthe- 
Joae the cellular structure of tho organs even in auch a caso may 
show changes, a fact which is important whon we consider the 
essential pathology of the disense, 

Experimental Inoculation, —Of the avimals 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 


tions, If a small of anthrax bacilli be injected into 
peedratien fa icere sand ween 
within twodays. Post mortem uround the site of i 





Piv, 100,~Portion of kiduey of » guines-pi dev of autheax, showing the 
Lacilli in the capillaries, expocially of the glomeralun 
Puraflin section j stained by Grams method and Waworck-bown, 4900. 


sometimes small hemorrhages, and their capillaries contain 

enormous numbers of tmcilli, as has already been deseribed in 

the case of tho ox (Fig. 109); the spk ulso shows & corre- 

sponding condition. Highly ensooptibl mals may be infeoted 

by being made to inhale the bacilli or their spores, and also by 

Being fed with spores, a general infection rapidly oceurring by 
methods. 





Anthrax a oe Human Sabject.—As we hove noted, man 
congiles & middle position in the scale of susceptibility to 
fothrax, It is always communicated to him from animals, ww 


ie = 


the hair follicles. A local condition 
called Ne malignant pustale” develops, which may lead to a 
gencral infection. ‘This variety occurs chiefly among butchers 
and those who work among hikes (foreign ones capoctaly ), 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 discase the site of infection ix 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 
Inistles, which have beon taken from aniinals dead of the disease, 
and which have been contaminated with blood or secretions con- 
taining the bacilli, thes having afterwards formod spores. 
variety is often referred to as “ woolsorter's disease," from its 
cecurting in the centres of the woolstapling trade (in England, 
chiefly in Yorkshire), but it also is found in places where thers 
are hair and brush factories. 

(1) Malignant Pustule.—This usually occurs on the exposed 
surfaces—the face, hands, forearing, and back, the last being a 
common site among hideporters. One to three days after 
inoculation a small red painful pimple appears, soon becoming a 
vesicle, which may eontain clear or blood-stained Muid, and ix 
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 vosiclos, th tum being surrounded 
by acongested arca, From this pustule as a contre subcutancous 
edema spreads, especially in the direction of the lymphatics; 
the neighbouring glands are onlarged, ‘There is fever with 
general malaise, On nsicroscopic 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 tho rete Malpighi, Beneath 
them and in their neighbourhood the cells zt ‘the latter aro 
swollen and cxdeimatous, the papille being eulargod and flattened 
out and infiltrated with inflammatory exudation, which also 
extends beneath the contre of the pustule. In tho tissue next 
the eschar necrosis is commencing. The subcutaneous tissue is 
also codematous, and often infiltrated with leucocytes The 
bacilli exist in tho periphery of the eechar and in the neigh 


ody, Tn on, the pai of entrances is through ets or abmuioas 
th 





g 






















ANTHRAX IN MAN all 


the 
the pustule. Sometimes the soley OCR no furth 
ese Sethe talent lnsioe pea 


pat 
puthological changes detailed with to the 
in cattle. In man the epleen ig usually not much 
and the organs generally contain few bacilli. ‘The 
cause of death is therefore the absorption of toxins, It 
may here be said that carly exeision of an anthrax pustule, 
especially when it ia situated in the oxtremitics, is followed, in a 
large proportion of cases, by recovery. 

(2) Woolsorter's Disease —The pathology ef this affection 
waa worked out in this country ospecinlly by Greenfield. ‘The 
Jocal 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 hamorrhage into them. The 
edematous, and the cellular elements are separated, 
usually little or uo necrosis, ‘There is enormous 
of the mediastinal and bronchial glands, and 
infiltration of the cellular tissue in the region, 

and pericardial effusions, and hwmorthagic 

beneath the serous membranes. The lungs show 

. There may be cutaneous aedeta over the 

neck, with enlargement of glands, and the patient 

idly dics with symptoms of pulmonary embarrassment, and 

@ varying degree of pyrexia. It ix to be noted that in such 

eases, though numerous bacilli are present in the bronchial 

ions, in the lymphatic glands und affected tismes in the 

thorax, comparatively few may be present in the various organs, 

such aa tho kidney, spleen, ete., and sometimes it may be im- 
fo find any. 

3) Infection occasionally takes place through the intestine, 

ly by ingestion of ‘spores as in tho case of animals ; 

‘thi condition is rare, In auch casos there in a local lesion 

fin the intestinal mucous membrane, of similar patare to that in 

the brouchial form, the central parts of the hemorrhagic areas 

r, sometimes necrotic and yellowish, and there is a 

‘corresponding affection of the mesenteric glands In a case of 

Kind, recently recorded by Teacher hwmorrhagte uvnlayitia, 


Hit 


i 








312 ANTHRAX 


associated with the presence of the bacilli in large numbers, 
ocourrd as & complication, 

‘The ‘the Bacillus Anthracis.—Various theories 
were formerly held as to the mode in which the anthrax baeillus 
prodnces ita effects, ‘One of the earliest was the mechanical, 
ea} to which it was supposed that the serious ae wore 
prod! by extensive blocking af the capillaries in the various: 

organs by the bacilli. According to another, it was any 

that the bacilli weed up the oxygon of the blood, thus to 
starvation of the tissues. The discovery of definite toxins wl 
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 investiguted this 
subject. This observer used alkali-allumin 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, coucentrated by evaporation 
either at 100° C. or in weno at 35° to 45° C., there were 
igolated proto.albumom, doutoroalbumose, and traces of peptoms, 
‘The albumoses differed from those which occur in 

a wstion, in being strongly alkaline in their rwaction. This 
ty, Martin hold, was duo to traces of an alkaloidal body 
o which the albumosce were tho precursors, and which were 
formed when the process of digestion of the alkwli-albumin by 
the bacillus was allowed to go on further, By the albumoses 
and the alkaloid, pathogenic effects were produced in animals, 
clocely similar to those produced by the bacilli themselves, 
Martin, to account for the symptotna of the disease, considered 
that the fever was mostly due to tho alburmoses, while the 
edema and congestion were due to the alkaloid which seted 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 
poionous bodies similar to those produced by the bacilli growing 
in this artificial medium. Hankin avel Wesbrook arrived at the 
conclusion that the bucillue anthracis produces a ferment whieh, 
diffusing out into the culture fluid, claborates albumoses from 
the proteids present in it. The bacilli also produce albumoses 
direetly without the intervention of a ferment, Marmicr, after 
cultivating the b, anthracis in peptone solution containing 
certain salts, removed all the albumoses from the resultant 
liquid, and from them, cither by dinlysis or extraction with 
glycerin, ijolated a body which gave no reactions of albuminoid 
iuatter, peptone, propeptone, or alkaloid. ‘This he considered the 











SPREAD OF THE DISEASE IN NATURE 313 


toxin. It killed animals sasceptible to anthrax by a sort of 
‘aobexia, and in suitably small doews could be used to immunize 
them against subsequent inoculation with virulent bacilli. Iv 
was chiefly retained within the lacilli when these were growing 
in the most favourable conditions, Unlike the toxins of 


Tt cannot be said that great light has been thrown on the 
pathology of the diseaso by these researches. ‘The effects of 
infection by the b, anthnicis arc those shared by all other 
organisms producing infammation, the tendency to adem 
pecitation ‘of an unwouted dogroe being the chief special 

tare and one with reference to which Martin’s work may be 
important, ‘That toxic offects do occur in anthras is undoubted, 
for frequently, while the bacilli are still ey confined, there 
may occur pyrexia and ocdoma spreading widely beyond the 
pustule, but we have no definite information as to how these 
effects are produced. In this disease we ans probably dealing 
with another example of the netion of intracellular texing, 
regarding which, as in other cases, little is known. 

‘The Spread of the Disease in Nature.—We have sen that 
the b anthmeis mrvly, if ever, forms spares 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 

jie form would rarely occur, For it has been shown by 

many observers that in the course of the putrefaction of such a 
earcase the anthrax bacilli rapidly die out, and that after ten 
days ora fortnight very fow remain, But it must be remembered 
that while still alive, an animal is shedding into the air by the 
Dicey exerctions from the mouth, now, and bowel, myriads of 
Dacilli which may rapidly spora, and thns arrive at a very ce 
sistant stage. ‘These lie on the surface of the ground and are 
‘wialed off by surface water. At certain seasons of the year the 
temperature is, however, suiliciently high to permit of their 
tion, and also of their taultiplication, ax they eu un- 

grow on the organic matter which oscurs in nature, 

tal again form spores. It is in the condition of spores 

that they are dangerous to susceptible animals. In the tacillary 
if swallowed, thoy will bo killed by the acid gustrie con- 

but ms spores they can pass uninjured through the 
and gaining an ontrance inta the intestine, ile Ws 





(im 


a 


alt ANTHRAX 


wall, and ultimately reach, and multiply in the blood. Tt is 
Known that in the great majority of casos 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 nattnal 
of the disease by bringing to the surface anthrax spores. 

och 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 carcasea have been buried is due to Regeren of 
spores on the surface which has been infected by the cattle when 
alive 


‘The Disposal of the Carcases of Animals desd of Anthrax.—It is ex- 
teemoly important that anthrax careases should bo disposed of in such 
way as to prevent their becoming future sources of infection. If anthrax 
bo suspected us the catiso of death no pest mortem examination ahowld be 
mais, but only n small quantity of blood removed from an auricular 
ein for bacteriological tnvestigation, If such a earcase be now buried 
in a deep pit 














srefaction, Tho danger of 
‘greater when un animal has 








sf youre of danger to 
cattle aubsoquently, however, proceeds from the infootion of fields, yarda, 
‘nd byron with tho offal and the digcharge from the mouths. of anthrax 
onimils, All material that can be recognised aa such should be trurmed 
along with the straw in whieh the animale havo lain. The stalla or 
buildings in which the anthrax oases have been mast be limewashed. 
Needless to say, the greatest care must be taken in the oase of men who 
handle the auiinal or its canase that they haye no wounds ow 
soon, ani hat they thoroughly disinfoct theroelves by woking thelr 
Minds, oto, in to 1000 solution of corrosive sublimatt, and that all 
clothes soiled with blood, ote., from anthrax animals be the 
Dolled af atoamod for halfan hone beara being washed. 














The Immuniging of Animals against Anthrax.Having 
ascertained that there was ground for believing that in cattle 
one attack of anthmx protected aguinst 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, Te 
found that the continued growth of anthrax bacilli at 42° to 
43° C. caused thom to lose their capacity of producing spor, 
and also gradually to lose their virulence, so that after twenty- 
four days they could no longer kill cither gaineapigs, rabbits, 
or shoop, Such culturoe constituted his premier waccin, and 










IMMUNISATION AGAINST ANTHRAX 315 


pay eet aie re riecees with bacilli which 
grown for twelve at same temperature, and 
tho attenuation of which bad therefore not been carried 40 far. 
Tho latter constituted the deuxidme vacein, It was further 


injected wi any 
This method was cable also to cattle and horses, about 
double the dose of vuccine being here necessary, Extended 
experiments in Francs 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 thal time tho method has been regularly in use, Tt is 


U 
after the first or second vaceination, or during the following 
vaceination, ne er nee ity is lost in 
about 40 — of ~ anit a ue th 
permanently efficacious the process wor we to be repew 
Farther, the immunity is much higher in degree 
‘the first and second vaccinations, an inoculation with 
it anthrax is performed. Everything being taken into 
aecount, however, there is no doubt that the mortality from 
natural anthrax is much diminished by this system. 


During the twolre years 1882-98 8,206,815 sheop wore vaccinated, 
with « mortality, either after the first or ecoond vocoination, or uring 
he mubsoquent twelve montho, of “OL per cont, as conteaated wi 

orealiy. fa all the’ docks. of the Sates 10 ar on ap 

‘the camo time 439,824 cattle wore vaccinated, eas 

‘of “4 per cent, As contrasted with a probable martality of & per cent {f 
they had beon saprottet 





meee by careful dosage with virulent cultures, Marehoux 
“in this way produced immunity, and found that the scrum of 
ateanes satrsle had a certain degree of protective and eurative 
‘getion. ‘Tho most successful attempts in this direction have 
‘those of Sclavo and of Sobernbeim, ‘Tho former observer, 
tying Various animals, came to the conclusion Yhak Yas 





316 ANTHRAX 


ass was the most suitable, He first employed a method sithilar 
to that of Marvhoux; later, however, after noting the effects 
of the scrum of an animal so immunised, he commenced the 
immunisation by injecting 6 to 16 cc. of this serum along with 
a slightly uttentuated culture of the bacilli. A few days later 
‘thin was followed up with injections of virulent cultures which 
could now be periodically introduced for many months, and a 
Nigh degree of immunity resulted. What was even more 
tafe the serum of such an animal hid strongly protective 
curative properties. Tt has been extensively used in the 
ee oe of anthrax in man, Ina case of malignant pustule 
30 to 40 cc. aro injected in quantities of 10 ce into the 
abdominal wall, and if necessary the injection is repeated on the 
following day. Tp cases treated hy Selavo himself the serans 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 ton deaths or 
about a fourth of the ordinwry mortality in Ttaly. Sobernhelm 
independently elaborated an almoat identical method of com- 
bining possive with aetive immunisation for the obtaining of a 
powerful uutiserum, 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 (Pastour's 
second vaccine) into the other side; the doses given are for 
cattle or horses 5 e.c. of serum and “5 ce. culture, and for sheep: 
4 cc. of serum and 25 ee. culture. The method bas been widely 
used in Germany and in Brazil, and its originator claims us its 
advantages simplification of application, in that one operation 
instoad of two is sullicicnt, loss risk of death following the 
immuniastion procedure, and higher degree and more I 
character of the immunity resulting, Whether this method is 
really more efficient than that of Pastenr future experience will 
show, but it might be proferable for developing bee in 
herds at a time when an epidemic was raging. During the 
development of active immunity it is likely in every ease (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 thao with the Sobermheim procedure, where the 
presence in the antmal’s body of the protective seram might tide 
it ever the stage when the action of the vaccine was lowering 
its resistance, 
‘The affects 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 pi 




















ll 








METHODS OF EXAMINATION at 


facts have long boon known ; ehemunilips Mani 
shows great natural resistance, the serum 
‘bnctericidal action, while in the susceptible abhi eee is 
presont @ serum capable of killing the tdomtertt ‘Such observa- 
tions have hitherto been without ex nee rele the 
ies of the serum of immune animals have been mach 
nssed, Sobernheim and others have been unable to detect 
in it ony trace of special bactericidal action. Sclavo found that 
the serum when heated to 55" C. did not lose its protective 
| aaa as the serum might have been lemented (see 
ity) by the soram of the animal into which it was injected, 
he simultaneously introduced an anticomplementary serum and 
found that the heated seram was still effectual. From this he 
deduees 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 tho 
Jeucocytos, but Clor hae 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 
apleen and bone-marrow aro richer in these than the blood fluide, 
Tn this connection an interesting fact may be mentioned, namely, 
that Roger and Garnier found evidence of the liver and spleen 
ha specin! capacities for killing anthrax bacilli ; an otherwise 
fatal could be introduced into the portal vein or the splenic 
artery withont renee death. 
Methods of Examination..These include (a) microscopic 
examination; (2) the making of cultures; and (c) test ine 
‘oenlation: 


i 

(a) Microscopic Examination. —In a case of suspected 
‘malignant pustule, film preparttions should be made from the 
fluid in tho vesicles or from a scraping of the incised or excised 
pustulo, and stainod with a watery solution of methylene-hine 
‘and alko by Gram’s method. By this method practically con- 
daxivo evidence may be obtained ; but sometimes the result is 
doubefal, as the bacilli may be very few in number. Tn all 
asa confirmatory evidence should be obtained by culture, 
Occasionally cil aro so scanty that both film preparations 
made from different parts and even cultures may give negative 
‘roualta, and yet a few bacilli may be found whon a section of 
the is examined. It should bs notod that the greatest 
are it to be taken in manipulating a pustule before excision, 
‘a& the diffusion of the bacilli into the surrounding tissues may 
“Be nided and the condition greatly aggravated. ‘Tho wxamnimathem 


ANTHRAX 





of the blood in cases of anthrax in man usually gives nogative 
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. 

(8) Cultivation.—A small quantity of the material used for 
microscopic examination should be taken on a platinum needle, 
and successive strokes made on tubes, which are then 
incubated at 37°C. At the end of twenty-four hours anthrax 
colonics will appear, and can be readily i their 
wavy margins by means of a hand Jena They should. also be 
examined microscopically by means of film propnrations, 

(c) Test Znocwlations.—A little of the suspected material 
po ane ne ne some sterile bouillon or bet} and 

jected aubcutanoously Into a guinea-pig or mouse, or it may 
be introduced into the subcutaneous tissue by means of « veton. 
Tf 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 _ 
TYPHOED BACILLUS 


OTNER NAMES,——-ENTERIC FEVER: GASTRIC FEVER. GBAMAN, 
TYPUUS ABDOMINALIS; ANDOMINALTYTHUG: UNTERLNIRS 
TYPHUS. FRENCH, LA MIRVRE TYPHOLDR, 


Introductory.— The organism now known as the bacillus 
typhosus was first described in 1880-1 by Eberth, who observed 
its microscopic ap, uces in the intestinal uloors and in the 
loen in cases fever. It was first isolated (from 
we spleen) in yin task be Gaffkys a and its cultural characters were 
thon investigated. In 1885 Escherich observed a bacillus, now 
known as the bacillas coli communis, which occurs in the normal 
intestine and which both microscopically and culturally elosely 
resembles the typhoid bacillus. Ordinarily the b. coli ik no 
doubt a harmless saprophyte, but under experimental conditions 
in animals and also natnrally in man it may manifest pathogenic 
Investigation bas shown that these two bacilli 
belong to a widespread group of organiams isolated from various 
disease conditions, which all bear close resemblances to one 
another and whow differentiation is often a matter of consider- 
able difficulty. Othor anembers of this group are the 
typhoid bacillus, the organism of bacillary dysentery, 


itidis of Gacrtoer, the paittaccals bacillun, sti saat 
Geran 


Bacillus Typhoans—Microrcopic Apperrancer.—Tt is some- 

times difficalt. to find the typhoid bacilli in the organs of a 

oid Pationt. Numerous sections of different parts of a 

for example, may be examined before a characteristic 

is found. best_tissues for examination are a Peyer's 

patch where ulcemition haa not yot commenced or whwce i a 
319 


ba — 














320 TYPHOID FEVER 


just commencing, the spipen, the live ora pei 
"Tho spleen and livor are anodes 

in the Inttor the prroscnee of the b, coli is more frequent. 
serapings of atch solid organs dried films may be 


stained for a few minutes in the cold by any of the atzo 
ataining solutions, eg. with carbol-thionin: re or with 
Neelsen oa 
five orn 


of ‘lated ‘ane Asw 
rule decolorising is not 
necessary, Fortheproper 
obecrvation of the ar 
mangement of the baci 
in the tixmos, 
sections should be pre- 
paired” ond stained in 
ear thioninblue for a 
Zoe ome rin ner 
notiyleoe bine 
rie "he ball 
fae lip the stain some. 


what slowly, and aa rps 4 
Pit, 110.—A lunge clump of typhoid baollt aT Mirra’ 


in yploco. ‘The individual Toscilli axe 
‘only sem at th 
(In this sploe 
typhoid baoilit 











periphery of tho ma, A ie ea 


nomons numbers of with advantage (wide ps 
shown by eultums 19 93). In aucl 











reparn= 

te present in a practically pare condition.) * preps 

Paraffin section ; stalned with earboltbiou- tions the charncteriatic 
Mae. «600, appearance to be looked 


for is the occurrence of 
groups of tucilli lying between the cells of the tissue (Fig. 110). 
ye Individual. bacilli are 2 eto 4p: Tong, with somewhat oval 
ends, and * pin thickness. Sometimes filaments 8 eto 10 
long may be obserred, though they are less common than in 
cultures, Tt is evident that one of the short oval forms may 
frequently in a ection be viewed ondwire, in which caso the 
appearance will be circular. ‘This appearance accounts for some, 
at least, of the coccus-like forms which have been described, 
~The bacilli aro decolorised by Gram’s method. 

Isolation and Appearances of Oultures.—To grow the 
organism artificially it is best. to isolate it from the spleen, aa it 
exists there in greator 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 


sal 


ISOLATION AND APPEARANCES OF CULTURES 821 


capsule is seared with a cautery to destroy all superficial con- 
tamionting onganiams, 4 small incision is Roads feta thelcgian 

with a storile knife, a little of the palp removed by a platen! 
aoe, and agar or gelatin plates are prepared, or successive 


strokes made on agar tubes, soe 3 eee Mt 


woe visible after twenty. 

or alghtly inewalar at eet fie 
vi “ ears oe ies in fe stbtnee of ‘of 

appear_as minute round points, When 
vient uae slew ‘ob- 

Hee the surface 
colonies are found to be 
vory bei (requir- 
ing @ 
for their detinition, ae 
granular in appearance, 
and with « very coarsely 
erupated and well-defined 






finely granular on the 


surface, and mn 
eaheritsl 
onaee 


Jon king - 
Fra M1L—Typhoid Qovilli, from» young 

cover: glass yc a a 
Rev carit are Modind Tyo.) een ee acme ee Ra aalias 


forme, 
present the same micro- Stained with weak earbol-fuohsin. x 1000, 
scopic appearances as ars 
observed in preparations from solid organs, except that there 
may bo a greater numbor of the longer forms which may 
almot be called filaments (Fig, 111), ‘The same is true of films 
made from young gelatin colonies, Sometimes the diversity in 
the longth of the bacilli is such as to throw doubt on the purity 
of the culture, Ita purity, of course, can be readily tested by 
Proparing plates from it in the usual way, Asa goncral rule in 
& young (wenty-four to forty-right hours old) colony, grown at 
uniform temperature, the bacilli are plump, and the protoplasn: 
stains uniformly, In old cultures, or in caltures which have 
Deen exposed to changes of temperature, the protoplasm stains 
‘only fn parts; there may be au appearance of irregular vacuola- 
tion either at the centre or at ‘the ends of the bari, "Crere 


ie * - | 





$22 TYPHOID FEVER 


e2hANe te no evidenos that spore-fortmtion occurs inthe typhoid 
i. 


Motility.— In tunging-drop proparatious the bacilli are found 
to be actively motile. ‘The smaller forms have a darti 
tolling motion, passing quickly across tho field, whilst some 
rapid rotatory motion. The filamentous forme have an wn: 
dalating or serpentine motion, and move more slowly. Hanging 
drop prepanttions ought to be made from agar or broth cultures 











Yio. 122.—Typhold cll thom a yoangealtrs on sgn sewing Magali 
Stnined by Van Ermengetn"e method, x 10 


not more than twenty-four hours old, In older cultures the 
movements aro less active. 

Flagelta.Ou being stained by the appropriate methods (wide 
p. 103) the bucilli are soon to posesa many lon 
which are attached all along the sides and to the oi - 11.2), 
They are more numerous, longer, and_more wavy thin those of 
the b, coli: 

Characters of Cultures, Stab cultures in peptone gelatin give 
a somewhat characteristic appeara On tie Surface of the 
medium growth spreads outwards from the punctit@@s a thin 




















APPEARANCES OF CULTURES 323 
or pellicle, with irregular! 


ly wary in (Fig. 113, A). Tt 
uae transparent and” of Bidieh:- whi tie atte Ultimately this 


surface growth ma rnc Se lla is taber Not infreguently, 
however, the surface growth is not well marked. Henussie the 


stab there is an 
oe, hes ea no i SF vin candin, and x0 


ga. plete hawt isa thin bluish-white 
Pa. 19, 


‘A. Saab ultore of the typhoid bacilve i satin, ie da 
AK. Ste etre of ue Eola 
© ah ealtarg of the baclbue colt ngwintin nine 
a Ne Lower part Owing te the fornmatho OF wes. 
















Fad aarp 


fin, but it does not spread to such an extent aa in the case of 
tho surface growth of a stab culture (Fig. 113, B). In gelatin 
lates leo tho superficial and deep colonies presout correspond 
diGerences. ‘The former are delicate semi-transparent films, 

ith wavy margin, and are inuch larger than the colonies in the 
eututance, which appear as amall round points (Fig. 114). These 
joes, which are wall seen on the third or fourth day, 
resemble those seen in agar plates, as already described in the 
method of ikolation; but on gelatin the surface coleniea axe 





3 TYPHOID FEVER 


rather more transparent than those on agar, Their characters, 
as seen under a low power of the microscope, also correspond. 

Jo stoke coltures an agar there ins Ullah geet 

wth, with fairly regilar margins, but without any 

je Teatures, This fila is loosely attached to the surface, and 
ean bo easily seraped off. 


The growth on potatoce in important, For several days (at 
iat ere) afer Tasatin there appenaitly 
no growth, Tf looked at obliquely, the surface appears wet, 


iid if it is scraped with the platinum loop, @ glistening track 





dull, some 

sirfaoe May appear, and 
thi may ‘oven navume 
4 brown appearance, 
‘Those charicteristic ap- 
pearances arv only seen 
when a fresh potato with 
an acid reaction has been 
used, 





In Louillon inenbated 
at 37° C. for twenty-four 
hours there is simply a 
‘one aorta an ree Seep in apelin Goes ees ey 

w supordetnl and three dee we 

fie Three dayy growth at rox ten COvS™HA preparations 

area eras made from sich some- 

times show filamentous 
forms of considerable length without apparent segmentation, 

Conditions of Growth, ete—The optimum tem 0 of the 
typhoid bacillus is about 37° C., though it also omar woll at 
the room temperature. It will uot grow below 9° C, or above 
42°C, Growth takes place in annerobie as well as in aerobic 
conditions, Its powers of resistance correspond with those of 
most non-sporing bacteria, It ix killed by exposure for half 
hour at a0, or for two or three Sealer 00° C. "Typhoid 
Ducillt Kept in distilled or in ordinary tap Water have usually 
been found to bi dead aftor three weeks (Frankland), 

Bacillus coli communis.—Thia bacillus is the ebief organiam 
present in the amall intestine in normal conditions, and, with 
many other bueteria, it also inbabits the large intestine. Daring 
typhoid fever, and othor pathological conditions affecting the 




















REACTIONS OF B TYPHOSUS AND B. COLI 325 


itestines, it is relatively and abheclately”enertacndly,inseseel 
in the latter situation, where it may Kom 
only bacillns ee Its rlations to various suppurative and 


se sageh it is usually somewhat shorter 
ai 115), nies is eae and possesses Interal flagella, which, 
however, are fewer in number.and somewhat shorter than these 
of the typh jus. It is casily isolated from the stools 
of men ri animals by 

any af the ordinary 

mathos._Aftos twenty 
four hours’ incubation at 


* C.on agar, there are 
colonies 
a deep colonies 


in thé plates; to the 
naked eye ie are 
denser and more gliaten~ 
ing ta those of typhoid 
when viewed by trate 
mitted light,and ratherof 
@ brownish-white colour, 
Under a low objective 
the colonies again a 

denser than those of the 











Fi, 118,—Bacillos coli commvanix, Film 
typhoid bacillasand more preparation from a young enlture on agar, 
granular. On orditiary stained with weak carbolfuchsin, © 1000, 


eae and agar media 

hoes Are similar to those of the typhoid bacillus, 
bat eth i wt, ticki nm op paque, and gives 

the impression of having greater vigour. Th ti of 
stub cult few bubbles sometimes develop in the 
medium (Fig. 113, Tapa it forty-Gght ours there 
jon diainct 1 film of growth of brow: tint and moist-looking 
as whieh rapidly spreads and becomes thicker, This con- 
a bameeeey with the colourless film of the b. typhowrs, 
tive Culture Reactions the B. typhosus 
axd ied ot Te importance of the relationships between 
the b, typhosus and the b. coli has euused great attention to be 
to their biological characters, in order to facilitate the 
of the one from the other. Some of these we have 
already noted. Of the cultural charmcters the growth 


—_ 9 









326 TYPHOID FEVER 


most important. As has been pointed out by 
Wathelet, aad also by Klein, differences exist in the growth of 
the two bacilli in ual gelatin, A’gelatin tube ix inoculated, 
and instead of being kept atthe room temperature, ix placed in 
the incubator at 37° C., at which temperature it is af course 
fuid. Tn sneh cultures, in the case of the be yphosy there ix 
general turbidity of the gelatin, while with the b. coli there 
are Targe foscalt alone ‘which float on the surface, It ia, 
however, 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 effecta, and it many be that in several 
apparently diverse reactions the same biological processes are 
really at: work. 

(1) The Fermentation of Sugars.—Of them one of the: most 
important is the effect on lactose as first pointed out by 
Chantemesse ond Widal, This ix usually demonstrated by 
using a | per cent solution of the sugar in peptone-salt solution 
placed in Durham's tubes (p. 76). If such @ medium be 
coloured with littans the production of acid and gas by the 
b. colican easily be demonstrated, Similar changes cansed hy 
this organism can also be observed in litmus milk and in 
Petrnschky’s litmus whey. 

Chantemesse and Widal first showed that the b 
docs not act on lactose in bouillon though decolorisat 
Utmns may cedur,” Thinay bo stated that undor most conditions 
of making Mie test an acid reaction does not rosult and there is 
never any formation of gaa This organism ia sald, however, to 
break’ Up lactose in lithiua mille 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 y-and if MacConkey's bile-salt Tactoae (huid 3 runs 
used, this organism and-its congoners ean be distinguished trom 
the b, typhosus, b, paratyphosus, and from the dysentery bacilli 
(s. iwfra), none of whsé colonies are crimson on this medium: 

‘The effects of the b. coli und the b. typhosus on other sugars 
is also of great importance. As media to which the sugars ta; 
be added, either peptonoealt solution or MacConkey's bile-ealt 
media are used (g.v.). To sum ap the general results it may be 
said that b, coli produces acid and gus in bilesalt glucose, 








— = 





REACTIONS OF B. TYPHOSUS AND B. COLI 327 


peptone-sult glucoss, lactose and mannite, but not in cane sugar’ 
while the b. typhosus produces acid without gas in bile-salt 
ghuense, recone sit Biases, and es but pen lactose or 
cane sugar. It can alao cause similar changes in arbbincse, 
galactose, and fructose. 

Gax_produstion by the b. coll can also be demonstented 
by means ko cultures As ordinary bouillon contains 
traced of glucose it is best to use peptone-ralt aolation to which 
an appropriate sugar has been added and which bas been 
converted into & 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 babbles of gas will form all 
throughout it, Th ordinary media inoculated with the b. coli 
bubbles of yas aro often developed along the needle track, 

An the caso of acid production by tho b. coli or b. typhosus in 
ordinary media the acid probably comes, as hus been ssid, fram 
the glucose developed from the muscle sugar, but there may also 
‘bo « subsidiary acid formation from the breaking up of the 
proteid clementa. 

In certain members of the coli-typhoid group it has been 


the fermentation of the Tactose of the milk and the throwing 
down of tie easwin ky the resulting lactic acid’; but the reaction 
sway bo a more camnplicatod ove, as milk ean be cundled by 
organisias which do not possess wcid-forming properties Tn any 
aso the observation of the reaction is important. "The txphoid_ 
bacillus ypealteae no visible change in milk. 

(3) dation on Media containing Neutral Red.— While, ax will 
have been already gathered, neutral-red is used as an indicator, 
ther is some evidence that an actual breaking up of the 
substance can take place by tho action of thocoli-typhoid group ; 
the evidence for this lies in the fact that when the effects of 
veld formation are olserved the tint of the medium cannot be 
bce hack by the addition of alkali, ‘The medints used here is 

containing an approprinte sugar and “6 por cunt of a 1 
per cent watory salution of Grubler's neutral-red, In the case of 
the typhoid bacillus no change ocours, but in the case of the b, 

is developed a bewatiful canary yellow witl a grooniah 


# A variety of the onganiem which does Germent cane sugar bas been 
‘under the name of the b. colt eormounlor. 


te | 


4g 





323 TYPHOID FEVER 


fluorescence, Fitz-Gorald and have shown that an 
important factor here is the reaction of the medium, and that the 
‘effects of the bacteria may be one of degroe,—under certain 
circumstances tho offeets ibed ws characteristic of the two 
onganisins imay be reversed. 

(3) Formation of Indot.—Among the bacterin capable of 
forming indol to bo classed the b coli, Indol can” be 
site in bouillon cultdres 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 alsa, 
Peckham, however, has found that if the typhoid bacillus be 

wn in peptone solution, after a few generat) ree 

js wach it may aequire the property of producing indol. e 
formation of indo! by an organism alter 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 Te 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 medinm,~ 

(4) The Media of Capaldi and Proskauer. —The first of these 
(*No, 1") is @ medium free of albumin, in which b. coli grows 
weil and frvely produces acid, while the typhoid bacillus hardly 
grows at all, and certainly will produco no change in the 
reaction, Its composition is as follows; asparagin “2 parts, 
mannite ~%, sodium chloride ‘02, magnesium sulphate -O1, 
calcium ebloride 02, potassium monophosphate 2, distilled 
water to 100 parte, ‘he second mediam (No, 2") contains 
albumin, and is such that the b. coli produces no acid, while 
the typhoid bacillus graws well and produces an acid reaction, 
It consists of Witta's peptone 2 parts, mannite “1, distilled water 
to 100 parte, After the constituents of each medium are mixed 
and dissolved, it is steamed for one and a half hours and then 
made neutral to litraus—the first medium, being uanally naturally 
acid, by aodinm hydrate, the second, being usually alkaline, by 
citrie acid, The mediam is then filtered, filled into tabes con- 
taining 5 ce. and thewe are sterilised. After inoculation for 
twenty hours the reaction of the medium is tested by adding 
litmus. 

(5) Lhe Application of the Agglutination Test in distinguish. 
sg & typhosus from B. coli.—The sope of the application of 








PATHOLOGICAL CHANGES 329 
yin Nn ei set St (sce Immunity), Eecwsoy 
._xusproted 


negative result obtained with a 
culture is of greater ate a 
ieee aaa rat ct eaters The tat hte bs 
‘err cunt we hor me of differentiating the 
two eigen (of. p. $40), 
will thus be seen that tho diagnosis between tho b. 
aoe and the b. coli is « matter of no small difficulty. 
‘Therv is no evidence that the one organism ever passes into the 
other. Great difficulties sometimes ae in Soe of a 
bacillus being found which, while a number of the 
characteristics of either one or the ‘af fll fails to give some of 
the charactoristie teats, or only gives them vory nae This is 
especially troc of organisms related to tho b coli, [t has 
a juently become common to speak of the es group 
coli group in order that such varieties may be included. 
eet in Typhoid Fever.—Hero wo confine 
our attention solely to the bacteriological pr, of the discase, 
The ry ano itt ulceration in the % 
the ae are the Pigs Tn the 
is produced an acute inflammatory condition, 
at Siot eatannite Joneoegti tie_emi jon and “sometimes 
with small Iemorthages. ante is ie typhoid bacilli are 
most nitnerous in Sa patches, groups bein caally found between 
the calls. The subsequent necrosis is crideatly 4 in chief part the 
Perpet the action of the toxic products of the bacilli, which 
sr disappear from their former positions, though they 
berm be found in the deeper tissues and at the spreading 
margin of the necrosed area. They also occur in the lymphatic 
ef the muscular coat, It is to bo remarked that 
the number of the ulcers arising in the course of a case bears 00 
relation to its severity, Small ulcers may occur iss the lymphoid 
follicles of the large intestine. 

‘The mesenterse glania corresponding to the affected part of 
the intestine are usually pete = mibaticaee to & Yory great 
ecient the whila trosenlary’ ‘being filled with glandular masses, 
Tn such glands ther may be acute inflammation, and occasionally 
necrosis in patches eccura. Sometimes ob section the glands 
are of « pale-yellowish colour, the contents being diffluent and 

largely of lencocytes. Typhoid bacilli may be isolated 
both from the glands and tho lymphatics connected with them, 
but = b. coli is in addition often present, 


print —on section usually of a fairly firm 
pink colour, and in a state of congestion. 





a 


830 TYPHOID FEVER 


Of all the solid organs it usually contains the in et 
Wate of local reaction 
i : nilaeahamp may sliver 





which, 9 fur as our experience goes, bacilli cannot be demon 
strated. The bacillus ix found, often in largo numbers, in the 
gall-bladder, where it may porsist for years, Clamps of bacilli 
may also occur in the kidney, 


Iu addition to these local changes in the solid organs there are also 
widesproa eelfular deyenerations in the solid organs which auggoxt the 
oiroulation of soluble poisons jn. ghabiced 

Tn the fasgr the ‘be bronehitis, patches ion and of 
acute hroncho-pneumonia, In these, typhoid bacilli may sometimes be 
CUaETWL- COL evideaay-of» toxic notion Geprosalng the yowers of rest: 
ance of the lung timue is found in the fuot thar the jueumococos 
froqanatly oecurs in auch complications of syphoid fever 

he nereous system shows little change, though meningitis aeociated 
eithor with the typhoid bacillus, with the b. coli, or with the streptococcus 
pyogenes has been observed. 

‘ne typhoid bacilli qwobably travel by the blood stream, and they ean 
be isolated from the blood much more readily than was formerly supponod. 
Considerable quantities of blood (say, 4 G0.) must of course be taken 
(e p. 68) They have bocn found in’ the rosolar spots whieh ocowr in 

Wot it cannot bo yet stated that sach spots are always due 
of the bacilli, The faot ehat the typhoid baoilli ary usual 
confined to certain organs and tissues shows that thoy probably hai 
selective action on certain theses. 




















To sum op the pathology of typhoid fever, we have in fia 
diseaso the contro of which Tex in the lymphoid tise in_and 
ineeted with the intest Tn this situation we must have 
iant, against which the inflammatory reaction is set up, 
and whteh in the fntestine i sufficiently powerful. to eatise 
necrosis. ‘Tho affections of the other organs of the body siggeat 
the circulation in the blood of poisonous substances capable of 
depressing cellulae vitality, and producing histological changes. 
‘The oovurrence of bacilli mn the blood and organs. 
fever with seplicemic processes, 

Suppurations occurring in connection with Typhoid Fever.— 
With regard to the relation of the typhoid bactllug to aueh 
conditions, statements us to its igolation from pis, ete, can be 
accepted only when all the points available for the diagnosis of 
the orgunism have been attended to, On this understanding 
the following summary tay be given :—In a small peopartion 
of the cases examined the typhoid bacillus has been the only 
































PATHOGENIC EFFECTS OF B. TYPHOSUS 931 
organism found, This hos been the ease in subeutancous 





aliscesses, in suppurntire periostitis, ion in the parotid, 
abscesses in te Rang oe ote., and. “oro aeay vaso i yaad 
cases of ulcerative ondocarditix. But in the majority of cases 
itr ec a the at * 
micrococe:, have been obtained, the typhoid bac bacillus having 
searched for in vain, Tt has, moreaver, 
shown, notably by Dmochowski and lige that io ss 
can be experimentally produced by injection in animals, expecially 
in rabbits, of pure cultures of the typhoid bacillus, ee iaraee 
of ot ep tion being favoured by conditions of deprossed vitality, 
"These observers also found that when typhoid bacilli were 
Injected along with pyogenic staphylococei, the former died out 
in the pus more quickly than tho latter. Acoordingly, in clinical 
arnnaretse typhoid bacillus is present alone, it is improbable 
tate other organisins were present at an earlier 
Pat Effects produced in Animals by the ‘Typhoid 
Bacillus, here is no diseaso known to veterinary acience 
which can be said to be identical with typhoid, nor is there any 
evidence of the occurrence of the typhaid hacilluk tinder ordinary 
Pathological conditions in the bodies of animale Attempts 
to communicate the clisease to animals feeding them on 
typhoid dejecta have been unsuccesaful, and though yuthogenie 
effects have been produced by introducing pure cultures in 
food, the diveaze has neually borne no resemblance to human 
typhoid. The most successful experiments have been those of 
Remlinger, who, by continuously feeding: rabbits on 
soaked in water containing typhoid bacilli, produced in certain 
‘cases symptoms. Teaubsbliny those of typhoid ferer (diarrhea, 
remittent pyrexia, ete.) An agglutinating action was observed 
in the serum, and post mortem there was congestion of the 
Peyerian patches, and typhoid bacilli were isolated from 


While feeding experiments are thus rather unsatisfactory, the 
same may pei a of the results of subcutaneous or intmperitoncal 
infection, Here, again, pathogenic effects can easily be produced 

the ‘yphad bacillus, but these effects are of the nature of a 
‘acute illness characterised by pyrexia, mpid loss of weight, 
inability to tako food, and frequently ending fatally in from 
twenty-four to forty. ight hours. The type of disease as very 
different from what occurs naturally in man. fn such injection 
it the results vary considerably, sometimes scarcely 

‘aay effect being produced by w large dese of a culture. ‘This 
tx to doubt due to the fact that different strains of the bacitlus 














ale _ 


332 TYPHOID FEVER 


vary much in virulonce. Ordinary laboratory cultures ure often 
almost non-pathogenic. They can, however, be made virulent 
in various ways. Sarurelli used the method of injecting 
aterilised cultures of the b. coli intraperitoneally at the same 
time as the typhoid bacillus was introduced enbcutancously, 
After this procedure hud been repeated through a series of 
animals a culture of typhoid wns obtained of exalted Hane: 
Sidney Martin has obtained virulent cultures by pussy bers 
derived directly from the spleen of a person fare typ 
fever, through the peritoneal cavities of n series of guinempigs 

Sanarolli, studying the effects of the intmperitoneal 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 puralent, and 
that thoy contained typhoid bacilli, as also did the moscnteric 
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 goneral results may be outlined, We may state that there 
exist in the bodies of typhoid barilli toxie substances, that in 
artificial cultures these do not yas 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, apie Sidney 
Martin found that the bodies of bacteria killed by pit 
form vapour were very toxic,—more so than filtered caltures 
Diurrhos, was 4 constant eymptom after injection, but no change 
in the Peyerian patches was obscrved. 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 whore toxie 
wffct he attributed to the presence of the intracellular poisons. 

The Immunisation of Animals against the Typhold 
Bacillus.—Karlier observers had been successful in mie 
mice to the typhoid bacillas by the sncocesive injections of small 
and gradually increasing doses of living cultures of the bacillus, 
Later, Brieger, Kitasato, and Waasermann found that the bacillus 
when modified by being grown in a bouillon made from an 
extract of the th 
pigs. 
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. COLT 393 


which it would naturally suecumb. Chantemesse and Widal, 
‘Sanarelli, and also Pier, wacceeded in it Ruincapiga 
against the subsequent intraperitoneal injection of virulent 
ting typhoid bacilli, by roped aad eaten igcee | 
or subcutaneous doses 
ine bouillon, Experiments performed beige serum di 
typhoid patients and ronvalescents indicate that similar toa 
occur in those who have successfully resisted the natural disease, 
Tho scram of such paticnts ret antilacterial powers, but 
there is no evidence that it contains any antifarie bodies (see 
chapter on Immunity), Pfeiffer, for example, found on addin, 
eorum from typhoid convalescents to the lies of bat 
bacilli killed by heat, and injecting the mixture into guinea- 
pigs, that death took place as in control animale whieh had 
received these toxic agents alone, Pfeiffer also found that by 
using the serum of fmmunised outs, he could, to a certain 
‘extent, protect other animals mers the subsequent ie 
‘of virulent living typhold bacilli, On trying to use the 
in a curative way, te. injocting it only after the bacilli 
begun to produce their effects, he got little or no result. 


mee of the B, coli and fis Relation to that of the 
ius. —We have already sean that the b. coli is probably 
pe Hoag rhe ceourrence a co nies ey 
vil Teves wisi apparen cause of 901 

ult Kholere. noeleasy of Tyla deer of some foe 
Tes nurobers in the intestine are Fu increased during 
weer, and also during any aes ition alfesting the 

Intaitins. Intraperitoneal snjection in guined-jige i aMen fatal Su 


taneous Injection may reynt loa Feleods Sa tecsstaie death 
Sanareli found that the be coll Snolated from typhoid 


yas wuch more virulent then whesDotate” 
ic Ga "HC SSN TRa the inavsuas fr vietooos bras Tate 
typhoid toxins. This increased virulence of the . ooli in the 
Syphoid bitestine makes it possible that sorve of the pathological changes 
ta typhotd may te duc, not to the typhold teellas bat to the bet 
Some of the geonral symjtoma tus bo intemal! by the abmarption of 
fondo products fered by it nord by other oryaninmas I ie to be uoted 
that lesions produced in guinea-pigs aro very similar to these of the bk 
heaus Differences of Webaviour of tho two baelll in connection with 
pathological effets have boen brought forward as confirmatory of 
the fee Foot their boing distinot specie, “fvos Sanatelli wocstemecl te 
intestinal mucous membrane of guinea: a to toxins derived from an old 
culture of the b, ex, by futraducing day by day small yuantitice of the 
Intter into themtomach. Whe tively lange dene could be tol 
feipes vend that tho itredoation in toe enna vay of # small quantity 
of Syplield toxin was sil followed by fatal rewlt. | PYsiter also foun 
‘while the serum of convalescents from typhoid paralysed the 
d bacilli, it had no more effect on kimilar numbers of & coll than 
serum of healthy men. 


——— vil 






























334 TYPHOID FEVER 


General View of the Relationship of the B. typhosus to 
‘Typhoid Fever.—1. We have in typhoid 
its centre in and about the intestine, ai 
niany othe “Tn 
inuiways"@ bocilta opi 
bacilli, copocally the b. coli which is a normal inhabitant of 
the animal intestine. ‘This bacillus can be isolated from the 
charucteristic lesions of the disease and from other parts of the 
body ou described, and further, it ie found by vulture and scram 
reactions to differ from are organisms. Here the important: 


point ix that a tacillus gi all the renetions of the t; id 
lias hin beett an la ~exmpt from cone opi 


fever, or under circumstances that make it oe 
bacillus in question to have heen derived fran case Scotian 
fever. 
2. A difficulty in the way of accepting the etiological ration: 
ship of the b. typhosus lies in the comparative failure of att 
to cause the dixeuse in aninuls. We have noted, however, aiae 
iW nature aniinale donot euifer from typhoid fever. 

3, The observations of Pfeifer and others on the protective 
[sis against typhoid bacilli shown, on testing in animals, to 
polong to the serum of salad patients and convalesconts, and 
the peculiar nction of such scrum in immobilising and causing 
clumping of the bacilli (vide infra) are alto of great. importance 
aa indicating an etiological relationship between the bacillus and 
the disease, Additional important evidence is found in the fit 
that vaccination by means of the dead bacilli (wide infra) has a 
marked eifet Ti preventing the disease from acising [na popula 
tion exposed to infection, and alao “Ta lowering” Cio-amGrtATTy 
when the fover atiadeF those who have been inoculate eae 
fucts may thus be accepted aa indirect but practically conclusive 
evidence of the pathogenic relationships of the typhotd bacillns 
to the disease, 

According to our present results we must thus hold that the 
b. typhosus constitutes a distinct Tee of bacterium, and 
that it is the cause of ty) (fe id fover, lence of an itportant 
nature confirmatory of this view ia, we tite found in the fact 
that cases have occurred where bacteriologists have wecidentally 
infected themselves by the mouth with pnre cultures of the 
typhoid bacillus, and after the usnal incubation period has 
developed typhoid fever, Several cases of this kind have been 
brought to our notice and are not, we think, vitinted by the fact 
that other similar instances have oourred without the subsequent 
development of illness. ‘These latter would be accounted for by 

































PARATYPHOTD BACILLUS 335, 


a low degree of susceptibility on the of the individual or to 
2 rato angst he S 





any serum clumps both the paratyphoid and the typhoid bacilli 
the more closely the maximal clumping dilutions correspond, the 
more likely {# tho case to be typhoid fever the other hand, 
if a high dilution will clunp tho yaratyphoid bacillus, while 
a low dilution is necessary for the typhoid bacillus, then 


imi va 














336 TYPHOID FEVER 


the caso is likely to be paratyphoid fever. With regard to the 
cflects of other scra on the paratyphoid bacillus, it may bo wid 
that usually a typhoid serum will require to be used in greater 
concentrations to clump this bacillus than arc necessary to obtain 
fan effect with the typhoid bacillus itsolf. Similar effocts are 
‘observed when the ser of animals immunised against Gaertuer's 
bacillus or the bacillus of psittacasis arm usd. Tn all sorum 
testa the essential point is that deductions should alone be based 
‘on comparative observations of the highest dilutions in which 
x clumping effect is produced with any series of organisms 


has boon indicated, a discase resembling typhoid fover is 
not the only condition originated by the parstyphoid bacillus. 
‘Tho organism has been isolated from eases of bone abscess, from. 
orchitis, and in Widal's ease from a thyroid abscess, and in auch 
cases the history of « previous typhoid-like illness may not be 
elicited. It as also been found in ordinary feces, Tn animal 
experiments it produces in rabbits and guinea-pigs a fatal ill- 
‘nesa of a aopticwmic type with serous inflammations, 

Bacillus Enteritidis (Gaertner).—In 1888 Gaertner, in 
investigating a number of cases of illness resulting from entinge 
the flesh of a dizeawed cow, isolated, from the meat and from the 
spleen of a man who died, bacillus closely resembling the 
typhoid bucillus. Since then a great number of outbreaks of 
gistro-enteritis doe 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 
yaratyphoid organisin,—indol is not produced, and generally 
speaking the fermentations of sugars ale correspond. With 
regard to the latter it may, however, be said that, according to 
some, lactose ix 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. Tt has also been found that the serum of persons suffer 
ing from meat poisoning sometimes clumps the typhoid bacillus, 
thongh a higher concentration is required than in the case of 
Guertoor’s bacillus. ‘The Gaertner group of organiama i very 
pathogenic for laboratory animals. Ofteu, whatever the channel 
of infection, there is intense hiemorrhagic enteritis, and very 
usually thero isa septicemia with the occurrence of serous inflam- 
mations; the bacilli are recoverable from the solid organs and 




















: PSITTACOSIS BACILLUS 337 
often from the blood. erent pelea arsartapthen malted 


membrane, some- 
base robe with haemorrhage into it; evidence of Siti eae 
Hon sy also exist. Infection may take aa by the 
foals itself whore moat has been insufticion' 


fn. ta an 
‘to the action of toxins; here it is important to note that the 
poisons formed by this group of organisma are relatively heat 
resisting, 80 that boiling for a time does not destroy the toxicity, 
Tn cases of Gaertner bacillus Polaoning, the animal whose 
has usually to have been itself 
suffering from the action of the bacillus, but cases of meat 
pottoning aleo occur where the meat of « healthy animal becomes 
Infected subsequently to slaughter with organisms pathogenic to 
man. In such cases these organisms are often varieties of the 
bk coli group, and indeed the b, coli itself may be the causo of 
meat poisoning. 


La 
tro} 


lo to ite action, = een ce oes a 
inea-pige, rabbits, mice, pigeons, 
te cl the alld olgtocs Poca 

pyar te bbe read arenes 

protable, from tho frathers being sotied 

eral sinall epidemics hare been 
vestigated After about ten days’ incubst 
ihn aoe followed by great rostlesstess, 

Aaron, and albuininurie: Frequently branch pomnnonia 
irene andl x fatal result hiss followed tn abont = third of the cases 
Gidarved "the organ haa been lola from the blood of tho rere 
he pinta lwctllus is evidently ue of the typhoid group, « fi 
“a further borne out bs baaevation that it ia clumped by a 
Beta tty at (asronl means haviag vo rerat).- The Sexytog 
fo ts incomplete, asthe bail between the clump may 
It differs from the typhoid taaillus in its grovt! 








333 TYPHOID FEVER 





Bix eats is Cae on ve a ‘that living and aes motile 
motility a oo a ay is 
of cansing ap Ln 
The principles on which the possession of the faculty lip and’ 
ulso its significance, are obsenre, and in the cage 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 bo applied have already 
boon described (p. 109), 

(1) It will bet thore acon that the loss of motility and elamping 
may be observed microscopically, Tf a preparation be made by 
the method detailed (typhoid serum in a dilution of, say, 1: 30 
having beon eraployod), and examined at onca under the miero- 
scope, the bacilli will usually be found actively motile, darting 
about in all directions. Tn 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 aim with 
difficulty be recognised, In a preparation similarly made with 
non-typhoid serum the individual bacilli can be observed separnte 
and actively motile for many hours. 

(2) A corresponding reaction visible to the nuked eye 
obtained by the “sedimentation test,” the method of sine 
which hus also been described (p, 111). Here at the end of 
twenty-four hours the bucilli form a nase like a preeipitute at 
the bottom of the mixture of bacterial emulsion and dilated 
typhoid scrum, while the upper part remaina 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 mieroecopic method, 
but useful as a contro! ; in nature it is similar, 

Such ix what occurs in the case of a typical reaction, The 
value of the method as a means of dingnosis 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 
suseoptibility depends, A race must therefore be sclocted 

















SERUM DIAGNOSIS: 339 


whieh gives the best result in the greatest number of undonbted 
casos acapella gives as little reaction as 
ible with normal sera or sera derived from other discases, 
latter point is important, as some mices react very readily to 
non pid be Again, cure must be taken as to the slate of 
the culture naod, The anitability of a culture may be impai 
by the conditions of its growth. Continued of 
race at 37° C. makes it leas suitable for use in the test, as the 
bacilli tend naturally to adhere in clumps, which may bo 
mistaken for thoes produced by the reaction, Wyatt Johnson 
recommended that the stock culture should be kept growing on 
Agar at room temperature and maintained by agar subcultures 
made once a month. For use in orplying the test, bouillon 
sob-cultures are made and incubated twenty-four hours at 
37° C. The relation of the dilution af the serum w the 
occurrence of es Fe is most important. It has been found 
that if the degree of dilution be too small a no: old serum 
may cause clamping. If possible, observations should always be 
nude with dilutions of 1:10, 1:30, 1:60,1:100, ‘To speak 
generally, the mors dilute the serum the longer time is necessary 
for a couplete reaction. Some typhoid sera have, however, 
very powerful agglutinating properties, and may in a com 
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 cither 
have been ered motionless or they may still be motile. No 
dis is is conclusive which is founded on the occurrence of , 
mae incomplete clumping alone. Seoing 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 indicutos a positive reaction. The general consonsis 
‘of opinion, with which our own experience agrees, ia that when 
4 serum in a dilation 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 fever, Suspicion should be entertained as to 
the a ag if « lower dilution is required, or if # longer time 
i" 


is 

"Tbe. fesetion given by the serum in typhoid fever usnally 
begins to be observed abont tho seventh day of the disease, 
though occasionally it has been found as carly as the fifth day, 
and sometimes it does not appear till the third week or later, 
Usnally it becomes ually moro marked as the disease 
advances, and it is still given by the blood of convalescents fre 





" A 





340 TYPHOID FEVER 


typhoid, but cases occur in which it may permanently disappear 
before convalescence sets in, How long it lasts aftor 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 
Ww a certain point, the renction is more marked where the fever 
is of & pronounced character, whilst in the milder cases it in lesa 
ee Tn certain graye cases, however, the reaction has 

n found to be feeble or almost absent, and accordingly same 
hold that a feeble reaction when the disease is manifestly severe 
is of bad omon, In some cases which from the clinical symptoms: 
were almost certainly typhoid, the naction has apparently been 
found to be absent. Snch casea should always be investigated 
from the point of view of their poaibly being paratyphoid fever. 

It has been found that the reaction is not only obtained wit 
living bacilli, but in certain cirenmstancea also with bhaeflli 
that have beon killed by heating at 60° C. for an hour,—if 
@ higher temperature bo weed, scnsitivences to agglutination ia 
impaired. ‘The capacity is also still retained if a germicide be 
employed, Here Widal recommends the addition oro 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 
ia given in cas 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 w fetus may have little 
agglutinating effect though that of its mother may have given 
a well-marked reaction ; sometimes, however, the fatal blood 
gives a well-marked reaction. [t 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. Typhowus 
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 maces 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, Tt bax also been found that other 
organisms belonging to the typhoid group (». p. 335) react ina 
lax way, The reaction as a mothod of distinguishing between 
these forms is thus not absolutely reliable, but in certain cases it 
ia of great value in giving confirmation to other tests The im- 
portant point here is the determination of the highest dilation 




















SERUM DIAGNOSIS 341 
with which clumping ts obtained. 


perenne 


ents ‘out thata 
or from those 






may occur, The most important+of these sources of orror is 
that with which diseasos caused by altiod nisms are 
concerned, as it ix | Saag that all the forms which theeo take 
in men have not mised, The very wide: application 
of the reaction has ‘licited the fact that it is given in many: 
case of Aight transient, and ill-defined febricule, which occur 
especially when typhoid fever is provalent. Some of these may 
beaborted typhoid, some may be paratyphoid. ‘There is no doubt 
what, if all the facts are taken into account, the cases where the 
reaction gives undoubtedly correct information #0 far outnumber 
thoge in which an error may be made that it must be looked on. 
as a most valuable aid to diagnosis. In conchision herw we may 
say that the fact of a typhoid serum clumping bacilli in 
mo way, 50 far as our present knowl goes, justifies doubt 
being cast on the specific relation of the typhoid bucillus to 
typhoid fever. 
Tx connection with the phenomenon that a scrum cither from 
normal person or a typhoid patient may elump several varieties 
of bacteria, some points arise. The theoreticnl consideration of 
tination ix reserved for the chapter on Mage ‘but here 

it may be said that agglutinating propertics may be 
















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 eapable of 
acting on that bacterium aj rin the esl but the serum 
become capable of agglutinating other, and especially kinds 
bacteria ; further, any normal ngglatinins for the infecting 
bacterinm present in the serum may be increased in amount. 
‘The agglutinin acting on the infecting organism has hoen called 
the primary or homologous agglutinin, while the others have been 
called the secondary or heterologous agglutinins. But besides 
‘what we know to bea fact, that infection by a single bacillary 
‘species can originate aggtatinins acting both on itself and on allied 
species, we must consider the possibility of infections by more 
‘than one species occurring in an animal, ¢g. b. typhosus with b. 
coli or b. parntyphoous, In such a case cach organism 
may Sate its primary agglutinin so that the prosence of 
multiple agglutinins in a rerum may really be an indication of 
a mixed infection, Some attention haa been directed to the 
diagnosis and differentiation of these conditions, Cuatellani 
has introduced « method for their Investigation, ‘This depends 
on the capacity manifostod by bacteria of absorbing the ag- 
qlutinins from a vorum. A small quantity of the saphatiaeieg 
serum, say “O 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, tho mixture is well shaken and est at 87° C. for 12 
hours. Clamping of course occurs, and the clumps fall to the 
bottom of the tabe, ‘The supernatant luid is pipetted off and 
is available for further teeta Castellani studied the primary and 
secondary agglutinins produced in infections in rabbits; he found 
that when an animal had been infected with b. typhosux this 
orgunisi would absorb from its serum not only the primary 
typhoid agglutinins but also euch secondary agylutinins ax thoes 
wcting on the b. coli, If, however, an animal had un 
infection with, say, both the b. typhosns and the b. coli, then the 
b. typhosus could not absorb from its serum the b. eoli (primary) 
agglutinin, Castellani thus put forward the view that by thi 
means primary could be differentiated from secondary petite 
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: Sale 
deductions can only be drawn when any serum is tested with 
several species of faitly 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, Tf 











VACCINATION AGAINST TYPHOID 343 


such precautions be adopted the absorption method ean be ntil- 
Tred fr Use dieroniiabinr of se Bokeel ead oe id organ- 
isms and their infections and for similar in’ 

Vaccination againat yptold—The petnciples of ‘tha, in. 
munieation of animals against typhoid i have i 
by Wright and Semple to man in the following way. 
bacilli are obtained of such virulence that a quarter of a twenty~ 
four hours’ old sloped agar culture when administered 
dormically 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 ix 
injected hypodermically, usually in the flank. ‘The vaccine now 

however, actually consists of a portion of a bouillon 
culture sini treated, The offects of the injection are some 
tenderness locally and in the adjacont lymphatic glands, and it 
may be local swelling, all of which come on in a few hours, and 
may be accompanied by a general focling of restlessness and a 
riso 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 lutination 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 tho vaccinated person possesses a degree of 
immunity against the bacillus, and this conclusion ix borne out 
by the results obtained in the use of the vaccine as a prophy- 
Inctic against typhoid fewer, Extensive observations have been 
mode in the British army in India, and in the South African 
War the efficacy of the treatwent was put to test, Though 
in faohated cases not much difference was ol Among 
those treated as compared with thow untrusted, yet tho broad 
general result may be said to leave Little doabt’ that on the 
one hand protective inoculation diminishes the tendeney for the 
individual to contmet typhoid fever, and on the other, if the 
dissase be contmeted, the likelihood of its having a fatal result 
is diminished. Thus in India of 4502 soldiers inoculated, “93 
per cont contracted typhoid, while of 25,851 soldiers in the 
tame stations who were not inoculated, YS4 per cent took the 
dimase. In Ladymnith during the siege there were 1705 
soldiers inoculated, among whom 2 per evnt of eases occurred, 
and 10,529 uninceulated, among whom 14 per cent suthered 
from typhoid. Weight has callceted statistics dealing in all 
with 49,600 individuals, of whom 8600 were inoculated, awl 











= 





Suh TYPHOID FEYER 


showed a case incidence of 2-25 per cont, with a case mortality 
of 12 per cent; in the remaining 41,000 uninoculated the case 
incidence was 5:75 per cont and the cane mortality 21 per cent. 
‘The best results seemed to be obtained when ten days after the 
first inoculation a second similar inoenlation 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 = 
temporary increased susceptibility to the disease, Ho therefore 
recommends that when possible the vaccination should be carried 
out some time previous to the exposure to infection, There can 
bo vory little donbt that in this method an important prophy- 
lactic moasure has boon discovered, 

Antityphoid Serum, Chanternesse hos immunised animals with dead 
cultures of the typhoid bacillus and having found that thelr vers bud 

otvetive and curative olfests in other auimals, ak ured euch sera 
a Inman aon of tyioid with apparent good result, In the bands 


of others, however, such a line of treatment has not heen equally 
successful. 


Methods of Examination...The methods of microseopic 
examination, and of isolation of typhoid bacilli from the spleen 
pot mortem, Wave ulready been deseribed. They may be isolated 
from tho Peyer's patches, lymphatic glands, ote. by a similar 
method. 

Daring life, typhoid bacilli may be obtained in eultare in the 
following ways — 

(a) From the Spleen.—Thia is the most cortain mothod 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, 
Tn a large proportion of eases of typhoid the bacillus nay be 
thus obtained, failure only occurring whon the noedle does not 
happen to touch a bacillus Numerous obesevations have shown 
that, provided the needle be not too lange, the procedure is quite 
safe, Tts use, however, is scarcely called far. 

(8) 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 chielly when there are groups in the kidney 
wubstance. For mothods of examining suspected urine, ste 

». 69. 

E (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 methodé—preferably in MacConkey's lactose bile 











METHODS OF EXAMINATION 345 


alt neuteal-red agar, or in the medium of Dri and Conrad 
( £2): After that period, thongh the con infectiveness: 

ee 
very difficult, We have seen after ulceration is fairly extab- 


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 aleers in a case of typhoid may be very few in 
number, it vident that there may be at no time very many 
typhoid tucilli in the intestine. ‘The microscopic examination 
‘of the stools in of course useless a8 a means of diagnosing the 
presence of the typhoid bacillus, 





Lrotation from Water Supp) great deal of work has been done 
on this subject. It is evident that if it is difficult to isolate the bacilli 
frou the stools it must « fortiori bo much more diflieult to dos when 
the latter are enormously diluted by water. The b. typhoaus hina, how- 
ever been isolated fom water daring opidemion ‘This was dene by Kien 
in the outbreaks in recent years at Worthing and Kotherbam. “The b. 
colt fs, at might be expected, the organiaa most common! 

such ciroumstances, 














toa C, betre 
10 ce. of 1 ger 0 
12hoursand then 


F proceed 
‘Oo the whole there is to be gained from this attempt to isolate the 
ecillus from water in any partiowlar caso, and it is much more 








B46 TYPHOID FEVER 


Bacrexta ty Dysesreny. 


Dysentery has for long been recognised as including « number 
of differont pathological conditions, and within more recent times 
ambic and non-amabie forms have boon distinguished, Of the 
latter bacteria have boon believed to be the caueal agents, and an 
organism described by Shiga in 1898 has almost certainly been 
established ax the canse af a large proportion of cases, Shiga's 
obsorvations were mado 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 Daval in tho United States, It is now further recogalsed 
that the epidemics of dysontory which from time to time occur 
in lanatic asylums are usually due to bacilli of this ee and in 
America the organiam has been demonstrated in the summer 
diarchosa of children. ‘The evidence for the relationehip of the 
organism {0 the disease consists chiefly in the apparently con- 
stant presence of the organism in the dejects in this form of 
dysentery, and the agglutination of the organism by the geram 
of patients sufforing 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 powscas common characters and 
are undoubtedly closely related to one another, The various 
strains resolve themselves into two ehief groups, whose differences 
lie in their behaviour towards cortain sugars, in their capacitios 
of originating indol and in their agglutinating reactions, The 
relation of amabw to dysentery will be discussed in the 
Appendix, 

Bacillus Dysenteriz: (Shiya).—Morphological Character, — 
This bacillus morphologically closely rescinbles the typhoid 
tucillus, but ixon the whole somewhat plumper, and filamentous 
forms are comparatively rare, Tnyolution forms sometimes ocour, 
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 Kemengen’s 
process, demonstrated in the case of one strain the presence of 
humerous lateral flagella, which ar of great finevess, but of 
considerable length. No apore formation occurs; the organism 
is stained readily by tho ordinary dyes, but ia decolorised by 
Gram's method. 

Cultural Characters, —n these wlxo considerable resemblance 
is presented to the typhoid bacillus. In gelatin a whitish line of 

growth occurs along the puncture, but the superficial filmlike 














== 





BACILLUS DYSENTERLAE ‘B47 
Siar te pra ont af an sor 


es at Oe the 

age, ‘ocoura as a smooth film wi ee em 
after two or three days, expecially if the eusfars be molar Volder 
and Duval describe an outgrowth of offehoota on tho 
surfuee of the medium. On agar plates the colonies resemble 
shoas 6 ts ee Bp big ee 


placed i 
th eg the may be classified into the Shigu-Kruse sup 

and the group. All produce acid in peptone glucove ani 
in taurocholate Eiyte eed none produce clunge in lactose or 
canemagar. Tha ha shige group do not produce acid in maltow or 
mannite, while ner group de, and, generally speaking, the 
former do not produce indol while the latter ae vorms inter: 
te betwoen the two groups occur, There ix never any 
SSR gaa observed in sugar media. In litmus mille thore 
is developed at first a alight degree of acidity, which is followed 
ly @ phase of ioral alkalinity ; neo coagulation of the milk 
ever foci, Anes the ‘onganiem forms & transparent or 
ee Joy fed, Rewer 3 in the course of  fow days assumes 
shes or dirty grey colour, with sore discoloration of 

ree potato at the margin of the growth, 

ition to the Diseore.—The organiem has been found in 
large numbers in the dejecta, especially in the acute cases, where 
be present in almost pure culture. In the thirty-six cases 
rt ined, Shiga obtained it in thirty-four from the dejecta, and in 
the two others post mortem from the intestinal macous membrane. 
‘The organism does not appear to spread deeply or to invade 
the general circulation. In the more chronic eases it is difficult 
to obtain on account of the large number of the bacillas coli and 
other bacteria present. Vedder and Duval found agar plates to 
be the best method of culture, these being inculated at the 
blood temperature. They also found that if the colonies whieh 
appeored at twelve hour were marked with a pencil, there was 
‘8 greater profubility of obtaining the lucillus of dysentery from 
Usose which appeared Inter, most of those appearing early being 
colonies of the bacillus coli. MacConkey's agar medism with 
Tnctose added may be used for isolation from stools. A Wthe & 


348 TYPHOID FEVER 


the feces is rubbed up in broth and some of the mixture stroked 
‘onthe medium. ‘The formation of acid by the coli colonies enables 
them to be excluded, and, therefore, as the b, dysenteria is not 
‘a lactose fermenter, the colourless colonies which develop after 
twenty-four hours ane picked out for further investigation. 

Asulready stated, both acute and chronic cases are 
by the presence of this organism. In the former, where death 
may occur in from one to mix days, the chief changes, according 
to Mlexner, are a marked swelling and corrugation of the mucous 
membrane, with hamorthage and pseudo-membrane at places, 
‘There ix extensive congulation-necrosi# with fibrinous exude 
tion and abundance of polymorpho-nuclear leucceytes, and the 
structure of the mucous membrane, os well as that of the 
muscularis mucosa, is often lost in the exudation, There ix 
also great thickening of the sub-mncom, with great infiltration of 
lougoeytes, these being chiefly of the ehameter of “plauma cells.” 
In the more chronic forms the changes correspond, but arc 
more of a proliferative character. The mucous membrane is 
gmoular, and superficial arcas aro devoid of epithelium, whilst 
ulceration and pscudo-membrane arc present in varying degree. 
A featare of bacillary dysentery is the fact thar abscess of the 
liver does not occur as a complication, 

Agalutination,—Al the above-nentioned observers agree re- 
warding the agglutination of this bucillus by the serusm—that is, 
in the cases of dysentery from which the organism can be eul- 
tivated. ‘The raction may appear on the socond day, and is 
most marked after from six to even 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 eomotimes much bighor dilutions 
give a positive result, In the more chronic casce the reaction 
is low marked, and here the sedimentation method is to be 
preferred. It is difficult to make any general atatementa with 
regard to the effects of dysenterie sera on the different strains 
of the bacilli, but it may be said that generally # serum 
agglutinates the strain which produced it and the other strains 
of the same group in higher dilutions than it docs the strains of 
the other group. Many observers bave found that the serum 
from a case associated with strains of the ShigacKruse group 
have not agylutinated strains of the Flexner group, and 
corresponding observations have been made in cases associ 
with the Flexner group, Often the sera of animals immunised 
with bacilli have been used for such tests, but apparently great 
caro must be exorcised in basing diagnoses on such observations, 
for some species of animals when treated with » particular atrain 








BACILLUS DYSENTERLA 349 
will yield a serum which is active against many strait 
ait olor mpectan will) whan "Seomouised “with fot tani! 
ee ee ee has not been obtained with serum 


Pathogenic Propertics.—The organism is pathogenic in guinea 
pigs and other Intoratory simaie bnt, in these, charucteristic 


obtained lly 

— when the virulonce of the organiem has been previously 

ted by vitoneal passage. In two cases, Larne tigicr 

well in laeaarcg ea a dysenterie condition haa followed in the 
human subject from ingestion of pure cultarvs of the organixm, 

Be Sable: that’ in the action of the bacilku a toxin is 

concerned. Te has been found that the filtrate from three weeks! 





the surface of the mucous membrane. According to some obser. 
vers th toxin is more readily obtainable from the Shiga-Kruse 
stmins than from the Flexner strains The toxin is fairly resist- 
ant to heat, standing temperatures up to 70° €. without being 
From the fact, that by the maceration of cultures 

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. 
Iimuniation Erperiments,—Both large and small animals 
have boen immunised against the bacillus and also against its 
tore filtrates, In the former case the immunisation has been 
commenced either with non-lethal doses of living cultures or with 
enltures Killed by heat. Tho nature of the immunisation ix 
complex. When cultures have been used, a bactericidal 

“rum, which iminuine bodies and complements («ae Emmunity) 
are conosrned, ixdeveloped. Whea tho toxin is used for immun- 
iration a serum protecting against the toxin is produced. Ac 





cording to some results animals immunised with cultures are 
immune and vice vera. All races of animal 
do not imunisation. Large animals (horses, 





goats) have been immunised with the toxin with » 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 
haa been produced by the subcutancous ailministration of the 
serum, ly in early eases of the diseaso, 

Tt will be soon that the evidenco furnished ia practically 
conclusive as to the causal relationship between this baci/los and 
one form of dysentery, a form, moreover, which ix both wide- 
spread and embraces a large proportion of cases of the disease ; 
and cspecially of importance is the fact that observations made 
independently in differenb countries have yielded practically 
identical resulta on this point. 


Bacillus (Ogata) Ogata obtained this tues ix an 
extonsive epidemic in Japan in which no amebe wore prosent. Ho 
found in xections of the atfected tixeies enormous numbers of small 
Dactili of about the same thickness ax the tubercle bucillns, but very 
much shorter, ‘Thoxe bacilli were sometimes found in a protically pure 
condision. ‘They wore actively motile and could bw stained by 
method. Te also obtained pure caltures from various cases and tested 
thelr pathogento offcts, They grew well on gelatin, at the ordinary 
fomperaturo producing liquefwstion, the growth aomowhat resembling 
that of the cholera «pirilium, By injection into cats and guinea. Figen 
well as by feeding them, this organigm was found to have distinet 
pathogonie ffcia: these were chiefly confiued to the large intestine 
wnorrhagic inflammation and ulveration beng produced, It still 
remains to be determined whether this organism has a causal relation: 
ship to ono variety of dysentery. 














Bactunws Esrentrims Sronocrsrs, 


This organism was first isolated by Klein from the evacuations in an 
outbreak of diarrhow following the ingestion of milk which contained 
the microbe, and it was aubscquently found by him in certain cases of 
infantilo diarthaa and of sumer diarrhea, in certain instances in milky 
‘and as a constant inhabitant of sewage (soo Chap, 1V,). In films made 
from the stools in diarrhcos eases wiere it is present it ean be miloro- 
scopioally rocognikod ax a bacillus 1°6 u to 4°84 in length and °8 w 
Ureadely, staining by ordinary stains aud retaining the dye in Gram's 
method. It often contains « spore near one of the ends, or sometimes 
neater the centre. It is slightly motile, and in cultures can be shows to 
Powwose a wmall number of terminal flagella, It grows well under 
Anncrobi¢ conditions in ordinary media, capecially on thove containing 
reducing agonta. On agar the colonies are ciroulst grey, and translucent, 
and onder a low power are seen to have a granular appearance. On ti 
medium apore formation does not occur, but ia casily obtained if the 
orguniom is grown on solidified blood serim, which, further, i liquefied 
during grovth, Qu gelatin plates liquefaction commandos after twent 
four hours at 20°C, “It produces aoid and gas in bile-salt glacoes media, 
and in poptone salt solution containing glucoss or mannite. Spore 
formation can b# seen to take place in 2 per cont doxtrone golatin, bOE 
the degree seems to be in inverse ratio fo the amount of gas formation. 
Very typical ix the growth on milk, and it is by this mediom that 
fyolation can bo best olfected. A ‘small quantity of the material 















































SUMMER DIARRHEA 351 


ted to contain the bacillus ie placed in 15 to 20 o.¢, of sterile 
re ehih ie thn hosed for ten minutes at 80° C. to destroy all 
tive a 4 the ae is cooled, placed under aa con 


ei 
1 the tacllus bo preweat there is abundant us formation, ire 
‘complete ‘of tho curd from the whey takes place." The former 
ies of ths tuba i ahenis Inge masse gather with 
top of the fluid, all being torn by the gas evolved, 
slightly turbid and contains numerous q 
ar of butyric acid. Ifa small quanti (any 
hey br Infos eiuea-pig, the euttisl Bocas 
aud dies in rer. four hours. At 
shinend subestaneous tsonoy and vometines 
fare greed and gengrenone and vvilcmelling, there da considerable redone, 
and there may also be gas formation. Tho exudation t crowded with 
boul, whieh Howorv,arw not gwuerally atribaed in any tubes 
thronghout the body.’ ‘These pathogenio projertios of the bacilli 
futerttdis sporogencs are important In its recagntion, for it culture 
eset are very sianilar to those of the bacillus butyriens 
itt, 










Susann Drarniaa, 


‘As hns boon already stated, both tho bacillus of dysentery, b. coli 
and the b. enteritidis sporogenes have been found associated with 
epidemics of this disense, This indicates that the condition may 
be originated by a varicty of organisms, and it ia 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 diseaso in 
Britain, bas been unable to find evidence of the dysentery 
bacillus being prosent. He has, however, very constantly found 
in the stoals and intestine a bacillus (* Morgan's Na. 1 bacillus") 
which isa motilo Gram-negative crganisw producing acid and 
alight gas formation in glucose, levulose, and galactose, and no 
change in mannite, dulcite, maltose, dextrin, cane sugar, atest 
fnulin, amygdalin, salicin, arabinose, raffinose, sorbite, o 
thrite; it further causes indol formation, and in Litmus mil ilk 
slowly’ Originates an alkaline rewction. Tt produces diarrhoea and 
death in young rabbits, rats, and monkeys when these animals 
are fed on cultures, It is thua possible that in thia bacillne 
wwe hays till another cause of the dseus 











GHAPTER XY. 
DIPHTHERIA. 


Tue is no better example of the valuable contributions of 
bacteriology to scientific medicine than that afforded in the ease 
of diphtheria, Not only haa research supplied, oa in the ease of 
tubercle, 4 means of distinguishing trac diphtheria from condi: 
tions which nyaomble it, but tho study of tho toxins of the 
bacillus has explained tho manner by which the pathological 
changes and characteristic symptoms of the disease are brought 
aboat, and his led to the discovery of the most efficient means 
‘of troatinent, namely, the anti diphtheritic serum, 





Bistorical.—The first secount of the bacillus now known to be the 
couse of diphtheria was given by Klebs in 1863, who described its 
characters in the falas membrane, but made no cvltivetions. It was 
firwt cultivated by Lofflor from a numbor of cases of diphthoria, hie 
observations being published in 1884, and to him we owe the first 
acconnt of its characters in cuitures and of some of its pathogenic effects 
on animalx The organism in for these rrasons known as the Klebse 
Uitier incillus, or simply ox Lafer’s bacillus, By oxporimental: in- 
tion with the oulures obtained, Tiitfler was able to produce false 
membrane on dainaged mucous surfaces, bat ho hesitated to eonelude 
dofinitoly that this organisin wax the cause of the disease, for be did 
not find it in all the caves of diphtheria examined, he was not able to 
reduce paralytic phenomena in animals by ite injection, and, farther, 
ie obtained the same organism from the throat of a healthy child. | This 
organism became the subject of auch inquiry, but ite relationship to 
the disease may be said to have bean definitely establiehed by the 
brilliant rescarches of Roux and Yersin whieh showed that the most 
important features of the disoase could be produced by mosne of the 
soqurated toxins of the organiatn, Their experiments wore putilishod in 
1888-90, Further light ts boen 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 verve poisons, and 
aleo produce other phenomena mot with in diphtheria, 


General Facts.—Without giving a description of the patho- 
Jogica) changes in diphtheria, it will be well to montion the out- 


aoe 



















































BACILLUS DIPHTHERLA ‘ 353 


standing features which onght to be considered in connection 
with its bacteriology. In addition to the formation of falae 
Sean may prove fatal by Beery only effects, the 





ing paralyses wl ‘occur in disease, 
and which affect the muscles of the 


8 ess male ing rometinnen of 
paraplegic ), these ing grouped together under tho 
term th * “Tt may be stated here that all 


these conditions have been experimentally reproduced by the 
action of the bacillus of diphtheria, or by its toxina, Other 
bacteria aro, however, concerned in producing various secondary 
inflammatory complications in the region of the throat, such as 
uuleoration, gangrenons epi ‘and suppration, which may be 
accom by symptor roan ERR sabe ‘The dotec- 
nee tt La he a ibranc or secretions 
mouth is to as supplying the only certain 

means of en of apn en a 34 
—f a film 


Microscopical Characters. 
preparation ieee tee from ¢ a piece of diphtheria membrane (in 
the manner deseribed below) and stained with methylene-bh 
the bacilli are found to have the following ebaractors. They are 
slendor straight or slightly curved, and usnally about 3 « 
in length, thefr thickness: being @ Tithe greater than that of 
the tubercle becillua. Tho im, however, varies eomewbat in 
different cases, und for this reason varieties havo boon distin- 
guished as smal! and large, and eveo of intermediate sive, It is 
sufficient to mention here that in some casea most are about 3 je 
im length, whilst in othors they may measure fully 8 gp, Corre- 

differences in size are found Een ee They stain 


sponding 
dee ith = blue, sometimes ee formly-solaused, bat 
an i their substance, aa 
» 1k dotted br bead ‘appearance ts 
Sometimes the guds ars swollen and more darkly stained. than 
the rest ; often, however, they are rather tapered off (Fig. 116). 
In somo cases the terminal ewelling le very marked, so aa to 
amount to clubbing, and with some specitnens of methylone-bluo 
these swellings and granules stain of a violet tint Distinct 
as however, is Joes frequent than in cultures, ‘There fs a 
‘uniformity in the appearance of the bacilli when com- 
Fast cabs by side, They usually lie rly scattered or in 
clusters, the individual bacilli being a Thal directions. 
Some may be contained within leucocytes. They do not Corum 
co 








304 DIPHTHERIA 


chains, bnt o forms longer than those mentioned may 
Tl Aa Goa eli eee tae prec area 
fibrin as seen in 








Fi, 116,—Film proparation from diphtheria membrane ; showing mumerons 
Aiplitheris bacilli, One or two degenerated forms axe seen tear the 
contre of the uli, (Cultures made from the sume piece of membrane 
shores the cepa to Ne Gren a psstclly ae endian) 

Stained with Cabylene. i 


It may be mentioned that distinctions formerly drawn between 
true diphtheria and non-diphtheritic conditions from the appear- 
ance and site of the membrane, an no selentitic value, ih 


trne criterion being the dipht! 
Decurrence of a membranous Taroalia produced by streptococci 


Is already beon mentioned (p. 184), 

Tn diphthoria the membrane has a somewhnt different 
structure according as it ia formed on the surface covered with 
stratified squamous epithelium as in the pharynx, or on a surface 


DISTRIBUTION OF THE BACILLUS 856 


covered by ciliated opithelium us in the trachea. In thé former 
situation necrosis of the epithelium occurs cither uniformly or in 


reaction in the connective tissue beneath, attended by abundant 
fibrinous exudation, The nocrosed epithelium becomes raised 
‘up by the fibrin, and its interstices are also filled by it, The 





ig, 117,—Seotion through a diphtheritio membrane in trachea, showing Leet 
‘theria bacilli (91 daxk 1 as 8 ees 
fibrin, Some atroptecocel are also shown, towards the surface aa a 


Strined by Gram's method and Biemarck-brown, x 1000, 


fibrinous exuilation aleo occurs around the vessels in the tissue 
beneuth, and in this way the inembrane is firmly adherent. In 
the trashos, on the other hand, the epithelial cells rapidiy 
tecome shed, and the membrane is fount to prong most 
exclusively of fibrin with leucocytes, the former arranged in a 
reticulated or somewhat laminated manner, and varying in 

density in different parts, ‘The mombrane lies upon the base: 
ment meinbrane, and is less firmly adherent than in the case of 
the pharynx. 


or i 


lar the 
a 7 
(Fig. 117). here they Woy in a practically pure ion, 
thongh streptococe! and stn: 
be it wlong with them, They may occur also deeper, but 
ive rarely” Taint Ti the fibrin around the blood vessels. ao the 


surface of the membrane they smay-be aleo- sen I 
numbers Foacumvay care 


organisms. “Optacianally. sem alll bags Cae aedostod fo ts 
Iympbetic gland Ax Toffler ‘fi described, they may be 
found after death in pneumonie patches in the Inng, these 
Being duo ton aseondary extension by the si They 


have also been oceasional leon, liver, and 
other organs after death. ae Ba 1s prol 

be ex] SAS) ‘an entrance into the blood stream erly 
before death, similar to what occurs m the ose of other 
organinns, eg. the bacillus coli communis, The diphtheria 
tmeillus may also infect other mucous membranes. Tt is found 
in true diphthoria of the conjunctiva, and May also occur 
in similar affections of the vulva _and vagina; some of these 
cases have been treated successfully with dh with Mphtheria antitoxin. 
‘Tho paeudodiphtheria bacillus, however, may also occur in those 
situations, 


Aascciation with other Organiems—The diphtheria organism ix 
sometimes present alone in the membrane, but more_frequently 


is ogsooiated with some pyouenic organism, the atrepto- 
cgocua pyogenes being the err ene atapbylococed, api 
cccaxionally the pneumococens or the bacillus eoll, may. be 
preaont in some casea, Streptococei are often found lying wide 
y side with the diphtheria bacilli in the mombranc, and alee 
penetrating more deeply into the tienes Ta some cass of 
tracheal diphtheria, we have found streptococe’ alone at a lower 
lovel in the trachea than the diphtheria becilli, where the 
membrano was thinner and softer, the appearance in these eases 
being as if the atreptococel acted as oxciters of inflammation and 
prepared the way forthe bacilli, It iv atill a matter of dixpate 


as to whether the association of tho diphtheria bucillus with the 
pyogenic ongunigus is « favourable sign or the contrary, themgh 


on experimental grounds the latter is the more probable, We 
know, howerer, that some of the complications of diphtheria 
may be dae to the action of pyogenic organisms ‘The extensive 


swelling of the tissnes of the neck, sometimes attended by 

















CULTIVATION OF THE BACILLUS So7 
ition in the glands, and also various haemorrhagic con- 


suppurat 
ditions, have been found to be as- 


sociated with their presence ; 1 THe, 
in some cases TES Tiphtlsritic lesion 
eeabien thea get a foothold in 
the times, where they exert their 
usual action and may [oud to exten 
sive suppurative change, to septic 
poisoning or tosepticwmia. In cases 


ng 
ane ai uc ome 
tion produced by other organisms 
anti-dy a no 


fa 
Cultivation — ‘The diphtheria 
bacillus grows best in oulturesat the 


deny re fe body; growth 
Ril ake prea TC Dut nme 
about 20°C, The best media aro the 
followin, Syatters original median 








Fu. 118,—Cultures of the 
Aiphitheria becilius oa au 
spr plate; tweaty-aix 

hows’ growth. 

(6), Tye pacar tree 0 

‘counion frat the athe 





Lrg 


ze: 
{p. 40), solidified blood serum, alkaline blood serum (Lorrain 


ud. 11 G—dviphtheris bactllt from s twenty 
four 








Smith), blood agar, and 
the ordinary agar tmodia. 
Tf inoculations be made 
on the surface ef blood 
serum with a piece of 
diphtheria membranes, 
colonies of the bacillus 
may appear in twelve 
‘hours and are well formed 
within twenty-four hours 
often before any other 
growths are visible, The 
colonies are small circular 


Tees of opaque whitish 
golour, their contre being 
thicker and of darker 
greyish appearance when 
viewed by transmitted 
light thin the periphery. 
On the second or third 


day they may reuch 3 im. in size, but when numorous they 





358 DIPHTHERIA 
remain smaller. On leila og oper 


the same appearance (Fig. 
117)bi Tes 
n) at grow ed 


nes. In stroke eul- 
tures the growth forms a 
continuous layer of 

same dull whitish colour, 
the margins of which often 
show single colonies partly 
car completely xeparated. 
On gelatin at 29° Ca 


(paearrere 
ine of 
Fig, 120.—Diphtheria bacilli of larger size ne wi at 
Sais le ae Ce Taal 
of protplan re 
Desatiee sestiaiees “rather thicker in the 


Stained with weak carbol-fuchain, . x 1000, pee Tn Sarg 
an secur. In bouillon the organism luces a turl 
which soon sottles to Mee sapiens 
bottom and forms a 
tay ay oti ell 
tho vessel. By starting 
the growth on the surface 
and keeping the flasks at 
rest a distinct seum forms, 
and this ia capecially suit- 
able for the development 
oftoxin, Ordinary bouillon 
becomes acid during the 
first two or three days, 
and several dayw later 
again sequires an alkaline 
reaction. If, however, the 
bouillon is’ glucose: free rarity fe 
© acid reaction ig, 121.—Involution forms of the diphtheria 
(p79) the aid rection, 12— natin te of he ih 





4 cy; days growth, 
Inthesemediathe bacilli geained with earbol-thionin-blue x 1000, 
show the same charactors 


asin the membrang, but the irregularity in staining is more marked 





|__sie 


POWERS OF RESISTANCE OF BACILLUS 359 


119, 120). They are at first fairly uniform in size and 
Dut later involution forms are Many are 


‘eng 
stained line, 
iming.—They. take uy o ie eg. mothy- 
Tentise watery solution, ie og ae 
Ba Senet beg Ne Nip geen ght 
deseril They also rot Sho colour in Grams mothods 
in 


almost tae, 





iecipersture of 96°C foran hour, Dried dipkthera membrane, 
kept in the absence of light and at the room temperature, has 
boon proved to contain diphtheria bacilli still ae and viralont 
st tho end of several montha. ° The presen 






Corresponding Tascies 
oakliarie andiboers on died: taal” Thees aa Passi 
pene es aera ae of A importance, as they show that 


contagium of diphtheria may be preserved for a long time 
in the dried membranc. 

Bifects of Inoculation.—In considering the effects produced 
in animals by experimental inoculations of pure cultures, we 
have to Koop in view the local changes which occur in diphtheria, 
and also the symptoms of general poisoning. 

As Liifflee stated in his original paper inoculation of tho 
healthy mucons membranes of varions animals with pure cultures 
causet no lesion, but the formation of false membrane may 
result when the surface is injured by scarification or otherwise, 
Se similar result may be othalned when the trachea is inoculated 

my has been performed, In thie case the 
phate tissues may become the scat of a blood-stained 








360 DIPHTHERIA 
cadens, and the Iymphte lenis become thi 
e tymp! doy ene ve general 


ve el eka a is usally las 


negative results, though sometimes a few colonies may be 
obtained. If a habia dose of a culture be injected, a local 
necrosis of the skin and subcutancous tissue may follow at the 
‘site of inoculation. 

Tn mbbits, after subeutancous inoculation, results of the mame 
nature follow, but these animals are less susceptible than guinea 
pigs, und the dose requires to be proportionately larger, Roux 
und Yersin found that after intravenous injection the bacilli 
rapidly dimppeared from the blood, and when 1 cc, 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 toxwmia, 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 bigh degree of immunity. 





Klein found that cats alo were susceptible to inoculation, The 
snimals usually div after a few days, and post mortem there Is well-marked 
nopliritix, Ho aleo found thot after subeatancous injection in cows, « 

lar eruption appeared on tho teate of the udder, the Maid in which 
ned diphtheria hacillf. At the time of death the diphtheria bacilli 
‘wore still alive and virulent at the site of injection. ‘The moat striking 
result of these experiments x that the dipht 
circulation and were present in the eruption on the udder, Te consi 
that this may throw light on certain opidemice of diphtheria in which 











pal 


THE TOXINS OF DIPHTHERIA 361 


the contagion was the milk, Other observers 
have, however, faite to obs Hal ele aeale es an Todd, in 


renigaling an oothresk of diphthorka 

ieraad Spenco cretteon on vor eeie nee erin in which nic pie is 

Saellt wero prsent. They, however, ome ceabetory 

‘cll are nok the cause of the hat Sere tie ant seas 

secondary contamination, (wich fem the: vee eal of the milkers. | The 
the diphtheria bacillus in the horse 


Pid sox ot Dobe in a shore experiments 
symptoms: poisoning: ultimat a fat It occur 
eerie nels diminishing in number, or even after they 
have practically disappeared, Roux and Yersin on Bat fe tho 
ied ome a bso sok Reet 
to be correct. we it 
th fread fromm blll by filtration ies 


tee The ats when 
epeetan gm se ates an ie living boss 
is fibrinows exudation but a considerable amount 








phenomena 

-may occur. Tho hind limbs are aan! nifeeted first, the 
paralysis afterwards oxtending to other sometimes 
the fore-timbs and neck ‘int show ee Sometimes 
symptoms of paralysis do not appear till two or thee wecks 
after inoenlation, After paralysis hax appeard, a fatal result 
usually follows in the elle animals, bat in doga recovery may 
take There is evidence that these paralytic phenomena 
are produced by toxone (p, 171), ax they may occur when there 
injected along with the toxin sufficient antitoxin to neutralise 
the more rapidly acting toxin proper. This toxone is snyy 
by Ehrlich to have a different toxic action, ie a di it 

orows group, from that of the toxin, and to have a weaker 
alfinity for antitoxin ; much of it may thus be left unneutralieed, 
Tt ls to be noted ix this connection that paralytic symptoms are 
of not uncommon occurrence in the human subject after treatment 
with antitoxin, the explanation of which occurrence ie probably 
the same os that just given. One point of much interest is the 
set degree of resistance to the toxin posmsed by mice and 

Roux snd Yersin, for example, found that 2 ec. of toxin, 
Thich was sufficient to kill a rabbit in sixty hours, had no effect 











362 DIPHTHERIA 


on & mouse, whilst of this toxin even yy c.c. produced extensive 
pened the akin of the guinea-pig. 

Preparation of the Toin.—Tho obtaining of very active 
toxin in large quantities is an essential in the ration of anti- 
diphtheritic serum. Certain conditions favour the development 
of a high degree of piety, Vie 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 f favoured. This expedient ix 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 glacose—an 
allimportant point—may be attained by the method described 
above (p, 75), or by nsing for tho preparation of tho meat extmict 
flesh which is just commencing to Patrefy (Spronck). L, Martin 
noses medium composed of equal parts of fresbly prepared 
ee (by {oy digeting Pigs’ stomachs with HCI at 35° ca and 

bouillon. By this medium he has obtained « 
an ‘yky 60. 18 the fatal dose toa guinea-pig of 500 
grma Ho finds that glucose, glycerin, saccharose, and 
Tead to the production of an acid reaction, whilat glycogen docs 
not, The Jatter fact explains how some observers have found 
that bouillon propared from quite fresh flesh is suitable for toxin 
formation. ‘There ia in all cages a period at which tho toxicity 
reaches a maximum, usally in 2-3 weeks, but earlier if the toxin 
is rapidly formed ; later the toxicity diminishes Martin found 
that to his mediaim 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, aud 
that inany may require to be tested ero one suitable is obtained, 
Properties and Nature of the Tovin.—Tho toxic eubstanee it. 
filtered cultures is a relatively unstable body, When kept in 
sealed tubes in the absence of light, it may preserve ite 
little altered for several months, Bat “outa other ay 
gridually loses therm when exposed to the action of light and 
air. As will be shown luter (p. 473), the toxia probably does not 
“Become destroyed, but ita toxophorous group suffers a sort of 
deterioration so that a toxoid is formed which has still the 
power of combining with autitoxins, Heating at 58° ©, for 
two hours destroys the toxic properties in great part, but not 
altogether. When, however, the toxin is evaporated to drynenms, it 


NATURE OF THE TOXIN 363 


has much greater resistance to heat, One: fact, discovered 
‘by Roux and Yersin, is that after an organic: ‘such as tartaric 
pal epapeoteer perience tant herr perp 
can restored by again Inid alkaline, 
shows Ons taste wes forud ty the baci when 


ener 
a proteid, it may be formed by synthesis within the bodies of the 
tweilli, Brieger and Boer have separated from diphtheria cultures 
1 toxic body which gives no proteid reaction (vide p. 166). 
‘Toxic bodies have also been obtained from the tissues of 
who have died from diphtheria Roux and Yersin, by using 
watery extrict from the spleen from very virulent cases 


urine, ‘The snbject of toxic bodies in the tismes haa, 
ees specs worked out by Sidney Martin. Ho 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 tho latter), and an organic acid. ‘The albumowes 


when injected into rabbits, especially in 1 doses, produce 
fever, diarrhea, paresis, and loss of with ultimately a 
fatal result. As in the exporiments with the toxin from cultures, 


the posterior limbs are first affected ; afterwards the respiratory 
mnsclos, and finally the heart, are implicated, Ho further found 


‘that this ia is due to well-mar! Sa ee 
The tooditlary abcoth fist become affected, breaking up into 
3 tee ‘the axis cylinders are aie and may 

© that tion occurs in peripheral 
i foatety tines Beal changes occur irrogalarly in 
sensory and motor fibres being alfected. Fatty 
place in the associated muscle Gliwe ‘There may 
‘a similar condition in the cardiac muscle. The organic 
4 similar but weaker action. Substances obtained from 
membrane have an action like that of the bodies 
1 Uschinsky's modiam hes the following composition : water, 1000 parts ; 
‘30-40; sctiam chloride, 5-7 ; calcium chloride, ") ; magnesia 

“24; Gipotaxsium phosphate, “2°25; ammonium lactate, G-T ; 

sparagiuste, 3-1. 


— Pe 


ii 


H 
E 


i 
E 


t 





364 DIPHTHERIA 


obtained from the spleen, but in higher degree. Martin con- 
widers that this is due to the presence in in the membrane of an 
enzyme which has « proteolytic action within the body, resulting 
in the formation of poisonous albumnoses, According to. this 
view the actually toxie bodies are not the direct product af the 
bacillus, but are formed by the enzyme which is produeed by it 
locally in the membrane. Cartwright Wood has also found 
when diphtheria cultures in an albumin-containing mediun are 
filtered germfree and exposed to 65° C. for an hour (the mpposed 
forments being thus destroyed), there still remain albumoses 
which produce febrile reaction and ure active in developing: 
immunity. In the present state of knowledge we are not in a 
position to give an interpretation of such experiments, and wo 
cannot even say whether the proteids obtained by precipitation 
from cultures und from the tissues ure in themselves toxie, or 
whether the true toxie bodies are carried down along with 
thom. 


Immunity.—This is described in the general chapter on 
Tmmunit It is sufficient to state here that a high degree of 
immunity, againet both the bacilli and their toxins, can bo 
produced in various animals by gradually increasing doses either 
of the bacilli or of their filtered toxins (rite 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 tho bacilli were grown at an abnormally high temperature, 
namely, 39°5" ©, and in a current of air, the virulence diminished 
so much that they became practically insocuoux. When the 
virulence was much diminished, these observers found that it 
could be restored if the bacilli were inoculated intoanimals along 
with streptococci, invculation of the bucilli alone not being 
successful for this purpose. If, however, the virulence had fallen 
yery low, even the presence of the streptococci was insufficient 
to restore it. As a rule, the cultures most virulent to guinear 
pigs are obtained from the gravest eases 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 
whieh 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 wooks, though they often 
quickly disappear, Roux and Yorain found, by making cultures 











BACILLE ALLIED TO DIPHTHERIA BACILLUS 965 


at various stages after the termination of the diseass, 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 cc. of a 24 hours’ 


Bavilli allied to the Diphtheria Bavillus, 
Bacteriol: examinations carried on within recent times 


“ paeado-diphtheria bacilli” and ‘*diphtheroid bacilli have been 
applied in a loose way to organisms which resemble the diphtheria 
bucillns microscopically, expecially as reyards the headed ap- 
pearance. Such Sine iar, Shei eteaet FEA pi 
‘nose, skin, organs, and even from the blood in certain 
dissases, oan to be suet with sometimes in conditions of 
health, and they have ‘been obtained from many diverse morbid 
conditions—from ukin diseases, from coryza, from leprosy, and 
‘even from gencrul paralysis of the inmano, As bas beon found 
with other groups the differentiation is a matter of considerable 
difficulty. Some are practically identical with the diphtheria 
becillus both morphologically and culturally, and a fow oven kive 
the charmeteristic reaction with Neisser’s stain; others again 
differ in essentin) particular The fermentative action on 
sugars? has also been called into requisition as a moans of distin- 
‘them, but the results obtained cannot be said to be of a 
jefinite charucter, and further work is necessary, Tt may be 
stated, however, that most observers have found the diphtheria 
becillus of all the members of the group to be the most active 
acid-producer, though here the difference seems to be one of 
rather than of kind. The absence of the power of 
fermunting certain sugars, notably glucose, may, howover, be 
excepted in any particular case as sufficient to exclude the 
+ Vole o paper by Geshain Smith, Jewrnnl of Hygiene, vi 296. 


= 7 


DIPHTHERTA 
organism from being the diphtheria bacillus, From thee 
and from what 2h been stated with regard to 
bacilli, it will be seen beeper iene asf 

suspected: may in some 
practical impossibility, Itmay ‘hat sona af the 'diphtherstd® 
Cee cultivated have really been uon-virulent diphtheria 

of this on the practical means of dlagnodit 
wi be 


ieee 
‘The term “ Poadodiphiberis bacillus” is often restricted by 
wat writers to an organixm frequently met with in the throwt. 
Tie hie organist, which isaleo known as Hofmann's bacillus, morits 
a separate description, 
Hofmann’s Bacillus —Pseudo-Diphtheria Bacillus. —This 
organism, described by Hofmann in 1888, is probably i 









previous 
yeur, and regarded by him 
as being a distinet 
from ‘the ‘iphtheria nl 
Jus The organism is a 


diphtheria Tatas, i with 
usually a single unstained 

septum running across it, 
though sometimes there 
Oy. 122). the typ 
(Fig. i 
Tadd uppoerance isvarel) 
ween, and the reaction wil 
Neisser's stain is not given. 

Pio, 199,—Peendo-diphthoria bacillus Tt grows readily on the 

(Hotmann’s). Young agar onitare. same modia as the dij 

‘Stained with thioniatiae, «1000. theria bacillus, bat the 

colonies are whiter and 
more opaque. Tt does not form acid from glucose or other 
sugars, and ix 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 organiam from the 
diphtheria bacillus. 

Hofmann's bacillus ia 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 classea, Cobbett found it 167 times in an 
examination of 692 persona examined, of whom 650 were not 





The opinion is that the two organisms are distinet species 
which are comparatively easily oan charactors. 


jak—Thie term hae fan organism int 
Pocgt rKeuhber iad Wobwer fa careels nt ths qinjSantin, aed 
which has been since found in many other affections of the Sea 





salight extent ; ay tan abe diet Si 
pa. Lieadcphoingtal marie es in Fig. 123, 


Foo, 125—Kereals bcilne from a yong 
ager culture, x 100 


the 

hence axlema and tendency to hemorrhage are produced 5 
thia action on the vessels iz also exemplified by the general 
congestion of organs, The hyaline change in the walle of 
arterioles and capillaries so often met with in diphtheria ix 
another example of the action of the toxin. The toxins have 








368 DIPHTHERIA 


also 4 perniclons action on highly-developed cells and on nerve 
fibres, ‘Thus in the kidney, ch eit awelling occurs, which may 
be followed by actual necrosis of the wil Ds cells, and along, 
with these changes albuininuria ts present, aetion Js alao: 
woll seen in the case of the muscle fibres of the heart, which 
roay undergo a sort of hyaline change, followed by. ir it 
ion or by an actual fatty degeneration. 
are of great importance in relation to heart failure in the disease. 
Changes of & somewhat similar nature have beon oily 
observed in the nerve cells of the central nervous system, those 
Iyin near the capillaries, it is sald, being affected first, There 
iso the striking change in the pa ipheral nerves, Which ia 
ie first by the disintegration he medullary sheaths as 
already described, It is, however, still a matter of dispute to 
what extent these nerve lesions are of primary nature or 
acoondary to changes in the nerve calla, 

Methods of Diagnosis.—The bucteriological diagnosis of 
diphtheria depends on the discovery of the bacillua As the 
bacillus occurs in largest numbers in the membrane, a portion of 
this should be obtained whenever it is possible, transferred 
too sterile test-tube. (The tube can be readily sterilised by 
boiling some water in it.) Tf, 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 tho 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 
steriline, In use tho wire and plug are oxtmoted in one pices, 
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 ameared over the surface of n serum tube 
to obtain a culturo, This mothod of taking and treating #waba 
is that usually employed in routine public health work. ‘The 
results obtained ordinarily suffice for the diagnosis of casos 
suspected to be diphtheritic in nature. 

Phe means for identifying the bacillus are (a) By micro 
scopical examination.—For microscopical examination it is 
suflicient to tease out a pice of the membrane with forcops and 
rub it on @ cover-glass; if it be somewhat dry a small drop of 

















METHODS OF DIAGNOSIS 369 


‘normal Le aiare reaps ‘Tho films are then dried in the 
usual way stain any ordinary basic stain, though 
methylene-bluc is on the whole to be preferred, used either as 
‘a saturated watery solution or in the form of Léfiler’s solution, 
After staining for two or three minutes the films are washed in 
water, and mounted. As a rule no decolorising is 
necessary, a3 the blue does notoverstain. Neisser's stain (p. 108) 
may also be weed with advantage. Any wceretion from the 
pharynx or other part is to be treated in the same way. Tho 
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 sufficiontly 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 mime picce of membrane was non-conclusive As ao 
mid, br lett microscopical examination alone is more relinblo 





The reaction should be tested after one and after two days’ 
growth. If it mains alkaline, the diphtheria bacillus may be 
u 


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). E 


CHAPTER XVI 
TETANUS? 


SYNONYM —LOCKIAW.  GREMAN, WUNIBTARIKRAMPR, 
FRENCH, TREANOS. 


Introductory,—Totanns is a disease which in natural conditions 
affects chiefly man and the horse, Clinically it is characterised 
hy the gradual cnwt of general stiffness and spasins of the volu 
tary museles, Summons in those of the jaw and the back of th 
neck, and extending to all the muscles of the body. “aaa 
are of @ tonic miture, and, as the disewse advances, 5 each 
other with only a alight intermission of time. ‘There are often, 
towards the end of a case, fever and rise of respinition and pale 
rate. The diseuse is usually associated with a wound received from 
four to fourteen days previously, and which has been defiled by 
earth or dung. Such a wound mnay be very small. ‘The disaase 
‘is, in the majority of cases, fatal. Post mortem there is little to 
be observed on miked-eye examination. The most marked 
feature is the occurrence of patches of congestion in the spinal 
1d expecially the medulla. 


Wistaroal.—Tho general association of the development oftetanas with 
the presence of wounds, though theae might be very small, suggested that 
some infection took place through the latter, but for long nothing wax 
knowns to the nature of this infection, Carle aud Rattone in 1884 
snnonnoed that thoy had produced the disease in w number of animals by 
inoculation with material from a wound in tetantia They thus deman- 
trated tha transmissibility of the diseas,  Noeolaier (1885) infonted micr 
and rabbics with garden earth, aud found that many of them devoloped 
tetanus, Suppuration ewurred in the neighbourhood of the point of 












§ ‘This disease is not to te confused with the“ tetany" of infant which In 


om 





dl 


372 TETANUS 


inoculation, and in this pus, besides other organisms, there was always 
present, when tetanus had occurred, a bacillus haying certain constant 
microscopic characters. Inoculation of fresh animals with such pus 
reproduced tho divense. Nicoluier'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 scrum, but always in 
mixture with other organisms, Infection of animals with such a culture 
produced the disease. ‘Theso results wero 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 ont, as charactoristic of the bacillus, its development of terminal 
spores. In 1889, Kitasato succeoded in isolating from the local suppura- 
tion of mico inoculated from a human case, several bacilli, only one of 
which, when injected in pure culture into animals, caused the disease, 
tnd which wae now named the b. tatuni, This organist Is the same ah 
that observed by Nicolaier and Rosenbach. itasato found that the 
causo of earlier culture failures was the fact that it could only grow in the 
absence of oxygen. Tho pathology of the disease was further elucidated 
by Faber, who, having isolated bacterium-frev 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, howover, the tetanus bacilli have not formed 
spores, they appear as somewhat slender rods, without present- 
ing any characteristic features. ‘There is usnally present in such 
pus a great variety of ‘other organisms—cocci and bacilli. The 
characters of the bacillus are, therefore, best studicd in cultures. 
It is then seen to be a slender organism, usually about 4p to 5 
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 anacrobie 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 may be of consider- 
able length, are usually curled up close to the body of the bacillus. 
The formation of flagella can be hest 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 873 


very characteristic . The x ‘are round, and in 
plies may be sic tacperetiey the thickness of the bacilli, 
They are doveloped at one end of bacillus, which thus assumes 
what is usually described as the dramstick form (Figs 124, 127). 
Ina men stained with a watery solution of gentian-violet or 
methylone-blue, the spores are uncoloured except at the periphery, 





Pro, 124-—Film preparation of discharge from wound ino case of tetanus, 
several tetanas bacilli of *drumatick” form, (The thicker 
Yexillag with eval and not. quite termina spony in the upper prt ofthe eld 
towanls the right wide, ie not x tetanns bacillus but « putrefsative 
anaorole which was obtained fn pure culture from the wound.) 
Stained with pentian-violet, x 1000, 











so that the appearance of a small ring is produced ; if a poworful 
stain auch as carbol-fuchsin be applied for some titnc, the sporce 
become deeply coloured like the bacilli, Further, especially 


ifthe ion be heated, many spores may become free from 
‘the bacilli in which they wore formed. 
Tsolation.—The isolation of the tetanus becillus is »omewhat 





diffealt. By inoculation experiments in animals, its natural 
habitat has been proved to be garden soil, and especially the 


374 ‘TETANUS 
a ee 





weiatance, function ag a aap 

trun atrlyogxertaly proltendit has bad 
or au 

isolated by means of the methods 4 "for 


Pet. ‘The beat methods for dealing with such pus are as 
(1) The principle is to take advantage of the resistance of 


~ 





Litt 135. Tetarne Vacilll, ahowing fapalls 
sect by Hal. Muie's method. x 1000, 


the spores of the bacillus to heat, A sloped tube of inxpisaated 
serum or a deop 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 haeilli can often be o 

microscopically. ‘The culture ix then kept at 80° C. for from 
three-quarters to one hour, with the view of killing all organisms 
execpt those which have spored. A loopful is thon added to 
glucose gelatin, and rolltube cultures are made in the usual 
way and kept in an atmosphere of hydrogen at 29° C.; after 
five days the plates are ready for examination. Kitasato eom- 





ISOLATION OF THE BACILLUS 375 
Fae ee ote plates to those of the b. subtilis, 


el 
i 
isl 


i 
iy 


u 
| 


i 
E 
3 
a 





than thetetants bacillus, —y,,. 199, spiral composed of nameroux 


ts etal tages ofthe causa tect, 
in any diss Stalnot by Kd. Muir's snethot, 1000. 


adoy Tnoeula- 
tions sit ie 
material are in 


half a dozen deep tubes 
of glucoss agar, provi- 
ously melted and kept at 
& temperature of 100" 
Lo inoculation 

are in 
boiling water and kept 
for vat Himes, say 
for fatigeres for 
cone, three, four, five, 





incubator at 37° C., in the hope that in one 
or other of the tubes all the orgenisins prevent will have been 








376 TETANUS 


killed, except the tetanns spores which can develop in pare 
culture, 


(3) Some method of anaerobically making plates, such as 
cine Balloch, may be employed. ‘The laclaifin of chosatare 
bacillus i in many cases a difficult matter, 
and various expedients require to be tried. 
Characters of Oultures.—Pure cultures 
having been stele) sub-cultures can be 
made in deep upright glucose gelatin or 
agar tubes. On glucose gelatin in such a 
tobe 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 ont into the modinm (Fig. 
131, a). There is slight formation of 
but, of course, no liquefaction. Growth alo 
occurs in blood serum and alao in glucose 
bowilfon under auacrobic conditions, The 
latter is the medium usually employed for 
obtaining the aoluble products of the or 
gonism. There is in it at first a slight 
turbidity, and Inter a thin layer of a 
powdery deposit on tho walla of the Yox#el. 
_ All the cultures give out a peculiar burnt 
. odour of rather unpleasant character. 
Conditions of Growth, etc.—The b. 
# totani grows best at 87°C. The minimum 
growth aperature is about 14’ ©. and 
(Kitt) sata below 22° C. growth takes place very slowly. 
Growth takes place only in the absence of 
gen, the organism being « strict awzerote, Sporulation may 
commence at the end of twenty-four hours in cultures grown 
at 37° C.—auch later at lower temperatures Like other 
spores, thowe of tetanus are extremely resistant, ‘They ean 
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 
wntiseption 

















PATHOGENIC EFFECTS 377 


Pathogenic Effects.—The proof that the b tetani is the cause 
of tetanns ia complete, It ean be izolated 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 diseuse, 
bat the cause of this very probably is the small nambers in 
which it sometimes occurs, 

(a) The Disease ax arising Natwrally—The discase occurs 

;, chiefly in horses and in man. Other animals may, 
however, be affected. There is usually some wound, often of & 
ragged character, which ns cither 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- 

though this may be absent. Microscopic examination 
‘sections may show at the edges of the wound necrosed tissue 
in which the tetanus bacilli may be very numerous Tf a 
jing from the wound be examined microscopically, bacilli 
feeibling the tetanus bacillus may be egiied If these 
have spored, there can be practically no doubt as to their 
identity, as the drumstick appearance which the terminal 
gives to the bacillus is not common among othor bacilli, ee 
must be taken, however, to distinguish it from other thicker 
bacilli with oval spores placed at a short distaneo from their ex- 
tromities, such forma boing common in earth, ote., and also met 
‘with in contaminated wounds (Fig. 124), Tt is important td note 
that the wound through which infection has taken pluos may be 
very small, in fact, may consist of a mero abrasion, In some 
in tho tropics, it may bo merely the bite of an 
ice of & ite channel of infection has given 
a “idiopathic” totanns, ‘There is, however, 
bt that all such cases are true casos of tetanus, 
of them the cause is the b. tetani. The latter 
wind in the bronchial mucous membrane in some 


a 


i 
Fy 


Fig? 
rege 
BEE 


no 


Li 
Fie 
z 

z 


pathological changes found post mortem are not strikin; 
uemorrhayes in the muscles which have been the 
the spasma ‘These are probably due to mechanical 
atarally it is in the nervous system that we look for 
in lesions. Here there is ordinarily a general 
matter, and the most striking feature is the 
patches of slight congestion which ure not 
particularly to grey or white matter, or to any tract of 
Tater. These patches are usnully best marked in the grey 
‘of the medulla and pons. Micreacopically there is little 


(ttl 
any 


Hi 


(ll 


378 TETANUS 


of a definite nature to be found. There is congestion, and there 
may be minute hemorrhages 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 ix, however, 
probably the case that very rarely, if ever, are the organisms 
found anywhere except in the local lesion. 

(b) The Artijicially-produced Disease. —The disease can be com- 
municated to animals by any of the usual methods of inoculation, 
but docs not arise in animals fed with bacilli, whether these 
contain spores or not, Kitasato found that pure cultures, 
injected subcutancously or intravenously, caused death in mice, 
rata, 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 subcutancously 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. After 
death there is found slight hyperemia 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 aoc, was suilicient to destroy the activity of the toxins 
Loeren splinters bee Jia Sore tenp de a reel 
development of the spores wl oy carry. In this way 
hho comploted the proof that the bacilli ty themselves can form 
toxins in the body and produce the disease, Further, if a small 
eey of gerden earth be placed under the skin of a mouse, 
from tetanus takes place in & grat many easen [Some- 
times, however, in such circumatonces death occurs without 
ee and is not due to the tetanus bacillus but to 
lias of malignant oedema, which also is of common. 
courrence in the ecil (wide fyfra).] By such oxporimenta, supple- 
mented by the culture experiments mentioned, the natural 
itats of tetani, as given above, have become known. 
‘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. conten with the work on diphtheria was tho 
Fee oe eo ee ee 
Uy asppotiog tbat the bacillus could’ excrote salable poison, The 


was wverod by Faber in the same year, Brieger auil 
‘onaisted practically of an alooholic precipitate from. 
Gltered cultures in bouill wna undoubtedly toxic. Within recent 


Mevteanky acmncian sxlbeta (6c, 167) 


‘The toxie properties af bucterium-free filtrates of pure cultures 
of the b tetani wore investigated in 1891 by Kitamito. Thin 
observer found that when the filtrate, in certain doses, wns 
injected subcutaneously or intmvenously into mice, tetanic spasms 

first in muscles contiguons to the site of inoculation, 
and later all over the body. Death resulted. He found that 

i jigs Were more susceptible than mice, and rabbits lows so. 
asia dass strongly toxie bouillon be produced, it must 
een have been either neutral or slightly alkaline. Kitasato 

found that the toxin waa casily injured by heat, Exposure 

for a few minutes at G5" C, destroyed it, It was also destroyed 
minutes’ exposure at 60° C., and by one and a half 
pn Be tiertrg hadi 11a, aves, 10, pany, baworer, 
destroyed by various chemicals such as pyrogallol and also by 


a 





380 TETANUS 


sunlight, Behring has moro recently poit out that after the 
filtration of cultures containing toxin, i¢ latter may very rapidly 
Jose its power, and in a few days may only possess 425th of its 


original toxicity, "This he attributes to such factors as temperature 
and light, ani especially to tho 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 diphthoria toxin, and it is also 
true ai ad the ae while fon their ee vy still 
retain their power of producing mnity against the it 
toxin, Further, altogether apart from the occurrence ‘die by 
sido in tho crude toxin of strong and woak poisons, it bas been 
shown that such crude toxin contains toxic substances of probably 
ae adifferent nature, Ehrlich has shown that besides the ia 

lominant spasm-producing toxin (called by him tetanospasmin), 
there exists in crude toxin a Aen capable of producing the 
solution of certain red blood corpuscles, ‘This hatuolytic agent 
he calls tetanolysin. It does not occur in all samples of crude 
tetanus toxin, nor is it found when o bouillon culture of the 
bacillus is filtered through porcelain. ‘To obtain it the fresh 
culture must be treated by ammoninm snlphate, as described in 
the method of obtaining concentrated toxins (p. 167). This 
substance also has the power of originating an antitoxin 20 that 
certain antitetaniec sera ean protect red blood corpuscles against 
its action, Madsen, studying the intornctions of thix antix 
tetanolysin with the tetanolysin, bas shown that the phenomena 
can be demonstrated similar to those noted by Ehrlich on 
occurring with diphtheria toxin, and which he intorpreted am 
indicating the presence of dogencrated toxins (toxoids) in the 
crude poison, With tetanus as with diphtheria toxin the action 
of an acid is to ennao an apparent disappearance of toxicity, but 
if before a certain time haa elapsed the acid bo neutralised by 
alkali, then 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 
Dacillus ca produce its toxin whon growing in a Buid containing 
no proteid matter. The toxin may is ‘be formed independently 
of the breaking up of the protelds on which the bacillus may be 
living, though the latter no doubt has a digestive action on 
these. Tho 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. Tt 
iy probable that there is an independent poptic ferment which 
will, of course, also pass through 4 filter. For if equal portions 





in those kept longest. There ix thus no fresh development of 
toxin tas the advancing liquefction of the gelatin. Thns 
ie digestion and toxin formation are apparently due to 
if it vital ‘on the part of the tetanus bacillus. 
rer the nature of the toxin is, it in undoubtedly one 
most powerful poisons known, Even with a probably 
toxalbumin Brioger found that the fatal dose for a 
was -0005 of a milligramme. If tho susceptibility of 
the same as that of a mouse, the fatal pee einn 
have beon “25 of @ milligramme, or about sap 
Animals differ vory much in thelr roi 
of tetanus toxin, According to v. Lin, ity if 
imal Jothal dose per gramme weight for a horse be takea 
1 for tho guinea-pig would be 6 timos the amonnt, 
12, the goat 24, the dog about 500, the rabbit 1800, 
the goose 12,000, the pigeon 48,000, and the 


A striking feature of the action of tetanus toxin is the 
ocenrrence of a definite incubation period between the introduc: 
tion of the toxin into an animal's body and the appearance of 
aymptoms. ‘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 cighteen hours, in the horse five days, 
and the incubation is shorter when the poison ik introduced into 
fa yein than when injected subcutaneously. In man the period 

the receiving of an injury and tho appearance of tetanic 
sym is from two to fourteen days. 


i 


Hey 





reeeegt 
eli 


calls in the spinal cord. The motor cells in the pons and 
modula are as affected, and to a nmch greater degree than 
in the cerebral cortex, When injected subcutaneously 
fs absorbed into the nerves, and thence finds its way 
part of tho spinal cord from which these nerves spring. 
Hains the fact that in some animals the tetanic spasms 
in the muscles of the part in which the inoculation 
taken place. This is not the caso with man, In whom usually 
first eymptoms appear in the nock, In artificial injection of 
finds its way into the blood stream, and if infected 
be killed daring the incubation peried there is often 
toxin in the blood and solid organs. Tn the guinea- 


if 


Has 





382 TETANUS 


pig there fs little doubt that tetanus toxin has an affinity solely 
for the nervous system. In other animale, such ns the rabbit, 
an affinity may exist in other organs, and the fixation of the: 
poison in snch situations may give rise to no 
symptoms. In such an animal as the alligator, it ix possible 
that while some of its organs have an affinity for tetanus toxin 
its nervous system has none. These facts are of great scientific 
interost, and a possible explanation of them will be disenssed in 
the chapter on Fmimatey Tf tetanus toxin be introduced into 
the stomach or intestine, it is not absorbed, but to # large extent 
Paes through the intestine unchanged, Evidence that any 
truction takes place ia wanting, 
Within recent years some important light has been shed on 
the mode of action of tetanus toxin, Marie and Morax stndied 
oa path of absorption when the toxin was injected into the 
les of the hind limb. "Tho sciatic nerve in a rabbit was cut 
war the spinal cord and toxin introduced into the muscles of the 
ane side; after some hours the nerve was excised and introduced 
into a mouso—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 sime 
experiment were performed and an excess of toxin injected into 
the other limb, still only the nerve which wus left in page a | 
with the muscle showed evidence of containing toxin. From 
this it was deduced that the toxin was absorbed by the end« 
plates in the musele and not from the lymphatics bie 
the nerve. Tt was further shown that a nerve in the 
degeneration following section did not absorb toxin Fitton t the 
manner of « normal nerve. By a similar method it was showa 
that the absorption by the nerve was fairly rapid, as one hour 
after injection the toxin was prosont in it, and from other 
experiments the view was put forth that the toxin was centripetal 
in its flow and did not pass centrifugally ina nerve to whieh it 
artificially gained access, Farther observations have been made 
on this subject by Meyer and Ransom, ‘These observors found 
evidence that toxiu is only absorbed by the motor filaments of a 
nerve, for while tetanus conld be produced by injection inte a 
mixed nerve like the seiatio the introduction of « lethal does into 
such a sensory perve as the infra-orbital was not followed by 
disease aymptoms. If a small dose of toxin be injected into the 
sciatic nerve it reaches the corresponding motor cells of the cord, 
and a local totanus of the muscles supplied by the nerve result. 
With a larger dose the poison passes across the commissure to 





TOXINS OF THE TETANUS BACILLUS 383 


the cells of the other side, and if still ee 
piece up tho cord to higher centros. 

affection of such Saar ck Ge orertoaite COTE 

the cord, Meyer and Ransom hold that when toxin is injected 

snbentaneously Saray; it aly ae ie eee 

ze ini in miuacles and thence passe to 

lee A Jatned Uy the 

this extended pasaage to occur. Tn this connection 

~ ls, where the nerve 

Me an yt ron long. Like 

believe that absorption of toxin by its 

Roche oes nervous btructures does 

Eri ae hei in euy they bring forward the 
observation that see intravenous injection ix i 


tion of the normal tonus, which accounts for the continous 
stiffness ee cues, and Pee ee rs inervase in reflex 
irritability, isa inent factor in the recurrin, 

While no absorption of toxin takes place by sensory Set 
they have found evidence of sensibility af the veo tl a bus 


‘in the occurrence of what they call tetamus doloroeus, ina 
‘great joaia and a alee which can be 
caused ‘ing toxin into the spin card or into a sensory 


_ death of the animal was not Pieused! This they attribute 
to the fact that antitoxin can only neutralise the textn which ix 
still circulating in the blood. This in a very far-reaching 
conclission, as it throws doubt on what has been held to be a 
ponsibifity, namoly, that toxin can be actually detached from 
cells in which it is already anchored. But a still more 

observation was made, for in ono case of an animal 
‘sctively immunised against tetanus, and which contained in its 
eerum a considerable quantity of antitoxin, the injection of toxin 


(ll 


S84 TETANUS 





into the sciatic nervo was followed by tetanus. ‘This would 
‘appear to militate againat Hhrlich's position that antitoxin ix 
manufactured in the cells which wre Jensitive to the toxin (see 
Tmmanity). 

Roferenco may hore bo made to the effects of injecting tetanus 
toxin into the brain itself, as investigated by Roux and Borrel. 
Tt was found that tho ordinary type of the diseaw was not 

roduced, but what thosp observers called “cerebral tetanus,” 

Whi euoipisls APs panerellencosiy eparctomas) ctw aaa 
character (apparent hallucinations, fear, ete.), and opileptifonn con- 
valsions, Death occurred in from twelve to twenty hours without 
Any true totanic epaama Ln thie manifestation of totanus the 
incubation period waa much shorter than with subcutaneous 
injection, and the fatal dose was one twenty-lifth of the minimal 
subentancous does, Further, the injection of antitoxin forty- 
to ninoty-six hours previously did not preventan animal from suc- 
cumbing to the intracerebral inoculation, In the light of what bas 
‘been already said these results would seem to indicate a 
effect of the toxin when brought into direct contact with the 
protoplasm of the brain cells 

We have seen that unless suitable precantions are ad in 
experimental tetanus in animals death results not from inocula- 
tion but from an intoxication with toxin previously existent in 
the fluid in whieh the bacilli have been growing, According ta 
Vaillard, if spores rendered toxin-freo, by being kept for a 
sufficient time at 80° C., are injected into an animal, death does 
not take place, Tt was found, however, that such spores ean be 
rendored pathogenic by injecting along with them such chemicale 
aa Inctic acid, by injuring the scat SL inooulatlon 6 as 9 cause 
effusion of blood, by fracturing an adjacent bone, by ee 
a mechanical irritant such as soll or a splinter af wood (aa in 
Kitasato’s experiments), or by the simultancous injection of 
other bacteria such as the staphylococeus pyogenes aureus 
facts, especially the last, throw great light on the disease aa it 
oceurs naturally, for tetanus resulta especially from wounds 
which have beon accidentally subjected to conditions such ox 
those enamersted. Kitasato now holds that in the natanal 
infection in man, along with tetanns spores, the presence of 
foreign material or of other bacteria is necemary. 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 Belieing and Kitasato in Germany, and of ‘Tizeoni 
and Cattani in Italy. former found that such an 
immunity could be conferred by the injection of very small and 
tswrsaly increasing doses of the tetanus toxin, ‘The degree 
of immunity attained, however, was not high. Subsequent 
work has shown that the less rich # crude toxin is in modifica 
tions of the tro toxin the lost useful ft js for immunisation 
procedures, In fact it is doubtful if small animals can be 
immunised at all by fresh filtrates. In sore cases it hos been 
found that the injection of nonlethal doses instead of commenc> 
ing an immunity actually increases the susceptibility of the 
animal. More «uceersful ans tho methods of accompanying the 
early injections of crude toxin with the subcutaneous introduction 
‘of sriall doses of iodine terchloride, or of using toxin which has been 
neted on with todine terchloride or with iodine iteelf. Tizzoni 
and Cwttani have also used the method of adiwinistering prov 
gressively increasing doses of living cultures attenuated in 
various ways, ¢g. by heat, By any of these methods susceptible 
onimals can be made to ‘ire great immunity, net only 
against many times the fatal dose of tetanic toxin, bat also 
against injections of the living bacilli. The degree of immunisa- 
tion nequired by au animal remaina in existence for a very Jong 
time. Not ouly so, but when o high degree of immunity has 
Keen produced by prolonged treatment, it is found that the 
sera of immune animals possesses tho capacity, when injected 
into animals susceptible to the disease, of protecting them 
against a subsequent infection with a fatal dose of tetanus 
bocilli or toxin, Furthor, if injected kabsequently to smck 
infecting, the serum can in certain cases prevent a fatal reanlt, 
even when symptoms have begun to appear. The degree of 
smnceeas attained depends, however, on the shortness of the time 
whieh has olapaed betwoon the infection with the bacilli or toxin 
and the injection of the seram, In animals where symptoms 
have fully manifested thomaclves only small propartion of 
cuss can be saved, As with othor antitoxins there is no 
evidence that the antitetanie scrum has any detrimental effect 
on the Imcilli, Tt only neutralises the effects of the toxin. 
‘The standardisation of the antitetanic sera is of the highest 
importance. Behring recommends that for protecting animals 
a sorum should be cbtuined of which one gramme will protect 
1,000,000 grammes weight of anice against the minimus fatal 
doee of the bocillus or toxin. A mouse suighing twenty 
grammes would thas require O02 ine of the aeram to 
protect it against the minimum etka dass Ta the injection 
% 


386 TETANUS 


of such a serum subsequent to infection, if symptoms have 
fan te open 1000 ans this dose would be necessary 5 a 


‘As the result of his experiments, Behring aimed at obtaining 
a curative offect in the nataral disoase occurring in man. For 
this purpose, as for his Iuter laboratory experiments, he obtained 
serum by the immunisation of such large animals ax the horse, 
the sheop, and the goat, by the injection of toxin accompanied 
at first with the injection er iodine terchloride, Tt was found 
that the greater the degree of the natural susceptibility of an 
animal to tetanus, the easier was It to obtain a sernm of @ 
antitetanice potency, ‘Tho horse was, therefore, the most 
suitable animal, it now we take for granted that the relative 
anaooptibility of man and the monse towards tetanus = 

tial, A man weighing 100 kilogrm. would require 
Pe ean mentioned above to protect hin ean fnoslatled 
with the minimum lethal dose of bacilli or toxin. If symptoms 
Tad bogun to appear, 100 c.c, at once would be necessary, and 
os the injection of such a quantity might be inconvenient, 
Behring recommended that for man a more powerful serum 
should be obtained, viz, a seram of which one gramme would 
protect 100,000,000 grammes weight of mice. The potency in 
maintained for severe! months if precautions are taken to 
avoid putrefaction, exposure to bright light, ete. To this end 
“5 per cent carbolic acid is usually added, and the serum ix 
kept in the dark. In a case of tetanus in man, 100 ce, of 
such a serum should be injected within twenty-four hours in 
five dows, each at a different part of the body, and this 
followed up by further injections if no improvemont takes 
place, Intravenous injection of the antitoxin has also been 
practised, and, in cases which we luwe seen treated in this way, 
has seemed to give better results than those obtained by the 
snbentancous method. The seram 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 e.c. can be injected every fow 
and in all 100 c.c. should be given in as short # time as possil 
Henderson Smith has shown that when antitoxins to toxins of 
the tetanns group are injectod intravenously a high concentration 
in the body fluid in maintained for some time and the op- 
portunity for neutralisation of toxin ix thus great, He sugwests 

‘The antitetanie serum sent oat by the Paxtour Institate In Prarie bas w 


strength of 1 1,000,000,000, Of this it is recommended thut 50 to 100 ee, 
should be injected in one ar two doses. 














IMMUNITY AGAINST TETANUS 387 


that both intravenous and subcutaneous injections should be 
simultaneously practised. The former gives the quickly attained 
concentration which is desiruble, and when the antitoxin injected 
intravenously is beginning to be climinated that introduced 
hypodermically comes into the cirenlation and the concentration 
is maintained. The antitoxin has also heen introduced intra- 
cerebrally, very slow injection into the brian substance being 
proctived, but no better remulta pve been obtained: thaa by the 
subeutancous method, 

Many cases of homan 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, asa 
matter of fact, we have not the opportunity of recognising the 
presence of the tetanus bacilli till they have begun to manifest 
nisiergores effects, In diphtheria we have a Nps 
clinical feature which draws attention to the probable presenc 
‘of the bacilli—a presence which can be readily phat 
the curative agent can thus be early appliod. Tht totanus the 
wound in which the bacilli cxist may be, as we have seen, of 
— most trifling character, and even when a well-marked 

exiata, esearch for the bacilli may be a matter of 
Bfiealy. Still it might be well, when pructicable, that every 
a ae wound, especially when contarninated 
SE istinah ees poloene ioe wee 
bacteriologically, In such cass, undoubtedly, from time to 
time cases of tetanus would be detected early, and their treat- 
tment could be undertaken with mare hope of snecess than at 
present, However, in the existing state of matters, whenever 
ire iret \ptoms of tetanus appear, lange doses, such as those 
above ind |, of & seram 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 
Bebring, chief reliance ought to be |, are the presence or 
absence of interference with rexpiration, and the rapidity with 
which the groups of muscles usually affected are attacked. If 
dysprees or irregularity in respintion cores on soon, and if group 
after group of muses is quickly involved, then the outlook Ix 
extremely grave, In addition to these points the duration of the 
ineubation period is of high importance in forming » prognosis 
The shorter the time between infliction of « wound and the 
‘Appearance of kymptoms the graver ix the outlook. 






388 TETANUS 


‘Tho theory as to tho nature of antitoxic action will be 
discusacd later in the chapter on Lawanity, 

Methods of Examination in a case of Tetanus —'lh 
routine bacteriological procedure in ease prsentog ‘the clinical 
features of totanns ought to be as follows :— 

(a) Microscopic—Though tetanus is nob a disease in which 
the discovery of the hacilli ix wasy, still microscopic ae 
should be undertaken in evory case, Krom every wouni 
abrision from which sufficient discharge can be tad ‘im 
preparations ought to be made and stalied with any of the 
ordinary combinations, ¢g. carbol-fuchsin diated with five parts 
of water. Drumetick-shapod spore-bearing bacilli ant to be 
looked for, The presence of such, having charactors corres 
sponding to those af the wtantx lacilli, hough not absolutely 
conclusive proof of identification, ix yet sufficient for all practical 
purposes. If only bacilli without spores resembling the tetanus 
bacilli are seen, then the identification ea only be provisional, 

‘The microscopic examination of wounds contaminated by sel, 
ete., may, as we have said, in aome cases lead to the anticipation 
that tetanus will probably result. 

(6) Cultivation.—The methads 16 be employed in isolating 
the tetanus bacilli have already been detoribed (p. 373). It 
may be added, however, that if the charicteristic forms ane 
not sen on microscopic exatnination of the material fram the 
wound, they may often be found by inoenlating a deep tube af 
one of the glucose media with such material, und incubating for 
At the end of this period, spone 
Tearing tetanus bacilli may be detected microscopically, thongh 
of course mixed with other onganisms 

(c) Froculation,-Mice and guinewpigs are the most saitable 
animals Tnoculation with the materi from » wound shoul 
be made subcutancously. A loopful of the discharge intreduead 
‘ot the root of the tail in a mouse will soon give riso to the 
charneteristic symptoms, if tetanus bacilli are present, 





























Martonanr Rpxma (Septieemie de Pasteur), 


The organism now usually known as the bacillus of maligoant 
axdema is the same as that first discovered by Pasteur and 
named wibrion septique. He described ite chameters, distin- 
guishing it from the anthmx bacillus which it somewhat 
resonbles morphologically, and also the lesions produced by it 
He found that it grew only in annerobie conditions, bmt was 
Able to cultivate it merely in an impure state, It was mony 















MALIGNANT G2DEMA 389 


fally studied by Koch, who called it the bacillus of malignant 
code, and pointed out that the disease produced by it is not 
realy of the nature of a septictnia, as immediately after death 
the i. 





Pos, 12%.—Fily froen the adfected tierees to of wadignant 
toiowe fs tho Loots euipec, showing the npore-bearig el 
Geuten-vielet, 1000. 


uuud subjoceut parts, Iu many cases of this nature the bacillus 
of malignant oxdema is preset, associated with other onganisins 
whieh aid its spread, whilst in others it may be absent, Ono ef 
ms has, however, observed a caso in which the bacillus was 
‘Present in pure condition, Here there occurred intense edema 
‘with swelling ani induration of the tisars, and the formation 
of vosicles on tho skin, Those charges wore attended with a 
reddish disoloration, aftorwards becoming livid. Limphysoma 
‘Was pot reosgnisable until the limb was incised, when it was 
detected, though in small degree. Farther, tbe tissues had « 


<_ 





390 MALIGNANT (DEMA 


peculiar heavy, but not patrid, odonr. ‘The bacillus, which was 
losis in culture, Was present in enormous numbers in. 
times, attended by cellular necrosis, serous 
caer and at places much Ieucocytic emily a The 
picture, in short, corresponded with that seen on inoculating a 
guinea-pig with a puro culture, ‘The torm “malignant codema™ 
should Le limited in its application to cases in which the 
\ bacillus in question is present, In most of these therm is a 

mixed infection ; in some this bacillus may lee 

This bacillus has a ei a Se tibutin. te 





substances that the 


is usual in the 
Toman nies. Malgaoat 
ee ne ee 


by inoculating as- 
oie er such as 
ined~ wi 
Ei The baci ars 
often present inthe intestine 
of man and anitnals, and Ties 
boon described as being 
Pia, 190.—Haeilas of nalignant stems, present in some gangrenous 
bowing spore From n eultare It Honditions gna pe 
ee ee Ineubated for three daye connection with the ins 
Stained with weak carbel-fuchain, «1900, testine in the human sub- 


ject. 
Microscopical Characters.—'The bacillus of malignant wdema 
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 pz to 10 x in length, but both 
in the tissucs and in cultures in fluids it frequently grows ont 
into long filaments, which may be uniform throughout or 
segmented ot irregular intervals, In cultures on solid media it 
chiefly occurs in the form of shorter rods with somewhat roundéd 
| onda, ‘Tho rods are motile, possessing several Intorally pla 
flagella, bat in a given specimen, as a rule, only a fow baeilli 
show active movement, Under suitable con ons they form 
xpores whieh are araly near the centro of the rods and have an 
oval shape, their thickness somewhat exceeding that of the 








CHARACTER OF CULTURES 391 





bacillus (Figs. 129, 130). The bacillus can be readily stained 
y 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 anderohic conditions, In 
a puncture culture in a deep tube of glucose gelatin, the growth 





B 
five days’ growth at 3 Natural size. 


A. Trtan Vacillon, B Bacillus of malignant wsleina, C. Bacillun uf 
aquarter.evil (Rausehtieand). 





appears as a whitish line giving off minute short processes, the 
growth, of course, not reaching the surface of the medi 
Soon Ti ion occurs, and a long fiuid funnel is formed, 
turbid contents and floceulent masses of growth at the bottom. 
At the same time Iubbles of gas are given off, which may split 
up the gelatin. The coloniex 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 O3DEMA 


liquefaction occurs around the colonies, and spheres with turbid 
contanta result ; ges ia developed around the colonies, 

Tn deep tubes of glucose agar at $7" CL, growth is extremely 
rapid. Along the line of puncture, geowth appears as @ some 
What broad white line with short lateral projections hore and 
thore (Fig. [31, 3). Here also gas may be formed, but this ia 
most marked in a shake cultures, in which the medium becomes 
cracked in varions directions, and may be pushed upwards so 
high a2 to displace the cotton wool plug. ‘The cultures possess 
# peculiar heavy, though not putrid, mick 

Spore formation oceure above 20° C., and is nsnally well 
soon within fortycight hours at 37°C. The spores have the 
usual high powers of resistance, and way be kept for months in 
the dried condition without being Killed. 

Experimental Inoculation.—A considerable numberof animale 

the guinew pig, rabbit, sheep, and goat, for example—are 
surceptible to inoculation with this organinm. The ox is said to 
he quite immune to experimental inoculation, though it “eam, 
under certain conditions, contract the discuss hy natural chant 
‘The guinea-pig is the animal most convenient for experimental 
inoculation. When the disease ix set up in the guineaypig: by 
snbentancous inceulation with garden soil, death usnally oceans 
in about twenty-four to forty-cight hours. | There is an intense 
inflammatory cdema nround the site of inoculation, which 
oxtends over the wall of the abdomen und thorax. The skin 
and subcutancous tissue are infiltrated with a reddish brown Suid 
and softened ; they contain bubbles of gas and are at places 
gangrenous. ‘The superficial museles are also involved. These 
yarts have a very putrid odour, ‘ho internal organs are con- 
xested, the spleen soft but not much enlarged, In such con. 
ditions the bacillus of maligaant wdema, both in short and long 
forms, will he found in the affeeted tissnes along with various 
other orguniama. Spore may be present, especially whea the 
examination is made 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 plourm, usally in 
the form of Jong motile flamonta, but they are almost invariably 
abyent froin the blood. A short time after death, however, they 
syread dirvetly into the blood and various organs, and may then 
be found in o 

Subcntanoous inoculation with pure cultures of the bacillus of 
malignant edema produces chiefly a spreading bloody cedems, 
the muscles being softened and partly necrosed ; but there ig 
ittly Jormation of gas, and the putrid odour is almost absent, 




































asidorable numbers, 








BACILLUS BOTULINUS 398 


When the bacilli are injected into micc, however, they enter 
and multiply in the blood stream, and they are fousd in con 
sidernble numbers in the various organs, so that a condition not 
re that of anthrax is found, The spleen also ix much 

The virulence of the bacillus of malignant cdema varies com 
siderably in different cases, and it always becomes diminished in 
cultures grown for some time, To produce m fatal disease, a 
relatively large number of the organisius is necessary, and these 
twust be introduced deeply into the tiasues, inoculation hy scart 
fication being followed by no result, A smaller dose produces a 
fatal result when injected along with various other organism 
(bacillus prodigiosus, ote.) 

Tmmunity.—Malignant codoma was one of the first discases 
against which immunity was produced by injections of toxins, 
‘The filtered cultures of the bacillus in sufficient doses produce 

death with the sumo symptoms aa those cansed hy the living 
organisms, but arolatively quantity ie necestary, Chamber 
land and Roux (1887) found that if guinea-pigs were injected 
with several nonfatal ee of cuJtures sterilised by heat or freed 
from the bacilli by filtration, immunity aguinst the living onganiam 
could be developed in a comparatively short tims. found 
thst the filtered serum of animals dead of the disease is more 
highly toxic, and also gives immunity when injected in small 
dors These experiments have been confirmed by Sanfelice, © 

Methods of .—In any case of supposed malignant 

Sco § the fluid from the affected tismmes onght first to be 
exanined microscopically, to ascertain the characters of the 
erganiame present. Thongh it is not possible to identify abe 
solutely the bacillus of mulignant edema without cultivating it, 
the presence of spore-bearing bacilli with the chamcters deseribed 
ubore is highly suspicious (Fig. 129), In such a case the fuid 
ining the bacilli should be first exposed to w temperature 
‘of 80°C. for balf an hour, and then a deop glucoms ugar tube 
klvuld be inoculated. In this way the apore-free organistas are 
Killed off. Pure cultures may be thus obtained, or this procedure 
amy mequire to be followed by the roll-cube method under 
anaerobic conditions. An inoculation experiment, if available, 
may aleo be mado on 6 guinea-pig. 


BactLus Borviixus. 


The term “meat-poisoning” embraces a number of conditions 
produced by different agents, and the relation of the beecillas of 


= 





504 BACILLUS BOTULINUS: 


Gaertner to one class of ease has already been disenised. Another 
group was shown by yan Ermengom in 1806 to be caused by am 
woaerobic bacillus to which he gave the name baeslles 

He cultivated the organism from n sample of ham, the ingestion 
of which in the raw condition had produced a number of casce 
‘of poisoning, some of them followed by fatal reeult. The 
symptoms in these cases clusely corresponded with those occur- 
ring in the so-called * samage poisoning” met with from time to 
time in Germany and other countries where maumges and ham 
are eaten in an imperfectly cooked condition, Such uses form 
a fairly welldefined group, the «ymptoms in which are chiefly 
referable to an wetion on the medulla, and, as will be detailed 
below, similar symptoms haye been experimentally produced 
by means of the bacillus mentioned or its toxins. The ehief 
symptoms of thie variety of botulismus, us detailed by van 
Ermenget, are disordered seerction in the mouth and nose, more 
or less marked ophthalnoplegia, externa and interna (dilated 
pupil, ptosis, ete), dysphagin, and sometimes uphagia with 
aphonia, marked cemstipation and retention of urine, and im 
fatal cases interference with the cardiac and respiratory centres, 
Along with these there is pr ly no fever and no interference: 
with tho intellectual faculties, The symptoms commence at 
carlicat twolve to twenty-four hours after ingestion of the poison, 
From the ham in question, which wae not decomposed in the 
ordinary sense, van Ermengem obtained numerous colonies of this 
bacillus, the leading charnetors of which aro given below. Lt may 
be added that Romer obtained practically the same results as van 
Ermengem ina similar condition, and that the bacillus botulinas 
has bewn caltivated by Kempner from the intestine of the pig, 

Microscopical and Cultural Characters,The organiemn is @ 
bacillus of considerable siz, measuring 4 to 9 «in length aad 
9 to 1-2. x in thi ; it has somewhat rounded ends and 
sometimes is son in a spindle form, It ia often arranged in 
times in short threads, Under ce: conditions it 
-s which are oval in shape, usually terminal in position, 
nd @ little thicker than the bacilli, It is a motile organism 
and has 4 to 8 lateral flagella of wavy form, It ataina readily 
with the ordinary dyes, and also retains the colour in Gran 
method, though eare must be employed in decolorising, 

‘Tho orginiam can be readily cultivated on the ordinary 
media, but only under strictly anaerobic conditions, Tn glucose 
gelatin a whitish line of geowth forms with lateral offshoots, 
but liquefaction with abundant gas formation soon occnra, Tn 
tin plates the colonies after four to six days are somewhat 





















































MICROSCOPICAL AND CULTURAL CHARACTERS 595 


appear to the naked eye as small simi- 
Pi opine’ (getter ie ear 
power of the microscope have a yellowish-brown colour and are 
seen ta he composed of granules which show a streaming move- 
ment, especially at the periphery. Cultures in glucose agar 
resemble those of certain other anacrobes; there is abundant 
development of gas, and the medium ix split up in, various 
directions. ‘The cultures have a rancid, yh not foul, odour, 
due ehiofly to tho development of butyric acid. The Ma 
temperature is below that of the body, viz, between 20° and 
30° CL; at the body temperature growth is slowor and less 
abundant and formation does not ovcar. 

Pathogenic —Like the tetanus bacillus the bacillus 
botulinus has little power of flourishing in the tissues, wherens it, 
produces a very powerful toxin, Van gem found that the 


by bo gripable watery extracts of the infected ham or cultures: 
either by tho alimentary canal or by subcutaneous injection. 
Hero also there i4 4 period of incubation of not less than six to 
twelve hours before the pellet appear, and when the dose is 
amall 4 somewhat chronic condition may reante in which local 
a area a ee feature. ‘The characteristic effects: 
aay eet ot the filtered toxin by either of 
the eset 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 tetanns poison, the Miers tps toxin is 
remarkable, the fatal dow for a guinea-pig of 260 gran. weight 
being in some instances 0000 c.c: of the filtered toxin. En cases 
of poisoning if the human subject the effects would  toorlngly 
produced itecspticn ‘of the toxin from 
dieses canal; it is ouly after or immediately before death 
that a few bacilli may voter the tisues Van m 
obtained a few colonics from the spleen of a patient who 
died from bam-poisoning. ‘The properties of the botulinus toxin 
hare been investigated and have been found to correspond 
closely, as regards relative instability, conditions of precipitation, 
ete, with the toxins of diphtheria and tetanus. An antitoxin 
hax dlso been prepared by Kempner by the usual methods, and 
has been shown not only to have the neutralising property but 
to lmve 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 


and Sehepilewsky by the kame methods ax Wassermann 
Ba aes employed in the care of the tetanus toxin. The 








396, QUARTER-EVIL 


condition of the norve cells in experimental poisoning with the 
Lotalinus toxin has been investigated independeutly by Mariaesce 
and by Kempner and Pollack, and these observers agree ax to 
the occurrence of marked degenerative chav, ially in the 
motor cells in the spinal cord and medulla, Marinesoo also 
observed hypertrophy and proliferation of the nourogkia’ cell 
around ther, 

These clwervations, therefore, show that in one variety of 
meat-poisoning the symptoms are produced by the absorption of 
the toxins of the bacillus botalinus from the alimentary eamaly 
and, as vou Ermongem points out, it is of special im to 
note that the meat say be extensively contaminated with this 
tucillas and may omntain relatively large quantities of its toxins 
without the ordinary signa of decomposition being prosert. 
‘The production of an extracellular toxin by this organism, with 
extremely potent action on the nervous system, is fact of 
scientific interwt and has a bearing on the etiology of onher 
obscure nervous affections, 











QuanrmenyiL (Genmas, Baveciauaxp; Funvow, Coamnox 
SyMrroMaTIQuR). 
The characters of the bacitlus need be only brietly deseribed, ws, a0 
far as ix kuown, it never infects the human subject, ‘The natural disease, 
which specially oooars in certain localitics, aifects chiefly ab 








1 
uid gout Iofection takse place by sows wound of the Hurliey, rl 
there «presdy in the region around, inlanunatory ewolllug attended by 





bloody wdema and omphysema of the Tho prt becomes greatly 

Hien, and of a dark, almost black, colour. Hence the name ' blaoke 
leg” by which the disease ia sometimes knowa, ‘The bacillus which 
produces this comition is present iv large numbers iu the affected Usaties, 
mocciated with otber orgauians, aud alo occurs in small mumbers im 
the blood of intornal organs 

The baotiiue morphologically closoly resoinbles that of xalignant 
nema. Like the latter, also, {t is a strict anasrobe, and 4ta conditions 
of growth as rogards temperature are alsu similar. Th is, however, some 
list thicker, aud does not usually form such long filaments, Moreorur 
the spores, which are of oval shape and brosdor than the bedillus ar 

































sliost invarinbly sitoated oles to one extremity, though not aetuall 
torminsl (Fig. Tz). ‘The characters of the cultures, alao, resem 
those of the bacillus of malignant adema, but in a stab oultune 1 





glucos agar thore are suore numerous and loner Tnteral offshoots, the 
growth being also more luxuriant (Pig. 131, ch ‘This bacillus iswatlvely 
Tuvtile, and possess sumerons lateral tay 
The disesoo cau be readily produced in various animals, e.g. nine 
pigh, by inooulation with the affected tissues of diseased animals, and 
also hy means of pore cultures, though an intramuscular Injection of & 
iderable amount of the latter is sometiines uccewary. ‘The condition 
produced in this way closely resembles that in malignant cuema, though 














BACILLUS MROGENES CAPSULATUS bie 


there is Kai to be more formation of gas in the tisttes Rabbits are 
immuno against this disease, whilst tae are comparatively susoey 
tible to malignant oxdema, Ax in the case of tetauns, Taooulstlon wi 
Tying which have ser dey red of adherent toxin by heat does 
it produce disease. m con be Ca gent ‘by filtration from 
‘bouillon cultures, Lf in tutly resistant to hea 
at 70.75" C. without being 


ae rf int ita sction, being 
Sitting apts 


"Phin dinadse is ona against 
which immunity can be 





Fou, 13%, —Bacilius of qnarter-evil, showing 
~ colters burg pried via contains: mer 


See pee ‘uy heat and by the products of asks ieatn| 
by filtration of caltares. Pair nea pest preduced ayainst the 
taxina of the bari/Ios, ad a method of protection in which the antien 

‘of thik antitoxin is combined with that of the viror has bee ase 
‘The antitoxin ie sail to increase the chemotactic propertion of the 
Teaoon ytes, 


Bactivcs Brouexes Caraviares. 


This bacillus, though sometimes siding in the laction ef pathe- 
Joghea hangs, is chiefly of interest ou secount ef the stenadre gases 
whiied: fig tet the tisstion post Tt won 

Ruttall fx 1602; Ut fe how reeogieed wa etn 

an organssan found im gastuns phiegimen by K. Fraenkel, 
er a i tie bwotlins testa Sot» , The = m 
a comparatively barge ons, in 106 » in Tength and levi 
Sr abcint the same ay Unt of tho euturax Vecillae, ite « 
ew rounded (Fig. 13). Tt often oceursin pai 
ehains ; oecasionally filamentous forms are met vith. It usual 
irked ivooped bence the mame; it Gs noo-motile and does not 
form re It stains readily with the basic aniline dyes amd retains 
fi Grara's wethed, It grows readily on the ordinary media, 




















398 BACILLUS AROGENES CAPSULATUS 


Unt only under anderoble conditions; the optimam temperature is that 
‘of the body, growth at the roo. Lerpera tre being comparatively alow, 
In Ree amare eee of 
with somewhat indented mai mi the eal 

Besasnint arooal ras Tusa tapesatioy ox i 
ASeaph i sistent besten Soearhad channel ean LNs rows 
Tn all cases thore is « tendency to abundant evolution of 

coultaros, aud thir ix ae 
ally marked when ferment, 











bw the mom frequent se 
of rapid 
wat i ial 2d 
: 
a “— an invaKion 
eter lies 
‘beforedeath. In such oases, 
even within twenty + four 


hours under a a 
len 





Pio, 198, — Bacilluy nex cnpouilatan 
tlm preparation tron boue-marrow in x Ere, 
case Where memati were present io the (Hix organiaw in snet 
organs from and ‘to time in by oe 

censsotion with sleerative or gangresou cenditions of” the foreciney 
the bacillus ix also found roquoutly in the peritoweam in 
eases of perforation. Although the striking ehanges in the organs 
are due t & post-mortem development of the bacillus, there is mo 
doubt that its entrance Into the blow stream often hastens 

ant tuay in some instances bo the cause of Aw already stated, the 

orgenisin is aleo met with in vomne casos of «preading adema with 


omphyeerns us 0 Ieling feature, 

hon tostod experimentally the bacilias by itself is found to have 
little pathogenic action, Injection of pure cultures in mbbite aud 
guinea-pigs may be followed by little result, but eomotimen in the latter 
animal eel phlegmon "iy produced, without suppuration wales 
other organiame are present. Ifa small quantity of oulture be i 
intravenously, ep in a rabbit, and then the animal billed; bubbles 
of gas are rapidly produced in the blood and organs, the pictite sorte 
sponding with thet In the human cases. 























CHAPTER XVII. 
CHOLERA. 


Introductory:—It is no exaggertion of the facts to my that 
previously to 1883 practically nothing of value was known regurd= 
ing the nature of the virus of cholera. In that year Koch was 
sent to Ezypt, where the disease hud broken out, in charge of a 
Commission for the purpow of investigating its nature, In the 
course of his ressarches he discovered the organism now generally 
known as the “comma bacillus” or the “cholera epirillum.” 
He also obtained pure cultures of the organism from a large 
namber 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 
Enrope in 1392.93, spirilla have been cultivated from cases of 
cholera in a great many differont localities, and though this 
extensive investigation has revealed the invariable presence in 
tme cholera of organisms resembling mone or less closely Koch's 

‘rillum, certain difficulties have arisen. For it has beon found 

the cultures obtained from different places have shown can- 
siderable variations in their characters, end, further, spirilla 
which closely resemble Koch's cholera spirillum have been 
cultivated from sources other than cases of tras cholera, ‘There 
has therefore been much controversy, om the one hand, as to the 
signitication of these variations, —whether they are to be regarded 
a& indicating distinct species or mercly ‘urictios of the same 
spoties,—and on the other hand, as to the means of distinguishing 
the cholors spirillum from othor species which resemble it. These 
queations will be discussed below, 

In considaring tho 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, ancl 


300 





400 CHOLERA 


often by vomiting, there are also symptoms of general systemic 
disturhance whic! assert be accounted for pales: ‘by the with- 
drawal of water and certain substances from the yatetns ‘Sueh 
symptoms include the profound general prostration, cramps in 
the muscles, extreme cardine depression, the cold and clara 
condition of the surface, the subnormal Meroe sci suppression: 
of ot ote. These pees an Pairs birety aa indi ieatiocn a a 
general poisoning in he cireulatory an Leemonegalatey 
nechaniams cael involved. In sore, thongh rare, cases 
known a4 cholere sieor, general collapae occars with re 
suddennoss, and i rapidly followed by « fatal result, whilst there 
is Tittle of no evacuation from the bowel, though port mortem the 
intestine is distended 
with fluid contents. Aw 
the characteristic ongane 
jams in cholera are found 
only in the intesting, the 
general disturbances are 
to be noganted as the 
result of toxi¢ substaneos 
absorbed from the bowel. 
Tt is alxo to be noted 
that cholern is a disease 
of which the onset and 
course are meh more: 
mpid than is the case in 
most infective disoaana, 
such as typhoid and 
Fou, 184.—Choleta epirsla, from culture on “iPhtherin: and! thet 
‘agar of twentyefour hours’ growth, recovery also, when it 
‘Btained with week carbol-fuchein, % 1000, takes place, does ao more 
quickly. ‘The two factor 
to he correlated to those facts ane (a) a rapid multiplication of 
organiams, (5) tho production of mpidly acting toxins, 

‘The Cholera Spirillum. Microscopical Characters.—Tho 
cholera spirilla ax fonnd in the intestines in cholera are amall 
organisms measuring about 15 to 2 in length, and rather bese 
than *D in thickness. ‘They are distinctly curved in one direction, 
hence the appearauce of a comma (Fig. 134) ; most ocene singly, 
but some aro attached in pairs and curved in opposite directions, 
40 that an Sehapo results, Longor forma aro rarely soon in tho 
intestine, bat in cultures in fluids, as is expecially well seen in 
hangingdrep preparations, they may grow into longer spiral 
filaments, showing & large number of turns. In film preparations 




















THE CHOLERA SPIRILLUM 401 


made from the intestinal contents in real cates, Sten orga: 
isms are present io enor: 

mons numbers in aia 
poro enlture, most of the 
apirilla with their 
Tong axes in the same 
direction, 6 a8 to give the 
Appearance Koch 
com toa number of 
fish in a streazn, 

"Thoy poses voryactire 
soli whi aries is most 
marked in thesingleformn. 
When stained by the euit- 
oie methods they are seen 
to be fagellated. Usual! 
a a ring terminal phere 
Pri 186.—- Cholera iar walped to show if prossnt at one end 

«1000, aly (El (Fig. 135). It is 

weydelicate, and measures 

four or five times the length of the organism, In some varieties, 
howerer, there mmy be a flagellum at both ends, or more than 


one may be bs cule 
tures: pobialned af different 
places have shown con- 
siderable variations in this 
t, Cholera spirilla 
ilo not form spores. Tn 
old caltures the eeealeaie 
present great variety 
tha an shape, Som 
are irregularly twisted fila- 
ments, sometimes globose, 
somietinie clubbed at their 
extromitins, und alse show- 
inginragulr swellings along 
Others ace 
a find. thiek, and may 
eespenerresrsnte of argo oly 
eerenree maining flatly, te ‘tcxergs hlaty alan 
Allthose changes in appenr- IG bales —tnvolution forme 
ance are eed See we tained with fuchsin, 1000, 
lution forma. 
Cholera spirilla stain reatily with the usual basic 








an ald agar 









= 


402 





aniline stains, though Léfler’s methylene-bluc or weak carbol- 
fuchsin is specially suitable, ‘They lose the stain in Gramn's 
method, 


Distribution within the Body.—The chief fuet in this con- 
nection is that the spirilla are confined to the intestine, and are 
not t in the blood or internal organs. ‘This was determined 
by in hia eartior work, and his statement has been 
confirmed. In cases in which thero is the charwcteristic " 
water” fluid in the intestines, they oceur in enormous numbers 
~-almost in pure culture, ‘The lower half of the «mall 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 celle and mucus, amon which are 
numerous spirilla, ‘Tbe spirilla aluo penetrate the follicles of 
Lieberkithn, and may be seen lying between the basement 
mombrine and the epithelial ining, which becomes loosened by 
their action, ‘They are, however, rarely found in the connective 
tise beneath, and never penetrate deoply, Along with these 
changes there ix congestion of the mucosa, expecially around the 
Poyor’s patches and solitary glands, which aro somewhat ewollon 
‘and prominent. In some very acute cases the mucosa may show 

neral aente congestion with a rosy pink colour but very little 

losquamation of epithelium, the intestinal contonts being a eom- 

ratively clear fluid containing the spirilla in large aumbera 
In other cases of a more chronic type, the intestine may show 
more extensive necrosis of the mucosi and a considerable 
amount of hamorrhage into ite substance, along with formation 
‘of false membrane at places. Tho intestinal contents in such 
cass are blood-stained and fonbsmelling, there boing « 
aaa of otbor organisms present besides the cholora spirilla 
(Koch). 

Cultivation.—(For Methods, see p. 419.) 

‘Tho cholera spirillum grows readily on all the ordinary media, 
and with the exception of that on Hea growth takes place at 

rdinary room temperature, The most suitable temperatury, 
wever, is that of the body, and growth usually stops about 
16° CG, though in some cases it has been obtained at a lower 
temperature. 

Pantone Gelatin —On this medium the organism grows well 
and produces liquefaction. Tn pnneture cultivations at 29° C, @ 
sh line appears along the sioedle trek, at the upper part of 

h 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 iz at the snrfaee the bnbble-shaped de 
frreaaion ; below this there ia a {annol-shaped area of liquofaetion, 
the Muid being only slightly turbid, but showing at its lower end 
thick masses of growth of & maru or leas spiral shape (Pig. 137), 
‘The liquetied portion gradually tapors off downwards towards the 
noodle track. (‘This appearance i, however, in some variotive not 
produced till much later, especially when 
the gelatin ia 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 
tho tube, 
In gelatin plates the colonics are come: 
what charmeteristic. ‘They appear as minute 
whitish points, visible in twenty-four to forty- 
Gight hours, the surface of whieh, under a low 
powerof the microsoope, is iregularly granular 
‘or farrowed (Fig. 138, A), and later has an 
appearance which has been ccmpared to 
fmgments of broken glass. Liquefaction 
cocours, and the colony sinks into the small 
cup formed, the plate then showing small 
sharply -tarked rings around the colonics 
Under the microscope the outer margin of 
the enp is cirewlar and sharply marked. 
Within the cup the liquefied portion forme 
a-ring which has a more or granular 
Appesranes, whilst the mass of growth in the 
centre is irregular and often broken up at its 
marging (Pig. 148, B), The growth of the 
colonics in gelatin plates constitutes one of 
the most important means of distinguishing 
the cholera spirillum from other organisms PH. 157, —- Puetnre 
On the surface of the ayer moda w thin  s#itumofthe chews 
almost transparent layer forms, which pre  SUyiiyin ls Petites 
sents no special characters, On soliditicd growth. Naturalsine 
Mood serum the growth has at fret the same 
appearsace, but afterwards liquefaction of the medium occurs 
On agar phates the superticial colonies under low power are 
eirvular dises of brownish-yellow colour, and more transparent 
than thore of most other organisms. On potato at the endinary 
th does not take place, but on incubation at a 
ture of from 30° to 37° C., a moist layer appears, which 
assumes a dirty brown colour somewhat like that of the glanders. 








== 





404 CHOLERA 


bacillus; the ance, however, varies aomewhat in differont 
varieties, and also on different sorts of potatoes. 
Tn bomitfon with alkaline reaction the organiam py) 





readily, there occurring in twelve hours at 37° OC, a 
turbis ity, while the surface shows « thin pellicle composed of 
apitilla ina very actively motile conditic Growth takes place 
under the sane conilitions equally ray Le ho olution 
(1 per cont with °S per cent sodinm eh bride ). 
Tn mit also the organi grows well aud produces 10 cowgas 
Jation nor any change in its appearance, at least for soweral days 
On all the media the growth: of the cholera @picillam ina 
rolatively rapid one, and especially is this the case in peptoue 


Faw, 133, Peat the cholera «pirillam in a art plate ; three days! 


ir A shows the grannlar «urfacr, liqueliotion just commenclig ; i B 
iquofastion is well mvrce. 








solution and in bouillon, 4 ciroumstance of importance in relation 
to its sepuration in eases of cholera (wile p. 413). 

The cholera organism ix one which grows much moro rapidly 
in the presence of oxygen than in anacrobie conditions; in the 
complete exclusion of oxygen very little growth occurs, 

Cholert-reed reaction.—This is one of the most i 
in the dingnosis of the cholera organism. Tt ix always given by 

a true cholera spiritlum, nnd though the reaction is not peculiar 
to it, the number of organisms whieh give tl 
the conditions mentioned are comparativ 
made by adding a few drops of pure sulphuric acid to « enltuns 
in bouillon or in peptone solution (1 por cent) which has been 
incubated for twentyfonr hours at 37° C,; fn the cise of thie 
choler spirillum a reddish-pink colour ix produced. ‘This i dae 
to the fact that both indol and a nitrite are formed by the 
spirillum iu the medium, ‘The addition of sulphurie actd | enki 
a nitroso-indol body to be produced from those, and thik gives 

















POWERS OF RESISTANCE. 405 


the rel colour. Here, as in testing for the roca of indo 
bby other bactoria, it is found that not every specimon oaume 

is suitable, and it is adyiaable to select a peptone whic 

the clutacteristic rietion with a known cholera onganism, ute] 
to use it for further tests Tt i also essential that the sulphuric 
acid abould be pure, for if traces of nitrites are present the 
reaction might be given by an organism which had not the 
power of farming nitrites. 

Hamolytic Test—This mothod introduced Kraua is 
performed by moans of agar plates, a small quantity of sterile 
dofibrinated blood being added to the agar and. thoroughly 
diffused ; if any organism has humolytic properties a clear zone 
or areola forms around each colony by asion of hrctno 
globin. In no instance has an undoubted cholecs organisa: been 
found to produce hemolysis, whereas many species of splrilia 
closly resembling it possess markod hemolytic action, This 
teat may accordingly be appliod along with the others in 
stern th the identity of a supposed cholera organism, 

Powers of Resistance. —In their resistanen againat Iwat, 
cholera apirilla correspond with :oat spore-free ongankenes, and ane 
killed in au hour by « temperature of 56° C,,and much more rapidly 
ut higher temperatures. They have eomparatively high powers 
of resistance againat great cold, and have been found alive after 
being éxpoeod for several hours to the tempeentare of —10" C, 
Thoy arv, however, killed by boing kept in ioe for n. few days. 
Against the ordinary sntisnptios they have comparatively: low 
powers of resistance, and Pull found that the addition of Time, 
in the proportion of | por cent, to water containing the cholera 
ongwnlsms was sufficient to kill them in the course of an hoar, 




















ootgrown by putrefactive Laeteria, but in exeuptional eases they 
have beer found alive even after two or three months, In most 
experiments, however, attempts to cultivate them even after a 
mach shorter time have failed. The general conclusion ray be 
drawn from the work of various observers that the «pirill 
ralsiply frocly in ordinary sewage water, 
reenait alive for a considerable period of 
we Kock showed, they can flourich vory rapidly. When the 
organisms are grown alosg with other onganiams 

in fluids at a warm temperntare, it is found that at 
may multiply more mpilly than the others, but that after 
vertaln time they are outgrown by some of the organisms yoosews. 
















406 CHOLERA 


gnidually diminish in oumber, and ultimately disappear, It 
must not, however, be inferred from such experiments that # 
similar result will necessarily follow in nature, os any particular 
sapmphytic organism requires a special hnbitat—that is, certuin 
suitable conditions for its growth in competition with other 
organisms, Though we ean state generally that the conditions 
favournble for the growth of the cholera spirillam are a warm 
temperature, moisture, a good supply of oxygen, and a consider- 
able proportion of organie material, we do not know the exact 
cireumstances under which it can flourish for an indefinite period 
of time as a saprophyte, The fact that the are 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 bax 
there is no doubt that in ordinary conditions it ean live a 
autliciont time outside the body and multiply to a sufficient 
extent, to explain all the facts known with regard to tho por 
sistenco and spread of cholera epidemics. 

Numerous exporimonta show that the cholera organisms are, 
as a ralo, rapidly killed by drying, usually in two or three 
minates when the drying has been thorough, and it is inferred 
from thix that they cannot be carried in the living eonditien for 
any great distance through the air, a conclusion which ie well 
supported by obserrations on the spread of the disease, Oholens 
is practically always transwnitted by means of water or food 
contaminated by the organism, and there is no doubt that eon 
tamination of the water eupply by choleraic discharges is the 
chief means by which areas of population are rapidly infected: 
[t has been shown that if fies are fed on material containing 
cholera organinns, the organivme may be found alive within their 
bodies twenty-four hours afterwards, And further, Haffkine 
found that sterilised milk might leeome contaminated with 

cholera organisms, if kept in open jars to which flies hud free 
aceess, in a locality infected by cholera, It is quite posible 
that infection may be curried by this agency in some casen. 

Experimental Inoculation.In considering the effects of 
inoculation with the cholera ongenism, we are met with the 
difficulty that none of the lower animals, so far as is Known, 
sniffer from the diswue under natural conditions Even in places 
where cholera iy endemie, no corresponding affection has bean 
observed in auy animals. And further, before the discovery of 
the cholens organism, various efforts had becn made to induce 
the disoase in animals by feeding them with cholera dejecta, bat 
without success, It is therefore not surprising that the earlier 















































EXPERIMENTAL EXOCULATION 107 


‘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, ts to induce 
‘their multiplication within the intestine of animals by artificially 
arranging favouring conditions, have occupied a prominent place 
in tho experimental work. We shall give a short account of 


much experiments, 


icati and Ristech worv the fst to injet the organisms dirotly into 
the duodenum of doge an rabbits, and they at producing, in 
considerable proportion of the animals, a Sholertecoulition of tha 
intestine; in theer earlier experiments the cominon bile duct wns 
Haatured, but the later were performed without this operation. ‘These 
sxperimiente were contired by eth observers, including Koch, ‘Think- 
ing that probably tho © when intecduoed by the mouth, is 
devteoed by the vatian o md ‘hgttroehorie cid of the guatele anert{on, 
nipatralised this ama Ty muainstatog to palsasrnignS oe 
of & 5 por cent solution of carbonate of soda, ime afterwards 
Sntroduced « pure culture into the pipe! iy wenns of « tube an le 
ethod tiled ts pie positive results he tried the effect of artificially 
faterforing with tho intestinal peristalsis Vy injecting tivoture of oyun 
into the (Lec, per 200 grm. weight), in addition to Does 
fainy ax before with the earbonsto of sodium solution. Tho result 
roma ne thirey out of thirty-five anisoals temted Glad ‘The 
sulmale iufcted Ly this method show rigns of geucral prosiration, their 
posterior extremities being especially meake: the ven becomes 
tami, tio hans ection weak, and the. surface cold, 
Death’ oscars after a few hours. sorter the small intestine is 
distended, its mucous membrane congested snd it contains 8 solourens 
fluid with amall Hocenik and the eh nisms in practically p 
saltaren, These sxprrineats, which have becu repeatelly con fed, 
demonstroted that tho colere organisms cond, under certain 
Bo Pa in some respects res lity 
h, However, ound: that ‘bea The splela of Winkler an an 
Prior, of Denoks, and of Miller (rise fra), wore employed by thi 
method, « certain, though muoh smaller, proportion of = aaimala “tied 
from an fntestinal Inneetian. ‘Though the changes in Une cape ware 























‘Bot 9 characteristic, wore sullicient to prevent the results obtalned 
with the cholera ox from ae a donmonatration of the 
apecitio relation sa to the disensn, 


Within later years some additionol fects of high interest have been 
= WEAR expand to eholarao tftation of snlmals. For example, 
ese fac oe that in the meroct aa tntesttnalRafeetion readily takes 
feeding with the organism, there resulting the usual 
Itc sometimes with hemorrhagic peritonitis, the ongau: 
ing presont also in the blood. It was found by Insetf 
and ssh young rabbits could be infectnt by merely nottralicing 
Whe gesteio soetetion with sedium carbonate, the use of opium bein, 
Parra i of pple: Interest ea De, ‘iiscoreresd by Metclnikotl 
a tla foang uLMia ahoetly afer rth olan opwrtion 
die of obs tion when the orgauinus aro simply introduced 
ote aie ‘as uiay be done by infecting the tests of the 
Pertisr, from these outwale thus infected the disesan wary Ym 














Pi 


408 CHOLERA 


brausinitted to others by a watural mode of infection. La thls affection 
of young rabbits many of the aymptoms of cholera are presaut, 
orgonisns occur in largo numbers in tho intwting, and in woMo OaMnd a 
fay may bo found inthe bloc, sod especialy in the gall Madden.” Mang 
Af those exprtmanta were performed with the vjurio o¢ Maseomalh, w 

ty now wimitted not to be a true cholera ongauiam, others with « 
cholera vibrio obtained from the water of the Seine, 

Tt will be men fram the above account that the evidence 
obtained from experiments on intestinal infection of animals, 
thongh by no moana sufficient to establish tho specific relation: 
ship of the cholem organism, is on the whole favourable to this 
view, expecially 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, aud, 
ultimately, profound collapse. There is peritoneal effusion, 
which may be comparatively clear, or may be somewhat: turbid 
und contain takes of lymph, according to the stage at whieh 
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 thoy are practically confined to 
the peritoncum. Kolle found that whon the suinimum lethal 
doe was used in guinew-pigs, the peritoneum might be free from 
organisms at the time of desth, the fatal result having taken 
place from an intoxication (of. diphtheria, p. 360), ‘Those 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 thewen is 
not a marked o On the other hand the symptoms of general 
intoxication are always pronounced. Henes arise questigns a8 
to the nature and mode of action of toxic bodies preelaced by 
the cholera organism. 

Toxins.——Though there is no doubt that there are formed by 
Koeb’s spirillum toxie bodies which produce many of the 
symptoms of cholora, thore is at prevent very little satisfactory 
Knowledge regarding their chemical nature. ‘The following 
summary may be given, 

It has been shown, especially by I. Pfeiffort that toxio 
phenomena can be produced by injection of the dead spirilia 
into animals. A certain quantity of a young culture on agar 

1 Pfoitfer obtainod his cartier nosults ‘ith « vil from Massowul, skied ie 


now Iaown (as mentioned above) nut to be a trie cholera organinm. ‘This feat 
alums that the ellests described are not spesitio to the lnster. 



























































TOXINS OF KOCH'S SPIRILLUM 409 


killed by oxpesuro to the vapour of chloroform, whet injected 
intraperitoneally into a guinea-pig, may cause death in from 
eight to owolve hours, Thore is extreme collapses, sometimes 
clonic spasins occur, and the tomporature may full below 30° ©, 
before death. Pfeilffer considers that the toxic substances are 
contained in the bodies of the onganisms—that is, they are ia 
truvelintor,—and that thoy ary only sot free by the disintegration 
of the latter, This opinion is grounded chieHly 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 
ongaiism, and then some time later anti-cholera serum which 
produces bacteviolysis is injected, rapid collapse with w fatal 
result may ensue, opperently due to the Hborston of the intew 
cellular toxins The dead caltures administered by the mouth 
produce no effect unless the intestinal epithelium is injured, in 
which case poisoning may resolt. He considers that ‘the 
desquamation of the epithelium is on essential factor in the 
production of the phenomena of the disease in the human 
wabject. Pfeiffer found that the toxic bodies were to a great 
extont destroyed at 60° C,, but even after heating at 100°C. a 
small proportion of toxin remained, which had the same phyalo- 
Togieal action. Recently A. Macfadyen found that the product 
obtained by grinding up the spirilla frozen by moans of liquid air 
lind a very high dogree of toxicity when injected inteuvenoualy. 
Like Pfeiffer he found that the “endotoxin” was in great part 
deatroyed at 60" CL 
On the other hand, other olservers (Petri, Rantom, Kleitn, 
wod others) have obtained toxic bodies from filtered cultures, 
Metchnikotf, % Roux, and TanrelliSalimbeni have demon- 
strated the formation of such diffusible toxic bodies in faid 
media. By means of cultures placed in collodion saca in the 
feritonenn of animals, they found that the living organisms 
prodwce toxic bodies which diffuse through the wall of the su 
and produce toxic symptoms By greatly ineressing the 
virulence of the organistn, then growing it in bouillon and 
filtering tho cultures on the third and fourth day, they obtalnod 
a aid which Pier Bee? toxie to guinea-pigs (the fatal dows 
ually being Les per 100 gem. weight), If the dow of the 
hee death follows in an hour orever leas Theo 
remies 3 josely resemble those obtained by Pfeiffer, the rapid 
fall af temperature being a striking feature. ‘They found that 
the pencity cof the filtcate was not altered by botling; appar 
ently she toxic substance is different from Pfeiffer's enubatersin. 












410 CHOLERA 


Attempts to investigate the chemical nature of the toxic bodies 
have not led to definite results. 

on the Human Subject.—xporiments have alee 
been performed in the ease of the human subject, both intention 
ally and accidentally. Tn the course of Koch’s earlier work, one 
of the workers in hix laboratory shortly after leuving was seinsd 
with severe choloraic symptoms ‘The stools were found to 
contain cholera spirilla in enormous numbers, Recovery, howr 
ever, took place. Tu this case there was no other posible 
source of infection than the cultures with which the man had 
been working, a9 no cholera was present in Seer, at tho time 
Within recent years a considerall 
been performed on the human subject, which eretaly show thit 
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 ease, for example, with Emmerich and 
Pottonkofer, who made experiments on themselves, the former 
especially becoming seriously ill, In the ease of both, diarrhea 
was well marked, fous cholora apirilla were present in 
the stools, though toxic symptoms wore proportionately fittle 
pronounced. Metehnikoff also, by experimonts on himeelf aud 
others, obtained results which convinced him of the if 
rolation of the cholera spirillum to the disease. Lastly, we may 
mention the eae of Dr, Orgel in Hamburg, who contracted the 
diseaso in the course of experiments with the cholérs and other 
spirilla, and died in spite of treatment, Tt is helieved that in 
sucking up vome peritoneal fluid containing cholem spirillay & 
little entered his mouth ond thus infection us produced. hia 
took plue in September 1894 at a time when there ane 10 
cholera in Germany. On the other band, in many eases the 
experimental ingestion of cholera spirilla by the human subject 
has given negntive results. Still, as the result of observation of 
what takes place in a cholera epidemic, it is the general opinion 
of guthorities that only a certain proportion of people are 
susceptible to cholera, and the facts mentioned above aré, in Our 
opinion, of the greatest importance in establishing the relation 
‘of the organism to the disease. 

Immunity,—Ac this subject is digcussod later, only a few 
facts will be here stated, chiefly for the purpose of making clear 
what follows with ryurd to the means of distinguishing the 
cholera spitillum from othor organinus, ‘The guinea-pig or any 
other animal may be easily immunised against the choler 
organism by repeated injections (conveni into the 
peritoneum) of non-fatal doses of tho spirilla, It is better to 






































IMMUNITY 4 


comnenee the Wisp gehete grees bapa by 
the vapour of chloroform or by heat, the doses being 
tg pea: UD is wa a igh Scns cline ata 
i ils way a of it 
ie iam is dovol hp entegre preteen 
evimel thos in (uti-cholera serum) has marl pro 
tective power when injeetod, even in a small ane ae is 
Sy ae OE 
© living organism. Under these circumstances the 
undergo s gravalar transformation and, ultimately, solution ; a 
pocdeoedeat it generally known as Pfeiffer’s reaction, and waa 
‘applied by him to distinguish the cholera, svirilam from organistas 
resembling it. ‘The following are the details — 


Lyeitier's Beoction.—A loopful (2 mgrm.) of recout agar culture of the 
iain to be tested addled to 1 cr. of urdinary bouillen oenlainiag 
eve of eatiahelers ern The misture is then fe tes BH 
itoneal cavity of « young guinea-pig (ahont 200 1m weight), am 
Fh peritoneal iid of thle stil Getventently stained by acene af 
conillary wis tales inverted tata the peritoneum) examined mlero- 
alter a fow minutes. Lf the spirills injected Rares 
spill, Ht will be found that they become motionless, gvell up 
globales, and ultimately troak down and disappear— if 
Gisy ere, feud mative ad sotiin, then the pouafbihty of Lustt being, 
true cholera spirille may be excluded—negaiene reswit. In the former 
raze godtive result) there sy howaver, ail! the posaltllty, tht, the 
ongauiom is deruid of pathogenic propertion aud baa been dos ¥ 
Hho! mormal peritoneal fiid. A control experiment wiould scoordingly 
be made with “001 0,6 of normal eeram in place of the anti-ebolera serum. 
1 io alteration of the organism ccours with ita use, thom it ts to be 
concluded that a trae Fraction has been givon, 


‘The serum of an animal immunised by the above method has 
also marked agglutinative action against the cholem spirillum, 
and this property closely corresponds with Pfeiffer's reaction as 
regards specificity. Such a serum has, however, little protective 
i je action of the dend spirilia, On the other 
hand, Macfadyen by injecting the endotoxin derived from the 
spirilla by grinding obtained a sorum which had antitoxic as well 
a Jutinative aud bacteriolytic propertics (vide Immunity), 
Metchnikoft and others have also obtained antitoxie sera which 
wet on the extra-cellular toxins obtained by filtration. 

‘The serum of cholene convilescents bas been found to possess 
Bespin shnilar to those of immunised animals; that is, it 

tion against the cholern spirillam and may also 

i tire Beefs reaction, ‘These propertics of the scrum may be 
ae eight or ten days after the attack of the disease, bat 
Bre most. marked four weeks after ; they then gradually becouvs 














42 CHOLERA 
weaker and disyppear in two or three mouths (Pfeiffer amd 
Tsea) 


Specific agyluteantive properties lve, huwerer, been detected 
in the scrum of cholera pationts at a much earlier date, in nome 
cases even.on the first day of the discuse, though usually «day 
or two Inter. The dilutions used were 1:15 to 1: 120, and these 
had no appreciable effect on organiams other than the cholenm 
spirillam (Achard and Bensande), Needless to aay, suck fuets 
apply strong wdditional evidence of the relation of Koeh's 
spirillum to cholera, 

Anti-Cholera Inoculation. Haftkine’s method for ineculation 
against cholera exemplifies the above principles, It depends 
upon (@) attenuation of the virus—thot ix, the cholera onganiam, 
end (4) exaltation of the viru, The virulence of the organism 
is diminished by passing a current of sterile air over the surface 
of the cultures, er by varions other methods. 
eoalted by the method of passrge 
organism in the peritoneum ina 
latter mothod the virulence after a time is increased twenty-fold 
—that is, the fatal dese has been reduced to a twentieth df the 
eriginal, Culture tod in this way constitute tho wirue enalté, 
Subcutaneous injection of the virus eeulté produces a leral 
necrosis, and may be fallowed by the death of the animal, but if 
the animal be treated first with the attenuated virus, the abe 
tion af the virws eeelté produces only a local eedema, 
alation first Ly attenuated and afterwants by exalted 
virus, the guinea-pig has aequired a high dogree of immunity, and 
Haffkino bolieved that this immunity was effoctive in the eee 
ef every method of inoculation—that is, by the mouth as well as 
by injection inty the tissnes After trying his method on the 
human subject and finding it free from risk, he extended it in 
pmuctice on # large scale in India in 184, and these experiments 
are still yoing on. So far the results are, on the whole, distinelly 
encouraging, In the human sabject two or sometimes three ite 
culations wore formerly made with attenuated virus before the 
wirus exalté was used; now, however, a single injecti 
latter is usually practived, Wassermann and 
Kloin, have found that gain 
method are not immunised against inte 
animal is treated by Koch's method (mee p, 407). Nobwith 
standing this fact Hatfkine’s method may still have a beneieial 
1, though it may not be preventive in all cases. 

‘Methods of Diagnosis. —In the first place, the stools 
to be examined microscopically. Dried film preparations should 









































f 


METHODS OF DIAGNOSIS 415 


he tandb ond stained by any ordinary stains, thongh earbol-fuchsin 
diluted four times with water ix xpecially to be recommended. 
Hanging drop a Hut eae with or without the addition of a. 
tion of gentinn-violct or other stain, should also 
big made, eegrid which method the motility of the organism can be 
retdily soon, Ry microscopic examination the presence of spirilla 
will be ascertained, and an idea a» to their eiatae ‘obtained. 
Ih some eases the cholera spirilla are so numerous in the stools 
tht m picture is presented which ix obtained in no ather con- 
dition, and a microscopic examination may be snfficient for 
Practical purposes. According to Koch, a diagnosis was made 
tm_50 per cent of the cases during the Hambarg epidemic by 
microscopic ewamination alone. In the ease of the first nppenr- 
ance of a cholermlike disease, however, all the other teste 
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 
hangdrop preparationy are made, each consisting of a small 
portion of mucus from te suspected stool broken up in, peptone 
wolution, To one a di f a 50-fold dilution of normal serum 
is added, to the other a as of a 500-fold dilution of an active 
cholera serum, If the aes present arc cholera organisms 
thoy retain their motility in the firat preparation, while they Jose 
it and then become aggtatinated 4 in the second, By thie method 
a dingnosis may sometimes be givon ina few minutes, 

Tf the organisms am very numerons, gelatin or agar plates 

at ‘be made at once and pure enltures obtained, 

{ tho spirilla occur in comparativoly small numbers, the best 
methedl is to inoculate peptone solution (1 percent) and incubate 
for from eight to twelve hours. At the end of that time the spirilla 
will be founil on micescopic examination in enormous mambere 
at the surface, and thereafter plate cultures can readily be made, 
Tf the xpirilla are very few in number, ot if a suspected water is 
fo be examined for cholera organisms, the peptone elution 
beaestl hus been inceulated ehould be examined at short intervals 

illa are found microscopically. A second flask of 

mone solution should ther be inoculated, and possibly again 

from the #cond. By this method, properly carrmed unt, 

& ealtare may be obtained which, though impure, contains a 

large proportion of the spirilla, and then phite enltures may be 
male. 

When a spirillum has been obtained in pure condition by 

thee methods, the appearance of the colonies in plates should 


be specially noted, the test for the cholenred reaction ai 














44 CHOLERA 


be applied, and in many cases it is advieable to test the effects 
of intraperitoneal injection of « portion of w recent agar culture 
in a guineaspig, the amount sufficient to canse death being also 
ascertained. The agglutinating or sedimenting properties of the 
serum of the patient should be tested against a known cholers 
organism, and against the spirillum cultivated from the ease. 
The action of an anti-cholera serum, ce. the serum of animal 
immunised against the cholera spirillum, should be tested ia a 
similar manner, 

Up till recent timex there had been cultivated from sources 
other than cholo casca, no organism which gave all the cultmril 
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 orguniams were obtained at El Tor from $he 
intestines of pilgrims who had died with dysenteri¢ symptoms, 
and there were no cases of cholera in the vicl: ‘The organisms 
in question, however, differ from the cholera organiom in having 
marked hemolytic action, and also in producing a rapidly meting 
extra-cellular toxin, There has been diversity of opinion 
with regard to the nature of these organisms, for while same 
consider that they are a different spociea from the cholen 
organist, others regard them as true cholera spirilla which fad 
toon carried by the patients, although no symptoms of cholen 
resulted from their prosence. Lf they are not to be regarded at 
cholera organisms, we have the stciking fret that they oo pound 
in the immunity teactions. This instance exemplifies well the 
great ditticulty which may surround the identification of « 
particular organiain obtained from non-cholera cases, and one 
can hardly doubt that if cholera-like symptoms had been 
present in the EU'Tor cases, the spirilla would have heen ascepted 
us varieties of the cholera organism, though differing im their 
honolytic action, Nons of the facts ascertained, however, really 
affect the question as to the causal relationship of Koeb’s spirillusa 
to cholera, although they indicate the difficulties which may 
attend the bacteriological diagnosis in isolated cases of the 
dinenow, 

General Snmmary.—We may briefly summarise ss follows 
the facts in favour of Koch's epirillum being the cause of chelora. 
Firs, there is the constant presence of spirilla in true easea Of 
cholera, which on the whole eonform closely with Koohw 
description, though variations undoubtedly occur. Moreover, 
the facts known with regard to their conditions of growth, ete, 
are in conformity with the origin and spread of cholo epidemiex: 

































































SPIRILLA RESEMBLING CHOLERA SPIRILLUM 415 


Sreondiy, the experiments on animals with Koch's hum or 
ite toxins give us definite resulta aa one sao semmettegen 
in view of the fact that animals do not suffer naturally from the 
iliwase, ‘Thirdly, the experiments on the human subject and 
the results of accidental infection by means of pure cultures ary 
also strongly in favour of this view. FourtAly, the agglutinative 
and protective properties of the serum of cholere patient and 
convalescents constitute another point in ite favour. Jiyily, 
Racterielogical methods, which proceed on the assumption that 
Koch's spirillam is the cause of the disease, have been of the 
greatest valne in the diagnosis of the disease. And lastly, the 
resalts of Haffkino’s method of proventive inoculation in the 
human subject, which are on the whole favourable, also supply 
additional evidence, Tf all these facts ane taken We 
consider the conclusion muet 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 probwbilivy of an important 
part being played by conditions of weather and locality, though 
such aro very imperfectly understood. Pottenkofor, for cxasaple, 
recognised two main factors in the causation of epidemics, which 
he designated z and y, and considered that these two must be 
preeent together in order that cholera may spread. The » is tho 
direct cause of the disease—an organise which he admitted 
to be Koch's spirillam ; the y ineludes climatic and local con~ 
ditions, eg. state of ground-water, ote. 

Other resembling the Cholera Organism. —Thwwe 
have been chieily obtained either from water contaminated by 
sewage or from the intestinal discharge in cases with choleraic 
symptoms. Some of them differ 0 widely in their cultural and 
other characters (some, for example, are phosphorescent) that no 
one would besitate to chiusify them as distinct species. Others, 
however, closely reseanble the cholera organiam. 


The ethic brrolincnsis, caltivated by Neiwer from Berlin sewage 
water, dilfers from the «holera organist only in the appearance of tte 
celonios fn gelatin plates, te weal action, and its plying « 
negative reamlt with Pieifer’s test. It, however, given the cholera eed 
renotion, The eiirio Ddunubens, cultivated by Helder (rem eanal water, 
also didfers im the ace of ite colonies in plies, and also resets 
negatively 00 teat ; In mot respecte it cheely resesbter the 
naeiitieslan! dnster’ ilies: te? Tematgy es paironed a 
Ivanell fream the stools of 4 typhoid patient after theme had tern ddatel 
with water, The orgs < somewhat im the ppearanew of ite 

fm its great tembeney to grow out Im th of ley, 
Uk Pieides found thai it reacted tc Ms teat tn the sane way a 
the ‘orpanieat, nnd be considered that tt was rally a variety of 
teechslers sopmniann. Io mplrile could be found micrunpically tn’ te 












416 CHOLERA 


stool in thik case, and Pfeiffer te of the opinian that the eee 

gained entrance accidentally. These examples will show how di 

of opiniou, even auongat experts, aight arise ax to whether « vertain 

sui were teally tho cholera orgasiiom or a distinet species renem= 
ng it. 


A few examples may also be given of organi» cultivated 
from eases in which cholora-like symptoms Were yireaent, 





quale from a ease dui 
esol, resembles Koch's 





Pestwua and Bettencourt also 
number ofear during an opid 

ro were aymptomis of gustro-enteritis, although only in a few instances 
did the disease resomble eholora, They alio cultivated the same 
conganism from the driuking water. It differs from the elolers organi 
in the appearanco of itx colonies and of pamneture cultures in gelarsh. It 
bas very feeble pathogenic elfects, and gives a vers faint, or no, choles: 
red reaction. To Dfelffer's test it also rests negatively. Awother 
spitillun (+. Komonus) was obtained by Celli and Saatork from twelve 
cut of forty-four cases where thery were the aymptoms of mild choler 
‘This organism does not give the cholora-ted resection, nor is it pathogenie 
for animals. ‘They look upon itas n “transitory variety" of the cholera 
‘organiam, though snfliciont evidence for this viaw fs not adduced. 




















We have mentioned these examples in order to show some of 
the diiiculties which exist in connection with thia subject. Tt ix 
important to nolo that, on the one hand, spixilla which have 
boon judged to be of different species from the cholera organise, 
hav ivated from cases in which cholenlike aymptoans: 
nd, on the ether hand, in cases of apparently true 
cholera 

peciall 














preser 





Jerablo variations in the characters of the 
organisms have been found. Such variations have 

heen recorded by Colonel Cunningham in India, It ie there 
fore quite an open question whether some af the 

in the former class may not be cholera spirilla which have ander 
variations x a resnit of the conditions of their growth, 
That euch variations ar we have « considerable amonwt 
of evidence, ‘The great bulk of evidence, however, gocs to slow 


cholera con: 













METCHNIKOFFS SPIRILLUM Like 


that Asiatic cholera always spreads as an epidemic from places 
in India where the disease is endemic, and {ta direct cana 
is Koch's spirillum, It is sufficient to bear in mind that eholoraic 
elie rhe Pa peaecee by other causes, and that in some 
at ae which have some resemblance to Koch's 
cael serge de Be eT 


large oun) 

A sumber of other spirilla have ies eal reset, which are of 
peeton cea Be Solent onde 
101 ‘ol ly luce no 10 

ep aa Hienast bainees 7rd sie 


Motchsikoffs Gpiritlum (vibrio Metchniiov{) —This ob. 
tain by Gaul tem an opine Alataueat fowls Oto tnd lal 
ipectal tareat on accoutt of x ove fraent cegealars. 
Th the natural dineaan, whi hich te shag « 





a 139). It is actively Fro, 189.—Metchuikott’s spirillam, both in 
motile, and has the same — curved and straight forms ; from an mgar 
taining reactions. Ite culture of twenty-four ours’ F 
growth in peptone gelatin Stained with weak earbol-fuchxin, 1000, 
leo Sealy. meen rosamibilos that 
of the cholera organism, though it produces liquefactioa soore rapidly 
(Fig. 140, 8). is gelatin plats the young colonies azo, howover, 
smoother and more circular, After liquefaction oseure, some of the 
colonies are almost identical Px Sppearance ‘with those of tho cholers 
@ibrio, whilst others ahow more uniformly turtid contents. In puretare 
caltares: the awth takes place more rapidly, but in appearance 
sort Ie jes that of the chelors orgavinm & few days older. Its 
in wi peptone solution too is closely similar, and it also gives the 


aver, be swadily dtitingulabed trom, the 
fects of ineculation on animals, especially on 
inoculation of small quantities 









418 CHOLERA 


of puro culture in pigeons is followed by septicmmis, which * 
fatal Tosult usually within twonty-four hours. Inoculation with the same 
uontity of cholera culture jncen a, no result ; even with 
Hinge, qusniitian act’ bn Surely” prosao he vibrio Motehnikei 
prodacm somewhat similar effcta in the guinea-pig to thos in the 
pigeos, subeutanvous inoculation 
ing followed by extensive hemor 
rhogio adema and « repidly fatal 
septionmis, Young fowls can. be 
infooted by fvoding with viralont 
cultures, We have evidence from 
‘the work of Gamalela that the toxins 
of this organism have somewhat the 
same action as those of the cholera 

organism. 

‘he organiem is therefore one 
which vary closely resembles the 
cholera organism, the rveults on in- 
oculating the pigeou offering the most 
ready means of distinction, It gives 
s nogative reaction to Pfeiffer's teat 
—that is, the propertion of an anti- 
cholera serum arenot exacted apainet 
it, Tt may also be mentioned that 
au organisin which is apparently the 
sameas the vibrio Motolnikov) was 
cultivated by Pfubl from water, and 
named , Nordhafen, 

Finkier and Prior's Spiriltum.— 

‘Those observers, shortly after Kooh's 

discovery of the cholera organism, 

separated a spiritlum, in a case of 

cholera nostras, from the stools after 

id been allowed to decom pase 
ral days. ‘Thore is, however, 
5 ry no evidence that the spirilium Bes 
























ho, 110,—Puneture ont eee te i eat 
poptone-gelatin. reat logically it closely resembles 
A. Moichuorsapieliom. Hive Koch's peels, nurk easaot be tee 





tinguished from it by ite micro 
Four daye' growth, Natarsl wlae, scoplon! characters, although, on the 
whole, it, tends to"be rather thicker 
in tho contre and more pointed at the ends (Fig. 141). In oulttres, 
however, it prosenta marked difforonces. Tu punctare cultures oh 
gelatin it grows much more quickly, and Nigualection is geoorally 
visible within twenty-four hours. ‘The liquefaction spreads mmpidly, 
and usually in forty-eight hours it has prodnoed a funmél - aha 
tube with tarbid contents, denser below (Fig. 140, B). Tn 
cultures the growth of the colonies ix proportionately rapid. Before 
they have produced \iquefaction around Yhesn, they apposr; unlike t Roun 
of the chcista otgeaiies, as miiaste epheroa with ancolt RiaREAenaaEm 
Tiguefsotion occurs, thes appear ae little sphores with turbid soutenty 
which rapidly inofoase’in size ; ultimately general liquefaction oceurs. 
Onjpotatoer this organism grows well at the onlinary temperature, and 

















DENEKE'S SPIRILLUM 419 





bat ite 


o 
fi Fi, LAL. —Finkler and Prior's epirilium 3 from 
virnlence & of « muck lows 


_ . culture of twenty « four 
‘Anot onltivatedby — Rrowth. 
acitor(EMitorespisiinn’)—‘Maived with catboluchsn, <1000, 


‘Doneke's Bpirillum —This organism was obtained from old cheese, and 
jaaleo Bacwa an the epirilinm iyregrnee, It cloely, plc ad 
spina in iterosopio appara, though x i rather inner and 
smaller. Ite growth in gelatin is also somewhat similar, tt liquefhction 
more rapidly, and the bell-shaped depression on the surface 1a 
‘and shallower, whilst the growtht has « tore distinetly yellowish 
‘The colonice in plates also show pointsof rvsctablanen, thongh the 
‘colonies aro rather smoother and more regular on the surfhes, and 
ipeeftin ‘oecure more rapidly than iu the case of the cholors arganiam. 
colonies have, on naked-aye exarnination,a distinctly yellowish colonr, 
Tho orgatiiem does not give the clolers-red reaction, and on potato tt 
forms's thin yellowish layer when incatated above 30°C. When tested 
by ltraperitoneal injection and by other methods it ie found to poseas 
very feeble, or alinost ne, pathogenic properties, Koch found that this 
orgationy, when advninistered through the stomach in the samo way a 
the cholera , cnet 8 fatal result in three cases out of 
fifteen, Sa xpirillum is usually regarded aa a comparatively 
harmless saprophyte. 


CHAPTER XVIL. 


INFLUENZA, PLAGUE, RELAPSING FEVER, 
MALTA FEVER, YELLOW FEVER. 


Txrtursza, 


THE first accounts of the organism now known as the influenss 
bacillus were published simultancously by Pfeiffer, Kitasato, and 
Canon, in January 1892. The two first-mentioned observers 
found it in the bronchial 
apatum, and obtained puro 
cultures, and Canon ob 
served it in the blood ina 
fow casos of the disease, 
Tt ix, however, to Pfeiffer’s 
work that we owe most of 
our knowledge. roga 

ita characters and action, 
Tis rvsults aye beea 
amply confirmed by these 
of others in various epi- 
domica of the disease, and 
this onganixm hae been 
generally accepted as the 
Fu, 2—Tataeum tlt foo acme {home alolute eek ie 


on blood agar. 
Stained with carbol-fucsta, 1000, #4411 wanting. 

Stained with carbol-fuch Mi 

acters.—The influenz bacilli as seen in the sputum are very 
minute rods not exceeding 1:5 » in length and 3 in thickness 
‘They are straight, with rounded ends, and sometinnes stain more 
deeply at the extremities (Fig. 142). ‘The bacilli ecour singly 
or form clumps by their aggregation, but do not grow inte chains 
They show no capsule, They take up the basic aniline stains 


420 











CULTIVATION OF BACTLLL 421 


somewhat fecbly, and are best stained by a weak solution (1:10) 
of carbolfuchsin applied for 5 to 10 minutes, 
stain in Gram's |. They are non-motile, and do not form 


spores, 

Tn 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, oven whon the symptoms 
are sey this may be duc to the restriction of the organisms 
to som not readily accessible, or it may be that they 
actually die out in areas part while the effects of their toxins 
persist. It has also been observed in recent epidemics, in which 
the disease has been lest widespread and on the whole less 
severe, that the period during whieh the bacilli have been readily 
demonstrable in the secretions has been on the average shorter 
thaw in the previous epidemics, 

Cultivation.—The beat medium for the growth of the 
intluonga bacillus ie blood agar (soe 38), which was intro 
duced by Pfeiffer for this purpose. He obtained growths of the 
facili on agar which had been smeared with influenza sputum, 
bat he failed to get any sub-cultures on the agar media or on 
seram. 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 agnr with drops of blood before making the inoculations. 
In this wny ho completely succeeded in attaining his object, 
The blood of the lower animals is suitable, os well as buman 
blood ; and the favouring influences of the blood would appear 
to be due to the himoglobin, as a solution of this substance is 
eqnally effective. The colonies of the influenza bacilli on blood 
bat incubated at 37° C. within twenty-four hours, in 
the form of minute circular doto almost. transparent, like drops 
of dew. When numerous, the colonies are scarcely visible to 
tho nuked eye, but whon sparsely arranged thoy may tvach the 
size of a small pin's head. ‘This size is generally reached on tho 
second day, The bacilli die out somewhat quickly in cultures, 
and in onfer to keep them alive sub-cultures should be made 
every four or five days. By this mothod tho 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 moro marked whon other organisis aro 
present, that is, ia favoured by aymbiosia, Noisser, for example, 
was able to cultivate the iniluenza bacillus on plain 

several generations by growing the xerosis bail 





422 INFLUENZA 


along with it; dew cultures of the latter had not the same 
favouring effect. A very small amount of growth takes place 
in bouillon, but it is more marked when a little froth blood is 
added. ‘The growth forms a thin whitish deposit at the bottem 
of the flask. The limits of growth are from 25° to 427 G., the 
optimum temperature being that of the body. The influenza 
bacillus is a strictly aerobic organiam, 

‘The powers of resistance of this organism are of a low order. 
Pfeiffer found that dried cultures kept at the ordinary tempers 
ture were usually dead in twenty hours, and that if apatum were 
kept in a dry condition for two days, all the influenza bmcilli 
were dead, or mther, cultures could be no longer obtained. 
‘Their duration of life in ordinary water is also short, the bweilli 
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 @ short time, 
‘Tho mode of infection in the disease he necordingly considers to 
be chiefly by means of fine particles of disseminated sputum, ete, 

Distribution in the Body.—The tucilli are found, chiefly in 
tho respiratory passages in influenza, They may be present in 
larg: numbers in the nasal secretion, erally mixed with a 
considerable number of other organisms, but it is in the small 
mastes of greenish-yellow sputum from the bronchi that they 
occur in largost numbers, and in many casca almost in a state 
of purity. They occur in clumps which may contain as sauay 
us 100 Imeilli, and in tho carly stages of the disease are chiofly 
lying froe, As the diseaso advances, thoy may be found in 
considerable numbers within the leucocyte 
end of the disease a large proportion he 
a matter of considerablo importance, however, that they may 
persist for weoks after symptama of the discase have disappeared, 
and may still be dete in the sputum. Especially is this the 
ease whon thore is any chronic pulmonary disease. They also 
occur in la pumbers in the capillary bronchitis and eatarrhal 
pucumonia of influenza, os Pfeiffer showed by means of sectiens 
of the affected } In these sections he found the bacilli 
lying amongst the leucocytes which filled the minute bronchi, 
and also penotrating between the epithelial cella and inte the 
superficial parts of the mucous membrane, Other ongenisme 
also, especially Fraenkel’s pneumococcns, may be concersed in 
the pncumonte conditions following influenza. Tn some oases 
influenza occurs in tubercular subjects, or is followed by tabercalae 
affection, in which cases both influenza and tubercle bacilli may 
be found in the xputum, In such a condition the progmonis i 





































— 





DISTRIBUTION OF BACILLI 423 
the 


presence of influenza bacilli in 
Se heniieeaae le following influenza, much in- 
in tho foci of sup- 


a a at ncay iy pts os be hi 

Rader poieersaee on a large series of ere 
tho organion was rarely prosont blood — 

ta Msc ie, actanrence’ there 1eMa\ Tx locked fsa 
onal. The conclusions of other observers have, on the 
confirmed this statement, and it is 


in some of the le mee Ae Preitfer found 
Le Pico rieneateenelimraateh but in a case of mi ae 
fi hnverness nkel’s diplecoceus was ibe 

poate of mentngitn Tsievers this kudha Becta an ead 


sometimes alone, sometimes along with pyogenic eocei 

(Pfahl and Walter, Cornil and Durante); Pruhl considers that 

in chess the path of infection is usually a direct one through 

nasal cavity. This observer also found post 

iy fntal case with profound genoral symptoms, 

various both within and outside of 

the vessels. Tn a few cases also the Wueilli have been found in 

the brain and its membranes with little time change in the 
around, 

Extensive observations on the bacteriology of the respiratory 
system show that influonzalike bacilli may be present in a great 
ariety of conditions ; we havo, in fact, once more to do wi 
group of orgnnisms with closely allied characters, of which 


influenza” bacilli have been obtained from the 

fruzces, bronchi, and lungs in inflammatory conditions, and aleo in 

‘various specific fevers. ‘To this group belongs the bacillus which 

has beon cultivated from casos of whooping-congh by Spengler, 

Jochmann, Davia, and others, and which is present is considerable 

pas perycetion of cases of this disease, Wollstein 

agglutinative reaction on this organism 

the soram of whooping-congh pationta, all the sera examined 

A positive reaction in « dilution of at least 1;100 on all 

atrains of the organism isolated ; on the other hand, clumping 
was nover obtained with a normal serum in a greater 





424 INFLUENZA 
= wen 10, Davia enn the fauces of 


eee = iieeee 
oti re to the disease, 
be cme sottled. Meller “trachoma bacillus 


times 4) Some examples: 

are a little larger than influenza-bacillus, and tend to form 

short filaments, but others are quite indistinguishable, All of 

them also seem to have very feeble 

the lower animals, At present it can scarcely be claimed as 
possible to identify Pfeiffer's bacillus by: its Diaceghaell 

Talal charieters, 

‘Exporimental Inoculation.-'There is no watisfuctory evidence 
that any of the lower aninuls suffer from influenza in natural 
conditions, and accordingly we cannot look for very 
resulte from experimental inoculation. Pfeiffer, by 
living cultures of the organism into the lungs of monkeys, itt 
three caxex produced a condition of fever of a remittent 
‘There was, however, little evidence that the bacilli had 
gone multiplication, the symptoms being apparently produced 
by their toxins. In the case of rabbits, intrevenous injection of 
living cultures produces dyspnoea, muscular weakness, and 
slight rise of temperature, but the bacilli rapidly cisco in 
the body, and exactly similar symptoms are os 
injection ‘of cultures killed by the vapour af chi 
Pfeitfer, therefore, came to the conclusion that the influenm 
bacilli contain toxic substances which can produce in animals 
some of the substances of the disease, but that animals are not 
Finble to inyeenion, the bacilli not having power of multiplying 
to any oxtent in their tismes. 

Cintani 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, after * infecting the substunce 
of the cord, An acute encephalitis was thus produced, and 
sometimes a purulent condition in the lateral ventricles, The 
lncilli were, however, never found in the blood or in other 
Similar xymptoms were also produced by injection of 
cultures, though in this eaae the dose required to be five or 

















METHODS “OF EXAMINATION 4 


tisaee ngs: Cantani therefore concludes that the brain 
is the most suitable nidus for their growth, but 
agrees with Pfoiffor in believing that the chief symptoms are 
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 cermbral tissue, 
evidence, accordingly, that the influenza bacillus is the 
ith of the discase ae iefly on ee {net that 
it is always present in the secretions of the respiratory tract in 
shld Pree emice large numbers The 
sie relationships Tae ‘tho organiam to lesions in the Scie 
elsewhere leave no room for doubt that it is possessed of 


iu 


certain. eid of dered evidence haa wupplied by 


the results of experiment. 
‘Methods of Hxamination—(a) Micrusopiz.—A portion of the 


greenish-yellow purulent material which often oevurs im little 
round maeses in the spatum should be selected, and film prepara 
tions should be made in the meual way. Films are best stained 
fs ZichLNeelsen cirbol-fuchsin diluted with ten parts of water, 

i¢ films being stained for ten minutes at least. In sections of 
the tikes, such ax the lungs, the Incl are best broaght out, 
acconding to Pfeiffer, by staining with the same solntion as above 
for half an hour. The sections are then placed im aleohol 
containing a few drops of acetic acid, in P hich they are 
dehydrated and slightly decolorised at the same time. They 
should be allowed to romain 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, 

(8)! Culture—A suitable portion of the greenish-yellow 
material having been selected from the sputum, it should be 
washed wll in sovoral changes of sterilised water. A portion 
should then bo 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, usally within 

four hours, These should give a negative result on 
ion on ordinary agar media. 


Praqun 


The bucillus of oriental plague or bubonic post was discovered 
independently by Kitasato and Yersin during the epid 


— 


426 





susceptible animals by inoculation of pure cultures, It is 
be noted that during an epidemic of plagne, even 
procoding it, a high mortality haa boen pe seis amonget certain 








/2.—Film proparation from a plaguc babe showing enortnous tiers 
‘of bacilli, moxt of which show well-marked bipolar statiig, 
‘Stained with weak goutian-violet. 1000, 


animals, especially rate and mice, and that from the bodies of 
these animals found dead in the plague-stricken district, the 
tame bacillus was obtained by Kitasato and also by Yorsin, 
Bacillus of Plague—AMicroscopical Character,—Ae seen in 
the affected ghinds or buboes in this disease, the bacilli ane 
small oval rods, somewhat shorter than the typhoid bacillus, 
and about the same thickness (Fig, 143), though considerable 
variations in size occur. y have rounded ends, and in 
stained preparations a portion in the middle of the bacillus is 


al 

















BACILLUS OF PLAGUE 497 


often left uncoloured, the so-ealled “polar staining." In 
films from the tissues vos 
are found scattor 


1 singly, 
ton ch Pian pos 
he other bind, in eul- 


length tho) form town 
fog (Fig. 143). Tn ae 


size, and polar staining is 
Teas marked than. i 
tissues: sometimes forms F% 14.—Milia o plagoe from a young 
of considerable Tongth are guinot with weak cxebol fackain. 51000. 
present. After a time in- ; Riper 
olution forme appear, expecially when the surface of the 
is dey ; but the formation 
of is much more 
rapid and more marked 
when 23 per cent of 
sodiam chloride is added 
to the medium, constitut- 
ing the so-called “salt. 
agar” (Hankin and Leu- 
eae On ie ees 
eapecially with the higher 
pasenisan Seas 
forms weumme a great size 
and a striking variety of 
shapes, large globular, 
|, or pte bodies 
resulting (Pig. 146); with 
Yoo, 148-—Tarllas of place In caine show- aout 2 per cent sedinm 
Ing polar daulog. “Frome youngcultary chlorideyaftertwenty four 
Fined with thionin Woe. 1000, hours’ incubation, the 
most striking feature ix a 
Keneral 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 moss common. They do not form 











428 PLAGUE 


Gordon, who has found that they possess flagella 
however, stain with difficulty, states that they are 
‘Most observers, however, and with these we 

have failed to find evidence of true motility. They stain readily 
with the basic aniline stains, but are decolorised by Gram’s 
method. 

Oultivation.—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 
and sinooth, 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 
Fla. 146.—Culture of the bacillus of plague ST°Wth with more opaque 

ro, M6 Cattar of the tcl of ee¥e Sipearance 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 surfuce 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 BACTLLT 429 
ux Ef oil or melted butter is added to the bouillon 


the fat globules and reli downwards in the form of 
Reossions ike masses. ‘These masses are exceodingly 
iclicate, and lily break off on the slightest shaking of the 
flask ; accordingly during their formation the culture must be 
kopt absolutely nt 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 a 
varies from time to time with the sume race. 
flourishes best in an abundant suy aie of oxygen; in oe 
anaerobic Soe almost no woh ke se 2 
‘The organism in its powers reas cocoa 
other ree bacilli, and | is readily killed by heat, an exposure 
for an at 58° ©. being fatal. On the other hand it has 
remarkable powers of resistance against cold ; it has been exposed 
to a tem several degrees below froczing-point without 
being killed. Experimenta on the effects of drying vlads eee 
somewhat diverse results, but as a rale rate a! 
found to be dead atter being drid for from six to eight days, 
though sometimes it has survived the process for a lone period period ; 
bad Ke to direct sunlight for throe or four hours kills 
cul 


tivated outside the body the organism often loses its aay 
‘but some races romain virulent in cultures for a long period of 
time. 


Anatomical Changes and Distribution of Bacilli—The 
dimes occurs in several forma, the Mubonéc and the pifmonary 
10 theae may be added the eeptiommin. 
ing feature in the éusonic form is the affection of 
the lymphatic glands, which undergo intense inflammatory 
swelling, uttended with Rando i ‘and generally endi 
@ greater or less degree of nocrotic softening if the patient lives 
long . ‘The connective tissue around the glands is 
similarly affected. ‘The bubo is thus usually formed by a 
colloetion of enlarged glands fused by the inflammatory swelling 
‘True suppuration is arc. Usually one group of glands 
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 theso changes there is great swelling of 
pesertctan | intense cloudy swelling of the cells of the 
Mase ten acd other organ. There may also occur 





430 PLAGUE 


areas of hemorthage 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 





Fio. 147.—Section of a human lymphatic gland in plague, showing the 
injection of the lymph paths aud siunses 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 ells 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 ix 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 
hemorrhage ; the bronchial glands show inflammatory swelling. 
Clinically there is usually a fairly abundant frothy spatum often 
tinted with blood, and in it the bacilli may be found in large 
numbers. Sometimes, however, cough and expectoration may 
be absent. The discase in this form is said to be invariably 
fatal. In the septiceemic 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- 
cemia ; 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 septicemic 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 infammatory swelling of the corresponding 








432 PLAGUE 


lymphatic glands, and thoreatter by a 

nea a) ed embatie pcan ea 
characters with in tho human subject, 
wt the time of death they have not 


A 
r 


i 
d 
i 







By this method of inoculation mi ness 13 days, 

slices nd tin, 2a ae in 47 
f changes, in uddition to the 

enlargement, are pnp of internal organs, with 

homorrhages and enlargement of the spleen; the bacilli ar 

numerous in the lymphatic glands and usually in the spleen 

Fi, ), and alsa, 

ee 

Tnfection can 

also be prodneld by 


Paoumocoscs.. 
Pua. 148 —Plm preparation of spleen of rat Rats and mice oan aleo 
after inoculation with the bacillus of plague, be infected hy feeding 


showing numerous bacilli, movt of which 4 ! 
fare somewhat plump, either with pure cultures 


Stained with carbol-thionin-bina «1000, bed with pieces of organs 


though in this case infection probably takes place Sion the 
mucous membrane of the mouth and adjacent parts, and only 
to limited extent, if at all, by the alimentary canal. 
also are highly susceptible to infection, and it bas been shows 
in tho case of thoso animals that when inoculation is made on 
the skin surface, for example, by means of a spine eb 
the bacillus, the glands in relation to the part may the 
characteristic lesion and # fatal result’ may follow withont ther 
being any noticeable lesion at the primary seat, ‘This fact 
throws important light on infection by the skit in the human 
wubject. ‘The disease may also extensively affect monkeys by 
natural means during an epidemic, 

Paths and Mode of Infection—Plaguo bacilli may enter 


PATHS AND MODE OF INFECTION 433 


by the skin surface through small wounds, eracks, 
al ete. and in such cases there is usually no reaction 
at the site of entrance. This Inst fact is in accordance with 
what has been stated above with regard to experiments on 
‘The path of infection is shown by the primary 
which are usually in the glands through which the 
skin is drained, those in the groim being the commonest site, 
Absolute proof of the possibility of infection by the skin is 
See eed Some Bie the disease has boon acquired 
it post-mortem oxarminations, the lesions of the sae surface 
being in the majority of these of trifling nature; in only twe 
‘was there local reaction at the site of inoculation, Tn most of 
these cases tho poriod of incubation has boon from two to thro 
days; under natural conditions of infection the average period 
is withp pve days While infection may oocnr by accidental 
ion through amall wounde of the skin surface, it appeers 

Tad sgist aoa aks pla by means of the 
‘of fleas, For some time it bad been known that phaygue bacilli 
might be found for some time afterwards in the stomach of tlear 
allowed to feed on animals suffering from plague, and some 
observers, for example Simond, had succeeded in transmitting 
the disease to other animals by moans of the infected insects. 
Most observers, however, had obtained negative results, and it 
was only by the work of the Advisory Committce appointed by 
the Seeretary of State for India in 905% that the importance 
of this meuns of infection was established. By carefully planned 
experiments the Committee showed that the diseasc could be 
transmitted from a plague rat to a healthy rat kept in adjacont 
eages when floas wore presont ; whoroas this did not occur when 
means were taken to prevent the access of fleas, though the 
Bp ae for eons infection were the same. The disease can 











‘inew-pigs are placed amongst healthy guinea-pigs 

few of the Natear acquire the disease when fleas 
are absent or scanty; whereas all of them may dic of plague 
when fleas are uumeroun This result demonstrates the com- 
part played by direct contact, even when of 





a clos ter. Important reeults were ale obtained with 
fogard to the mode of infection in houses where there had been 
cases of | Tt was found possible to produce the discase 
in animals by means of fleas taken from rats in 


# See Journal of Hygienr, vi, 421 ; vii. 328 





134 PLAGUE 


plagno house, When animals were plaend in plague houses 
and officiently protocted from flcas they pis Sei 
whereas they acquired the discase when the cages were free 
to the access of ileas in the neighbourhood, 


‘The following are some of tho experiments which were couduoted. A 
sorics of nix buts were built which only differed in the stracture of thelr 
roofs, In two the roofs wero made of ordinary native tiles in which rate 
frecly lodgo ; in two others flat tiles wero vied in which mate Hive, but in 
which they have not each facilities for movement as in the firet set, and im 
the thint pair the roof was formed of corrugated iron. Under the roof tn 
bach cass was placed & wire diaphragm which yrevented rate or thele 
droppings having nosom to the hut, but which would not prevent floss 
falling down on to the floor of the hut, ‘Tho huts were left sufficient 
timo to become infoeted with rats, and then on the floor in sach ase 
healthy guinea-pigs mixed with guinoa-pige artificially infected with 
plague wero allowed to ranabout together. — Tn the first two ants of huts 
fo whioh teas bad nooes the healthy guinepigs contracted 
while in the third set they remained unatlected, though they were freely 
liable to contamination by coutact with the bodies and excrets of the 
diseased animale. In thé third «vt of hute no infoetion took place ax 
Jong as teas were excluted, but whon accidentally these inasets 4 
admission, then infection of the uninoculated animals commenced, 
Othor experiments wore also performed. Tn one case healthy guineaspiiee 
were suspended in a cage two inches abore » floor oa whieh infeated and 
ffca-infested animals were ranning about. Infection occurred in theese, 
but if the latter wore snspended at a distance above the floor higher than 
a flea could jump, then uo infeetion took place. Again, tua but in whieh 
guinoa.pige had died of plagas, and which contained infhoted fleas, two 
cages were placed, each containinga monkey. One cage was surrounded 
hy a zone of sticky mnaterial Iroaler than the jump of a flea ‘The 
monkey in this cage remained unaffected, but the othor mankey eon 
tracted plagne, 

































Other experiments showed that when plague bacilli were 
placed on the floors of houses, they died off in a comparatively 
short period of t After forty-eight hours it was not fond 
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 Tn all these expert 
monte the common rat-tea of Indin—puler cheopis (Rotheshilél) 
—was used, but it has been shown that this flea, when a rat is 
not available, will bite aman, ‘These results are manifestly of 
gruat practical importance. They show that direct infection by 
dust and other material through small lesions of the skin qilays, 
probably, a comparatively mall part in the spread of tho dlisgisg 
fleas apparently being in the majority of cases the carriers of ine 
fection. ‘They also point to important proventive mementos, 
which will no doubt be put to a practical text before long. 

















TOXINS, IMMUNITY, ETC. 435 


Tn primary plague pneumonia, from a consideration of the 
anatomical changes and the elinieal facts, the disease: may be 
mid to be produced by the direct pasmge of the bacilli into the 
passages. Nevertheless there must be certain factors, 
imperfectly understood, which determine the incidence of 
this form ; as in somo 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 cas of plague pneumonia is of great infectivity in 
producing other eases of plague pnoumonia. If wo excopt 
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 
the bacilli are not discharged from the unbroken eurface 
body, and are only preseat in the secretions in severe cases, 
pi ged isease in rata was early recognised, and 
there ie no doubt that it plays a very important part in the 
spread of epidemics The discase in these animals has, in fact, 
been the means of rapidly distributing infection over wide areas 
of a town or district, ‘Thix has been abundantly proved in 
the case of Bombay, where observations have shown that the 
migration of phague-infected rats to quarters comparatively free 
from the disease, has been followed by extensive outbreaks in 
theee places. The fucts stated above show how tho disease is 
‘among thexe animals by fleax and how it ix conveyed 

by them to the human subject, 

Toxins, Immunity, etc. —As ix the case with most organixms 
which extensively invade the tissues, the toxins in plague cultures 
are chiefly eontained in the bodies of the bacteria. Injection of 
dead cultures in animals _prodncee distinctly toxic effects ; post 
mortem hemorrhage in tho mucous membrane of the stomach, 
areas of nocrosis in the liver and at the site of inoculation, may 
be pel Sasa ‘The toxic substances aro comparatively resistant to 

heat, being unaffected by an expcanre to 69° ©, for an hour, By 

the injection of dead cultures in suitable acertain degree 
of immunity against the living viru! welll is obtained, and, 
as firet shown by Yersin, Calmotto, and Borel; tho serum of 
wach immunised animals confers a degree of protection on 
small animals such as mice, On these facts the principles of 
Prec peste and serum treatment, presently to be 
dopend. It may aleo be mentioned that the filtrate 


ae anes ture ean uw very slight toxic action, and the 












Commission found that such a filtrate has practi- 
in the direction of conferring immunity. 





436 PLAGUE 


1. Preventive Incoulation—Hafkine’s Method.—To prepare 
‘the preventive fluid, cultures are made in flasks of bouil 

drops of oll on the surface (in India Haikine a 
medium by digesting goats’ flosh with 

acid at 140" C.and afterwards neutralising with 


exposing the contents of the finks to 65° C, for an 
thoreafter curbolic acid is added in the proportion of % 

‘The contents are well shaken to diffuse: thoroughly the sediment 
in the fluid, and are then distributed in small sterilised bottles 
use. The ‘ive fluid thus contains both the 

of the baci 


Ion 


of cases of disease and the mortality amongst the two classes 
The resulta of inoculation, as attested by tho first i 
Commission, have been distinctly satisfactory, For 
absolute protection is not afforded by inoculation, both 
proportion of cases of plague and the percentage mortality 
amonget these cases have been considerably smaller in the 
inoculated, ns compared with the uninoculated, 
ig not established till some days after inoculation, and lasts fer 
a considerable number of weeks, possibly for several months 
(Bannerman). In the Punjab during the season 1902-3 tho case 
incidence among the inoculated was 1°S per cent, the 
uninoculated 7°7 per cent, while the case mortality waa 23° and 
60-1 per cent respectively in the two classes, the statiation 
taken from villages where 10 per cent of the population 
upwards hnd been inoculated. 

2 Auti-plague Sero.—Of thew, two have beon weed ae 
agents, wiz, that of Yerein and that of br ‘Yersin’s seranr is 
propared by injections of inereasing doses of plague bucilii freto 
horse. In the early stages of immunisation dead bacilli am uf 
subeutancounly, thervafter into the veinm and, foal} living ore 
injooted intravenously, After a suitable time blood ta drawn off 
Ue scram ia preserved in the usual way, Of thie serum - 




















METHODS OF DIAGNOSIS. 437, 


faved, am ajo are oral, peed on aubmeqent days 
Taetig'oi penis aa Tytetice a hae a ea 
incrossing ofs 100 derived from the bodies of egae alte 
pro in great jar wacc-proted Mawes of grow 

the su! cultures, and are eokecenan and dlevalvedt ina 
1 ant solution of caustic potash. The solution is then made eli 

Wy. hydrochloris acid, when a bulky precipitate forms ; 

sale on a filter and — ce. Por use a wel amount is netted 
lation of of soda and thi 





“1 plague, ‘im certain tnstances 
‘The Indian Com- 

sus ar, eazae to the covelusion *' that, en the whole, a certain 
mnt of advantage soerued to the patients both, in ous, of those 
injected with Yersin's scrum and of those injected with Lustig's serutu.”" 
abe be mentioned that the Commission ands ae the result of 





riably present, the as of 
carrying out of the teat ia com: 
= ented Wy the natural teudency of the bacilli to coberw in clamps. For 
be last reason rasnopic (mdimentation) method 1s be prafeered 
to iaesemes (qi. A suspension of plagne bac fs Tinds 
Ureoking rat aa, caltare in 78 par cent, sodican ob 
solution; tho larger Soc 
fino, supernatant ‘staan ta om esl in the usual way. According to 
the restlta of tho Gorm Gomatinon and the obaevations of 
demic, it tay bo wai that the 
ms of the serim of from 1:10 to 
found thas the date of its appearance ts about a week 
After tho onset of isis, aad that it uavally facrenses tll about the end 
of the ee ee fading off. 14 ix most marked in severe 
eases ul 









Uy an carly and favourable crisis, lees marked in 
ere cases nltiastaly proving fatal, whilst im vory mild cases tba 
feoblo oF may by abwnt.” The mothod, if carefully appliai, may be of 

certain conriitions; bat it will be seen that its use ava 
means of diagnosis is somowhat restricted. 


Methods of Diagnosis.— Where a bubo is present n little of 
the juics 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 
id the wucceasive stroke mothod. The cultural and morpho- 

characters are then to be investigated, the most important 

the involution forms on salt agar and tho stalactite 
Lit in bouillon, though the latter may not always be obtained 
twcillus: the pathogenic properties should also 

@ guine-pig being on the whole most uitalie Sor 


= 





subcutaneous inoculation. In many eases a diagnosis 
made by microscopic examination alone, ws in no oth 
onion than plague do bacilli with the morphalo 

actors of eta 






‘opinion is given. 

Tn a caxo of suspected plague 
microscopic examination of tho emit, 
methods along with animal inoculation with the spntam. 
be carried out; sulicutaneons injection in the guinea-pig 
smonring the nasil mucous membrane of the rat may be 
mended, Here @ positive diagnosis should not be att 
mlcroscopic: oxamination alone, especially in « 










trict, as bacilli morphologically resembling the pl 
may oceur in the sputum in other conditions, 


Tearsixo Feven avy Arnicax Tick oe 


of velapsing ao He described. ite peels chanacters, 
and found that its presence in the blood had @ definite relation 
to the time of the fever, as the organism rmpidly dimppeared 
about the time of the crisix, and nsyppeared when Ae 
occurred, He failed to find such an organism in 

disease, His observations wero fully confirmed, and rd views 
‘as to its causal relationship to the discaxe were generally, 

Later, tho disease was produced in the human subjest by inootlae 
tions with blood containing the orgunisms, and a similar eon 
dition has been produced in apes. 

Recently it has boon shown that the so-called tick fever” 
provalont in Africa is also due to a spirochmte of similar 
charucter, and results of the highest importance have been | 
established with regand to the part played by tieks in the team 
mission of the disease, Doubt still obtains as to the 


of the organisms of the two diseases, but all are agreed that thoy: 














CHARACTERS OF THE SPIRILLUM 439 


are closely similar, if not identical. Asa matter of convenience, 
and in accordance with the history of the investigations, we shall 


are bape ane in the animal Engi 
infections have Leen deseribed in geous et 
Marchoux and Salimbeni, in oxen and 

bats by Nicolle and Comte, and it is presto ayes nate. oe 
the case of the Sette ae ‘oxen and fowls the infection is 
tranemisaible bs i fine 

The work Shatin p. 548) hs led to these spirochate 
Boe oui by various authorities ax members of the protazoal 

however, 
netadinal division has 
nob soe bl satisfactorily 
observed and no cycle of 
development has been 
determinedamongat ther, 
we are not justified at 
present in removing them 
from the class of bacteria, 
of the 
Spirillum.—The orgin- 
jams as seen in the blood 
during the fever are deli- 
eato spiral filaments which 
havo a length of from two 
ui F) Mood cor mace, 

‘2 ap Pid, 149-—Spirilla oF relapsing fever in 
‘They are, however, exceed: human blood, Film preparation, (After 
ingly thin, their thickness Koch.) abont 1000, 

‘being inuch less than that 
‘of the cholem spirillum. They show several regular sharp curves 
‘or windings, of number varying according to the of the 

irills, and their extremities are finely pointed ( 149). 
Pies ach often to be seen in the pals portions wi ck are 
thinner and loss deoply stained than tho rest, and which suggest 
hee occurrence of trannverse division, Thoy are actively ey 

and may be xeon moving quickly across the microscopic fic 
with @ peculiar movement which ix festly twisting and partly 
, and disturbing the blood corpuscles in their coures, 
Novy and Knapp hare found that thoro is « single flagellum as 
‘one end of the organ 
‘They stain with watery solutions of the basic aniline dyes, 














RELAPSING FEVER 


eer, 
ie eae eet of ia madtioninanen 


Tn blood ont crepes Bache: 
degree of vitality, and when kept in scaled tubes 
found alive and active after many days. 
killed at @ tomperatnre of 60° C,, but may be 


of incubation thers occurs a rapid 

lasts for about five to seven days, 

crisis occurs, the temperature quickly 
course of about other seven days a sharp rise er 
aguin takes place, but on this occasion the fever 
time, again suddenly disappearing, A second 


relapse may occur wfter a similar interval. The spirilla 
appear in the blood shortly before the onset of the 
during the «rise of REE 
‘They are very numerous during the 
often present in every field of the rio 
examined at this stage, They begin to disay 
the cri after tho crisis they are entirely absent 
circalating blood. A similar relation between the presenos 
tho splrilla in the blood and tho fever is found in the case of the 
rolapses, whilst botwoon those they aro ontirely absent, Miineh 
in 1876 produced the disease in the human subject by 
blood containing the spirilla, and this experiment has been 
suveral times repeated with the éamne remult, Additional proof, 
thut the organism is the cause of the disease has been afforded by 
experiments on animals. Curter in 1579 was the first to show that 
the disease could be readily produced in monkeys, re his 
ments wore coined by Koch, in auch exper th Me 
taken from jatients and containing the spirila saat 

taneously, In the diseass thus produced there is an 
period which usually lasts about three days. At the end oat 
time the spirilla rapidly appear in the blood, and shortly after 
wards the temperature quickly rises. The’ period of pyrexia 
usually Insts for two or three days, and i followed by ae 
crisis, As a rule there is no relapse, but oceasionally one 
short duration oce White mice and rats are also susceptible 


+ Norris, Pappeubicimer, aud Flourauy, in their experiments om pa 
In Americ. ind that several relapses occurtwl, 





























IMMUNITY 4a 


to infection. In the former animals the disease is characterised 
by several relapses, in the latter there is, however, no rel 
Numerous attempts to cultivate this organist ot the 
body havo all been attended with failure, and it has beon 
abundantly shown that it does at a on any of the media 
ordinarily in use Koch found wt on blood serum the 
filaments of the apirilla increased somewhat in length, and 
formed « sort of felted mass, but that no multiplication took 





‘Hiv, 150.—Spirillum Obermederi Iu blood of infected mouse. x 1000, 


place. Recently Norris, Pappenbeimer, and Flournoy have 
found that a considerable amount of multiplication may take 
place in the citrated blood of man and the mt. 
Immunity.—Metchnikoff found that during the fever the 
spirilla were practically never taken up by the leucocytes in the 
eculating blood, but that at the time of the erisia, on dis- 
Sppenring from the blood, they accumulated in the spleen and 
were ingested in large numbers by the microphages or poly- 
morphoauclear leucocytes. Within these they mpidly under 
‘went degeneration and disappeared. It is to be noted in this 
connection that swelling of the spleen isa very marked feature 
in relapsing fever. These observutions were entisely euuiewoa | 


442 RELAPSING FEVER 


by Soudakewitch, who also produced the disease in two suonkkeys 
(corcocebus fuliginome) from which the spleen bad been previously 
removed, the animals having leon allowed to recover completely 
from the operation, and found that in these cases the spirilia 
did not dissppear from tho blood at the usual time, but rather 
increased in number, aud a fatal result followed on the eighth 
and ninth days respectively. Recent observations, however, in- 
dicate that, as in the case of eo 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 (rucacus 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 scrum. He found, for example, that 
in vitro the seram of an immune animal brought the movements 
of the spirilla to un end, clamped them, and caused their dis- 
intogrution ; and further, that when the spirilla and the immune 
scrum were injected in one case into a fresh monkey no disease 
developed, In opposition to Soudakewitch, Lamb found that 
with « monkey from whieh the spleen had been removed death 
did not occur after it was inoculuted with the spirilla, Baw 
schenko and Milkich found that there are developed during the 
disease an imamune body and an agglutinin, while Nevy and 
app in their recent important work distinguish germictdal, 
unising, aod agglutinating substances, They found that 
the blood of the mt has no germicidal properties during the 
onset of the disease, but that thea» appear and become well 
marked during the decline, hey produced « “ hyper-immunity™ 
in ruts by repeated injectious of blood containing the spirilla, 
and found that the serum of sacl animala had & markedly eum 
tive effect, and could cut ahort the disease in ratty inies, 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 proubly rather the ease 
that the immunity conferred ia of very short duration, The 
course of events in the disoxse might be explained by supposing 
that immunity of short daration is produced during the frst 
period of pyrexia, but that it does not last until all the spirilia 
have boon dostroyed, 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 43 


ius reappear in the blood, the mlapse being, however, of 
shorter duration and Jess severe than the first attack. This is 
repeated till the immunity lasts long enongh to allow all the 
organistns 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 mot with 
in the human subject will probably be emincatly amenable to 
serum therapeutics. 

‘The fact that other like spirillar discases may be conveyed 
by the bites of insects makes it extremely probable that relaps- 
ing fever may also be transmitted in this way, and # number of 
facts point to the bed-bug as the means of transmission. The 
presence of the spirilla within the bodics of bugs has been 
demonstrated, and it has also been shown that they may be 
present fora ‘iderable time after the insets have sucked the 
blood,—aecording to Karlinaki for forty daya. ‘Tictin, by injoct- 
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 ertelal experiment 
of infecting man by means of the bites of these ineeots hae not 
yet been successfully carried ont, 




















African Tick Fever. 


‘The disease long known by this nv 
has also boen shown to be caused by a spirillum or spiroehaste. 
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 whieh led 
them to the conclusion that tick fever was due to a spirochwte. 
It ia, howover, chiefly owing to the work of Dutton and Todd in 
the Congo Pree State, on the one hand, and of Koch in German 
Kast Africa, on the other, that our knowledge of this disease has 
boen thoroughly establiehed, ‘The former gave a full account of 
the organisin, 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 orguniams to 
the young, and also important facta with regard to the extent to 
which wore infected in certain districts. 

The following are the chicf facts regurding this fover. 
Clinically the fever closely resembles relapsing fever, but the 
periods of fever are somewhat shorter, rarely lasting for more 
than two or three days [tis rarely attended with a fatal result 





as prevalent in Africa 





















4a AFRICAN TICK FEVER 


unless in pationts debilitated by other causes, The spirilla are 
considerably fewer in the blood than in the European relapsing 
fever, and sometimos a careful search may be necessary before they 
are found. Morphologically they are eaid to bo. prac 
identical, although Koch thought that the organisms in 
fever tonded an the whole to be slightly longer; the average 
Jongth may be said to be 19 to 36 4, Dutton and ‘Todd showed 
that it was possible to transmit the disease to cortain monkeys 





Fra, 151,—Hilm of human blood containing spirllum of tick fever, % 10004 


(coreopitheci) by moans of ticks which had been allowed to bite 
patients suffering from the di: ymptoms in these 
animals appearing about y ation. The diseass 
thus produced ia characterise val rolapsos, and often 
loads too fatal result, In o: 
by means of young ticks hateb 
had been alle to suck the blood of fever patients, and they 
camo to the conclusion that the spirilla were not simply carried 
mechanically by the ticks, but probably underwont some cycle of 









+ We are fnilebted to Colonel Leishinas, R,AM.C. 
from which Fign, 150. rw taka, 





the preparations 





AFRICAN TICK FEVER 45 


development in the tissues of the latter. The species of tick 
concerned is the ornithoderus mowbate, These results were con 
firmed and extended by Koch. He found that after the ticks 
had been allowed to auck the blood containing the organisms, 
these could be found for a day or two in the stomachs of the 
insects, After this time they yradually disappeared from the 
stomach, but were detected (n large numbers in the ovaries of 
the fomale ticks, where they sometimes formed felted mnsses. 





Pw, 162,—Spirillum of human tick fever (Bpirilium Duttoni) ta blow! of 
infected mouse, x 1000, 


Ho 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 

meration ; in the process of tranamission, however, the spirillar 
form was always olwerved, and there was no evidence that the 
organism went through a cycle of change. Koch also made 
‘extonsive observations on the ticks in German East Africa, and 
found that of over six hundred examined 11 per cent of these 
inscots along the main caravan routes contained spirilla, and 
im some localities almost half of the ticks were infeeted. In 


=a 














AMG MALTA PEYER 


Sahiba main lines of commeres ho still: 
them, though in amallor number, It haa alao been: 
that in some places the ticks are found to be 
spirilla although the inhabitants do not suffer 
eircumatance which is probably duc to an 
against the disease, 
‘Although onr knowledge regarding the 
to other apirilla i js far from complete, cortain differences: 
the organisms of Earopean relapsing ee) and of 
fave tare wen established.  Zebinow, 
that the organistn of tick fever ‘posseases AEMEPONS 
Hagella, wherens, aa already stated, the ap, Obermeieri 
single terminal Hagallam. ‘Thia obeeryntion, 
yet been confirmed, Differences are also brotyght out 
inoculation, In addition to the more savers illness 
the spirillum of tick fever in monkeys, it has heen found 
Bruin and Kinghorn that a considerable uumber of 
are susceptible to the Afsoan spirllam, inetd rabbits 
guinowpigs, which appear to be refractory to _onganism: 
relapsing fever, Breinl also compared the immunity conferred 
by the sp. Obermoieri and by the tiek fever spirillum, and found 
that each conferred a relative active immunity against iteelf, but 
not against the other. It is thus highly probable that they 
represent two distinct species. Spirillar fover has also been 
found in India, but its relations to the European and African 





ee 


Hit 


fevers have not yet been fully worked out, 


Mata Fever. 


Synonyms—Mediterranean Fever: Rock Fever of Gibraltar = 
Neapolitan Fever, ete. 


‘This dimase is of common occurrence along the shores of thie 
Mediterranean and in its islonds. Since its bact hus 
been worked out, it has been found to oecur also in India, 
and in some parts of North and South America, ite distritmtion 
being mach wider than waa formerly supposed. Although from 
its symptomatology and pathological anatomy it had been re 
cognised as a dit 
names, its precise etiology was unknown till the publication: 
of the remarehes of Colonel Bruce in 1887. From the spleen 
‘of patients dead of the disease he cultivated characteristic 
orgunismn, now known ax the micrococeus melitensis, wad ly 








affection, and was known under various 























MICROCOCCUS MELITENSIS aa 
means of inoculation experiments established its causal relation- 


ship to the disease. ht and Semple applied the agglutina- 
tion test to the di of the disease, while within recent 
the mode of spread of the disease has been studied 


xy & Commission, and it has been demonstrated that goat's milk 
is the chief means of infection. 

‘Tho duration of the disease is usually long—often two or 
hice metas Ihoudlo wiorior andl masel lege ens ei 
with. Its course is very vuriuble, the fever being of the eon- 
tinued type with irregular remissions. In addition to the ueual 
symptoms of pyrexia, there occur profuse perspirations, pains 
und sometimes swellings in the joints, occasionally orchitis, 
whilst constipation is usually a marked feature. ‘The mortality 
is low—about 2 por cent (Bruce), 

In fatal cases the most striking post-mortem change is in the 
spleon. This ongin is enlarged, often weighing slightly over a 
pound, and in a condition of acute congestion; the alps soft. 
and may be diffluent, and the Malpighian bodies are swollen and 
indistinct. In the other organs the chief change is cloudy 
swelling; in tho kidneys thore may be in addition glomerular 
nephritis, The lymphoid tissue of the intestines shows none of 
the changes characteristic of typhoid fever. 

Micrococcus Molitensis.— This is a smal), rounded, or slightly 
oval organiam about 4 «in diameter, which is specially abundant 
in the spleen. Tt usually occurs ‘singly or in pairs, but in 
cultures short chains are also met with (Rig. 193), (Durham 
has shown that in old cultares kept at the room tomperature 
bacillary forms appear, and we have noticed indications of such 
in comparatively young cultures; the usual form is, however, 
that of  cocous) It stains fairly readily with the ardit 
basic aniline stains, tut loses the stain in Gram's method, Tt is 
generally said to be a non-motile organism. Gordon, however, 
is of & contrary opinion, and has recently demonstrated that it 
possesses from one to four flagella, which, however, are diffieult 
to stain. In the spleen of a patient dead of the disease it occurs 
irregularly sattered through the congested pulp ; it may alao 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 axes; for this purpose 5-10 ec. of 
blood should be withdrawn from @ vein and distribated in «mall 
flasks of bouillon. 'The micrococeus was found by the members 
of the Commission in the of Malta fever patients in 10 per 
cent of the cases examined ; it was sometimes scanty, but sone 
times present in large numbers. 


















4 


448 MALTA FEVER 


Cultivation, —This can usually readily be effected by 
stroke cultures on agar tubes from the spleen pulp and incu 
ating at 57°C, The colonies, which are usually not visible 
before the third or fourth day, appear as small round disos, 
slightly raised and of scmewbat transparent aj ‘The 
maxiinum size—2-3 mm. in diameter—is maara about the 
ninth day ; at this period by reflecwed light they appear pearly 
white, while by tranumitted Hebt 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 — margi Old 
cultures assume a buff tink, 
‘The optimum temperature 
is 37° C., but growth still 
ocenrs 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 delleate 
line of growth along the 
needle track and a small 
flat expansion of growth 
on the surface. ‘There ix 
no liquefaction of the 
z wis, from Medium. Tn bouillon there 

two days’ calturo on agar at 37" ¢,O¢curs a general turhi 

Stained with fuchsin. «1000, with flocenlent deposit at 

the bottom; on the surface 

there is no formation of w pellicle. The reaction of the media 

ought to be very faintly alkaline, as marked alkalinity interferes 

with the growth, On potatocs no visible growth tales place 

even at the body temperature, though the organism anultiplios 

to a certain extent. Ontside 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.—Thore is in the first place ample 
from examination of the spleen, both post martem and 
hat this organisin is always present in the disease, 
‘The experiments of Brace and Hughes first showed that by 
inoculation with even comparatively small doses of pare cultures 
the disease could bo produced in monkeys sometimes with « 
fatal result. And it has now been fully established that inoeulae 







































i 





MODE OF SPREAD OF THE DISEASE 449 


tion by the perma herety Durham, by en te ee 
cerebral method of inoculation, has, however, succeeded in 
raiking the virulence so that ths organiam is enpable of prodie- 
ing in guinea-pigs on intra-peritoneal injection illness with some 
times a fatal result many weeks afterwards. An interesting 
point brought out by ‘Tae experiments is tht, in the ease of 
animals which survive, the micrococens may be cultivated from 
the urine several months after inoculation. 

Mode of Spread of the Disease.—The work of the recent 
Commission has sonal in a paablishion fost of ‘of the highest 
importance with regard disease, In the 
course of pleat shy Coskty found ura the blood of many 
of the gots agglutinated the micrococeus melitensis, and 
Horrocks obtained cultures of the organism from the milk. 
Further observations showed that lutination was given in 
the case of 50 per cent of the goats in Malta, whilst the organism 
was prosent in the milk in 10 per cent. Sometimes the organiam 
was present in enormous numbers, and in these eases the animal 
usually appeared poorly nourished, whilst the milk had a some- 
what serous character. In other cases, however, the organiam 
was found when the animals appeared healthy and thery 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 infoeted 
avimals, and this property was always present when the micro- 
cocens was found in the milk. Tt was, moreover, found that mon- 

a and geats could be readily infected by feeding them with 
milk containing the micrococcus, the disease being contracted 
by fully 80 per cent of the moukeys usd. It was therefore 
rondored 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 

troops In Malta haa 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 

thousand in the corresponding part of the preced 
The various facts with regard to the epidemiology of gece 
have thus been cleared up. For example, it is more prevalent 
in the summer months, w! yen more milk is consumed, and there 
is a larger proportion of cases amongst those in good social 

2 











MALTA FEVER 


ion | the eee gehts for example, Germs oe as ba 
meats that the disease has Sealey aan starred 
Bees since the practice of importing goats fro 


sto) 
j¢ work of the Commission, so far ns it has gone, 

been to exclude other modes of infection us begat € practical ; 
importance, by dust, by the bites of mosquitoes, ete., and if it 
in convoyed by contact at all this is onty when’ the dere: 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 caullceae 

Agglutinative Action of Serum.—The blood serum of patients 
suffering from Malta fever possesses the powor of 
tho micrococeus melitensis in a manner analogous to what has 
been described in the case of typhoid fever, The 
appears oopescaely early, often about the fifth day, and ay 
be present for a considerable time after recovory—somotisnes 
for more than « year. Distinct agglutination with a 1s 
dilution of the serum in half an hour may be taken a a positive 
reaction, antficient for diagnosis, Tho reaction ix, however, 
nisnally given by much higher dilutions, «7 1:500, and even 
higher, “It is to be noted that normal serum diluted 1:2 
produce some agglutination, As rogarda relation to Iroguin 
tho obsorvations of Birt and Lamb and of Bagsett-Smith has 
given results analogous to those obtained in typhoid (p. $40), 

The Commission has recently found that vaccination with 
dead cultures of the micrococens confors a certain degree of 
protection amongst those exposed to the disonse, Asa rule two 
injections were made, 200-300 million cocci being the dose of 
the first injection, and about 400 million of the second. 
use of vaccines has also been carried out in the treatment of 
the disease, but the observations are not sufficiently namerons: 
to allow a definite statement to be made ms to ite valne. 

Methods of Diagnosis.—During life the readiest moana of 
diagnosis is supplied by the agglutinative test just described 
(for technique, wide p. 109). 

tures are most aay obtained from the either 

during life o pot mortem, Inoculate a number of agar tubes 
by successive strokes and incubate at 87°C. Film 
should also be made from the spleen pulp and stained with 
carbol-thionin-blue or diluted carbol-fuchsin (1 ; 10). ‘ 


































YELLOW FEVER 451 


Yetrow Fryar, 


Yellow fever is an infectious disease which is endemic in the 
Weat Indies, in Brazil, in Sierm Leone and the adjacent parts 
of West Africa, though it is probable that it waa from the 
first-named region that the others were originally infected. 
From time to time serious outbreaks occur, during which 
neighbouting countrics also suffer, and the disease may be 
carried to other parts of the world. In thix way epidemics 
havo occurred in the United States, in Spain, and even in 
England, infection usually being carried by cases occurring 
among the crews of ships, In tho parts where it is endemic, 
though uanally a few eases may secur from time to time, there 
is some evidencs that occasionally tho discase may remain in 
abeyance for many years and then originate de nove, ‘There ix 
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 occarrence 
of amild type of the disease which may be grouped with the 
“ bilious fevers” prevalent in yellow-fover regions, This would 
explain the degiwe of immunity which is ehown during a serious 
epidemic by the older immigrants. 

Otent. vartationn are: obpecved kn five olfntead types under 
which the disease presente itself, Usually after from two to 
six days’ incubation » sudden onset in the form of a rigor 
occurs The temperature rises to 104-105" F. The person ix 
livid, with outatanding bloodshot eyea. ‘There are present groat 
prostration, pain in the back, and’ vomiting, at first of mucus, 
later of bile. The urine is idhed and contains albmmin, 
About the fifth day an apparent improvement takes place, and 
this may load on to recovery. Frequently, however, the remission, 
which may last from a few hours to two days, is followed 
hy an aggravution of all the symptoma. The tempernture rises, 
jaundice is obverved, hemorrhages occur from all the mucous 
surfaces, causing, in the case of the stomach, the “black vomit” 
—one of the clinical signs of the disuse in its worst form. 
Annria, coma, and cirdise collapse usher in a fatal issue, The 
mortality varies in different epidemics from about 35 t 
per cent of those attacked. Both white and black rac 
susceptible, but those who have resided [ong in a count 
lees 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 g 
and contains much altered blood derived from hiwens 

























452 YELLOW FEVER 


which have occurred in the mucona 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 lese degree, but often 
resembling the condition found in p poisoning. The 
kidneys are in a state of intense glomerulo-nopliritis, with fatty 


degeneration of the epithelium. ‘There is tion of tho 
meninges, expecially in the lumbar region, and hemorrhages 
may ocour, The other organs do not show much — 
though small hamorhiges under the skin and into all 
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. 





now chiefly a historical interest, as it is now known that 
cansal agent is not one of the ordinary bacteria, but belongs to 
tho group of pli sseroeogla organisms.’ A mosquito acts ns 
the intermediate host, and facts detailed below point to the: 
organism yassing through some eyelo of development. int the: 
body of the insect, The analogy of malaria makes it extremely 
probable that the organiam is a protozoon, but as this has not 

yet been completely proved we have not felt justified in 

the position of the ooo nad placing it amongst the | 
infections, As bacteriologieal work led up to etaniatnsent 

of our knowledge regarding the nature of the disease, some 
reference must be made to it. 

A very fall researvh into the bacteriology of yellow fewer 
was that of Sternberg, the result of which was that of the 
varied organisms isolated, one which he called the bwcillus a 
appeared possibly to have some relationship to the disease 
Sanarelli in 1897 obtained cultures of an organiem which he 
called bavillus ictervides, and which he considered to be the 
catise of yellow fever ; it is probably identical with the taille 
x of Sternberg, Subsequent observations made by others 
gave conflicting results, some finding this hacilay others 
failing to do so. The bacillus icteroides, ax deseril 
Sanarelli, belongs to the paratypbold group, possessing bat 
flagella, growing on gulatin without liquefaction, and fe 
glucose but not lactose. Reed and Carroll found that it was 
pmotically identical with the bacillas of swine cholerm Tt 
































1 Tn several disoasoe the existence of such cansal factory 4 suspested. 
Other examples aro foot ant mouth diwease, South African horeiekzess, and 
the contagious plearo-pueumonis of wattle. a 





ETIOLOGY OF YELLOW FEVER 403 


must now be considered merely as an organism which may 
occur in the organs and tlasnes in yellow fever as a secondary 
infection, but without any otiological 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 ita work in 
IU The members of the Commission first dirveted their 
inquiries towards determining whether the bacillus icteroides 
was present in the blood during life, and a series of cases were 
investigated bactoriologically, with entirely nogative rosalte in 
bree meg fee resolved to test the h; of 
Dr. Carlos Fin wami, promulgated years pre- 
viously, that the diseaso was carried by mosquitoes ” Selecting 
mosquitoes which they reared from eggs, they allowed them to 
Vite yellow fever patients and then to bite healthy men. Of 
several experiments of this nature two were successfil in the 
firet instance, the first individual to be infected in this way 
being Dr. James Carroll, » member of the Commission, who 
passed through a severe attack of typical yellow fever. Experi- 
ments were en pestered on a larger scale, with completely 
confirmatory ita, ws to the conveyance of the disease by 
mosquitoes. Of twelve non-immunes living under circumstances 
which excluded natural means of infection, ten contmeted 
yellow fever after having been bitten by mosquitocs which had 
previously bitten yellow fever patients; happily all of these 
recovered. Two of the men who were thus infected had been 
previously expoved to contact with fomites from yellow fever 
patients without results, These results were confirmed by 
Guitéms, whose investigations were carried out along similar 
linos; of seventeen individuals bitten by infected mosquitoes, 
tight took yellow fever, and three of these died, 

The species of mosquito used by the American Commission 
was the Steyomyia fasciata, and up to the’ present time no other 
species has been found capable of carrying the infection, It has 
aloo beon determined that a certain period must elapse after the 
insect has bitten « yellow fever patient before it becomes infec- 
tive to another subject. In summer weather this period ix about 
twelvo days; at a lower temperature somewhat longer. This 
probably means that, ox in the case of malaria, the paraite 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. Tnfectod mosquitoes, however, retain the power of 
infection for a considerable time afterwards, probably as long «8 
dixty days It has also been shown that mosquitoes may become 


f 








wil 


seven men were yaxponed to the most intimate 
fomites of yellow fever patients for a period of pesto <. 
oe soiled garments worn by the patients being in some eases 
slept in by these men ; the result wax that not one of 

one ius exposed contracted the discaso, ‘The conelusions 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 quantitios of 
blood or of scrum taken from a yellow fover patient at any 
period up till the thind day- of the disease. ‘The period of 
incubation in this ease is somewhat vhorter than when the diseasy 
is conveyed by tho bite of mosquitoos, the average duration in 
the former case being about three days, and in the the latter about: 
four days, though timea may be considerably exceeded. 
Tr is also interesting to know that in these experimental injeo- 
tions the blood or scrum used wos found to be free from bacteria, 
Up till the present time, we know of only these two methods of 
infection; namoly, indirectly by the bite of a moxquito Infected 
with the yollow fever germ, or directly by the injection of some 
of the blood from a yellow fever patient. In these respects 
tHiore\fe a striking almtlarity: to: what has’ beon entailed aii 
ease of malarial rea 

Experiments with regard to the nature of the yellow fever 
organism wore carried ont by Reed and Carroll, and interest 
ing results were obtained. ‘They found that the organism of 
tho disewso was very easily killed by heut, ax blood from aoe 
yellow fever patient lost its infective power on being heated 
Ww 55° C. for ten minnter. On the other hand, blood 4 serum 
was found to be still infective after having boon passed 
a Berkefeld filter. This has been confirmed by the French 
Commission, with the additional result that the virus jomasee 
through a Chamberland F filter, but not through a Chamber: 
land B. ‘These facts would show that the parasite ia of 
extremely minute size, and apparently belongs to the group: 
ultra microscopic organisms, Up till the present time a 
























ETIOLOGY OF YELLOW FEVER 405 


attempts to find by microscopic examination the yellow fever 
parasite, cither in the blood of patients «nfferinig 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 ulready obtained have 
supplied the basis for preventive measures against the disease, 
those being directed towards the destruction of mosquitocs and 
the protection of those suffering from yellow fever, and also the 
healthy, against the bites of these insects, Already a striking 
dogree of success hag been obtained in Havana, Such meagures 
came into force in Febroary 1901, and in ninety days the town 
was free of yellow fever and for fifty-four days later no new 
cases occurred; and although subsequently the diseaso 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 diseaso may 
be said to be practically eradicated from Havana. In other 
places also successful results have beoo 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-suscoptibility to a 
given disease or to a given organiam, either under natural 
conditions or under conditions experimentally B cee The 
term is also used in relation to the toxins of an 

Immunity may be possessed by an animal naturally, and i then 
usually called natura? immunity, or it may be acquired by an 
animal either by passing through an attack of the disease, or 
artificial means of inoculation. It is to be noted that man an 
the lower animals may be exempt from certain diseases 
natural conditions, wud yet the causal organisins of these diseases 
may produce pathogenic effects when injected in gufficient 
quantity. Immunity is, in fact, of very varying di ‘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, tho resistance muy in certain cases be overcome by 
a very large dose of the toxic agent, For example, the commen 
fowl may be able to resist as much as 20 cc, of powerful tetaiias 
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, theas ean 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 « study of the natural diseases which affect 
the human subject, First, in the case of cortain distases, one 
attack protects agninst another for many years, sometimes 

ically for a lifetime, eg. smallpox, typhoid, srarlet fever, 

dl 





in the case of other discasos, ag. erysipelas, 
acnza, and pneumonia, a pationt may & from 
several attacks, In the casoof the diseases of the secund group, 


Ao) 




















ARTIFICIAL IMMUNITY 457 


however, experimental research has shown that in many of them 
a certain degroe of immunity does follow; and, though we 
sored highly probble tt the ping trwugh a attack fan 
i ‘ighly probable that the passi an at ofan 
acute disease produced by an organism, confers immunity for a 
longer or shorter period, ‘The immunity is not, however, to be 
regarded aa the result of tho disease per a, but of the bacterial 
jucts introduced into the system ; ax will be shown below, 
y Suitable yradation of the doses of such eens or by the 
use of weakened toxins, o high degree of ity may be 
attained without the occurrence of any symptoms whatever, 

‘The facts known regarding vaccination and smallpox 
La oe principle. ‘© may take it ae practically: 
proved ‘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 ix less complete and lasts a shorter time than that 
poet by the natural disonse. Again, inoculation with lymph 
rom a smallpox pustale 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 occupics an inter- 
mediate position. 

Sormunity and Recovery from Disease.—Recovery from an 
acute infective disease shows that in natural conditions the virus 
may be exhausted after a time, the period of time varying in 
different diseases. How this ix accomplished we do not yet 
fally know, but it has been found in the case of diphtheria, 
typhoid, cholera, pneumonia, etc., that in the coume of the 

isease certain substances (called by German writers Antikirper) 
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 immunimtion would appear to be going on during the pro- 
grees of the disease, and when this immunisation has reached a 
cortain height, the disease naturally comes toan end, It cane 
not, however, be said as yet that such antagonistic substances 
are developed in all cases; though the results already obtained 
make this probable. 

Averieias, Toeroxrry 


Varieties —According to the means by whieh it is produced, 
immunity may be mid to be of two kinds, to which the terms 








458 IMMUNITY 


active and passine are generally applicd, or we may speak of 
immunity dirvetly, or indirectly, pronucet. We shall first give 
an account of the established facta, 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 desea, or (4) 
by sublethal doses of their products, te of their “ toxins,” the 
word being used in the widest sonse, By repeated injections 
at suitablo intervals the doxo of organisms or of the products 
van be gradually increased ; or, what practically amounts to the 
same, an onganism of greater virulence or a toxin of greater 
strongth may be used, A proportionate degree of resistance or 
immunity can thus be developed, which degree in course of time 
ay reach w very high level. Such methods constitute the 
means of preventive inoculation or wiccination, Immunity of 
this kind is comparatively slowly produced and lasts « 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 ease may be. Such a serum, gonerally 
known os un antiserum, 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. Tho serum is also preventive, ie protects an animal 
from subsequent infection, but the immunity thus conferred 
lasts a comparatively sh ime. These facts form the basis of 
serum thempeuties. When such a seram has the power of 
neutralising a toxin it in called aatitowie; when, with little oF 
ho antitoxic power, it protects against the living bacterium in & 
virulent condition, it ix called antimicrobie or antibacterial (wile 
fra). 

Tn the accompanying table a sketch of the chief methods by 

which an immunity may be artificially produced is given. Tt 
has been arranged merely for purposes of convenience and t 
aid subsequent description; the principles underlying all the 
methods are the same. 



































Anrurictan TIamestrr 
A. Active Immunity produced in an animal by aa ine 


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 organiema. 
(a) Attenuated in various ways. Examples :— 
(1) By growing in the presence i oxygen, or in a 
current of air, 
(2) By passing through the tissucs of one species 
theses (becomes attenuated for another 


es). 
(sili eccetee etatenemnl pera tenanrte 
(4) By growing in the preseace of weak antiseptics, or 
‘by injocting the latter along with the organism, 
ote. 


(6) Tra viralent condition, in nonlethal doses 


2. By injection of the dewl orguninms, 

3. By injection of filtered bacterial cultures, te, toxins ; or of 
chemical substances derived from such filtrates, 

‘These methods may also be combined in various ways, 


B. Passive Immunity, ie,  producnd in one animal by econ 
of the serum of another animal highly immunised by the 
methods of A. 


1, By antitoric serum, ie. the serum of an animal highly 
immunised against @ particular toxin. 

2. By antibacterial serim, i¢. the serum of an animal highly 
immunised against » porticular bacterium in the living 
and virulent condition. 


A. Active Immunity. 


1. By Living Cultures.—(x) At(enuated.—In the earlier 
work on immunity in the case of anthrax, chicken choles, swine 

a@, ote, the investigators had to deal with organisms of 
is virulence, which had accordingly to be reduced before the 
onganisnis could be injected in the living state. It is new found 
most convenient axa rale to start the process of active immunisa- 
tion with the injection of dead enltures, 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 a8 
vacemner, ‘The virulence of an organism may be diminished in 
yurious ways, of which the following examples may be given. 

(1) Tn the first place, pructically every orguniam when eulti> 
vated for some time outside the body, lows ite virulence, and 
in the case of some thia is very marked indeed, eg. the 





atin: 
animal. Duguid and Bi 
of the anthrax bacillus for bovine animals was lessened by its being 


Acsimilar principle was applicd in the case of swine 

Pasteur, who found that if the organism yroducing this dit 

was inoculated from rabbit to rabbit, ite virulence was 

for rabbits but was diminished for pigs. The method of vaceina- 
tion against smallpox depends upon the same principle. There 
is als evidence to show that the virulonee of the tubercle 
baci becomes modified according to its host, being often 
diminished for other animals, 

(3) Many organisms became diminished in virulence when 
grown at an abnormally high temperature. ‘The method of 
Pastour, already described (p. 314), for producing inne in 
sheep against anthrax bacilli, depends upon this fact. A vi 
‘organism may also be attenuated by being exposed to an elevated 
temperature which ie insufficient to kill it, as was found by 
‘Toussaint in the case of anthrax. 

(4) Still another method aay be mentioned, namely, the 
attenuation of the virulence by growing the organism in the 
presence of weal antivoptica Chamberland and Rewx, for 
example, succeeded attenuating the anthrax bacillus by 
growing it in a medinm containing carbolic acid in the 
tion of 1 = 600, 
Thea examples will serve to show the principles 
There 










































attennation of the virulence of an organi 
over, still other mothods, most of which consist in 
organiam in conditions somewhat unfavourable to ite 
under compressed air, ete. 








BY LIVING CULTURES 461 


(0) Lmmunity by living Virutent Cultures in Non-tethal 
—Tmmanity may a bo produced by employing virulent 
cultures in small, that ix non-lethal, doses. In snbsequent 
inoculations the doses may be increased in amount. essere 
immanity may thus be obtained in rabbite against the bacil 
pyocyaneus. Sueh a method, however, has bad a limited 
application in the case of viralent onganiams, ax it bax been 
found more convenient to commence the process by attenuated 
caltures, and then to continue with living cultures. 
Exnitation of the Virulence. —The converse 
attenuation, i the exaltation of the virulence, is Deals 
chiefly by the method of cultivating. the he orgie from ‘animal 
to animal—the method of passage discovered by Pasteur (first, 
we believe, in the ease of an onguniam obtained from the saliva 
a hydrophobia, though having no ciueal rolationship to that 
fisease). ‘This is most conveniently done by dobepertaesl 
infeatoay as there is less risk of contamination, ‘The orgunisins 
in the peritoneal flaid may be used for the subsequent injection, 
or a culture may be made between each inoculation. The 
virulence of a great rmmber of organisms can be increased in 
this way, the animals most frequently wsed being mbbite and 
This mothad can he applied to the orguniama of 
foal clctares Forcaca to streptococci and staphylococci, 
thos: organisms generally which invade the 
uae 
‘The virnlonce of an organism, especially when in a relatively 
attenuated condition, can also be raised by injecting along with 
it m quantity of a culture of another organisra either in the living 
or dead condition. A fow examples may be mentioned. An 
attenuated diphtheria cultare may have its virulence raised by 
being injected into an animal ulong with the streptococcus 
yogones ; an attenuated culture of the bacillus of malignant 
pecs by being injected with the bacillus prodigiosus; an 
attenuated streptococcus by being injected with the bacillus coli, 
ete, 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 necompanying in- 
jection enables the attenuated onganism to gain a foothold in the 
‘tisaues, and it may be stated us a general rule that the virulence 
of an organiam for a particular animal is raised by ite 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 


afterwards more virulent, and by increasing the doses, a high 
degree of immunity may bo obi 4 
oy unity by Dead Cultures of Bacteria.—In some cases 


if 

this method the no-called endotoxins will be injected along with 
the other substances in the bacterial protoplasm, but the resulting: 
immunity is chietly directed aguinst the vital uetivity of the 
‘organisme—is antibacterial rather than antitoxic (wade preted 
‘The cultures when dead produce, of course, lees 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 extemal 
ured for experimental purposes, and is that adopted in 
Plague and anti-typhoid inoculations. 

). Immunit 


« fluid medium for w certain time, and the fluid is then filtered 
through a Chamberland or other porvolain filter, ‘The filtrate 
contains the toxins, and it may be used unaltered, or be 
reduced in bulk by evaporation, oF tay be evapo to 
dryness. ‘The process of immunisation by the toxin is started 
by smal! non-lethal doves of the strong toxin, or by largor doves 
of toxin the power of which has been weakened by various 
mothods (wide infra), Afterwards the doses are grachinily 
increased, ‘This method was carried out with a great degnes 
‘of success in the case of diphtheria, tetanus, malignant edema, 
etc, Ib appeare capable of general application in the case of 
organiama where ft is posalble to get an active toxin from the 
filtered onltures, Tt haa also boon appliod in the case of snake 
poisons by Calmotte and by Fraser, and a high degree of im= 
munity has been produced. 

Immunity may also be obtained by means of certain chemical 
substances deparuted from filtered bacterial cultures, though: 
these substances are generally in a more or lest impure 
condition. Hankin was the first to obtain this result by em 
of an albumose separated from anthrax cultures 

The following may be mentioned os some of the most 
important examples of the practical application of Tee Re 
of active immunity, he, of protective inoenlation 4 
tion of shecp and oxen against anthrax (fastoas)(p ne aH; Ae 


Jennerian vaccination against smallpox (p. 503); (3) 











BY BACTERIAL PRODUCTS OR TOXINS 463 


cholera inoculation (Haffkine) (p. $12); (4) Ai pines 
inoculation (Haif kine) (p. 434) ; As Anti-yphoid inoculation 
(Wright and Semple) (p. 343); (6) Pasteur's mothod of inocu. 
lation against hydrophobia, which Vein esentially the eame 
principles (p, 616). 

Vaccines as co Method of Treatment-—Up till recently the 
principles of active immunity hod 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 
immunization in such circumstances i not only possible, but ix 
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 
Dacterial infections by carrying on an active immunisation 
against the causal agents by the injection of dead cultures of 
the latter, ‘The justification for such w procedure lies in his 
contention that in many cases infections are to be looked om ns 
practically entirely localised, eg. the cases of an acne pustule, 
or a boil, The reason for the local growth of bucteria in the 
part of the body affected ix thut there ix for unknown canses a 
doticioney of the opsonie power (wide p. 483) of the body fluids, 
which is essential for the phagocytosis of the invading bacteris. 
Still more marked in such cases is the deficiency in the opsonic 
qualities of the fluids in the actual site of infection, An 
procedure which will raise the opaonic power ef the body fluids 
aaa whole, and therefore of the fluids in the focus of infection, 
will aid the destruction of the bacteria by sensitising them to 

phagocytic action, Sueh a procedure is found in the active 





pate kos which results from the injection of @ vaccine 
consisting of a dead culture af tho causal bacteria. ‘The appli- 
cation of « saceino of thia kind must, however, be controlled by 
constant observation of the oponic index of the patient's serum 





what won calla the occnrrence of a nogative phase. In wease 
where the | treatment is eneceatul, negative phase is succeeded 
deh in the opeonic index re its original level,—ocurrence 
itive phass,—and with this reaetion there is an improve: 
ae in the local condition. Usually in such cases repeated 
injections are required to effect @ curs, “and the important point, 
according ht, is to avvid giving an injection when a 
negative phase is in progress. If this point is not attended to 



















464 
only an rr foe of symptoms is to be looked for (wile 


and killed hy steaming for a sufficient time—eay pee. L hours, 
‘The efficiency of the steritisation is tested by inoculating tubes 
of uppropriate media. ‘The strength of the che aaa is then 


the measurement of ‘this quantity and in its pirat every 
aseptic precaution must, of course, be adopted. Such vaccines 
have been used extensively in the treatment of acne, boils, 
syeosis, infections of the genitourinary tract by the b, coli, 
infections of joints by the gonococeus, and in many cases con- 
siderable success haa followod the treatment. | 

Active Immunity by Feeding.—Dhvlich found that tice 
could be gradually immunised against riein and abrin 
them with increasing quantities of these Papers pe ee 
Tn the course of some weeks’ treatment in this way the 
immunity was of so bigh a degree that the animals ould tee wea 
‘on subcutaneous inoculation 400 times the dose 
Fraser alao found in the case of snake venom that ert coal 
be immunised, by feeding with the poison, against several times 
the lothal dose of venom injected into the tissues, 

By focding animals with dead cultaros of bacteria or with 
their separated toxins, a degree of immunity may in some enses 
be gradually developed. But this snethod is so much less certain 
in results, and #0 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 aa specisic, that is, is exerted 
towards the organism or toxin by means of whieh it has been 
produced. A certain degre of immunity, or rather of inerensed 
general resistance of parta of the body (for example the 
toncum), can, however, be produced by’ the injection of va 
substances—bouillon, blood serum, solution of nuclein, ete 
(Teseff). Also increased resistance (o one orgunisin aun be thas 
produced by injections of another organism, Immunity of this 
kind, however, never reaches a high degree, 


B, Passive Immunity. 


Action of the Serum of HighlyImmunised Animals,—1. 
The serum of an animal A, treated by repeated. and f 








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 lntter, or a short time before it, As 
would be expected, it has loss effect whon injected some time 
afterwards, bat even then within ecrtain limits it has a sures 
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 scram of on animal A, highly immunised against a 
Twcteriam by evpeated and gradually inereasing doses of the 
erp, may protect an animal B against an infection hy the 
living organi«m when injectod under conditions similar to the 
rae ‘This serum is therefore antimicrobic, or antibacterial, 

ntive against invasion by a particular organism. (1 
sdiltion to the preventive or protective action in vive, such a 
serum may exert cortain recognimble elfects on the corryonding 
organism in eftro, Thus (a) it may lead to the death or solution 
of the ooganiams— bastriddal ‘or fysogenie ation ; (b) it may 





prodace an increased tibility to ingestion by 
opaonic action ; (c) it may lead to the clumping of the organism: 
—aygtutinative action.) 


‘These tw kinds of anti-sora — antitoxic and antibacterial — 
exert their effect when injected along with the toxin or organism 
tively or some time proviously; as would be expected, 
they have less effect when injected some time afterwards, though 
even then they may have « 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 vera is differont, A serum may, however, possess 
Doth properties in varying degrev, ‘Tho 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 subs 
stanoos to the toxin or bacterium as the caso may be,—theve 
being generally spoken of + anti-substances. It will eccordingly 
be convenient to speak of anti-sutetances in general, 
pinent of anthanhstances, first observed in the came 
of the injection of taxing, is found to occur when & great many 
different substances are introduced into the tismes of the Living 
body. We can, in fact, divide organic molecules into two clases 
—thows which give rive to the production of anti-snbstances, and 
those which ave not this property, Amongst the former aw 
20 


=. 














forms: ‘ical or physical union wit 


smbatanen which has led to its development, 
‘the evidence for this later, Furthermore, the #1 
‘apparently a specific combining group which fits, 


frou in the mding substance, the tw 
compared ta a 


specificity is not invalidated, The number of 
substances, as judged hy their combining propertics, 
appear to be almost ieollnied, a fact whtshy HEGreneia 

‘on the complexity of the structure of living matter. When anti- 
substances are studied a4 dé their action on the substances: 
with which they combine, they may be conveniently 

in three classes corresponding to Ehrlich's three clawes of 
receptors (idle p. 491). In the first place, the antimubstance 
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 
antisubstance, in uddition to combining, may produce some 
recognisable physical alteration. In other words, it possesses 
an active or zymotoxie group us well at a combining grap. 
‘The agglutinins may be mentioned as examplox of thix group. 
In the third place, the anti-substance after combination leads to 
the combination of another body mally present in serum 
called complement or nlexine, and this Intter, whieh has a ¢on- 
stitution very similar to that of a toxin, may lend to 

change, for example, death or solution of a cell. Antiaul oes 
of this claws are own as immune<bodies or 

(Ehrlich) or as sensitixing substances—audatances seuribitieatriogs 
of French writers, Antisubstances of the second and thin 
groups are met with especially, though not exclusively, 
formed elements such as bacteria, red corpnscles, or tissue 
etc, aro injected, the autiscram developed posscading: 



























ANTITOXIC SERUM 407 


tinating, solvent, or other propertios towands the particular 
snbstance, 

Aftor this preliminary statement in explanation we shall 
consider tho actual properties of the two classes of soram, and 
later wo shall resume the theoretical consideration. 

Antitoxic Serum.—Tho best examples are the antitoxic 
sera of diphtheria and tetanus, though similar principles and 
mothods aro involved in the case of the anti-sera to mein and 
abrin, and to snake poison. We shall here apeak of diphtheria 
and tetanus, ‘The steps in the process of preparition may be 
said to be the following; First, the preparation of a powerful 
toxin. Second, the oatimation of the power af the toxin, Third, 
the development of antitoxin in the d of a suitable animal 
by gradually increasing doses of the toxin, Fourth, the estima- 
tion from time to time of the antitoxie power of the serum of 
the animal thus treated, 

1. Preparation of he Torin.—The mode of preparation and 
the conditions affecting the dovelopment of diphthoria toxin 
have already boon deseribed (p, 362). In the ease of totanna the 
growth takes place in glucose bouillon ander an atmosphere of 
hydrogen (wide p60). In either ease the cultore is fil 
through & Chamberland filter when the maximam degree of 
toxicity has been reached. The term “toxin” is asually applied 
for convenience to the filtered (i. bucterium-free) culture. 

2, Estimation of the Toxin.—The power of the toxin is 
eotimated by the subcutaneous injection of varying amounts in 
a number of guinea-pigs, and the minimum dese which will 
produce death is thus obtained. This, of course, varies in 
proportion to the weight of the animal, and is expressed accord. 
iny ie In the case of diphtherio, in Ebrlich’s standard, the 
auinunum Jethal doss—known as M.L.D.—is the sinallest amount 
whieh will certainly canse death in a guinea-pig of 250 Var 
within four days Behring nees the term “normal diphtheria 
toxin of simple strength" (DTN'), as indicating a toxin of 
which ‘01 c.c. is the mini 1 lethal dose under these conditions, 
A toxin of which the minimum lethal dose is 02 will be of half 
normal strength (DTN * soon, The testing of a toxin 
direetly is a tedious process, and in actual practice, where many 
toxins have to be dealt with, it ix found mons convenient to test 
them by finding how much will be neutnslived by a certain 
amount of a standard antitoxie serum, viz, an “immunity unit" 
(p. 468). 

3, Development of Antitorin.—The earlier experiments on 
tetanus and diphtheria were performed on sniall animals, sucks 












468 IMMUNITY 


as guinca-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 uae in the first stages of the process either « 
weak toxin or a powerful toxin modified by certain methods. 
Such methods are the addition to the toxin of terchloride of 
jodine (Behring and Kitasato), the addition of Gram's fodine 
solution in the proportion of one to three (Roux and Vaillard), 
and the plan, adopted by Vaillard in the case of tetanus, af 
using @ series of toxins weakened to varying degrees by bn 
exposed to different temperatures, viz, 60°, and 56", and 50° 
Th the cave of large animals immunisation is sometimes started 
with small doses of unaltered toxin ; and the doses are gradu 
ally inereased, ‘The toxin is at first injected into the sithe 
cutancous tikened, later into a vein, Ultimately 300 00, oF 
more, of active diphtheria toxin thas injected may be borne by 
horse, such a degree of resistance being developed after the 
treatment has been carried out for two or three months. Tn all 
cases of immunising the general health of the anisnal onght not 
to suffer. If the process is pushed too rapidly the antitoxie 
power of the serum may diminish instead of inereasing, und # 
condition of marasmus may set in and may even lead to the 
death of the animal (». p, 494). (In immunisation of small 
animals an indication of their general condition may be obtained 
hy weighing them from time to time.) 
4. Estimating the Antitorie Power of, 
Serwm.—This ix d 
bine sikurn’ of the tinevuniatd 














“ tandardising,” the 
of various quantities 
imal against « certain amount 
of toxin. Various standards have been used, of whieh the two 
chief are that of Ehrlich and that of Roux. lich has 
adopted as the immunity wnit the amount of untitoxie serum 
which will neutralise 100 times the minimum lethal dose of 
toxin, serum and toxin being mixed together, dilated up to 
4 cc, and injected subewtancously into a guinea-pig of 250 
grms. weight, the prevention of the death of the animal within 
Your days being takon as the indioetion of noutrilaaltacetiaant 
standard in testing, Ehrlich employs quantities of serum of 
known antitexic power in a dry condition, preserved in « yaeuam 
in a cool place, and in the atwonce of light, A thoroughly dey 
condition is ensured by having the glaas bull containing the 
dried serum connected with another bulb containing anhydrvus 
phosphoric With such a standard testserum any newly 
prepared scrum can readily be compared. A ‘normal " antitexie 
serum Is one of which 1 éc, contains an immunity unit, 1 Ge 
of a serum, of which °02 cc, will protect from a hundred times 





























USE OF ANTITOXIC SERA 469 


the Icthal dose, will possces 50 immi masa genie fi 
this serum 1000 Gonna units Sera boon prepared of 
which 1 ee. has the value of 800 units or even more. 


Roux odopts « standard which represents the animal 
ramnuen protected by 1 ot of era againat the dost of vaateat 
iethal toa control betel in thirty hours, 0 
twelve hottrs Finley fray. Eu, 46 01 enc. of a sorum will foes 
guinen-pig of 500. inst the luthal dose, 1 ec. (1 yrm., Rea tw 
3,000 prise. 6f getnenspie, anid the-veltie oF tie werua will 


During the prove of development of antitoxin a stall 
quantity of the lood 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 antitoxio power his 
been reached the animal is bled un Cae 

‘the serum is allowed to separate in the usual manner. Tt is then 
Phecs ie cgi mano y poser rater ee 
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. S84. 

Use of Antitoric Sera—n all cases the antitoxie serum ought 
to bo injected as carly in the disease aa 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 odvisability of using larger doses has 
gradually become more and more evident, iney Martin 
recommends that as much as 4000 units should be administered 
at once, and that if necessary this quantity shonld bo ropeatod. 
‘A atrong serum prepared by Behring contains 3000 unite in 
5-6 ce, but even stronger sera may be obtained. Even very 
large doses of antitoxic serum are without any harmful effects 
Leyond the occasional production of urticarial and erythematous 
rashes. Whero large quantities of seram require to be ad- 
tinistered, as isalways the case with antitetanic serum, injections 
must be made at different parts of the body ; proforsbly not 
more than 20 ¢.¢, should be injectod atone place, The immunity 
conferred by injection of autitoxic serum lasts « comparatively 
short time, usually a few weeks at longest, 

Sera of Aniviale immunised againet Vegetable and Animal 
Poieana,—It wna found by Ehrlich in the exse of the vegetable 
toxins ricln and abrin, and also by Calmotte and Fraser in the 
case of the snake poisons, that the serum of animale immanisod 
ae respective substances had a protective effect when 

aan tee them into other animals Ehrlich found, 
for example, that the serum of « mouse which had been highly 





470 IMMUNITY 


immunised against ricin by feeding as described above, could 
protect another mouse aguinst forty times the fatal dese of that 
wubstance, He considered that in tho ease of the two poisons, 
antagonistic substances—" anti-ricia™ 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 poieon. 

‘Theae investigations are reap instructive, as sueh 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 bucteria, 

Nature af Antitexic Action.—This subject is only part of the 
general question with regnrd to the relation of antisubstances 
to their corresponding substances, but it is with regard to anti- 
toxic wction that most of the work has been done. We have to 
consider here two points, viz (a) the relation of antitoxin to 
toxin, and (4) the souree of the antitoxin, With rogurd te the 
former subject there lins been much diversity of opinion, but 
the evidence now available goes to show that the 
between toxin and antitoxin ix not a physiological ono but that 
the two bodies unite in vitro to form a compound inert towards 
the living tismos, there being in the toxin molecule an atom 
group which has a spocific affinity for the antitoxin molecule or 
part of it. We shall consider the facts in favour of this view, 
aad 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 witro it 
can be proved that their behaviour towards cach other resembles 
what ia observed in chemical union, Thus it has been found 
that a definite period of time elapses before the neutraliaation 
of the toxin is complete, that neutralisation takes place mons 
rapidly in strong solutions than in weak, and that it ix hastened 
by warmth and delayed by cold. C. J. Martin and Cherry, and 
alao Brodie, have shown, that in the case of diphtheria toxin and 
in that of an Australian snake poison tl molecules will 
pase through a colloid membrane (f. 166), whilst thase of the 
corresponding antitoxin will not, Now if a mixture of 
equivalent ports of toxin and antitoxin ia 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 filtration 
the less texin passes, till a time is reached when no toxin is 
found in the filtrate. Further, if the portion of fluid which at 
this stage bus not yassed through the filter be injected into an 


























NATURE OF ANTITOXIC ACTION 471 


animal no aymptoms take place; this shows that after a time 
neutralisation is complete, Again, in cases when the toxin has 
somo definite physical effect demonstrable in vitro, ¢g. lysis, 
agglutination, coagulation, or the prevention of cougwlation, ite 
action can be annulled by the antitoxin ; in such cireumstances 
manifestly no physiological action of antitoxin through the 
modium of the cells of the body can come into . These 
facts are practically conelusive in favour of antitoxin action 
depending upon a direct union of the two substances concerned. 
‘Tho evidence usually brought forward against the direct unton of 
toxin and antitoxin reats ebioly on tale cheetaiion te Calmette, 
who found that the antitoxin toa snake venom was more easily di 
‘by heat than the toxin, and stated that when # neutral mixture of the 
two was heated at a temperature sufficient to destroy free antivesin, the 
toxie Bropertine fa. part ralurned... Hance: he ooosluded that tha tee 
bodies existed in an unoombined condition in the mixture, Martin and 
Cherry, however, on repeating theas experiments, found that the above 
result was not obtained if sufttent time for complete combination was 


they wonld only prove that the toxin has not jeatroged. 
two complicated chemical compounds of uneyaal stability sre in ivoww 
chomical union, it is quite concsirable that the less siable may be 
destroyed (ey, by heat), whilst the moro stable weapon 


Although practically all authorities are now agreed as to the 
direct combination of toxin and antitoxin there is still much uncer- 
tainty ux to the exact nature of this union, Controversy an 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 erude taxin the largest amount of 
toxin which could be neutralised completely, so that no. 
resulted from an injection of the mixture, This amount he 
called the fimes null dose, expressed ax Ly. He then investigated 
the effocts of adding larger amounts of toxin to the insanity 
unit and observed the quantity which was first sufficient to 
produce « fatal result, that is, which contained one M.L.D. of 
toxin; this amount he called the fimes tidtlich, fatal limit, 
oxprouied as Ly. Now if, as be supposed, the union of toxin and 
antitexin reeemblod that of a strong acid and bese, Ly —L, 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 Sivaye considerably more than ono M.L.D, For example, 
in the case of one toxin, M.L.D. «0165 gr., Iy=126 gr., Ly= 


472 IMMUNITY 


“9 ge. ; differonce = "36 gr, 19 MID. This, in brief, is 
what i¢ known as the "Ehrlich phenomenon,” and it has been 
explained by him as the result af the presence of toxoids (wide 
». 171), ie. toxin molecules in which the toxophorons has 
mnie degenerated, Ho distinguishes three possible var 
of auch bodies according to the allinity of the hay OTOL 
group, namely prototacoid with more powerful affinity than dhe 
toxin molecule, epizaroid with less powerful aftinity, and aymtoxroset 
with equal atfinity, ‘The presence of epitoxeids would manifestly 
explain the above phenomenon, The L, dose would re) it 
toxin +epitoxoid molecules all united to antitoxin molecules and 
the addition of another ML of toxin would got resnlt in 
‘there being a froo fatal dove, but in the added toxin taking the 
place of epitoxoid, Several lethal doses would need to be added 
before the mixture was enifieiant to produce a fatal result; that fs, 
Ly ~ Ly would equal soveral 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, 
40 that in one case observed the M.L.D. was three times the 
original fatal dove, and still the amount of antitoxin necessary to 
neutralise it completely was the same ax before. Ebrlich also 
investigated the effeots of partial neutralisation of the L, amount 
of toxin, that is, he added to this amount different fraetions 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 distinet bodies 
present with different combining affinities—the graphic repre: 
sentation of the mixture not being « curve but o stepstair line. 
Thus he distinguished proto. deutero., and tritotoxins (with 
corresponding toxoids). Lt will thus be wen that Ehrlich regards 
the combination toxin-antitoxin to be a firm one, and that the 
nentralisation phenomena are to bé explained by the compliented 
constitation of the crude toxin. 

The chief criticism of Ehrlich» views has come from the 
important work of Madsen and enius Their main cone 
tention is that the toxinantitoxin combination ix not « firm ene 
bat a reversible one, and ix governed by the laws of physical 
chemistry, For example, in the case of a mixture of ammonia 
and horacic 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 combinat is reversible, so 
that if some of the froe ammonia were romoved # certain amount 
of the combined ammonia would become dissociated to take its 
place; further, Uf to the mixture, in a state of oq 

































MODE OF PRODUCTION OF ANTITOXINS 473 


more ammonia or more bonele acid were added, part would 
remain froe while part would combine, Accordingly, if tesla 
and antitoxin behaved in a similar manner an explanation of 
Ehrlich phenomenon would be afforded, Madsen and enone 
have worked out the question in the case of a great many toxins 
and find that the graphic representation of DpotralieaGon 3a 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 caso ryprosont a single 
sul ce or several! (2) What is the nature of the combination 
of any one constituent substance and its anti-substanco—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 sone must in our opinion be supposed. ‘Thon there ix 
an important fact ‘atablishod by Danysz and by y. Dungern, 
namely that the amount of toxin neutralisable by a given amount 
‘of antitoxin is different according as the toxin is a a aoe 
tnoiotios or all wt once—in the latter case the amount pips cK 
neutralimble is greater. There seems no explanation of this 
to the view of Maden and Arrhenius as the same state 
of pe ‘ought to be reached in the two eases, 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 
antisubstance being reversible—the example of a hiwmolytic 
immune-body may be cited (p. 481)—and there is no doubt that 
thers are varying degrees of firmness of the unic It is quite 
evident that if there should be several toxic bodies ina “toxin,” 
and that if the union of some of these with antitoxin should be 
reversible, the problem becomes one of extreme complexity. 
‘There ns 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 «maller 
toxin molecule becomes entangled, ax it were, in the larger 
autitoxin one, very much as a dye becomes attached to the 
structure of a thread, Rordet has long maintained a theory of 
this nature and gives reasons for believing that there is no 
definite quantitative rolationship in the combination of the 
molecules of the two substances, different amounts of antitoxin 
affecting in varying dogroe all the molecules of a given amount 














prol 

taing different toxic bodies with varying affinity ; 

fow instances the combination has boen proved to be phased 
‘but to what extent this is the case remains still to be 

‘The next question to be considered is the xoune of antitoxin, 

‘Tho following throo possibilities prosont thamealves : (2)asitenin 


may be formod from the toxin, ie, may be « “ modified toxin" 5 
(6), antitoxin may. be the renult of en increnssd) formation! of 
molecules normally present in the tissnos ; (c) antitoxin may be 
an entirely now product of the celle of the » Ikcan now be 
stated that antitoxin is not a modified toxin. It hus been shown, 
for example, that the amount of antitoxin produced by an animal 
af be many times greater than the equivalent of toxin inj of] 
and further, that when an animal is bled the total amount 
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 ‘ter circumstance 
shows that antitoxin is formed by the cells of the body, If 
antitoxin isa product of the cells of the body, we are almoxt eom- 
polled, on thooretical grounds, to conclude that it is not a nowly- 
manufactured substance, but a normal constituent of the living 
cells which is produced in incrensod quantity. We have, however, 
direct evidence of the presence of antitoxin under normal con- 
ditions,—the presence of such being shown by its uniting with 
toxin and rendering it inert. Normal horse serum, to mention 
an example, may have a varying amount of antitoxie action to 
iphtheria poison, ox-bile his a sitnilar action to snake poison, 
whilst in the case of other anti-substances—such as agglutinins, 
bacteriolysins, hnmolysins, etc.—whose production ix 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, Tn the first place, 
wo have ovidence that in the living body bacterial toxins enter 
into combination with, or, as it is often expressed, aro fixed by 
the tissues—presumably by means of certain combining affinitles: 
‘This has bean shown by the experiments of Dénitz and of 
mana with totanus toxin, We have, in sich cases, however, 
ids as to where the toxin ia fixed beyond that suppli 
iy urrence of symptoms, Another line of research which 
has been followed ia to bring emulsions of various organs into 
contact with a given toxin and observe whother any of the 
toxicity is removed. ‘This was firet carried out by Wassermann 


al 











CHEMICAL NATURE OF ANTITOXINS 475 


and Takaki, who investigated the action of emulsions of the 
central nervous system of the useptible guinca-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 ec. of emulsion of brain and 

inal cord was capable of protecting 9 mouse against ton times 

fatal dose of toxin. ‘These observations have been confirmed, 

though their significance bas been variously interpreted. Tt 
would, however, bs out of place to discuss at length the opposin, 
views, and we accordingly simply state the facts ascertai 
‘We may note, however, that it is not a serions 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. 

Tt 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 

jisoning, may act as an antitoxin when fre in the blood, 
This wil be discussed below in connection with Ehrlich’s theory 
<3 etidbers tea We aay conclude by saying that anti- 
toxin ly represented by moleenles normally present in 
tho colls or (more rarely) in tho fluide of the body. 

Of the chemical nature of antitorins we know little From 
their experiments ©. J, Martin and Cherry deduce that while 
toxing are probably of the nature of albamoses, 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 procipitate with magnesinm sulphate from anti- 
diphtheria seram contained practically all the antitoxin, and that 
any snbstance obtained: which had an antitoxic value gave all 
the reactions of a globulin. Thay also found that the per- 
centage amount of globulin precipitated from the serum of the 
horas increased after it was treated in the usual way for the 
production of antitoxin. Such a supposed difference in. the 
tise of the molecules might explain the fact, observed by 
Fraser and alo by ©. 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 


BE 


‘bactorial Pera Te stages in 


cei ae ap to these in 

vera, but living, or, in the early dew 
instead of toxin separated by filtration, and in 
obtain @ serum of high antibacterial power a very 
calture in large doves must be ultimately tolerated: 
animal, For this parpose a fairly virulent culunre ts 
fresh from a case of the particular disoase, and its vi 
may be further incressed by the method of 
method of obtaining a high degree of tinmunity against 


rachel 


microbe is specially applicable in the case of those organisins 
whieh invade the an and multiply to a great extent within 
the body, aud of whick the toxic effects, though always existent, 
are proportionately small in relation to the number of organisnus 
proeoat The method has heen apple in the ets of the 


and cholera organisms, the bacillus of bubonic plague, 
the bacillus coli communis, the pneumococous, sl 
capable of very 


development of the immunity is accompanied by the 6] 
in the blood of protective substances, whieh can 
another animal. ‘The law enuneiated by Behring regarding 
immunity agninst toxins thus holds good in the case of the 
living orgnnisins, as was first chown by Pfeiffer, ‘The Intter 
found, for example, that in the case of the cholem organism, s0 
high a degree of munity could be produced in the yuit 
that -002 cc. of its scrum would protect another gninen 
against ten times the Icthal dose of the organisms, when io- 
fected along with them, Here again is presented the remarks 
able potency of the antagonising substances in the serum, 
which in this case lead to the destruction of the corresponding 
microbe 

‘The anti-streplococelc scrum of Marworek may be brielly described, » 














PROPERTIES OF ANTIBACTERIAL SERUM 477 


aaa analy ie trviones of enuopevanoes le erscing sltaneany 
ein ° r 
sieeoeaea ‘alin istry eed 
strum a owilon (oie. 


resistance in aye Perel mas continued over & con- 

oi adiile Tai of Gime, an tho "ent eek Sn 
ha cortain dove of the virulent 

font ing ie area ore Righty ad 


i nay cae of 
Sie limaay antics > Mareaeel, Tovever, fo rere armas 
Little antitsate power, that & could only protect from n comparatively 
senall dow of toxin obtained tyr ltration ‘of cnitares, 


Antityphoid, anti-cholera,! antipneumococcic, anti-plague, 
wad other sera are all prepared in an analogous manner. 
of Antibacterial Serum. 


consider the three main actions mentioned above, viz (a) 
bactericidal and lysogenic action, ($)-opronic action, and (c) 


—ieiffer found that 
illum, were injected 
into the peritoneal cavity of a guinewpig highly immunised 
against these organisms thoy at sie eo almost immedi- 

ually became granular, swollon, and thon disappoarod 

at ier floss changes constitute “ Pfeiffer’s phenomenon.” 
Farther, he found that the same phenomenon was witnessed if 
‘@ minute quantity of tho antiserum was added to a certain 
quantity the corresponding organisms, and the mixtare 
injected into the peritoncal cavity of a non-treated animal, 
Pfeiffer found that the sernm of convaleseant cholera patients 
gave the same reaction aa that of immanieed animale, He 
‘obtained the same reaction also in tho case of the typhoid 
bacillus and other organisms From his observations he con- 
cluded that the reaction was specific, and could be need as a 
means of distinguishing organiemna which revemble one another. 
peeeney considered thats specific substance was developed 
Ce eee of immunisation and that this was rendered 
sdeay icidal by the aid of the living cells of the body. 
Tt was ‘chapped shown, however, by Metchnikoff and by 
Bordet that lysogenesis might occur outside the body by the 


© A trie anfitonke obolers serum has been prepared by Metehathett, B. 
‘Rows, and Taurelli-Bolinnbont, 









—— 


AT 





immunoeerum. Pfeiffer alvo found that an antieerum 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 aguinst a bacterium « substance 
appears in its serum with combining allinity for that 

organism. ‘This substance which is generally known as the 
immunobody, ar (Ehrlich), or eudstance sensibilieatrice 
(Bordet) is comparatively stable, resisting usually a temperature 
of 70° C for aun hour. Tt_cannot produce the destructive effect 


plement is fees swat being mpl destroyed by a 
temperature of 60° C., and it is not increased in amount 
the process of immunisation, Though fermentlike in its in- 
stability, it differs from a ferment in being fixed or used up in 
ane ‘quantition 

The phenomenon of lysogenesis is, however, only seen in the 
ese oar certain organisms when an animal is highly immunised 
against thom ; the typhoid and eholora group are outstai 
examples, It ia alao to be noted that it sometimes ia seen in the 
case of « normal serum (vide Natural Inumunity), Tr other cases 
the hactoricidal effect of a serum may oceur without the rapid dis- 
solution characteristic of Lysogenosia though dthor 
changes may be produced, In still other cases a bactericidal 
effect may be wanting ; nevertheless it may be shown that an 
inmuno-body ie developed by the procoss ef immunisation. This 
may be done by observing the increased amount of complement 
which is fixed through the medium of the antiserum (immune 
body), sensitised red corpuscles boing used as the test for the 
presence of free comploment, The following achome will show 
the modo of experiment, which is carriod out in a series of 
sina] test-tabes -— 





in } Iumune-body(anti-seram hoatedat 55°C.) + complement, 
amount of bacteria and immuno-body in each tube, Yarying 
of complement in different tubes.) 
Incubate at 47° C. for one and « half hours, 

(3) Add co each tube red corpuscles treated with the corresponding: 
immune-body, and incubate for another hour. 


‘The ne-body'” ia in each eas the antiserum deprived 
‘of comploment (by beating at 55° C,), obtained from ae 
injected with the bacteria and red corpuscles respectively. 
















HEMOLYTIC AND OTHER SERA 479 


control ix got by substituting in another experiment the same 
amount of heated normal serum for the antiserum. If there is 
free complement left there will be corresponding lysis of the red 
corpuscles ; iran siesec Has clon fal ieee eine 
lysix. To takean example from Muir's experiments, — it was found 
that an emulsion of the bacterin alone took up -03 cc, of guinea 
Pig's complement, whilst the sme amount of bacteria treated 
with immune-body took up 13 cc. ‘The allimportant action of 
the immnno-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. 

Tt 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. 
Thin result, which is generally known as the Neisser- Wechsberg 
phenomenon, has becn the subject of much controversy, and 
cannot yet be said to bo satisfactorily explained. It would 
accordingly be out of place to discuss here the different viows 
with to it. (Regarding some theoretical considerations 
aa to therapeutic applications of antihactorial sera, wide 


489.) 
g The Jaws of lysogunesis ars, however, not peculiar to the 
case of solution of bacteria by the fluids of the body, but, as has 
been shown within the last fow years, hold also in the caso of 
other organised substances, rd corpuscles, leucocytes, ete., when 
‘these are Introduced into the tissues of an animal as in a process 
of immunisation. Of such sera the hamolytic have been most 
fully studies, and owing to the delicacy of the raction and the 
caso with which it can be observed, have been the means of 
Uhrowing 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, 

Hemolytic and other Sera.—Tt has been known for some time 
that in some instances the blood serum of one animal bas, 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 scram of the former 
acquired « marked lwemolytic property towards the corpuscles of 
the Intter, the property being demonstrated when the seram is 
wided to the corpuscles. Bordet also found that the haemolytic 


scrum while the complement is left. Thay ene ee 
clusion that immune - body arent the complement. 
though the combination was less 
at a higher temperature—beat about 7 CG. They therefore 
consider that the imrnune-body acte as a sort of connecting link 
between the red corpuscles and the cory ee hence the term: 
“amboceptor” which Ehrlich after pplied, Te may be 
etated, however, that the direct union St: complement and 
immune-hody has not been conelnsively demonstrated. Bordet, 
on the other hand, holds that the immune-body nets merely ax 
4 sensitising ugent—hence the term substance sensibilisatricg— 
and allows the fermentlike complement to act. It is quite 
evident from his writings, however, that he docs not mean, as 
ix often assumed, that the immune-body causes some Tesion in 
the corpnscle which allows the complement to act, but sim 
that it produces in the molecules (receptors) of the red. 
an avidity for compl t. All that we can ty definitely at 
present is that the combination of reeeptor + immune-body takex 
up complement in firm union while neither docs 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 
loked with water tho recoptors aro not destroyed: Muir and 
Ferguson have shown that they can still take up immune: 
body and, through its medium, complement, just ax the intact 
corpuscles do, Ehrlich and Morgenroth showed that in some 
eases the red corpuscles enn take up much more immuane-body 
than is necessary for their lysis, and Muir found in one case: 
studied, that each further dose of immune-body Ted to the fixation 
‘of more complomont, so that as many as ten times the hwmelytic 
dose of complement might thus be used up. Te is matter of 
consi lc importance that the union of Immuane-body and red 
corpuscles can bo shown to be a rorersible action. eae, 
found by Morgenroth and Muir indopondently, corpuselea 














HAEMOLYTIC AND OTHER SERA 48t 


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 satficient immuye-body will pass 
from the former to the latter, so that all Deane eae on 
the addition of sufficient cory goers ‘The combination of 


sveral 

simgleeate in # pie serum with different Seer to 
ienbs 7 for which Ehrlich and his co-workers 

vo nae forward & large amount of evidence, or whether, 
as Bordet holds, there is a se cents 
ever, show slight variations behaviour towards. different 
immune-bodies There is at least no doubt that all the com~ 
a Workers of 


the free condition inthe blood, bt is berated. from the 
leucocytes when the blood is ehed; though this cannot be beld 
as proved, there is cvidence that the amount of free complement 
inerwases after the blood is shed and some time later gradually. 
diminishos 


‘Tho hemolytic action of a wormal scram can be shown in 
many cases to be of the sume nature ux that of an immune- 
sorum, that is, complement and the homaloguo of an immunc- 
body can be distinguished, For example, the gaines-pig’s eerum 
is luemolytie to the ox’s corpuscles; if « portion of serum be 
heated at 55° C, the complement will he destroyed ; if another 
Ea be treated with ox’s lex at OC, tho natural 

mmune-hody will be removed only complement will be left, 
Neither portion ik in itself hwamolytic, but this property becomes 
manifest again when the two portions aro mixed. Hawmolytic 
‘sera aro 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 
ome action on those of an allied apeciea, that is, some receptors 
are common to the two species. This fact can be readily shown 
by the natal absorption tests, for example, in the case of an 
anti-ox serum tested on shoop's corpuscles, A clos analogy 
holds to what has been established in the case of agglutinins. 
Tt is further of great interest to nove that by the injection of red 
corpnseles into an animal ite serum not only becomes hemolytic, 
nut in many eases when heated at 55° C. possceses also ogglu- 
inating and opsonio properties towards the red corpuscles used. 
And further, it would appear that in some cases at lenst the 
immunsbody, bemagglutinin, and hemopenin are distinct 

a 


—————— 





482 IMMUNITY 


substances, ‘These facts abundantly show how close an analogy 
obtains between anti-bacterial nnd huemolytic sem, and how 
important a bearing hemolytic studies have on tho questions of 
immunity in general. 
To addition to hemolytic sera, anti-sera have been obtained 

by the injection of leucocytes, spermatozoa, ciliated epithelium, 

ver colls, norvous tissuc, ete, The laws governing tho pro- 
duction and properties of these ave identical, Chat is, each serum 
exhibits « apecific property towards the body need in its produc 
tion—i«. diseolves loucocytes, immobilises spermataza, ete. 
The specificity is, however, not eo marked as in the case of 
sera produced against rd blood corpuscles; thus a seram 
duced against tissue cells is often hwmolytic; thie ix 
due to various cells of tho body having the same 
Here again when the apti-soram produces uo destructive effect 
on the corresponding cells, the presence of an immune-hody: 
be demonstrated by the increased amount of complement whis 
is taken up through its medium. It may also be mentioned 
‘that each anti-sermn usually exhibits toxic ies towards 
the animal whose cella have been teed in the injections, eg. a 
luemolytic serum may produce a fatal result, with signa of 
extensive blood destruction, hwmoglobinurin, eto, we it ix 
hwmotoxie for the particular animal; n serum prepared by 
injection of liver colls has been found to produce on injection 
noorotic changes in the liver in the specics of animal whose liver 
cells were used. These are mentioned as cxamples of a very 
large group of specific activities. 

“Ayah regard to the sites of origin of immunc-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 fn 
the spleen, lymphatic glands, and bone-marrow, According to 
certain workers of the French school, the chief source of anti. 
substances acting on cella such as ted blood corpuscles is the large 
mononucleated leucocytes, whilst those acting on fa are 
chiefly derived from the polymorpho-nuclear Jencocytes (vide 

495), Another view is that immune-bodios are chiefly formed 

the large mononucleated leucocytes, whilat 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 
overlooked. As yet, definite statements cannot be made on t 


point. 


‘Methods of ‘Tonts. sora ie usuall 
Py injoare terre corpaeclics esas Bey the peritopecas 
Fran animal of diferent corpuscles of most 
frequently used, and the rabbit & the mast suitable animal for Injection. 
Tho corpuscles ‘ought to bw completely feeed of serum by 
‘washing them in sterile salt so! ‘An injection 
of the eorpuseles of 5 e.¢. of o's blood followed by two injeotions, exch 
of 10 0.c, at intervals of ton days, will usually give an active serum. 
Tha anim should Lill Ly Mecding it, maptzaly a tr a4 pouble, 
Seven to ten dave after the Test injectiou 5 the serum which separates 
ay be collected te suitable lengths of quill glase-tubing drown out at 
the onds, which are afterwards sealed in tho ‘To onsure sterility 








cent puacles in °$ per cent sodinm chloride solu 
that is, the ies of 5 0,0. blood are completely freed of soram by 
rey in salt solution, ond then salt solution is added to 
make wp 100 cc.” In any invosti it fe a to obtain the 
minimom haemol LD.) of the immune-body and of the 
complement to be used. (It is to be noted that as complement does not 
i cr tion, the hemolytic dose of the fresh 


‘will come far short of ropresenting the annount of Imuune-body present.) 
In testing the dose of gee the fresh seram to be ma com: 
Plement mut be devoid of hamolytic netion (in the present instance 
rabbit's sermn will be found suitable) aud more than sutheient fo produce 
Iyxia with immune-body is added to each of a weriex of tube Varying 
amounts of immune-body are added to the tubes, the contents are 
shaken, made up to 1°S o.c. and Incabated for two hours ‘The amount 

then noted and tho t ed in a cool chamber till 
1, The smallest amount of 
immune-body which giver complste lysi« ix of coures the MHD, ¢ 





corpuscles, When further observations are to be continuel an the sane 
day, the reading after incubation must bo taken ss the working 
standard To eatimate the MILD, of complements proceed in 

ing manor; to cach of a active of tubes add several doses of 
‘and thon to the several tubes ditforent amounts of com- 
activity of a serum as complement ratios considerably, 
pls 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) Opwmic Action —The presence of a substance in an 
immunes#rum whieh makes the corresponding onanism sensitive 
So pgogioate was firet demonstrated by Denys and Leclef in 
1895, in the case of am anti-streptococcal serum. They also 
showed that thes seram produced this effect by acting on the 


‘orguaian, not on the leueocytes. It ix, however, chiefly to the 
rescarehes of Wright und his co-workers that this subject hus 







come inte special prominence. Wright and Douglas in their 
first paper shows that the phagocytosia of staphylococe’ by 





pp. 194, 261), There i 


washed in salt solution ; normal serum heated to 55°C. ix also 
without effect in inducing this phenomenon, They eould not 
demonstrate any effect of the opsonin on the leucocytes. On the 
other hand, if bacteria be exposed to the fresh serum, ond they 
bo freed from the excess of serum and then exposed to phagss 


eytos also washed free from serum, thoy will be readily 
up hy the cells, It has been aopitinte een tl 
action of the serum nL ox hy tne. of Lave 
inst: an organism, and the opsonic index represents 
immunity in one of ite aspects ax alroady explained (p, 
The matter has, however, become complicated by the cit 
stance that in an immune-serum an opsonin may still be 
after the serum is heated at 55° C., ax has bon shown by 
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 hamolytic sora. Wo are, however, probably safe in 
saying that the thermostable opsonin of an immunesertin ix a 
trie anti-anbstance, possessing the specific characters of anti 
substances in general and comparable in this respect and im ite 
mode'of production with an agglutinin. Muir and Martin have, 
however, found that the thermolabile opsonin of a nortwal serum 
hos different characters For example, when & normal serum ix 
tested on o purticular bacterium, the epsonie effect on that 
ium may be removed by treating the serum with other 
other words, the thermolabile opsonin of normal 
serum docs not posess the specific character of the opsonin 
developed in the process of immunisation. They have alvo 
found that various substances or combinations of substances 
which act as “complement-nbsorbers" also remove the i 
property from a normal scrum, while they have no effeet on an 
immuncopsonin. According to this view the opsonic effect of 
the unheated serum of an actively immunised animal er person 
would represent the sum of the effects of the two kinds of 
‘opsonin, 
Pe urther stndy will be necessary befone the exnct. relati 
of these substances are fully understood, and other questions with 
gard to them have as yet scarcely been touched upon, 
Increased phagocytic action had long beou known by the work 
of Metchnikoff to be associated with the development of active 


HE 


EI 


il 


























AGGLUTINATION 485 


sore Gr at Mes oak oe tinea Ea arti ae 
Tan we work ot a 

eee pecan the other dirvetion, and points to Pati 

development of anti-substances in the seram as the all-important 


an animal immunised aguinet th rg the er the growth formed 
a deposit at the foovet tha ‘the vessel 5 growth in no1 
serum produced a uniform turbidity. (Ciraber ad Trisha i 
investigating Pfeiffer's reaction, found that when a «mall quantity 
of an antiveram is added to an emulsion of the corresponding 
tucterium, the organisms become agglutinated into clumps, 
this phenomenon depending upon the presence of definite bodies 
in the serum called aggtutentne. 
Tt 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 {nets experimentally 
established, it appeared a natural proceeding to enquire whether 
sach serum Possessed ant bo epee netion and at what stage 
of the discaso it appeared. The result, obtained indopend- 
ently by Griintaum and Widal, but first pablished 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 varicty of anti-substance can be demonstrated at 
an carly stage of the disease, Agylutination is also observed in 
the case of cholera, Malta fever, bacterial dysentery, glanders, 
plague, infection by Giirtner's bacillus, ». coli, ete. Furthermore 
the phenomenon i# not peculiar to bacteria; it ia seen, for 
exemple, when an animal is injected with the ted corpuscles of 
anothoe species, haemaygintining appearing in the serum, which 
have a corresponding specificity, 
The physical changes on which agglutination depetids earmot 
aa yet be anid to be fully understood. Graber asd Durham 
considered that tho agglutinin produced a chasge in the envelope 
of the bacteriutn, causing it to ewell up and become viscous, and 
the facts first established appeared to be in favour of this view. 
On the other hand, this ix not the fall 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 cormsponding antiserum produces a sort of granular 
precipitate in it, and that when, as in the agglutination af bacteria, 
















486 
weds 


animal or person Hees to the prema of the Fela 
Joos has found in the case of the typhoid bueilins that 
there are two agglutinable substances which differ in their 
resistance to heat—a- and S-agglutinogen, and piece ive rise: 
to corresponding agglutinins, Further, a com. 
parative study of the agglutinins of a a bane a eon 
variety of the hog cholera bacilIng Theobald Smith hns come | 
to the conclusion that there is an agglutinin which is poo 
hy und acti on the flagella and another which is similar! 

to the hueterial bodies. ‘The former acts in very much higher 
dilutions than the latter, and this is reyarded as an 

of the fact that in the case of non-motile organisms the 


bacteria are freed from salt by washing in ma water they: 
become resolved, and that on the addition of some sodium 
chloride they are formed again, and Joos has alao 

forward striking confirmatory evidence as to the 

the presen . It i thus probable that in the 

menon of agglutination aa ordinarily understood mora Leen one 
factor is concerned, and it is possible that in part it me 

on some altered molecular relationship of the so toca a 
surrounding fluid analogous to altered surface tension, 

AAs stated above, the agglutinins are usually placed in the 
second order of anti-substances, and are regunied us possessing 
a eombining group and an active or agglutinating group, “The 
constitution would thus be analogous to that of @ toxin, and in 
conformity with this view Risenberg and Volk consider that the 
agglutinating group may be destroyed while the combining: 
group nomaing, tho reault being an agglutinoid. "The evidences 
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 mid to be Gilet pedis cif 


power does not vary paré passu with the degree of immunity— 
aserum may be Rroogly ugglutinative and feebly bactericidal 
and viee versa. But while probably as a rule the two substances 
are distinct, it would not bo 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, agglutinine and 
immune-bodies are the products of corresponding reactive pro- 
cesses, and their formation is governed by corresponding Jaws. 
gpa 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 tho amount necessary to their 
agglutination—a corresponding faet to what has been established 
with regard to immune-bodics of haemolytic sem The 
aed are ifie in the sense which has been explained 
above (p 466), It can be shown by the method of absorpti 
in an sgglutinating sernm there may be several agglutinins 
‘with different combining ps some of which may be taken ap. 
by organiams allied to that which has given rise to the anti- 
serum. Whether or not the combination of an agglutinin with 
the bacterial roceptors is @ reversible action urust be left ae open 


Besides those stated above, other phenomena have been 
observed in the interaction of anti-sera and the corresponding 
Por example, it has been shown that when certain 
bacteria—eg. the typhoid bacillus, b. coll, and b. proteus—are 
im bouillon containing a «mall proportion of the horo- 
serum, their morphological characters may be altered, 
growth taking place in tho form of threads or chains which are 
not obenrved in ordinary conditions. In other instances a seram 
may inhibit some of the vital functions of the corresponding 
bacterium, 





—Thisx subject deos not strictly bel to bacterh r 
Hee tie pemeetlchaccennss arco clonaly allicl te the june desrieed 


fi 








Jasts Lea aan speed ehurera te eleoae 
the 301 lal ve. Le i. 
theserurn of the speelse aed Su the furtuntation 0 





fssconiing to tha ubove method, amd thet a maall eantity of complement, 


say fresh guinea-pig serum, be added, it will be found that the eomple- 
iment becomes almurbod, as uiny be shown by subsequently adding «test 
amount of sensitised rod blood corpuscles, ‘This derintion pl nob 
is oven « more delivato resction than the procipitin test, it being often 
posible to doinonsteate by its uso from a tenth tom hundredth of the 
fuallost amoant of serum which will give a poreeptitle procipitate ; at 
‘also is spocifio within tha same limits.” 


Therapoutic Bifocts of Anti-Sora—As will have been 
vathered, the chief human disesses treated by antisera are | 
diphtheria, tetanus, streptococens infection, pneumonia, plague, 
and snake bite, Of the results of such treatment most is known 
in the case of diphtheria, Hero a very great diminution inthe 
mortality has resulted. The diphtheria antitoxin came into 
general use about Oetober 1894, and the statintios published by 
Rehring towards the end of L805 indicuted results which baye 
since been confirmed. In the Berlin Hospitals the a) 
mortality for the years 1891-93 was 36° per cent, in 1894 i 
was 21°] porcent,and in January-July 1895, 149 percent. The 
objection that in some epidemics very mild type of disease 
prevails is met by the fact that similar diminations of mortality 

* For an seconut of prvetpiting wide Nuttall, * Blood Comunity and 
Relstionsiips,” Corbridge 1904; and of complement deviation, Muir and 
Martin, Jowrn. of Hyg. vo, 1996, pe 26%. 


=" S 





THERAPEUTIC EFFECTS OF ANTISERA 489 


have occurred all over the world. Loddo collected the results 
of 7000 cases in Europe, America, Australia, and Japan, in 
cal titi itd Dinetegt belle hire plein 
ity in the same hospitals of 44 per cent. It has aleo been 
‘that if during an epidemic embed ei 
haimortaiy ot once rises; and in two instances recorded it 


feed ob ts cares knowledge the diagnosis of diphtheria 
ig now much more accurate than formerly. Another 

‘the antitoxic treatment has been that when paprtanac ks 
necessary the percentage of recoveries is now much higher, being 


584 as compared with 32-1 cent previous to the intro- 

toxin. ia 10 most striking results obtained 
in the same heapitals is « reduction of the death-rate in post- 
scarlatinal diphtheria dais 50 per cent Wa + per cont 


Behring showed that in cases treated on the firet and seoond 
days of the disease the mortality was only 7-3 per cent, and this 
has been ly confirmed, whilst after the fifth day it was of 
Nethe to apply the treatment. In order to obtain such 
it cannot be too strongly insisted on that attention 

should be given te the domge. When bad results are obtained 
it may be strongly suspected that this precantion has not been 
observed. In the treatment of acute tetanus by the antitoxin 
the improvement in rewults has not been marked, but some 
chronic eases have been benefited, and ax already stated (p. 330) 
results are obtained in acute cues if intravenous in- 
jection be pmetised. In the case of Yersin's anti-plague serum, 
benefit has appeared to follow its use, experience 
es its effects has been too limited to enable a judgment 
be formed. ‘Tho same may be said to be true of the anti- 
seem ‘and anti-pneumonio sera, though in the case of the 
first mentioned numerous cases of apparently successfal resule 
Aue been recorded. With regard to anti-venin, Lamb has shown 
ion with full glands bites a man, many times the 
wi Lethal dose: are probably injected. Tn eases of alight 
bite, however, benwfit may accrue from the use of the anth 
serum, 


Fr 








action a suiiciency of and ax these 
ey en abies the y 
results with this class of sera may be toa iency of 
complement. Oe i say baie Erlity ba engaee eee 


nt niturally existing in haman scram doce 
the immune-body in the anti-serum—that is, is not taken up 
through the medium of he latter and brought into combination: 


with the bacterium, And thers ie still the further possibility: 

that even thonzh the complement should be taken 

Nib one group of the latter is not sufliciently active towards: 
bacterium to effect its death, In both eases it : 

‘that an extrnoellular bacterici action cannot tires by 


ery case. 


Thecwies a4 to aes Tri 


Pee rica eae nak he aaeae te ne 
artificial immunity the m aay) 
be snes have ieribonata the insufticiency of various 
thors which had been propounded. Only a ahort ference, 
ste need be mule to these. The theory Lb distant 
tour's name is wssociated, supposed that in 
o for the exietenes: 


living animal there are substances necessary 
4 particular organism, w ch become used tp during’ thei sqjemiatay 
‘of that organism in the tissnes; this pabulum belng cohaaaay 
the organixme die out, Such a supposition ix, of sme eee 
disproved by the facts of passive immunity. “Accritngeo 

theory of retention the bacterin the body an 
to produce substances which are inimical to their growth, #0 

they die out, just as they do in a test-tube culture before | 
medimm is really exhausted, Such a theory only survives 

in the that antitoxins are modified toxins, the 

against which has already been Sesires Ln 470), There th 
eame the humoral theory an 
neither of these is tenable in it 
botwoen them x 
any substance specific property in the serum must 
Froiact of cellular activity and on the other hand, 
th regard to passive immunity go far beyond the 
ae digestive properties of phagocytes, though these 
























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 Metchnikoif’s phagocytic theory as further 
developed. These will now be discussed, and it may be noted 
that the ground covemd by cach is not coextensive. For the 
former doals chiefly with the production of antisubstances and 
its biological significance, the latter dealx the defe 
properties of cella, either directly by their agoertc activity 
or indireetly by subatances produced bptaaatee the manner 
of digestive ferments, It will be seen, however, that each bas 
a normal as its basis, viz, that of nutrition. 

si i ids eter ae may be said % boan 
‘application views regurding nourishment of proto: 
plasm. A molecule of protoplasm (in the general sense) may be 
regarded aa composed of a cont atom or executive 
contro (Leistungskern) with & large number of side-chains 
Os ertaal fe atom groups with combining allinity for 
food-stafla, It ia by means of these latter that the living 
molecule is ine in the process of nutrition, and hence 
the name receptors given by Ehrlich is on the whole preferable, 
‘These receptors are of thres chief kinds corresponding 10 the 
classes of anti-substances described (j. 466); the hws a 
single unsatisfied combining group and fi lecules. of 
i itution—reeeptor of the first order; 
has a combs group for the food molecule, and another 
active or zymotoxic group, which leaila to some physical change 
in it—receptor of the seoond order; the third las two cum: 
init one -another_ which 


fixes w nt eal Jpotiumaxpund—receptor of the 
third order or amboceptor, ose latter receptors come into 
action in the case of larger food molecules which meanire to be. 
broken up by ee of the cell 
peaks UP coondaring ‘application of thie idea. to the 
facts of passive immunity, {t must be kept in view that all the 
to which anti-aubstances have been obtained are, like 
proteids, of unknown bat undoubtedly of very complex chemical 
constitution, and that in apparently every case the anti-eubstance 
enters into combination with its corresponding substance. The 
daul constitution of toxins and kindred substances, as already 
described (p, 170), is also of importance in this ‘connection, 
Now, to take the case of toxing, whea these are introduced into 
the system they are fiver, like food-stuffs, by their haptophorous 
foe to the receptors of the cell protoplasm, but are unsnitable 
assimilation, If they an: in suficently largo amount the 








of 
poisoning. If, however, they are in smaller dose, as in the 
carly stages of immunisation, fixation to the protoplasm scours 
in tho mme way; and as the combination of receptors with 
toxin is supposed to be of firm nature, the are lost 
for the purposes of the cell, and the combination RP. {emia 


+toxin) is shed off into the blood. The thus 
become Inced by new ones, and when litional toxin 
molecules are introduced, these new res are used up in 


or-rogeneration, 
wad the receptors formed in excess appear in the free condition 
in the blood stream and then comstitute antitoxin molecules, 
‘There arv thus three factors in the process, namoly, (1) fixation 
of toxin, (2) overproduction of receptors, (3) setting free of 
receptors prodaced in excess, Accordingly 
which, when forming part of the cell protoplnam, anchor the 
toxin to tho cell, and thus are casential 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 throe orders of pinta ho 
separated from the cells, thus give the three kinds of anti 
ices. Ehrlich does not state what cells are specially 
concerned in the production of antisubstances, but from what 
has been stated it is manifest that any ovll which fixes a toxin 
molecule, for example, is potentially a source of antitoxin. 
Cella, to whoxe disturbance, resulting from the fixation of toxin, 
chareteristic symptoms of poisoning are due, will thus be 
sources of antitoxin, eg. cells of the nervous system in the case 
of tetanus, though the cells not so seriously affected by toxin 
fixation may uct in the same way. The experimental int 
tion of the source of antitoxins haa, however, yielded little 
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 thore is much in ite 
favour, In the first place, it explains the difference between 
active and jmssive inununity, eg. difference in duration, ete. in 
the former the cella have acquired the habit of discharging dmti- 
substances, in the latter the antisubstances are simply present 
as the result of direet transference. It is also in bu with 
the section of antitoxing, ote., ns detailed abowe, and sapelally it 
affonla an oxplination of the maltiplicity of antisubstances. 
For, if we take the case of antitoxins, we sec that this depends: 


i 

















er 


EHRLICH SIDE-CHAIN THEORY 493 


race seen simply that of over 
Teeealen whe ioe Ie wld aya key a th integrity 
of the executive centres of the protoplasm molecules would be 
essential to the sutisfactory production of side-chains, and this 
would Pieced sted in necordance 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 sport _f io Sp apart fon the presence of and abana te 
may to withstand @ 


ese aenit of toxin than could be neutralised by the 
total eae ‘antitoxin in its scrum. This might theoretically 
be explained by supposing a special looseness of the cell re- 
pret ai the toxin-reeeptor combination became readily east 
‘The question, however, arises whether there may not be really 
an increased resistance of the cella to the toxophorous affinities, 
An observation recently made by Meyer and Ransom (v, p. 383) 
{a also difficult ef explanation according to the view that antitoxin 
fa formed by tho cells with which the toxin combines and on 
hip ‘They found that in an animal actively immun 
ised against tetanus with antitoxin beginning to appear in 
j single M.L.D, of tetanus toxin into w 7 
peri nerve brought about tetanus with a fatal result, On 
the other hand, the injection of antitoxin into the sciatic nerve 
above the oo a injection of toxin prevented the latter from 
reaching the con One can scarcely imagine an 
esas ibe facts if antitoxin molecules were in process 
ing shed off hy the cells of the nervous system. Further, 
when the serum of an animal contains « large amount of anti. 
toxin, how does the additional toxin injected rench the cells 
in onder to influence them as we know it doest 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 supersensitivencss of the nerve-cells of an 
animal to tetanus toxin, sometimes observed even when there ik 
a large amount of antitoxin in the serum, has been often brought 
forward a4 an objection, But this also may perhaps be explained 
Dy there having ocenrred a partial damage of the cell protoplasm 
by the torophurous action in the process of immuniaation—an 
‘explanation which, of course, demands that in some way the 
freshly introduced toxin may reach the cells in spite of the anti- 



















r 


494 IMMUNITY 


toxin in the blood. Further investigntion alone -will settle these 
and various other disputed points, and may remove many of the 
apparent objections. At present wo may say, however, that 
Ehrlich's theory is the only one which even attempts to explain 
tho cardinal facts of this nspect of immunity. 


Tn connoction with the condition of aupersenaitivenass refined. to 
hors, au interesting phenomenan, has recently been described 
‘Theobald Smith, and ts now generally known as “serum axis, 
or Theobald Stuith’s phonomwenon.” It is briefly the following ;—If 3 
ines pig be injettod with » quantity, say 5 cc of laree serum, mo 
rorbuoee follawa ;_(%, howvrer, nal epee 
say fourteen days hhofors, with a ‘ey om small quantity of horse 
6 (aren Lem a sulelent), ant ten the 6 eof eran bf 
fhe animal uyually dies within an honr sity 
Tho gaperal lesions ar of lammorrbi a in ath and 








ing to Gay and Southard the phenomenon dey aaubstance in 
fhe ores serant which they call anaplplactin, mt a fora 
loug period of time in the blood of the guines-pig. Thi body they 


is cone 
sider to act asm slight irritant to the cells of the guinea-pig, and to 
ce an fucreesed alfnlty fer the molecules in the hotee sorta, 
serdingly when th seo faction i tds the rapid oom binaton of Ea 
Dbstances with the cells result in the disturbances referred to, 
wy be the explanation, the phenomenon is of extreme ier 
ng the profouml alterations ia metabolinn whieh may be 
Uy 6 minute quantity of serum of « normal enimal, 





The facts relating to hypersensitiveness mise the question of 
whether in any immunisation procedure an injury may not be 
constantly dono to the colls forming tho anti-aubstances, We 
have already drawn attention to the occurrence of wl i 
has called the neyutive phase in the course of the inersae of the 

mio power of the serum aimed at in a bacterial vaccination, 
There & is evidence that such negative phases are commen i all 
immunisations, Chey have been also noted in the formation of 
antitoxins, of immune-bodies, and of agglutinins. ‘Thus in the case 
of the first, Salamonsen and Madson ehowod that the fall in the 
content of an anitnal’s serum in antitoxin after a fresh toxin injec 
tion waa greater than could be accounted for by the neutralisn 
tion of the free antitoxin in the blood by the toxin imtroduced, 
and they attributed the occurrence to an injury to the prod 
cells temporarily diminishing the productive activity. ‘The 
normal course of every immunisation may be said to consist iy 
‘a succession of positive and negative phases, and an effective 
immunisation ix one where each succeeding positive phase brings 


1 Vide Gay and Southard, Journ, Med, Resewreh, xvi, 1907, 148, 






















THE THEORY OF PHAGOCYTOSIS 495 


sort f ego 
as not possible to raise opsonic power of # serum 
than & not very great multiple of ite Ale 

content. On the other hand, when we are dealing wi he 
reaction agaiuat bweterial toxins we find that tho Seana? 
producing antitoxin can react in an ext cist 
& sermm many thousand times stronger than that produced 
diving the early days of immunization may ultimately be 
aMtaiieL ‘The nuima | body also exhibits grent power of forming 
aggintining and the cagmeity of forming immune bodies seems 
to coeupy an intermediate position between the opsonic reaction 
and the antitexin reaction. But even in the antitoxin reaction 
‘a time comes ina high immunisation when evidence of exhaue 
tion of the producing mechanism is manifest, so that the injection 
of fresh toxin is no longer efficient, and the negative phase in 
not followed by a positive phase. From the practical stand- 
point it is the nim 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, Bat such facts 

what wo have said aa to tho possibility of every 
immuinisation entailing the infliction of an injury on come bodily 
mechanisem, 

2. The Theory of Phagocytosis.—Thix theory, brought 
forward by Motehnikot! to explain the facts of natural and 
acquired immunity, has been of enormous influence in stima- 
lating research on the subject. Looking at the subject from the 
standpoint of the comparative anatormst, 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 disearc, he 


animal against tacteria depended on the activity of certain cells 
called phagecytes. In the human subject he distinguished two 
chief varieties, namely, (a) the microphages, which are the 

japho-nuclear” finely granular leucocytes of the blood, 
and (2) the macrophages, which include the larger hyaline 
Teucocytes, endothelial cells, connective tissue corpuscles, and, iM 
short, any of the larger cells which have the power of ingesting 
bacteria, Insnacoptibility to a given disease is indicated by a 
rapid activity on the part of the phagocytes, different vurietion 





496 IMMUNITY 


being concerned in different casex—an activity which ey 
rapidly destroy the bacteria and prevent even local 
the organiwns are introduced into the tissues of am 
suscoptible aniraal, there occurs an inflammatory reaction wii 
Toval loncocytosis, which results in the intracellular destruetion 
of the invading organisms, Phagocytosis is mgarded by 
Metchuikoff as the essence of inflammation. Ha ales’ shawal 
that. the bacteria may he ina living and active state when they 
tare ingested by leucocytes. On the other hand, hy found that 
in w anaceptible animal phagocytosis did not occur or was only 
imperfect, He also showed that when « naturally satcepitia 
animal was immunised, tho process was accompanied by the 
appearanos 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 ix known that 
amabw and allied organise have digestive properties which 
aro specially active towards bacteria, and from what can be 
dirvotly olwerved, as well as indirectly inferred, thers ean be no 
doubt that euch a faculty 8 also possessed by the phagocytes of 
the body. Thus bucterla within these cells are in a position 
favourable to their destruction and do in many instances 
become destroyed. In fuct, observations on phagocytosis in 
vitro show that such destruction may in tho ene of tome 
orgnnisms oceur 40 rapidly that the actual number observable in 
the leneoeytes is no indication of the activity of the process. 
Tn othor instances, «.¢. in gonorrhe, the ingested organisms 
would appear to survive a considerable time without undergoing 
change. Undoubtedly phagocytosis is of the highest importance 
in active immunity, as hy ite means organisms which would not 
undergo an oxtra-cellular death may be killed off. Tn the process 
of immunisation of a susceptible animal we see a negative or 
nontral chemiotaxis becoming replaced by positive chemlotaxix 
‘This has beon explaincd by Metchnikoff aa due to an education 
or stimulation of the phagocytes, The recent work on opsonins 
shows, however, that this is not the case, a= 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 immane animal ‘Thos this 
phase of immunity comes to be merely a part of the subject of 
anti-substances in gon 
‘The digestive forments of phagocs 
to Metchnikoff, retained within che cells uader normal conditions, 
but are set free when these cells are injured, for example, when 
the blood is shed. They thon become free in the serum by 

































os or cotaees are, according 











THE THEORY OF PHAGOCYTOSIS 497 


psa len oye hehe aE). sie 


iready. 
are probably two kinds—one called macrorytase, contained in 
the macro lich is specially active toward the formed 
mr iis pre ate eet eae 
microcytase, contained within the palymorpho-anel 
which has a ‘inl digestive ala bens ties Tt is the 
microcytase which givos blood serum its bactericidal propertios, 


ppears to us, howover, that Motchnikoif has gone too far in 
distinguishing the setivities of the two classes of cells so much 
as ho has done, 

When the properties of antibactorial sora, as above described, 
are considered in relation to phagocytosis, Metchnikoff gives the 
following explanation, He admits that the imune-body is 
fixed by the bacteria (or red corpuscles, as the case may bo}, 
though he does not state that a chemical combination takes 
place; hence he calla it a fixative (fixateur). The finmune-hodies 
are to bo regarded asansiliary ferments (fermente adjuvants) 
which aid the action of the alexine, Unlike the latter, however, 
they are formed in excess during immunievtion and set free in 
the serum. He compares their uction to that of enterokinass, a 
ferment which is produced in the intestine and whieh 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 hus been set free by phagolysis, 
He, however, nintains that extracellular digestion or lysogeneais 
docs not take place without the cecurrence of phagolysix. The 
soureo of immuno-bodies ia, in all probability, also the leucocytes, 
as these substances are specially abundant in organs rich in 
aneh eells—splean, Iymphntie wands, ete. ; here aguin the mono~ 
nucloar tou 









muno-bodies are usually sot fro in the serum, th 
ease ; sometitnes they are contained in the coll, and this probably 
oceurs when thore is a high degree of ective immunity against 
hacteria without a serum having an antibacterial action, 1! 

urs of intracellular digestion being in auch cases increased, 
PB this way the facts of immunity can be explained eo far us 
these conourm the destruction of bacteria. 

Metolinikothe work has Joas dirvet bearing on the production 
of antitoxins He m of the toxin by the anti 
toxin to form a & he spparently considers 





498 IMMUNITY 


that leucocytes may nleo be concerned in the production of 
antitoxina. Apart, however, from antitoxin formation, he con- 
siders the acquired resistance of the cells themselves of high 
importance in toxin immunity. 

When we consider Metchnikoif's theory ns thus extended to 
cover recently catablished facts, it must be admitted that it affords 
a rational explanation of a considernble part of the subject, 
though the elucidation of the chomiotactic phenomena during 
immunisation as explained above detracts from the importance 
which he attached to the leucocyte, Tt, however, does not afford 
explanation of the multiplicity and specificity of antitoxina as 
Ehrlich’s doca ; 6n tho other linnd, it ia more concerned with the 
cells of the body as destroyers or digesters of lucteria, As 
regarda the subject of antibacteeial sera, the results of theae two 
workers may be said to be in harmony in somo of the funda- 
mental conceptions, And it is of interest to note that Metchni- 
kof, starting with the phenomena of intracellular digestion, hus 
arrived at the giving off of specific ferments by phagocytes ; rehillat 
Ehrlioh, from his first investigations on tho constitution of toxins, 
has arrived at an explanation of antitoxins and inumune-bodies 
also with u theory of cellnutrition as its basis. Within the last, 
few years marked progress has thus been made towards the 
establishment of the fundamental laws of immunity, 














Naronan Inmoniry. 


We have placed the consideration of this subject after that of 
acquired immunity, as the latter supplies facta which indicate in 
what direction an explanation of the former may be looked for, 
Thore may be said to be two main facts with regard to natural 
immunity, The first is, that thems is a hu uinber of bacteria 
—the so-called non-pathogenic organikins—which are practically 
incapable, unloes perhaps in very large doses, of producing patho- 

nic effects in any animal; when these are introduced into the 
body. they rapidly die ont. This fact, accordingly, shows that 
the animal tissues generally have a remarkable power of destray- 
ing living bacteria. The second fact ix, that there are other 
Jwcteria which are very virulent to some species of animals, 
whilst they are almost harmless to other specios; the anthmx 
bacillus may be taken asan example. Now it ia manifest that 
natural immunity against such an organism might be due to @ 
special power possessed by an animal of destroying the onganiams 
when introduced into its tisenes, It might also possibly be due 
to an insunceptibility to, or power of neutralising, the’ toxihall 

























NATURAL BACTERICIDAL POWERS 499 


the organism. For the study of the various diseases shows that 
the taxing (in the widest sense) are the weapons hy which morbid 
changes are produced, and that toxin-formation is a property 


common to all pat bacteria. ‘There ix, moreover, no 
such thing known as a rium multiplying in the living tixsnes 
without Jocal or general ol 


thee atigh t be. Sie aaa af tech ear Sater aa 
is in most cases one against infection, ir. consists in a power 


soparately. 
1. Fariations in Natural Bacterievdal Powers.—The funda 
mental fact here is that a given bacterium may be rapidly 
destrayed in one animal, whereas in another it pay mpidly 
multiply and produce morbid offocts. "The special powers of 
destroying organisins in natural immunity have been ascribed to 
(a) phagocytosis, and (2) the Val ay the serum, 
(@) The chief factors with regat oy Beth have been 
given above. The bacteria Mis a eal une animal, for 
example, the anthrax bacillus in the tissues of the white rat, are 
undonbtedly taken up in large numbers and destroyed by the 
phagocytes, whereas in 4 anteeptible 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 
‘wuxeuptible animal. Variations in phagocytic activity are found 
to correspond more or less closcly with the degree of immunity 
prewot, but are probably in themselves capable of explanation, 
‘The fundamental observations of Wright and Douglas show that 
in many cases at Icast, leucocytes do not ingest orgnniame in a 
neutral saline solution, and that this is not due to the medium 
in which they ar, is readily shown by subjecting the organisms 
to the action of frosh serum and then washing them ; thereafter, 
they are rpidly taken op by the Jencocytes in salt solution. 
Th most cases this result is due to the labile opaonin of normal 
sorum which haa combining affinities for a great many onganixmns 
as already stated. In other cases more specific substances may 
be concerned. But the all-important fact is that whether 
eyteals occurs OF Net, appears to depend upon certain bodies 
forum. As yet we cannot say whother the phagocytosis 
ian given serum, observed according to the opsonic technique, 
always rans jarallel with phagocytosis in tho tixsnos of the 
‘animal from which the serum has been taken. This is » subject 


‘on which extended observations are necessary. But whether or 
— co 4 





500 IMMUNITY 


not phagocytosis in vinw corresponds with that in miro it is 
probably to be explained in ie mae way; that fa, it probably 
dopends upon the content of the aorum, ‘The compeeition of the 
latter, no doubt, is the result of cellular activity, and in this 
the lencocytes themselves are in all probability conrarned, tut 
the movements and phagocytic activity of these cella seam to 
be chiefly if not entirely controlled by their environments, 
Thavstion 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 pro! 
varies in the case of leucocytes of different animals, but regarding 
this our knowledge is deficient, and, farther, acteria may somes 
times survive the cells which hnve ingested them. 

{4) When it had been shown that normal scram 
bactericidal powers against different organisms, the question 
naturally arose as to whether this bactericidal power varied in 
differcnt animals in proportion to the natuml 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 aorum of the whito rat, which has « romark- 
able immunity to anthrax, had greater bactericidal powers than 
that of other animals Investigated, Further investigation, how- 
ever, has shown that this i not an example of a general law, 
and that the bactericidal action of the serum does not vary part 
posen with the degree of immunity. Tn many cases, however, 
non-pathogenic and alto attenuated pathogenic bacteria can be 
wen to undergo mpid solution and dippear when placed ina 
drop of normal scrum, The bactericidal action of the serum 
was specially studied hy Nuttall, and Inter by Buchner and 
Hankin, who believe that the serum owes ite power to certain 
substances in it derived from the spleen, lymphatic glands, 
thymus, and other tissues rich in  lenene To these 
mbstanees Ruchticr gnve the name of aleriner; as already 
explained, they correspond with Motchnikoffs qytmsa 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 fermeats. Variations in bactericidal power of the 

rum as tested én nitro, howerer, do not explain the presence 
or abeonce of natural immunity against a living bacterium. Tn 
some casex, for example, it has been found to be considerable, 
while the onganiame flourish in the body, and the animal has no 
immunity. La such a caso Metchnikelf says that thore occurs in 
the living body no liberation of aloxines by tho phagocytes, and 
















































a! 





NATURAL SUSCEPTIBILITY TO TOXINS 501 
henee no bactericidal action such as occurs when the blood is she, 
In the case of the hemolytic action of a normal serum, it has 
perme fiver erry cle ear ee rok 
natural papa ie) Riegel 481), and ers rs 
appear to rale 5 to what 
aan a the cane! of inn vatldidally develope Iemolytic serum, 
Tn certain instances an analogous condition appears to obtain in 
anormal bactericidal serum. For example the dog's serum heated 
ut 58° C. contains a natural immune-body to anthrax which ean 
be activated by the addition of normal guinea-pig’s serum so as to 
produce a bactericidal nction, though the latter is by iteelf with- 
out any such effect, At present, however, the possi of 
bactericidal action by complement alone cannot be excluded, as 
it appears to combine with many bucteria without any inter: 
amediary. 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 Netwral ibility co Toxine —We must 
hore 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 Im every case a certain dose must be 

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 animale. 
‘The great resistance of the common fowl to the toxin of the tetanus 
bacillus may be here mentioned (e. p. $81), and large amounts of 
this poison ean be injected into the scorpion without producing 
any effects whatever; the high resistance of the pigcon to 
morphia is a striking example in the case of vegetable poisons. 
This variation in resistance to toxins applies alxo to those which 
produce Local offects, us well as to those which eauso sym 
of geneml poisoning, Inatances of thie are furnished, for 
example, by the vegetable poisons ricin and abrin, hy 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 Bhrtich's view of the constitution of toxins, it 
might be doe to the want of combining altinity between the 
tissne cells and the haptophorous group of the toxin ; or, on the 
other hand, supposing this aflinity to exist, it might be dae to 
an innate non-suseuptibility to the action of the toxophorous 
con Certain investigations have been made in order to 

ine the combining affinity of the nervous system of the 


a 


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 


to the toxophor 
very im 
ing by means” 


part. Mui 

“hemolytic 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, 


Smantrox isa 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 ut the 
day it in difficult to realise,—and on the other hand, to the 
controversies which have arison in connection with the active 
immunisation against it introduced by Jenner, Though there ix 
little doubt that a contuypium eieune is concerned in ite 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 iedieray which is now 
enabling us to understand the principles which underlie the 
treatment, and which’is furnishing methods whereby, in the near 
futury, the vexed questions concerned will probably be satis- 
factorily settled. We cannot here do more haw touch on some 
of the results of investigation with regard to the disease, 
Jennerian Vaccination. ('p to Jenner's time the only means 
adopted to mitigate the dieoase had been by inoculation (by 
scarification) of virus taken from a smallpox pustule, especially 
from mild case. By this means it was shown that in the grewt 
nu\jority of cases a mild form of the disease was originated. It 
had previously beon known that one attack of the dircase 
protected against future infection, and that the mild attack 
Leyregrte | inoculation also had this effect. This inoculation 
method had long been practised in various parte of the world, 
had considerable popularity all over Europe during the 
cighteenth century, Ita disadvantago was that the resulting 
disease, though mild, was still infections, and thus might be the 
starting-point of a virulent form among amprotected persona, 
Jenner's discovery waa published when inoculation was still 
considembly practised. It was founded on the popular belief 
‘that those who had contracted cowpox from an affected animal 
sos 











BOL SMALLPOX AND VACCINATION 


went inansceptible to subsequent infuction from smallpox, Solar 
homme tliere occury a disease known ax horsepox, os 
ing to arise in wet cold springs, which consists in au preetiens 
condition abont the hocks, giving rise to ulceration. Jenner 
believed that the matter from these uleors, when transferred by 
the hands of men who dressed the sores to the teats of cows 
Die velel milked by them, gave rise to cowpox in the latter. 

This disease was thus identical with horsepox in e] of 
which it had ita origin, Jenner was, however, probably in error 
in confounding borsopox with another disease of horses, namely, 
grease. Cowpox munifests itself usa papular omption on the 
toute; the papules become pustules; their contents dey 
form sexbs, or more of less deep ulcers are formed at their sites, 
From such a lesion of the milkers may become infected 
through abrasiona, and a similar local eruption occurs, with 
gonerol symptoms in the form of slight fever, malaise, and loes of 
appetite, It is this illnoss which, according to Jenner, gives rise 
to immunity from amallpox infection, He showed experimentally 
that persons who had suffered from such attacke did not reaet 
to inoculation with smallpox, and further, that persons to whom 
he communicated eowpox artificially, were similarly immune. 
The recults of Jentict’s observations and experiments were 
published in 1798 under the title An Znguiry into the Comses cenel 
Effects of the Variola Vaceine, Though from the first Jennerian 
Vaecinution had smany opponents, it gradually gained the eon- 
fidence of the unprejudiced, and became extensively practised all 
over the world, ax it is ut 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 iwaintained with very Little variation in 
strength for a long time by possage from calf to ealf, and ae 
calves aro now the usual source of the lymph wed for human 
vaccination, When lymph derived from them is wad for the 
latter purpose, immunity ayainst smallpox is conferred on the 
vaccinated individual. It has boon objected that some of the 
lymph which bas boon ured has been derived from calves 
inoculated, not with cowpox, but with human smallpox, Te ix 
possible that this may have occurred in some of the straing of 

wortly aftor the publ 

















derived originally from cowpox. ‘The most striking evidence in 
favour of vaccination is derived from its effects among the stafiy: 
of smallpox hospitals, for hore, in numerous instances, it is only 
the unvaccinated individuals who have contracted the disease, 


| 





if immunity in to continue; and where this is done in any 
population, smallpox becomes a rare discase, as has happened i 
the German army, whore the mortality ix practically nil. The 
whole question of the efficacy of vaccination was investignted in 
this country in 1896 by « Royal Commission, whose general 
conelusions were as follows Vaccination diminishes the liability 
toattack by smallpox, and when the latter doos occur, the 
disease is milder and less fatal, Protection against attack is 
greatest daring nine or ten years after vaccination, It is still 
etlicacious for a farther period of five years, an Grated newer 
wholly cones, The power of vaccination te modi iy an attack 








(Vaccinia). This ix the question regarding which, since the 
introduction of yaceination, the greatest controversy has taken 
place; a subsidiary point lins been the inter-relationships within 
tho group of animal disnses which ineludes cowpox, horsepox, 
abeop-pox, and cattleplagns, With reference to smallpox and 
cowpox the problem has been, Are they identical or not? There 
is no doubt that cowpox ean be com: ited to man, in when 
it produces the eruption lisuited to the point of inoenlation, and 
it 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 snatter from it is introduced into an abrasion, 
‘The loss of infeotiveness by transmission through the body of a 
relatively intuscoptible anitnal is @ condition of which we have 
already seen many instances in other diseases, and the uniformit: 
‘of the type of the affection resulting from vaccination with 
lymph finds a parallel in euch a divecaw as hydrophobia, where, 
after passage through n verics of monkeys, a virus of attenuated 


ft 











506 SMALLPOX AND VACCINATION 


but constant virulence cn be obtained. We have seen that 
there are good grounds for believing that the virus of ealf lym gh 
confers immunity against human smallpox. In eonsidoring 
relationships of cowpox and smallpox, this is an important 
though subsidiary point; for at present it is questionable 
whether there ore any well-authenticated instances of ono 
discase having the capacity of conforring immunity against 
another, The most difficult question in this connection is what 
happens when inoculations of smallpox matter are made on 
cattle. Chanveau denies that in such cirenmstances cowpox is 
obtained, He, however, only experimented on adalt cows, The 
transformation has been accomplished by many observers, 
including, in this country, Simpson, Klein, Hime, and Copeman, 
‘The general result of these exporimonts haa been that if @ series 
of calves is inoculated with variolous matter, in the first thers 
may not be much local reaction, though redness and swelling 
appear at tho point of inoculation, and some general symptoms 
nuwnifest themselves, On aqueceing some of the lymph from 
auch reaction as occurs, and using it to continue the 

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 tho lymph from such yariolated calvos, results 
indistinguishable from those produced by vaccine lymph ans 
obtained, and the transitory illness which follows, unlike that 
produced in man by inoculation with smallpox lymph, ia no 
longer infectious, Tn fact, mauy of the strains of lymph in uss 
in Germany at present have been derived thus from the variola- 
tion of calves, he criticism of these experiments which has 
bon offered, namely, that since many of them were performed 
in vaccine establishimente, the calves were prolubly at, tho sina 
time infected with vaccinia, is not of great weight, as in all the 
recent casos at loast, vory claborato precautions have ‘been 
adopted against such a contingency. And ab any rate it would 
be rather extraordinary that this accident should happen to 
ocour in every cass, We can, therefore, aay that at present 
there is the very strongest ground for helding not only that 
yueinia confers immunity against variola, but that variola 
confers immunity against ina. The experimentum erucia 
for establishing the identity of the two diseases would of course 
be the isolation of the same microorganism from both, and the 
obtaining of all tho results just detailed by means of 
cultures or the producta of such. In the absence of this’ 




















































all 





evidence wo aro at ih veesthrert mt amnee! 
atrong reason for og vuceinia and variola are the 
disease, and that the between them result from the 
relative susceptibilities of the two species of animals in which 
sy ih; rege to. the relation. af horsepox, 

i to the relati to it is 
SR aire agg mn 
pldansion of be foemer Dave ariginated ftten tha hoses, bat id 


‘anittomy, resembles very closely homan smallpox. ‘Though 
each of the two discascs ig extremely infections to its appropriate: 
animal, there is no record of cattle-plague giving rise to small- 
pox in man or vice versd, When matter from « cattle-plague 
pastule is inoculated in man, a arecicheeers Repimaee 


have looked on the latter as Bank: roali it COWPOX. meri 
pos also ins many, liical and ps I analogies wit 
umn smallpox, and facts as to its relation to cowpex yaceina- 
tion similar to those obwerved in csttle:plugue have been 
reported. Smallpox, cowpox, cattle-plague, horsepox, aud 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 lator stages of the latter, many of the pyogenic 
organiams are always present, eg. aaphylococcus aureus and 
staphylococcus cereus flavus, and many of the ordinary skin 
saprophytes also aro often present, but no organism has ever 
been isolated which on transference to avimals been shown 
to have any specific relationship to the disease. Streptococci 
have also been described as agglutinable by the sera of smallpox 

tients and of vaccinated pereons ; such eer it may bo said 
Pad no effect on other strains of streptococei. Calmette and Guerin 
have described very minute granules in the lymph which could 
not be cultivated Wut which persisted after all the bacteria had 
been removed, (The method by which the latter was accom- 
plishod was by exciting « leucocytasis in a rabbit's peritoneum and 


508 SMALLPOX AND VACCINATION: 


then introducing t yuccinal lymph ; the leucocytes phagocyted 
the bacteria so that the lymph no longer gave cultures oe ordinary 
media. Tt was, however, still potent to produce vaccinia.) 


Klcin and aleo, independently, Copeman, have observed an 

in lymph taken from w vaccine pustule in calf on tho fifth and sixth 
days, in uman vaccine lymph on the ojghth day, and in Iymaph from 
® anwlipox pustuly on the fourth day, ‘To demonstrate the bapitit, 
‘corsrglaas flims are dried and placed for flve minutes in acetic acid (i 
in 2), wash in distilled water, cried, aud placed i aleaolis gout 
violet Yor from twenty-four to forty-cight hour after which they are 
washed in water and mounted, Copoman and Kent alo found tho 
Lasilli in sootions of vaooins pustules stained by Lottler's mothylene-blt 
or by Gram's method. ‘The organisms are “i to 78 q in length, am 
one-third to lulf of this in thickness. ‘They ato youerally thinner and 
stain better at the ads than wt the middle. ‘They ovour In groups of 
from throe t> ten in both the lymph and the tissues, In the centre of 
their protoplasm there is often u clear globule, which ia looked on as & 
kpore. ‘They to rosivted the o} i . 
fact which ie rather in favoar of their 

























Various observers have described appearances in the epithelial 
cells in the neighbourhood of the xuullpox or vaccine pustules, 
which they have interpreted ox 1 
and Plimmer describe as occurring in clear vacuoles in the cells 
of the rete Molpighii at the edge of the pustale, in paraffin 
sections of vaccine and smallpox pustules carefully hurdenod in 
aleohol, and stained by the Ehrlich-Biondi mixture, small round 
bodies of abont four times the size of a staphylococcus 
coloured rd by the ucid fuchsin, somotimes with a. pete 
stained by the methyl-groon, ‘Theso are described as multiplying 
by simple division, and in the living condition exhibil 
amaboid movement. Similar bodies have been deseribed 
Reed in the blood of smallpox pationts and of vaccinated 
children and calves 

‘These are probably the bodies described by Guarolerl and to. 
which considerable attention has been paid. They are from 
13 in diameter, aro round, oval, or sickle-sbaped, and stain 
by onlinary nuclear dycs, They lio in the colla de 
often near the nucleus, and emonetrable in vaccine 
pustules and also in the oxperimental lesions whieh can be 
produced in the rubbit’s cornea, the larger bodies being defined 
in the cells towards the centre of the | 

































~ 4 <1, 
NATURE OF VACCINATION: 


prance dle areca 
stated that mull seen occurring in 
fresh lymph, bat asap ptregreserly cen eh forward 
strong evidence for the appearances being due to nuclear 

Still tho question of the specificity of these changes to 

lesions remains, and here it may be eid Wasieloweki haa chown 
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 


bodies in the epithelial colls 14 jc in size, ae contoured 
and having mgged 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 longitudinal division. 
Prowazek has also seen these “lymph-bodies” as he has called 
them in the lymph, and he ptetee the idea that they may be 
protozoa, Bonhof and also Carini have described 

as occurring in variolous lesions, but this has not been confirmed, 
Future investigations must show what siguificance is to be 
attached to these varions observations, 

The causal organism of smallpox is probably very small aa, 
though there hus been some difference in opinion ou this point, 
‘there is little doubt that it will pass through the coarser porcelain 
filters, 
norant of the cause 
of smallpox, we ean only conjecture what the nature of vaccina- 
tion is. From what we know of other liko 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 ix maintained after vaccination, 
wo do not know much. Some, including Béclire, Chambon, 
and Ménard (who jointly investigated the subject), maintain that 
in the blood of vaccinated animals substances exist which, when 
traneferred to other animals, can confer a certain degres of 
yasive immunity against vaccination, and which have also a 

‘of curative action in animals already vaceimated. Beumer 
and Peiper, on the other hand, could not find evidence of the 
‘existence of such bodies. 











MO 


el 


re 


2 








PATHOLOGY OF HYDROPHOBIA Bil 


more common. After a period of incubation ef from throe to 
six wocks, 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 bas a peculiar 
high-toned bark. Spasms of the throat muscles come om, 
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 convalsions, iy 
snd 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 
usnally about forty days. When symptoms of rabies are about 
to appear, certain prodromata, such a4 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 
ieritability, during which ali the teflexes are angmented—the 
victim starting at the slightest sound, for example, ‘There are 
spasms, especially of the mascles of deglutition and respiration, 
and cortical excitement evidenced by delirium may occur. 
this follows « period in which all the reflexes are diminixhed, 
weakness and _kamalyis are obverved, convulsions cocut, an 
finally coma and death supervene. The duration of the acute 
illness ix usually from four to cight days, and death invariably 
resulta. The existence of paralytic rabies in man his been 
denied by some, bat it undoubtedly occurs. This ix usually 
manifested by paralysis of the limb in which the infection has 
been reccived, and of the neighbouring parte; but while in such 
cases this is often the first symptom obscrved, during the whole 
of the illness the occurrence of widespread and progressive 
paralysis is the outstanding feature, In man thon also occur 
cases where the cerebellum and also the sympathetic ayster seem 
to bo 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 
xymptome aro naturally referred, are comparatively unimportant, 

jeye examination, congestions, and, it yay be, minute 
henorrhages in the central nervous aystem, are the only features 
noticeable. Microscopically, leucocytic oxidation into the peri- 





512 HYDROPHOBIA 


vascular Vag b callin in the nerve centres has been observed, 
and in the cells of the antarior cornua of the grey matter In the 
apinal cord, and also in the onelei of the erninl nerves, various 
degenerations have been described. Round the nerve cells in 
the grey matter of the cord and medulla Babés described 
Accummiations of newly-formed cells,and Van Gehuehten observed 
& 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 cao, In the white matter, cepecially 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 
h infection has come, In the nervous aystem also some 
have seen minute bodies which they have considered to be eoecel, 
but there ia no evidlonco that they are roally of this nature, ‘The 














wl pathology confirma the view that the nervous 
system is tho contre of the disoase 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 
anade by subentaneous i of material from variows parts 
of animals dead of rabies had not given uniform results, nk 
whatever was the source of the material, the disease was not 
invariably produced. Pastent’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, hy this method, of emulsions of any part of the central 
nervous system, of the cerebrospinal fluid, or of the saliva, 
invariably gave tise to rabies, and also that the natural period of 
incubation was shortened. Further, the identity of the furious 
and paralytic forms was proved, az sometimes the one, sometimes 
the other, was produced, whatever form had been present in the 
original case, Inoculation into the anterior chamber of the 
is nearly as efficacious aa subdural infection, Infection with the 
blood of rabic animals doce not reproduce the diseaeo, ‘Phore da 
evidence, however, that the poison also exists in such glands as 
the pan nd mamma, Subcutaneous infection with part of 
the nervous éystem of an animal dead of rabies usually gives 
rise to the dise 
Tn 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, Gaming 















































THE VIRUS OF HYDROPHOBIA 18 


entrance by the infected wound, it carly manifesta ite affinity for 
the nervous tissues. It reaches the central nervous system 
chiefly by spreading up the peripheral nerves. This ean be 
shown by inoculating an animal eubeutanoously in one of ite 
limbs, with virulent material. If now the animal be killed 
hefors symptoms have manifested themselves, rabies can he 
produced. by subdural inoculation from the nerves of the Timnb 
which was infected, Further, rabies can often be produced wes 
such a cuss by subdural infection with the part of the 

cord into which these nerves pass, while the reali parts of _ 
animal's nervous system do not give ries to the disease. Thin 
explains how the initial symptoms of the ditewe (pains along 
nerves, paralysis, ete.) so often appear in the infected part af the 
body, and it probably also explains the fact that bites in sueh 
richly nervous parts as the face and head are inach more likely 
to be followed by hydrophobia than bites in other parts of the 
body. Again, injection into a peripheral nerve, such as the 
seintic, is almeat ns certain a method of infection as injection 
into the subdural spnec, and gives rise to the mune type of 
symptoms as injection into the corresponding limb.  Intravononis 
injection of the virus, on the other id, differs from, the other 
modes of infection in that it more frequently gre rise to 
paralytic rabies, This fact Pastenr 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 allinity for the walivary glands, as well as for 
the nervous syetem. Roux and Nocard found that the saliva of 
the dog became virulent threo days before the frst. 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 treed, all attempts to determine the actual morbific canse 
have heen unsatisfactory. Tn this conneetion varioux organismas 
have been described as being associated with the disease. 


Thus Mommo has isolated an organism which resombles a yeast, but 
rele places amongpt the Mautomycetes, aud with whic he eaten 
be hax laced both types of rabies in rabbite and doge Brurehottind 
also, wmans medix containing brain substance, has grown « bacilius 

iplitheria group, and 
jen iu rabbite, In 
there been com- 








tie care i the af 
firmation from indepowdent sources, and in neither ease se evidence 





ofthe crucial teat having been a 
inst the ordinary hydrophobic virus by means of 


ca the alloged causal organinm, With regard to other possible 


ied, namely, th 


ols HYDROPHOBIA 


causal agents, Grigorjew thinks auch may be found ia a protozoa whieh 
te has sonstaatly cbvareed efter tuoediasiou fa tbe osmaas.” 


Tn 1903 Nogri described certain bodies as occurring in the 
nervous gyatom in animals dying of rabies to which considerable 
attention bas einee boon devoted, and regarding the significance 
of which opinion is still divided. It may be said that Negei’s 
observations have been generally confirmed, and as it is probable, 
whatever the Anal opinion as to the nature of the bodies 
may be, that their occurrence ia specific to the disease and hence 
may be used for dingnosis, we shall describe the methods for 
their demonstration, In doing so we shall chiefly follow the 
work of the Amorican observers, Williams and Lowden, who, 
more than any others who have confirmed Negri, baye used 
methods widely employed in the investigation of similar 
appearances, 


‘Their chief method ix to take a piece of the brain tiene, to squeene it 
bovwee @ slide nad coversglass, and, sliding off the latter, to make » 
suiear which is then fixed in motly! aleohol for five minutes and stained 
by Giernsa’s wtain ?. 107) for half an hour to three hours; the prepara- 
tion ia then waahod in tap water for ‘st min, and dried, Wor rapid work, 
after fixntion, equal parts of distilled water and stain are wsod instead of 
the more dilute mixture, 

For sections the Lissues ar at's fluid? for 8-4 Bours, thin 
placed in tap water for fi 80 per cont aleohol with enough 
iedine added to givo it a port wine colour for 24 hours ; 05 per cent 
Jeohol and jodino, 24 hours; absolute aleohol, 4-6 hours; cleared with 

dar ofl and ernbodided in paraffin of molting point SY" C,; sections shonld 
%&6 m thick, For staining, Mallory’s mothylene-bine e is 
commended ; the steps are as’ follows : xylol; absolute alcchal ¢ % 
¢ cent alcohol and iodine, } hour ; 95 per cent alcobol, & hour; 

hol, 4 hour ; eosin solution (5:10 per oent aquooue solution), 
2) minntes; rinso in tap water) Unna’s polywhrome soethylene-biue 
solution diluted 1-4 with distilled water, 15 minute, ifferontiation in 
6 per cent alcohol for 1-6 minutes (the proparation being Kept fa 
motion and its progress watched with » low power) ; rapid and oareftl 
dehydration and clearing, 









































‘The bodios vary much in size, measuring from 3 p to 25 jo 
‘They are round, oval, or angular in outline. ‘They are foand im 
the protoplasin of the nerve cells and of their processes, ‘They 
hove a hyaline appearance with a sharply.defined outline, apd in 
their substance they contain granular material. Taking for granted 
their cellular structure we may say that with the Giemsa mixture 


position : potassium blelramabe 











4 Zeuker’s (uit is of the followin 

2-5 grey sodium aulpbato 1 gr, porchloride of mercury & sry elaalal poetic 
aay water to 100 oc. Dinwolve the perehiloride of mereary wail thi 

to of potaminm in the water with the aid of heat and add: thy 














THE VIRUS OF HYDROPHORIA 515 


their cytoplasm staina blue and the granules a buered,—by 
Mallory's stain the cytoplasm is magenta and the granules a 
deep bine ‘The cytoplam is homogenoona, and in it ism 
nuclous-like body whose chromatin particles in tho largor 
individuals aro arranged round the periphery, there being « 
clear centres containing a nucleolus; in the smaller forms the 
nucleus is @ 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 pnre chromatin stain as the nuclens There is 
evidence of division of the nuclens, and sometimes there may 
apparently be three or four nuclei in one body without division 
of tho protoplasm having occurred. Sometimes the chromatin 
appears te fmgment 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 reanit that some very small forms may 

‘De proruced, these sometimes appearing in mulberry musscs. 
The Negri bodies have been found in nearly all cases of 
street-mbics examined by many observers, and have never been 
found in other conditions of brain disease, Thoy occur in all 
parts of the central nervons system, but are said to be most 
abundant in the cella of the cornu Ammonis, as are 
apparently not 40, aon found, at least in their larger forme, 
in animals dying from the inoculation of virus sine What the 
significance of these bodies is, it is at present; impossible to xay ; 
but whatever may be their nature, there is now considerable 
evidence that their presenco 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 locked on thees bodies as protozoa, 
and their appearance is not inconsistent with such a view. Tho 
objection which hax been mixed, that if they were protozoa 
they could not pase throngh a porcelain filter (vide infra) ax 
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 Ej, The occurrence of 
minute ferms would also account for the non-recognition of the 
ae in tho more acute forms of the disease where there had 
an active vegetative condition, and thus no time for the 


Targor far to dvolop, 
is mo doubt that betwoon 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 
4 micro-organism being the cause, 'The organism, whatever it is, 
ia, in its infective form, probably very mall, as it eam pmax 
through the coarser Berkefeld filters, and also occasionally 
throngh the coarser Chamberland candles, Evidence that it ix 
the erganiem iteolf which passes through, is found im the fact 
that when on animal dies from infection with the filtrate, a 
small portion of its central nervous system will originate the 
disease in a froeh animal, Judging from our knowledge of 
similar diseases we would strongly suxpoct that it is actually 
present ino living condition in the centeal nervous system, the 
saliva, ete, which yield what wo have called the hydrophobic 
virns, for by no more toxin could the disease be transmitted 
through a series of animals, as we shall presently soe can. be 
done. A toxin may, however, be concerned in the production 
of the pathogenic effects Remlinger found that death with 
Tsralytic symptoms sometimes followed the injection of filtered 
virus, but that the nervous system of the dead animals did net 
ryproduce rubies. He explains this occurrence by anppesin that 
the filtrte contained o toxin but not the actual infective agent, 
The resistance of the vin to external agents varies. Thus a 
nervous system contail virulent till destroyed by putre- 
faction ; it can resist the sie application of a temperature 
of from = 10° to =20° C., but, on the other hand, it is rendered 
non-virulent hy one hour's exposure at 50° C. Agni, ite 
potency probably varies in nature according to the souree. 
hus, while the death-rate among persons bitten by mad dogs i 
about 16 per cent, the corresponding death-rate after the bites 
of wolves is 80 por cont. Here, howover, it must be in 
view that, as the wolf is naturally the more savage animal, the 
number and extent of the bites ie. the number of chawnols of 
entrance of the virua into the body, and the total dose, are 
of persona bitten by doga. As we shall 
potency of the virus can certainly be 
























greater than in the e 
see, alterations ij 






The Prophylactic Treatmont of Hydrophobia.—Until the 
publication of Pasteur’s researches in 1885, the 
adopted to prevent the development of hydsophobia 
bitten bya rabid animal had consisted in the catiterisstion of 
the wound, Such a procedure wna ndoubtedly not without 
Tt has been shown that cauterisation within five minuter 
of the infliction of a mbic wound prevents the disease fron 
doveloping, and that if done within half an hour it «nves 
proportion of the cnscs, After this time, canteriantion only 




















PROPHYLACTIC TREATMENT OF HYDROPHOBIA 517 


lengthens the period of incubetion ; ie “ wo shall see 
presently, this is an extremely important effoe 
‘The work of Pasteur Had lerescerynevaieinaioel te whole 
treatment of wounds inflicted by hydrophobic animals, Pasteur 
started with the idea that, sea ‘the period of incubation in the 
cam of animals infected sabdarally from the nervons ras Of 
mad dogs is constant in the dog, the virus has been from time 
immeniorial of constant strength Such # virus, of what might 
‘be called natural strength, is usually referred to in his works as 
the virus of Za nage des rues, in the writings of German authors 
as the virus of die Strasswuth. Pasteur found on inoculating 
monkey sabdurally with such a viras, and then inoculating 
a second monkey from the first, and so on with a serice of 
monkeys, that it gradually lost its virulence, as avidenced by 
lengthened periods of incubation on subdural inoculation of 
dogs, until it wholly lost the powor of producing rsbioe is in dogs, 
hen introduced  subcutancously, this point had 
ts virulence was not “fiminished by further tls 
through the monkey. On the other hand, if the virus of fa 
rage dee rues wore similarly passed through a series of rabbits 
‘or gnines-pigs, its virulence was increased till » constant strength 
(the virws fire) was attained. Pasteur had thus at command 
three varieties of virus—that of natural etrength, that whieh bad 
been attenuated, and that whieh bad been exalted. He further 


found that, commencing with the subcutaneous injection of a 
weak virus and arenas up with the injection of the 


stronger varieties, he could ultimately, in a vory short time, 
immunise dogs against subdural infection with a virus which, 
under oniinary conditions, would certainly have caused a fatal 
result, He also elucidated the faet that the exalted virus con- 
tained in the spinal cords of mbbits such as those referred to, 
could be attenuated 40 as no longer to produce rabies in dogs hy 
subcutaneous injection. This was done by Lig. | the conis in 
nie over caustic potash (to absorb the moisture), the diminution 
of viralonce being proportional to the length of time during 
whieh the cords wore 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 differvot strengths Pasteur 
at once applied himself to find whethor the comparatively feng 
period of incubation in man could not be taken advastage of to 
“ vaccinate” him against the disease before ite gravest manifesta 
tion took piace, The folk 
‘Tho techy 















518 HYDROPHOBIA 


peed pecker Lact i Tie Aen aie 
made with a cord fourtosn days 


w very” 
An subsequent injections the of the virus was 
copie Gres a 


Suly 7, 1885, 8 Ad, cord of J fe 1d days old. 
ae or sa 2 ie 
a 








OTe. 9 

11 Ais, cord of July A 
meee i 6 
” ” ” 6 
” ” ” 4 
4 ” 2 
” ” ” 2 
” ” » 1 





‘The patient newer manifested tho slightest sym) 
phobia. Other similarly favourable results pe ts 
Peptic treatment of the disease quickly 

idence of the selentitie world, which rs still Bris (the 


ene is, of course, i a os ey 


of active immunity against 


‘The ouly modification which the method thas undergone 
the treatment of serious cases, stich ax multi 
extonsive bites about the hosd, 

‘under treatment at a late pariod of t 

the wounds have not cleatriaad. 

mont ary conde 

and or. 








are also proparcd. As we have said, ‘him 

was 16 por cont of all persons bitten. Dur ng tl 

17,587 cases wero troated, with'a mortality of 

alfaged that many people are treated who have baer bitten by dope 
re not mad. ‘This, iowever, is not wore true of the cases treated 
mteure method than it was of thove ou which the, onlay: 

of 16 per cent was baved, aud care is taken in maki ing pee 

to distinguish the eases into throe classon, Clase A inc 


bitten by dogs proved to have lad rabies, by sceaiatien Be in healt! 
animals of parts of the conteal nervous system of the diseased an 
Class B includes hose bitten hy dogs that  Soompaten veterinary 

has pronounced to be mad, Class © includes sll other cases. 

1896, 122 cases belonging: to Claas A wore treated, with no d 
belocging to cleo Br with two destha; oud 440 tolonglng. 


r 





METHODS 519 


with no deaths, Bosides the Institute in Paris, similar institutions exist 
im other gurte of France, in ftaly, and es) in Russia, as well as 
Jn other part of the world); and in uheag inilar succes has Been 
exporienced. 1 mny tow taken aa established, ata gmve 
Teapousibility wets eo thoes ouoeraecs Af, paean bitten, Wyre cra 

imal is not sul ted to tho Pastour treatment. Sometimes during or 
afer sreataent there spgear slight pauniy is ayauplonss with abate 
constipation apd it may be retention of urine, but thew par off within 
‘a few weoks ond Josve behind tio ill effects. 









Antiralde Serum.—In the early part of the nineteenth century 
an Italian physician, Valli, showed that immunity against rabios 
could be coufberad by edkafnltaclag. Uirough Sho stomach pre 
grevavely increasing doves of hydropliotie virus Following up 
this observation, Tizzoni and Centanni have attenuated ribi 
virus by «ubmitting it to peptic digestion, and have immunised 
aniinals by injecting gradually increasing strengths of such virus. 
‘This method ia usnally referred to as the Italian method of 
immunisation, The latter workere showed from this that the 
serum of animals thus immunised could give riso to passive 
immanity in other animals: and further, that if injected into 
animals from seven to fourteen days after infection with the 
virux, it prevented the Intter from producing its fatal effecte, 
even when symptoms bid begun to manifest themselves. They 
further succeeded in producing in the aheep and the dog an 
immunity equal to from 1-25,000 to 1-50,000 (ride p. 454), and 
they recommended the usc, in severe cases, of the serum of such 
animals in addition to the treatment of the patient hy the 
Pasteur method. A like seram has been obtained from animals 
treated by the ordinary Pasteur method, 

Methods. (a) Dimnosis.— When a person is bitten by an 
animal suspected to be mbid, the latter must under no circam- 
stances be killed, Much more can be learned by watehing it 
while alive than by portvmortem examination. In the latter case 
only such things as the occurrence of broken teeth, marked 
congestion of the fancea, or the presence of unwonted material 
is te stomach throw any light on the condition; nothing of a 
positive nature can be learned from exunining the nervous 
ayatem. On the other hand, in tho living animal tho develop 
ment of the characteristic symptoms can be watched, and death 
‘will occar in not moro than five days. If the suspected animal 
jus been killed, then « amall piece of its medulla or cord must 
be taken, with all asoptic precautions, rubbed up in a little 
sterile 74 per cent mela chloride solution, and injected by 
means of a syringe beneath the dura mater of a rabbit, the latter 
having boon trophined over the oerebrum by means of the small 


sa fl 


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. 

() 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. 


I has now beon conclusively proved that the eanse of malaria 
fever is a protozoan of which there are severnl spocios ‘They 
belong to the hamoxy ia (a sub-class of the sporozca) which 
are blood parasites, infecting the red corpuscles of inammuls, 
roptilos, and birds, ‘The parasite was formerly known as tho 
huematocoon or plasmextivn matariee, although the use of the 
Jatter term is incorrect ; the term Acemamesba ix, however, now 
generally employed, The parasite was first observed by Laveran 
in 1880, and his discovery received confirmation from the in- 
dependent researches of Marchiafaya and Celli, and later from 
the researches of many others in various parte of the world, 
Golgi supplied valuable additional information, especially in 
relation to the sporulation of the organism and the varieties in 
different types of mulurial fever. In this country valuable work 
on the subject was done by Manson, and to him specially belongs 
the credit of regending the exflagellation of the organism as 
a preparation for an extracorporeal phase of existence. By ine 
duction be arrived at the belief that the eyele of existence outside 
the human body probably took place in the mosquito, Lt was 
specially in order to discover, possible, the parasite in the 
mosquito, that Ross commenced is 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 pigenent in these bodies was exactly similar to 
that in the malarial parasite, and he excluded the possibility of 
their representing anything el than a stage in the life cycle of 
the organism. Ho confirmed thia discovery and obtained eor- 
respotding results in the case of the proteosotun infection of 
Winds, whore the parasite is closely related to that of malaria, 
21 

















= 


522 MALARIAL FEVER 


Tn 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 tho time when it settled ina special 
form in the salivury glands of the insect. Ross's results were 
published in 1898. Feactly corresponding stages were uftery 
wirds foand in the cate of the different species of the human 
parasite, by Grossi, Bignami, and Bastianclli; and theee with 
other Italian observers also supplied important information 
regarding the transmission of the disease by infected mosquitoes, 
Abundant additional observations, with confirmatory result 
were supplied by Koch, Daniets, Christophers, Stephens, a 
others. ‘herever malaria has been studied the result has been 
the same, Lastly, we may mention the striking experiment 
carried out by Manson hy means of mosquitoes fed on the blood 
of paticnts in Italy suffering from tild tertian fever, 
insects, after being thus fed, were tuken to London and allowed 
to bite the human subject, Manson's son, Dr. P. ‘Thurburn 
naon, offering himself for the purpese. ‘The result wus that 
infection occurred ; the parasites appearod in the blood, and 
were associated with an attack of tertian fever, Ross's discovery 
haa not only boon # moans of elucidating tho mode of infection, 
but, as will be shown below, has also supplicd the means of 
successfully combating the disease. 

From the zoological point of view the mosquito ix regarded 
a8 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 eyele in 
secondly, that in the moequito, Vari 
wpplied to the various stages, but we shall give thowe now 
generally wed. 

The Cyclo in the Human Subject.—With regard to this 
cyele, it may bo stated that the parasite is conveyed by the bite 
cof the mosquito in the form of a small filamentoos cell 
sporowite or exotospore, which penetrates « red corpusole and 
becomes 4 small amecboid organiem or ameebula. ‘There is then 
aw regularly repeated asexual cycle of the parasite in the bloody 
the length of which cycle determines the type of the fever. 
Daring this cycle there is a growth of the ametbulie or 
trophozoites within the red corpuscles up to their complete 
development ; sporulation or schizogony then occurs. The onset 
of the febrile attack corresponds with tho stage of sporulation 
and the setting free of the spores (onhurosporcs oF meraacites), 
fe with the production of a fresh brood of parasites, These 
spores soon become attached to, and penetrate into the interior 


2 


? 























FORMS OF THE MALARIAL PARASITE 538 


of the red corpuscles, becoming intra-corpuseular amecbulie ; the 
eyele is thus completed. The parasites are most paiene 
the blood during the development of the pyrexia, and, ry 
they are also rail 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. 

Tn addition to these forma which are part of the 
asexual eyele, there are derived from the amebule other forms, 
which are called gametocytes, or sexual eclls. ‘These remain 
unaltered during succossivo attacks of pyrexia, and undergo no 
futher change eotll the blood la removed roca tn ama body 
In the simple tectian and quartan fevers (vide fayra) the 
cytes resemble somewhat in appearance the fully devel 
ameebulie before sporulation, whores in the malignant type 
havea characteristic crescontlike or sxusage-shaped form ; hence 
they ar often spoken af as * crescentic bodies,” 

‘The various forms of tho parasite seen in the human blood 
may now be described more in detail. 

1. The Enhemoyares (Lankester) or Merosoites are the 
youngest and smallest forma resulting from the segmentation of 

jult amarbula—sporocyte or achizont, ‘They are of round 

or owil shape and of stl! size, usually not exceeding 2 yc in 
diameter; the size, however, varies somewhat in the different 
types of fever. A nucleus and peripheral protoplasm ean 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 
Romanowsky method; the peripheral protoplasm is coloured 
fairly deoply with methylene-blue. ‘The spores show little or 
noameboid movement ; at first free on the plasma, they soon 
attack the red corpuselos, where they become the intra corpuscular 
amocbules, If the bload, say in a mild tertian case, be eximnined 
in the early stages of pyrexia, one often finds at the same time 
Teed corp forms, free spores, and tho young amebula within 

red corpuscles, 

2, Iutra-corpurcular Auurbuler or Trophesoites. —Thewinelude 
the | Parasites which have attacked the red corpuscles; they are at 
firet situated on the surface of the latter but afterwards penetrate 
their substance. ‘They usually occur singly in the red corpuscles, 
but sometimes two cr mom may be present together, ‘The 
youngost or «mallost forms appear as minute colourless 
of wlout the same size as the apores, As sesh in frech blood, 
they exhibit more or loss active amaboid movement, ehowing 
marked variations in shape. The amount and character of the 


ou MALARIAL FEVER 


amoboid moverncnt varies scmewhat in different types of fewer, 
As they increase in xize, pigment appears in their interior as 
minute dark brown or black specks, and gradually becomes 
more abundant (Figs. 165, 166), The pigment may be 
ewattered through their substance, or concentrated at ene or 
more points, and often shows vibratory or oscillating movements. 
Thie pigment is elaborated from the hemoglobin 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 swollon and pale (tertian 
fever), or somewhat ebrivelled and of dorker tint (malignant 
fever). In stained specimens a nucleus may be seen in the 
panisite as a pale spot containing chromatin which may be 
arranged as n single concentrated mass or as several separated 
granules, the chromatin being coloured a deep red by, the 
Romanowsky sucthod. ‘The protoplasm of the parasite, whieh 
is coloured of varying dopth of tint with mothylene-blue, shows 
great variation in configuration (Fig, 156), The young parasites 
not unfrequently present a “ying-form,” a portion of the red 
corpuscle being thus enclosed by the parasite. These ringforms 
aro met with in all tho varietios of the parasite, but thoy are 
ially common in the case of the malignant parasite, where 
they are of smaller size and of more symmetrical form than in 
tho others (Fig. 160); the pigment is usually collected in a 
tall clutap at one sid 

Within the ced corpuscles the parasites grudually Increase 
in size till the full adult form is reached (Fig. 157), In this 
stage the parasite loees ite amaboid raovement inore or lee 
completely, his 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 #0; some 
become degenerated and ultimately break down 

3, Sporocyter or Schizonte—In the process of sebizogony 
the chromatin becomes divided into a number of daughter 
nuclei which are seattered through the protoplasm; the latter 
then undergoes corresponding segmentation and. the small 
merozoites or enhwmospores result. The pigment during the 
process becomes uggregnted in the centre and is surrounded by 
asmal) quantity of residuary protoplasm. Schaudinn has found 
in the caso of the tertian parasite that schizogony begins by m sort 
of primitive mitosis, which ia then followed by simple multiple 
fission, ‘The spores or merozoites are of rounded oraval shape, 
as above described, and are set froe by the raptuns of the 




















































Pro, 168, Fra, 15%, 
‘Pron. 164-159.—Varioms phases of the benign tertion parnsite, 





FORMS OF THE MALARIAL PARASITE 527 


tnd tay be ten up Oy cnnber ant arg: 
fon up by aay and arrange- 
cateian epores within the sporooyte vary in the es 
types. In the quartan there are 6-12, and ‘the 
ina radiate manner, giving rise to the characteristic pane 
appearance; in the tertian they number 15-20 or more, and 
havo o somewhat roscttetike arrangement (Fig. 158); in the 
malignant there ure usually 6-12 spores of small size and 
somewhat irregularly arranged. ' 
Gametocyter.—As stated above, these are sexual colle which 
are formed from certain of the amabule, 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 amecbuli, The fernale cells, itocytes, 
are of large size, measuring up to 16 4 in dinmeter; they con 
tain coarse grains of pigraont, and the protoplasm stains event 
deeply with methylene-blue. The male cella, microga 
are smaller, and the protoplasm stains faintly ; the muckun 
[ icp in the centre, is rich in chromatin. Tn the mali 
levers the gametocytes have the special crescentic form pe 
above. They measure 8-9 in length, and occasionally a fine 
curved line is seen Joining the extremities on the coneave aspect, 
which represents the envelope of the red corpunal (Fig. 161). - 
‘They are colourleas and transparent, and aro em closed by a 
distinct membrane ; in the central part there ix a collection of 
pigment and pace of chromatin. Tt 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 saattered 
throagh the cell, and there are several granules of chromatin ; 
in the Inter the pigment is dark and concentrated, often in a 
staal ring, and there are one or two masses of chromatin in the 
centre of the crescent. According to the Italian observers the 
carly forms of the crescents are somewhat fusiform in shape and 
are produced in the bone-marrow. The fully developed eresconts 
do not appear in tho blood till soveral days after the onset of 
the fever, and thoy may bo found a considerable time aftor the 
disappearance of the areal attacks. ‘They aro aleo little, if at 
all, influenced by the administration of quinine. 
Te ix well known that after a =r Oo apparently recovered 
from malarial fever a relapao may take place without fresh 
infection occurring, and Schandinn bes published interesting 
observations Ene is en point. He has found that the 
‘macrogamotocyte of tertian fever may by a process of partheno- 
goneaia give rise to merozoites, which in thee t ‘turn infect the red 











598 MALARIAL FEVER 


es oyolé again, As dosoribed and fi 
fat cone 








ng 
chromatin long with part of the protoplasm breaks up 


dina 
Oycle in the Mosquito.—As already explained, this 
starts from the gametocytes. After the blood is shed, or after 
it is swallowed by tho mosquito, two im phenomena 
occur, viz, (@) the full development of the nexual cella or 
gamotocytes, and (2) the impregnation of the female, Tf the 
Blood from a case of malignant: infection ba examined in a moist 
chamber, preferably on a warm stage, under the mi 
both male and female gametocytes may be seen to become oral 
and afterwards ronnded in shape (Figs. 162-164). Thereafter, 
in the case of the male cell, a vibratile or dancing movement of 

pigment granules can be seen in the interior, and soon 
several flagellalike structures shoot out from the periphery 
(Fig, 165). They are of considerable length but of groat fineness, 
and often show a somewhat bulbous extremity. By the 
Romanowsky method they have been found to contain « delicate 
core of chromatin, which ik covered by protoplasm. They 
represent the male cells proper, that is, they are sporm-cells or 
‘spormatozca ; they are also known as imicrogametes. They 
become detached from the sphere and move away in the 
surrounding fluid. The fomale cell also assumes the rounded 
form, and maturation takes place by the giving off of part of 
the nuclear chromatin. Tmpreguation occurs by the entrance of 
a microgameto, tho chromatin of the two cells afterwanla 
becoming fused, Impregnation was first observed by MacCullara 
in the case of halteridiu d he found that the female cell 
aftwewards acquired the power of independent movement or 
became A “travelling vermicale." He also observed the 
impregnation of the malignant parasite, The fertilised fernale 
cell is now generally spoken of as a sygote or otjbinete 

Te has boon establ that tho phenomena just dessribed 
occur within the stomach of the mosquito, and that the fertilised 
coll or zygote penetrates the stomach wall and settles bebween 
tho musclo fibres; on the second day after the mosquito Imus 
ingested the infected blood small rounded cells about 68 pw in 
diameter and containing clumps of pigment may be found iy 
this position, (It was in fact the character of the pigment 






















place, 
fect ind Aiea of the stomach into the body cavity, 
dividos into a number of cells called blastophores or 
nd these again divide and form @ large number of 
filiform cells which have a radiate arrangement; these were 
callod by Ross “germinal rods," but aro now usually known aa 
spo orerotospores (in contradistinction to tho \OepOres 
of the human eyele). The full development within the sporoeyat 
ocenpies, in the caso of proteosoma, about seven days, in the 
cam of the malarial parasites a little longer, wn fully 
devel the cyst measures about 60 j in diameter, and 9) 
with sporozoites. Tt then bursts, and the latter are sot 
free in tho cavity. A large number sottle within the 
large veneno-alivary gland of the insect, and aro thus in a 
ine vag injected ree with ee my nS tame 
wal 1 Bporozoltes enter corpmaces an me 
amecbulw as above described. Daniela found that in the ease 
‘of the malignant parasite an interval of twelve days at least 
intervened between the time of feeding the mosquito and the 
appearnnce of the sporozoites in the : 

Tt will thus be seen that in the human subject the parasite 
passes through an indefinite number of regularly reeurring asexual 
bie! with the giving off of collateral sexual cells, and that in 
the mosquito there is one cycle which may he said to start with 
the impregnation of the female gamete. 

Varieties of the Malarial Parasite —Tho view propounded 
by Laveran was that there is only one species of malarial pamusite, 
which is polymorphous, and presents slight differences in 
structural character in the different types of fover. It may, 
however, now be accepted as proved that thore are at least three 
distinet 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 
re for the quartan and tertian types respectively ; whilst 

0 other includes the parasites of the severer forms—*' wstivo- 
autumnal” fovers, malignant or pernicious fevera 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 Ttalian writers distinguished (1) quotidian ; (2) 
‘non-pigmented quotidian ; and (3) 4 malignant tertian parasite, 

" 


— 


as 
parasites, 
scheme, 





Farnily : Hascascaentom (Wastelowelt). 


Genne I, Hemamebe. The matare gamotes resemble in form the 
Achizonts before segmentation has ocourred. 


Species 1, Marmamaba Dunilewaki or halteridiwin. 
Parasite of pigeons, crows, eto, 

Species 2 Hirmamoba velieta or proteosoma. 
Paraaite of sparrows, Inky, eto. 


Bpecios 3. Hy malaric, 
? fragt ces eae ae 


Species 4. xmamets vicar. 
Fasaaltd of tevtian fever of Guan, 


‘ Genus Ll. Homomenna Tho gametocytes have a special crescontic 
ror. i 


Bpecies ; Hama: proces, 
, Peer maliguant orestiro-outumosl fersrof mans 


In addition there are other species belonging to the same 
family of blood parasites, which infect froga, lizanda, bata, ete., 
specially in malarial ryiona. 

We shall now give the chief distinctive characters of the 
three human parasites. 

1, Parasite of Quarian Fever,—Tha eyelo of development in 
man is seventy-two hours, and produces pyrexia every third day ; 
double or triple infection may, however, occur. In fresh speci 
mensof blood the outline is more distinct than that of the tertlan 
yarusite, and ammboid movement is Joe marked. Only 
smaller forms show movement, and this is not of active character, 
‘The infected red corpuscles do not become altered fn size or 
appearance, and the pigment within the parasite is in the form 
aia granules, of dark brown or almost black colour, 


F 


F 





VARIETIES OF THE MALARIAL PARASITE 531 


appearance, 
tation into six to twelve 
= |e igcerperge ay ok 
eee ean ppe a 
ment is sight hours, thot a 

of fever may be prod sagen double infection. The epee 

Kein mci en ot rin a 
thus leas casily distinguished in the 

movements aro, however, much more active, while longer and 

more slender processes nn gi riven off. ‘The infected corpuscles: 


develoyadsohitont as a diy ba 
2S et ae 


by 

ment within the parasite is fine and of, yellowish-brown tint. 
‘The matare schizont is rather larger than in the quartan, has 
a rosotte nppearance, and gives rise to fifteen to twenty merozoite 
thongh sometimes even more occur; these have a somewhat ove 

sl 

Tn both the quartan and tortian fovers all the stagex of 
development can be readily observed in the peripheral blocd. 

3. The Pamsite of Malignant or dstivo-outumnal Fever, or 
Tropic’ Matario.—The cycle in the homan subject probably 
occupies forty-cight hours, though this cannot be definitely stated 
to be always the cam (ide supra). ‘The amobule in the red 

are of smal} size, and their amesboid movementa are 
wery active 5 radon often, however, pass into the quiescent ring 
form (160 The pigment granules, even in the larger 
forms, are few in number and very fine; the infectad red 
corpuscles have a tendency to shrivel and aseume a deeper or 
tint, The fully developed schizont occupics less than 
half the ned corpuscle, and gives rise to usually from six to 
twelve merozoites, somewhat irrogularly arranged and of minnte 
nize, potshe perma ge takes place almost exclusively in the interfial 
spleen, etc. so that, as a rale, no sporocytes canbe 
iommensha:tieod taken in tha wraal way. ‘The proportion 
of red corpusclos infected by the amavbnlio.in also much larger 
in the internal organs. The gametes have the crescentic form, 
as already described. 

Cases of infection with the malignant parssite somotimes 
fuesume a pernicious character, and then the number of onganistns 
in the interior of the body may be enormous In certain fatal 
Piscean net the cerebral capillaries appear to be almost 

i Parnsites being in proceas of sporulation ; 
and in so-called cates, characterised by great collapse, a 
similar condition been found in the capillaries of the 


i (a 





tho moaqui 

inet ie Mites of this insect, have, 

othors, aa detailed above, become established selentific fhets 
‘These facts, moreover, 

vention of spiecony wi 


parasite belong to the genus anopheles; of these there ure a 
large number of species, and in at loast cight or nine 
masite has been found. Some of these anopheles occur in 
England, specially in rogiona where mualarin formerly prevailed, 
The opportunity for infection from eases of malaria 
from the tropics to this country thus exists, and such infection 
has ocenrred. The breoding-ylaces of the insects are chiefly in 
stagnant pools and other collections of standing water, 
accordingly the removal, where practicable, ee dminage of 
Few ecdiacion cite vicinity of contres of population, and 
the killing of the larvw by petroleum sprinided on the water, 
have constituted one of the most important measures, ‘This 
procedure has been carried ont in varions places with marked 
snecese, Another measur ia the protection against my 
bites by netting, it being fortunately the habit of the 
to rarely become active before sundown, The experiments of 
Sambon and Low in the Campagna proved that individuals 
using thess means of meta ae may fre in a highly malarial 
district without becoming infec! The administration of 
quinine to persons living in highly malarial regions, in order to 
prevent infection, has also been recommended and carried out, 
Tn the tropics the natives in large proportion suffer from malarial 
infection, und 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 eome distance from the native 
centres of population, 
‘So far as is known nono of the lower animals have been found 


yot definitely excluded. On the death of infected mosquitoes 
the or sporozoites will become set free, and therefore 


Tt may also be mentioned ws a scientific fact of some interest, 
‘though net bearing on the natural modes of infection, that the 
disease can also be communicated from one person to another by 

the blood containing the jurusites Several i 


is an incubation period, uanally of from seven to fourteen days, 
after which the fever occurs; the samo type of fovor is re 
produced ss was present in the patient from whom the blood 
was taken, 

‘The Pathology of Malaria—While much work hos been 
done on tho malarial pari relatively ens attention has been 
directed tw the processes by which it produces its pathogenic 
effects. It may be said that the organiama are not always 
equally prevalent in the ciecelating lood, and probably at 

stages tend to be confined in the solid ongans ; thus foes 

be scanty at the height of the paroxysm. Some of the 
effects are probably associated with particular stages 

in the life cycle, Thus the pyroxin occurs when the stage of 
sporulation is actively in progress No opinion can be stated, 
however, a8 to the cane of the fever,—whether it is due to a 
toxic process or to general disturbance of metabolism. We can 
better explain the amemia 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 parusite in its sojourn 
im 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 
hy 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 


qd 





clear coils (due to phagocytonis Pigment parson), 
fee pe ale malaria, 


whe aster the possibility of alario. 
question poseil immunity to malari head 
developed naturally arises and this is specially intoresting 
ue light ae Tromcois reaction ae have seen must be 
a on a an element in ee eore fa infoction, 
to Europeans developin, man difficult to. 
speak rin ache. malarie otrlsbon region a thle Wosbee a 
Africa the death-rate in residents of more than four years” 


to the survival of the more resistant Lae tes Bat 
‘be little doubt that malaria in the negro is a much Jess serious 
condition than in the Kuropean. Koch from is observations in 
New Guinea attributes this to the infection of the native atin 
leading to the development of immunity in the adult community, 
He found, what hag been independently noted at fess reps 
Christophers i in West Africa, that the of 
children harboured malarial parasites in their ‘blood, The wide 
spread presence of parasites in children might appear to 
the immunity of the adult being due to survival of moat 
resistant, but the infant mortality in these regions may be very 
high, and such « survival may be the real explanation. On the 
other hand, Keeh states that while an imrunity appears to exist 
in native adults in malarial districts, this is only true of those 
born in the locality; natives coming from neighbouring nou 
mularial districts into the malarial region being liable to contract 
the disease. At present it must be held that tho fuets available 
do not enable us to determine the relative parts played the 
development: of artificial immunity on the one hand, 
existence of a natural immunity on the other, in apparent b> 
susceptibility to malaria, 

Our knowledge on the relationship of blackwater fever to 
malaria is also in au unsatisfactory condition. Blackwater fover 
is condition often occurring, especially in Europeans, in tropical 














| METHODS OF EXAMINATION 535 


countries, It is characterised by pyrexia, darkly-coloured urine, 
Sain esivur teing dis toahered ba-mgiohid igment, delirium 
frequently ending in coma and death. By some 
termionl stage of a severe malaria, With to the 
vie yecial parasite has yet been demoustrated. 
up the evidence for the second view by saying 
from the occurrence of blackwater fever, is a 
non-fatal disease, that in the great majority of cases 
lireet or indirect evidence of the subject of the condition 
suffered from repeated attaeks of malaria, that while in 
re must be an agent ut work causing hemolysis, 
evidence that in many casos there is the possibility of 
being quinine, This last point is of great interest. 
shown that in cortain individuals the taking of this 
sometimes followed by haemoglobinuria. The conditions 
which this occurs are unknown, and in the ease of black- 
jonts, neither is the serum hemolytic for normal 
nor do the red corpuscles sccm to be specially 
sensitive to hemolysis by quinine, in fact, the latser do not 
cidero ited from ontinary red cella. The whole subject 
‘of the pat ete the condition ia thus very obscure, 

Methods of Examination —The jarasites may be studied by 
examining the blood in the fresh condition, or by permanent 
| asst In the former case, a tlide and cover-glnes having 

thoroughly cleaned, a stall drop of blood from the finger 
‘or lobe of the ear is caught by the coverglass, and allowed to 
irencl cut between itand the slide. Tt ought to be of such a 
‘that only a thin layer is formed, A ring of vaseline ix 
round the edge of the cover-glass to prevent evaporation. 
satisfactory examination an immersion lens is to be preferred. 
The daar eater ents are visible at the ordinary room 
. though they are more uctive on a ward & 
RNpauyAbie erntenase a. cull uperture of the Catal 
‘shonld be used, 

Permanent preparations are best made by means of died 
films. A small drop of blood is allowed to spread itself ont 
hetween two cover-glasses, which are soparated by sliding the 
‘ene on the other. ‘The films arv then allowed to dry. A very 
good mi is that of eee who catches the drop of blood 
on a pisce af gutta-percha tissu (a ploce of cigarette-paper also 
docs well), SotMibscinaien 6° fiche ales cise by drawing 
the blood over the surface, The dried films are then fixed by 
one of the methods already given (p. 57), or by placing in 


tAgTETNETEETTE 
Ht a 


he 





536 MALARIAL FEVER 


They 

thionin-blug, sharper results are obtained by in 
aleahol and clearing in xylol te mounting. aha 
‘ard, however, obtained by one of the Romanowaky methods a8 
described on p. 106, 

‘The fact that in many cases the parasites may be 
number Sa a De aa their recognition 
more easy by using blood films of unusual thickness, Here 
about as ian much bette 


dye (vide p. 106). ‘This is allowed to aot for about m quarter 
of an hour, and then very gently washed off with distilled water, 
‘The Romanowsky methylene-blue solution is then applied for a 
fow seconds and ulso carefully washed off, and the proparation 
dried and mounted. The hemoglobin of the red 

washed out by the cosin solution, and the smaller forms of the 
malarial parasite stand cut as round circles containing the char- 
actoristic chromatin dots; and in consequence of 

number present in an area of unit size as compared with an 
ordinary preparation their recognition is very easy. For the 
largo forms of the parasite Ross has found it useful to make 
sich a film aud, homolysing the red coll with distilled water, 
to examine it unstained. The presence of pigment in the para- 
sites enables these to be readily seen, 





APPENDIX D. 
AMUEBIC 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 ax caneil agents 
have boen there discussed (vide p. 346). We shall here consider 
that varicty of tropical dysentery which is believed to be due to 
an amerba, and hence often kuown as amsbie dysentery. 
Amongst the early researches on the relation of jisms to 
dlysontery probably the most important aro those of , who 
noted the presence and described the characters of Fae in 
the stools of a person anfforing from the disease, and considered 
‘that they were probably the causal agents, Further obeervations 
‘on a more extended scale wore made by Kartulis with confirma 
tory results, this observer finding the same onganiams also in 
liver abscesses asvociated with dysentery, Councilman and 
Lafleur, working in Baltimore, ehowed that this variety of 
dysentery can be distinguished from other forms, not only by 
the presenee of ammba but also by its pathological anatomy, 
‘The intestinal lesions, to which reference is made below, are of o 
grave character mortality is relatively Mich, and recovery, when 
i ct extensive tissue 
changes. Thies subject wan, however,” complicated by the fret 
that a similar organism—tho amaha cole—had been previously 
found in the intestine in normal conditions and in other 
diseases than dysentery (by Cunninghain and Lewis and others), 
and additional research confirmed these results. It may now be 
regarded as established that the amcaba of dysentery and the 
common amaba of the colon are two distinct species ‘This has 
ially been shown by the researches of Schaudinn, who has 
given the terms enfamata histolytica and enfamata coli to the 
two organisins, 
Evitamaba histolytica wx soon in the dysenterio stools occurs 
ost 











« 8 
Po. 166, —Auvetee of dyyentery. 


sand, amorten as seon Jn the fresh stools showing blunt, anexboal of 
cloplsan! “The endeplann UE shows a ner eof eorgvees a pment 






kof, imerons Fel corpaledtes ant few tmewien 
as soon th a tand lin preparation, showing a small ronulod nuclei 
Vanquale). 00, 


of an active kind and locomotion may be fairly rapid; not 
infrequontly red corpuscles, bacteria, cells, ote. may be seen in 
the interior, The organism usually dies and undergoes disinte- 
gration in 4 comparatively short time after being removed from 
the body ; the stool ought therefore to be examined in as fresh 
a state as possible, Multiplication takes place by simple amitotic 
division aud also by budding. The entarmeba colt is an onjpnisin 
‘of about the aame size, When at evst it shows no differentiation 
into vetoplasm and ondoplosm, and the nuclous, usually situated 
in the contre, shows « highly refractile membrane with chromatin 
muses seattered in the interior, During amaboid movement 


some delicate processes of ectophim come into view, 


ee 





DISTRIBUTION OF THE AMCBAz 539 


Both organisms have now been shown to pass into a resting 
Reiiinaain tree tee stents eed nace ok forma 
tion of which une markedly different in the twormaes. ‘The eyst 


asses. 

Around these buds concentric striation can be seen, and then 
cyst wall is formed, which is highly refractile in 
chamcter, The eyst then becomes separated from the rest of the 


goes disintegration, These cysts, as will be shown below, repre- 
sont a resting stage with high powers of resistance to external 
agencies, and are congorned in producing infection of another 

subject. ‘The cellular wi oy encysting of the entamecba 
> evel alegre out by Schandinn, They are of 


ie in ee material used for inoculation. The plates were 

uae the oped position, and the inoculations were made in 

lower part ; the ambi moved to the upper part, where they 

‘wero got in pure condition. He succeeded in obtaining eultures 

‘in seven out of thirty cases, and in some instances cultivated the 

‘organisms for more than sixty generations. ‘The amebe multi- 

Tet Cesare division, and in certain cases produced 

‘These cysts, us described and figured by him, 

ot in all important respects with the changes observed 
wudina in eae stools, 

the Amabw—As already stated, they are 





usually of the 
intestine, in tropical amosbie dysentery. ‘They alta, or, 
penetrate into the tissues, where they appear to exert a - 
markedaction, In this disease the lesions are chiefly in the large 
intestine, especially in the rectum und ut the flexures, 

they may also be present in the lower part of the ileum, 

first there are seen local swellings on the mucous Renee on * 
duo to a sort of inflammatory gelatinous oedema fittle 
leucocytic infiltration ; soon, however, the mucous mombrang 
becomes partially ulcerated, more or leas extensive necrosis of 








Fa, 187.—Section of wall of liver abso, deeply into the submiacotin, 
‘shoving an aroata ofepheriod form with. aed'evan pens 
faetolatet protoplasm. From a ease AMA eve 
published by Sarguon-Mujor Dp, G. Coate. In these positions 
arsiall. x 1000, they may be unattended 


by 

and the tissues around them show ar swelling and more 
or leas necrotic change without much accompanying: collular 
reaction, beyond a certain arnount of swelling and proliferati: 
of the connective-tissue cells, This action of the amesba on 
tho tissues oxplains the character of the wleera ws just de 
scribed. ‘These lesions are considered to be characteristic of 
amcvbie dysentery. 

As & complication of this form of dysentery, liver abscesses 
are of comparatively common occurrenos. They are 
single and of large size, sometimes there are more than one, am 
oceasionally numerous small ones may be present. The contents 
are usually a thick pinkish fluid of somewhat slimy consistence 
nnd are lurgely constituted by necrosed and liquefied tinsac with 


ibably explains a fact pointed out by Manson, that examination 
oe tho contents first removed may oo 1 negative ms while 


little or no ered change. ‘The amesbae bave also been 
found in the sputum when « livor abscess has ruptured into the 
Tung, as not very infrequently happens Kartulis records two 
cases of brain abscess occurring secondarily to dysentery in 
which numerous amolwe were present. 

Inoculation. —The anatomical changes in 
dysentery, as above described, gives strong presumptive evidence 
‘as to the causal relntionahi of the amabre, and practically con. ° 


howuver, found to he more suscoptible, especially young animale 
teric changes have been produced in this animal by 
Kartulis, Krase a Pasquale, and others, The method generally 
adopted is the introduction of a small quantity of mucus from 
a dysontoric caso into the rectum, The resulting disease ix of 
an acute character, and sometimes leads to fatal result, Tho 
changes in the large intestine resemble those found in the 
Imman disease, and microseopic examination shows the ameoba 
[eos the wall of the bowel in tho characteristic manner, 
Pasquale obtained corresponding results when the 

material from a liver abseess, containing amceba without any 
‘other organisms, was injected. Quincke and Roos obtained no 
pclcathat the amosba wore administered by the mouth, but 
they obtained « fatal result in two out of four cases when the 
forms were given, They ales found that the cyata, 

wulike the amochw, 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 haa 


—— 


short period of time. It accordingly appear 

both organisms it is the cysts alone which give rise to infection. 
From the above facts, all of which have received lo 

confirmation, there can be no doubt that tho amecba i 







dysentery ought to be examined microscopically a4 soon as 
possible after being passed, as the amerbe disappear mupi 
capocially when the reaction becomes acid, A is 

‘on a slide without the addition of any reagent, a cov 
placed over it but not pressed down, and th i 
examined in the ordinary way or on a hot stage, preferably: 
the latter method, a the movements of the amcebar become more 
active, and it ia difficult to recognise them when they are at rest. 
Hanging.drop preparations may ulso be made by the methods 
described, Dried films nre not suitable, ax in the prepartion of 
these the amabe become broken down ; but wet films may be 
fixed with corrosive sublimate or other fixative (vide p.88), Tn 
soctions of tissue the amoxbar may be stained by mothylene: 

by safranin, by hwmatoxylin and.eosin, ote, 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 4 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 amebe are coloured red (like those of the tissue cells), 
the protoplasm being of a purplish tint. 


APPENDIX K, 
TRYPANOSOMIASIS—K ALA-AZAR—PIROPLASMOSIS. 


Tux Parnocesi Tryranosomes, 


Tre 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 ux living in the blood 
and tissues in various animals and as avusing important disease 
conditions As long ago as 1878 the T'rypanoxoma Lewisi 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 Burra, which occurs in 
cattle, horses, and camels in Tndia, and which is associated with 
the 7'r, Bransé ; Dourine, a condition affecting horses in especially 
the Mediterranean littoral (7'r. equiperdum or Rougeté); Mal de 
Caderas, a disease of South American horses (7. equtnum or 
Elmarsiani); ‘Tstao Fly Disewso or Nagana, atfbeting horses 
and herbivora in South Africa (7'r, Brucet); trypanosomiasia of 
AMrican cattle (Z'r. Theiler) ; and—most important from the 
human standpoint—the trypanosomiasis and sleeping aickness 
of West and Central Africs associated with the 7’, gambiense 
and 7'r, wyandene, 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, 
anwmia, fever often of an intermittent type and irregular esdemas, 
and often lead to a fatal result. In many cases the infective 
agent is conveyed from a diseased to & 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 
pases into a pointed flagellum, In the living condition the 
trypanosome is usually actively motile by an undulatory mavi- 

wa 


























‘THE PATHOGENIC TRYPANOSOMES 5 


ore egy ai arate of the Hum. ‘The size 
varies, but mentioned lover son 50 fog an about: 


progroesion the flagellum 
is in front, the flagellated end ix denaminated the anterior end 
‘of the organist. It ix stated that the method of examining 

rah blood merely allowed to spread iteclf 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 


seer, taining trypenosoriata (or the. Leishman-Denovan bodies) én 
sections 90 a8 to bing ont th chromatin structures, Laishman recom. 


carefully Gxed on oh a 7 Ghoroughly removed. b 
Faating it fire. apping the first ae ah ‘and then ‘yas vk 
ree or four times. 
oabol Coeeushi ‘washed olf by ytd Sam bel aoe 
‘water ja removed with eigarette papor. A drop of fresh blood wwrum in 
ucpcy sect tevoessoaoe wad allowed tock tb for fraatinber 
od by blotting. and the romalnder i atlowed £0 
on the section, which i now treated with « ge r me a 
Of Leishraivs slain and thece of distilled water, and 
Rt oe Utecad uoure Tie yreparotion ervey secktr eunal, the 
lel ona‘ sonlorinnion a4 aiteoetaties. ane 





essen of the method fs the 
applieation of tho blood serum, though what clfost thie hax in not 
Known; Laishman suggests that it restores the normal alkalinity of tho 


Tn preparations stainod by the above methods the protoplasm 
‘of trypanosomata stains blue, and in some species some parts are 
more intensely eolonrvd than others. Sometimes it contains 


thin body represents tho centrosome ix strongly bold by 

avoraa from the analogy of appearances in certain spermatozoa 
which closely resemble trypanosomes in structury). This miero- 
micleus ix often surrounded by an unstained halo, and in ite 
teighbourhood, in cortain species, a vacuole has been described 
85 


_ 





TRYPANOSOMIASIS: 


ts exintng; this fas been considered yy amo to be 
to the contractile vacuole Horna octane 


= aspect or an peetertes| — arizes on 
t structure in tho trypanosome, — undulatory 
patos This is of varying breadth, bas a eee 
free margin, and surmounts the protoplasm of the organism 
like & cock's comb; it narrows towards the anterior end 
where it into the flagellum. Motion is chiefly effected by 
the undulations of this membrane and of the flagellam, The 
latter ia continous 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 
ot trypanosomes variations occur in shape, in length, in breadth, 
sateen of the micronucleus Mand 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 ix sometimes blunt, sometimos 
sharp, and in the presence or abrence of free chromatin granules 
in the protoplasm, 

Multiplication in the body fluids ordinarily oceure by 
longitudinal, amitotic division (sco Fig, 168). Firat of all the 
micronucleus divides, sometimes transversely, sometimes longitnd- 
nally, thon the nucleus and undulating membrane, ts tly 
the protoplaam, Tn some species tho root of bes a 
divides so that in the young trypanosomes the 
short and subsequently increases in length (Tr. Le 
the whole eqalticd takes part in the general splitting of eid 
organism. 

Tu the cases of several of the trypanosomata it bas bean 
found possible to caltivats them ontside the body, the chief 
work here having beon dane by Novy and MacNeal, who have 
succeeded with the Tr. Lewisi, Ir. Evansi, and Tr. Brucei. 
‘The most suitable medium ix made as follows :— 

















Selume of dotbvinsted cabbit blood, whieh has boom iit 
aseptic prooantions ; the tubes are allowed to sot in the molined maa 
In inconlating such tubes they aro placed in on upright position for m 


fow minutes and then the infective material is introduced, 


- 


PATHOGENIC TRYPANOSOMES 517 





which are formed by a number of individuals arranging them- 
solves in a circle with the flagella dirocted towards the contre 
of the agglomeration. By repeated subcultures several of the 


oie oa Lisa tion. 

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 existenco of ach has 


Such differences have been described in 

‘Tr. Tawisi and Tr. Beneet by Prowazk, and in ‘Tr, ngandense 
Minehin, and have been made the bnais of a classification into 
typos which are looked on as representing male, female, and 
indifferent individonls. ‘The male typo is rather slender both in 
Ba ee een ee 

and the protoplasm is free from granules ; the female 
we its nucleus is larger and rounder, the andulating 
membrane narrower, the free part of the flagellum is shorter 

than the body, and the protoplasm contains many chromatin 
granules, which are Tooked upon as reserve food material, ‘The 
indifferent individuals present intermediate ehameters All 
‘multiply by fission as described, and the indifferent individuala 
ean on ocemsion become differentiated into male or femule 
forms, The females are the’ most hardy, and next come the 
indifferent individuals. If all tut the females die ont, these 
con undergo parthenogencsia, and representatives of all thro 
fare can be again rproduced. ‘The sexual cycle ix represented 
fn the invertobeato host. In Tr. Lowisi, according 

i Prowawk, this ie found in the rat lonse, Acrmatopinus 
When this insect sucks the Mood of an infected 

ition occurs by the male trypanesome entering the 

fernale near tho micronucleus and the various parts of the two 
individuals becoming fused. A Ro fagclated oltkinete results, 
through a spindleshaped gregarine-like stage, 

ign “tate a trypanosome in the stomach of the jouse 
a featingslage in an immaturo trypanosome-like form is 
as ocenrring between or attached to the intestinal 


ba 4 


bis TRYPANOSOMIASIS 


of 
while the nuperiluous nuclear structures along with the 
out of the call, In these obkinetoe a ditfrrentiation 
and inditferent forme can bo rocogninr pt ley 
Irypanoacme, In 
wif by a wort of 
the te nee cpa te reste 
of ‘the oscme and gives origin to tern 
isnt Slvsion ofthese forme tthe tmosquito's intestine may 
ooour, and eecser dtenre that Schaudinn differs from other observers 
in bolling that on division the membrane does not split, but that one of 
tho individuals gots its membrane by this being Jaid down along the root 
of that already in existenos, Further, a reatiog-atage of the try| 
ruay oreu, in which it Becomes attached te the intestinal « 
and losing more or lew its agellate form, may reeemble w gregaxine, 
‘The fermale ookinete is plumper and contain more chromatin 
in ite protoplasm than the male and indifferent forms, givew rise to a 
trypanieome after mary complicated division of tts muclety is ess 
motile, does not reproduce itself Ly longitudinal fusion, soom attaches 
If to the intestinal epithelia, is in vistue of the reserve snaterial 
its protoplasm much more rosistant than the malo or 
forme ; whon thove die out, ax they de when, for inatanoe, the insect ix 
starvod, it reprodnoes all threo forms by a process of parthe 
Tn the female ovkin the amaller ucleus which goes to form the 
Mlepharoplast of the indilferent trypanomuave divides into eight amall 
nuclei, all of which perish, and the blepbaroplast and mem 
formed by a fresh division in tho large nvoleus remaining, In the male 
obkinete, which differs from the female in the cloarnesso 
“pad aed mitoala takes place, and again rep vena 
produced, Those ovidently ropresent. the exentially male element, 


for sist, and eaoh, 9) isting to itself « portion of the 
da raha detaches ite that it small ‘shapes are 
‘budded off from the ookinete. This male ookinete Schaudion 
bo homologous with the microgamotocyte ooourting int} 
owl, and the «mall trypanosomes aro ctelarhy Roca 
formed when the blood of the host renches the stomach 
cmputta, These soall trypancarns ais) the lg wy 
somes readily die, probably becaune, by juction 
‘mucous 


neal, the assimilative powers of the larger have M 
ihn ‘the intentin 


dinninishod. In degenerating they often are found in 








THE PATHOGENIC TRYPANOSOMES bit 


tmst of the mosquito in rossttos, with sometimes the anterior 
sometimes the onda, directod towards the centre of the 
tage of derolontuont take place when tho Inrasiten 
the morgue’ Bite reach the Blood of the vertebrate host, and it is 
with this otage that Schoudinn's obscrvations are 
farcreaching. The iudiforent frre by this tne, by repeated 
Hear STERATDInO.Sorprcnheny whlch: Sey peawtrass, 22 se 
ives tO cor) les, iol trate, wasnt a 
falteridlam form, growin eae for twenty-four hours; ty te ive 
thecal lana gn ane the fae Gn, ers ely i the 









bod tl ee no el Mn hey again vain fh ol Thiele 
ie repeated ax ita, ining in sie with eao! jour 
fn ra Whee'the full nine ete ai 
tw (vision osctirs, and when the mnallest forms atw again reached! 
the intense Th i 

al caubot a hb the m ito"s 

forats féseh the owl they enter the red corpuslon, 


form, and ultimately their capacity of 
found Tying surronns by the remains of the Inst cell they entered. 
The c 


the miorogametorytes are ab ited 
or frou parton gona of oust fms 
‘that 


through this, and arm in a position to be njected when next 
the tanec Hite © : : 


‘Such are the views put forward by this observer on the eyele 
of life-history of the Tr. noctum. It will be recognised that 
the essential point i# the occurrence, both in the vertebrate and 
invertebrate host, of halteridium stages alternating with those 
in whieh the trypanosome form is assumed. It is evident that 
if this were substantiated important effects would follow in our 
Views a8 to the morphology not only of the trypanosome group, 
‘but aleo of that to which the malarial porusites belong. 





Certain criticisms of these resulte hay: ly by 
Novy and MacNeal, who songht confirmatory evidence by means of thoir 
eulture method, ‘These observers aro of opinion that the appearane 

Soluudinn were due to his dealing with a mixed infection 
of the owls by tryyancoomes on the one hand and luewamabe of the 
halterhlfam type on the other. They examined a very large number of 

nd established the fact that infection with 
haltoridium paresites on the one hand and with trypanosomes on the 
ther ts axtremely common, and further, that th thi hood of the same 
Bind both halteridium and trypanowme infoction could be olmorved. 
‘The bird jes could be readily oultlvated, it was observed 
that mp cultures wero obtainable from birds in which halteridia were 
alone found, and further, that when a trypanosome inolated from # ease 
whore both forms of parasite hud beow seen was injected into a freab 


























gl 





TRYPANOSOMIASIS 


teyyannome forms wore ound to dovlo nny. These 
ae eee sianot suseyele Is ths pe 

13 cont 
mee scent bee that botkewith ‘Tr Braces 


atten 
‘eco st ‘poe have res sae ot ont CE 
fafecion took plage in nagana by means of thw inet ‘the 
1a the tube of it yroboucl, whero fe observed hen to be 
motile i fry hours after the tuseot bad fod, and with 


free 
$ogand to the sleping 3 rgauiee Minchta held a similar epfales, 
showod that if gloosine bit an infotod animal, and then in smooes: 
sion two healthy anitaals, only the first of tho lator would contrast the 
Wisenso—tho probosets belng apparently cloaned by the biting, process. 


Reference may here be made to the views put forward by 

Schaudinn penta the relationship of certain Y eptclle to the 
eu, In the athene noctua, besides the Tr. noctum 

already referred to, there is a protozoal parasite infesting the 
leacocytes known a4 airillum Zécmanni or leucocytawon 
Ziemann, whose invertebrate host is also the cule pipiens, 
Ziemann had described the male and female forma in the owl, 
and microgawmotes had been observed forming from the miero- 
gametocytes. Sclaudinn observed the formation of at. obkinets 
iW the mosquito ; in certain cases this odkinete elongates, and 
the ricmioule rolla iteolf up into a ball with grea’ proberations 
of the nucleus. Each little nucleus attaches to itself a portion 
of the protoplism, and becoming a miniature trypanosome, 
swarms off and becomes free, These minute ahen 
elongate und develop into typical spirilla by rolling their ribbon- 
shaped bodies spimlly along their longitudinal axes, the 
individuals possessing male, fomale, or indifforent cha 
just os in Tr. noctuw. These spirilla multiply by } 
division, and often after fission the two individuals remain 
attached to each other by their posterior ends, and in thi way 
there in made possible what is often seen in spirilla, namely, a 
capacity to move in either direction, ‘The spirilla often divide 
ao frequently that ultimately the [ndividuals become invisible uy 
means of the microscope and can only bo seen when 
clumps. Tn this stage Schaudinn ‘ita the organism 
able to pass through a Chamberland filter, and this may be a 
vory important observation, as throwing light on the otiology of 
cortain dine such as yellow fever, in which no visible ae 
have been found. 


Schaudinn's views on the: iF feet leegregiver ee 
have mised important quostions regurding the 
similar forms which have bean Joo 


tniltar, auch ax Sp. 
and also of the Spirockmte pallida which Schaudinn himaelf @iswovmred. 











a 





? 
TRYPANOSOMA LEWISI 


‘Tt is a yot too soon to any opinion on the ultimate effect of 
Stan eetsc es ee ee 
: 


‘551 


sometimes seen in the course of Thoth all apirilla 
have this stracture must Wee are to empiee 
Difficulties egal u ilficance latory 
nal flagelltan which set a al sel 

0 WI sadinn himself found in pallida, 

and which he hed ht did not, ii la. 
Trib pendbls tint Ure grote of Segnninnn have litho been Hesed 


of 6 
under tho nate spirillumy and that ‘one o€ those may all 
‘have to bo placod with the bacteria, 

‘Trypanosoma Lowisi.—In 1878 Lewis doseribod in rate in 
Tadia 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 rata 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 
& very dofinite cours, it is very rarely fatal; in fact, many 
observers have been unable to produce death by infecting evou 
large series of animals. here is, however, little doubt that a 
fatal issue does ocenr sometiines in young individuals, expecially 
when those are infected with strains of the orgunism imported 
from other localitics. The trypanosome, which is actively 
‘motile, is of the usnal length bat is somewhat narrow, and its 

lagm does not contain any granules It mmltiplics by 
Tinion, of which Lavoran describes two varicties. In one, the 
organisa splits longitudinally and gives rise to smaller individuals 
than the parent, In the other, the trypanosome lowes its 
ordinary and becomes more oval; nuclear division, 
which is often aap tukes place, and on subsequent 
division of the protoplasm a number of amall flagellate organiams 
remult; those Inet may attain the full form and size before 
ivi or they may divide when still small, When a 
rat Is Infected by injection into the peritoneum active multi- 
Sea ateaitenl tn tha cavity Sor a for daye and then comea'to 
anond. Very soon aftor infection the organiams Login to appear 
in the blood and there rapid multiplicstion occurs, the extent 
of which is sometimes so great that the trypanosomes may seem 
to equal the red blood corpuscles in number, ‘The animal 
: ‘shows no symptoms of illness. ‘The infection goes on 
two months, and then the organisms gradually dis- 
appear from the blood. In the great majority of cuss the rat 
‘ig now immune against fresh infection. If trypanosomes be 








Fy 






a 









bbe TRYPANOSOMIASIS 


introduced into itx peritoneum they are, aceording to 
takon mononucleate and destroyed. 
pe aero peas shows ti 
Sipeendetag i apnad meearel eper ee 
ate in rosettes in which the flagolla are directed 
the serum of immune rate has a cortain degree 


me rupli. vie Bea from ‘an infected 
orgenism multiplies at the body temperat 
temperature is preferable, and at 20° C. Novy and 
in carrying a growth through many aub-eul 
trypanosome i# very resistant to cooling, and has boon: 
for fifteen ininutes to the temperature of liquid air (—19) 
without being killed. The teans by which the rst becomes 
infected naturally is not known, but probably this comes about 
by the bite of « flea or louse. 

Nagana or Tse-tse Ply Disease. —This is a disease affecting 
under natural conditions chiefly horses, cattle, and dogs ; it is 
prevalent especially in cortain regions of South Afri 
it probably may occur elsewhere, In the horse 
symptoms are the following :—The animal is obswrved to be 
ont of condition, its coat stares, it has a wat i 
from the eyes and nose, and the temperature ia clevat 
swellings appear on the under surface of the abdomen and in 
the legs, it gradually becomes extremely emaciated and 
anemic, and dice after an illncas of from two or throe weoks to 
two or three montha. Tp other animals the symptoms are of 
‘the same order, though tho duration of the disease varies mueh ; 
thus in the dog tho illness dees not last more than one or 
weeks, while in cattle it may continue for six months, It 
doubtful whether a domestic animal attacked by the 
ever recovers, The popular idea regarding the ettology of 
disease was that it was contracted by animals 
cortain rather restricted and sharply defined areas or 
characterised by heat and damp, usually lying beaide 
and always infested by the teo-tee ily (glosbina morsitiena), 
bite of which the disease was attributed ; in this connecti 
is important to note that though man is frequently bitten 
the teo-two fy he does not contmct nagana. Modern 
ledge on the subject dates from the discovery made by 












i 


& 
Ee 


A 


eet 


i 






F did not occur, key for a foe days and 
te suscoptible animals, tter did not contract 
result showing that it was not, as had been snpposod by 
tome, i i 
fly was allowed to bite a dog suffering 
to bite o healthy dog, the lattor 


kept moist, then it retained its infectivencss up 
7 days; up to 46 hours living trypano- 
necu in the tube of the fly's proboscis, 
roughly with what was found regarding the 
the infectiveness of a fly, in that 24 hours after it has 
on an infected animal ite bite is usually innocuous, 
Bruce showed that infection did net occur by any food 
ioe of by an animal while going through w fy 
took horses throngh such a region without allowing 
or drink, and found that they still contracted the 

infection, if during their few hours’ journey through the belt 
had been bitten by the tae-tae ‘ity. Finally, he showed 
‘flice were taken from an infected area to a healthy one 
'tniles off nod allowed at once to bite infected animals, the 

i 





He 





latter contracted nagar, 
‘those experiments it wax thus determined that nagana 
mieten hy the blood of the infected animal, that 





Oot TRYPANOSOMLA 





is, without the agency of tho 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 urea harboured the parasite and were thus infective, 
‘The question remained as to how the flies might beoomo infected 
in nature. It had been observed that in districts where the 




















168.1 Brace! trom blood of 
the organteme commencing division of micront 
wnbrane, — * 1000, 





«aod undulating 





tuo-tse fly lived tho provalence of the disease in imported animals 
was rolated to tho prosence in the locality of wild herbivorm 
Brace now found th amounts of the blood 
of the latter were taken to another locality and injected into 
dogs, these in « 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 infeotion. further and as yet unexplained 
fact was that other blood-sucking flies besides the tsetse 

















short exporars to temperatures 
ickly killed at 4445" ©, Ne 


coltivating this trypanosome also, 
fitout obtain a firat 
wt medinm ; nee started, however, 
many subcultures, the. opti 


rosette formation with the flagella directed outwards ; agglutina- 
tion phenomena are also observable in defibrinat blood. 
Under unfavourable conditions involution forms occur, the 

dividing frequently to form round #agellated 


ividuals, 
Nearly all laboratory animals are susceptible to infection, 
the duration of the illness corresponds to what has been 
in tho natural infection of these animala Tho rat 
largely used for experiment and usually succumbs in 
there being very few symptoms up till a few 
before death. A very important fact has been observed 


gh infection with ‘Tr. 
ible to the Tr. Brucei; from this it has been 
two organisms are to be looked on ns distinct 


jp 





of Sleeping Sickness.—Sincs the yoar 1800 
dimaso called slecping sicknoss, sleeping dropay, or negro 


be: all 


r 


TRYPANOSOMIASIS: 





brn Oy isomer bole Kaper 


‘the woast between these regions Tt has 
Sola ani Seep ony har a reir eck 


Seapjenhin ibe Ups ‘Protectorate, and it is in that 
oe the mugs ot is have. ae carried on which have led 


enlarged, Ina mpid case the lethargy Yee 
fine tremors, expecially of the tongue and arms, develop 

ressive emaciation occurs ; blood changes appear, ofa 
Laroche diminution of the red cells and of the vie eng 8 
and of a lymphocytosis in which the perventage of both the 


the drowsiness increases till it deepens into a coma from which 
the individual cannot be roused. Often during the disease there 
occur irregular adematous patches on the akin and sometimes 
erythematous eruptions, and effusions into the serous cavities, 
Not every case runs a progressively advancing course. Some- 
times along with enlargoment of glands the chiof early feature 
is the occurrence from time to time of attacks of fever which 
may be mistaken for malaria, and from these sppareatiy com 
plete recovery may take place; recurrence, however, 

as a rule, and ultimately the typical terminal phenomens 
may commence. Such cases may yo on for years and it ix 
probable that many patients die of pneumonia without 

typical manifestations of the malady from which they 
suffer, ‘The disease is an extremely fatal condition, and prol 

‘no case where the actual lethargy is developed ever recovers, 

On considering the disease from the stand; thee ‘of patho: 
logical anatomy there is little to be said. As loscribed, 
the most striking feature is the presence of a ane meningo- 
encephalitis and meningo-myelitix. The Lppeear is somo. 
times opaque and slightly thickened and may be 
to the brain, and its voswls usually show some ple! 








‘TRYPANOSOMA OF SLEEPING SICKNESS 557 





Fig, 168, —Trypanceoma gumblense front blood of gainewpig. x 1000, 


are related to the sub-arachnoid space and the perivascular lymph 
spaces, with accumalation and probably proliferation of lympho- 
‘eytea in the meshwork. He further points out that the changes 
in the Iymph glands are of similar nature and resemble the in- 
filtmtion of the perivascular lymphatics of the central nervous 
system. ‘Those are specially significant in view of the 

present in the blood, which hae already beon 
‘noted, and which so often occurs in protezoal infections, In the 
Mervous structures thero is comparatively little change, there 
being merely, acconding to Mott, some atrophy of the dendrons 


— a” a 









bbe TRYPANOSOMIASIS: 


of the nerve cells, a diminution of el 
oxcentricity of the nucleus, 


Haropens then ieee Baers the Gambin. 
of the disease wae hero very slow, and was by 
eer wasting and weakness, irrogular rinos of 
Ra, Sones Been neR eee 
and increased frequency of pulse and 
‘a year aftor the case came undor obsorvation ees an access of 
fever, and @ striking fact was the absence of any gross causal 
lesion, Daring the time the patient was under observation 
osomes wore repeatedly demonstrated in the peripheral 


3 mniries, Dutton and Todd demonstrated sill paraies in other 


‘uropeans and in several natives in tho whilst 
cell region eg ieee ppt ene 


CS eget nema are Tt thus came to be 
recognised that in man there occurred a disease having eharneters 
somewhat resembling nagana and in which trypanosomes: 

be demonstrated in the blood, and this was usually referred to ax 
human trypanosomiasis, or trypanosoma fover,—the trypanosome 
being named the Tr. gambicnac, 

Relation of Trypanosomes to Sleeping Sickness, — Several 
views ns to the etiology of this disease had been advanced, A 
Portuguese Commision in 1902 doscribed a diplococeus 
to grow in chains which they isolated from the corel 
flnid taken from casex during life, and to which were 
inclined from tho constancy of its occurrence to 
canml role, The seriousness of the epidemic in Uganda had led 
the Royal Society of London in 1902, at the instigation of ee 
Foreign Office, to despatch a Commission to investigate the 
condition on tho apot, Soon after commencing work, Dr. 
Gstellant found some on is sxtebrospinal ‘aid, 
ospecially when this waa centels sed ing tt 
atest the ‘Tr. gambiense ; he slso found in 80 par cont 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 
a8 accidental, but Colonel Bruce on going ak 9 with Nabarro: 
and Greig in 1903 to parsne the work of the Commission 











the starting-point 


ble from sleeping sickness and with the para- 
siete inetd vcs uence fue 
end of Victoria: wi i 
pcre eto Ne Nya whee ecg 


‘The trypanosome of sleeping sicknosa is 17-28 je long and 
1-4-2 ps broad (Pig. 109) (when about to divide it is both if 
Paiairalael A ccocding 'tor"Lavorun tis ‘five part of the 
flagellam often equals a fourth of the whole length, but occasion: 
ally ‘the bod: asm extends quite to the end of the 
organism, julating mombranc is narrow, and the 
posterior end may be either sharp or blunt The trypanosome 
contains the macro- and micronuclons characteriatic of the group, 
and tho protoplasm often shows chromatin granules. Castellani 

portance to a yacuole often seen in the neigh: 

micronuclens, but, as stated above, Laversn 
hold this to be an artefact. The organism divides longitadin. 
aily in the staf manner, and often two can be seen to approach 
and lie more or less side by side, but whether this 

njugation or not is not known, The organism docs 
‘not usually Jong eurvive removal from the body, but it has boen 
found to be motile for nincteon days when kept on rabbit-blood 
ager nt 22° CAs we have maid, when Tr, wgundenso ix in- 
eeulated into monkeys they often contmet an illness which 
nltimately presente the features of typical sleeping sickness, 


‘in 
ere 


| 


i 





560 TRYPANOSOMIASIS 


= cov 
ace the dae 
ithe the droplet of uid in 
Bria crac Pare vote bro-spinal fluid the 
of the avol % corel 
trypescemnen can be seen to be actively motile; the tmumber in 
they occur varies very much, and the same is true to « 
greater deyres of the blood, in which they ars, however, usually 
very scanty. With regard to the examination of the blood 
Bruce and Nabarro state that it is difficult by ordinary centei- 
fagalisation to concentrate the orgunisms, as they are not readily 
procipitated. They nccondingly recommend that the blood be 
mixed with citrate of sodium solution (equal parts of blood and of 
one per cent citrate solution) and centrifugalised for ten mit 
that the plasma he removed and centrifagnlised afresh for 
tho samo time, and that this be repeated three times, the depesi 
from cach ecntrifugalisation after the first being 
examined, Greig and Gry have insisted that the examination 
of the glands in a suspectod caso forms the most ready means of 
arriving at a diagnosis, and this opinion has found strong: 
pasha from the work of Dutton and Todd, The method is to 
h a hypodormic needle into the gland, suck up n little of on 
toe and blow it out on to a slide. In all eases where filing 
‘aay kind are to be prepared the staining methods of Leah 
or Gleinsa are to be recommended. Often in cerebrospinal fluid 
and gland juico the staining of the chromatin is difficult, but 
oo preparations aro obtained by the provedure 
by Leishinan for studying the parasite in sections (p. 545), 
Greig and Gray have studied the trypanosome in the 
of the glossina, They found evidence of its multiplying in the 
stomach of the insect, and it also was scen to under 
not elsewhere observed. These consisted in alterations in 
position of the micronuelens, which often became anterior to the 









“TRYPANOSOMA OF SLEEPING SICKNESS 561 


‘macromelona; there also eccurmsd rosettes, consisting of from 
four to twenty individuals attached ty their | aie ane oe 

wore aleo observed. Tt was that monkeys 
‘not be inoenlated with the trypanosomes from the stomach 


‘investigations the question arose ax to 
‘of slooping sickness was different from 


hy 


‘Tr. gam! This was forced on the inquirers by the fact 
that a very large ion of the natives in the sleeping 
‘sickness area wore found to harbour josomes in their 
blood, although not apparently suffering from the discase. 
Several cases were carefully examined in which trypanosomes 
‘wore present in the blood, but in which the pationts 
from time to sufferod from fever, and during these pyrexial 


‘jods trypanosomes were found in the cerebro-spinal fluid, 
‘Waa suggested that these cases wore on the way to develop 
‘A wry important obscrvation was that 
in Soe eter areas a large proportion of the native 
trypanosomes, this was not the case 
ing sickness did not occur. Furthor, it was found 
trypanosomes fram the cerebro-spinal fuid of sleeping 
ess cases and from the blood of persons harbouring try- 
but not suffering from disease symptoms, gave rise 
in monkeys to the same group of chronic effects which resembled 
the Jast stages of the discase in man. Those facts led the 
Commissioners to incline to the idea that trypanosoma fever 
and aloeping sicknoes aro duo to the same cuuse, and represent 
stages of the samo disease. Tt has already been 
‘ont that a fatal termination can occur in trypanosoma 
‘fan acute fobrilo attack or from intercurrent disease, 
the terminal lothargic stago may only develop in a 
tion of cases. The view of the identity of the 
has continued to gain ground. The best 
are agreed that morphologically no difference 
two organisms can be recognised, and the con- 
olwarvation of prolonged ensss of trypanosoma fover, 
in Uganda by Greig and Gray and in this country 
has shown that sometimes the termination of » 
1@ onset of typical sleoping nicknoss. 
of trypanosomes in the blood of apparently 
has raised the question of the possibility of 
ting and of immunity being established. It is 


ft 


i 


ze 


i 


he 


tit 


i33 
he 


Pi 





562 TRYPANOSOMIASIS 


that both phenomena occur, that not every infection 
Fula in mulation ion of the parasite in the body of the 
victim, and that in certain cases where multiplication docs 
‘oveur a resistance is developed which onnbles the body to kill 
the parisites. ‘The occurrence of the mononuclear reaction ix 
here significant ; it has been suggested that, when this resist- 
ance is weak, the orguniem gains entrance to the spinal eanal, 


Cnc and a riouiiae tiie ie by such 
the strongest evidence may be maid to cxist that the ‘Tr, 
apace is the canso of sloping sickness. 
Other Pathogenic Trypanoromata.—It is beyond the 

of this work to deal at Tomgeh with the other diseascs of 
canaed by trypanosomes, The chief of these hare been 
mentioned in the opening Pangea but it pies be percrr. 
many others have been described in various species of mammals, 
birds, and fishes, and that these are spread cither by flies or hy 
Jeoches, ‘Thy most interosting of thoxo mentioned is Dourine, 
@ condition resembling in as ways niugana. It, howover, 
presents this peculiatity, that in! lection does not take place by 
an intermediate host, but apparently directly through coitus, as 
it occurs only in stallion# and in mares covered by these, 

In several of the trypanosomal infecti 
appears ax if, us in the cae of Tr. Lewis, the animal suffers 





organism without being affected “o its presence more, 
cxample than is the mt by Tr, Lewis, Though no opinion 
a be expressed on this point, it is necessary to bear the fact 
x uitadhs hat (pidhestriateaal oe accnired immunity ean exist 
against such protozoa. Not only is this important from the 
point of view of the investigation of the conditions under which 
ch tolerance arises, but also from the bearing whieh the 
istenco 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 arg, however, 
aa 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, 














Kins Azan, 


na: Cachectir Fewer, Dum-Dum Fever, Non-malarial 


Reniittent Fever.) 


"Leishman noticed in several soldiera invalided from India for 
romittent fever and cachexia that the moat careful examination 
to reveal the presence of the malarial 
parasite, From the fact that such patients had almost invari- 
been quartered during their service at Dam-Dum, an 
cantonment near Calcutta, he ted he bad 
a ieatnerert ue he noticed in 
spleen ‘8 case peculiar bodies which, from comparison 
shige epyarens ound i deer one ‘a te 
Brucei, he suggested might panosomes, and on jrablie! 
his observations in 1903 (ibe forward the view that 
is might prevail in India and account for the 
eases of cachexial fever met with there. Soon after 


a 
& 
! 
. 
: 


They were found by Bentley, and later by Rogers, in 
disease known in Assam as Kiila-izar, the pathology of 
Jong puzzled those who hnd worked at it, from 
while it resembled malaria in many ways, no 
be demonstrated to occur in those suffering 


F 


é 
as 
et 


cl 
ae 


(or “black disease,"—to called from the bue 
chocolate-coloured patients suffering from it) has 
jown since 1869 aga eae opidemic disease in Assam, 
has spread from village to village up the Brahmaputra 
‘The disease is ior ett to occur in various sub- 
centres south of the forty-ninth parallel—easea wher» 
‘Loiehman bodies havo been found having been mot with in 
of India, China, North Africa, and Arabia, ‘The 
characterised hy fever of v very irregular type, 
ive cachexia, and by anwmia associated with 
of the spleen and liver, and often with ulcers of 
the skin and dropsical swellings, Rogers has pointed out that 

‘ceours 4 lencopania which differs from that of malaria in 
that it is almost alwnye more marked, the lencocytes usually 
numbering less than 2000, and further in that they are always 
‘reduced in greater ratio thin the red corpuscles, which condition, 
‘again, doo# not occur in malaria. The disease is elronic, 


a 





it 


TE 
: 


i 


= 


| 


3 
2 





82 
rf 
i 
; 





Faq, 170.—Leishman-Donoran bodion from spleen sincar, 1000, 


stain, the characteristic bodies can be readily demenntrated 
(Pig. 170), They are round, oval, on as Chri has 
pointed out, cockleshell shaped, and uxnally 25 to 35 ye im 
diameter, though smaller forms oconr, ‘The protoplasm stains 
pink, of sometimes slightly bluish, and contains two bodics 
taking on the bright red colonr of nuclear matter when stained 
by the Komanowsky combination. Ths larger ataina Toss 
intensoly than the smaller, is round, oval, heart-shaped, or 
bilobed, and lies rathor towards the periphery of the: 

the region of the “hinge” in the cockle-shaped i 


al 


KALA-AZAR 565 


via | ig that on their entoring the 

ead mononticlear leucocytes and by such cells ws 
opal ial lining of the splenic sinuses or by those lining 
capillaries or lymphatics, that in these cells multiplication takes 


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: ited. ~The 
clusters of bodies some- 


stably held together 
‘the remains mp 


cy 
Man neni 
cl, ler “pagcestig 
robably be- 
mate bas rt from the 
wall, as th > 

See ft tered fron 2 Sebe Dre ein 

in the lumen of the : 
vessel, —this being well seen in the hepatic capillaries, 
Im the body generally the parwsites are found in greatest 
istidsued Sx the spleen, liver, and bone marrow, and also in 
mesenteric glands, especially in thow draining one of the 
intontinal ulcers ; less frequently they occur in the ekin ulcers, 
and in other parts of tho body. Whether they can be demon. 
stnited microscopically in the blood is disputed. Donovan 
them as occurring in the blocd, and also as being 
present within red blood corpuscles, but though Laveran agreed 
with Donovan's description, the cbscrvation has not been 

confirmed by other observers. 

Th tho body tho parasite multiplios by simple fission, both 
nuclei dividing amitotically, two now individuals being 
formed ; but sometimos a multiple division takes place, each 











os 





nucleus dividing several times within the protoplasm AAS 
number of new parisites resulting. 

Jn view of Leivhman’s original opinion an ext extremly opera 
discovery was made by Régeed not i later confirmed by Leishman 
Aimeself, to the effect that in cultares a flagellate 
develo) from the Leishman-Donovan body. Cultivation waa 
el by taking spleen juice containing the parasite, placing it 
in 10 per cent sodium citmite solution and keeping at 17-24" G. 

Under such conditions there occurs an ment of the 
organinm, but especially of tho larger nucleus. This is followed 
by the appearance of a pink-staining vacuole in the neighbourhood: 
of the smaller nucleus. Along with these changos, 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 cay 
formed seoms to take its origin from the neighbourhood of 
small nucleus, The body of the parnsite ia now from st 
Tong and 3-4 « broad, with the Hagellum about 22y¢ long. The 
whole development occupies about 96 hours. The formation of 
an undulating mem yn not obwerved an although the 

ated organist mo’ lazellum firet, like a tr; ae 
3 evident that here the relationship of the al 
different as this structure lies anterior to the Hae 
In his cultures, which kept alive for four weeks, Leishman made 
a still further important observation. Th certain of the flagellate 
forms he saw ehromatin grinules develop in the protoplasm often 
in couples, a larger and w smaller, There then occurred a very 
unequal longitudiual division of the protoplasm, and a hair-like 
undulating individual containing one of the pairs of chromatin 
granules would be eplit off, At first there would be non 
flagellate, but later a red-staining Hagellum would appear at one 
end. The analogies between these observations and these of 
Schaudinn (v. p. 550) on the relations of spirochates to 
trypanosomes will be at once apparont; the further develop 
mont of these spirillary forms in Leishman's organism 
not, however, be tra 

‘The facts just detailed have caused considerable discussion 
to the classification of the arganism, which now usually gous by 
the name Leishmania donovani, originally given to it by Ross. 
According to one viow, it is to be looked on aa a tr 
and although, as we have noted, its Aagellated form 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 dondeaans of the 















KALA-AZAR 567 


‘presently to: 
‘The question arises, given that the Leishmania donovani is 
of kdladmr, how is infection spread? On this we 
have as yet no certain information, The fact that in some 


On the other band, the possible relation- 

the organism to the trypanosomata naturally suggests 

idea ef an insect as an intermediary, and Rogers has brought: 
forward some evidence that the bed-bug is the extrehuman host, 


but the organism has not as yet been demonstrated in the body 
cof this insect. [thas been objected to the theory of an insect 
ccurrivr that tho organism probably does not occur in the blood, 
but it has been pointed out that’ invisible spirillary forms may 
instruments of infection, and that sach may exist in the 
It may be said here that all attempts to communicate 
the disease to animals have been hitherto unsuccessful, 








fetheds of Bexmination—The Leishmania donovani can 
be readily aoen in films or sections of the organs in which we 
havo mentioned its occurrence, These should be: stained by the 
Romanowsky stains. Fluid taken from the enlarged spleen during 
Tife may be examined, but it is probable that in this disease 
tare of the spleen may not be a very efe operution, as 
from hemorrhage from this organ is a not uncommon 
natural terminal event. During life the main points on 
‘A fathological diagnosis may be basd aro tho al 









568 KALAAZAR 


malarial parasites from the blood, and the features of the 
oh Be Sapo ane sein 
-—In various tropical and sub-tropi 

there is widely provalent a variety of very intractable 
ulceration which goos by various names in different oer 

tho world, Delhi sore, tropical uloor, Aloppo boil, ote. Variows 
views haye been held as to the ae of the condition, but 
the work of J. EL Wright, whic 0 confirmed by other 
observers, makes it extromely voluble that » protozoal parneite 
is concerned in ite etiology. irs the discharge from the ulcer 





if 


and in sections of a portion of tissue excised from n aise coming 
from Armenia, Wag here great numbers of round or oval, 
sharply defined 24 p in diameter, When stained by = 


portion coloured a pale blue and a centril portion tending to be 
unstained ; there were also two chromatin bodies, one larger, 
occupying a fourth or a third of the whole and situnted in the 
periphery, another smaller, round or rod-shaped, and of a. ses 
colour than the Ja muss. It wow found that the 
were usually intracellular in position in the lesion, a8 many as 
twenty being in one cell, and that the type of cull containing: 
them waa, aa in kila~izar, that derivable from endothelial tieeues, 
There can be little doubt that these bodice aro of the same: 
type as those occurting in kiladaar, and the question of the 
identity of the two parasites has been raised. At present the 
sailors is to regard thom as distinct, As wo have seo, 
although skin ulcers are common in kila-dzar, it is difficult to 
find the panwite in this lesion of the disease, while, on the 
other hand, in Wright's case at least the number of 
present in the ulcer was enormous. Provisionally Minchin 
calls this purisite the Leishmania tropiea and include it as the 
second species of the gunus Leishmania, 





Pinornasmosis, 


Up to tho present no human disease has been proved to be associated 
with the pres of pee plasmata. The observations of Donovs 
Wich seuudod to Iudioate that the paraadte of hdle-deat mighl bx 
Within the red blood corpuscles, and which Ted Laveran tor denorafute 
the Leishmania donovant the piroplaema donovani, have, ai 
indissted, not boon confirmed ; the sutne fa tena of the asesolation oft 
iroplasis with, the eoourrence of the Rocky Mountain spotted. fovar 
Fometimes provalont in Montana. But sevoral impartant di of the 
Tower anna ry aot tally cated by rosa partes of Ye 
group, and a short account of te oncanintss mney bo gh 

"Tho piroplasnunta aro pear-shaped nicallular orguians about 260-6 pn 


a 














PIROPLASMOSIS 569 


i 
‘is a woll-stainit 
eepratsiaped 








‘pevudopodial 
coaamase bend 
inf 


Jmrocoases sprout out from the broad end of the ya 
Bot tt occurrence of comfugution of two such fodivideale Lyt 





harboured by it may come in oontact. 

‘The following aro tho ehiof pitoplasmate causing disease in aniznals »— 
Q) PH rminun. This waa firxt described by Theobald Senith 
and is the cause of Tesas or red-water fovur, a febrile condition associated 
with hemoglobinuria, which occurs in the Southern States of America, 
the Argentine, South and Contral Africa, Algeria, various parte of 
Northern Europe, and in Australia. ‘The organism gots ite name of 

Ageminu from the fact thst it ix often prevent in tho red cols in pairs 
which may bo attached to one another by a fine thresd of protoplasm ; 
this probably results from the complete separation of two individuals 
‘Doing delayed after division has occurred, Infection is herv spread by 
the tick boophitus boris, and some of the clracteristios of the disease 
* ily aro explained by the fsot that this insect goes through 
all its meetings on the same individual host. (2) Firgplanma parm. 
Whis organiem wax discovered by Thoilor in tho blood of cattle waffering 
fiom Affican East Coast, fever, ¢ discaao closely resembling Texas fever, 
whioh prevails endemcally in & narrow strip along « long extent of the east 
comat, aud which occurs cpidomically inland, Av ite designation implios, 
the organism is small, and itis also attenuated, Its insect hont is the 
rhipicephatus appendieulatur, and it may be noted that this tick drops 
of tho anita! ou which it may be feeding when it te about to go dhrough 
one of its wereral monltings Tt eam thus carry an {ufection much more 
qulokly and widely through » hord than can the carrier of ordinary red 
water fever. It tuay be sald that in England thove occurs « rod-water 
fevor also associated with the presence of a piroplasm in the bleod, but 
therulationship of this organism to the otlior varieties has uot yet been 
fully worked out. (3) Fireplasma equi. This organisa gives rise to 
biliary fever in horton, another South African disease, and it & carried 





























570 PIROPLASMOSIS 


by the tick rhipicephalus evertsii, In this disease Theiler made the 
interesting observation that when the blood of » donkey which had 
recovered from the disease was injected into a horse the latter sutfered 
slight illness only, although the organiams were present in the blood 
injected. Such a fact is of importance, as attenuation of virulence in 
pathogenic protozoa svems, 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 
chauges originated. 








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Wa 4, “Bolte, 2, Morph, w 


1888 + Bay ester, Quart, Journ, Micr, 
876), xvi 47, 278, Nirnurriva Onaastans. 
- (ere Fosteur, iy. 218, 267, 700; ¥. 92, 577, 


7 
bs 
s 


a 
reek 
ue 


4 
ni 
uy 


EL 


CHAPTER IL—Mernons ov Cuntivattox ov Bacrnuta. 


For GxxeKat Prancietes. —Pastour, 





sande. 1.1; Roux ct Nocand, Aw slo Put. Pasteur, te 

28; Marmot, ibid. ix. 08; Kitasato and. Weyl op, et. supra: PL 
and Ms Rev Frankland, * Micro-organiema in or,” London, 1804. 
i 








“Amer, Puke Health Ave, xx. 381, Theobald Sinith, 

. f. Bakteriol. u. Poresitenk. vil 502; xiv. 864. Durham, 

Brit, Med, Journ, (1898), i, 1387. Report of American Committee on 
Bacteriological Metheris," Conconl, 1808. MacConkoy, Thampron- 
Yates and Joknston Lab. Rep, vol, iii, pt. iii. 181; vol iy. ph be p. 1615 
Journ Hyg, 7. 193. Drigaloki and Conradi, Ztecbr, f. Hyg. xxxix. 
233, : 








OHAPTER ITL—Mtonoscoric Mernopa, ere, 










‘Consult text-books, especially Klein, Kanthaok and Dryslale, Hueppe, 
Oliather, Heim ; also Toles Tes, Tho Microtomi Vad 
London, 1905 (this is tho scst complete trea 


toxt; Koch, MUA. a. de te Gsmlht 

‘ vf Alin. 53; fi. Tl Gram, Forteckr, od 
‘Méd. (1884), ii. No. G; Nioholle, Awa de (inst. Pasteur, ix. 6665 
iit 





lf. Bakteriol Itebant Muir 
Poth, wad Bacteriol. ¥. S74; Mann, “Physiological Histol 


Ay 





vis. 138. 

‘AOOLWTELATION.—Doliping, Brit. Med, Journ. (1907), ih 

Widal and Sieardy ieiepsees =, a 
Journ. (1807), 1504 (1808), 586. 


CHAPTER TV.—Pactraua ov Arm, Soin, Watnt Arrisnrrios 


Arm, Sor, axo Waren.—Potri, Zuche, f Hyg. Wi 15 vi 283. 
Wid, Ee fo Sticher wk Wid, 3XK. fie! Handbuch 


Mek. Off. Loot Gow. 

8) aEt Si (1898-09) 00) 418 aos a 467 xxix. (1800- 

Sidney Martin, bid xxvi. "(1896 xu. 
189N-99) 38% Horrocks, * 







1397, Health, Mass., 
ae a tg. bie the Bocterto el Bad ot ‘of Water Supplies, 

7 1906, ls, loal, und Walker, Journ. Hoy. San. Jnat. 

(2908), xxiv. 424. 
Awrinnerion =R, Koch, Aftéti. ad, k Ganithtsamte, 

Rschr. f. Hyp ix, 3%.’ Ritwhie, Frans, Poth. Soe. Commas 

Rideal, “' Disinfection and Disinfectants,” ata 1898, 





CHAPTER V.—Ranations oy Bacreata To Dreeisx, ero. 


Ax the observations on which this chapter is based are 
through the rest of the book, the seferencoe to thems will ‘be found a 
the different diseases, 


CHAPTER VL—IsriamMarony avo Scrrvnartye Coxprriowa. 






Ogston, Frit, Med. Journ. (1881), i. 269, Rosenbuoh, * Milkro- 
xganisman bei don Wandiofeetionskrankhelten dex Mansa, What: 
ny TAB. Pam, Fortschr. cd. Mad. 11886), Now. @ and. 22. We 
Watson Chey ‘Suppuration and Septic Diseases,” Edint 1880. 
Grawitz, Virchow's Archie, cxyi, 110; Dewtsche med. Welnsohr: (1889) 
No, 28 Steinauy Ziske. f, Hy. v., 618 (mierovccous 
“Die Actiologie der acuten’ Kiteru Leipzig, 1889. 
Dirckinck-Holmfeld, "' Rooherobes exporimontales eur In #1 
Muir, Jowrn, Path and Saeteriot. vii, 1615 
bs Paraohr.  aat, (ESE), No, 6, 
ky, Ztachr, 


a Potrw 
Balt 4185 xxiii, 142 iat ie Kooh, xxiii, ath TE Tilhbert, q 
Itpilzuntersuchung," Witreburg, 1886, Krause, Fortec, ie 
esi’ Now. 7 and 8.” Ribbort, Horley. d. Med. (1858), No. 1. 
and Houangon, Ann, ole I'Tnst. Pastewr, ingelahoim, 





BIBLIOGRAPHY oh 


eee Bohring, Centralbl, f. Bakteriol. «. Pucusitenk. 
inot et Maslin tn de med, (1894), 449. Orth and 
ina mel. (0585), 677. Netter, 
‘path. (1886), 106, Weichnelbatim, iFiew, mek 
ss) ii Kon 2839; Conrail 


Fcktes Deatache stad 7% 
Rebard, donde Efut. Pesteur v- a09." Fehletara, “Dis A 
des Eryaipels," Boslin, 1883, Welch, im. Mat. Journ. Sc. Se (1891), 4 
Uamoine, iim, de Pfu. Pasteur, is. te Kurth, deb. 


289, fH, it all 
Lame (1806). 985, 210. Bedok civ. de P Fas Pasteur, 3h 177, 


Serer tore enteritis), Johue He i, 15a, 
Hirsh, sh, Goutal ry Piru h Son Liban, bad 
regi Roy. Soe, Lona, Isle, a 


ay cari 
lise, Jowrm Beper. Med. vi. 917. — Schottmuller, 
Miinchen, ued. Webnsche. (1908), Goril Med. Ofleer 
Toeat Gor. Beard (1905), 358; Lancet (1905), ii, 1400. Andrews and 
Horder, Lancet (1908), ‘fi. Ruwdigor, Journ, Tajeci. Dis, ii. 785. 
Breradis Bull de Inat, Pasteur, i 
—Morux, Ann. sie U'Tuat, Pasteur (1890), x. gp ie 
‘sir “fa ‘Tew "s Gphtial Rape Toate int Taber 
¢, Trans, im. Sie. i fold, in 
erase Pe Krgebniveo der atlgem. Pathol. uw Path. Anat,” 1901 ; 
"pw ila odor AugandolTkce,” 1907 (ful reference) 
Tribonlet and Cayon, Mull, Soo. mak at, hop. oe 
Pet Coy a cs West ‘Wassermann, ‘and Malkoif, Berl. kein 
Weknsehr. ( tan Paine, Lancet (1900), fi, 801, 932 
fe Trane, Soe, Loved. (1909), Titi, 981 5 
alker, Brits Med, sFourn. (1009), i. 297. 
Shaw, Journ. Fath. and Bactersot. (1908), ix. 158 Benttlo, sd. ix. W725 
Journ. Mec. Research, xiv. 399; Journ. Exper, Med. ix. 186. Cole, 
Journ. Infect. Dis. i. 74. 


















CHAPTER VII. —Isruawxatony axp Serrvnatiyn Comprrtexs, 
Corrie: AcoTk Pxrevxowtas, Evromwro Craenno-Srixan 
Mexpvarris, 


Fricdliindor, Porter. d. Mat. & No, 22; ii, 287; Vivehow's Archie, 
Texxvii. 319. A. Fraenkel, 2tshr. Alin, Med. (1886), 401. Salvioti 
and Zasloin, Centratil. f. Wissensch. (1883), T2L. Zieh, wid. 

: ‘Tuxgonsen, Berl, kim, 
854), 272, 292, Senger, Aah. 

Weiolisetboum, Wien. svat. 
she. J. Ohrenh. (1885), Now. 
atv SR Gamaléis, Aan, 
ev, Aeon, met. stk Roa 
J. Hoy. xh 9. Be 
“Sanarell 









akteriot. wv, Pars 

40. Guarnieri, 

Krav ard Passini, 290i 
4 














a gue, Gaz a 91), 
wath x ie ors (3889) f 
7 Compt. rend. Mol, lexxvil 3, 


Gononunar,—Noiasor, Conéralhl. fe, med 
sche mak, Wohnschy. (1882), 279 ; (1891), 386. Bum, 
jams der gonorthoischon eaictbautes kro 
1 an od, 1487 | Anshan, mad, HF nach 
Nos. 60 and 61 Contrath,/, Oynd. (1 
801), No. 4.” Bockhart, Monatsh, 
No. 19. Stetosshnalter, ‘Hérk kiva, Wehnsehe, 
3 Verhandt. d. deutsche derma. Gesottsch. 
mo Blan. Min, Weta. 
ae is Ke at) sea, Fee aos Wien 
i, Charité. (8, an 
‘otteduat Wid. (18h, 48 Comnellsany nc “in J 
Finger, Ghon, and Schlageuhianfor, Arch. fe 
Lang, thi, (1802, 1007 5, Wem med 
Dee’ Vensrlache, Katarzh, deseen 
‘Thorapio,” Wiesbaden, 4903, Klein, Momntior. f- 
(1805), 33, Michnolis, Htschr, é lin, Med. xxix. 556. Heiman, 
196), a 1896), Dee. 12. ari | rane 
Inst. Preven. 40. Do Christmas, tun. de U 
809. “Nicolayaon, Centraldl, f, Bakteriol, 
iin Webuathe, (1808), 481. Wi 
nchen, mad. Welnachr. (1901), No. AG; Si: 


(1807), 1 
i Konig, feel, Rin, bt tr ae j 
dan se EF Postar (190) 3 lasts Dente mats 
Not. unto, Contras f. Bateriole Parouind, 
bit sir 965, Brome, A 


Calon 
(008 No. 13, Muller, 


4 


ae: 
# 


ais 
2 
ee 


4 


a 





vend. Soe. Biol (1902), Tamasconwaki. Zische. f- Hi. 
BA 49, p. 227. Davin, Jourw. of Mud. Reaserch (1003) bs 
Seraitets, — “onegntn Wien, ‘med. Wedwochr. (1888), No, 43, 
eat and Sohiite,” Leutsche mec. Wehnshr. (3885), No, 19. 

Fortschr. i Med. (tsk Xo 16. De Miele and Radio, 


faternas. dé se. red. | Sabouraud, Ann, da [Jnat, 

Rosier iar gat ph (884), 1 
fe Darina. . Syphe (883), No. 3: Van 

C tala. Pores tchnikoll and Rous, 


Ann. de F Fast. Pasteur, xvii-xix. Larear, Beri. Min, Wekmachr. (1903), 
VIS9 Nelecor, Deutsche med, Wohnsehr. (E904), 1269, 148}, Schaudinn 





Dentache med. We 

= 8B, Sehasitinn, Dewtrchs mal Wehnschr, (1006). eo ae 2 

ann, Berl. Elin, Webwschr. (1008), No 165 “Selectol Boas 
. Sm x," New Sydenham Svetety, 1906, Motchiaketf, 
Seraine nat 1905), 234. Lowaditi, iid (1908), aa hy 
mat, Ahik, (1908), 
1897, Shennan, Lancet (1906), 1. 663, 71 nan, rit. Med. Journ. 
(1908), i, 1000. Leraditi, ann. de inal, pe ir (1808), 41 





CHAPTER 1X. —Tvnencvroste. 


ag. Btenaks Untersuchungen and Brfabrangen im Gabel der Anatomie 
ig, 1843. Villeruin, ** De In virulence et de ta spéciticlté de 
Ta taberetioe Para, Leuk Cottier and Fresnel, “ Recprertinentalde 
Wiitersachungon tibor der Ubertragbarkeit der Tuberculove auf Thicre.” 
Gohahein, “Dic Tuheroaloso vom Standpankt ser Tnfectionslehsa,”” 
Varions Authors, *Dinoussion sur tm tubervuloss,” Dull, dem, 
Aeredl: ne, srs, xxi Armano!, ¢ Novinente med chip, 
Naples, 872. "Bsnngarten, “Lohirb. d._ path 
“Ta teberonlose et son hacillo,” Paris. 1599. 
Wedinachr. (1852), 221 
mint. Weknachr, (1890), No, 46a, ; (1891), Now 3 and 
Ballosh, Lanoet (1901), ti, 268. Nooard, Tho At al 
{trent-h London, 198, Cornet, Zackr. J. Hy 
) Ann. de’ Eins. Mostewr, i 19. reninon “chek. i 208, 
Sander, trek. f. Hy. 


























{23% ‘Coppon Jones, Centrally, J. Huktertak. 
ne York Mok Ras. 
‘Straus aud 









iden wud Hodenpyl,. 2 








(1802), 742, 966, ,, Lancet (1883), & S12 
Pawlowsky, Ann. de I'fnst. Pasteur, vi. 116. Maffuooi, “SalP azione 
Lr 








tenk. ‘xii 587. ith, 7 
Beyer. Med, iti, 451. Keoh Brie Med Journ (1902), iis 1895 
rmternetle uber es, Lerten nk, ae ine, Dirit, 


ph rend ‘Soc. de biok. 1, i tae Bae 
13, 129, Ravenel, Mot, Bull 

4902 Koch, Deutsche med, ¥chnachr. ‘aoa. Ne a8. 
Sento and Micsnner, Ztechr, f. Hyg. 61, 300. De Jong, Cenbradbl, 

Bokterdot. w, Purusitenk. 98 (Orig.), 146. Ravenel, Tele a Paseo 

Med, Ruttetin, 1902, Kossel, Weber, and Hours, 

taunt, Kaiser, Gendhtemte, Berlin, 190: 
1905. Salmon and Smith, * Tubereulosix,” U8. 

culture, Washingtow, 1904. Wolbach and Ernst, Journ. Me. 





LH 








x. 314. Interim Reports of te Royal Commission on 
Loudon, 1904, 1967, Wer al Douglas Pro. Ky, So, Lond, bax 
fournat, Now, 9, + May 15, 1906 


10. Wright, CHnioal 
Med. 


Ohir. Trans. Ixxxix. (1906). Wisight bel Rel Proc. Roy. Soe, 
Tonal. Iwxvii, 194, 





m1. 
faut baci Moen, Place etd. Webmaster C1808 378. 
@ Bakteriol. u. Parasizenk. xxv. 369; wei 513. Petri, 












mnt — nent. 
nach (A807), Now 1900), No. 16 Zur fe Tyo, xxvl, 90. 
Korn, Arch, i 87 5 Centra ddl. 





4 l. f. Bakteriol. w, 
Parnsstonk, xxix, 425,  Potet, " fo Tow "pacttion ites © Pasi 
philes,’" Paris, 1902" Abbott and Gildersloove, Pennayte, Mak. Bw 

Fat 1902, “Johne and Frothingham, Dentathe Stach. f. Thversned 898% 
498. M‘Fadyean, Journ. Compar, Path. xx. (1907), 48, 





CHAPTER X.—Lxrnosy, 


Hanuon, Ni 
2) x0. 2 





Largeritensh.. 1874 ; Pérchea'e Archiv, lexix. 
| Firchow's Festschr, (1899), iii, Bee papers, by: 





al 





Investigation Com 
fe pak Derm wat (1601) 5" 2a Wesauer, Roars 
rl I. > ‘eaouser, 

Pararitenk. it. 450;' M@nchen. med. We ae 





Pabteriat.” i 
passer Dass, Tears wd 98. Babes in *” Ei bet 

axis. 1, Journ. ) ¥. 88. Bat in nent eel 
UKeilcund Wossermuna's Hantoush der Poihayenen Afltroongsetena 





Comil and Alvarer, Arch. de phystol. worm, ef potb, (1895), rd serion, 
Wi, 11.” Paltanf and Kiselsberg, Fortschr. df. Mot. (1886), Nos 19, 20, 
Wolkowttseh, Central. fds nad. WFissons» 1886, Dittrich, Zehr 
Se Meith, vill, 251, Babos, Centraibl, f. Bakteriol. w. Parnsttent. 11, 

. Pawlowski, (Md, ix. 742; "Sur Métiologio et Is pathologie da 
Fhibowslieomo,” Berlin, 1501. Paltauf, Wien mod Welnachr. 1801}, 
Now, £4, 58;'(1892), Nos. 1, 2 Wildo, Semaine mit. (1896), 396 
Klewpérer anil Seheier, Zteckr, J. tiie, Met. xiv. Heft 1-2 Lanai, 
Centralil. f. Badtervol, ». Purastienk. Refer. xxxiy. 027.  Sehablowski, 
Wid, xxxvill, TH. 











CHAPTER XIL.—Actixomyoouts, rere, 


Bellingwr, Centrwtl fs, vant. Winenseh., 1877. J. Larael, Pirchow's 
Archi, Ixxiv, 15; Ixxvili. 421. Ponfiek, Breslow. oertsl, Ztscke., 1879 ; 





__ <a 


580 BIBLIOGRAPHY 
moe aon 





leilr. i 
Virwhow's ot i i Beitrigo sur Klinile der 
abe hom vt Ween, Yo Grasiger Siovurt and Mair, lin. Howp. 
alent 18 1894. Centrattl. §. Bakteriol a 


Parasitenk, xv. ti, tries Beltre me ‘iia, Chir, xiii, No. 8. Taw. 
lowsky and Maksutof, dum, de UInat. Pasteur, vil. S44 MNeukiroh, 
Ueber Stroh ), Strassburg, Doephe, Manchon. ined. W¢ 
1902. Sillurschmidt, Zuker. f Hyg. xxvii. 345. J. Homer Wei 
Publications of the Muxsochuseite General He 

Ailes. Streptotlerices.—Nooart, Ann, del 
Eppinger, Beitr. 2. path. twat. w. & ally. F 
Ostertay, “ Ergebnisse Saige Path.” 
Hyg. xxiv. 470, _ Berentnow, 
Flexner, Journ, Einper. Med. iii ean, 
(1900), 26. Birt and Leishman, owen. Of Hyg. th Tao! 
Ceniratbl. f. Bakteriol. . Faraxitent. szix. 691. Youlorton, Trans. 
Path, Soc, Louden (192), 56. M*Donald, Trans, AMed.-Olie. Soe, Balin, 
xxiii, 191. Norris and Larkins, Journ, Kaper. Med. y. 1%, Buttery 
field, Journ. Infect. Diseases, vi. 421. 

Manoa Diszase.—Carter “On Myootoma or the Fungus Disease of 
India,” London, Bassini, Ref. in Central f: Bakteriol, «, Parapitent, 
fy. 052. Lewis and Cunningham, 11th Ann. Hep. Son. Com, tncin, 
Kobner, Mortachy. d, Meal, (1888), No. 17. Kanthack, Journ. Fath, amd 
Sinctersot, i, 140. Boyow and Surveyor, Proc. Rony. See, Lowdom, 1808, 
Vandyke Carter, Zrans, Path. Soo Loudon, 1986. Vinowby 
129, Wright, J. H., Journ. Beper. Med, lis 431, 
i, Dermal. 1. Syph. 1xxi. 209, 
















t Bostent, il 
Opponhoim, Arch, 





CHAPTER XI. —Awritnax, 





Rollinger in Ziemssen’s “Cyclopedia of Modicinn.” Greenfield, 
“eMaliguant Pastole” it Gunn sa ee ree ret: 
1804, Pollenier, Vrétjvehr. f geriehtt. Med. vill. « Davi 
rel, Acad, d, ae. Wii. 220, 361, 296 ; Tix, 903. Koeh, Cohu 
4. Bftons. (A876), ii Heft 2 Milth. a, dk, 
aur, Compt. vend.’ dean. d. se. xi. 

Buchner, Pérehou's Archie, xcl. Chamberland, 
viii. 163. Chauves, Compt, rend, Aewd, d. 2€, xe 
Goaplowshiy Deir.’ = path Anat x vie Polk, vi at, 
swaltins day de. Unt. Pastewry i 617. Marshall Ward, Pris 
wuton, Fob, 1898. Potrusoh 

367. Wayl, Ztechr. f. Hyg. xi. 38 ing. iit. 
Ondorna, dréh. f. yp. xt 01. Rows, Ara CIne 
Hankin, Brit, Mei. Jowrn. (1889), li, 5 peney, iL 65, 
Hankin and Wesbrook, Ann. ole fet. Funtour, vi. oan Winey Marti 


Rep, Med, Off. Loe. Gort, Booed (1800-03), 285. ara a 



































BIBLIOGRAPHY 58 


i 599, Ti. Muir, Journ. paneer fights © Belaro, 
Iyiene € Sannita x rasttics, joa. ‘Sulla state 
Inerr bonchian. Potso, 1008 903 (wee 


ella aioe tela ron, 
Fey Yate 180) 1) ros Aes Sade Sober ole ond panes 


7 Centeasth. 
steak, (Orig.) x1. 241, Bail, eid. xxxili. 345, 610. Sanfollce, ist. 
xxxiii, 61. © anil ‘ail Garnier, Compt, rewk” Sr." Blok vid’ BSD. 
Torshor, Limeet (1906), i, 1906, 





CHAPTER XIV.—Trenow Frvisr #10. 


Eberth, irekot's cna A res 58; Ixxxii. 488. Koch, Mfith. 
od, &, Gsuthisamte, i 46, 80, is ck Jere 
Fath, u. Pharmutel, xin 2915 tects Booherich, Pertoatr. ad. 
1885), Nos. 16, 17. Emimerioh, trek. f. Hyg. ill. 201. Rodot and 
Are de md. oe # d'anat. path. iv. 317. siohee aay 





Huy 
12. hich Michi, Pra on vil. 
i. f. Bakteriol, , Porosilent, ix. 953, Pfuhl, 
ibid, i, 762. "trasehiy bit, vi, 660, ‘Hunter, Lancet (1901), 4. 618° 
Kitusato, Ztsobr. 7. Hyg. vil. 516. Chantomosse and Widal, Bult. sre. 
sey, No, 82; dans 0s iT kust, Paster, vi. 1555 vii. 

Clin, Faxon, Tem Nelaer, Bischr. 1. klin. Me 








Sohal 4 Hea. Pasteur, vii. 958. | Quincke and, Sth, 
Berks this Weinsc. (1801), 46%. "A. Prucukel, ‘Bh, f. Rin. Med, 
No. 10. Prorakel and ‘Sinimonds, id. (1880), No. 30: 


qu 
eating Seminius andi. (1890), No, 27. Grawits, Charite.d 
228 Kemer and oe Centratbl, 7. tin, Mul. (1857), No. 
i i iid, i, 408, KR Pfeilfor and 
foilfer, Deuteche wd. Wohmachr. 
os TIvak, Pastews, vi, 72) ; wil, 198, 968: 
Seed Froenkel, Lert, bike, Wobnsehr, (1500\, 211, 265: Brieger 
Kitasato, aud Wassormana, Ztachr, 7. Hyp. xit. 197. Widal, Semone 
exdl, (1896), 295, 303, Laut, 
Sept, 1803, Delépine, frit. Med Journ. (1897), £ O29, 96T ; Lancet, 
Dee. Deer I806, Remllnger ont Bebneider, nn, de Ena. astesr, xl, 55; 
a 





a 

















Per are 120, ig 
Tiers, (1897), © 286. Wrigat and Lamb, Lancet { 
mec (G00) tha 68 5. '(r000), 000) 58, 17, 

je ALOT ; sbi, (30025, ii, 61 ; Brit, Mod, Journ. (1000). 








AOL), i. 043, 771. Wright, and Leishman, sii. (1900), i, 
Ses aloo disonssion at the oie Lymtan, Brit. Met. Sours, 
(ion 1342 Sidney Martin, shed. (1898), i, 1560, 1644; ih. M1, 73, 
ham, Qruas. Foth. Soe. London (189%), xlix, 373, Macfadyen, 
Proc. Hoy See. Tandon, Bo ixxvii. 548. ML and Rowland, 
Cent Se Bakteriol, wu Parasitenk. (Orig) xxxiv. 618 780, 
Ghantetneess and Widal, ann. de (Just. Pasteur, vi. 705. Christopher, 
Brit. Mod, Jowrn, (1573), i. 71 Hemy, Ane. de Plast, Pasteur, xiv, 
705 Wyatt Johnson, Brit. Med Journ. (1807), L231; samt 
fh Hi. 1248. © Darhain, Lancet (1808), {. 154; iil “il. 44, 

















582 BIBLIOGRAPHY 
Earmin Bnihy'und econs ae Mod. Journ. (1999), & 198. Gonken, 


Soura, Path, awd Bacteriol iv. Castoll a ~ 
eee 
39. (Bacillus Buteriti = 
bericht, iv. 2495 nget, in Alle 


Wasserman, Hawibuch, vol, ii, (Peittacosi 
Kericht sil. 490. "(Bucllan ateritdie Spor 
Og. Loowk Gort. Board, xxv. 171 ; xxvii. 210. 
Brorentar Dysuxrkny,—Shiga, Centealbl, f. Bakteriol, w. 
xalii, 90; xxiv. S17, 870, 913, — Kruno,  Adimacker. 
(3000) ), BOT. Floxner, Bull, Johus Hopkins Hogp, (1900), xi. 9, 202 5 
Med. Fourm (1900), th. 217. Strong and, Musgrave, Jour, dns 
Mec. Aesoe, (1900), xxxv. 493. Vedder and Duval, Journ, Raper, Met. 
(1902), vi, 181, Ogata, ‘Onntratbl. f. Hakteriol, u, Parasitenk, xi, 2H. 
Soo variowts authors in Studies from the ‘Dotkefeiter Hastitute Medical 
Hesearch (1904), vol. iL Park, Collins, and Goodwin, Jowen. Mal. 
Research (1904), xi. G53, Hiss, ibid. (1905), aii. 1, Torrey, Jowrn. 
oer. ra £3908) ‘vii, 268, _ Weaver, Tunniolilfe, Heinemann, and 
Michael, Jew ‘haf Din i 0) "Doe Det Dypontoradaatoe 
aa, 1907, 


Sumuer Diannmaa.—Morgan, Brit, Met, Journ. (1908), £908; 
(2907), ii, 16, 





CHAPTER XV.—Divaranesa. 


Klebs, Verkandi. d. Cog, f, innere Mek (1809), ie Uiller, Mi 
a the ke Orndhtaanite, (884), 421. Roux an Ann; de PEt, 
Fastewr, ih. 620; Wi. 735 Av. 385. 
Wernach (1805), 241, 268, Sprauck, Cradrall all. Futh. a. 
i. No, 1. 

Botks, 1304. “Dnelng and Werulaker Beabr. fe "Hyg. xii, 10, 
Gontradbl. f. Badieriot. ou. Purasitent. i. 

mad, Wehnschr, (1888, pay 
Path, and Bactersol, iv. W3. Porors, sbid. ix, 18) Wr 1) 
Mat. whd 8, Journ. (1894), 220, 367.’ Kanthack and 8 

Pach, and facteril, ws 48. Kinin, rit Ao Journ (hod) le 
(1890), 4. 100, Rep, Made Of Loc. Govt Bouril (1800-91), 2195 (1E91- 
1. Guinochet, Compt. rend, Sic, de Biot. (1802), 480. 

, Ann. do Bust. Pasteur, vil. 60% Cartwright Wood, Lancet 













), 1. 980, 1078 ; di. 1 di) istontan Lectures,” 
y “0, Mat. Off. Loe. Cont. 
i We. mot We 
pes a 
chr (1893), Noa. 21, 22, 2% 
hichto der Diphtheria” Leipelge 1809; Sige © atiol. 
t, Krankh,,” Leipaig, 1808 5 " Bokampfang der Tufeotions 


Leipeig, 1894. “Ehrlich and Wassermoann, Zisehr. Jc Mays. 
i, 28 Ehrlich and Kowwl, did. xvi. 486. helio, Kossel, aml 

Wassermann, Deutsche med, IFchnschr. (S94), 363, Funck, #tseler, 
Hyp xvi 461. Prochaska, 3M eid, xxi 

thd. xxiv. 443. 1, Pasteur, 
Balomonsen aud Madara, (063, rit. Meth, Journ. 
(1898), fi, $93 5 Zep, Mere “dsyl. Métin, Ann. de 
and Todd, 
















id. 
Elnst.’ Pasteur, xit ison, tid. siti, 568, S01. Dean 





BIBLIOGRAPHY 583 


onl A oi ii, 104. Cobbett, ibid. i, 486, Ce antag tel ig 134 
258 5 vi, 286, Leal rid, 5 134, 
(1903), No. 25. Berl. klin. bic eo 
Yonee’ Wed ieasseck (1004 1 478, Morgunrotbs Hache 

177. Boltou, Lencet fisosk i ut ‘Smith, Se 
Besearoh, (1908), sii, 941, ott, Journ. of yy. v. 223, 





CHAPTER XVL—Txranve, ere. 


Neoafer, “ Meltnige rar Aeiologe do Wundetartkrampfes,” Iuaug. 
Roseubach, Arch. fe Alin, Chir. xxsiv. 00, 
Sach sed bat Gior. dy r. Accouk ah med a Torino, 1934. Kit 


cry, x ee au 286, ao pel We 
we tis (triad Aun, de 
eae wind ot 








in, Hep, Mod. Off deve. Govt, Board (1800-94), 40 
be ie Bebierieh w, rk a, 318. 





inst, Bastewr, 

Bilk 166, | Marle and Moras, lun. de Fnit, Puen Es xvi. 18 
Prve. vy. Sos. London, Lexii, 28; Ared. f. exper. 

e Pharmakl, Deipsign alls, 960. Roux end Botrwl, dwar de 

Efast, Pasteur, Paris, i203); Henderson Sinith, Joven. Afys. ei XS, 
Kite, ma ref, ‘in Centratbi. 7.’ Buktoriot, u. Parasitend. Jena, eferate, 


“Slasioans Givewh.—Pasteur, Dull, donk. de mal, 1861, 1887. 
Koob, Mitsh, &, Gendhteomte, i. G4. Kitt, Jahres. de b, Contr, 

















0%, ante, ri f Hye 1. Tid. Rows and " Ghamtvrlat, 
hi 


th ir, rin and Compt. rend. Src, 
rae diok, 87D, sr ms "vol iv. 408. ae Pracukel, Ztackr, 
Banfelice, iid, xiv, 339, 
de 0 f Pastner, xiv, 209,590, 

Bacive Borvuixes —v. Krmengem, Centraitt f. Bableriad a. 
Parasitent. xix. 443; Ztechr. f. Hyy xxvi. 1, Kemjner, Wl. xvi 
folie (Remipesr and Schetilowsky, iid. exvil, 912. Nempner and 
Pollsok, med. Woenschr. (1807), No. 32 Drioger and Ki 
se (2607), Na. 95. Marincsco, Compt, rene. Soe. de bol, (180 

Sehneidemihl, Cen J Bakteriol. 0, Porasitenk, xxiv. 


















QUARTER-Rvii.—Ner Novard and Leolainoho, ** Tox maladies exbera- 
bienues des animaux,” Paris, 1896. Arioing. Cornevin, et ‘Thomas 
eerie hea — da beef,” , 1887. Nocard and Rows, 


ans, de stew, i, it 40. See also Journ. 
cd Therap, ii, 


(eiLtes Anoonxns Careeiarve.—-Wolch wd Nuttall, Jil, Jabas 
MapRine Hoxp. (189%, 31. Woich and Flexner, Journ, Exper. Afed. & Os 

















534 BIBLIOGRAPHY 


K, Frownkel, Ceatroley. f Bobteriol, i, Parasites. xiii, 18, Durham, 
uti, ae Hophins Hosp, (1887), 6 Norris, tm. Joura. Mal. Se 
oxvii, 172. 





CHAPTER XVIL—Cuounna. 


Kool, Rep. of Lat Cholera Conference, 1881 («. ‘* Moropurasites inn 
Disease,” New Sjplenam Sve, 1880), Nikati anil Rictech, Compt rena 
ond, de ve, xcix. 998, 1146, Bonk, Ann. de I'Enet Pasteur, i 507. 
Pottonkofor, Afiinchen. med, Wehnechr, (1892), No. 40 5 (1804), Ne 10, 
Sawtschenko, Cleatralbl. f. Naktertol. u. Porasitenk. xi. $93. Piatt 
Bache. 4. Hy. x1. 398 Kalle sid. xvi. 29. Vou and Rolle, tad 

17. Wassermmaun, iid,’ xiv. 39. Soberubeim, Wit. xix, 485. 














avi 
Metchnikot!, Anu, de @Znst, Pustewr, vil, 400, 602; vill. 529, 
Fracukel aud Soberaleim, 2 7. Duar, str. ad de 









Gmdhtaonte. tx. 379. Pleilfor and Wassormann, Zisehr. J. Hay, aie 
Wosbrook, dn. del dual, Pasteur, vii. 318. Scholl, Ber! 
(1800), No.1, Griberand Wonee, Avch.f. Hyp, xv. 961, Cunedogham, 
Seient. Mew, Med. Off. Eutin, 1890 und 1804. Musppe, Dewtache wed. 
Wehnschr. (1889), No. ilomporor, anid. ($804), 456 > Bert, ktine 
Webnachr. (1892), 969. id. {i02), 1071, 

mat. Wehnsahr. 
Centrale, Bakteriol Parattenky 
Gintralbl 7. Bodteriol, w. Paranitenk. xr 401. Dieudound, at. xiv. 
825, Colifand Bantori, (bid. xv. 28% Noisser, ibid, xiv. 666, Sanarelli, 
stn. de Cinst, Pasteur, vi. 693. Lvauoll, Zushr. f. Hyg. x¥. 486, 
Tnvgelly Sid, avi. 280, “Pful, ibid. x 510, Rurapel, Deutache wo 
j, 180. Klein, ep. Mot. Of. Lor. Govt. Bourd, 1898 
“ Mioro-organiama and Disease,” Lendov, he iMaltkine, Bre Made 
Journ. (1895), Si. 1541 ; Indian’ Med. Gas.’ (1895), No. 1; Anti-cholers 
Inoculation,” Kep. San dia, Caloutta, (895. Peltor im 
Filiggs, "Div Miksoorgeniaiwen,” Ard’ od,. 1890; Gamaléia, dna de 
Flust. Puatour, ii, 482, 662. Achani and Tensande, Semaine sel, 
(1897), 161. Rumpf, “ Dio Cholers Anlatica und Nustras,"" Jena, 1803, 
Kraus and Pribram, Ceafraddt. f. Bukieriol. xi. (Orig), 16 156. a 
and Prantichoff, bid. 877, 480. A. Mactadyon, ibid ‘xlit. (Orig, 

38, Gotschiich, Setenttte Ieapx. Senilory,, Mariitng, and Guavenion 
Counetl of Kyypt, Alexandria, 1905, 1906. Yor discussion, vice Suyeplen 
fentrulbt. f, Bakteriol, Referate, xxxviti, 64. Dunbar, Ber 


































1 


























she we, Webuser, 












IxrLuRNes.—PI Kitasato, and Cano 
xviil, 28 and Zvit, Med. Journ. (1992), 4 128. Babes, Zeutiche wand, 
Weinschr. xviii. 118, Pioilfer and Heck 465. Peahh, 
Centraldi, f. Buktes u, Parasitonk, xi, 897 fap, Med. Of. 





Meitfer, tschr. #. Hyg. silk $67 Huber 
i, Kruse, Detiteche wad. Webnache, (1898), 51% 


Lavi. Govt. Boowrd (189 
=f. Hyg. xv. 
















Policko, Bort. clin. Wehnsehr. (1804), 621. Pfuhi and Walker, Dewtache 
set. Wehnschr. (1890), $2, 106, Cantani, Zteie. . Hy. xxiil, 256, 
Ptaht, Zise f Hyg. xxvii 112. Wassermann, Deutsche med. Welnacker, 






1900), No. 28 Clemens, Munchen, met, Weknachr, (1900), No, 27. 





BIBLIOGRAPHY 585, 


Ww) Journ, Med. Asm, ¥ 1903. Neiever, Deulache reed. 
Wansst {1903}, No, 26. ‘Aiartaths Ththe f rs. (104), sri 289%. 
W Arch f. blin Mad. 70. 








he Se 
He B10. Cairns, Loncet (1901), L ‘iris, Moutenogro, he: Bobonie 
" Landon, 1900. Netter, Fe Pe ee a " Paris, 1900, 
Hide der Destichen Pest Routnimlny Devicke med, Weiner 1807) 
Koa. 17,19, 31,82. «' Report of the Indian Plague Commission (1998- 
99)," Tendon, 1900-1901. Also numerous papers in the Lancet and 


Severn, 1897-1901, mul lasgow epidemic seo ibid. 00h 
di. ** Reports oa Plague Tireigtionte U in Tt Sours. Hyg. (1908), 
wh 422 ; (1907), vii. 3: 


4x0 Fev en,—Obormeler, let, foals 8 di. med. Wissenseh, 
(187) 55 and Het. din, Welnwchr, (1879), No. 35. Muneh, 
Gemtratal, fs. med, Wisseusch,, 1876, Kosh, Deutsch 

ky, Lieuteohes Arch. J. 

Trans, London (1 
i |. Motchuikolf, sid. cix. 
“Inst, Poxtcwr, ¥. 645, Latby Solent, Mem. Mod. Of. 

Sawtschenko and Melkiol, 
Centrothi, f. Bakteriol. xxi. 179. Karlinslci, 

7. Bakteriol. (1 686. Gabritschowsky, Ztsckr 
f, Bis, Med. 1905), Bd, 56, ppenheiwer, Flournoy, Jewrn, 

: Wid. 291. Zettuow, diechr. fe 

906. 













ce Koch, ogee wert. Wehnathr , 1005 ; Berl. bi. lcouschy, 1986, 
son rit, Mad. Journ. (1905), 71a. Broull and xia _ 
608, ‘reall 1 Lanes (1906), i. 1800 Lavadith, Glmyst. ren 


is Vevun.—Druco, Proditioner, xxxin. 100) sh 241) nw de 

201.’ Bruce, Hughes, and Westcott, Zirit. Aled. 
on, de Inst. Pastewr, wil. 628; Lancet 
mpl, Brit. Mat, Journ. (1897), 1. 1214. 
Hd. (1897), 1. VL; Lanert (897), i. 636. Welch, 
Ganon, ibid. (1899), 1. 683. Durhen, J 
Path, and Bacteriol. +, 877. Broce in Davidwou's “ Hygiene and 
Disossos of Warm Climates," Edinburgh and London, 1893, Birt aad 
Lamb, Lancet (1899), ii, 701, Brunner, Hiem aim Wobnsedr. (1900), 
Wo. 7. Bruce, Journ. hoy. hemy Med. Corps (1904), if. 487, 731; (1907), 
with, Hortooks, Pros. Koy. Soc. London, Setios B (1006), Ixxel, 















Gena fre” gra Med Corp). Ln kyr ta Kala od 
1006, 


‘erLow Fern Ave Pub. Health Ass 178. 
Sanatsll tans de Theat acl ei. 08, S87 B,D 


sou, art. in Clifford Allbutt’x “System of Medicine,” wal. 
yt tagtn e Ae 
3. id Car ‘News, Apeil 


1899. of Hyg. fi references). 
Thompaon- Yates 1902), ir. 435, Gorgas, Lancet, 
300 Bo roar Math ab" Manboutt aaltnbenls seal Steed 
fam. ele Final. Pasteur, axel, 6655 
; (1904), sive SI. 
3. Eeed,, Ourrall, Agtsaonts, lasehtsJros, dear: 4 
oh Journ Amer. Met, Ass., Yev. 1901. Carroll, New York Med. 
Journ., Feb. 1004; Amer. Medicine (1906), xi. 983. 








CHAPTER XEX.—Daxwxin 


Yor carly inoculation methods (6.9. against eres chicken yea 
ete.), see Microparasites in Disease,” New Syd. Soe. 1886. 1360, Dugutd 
and Banderon, Journ. Koy. Agric. Soe. (1880), 207. “Greenfield, aiid 

(1880), 73 ; Broo. Rope. She j Hore ‘June, 1440.) “Tose 
end. “ond. od. ne. xoi. 136, Halting, Brit. Mod Journ. (1801), i. 
1278, “Kivin, si. (1893), 1. 682, 639, 651. Klompervr, Arch. f- exper. 
TX: ta Phdenakol eek 86 Bichnwe, Aachen. tad. IPA 
(1889), 449, 480, Ehrlich, Deutsche med. Wehmschr. ‘apy, 976, 1218. 
- Meliter, Ztsher. f. Hy. xvi. Lp xe, 106 Pieilfer and Kolle, iat. 
xxi 203. 'Bombot, dn. de UZnst. F 4 
kolf, Virchou's Avehiv, xovi. ¥ 
Ann, de UTast. Pasteur, iti, 289; iv. ri 
289; vit, 402; vil, vill. 429; ix. 48%, Calmetéay, dann, 
de TInt, Pasteur, vill, 276: xi. 95. Fraser, Pro, Soy. See. Bilin Xx, 
4, Marmorek, tun. de U'Tunt. Pustewr, ix, 69%, Motchnikoff, Roux, 
d Twurelli-Salimbeni, sid. x. 267. Charrin and Roger, vend. 
‘Soe. de biok. (188 
(1398), Mareh. 





















7), 667. Gviiber and Durham, Mianchon. mek. Wehnacher. 
Darks, Sours. Pash. ange =e 13, Cast: 







eoturei 
sion on 










iets ‘Path, 
Deutsche ried 


Soc, Pees 
aonechr, (867) 
Nicholle, 














umenthal, Deutsche wid. re 
Ransom, ‘ébid. xxiv. 117, Meade Bolton, Journ, Beper. 
. B43, Fragor, T. R, Brit. Mad, Pearcy eri B41, 
, i. 957 2 (1896), Hi, 910; (1897), H, 128 SOR Cooma, 
de Ulnat. Pasteur, vi. 160, 601 ii. 2755 ix. W295 x. OTS 

ii, 343. .C. J. Martin, Journ, Physiol. xx. 301; Pree Rey Soe 














Ete abs 
Seblussbet cogs rlioh and Mi Bert, ling 
ees KERVie A ay P= Se 463, 6815 ist xxviii: uh, 
* (1897), fw. J iin w i, 1890) Morgen- 
: peo ri nen ah aah Bs Trams. 





Sir 00 30.8, Gongon, ilud. xv, 222’ Sawtsobonko, 
itehte, Journ of Hyp. sh 215, 01, a2 (with ful 
Pathol Infecton,” inci -Aubute', 

od 1006, ol Notclanikot 


Hi, pik pe i 
” 1901 ‘echal 
1001, No. '18., Von oh (18 
1458 ; (1900), 677, 97%. Ehrlich, Caleta Stadten oo Zoumn 
Cent oberg and. Yolk, 
i Whoa and Vol 
Biche. J. yy. stise i ga Blake, Jowrn, Path. ond Bacteriol, 
Blood Tnmvnily art Bloud Relationship, 


sora Dinge and Leolef, La en Neil ii igen 
aD Peoe, 






def inat, Pasteur, 1902, 108. Wright asl 
367 ; exit, 128 ; Ixxty. 147. 
aatal Atkin, (ad, Lexie. 
sufeld and Riznpau, Dewtache 
Ruediger, Journ Infect. Diseases, U 
rans. Poth. Soe Lore 1008. ‘Mute asd Martie, Brsk, Mat 
Toure: 1906, ti; Pree, Sey. Soe, Londen, xxix. 187, 















Arrmxprx A,—SMattrox, 


Jonner, “An Inquiry into the Cause and Rflvote of the Vasioia 
Veorine,"' London, 1r08. Creighton, art. ** Vaccination” iu ney. Jrit., 







hed. “Crookshank, *' Bacteriology and Infoctive Diswasos” M'Vail, 
“Vaccination Vindieated,” Chauvean, Yi ) wceine 
‘et variole, nourelle ctade expeérin: ‘ideutité de 


‘oot denx affections,” Paris, 1866, 
(180293), 991 5 (1999-04), 498, 
631; Journ. Path, and ‘Bacteriol, ii. 407; 
Hiyama of Medici, vol. 1 Plo, “Die Protea al Krank 
» Jona, 801, “Rulfer, Brit, Met, Jowrn, (184), June 30. 
Béslern Chabon, and Ménard, alin. de fost, Paster, x. 1's xii, 837. 
““Vaocination,” London, 1899. Calmette wed Geerin, fn 
We Cini. Pasteur, xy. 61. Guarnieri, Central. J. Bakteriol, 
fiend. xvi, 200.' Ewing, Journ. Med. Sesenreh, xily 233, ewe 


_ 








588 BIBLIOGRAPHY 


Arh, a, & haieerl, Gevindbettonmte, xxii, 608 ; xaisi. 126. Waslalowalely 
Bef f Hyg. xxxvili, 212, ‘Douhoff piel Bie. Wednseheft. ¥ 
pI Carind Coutralbi. f. Bakteriol. u. Parasitest. (Orig.)xx3ix, 


Arvesnix B.—Typnornonia, 


Pasteur, Compt, rend. Aond, ol. ac. acti, 1260; xev. 1187; xoviil 457. 
12295 ol. 765; cll. 459, 688; oli 777. Schaller, Ann. de Mnat, Paatewr, 
AL aid, ° Fleming, Zrns 7th Jnternat, Cong. Alyy. anal Deg, ite 6: 
Helian, As de Coat, Patewr i 274 ; i 18.” aos aud Lapa si 
iii, 484.) “Nosand and Roux, cud, 4. 87; 478. 
Brosabettini, Cenivatbl. f, Bakteriol. . Forwsitent, xx, ‘14; xxi 208, 
Meuimo, sist, xx. 2005 xxi. O67. Frantsius, sid xxllie 7 
G1, Romlinger, Asn, de nal, Baslewr, xvii. 88 xix. 
$28, Nori, Zehipt faa. w Infectinaeranthy si, 6075 XUV. BID. 
Williams wtid Lowson, Jowen. Linge Dis. ite 46% 














Avreypix C.—Maranian Fuyen 

Laveran, Hull, doa. de mdf, (1880) aér. ii, vol. ix. 1046; “Traitédes: 
fiivree palustres,” Paris, 1881; "Do paludisme ot de on Leaabosealen © 
Paris, 1901. Marchiafava and Celli, Fortechr, d. Mek, 1838 aud 2 
also in Virchow's Featechrift. Golgi, dreh. per le ec. mad, 1896 and 1880 
Fortachy, &, Mad. (188), No.3; £tavkr. f. Hyg, x. 186; Dewteche mat 
Welmschr. (1882), 663, 085, 
xalx. No. 18, James, hil. xx3i 
(1688), Nos. Onlur, vu 
Grane: and Felotti, Biforma mesh, 



























i Soc. Pitocelpivies, xin wily 
1500), li, No. 60, Canalis, Montauk a 
Med, (1800), Nos. 4,0. Danilewsky, Ann. de Ulnat. Pasteur, 2h 76K 
© Parasites of Malatial Fevers,’ Syi. Soe, 1891 (Monographs by 
Marvhiafava and Bignami, aod hy Mansoberg, with Bibiogsphy 
Manson, Brit, Met, Journ, (1894) |. 1262, 1907 ; Lancet (1895), 1h. 302 5 
Beit, Mot. Journ. (1808), it, 819; Koch, Berk, tin, Wohmsehr. (1899), 
60. Rows, Indian Med. Gaz. xxxiii, 14, 133, 40), 448 Nuttall, 
Centraibl. f. Bak Parositenk. xxv. 877, 003) Xavi, 140] xEviie 
193, 218, 260, 428 (with full Literature). ‘Manson, Lancet (1900), 
i. 1417; (1900) ti. 151. Gray, Ait. Mee Journ. (1909) 1 2 
Letshman, tid. (1901), 1, 635; i. 757, Dantol, iid, (1903), b 
alli, did. (1902), i, 1080, Nuttall and Shigloy, Journ. of i 
469, 462 (with literatu: Ross, Vuture, Ix. 622; " Mosquito 
sud how to organise them,” London, 1902 Celli, Malaria,” frat, 
Kyre, London, 1900. Lankester, Brit, Mat. Journ, (1002), i, 
Kiwing, Jowrn.’ Riper, Med, ¥, 429; vi, 119. Sohwwdinn, Arte, Gnas 
Raiser’. Geruwthettsumie, xix; Argutinty Archiv méeroaup. Adame. 
Ixi. 331. Rage in Kolle and Wassermann's Homullnich ity 
Mikroorganismen, Bryinsungsband, 1907 (full literature): 
ws, Lancet (1903), i. 80, Minchin, **The Sporzoa,” Lendony 1909. 
Stophous, art. Blackwater Fever," in Allbutt's "System of Mi 
Vol. th. pt ik, London, 1907. 


eh al 












































—= 


BIBLIOGRAPHY 589 


AvpENpix D.—DysENTERY. 


Lisch, Virchow's Archiv, Ixv. 196. Cunningham, Quart. Journ. 
Micr. Sc., N.S. xxi, 234. Kartulis, Virchow's Archit, ev. 118 ; Centralbl. 
7, Bakteriol. u. Parasitenk. ii, 745; ix. 365. Koch, Arb, a. d. k. 
Gsndhtsamte. iti. 65. Councilman and Lafleur, Johns’ Hopkins Hosp. 
Rep. (1891), ii. 395.’ Maggiora, Centralbl. 7. Bukteriol. x. Parasitenk. 
xi. 173. Ogata, ibid. xi, 264. ‘Schuberg, sid. xiii, 598, 701. Quincke 
and Roos, Berl. Klin, Weknschr. (1893), 1089. Kruse and Pasquale, 
Ztschr. f. Hyg. xvi. i.’ Ciechanowski Centralbl. f. Bakteriol. 
u. Parasitenk. xxiii, 445. Howard and Hoover, Ai. Journ, Med. Se. 
(1897), cxiv. 150, 263. Harris, Firchiac's Archiv, clxvi. 67. Schaudinn, 
Arbeit. ans d. Raiserl, Gsndhtsamte. (1903), xix. 547. Lesage, Ann. 
de Inst. Pasteur (1905), xix. 9. Kartulis in Kolle and Wassermann's 
Handbuch a. pathog. Mikroorganismen, Erginzungsband, 1906 ; Cen- 
tralbl. f. Bakteriol. (Originale) 1904, xxxvii, 527. 





















Arrespix E.—TRYPANosoMIASIs, ETC. 


Gexenat.—Laveran and Mesnil, Trypanosomes et trypanosomiasis, 
Paris, Masson, 1904, Minchin, iu Clifford Allbutt's “System of Medicine,” 
2nd ed. vol. ii, pt. ii. p. 9 London, Macmillan, 1907. Schaudinn, 
‘Arbeit. w. d. kuiserl. Gesunudheitsunte, xx. 387. Mense, Handhuch der 
Tropenkrankheiten, Leipzig, 1906, Barth. Novy and MacNeal, Journ. 
Inf, Dis. ii, 256. ‘Taishan, Journ. Hug. iv. 434. 

SuRErING SickNess.—Mott, Reports of the Sleeping Sickness Com- 
mission of the Royal Socicty, pt, vii, No. 13, London, Bale, Sons and 
Danielsson, 1906, Dutton and Todd, Brit. Mel. Journ, (1903), i. 
Dutton and Todd, Thompson-Yates Lab, Rep. v. pt. ii. i. v. phe 
Dutton, Todd, and Christy, iid. vi. pt. i. p. 1. Manson and Daniels, 
(1903), i. 1249. Idem, ibid. (19 Low and Mott, sid. (1904), 
1000. ' Bettencourt, Kopke, Re: d Mendes, sed. (1903), i. 
Gastelani, Zepurts of the, Sleping Sickness Comunision of the Royat 
Society, No. 1, London, “Harrison and Sons, 1903,” Brnee and 
eo bal! (1903), Nor dy te te, Truery Nalarro, and Greig 

1 (1003), No. 4, viii. 3. Greig and Gray, ibid. (1905), No. 6, ii, 
3. Leishman, Journ. Hyy. iv. 484. Minchin, Gray, and Tulloch, 
Repurts uf the’ Nleping Sickness Commission of the Kagal Sweiely, No. 
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Med. Joncn. (103:, ii. 1219, 1461, See Discussions at British Medieal 
Association, Srit. Med Journ, (1903), On, Thomas, 


monte da em vi yt HY 
Med. sdonen, (103), 4 


“aud ed. vol. ii. | 
ch der Tropenkeankheiten, iii 















































Hew 





















Mac . Tem, Mense, Hawll 
591, Leip , Barth, 1908, Leishman amd Statham, Journ. af 
Ariny Med. Corps, iv. 821. Donovan, frit, Mad. dowrn, Vis), 


Idem, Bret, Med. Journ, 
ine, Ixdvii, 281. Bentley 





Juurn, Mier, Sur, xl 


Roge 
4) Pro, Row. 


eae ii, G4 Id 








590 BIBLIOGRAPHY 


Brit, Med, Journ. (1904), i. 658 5 did, (1906), 1. 706. | Christophers, 
Scientif. Mem. by Off. of the Med. and Sun. Dept. of the Govt. of India, 
Nos. 8, 11, 15. Ross, Brit. Med. Journ. (1903), ii, 1401. See discussion 
at Brit. Mod. Assoc., Brit. Med. Journ, (1904), ii. 642. 

Devut Sone.—Wright, J. H., Journ, Med. Research, x. 472. 

Prnortasmosts.—See Minchin, loc. eit. supra. Koch, Deutsche. med. 
Weknschrft, (1905), No. 47 ; Ztschrft. f. Hyg. u. Infektionskrankh. liv. 
Nuttall, Journ, Hyg. iv. 219. Nuttall and Graham-Smith, iid. v. 
237; vi 586. 











INDEX 


— 


INDEX 





Abrin, 169 Agelutinins, primary (homologous), 
immunity against, 464, 469 342 
seconilary (heterologous), 342 
Agglutinogen, 486 
in dysentery, 540 Agglutinoids, 486 
Absolute aleohol, fixing by, 88 Aggressing, 164 
‘Acid-fast bacilli, 239, 252 | Air, bacteria in, 126 
stain for, 100 | exainination of, for bacteria, 126 
Acid formation, observation of, 44,77 ' Albumose of anthrax, immunity by, 
Acquired immunity in man, 456 312 
i Albumoses, 1 
in diphtheria, 363, 
Alcohols, higher, fermentation of, 75 
tivation of, 292 Aleppo boil, 568 
jeculation with, 296 Alexines, 478, 500 
varieties of, 204 , Amboceptors, 480, 491 
| Amoebic dysentery, 587 
Amobule of malaria, 523 
nosis of, 296 | Anaerobes, 17 
ns in, 290 cultures of, 60 
separation of, 57 
toxins of, 60 
Anaerobic Esmarch’s tubes, 60 
‘Anaerobic fermentation tubes, 60 
‘Anaerobic plate cultures, Bulloch’s 
ial fevers, 529 apparatus for, 58 
ia (r, also Culture mestin), 35 leprosy, 269 
ration by, 55 , slehydrating by, 93 
sdntinable substance, 486 ater, 98 
Azglutination by sera, 485 Aniline stains, list of, 94 
Japsing fever, 442 ‘Animals, autopsies on, 123 
thorls, 109 inoculation of, 120 


































of ls, mallei, 282. Anthrax, 300 

of b. typhosus, ete, 337 antiserum, 315 

of cholera vibrio, 412 | bacittus, 361 

of m. melitensis, 450 biology of, 304 

of plague bacillus, 437 cultivation of, 302 
of red blood corpuscles, 481, 485 inoculation with, 308 
theories regarding, 485 toxins of, 312 


38 593 


Bo 


Anthnx, diagnosis of, 817 } 
i aninanls, 106 
in man, 309 
protective inoculation, 314 
spread of 18 
Antiabrin, 470 
Anti-anthrax saruxm, 915 
‘Anti-bactorial eura, 476 
properties of, 477 
Anti-cholera vaccination, 412 
Antieiiphtheritic serum, 467 
Antikticpen, 487 
‘Antirpiaguo indentation, 488 
“Anti-plague sera, 436 
Antipmenmococeie sarum, 210 
Antimabic serum, 619 
‘Antl-ricin, 470 
Antiseptics, 141 
actions of, U4 
dtandardisation of, 148 
testing of, 142 
Antisera, therapeutic 
488 
Antistreptocoools rerum, 489 
Antitetanic serum, 384 
preparation of, 467 ef so 
Antitoxie action, nature of, 470 
Dodios in normal tisueg, 474 
ser, Use of, 469 
wwram, 467 
cholera, 411 
standardleation of, 468 
al natare of, 








action of, 








origin of, 474 
Antitnbercular sernm, 264 


Antityphold serum, 844 


Appendicitus, 18 





Arthroxpores, question of ooourrence | 


of, 7 

Axtificial feomnnity, variation of, 
487 et sey. 

Attenuation of riralenos, 457 

Autoclave, 29 


‘Autolynis of bacteria, 102 
‘Autopuies on aniwale, 
Avian taberonloais, 360 





Bacilli, acid-fast, 289, 262 





aerogenes capwalat 
anthracis, 201 
hotulinns, 3! 


188, 307 








INDEX 


|} Bacillus coli communi lewione cxtien 


hy, 18S ef seg. 
cherieters of 324 
ccotapatison with b, typihonus, $2 
coll ia soil, 188, 184 
pathogenicity of, 888 
Aiphtherix, 358 
dpsertoriay Rhign-Florner, 246 
ontaritidis (Gaertner), 36, 
enteritidis sporogenes, 350 
in soil, 139, 136 
of glanders, 277 
icteroides, 482 
of bnflowuns, 420 
Koeh-Woels, 191 
notin aerogerien, 180 
Jacoumtus, 193 
of leprosy, 260 
of malignant cnferna, 300 
Muller's, 192 
mycoides in soil, 132 
onaram, 285 
yarstyphorus, 386 
of plage, 426 
poeumonise, 190 
prondo-diphthoriour, 166 
of prittacosis, 337 
pyoeyanens, 180 
agglntination of, $85 
occurance of, 185 
nyogenes fortidus, 174 
of quarter-evil, 96 
of eaincecleronia, WBA 
of wmogmn, 254 
of natt sore, 227 
subnilis, G7 
of ayplilin, 229 
tetoni, 
of Timothy grass, 268 
of tulwrele, 297 
of typhoid S19 
Aiflorentiation from b. eal, 34 
of seronis, 867 
Bacteria, action of dead, 104 
aarobho (et Ateobea); 17, 
anaerotde (e Anaerobes), 17 
biology of, 18 
coapaulated, 3 
chemlvat nctlon of, 1 
composition of, 8 
clansification af 11 
cultivation of, 25 
oath of, 141 
edfecta of light on, 19 





























Bacteria, food supply of, 16 


igher, 14 
Jower, 11 = 
mori " : mations 0 2 


saprophytic, 2) 

mparation of, 51 

species of, 23 

sors Semation bs (alo Ere 


stractrire of, 3 
solphuroontaining, 
temperature of growth of, 18 
foxtns of, 16 
variality among, 23 
virulenee of, 150, 461 
Bacterial fermente, 22, 16S 
pigment, 10 
preetoplaam, structure of, & 
teraktaent of sewage, 139 
Bactericidal powers of rerum, 
Bunter gs u 
tingnoia, 118 
exataination of discharges, 118 
Heer wort agar, 44 
08, 16 
tig Ou Ieamaunity, 385, 408 
Bieralt media, 43 
Bismarok-beown, 96 
Bisckeg, 306 
Bluckwater Sever, 534 
Blastophares (materia) SrA 
Mood-sear (¢. alo Cotture mits) 





Blood, examination of, 38, 58 

‘im ronlsrial fewer, 521 

tn relapsing fever, 438 

serum, coagulated, a retin, 40 
Bone-marrow in lenooeytosis, 156 
Wordet’s phenomencn, 477 
Botalism, bacillus of, 203 
BoaiTton (e. also Culinre media), 32 
Rovine tebervalonis, 248 





595 
Bubonie pest, 429 
Bochnar on alnxinen, 
Bulloch’'s apparatus for annercble 
ccultnrr, 68 


Butechli on bacterial structare, 9 
Butter baeilll, noid fant, 283 


Calmotse, 435, 48% 469, 472 
Canon on influenza, 420 
Cantani on influenza, 424 
Capaldi sind Preskaner, media of, $98 
Capeullas, staining of, 102 
Carbalfoohsin, 
-taothylene-bine, 95 
“thionin-blay, 98 
Carbolic acid wx antiveptic, 147 
Carrelt's method of making anawetie 
cabvures, 60 
Carter on relapsing fever, 440 
Cattle plague, 507 
Cerebrospinal fluid, exumination by 
Tumbar puncture, 68 
Chamberland and Roux, attonsation 
of b, anthracis, 460 
‘Charedorland’s filter, 79 
Chemintasis, 20, 495 
Chlorine as antixeptio, 146 
Cholera. 399 
jiky againal, 410 
tion of man with, 410 
methods of diagnosis of 412 
preveative inceniation apainst, 412 
red reaction, 404 
Cholers spirillum, 400 
distribution of, 403 
inoculation with, #06 
of resistance of 405 
talations to divenses 414 
toxins of, 408 
Cladothrices tn soil, 182 
Cladathris, 16 
astorultes, 296 
‘Clute in actisomyees, 259 
Goole characters of, 11 
Collodien capanles, preparation of, 
123 
Colonies, counting of, 65 
‘Comma hciltas, 340 
Commissiou ow taberoulosis, 248 









‘Ceajanetivitie, 191 
(Coaradi-Drigalias medians, 42 


ETT 


Sopeman on tools, 608 

Cornet's forceps, 

Corrosive films Of blood, eta., 80 

Corrosive sublimate, as antiseptic, 145 
xing by, 90 

Counelnan Laflour on dysentery, 





687 
Counting of eclonios, 65 
dead tuctoria in a cultare, 67 
tiving bacterin in a oniture, 68 
Cover-giases, cleaning af, &7 
Gowpos, relaiion to smallpox, 605 
Grescoulic bodies in malarin, £28 
Cultivation of anaerobes, 57 
Culture medix, preparstion of: agar, 
36 





alkaline blood serum, 41 
hood ayars 58 
ceria, 39 
bouillon, 32 
Wren paate, 47 
glicow npar, 37 
rath, 
gelatin, 35 
glyoorla agar, 97 
roth, 3% 
Titiaus wbey 44 
Laflur’s mars modi 
Marmorek’s aarum media, 4 
meat extract, 31 
peptone gelatin, 36 
rolution, 38 
potatoes, 44 
serum ager, 38 
Calturas, devtruction of, 88 
filtration of, 69 
from orguns, 117, 124 
langingdrop, astobie, 63 
anaerobic, 64 
inoubation of, 79 
mteroseopie examination of, 86 














pu 

* shakey 
Cutting of se 
Cywtitin, 185, 
Cytaue, 475, 496 
Cytolytic sara, 482 














De Bary, definition of species, 28 
Decolorining agents, 97 

Deep cultures, BO 

Delhi sore, 568 





INDEX 


Dehylration of cestions, 99 
Deléping, 110 
Deneke's spiriltiem, 419 
‘Dextrose-free bonilion, 75 
Dinynowis, bacteriological, 116, 118 
“a itheria, B52 
agnosis of, 868 
itomunity again, 407 
‘origin anid spread’ of, 368 
paralyed in, 458, 262 
Tosults of treatment, 488 
Diphtheria bavillus, astion of, 968 
Dacilli allied to, 965 
characters of, 353 
dletribtion of, 864 
inoculation with, 309 
isolation of, 365 
powers of resatanoe of 859 
staining of, 108, 350 
toxins of, 163, 881 
variation in virulence of, 864 
Dipto-hnsiline of conjanetivitie, 19 
Diplocossus, 12 
catarthalis, 217 
crassus, 217 
endocanlitidis encapanilatun, 188 
intracelluluris meningitidis, 213 
pneumoniin, 199 
Disturtwnoes’ of _raetabolisen 
bacteria, 159 
Drigalsk!-Couradi medium, 42 
Drying of sera, otc., in vacuo, TS 
Dacroy’s bocillns, 227 
caltivation of, 228 
Dum-Dum fever, BOS 
Durham's fermentation taba, 76 
Dysuntery, amedic, O87 
Vacteria in, 940 
characters of ameoba of 687 
Dyxontery, methods of exarnination 
tn, 347, O42 








by 





East coast fover in cattle, 569 
Kberth's tnoillus, 819 
Ehrlich on rioin and ebrin, 464, 469 
‘on toxins, 170 
side-chain theory of antitasin fore 
tontion, 411 
Hisner's medium, 46 
Jing in pasallin, 92 
ona, 205, 423 
Endocarditis, bacterin in, 188 
(materia) 623 
37 











INDEX 


Butanceba histolytica, 697 
cultivation of, 689 
Enteric fever, 319 | 
Enteritis, dyseuterie, 917, 640 | 
Eplaaleoerbeephunl “meningig, 
3 
Eppinger’s streptothrix, 205 
Be ou botulina, 304 
infor age, 104 
Rryeipelon 101 
Kxcharioh’s bacilins 319 
Bsmarch’s roll-tabes, £5, 69 
anasrobic, 60 
Esaltation of virulence, 461 
Examination of water, 195 
Eshaust-purp, 70 
Eotorpores (innlaris), G22 





Fal mermbrove, 184, 969 

Parey, 276 

Feeding, imiunity by, 464 

Fermentation by pnoumo- baciltis, 

4 

by tivcitins cot, 826 
methods of observing, 
‘of sugars by bacteris, 
test of twcterial action, 74 














Porrsimte forrved! 
68 
ju diphtheria, 364 
tu tetanus, 380 
Fover, 168 
Film preparations, ary, 
wet, 8 
stalning of, 96 
Filter, porcelain, yelatined, 106 
Vikteetion of oaltares, 69 
Hinkler aud Prior's spiridlum, 413} 
Fish, tabercalosis in, 251 | 
Fizatears, 497 | 
Fization of tissues, 89 | 
Flagella, uature of, 8 
sealuiing of, 108 
Plagellated organisms in molaria, 528 
Fligee, 14 
Force for cover-gisases, &7 
Rorsnalits ox antiseptic, 145 
Roth's dire maldein, 263 | 
| 


by bacteria 22 














Fraeukel's pueumocovens, 192, 198, 





190 
‘tae for tubarete, 101 | 


597 


Frambesin, xpirochibes tay 234 

Frankland, on water bacteria, 187 

Frower, T.'R., 462, 460, 175 

Frietlinder’s’ pooumobacillus, 10%, 
208 

¥rlseh om rhinosnteronm, 254 

Puchehn, carbol-, 99 





Gongrenone emphysema, $89, 89% 
‘puevmonta, 423 
Gas formation, olnervation al, 44, 76 
Gas-regulator, 80 
Golssler’s exhaust pump, 70 
Golatin media, 25 
pheticlated, 345 
separation by, £2 
Gelntined porcelain filter, 106 
Geatina- violet, 98 
Germicides, M1 
Geryk pump, 78 
Gietea's stain, 107 
staln for spirochaetes tn tims, 107 
Glanders, 275 
lagnosls of, 283 
in horse, 276 
in man, 276 
Jesiows in, 281 
Glanders bacillus, 277 
wlutiaation of, 282 
inoculation with, 280 
dna morwitans, 6&2 




















G 





palpaliy, 589 
Gliacove ties 
Glaco 


ia, 35 of sey. 

ides, fermentation of, 75 

morta, 25 ef ag. 

potato as culture mediwm, 46 

Golgi on rmslacia, 21 

Gonidis, 15 

Gonoooeeus, sharnctors of, 219 
inooulation with, 
toxin of, 223 














Grwentleld on astheax, 313, 446, 400 

Grater sul Durham's phesomenon, 
435 

raieri bodies in amall pox, 





a 





soe 


598. 


Gulland (methods), 89, 92 
Hemamaba Danilewski, 580 


Hiametcanon malar, 520 
i sors, 4 
Hnmolyte tert, methods of, 488 
Haffkine on anti-cholera inocula 
tion, 412 
‘Haffkine's inoculation method against 
plague, 436 
Halteridium, 628, 580 
Hanging-drop cultures, 63 
‘examination of, 85 
Hankin, 812 
‘Hansen, leprosy bacilli, 269 
Hesse’s tube, 127 
‘Hiss's serum water media, 41 
‘Hofmann's bacillus, 866 
‘Horsepor, 504 
Houston on bacteriology of soll, 181 
Hueppe, 7, 14 
Hydrogen, supply of, 58 
‘Hydrophobia, 510 
dingnowis of, 519 
Negri bodies in, 514 
prophylactic treatment of, 516 
the virus of, 513 
Hypodermic syringes, 121 


Immune-Lodies, 478 
of, 482 
Immanity (v. also Special Diseases), 
456 


acquired, theories of, 490 
active, 458, 459 
artificial, 457 
by feeding, 464 
Uy toxing, 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 
teria, 157 
Influenza, 420 
bacilli, pseudo-, 423 


due to 














INDEX 


Inffuensa, bacillus, cultivation of, 421 
‘bacillag, inoculation, 424 
Joslona in, 422 
sputum in, 432 

Inoculation, methods of, 120 
of animoais, 120 

separation by, 56 
ve, 462 of aog. 
taleateal changes in cholera, 402 
amebic dysentery, 589 
‘bacterial dysentery, 347 
typhoid fever, 329 
Intestinal infection in cholera (ex- 
perimental), 407 

Involution forms in bacteris, 4 

Todine solution, Gram’s, 99 
terchloride, 468 

‘as antiseptic, 145 

Todofotm as antiseptio, 148 

Tesaof, 464 

Ivanoff's vibrio, 415 


Japanese dysentery, 350 
Jenner on vaccination, 503 
Jenner's stain, 106 
Johne's bacillus, 254 
Joints, gonococe! in, 225 


Kile-dzar, 563 
Kipp's apparatus, 58 
Kitasato on bacillus of influenza, 420 
of plague, 425 
of tetanun, 872 et seq. 
Klebs-Lffier bacillus, 852 
Klein, 345, 508 
Klemperer on pneumonia, 210 
Koch on avian tuberculosis, 250 
‘bacillus of malignant codema, $83 
bovine tuberculosis, 248 
cholera spirillum, 399 
cultivation of b, anthracis, 301 
leveller for plates, 53 
tubercle bacillus, 235 
tuberculin, 268 
“tuberculin Q,” and “R,” 260 
Koch-Weeks bacillus, 191 
Korn’ ast bacillus, 253 





m itinus solution, 
thylene-blue, 98 
moni of Gram's method, 100 
Lamb on relapsing fever, 442 
larial parasite, 621 
Leishmau-Douovan bodies, 563 


. INDEX 


‘Leishman-Donovan bodies, cultivation 
of, 566 
Leishman’s opsonic technique, 111 
serum method for staining try- 
Panosomes, 545 
stain, 106 
ishmania donovani, 567 
tropics, 567, 568 
Lenses, 85 
Lepra cells, 269 
Leprosy, 267 
bacillus, 269 
distribution of, 271 
staining, 100, 270 
dingnosis of, 274 
etiology of, 272 
Leprosy-like disease in rats, 273 
Leptothrix, 15 
Lesions produced by bacteria, 155 
Leucocidin, 165 
Leucocytosix, 156, 495 














Lencowaines, 161 
Levaditi's method for staining spiro- 
chates, 104 
Litmus solution, Kubel-Tiemann's 42 
whey, 44 


Liver abycess in dysentery, 540 
Lockjaw, 871 
Lofiler's bacillus, 352 
methylene-llue, 98 
serum medium, 40 
and Schutz’ glanders bacillus, 275 
Linch, amerba of, 537 
Lumbar paneture, 68 
Lustgarten’s bacillus, 229 
Luustig’s anti-plague serum, 436 
Lyinph, vaccine, 506 
Lymphangitis, 184 
Lysogenic action of serum, 477 
towards blood corpuscles, 479 














MacCoukey’s hile-salt media, 43 
meilium, use of in dysentery, 317 
in examining water, 186 
in paratyphoid fever, 385 
‘M-Fadyean on glanders, 282 
Macrocytaxe, 497 
Macrophages, 495 
Maura disease, 207 
alaria, eyele in man, 522 
mosquito, 528 
pathology of, 533 
prevention of, 532 
question of intinunity against, 534 




















599 


‘Malarial fever, examination of blood 
in, 535 
malignant, 523, 531 
mosquitoes in, 532 
Malaria! parasite, 521 
inoculation of, 522 
staining of, Leishman’s method, 
106 
Romanowsky methods, 106 
varieties of, 529 
Malignant edema, bacillus of, 388 
diagnosis of, 393 . 
immunity against, 393 
Malignaut pustule, 310 
Mallein, 283 
Malta fever, 446 
methoils 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 
21 
Marmorek, on streptococci, 183 
antistreptococeie serum, 476 
Marmorek's serum media, 41 
antitubercular serum, 265 
Martin, Sidney, on albumoses, ete., 165 
on anthrax, 312 
on diphtheria, 363 
Martin, C. J., om toxins, 166 
‘on autitoxins, 475 
Massowah vibrio, 416 
Measuring bacteria, 119 
Meat extract, 31 
Meat-poisoning by bacillus botulinus, 
393 
by Gaertuer's bacillus, 336 
Mediterranean fever, 446 
Meningitis, bacteria in, 217 
epidemic cerebro-spinal, 174, 213 
in influenza, 423, 
pneumuococei in, 205 
posterior hasal,’218 
Mercury perchloride as antiseptic, 145 
Metabolism, disturbances of,” by 
‘bacteria, 159 
Metachromatic granules, 8 
Metchnikoff on cholera in rabbits, 407 
relapsing fever, 441 
MetchnikofTxphagocytosistheory, 495 
spirillum, 417 
Mothylene-hue, 95, 98 
































600 


Methyl-violet, 94 
Meyer and Ransom on tetanus toxin, 
382 
Micrococei of suppuration, 174 
Micrococens, 12 
of gonorrhees, 219 
melitensis, 447 
pyogenes tenuis, 174 
tetragenus, 181 
lesions caused by, 185 
ures, 20 
Microcytase, 497 
Microphages, 495 
Microscope, ure of, 85 
Microtomes, 90 
Migula, 12 
Mikulicz, cells of, 284 
as culture medium, 46 
Miller's stain for spores, 102 
Moeller's Timothy-grass bacillus, 253 
Morax, bacillus of, 192, 193 
Morlants, 97 
Morgan's bacillus, No. 1, 351 
Mosjuitoes, in malaria, 528, 532 
rile in yellow fever, 453, 
Moulds, media for growing, 44 
Muencke’s filter, 72 
Miller's bacillus, 192 
Myvetoma, 297 
Myclocytes, neutrophile, 156 








Nagana, 552 

Natural immunity, 498 

Neelsen’s stain for tubercle, 101 

Negative phase in immunisation, 282, 
494 

i bodies in rabies, 514 

Neisser's gonococens, 219 

stain for b, diphtheri«, 108 
Nencki, 10 








Neutral-reil as indicator for media, 43 
use of, 38 r 
with b. typhosus, 827 i 
Neutrophile leucocytes, 156 f 
| 





myelocytes, 156 

Nivulaiety tetanus bacillus, 371 

Nicolle's’ modification of |G 
metho. 100 

Nikati auc Rietsch on cholera, 407 

Nitvitying bacteria, 23 

Nitroso-indol bealyy 73 

uwten vibrio, HS 

Novy aud) MacNeal, for 
culture of trypanosomes, 515 








medline ' 


INDEX % 


Overimeier’s spirillum, 438 
GQedema, malignant, 388 
Ogata’s dysentery bacillus, 350 
Ogston, 174 
Oil, aniline, for dehydrating, ete, 93 
Oil immersion lens, 85 
Odkinete, 528, 547, 548 
Opsonic action, nature of, 483 
‘technique, 111 
Opsouins, 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 
Ozona bacillus, 285, 








Para-colon bacillus, 385 
Paraffin embedding, 91 
Paratyphoid bacillus, 335 
461 
munity, 458, 464 
Pasteur on exaltation of virulence of 
vucteria, 461 
on hydrophobia, 516 
on vaccination against anthrax, 314 
septicémie de, 388 
Pathogenicity of bacteria, 149 
Peptone gelatin (v. Culture media), 35 
solution, 38, 404 
Periostitis, acute suppurative, 190 
Peritonitis, 184, 224 
Perlsueht, ‘236 
Pestis major, 431 
tuinor, 431 
Petri’s acid-fast bacillus, 253 
capsules, 52 
sand-tilter for examining air, 128 
Petruschky's 
Pettenkofer ou cholera, 
Petter, 20 
Pleitfer om anti-serum, 477 
cholera, WT 
intlueuza, 120 
typhoid, 333 
Picitler's phenomenon, 11, 477 
Magneytes. 18 
Phagoe} tosis theary of Metehnikelf, 
AUS 


Phenol broth, 247 



























INDEX 


Phenol-phthalein as indicator, 38 
Phenomenon of Bordet, 477 
Gritber and Durham, 485 
Pfeiffer, 411, 477 
Pigments, bacterial, 10 
Pipettes, 66, 108, 110, 116 
Piroplasmata ax causes of disease, 569 
plasmosis, 568 
Pitfield’s flagella stain, 103 
Plague, bacillus of, 426 et seg, 
‘Haffkine’s inoculation against, 436 
inimunity against, 435 
infection in, 432 
involution forms 427 
part played by rat fleas in the 
spread of, 433 
preventive inoculation against, 436 
serutn diagnosis, 437 
stalactite growths of, 429 
varieties of, 431 
Plasmolysis, 9 
Plate cultures, agar, 55 
gelatin, 51 
gouccoveus, 222 
Platinum needles, 49 
Pueumobacillus (Friedlinder's), 199, 
203 vt seg. 
Pneumococcus —(Fraenkel’s), 
201 et seq. 
immunity against, 210 











199, 








Pneumonia, bacteri 
saungrenous, 423 
in influenza, 422 
methods of examination of, 212 
neptic, 197 

arieties of, 196 
Polar granules, 8 
Positive phase in i 

494 
Potassium permanganate ax antiseptic, 
M7 
Potatoes ax culture material, 44 











unisation, 262, 











Poynton aul Payne on acute 
rheumatism, 193 

Precipitins, 48 

Preparations, impression, 113 





Protective inoculation, 482 ef sy. 
Proteosona, 530 
Protozoa deseriled in hydrophobia, 
513 
smallpox, 508 





601 


Pseudo-diphtheria bacillus, 365 
-tuberculosis streptothricea, 296 

Pasittacosis bacillus, 337 

| Peomaines, 161 
Puerperal septicaemia, 184 

Pus, examination of, 87, 195 

Pustule, matignant, 310 

i Pyeemia, 184 et seq. 

! “nature of, 173 


| Protozoon malaria, 521 


Quartan fever, 580 
| Quarter-evil, bacillus of, 396 
Quotidian fever, 529 


Rabies, 510 
Rabinowitch's acid-fast baclllus, 258 
Rauschbrandl bacillus, 396 
Ray-fungus (actinomyees), 286 

* Reaction of media, standardising of, 

33 

| Receptors, 491 

| Recovery from disease, 457 

1 Red stains, 05 

| Red-water fever in cattle, 569 

| Reichert's gus regulator, 80 

Relapsing fever, agglutination 

spirillum, 442 
bactericidal serum in, 442 
spirilun of, ete, 438 

. Reversibility of toxin-antitoxin reae- 

i tion, 472 

j Rheumatism, acute, 193 
Rhinoscleroms, bacillus of, 284 
Ricin, 169 

nimwunity against, 464, 469 


of 








Rivers, hacteria in, 137 
Robin, 169 
Rock fever, 448 


Roll-tubes, Esmarch’s, $5, 59 
Romanowsky stains, 105 
Roseubach (bacteria in suppuration), 
174 
Ross, on malaria, 
thick film 


p21 


method for malarial 





Salt-agar ay medium for b. pestis, 427 
Sanarelli (typhoid fever), 382 
| Sanderson, Burdon, 460, 507 





= 


602 INDEX 


Baprophytes, 149 
Sarcina, 12 
Sansage poisoning, bacillus botulinne 
‘in, 304 
Shanahan onbilogy of tyranny 
Sal 
00 amoctee of dyyentery, 687 
‘on worphology of spirilla, 660 
on spirochwete pallida, £20 
on spirilluw Ziemann, 650 
Mebimomyeeton, 3 
‘Schimophycem, 3 
Sehizophyta, 3 


Sebilluer's dots 106, S31 
Solavo's anti-authrax serum, 315 
Scorpion poison, 160 
Soction-cutting, 90 
Seetions, dehgiration of, 98 
‘Solimeutation methorts, 109 
teat for typhot, 338 
Beiteaketten, 491 
Soptioamis, mat 
puerperal, 18 
spatiun, 197 
Soptiedaaie dy Pasteur, 398 
Septic pneumonia, 197 
Hera, luemolytic, 479 
Soeur agar, 88 
Sorum, agglutinative action of, 485 
soaphylaxis, 494 
antibacterial, 476 
anu cholera, 
antidiphtheritic, 407 
auti-plague, 490 
autipuoumpeoeeie, 20 
antirabio, 619 
auilictroptonnocte, 476 
antitetanic, 54 
Antitosie, preparation of, AGT et sep. 
itubercular, 264 
antityphoid, 314 
bacterioldal nation of, 477 
Inloort (#. Culture media), 30 
Liuguonts, 485 
wuwthodsy 109 
of typhoid, 897 
inapiseator, 30 
Iysogenic ation of, 477 
towards titood carpuacles, 479 
Serum medin, 39 
Sorum-water media, 41 
Sownge, bacterial treatment of, 139 
contaroination of water by, 196 
Shake valtures, 77 


of, 178 
































Sbeop-por, 507. 
Shiga’s bacillus, #6 
Side-chain theory, Ehetieh's, 491. 
ickwest, 365 
Slides for banying-dropa, 63, 64 
Sloped onltuires, aerobic, #8 
‘auastobio, 2 
Snuallpos, 608 
bacteria in, 507 
Gourniert bodies in, 508 
Bunga tang 34 
Smith's Lorrain, serum sultan 4 
Smith, Thoobald, phenomenon of, 
404 
Stake poisons, 160 
netivating of, Uy seruta, 170 
constituents af, 169 
fmumnunity again, 462 
Sobecnhelm's ant-antirax seram, 316 
Soft sore, 227 
Boil, uxamination of, for bweteriay 181 
Sondakeritoh on relapsing fore, 4 
Splual cond estons Dy” pyogwalle 
ongunisans, 183 
Spirits, chararters of (1. also Wibeto), 
14, 550 
{ike elit spirit 427 
Spirillonis in animal, 430 
Spirillam Metohmikovi 417 
‘of chalers, 400 
Dencke, 419 
Fiulkler and Prioe, #18 
Miller, 419 
relapilug fevée, dnpenlation with 
hoy 438 
Spiroclute, 14, 229, 660 
pallida, 29 














id 
refrin 230 
Spirochates ta oy pbllin 228 
In yaws, 284 
staining of, in fils, 107 
sala of, iu sect, 204 
Splropeua palliduns, £29 
Splenic fever, 800 
pore fercualten, 
walegenont, 
tn te anthracis, 204 
por 
Sporvbas 
Sporveyat (ianlatial B29 
Sporocytes, in malaria, 524 
Sporoaoltes, 620 











INDEX 


Sporulation of malarial parmite; 622 





in plague, 238 
in pneumonia, 200 
phthisieal, 241, 255, 265, 





nia, 197 
Staining methods, 94 et sey. 
of capsules, Welch's method, 102 
Richard Muir's method, 102 
of flagella, 103 
of leprosy’ havilli, 270 
of spores, 102 
of tubercle bacilli, 100 
Principles, 94 
aniline, 94 








of media, 33 
caused by, 184 





Staphylococcus, 12 
cereus albus, 176 
wus, 178 
pyogenes albus, 176 
aureus, characters of, 174 
inoculation with, 182 
citreus 174 
Steam steriliser, Koch's, 27 
Stegomyia fascinta, 483 
Sterilisation by heat, 26 ef su. 
at low temperatures, 29 
by steam: at high pressure, 
Streptovoeci in diphtheria, 3: 
in false membrane, 184 
lesions caused by, 184 
rieties of, 178 
Streptococcus, 12 
anginosus, 179 
brevis, 178 
conglomeratus, 178, 17 
equinus, 17 




















inoculation with, 182 
in air, 130 
in soil, 133 
salivarius, 179 
Streptothrices allied to actinomy 
294 











603 


Streptothrix, anaerobic in actino- 
mycosis, 293 
madure, 297 
Subcultures, 49 
‘Sugars, classitication of, 74 
fermentation of, 74 
Sulphnrous acid as antiveptic, 147 
Suinmer diarrhoea, bacteria in, 351 
Suppuration, bacteria of, 174 
gonococci in, 223 
‘methods of examination of, 195 
nature of, 172 
origin of, 186 
pneumococei in, 205 
phoid bacillus in, 380 
mptoins caused by bacteria, 161 
philis, bacillus of, 220 
spirochaete pallida in, 229 
transmission to animals, 233 
Syringes for inoculation, 120, 121 














Tales meveuterica, 257 
‘Taurocholate media, 43 
Tertinn fever, 530, 531 
‘Test-tubes for cultures, 47 
‘Tetanolysin, 380 
‘Tetanospasmin, 380 
Tetanus, 37 
auti-serum, of, 884, 467 ef seq. 
intraveuous injection of, 386 
cerebral, 384 
dolorous, 383 
unity against, 34 
ination in, 388 














‘Tetanus bacillus, 372 
inoculation with 
isolation of, 373 
spores of, 3 
toxins of, 153, 379 

Tetras, 12 

‘Texas fever, 569 

‘Theory of exhaustion, 490 

cytosis, 495 

of retention, 490 














‘Thermophilie bacteria, 18 

‘Thermostable opsoniny, 484 

‘Thiouin-bine, 95, 98 

‘Thiothrix, 15 

fever, African, 443 

‘Timothy-srass bacillus, 153 

‘Tissues, action of bacteria on, 155 
fixation of, 89 














604 


Tizzoni and Cattani on tetanus, 385 
Toxalbumins, 162 
Toxic action, theory of, 170 
Toxicity, estimation of, 467 
‘Toxins, concentrated, method of ob- 
taining, 167 
constitutiow of, 491 | 
early work on, 161 | 
effects of, 158 | 
I 








immmnisation by, 467 
intra. aud extra-cellular, 162 
nature of, 165 
non-proteid, 166 
of anthrax, cholera, 
Special Diseases) 
production, 154 
susceptibility to, 491 
vegetable, 169 | 
‘Toxoids, 171, 472 
Toxones, 171 
‘Trachoma, bucteria iu, 192, 424 | 
Trichophyta, media for growing, 44. | 
‘Tropical ulcer, 568 
‘Trypanovoma gambiense, 558 
Lewisi, 544, 551 
noctua’, 548 
of sleeping sickness, 555 ' 
ugandense, 544, 558 
ugandense, relation to Tr. Gam- 
Dbiense, 561 
‘Trypanosomata associated 
various diseases, 544 
culture of, 546 
morphology of, 544 


ote, (vide 





with 











tse fly disease, 552 
‘Tubercle bacillus, 237 
action of dead, 255 
avian, 250 


cultivation of, 239 


distribution of, 243 





powers of resista 
iy sputum 
toxins of, 
stains fe 
ist eells, 
tnethods af examination of 
Tabereles, structure of 242 
Tudervular leprosy, 2 
Taberenling 258 
** Bazillenemulsion ” 260 1 










INDEX 


Tubereulin, “0” and “R," 260 
Tubereulosis, 235 
aviau, 250 
dovine, 248 
its relation to human, 248 
diagnosis by tuberculin, 259 
‘Tuberculosis, in animals, 236 
in fish, 251 
modes of infection, 266 
precautions in diaguosis of, 255 
Tubes, cultures in, 47 
‘Typhoid bacillus, 819 
comparison with b. coli, 325 
examination for, 344 
immunity against, 382 
inoculation with, 381 
isolation from water supplies, 345 
toxins of, 332 
serumn diagnosis, 337 
suppurations in, 380 
vacciuation against, 343 
‘Typhoid fever, 319 
pathological changes in, 329 


Ulcerative endocarditis, 188 
experimental, 190 
gouococet 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 hydrophobin, 516 
against typhoid, 343 
for infection by pyogenic bacteria, 
194 

















nature of, 509 

Varioln, 505 et seg. 

Veuins,’ 169 

Vibrio (see also Spirillum), 14 
berolinensis, 415 
of cholera, 400 
Dannbiews, 415 
Deueke's, 419 
Finkler anil Prior's, 418 
Gindha, 416 
Want, 445 
Massiwah, 408, 416 
Metehnikovi, 417, 
Nondhaten, 418 
of Pestana and Better 
Romans, 416 

Vilrrion septi 














court, 116 





, 338 





INDEX 


Virulence, attenuation of, 459 
exaltation of, 461 
of bacteria, 150 


Water, bacteria in, 135 
‘contamination of by sewage, 188, 
examination of, 185 
supplies, typhoid bacilli in, $45 

Weichselbaum on pneumonia, 198 

Weigert’s method of dehydration, 98 
modification of Gram’s method, 100 

Wertheim’s medium, 220, 226 

Whooping cough, bacteria in, 423 

Widal on serum diagnosis, 485 

Wiilal’s reaction, synonym for aggla- 

tination of b, typhosus, q.r., 
109, 337 

Winogradski, 23 

Winter-spring fevers, 529 

Wolff and Israel's streptothrix, 295 

Woorlhead on tuberculosis, 257 

Woody tongue, 202 





Woolsorter's disease, 311 
Wright's, A. E., calibrated pipette, 
108 
diluting pipette, 66 
method of counting deal 
bacteria, 67 





605 


Wright's, A. E., opsonic technique, 
112 
vaccination against, tuberculosis, 
261 
vaccination treatment of pyo- 
genic infectious, 194 
Wright, J. H., on anaerobic strepto- 
thrices, 293 
Romanowsky stain, 107 


Xerosis bacillus, 367 
Xylol, 93 


‘Yaws, spirochetes in, 284 
Yellow fever, 451 
‘bacteria in, 452 
etiology of, 452 
mosquitoes in relation to, 458 
Yersin (r. also Roux), on plague, 
435 ef seq. 
Yersin's anti-plague serum, 436 


jemanni, spirillum, 550 
iehl-Neelsen stain, 101 
Zongliea, 3 

Zygote (malaria), 328 





om 














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