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CORNELL UNIVERSITY. Convers | 

THE | 

Koswell P. Flower Library 
"THE GIFT OF 


ROSWELL P. FLOWER 
FOR THE USE OF 


THE N. Y. STATE VETERINARY COLLEGE. 
1897 


text-book of bacteriotog 


ete i eal ea ia RE 


A TEXT-BOOK 


BACTERIOLOGY 


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


= 
SURGEON-GENERAL U. 8S. ARMY 
EX-PRESIDENT OF THE AMERICAN MEDICAL ASSOCIATION ..ND OF THE AMERICAN PUBLIC HEALTH 
ASSOCIATION; HONORARY MEMBER OF THE EPIDEMIOLOGICAL SOCIETY OF LONDON, OF | 


THE ROYAL ACADEMY OF MEDICINE OF ROME, “¥ THE ACADEMY OF MEDICINE OF 
RIO DE JANEIRO, OF THE SC CIETE FRANCAISE D’HYGIENE, ETC., ETC. 


¥ 


if 


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


Second Revised Codition 


NEW YORK 


WILLIAM WOOD. AN) COMPANY 
MDCCCCI 


WYO puget 
COPYRIGHT BY 
WILLIAM WOOD & COMPANY, 
1901. 


Qk 
Y | 
S$5 
\Qo| 


PRESS OF 
THE PUBLISHERS’ PRINTING COMPANY 
82-34 LAFAYETTE PLACE 
NEW YORK 


PREF ACH. 


ize writer’s Manual of Bacteriology, published in 1892, has been 

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

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

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

1896. 


Cornell University 


The original of this book is in 
the Cornell University Library. 


There are no known copyright restrictions in 
the United States on the use of the text. 


http:/www.archive.org/details/cu31924000226013 


PREFACE TO SECOND EDITION. 


te the request of the publishers the author has again undertaken 

a revision of his Manual of Bacteriology, published in 1892. 
This is practically a third edition of that work, although the title 
was changed in 1896, and it now appears as a second edition of a 
TEXT-BooK OF BACTERIOLOGY. 

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

Wasnineton, May 27th, 1901. 


TABLE OF CONTENTS. 


PART FIRST. 
CLASSIFICATION, MORPHOLOGY, AND GENERAL BACTERIOLOGICAL 
TECHNOLOGY. 
PAGE 
. HISTORICAL, . ‘ : i é : 3 ‘ : » 8 
. CLASSIFICATION, . : é é ‘ ‘i : . F . 10 
. MorPHOLoGY, . : ‘ . . é ‘ ‘ . 20 
. STAINING METHODS, ‘i , F A ‘ : ; . 25 
. CULTURE MEDIA, . ‘ : - : é F g 3F 
. STERILIZATION OF CULTURE “Meta, 5 ‘ : 3 3 . 52 
. CULTURES IN Liguip MeEpia, 5 2 2 F 7 . 62 
. CULTURES IN SoLID Mep1a, : ‘ i , g . 69 
. CULTIVATION OF ANAEROBIC BACTERIA, ; 3 F : . 80 
. INCUBATING OVENS AND THERMO- -REGULATORS, : - . 88 
. EXPERIMENTS UPON ANIMALS, . : é , , - 96 
. PHOTOGRAPHING BACTERIA, F . : : ° $ - 103 
PART SECOND. 
GENERAL BIOLOGICAL CHARACTERS. 

. STRUCTURE, Motions, REPRODUCTION, : 2 ; . . 117 
; Conprtions OF GrowTH, é ‘ . 4 F . 125 
. MODIFICATIONS OF BIOLOGICAL CHARACTERS, F ; : . 129 
Propvucts oF ViTaL ACTIVITY, . 2 ‘ 2 i é . 133 
. PTOMAINES AND TOXALBUMINS, : , ‘ 3 : - 146 
. INFLUENCE OF PHYSICAL AGENTS, . , . 153 

. ANTISEPTICS AND DISINFECTANTS (GENERAL Acoourr OF THE 
ACTION OF), . - 164 

. ACTION OF GASES AND OF THE chepony ELEMENTS 1 UPON “Bao. 
TERIA, 5 F - , zs . 172 
. ACTION OF Acts 4 AND ‘Antanas, “ : F : : . 180 
. ACTION OF VaRIOUS SALTS, . ; 5 . . 186 
. ACTION OF COAL-TAR PRopucts, ESsEnTraL Ons, ETC., . 197 
. ACTION OF BLooD SERUM AND OTHER ORGANIC Liqurps, . 208 


XL, 


PRACTICAL DirecTIONS FoR DISINFECTION, : ‘ , - 214 


INDEX, 


. Mopgs or ACTION, 

. CHANNELS OF INFECTION, 

. SUSCEPTIBILITY AND IMMUNITY, 

. PROTECTIVE INOCULATIONS, 

. PYoGENIC BACTERIA, 

. BACTERIA IN CROUPOUS PNEUMONIA, 
. PATHOGENIC MICROCOCCI NOT DESCRIBED IN SCrIoNs Vv. 
. 410 
. 422 
. 431 
. 449 
. 463 


. THE BacILtus OF AnTHRax, 

. THE BACILLUS OF TYPHOID FEVER, 

. BACTERIA IN DIPHTHERIA, 

. BACTERIA IN INFLUENZA, 

. BACILLI IN CHRONIC INFECTIOUS Diseases, : 
. BACILLI WHICH PRODUCE SEPTICEMIA IN SUSCEPTIBLE wee 


. BACTERIA OF THE STOMACH AND “InrEstines, 
. BACTERIA OF CADAVERS AND OF PUTREFYING Marmeiat; 3 FROM 


TABLE OF CONTENTS. 


PART THIRD. 
PATHOGENIC BACTERIA. 


AND VI, 


MALS, 
. PATHOGENIC Agronio Bacrta NOT DESCRIBED IN ‘PREVIOUS 
SECTIONS, : 
. BACTERIA IN PLANT DISEASES, : : : Nf 
. PATHOGENIC ANAEROBIC BACILLI, . : é é : s 
. PATHOGENIC SPIRILLA, : é é . - F . ° 
PART FOURTH. 
SAPROPHYTES. 
. BACTERIA IN THE AIR, 
. BACTERIA IN WATER, 
. BACTERIA IN THE SOIL, 


Mucous MEMBRANES, 


VARIOUS SOURCES, . i ‘ ; ‘ 3 


. BACTERIA IN ARTICLES OF Foop, : . j : ‘ % 


PAGE 
. 221 
. 229 
. 233 
. 272 


371 


. 396 


. 467 


. 498 


. 528 
- 571 


578 
590 


- 613 
- 626 
- 642 
. BACTERIA OF THE SURFACE OF THE Bose AND OF ” Expose 


- 648 
. 658 


664 
667 


673 


PHIM aPowee 


LIST OF ILLUSTRATIONS. 


Staphylococci, 

Zobglea, . 

Ascococcus, 

Streptococci, 

Tetrads, 

Packets—sarcina, 

Bacilli, . : 

Tnvolution forms, : 

Chains formed by binary divisor 

Spirilla, 

Cladothrix, 

Flagella, 

Platinum wire in glase handle, 

Flask for drawing off blood serum, 

Method of forcing blood serum into test tube, 
Suction pipette, 

Hot-water funnel, 

Karlinski's agar filter, 

Glass dishes for preserving potato cultures, 
Test tube for sterilizing potato, 

Shape of potato for test-tube culture, 

Hot air oven, 

Koch’s steam sterilizer, 

Koch’s steam sterilizer, . ; : ‘ 
Arnold’s steam sterilizer, 

Miincke’s steam sterilizer, 

Koch’s apparatus for coagulating blood serum, 
Mincke s steam sterilizer and coagulator. j 
Pasteur Chamberlain filter, 

Pasteur Chamberlain filter without stat case, 
Moditied Pasteur-Chamberlain filter, 
Erlenmeyer flask, ; 

Flask used by Pasteur, 

Platinum wire loop, 

Platinum needle, . 5 3 . 
Sternberg’s bulb, . , : ‘ : 
Fermentation tube, . 

Method of making stick culture, 


PAGE 


22 


59. 


LIST OF ILLUSTRATIONS. 


Sloping surface of culture medium, ‘ : 


- Growth of non-liquefying bacteria in gelatin etiote cusitianaa, 


Growth of same along line of puncture, 
Growth of liquefying bacilli, . 

Colonies of bacteria, 

Apparatus for gelatin plates, 

Esmarch roll tube, . 

(See Fig. 15). 

Mode of development of a facultative anaérobic bacillus, 
Mode of development of strict anaérobic in long stick culture, 
Exhausted-air flask for liquid media, . 
Method of displacing air with hydrogen, 
Salomonson's tube, . : 
Frinkel’s method of daltivation, 
Sternberg’s method of cultivation, 
Sternberg’s method of cultivation, 
Buchner’s method of cultivation, 
Hydrogen generator, 

Hydrogen apparatus for plate eistbares; 
Incubating oven, 

Thermo-regulator for eas, 

Moitessier’s pressure regulator, 

Mica screen for flame, 

Koch's device for cutting off fl bat 
Reichert’s thermo revulator. 

Bohr's thermo-regulator, 

Miincke’s thermo-regulator 

Sternberg’s thermo-regulator, 


Gas valve for the same, . Z a . 
D’Arsonval’s incubating apparatus, : ; ‘ : 2 
Roux’'s incubating ovea and thermo- eegunlater, ‘i ‘ : ‘i ° 
Roux's thermo-regulator, : ‘ ; p a 5 ° 
Koch's syringe, ‘ 3 : ; ‘i : ‘ . . 
Sternberg’s glass syringe, : ‘i F 


Pringle’s photomicrographic apparatus, 

Sternberg’s photomicrographic apparatus for gas, 

Spores of bacilli, ; 

Method of germination of spared ‘ ‘ ‘ ‘ 

Apparatus for cultivating anaérobic bacilli, . . z 

Bacillus of mouse septicaemia in leucocytes from blood of mouse, 

Staphylococcus pyogenes aureus, 

Gelatin culture of Staphylococcus pyogenes aureus, 

Vertical section through a subcutaneous abscess caused by inoeulation 
with staphylococci in the rabbit, 3 - 3 

Pus containing streptococci, , 

Streptococcus of erysipelas in nutrient actin . 

Section from margin of an erysipelatous inflammation, showing sirepti- 
cocci in lymph spaces, 

Gonococci, 

Gonococcus in gonorrhea mae 

Gonorrheeal conjunctivitis, second day ae genes, 


LIST OF ILLUSTRATIONS. 


Friedlander’s bacillus, . ; 

Friedlainder'’s bacillus; stick’ ealiare in reldiin, 

Micrococcus pneumonie croupose, 

Micrococcus pneumonie croupose, : : ; 

Micrococcus pneumonize croupose, . ‘ 

Micrococcus pneumonie croupose, showing capsule, ' ‘ 

Single colony of Micrococcus pneumoniz croupose upon agar slate, 

Micrococcus pneumonie croupose in blood of rabbit inoculated with 
sputum, ; ; : é : Bee a é , . 

Micrococcus tetragenus, . 3 ‘ , . 

Streptococcus of mastitis in cows, ‘ 

Bacillus anthracis, showing development of ihe ee in convoluted 
bundles, . ‘ ‘ 7 : é : ‘ ‘ 3 2 fi 

Bacillus anthracis, showing formation of spores, 

Culture of Bacillus anthracis in nutrient gelatin, 

Colonies of Bacillus anthracis upon gelatin plates, . 

Bacillus anthracis in liver of mouse, . : * ‘ 7 . 

Bacillus anthracis in kidney of rabbit, 

Bacillus of typhoid fever; colonies in stained pectin of sileeks 

Bacillus of typhoid fever; colonies in stained sections of spleen, 

Bacillus typhi abdominalis, . i : ; ‘ z : 7 

Bacillus typhi abdominalis, . s ‘ ; ' j 

Bacillus typhi abdominalis, showing flagella, 

Single colony of Bacillus typhi abdominalis in nutrient aden, 

Bacillus typhi abdominalis; stick culture in nutrient gelatin, . 

Section through wall of intestine, showing invasion by typhoid bacilli, 


Bacillus diphtheriz, ‘ A i 
Colonies of Bacillus diphtheris { in nuirlont ava: 
Bacillus tuberculosis, : : : ‘ . 


Bacillus tuberculosis in cepiituarey 4 P 

Section through a tuberculous module ts in ie lung at a cow, sowie 
two giant cells, . . ‘ . . 

Tubercle bacilli from surface of eiltiire upon ‘loa serum, . 7 : 

Culture of tubercle bacillus upon glycerin-agar, . é : . 

Limited epithelioid-celled tubercle of the iris, 

Section of a recent lepra nodule of the skin, 

Bacillus mallei, 5 . . 

Section of a glanders nodule, ‘i ; : 

Section through a glanders nodule in liver of field 1 mouse, 

Migrating cell containing syphilis bacilli, : 

Pus from hard chancre containing syphilis bacilli, : A . 

Bacillus of rhinoscleroma in lymphatic vessels of the superficial ware of 


tumor, ‘ . . ‘ 
Bacillus sepiiennile leemordlinglen | in blood at a vabibit, 2 . 
Bacillus septiceemiz hemorrhagice; stick culture in nutrient gelatin, 
Bacillus of Schweineseuche, . ‘ js z : . F F . 
Colonies of bacillus of swine plague, . F ; : ; 5 - 
Bacillus of Schweineseuche in blood of rabbit, . . ‘ . ‘ 
Bacillus of hog cholera, . ‘ . . 3 


. Bacillus of mouse septicemia in letemerves ome ibaa of mouse, . 


Bacillus of rouget, 


LIST OF ILLUSTRATIONS, 


Bacillus of mouse septicemia ; culture in nutrient gelatin, 


Bacillus of mouse septicemia; single colony in nutrient gelatin, 
Section of diaphragm of a mouse dead from mouse septicemia, 


Bacillus cavicida Havaniensis, ‘ 2 . 

Bacillus crassus sputigenus, . 

Proteus hominis capsulatus, 

Bacillus capsulatus, 

Bacillus hydrophilus fuscus, ‘ 

Culture of Bacillus hydrophilus fuscus in iguuitient gelatin, 
Bacillus coli communis, . F 

Bacillus coli communis in nutrient aelatie, 

A portion of the growth shown ii Fig. 147, . 

Bacillus lactis aérogenes, . z 

Bacillus acidiformans, . ? 
Culture of Bacillus aciiitontiana’ in nattient pelitin, 
Bacillus cuniculicida Havaniensis, . ‘ ‘ 

Colonies of Bacillus cuniculicida Havaniensis, 

Colonies of Bacillus cuniculicida Havaniensis, 

Bacillus pyocyaneus, : : 

Proteus vulgaris, . ‘i 

“Swarming islands” from a autnare of Broieue mairabilts, 
Spiral zodglea from a culture of Proteus mirabilis, 
Bacillus gracilis cadaveris, 2 

Colonies of Bacillus gracilis cadaveris, . 

Tetanus bacillus, : ‘ ‘ 5 ‘ 
Tetanus bacillus, . . . . . . 
Culture of Bacillus tetani in nutrient gelatin, 

Bacillus cedematis maligni, . 

Bacillus cedematis maligni,  . . ‘ 
Cultures of Bacillus cedematis isieia 2 in eawient gelatin, 
Bacillus cadaveris, . ‘i , ; : ‘ ‘ ‘ 
Bacillus cadaveris, 

Bacillus of symptomatic nabtinas, 

Bacillus of symptomatic anthrax, é F i 
Culture of bacillus of symptomatic anthrax, . . : 
Spirillum Obermeieri, 

Spirillum Obermeieri, . ‘ 

Spirillum cholere Asiatice, 


Spirillum cholere Asiatice, . ; . i : 
Colonies of Spirillum choler Asiatice. . 
Spirillum cholere Asiatice, . ‘ . 7 


Cultures of Spirillum cholere Asiaticee in nai mien gelatin, 
Spirillum cholere Asiatice, 


Colonies in nutrient gelatin of Syiritiom eolare, Action, Spiriifam 


tyrogenum, and Spirillum of Finkler and Prior, 


Section through mucous membrane of intestine from eben etna. 


Spirillum of Finkler and Prior, ‘ P 
Colonies of Spirillum of Finkler and Prion: 
Spirillum of Finkler and Prior ; culture in gelatin, 


. Spirillum tyrogenum, 
. Colonies of Spirillum resents 


FIG. 


185. 
186. 
187. 
188. 
189. 
190. 
191. 
192. 
193. 
194. 
195. 
196. 
197. 
198. 


LIST OF ILLUSTRATIONS. 


Spirillum Metschnikovi, . ; j 5 A m . 
Penicillum glaucum, : 
Miquel’s aéroscope, . ; é 4 : qi 

Hesse’s aéroscope, . ‘ ; x . i . 
Miquel’s flask, . : é . é . . 
Straus and Wiirtz’s soluble tiger: 5 - - 
Petri's sand filter, . . ; F . 

Sugar filter, . : . . . 


Sedgwick and Tucker’s spring: : 

Sternberg’s vacuum tube, 

Lepsius’ apparatus for collecting water at various depths, 
Koch’s plate method, é 
Smear preparation from liver of wallow: fear daduvrer, < 
Bacillus cadaveris grandis, 


“PART Finks 


CLASSIFICATION, MORPHOLOGY, AND GHNERAL 
BACTERIOLOGICAL TECHNOLOGY. 


I. Historicau. II. CuassiricaTion. JII. MorpHotocy. IV. Srarnina 
MetruHops. V. CuLTuRE Mepia. VI. STERILIZATION OF CULTURE 
Mepia. VII. CuLturrs in Liguip Mepia. VIII. Cuitures 
IN Sotip Mepra. IX. CuntTivation of ANAEROBIC Bac- 

TERIA. X. INCUBATING OVENS AND THERMO-REGU- 

LAToRS. XI. EXPERIMENTS UPON ANIMALS. 

XII. PHOTOGRAPHING BACTERIA. 


PART FIRST, 


I. 


HISTORICAL. 


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

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

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

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

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

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

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

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

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

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


4 HISTORICAL. 


isms, nearly allied to the alge. His classification will be found 
in the “‘ Dictionnaire Encyclop. des Sciences Médicales,” art. ‘* Bac- 
téries” (1868). This view is also sustained by the German botanist 
Cohn and is now generally accepted. 

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

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

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

Helmholtz, in 1843, repeated the experiments of Schwann with 
calcined air, and arrived at similar results—7.e., he found that the 
free admission of calcined air to boiled organic infusions did not pro- 
duce fermentation of any kind. 

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

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


HISTORICAL. 5 


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

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

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

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

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

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


6 HISTORICAL. 


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

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

Since it has been demonstrated that certain infectious diseases of 
man and the lower animals are due to organisms of this class, phy- 
sicians have been especially interested in bacteriological researches, 
and the progress made during the past fifteen years has been largely 
due to their investigations. It was a distinguished French physi- 
cian, Davaine, who first demonstrated the etiological relation of a 
micrvérganism of this class to a specific infectious disease. The an- 
thrax bacillus had been seen in the blood of animals dying from this 
disease by Pollender in 1849 and by Davaine in 1850, but it was sev- 
eral years later (1863) before the last-named observer claimed to 
have demonstrated by inoculation experiments the causal relation of 
the bacillus to the disease in question. 

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

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


HISTORICAL. v4 


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

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

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

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

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

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

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

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

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

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

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

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

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

The same year (188+) Loffler discovered the diphtheria bacillus. 

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


8 HISTORICAL. 


nitely the characters of the various microérganisms found in pus 
from acute abscesses, ete. 

The tetanus bacillus was discovered in 1854 by Nicolaier, a stu- 
dent in the laboratory of Prof. Fliigge, of Gottingen. That this 
bacillus is the cause of tetanus in man has been demonstrated by the 
subsequent researches of numerousinvestigators. For anexact knowl- 
edge of its biological characters we are especially indebted to Kitasato. 

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

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

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

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

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

The Annales de l’ Institut Pasteur is a monthly journal which 
has been published since 1888. Itis edited by Duclaux, and contains 
many important papers and reviews. «+ well as the statistics of the 


HISTORICAL. 9 


Pasteur Institute relating to preventive inoculations against hydro- 
phobia. 

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

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

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

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

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


IL. 


CLASSIFICATION. 


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


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

1. Bactertum—filaments linear and inflexible. 

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

3. Spirillum—filaments spiral, inflexible. 

4, Sptrochete—filaments spiral, flexible. 

Dujardin (1841) united the two genera Spirillum and Spirochete of 
reer and added to the description of the generic characters as fol- 
ows: 

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

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

3. Spirillum—filaments spiral, movement rotatory. 

It will be seen that this classification leaves no place for the motionless 
bacilli, such as the anthrax bacillus and many others, and does not include 
the spherical bacteria, now called micrococci. 

The classification of Davaine (1808) provides for the motionless, fila- 
mentous bacteria, but does not include the micrococci. This author first 
insisted that the vibrioniens cf Ehrenberg are truly vegetable organisms, 
allied to the algae. He makes four genera, as follows: 


Filaments straight or bent, ( Moving spontane- | Rigid Bacterium. 


but not in a spiral, ously, Flexible Vibrio. 
Motiouless, . Bacteridium. 
Filaments spiral, . : ‘ 3 : : ; Sprrillun, 


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

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


CLASSIFICATION. 1d 


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


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

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

1. The Mucorini, or mould fungi. 

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

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

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

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

2. NemMaTOGENES—cells disposed in filaments. 

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

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

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


THALLOPHYTES. 


Forms with chlorophyll. Forms without chlorophyll. 


Class I.—Protophytes. 


A. Cyanophycez (Oscillatoria- A. Schizomycetes (Bacteria). 
cee, etc.). 
B. Palmellacez. B. Saccharomycetes. 


12 CLASSIFICATION. 


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


Genera, 
Streptococcus, 

1. Coccocrea# —Up to the pre- Merismopedia, 
sent time, only known in the form of Sarcina, 
cocci. Micrococcus, 

Alscococcus. 

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

8. LEPTOTRICHES. — Spherical, . 
rod-shaped, and filamentous forms; ene 
the last show a difference between the ¢ pies midiothriz. 
two extremities ; filaments straight Te brie 
orspiral; sporeformation not known. D . 

4, CLADOTRICHE®. — Spherical, ) 
rod-shaped, filamentous, and spiral | 
forms ; the filamentous form pre- | Cladothriax. 
sents pseudo-branches ; spore forma- | 
tion not known. 1 


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

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


The classification of Baumgarten (1590) appears to us to have 
more practical value, and, with sligit modifications, we shall adopt 
it in the present volume. This author divides the bacteria into two 
principal groups, as follows : 

Group I. Species relatively monomorphous. 

GrovupP IL. Species pleomorphous. 

The first group includes the micrococc?, the bacilli, and the 
spirilla; the second group the spirulina of Hueppe, leptotrichece 
(Zopf), and cladotrichee. 

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

The Cocct, in the classification of Baumgarten, constitute a single 
genus with the following subgenera: 1, Diplococcus ; 2, Strepto- 
coccus ; 8, Merismopedia (Zopf)—‘‘ Merista” (Hueppe); 4, Sar- 
cina (Goodsir) ; 5, Micrococcus (‘‘ staphylococci”). 

The BacILui are included in a single genus embracing all of 


CLASSIFICATION. 13 


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

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

This simple morphological classification of the monomorphous 
group of Baumgarten corresponds with the nomenclature now gene- 
rally in use among bacteriologists. We speak of the spherical bac- 
teria as cocc2 or as micrococci, of the rod-shaped bacteria as bacilli, 
and of the spiral bacteria as spir7lla. 

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

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

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


14 CLASSIFICATION. 


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

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


CLASSIFICATION. 15 


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

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

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

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


16 CLASSIFICATION. 


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

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

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

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

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

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

' An account of this mode of reproduction is given ou page 19. 


CLASSIFICATION. 17 


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

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


MICROCOCCL. 


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

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

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

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

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

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

Association in pairs is common to all of the micrococci, inasmuch as 

‘they multiply by binary division. When such association has rather a per- 
manent character, it is customary to speak of the microdrganism as a diplo- 
coccus, but we doubt the propriety of recognizing this mode of association 
as a generic character. 

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

SaRcInA.—Division in three directions, forming packets of eight 

2 


18 CLASSIFICATION. 


or more elements, which remain associated in more or less regular 


cubical masses. 
BACILLI. 


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

BacILuus.—Characters as given above. 


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

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


SPIRILLA. 


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

SprRILLUM.—Characters as above. 


Spirochcete.—Flexible, spiral filaments; movements rotatory. 

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

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


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

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


CLASSIFICATION. 19 


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

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

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

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

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


Ti. 


MORPHOLOGY. 


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

The standard of measurement used by bacteriologists is the micro- 
millimetre, or the one-thousandth part of a millimetre. This is 
represented by the Greek letter 4. One yu (micromillimetre) is equal 
to about one-twenty-five-thousandth of an English inch. 

The spherical bacteria, or micrococci, differ greatly in size, and 
also in the mode of grouping when, as a result of binary division, 
they remain associated one with another. The smallest may mea- 
sure no more than 0.14, whilesome of the larger species are from 
one to two in diameter. The enormous number of these minute 
organisms which may be contained in a small drop of a pure culture 
may be easily estimated in a rough way. Compare a single micro- 
coccus, for example, with a sphere having a diameter of one-twenty- 
fifth of an inch. If our micrococcus is one of the larger sort, having 
a diameter of one , it would take a chain of one thousand to reach 
across the diameter of such a sphere, and its mass, as compared 
to the larger sphere, would be as 1 to 523,600,000. 

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

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

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


MORPHOLOGY. 21 


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

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


Fig. 1, 


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


Fig. 3. Fie. 4, 


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

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


22 MORPHOLOGY. 


remain united in chains (Fig. 4, a), they are described as streptococct, 
and are sometimes spoken of as in chaplets or in torula chains. In 
such chains we frequently find the evidence of recent division of the 
cocci, as shown by the grouping of the elements of the chain into 
pairs (Fig. 4, 0). 

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


8% 8B an 
8 OO 
eis b 
O8 
Fie. 5. 


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

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

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


MORPHOLOGY. 23 


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

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


COO Sa Of: oO y 
Saf 
eS C o 
Fic. 7. Fie. 8. 


a large central spore—‘‘clostridiwm”’—or one end may be dilated 
from the formation of a large terminal spore. 

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

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


= \ 
= Jer 
Fie. 9. 


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

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

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


24 MORPHOLOGY. 


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

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


Fie. 10. Fig. 11. Fig. 12. 


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

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


IV. 


STAINING METHODS. 


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

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

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


oe 


Fie. 13. 


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

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


26 STAINING METHODS. 


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

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

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

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

Most bacteriologists make their preparations upon the cover glass, 


STAINING METHODS. 27 


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

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

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

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


28 STAINING METHODS. 


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

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

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

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

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

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

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

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

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

If the preparation is not intended for permanent preservation, 


STAINING METHODS. 29 


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

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

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


1. Aniline-Gentian-Violet (Ehrlich). 


Saturated alcoholic solution of gentian violet, 
Aniline water, . ‘ : ‘i ‘ = : 100 cc. 


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


Saturated alcoholic solution of methyl violet, ‘ 11 ce. 
Absolute alcohol, . , : j : A 19 ee. 
Aniline water, ‘ : : : . j ‘ 100 ce. 


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


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


Fuchsin, ; ‘ ‘ 4 : ; . ‘ - igm. 
Alcohol, 4 . i : 4 3 : ‘ 10 ce. 


Dissolve and add 100 ce. of a fiv 2-per-cent solution of carbolic acid. 


4. Alkaline Blue Solution (Léffler’s solution). 


Saturated solution of methylene blue, ; . ‘ 30 ce. 
Solution of caustic potash of 1:10,000, : 5 100 ce. 


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

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

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


30 STAINING METHODS. 


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

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

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

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

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


Acid, nitric, pure, ‘ : : : 5 ce. 
Alcohol (eighty per cent), : ; “to 100 ce. 


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

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


STAINING METHODS. 31 


earbol-fuchsin solution (No. 3) the cover glass is placed for one or 
two minutes in a solution containing: 


Sulphurie acid (twenty-five-per-cent solution), : . 100 ce. 
Methylene blue, . Z . P 4 3 2 gms. 


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


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

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

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


32 STAINING METHODS. 


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

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

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

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

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

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

Léffler’s Method.—The following solution is used as a mordant: 


No. 1. 
Solution of tannin of twenty per cent, : A 10 ce. 
Saturated (cold) solution of ferrous sulphate, . ‘ . Bee 
Aqueous or alcoholic solution of fuchsin, 1ce. 


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


STAINING METHODS. 33 


No. 2. 
A one-per-cent solution of caustic soda. 


No. 3. 


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


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

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

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


‘Prepare the following solutions : 
3 


34 STAINING METHODS. 


A.—THE MoRDANT. 


Tannic acid, ten-per-cent aqueous solution, filtered, é 10 c.c. 
Corrosive sublimate, saturated aqueous solution, : . 5¢.¢. 
Alum, saturated aqueous solution, . : ‘ 5 ¢.c. 
Carbol-fuchsin solution,* ; i 5 . 5c. 


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


B.—THE STAIN. 


Alum, saturated watery solution, .  — . : ‘ 10 c.c. 
Gentian violet, saturated alcoholic solution, . ; . 26.6. 


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


METHODS OF STAINING BacTERIA IN TissuES.—The solutions re- 
commended for staining cover-glass preparations are also used in 
staining bacteria in thin sections of the various organs, in which 
they are found in certain infectious diseases; but, in general, a 
longer time is required to stain sections, and it is best not to hasten 
the process by the use of heat. To obtain good thin sections, the 
material, cut in small cubes, must be very thoroughly hardened in 
absolute alcohol. The piece selected for cutting may be attached to 
a cork by the use of melted glycerin jelly, which is hardened by 
placing the cork and attached piece of tissue in alcohol. This an- 
swers for well-hardened pieces of liver, kidney, etc., but the hollow 
viscera and tissues of loose structure will require embedding in 
paraffin or celloidin. Any well-made sledge microtome will answer 
for cutting the sections, if the knife is properly sharpened. The sec- 
tions should, of course, be cut under alcohol, and they can scarcely 
be too thin when the object is to demonstrate the presence or ab- 
sence of bacteria. Very thin sections may be cut dry by embedding 
in paraffin having a melting point of 59° C. In this case the knife 
is set at a right angle to the material to be cut, and the sections 
are spread out upon and attached to the glass slide for staining. 

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


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


STAINING METHODS. 35 


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

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

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


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


36 STAINING METHODS. 


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


The following method of staining sections for the puposs of demon- 
strating bacteria present in the tissues is recommended by reel (1891) asa 
substitute for the method of Kiihne. The results are said to be excellent, 
and it is much simpler and more expeditious. 

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

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

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


Vv. 


CULTURE MEDIA. 


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

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

NatursAL CuLTURE Mep1a.—The most important natural cul- 
ture medium is blood serum, which may be obtained from one of 
the lower animals—preferably from oxen or calves. This is to be 
collected in a sterilized jar, with every precaution to insure cleanli- 
ness, at the moment of slaughtering the animal. Or the blood of a 
calf, sheep, or dog may be collected at the laboratory by a carefully 
conducted operation, in which the femoral or carotid artery is con- 
nected with a sterilized glass tube leading into a sterilized receptacle, 
such as a Woulf’s bottle, into one neck of which a cotton plug has 
been placed to permit the air to escape as the bottle fills with 
blood through a tube which is secured in the other neck. When 
blood is passed directly from an artery into a sterilized receptacle 
the serum will not subsequently require sterilization. The writer isin 


38 CULTURE MEDIA. 


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


Fia. 14. Fia. 15. ihe, 16. 
as soon as it is withdrawn. The sterile blood serum may be pre- 
served indefinitely in this way, and may be used as a liquid culture 
medium in the little flask, or it may be transferred to a test tube 
and solidified by heat whenever a solid blood-serum medium is re- 
quired. The advantage of preserving blood serum and other liquid 
media in these little flasks is in the fact that they may be preserved 
indefinitely without becoming contaminated or drying up, and that 
they are easily transported, while a liquid medium in a test tube 
must be kept upright. The contents of one of these flasks are readily 


CULTURE MEDIA. 39 


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

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

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

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

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


40 CULTURE MEDIA. 


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

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

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

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

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

Liggs, sterilized by boiling, have been used by some bacteriolo- 
gists, especially for the cultivation of anaérobic species. 

ARTIFICIAL CULTURE MEp1a.—A great variety of liquid media 
have been employed by bacteriologists, the most useful of which are 
infusions of beef or mutton, with the addition of a little peptone. 
But Pasteur has shown that some species of bacteria will grow in a 
medium which does not contain any albuminous material, nitrogen 
being obtained from salts containing ammonia. 

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

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

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


CULTURE MEDIA. 41 


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

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

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

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

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

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

Sotip CuLrurE Mep1a.—The introduction of solid culture 
media, and especially the use of gelatin and agar-agar, as first 
recommended by Koch (1881), for the isolation and differentiation of 
species, was a most important advance in bacteriological technology. 
We are concerned here only with the composition and preparation 
of these media. 

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


42 CULTURE MEDIA. 


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


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

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


CULTURE MEDIA. 43 


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

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

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

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

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


Ad CULTURE MEDIA. 


Agar-gelatin, a medium which has recently come into favor and 
is said to be very useful, as it resembles gelatin in transparency and 
has a considerably higher melting point than ten-per-cent gelatin, is 
made by adding fifty grammes of gelatin and 7.5 grammes of agar 
to a litre of flesh-peptone solution. Care should be taken not to cook 
this longer than is necessary. 

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

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

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

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

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


CULTURE MEDIA. 45 


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


4--- SSS 


roy 


= 


Fie. 18. 


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


46 CULTURE MEDIA. 


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


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


For special purposes various substances are added to the above- 
described solid and liquid media. A favorable addition for the 
growth of a considerable number of bacteria is from one to three per 
cent of glucose. The phosphorescent bacteria grow best in amedium 
containing two to three per cent of sodium chloride. The addition 
of three to four per cent of potassium nitrate is made in conducting 
experiments designed to test the reducing power of certain bacteria, 
by which this salt is decomposed with the production of nitrites. 
Acids are also added in various proportion to test the ability of 
bacteria under investigation to grow in an acid medium. From 
1: 2,000 to 1:500 of hydrochloric acid may be used for this purpose. 
The addition of [itinus to milk or other culture media is fre- 
quently resorted to for the purpose of ascertaining whether acids or 
alkalies are developed during the growth of bacteria under investi- 
gation. The addition of aniline colors which are variously changed 
by the products of growth of certain species has also been resorted 
to in the differentiation of species. Various disinfecting agents, such 

' Abbott’s “Principles of Bacteriology ” Fifth edition. pp. 100 and 101, 


CULTURE MEDIA. 4” 


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

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

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


Ammonium sulphate, : : ; 0.4 gramme 
Magnesium sulphate, . 0.05 s 
Potassium phosphate, : 4 : 0.1 ~ 
Calcium chloride, : : ; a trace. 
Sodium carbonate, ‘ ; 0.6 to 0.9 gramme. 
Distilled water, : P : e 4 100 grammes. 


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

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

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

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

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


48 CULTURE MEDIA. 


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

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

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

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


CULTURE MEDIA. 49 


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

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


e eT 


‘titi iil i= 


rar i 


i 


Fie. 20, Fie. 21. Fie. 22. 


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


50 CULTURE MEDIA. 


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


This indicator is prepared by dissolving five grammes of commercial 
phenolphthaleinin one litre of fifty-per-cent alcohol. It is not feasible to use 
this indicator on strips of paper as the alcohol quickly evaporates, leaving the 
powder. The colorless liquid, however, may be added in small quantities to 
solutions of an acid or neutral nature without any change of color, but alka- 
lies quickly change it toa purple red. This change from no color to one of 
purple red makes the indicator a very satisfactory one, owing to the ease 
with which the eye detects the so-called end-point. ale 

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

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

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

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

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


CULTURE MEDIA. 51 


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

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


VI. 
STERILIZATION OF CULTURE MEDIA. 


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

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

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

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


STERILIZATION OF CULTURE MEDIA. 53 


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

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

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

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


54 STERILIZATION OF CULTURE MEDIA. 


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

It must be remembered that a much higher temperature is re- 
quired for the destruction of microédrganisms when dry heat is em- 
ployed than is the case with moist heat. The experiments of Koch 
and Wolffhiigel (1881) show that a temperature of 120° to 128° C. 
(248° to 262° F.) is required to destroy the spores of mould fungi, and 
micrococci or bacilli in the absence of spores. For the spores of ba- 
cilli a temperature of 140° C. (284° F.), maintained for three hours, 
was required. 

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


(302° F.) for an hour or more; and it is customary to sterilize all 
test tubes and flasks, which are to be used as receptacles for culture 
media, in the hot-air sterilizer. This procedure could no doubt, how- 
ever, be dispensed with in many cases and reliance be placed upon 
the sterilization of the flask, together with its contents, in the steam 
sterilizer, especially with such culture media as are not injured by 
long exposure to a boiling temperature—e.g., bouillon and agar-agar. 

When we propose to cultivate aérobic bacteria, or such as require 
oxygen for their development, a cotton air filter is placed in the 
mouth of each test tube and flask before it is sterilized in the hot-air 
oven. This is a loose plug of cotton, pushed into the neck of the 
flask for an inch or more, and projecting from its mouth for a short 
distance. These cotton filters should fill the tube completely and 


STERILIZATION OF CULTURE MEDIA. 55 


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

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


Hite 


Fie. 24. Fie. 25. 


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

Fig. 25 is a sectional view of this sterilizer. 

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


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


56 STERILIZATION OF CULTURE MEDIA. 


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

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


Mm 


Fie, 26, 


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


STERILIZATION OF CULTURE MEDIA. 57 


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

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

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


Fia. 28. 


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


58 STERILIZATION OF CULTURE MEDIA. 


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


Fic. 29, 


may remark that since it has been shown by Roux and Nocard that 
the tubercle bacillus grows very well in agar-agar jelly to which 
five per cent of glycerin has been added, blood serum is not so 
largely used as a culture medium in bacteriological laboratories. 
Sterilization by Filtration.—This method is especially useful 
for separating the soluble substances contained in a liquid culture of 
bacteria from the living cells. It has been demonstrated that several 
of the most important pathogenic bacteria produce toxic substances 
during their growth which may cause the death of susceptible ani- 
mals independently of the living bacteria ; and this demonstration 


STERILIZATION OF CULTURE MEDIA. 59 


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


wc 
= 


ie pe a ey 


si 
SSS 


con 


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

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


60 STERILIZATION OF CULTURE MEDIA. 


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


Fie. 31. 


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

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

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


STERILIZATION OF CULTURE MEDIA. 61 


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


Fia. 32. 


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


VIL. 


CULTURES IN LIQUID MEDIA. 


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

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

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


CULTURES IN LIQUID MEDIA. 63 


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

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

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


Fia, 33. Fig. 34. 


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


64 CULTURES IN LIQUID MEDIA. 


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

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

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

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


Fig. 35. 


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

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


CULTURES IN LIQUID MEDIA. 65 
of culture fluid, attached to a thin glass cover and suspended over a 
circular excavation ground out of a glass slide, is very useful. 
Such a drop culture may be left under the microscope and kept 
under observation for hours or days. 

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

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


Fie. 36. 


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

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

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

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

5 


66 CULTURES IN LIQUID MEDIA. 


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


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


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


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


CULTURES IN LIQUID MEDIA. 67 


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

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

To inoculate the liquid contained in one of these little flasks with mi- 
cro6rganisms from any source, the end of the tube is first heated to destroy 
germs attached to the exterior; the extremity is then broken off with steril- 
ized (by heat) forceps; the bulb is very gently heated, so as to force out a 
little air, and the open end is plunged into the liquid containing the organ- 
ism to be cultivated (or into a vein, or one of the solid viscera of an animal 
dead from an infectious germ disease, such as anthrax). 

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


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

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

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

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


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


68 CULTURES IN LIQUID MEDIA. 


mal death-point, consists in the application of heat and thus destroy- 
ing all except the most resistant species. This method is especially 
applicable when one of the species, only, forms spores. By subject- 
ing the mixed culture to a temperature which is sufficient to destroy 
all the vegetative cells in it, the more resistant spores are left and, 
under favorable conditions, may subsequently 
vegetate and give us a pure culture of the 
species to which they belong. 
Fermentation.—The development of certain 
bacteria is attended with an evolution of gas, 
especially in media containing grape sugar or 
glycerin. For the determination of the quantity 
and kind of gas produced by a given micro- 
organism the fermentation tube recommended 
by Theobald Smith has special advantages. 
This is a bent tube (Hihorn’s) supported upon 
a glass base as shown in the accompanying 
figure taken from the catalogue of Himer & 
Amend. The graduation shown upon the up- 
right arm is not essential for ordinary labora- 
tory work. A liquid culture medium containing 
one to two per cent of grape sugar is usually 
AGE es used. This is introduced into the upright arm 
of the fermentation tube, where it is held by atmospheric pressure. 
A cotton plug is placed in the opening of the short and bulbous arm 
of the tube, which is intended asa receptacle for the culture liquid 
when it is forced out of the closed arm by the accumulation of gas at 
its upper extremity. 


“umn 


VIII. 


CULTURES IN SOLID MEDIA. 


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

Koch’s flesh-peptone-gelatin, which contains ten per 
cent of gelatin, is a transparent jelly which liquefies at 
from 22° to 24°C. It isa favorable culture medium for 
a great number of bacteria, and many species show de- 
finite characters of growth in this medium which serve to 
differentiate them. One of the most prominent of these 
characters depends upon the fact that some bacteria liquefy 
gelatin and others do not. This is made apparent when 
we make “stab cultures.” This is the usual manner of 
inoculating a solid culture medium, and is illustrated in 
Fig. 39. <A platinum needle, consisting of a piece of 
platinum wire inserted into a glass rod which serves as a 
handle, is passed through the flame of an alcohol lamp to 
sterilize it. When cooled, which occurs very quickly, the 
point is introduced into the material containing the bac- 
teria to be planted in the gelatin medium. We may ob- 
tain our seed for a pure culture from a single colony, from Fic. 89, 
another stab culture, from the blood of an infected animal, 
etc. The point of the needle is then carried into the sterilized jelly, 
as shown in the figure, care being taken to introduce it in the central 
line and in a direction parallel with the sides of the tube. It is best 


70 CULTURES IN SOLID MEDIA. 


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

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


Fia. 40. 


sloping surface, as shown in Fig. 40, We may then draw the nee- 
dle over the surface in a longitudinal direction, and by this means 
distribute the seed in a line along which development will take place. 

The characters of growth in these stab cultures in gelatin are 


Fria, 40a. 


very various. Non-liquefying bacteria may grow only on the sur- 
face, as at a, Fig. 404; or both on the surface and along the line 
of puncture, as at b; or only at the bottom, as atc. In the first 
case the microdrganism is aérobic—that is, it requires oxygen, and 
grows only in the presence of this gas. In the second case it is 
not strictly aérobic, but may grow either in the presence of oxygen 


CULTURES IN SOLID MEDIA. V1 


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

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


“2 
os 
‘ iF 
é 4 
> t 4) 
4 : 
i 
0 Zi 2 
i 
; a 
& 
b 
ce J WS 
a b 
Fre. 41 


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

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


72 CULTURES IN SOLID MEDIA. 


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

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


¢ d 


Fie. 42. 


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


CULTURES IN SOLID MEDIA. 73 


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

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


Fie. 48.—Colonies of Bacteria. 


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

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


74 CULTURES IN SOLID MEDIA. 


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

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

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


CULTURES IN SOLID MEDIA. 75 


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


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

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

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


76 CULTURES IN SOLID MEDIA. 


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

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

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

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


~ 
= 


= 


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


CULTURES IN SOLID MEDIA. V7 


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

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

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

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

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


78 CULTURES IN SOLID MEDIA. 


neck of the little flask along the side of the cotton filter (see Fig. 46) 
and applying gentle heat to the bulb. The slender neck is first ste- 
rilized by passing it through a flame, and the point is broken off 
with sterile forceps. After inoculating the liquefied medium in the 
test tubes in the usual manner we may make plates or roll tubes. 
Cultures on Cooked Potato.—The method of preparing pota- 
toes for surface cultures has already been given (page 48). It was 
in using them that Koch first got his idea of the importance of solid 
media, which led to his introduction of the use of gelatin and agar- 
agar and the invention of the plate method. By means of streak 


ae 


Fic, 46. 


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

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


CULTURES IN SOLID MEDIA. 79 


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

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

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

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

Kruse (1894) pours the liquefied gelatin or agar into Petri dishes, 
and after it is solidified brushes the surface with a sterilized camel’s- 
hair brush which has been dipped into water containing in suspen- 
sion—properly diluted—the bacteria to be studied. By this procedure 
surface colonies only are obtained. Von Freudenreich (1894) prefers 
to pour the contents of the test tube upon the surface of the sterile 
medium, in Petri dishes. The fluid is allowed to run off by placing 
the Petri dish in a vertical position, and this is subsequently placed in 
the incubating oven in an inverted position—7.e., with cover below. 
To obtain satisfactory plates with well-separated, superficial colonies 
it may be necessary to use two or three dilutions, made in sterilized 
water in the usual way—17.e., from one tube to another, by means of 
the platinum wire having a loop at its extremity. 


IX. 
CULTIVATION OF ANAEROBIC BACTERIA. 


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


Fic. 48. 


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


CULTIVATION OF ANAEROBIC BACTERIA. 81 


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

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

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

The flask shown in Fig. 49, which can be made from a test tube, 
may also be used in connection with a hydrogen apparatus. In this 


case a slender glass tube is passed into the flask, as shown in Fig. 
6 


82 CULTIVATION OF ANAEROBIC BACTERIA. 


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


A 


Fie. 49, Fia. 50. Fic. 51. 


who are not skilful in the use of the blowpipe to use Salomongon’s 
tube, shown in Fig. 51. In this, hydrogen is admitted through the 
arm b, and escapes through the cotton plug a. The vertical tube is 
sealed at c while the gas is flowing, and then the horizontal tube at b. 

Frdnkel’s Method.—Instead of these tubes specially made for 
the purpose, an ordinary test tube may be used, as recommended by 
Frankel. This is closed by a soft rubber cork through which two 
glass tubes pass—one, reaching nearly to the bottom of the test tube, 


CULTIVATION OF ANAEROBIC BACTERIA. 83 


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

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


¢ 
b 
eee 
ay 
¢ 


= 


in 
LS Se 


A 


Fie. 52, Fie. 53. 


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

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

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


84 CULTIVATION OF ANAEROBIC BACTERIA. 


test tube for about half an inch, as shown at 6b, Fig. 53. The space 
above the cork is then filled with melted sealing wax, which I have 
found to prevent leakage better than paraffin, which contracts upon 
cooling. The test tube is inverted while hydrogen is passed through 
the tube c, and by reason of its levity the gas quickly passes through 
the cotton air filter and displaces the oxygen in the test tube (Fig. 
54). After allowing the gas to flow for a few minutes the outlet 
tube is first sealed in a flame and then the inlet tube. As the cotton 
filter is interposed between the rubber stopper and the culture mate- 
rial, no special precautions need be taken for the sterilization of the 
rubber cork and the glass tubes which it carries. 


Fie. 54. Fie. 55. 


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

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


CULTIVATION OF ANAEROBIC BACTERIA. 85 


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

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


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

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

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


86 CULTIVATION OF ANAEROBIC BACTERIA. 


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

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


Fia. 57, 


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


potash. 
Evidently plates prepared by Koch’s method, or Esmarch roll 


CULTIVATION OF ANAEROBIC BACTERIA. 87 


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

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

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


X. 


INCUBATING OVENS AND THERMO-REGULATORS. 


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

The ordinary thermo-regulator is shown in Fig. 59 as manufac- 
tured by Rohrbeck. A glass receptacle, shaped like an ordinary 
test tube, has an arm, c, for the escape of the gas, which enters by 
the bent tube a, which passes through a perforated cork and is ad- 
justable up and down. Tube a is connected with the gas supply and 
tube ¢ with the burner by means of rubber tubing. <A glass parti- 
tion extending downward as a tube, g, makes an enclosed space in 


INCUBATING OVENS AND THERMO-REGULATORS, 89 


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


Fia. 58. 


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


90 INCUBATING OVENS AND THERMO-REGULATORS. 


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

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


ese 


Fig. 59, Fig. 60. 


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

The regulator of Bohr, shown in Fig. 64, is more sensitive than 
that of Reichert, and rather simpler in construction than the usual 
form shown in Fig. 59. The thermometer bulb a contains only air, 
and the gas which passes through the tube f is shut off at the 
proper temperature by the mercury in the U-shaped tube c. The 
stopcock 6 is left open when the bulb @ is immersed in the water 


INCUBATING OVENS AND THERMO-REGULATORS. 91 


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


Fre. 61. : Fig, 62. 


Fig. 63. Fig. 64, 


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


92 INCUBATING OVENS AND THERMO-REGULATORS. 


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

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


b 


Fic. 65. Fie. 66, 


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

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


ce 


Whe 
- 


Fic. 67. 


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


INCUBATING OVENS AND THERMO-REGULATORS. 93 


Another platinum wire passes down the tube of the thermometer, b, 
and is adjustable for any desired temperature. The gas passes 
through a valve which is controlled by an electro-magnet. A 
simple form of valve is shown in Fig. 67. The bent tube @ is con- 
nected with the gas supply by a piece of rubber tubing. The up- 
tight arm of this tube is enclosed in a larger tube, b, having an out- 


PEGA T OLE LOC TET 


G AT 

i = 

Te TL Le Wy 
CES | 


i mm TTT Ta TTT 


nn we em i e 


7 

Fie. 68. : Fic. 69. 
let, e, which is connected with the burner under the incubating 
oven. The upper end of this larger tube is closed by means of a 
piece of sheet rubber, which prevents the escape of gas. When this 
is depressed by means of the lever c, the flow of gas through the 
valve is arrested. The lever c has attached to it the armature d, 
and is operated by an electro-magnet under the control of the regu- 
lating thermometer. 


94 INCUBATING OVENS AND THERMO-REGULATORS. 


When the thermometer is immersed in a water bath the tem- 
perature of which it is desired to regulate, and the proper electric 
connections are made, it acts as a circuit breaker. When the de- 
sired temperature is reached the mercury in the tube of the ther- 
mometer touches the wire b (Fig. 66), an electric circuit is com- 
pleted, and the valve is closed, shutting off the gas supply and 
preventing the temperature from going any higher. When contact 
is broken in the thermometer tube the valve opens and permits the 
gas to flow again. A small opening, 0 (Fig. 67), permits the con- 
stant flow of a sufficient amount of gas to prevent the flame from 
being extinguished. In practice, however, it is better to have a 
small side jet of gas, quite independent of that which passes through 
the valve, which burns constantly and relights the principal jet when 


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

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


INCUBATING OVENS AND THERMO-REGULATORS. 95 


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

In the absence of gas incubating ovens may be heated by a small 
petroleum lamp, and various devices have been invented for control- 
ling the temperature. Reichenbach describes an apparatus for this 
purpose in the Centralblatt fiir Bakteriologie, Vol. XV., p. 847, 
1894. Dr. Borden of the U. 8. Army has also invented a thermo- 
regulator to be used in connection witha petroleum lamp. In the 
absence of any regulating apparatus an incubating oven may be kept 
at a tolerably uniform temperature by personal supervision—adjusting 
the flame of the lamp and its distance from the bottom of the oven ac- 
cording to the changes in the external temperature. For most bac- 
teria a variation of several degrees is not important, so long as the 
temperature is not allowed to rise above 37° to 38° C. The typhoid 
bacillus, the diphtheria bacillus, the anthrax bacillus, the pus cocci, 
and most saprophytic bacteria grow at the ordinary room temperature, 
apd may therefore be cultivated without any form of incubating oven 
or thermo-regulator. 


XI. 
EXPERIMENTS UPON ANIMALS. 


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

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

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


EXPERIMENTS UPON ANIMALS. 97 


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

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


Fig. 71. 


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

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


Fig. 72, 


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


w 
‘ 


98 EXPERIMENTS UPON ANIMALS, 


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

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

In making injections into the peidionaal cavity care must be taken 
not to wound the liver or the distended stomach. The intestine is 
not very likely to be wounded, as it slips out of the way. By seizing 

‘a longitudinal fold of the abdominal wall and pushing the point of 
the syringe quite through it, and then releasing the fold and care- 
fully withdrawing the instrument until the point remains in the 
cavity, the danger of wounding the intestine will be reduced to a 
minimum. 

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


EXPERIMENTS UPON ANIMALS. 99) 


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

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

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

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

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

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


100 EXPERIMENTS UPON ANIMALS. 


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

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

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

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

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

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

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

(e) The quantity 7njected is evidently an essential point when 
the result depends largely upon the toxic products formed in the cul- 


EXPERIMENTS UPON ANIMALS. 101 


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

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

In making a post-mortem examination of an inoculated animal it 
is best to stretch it out on a board, belly up, by tying its legs to nails 
or screws fastened in the margin of the board. When the abdomen 
is dirty, as is usually the case, it should be carefully washed with a 
disinfecting solution. An incision through the skin is then made in 
the median line the full length of the body, and the skin is dis- 
sected back so as to expose the anterior walls of the abdomen and 
thorax. These cavities are then carefully opened with a sterilized 
knife or scissors, and the various organs and viscera examined. At- 
tention should also be given to the appearances at the point of in- 
oculation. To ascertain whether the microdrganism injected has 
invaded the blood, smear preparations should be made with blood 
obtained from a vein or from one of the cavities of the heart. It 
will be well also to make a smear preparation from a cut surface of 
the liver and spleen. In the various forms of acute septicemia the 
spleen is usually found to be enlarged. If but few microdrganisms 
are present in the blood and tissues they may escape observation in 
stained smear preparations, and it will be necessary to make cultures 
to demonstrate their presence. A little blood from a vein or from 
one of the cavities of the heart is transferred, by means of a plati- 
num loop (6se) or a sterilized collecting tube (see page 38), to a 
test tube containing liquefied nutrient gelatin or agar-agar, and an 
Esmarch roll tube is made. This is put aside for the development of 
colonies from any scattered bacteria which may be present. As a 
rule, it will be best to make agar cultures, as these can be placed in 
the incubating oven at 35° to 38°C. Stab cultures may also be 
made and will serve to show the presence of microdrganisms, but 
will not give information as to how numerous they may be. The 
roll tube also has the advantage of showing whether there is a 


102 EXPERIMENTS UPON ANIMALS. 


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

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

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


XII. 


PHOTOGRAPHING BACTERIA. 


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

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


1 The paper referred to is published in Cohn’s ‘‘Beitriige zur Biologie d. Pflanzen.” 


104 PHOTOGRAPHING BACTERIA. 


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

Those who have had no practical experience in making photo- 
micrographs are apt to expect too much and to underestimate the 
technical difficulties. Objects which under the microscope give a 
beautiful picture, which we desire to reproduce by photography, may 
be entirely unsuited for the purpose. In photographing with high 
powers it is necessary that the objects to be photographed be in a 
single plane and not crowded together or overlying each other. 
For this reason photographing bacteria in sections presents special 
difficulties, and satisfactory results can only be obtained when the 
sections are extremely thin and the bacteria well stained. Even 
with the best preparations of this kind much care must be taken in 
selecting a field for photography. It must be remembered that the 
expert microscopist, in examining a section with high powers, has 
his finger on the fine adjustment screw and focuses up and down to 
bring different planes into view. He is in the habit of fixing his at- 
tention on that part of the field which is in the focus and disregard- 
ing the rest. But in a photograph the part of the field not in focus 
appears in a prominent way which mars the beauty of the picture. 
In a cover-glass preparation made from a pure culture, when the 
bacteria are well distributed, this difficulty does not present itself, as 
the bacteria are all lying in a single plane; but the portion of the field 

which can be shown at one time is limited by the spherical aberra- 
tion of the objective, which the makers do not seem able to overcome 
in high-power lenses of wide angle, at least not without loss of de- 
fining power. 

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


PHOTOGRAPHING BACTERIA. 105 


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

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

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

To show bacteria in photographs in a satisfactory manner we 


106 PHOTOGRAPHING BACTERIA. 


require an amplification of five hundred to one thousand diameters ; 
and as it is often desirable to make comparisons as to the dimen- 
sions of microérganisms which resemble each other in form, it is 
best to adopt a standard amplification. The writer has himself 
adopted, and would recommend to others, a standard amplification 
of one thousand diameters. This is about as high a magnifying 
power as we can get with satisfactory definition, or as we require, 
and it is a convenient number when measurements are made from 
the photograph. The beginner, after having put his apparatus in 
position, should focus the lines of a stage micrometer upon the 
screen with the optical apparatus which he proposes to use ; then by 
moving the screen forward or back as required, and carefully focus- 
sing the lines, he will ascertain what is the position of the screen for 
exactly one thousand diameters. If the stage micrometer is ruled 
with lines which are one one-thousandth of an inch apart, it is evi- 
dent that when projected upon the screen they should be one inch 
apart to make the amplification one thousand diameters. But it 
must be remembered that any change in the position of the optical 
combination will change the amplification. If, therefore, the cover 
correction of the objective is changed, or the position of the eyepi2ce 
—if one is used—it will be necessary to again adjust the distance of 
the screen. 

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

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

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

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


PHOTOGRAPHING BACTERIA. 107 


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

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

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

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

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


108 PHOTOGRAPHING BACTERIA. 


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


Fie, 73. 


| | 
ait 
4 
I) 
I) 
i | 
| q 


a= 


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


We cannot in the present volume give full details with reference 


PHOTOGRAPHING BACTERIA. 109 


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


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

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

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

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

‘‘T am in the habit of using a yellow screen for my preparations stained 
with fuchsin or methylene blue, and have obtained very satisfactory results 
with the orthochromatic plates manufactured by Carbutt, of Philadelphia, 
and a glass screen coated with a solution of tropeeolin dissolved in gelatin, 

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

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

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


110 PHOTOGRAPHING BACTERIA. 


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


Pron 
oe) it] 


ELLE LTE CL EEO LL LDL ODEO LLL EH LLC LALLA ALLY LATTA TT ODL TERT LLU ET 


Fie. 74, 


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

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


I have recently (1895) seen a gaslight which I believe would prove 
to be a valuable substitute for ordinary street gas, and I judge that, 
owing to its superior brilliancy, a single jet would suffice to replace the 
five burners in a linear series which are shown in the above figure. 
The gas referred to is acetylene, which may now be obtained in a 
liquid form in strong metal cylinders. Reference has already been 
made to the use of an oil light, and for low powers an ordinary lamp 
with a flat wick may be used. That bacteria may be successfully 
photographed, with an amplification of one thousand diameters, by 
means of an oil lamp is shown by the beautiful photomicrographs 
made by Capt. W. C. Borden, Assistant Surgeon U. 8S. Army. 


At my request Dr. Borden has prepared the following detailed 
account of his method: 


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


PHOTOGRAPHING BACTERIA, 111 


DESCRIPTION OF APPARATUS FOR PHOTOMICROGRAPHY BY OIL 
LIGHT. 


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

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

Microscope.—The microscope is used in the upright position. I have 
used this position rather than the horizontal for several reasons. The 
microscope is used on the work-table in an upright position, and in working 
when an object is found which it is desired to photograph, the microscope 
without changing adjustments has only to be carried to the photomicro- 
' graphic apparatus, placed in position, correct adjustments of light made, the 
camera racked into contact and the exposure made. With a conveniently 
placed dark room the whole operation will occupy but a few minutes. The 
upright position is necessitated when liquid preparations, as colonies of 
bacteria floating on liquefied gelatin, are to be photographed, or when the 
microscope is used for clinical photomicrography, as in photographing uri- 
nary deposits in urine, blood corpuscles in Thoma blood counter, ete. In 
bacteriological work where the bacteria are stained on the cover and after 
mounting the balsam is not quite dry, the cover is apt to slip if the micro- 
scope is used horizontally, but this doesnot occur with the microscope placed 
vertically. The horizontal position and long extension of camera is neces- 
sary for certain work, particularly where large pictures (7.e., over four 
inches in diameter) have to be taken, or where it is desired to obtain high am- 
plification by extension of camera rather than by high eyepiecing, or in 
photographing test diatoms with very high amplifications. For practical 
work, however, up to amplifications of one thousand diameters, and for 
photographs for illustration or reproduction, which are seldom required of 
over three and one-half or four inches in diameter, the upright position is 
much to be preferred on account of its ease of application and its practical 
advantages. 

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

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


112 PHOTOGRAPHING BACTERIA. 


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

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

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

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

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

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


PHOTOGRAPHING BACTERIA. 113 


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

It is with these plates that I have found a color screen unnecessary, and 
since using them I have had no difficulty in photographing bacteria, for they 
are particularly sensitive to the yellow-colored oil light. 

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

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


No. 1. 
Water, .| 3 ‘ i : : : 10 ounces. 
Sodium sulphite,  . ; : 1 ounce. 
Potassium bromide, : ‘ ‘ : 10 grains. 
Hydrochinon, . : . 30 grains. 
Metol, 3 ‘ i ‘ ‘ 3 : 40 grains. 
No. 2. 
Water, , i ‘ ‘ : : . 10 ounces. 


Sodium carbonate, 


: ‘ 300 grains. 
Use equal parts of No. 1 and No. 2. 


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


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

8 


PLATE I. 


PHOTOMICROGRAPHS OF BACTERIA MADE BY GASLIGHT., 


Fia. 1.—Streptococcus cadaveris, froma culture in aguacoco; stained 
with fuchsin. x 1,000. (Sternberg.) 

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

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

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

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


PLATE I1. 


PHOTOGRAPHS OF COLONIES (IN ESMARCH ROLL TUBES) AND OF TEST- 
TUBE CULTURES. 


Fie. 1.—Colonies of Bacillus leporis lethalis, in gelatin roll tube, 
end of forty-eight hours at room temperature. x5. (Sternberg.) 

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

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

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

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

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

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

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


PLATE TI. 
STERNBERG’S BACTERIOLOGY. 


PHOTOMICROGRAPHS BY GAS LIGHT. 


PLATE IL. 
STERNBERG’S BACTERIOLOGY, 


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= fe e a 
= €€ oa ° 


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Fig. 2. 


Fig. 3. Fig. 4. 


Fig. 8. 


COLONIES AND TEST TUBE CULTURES. 


PART SECOND. 


GENERAL BIOLOGICAL CHARACTERS : 


INCLUDING AN ACCOUNT OF THE ACTION OF ANTISEPTICS 
AND GERMICIDES, 


I. Srructurze, Motions, REPRODUCTION. II. CoNDITIONS OF GROWTH. 
III. MopiricaTions oF BIioLOGICAL CHARACTERS. IV. Propucts oF 
VITAL ACTIVITY. V. PTOMAINES AND TOXALBUMINS. VI. INFLUENCE 
oF PHysicaL AGENTS. VII. ANTISEPTICS AND DISINFECTANTS 
—GENERAL ACCOUNT OF THE ACTION OF. VIII. ACTION OF 
GASES AND OF THE HALOID ELEMENTS UPON BACTERIA. 

IX. AcTION of ACIDS AND ALKALIES. X. ACTION OF 
VARIOUS SALTS. XI. ACTION OF COAL-TAR PRO- 

Ducts, EssENTIAL OILS, ETC. XII. Ac- 

TION OF BLOOD SERUM AND OTHER OR- 

GaNic Liquips. XIII. PrRactTicaL 
DIRECTIONS FOR DISINFECTION, 


PART SECOND, 


J 


STRUCTURE, MOTIONS, REPRODUCTION. 


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

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

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


118 STRUCTURE, MOTIONS, REPRODUCTION. 


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

In his address before the International Medical Congress of Ber- 
lin (1890) Koch says : 

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

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

The granules in bacterial cells which may be demonstrated by 
special methods of staining are of two kinds: metachromatic gran- 
ules and polar granules. The former lie in the protoplasm, and 
when properly stained may present the appearance of a short chain 
of cocci lying in the bacterial cell. To demonstrate their presence 
Ernst recommends the use of L6ffler’s solution of methylene blue. 
This is placed upon a coyer-glass preparation and heated over a 
flame until steam begins to rise. After washing in water the cover 
glass is placed for a minute or two in a watery solution of Bismarck 
brown. This shows the granules stained blue and the surrounding 
protoplasm brown. The polar granules are often seen in prepara- 
tions stained in the usual way with an aniline staining solution. 
Some observers have regarded these stained granules as spores, but 
this has not been demonstrated, and cultures containing them show 
no greater resistance to heat or to chemical agents than that estab- 
lished for the vegetative cells of the particular species in which they 
are found. It seems probable that the matachromatic granules re- 
sult from degenerative changes rather than that they are reproduc- 
tive bodies. 


STRUCTURE, MOTIONS, REPRODUCTION. 119 


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

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

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

It must be remembered that the molecular movement which is 
common to all minute particles suspended in a fluid is a vibratory 
motion 7 s?tu, which does not change the relative position of the 
moving particles. This so-called Brownian movement has frequently 
been mistaken for a vital motion, as has also the movement due to 
currents in the liquid in which non-motile organisms are suspended. 
The latter is to be distinguished by the fact that the microédrganisms 
are all carried in one direction. This movement due to a current, in 
connection with the vibratory Brownian movement, is very deceptive, 
and it is often hard for a beginner in bacteriological study to con- 
vince himself that what he sees is not a vital movement. But in 
true vital movements we have progression in different directions, and 
the individual microérganisms approach and pass each other, often 
in a most vigorous and active manner, passing entirely across the 
field of view or changing direction in an abrupt way. Sometimes 
the motion is slow and deliberate, the bacillus progressing with a to- 
and-fro motion, as if propelled by a trailing flagellum ; or it may be 
serpentine when the moving filament is flexible; or again it is 
a darting forward motion which is so rapid that the eye can scarcely 
follow the moving body. The spirilla have a rotary movement as 


120 STRUCTURE, MOTIONS, REPRODUCTION. 


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

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

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

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

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


STRUCTURE, MOTIONS, REPRODUCTION. 121 


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

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

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


Fie. 75. 


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


122 STRUCTURE, MOTIONS, REPRODUCTION. 


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

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

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


°"- OO@ €@= 
b-- 0 


Fie. 76. 


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

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


STRUCTURE, MOTIONS, REPRODUCTION. 123 


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

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

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

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

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


124 STRUCTURE, MOTIONS, REPRODUCTION. 


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

The chemical composition of the bacterial cells has been inves- 
tigated by Nencki, Brieger, and others. Putrefactive bacteria culti- 
vated in a two-per-cent solution of gelatin, and which produced an 
abundant intercellular substance connecting the cells in zodgloea 
masses, were found by Nencki to have the following composition : 
Water, 84.26 per cent; solids, 5.74 per cent, consisting of albumin 
87.46 per cent, fat 6.41, ash 3.04, undetermined remnant 3.09. 
The albuminous substance, according to Nencki, is not precipitated 
by alcohol, and differs in its chemical composition from other known 
substances of this class. He calls it myhoprotein and gives the fol- 
lowing as its chemical composition : C, 52.32 per cent; H, 7.55 per 
cent ; N, 14.75 per cent. It contains no sulphur and no phosphorus. 
The spores of the anthrax bacillus, according to Nencki, do not con- 
tain mykoprotein, but a peculiar albuminous substance which he 
calls anthrax-protein. Brieger analyzed a gelatin culture of Fried- 
lander’s bacillus, with the following result : Water, 84.2 per cent ; 
solids, 5.8 per cent, containing 1.74 per cent of fats. After removal 
of the fat the solids gave an ash of 30.13 per cent ; this contains cal- 
cium phosphate, magnesium phosphate, sodium sulphate, and sodium 
chloride. The amount of nitrogen in the dried substance after re- 
moval of the fat was 9.75. 


IL. 


CONDITIONS OF GROWTH. 


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

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

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

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


126 CONDITIONS OF GROWTH, 


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

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

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


PASTEUR’S SOLUTION. 


Distilled water, : ‘ ‘ A : 100 
Cane sugar, ‘ ‘ 5 ; 10 
Tartrate of ammonia, ; i a Z 

Ashes of one gramme of yeast, i ‘ F : 0.075 


COHN’S SOLUTION. 


Distilled water, 3 : . . : 100 
Tartrate of ammonia, : : ‘ : ? 1 
Ashes of yeast, . ‘ ‘ ‘ 5 ¥ é 1 


Many bacteria multiply abundantly in these solutions. 

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

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

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


CONDITIONS OF GROWTH. 127 


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

Miquel some years ago found a bacillus in the water-of the Seine 
which grew at a temperature of 69° to 70° C.; Van Tieghem reports 
having observed species in thermal waters capable of growth at a 
still higher temperature (74° C.) ; and Globig has more recently ob- 
tained from garden earth several species which multiplied at 65° C. 
Some of the species found by the last-named observer were even 
found to require a temperature of about 60° for their development; 
and yet this temperature is quickly fatal to a large number of the 
best known species. 

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

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

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

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


128 CONDITIONS OF GROWTH. 


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

Substances favorable for the growth of a particular species may 
restrain its development if present in too large an amount. Thus 
the phosphorescent bacilli multiply abundantly in a nutrient solution 
containing 2.5 per cent of sodium chloride ; but this amount would 
restrain the development of some other species, and a considerable 
increase in the quantity of salt prevents the growth of all microér- 
ganisms. In the same way the addition of two per cent of glucose 
to culture solutions is favorable for the development of certain spe- 
cies, and especially for the anaérobic bacteria ; but a concentrated 
solution of the same substance prevents the growth of all bacteria. 

The influence of one species upon the growth of another has 
been studied by various bacteriologists, and especially by Sirotinin 
and by Freudenreich. When several species are associated in the 
same culture one may take the precedence and the others may de- 
velop later ; or two or more species may develop at the same time ; 
or the growth of one species may prevent the development of an- 
other, either (a) by exhausting the pxbulum necessary for its growth 
or (0) by producing substances which inhibit the development of an- 
other species or destroy its vitality. 

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


IIT. 


MODIFICATIONS OF BIOLOGICAL CHARACTERS, 


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

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

9 


130 MODIFICATIONS OF BIOLOGICAL CHARACTERS. 


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

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

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

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

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


MODIFICATIONS OF BIOLOGICAL CHARACTERS. 131 


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

In the case of the anthrax bacillus it was found that the spores 
retain their full virulence for years, and that the production of an at- 
tenuated virus required the exclusion of these reproductive elements. 
Cultivations were consequently made at a temperature of £2° to 43° 
C., at which point this bacillus is incapable of producing spores. 
Cultivation at this temperature for eight days gave an attenuated 
virus suitable for use in protective inoculations. 

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

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


132 MODIFICATIONS OF BIOLOGICAL CHARACTERS. 


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

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

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

Pasteur had previously shown (1882) that the virus of 1ouget can 
be attenuated by passing it through rabbits. 

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

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


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


TV. 


PRODUCTS OF VITAL ACTIVITY. 


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

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

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


134 PRODUCTS OF VITAL ACTIVITY. 


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

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

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

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


STERNBERG'S BACTERIOLOGY, Plate Il. 


ake 


t 


Rea ats 


7 » ) 
Janes 2 


Fig 3 Bacillus pyocyanus, agar culture. 
Fig. Bacillus Havaniensis, potato culture 


PRODUCTS OF VITAL ACTIVITY. 135 


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

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

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

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

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

In a study of the ferments produced by bacteria which cause 
liquefaction of gelatin—“ tryptic enzymes”—made by Fermi, in the 


136 PRODUCTS OF VITAL ACTIVITY. 


laboratory of the Hygienic Institute of Munich (1891), the following 
results were obtained: 

The enzymes were ~ot obtained pure, and their isolation from 
other proteids present in the cultures was found to be attended with 
great difficulties, but their ferment action was studied and was found 
to be influenced by various conditions. 

All were destroyed by a temperature of 70° C., but the enzymes 
produced by various liquefying bacteria differed considerably as to 
the temperature which they were able to withstand. Some were de- 
stroyed by a temperature of 50° to 55° C.—Bacillus megatherium, 
Bacillus ramosus, Staphylococcus pyogenes aureus; some by a tem- 
perature of 55° to 60° C.—Bacillus subtilis, Bacillus pyocyaneus, Ba- 
cillus fluorescens liquefaciens, Sarcina aurantiaca; some by 65° to 
70° C.—Bacillus anthracis, Spirillum cholerz Asiatice, Spirillum of 
Finkler and Prior, Spirillum tyrogenum. 

These enzymes, like the previously known pepsin, trypsin, and 
invertin, do not dialyze. 

Only a few of these bacteria enzymes acted upon fibrin, and no 
action was observed upon casein or upon egg albumen. 

Their liquefying action upon gelatin was prevented by the action 
of sulphuric acid, and to a less degree by nitric acid, but was not in- 
terfered with by acetic acid. 

The liquefying bacteria, as a rule, only produce enzymes when 
cultivated in a medium containing albumen. 

These enzymes are not produced by a solution of the protoplasm 
of dead bacterial cells, but are a product of the vital activity of liv- 
ing cells. 

Among the numerous liquefying bacteria known to bacteriolo- 
gists we may mention the following species as deserving the student’s 
special attention: Staphylococcus pyogenes aureus, Staphylococcus 
pyogenes albus, Sarcina lutea, Sarcina aurantiaca, Bacillus anthra- 
cis, Bacillus pyocyaneus, Bacillus subtilis, Bacillus indicus, Bacillus 
prodigiosus, Spirillum cholerze Asiatice, Spirillum of Finkler and 
Prior, Proteus vulgaris. ; 

Fermentation.—The fermentation produced by various species of 
bacteria in culture solutions containing saccharose, glucose, or lac- 
tose constitutes a valuable character for the differentiation of species. 
While some bacteria give rise to fermentation in solutions contain- 
ing either of the carbohydrates above mentioned, others break up lac- 
tose, but have no effect upon glucose or saccharose, and others again 
are without any ferment action. The gases evolved are chiefly car- 
bon dioxide and hydrogen. Ferment action may be tested by adding 
one to two per cent of glucose to a solid culture medium—preferably 
agar-agar. This is liquefied by heat in the test tube containing it and 


PRODUCTS OF VITAL ACTIVITY. 137 


asmall quantity (one ése) of the microdrganism to be tested is intro- 
duced. The culture medium is then quickly solidified by placing the 
test tube in iced water. It is then placed in the incubator, and when 
colonies form bubbles of gas will be seen in their vicinity, if the bac- 
terium under observation is able to cause fermentation of glucose. 
For accurate observations as to the quantity and nature of the gases 
produced the fermentation tube should be used, as recommended by 
Theobald Smith (see Fig. 38). 

Production of 4ceids.—Numerous bacteria give an acid reaction 
to the media in which they are cultivated, and the acids produced 
are various—lactic, acetic, butyric, propionic, succinic, etc. 

The power to produce an acid is well shown by adding to neu- 
tral or alkaline culture media a solution of litmus. The change in 
color due to the formation of an acid may be followed by the eye, 
and comparative tests may be made to aid in the differentiation of 
similar bacteria. 

A considerable number of bacteria are able to produce lactic 
acid from milk sugar and other carbohydrates. One of these is 
considered the special lactic-acid ferment—Bacillus acidi lactici—and 
is the usual cause of the acid fermentation of milk. Pure cultures 
of this bacillus introduced into sterilized milk or solutions of milk 
sugar, cane sugar, dextrin, or mannite, give rise to the lactic-acid 
fermentation, in which carbonic acid is also set free. The process 
requires free access of oxygen, and progresses most favorably at a 
temperature of 35° to 40° C., ceasing at about 45°. In milk, coagu- 
lation of the casein occurs within fifteen to twenty-four hours after 
adding a small quantity of a pure culture of the lactic-acid bacillus. 
This is not due, however, to the acid fermentation, but to a ferment 
resembling that of rennet, which is produced by many different 
bacteria, some of which do not produce an acid reaction of the milk. 
Among the bacteria which produce lactic acid from milk sugar we 
may mention the staphylococci of pus, Bacillus lactis aérogenes, and 
Bacillus coli communis. 

The formula showing the transformation of sugar into lactic 
acid is usually stated as follows : C,H,,O, = 2(HC,H,0O,). 

Acetic acid is also produced from dilute solutions of alcohol by 
the action of a special bacterial ferment, which accumulates upon 
the surface of the fluid as a mycoderma, consisting almost entirely 
of the Bacillus aceticus (Mycoderma aceti). Free access of oxygen 
is required, and a temperature of about 33° C. is most favorable to 
the process. According to Duclaux, the ‘‘ Mycoderma aceti” oxi- 
dizes the alcohol, in solutions containing it, so long as any is present, 
and when it is exhausted it oxidizes the acetic acid previously 


138 PRODUCTS OF VITAL ACTIVITY. 


formed by oxidation of the alcohol, producing from it carbon diox- 
ide and water. 

The formation of acetic acid from alcohol is shown by the follow- 
ing formula : Ethyl alcohol CH,.CH,.0OH+ 0, = CH,.COOH+H,0. 

Butyric acid is produced by a considerable number of bacteria, 
one of which, named Bacillus butyricus, has received the special at- 
tention of Prazmowski. This is strictly anaérobic. In solutions of 
starch, dextrin, sugar, or salts of lactic acid, when oxygen is ex- 
cluded it produces butyric acid in considerable quantity, and at the 
same time carbon dioxide and hydrogen gas are set free. Duclaux 
gives the following formula of a solution containing lactate of lime 
in which the action of the butyric-acid ferment may be well studied : 


‘Water, . : ; 8 to 10 litres. 
Lactate of lime (pure), : . ; : 225 grammes. 
Phosphate of ammonia, : . 5 : 0.75 
Phosphate of potash,. : - ; 0.4 i 
Sulphate of magnesa, : 5 ‘ ‘ 0.4 ne 
Sulphate of ammonia, r : : 0.2 “ 


gaia 


: 


i] 
i 


ry 


i 


a 


This is introduced into a flask with two necks, such as is shown 
in Fig. 77. Having filled the flask with the culture liquid, the bent 
neck is dipped. into a porcelain dish containing the same. Heat is 
then applied both to flask and dish, and the liquid in each is kept in 
ebullition for half an hour. By this means the air is completely 
driven out of the flask. This is now allowed to cool, while the fluid 
in the shallow dish is kept hot, so that the liquid mounting from it 
into the flask shall be free from air. When the flask is full it is 
transferred to an incubating oven heated to 25° to 30° C., and the bent 
tube is immersed in a dish containing mercury. The little funnel 


PRODUCTS OF VITAL ACTIVITY. 139 


attached to the upright tube is then filled with carbon dioxide and a 
culture of the butyric-acid bacillus is introduced into the funnel. 
By turning the stopcock in the upright tube a little of the culture is 
admitted to the flask without admitting any air. Fermentation com- 
mences very soon, as is seen by the bubbles of gas given off. The 
liquid loses its transparency and the lactic acid is gradually con- 
sumed, butyrate of lime taking the place of the lactate. 

Aérobic bacilli capable of producing butyric acid in culture solu- 
tions containing grape sugar or milk sugar have also been described 
by Liborius and by Hueppe. 

Fitz has shown that in culture solutions containing glycerin the 
Bacillus pyocyaneus produces butyric acid in addition to ethyl alcohol 
and succinicacid. Bacillus Fitzianus also produces some butyric acid 
in solutions containing glycerin, although the principal product of the 
fermentation caused by this microérganism is, according to Fitz, 
ethyl alcohol, twenty-nine grammes of which may be obtained from 
one hundred grammes of glycerin. 

Botkin (1892) has described a “Bacillus butyricus” (No. 466) 
which he has not been able to identify positively with the butyric- 
acid ferment described by Prazmowski. It is a widely distributed 
anaérobic bacillus, which he was able to obtain from milk or water 
containing it by placing it in the steam sterilizer for half an hour. 
The spores resisted this temperature and subsequently grew in anaé- 
robic cultures, in a suitable medium, while all other bacteria and 
spores present were destroyed. 

The writer has described a bacillus which causes active acid 
fermentation in culture solutions containing glycerin. The acid 
formed is volatile and is probably propionic acid—see Bacillus acidi- 
formans. 

The Caucasian milk ferment—Bacillus Kaukasicus—produces 
a variety of products in the fermented milk which is a favorite 
drink among the Caucasians. The principal ones are ethyl alcohol, 
lactic acid, and carbon dioxide, but in addition to these small quanti- 
ties of succinic, butyric, and acetic acids are formed. The inhabi- 
tants of the Caucasian mountains prepare this fermented drink in a 
very simple manner from the milk of cows or goats, to which they 
add the dried ferment collected from a receptacle in which the fermen- 
tation had previously taken place. Fltigge gives the following di- 
rections for the preparation of this drink : 


‘Two methods may be employed. In the first the dry brown kefir-kér- 
ner of commerce are allowed to lie in water for five to six hours until they 
swell; they are then carefully washed and placed in fresh milk, which 
should be changed once or twice a day until the kérner become pure white 
in color and when placed in fresh milk quickly mount to the surface—in 
twenty to thirty minutes. One litre of milk is then poured into a flask and a 
full tablespoonful of the prepared kérner added to it. It is allowed to stand 


140 PRODUCTS OF VITAL ACTIVITY. 


open for five to eight hours; the flask is then closed and kept at 18°C. It 
should be shaken every two hours. At the end of twenty-four hours the 
milk is poured through a fine sieve into another flask, which must uot be 
more than four-fifths full. This is corked and allowed to stand, being 
shaken from time to time. At the end of twenty-four hours a drink is ob- 
tained which contains but little COs or aleohol. Usually it is not drunk 
until the second day, when, upon standing, two layers are formed, the 
lower milky, translucent, and the upper containing fine flakes of casein. 
When shaken it has a cream-like consistence. On the third day it again 
becomes thin and very acid. 

‘* The second method is used when one has a good kefir of two or three 
days to start with. Three or four parts of fresh cow’s milk are added to one 
part of this and poured into flasks which are allowed to stand for forty- 
eight hours with occasional shaking. When the drink is ready for use a 
portion (one-fifth to one-third) is left in the flask as ferment fora fresh 
quantity of milk. The temperature should be maintained at about 18° C.; 
but at the commencement a higher temperature is desirable. The kérner 
should be carefully cleaned from time to time and broken up to the size of 
peas. The cleaned kérner may be dried upon blotting paper in the sun or 
in the vicinity of a stove: when dried in the air they retain their power to 
germinate for a long time.” 

Fermentation of urea. The alkaline fermentation of urine is 
effected by various microérganisms, but chiefly by the Micrococcus 
ureze, the ferment action of which has been carefully studied by Pas- 
teur, Duclaux, and others. The change which occurs under the 
action of the living ferment was determined by the chemist Dumas 
as long ago as 1830, but it remained for Pasteur to show that this 
change depends upon the presence and vital activity of a living 
microérganism. 

The transformation of urea into carbonate of ammonia is shown 
by the following formula: COH,N, + 2H,O = CO, + 2NH, + 
H,O = (NH,),CO,. 

According to Van Tieghem, Micrococcus ures continues to grow 
in a liquid containing as much as thirteen per cent of carbonate of 
ammonia. Itmay be cultivated in an artificial solution of urea, with 
the addition of some phosphates, as well as in urine. 

The Bacillus ureee of Miquel has also the power of producing the 
alkaline fermentation of urine, but it does not thrive in so strong a 
solution of carbonate of ammonia. 

A different micrococeus—Micrococcus uree liquefaciens—nas also 
been studied in Fliigge’s laboratory which possesses the same power. 
According to Musculus, a soluble ferment may be isolated from urine 
which has undergone alkaline fermentation, which changes urea into 
carbonate of ammonia. He obtained it from urine containing con- 
siderable mucus, in a case of catarrh of the bladder. But Leube has 
shown that cultures of Micrococcus ures from which the micrococ- 
cus was removed by filtration through clay do not induce alkaline 
fermentation. The soluble ferment obtained by Musculus must 


therefore be from some other source. 


PRODUCTS OF VITAL ACTIVITY. 141 


Miquel has given special attention to the study of bacteria which 
produce alkaline fermentation in urine, and in addition to the spe- 
cies above mentioned has described the following : Urobacillus Pas- 
teuri, Urobacillus Duclauxi, Urobacillus Freudenreichi, Urobacillus 
Maddoxi, Urobacillus Schutzenbergi. 

Viscous fermentation. A special fermentation which occurs 
sometimes in wines, and in the juices of bulbous roots containing 
glucose, and in milk, is produced by various bacteria. One of these 
is a micrococcus which has been described by Conn under the name 
of Micrococcus lactis viscosus. The fermented juices become very 
viscous, owing to the formation of a gum-like product resembling 
dextrin; at the same time mannite and CO, are produced. The 
gum-like substance, called viscose by Beéchamp, is soluble in cold 
water and is precipitated by alcohol. Guillebeau (1892) has de- 
scribed a micrococcus and a bacillus which produce viscous fer- 
mentation in milk—Micrococcus Freudenreichi and Bacillus Hessi. 
A micrococcus producing viscous fermentation in milk has also 
been described by Schmidt-Miihlheim, and a bacillus by Léffler. 
Bacillus mesentericus vulgatus also produces a similar change in 
milk. 

Marsh gas, CH,, is produced by the fermentation of cellulose, 
through the action of microédrganisms the exact characters of which 
have not yet been determined. According to Tappeiner, there are 
two different fermentations of cellulose. The first occurs in a neu- 
tral one-per-cent flesh extract solution to which cotton or paper pulp 
has been added. The gases given off are CO, and CH, and small 
quantities of H,S. The second fermentation occurs when an alkaline 
solution of flesh extract containing cellulose in suspension is used. 
The gases formed are CO, and H. In both cases small quantities of 
aldehyde, isobutyric acid, and acetic acid are produced. 

Hydrosulphuric acid, H,S8. This gas is produced during the 
growth of certain bacteria. The conditions governing its develop- 
ment have been studied by Holschewnikoff, who experimented with 
two species, one isolated by himself and one by Lindenborn, named 
respectively Bacterium sulfureum and Proteus sulfureus. The first- 
mentioned bacterium, when inoculated into eggs, produced within 
three or four days an abundant quantity of H,5; the other did not. 
Upon raw albumin both species produced but little, and on the yolk 
of egg aconsiderable amount of this gas. Upon cooked egg the 
action was the reverse. In peptone-bouillon the evolution of H,S 
was abundant ; in the absence of peptone, very slight. 

Putrefactive fermentation. The putrefactive decomposition 
of albuminous material of animal and vegetable origin is effected 
by a great variety of microdrganisms and gives rise to the forma- 


142 : PRODUCTS OF VITAL ACTIVITY. 


tion of a great variety of products, some of which are volatile and 
are characterized by their offensive odors. According to Fligge, the 
first change which occurs consists in the transformation of the albu- 
mins into peptone, and this may be effected by a large number of 
different bacteria. Among the products of putrefactive fermenta- 
tion known to chemists are the following substances : Carbon diox- 
ide, hydrogen, nitrogen, hydrosulphuric acid (H,S), phosphoretted 
hydrogen (PH,), methane, formic acid, acetic acid, butyric acid, 
yalerianic acid, palmitic acid, crotonic acid, glycolic acid, oxalic 
acid, succinic acid, propionic acid, lactic acid, amidostearic acid, 
leucin, ammonia, ammonium carbonate, ammonium sulphide, tri- 
methylamine, propylamine, indol, skatol, tyrosin,neuridin, cadaverin, 
putrescin, cholin, neurin, peptotoxin, and various other volatile 
acids, ptomaines, etc. 

' The special products of putrefaction vary according to the nature 
of the material, the conditions in which it is placed, and the micro- 
érganisms present. One or the other of the bacteria concerned will 
take the precedence when circumstances favor its growth. Thus the 
aérobic bacteria cannot grow unless the putrefying material is freely 
exposed to atmospheric oxygen; the anaérobic species require its 
exclusion. Some saprophytic bacteria grow at a comparatively low 
temperature, others take the precedence when the temperature is 
high ; some, no doubt, thrive only in presence of products evolved 
by other species, and are consequently associated with and depend- 
ent upon these species ; some are restrained in their growth sooner 
than others by the products evolved as a result of their own vital 
activity or that of associated organisms ; some grow in the presence 
of acids and give rise to an acid fermentation which wholly prevents 
the development of other species. 

At the outset putrefaction is often attended with the presence 
of several species of micrococci and certain large bacilli, which are 
displaced later by short motile bacteria belonging to a group which 
includes several bacilli formerly described under the common name 
of Bacterium termo. 

The malodorous volatile products of putrefaction are to a consid- 
erable extent produced by anaérobic species. For this reason these 
odors are more pronounced when masses of albuminous material 
undergo putrefaction in situations where the oxygen of the air has 
not free access or where it is displaced by carbon dioxide. The 
body of a dead animal, although freely exposed to the air, furnishes 
in its interior a suitable nidus for these anaérobic gas-forming spe- 
cies, and they may give rise to products of one kind, while aérobic 
species upon the surface of the mass induce different forms of putre- 
factive fermentation. In the bodies of living animals these anaéro- 


PRODUCTS OF VITAL ACTIVITY. 143 


bic microdrganisms are constantly present in the intestine, and after 
death they quickly invade the body and multiply at its expense 
under favorable conditions as to temperature. The surface decom- 
position due to aérobic bacteria occurs later and is not attended 
with the same putrefactive odors, the products evolved being of a 
simpler chemical composition—CO,, HN, No doubt these aérobic 
bacteria, by consuming the oxygen and forming an atmosphere of 
carbon dioxide, help to make the conditions favorable for the con- 
tinued development of the anaérobics in the interior of the organic 
mass ; at the same time they find a suitable pabulum in some of the 
more complex products of decomposition occurring in the absence 
of oxygen. The gases produced in the interior of a putrefying mass 
are mainly CH,, H,S, and H. 

Many of the bacteria of putrefaction are facultative anaérobics— 
that is to say, they are able to multiply either in the presence of oxy- 
gen or in its absence. The products evolved by these differ, no 
doubt, according to whether they are or are not supplied with atmos- 
pheric oxygen. 

The anaérobic bacteria concerned in putrefaction have as yet 
received comparatively little attention. Among the aérobics and 
facultative anaérobics the following are best known: Micrococcus 
foetidus, Bacillus saprogenes I., II., and III., Bacillus coprogenes 
foetidus, Bacillus putrificus coli, Proteus vulgaris, Proteus Zenkeri, 
Proteus mirabilis, Bacillus pyogenes fcetidus, Bacillus fluorescens 
liquefaciens, Bacillus pyocyaneus, Bacillus coli communis, Bacillus 
janthinus. 

Soluble Ferments.—Several species of bacteria produce soluble 
ferments capable of changing starch into maltose, dextrin, etc. 
Hueppe has shown that the lactic-acid bacillus produces a diastase, 
and Miller obtained from the human intestine a species which dis- 
solves starch. Marcano, by filtering cultures of species capable of 
this ferment action through porcelain, was able to show that the 
effect is due to a soluble ferment, which must have been produced 
by the vital activity of the living microérganisms. Wortmann also 
obtained a diastase from culture liquids which was precipitated by 
alcohol and again dissolved in water; in slightly acid solutions it 
promptly converted starch into glucose. This is said to be produced 
in culture liquids only when these do not contain albumin. In the 
presence of albumin a peptonizing ferment was formed ; in its ab- 
sence, a diastase by which starch was dissolved to serve as pabulum 
for the bacteria present. These experiments were not made with 
pure cultures, and more exact researches in this direction are de- 
sirable. 


144 PRODUCTS OF VITAL ACTIVITY. 


A peptonizing ferment for gelatin is produced by a considerable 
number of bacteria, as stated under the heading “ Liquefaction of 
Gelatin.” The jellified albumin in cultures in blood serum is also 
liquefied by a peptonizing ferment produced by certain species of bac- 
teria. 

Some authors also speak of a soluble ferment capable of inverting 
cane sugar or milk sugar. According to Hueppe, such a ferment 
is produced by the Bacillus acidi lactici. A soluble ferment for cel- 
lulose is supposed by Fliigge to be produced by several species— 
among others by Bacillus butyricus and by Vibrio rugula. 

Several bacilli produce a soluble ferment capable of coagulating 
the casein of milk. 

Reduction of Nitrates, and Nitrification.—The researches of 
Gayon, Dupettit, and others show that certain bacteria are able to 
reduce nitrates with liberation of ammonia and free nitrogen. This 
is effected in the absence of oxygen by anaérobic bacteria, and, 
among others, by Bacillus butyricus. Certain aérobic bacteria also 
accomplish the same result. Thus Herzus obtained two species 
from water which reduced nitrates in a very decided manner. On 
the other hand, a number of species are known to oxidize ammonia, 
producing nitric acid. Schlésing and Miinz, as a result of numerous 
experiments, arrived at the conclusion that in the scil nitrification is 
effected by a single species. But it is doubtful whether they worked 
with pure cultures, and more recent researches show that several, 
and probably many, different bacteria possess this power. Accord- 
ing to Heres, the following species, tested by him, oxidize am- 
monia : Bacillus prodigiosus, the cheese spirillum of Deneke, the 
Finkler-Prior spirillum, the typhoid bacillus, the anthrax bacillus, 
the staphylococci of pus. The oxidation does not always go to the 
point of forming nitrates, but nitrites may be formed in the soil 
(Duclaux). Warrington states that certain bacteria which formed 
nitrates in a suitable culture medium produced only nitrites when, 
after an interval of four or five months, some of the culture was 
transferred to a solution containing muriate of ammonia. The same 
author states that the process of nitrification occurs only in the 
dark. 

The researches of Winogradsky, of the Franklands, and of Jor- 
dan show that the failure of earlier investigators to obtain the nitri- 
fying bacteria from the soil in pure cultures was due to the fact that 
these bacteria do not grow in the usual culture media. By the use 
of certain saline solutions the authors named have succeeded in iso- 
lating nitrifying bacteria in pure cultures, or nearly so. It is still ° 
uncertain whether these investigators have obtained the same bac- 
teria, but the microdrganisms described by them, and obtained from 


PRODUCTS OF VITAL ACTIVITY. 145 


widely distant sources, are similar in their morphological and bio- 
logical characters, and at least belong to the same group. In a com- 
munication made in 1891 Winogradsky arrives at the conclusion 
that the ferments which cause the oxidation of ammonia and pro- 
duction of nitrites are not capable of producing nitrates, but that 
other microdrganisms are concerned in the oxidation of nitrites. 
In sterilized soil to which a pure culture of his nitromonas was 
added nitrites only were produced, and the presence of various 
microédrganisms common in the soil did not result in the forma- 
tion of nitrates so long as the specific ferment was absent to which 
this second oxidation is ascribed (nitrifying bacillus of Winograd- 
sky). 

Phosphorescence.—Several different bacteria have been studied 
which, in pure cultures, give rise to phosphorescence in the medi- 
um in which they are cultivated. In gelatin cultures the light 
is sufficient in some instances to enable one to tell the time by a 
watch in a perfectly dark room, and such cultures have even been 
photographed by their own light. 

The phosphorescence is influenced by changes in the culture 
medium and by conditions of temperature, but we have no exact 
knowledge of the mode of its production. The Bacillus phosphores- 
cens from sea water in the vicinity of the West Indies gives the 
most striking results, especially when planted upon the surface of 
cooked fish and placed in an incubating oven at 30° C. Two other 
species have been studied by Fischer—one obtained from the water 
of the harbor at Kiel, and the other a widely distributed species 
called by Fischer Bacterium phosphorescens. Katz (1891) has de- 
scribed several species obtained by him from sea water and from 
phosphorescent fish in the markets at Sydney, New South Wales— 
Bacillus smaragdino-phosphorescens, Bacillus argenteo-phosphores- 
cens, Bacillus cyaneo-phosphorescens, Bacillus argenteo-phosphores- 
cens liquefaciens. 

10 


V. 


PTOMAINES AND TOXALBUMINS. 


Various basic substances containing nitrogen, and in chemical 
constitution resembling the vegetable alkaloids, have been isolated 
by chemists from putrefying material and from cultures of the bac- 
teria concerned in putrefaction, and also from -certain pathogenic 
species. Some of these ptomaines are non-toxic, and others are 
very poisonous in minute doses (toxines). The toxic substances 
sometimes developed in milk, cheese, sausage, etc., are also of this 
nature, and are doubtless produced by the action of microdrganisms. 
The pathogenic power of the bacteria which cause various infectious 
diseases in man and the lower animals has also been shown to result 
from the production of toxic ptomaines or of toxalbumins. Selmi first 
gave the name ptomaines to cadaveric alkaloids isolated by him, and 
Panum subsequently called attention to the fact that poisonous basic 
substances of this class are contained in putrefying material. LEx- 
tended researches with reference to the ptomaines have since been 
made by numerous chemists, the most important being those of Berg- 
mann, Schmiedeberg, Zuelzer and Sonnenschein, Hager, Otto, Sel- 
mi, Brieger, Gautier and Etard, and Vaughan. 

For a full account of the history and chemical composition of the 
ptomaines the reader is referred to the valuable work of Vaughan 
and Novy (‘‘ Ptomaines and Leucomaines,” Philadelphia, 1891). In 
the present volume we shall give a brief account only of some of the 
most important. 


NON-TOXIC PTOMAINES. 


Neuridin, C,H,,N,.—This is one of the most common of the al- 
kaloids of putrefaction and was isolated by Brieger in 1884. It is 
obtained most abundantly from tissues containing gelatin. Very 
soluble in water, but insoluble in ether and absolute alcohol. Has a 
disagreeable odor. 

Cadaverin, C,H,,N,.—Isomeric with neuridin ; has a very dis- 
agreeable odor ; forms a thick, transparent, syrupy liquid; is vola- 
tile, and can be distilled with steam without undergoing decomposi- 
tion. When exposed to the air the base absorbs carbon dioxide and 


PTOMAINES AND TOXALBUMINS, 147 


forms a crystalline mass. Is produced in cultures of the cholera 
spirillum and of the spirillum of Finkler and Prior which have been 
kept for a month or more at 37° C. 

Putrescin, C,H,,N,.—A_ base resembling cadaverin and com- 
monly associated with it. Obtained by Brieger from various sources, 
most abundantly from substances containing gelatin and in the 
more advanced stages of putrefaction. It is obtained in the form of 
a hydrate, which is a transparent liquid having a boiling point of 
about 135°. With acids it forms crystalline salts. 

Saprin, C,A,,N,.—Resembles cadaverin and is commonly as- 
sociated with it in putrefying material. Isolated by Brieger. 

Methylamine, CH,.NH,.—Obtained by Brieger from putrefying 
fish and from old cultures of the cholera spirillum. 

Dimethylamine, (CH,),.NH.—Obtained by Brieger from putre- 
fying gelatin and by Bocklisch from decomposing fish. 

Trimethylamine, (CH,),N.—Obtained from various sources, and 
by Brieger from cultures of the cholera spirillum and of the strepto- 
coccus of pus. 

TOXIC PTOMAINES. 

Neurin, C,H,,NO. —First obtained by Liebreich in 1865 as a 
decomposition product of protagon from the brain. Obtained by 
Brieger from putrefying muscular tissue. When crystallized from 
an aqueous solution it forms five- or six-sided plates ; from an alco- 
holiec solution it crystallizes in the form of needles (Liebreich). This 
base is toxic in small doses. In frogs the injection of a few milli- 
grammes produces paralysis of the extremities. Respiration is first 
arrested and the heart stops in diastole. Atropine appears to be a 
physiological antidote to the toxic effects of neurin. In rabbits it 
produces profuse salivation. The pupil is contracted by the direct 
application of a concentrated solution. 

Cholin, C,H,,NO,.—First obtained from hog’s bile by Strecker 
in 1862. Has been obtained by Brieger from various sources, in- 
cluding cultures of the cholera spirillum. It is also found widely 
distributed in the vegetable kingdom. May be prepared from the 
yolk of eggs by the method of Diakonow. Cholin is obtained in the 
form of a syrupy, alkaline liquid which combines with acids to form 
deliquescent salts. At first this base was not supposed to have toxic 
properties, but more recent researches have shown that in compara- 
tively large doses it produces symptoms resembling those caused by 
minute doses of neurin. 

Muscarin, C,H,,NO,.—This toxic principle of poisonous mush- 
rooms has also been obtained by Brieger from putrefying fish. It may 
be produced artificially by the oxidation of cholin. In small doses 
it kills rabbits and frogs. In the rabbit it produces lacrymation and 


148 PTOMAINES AND TOXALBUMINS. 


salivation, the pupil is contracted, and the animal dies in convul- 
sions. Frogs are completely paralyzed by the action of muscarin 
and die with arrest of the heart’s action in diastole. 

Peptotoxin.—The exact composition of this ptomaine has not 
been determined. Brieger obtained it during the early putrefac- 
tion of proteid substances and also from the artificial digestion of 
fibrin. It is very poisonous for frogs, which become paralyzed and 
die within fifteen or twenty minutes after the subcutaneous injection 
of a few drops of a dilute solution. Rabbits also are killed by doses 
of half a gramme to a gramme, the symptoms being paralysis of the 
posterior extremities and stupor. Peptotoxin is soluble in water, 
but insoluble in ether or chloroform. It is not destroyed by boiling. 

Tyrotoxicon.—First obtained by Vaughan in poisonous cheese, 
and subsequently by the same chemist and others in poisonous milk 
and ice cream. Chemically tyrotoxicon is very unstable. It is de- 
composed when heated with water to 90° C. Itis insoluble in ether. 
From sixteen kilogrammes of poisonous cheese Vaughan obtained 
0.5 gramme of the poison. The symptoms produced in man by eat- 
ing cheese or milk containing tyrotoxicon are vertigo, nausea, vomit- 
ing, and severe rigors, with pain in the epigastrium, cramps in the 
legs, griping pain in the bowels attended with purging, numbness 
and a pricking sensation in the limbs, and great prostration. 

JAethyl-guanidin, C,H,N,.—Obtained by Brieger from putrefy- 
ing horseflesh which had been kept at a low temperature for several 
months. This base was previously known to chemists, having been 
obtained by the oxidation of creatin. By Bocklisch it has been ob- 
tained from impure cultures of the Finkler-Prior spirillum which 
had been kept for about a month. It is obtained as a colorless mass 
having an alkaline reaction, and which is quite deliquescent. Brie- 
ger gives the following account of the toxic action as tested on 
guinea-pigs in a dose of 0.2 gramme: The respiration increases: in 
rapidity, the pupils dilate to the extreme limit, the animal has copi- 
ous discharges of urine and feces, the extremities become paralyzed, 
and at the end of about twenty minutes death occurs in convulsions. 

Mytilotoxin.—Obtained by Brieger from poisonous mussels. 
The toxic action resembles that of curare. 

Typhotoxin, C,H,,NO,.—Obtained by Brieger from bouillon 
cultures of the typhoid bacillus which had been kept for a week or 
more at a temperature of about 37.5° C. In mice and guinea-pigs 
this base produces salivation, rapid respiration, dilatation of the 
pupils, diarrhoea, and death in from twenty-four to forty-eight hours. 
It is believed by Brieger that the specific action of the typhoid bacil- 
lus is due to the production of this ptomaine. 

A base which is isomeric with typhotoxin has been obtained by 


PTOMAINES AND TOXALBUMINS. 149 


Brieger from putrefying horseflesh which was kept at a low tempe- 
rature for several months. Unlike it, however, the free base has 
an acid reaction, while typhotoxin is strongly alkaline. It differs also 
in its physiological action, being more toxic and producing convul- 
sions; the heart is arrested in diastole. Typhotoxin, on the other 
hand, does not induce convulsions and the heart is arrested in systole. 

Tetanin, C,,H,,N,O,.—Obtained by Brieger from impure cul- 
tures of the tetanus bacillus cultivated in bouillon in an atmosphere 
of hydrogen. (The tetanus bacillus is a strict anaérobic.) Obtained 
subsequently by the same chemist from the amputated arm of a pa- 
tient with tetanus. This base has been obtained, by crystallization 
from hot alcohol, in clear yellow plates which are not very soluble in 
water. The hydrochloride is a deliquescent salt which dissolves 
readily in alcohol. When injected into guinea-pigs or mice in rather 
large doses, tetanin first causes the animal to fall into a lethargic 
condition, followed by increased rapidity of respiration and tetanic 
convulsions. In guinea-pigs opisthotonos is induced, together with 
the characteristic tetanic convulsions as seen in animals suffering from 
tetanus. Three other toxic bases have been obtained by Brieger 
from cultures of the tetanus bacillus, which cause similar symptoms, 
One—tetanotoxin—is given by Brieger the formula C,H,,N. A 
second base, the composition of which has not been determined, is 
called spasmotoxin. 

Cholera Piomaines.—Brieger has obtained from pure cultures 
of the cholera spirillum several of the toxic ptomaines heretofore re- 
ferred to—cadaverin, putrescin, cholin, methyl-guanidin. In addi- 
tion to these he found two toxic substances which appear to be pe- 
culiar products of this microdrganism. One induces cramps and 
muscular tremors in small animals, the other diarrhoea and symp- 
toms of collapse. 

Toxalbumins.—Researches by Brieger and Frankel (1890) show 
that very toxic substances of a different nature are present in cultures 
of some of the pathogenic bacteria; these have been designated by the 
authors named “toxalbumins.” 

Roux and Yersin had previously shown that filtered cultures of the 
diphtheria bacillus contain a toxic substance which produces paralysis 
and death in guinea-pigs and rabbits. This substance has now been 
obtained in a pure state and its toxic action tested by the authors 
first named. It is destroyed by a temperature of 60° C., but remains 
in an active condition in cultures which have been sterilized by scve- 
ral hours’ exposure to a temperature of 50°, or in those which have 
been passed through a clay filter. It is not volatile, and differs exsen- 
tially from the ptomaines and also from the soluble ferments. It 
was obtained as a snow-white, amorphous mass which was ex- 


150 PTOMAINES AND TOXALBUMINS, 


tremely toxic in its action upon small animals. When injected into 
guinea-pigs in the proportion of two and one-half milligrammes to 
one kilogramme of body weight, it caused death after a considerable 
interval of time (from afew days to several weekx), during which 
the animal became emaciated and spreading abscesses and necrosis 
of the tissues occurred at the point of injection. This toxalbumin 
was obtained in a pure state by repeated precipitation from an aque- 
ous solution by means of alcohol. It is produced most abundantly 
in cultures containing albumin, and old cultures are more toxic than 
recent ones. Chemical analysis gave the following result: C 45.35, 
H 7.13, N 16.33, S$ 1.39, O 29.80. The authors remark, however, 
that the chemical characters have not yet been fully determined. 

The same chemists have obtained toxic substances of a similar 
nature from cultures of the bacillus of typhoid fever, of the tetanus 
bacillus, of the Staphylococcus aureus, and of the cholera spirillum. 
Hankin had previously obtained a toxic ‘“‘albumose” from cultures 
of the anthrax bacillus by precipitation with alcohol, drying, solu- 
tion in water, and filtration through porcelain; and Christmas had 
obtained an albuminous substance from cultures of Staphylococcus 
aureus which produced pus formation when injected beneath the 
skin of rabbits or into the anterior chamber of the eye. 

According to Brieger and Frankel, these toxalbumins are divided 
into two principal groups, one of which is characterized by solubility 
in water, as in that produced by the diphtheria bacillus ; and one in 
which the albumin is insoluble or but slightly soluble, as is the case 
with those obtained from cultures of the typhoid bacillus, the cholera 
spirillum, and the Staphylococcus aureus. 

The toxalbumin from cholera cultures, obtained as pure as pos- 
sible and suspended in water, when injected under the skin of a 
guinea-pig, caused its death in two or three days. It was not, how- 
ever, toxic for rabbits, even when injected in considerable quantity. 

On the contrary, the toxalbumin of the typhoid bacillus, which is 
dissolved with difficulty in water, was more poisonous for rabbits 
than for guinea-pigs. When injected subcutaneously into rabbits 
death usually occurred in eight to ten days. No notable pathologi- 
cal changes were observed at the autopsy. 

The toxalbumin of Staphylococcus aureus killed rabbits and 
guinea-pigs within a few days, and in some cases at the end of 
twenty-four hours. The post-mortem appearances were necrosis or 
purulent breaking down of the tissues at the point of injection, with 
swelling and redness of the surrounding tissues and general inflam- 
matory appearances. The toxalbumin of anthrax cultures resembles 
that of the diphtheria bacillus in being soluble in water. It was 
obtained by Brieger from the organs of animals recently dead from 


PTOMAINES AND TOXALBUMINS. 151 


anthrax. In a dry condition it has a grayish-white color and gives 
the reactions of albumins. 

The toxalbumin of the tetanus bacillus is also soluble in water. 
It is best obtained in bouillon cultures containing glucose. 

G. and F. Klemperer (1891) have announced their success in 
obtaining a toxalbumin from cultures of Micrococcus pneumoniz 
croupose (‘diplococcus pneumonie’); this they propose to call pneu- 


motoxin. 
Some recent authors prefer the name toxins for the poisonous 


products of bacterial growth designated by Brieger and others as 
“toxalbumins.” This avoids any definite statement as to their 
chemical composition, which appears to be still in doubt. The 
poisonous precipitates obtained from cultures of the tetanus or the 
diphtheria bacillus give the reactions of an albumin or albumose 
(Martin), but it is possible that the toxic substance is simply as- 
sociated with bodies of this class, and that they have not yet been 
isolated in a pure state. These toxins in some cases are intimately 
associated with the bacterial cell—intracellular toxins—and their 
toxic effects are exhibited when small quantities of dead bacteria 
are introduced into a susceptible animal. The extracellular toxins 
are better known, and may be obtained from filtered culture solutions 
by precipitation with strong alcohol. In this case they are associated 
with the proteids which may have been present in the culture. The 
fact that a considerable interval elapses—twenty-four hours to several 
days—after the injection of these toxins into a rabbit or a guinea-pig 
before death occurs, has given rise to the inference that these sub- 
stances are of the nature of enzymes or ferments. This view is also 
supported by the very minute quantity required to produce a fatal 
result. According to Vaillard a dose of 0.00025 gramme of the tet- 
anus toxin is sufficient to kill a guinea-pig. 

Indol Productton.—Numerous species of bacteria, as a result of 
their vital activity, give rise to the production of indol. This may 
be detected by cultivation in “Dunham’s solution” of peptone 
(dried peptone, 1 part; sodium chloride, 0.5 per cent; distilled water, 
100 parts). Upon adding a drop of yellow nitric acid to ten cubic 
centimetres of a culture in this medium the presence of indol will be 
revealed by the development of a rosy red color. The presence of 
nitrous acid in the yellow nitric acid is essential for the reaction, 
which, however, may be obtained with pure nitric or sulphuric acid 
if a small quantity of potassium nitrate is added to the culture—one 
cubic centimetre of a 0.2-per-cent solution. 

“Koch’s Tuberculin.”—This is a glycerin extract of the toxic 
substances present in cultures of the tubercle bacillus. Crude tu- 


152 PTOMAINES AND TOXALBUMINS, 


one per cent of peptone and four to five per cent of glycerin have 
been added. This culture liquid is placed in flasks and inoculated 
upon the surface with small masses from a pure culture of the tu- 
bercle bacillus. A tolerably thick and dry white layer is developed, 
which after a time covers the entire surface. At the end of six to 
eight weeks development ceases and the culture liquid is evaporated 
over a water bath to one-tenth its volume ; this, after being filtered, 
constitutes the crude tuberculin. By precipitation with sixty-per- 
cent alcohol Koch has obtained from this a white precipitate which 
has the active properties of the glycerin extract. This is soluble in 
water and in glycerin, and has the chemical reactions of an albumi- 
nous body. 

Zuelzer has (1891) reported his success in isolating a toxic sub- 
stance from tubercle cultures. The contents of tubes containing 
pure cultures of the bacillus are first treated with hot water 
acidulated with hydrochloric acid. This solution is filtered, evapo- 
rated, and then several times precipitated with platinum chloride. 
The double salt formed is decomposed by hydrosulphuric acid, 
after which the liquid is filtered and evaporated to dryness. <A 
white, crystalline salt is thus obtained which is soluble in hot water. 
This salt was toxic for rabbits and guinea-pigs in doses of from one 
to three centigrammes. Death usually occurred in from two to four 
days. In guinea-pigs one centigramme injected subcutaneously 
caused, within a few minutes, a greatly increased frequency of respi- 
ration, an elevation of temperature, and protrusion of the eyeballs. 

Mallein.—Kalwing, Preusse, and Pearson have obtained from 
cultures of the glanders bacillus a “‘lymph” which somewhat re- 
sembles the crude tuberculin of Koch. This was obtained by 
Preusse by treating old potato cultures of the glanders bacillus with 
glycerin and water. The extract was filtered several times and then 
sterilized in a steam sterilizer. This lymph injected into horses in- 
fected with glanders gives rise to a very decided elevation of tempe- 
rature, while in horses free from this disease no such result follows. 


VI. 
INFLUENCE OF PHYSICAL AGENTS. 


Heat.—We have already seen (Section II., Part Second) that the 
temperature favorable for the growth of most bacteria is between 20° 
and 40° C.; that some species are able to multiply at the freezing tem- 
perature, and others at as high a temperature as 60° to 70° C.; that, 
as arule, the parasitic species require a temperature of 35° to 40°; 
and that low temperatures do not kill bacteria. 

Frisch (1877) exposed various cultures toa temperature of —87° C., 
which he obtained by the evaporation of liquid CO,, and found that 
micrococci and bacilli, after exposure to such a temperature, multi- 
plied abundantly when again placed in favorable conditions. Prud- 
den has also made extended experiments upon the influence of 
freezing. He found that while certain species resisted the freezing 
temperature for a long time, others failed to grow. Thus Bacillus 
prodigiosus did not grow after being frozen for fifty-one days; Pro- 
teus vulgaris was killed in the same time, and a slender, liquefying 
bacillus obtained from Croton aqueduct water was killed in seven 
days. Staphylococcus pyogenes aureus withstood freezing for sixty- 
six days, a fluorescent bacillus from Hudson River ice for seventy- 
seven days, and the bacillus of typhoid fever for one hundred and 
three days. Cultures made at intervals showed, however, a dimi- 
nution in the number of bacteria. A similar diminution would per- 
haps have occurred in old cultures in which the pabulum for growth 
was exhausted, independently of freezing ; for bacteria, like higher 
plants, die in time—which varies for different species—as a result of 
degenerative changes in the living protoplasm of the cells, and con- 
tinued vitality in a culture depends upon continued reproduction. 

Repeated freezing and thawing was found by Prudden to be 
more fatal to the typhoid bacillus than continuous freezing. Cul- 
tures were sterilized by being thawed out at intervals of three days 
and again refrozen, after repeating the operation five times. 

Cadeéac and Malet kept portions of a tuberculous lung in a frozen 
condition for four months, and found that at the end of this time 
tuberculosis was still produced in guinea-pigs by injecting a small 
quantity of this material. 


154 INFLUENCE OF PHYSICAL AGENTS. 


In considering the influence of high temperatures we must take 
account of the very great difference in the resisting power of the 
vegetative cells and the reproductive elements known as spores, also 
of the fact as to whether dry or moist heat is used and the time of 
exposure. 

Dry Heat.—When microérganisms in a desiccated condition are 
exposed to the action of heated dry air, the temperature required for 
their destruction is much above that required when they are in a 
moist condition or when they are exposed to the action of hot water 
or steam. This was thoroughly demonstrated by the experiments of 
Koch and Wolffhiigel (1881), A large number of pathogenic and 
non-pathogenic species were tested, with the following general result : 
A temperature of 78° to 123° C. maintained for an hour and a half 
(over 100° for an hour) failed to kill various non-pathogenic bacteria, 
but was fatal to the bacillus of mouse septiceemia and that of rabbit 
septicemia. To insure the destruction of all the species tested, in 
the absence of spores, a temperature of 120° to 128° C., maintained 
for an hour and a half, was required. 

The spores of Bacillus anthracis and of Bacillus subtilis resisted 
this temperature and required to insure their destruction a tempera- 
ture of 140° C. maintained for three hours. This temperature was 
found to injure most objects requiring disinfection, such as clothing 
and bedding. But the lower temperature which destroys micro- 
érganisms in the absence of spores (120° C. = 248° F.) can be used 
for disinfecting articles soiled with the discharges of patients with 
cholera, typhoid fever, or diphtheria, as the specific germs of these 
diseases do not form spores. It is probable also that it may be safely 
used to disinfect the clothing of small-pox patients, for we have ex- 
perimental evidence that a lower temperature destroys the virulence 
of vaccine virus (90°-95° C.—Baxter). 

In practical disinfection by means of dry heat it will be necessary 
to remember that it has but little penetrating power. In the experi- 
ments of Koch and Wolffhiigel it was found that registering ther- 
mometers placed in the interior of folded blankets and packages of 
various kinds did not show a temperature capable of killing bacteria 
after three hours’ exposure in a hot-air oven at 133° C. and above. 

Moist Heat.—The thermal death-point of bacteria, in the ab- 
sence of spores, is comparatively low when they are exposed to moist 
heat. The results of the writer’s experiments are given below: 


“In my temperature experiments I have taken great pains to insure the 
exposure of the test organisms to a uniform temperature, and have adopted 
ten minutes as the standard time of exposure. The method employed 
throughout has been as follows: From glass tubing having a diameter of 
about three-sixteenths of an inch I draw out in the flame of a Bunsen burner 
a number of capillary tubes, with an expanded extremity which serves as 


INFLUENCE OF PHYSICAL AGENTS. 155 


an air chamber. A little material from a pure culture of the test organ- 
ism is drawn into each of these capillary tubes by immersing the open 
extremity in the culture, after having gently heated the expanded end. The 
end of the tube is then hermetically sealed by heat. These tubes are im- 
mersed in a water bath maintained at the desired temperature for the stan- 
dard time, The bath is kept at a uniform temperature by personal supervi- 
sion. At the bottom of the vessel isa thick glass plate which prevents the 
thermometer bulb and capillary tubes, which rest upon it, from being ex- 
posed to heat transmitted directly from the bottom of the vessel To further 
guard against this I am in the habit of applying the flame to the sides of the 
vessel, and a uniform temperature throughout the bath is maintained by 
frequent stirring with aglass rod. It is impossible to avoid slight variations, 
but by keeping my eye upon the thermometer throughout the experiment 
I have kept these within very narrow limits. . . . No attempt has been made 
to fix the thermal] death-point within narrower limits than 2° C., and in the 
table the lowest temperature is given which has been found, in the experi- 
ments made, to destroy all of the microdrganisms in the material subjected 
to the test. No doubt more extended experiments would result, in some in- 
stances, in a reduction of the temperature given as the thermal death-point 
for a degree or more. But the results as stated are sufficiently accurate for 
all practical purposes.” ! 


The results obtained in these experiments, for non-sporebearing 
bacteria, are given in the following table. The time of exposure 
was ten minutes, except for the cholera spirillum and the cheese spi- 
rillum of Deneke. 


THERMAL DEATH-POINT OF BACTERIA. 


Centigrade. Fahrenheit. 

Spirillum cholerze Asiatice#.......... cece eee ec eee ee 52° 125.6° (4 m.) 
Spirillum tyrogenum (cheese spirillum)............ 52 125.6 (4m.) 
Spirillam Finkler-Prior .... 0.0.0... c cee eee eee eee 50 122, 
Bacillus typhi abdominalis............. 0... ....008. 56 138.8 
Bacillus of schweine-rothlauf (rouget)... ........6- 58 186.4 
Bacillus murisepticus ... ........... a. % ete 58 136.4 
Bacillus Neapolitanus (Emmerich’s bacillus).......... 62 143.6 
BaCilusiCavicid asin: cisesisrieiaeiewad araaraaerseiss -clsleas 62 143.6 
Bacillus pneumonie (Friedlénder’s)........ ......-. 56 132.8 
Bacillus crassus sputigenus.... 1... 0.2... seven eeeee 54 129.2 
Bacillus: pyocyaneus: ~ esnewists ereoaergieewinawuisewisetea’s 56 182.8 
Baellus: indicus) 6" avs neetaime dagen seaiiony 58 136.4 
Bacillus prodigiosus .....,.. . Aisiasaeae a0 sSopeengoanh earn 58 136.4 
Bacillus cyanogenus.............. Lwuadeg sameaees 54 129.2 
Bacillus fluorescens: aiicnas x oeurwctariaasad <2 sp 54 129.2 
Bacillus acidi lactici..... cS Webs sua rrieomtue cuted 56 132.8 
Staphylococcus pyogenes aureus. .... .......6 ‘ 58 136.4 
Staphylococcus pyogenes citreus 62 143.6 
Staphylococcus pyogenes albus : 62 143.6 
Streptococcus pyogenes 54 129.2 
Micrococcus tetragenus 58 136.4 
Micrococcus Pasteuri ... 1.0... .. eee ee cece eee ee : 52 125.6 
Darina lta... c.asicce ap cieaeeaenass 64 147.2 
Sarcina aurantiaca 62 143.6 


The following determinations of the thermal death-point of path- 


1 Quoted from the Rzport of the Committee on Disinfectants of the American Pub- 
iic Health Association, pages 186 and 152. 


156 INFLUENCE OF PHYSICAL AGENTS. 


ogenic organisms have been made by the authors named : Bacillus 
anthracis (Chauveau), 54° C.; Bacillus mallei—the bacillus of glan- 
ders—(Léffler), 55° C., Bacillus gallinarum—micrococcus of fowl 
cholera—(Salmon), 56° C. ; Bacillus of diphtheria (Léffler), 60° C. 
In the writer’s experiments the micrococcus of gonorrhcea was 
apparently killed by exposure for ten minutes to a temperature of 


60° C. 


“Some gonorrheeal pus froma recent case which had not undergone 
treatment was collected for me by my friend Dr. Rohé in the capillary 
glass tubes heretofore described. A microscopical examination of stained 
cover-glass preparations showed that this pus contained numerous ‘ gono- 
cocci’ in the interior of the cells. Two of the capillary tubes were placed 
in a water bath maintained at 60° C. for ten minutes. The pus was then 
forced out upon two pledgets of cotton wet with distilled water. Two 
healthy men had consented to submit to the experiment, and one of these 
bits of cotton was introduced into the urethra of each and left in situ for 
half an hour. As anticipated, the result was entirely negative. For obvi- 
ous reasons no controi experiment was made to fix the thermal death-point 
within narrower limits. 

‘‘In connection with these experiments upon the thermal death-point of 
known pathogenic organisms, it is of interest to inquire whether the viru- 
lence of infectious material, in which it has not been demonstrated that this 
virulence is due to a microérganism, is destroyed by a correspondingly low 
temperature. Evidently, if this proves to be the case, it will be a strong 
argument in favor of the view that we have to deal with a microdrganism 
in these diseases also. We have experimental proof that a large number of 
pathogenic organisms are killed by exposure for ten minutes to a tempera- 
ture of 55° to 60° C. But, s» far as I am aware, this low temperature would 
not be likely to destroy any of the poisonous chemical products which might 
be supposed to be the cause of infective virulence, leaving aside the fact that 
such chemical products have no power of self-multiplication, and, there- 
fore, could not be the independent cause of an infectious disease.! 

“ Vaccine Virus.—Carstens and Coert have experimented upon the tem- 
perature required to destroy the potency of vaccine virus. In a paper read 
at the International Medical Congress in 1879 they report, asa result of 
their experiments, that the maximum degree of heat to which fresh vaccine 
virus can be exposed without losing its virulence probably varies between 
52° and 54°C. Fresh animal vaccine heated to 52° C. for thirty minutes 
does not lose its virulence. Fresh animal vaccine heated to 54.5° for thirty 
minutes loses its virulence. 

‘‘ Rinderpest.—According to Semmer and Raupach, exposure for ten 
minutes to a temperature of 55° C. destroys the virulence of the infectious 
material in this disease. 

“* Sheep-pox.—The authors last mentioned have also found that the same 
temperature—55° C. for ten minutes—destroys the virulence of the blood of 
an animal dead from sheep-pox. 

‘* Hydrophobia.—Desiring to fix the thermal death-point of the virus of 
hydrophobia, I obtained, through the kindness of Dr. H. C. Ernst, a rabbit 
which had been inoculated, by the method of trephining, with material 
which came originally from Pasteur’s laboratory. The rabbit sent me 
showed the first symptom of paralytic rabies on the eighth day after inocu- 
lation. It died on the eleventh day (March 2d, 1887), and I at once pro- 
ceeded to make the following experiment : 

‘CA portion of the medulla was removed and thoroughly mixed with 


1 Since this was written Brieger has isolated a toxalbumin from cultures of the 
diphtheria bacillus which is destroyed by a temperature of 60° C., but resists 50°. 


INFLUENCE OF PHYSICAL AGENTS. 157 


sterilized water. The milky emulsion was introduced into four capillary 
tubes, such as had been used in my experiments heretofore recorded. Two 
of these tubes were then placed for ten minutes in a water bath, the tem- 
erature of which was maintained at 60°C. Four rabbits were now inocu- 
ated by trephining, two with the material exposed to 60° C. for ten min- 
utes, and two with the same material from the capillary tube not so exposed. 
The result was as definite and satisfactory as possible. The two control 
rabbits were taken sick, one on March 10th and one on the 11th ; both died 
with the characteristic symptoms of paralytic rabies on the third day. The 
two rabbits inoculated with material exposed to 60° C. remained in perfect 
health. On the 26th of March one of these rabbits was again inoculated, 
by trephining, with material from the medulla of a rabbit just dead from 
hydrophobia. This rabbit died from paralytic rabies on the 8th of April. 
Its companion remains in perfect health. 

‘*A second experiment was made in the same way on the 14th of March. 
Two rabbits were inoculatéd with material exposed for ten minutes to a 
temperature of 50° C.; two with material exposed to 55° C.; and two con- 
trol rabbits with material not so exposed. One of the rabbits inoculated 
with material exposed to 50° C., and one of the control rabbits, died on the 
25th; the other rabbit inoculated with the material exposed to 50°, the other 
control, and one inoculated with material exposed to 55°, on the 26th. The 
second rabbit inoculated with material exposed to 55° died five days later 
with the characteristic symptoms of the disease. These experiments show, 
then, that the virus of hydrophobia is destroyed by a temperature of 60° C., 
and that 55° C. fails to destroy it, the time of exposure being ten minutes.”? 


The experimental data given show that the pathogenic bacteria 
tested and different kinds of virus are all killed by a temperature of 
60° C. or below; some, like the cholera spirillum and Micrococcus 
pneumonie croupose, failing to grow after exposure to as low a tem- 
perature as 52° C. for four minutes. By extending the time a still 
lower temperature will effect the same result. Thus, according to 
Chauveau, the anthrax bacillus is killed by twenty minutes’ exposure 
to a temperature of 50° C.; and Brieger sterilizes cultures of the 
diphtheria bacillus, to obtain the soluble toxalbumin produced in 
them, by exposure for several hours to 50° C. A temperature of 60° 
has been found to decompose the toxalbumin. The non-pathogenic 
bacteria tested have, as a rule, a higher thermal death-point—s8° C. 
for Bacillus prodigiosus, 64° C. for Sarcina lutea, etc. 

It is a remarkable fact that certain bacteria not only are not de- 
stroyed at higher temperatures than this, but are able to multiply at 
a temperature of 65° to 70°C. Thus Miquel, in 1881, found in the 
waters of the Seine a motionless bacillus which grew luxuriantly in 
bouillon at a temperature of 69° to 70°C. Van Tieghem has also 
cultivated several different species at about the same temperature, 
and more recently Globig has obtained from the soil several species 
which grow at temperatures ranging from 50° to 70° C. 

The resisting power of spores to heat also varies in different spe- 
cies ; but the spores of known pathogenic bacteria are quickly de- 
stroyed by a temperature of 100° C. (212° F.). In the writer’s experi- 


‘Report of the Committee on Disinfectants (op. cit.), p. 147. 


158 INFLUENCE OF PHYSICAL AGENTS. 


ments the spores of Bacillus anthracis and of Bacillus alvei failed to 
grow after exposure to a temperature of 100° C. for four minutes, 
and only a few colonies developed after two minutes’ exposure to this 
temperature. The thermal death-point of spores of the “‘ wurtzel ba- 
cillus” and of Bacillus butyricus (of Hueppe) was the same—100° C. 
for four minutes. 

Schill and Fischer, in 1884, made a number of experiments to de- 
termine the thermal death-point of Bacillus tuberculosis. They 
found that five minutes’ exposure to a temperature of 100° C. in 
steam destroyed the vitality of the bacillus in sputum in five min- 
utes. When the time was reduced to two minutes a negative result 
from inoculation was obtained in two guinea-pigs, but one inoculated 
at the same time became tuberculous. My own experiments and 
those of Yersin, made since, lead me to think that there may have 
been some cause of error in this experiment of Schill and Fischer, 
and that the thermal death-point of the spores of Bacillus tuber- 
culosis is considerably below the boiling point of water. I inoculated 
guinea-pigs with tuberculous sputum subjected for ten minutes to 
the following temperatures: 50°, 60°, 70°, 80°, 90° C. The animal 
inoculated with material exposed to 50° died from tuberculosis at the 
end of seven weeks. None of the others developed tuberculosis. 

Yersin exposed an old culture in glycerin bouillon, in which many 
of the bacilli contained spores—‘‘ trés nettes”—to the following tem- 
peratures : 55°, 60°, 65°, 70°, '75°, 80°, 85°, 90°, 100° C. ‘* At theend of 
ten days the bacilli heated to 55° gave a culture in glycerin bouillon ; 
those exposed to 60° grew after twenty-two days; none of the 
bacilli heated above 70° gave any development. This experiment, 
repeated a great number of times, has always given us the same re- 
sult.” Voelsh, who has studied the same question, reports as the 
result of his experiments that the tubercle bacillus in sputum was 
not destroyed by heating to 100° C. Further experiments will be re- 
quired to reconcile these contradictory results. 

While the spores of the pathogenic bacteria mentioned are de- 
stroyed by the boiling point of water within a few minutes, certain 
non-pathogenic species resist this temperature for hours. Thus 
Globig obtained a bacillus from the soil the spores of which required 
five and one-half to six hours’ exposure to streaming steam for their 
destruction. These spores survived exposure for three-quarters of an 
hour in steam under pressure at from 109° to 113° C. They were de- 
stroyed, however, by exposure for twenty-five minutes in steam at 
113° to 116°, and in two minutes at 127°. 

In the practical application of steam for disinfecting purposes it 
must be remembered that, while steam under pressure is more effec- 
tive than streaming steam, it is scarcely necessary to give it the pre- 


INFLUENCE OF PHYSICAL AGENTS. 159 


ference, in view of the fact that all known pathogenic bacteria and 
their spores are quickly destroyed by the temperature of boiling 
water ; and also that superheated steam is less effective than moist 
steam. When confined steam in pipes is ‘‘ superheated ” it has about 
the same germicidal power as hot dry air at the same temperature. 
This is shown by the experiments of Esmarch, who found that an- 
thrax spores were killed in streaming steam in four minutes, but 
were not killed in the same time by superheated steam at a tempera- 
ture of 141° C. 

Desiccation.—Cultures of bacteria kept in a moist condition re- 
tain their vitality for a considerable time, which varies greatly with 
different species. The writer has found that a culture of the typhoid 
bacillus preserved in a hermetically sealed glass tube retained its 
vitality for eighteen months, as did also Bacillus prodigiosus, Bacil- 
lus cavicida, and some others. According to Kitasato, the cholera 
spirillum may be preserved in a moist state for seven months ; other 
bacteria die out in a month or two, but, as a rule, vitality is preserved 
for several months at least. 

Spores in a desiccated condition preserve their vitality for a 
great length of time. But desiccation is quickly fatal to some of the 
pathogenic bacteria, and especially so to the cholera spirillum. Koch, 
in his earlier experiments, found that his “‘comma bacillus” did not 
grow after being dried upon a cover glass for three hours. Kitasato, 
in experiments made since, found that a bouillon culture dried upon 
a thin glass cover was incapable of development after three hours’ 
time, but that cultures in nutrient agar or gelatin survived for two 
days, probably on account of the thicker layer formed and the longer 
time required for complete desiccation. Pfuhl has found that the 
typhoid bacillus dried upon a cover glass retains its vitality for 
eight to ten weeks, and Léffler states that the diphtheria bacillus re- 
sists desiccation for four or five months. Cadéac and Malet pro- 
duced tuberculosis in guinea-pigs by injecting material from the 
lung of a tuberculous cow which had been kept in the form of a dried 
powder for nearly five months; at a later date the virulence was 
lost. 

Light.—Downes and Blunt, in a communication made to the 
Royal Society of London in 1877, first called attention to the fact that 
light has an injurious effect upon bacteria, and that cultures may be 
sterilized by exposure to direct sunlight. 

Tyndall, in experiments made in the clear sunlight of the Alps, 
verified the fact that the development of bacteria was restrained in 
cultures during their exposure, but failed to obtain evidence that 
vitality was destroyed. 

In 1885 Duclaux took up the subject with pure cultures of various 


160 INFLUENCE OF PHYSICAL AGENTS. 


bacteria, and showed that by prolonged exposure to direct sunlight the 
spores of various bacilli lose their capacity to germinate. About the 
same time Arloing published his researches upon the influence of 
light upon the development of anthrax spores. He found that the 
anthrax bacillus was not restrained in its growth by diffused lamp- 
light, but its growth was retarded by an intense gaslight. Spore 
formation was more abundant in darkness than in red light, and more 
abundant in red than in white light. When a screen was interposed 
between the culture and the source of light, consisting of an aqueous 
solution of heematoglobin, the growth of the bacilli and of spores was 
much more luxuriant than in white light. In yellow light it was less 
abundant than in red. The blue and violet rays were still less favor- 
able for the growth of the bacillus and the development of spores. 
The pathogenic power of cultures was not especially influenced by 
exposure to white gaslight. In subsequent experiments with sun- 
light Arloing found that two hours of exposure to the July sun suf- 
ficed to destroy the vitality of anthrax spores, but that a considerably 
longer exposure (twenty-six to thirty hours) was necessary when the 
spores had been allowed to germinate in a suitable culture medium. 
Cultures which were not exposed long enough to destroy the vitality 
of the bacilli were retarded in their growth, and subsequent exposure 
for a shorter time (nine to ten hours) completely sterilized them. 
Cultures which were weakened in their reproductive energy by ex- 
posure to sunlight were also ‘“‘attenuated” as to their pathogenic 
power and could be used as a vaccine in protective inoculations. Ac- 
cording to Arloing, the effect produced results from the action of the 
full sunlight and cannot be obtained by the use of monochromatic 
light. 

The experiments of Strauss seemed to give support to the view 
advanced by Nocard that in Arloing’s experiments spores did not 
really exhibit a less degree of resisting power than the vegetating 
bacilli, but that in fact they commenced to vegetate before they were 
killed. Strauss placed anthrax spores in sterilized distilled water and 
in bouillon, and found that, under the same conditions of exposure, 
the bouillon cultures were sterilized in direct sunlight in nine 
hours, while the spores suspended in distilled water grew when trans- 
ferred to a suitable medium. This was accounted for on the suppo- 
sition that the bouillon furnishes the necessary pabulum for the de- 
velopment of the spores and that distilled water does not. 

Arloing combats this view and has published additional experi- 
ments which seem to disprove it. He placed small flasks containing 
anthrax spores in bouillon in the direct rays of the sun in February. 
Some of the flasks were placed upon a block of ice which reduced the 
temperature to 4° C.; the others were not so placed, and the tempe- 


INFLUENCE OF PHYSICAL AGENTS. 161 


rature, in the open air where all were exposed, was 11° C. All of the 
spores failed to grow after an exposure of fourhours. When exposed 
in water the time of exposure was longer. 

Roux has shown that the light also has an effect upon the culture 
medium, and that sterilized bouillon which has been exposed to direct 
sunlight for some hours restrains the development of anthrax spores 
subsequently introduced into it, but not of the growing bacilli. His 
experiments show that access of oxygen is a necessary factor in the 
sterilization of cultures by sunlight. 

In the experiments of Momont (1892) dry anthrax spores were 
found to resist the action of light for a long time, but moist spores, 
freely exposed to the air, failed to grow after forty-four hours’ ex- 
posure to sunlight. In the absence of spores, anthrax bacilli in a 
moist condition, when freely exposed to the air, failed to grow after 
exposure to sunlight for half an hour to two hours; but in the ab- 
sence of air the same bacilli were not destroyed at the end of fifty 
hours’ exposure. 

Geisler (1892), in experiments made upon the typhoid bacillus, 
found that all portions of the solar spectrum except the red rays ex- 
ercised a restraining influence upon the development of this bacillus. 
The electric light gave a similar result. The most decided effect was 
produced by rays from the violet end of the spectrum. The restrain- 
ing influence appears, from the researches of Geisler, not to be due 
solely to the direct action of light upon the development of the 
bacilli, but also to changes induced in the gelatin culture medium 
employed in his experiments. 

In his address before the International Medical Congress of Berlin, 
1890, Koch states that the tubercle bacillus is killed by the action of 
direct sunlight in a time varying from a few minutes to several hours, 
depending upon the thickness of the layer exposed. Diffused day- 
light also has the same effect, although a considerably longer time of 
exposure is required—when placed close to a window, from five to 
seven days. 

Dieudonné (1894), in experiments upon Bacillus prodigiosus and 
Bacillus fluorescens putidus, found that direct sunlight in March, 
July, and August killed these bacilli in one and one-half hours, in 
November in two and one-half hours. Diffuse daylight in March 
and July restrained development after three and one-half hours’ ex- 
posure (in November four and one-half hours), and completely de- 
stroyed vitality in from five to six hours. 

Ward’s experiments (1892-1894) show that the blue and violet 
rays have decided germicidal power, while the rays at the red end of 
the spectrum are comparatively inert. This corresponds with results 
previously reported by Arloing. 

11 


162 INFLUENCE OF PHYSICAL AGENTS. 


In the writer’s experiments on the cholera spirillum (1892) test 
tubes, containing sterile bouillon inoculated with one or two ése of a 
pure culture, were sterilized by two hours’ exposure to direct sunlight 
(in December). 

Dieudonné (1894) found that the electric are light destroyed his 
test organisms (Bacillus prodigiosus and Bacillus fluorescens putidus) 
ineight hours. The same result was accomplished by the incandes- 
cent light in eleven hours. 

In view of these facts we may conclude, with Duclaux, that sun- 
light is one of the most potent and one of the cheapest agents for the 
destruction of pathogenic bacteria, and that its use for this purpose is 
to be remembered in making practical hygienic recommendations. 
The popular idea that the exposure of infected articles of clothing 
and bedding in the sun is a useful sanitary precaution is fully sus- 
tained by the experimental data relating to the action of heat, desic- 
cation, and sunlight. 

Electricity.—Cohn and Mendelssohn, in 1879, attempted to de- 
termine the effect of the galvanic current upon bacteria. Cultures 
were placed in U-tubes through which a constant current was passed. 
A feeble current was found to be without effect. A strong current 
from two elements, maintained for twenty-four hours, restrained de- 
velopment in the vicinity of the positive pole, but this was probably 
due to the highly acid reaction which the culture liquid acquired. 
‘When a current from five elements was used for twenty-four hours 
the liquid was sterilized, but this may have been due to the decided 
changes produced in the chemical composition of the culture liquid 
rather than to the direct action of the galvanic current. 

The same may be said of the similar results obtained in later ex- 
periments by Apostoli and Laquerriére, and by Prochownick and 
Spaeth. The last-mentioned investigators found that the positive pole 
had a more decided effect than the negative, and that the effect de- 
pended upon the intensity and duration of the current. A current of 
fifty milliampéres passed for a quarter of an hour did not kill Staphy- 
lococcus pyogenes aureus, but a current of sixty milliampéres main- 
tained for the same time did. The spores of Bacillus anthracis 
required a current of two hundred to two hundred and thirty milli- 
amperes during an hour or two. In these experiments the cultures 
in gelatin were attached to the strips of platinum serving asthe two 
poles, and these were immersed in a solution of sodium chloride. As 
chlorine was disengaged at the positive pole, the germicidal action is 
attributed to this gas rather than to the direct action of the current 
upon the living microorganisms. 

The more recent researches of Spilker and Gottstein, made with 
an induction current from a dynamo machine, are more valuable in 


INFLUENCE OF PHYSICAL AGENTS. 163 


estimating the power of this agent to destroy the vitality of bacteria. 
The current was passed through a spiral wire which was wrapped 
around a test tube of glass, containing the microdrganism to be tested, 
suspended in distilled water. In a first experiment Bacillus prodigi- 
osus, suspended in sterilized distilled water and contained in test 
tubes having a capacity of two hundred and fifty cubic centimetres, 
was subjected to a current having an energy of 2.5 ampéres X 1.25 
volts for twenty-four hours. The temperature did not go above 
30° C. No development occurred when the microdrganism tested 
was subsequently planted in nutrient gelatin. Further experiments 
gave a similar result. It was found that stronger currents were 
effective in shorter time; but in no case was sterilization effected in 
less than an hour. 

Pressure.—D’ Arsonval and Charrin (1894) submitted a culture 
of Bacillus pyocyaneus to a pressure of fifty atmospheres, under car- 
bon dioxide. At the end of four hours cultures could still be ob- 
tained, but the bacillus had lost its power of pigment production. A 
few colonies were developed after six hours’ exposure to this pressure; 
but after twenty-four hours no development occurred. 

Agitation.—Meltzer (1894) has shown that the vitality of bacteria 
is destroyed by protracted and violent shaking, which causes a molec- 
ular disintegration of the cells. 


VIL 


ANTISEPTICS AND DISINFECTANTS. 


GENERAL ACCOUNT OF THE ACTION OF. 


THE term autiseptic is used by some authors to designate an 
agent which destroys the vitality of the microérganisms which pro- 
duce septic decomposition, and others of the same class. We prefer 
to restrict the use of the term to those agents which restrain the de- 
velopment of such microédrganisms without destroying their vitality. 
The complete destruction of vitality is effected by germzcides or dis- 
infectants. Material containing the germs of infectious diseases: is 
infectious material, and we disinfect -it by the use of agents which 
destroy the living disease germs or pathogenic bacteria which give 
it its infecting power. Such an agent is a disinfectant. But we ex- 
tend the use of this term to germicides in general—that is, to those 
agents which kill non-pathogenic bacteria as well as to those which 
destroy disease germs. All disinfectants are also antiseptics, for 
agents which destroy the vitality of the bacteria of putrefaction ar- 
rest the putrefactive process ; and these agents, in less amount than 
is required to completely destroy vitality, arrest growth and thus 
act as antiseptics. But all antiseptics are not germicides. Thus a 
concentrated solution of salt or of sugar will prevent the putrefac- 
tive decomposition of organic material, animal or vegetable ; but these 
agents do not destroy the vitality of the germs of putrefaction. In 
a certain degree of concentration they are antiseptics and are largely 
used for the preservation of meats and vegetables. In the same way 
many mineral salts in solutions of various strengths act as antisep- 
tics, and some of these in still stronger solutions are disinfectants. 
Thus mercuric chloride, when introduced into a culture solution in 
the proportion of 1: 300,000, will restrain the development of anthrax 
spores, but to insure the destruction of these spores a solution of 
1 :1,000 must be used. As arule, the difference between restraining 
action—antiseptic—and germicidal power—disinfectant—is not so 
great as this. We give below some recent determinations by Boer 
which illustrate this point, the test organism being the bacillus of 
typhoid fever in a culture in bouillon twenty-four hours old : 


ANTISEPTICS AND DISINFECTANTS, 165 


Restrains. Kills. 
Hydrochloric acid... ...... 0.2. oe eee tas 1.2100 1: 800 
Sulphuric acid sees: wow sesiertveidine auneperice aes 1: 1550 1:500 
Sil Ver Wibrate: soe eaccsseansoetaus; oacd mao aia sees 1 : 50000 1: 4000 
Sodium arseniate........... ....0... eee eters 1: 6000 1: 250 
Carole AGids cice~ ie eased o8 se nhee Keg anadnind er 1 : 400 1: 200 


Method of Determining Antiseptic Value.—To determine the 
restraining or antiseptic power of an agent for a particular micro- 
érganism, the agent is dissolved in a definite proportion in a suitable 
culture medium, which is then inoculated with a pure culture of the 
test organism and placed in favorable circumstances—as to tempera- 
ture—for its growth. At the same time a control experiment is 
made by placing another portion of the same culture medium, inocu- 
lated with the same microérganism, in the same conditions, but with- 
out the addition of the antiseptic agent. If development occurs in 
the control experiment and not in the culture medium containing 
the antiseptic, the failure to grow must be attributed to the presence 
of this agent. Having made a preliminary experiment, we are 
guided by the result in further experiments to determine the exact 
amount required to restrain development under the same conditions. 
Or we may make a series of experiments in the first instance. The 
problem being, for example, to determine the antiseptic value of 
carbolic acid for the typhoid bacillus, we may add this agent toa 
definite amount of bouillon in test tubes in the proportion of 1 : 100, 
1:200, 1:300, 1:400, 1:500. In experiments with volatile agents 
the bouillon, in test tubes or small flasks, must be sterilized in ad- 
vance, and the antiseptic agent introduced by means of a sterilized 
pipette with great care to prevent the accidental contamination of 
the nutrient medium. In experiments with non-volatile agents it will 
be best to sterilize the culture medium after the antiseptic has been 
added. Next we inoculate the liquid in each flask with a pure cul- 
ture of the test organism. The flasks are then placed in an incubat- 
ing oven at 35° to 37° C. At the same time a control, not containing 
any carbolic acid, is placed in the oven. At the end of twenty-four 
hours the control will be found to be clouded, showing an abundant 
multiplication of the bacillus. Taking the result of Boer above given. 
we would expect to find all of the solutions clear except that contain- 
ing 1: 500. This too might remain clear for some days and finally 
‘break down,” for experience shows that when we pass the point at 
which a permanent restraining influence is exerted there may be a 
temporary restraint or retardation of development. For this reason 
we must continue the experiment for a considerable time—not less 


166 ANTISEPTICS AND DISINFECTANTS. 


than two weeks. Having found that 1:400 and below prevents 
development, and 1 :500 does not, we may make further experiments 
to determine the antiseptic power within narrower limits ; but this 
is hardly necessary from a practical point of view. 

In these experiments the result will be influenced by several cir- 
cumstances, as follows : 

(a) By the composition of the nutrient medium. This is a 
very important factor, especially in determining the antiseptic value 
of certain metallic salts. The presence of a considerable quantity 
of albumin, for example, reduces greatly the antiseptic power of 
mercuric chloride, silver nitrate, creolin, etc. The presence of a sub- 
stance chemically incompatible, as, for example, sodium chloride in 
testing nitrate of silver, will of course neutralize antiseptic action. 

(b) The nature of the test organism. Within certain limits an 
antiseptic for one microédrganism of this class restrains the devel- 
opment of all, but there are wide differences in the ability of differ- 
ent species to grow in the presence of different chemical agents. 
Some grow readily in the presence of a considerable amount of free 
acid, others are restrained by a slightly acid reaction of the medium 
in which they are placed. The Bacillus acidi lactici, for example, 
can thrive in the presence of a considerable amount of the acid 
which is a product of its growth, but there is a limit to its power of 
developing in the presence of this and other acids. So, too, Mi- 
crococcus ures, which causes the alkaline fermentation of urine, 
grows in the presence of a considerable amount of carbonate of am- 
monia, but is finally restrained in its growth by this alkaline salt. 
The following determinations by Boer show the difference in the 
antiseptic power of hydrochloric acid for certain pathogenic bacte- 
ria: Bacillus of anthrax (without spores), 1 : 3,400 ; diphtheria bacil- 
lus, 1:3,400; glanders bacillus, 1: 700; typhoid bacillus, 1 : 2,100; 
cholera spirillum, 1:5,500. It will be noted that the cholera spiril- 
lum is restrained in its growth by about one-eighth the amount of 
hydrochloric acid which is required to prevent the development of 
the bacillus of glanders. The typhoid bacillus has a special tole- 
rance for carbolic acid, ete. 

(c) The temperature at which the experiment is made. At 
the temperature most favorable for growth a greater proportion of 
the antiseptic agent is required than at unfavorable temperatures— 
lower or higher. 

(d) The restraining influence for spores is much greater than 
for the vegetative form of bacteria. 

Methods of Determining Germicide Value.—The disinfecting 
power of a chemical agent is determined by allowing it to act for a 
given time, in a definite proportion, on a pure culture of a given 


ANTISEPTICS AND DISINFECTANTS. 167 


microérganism, and then testing the question of loss of vitality by 
culture experiments or by inoculations of infectious disease germs 
into susceptible animals. 

The test by cultivation is the most reliable, but in making it 
several points must be kept in view. Naturally the conditions must 
be such as are favorable for the growth of the particular microér- 
ganism which serves as the test ; and we must allow a considerable 
time for the development of the test organism, for it often happens 
that its vital activity has been weakened without being completely 
destroyed, and that growth will occur after an interval of several 
days, while in the control experiment it has perhaps been séen at 
the end of twenty-four hours. Another most important point is the 
fact that some of the disinfectinz agent is necessarily carried over 
with the test organisms when these are transferred to a nutrient 
medium to ascertain whether they will grow, and this may be in 
sufficient amount to restrain their development and lead to the mis- 
taken inference that they have been killed. This is especially true 
of mercuric chloride, which restrains the development of spores in 
very minute amounts. Spores which have been subjected to its ac- 
tion in comparatively strong solutions, when transferred to a culture 
medium may fail to grow because of the restraining influence of 
the mercuric chloride carried over at the same time. For this rea- 
son liquid cultures are to be preferred in experiments of this kind. 
When the test organisms are planted in a solid culture medium the 
chemical agent is left associated with them; in a liquid culture, on 
the other hand, it is diluted, and the microérganisms, being distri- 
buted through the nutrient medium, have the disinfecting agent 
washed from their surface. In the case of mercuric chloride, how- 
ever, the experiments of Geppert show that the agent is so attached 
to spores which have been subjected to its action that ordinary 
washing does not suffice. Moreover, spores which have been ex- 
posed to the action of mercuric chloride without being killed are re- 
strained in their growth by a much smaller proportion of the corro- 
sive sublimate than is required for spores not so exposed—according 
to Geppert, by 1 part in 2,000,000. Geppert therefore proposes, in 
experiments with this agent, to neutralize the mercuric chloride 
which remains attached to the test organisms by washing these in 
a solution of ammonium sulphide, by which the sublimate is preci- 
pitated as an inert sulphide. 

With most agents simple dilution will serve the purpose of pre- 
venting an erroneous inference from the restraining influence of the 
chemical agent being tested. If we carry, by means of a platinum 
loop, one or two 6se into five to ten cubic centimetres of bouillon, 
the dilution will usually be beyond the restraining influence of the 


168 ANTISEPTICS AND DISINFECTANTS. 


germicidal agent ; but we may carry the dilution still further, to be 
on the side of safety, by inoculating a second tube containing the 
same amount of sterile bouillon from the first, carrying over in the 
same way one or two ése. We will still be very sure to have a 
considerable number of thé microdrganisms to test the question of 
the destruction of vitality. Instead of bouillon we may use liquefied 
flesh-peptone-gelatin, which gives us the same advantage as to dilu- 
tion of the disinfecting agent; and after inoculating two tubes -as 
above indicated, we may make Hsmarch roll tubes by turning them 
upon a block of ice. The development of colonies will show that 
there was a failure to disinfect; their absence, after a proper inter- 
val, will be evidence of the germicidal action of the agent employed. 

Koch’s Method.—In 1881 Koch published his extended experi- 
ments made to determine the germicidal power of various chemical 
agents as tested upon anthrax spores. His method consisted in ex- 
posing silk threads, to which the dried spores were attached, in a 
solution of the disinfecting agent, and at intervals transferring one 
of these threads to a solid culture medium. The precaution was 
taken to wash-the thread in distilled water when the agent tested was 
supposed to be likely to restrain development. In these experiments 
a standard solution of the disinfecting agent was used, and the time 
of exposure was varied from a few hours to many days. 

The Writer’s Method.—In the writer’s experiments, made in 
1880 and subsequently, a different method has been adopted. The 
time has been constant—usually two hours—and the object has been 
to find the minimum amount of various chemical agents which 
would destroy the test organisms in this time; and instead of sub- 
jecting a few of the test organisms attached to a silk thread to the 
action of the disinfecting agent, a certain quantity of a recent cul- 
ture—usually five cubic centimetres—has been mixed with an equal 
quantity of a standard solution of the germicidal agent. Thus five 
cubic centimetres of a 1: 200 solution of carbolic acid would be 
added to five cubic centimetres of a recent culture of the typhoid 
bacillus, for example, and after two hours’ contact one or two ése 
would be introduced into a suitable nutrient medium to test the 
question of disinfection. In the case given the result obtained 
would be set down as the action of a solution of carbolic acid in the 
proportion of 1: 400, for the 1 : 200 solution was diluted by the addi- 
tion of an equal quantity of the culture. 

Other experimenters have adopted still a different method. In- 
stead of using a considerable and definite quantity of a culture con- 
taining the test organism, they introduce one or two ése from such 
aculture into a solution containing a given proportion of the disin- 
fectant ; then after exposure for a given time the nutrient medium is 
inoculated. 


ANTISEPTICS AND DISINFECTANTS, 169 


These different methods give results which cannot be directly 
compared one with another, for to obtain corresponding results we 
must have identical conditions. 

Test by Inoculation into Susceptible Animals.—In testing the 
action of disinfectants upon anthrax spores and other infectious dis- 
ease germs, we may inoculate the microérganisms, after exposure to 
the disinfectant, into a susceptible animal. This method was adopted 
by the writer in a series of experiments in 1881, but he has not since 
employed it, for reasons set forth in his paper giving an account of 
these experiments. 

“First. The test organism may be modified as regards repro- 
ductive activity without being killed; and in this case a modified form 
of disease may result from the inoculation, of so mild a character as 
to escape observation. Second. An animal which has suffered this 
modified form of the disease enjoys protection, more or less perfect, 
from future attacks, and if used for a subsequent experiment may, 
by its immunity from the effects of the pathogenic test organism, 
give rise to the mistaken assumption that this had been destroyed 
by the action of the germicidal agent to which it had been sub- 
jected.”? 

In experiments to determine the value of an agent as a disinfec- 
tant, no matter by what method, the following conditions, which in- 
fluence the result, should be kept in view : 

(a) The difference in vital resisting power of different species 
of bacteria. As arule, the pathogenic species have rather less re- 
sisting power than the common saprophytes, and the micrococci 
have greater resisting power than many of the bacilli. The differ- 
ence in the vital resisting power of some of the best known patho- 
genic species is shown in the following table, which we have made 
up from determinations made by Boer—cultures in bouillon twenty- 
four hours old ; time of exposure, two hours. 


| | Chloride of Nitrate 

Hydrochloric Caustic Gold and of Carbolic 

| Acid. Soda. Sodium. Silver. Acid. 
Anthrax bacillus....... 1: 1100 1 +450 1:8000 1: 20000 1: 300 
Diphtheria bacillus..... 1:70) 1:3800 1 :1000 1 :2500 1:3800 
Glanders bacillus ..... 1: 200 1: 150 1:400 1: 4000 1 :3800 
Typhoid bacillus...... 1 :300 1:190 1 :500 1: 4000 1: 200 
Cholera spirillum...... 1: 14850 1:150 1: 1000 1 :4000 1:400 


(b) The presence or absence of spores. The reproductive ele- 
ments known as spores have a far greater resisting power to chemi- 
cal agents, as well as to heat, than have the vegetative cells. In 


1 Quoted from article on ‘‘ Germicides and Disinfectants,” in ‘‘ Bacteria,” p. 212. 


aa 
170 ANTISEPTICS AND DISINFECTANTS. 


practical disinfection, therefore, it is important to know what disease 
germs form spores and what do not. The following are known to 
form spores: The bacillus of anthrax, the bacillus of tetanus, the 
bacillus of malignant cedema, the bacillus of symptomatic anthrax, 
the bacillus of foul brood (infectious disease of bees). The following, 
so far as is known, do not form spores: The pus cocci (Staphylo- 
coccus pyogenes albus, aureus, and citreus, and Streptococcus pyo- 
genes), the micrococcus of pneumonia, the bacillus of typhoid fever, 
the bacillus of glanders, the bacillus of diphtheria, the spirillum of 
cholera, the spirillum of relapsing fever. 

Many agents which kill the growing bacteria are incapable of 
destroying the vitality of spores, and others only do so in much 
stronger solutions or after a long exposure to their action. 

(c) The number of bacteria to be destroyed, This is an essen- 
tial factor which has often been overlooked by those making experi- 
ments. To destroy the bacteria carried over to five cubic centimetres 
of distilled water by means of a platinum loop, is a very different 
matter from destroying the immensely greater number in five cubic 
centimetres of a recent bouillon culture. 

(d) The nature and quantity of associated material. The 
oxidizing disinfectants, like permanganate of potash and chloride of 
lime, not only act upon the bacteria, destroying them by oxidation, 
but upon all organic matter with which they come in contact, and at 
the same time the disinfecting agent is destroyed in the chemical 
reaction, which is a quantitative one. The presence, therefore, of 
organic material in association with the bacteria is an important 
factor, and if this is in excess the disinfectant may be neutralized 
before the living bacteria are destroyed. Other substances which 
precipitate the disinfecting agent in an insoluble form, or decompose 
it, must of course have the same effect. Thus the presence of sodium 
chloride in a culture medium would be an important circumstance if 
nitrate of silver was the agent being tested, as the insoluble chloride 
would be precipitated. And in the case of mercuric chloride and 
certain other metallic salts the presence of albumin very materially 
influences the result. Van Ermengem states that the cholera spiril- 
lum in bouillon is destroyed in half an hour by mercuric chloride in 
the proportion of 1: 60,000, while in blood serum 1:800 was required 
to destroy it in the same time. 

(e) The time of exposure is also an important factor. Some 
agents act very promptly, while others require a considerable time to 
effect the destruction of bacteria exposed to their action. Thus a 
solution of chloride of lime containing 0.12 per cent destroys the 
typhoid bacillus and the cholera spirillam in five minutes, and 
the anthrax bacillus in one minute (Nissen). On the other hand, 


ANTISEPTICS AND DISINFECTANTS. 171 


quicklime (milk of lime) requires a contact of several hours to in- 
sure the destruction of pathogenic bacteria. 

(f) The temperature at which the exposure is made has a 
material influence upon the result. This is shown by the experi- 
ments of Henle and of Nocht. Asa general rule germicidal activ- 
ity increases in direct proportion to the increase in temperature from 
20° C. upward. 

(g) The degree of dilution of the disinfecting agent is also a 
matter of importance. This is especially true of solutions of acids 
and alkalies. When a silk thread to which bacteria are attached is 
suspended in an acid solution the essential point is the degree of 
acidity, and not the quantity of acid in the entire solution. Butif a 
solution of permanganate of potash, or any other active oxidizing 
agent, is used, the principal question is not the degree of dilution, but 
the amount of the disinfecting agent present in the solution used. A. 
grain of potassium permanganate dissolved in two fluidounces of 
distilled water would probably kill just as many bacteria as if it 
were dissolved in half a fluidounce, although the time required for 
disinfection might be longer. 

From what has been said it is evident that the simple statement 
that a certain agent is a germicide in a certain proportion has but 
little scientific value, unless we are made acquainted with the condi- 
tions under which its germicidal action has been tested. 


Vill. 


ACTION OF GASES AND OF THE HALOID ELEMENTS 
UPON BACTERIA. 


Oxygen.—Free oxygen is essential for the development of a large 
number of species of bacteria—aérobics ; and it completely prevents 
the growth of others—anaérobics. Many bacteria, even when freely 
exposed in a desiccated condition to the action of atmospheric oxygen, 
retain their vitality for along time. The gradual loss of pathogenic 
power which Pasteur has shown occurs in cultures of the anthrax 
bacillus and the micrococcus of fowl cholera, is ascribed by him to 
exposure to oxygen, and as proof of this he states that cultures kept 
in hermetically sealed tubes do not lose their virulence in the same 
degree. But other circumstances may influence the result. Thus 
some of the products of growth which accumulate in culture fluids 
have an injurious effect upon the vitality of the bacteria which pro- 
duced them, and in time may cause a complete destruction of vitality. 
In cultures exposed to the air these products would be in a more 
concentrated solution from the gradual evaporation of the culture 
liquid. It must also be remembered that light in the presence of 
oxygen is a germicidal agent. 

The experiments of Friinkel show that the aérobic bacteria grow 
abundantly in the presence of pure oxygen, and some species even 
more so than in ordinary air. Micrococcus prodigiosus, however, 
appeared to be unfavorably affected by pure oxygen, inasmuch as it 
did not produce pigment so readily as when cultivated in ordinary air. 

Nascent oxygen is a very potent germicidal agent, as will be seen 
in our account of such oxidizing disinfectants as potassium perman- 
ganate and the hypochlorite of lime. 

Ozone.—It was formerly supposed that ozone would prove to be 
a most valuable agent for disinfecting purposes ; but recent experi- 
ments show that it is not so active a germicide as was anticipated, 
and that from a practical point of view it has comparatively little 
value. 

Lukaschewitsch found that one gramme in the space of a cubic 
metre failed to kill anthrax spores in twenty-four hours. The cholera 
spirillum in a moist state was killed in this time by the same amount, 
but fifteen hours’ exposure failed to destroy it. Ozone for these ex- 
periments was developed by means of electricity. 


ACTION OF GASES AND HALOID ELEMENTS UPON BACTERIA. 173 


Wyssokowicz found that the presence of ozone in a culture me- 
dium restrained the development of the anthrax bacillus, the bacillus 
of typhoid fever, and others tested, but concludes that this is rather 
due to the oxidation of bases contained in the nutrient medium than 
to a direct action upon the pathogenic bacteria. 

Sonntag, in his carefully conducted experiments, in which a cur- 
rent of ozonized air was made to pass over silk threads to which were 
attached anthrax spores, had an entirely negative result. The an- 
thrax bacillus from the spleen of a mouse, and free from spores, was 
then tested, also with a negative result, even after exposure to the 
ozonized air for twenty minutes at a time on four successive days. In 
another experiment several test organisms (Bacillus anthracis, Bacil- 
lus pneumonizs of Friedlander, Staphylococcus pyogenes aureus, 
Staphylococcus pyogenes albus, Bacillus murisepticus, Bacillus 
crassus sputigenus) were exposed on silk threads for twenty-four 
hours in an atmosphere containing 4.1 milligrammes of ozone to the 
litre of air (0.19 volumes percent). The result was entirely negative. 
When the amount was increased to 13.53 milligrammes per litre the 
anthrax bacillus and Staphylococcus pyogenes albus failed to grow 
after twenty-four hours’ exposure. The conclusion reached by Nis- 
sen, from his own experiments and a careful consideration of those 
previously made by others, is that ozone is of.no practical value as a 
germicide in therapeutics or disinfection. 

Hydrogen.—This gas has no injurious effect upon bacteria, as is 
shown by the fact that the anaérobic and facultative anaérobic species 
grow readily in an atmosphere of pure hydrogen. 

Hydrogen peroxide in solution in water is a valuable antiseptic 
and deodorant, but its value as a germicide has been very much 
overestimated. Miquel, in his experiments to determine the anti- 
septic value of various agents, places H,O, third in the list of ‘‘ sub- 
stances eminently antiseptic,” and states that it prevents the develop- 
ment of the bacteria of putrefaction in the proportion of 1: 20,000. 

In the writer’s experiments (1885) a solution was used which 
contained at first 4.8 per cent of H,O,, and five per cent of sulphuric 
acid which was added by the chemist who prepared the solution, te 
prevent loss of the hydrogen peroxide. At the end of a month the 
amount of H,O, was again estimated, and found to be 3.98 per cent. 
Five weeks later the proportion was 2.4 per cent. Tested upon 
‘“broken-down” beef tea, this solution was found to destroy the 
vitality of the bacteria of putrefaction contained in it, in two hours’ 
time, in the proportion of thirty per cent (about 1.2 per cent of H,O,). 
Anthrax spores were killed in the same time by a twenty-per-cent 
solution (0.8 per cent H,O,). Tested upon a pure culture of pus 
cocci, it was active in the proportion of ten per cent (0.4 per cent of 


174 ACTION OF GASES AND OF THE 


H,0,); a solution containing 0.24 per cent of H,O, failed to kill pus 
cocci. But the solution used in these experiments contained also five 
per cent of sulphuric acid, which by itself kills micrococci in the pro- 
portion of 1:200, My conclusion was that, unless the chemists can 
furnish more concentrated solutions which will keep better than that 
with which I experimented, we are not likely to derive any practical 
benefit from the use of hydrogen peroxide as a disinfectant. 

Altehofer more recently has experimented with a solution contain- 
ing 9.7 per cent of H,O,, and reports the following results: He added 
to ninety-eight cubic- centimetres of hydrant water two cubic centi- 
metres of a bouillon culture of the typhoid bacillus, and to this was 
added sufficient of his aqueous solution of H,O, to make the propor- 
tion present 1:1,000. At the end of twenty-four hours the bacillus 
was proved by culture experiments to be killed. Water containing 
the cholera spirillum, treated in the same way, was not entirely steril- 
ized, as a few colonies developed in Esmarch roll tubes ; but the gen- 
eral result of his experiments was that the ordinary water bacteria, 
and the pathogenic bacteria named (cholera, typhoid) when sus- 
pended in water, required for their destruction exposure for twenty- 
four hours in a solution containing one part of H,O, in one thousand 
of water. 

Carbon Dioxide.—The experiments of Frinkel show that certain 
bacteria grow in an atmosphere of CJ, as well as in the air ; among 
these are the bacillus of typhoid fever and the pneumonia bacillus 
of Friedlander. Other species are slightly restricted in their growth, 
e.g. Bacillus prodigiosus, Proteus vulgaris. Still others grow only 
when the temperature is elevated, including the pus cocci and the 
bacillus of swine pest. Most of the saprophytic bacteria failed to 
grow in an atmosphere of CO,, although their vitality was not de- 
stroyed by it. Certain pathogenic species were, however, killed by 
the action of this gas, among others the cholera spirillum, Bacillus 
anthracis, and Staphylococcus pyogenes aureus. 

Leone and Hochstetter had previously reported that certain bac- 
teria are injuriously affected by CO,. Frankel also found that the 
growth of strictly anaérobic species was restricted in an atmosphere 
of carbon dioxide. The aérobic species which failed to grow in pure 
CO, grew abundantly when a little atmospheric oxygen was ad- 
mitted. In the experiments of Frankland the cholera spirillum and 
the Finkler-Prior spirillum failed to develop in an atmosphere of 
CO,, and at the end of eight days were no longer capable of growth 
when the carbon dioxide was replaced with atmospheric air. 

Carbonic Oxide.—Frankland’s experiments show that an atmo- 
sphere of this gas is not favorable to the growth of the cholera spiril- 
lum or of the Finkler-Prior spirillum, although it did not entirely 


HALOID ELEMENTS UPON BACTERIA. 175 


prevent development, and after seven days’ exposure the spirilla were 
not all killed, although a comparatively small number of colonies de- 
veloped. Bacillus pyocyaneus failed to grow in an atmosphere of 
CO, but when air was admitted, at the end of seven or eight days, 
abundant development occurred. 

Methane, CH,—We have no exact experiments to determine 
the action of marsh gas in a pure state on bacteria, but the experi- 
ments of Kladakis upon illuminating gas may be taken as repre- 
senting approximately what might be expected from exposure in 
pure CH,. An analysis of the gas used in his experiments showed 
it to contain 37.97 per cent of hydrogen, 39.37 per cent of methane 
(CH,), 9.99 per cent of nitrogen, 4.29 per cent of ethene (C,H,), 3.97 
per cent of carbonic oxide (CO), 0.61 per cent of oxygen, and 0.41 per 
cent of carbon dioxide. As hydrogen and nitrogen are neutral, and 
carbonic oxide is shown by the experiments of Frankland not to act 
as a germicide after several days’ exposure to its action, the positive 
results obtained in the experiments of Kladakis may be ascribed to 
the presence of CH, (39.37 per cent) or of C,H, (4.29 per cent), or of 
both together. 

A large number of microérganisms were tested, and among these 
Proteus vulgaris alone grew in an atmosphere of illuminating gas. 
The others not only failed to grow in such an atmosphere, but were 
destroyed by it. Cultures of Bacillus anthracis, Staphylococcus pyo- 
genes aureus, and Spirillum cholerze Asiaticee were sterilized in half 
an hour by the action of this gas. The gas was also found to be un- 
suitable for anaérobic cultures. 

Nitrous Oxide, N,O.—The experiments of Frankland, made 
upon the cholera spirillum, the spirillum of Finkler-Prior, and the 
bacillus of green pus, gave results similar to those obtained with CO, 
viz., seven days’ exposure in an atmosphere of this gas failed to de- 
stroy the test organisms, but completely restrained the growth of 
Bacillus pyocyaneus and interfered materially with the development 
of the two species of spirillum without entirely preventing it. 

Nitrogen Dioxide, NO.—Frankland found that his test organ- 
isms were quickly killed by this gas (Bacillus pyocyaneus, Spirillum 
cholerz Asiatic, Spirillum Finkler-Prior). 

Hydrosulphurtc Acid, H,S.—In the experiments of Frankland 
this gas proved to be quickly fatal to the bacteria tested (Bacillus 
pyocyaneus, Spirillum cholere Asiaticee, Spirillum Finkler-Prior). 
On the other hand, Grauer found that this gas did not exercise any 
injurious influence upon the tubercle bacillus, the bacillus of anthrax, 
the typhoid bacillus, or the cholera spirillum, after the exposure of 
these microédrganisms in a current of the gas for an hour. 

It has been shown by the experiments of Holschewnikoff and 


176 ACTION OF GASES AND OF THE 


others that certain species of bacteria cause an abundant evolution 
of H,S as a result of their development in an albuminous medium 
(Bacillus sulfureus and Proteus sulfureus). 

Sulphur Dioxide, 8O,.—Very numerous experiments have been 
made with this gas, owing to the fact that it has been extensively 
used in various parts of the world for the disinfection of hospitals, 
ships, apartments, clothing, etc. 

In the writer’s experiments, made in 1880, dry vaccine virus on 
ivory points was disinfected by exposure for twelve hours in an at- 
mosphere containing one volume per cent of this gas, and liquid 
virus, exposed in a watch glass, by one-third of this amount. Sub- 
sequent experiments (1885) showed that pus micrococci were killed 
by exposure for eighteen hours in a dry atmosphere containing twenty 
volumes per cent of SO,, but that four volumes per cent failed. In 
the presence of moisture this gas has considerably greater germicidal 
power than this, owing, no doubt, to the formation of the more ac- 
tive agent, sulphurous acid (H,SO,). Butin a pure state anhydrous 
sulphur dioxide does not destroy spores. The writer has shown that 
the spores of Bacillus anthracis and Bacillus subtilis are not killed by 
contact for some time with liquid SO, (liquefied by pressure). Koch 
exposed various species of spore-bearing bacilliin a disinfection cham- 
ber for ninety-six hours, the amount of SO, at the outset of the ex- 
periment being 6.13 volumes per cent, and at the end 3.3 per cent. 
The result was entirely negative. 

But in the absence of spores the anthrax bacillus, in a moist con- 
dition, attached to silk threads, was destroyed in thirty minutes in 
an atmosphere containing one volume per cent. 

In another of Koch’s experiments the amount of SO, in the disin- 
fection chamber was at the outset 0.84 per cent, and at the end of 
twenty-four hours 0.55 per cent. An exposure of one hour in this at- 
mosphere killed anthrax bacilli attached to silk threads, in a moist 
condition; but four hours’ exposure failed to kill Bacillus prodigiosus 
growing on potato, while twenty-four hours’ exposure was successful. 
A similar result was obtained with Bacillus pyocyaneus. 

Thinot, asa result of experiments made in 1890, arrives at the 
conclusion that the specific germs of tuberculosis, glanders, farcy of 
cattle, typhoid fever, cholera, and diphtheria are destroyed by twenty- 
four hours’ exposure in an atmosphere containing SO, developed by 
the combustion of sixty grains of sulphur per cubic metre. This 
amount corresponds closely with that fixed by the Committee on Dis- 
infectants of the American Public Health Association on the experi- 
mental evidence obtained by the writer in 1885. But the committee 
insisted upon the presence of moisture and made the time of exposure 
twelve hours—‘‘ exposure for twelve hours to an atmosphere con- 


HALOID ELEMENTS UPON BACTERIA. 177 


taining at least four volumes per cent of this gas in the presence of 
moisture.” 

Chlorine.—The haloid elements are active germicidal agents, 
especially chlorine on account of its affinity for hydrogen, and the 
consequent release of nascent oxygen when it comes in contact with 
microérganisms in a moist condition. And for the same reason this 
agent isa much more active germicide in the presence of moisture 
than in a dry condition. The experiments of Fischer and Proskauer 
showed that when dried anthrax spores were exposed for an hour in 
an atmosphere containing 44.7 per cent of dry chlorine they were not 
destroyed ; but if the spores were previously moistened and were ex- 
posed in a moist atmosphere for the same time, four per cent was 
effective, and when the time was extended to three hours one per 
cent destroyed their vitality. The anthrax bacillus, in the absence of 
spores, was killed by exposure in a moist atmosphere containing 1 
part to 2,500, the time of exposure being twenty-four hours, and the 
same amount was effective for Micrococcus tetragenus ; the strepto- 
coccus of erysipelas and the micrococcus of fowl cholera were killed in 
three hours by 1:2,500, and in twenty-four hours by 1:25,000. The 
bacillus of mouse septiceemia and the tubercle bacillus were killed in 
one hour by 1 : 200. , 

In the writer’s experiments (1880) four children were vaccinated 
with virus from ivory points which had been exposed for six hours in 
an atmosphere containing one-half per cent of chlorine; also with 
four points, from the same lot, not disinfected. Vaccination was un- 
successful in every case with the disinfected points, and successful 
with those not disinfected. Koch found that anthrax spores failed 
to grow after twenty-four hours’ exposure in chlorine water. In 
the experiments of De la Croix to determine the antiseptic power of 
this agent, it was found that when present in unboiled beef infusion 
in the proportion of 1: 15,600 no development of bacteria occurred. 
Miquel gives the antiseptic value of chlorine as 1 : 4,000. 

Chloroform.—Immersion for one hundred days in chloroform 
does not destroy the vitality of anthrax spores (Koch). This agent 
is without effect on the virus of symptomatic anthrax (Arloing, 
Cornevin, and Thomas). Salkowski found that the anthrax bacillus 
in the absence of spores, and the cholera spirillum, were killed by 
being immersed in chloroform water for half an hour. Kirchner 
reports still more favorable results. In his experiments a one-per- 
cent solution killed the cholera spirillum in less than a minute, and 
a one-quarter-per-cent solution in an hour. But the typhoid bacillus 
required at least one-half per cent acting for an hour. 

Iodine.—In the writer’s experiments (1880) iodine in aqueous 
solution with potassium iodide was found to be fatal to Micrococcus 

12 


178 ACTION OF GASES AND OF THE 


pneumoniee crouposa in the proportion of 1 : 1,000, and to the staphy- 
lococci of pus in 1 : 500—time of exposure two hours. Iodine water 
was found by Koch to destroy the vitality of anthrax spores in 
twenty-four hours, but a two-per-cent solution in alcohol failed to 
destroy anthrax spores in forty-eight hours. In the experiments of 
Schill and Fischer twenty hours’ contact with a solution of the 
strength of 1 : 500 failed to destroy the virulence of tuberculous spu- 
tum, as tested by inoculation experiments. The antiseptic value of 
iodine is given by Miquel as 1 : 4,000. 

Bromine.—Fischer and Proskauer have studied the action of 
bromine vapor upon various microérganisms. They found that ex- 
posure for three hours in a dry atmosphere to three per cent does 
not destroy the tubercle bacillus in sputum or the spores of an- 
thrax. But when the atmosphere is saturated with moisture 1 : 500 
is effective ; and when the time of exposure was extended to twenty- 
four hours, 1:3,500. A two-per-cent solution destroys the vitality 
of anthrax spores in twenty-four hours (Koch). Bromine vapor is 
an active agent for the destruction of the virus of symptomatic an- 
thrax (Arloing, Cornevin, and Thomas). Miquel gives the antisep- 
tic value of bromine as 1 : 1,666, which is considerably below that of 
chlorine and iodine. 

Iodine Trichloride.—According to Behring, we possess in this 
agent a disinfectant which possesses the potency of free chlorine and 
iodine without having their disadvantages. As prepared by O. Rie- 
del it is a yellowish-red powder of penetrating odor. It remains un- 
changed for weeks in concentrated aqueous solution (five per cent). 
A. one-per-cent solution destroys anthrax spores suspended in water 
almost instantly, and a 0.2-per-cent solution within a few minutes. 
Anthrax spores in blood serum are killed by a one-per-cent solution 
in forty minutes (Behring). Langenbuch found that a solution of 
1:1,000 kills spores in a short time, and that when added to nutri- 
ent gelatin in the proportion of 1:1,200 it restrains the develop- 
ment of bacteria. 

JIodoform.—Numerous experiments have been made with this 
agent, which show that it has little, if any, germicidal power ; but 
it acts to some extent as an antiseptic. Tilanus reports that the tu- 
bercle bacillus will not grow in glycerin-agar cultures to which a 
small quantity of iodoform has been added, and that a pure culture 
of the tubercle bacillus was not killed in six days by exposure to 
iodoform vapor, but that after six weeks’ exposure it failed to grow. 
The experiments of Neisser and of Buchner show that while most 
bacteria are not injuriously affected by exposure to iodoform vapor, 
the cholera spirillum and the Finkler-Prior spirillum are restrained in 
their growth by such exposure. When plate cultures of the cholera 


HALOID ELEMENTS UPON BACTERIA. 179 


spirillum were placed under a bell jar beside iodoform powder 
no development occurred, but when they were removed colonies de- 
veloped, showing that the spirilla were not killed. 

Iodoform Ether, according to Yersin, is fatal to the tubercle ba- 
cillus in one-per-cent solution in five minutes. Cadéac and Meunier 
found that a saturated solution required thirty-six hours to kill the 
bacillus of typhoid fever. 

Jodol.—In experiments made by the writer (1885) this agent was 
found to be without germicidal power. Riedlin found it without any 
action, even upon the cholera spirillum. 

Hydrofluoric Acid, H¥1.—From a series of experiments made 
with this gas, Grancher and Chautard arrive at the conclusion that 
‘the direct and prolonged action of hydrofluoric acid upon the tuber- 
cle bacillus diminishes its virulence but does not kill it.” 

Sozotodol Acid,according to Drier, is a phenol, in which two atoms 
of hydrogen are replaced by two of iodine and one atom by the group 
HSO,. This acid and its salts with soda, potash, zinc, and mercury 
have been tested by the author named. The acid and its salt with 
mercury were found to destroy the cholera spirillum in two hours’ time 
in two-per-cent solution. A two-per-cent solution of phenol would 
have accomplished the same result and in lesstime. Tribromphenol, 
according to Drader, is less active than sozoiodol acid; and it appears 
from the experimental evidence on record that combinations of 
iodine, chlorine, or bromine with phenol are less active that the 
haloid elements alone. According to Karpow (1893) monochlor- 
phenol, tested upon anthrax spores attached to silk threads, proved 
to be decidedly more active than phenol. 

Nosophen (tetraiodphenolphthalein), according to Lieven (1895) 
contains sixty-one per cent of iodine. It is entirely insoluble in 
water. When added to nutrient gelatin in the proportion of one- 
quarter per cent it prevented the development of the anthrax bacillus 
and of Staphylococcus aureus, but failed to prevent the development 
of Bacillus pyocyaneus (Lieven). 


IX. 


ACTION OF ACIDS .AND ALKALIS, 


Sulphuric Acid, H,SO,.—The experiments of Koch (1881) 
showed that anthrax spores were still capable of growing after ex- 
posure in a one-per-cent solution of sulphuric acid for twenty days. 
In the writer’s experiments (1885) a four-per-cent solution failed to 
destroy the spores of Bacillus subtilis in four hours, and an eight- 
per-cent solution was found to be required for the sterilization of 
culture fluids containing spores ; but the multiplication of the bacte- 
ria of putrefaction was prevented by the presence of this acid in a 
culture solution in the proportion of 1:800. Pus micrococci were 
destroyed by exposure for two hours in a solution containing 1 : 200. 

The experiments of Boer show that there is a considerable differ- 
ence in the resisting power of different pathogenic bacteria. The 
time of exposure being two hours, cultures in bouillon twenty-four 
hours old gave the following results : 


Restrains Destroys 
development. vitality. 
Anthrax bacillus..........0. ceseeeeseeveeeees 1 : 2550 1:1300 
Diphtheria bacillus... 22... 0. ce eee eee eee eee 1: 2050 1: 500 
Glanders bacillus..........--eeeeeeceeceseeeoes 1:750 1: 200 
Typhoid. bacillus. :.:. si. assuvecsnaesrs ie eecess 1: 1550 1: 500 
Cholera spirillum ..........0 0c. eee cece eeu 1: 7000 1: 1300 


Leitz, in his studies relating to the bacillus of typhoid fever, 
reports the following results: The dejections of typhoid patients, 
mixed with an equal proportion of the disinfecting solution, were 
sterilized by a five-per-cent solution of sulphuric acid in three days. 
A pure culture was sterilized in fifteen minutes by two per cent, and 
in five minutes by five per cent. ; 

Sulphurous Acid, H,SO,.—In the writer’s experiments (1885) 
micrococci were destroyed in two hours by 1 : 2,000 by weight of SO, 
added to water. Kitasato found that solutions of sulphurous acid 
in the proportion of 0.28 per cent killed the typhoid bacillus, and 
0.148 per cent the cholera spirillum. De la Croix found that one 


ACTION OF ACIDS AND ALKALIES. 181 


gramme of SO, added to two thousand of bouillon prevents the de- 
velopment of putrefactive bacteria and after a time destroys the 
vitality of these bacteria. The writer found that pus cocci failed to 
grow in a culture solution containing one part of SO, in five thousand 
of water. 

Nitric Acid, HNO,.—In the writer’s experiments an eight-per- 
cent solution which contained 0.819 gramme of HNO, in each cubic 
centimetre sterilized broken-down beef tea containing spores, and 
five per cent failed todo so. Kitasato, inexperiments upon the chol- 
era spirillum and typhoid bacillus, obtained results corresponding 
with those obtained with hydrochloric acid—0.2 per cent destroyed 
vitality at the end of four or five hours. In these experiments the 
acid used contained 0.35 gramme HNO, in one cubic centimetre. 

Nitrous Acid.—In the writer’s experiments on vaccine virus (1880) 
exposure for six hours in an atmosphere containing one per cent of 
nitrous acid destroyed the virulence of dried virus upon ivory points. 

Hydrochloric Acid, HCl.—Anthrax spores are destroyed in ten 
days by a two-per-cent solution, but not in five days (Koch). Tested 
upon broken-down beef tea containing spores of Bacillus subtilis, it 
was effective in two hours in the proportion of fifteen per cent, but 
failed in ten per cent (Sternberg). In the experiments of Kitasato this 
acid destroyed the typhoid bacillus in five hours in the proportion of 
0.2 per cent, and the cholera spirillum in 0.132 per cent—the acid 
used contained 0.26 gramme HCl in one cubic centimetre. We give 
the more recent determinations of Boer in tabular form. Its germi- 
cidal power was tested upon bouillon cultures which had been kept 
for twenty-four hours in an incubating oven ; time of exposure to 
the action of the acid solution, two hours. 


Restrains Destroys 

development. vitality. 

Anthrax Dacilusieii cies eevee a's sesesne o¥ es 1:3400 1:1100 
Diphtheria bacillus....... 2... eee e eee eee eens 1:3400 1:700 
Glanders bacillus..........0ceee veces eeveeeces 1:700 1:200 
Typhoid bacillus si. s60 cacsaevrneseeeseeees 1: 2100 1:300 

Cholera spirillum ..........02 02 eee cent cece 1 : 5500 1: 1350 


Chromic Acid.—In Koch’s experiments a one-per-cent solution 
destroyed anthrax spores in from one to two days. In the propor- 
tion of 1:5,000 it prevents the development of putrefactive bacteria 
(Miquel). 

Osmic Acid.—A solution of one per cent kills anthrax spores in 
twenty-four hours (Koch). It is an antiseptic in the proportion of 
1:6,666 (Miquel). 

Phosphoric Acid.—Exposure for four or five hours to a solution 


18% ACTION OF ACIDS AND ALKALIES. 


containing 0.3 per cent destroys the typhoid bacillus, and 0.183 per 
cent the cholera spirillum (Kitasato). The acid used contained 0.152 
gramme H,PO, in one cubic centimetre. 

Acetic Acid.—-A five-per-cent solution failed to kill anthrax 
spores after five days’ exposure (Koch). In Abbott’s experiments 
glacial acetic acid in fifty-per-cent solution failed in two hours to kill 
anthrax spores, but micrococci were killed by two hours’ exposure to 
a one-per-cent solution. A solution of 1:300 of glacial acetic acid 
destroys the cholera spirillum in half an hour (Van Ermengem). In 
the proportion of 0.25 per cent it restrains the growth of the typhoid 
bacillus, and 0.3 per cent destroys its vitality after five hours’ expo- 
sure ; the cholera spirillum fails to grow in presence of 0.132 per cent 
and is destroyed by 0.2 per cent (Kitasato). 

Lactic -lc/d.—The bacillus of typhoid fever is killed in five hours 
by a solution containing 0.4 per cent, the cholera spirillum by 0.3 per 
cent (Kitasato). 

Citric Acid.—The bacillus of typhoid fever is killed in five hours 
by 0.43 per cent, the cholera spirillum by 0.3 per cent (Kitasato). 
The cholera spirillum is killed in half an hour by 1:200 (Van Er- 
mengem). 

Oxalic Acid.—The typhoid bacillus requires a solution of 0.36 
per cent, the cholera spirillum one of 0.28 per cent, to destroy vitality 
in five hours (Kitasato). 

Boracic Acid.—In the writer’s experiments (1883) a saturated 
solution failed to kill pus cocci in two hours. <A five-per-cent solu- 
tion failed to destroy anthrax spores in five days (Koch). The 
typhoid bacillus is killed in five hours by 2.7 per cent, the cholera 
spirillum by 1.5 per cent (Kitasato). According to Arloing, Corne- 
vin, and Thomas, the fresh virus of symptomatic anthrax requires 
exposure to a twenty-per-cent solution for forty-eight hours for the 
destruction of vitality. Boracic acid acts as an antiseptic in the pro- 
portion of 1: 143 (Miquel). 

Salicylic Acid.—In the writer’s experiments this agent was dis- 
solved by the addition of sodium biborate, which by itself has no 
germicidal power. A two-per-cent solution was found to destroy pus 
cocci in two hours. Dissolved in oil or in alcohol a five-per-cent so- 
lution does not destroy anthrax spores (Koch). Micrococci are de- 
stroyed by solutions containing 1 : 400 (Abbott). The typhoid bacillus 
is killed in five hours by 1.6 per cent, the cholera spirillum by 1.3 per 
cent (Kitasato). A one-per-cent solution destroys Micrococcus Pas- 
teuri in half an hour (Sternberg). Itis an antiseptic in the propor- 
tion of 1:1,000 (Miquel). A solution of 2.5 per cent kills the tubercle 
bacillus in six hours (Yersin). In the proportion of 1 :300it destroys 
the cholera spirillum in half an hour (Van Ermengem). 


ACTION OF ACIDS AND ALKALIES. 183 


Benzoic Acid.—According to Miquel, this acid restrains the de- 
velopment of putrefactive bacteria when present in bouillon in the 
proportion of 1:909. In the proportion of 1 : 2,000 it retards the de- 
velopment of anthrax spores (Koch). 

Formic Acid.—The typhoid bacillus is restrained in its growth by 
0.25 per cent, and is killed in five hours by 0.35 per cent, the cholera 
spirillum by 0.22 per cent (Kitasato). 

Tannic Acid.—A solution of one per cent kills Micrococcus Pas- 
teuri in the blood of a rabbit in half an hour (Sternberg). A five- 
per-cent solution failed in ten days to destroy anthrax spores (Koch). 
A twenty-per-cent solution failed in two hours to destroy the vitality 
of spores of the anthrax bacillus or of Bacillus subtilis (Abbott). 
Micrococci are destroyed by 1:400, and 1:800 failed (Abbott). A 
twenty-per-cent solution has no effect upon the virus of symptomatic 
anthrax (Arloing, Cornevin, and Thomas). A solution of 1.66 per 
cent kills the typhoid bacillus in five hours, and 1.5 per cent the 
cholera bacillus in the same time (Kitasato). It restrains the devel- 
opment of putrefactive bacteria in the proportion of 1: 207 (Miquel). 

Tartaric Acid.—A twenty-per-cent solution of this acid fails, 
after two hours’ exposure, to destroy the spores of Bacillus anthracis 
or Bacillus subtilis. Micrococci are killed by two hours’ exposure in 
a solution containing 1 : 400 (Abbott). 

Malic Acid.—This was found by Kitasato to correspond with 
citric acid in its germicidal power. 

Valerianic Acid.—A five-per-cent solution in ether failed in five 
days to destroy anthrax spores (Koch). 

Oleic Actd.—A solution of five per cent in ether does not destroy 
anthrax spores in five days (Koch). 

Thymic Acid.—In the proportion of 1 :500 this acid prevents the 
putrefactive decomposition of beef tea (Miquel). 

Butyric Acid.—Five days’ immersion in this acid failed to de- 
stroy anthrax spores (Koch). 

Arsenious Acid.—A one-per-cent solution destroys the vitality 
of anthrax spores in ten days, but failed to do so in six days (Koch). 
In the proportion of 1 :166it prevents putrefactive changes in bouillon 
(Miquel). 

Gallic Actd.—Abbott found this acid to destroy the bacteria in 
broken-down beef tea in the proportion of 2.57 per cent, but it failed 
to destroy anthrax spores in two hours in the same proportion. Mi- 
crococci were killed in two hours by 1 : 142, while 1 : 250 failed. 


ALKALIES. 


Potassium Hydroxide, KHO.—In the writer’s experiments a ten- 
per-cent solution of caustic potash was fatal to pus cocci, and an 


184 ACTION OF ACIDS AND ALKALIES. 


eight-per-cent solution failed—two hours’ exposure. Exposure for 
twenty-four hours to a ten-per-cent solution failed to kill the tubercle 
bacillus (Schill and Fischer). A solution of one per cent kills the 
anthrax bacillus, the bacillus of rothlauf, and several others (Jager). 
The addition of 0.14 per cent restrains the development of the typhoid 
bacillus, and 0.18 per cent kills this bacillus in four or five hours; the 
cholera spirillum failed to grow in cultures containing 0.18 per cent 
and was killed by 0.237 per cent in the same time (Kitasato). 

Sodium Hydroxide, NuaHO.—The experiments of Jager and of 
Kitasato show that soda has about the same germicidal power as 
caustic potash. Boer obtained the following results with bouillon 
cultures after two hours’ exposure: Anthrax bacillus, 1: 450; diph- 
theria bacillus, 1:300; glanders bacillus, 1: 150; typhoid bacillus, 
1:190; cholera spirillum, 1:150. In about one-half the amount 
required to destroy vitality the development of the above-named bac- 
teria was prevented. Inthe proportion of 1:56 it acts as an anti- 
septic (Miquel). 

Ammonia, NH,.—In Kitasato’s experiments the typhoid bacillus 
was destroyed in five hours by 0.3 per cent of NH,, and the cholera 
spirillum by about the same amount. Boer obtained the following 
results, the time of exposure being two hours: Anthrax bacillus, 
1:300; diphtheria bacillus, 1: 250; glanders bacillus, 1 : 250 ; typhoid 
bacillus, 1:200; cholera spirillum, 1:350. The growth of the an- 
thrax bacillus and of the diphtheria bacillus in culture solutions was 
prevented by 1 : 650. 

Calctum Hydroxide, CatHO.—According to Kitasato, the ty- 
phoid bacillus and the cholera spirillum, in bouillon cultures, are 
killed in four or five hours by the addition of 0.1 per cent of calcium 
oxide. Liborius had previously reported still more favorable results, 
but his bouillon cultures were largely diluted with distilled water. 
From a practical point of view the experiments of Pfuhl are more 
valuable. Calcium hydrate was added to the dejections of typhoid 
patients. When added in the proportion of three per cent steriliza- 
tion was effected in six hours, and by six per cent in two hours. 
When milk of lime containing twenty per cent of calcium hydrate 
was used the results were still more favorable, the typhoid bacillus 
and cholera spirillum being killed in one hour by the addition of 
two per cent of the disinfectant. The practical value of lime-wash 
applied to walls has been determined by Jager. Silk threads soaked 
in cultures of various pathogenic bacteria were attached to boards 
and the lime-wash applied with a camel’s-hair brush. Anthrax ba- 

-cilli (without spores), the glanders bacillus, Staphylococcus pyogenes 
aureus, and several other pathogenic bacteria were killed by a single 
application after twenty-four hours, but the tubercle bacillus was not 


ACTION OF ACIDS AND ALKALIES, 185 


killed by three successive applications. In the writer’s experiments 
(1885) the typhoid bacillus and Staphylococcus pyogenes aureus were 
killed in two hours by a solution containing 1:40 of calcium oxide, 
and 1:80 failed. Spores of the anthrax bacillus and of several other 
spore-forming species were not killed by two hours’ exposure toa 
milk of lime containing twenty per cent of calcium oxide. 

Potash Soap has been shown by Jolles (1895) to have considerable 
germicidal value. In experiments with a soap containing 67.44 
per cent of fat acids, 10.4 per cent of combined alkali, and 0.041 
per cent of free alkali, the following results were obtained: The 
typhoid bacillus was destroyed at 18° C. by a one-per-cent solution 
in twenty-four hours; by a six-per-cent solution in thirty minutes. 
The Bacillus coli communis required somewhat stronger solutions or 
longer exposure—eight-per-cent solution required thirty minutes. 
These experiments show that scrubbing with soap and water is a 
reliable method of disinfecting surfaces. Solutions of potash—com- 
mon lye—or of soda also are useful for certain purposes in domes- 
tic disinfection, and scientific researches justify the continued use of 
the cleansing methods which have heretofore been in use by careful 
housewives. 


XxX. 


ACTION OF SALTS. 


WHILE some of the metallic salts, and especially those of mer- 
cury, silver, and gold, have remarkable germicidal power, others, 
even in concentrated solutions, do not destroy the vitality of bacteria 
exposed to their action. For convenience of reference we shall con- 
sider the agents in this group in alphabetical order, but first we give 
Miquel’s tables of antiseptic value. This author recognizes the im- 
portance of experiments to determine the restraining power of chem- 
ical agents for various species of pathogenic bacteria, but says : ‘‘ As 
to me, faithful to a plan I adopted at the outset, I will treat the sub- 
ject ina more general manner by making known simply the mini- 
mum weight of the substances capable of preventing the evolution of 
any bacteria or germs. The method adopted is very simple. Toa 
liquid always comparable to itself it is sufficient at first to add a 
known weight of the antiseptic and some atmospheric germs or adult 
bacteria, and to vary the quantity of the antiseptic until the amount 
is ascertained which will preserve indefinitely the liquid from putre- 
faction. In order to obtain germs of all kinds in a dry state it suf- 
fices to take them, where they are most abundant, in the dust col- 
lected in the interior of houses or of hospitals; and to procure a 
variety of adult bacteria we may take the water of sewers.” 


SUBSTANCES EMINENTLY ANTISEPTIC, 


Efficient in the 

proportion of— 
Mercuric iodide, 1: 40000 
Silver iodide, 1: 33000 
Hydrogen peroxide, . 1: 20000 
Mercurie chloride, 1: 14300 
Silver nitrate, 1: 12500 

SUBSTANCES VERY STRONGLY ANTISEPTIC. 

Osmic acid, 1: 6666 
Chromic acid, 1: 5000 
Chlorine, 1: 4000 
Todine, : 1: 4000 
Chloride of gold, . 1: 4000 
Bichloride of platinum, 1: 3333 
Hydrocyanic acid, 1: 2500 


ACTION OF SALTS. 


Bromine, 
Cupric chloride, 
Thymol, 

Cupric sulphate, 
Salicylic acid, 


SUBSTANCES STRONGLY ANTISEPTIC, 


Benzoic acid, : 

Potassium bichromate, 

Potassium cyanide, 

Aluminum chloride, 

Ammonia, 

Zine chloride, , 2 : ‘ 
Mineral acids, ; : : ; : 1: 500 to 
Thymic acid, ; : ‘ ‘ 
Lead chloride, 

Nitrate of cobalt, 

Sulphate of nickel, 

Nitrate of uranium, 

Carbolic acid, 

Potassium permanganate, 

Lead nitrate, y 

Alum,. 

Tannin, 


SUBSTANCES MODERATELY ANTISEPTIC. 


Browhydrate of quinine, 
Arsenious acid, 

Boracie acid, 

Sulphate of strychnia, 
Arsenite of soda, 
Hydrate of chloral, 
Salicylate of soda, 
Ferrous sulphate, 
Caustic soda, . 


SUBSTANCES FREELY ANTISEPTIC. 


Perchloride of manganese, 

Calcium chloride, 

Sodium borate, 

Muriate of morphia, 

Strontium chloride, ‘ : 

Lithium chloride, . ; : : ‘ 
Barium chloride, ‘ 5 F 
Alcohol, 


SUBSTANCES VERY FEEBLY ANTISEPTIC. 


Ammonium chloride, 7 : : 
Potassium arsenite, . 2 z s F 3 
Potassium iodide, 

Sodium chloride, 

Glycerin (sp. gr. 1.25), 5 ‘ « 
Ammonium sulphate, 7 7 F ; 5 
Sodium hyposulphite, 


Hee eet 


Pe ee ee 


PE ee et a 


PE 


ee eet 
WER AW) 


: 1666 
: 1428 
: 1340 
21111 
: 1000 


: 909 
: 909 
: 909 
1714 
714 
: 526 
2833 
: 500 
: 500 
2476 
: 400 
7356 
: 333 
1285 
2277 
1222 
: 207 


2182 
: 166 
2148 
1143 
:111 
:107 
: 100 
: 90 

: 56 


:40 
125 
714 
213 
:12 
:11 
:10 
:10 


187 


188 ACTION OF SALTS. 


ANTISEPTIC AND GERMICIDAL VALUE OF VARIOUS SALTS, 
ARRANGED ALPHABETICALLY. 


Alwm.—Antiseptic in the proportion of 1 : 222 (Miquel). 

Aluminum Acetate.—According to De la Croix, this salt is an 
antiseptic in the proportion of 1:6,310, Kuhn found it to be anti- 
septic in 1 :5,250. 

Aluminum Chloride.—Antiseptic in the proportion of 1: 714 
(Miquel). 

Ammonium Carbonate.—When present in the proportion of 
1:125 it restrains the development of typhoid bacilli, and in five 
hours’ time it kills these bacilli in the proportion of 1:100; the 
cholera spirillum is killed in the same time by 1 : 77 (Kitasato). 

Ammonium Chloride.—Antiseptic in the proportion of 1:9 
(Miquel). A five-per-cent solution does not kill anthrax spores in 
twenty-five days (Koch). 

Ammonium Fluosilicate.—The bacillus of anthrax and of ty- 
phoid fever fail to grow in nutrient gelatin containing 1 : 1,000, and 
a two-per-cent solution kills anthrax spores in one-quarter to three- 
quarters of an hour (Faktor). 

Ammonium Sulphate.—Antiseptic in the proportion of 1:4 
(Miquel). A five-per-cent solution failed in two days to kill an- 
thrax spores, but was effective in five days (Koch). 

Barium Chloride is an antiseptic in the proportion of 1:10 
(Miquel). 

Calctum Chloride is an antiseptic in the proportion of 1 : 25 
(Miquel). A saturated solution does not destroy anthrax spores 
(Koch). 

Calcium Hypochlorite.—This is a powerful germicidal agent 
and has great value as a practical disinfectant. Good chloride of 
lime contains from twenty-five to thirty per cent of available chlo- 
rine as hypochlorite. The experiments made by the Committee on 
Disinfectants of the American Public Health Association in 1885 
showed that a solution containing 0.25 per cent of chlorine as hypo- 
chlorite is an effective germicide, even when allowed to act only 
for one or two minutes. In Bolton’s experiments a solution of chlo- 
ride of lime of 1:2,000 (available chlorine 0.015) destroyed the ty- 
phoid bacillus and the cholera spirillum in two hours. For the de- 
struction of anthrax spores a one-per-cent solution was required 
(available chlorine 0.3 per cent). Nissen found that the typhoid 
bacillus and the cholera spirillum are destroyed with certainty in 
five minutes by a solution containing 0.12 per cent, anthrax bacilli 
in one minute by 0.1 per cent, Staphylococcus pyogenes aureus in 
one minute by 0.2 per cent, anthrax spores in thirty minutes by a 


ACTION OF SALTS. 189 


five-per-cent solution and in seventy minutes by a one-per-cent solu- 
tion. Experiments made by the same author upon the sterilization 
of faeces showed that 0.5 per cent to one per cent could be relied upon 
to destroy the typhoid bacillus or the cholera spirillum in faeces in 
ten minutes, 

Chloral Hydrate.—Antiseptic in the proportion of 1:107 (Mi- 
quel). A twenty-per-cent solution destroys pus cocci in two hours 
(Sternberg). 

Cupric Chloride.—Antiseptic in the propertion of 1: 1,428 
(Miquel). 

Cupric Sulphate.—Antiseptic in the proportion of 1:111 (Mi- 
quel). Kills the cholera spirillum in the proportion of 1: 3,000 in 
ten minutes (Nicati and Rietsch). Destroys the cholera spirillum in 
bouillon cultures in less than half an hour in 1: 600, and in four 
hours in 1: 1,000; cultures in blood serum require 1 : 200 (Van Hr- 
mengem). A solution of 1:20 kills the typhoid bacillus in ten min- 
utes (Leitz). This salt failed, in the writer’s experiments, to kill the 
spores of Bacillus anthracis and Bacillus subtilis in two hours’ time 
in a twenty-per-cent solution. In Koch’s experiments a five-per-cent 
solution failed to kill anthrax spores in ten days. Kills pus micro- 
cocci in two hours in the proportion of 1: 200 (Sternberg). In Bol- 
ton’s experiments made for the Committee on Disinfectants of the 
American Public Health Association the following results were ob- 
tained: Recent cultures in bouillon, time of exposure two hours : Ba- 
cillus of typhoid fever, 1 : 200; cholera spirillum, 1 :500; Bacillus pyo- 
cyanus, 1 :200; Brieger’s bacillus, 1 : 200; Emmerich’s bacillus, 1 : 200; 
Staphylococcus pyogenes aureus, 1:100; Staphylococcus pyogenes 
citreus, 1: 100; Staphylococcus pyogenes albus, 1 : 200; Streptococcus 
pyogenes, 1:500. When ten per cent of dried egg albumin was 
added to a recent culture in bouillon of the typhoid bacillus the 
amount required to insure sterilization was 1 : 10. 

In the report of the Committee on Disinfectants of the American 
Public Health Association this agent is recommended in ‘a solu- 
tion of two to five per cent for the destruction of infectious material 
not containing spores.” The experimental data above given show 
that this is a liberal allowance for material which does not contain 
an excessive amount of albumin. In the experiments of Leitz the 
typhoid bacillus in cultures was destroyed in ten minutes by a five- 
per-cent solution. 

‘Ferric Chloride.—A five-per-cent solution failed in two days to 
destroy anthrax spores, but was effective in five days (Koch). 

Ferrous Sulphate.—In the writer’s experiments (1883) a solution 
of twenty per cent failed to destroy micrococci and putrefactive bac- 
teria. In amore recent experiment ten per cent failed to kill pus 


190 ACTION OF SALTS, 


cocci, but was fatal to Micrococcus tetragenus—two hours’ exposure. 
Koch found that a five-per-cent solution failed to destroy anthrax 
spores in six days. Exposure to a twenty-per-cent solution for forty- 
eight hours does not destroy the virus of symptomatic anthrax (Ar- 
loing, Cornevin, and Thomas). In the experiments of Jager immer- 
sion in a solution of 1:3 destroyed the infective virulence of certain 
pathogenic bacteria (fowl cholera, rothlauf, glanders), as tested by 
injection into mice, but failed to kill anthrax spores and tubercle ba- 
cilli. The antiseptic power of ferrous sulphate is placed by Miquel 
at 1:90. In the writer’s experiments 1 : 200 prevented the develop- 
ment of micrococci and of putrefactive bacteria in bouillon placed 
in the incubating oven for forty-eight hours. Leitz found that a 
five-per-cent solution required three days’ exposure for the destruc- 
tion of the typhoid bacillus. 

Gold Chloride.—Antiseptic in the proportion of 1 : 4,000 (Miquel). 
Boer has made extended experiments with the chloride of gold and 
sodium. We give his results below. In his disinfection experi- 
ments a bouillon culture which had been in the incubating oven for 
twenty-four hours was used, and the time of exposure was two hours. 


Restrains Destroys 

development. vitality. 

Anthrax bacillus 13 vas. she sieded eee eeiseaaess 1: 40000 1 : 8000 

Diphtheria bacillus............ 0c cs eeee rene eee 1: 40000 1: 1000 
Glanders bacillus: <2 20:35 icneceangeaevesge ees 1: 15000 1: 400 
Typhoid bacillus................ spine ese : 1:20000 1: 500 

Cholera spirillum..................00- ree 1: 25000 1: 1000 


Lead Chloride.— Antiseptic in the proportion of 1 : 500 (Miquel). 

Lead Nitrate.—Antiseptic in the proportion of 1 : 277 (Miquel). 

Lithium Chloride.—Antiseptic in the proportion of 1:11 (Mi- 
quel). 

Manganese Protochloride.—Antiseptic in the proportion of 1:40 
(Miquel). s 

Mercurie Chloride.—Koch’s experiments (1881) gave the follow- 
ing results: A solution of 1:1,000 destroys anthrax spores in a few 
minutes, and 1:10,000 is effective after a more prolonged exposure. 
The writer (1884) obtained similar results—1 : 10,000 destroyed the 
spores of Bacillus anthracis and of Bacillus subtilis in two hours. 
More recent experiments indicate that failure to grow in culture, so- 
lutions cannot be accepted as evidence of the destruction of vitality 
in the case of spores exposed to the action of this agent, unless due 
precautions are taken to exclude the restraining influence of the small 
amount of mercuric chloride which remains attached to the spores. 
Koch had ascertained that the development of spores is restrained by 


ACTION OF SALTS. 191 


the presence of 1 : 300,000 in a culture medium, and Geppert has re- 
cently shown that even so small an amount as 1 : 2,000,000 will pre- 
vent the development of spores the vitality of which has been reduced 
by the action of a strong solution (1:1,000). When this restraining 
action is entirely neutralized by washing the spores in a solution con- 
taining ammonium sulphide it requires, according to Geppert, a solu- 
tion of 1:1,000 acting for one hour to completely destroy the vitality 
of anthrax spores. Frankel found that a solution of 1:1,000 was 
effective in half an hour. The typhoid bacillus, the bacillus of mouse 
septicemia, and the cholera spirillum, in bouillon cultures and in 
cultures in flesh-peptone-gelatin, are destroyed in two hours by 
1:10,000; but in a bouillon culture to which ten per cent of dried 
egg albumin was added a one-per-cent solution was required to de- 
stroy the typhoid bacillus in the same time (Bolton). According to 
Van Ermengem, cultures of the cholera spirillum in bouillon are steril- 
ized in half an hour by 1 : 60,000, but culturesin blood serum require 
1:800 to 1:1,000. In experiments upon tuberculous sputum Schill 
and Fischer found that exposure of fresh sputum to an equal amount 
of a 1: 2,000 solution for twenty-four hours failed to disinfect it, as 
shown by inoculation experiments in guinea-pigs. The antiseptic 
power of mercuric chloride is given by Miquel as 1:14,300. In the 
writer’s experiments 1 : 33,000 was found to prevent the development 
of putrefactive bacteria in bouillon, but a minute bacillus contained in 
broken-down beef infusion multiplied, after several days, in 1 : 20,000. 
The pus cocci were restrained in their development by 1 : 30,000. 

In Behring’s experiments the anthrax bacillus and cholera spiril- 
lum were killed in one hour by 1:100,000 when the temperature 
was 36° C., but at a temperature of 3° C. the proportion required 
"was 1:25,000. The same author states that at 22° C. Staphylo- 
coccus aureus in bouillon is not always killed in twenty-five minutes 
by 1:1,000. 

Abbott (1891) has shown that a 1:1,000 solution does not always 
destroy Staphylococcus pyogenes aureus in five minutes. He says: 
“ Frequently all the organisms would be destroyed after five minutes’ 
exposure, but almost as often a certain few would resist for that 
length of time, and even longer, going in some cases to ten, twenty, 
and even thirty minutes.” 

According to Yersin, a solution of 1 : 1,000 kills the tubercle bacil- 
lus in one minute. 

We might add considerably to the experimental data given, but 
the results already recorded are sufficient to show the value of this 
agent as an antiseptic and germicide, and justify its use for general 
purposes of disinfection in the proportion of 1:500 or 1:1,000 for 
material containing spores, and in the proportion of 1:2,000 to 


152 ACTION OF SALTS. 


1: 5,000 for pathogenic bacteria in the absence of spores; due regard 
being had to the fact that the presence of albumin very materially 
reduces its germicidal potency, and that it may be decomposed and 
neutralized by alkalies and their carbonates, by hydrosulphuric acid, 
and by many other substances. 

The albuminate of mercury, as has been shown by Lister, is solu- 
ble in an excess of albumin, and, according to Behring, is just as 
effective as an aqueous solution containing the same amount of sub- 
limate when dissolved in an albuminous liquid like blood serum (?). 

In practice the addition of a mineral acid to sublimate solutions, 
or of sodium, potassium, or ammonium chloride, is to be recom- 
mended, to prevent the precipitation of the mercuric chloride by al- 
bumin in fluids containing it. Behring recommends the addition 
of five parts of sodium or potassium chloride to one of the subli- 
mate. Such a solution is more stable than a simple solution of sub- 
limate, and no precipitate is formed by the addition of alkalies or by 
albumin. 

The same result is obtained, according to La Place, by the addi- 
tion of five parts of hydrochloric or tartaric acid to one part of sub- 
limate in aqueous solution. 

Mercuric Cyanide, Hg(CN),, and the Oxycyanitde of mercury 
have been tested, with the following results : Staphylococcus aureus 
is destroyed in five minutes by 1:100, in one hour by 1:1,000, in 
two hours by 1:1,500 (Chibret). The development of Bacillus an- 
thracis in culture solutions is prevented by the presence of cyanide 
of mercury in the proportion of 1 : 25,000, and by the oxycyanide by 
1: 16,000 (Behring). 

Boer obtained the following results with the oxycyanide—cul- 
tures in bouillon, twenty-four hours in incubating oven, time of 
exposure two hours : 


Restrained Destroyed 

development, vitality. 
Anthrax bacillus) .0< ccsewse wewieisines a5 Qe neva. 1: 80000 1: 40000 
Diphtheria bacillus.......... 0. cece eee eee es 1: 80000 1: 40000 
Glanders bacillus.... 1: 60000 1: 380000 
Typhoid bacillus.. ... .. ... ... 1: 60000 1: 30000 
Cholera spirillum.. .. .............45 1: 90000 1: 60000 


Mercuric Iodide.—The antiseptic value of this salt is placed by 
Miquel at 1: 40,000, which is more than double that given by the 
same author to the bichloride. In the writer’s experiments upon the. 
antiseptic value of salts and oxides of mercury the following results 
were obtained : 


ACTION OF SALTS. 193 


Active. Failed. 
Biniodide of mercury.........0e-ceee cee eeeees 1.20000 1: 40000 
Bichloride ..........00..005 Jepad octet saasls 1: 15000 1: 20000 
Protiodid@sic ic cece vee ioiace 42a3t.esnanes 1.10000 1 : 20000 
Yellow’ oxid@isss cssaaevassseseevess saevsnes 1: 1000 1: 2000 
Black: Oxide... al aciead See. se nceee yess FARES 1: 5v0 1- 1000 


Morphia Hydrochlorate.—Antiseptic in the proportion of 1:13 
(Miquel). 

Nickel Sulphate.—Antiseptic in the proportion of 1:400 (Mi- 
quel). 

Platinum Bichloride.—Antiseptic in the proportion of 1 : 3,333 
(Miquel). 

Potassium Acetate.—A saturated solution of this salt failed to 
kill anthrax spores in ten days (Koch). 

Potassium Arsenite.—In the writer’s experiments Fowler’s solu- 
tion failed to kill micrococci in two hours in the proportion of four 
per cent. Miquel places the antiseptic value of potassium arsenite 
at 1:8. 

Potassium Bichromate.—A. five-per-cent solution failed in two 
days to destroy anthrax spores (Koch). Efficient as an antiseptic in 
the proportion of 1 : 909 (Miquel). 

Potassium Bromide.—The bacillus of typhoid fever and the 
cholera spirillum fail to grow in culture solutions containing 9 to 
10.6 per cent, and are killed in four or five hours by ten to twelve 
per cent (Kitasato). 

Potassium Carbonate.—The development of the typhoid bacil- 
lus and of the cholera spirillum is prevented by 0.74 to 0.81 per 
cent, and these bacteria are killed in five hours by 1 per cent (Kita- 
sato). 

Potassium Chlorate.--In the writer’s experiments a four-per- 
cent solution failed in two hours to. kill Micrococcus Pasteuri. A 
five-per-cent solution failed in six days to destroy anthrax spores 
(Koch). 

Potassium Chromate.—A five-per-cent solution failed to kill 
anthrax spores in five days (Koch). 

Potassium Cyanide.—Antiseptic in the proportion of 1 :909 
(Miquel). 

Potassium Iodide.—A solution of five per cent does not destroy 
anthrax spores in eighty days (Koch). Putrefactive bacteria in 
broken-down beef infusion are not destroyed by two hours’ exposure 
in a twenty-per-cent solution (Sternberg). The typhoid bacillus and 


the cholera spirillum do not grow in culture solutions containing 
13 


194 ACTION OF SALTS. 


eight per cent, and are destroyed by five hours’ exposure to 9.23 per 
cent (Kitasato). Antiseptic in the proportion of 1:7 (Miquel). 

Potassium Permanganate.—In the writer’s experiments (1881) 
a two-per-cent solution was required to destroy Micrococcus Pasteuri 
in the blood of a rabbit. In later experiments pus cocci in bouillon 
were killed by 1 :833—time of exposure two hours. One per cent 
was found by Koch not to destroy anthrax spores in two days, but 
five per cent was effective in one day. The glanders bacillus is de- 
stroyed in two minutes by a one-per-cent solution (Léffler). The 
experiments of Jager show that a one-per-cent solution is not reli- 
able for the destruction of anthrax bacilli and other pathogenic bac- 
teria tested, but a five-per-cent solution was effective. The tubercle 
bacillus was not, however, killed by exposure in a five-per-cent solu- 
tion. According to Miquel, permanganate of potash is an antiseptic 
in the proportion of 1 : 285. 

Quinine Hydrobromate.—Antiseptic in the proportion of 1 : 182 
(Miquel). 

Quinine Hydrochlorate.—Antiseptic in the proportion of 1 : 900 
(Ceri). Quinine dissolved with hydrochloric acid destroys anthrax 
spores in ten days in one-per-cent solution (Koch). 

Quinine Sulphate.—The writer found that in the proportion of 
1: 800 quinine prevents the development of various micrococci and 
bacilli. A ten-per-cent solution does not destroy the bacilli of symp- 
tomatic anthrax (Arloing, Cornevin, and Thomas). 

Silver Nitrate.—Miquel places nitrate of silver next to mercuric 
chloride as an antiseptic, effective in the proportion of 1 :12,500. 
Behring also places it next to bichloride as an antiseptic and germi- 
cide, and says that it is even superior to this salt in albuminous 
fluids. He reports that it prevents the development of anthrax 
spores when present in a culture liquid in the proportion of 1: 80,000, 
and in the proportion of 1:10,000 destroys these spores in forty- 
eight hours, We give below the result of recent experiments by 
Boer, in which the time of exposure was two hours: 


Restrains Destroys 

development. vitality. 

Anthrax bacillus........... 0... c eee eee eee ; 1: 60000 1: 20000 
Diphtheria bacilius . 1: 60000 1: 2500 
Glanders bacillus.... 1: 75000 1: 4000 
Typhoid bacillus.......... 1: 50000 1: 4000 
Cholera spirillum..............0.-e eee eee 1: 50000 1: 4000 


Silver Chloride.—A solution of chloride of silver in hyposulphite 
of soda is much less effective as an antiseptic than nitrate of silver. 


ACTION OF SALTS. 195 


Behring found that to prevent the development of anthrax spores a 
solution of 1 : 8,000 was required. 

Sodium Borate.—In the writer’s experiments a saturated solu- 
tion of borax was found to be without germicidal power. A twenty- 
per-cent solution does not destroy the virus of symptomatic anthrax 
(Arloing, Cornevin, and Thomas). A five-per-cent solution failed 
to destroy anthrax spores in fifteen days (Koch). Antiseptic in the 
proportion of 1:14 (Miquel). 

Sodium Carbonate.—A solution of 2.2 per cent restrains the 
growth of the typhoid bacillus, and of 2.47 per cent of the cholera 
spirilum. The first-named bacillus is killed by four or five hours’ 
exposure in a 2.47-per-cent solution, and the cholera spirillum by 
3.45 per cent (Kitasato). 

Sodium Chloride.—A. saturated solution failed in forty-eight 
hours to destroy the virus of symptomatic anthrax (Arloing, Corne- 
vin, and Thomas). A saturated solution failed in forty days to de- 
stroy anthrax spores(Koch). A saturated solution failed in twenty 
hours to destroy the tubercle bacillus in fresh sputum (Schill and 
Fischer). In the writer’s experiments a five-per-cent solution failed 
to kill Micrococcus Pasteuri in blood. Antiseptic in the proportion 
of 1:6 (Miquel). According to Forster, the bacillus of typhoid 
fever, the bacillus of rouget, and the streptococcus of pus are not 
killed by several weeks’ exposure in strong solutions of sodium chlo- 
ride, but the cholera spirillum is destroyed in a few hours. Cultures 
of the tubercle bacillus are not sterilized in two months by a satu- 
rated solution ; and tuberculous organs from an ox, preserved in a 
solution of salt, did not lose their power of infecting susceptible ani- 
mals inoculated with material from the diseased tissue. The flesh 
of swine which died of rothlauf was found by Petri to still contain 
the bacillus in a living condition after having been preserved in 
brine for a month. 

Sodium Hyposulphite.—In the writer's experiments a saturated 
solution failed in two hours to kill micrococci and bacilli. Exposure 
for forty-eight hours to a fifty-per-cent solution does not destroy the 
virus of symptomatic anthrax (Arloing, Cornevin, and Thomas). 
Antiseptic in the proportion of 1:3 (Miquel). 

Sodium Sulphite.—The results with a saturated solution of this 
salt were, in the writer’s experiments, entirely negative. 

Tin Chloride.—A one-per-cent solution acting for two hours de- 
stroyed the bacteria in putrefying bouillon, while 0.8 per cent failed 
(Abbott). 

Zinc Chloride.—In the writer’s experiments 1:200 destroyed 
Micrococcus Pasteuri in two hours, but a two-per-cent solution was re- 
quired to kill pus cocci in the same time ; spores of Bacillus anthracis 


196 ACTION OF SALTS. 


were not destroyed by two hours’ exposure in a ten-per-cent solution, 
but a solution of five per cent killed the spores of Bacillus subtilis in 
the same time. Koch found that anthrax spores germinated after 
being immersed in a five-per-cent solution for thirty days. The de- 
velopment of Bacillus prodigiosus is only slightly retarded by expo- 
sure for sixteen hours in a one-per-cent solution. Antiseptic in the 
proportion of 1 :526 (Miquel). 

Zinc Sulphate.—In the writer’s first experiments a twenty-per- 
cent solution failed to destroy in two hours micrococci obtained from 
the pus of an acute abscess. In later experiments a micrococcus from 
the same source resisted two hours’ exposure to a ten-per-cent solu- 
tion, but Micrococcus tetragenus was destroyed by this amount. 
Broken-down beef infusion mixed with an equal quantity of a forty- 
per-cent solution was not sterilized after two hours’ contact. In 
Koch’s experiments anthrax spores were found to germinate after 
having been immersed for ten days in a five-per-cent solution. 


XI. 


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


In the present section we shall consider the action upon bacteria 
of a variety of organic products, and for convenience will arrange 
them alphabetically. 

Acetone.—Anthrax spores grow freely after two days’ exposure 
to the action of this agent; at the end of five days their development 
is feeble (Koch). 

Alcohol.—In the writer’s experiments ninety-five-per-cent alco- 
hol did not destroy the bacteria (spores) in broken-down beef tea in 
forty-eight hours. Micrococcus Pasteuri was destroyed by two hours’ 
exposure in a twenty-four-per-cent solution ; pus cocci required a 
forty-per-cent solution. Koch found that absolute alcohol had no 
effect upon anthrax spores exposed to its action for one hundred and 
ten days. Schill and Fischer found that when tuberculous sputum 
was mixed with an equal amount of absolute alcohol its infecting 
power was not destroyed in twenty-four hours, but that in the pro- 
portion of five parts to one of sputum it was effective in destroying 
the tubercle bacillus, as proved by inoculation experiments. Yersin 
found that in pure cultures the tubercle bacillus is killed by five 
minutes’ exposure to the action of absolute alcohol. 

Aniline Dyes.—Recent researches have shown that some of the 
aniline colors possess very decided germicidal power. Stilling found 
that solutions of methyl violet containing 1:30,000 exercise a re- 
straining influence upon the development of putrefactive bacteria 
and pus cocci, and that these microérganisms are destroyed by solu- 
tions containing 1 :2,000 to 1:1,000. Methyl violet has been placed 
in the market by Merck under the name of pyoktanin. Jianicke re- 
ports the following results with pyoktanin : Staphylococcus pyogenes 
aureus was restrained in its development by solutions containing 
1: 2,000,000, Bacillus anthracis by 1: 1,000,000, Staphylococcus pyo- 
genes by 1: 333,300, Spirillum cholerze Asiaticze by 1 : 62,500, Bacil- 
lus typhi abdominalis by 1:5,000. In blood serum stronger solutions 
were required (1:500,000 for Staphylococcus pyogenes aureus). Sta- 
phylococcus pyogenes aureus, Streptococcus pyogenes, and Bacillus 
anthracis were killed in thirty seconds by 1 : 1,000, the typhoid bacil- 


198 ACTION OF COAL-TAR PRODUCTS, 


lus by the same amount in thirty minutes. Boer found malachite 
green to be still more effective than methyl violet. In his experi- 
ments upon bouillon cultures twenty-four hours old, with two hours’ 
exposure to the action of the disinfectant, he obtained the following 
results : 

MALACHITE GREEN. 


Restrains Destroys 
development. vitality. 
Anthrax bacillus .. 2... .. 2... cece eee ee 1: 120000 1: 40000 
Diphtheria bacillus ........ cece cece eee eee: 1: 40000 1:8000 
Glanders bacillus .......... 0... ce eee e eee e ee ene 1: 5000 1:300 
Typhoid bacillus 2. 6... . eee cee eee eee ee 1: 5000 1 :300 
Cholera spirillum........ 6.0. cc eee eee ee oe 1: 100000 1: 5000 


METHYL VIOLET (PYOKTANIN). 


Restrains Destroys 
development. vitality. 
Anthrax bacillus.......... cece eee ee cece eee 1 :'70009 1:5000 
Diphtheria bacillus....... .ceeeee cree ener e ees 1:10000 1: 2000 
Glanders bacillus... ... cece cece ee crete cer eeeee 1: 2500 1-:150 
Typhoid bacillus...... .....4. Sra tail tila Su mora races 1: 2500 1:150 
Cholera spirillum........ eee ee eee ee ee cee : 1: 30000 1:1000 


Aniline Oil.—According to Riedlin, the addition of 1:5 of ani- 
line water prevents the development of all bacteria in nutrient gelatin. 

Aromatic Products of Decomposition.—Klein has tested the 
germicidal power of phenylpropionic and phenylacetic acids. He 
finds that anthrax spores resist both of these acids, in the proportion 
of 1:400, for two days, but in the absence of spores anthrax bacilli 
are quickly killed by a solution of this strength. Certain non-patho- 
genic micrococci were not killed by exposure for twenty-five minutes 
to 1:200. The caseous matter of pulmonary tuberculosis infected 
guinea-pigs after exposure for ninety-six hours to 1 : 200, 

Aseptol,—A ten-per-cent aqueous solution kills anthrax spores in 
ten minutes, and a three- to five-per-cent solution is a reliable disin- 
fectant in the absence of spores (Hueppe). 

Benzene, C,H,.—Exposure in benzol for twenty days failed to 
destroy the vitality of anthrax spores (Koch). 

Camphor.—Alcohol saturated with camphor has no effect upon 
the virus of symptomatic anthrax (Arloing, Cornevin, and Thomas) 

The experiments of Cadéac and Meunier show that camphor (oil 
of, or tincture?) has but little germicidal power. The typhoid ba- 


ESSENTIAL OILS, ETC. 199 


cillus and cholera spirillum were only destroyed after eight to ten 
days’ exposure to the action of camphor (‘‘ essence”). 

Carbolic Acid.—Tested upon anthrax spores, Koch found a one- 
per-cent solution to be without effect after fifteen days’ exposure ; a 
two-per-cent solution retarded development but did not completely 
destroy vitality in seven days; a three-per-cent solution was effec- 
tive in two days. In the absence of spores Koch found that a one- 
per-cent solution quickly destroys the vitality of anthrax bacilli. 
He recommends a five-per-cent solution for the destruction of the 
““comma bacillus” in the discharges of cholera patients, and a two- 
per-cent solution for the disinfection of surfaces soiled with such dis- 
charges. In the writer’s experiments 1: 200 destroyed Micrococcus 
Pasteuri in two hours ; and pus cocci were destroyed by 1: 125, while 
1:200 failed. Davaine showed by inoculation experiments that an- 
thrax bacilli in fresh blood are destroyed by being exposed to the 
action of a one-per-cent solution for one hour. A two-per-cent solu- 
tion destroys the dried virus of symptomatic anthrax in forty-eight 
hours (Arloing, Cornevin, and Thomas). Solutions in oil or in alco- 
hol have been shown by Koch to be less effective than aqueous solu- 
tions. Thus a five-per-cent solution in oil failed to destroy anthrax 
spores in one hundred and ten days, and the same solution failed to 
kill the bacilli, in the absence of spores, in less than six days. A 
five-per-cent solution in alcohol did not destroy anthrax spores in 
seventy days. Schill and Fischer found that a three-per-cent solu- 
tion destroyed the infecting power of tuberculous sputum, as shown 
by inoculation into guinea-pigs, in twenty-four hours, while solutions 
of one and two per cent failed. Bolton’s experiments gave the fol- 
lowing results, the test organisms being in fresh bouillon cultures 
and the time of exposure two hours: The cholera spirillum, the 
bacillus of typhoid fever, the bacillus of schweinerothlauf, Brieger’s 
bacillus, the bacillus of green pus, and the pus cocci (Staphylococcus 

“pyogenes aureus, albus, and citreus, and Streptococcus pyogenes) 
were all killed by a solution of one per cent, while in a majority of 
the experiments a one-half-per-cent (1: 200) solution failed. Cul- 
tures of the typhoid bacillus in flesh-peptone-gelatin gave the same 
result (1 : 100 with two hours’ exposure), and the addition of ten per 
cent of dried egg albumin to bouillon cultures did not influence the 
result. 

The experiments of La Place show that the addition of hydro- 
chloric acid to a disinfecting solution containing carbolic acid greatly 
increases its germicidal power for spores. Thus it is stated that 
“two per cent of crude carbolic acid with one per cent of pure hydro- 
chloric acid destroyed anthrax spores in seven days, while two per 
cent of carbolic acid or one per cent of hydrochloric acid alone did 


200 ACTION OF COAL-TAR PRODUCTS, 


not destroy these spores in thirty days. A four-per-cent solution of 
crude carbolic acid with two per cent of hydrochloric acid destroyed 
spores in less than an hour; four per cent of carbolic acid alone did 
not destroy them in twelve days. Wan Ermengem reports that in 
his experiments the cholera spirillum in chicken bouillon was killed 
in less than half an hour by 1: 600, and that in blood serum 1 : 400 
was effective. Nicati and Rietsch fix the germicidal power for the 
cholera spirillum as 1 : 200, the time of exposure being ten minutes ; 
Ramon and Cajal, 1:50. Boer gives the following results, the time 
of exposure being two hours, cultures in bouillon twenty-four hours 
old : 


Restrains Destroys 

development. vitality. 
Anthrax bacillus... .. cece cece ee eee cee eee os 1: 750 1:300 
Diphtheria bacillus. .........c ccc e esc e cere ences 1:500 1: 300 
Glanders bacillus. .....cceeccerecesseees nites aad 1:500 1 :300 
Typhoid! Dacilus. «saws sieigsc:cies vais o eonisieisinendes ems 6 ere 1:400 1:200 
Choleraspirilum: 5 vas isssees  deleas ci ee aaie ea vene 1: 600 1:400 


Leitz reports the following results: The dejections of patients 
suffering from typhoid fever, mixed in equal quantity with the disin- 
fecting solution, were sterilized by a five-per-cent solution of car- 
bolic acid in three days. Pure cultures of the typhoid bacillus were 
sterilized in fifteen minutes by a five-per-cent solution. 

In the experiments of Nocht upon anthrax spores it was found 
that while at the room temperature these spores were not destroyed 

. by several days’ exposure in a five-per-cent solution, they were de- 
stroyed in three hours by the same solution at a temperature of 37.5°. 

Carbolic acid prevents putrefactive changes in bouillon when pre- 
sent in the proportion of 1:333 (Miquel). The tubercle bacillus is 
killed in thirty seconds by a five-per-cent solution, and in one minute 
by a one-per-cent solution (Yersin). 

Coffee Infusion.—Experiments have been made by Heim and by 
Liideritz on the antiseptic power of an infusion of coffee. The first- 
named author found that anthrax bacilli no longer developed after 
three hours’ exposure in a ten-per-cent solution, but spores were not 
killed at the end of a week. Streptococci in a bouillon culture re- 
quired twenty-four hours’ exposure, and the staphylococci of pus were 
not destroyed in this time. Liideritz found that a three-per-cent in- 
fusion restrained the growth in nutrient gelatin of the typhoid ba- 
cillus, and a five-per-cent infusion killed the bacillus in two days ; 
the cholera spirillum failed to grow in presence of one per cent, and 
a solution of this strength killed itin seven hours ; Staphylococcus 


ESSENTIAL OILS, ETC. 201 


pyogenes aureus was prevented from developing by two per cent, 
and was killed in six days by a five-per-cent solution ; Streptococcus 
pyogenes was prevented from growing by one per cent, and killed by 
a ten-per-cent solution in one day ; Proteus vulgaris did not grow in 
presence of 2.5 per cent, and was killed in two days by ten per cent. 
The question as to what constituent of the infusion of roasted coffee 
was the active germicidal agent was not determined, but the authors 
referred to agree that it was not caffeine. 

Creolin.—This is a coal-tar product which resembles crude carbolic 
acid in appearance, but smells rather like tar than like phenol. It 
makes a milky emulsion with water, which has been proved by nu- 
merous experiments to possess very decided germicidal power, being 
superior to carbolic acid. The first careful test of the germicidal 
power of this agent was made by Esmarch, who found that a solu- 
tion of 1:200 killed the cholera spirillum in a minute, the typhoid 
bacillus at the end of several days. Anthrax spores were not de- 
stroyed in twenty days by a five-per-cent solution, but this solution 
killed the tubercle bacillus attached to silk threads which were im- 
mersed in it for ashort time, and also disinfected tuberculous sputum. 
Behring has shown that in albuminous liquids creolin is less etfective 
than carbolic acid. In blood serum 1:175 was required to restrain 
the development of staphylococci, and 1:100 to destroy the same in 
ten minutes. Van Ermengem, as a result of numerous experiments, 
arrived at the conclusion that creolin is a cheap and useful disinfect- 
ing agent, in a five-per-cent solution, for various pathogenic organ- 
isms. Kaupe reports that in his experiments a ten-per-cent solution 
killed anthrax spores in twenty-four hours. According to Boer, a 
solution of 1:5,000 destroys anthrax bacilli in bouillon cultures in 
two hours, 1:2,000 diphtheria bacilli, 1: 300 the glanders bacillus, 
1 : 250 the typhoid bacillus, and 1 :3,000 the cholera spirillum. 

Creosote.—This agent was found by the writer to be fatal ‘to 
micrococci in the proportion of 1: 200. In the proportion of one per 
cent it failed, after twenty hours’ exposure, to destroy tubercle ba- 
cilli in sputum (Schill and Fischer). A saturated aqueous solution 
does not destroy the tubercle bacillus in cultures in twelve hours 
(Yersin). Guttman, in extended experiments upon various patho- 
genic organisms, found that development was prevented by 1 : 3,000 
to1:4,000. A solution containing 1 :300 killed Bacillus pyocyanus 
and Bacillus anthracis in one minute, Bacillus prodigiosus in two 
minutes, and the Finkler-Prior spirillum in one minute in the pro- 
portion of 1 : 600. 

Cresol.—This is a dark, reddish-brown, transparent fluid, some- 
what thinner than creolin, and, like it, having an odor of tar. It 
forms an emulsion with water, which is not so stable as that formed 


202 ACTION OF COAL-TAR PRODUCTS, 


by creolin. Of the three cresols, ortho-, meta-, and paracresol, the 
second was found by Frankel to be most active. This author states 
that the addition of sulphuric acid adds greatly to its germicidal 
power. A four-per-cent solution, containing equal parts of cresol 
and H,SO,, killed anthrax spores in less than twenty-four hours. In 
Behring’s experiments a solution containing ten per cent of each killed 
anthrax spores in eighty minutes, and five per cent of each in one 
hundred minutes, while an eighteen-per-cent solution of sulphuric 
acid alone did not kill them in twenty four hours. In the experi- 
ments of Jager a two-per-cent solution destroyed the tubercle bacillus 
in cultures and in sputum. Asa result of his experiments Behring 
concludes that cresol has no advantage over carbolic acid as a ger- 
micide for the destruction of spores. Tested upon Staphylococcus 
aureus, Streptococcus erysipelatos, and Bacillus pyocyanus, Frankel 
found that a solution of 0.3 per cent destroyed these microdrganisms 
in five minutes, while a two-per-cent solution of carbolic acid re- 
quired fifteen minutes’ contact to accomplish the same result. 

Trikresol (Schering) has been tested, with favorable results, by 
several bacteriologists. According to Hammer! it is about twice as 
active a germicide as carbolic acid. 

Diaphtherin (oxychinaseptol) has considerable antiseptic power, 
as shown by the experiments of Rohrer and others. Two to four 
drops of a one-per-cent solution was found to prevent the develop- 
mentof test organisms (Staphylococcus pyogenes aureus and Bacillus 
anthracis) in twelve cubic centimetres of bouillon. Stahle (1893) 
also finds that as an antiseptic it is far superior to carbolic acid or lysol, 
and that it has the advantage of being non-toxic. Tested upon an- 
thrax spores it was found to be comparatively inactive asa germicide. 
A fifteen-per-cent solution destroyed anthrax spores in three days. 

Disinfektol.—This is a coal-tar product similar to creolin which 
has been recommended in Germany for disinfecting purposes. It is 
an oily, dark-brown fluid having a specific gravity of 1.086. Itforms 
an emulsion with water, which has a slightly alkaline reaction. It 
has been tested upon typhoid stools by Uffelmann and by Beselin. 
The last-named author gives the following summary of the results 
obtained : An emulsion of five per cent of disinfektol equals in value, 
for the disinfection of the liquid discharges of typhoid patients, 12.5 
per cent of creolin, thirty-three per cent of hydrochloric acid, five per 
cent of carbolic acid, 1 :500 of mercuric chloride. 

Ether.—Anthrax spores may germinate after being immersed in 
sulphuric ether for eight days (Koch). The tubercle bacillus is de- 
stroyed by ten minutes’ exposure to the action of ether (Yersin). 

Essential Oils.—Chamberlain has made an extended series of 
experiments to determine the antiseptic power of the vapor of vola- 


ESSENTIAL OILS, ETC. 203 


tile oils. A large number of essential oils tested were found to pre- 
vent the development of the anthrax bacillus, while a few did not. 
At the end of six days the tubes were opened and the oil absorbed by 
the culture liquid allowed to evaporate. Cultures were now obtained 
from all except the following, which, it was inferred, had destroyed 
the vitality of the spores: Angelica, cinnamon of China, cinnamon 
of Ceylon, geranium of France, geranium of Algeria, origanum. 

Cadéac and Meunier have also made extended experiments upon 
the typhoid bacillus and the bacillus of glanders, for the purpose of 
determining the germicidal power of agents of this class. Their 
method consisted in the introduction of a sterilized platinum needle 
into a pure culture of the test organism, in immersing it in the 
essential oil for a certain time, and then making with it a puncture 
in a suitable solid culture medium. Their results are given below 
for the typhoid bacillus. 

Essences which kill the bacillus after a contact of less than 
twenty-four hours: 


At the end of— 
Cinnamon of Ceylon, . ‘ ; j . 12 minutes. 
Cloves, : 3 - : . : 25 ie 
Eugenol, . ‘ é 3 - ‘ . 80 mn 
Thyme, j : a 7 : 35 oi 
Wild thyme, 5 . 7 - . . 85 im 
Verbena of India, . : ‘5 . , 45 ee 
Geranium of France, . ‘ é . . 50 wt 
Origanum, : . : . : 75 es 
Patchouly, ae . ‘ ‘ : . 80 as 
Zedoary, : ‘ A 3 ; ‘ 2 hours, 
Absinthe, . F : F : 3 : Ly x 
Sandalwood, . : Fi 3 A z 12 


The following were effective in from twenty-four to forty-eight 
hours: Cumin, caraway, juniper, matico, galbanum, valerian, citron, 
angelica, celery, savin, copaiba, pepper, turpentine, opopanax, rose, 
chamomile ; the following required from two to four days: Illicium, 
sassafras, tuberose, coriander; the following from four to eight days: 
Calamus, sage, fennel, mace, cascarilla, orange of Portugal; the fol- 
lowing in eight to ten days: Mint, nutmeg, rosemary, carrot, mus- 
tard, anise, onion, marjoram, bitter almonds, cherry laurel, myrtle, 
lavender, eucalyptus, cedar, cajuput, wintergreen, camphor. 

Riedlin reports as the result of his experiments that the essential 
oils which have the greatest antiseptic value are oil of lavender, eu- 
calyptus, rosemary, and cloves. 

Eucalyptol.—Chabaunes and Perret found that a five-per-cent 
solution of eucalyptol is without effect upon tubercle bacilli in spu- 
tum. According to Behring, eucalyptol is about four times less ac- 
tive as a disinfectant than carbolic acid. 


204 ACTION OF COAL-TAR PRODUCTS, 


Huphorin (Phenylurethan) has been tested by Colasanti (1894), 
who finds that it has rather feeble germicidal activity. 

Formaldehyde (formol, formalin) has very decided germicidal 
power. According to Pottevin (1894) in the absence of spores a solu- 
tion of 1:1,000 kills bacteria, in comparatively small numbers, in from 
fifteen minutes to several hours. For the destruction of spores a 
much strorger solution is required—a fifteen-per-cent sciution at 
15° C. killed anthrax spores in one and one-half hours, and spores 
of Bacillus subtilis in twenty hours. At higher temperatures the 
germicidal action is more energetic, and microérganisms exposed to 
the vapor of formol are very quickly destroyed. Vanderlinden and 
de Buck (1895) find that solutions of formalin are decidedly inferior 
to corresponding solutions of carbolic acid, creolin, or solvéol, and are 
too irritating to be used in surgical practice. They report that a 
solution of five per cent failed to destroy their test organisms— 
Bacillus coli communis, Bacillus typhi abdominalis, Staphylococcus 
pyogenes aureus. Experiments made by Reed, at the Army Medical 
Museum in Washington, show that the diphtheria bacillus and other 
test organisms are quickly killed by formalin vapor. 

Glycerin has no action upon the virus of symptomatic anthrax 
(Arloing, Cornevin, and Thomas), and is inert as regards the spores 
of anthrax (Koch). Glycerin prevents putrefactive decomposition in 
bouillon when present in the proportion of 1:4 (Miquel). Roux has 
shown that the addition of five per cent of glycerin to a culture 
medium is favorable to the growth of the tubercle bacillus; it is also 
appropriated as pabulum by various other species. 

Guatacol.—Kuprianow, as a result of extended experiments with 
this agent (1894), reports that it ranks below cresol and carbolic acid 
asa germicide. In the proportion of 1:500 it restrains the develop- 
ment of the cholera spirillum, and the author named suggests its in- 
ternal administration in this disease on account of its non-toxic and 
non-irritant properties. 

Hydroxylanin.—Heinisch found that the development of the 
anthrax bacillus is prevented by 1:77 of hydroxylamin hydro- 
chlorate, and of the diphtheria bacillus by 1:75. In these experiments 
a solution of soda was added to release the hydroxylamin. Marp- 
mann found that 1:100 preserved milk without change for four 
to six weeks, and that alkaline fermentation of urine was prevented 
by 1:1,000. 

Ichthyol.—Latteux (1892) reports that the various pathogenic 
bacteria used by him as test organisms were killed by a five-per-cent 
solution (time ?) with exception of Streptococcus pyogenes, which 
required a six to seven-per-cent solution. The more recent experi- 
ments of Abel (1893) gave less favorable result, but the agent was 


ESSENTIAL OILS, ETC. 205 


shown to have considerable antiseptic value—1: 2,000 restrained the 
development of streptococci; 1: 500 of the diphtheria bacillus; 1: 20 
of Staphylococcus pyogenes aureus; 1:33 the bacillus of typhoid 
fever. Streptococci and diphtheria bacilli were destroyed in twenty- 
four hours by a solution of 1: 200; Staphylococcus aureus, subjected 
to the action of pure ichthyol, was destroyed in five hours—in a five- 
per-cent solution it survived for four days. Cultures of the typhoid 
bacillus mixed with a fifty-per-cent solution were not completely 
sterilized in thirty hours; a small number of bacilli in bouillon were, 
however, destroyed by a three-per-cent solution in forty-eight hours. 
Anthrax spores on silk threads were not destroyed by a fifty-per-cent 
solution at the end of one hundred and forty days. 

Indol.—When added in excess to water this agent failed to de- 
stroy anthrax spores in eighty days (Koch). 

Izal is a coal-tar product which has recently been introduced as 
a disinfectant. Klein (1892) reports that in the strength of ten per 
cent it kills anthrax spores in fifteen minutes. In the absence of 
spores various pathogenic bacteria were killed in five minutes by a 
solution containing 1: 200. 

Lanolin.—According to Gottstein, various microérganisms tested 
by him failed to grow in cultures after having been in contact with 
pure lanolin for five to seven days. 

Loretin.—Korff (1895) claims for this agent that a two-per-cent 
solution is superior to corresponding solutions of lysol, metakresol, 
or phenol, and that it has the advantage of being non-toxic, odorless, 
and non-irritating. 

Lysol.—Weiss (1895) has tested this product and reports that a 
solution of three-fourths per cent destroyed his.test organisms (pus 
cocci, typhoid bacillus, Bacillus coli communis, etc.) in five minutes. 
Anthrax spores were destroyed by the same solution in one hour. 

Naphthol.—In the proportion of 1: 10,000 naphthol prevents the 
development of the glanders bacillus, the anthrax bacillus, the typhoid 
bacillus, the micrococcus of fowl cholera, of Staphylococcus aureus 
and albus, and of several other microdrganisms tested by Maximo- 
vitch. The same author states that although insoluble in cold water, 
water at 70° C. dissolves 0.44 in one thousand parts. When urine is 
shaken up with naphthol in powder it does not undergo fermenta- 
tion. 

In the experiments of Foote hydronaphthol was found to show 
some germicidal power in the proportion of 1: 2,300, but the conclu- 
sion is reached that a saturated aqueous solution (1: 1,150) does not 
equal a one-per-cent solution of carbolic acid or of creolin. 

The writer, in 1892, obtained the following results in experiments 
with naphthols upon the cholera spirillum. 


206 ACTION OF COAL-TAR PRODUCTS, 


Alpha-naphthol and beta-naphthol have about the same antiseptic and 
germicidal value. In the proportion of 1: 16,000 both prevent the develop- 
ment of the cholera spirillum in peptonized beef-tea, while 1 : 24,000 fails to 
prevent development. In the proportion of 1 : 3,000 both destroy the vital- 
ity of the cholera spirillum in bouillon cultures, twenty-four hours old, 
after two hours’ contact, while 1: 4,000 fails to destroy this microdrganism 
in the time mentioned—two hours. 

In experiments made with a solution of 1:1,000, added to an equal 
quantity of a twenty-four hours old bouillon culture—making 1 : 2,000 after 
mixture—and in which the time of contact varied from five to thirty minutes, 
alpha-, beta-, and hydronaphthol were found to destroy the cholera germ by 
fifteen minutes’ exposure, but to fail after ten minutes’ contact, so that the 
germicidal value of each of these is similar, or nearly so. : 

In all these experiments the line was sharply drawn between success and 
failure. No development occurred and the bouillon remained transparent 
in those experiments in which the germicidal action was complete, and a 
characteristic development occurred within twenty-four hours in those ex- 
periments in which there was a failure to destroy the spirillum. 

Benzo-naphthol has no germicidal power, probably because it is insoluble 
in water. At least this is my inference from the experiments made. One 
gamme was added to one thousand cubic centimetres of distilled water, and 
after vigorous shaking was placed in the steam sterilizer for half an hour. 
At the end of this time the greater portion, at least, of the benzo-naphthol re- 
mained undissolved at the bottom of the flask. The saturated solution (?) 
was then filtered and added to recent bouillon cultures of the cholera spiril- 
lum in the proportion of 1:1, 1: 2,1:4,and 2:1. At the end of two hours 
sterile bouillon in test tubes was inoculated from each of these and placed in 
the incubating oven. At the end of forty-eight hours a characteristic devel- 
opment of the cholera spirillum had occurred in all of the tubes. 


Olive Ovl.—Anthrax spores germinate after having been im- 
mersed for ninety days in pure olive oil (Koch). 

Owl of Mustard.—Koch found that the development of anthrax 
spores is prevented by 1:33,000. 

Owl of Peppermint.—A five-per-cent solution in alcohol failed in 
twelve days to destroy anthrax spores, but the development of these 
spores is restrained by 1: 33,000 (Koch). 

Oul of Turpentine destroys anthrax spores in five days, but failed 
to do so in one day (Koch). The development of anthrax spores is 
prevented by 1: 75,000 (Koch). The addition of 1: 200 to nutrient 
gelatin prevents the development of bacteria (Riedlin). An excess 
of oil of turpentine added to a liquefied gelatin culture of Staphylo- 
coccus aureus does not destroy this micrococcus in five hours (v. 
Christmas-Dirckinck-Holmfeld). 

Saprol.—Laser (1892) recommends this agent for the disinfection 
of the excreta of cholera and typhoid patients. He reports that in the 
proportion of 1: 100 it sterilizes liquid feeces in twenty-four hours. 

Skatol in excess in water has no germicidal power, as tested upon 
anthrax spores (Koch). 

Smoke.—The researches of Beu show that meats which have been 
preserved by smoking commonly contain living bacteria capable of 
growing in culture media; and Petri has shown that pork which has 


ESSENTIAL OILS, ETC. 207 


been salted for a month and then smoked for fourteen days may still 
contain the bacillus of rothlauf in a living condition, as shown by in- 
oculation experiments. It was not until about six months after smok- 
ing that the bacillus failed to give evidence of vitality. 

Thymol.—A. five-per-cent solution in alcohol does not destroy 
anthrax spores in fifteen days, but the development of these spores 
is retarded by a solution of 1:80,000 (Koch). The anthrax bacillus 
and staphylococci fail to grow in culture media containing 1 : 3,000 
(Samter). The tubercle bacillus is destroyed by contact with thy- 
mol for three hours (Yersin). Thymol has about four times less 
germicidal power than carbolic acid (Behring). Antiseptic in the 
proportion of 1: 1,340 (Miquel). 

Tobacco: Smoke.—Tassinari found that tobacco smoke restrains 
the development of bacteria, and that certain species failed to de- 
velop after exposure for half an hour in an atmosphere of tobacco 
smoke—spirillum of cholera and Friedlander’s bacillus. 


XII. 


ACTION OF BLOOD SERUM AND OTHER ORGANIC 
LIQUIDS. 


Blood Serum.—Bacteriologists have long been aware of the fact 
that many species of bacteria, when injected into the circulation of a 
living animal, soon disappear from the blood, and that the blood of 
such an animal a few hours after an injection of putrefactive bacte- 
ria, for example, does not contain living bacteria capable of develop- 
ing in a suitable nutrient medium. Wyssokowitsch, in an extended 
series of experiments, has shown that non-pathogenic bacteria in- 
jected into the circulation may be obtained in cultures from the liver, 
spleen, kidneys, and bone marrow after they have disappeared from 
the blood, but that, as a rule, those present in these organs have lost 
their vitality, as shown by culture experiments, in a period varying 
from a few hours to two or three days. According to the theory of . 
Metschnikoff, this destruction of bacteria in the blood and tissues of a 
living animal is effected by the cellular elements, and especially by 
the leucocytes, which pick up and digest these vegetable cells very 
much as an amceba disposes of similar microérganisms which serve 
itas food. Some such theory seemed necessary to account for the 
disappearance of bacteria from the blood before the demonstration 
was made that the serum of the circulating fluid, quite indepen- 
dently of its cellular elements, possesses very decided germicidal 
power. 

Von Fodor first (1887) called attention to the fact that anthrax ba- 
cilli may be destroyed by freshly drawn blood ; and Nuttall (1888), 
in an extended series of experiments, showed that various bacteria 
are destroyed within a short time by the fresh blood of warm- 
blooded animals. Thus the anthrax bacillus in rabbit’s blood was 
usually killed in from two to four hours when the temperature was 
maintained at 37°-38° C., and the same result was obtained with 
pigeon’s blood at 41° C. But when the blood was allowed to stand 
for a considerable time, or was heated for forty-five minutes to 
45° C., it served as a culture fluid, and an abundant development of 
anthrax bacilli occurred init. Bacillus subtilis and Bacillus mega- 


ACTION OF BLOOD SERUM AND OTHER ORGANIC LIQUIDS. 209 


therium were also destroyed in two hours by fresh rabbit’s blood, 
but it was without action on Staphylococcus pyogenes aureus, which 
at a temperature of 37.5° C. was found to have increased in num- 
bers at the end of two hours. Further researches by Nissen and 
Behring show that there is a wide difference in the blood of dif- 
ferent animals as to germicidal power, and that certain bacteria 
are promptly destroyed, while other species are simply restrained for 
a time in their development or are not affected. Thus Nissen found 
that the cholera spirillum, the bacillus of anthrax, the bacillus of 
typhoid fever, and Friedlander’s pneumococcus were killed, while 
Staphylococcus pyogenes aureus and albus, the streptococcus of ery- 
sipelas, the bacillus of fowl cholera, the bacillus of rothlauf, and 
Proteus hominis were able to multiply in rabbit’s blood after having 
been restrained for a short time in their development. In the case 
of the cholera spirillum a period of ten to forty minutes sufficed for 
the complete destruction of a limited number, but when the number 
exceeded 1,200,000 per cubic centimetre they were no longer de- 
stroyed with certainty, and after five hours an increase occurred. 
The anthrax bacillus was commonly destroyed within twenty minutes 
and the typhoid bacillus at the end of two hours. In the experi- 
ments of Bearing and Nissen it was found that the most pronounced 
germicidal effect upon the anthrax bacillus was obtained from the 
blood of the rat, an animal which has a natural immunity against 
anthrax ; while the blood of the guinea-pig, a very susceptible ani- 
mal, had no restraining effect and served as a favorable culture 
medium for the anthrax bacillus. And the remarkable fact was de- 
monstrated that when the blood of a rat was added to the blood of 
the guinea-pig in the proportion of 1:8, it exercised a decided re- 
straining influence upon the growth of the anthrax bacillus. Later 
researches have shown that cultivation in the blood of an immune 
animal causes an attenuation of the virulence of an anthrax cul- 
ture (Ogata and Jasuhara) ; and also that the injection of the blood 
of a frog or rat—naturally immune—into a susceptible animal which 
has been inoculated with a virulent culture of the anthrax bacillus, 
will prevent the death of the inoculated animal. 

Buchner has shown that the germicidal power of the blood of 
dogs and rabbits does not depend upon the presence of the cellular 
elements, but is present in clear serum which has been allowed to 
separate from the clot inacool place. Exposure for an hour to a 
temperature of 55° C. destroys the germicidal action of serum as 
well as of blood ; the same effect is produced by heating to 52° C. for 
six hours or to 45.6° C. for twenty hours. The germicidal power 
of blood serum is not destroyed by freezing and thawing, but is 


lost after it has been kept for some time. Buchner’s experiments led 
14 


210 ACTION OF BLOOD SERUM AND OTHER ORGANIC LIQUIDS. 


him to the conclusion that the germicidal power of fresh blood 
serum depends upon the presence of some albuminous body present 
init. This view is sustained by the researches of Ogata, who has 
obtained from the blood of dogs and other animals a glycerin ex- 
tract of a ‘‘ ferment” which is insoluble in alcohol or in ether and 
which has germicidal properties. 

According to Emmerich and Tsuboi (1893), when the serum- 
albumin is precipitated by alcohol, dried in a vacuum at 40° C., and 
dissolved in water it has no longer any germicidal activity. But if 
the precipitated and dried albumin is dissolved at 39° C. in a weak 
solution (0.05-0.08 per cent) of soda or potash it recovers its original 
germicidal value. 

It has been demonstrated by several experimenters that other 
albuminous fluids possess asimilar germicidal power. Thus Nuttall 
found that a pleuritic exudation from man destroyed the anthrax 
bacillus in an hour, the aqueous humor of a rabbit in two hours. 
Wurz has experimented with fresh egg albumin, and found that the 
anthrax bacillus failed to grow after having been exposed for an hour 
to the action of albumin from a hen’s egg; other bacteria tested 
were not killed so promptly, but a decided germicidal action was 
manifested. Prudden has shown that the albuminous fluid obtained 
from a hydrocele, or from the abdominal cavity in ascites, possesses 
similar germicidal power ; and Fokker has demonstrated that fresh 
milk destroys the vitality of certain bacteria which induce an acid 
fermentation of this fluid. 

The results heretofore referred to induced Hankin to experiment 
with cell globulin obtained from the spleen or lymphatic glands of a 
dog or cat. This is extracted by means of a solution of chloride of 
sodium, the solution is filtered, and the globulin precipitated by the 
addition of alcohol. The precipitate is washed and again dissolved 
in salt solution. The result showed that this cell globulin possesses 
germicidal power similar to that of blood serum. 

Mucus.—The experiments of Wurtz and Lermoyez (1893) show 
that nasal mucus has germicidal properties, especially for the anthrax 
bacillus. Walthard (1893), in experiments with mucus from the cer- 
vix uteri, was not able to demonstrate any germicidal action, but 
arrived at the conclusion that it prevents the development of bacteria 
simply because it is an unfavorable medium. Various bacteria were 
planted upon the surface of cervical mucus in Petri dishes, and placed 
in the incubating oven, but all failed to grow. 

Nucleins from animal and vegetable cells have been shown by 
Professor Vaughan and his associates (1893) to possess considerable 
germicidal power. The nucleins of animal origin were obtained from 
the testes of dogs and rats. Dissolved in a 0.5-per-cent solution of 


ACTION OF BLOOD SERUM AND OTHER ORGANIC LIQUIDS. 211 


caustic potash and then diluted with four volumes of physiologic salt 
solution the germicidal activity was shown by the facts that Staphylo- 
coccus pyogenes aureus, and the anthrax bacillus without spores, 
failed to grow after twenty minutes’ exposure. Kossel (1893) has 
obtained similar results with nucleins from the thymus gland of the 
calf. 

Urine.—The experiments of Lehmann show that fresh urine has 
a decided germicidal power for the cholera spirillum and the anthrax 
bacillus, and no doubt for other bacteria as well. To what constitu- 
ent of the urine this is due has not been determined, but it may be 
due to the uric acid present. 


XIII. 


PRACTICAL DIRECTIONS FOR DISINFECTION. 


THE Committee on Disinfectants of the American Public Health 
Association (appointed in 1884), after an extended investigation with 
reference to the germicidal value of various agents, in a final report 
submitted in 1887 submits the following ‘‘ Conclusions”: 


The experimental evidence recorded in this report seems to justify the 
following conclusions: 

The most useful agents for the destruction of spore-containing infectious 
material are— 

1. Fire. Complete destruction by burning. 

2. Steam under pressure. 105° C. (221° F.) for ten minutes. 
3. Boiling in water for half an hour. 

4, Chloride of lime.’ A four-per-cent solution. 

5. Mercurie chloride. A solution of 1: 500. 

For the destruction of infectious material which owes its infecting power 
to the presence of microdrganisms not containing spores, the committee rec- 
ommends— 7 
Fire. Complete destruction by burning. 

. Boiling in water for ten minutes. 

Dry heat. 110° C. (230° F.) for two hours. 

. Chloride of lime. A two-per-cent solution. 

. Solution of chlorinated soda.? A ten-per-cent solution. 

Mercuric chloride. A solution of 1: 2,000. 

. Carbolic acid. A five-per-cent solution. 

. Sulphate of copper. A five-per-cent solution. 

Chloride of zinc. A ten-per-cent solution. 

Sulphur dioxide.* Exposure for twelve hours to an atmosphere con- 
taining at least four volumes per cent of this gas in presence of 
moisture. 

The committee would make the following recommendations with refe- 
rence to the practical application of these agents for disinfecting purposes: 


/ 
FOR EXCRETA. 


SOON AUP WwWWOh 


ry 


(a) In the sick-room: 

1. Chloride of lime in solution, four per cent. 
In the absence of spores: 

2. Carbolic acid in solution, five per cent. 

3. Sulphate of copper in solution, five per cent. 


1 Should contain at least twenty-five per cent of available chlorine. 
2 Should contain at least three per cent of available chlorine. 
3 This will require the combustion of between three and four pounds of sulphur 


for every thousand cubic feet of air space. 


PRACTICAL DIRECTIONS FOR DISINFECTION. 213 


(6) In privy vaults: 
1. Mercuric chloride in solution, 1: 500.! 
2. Carbolic acid in solution, five per cent. 
(©) For the disinfection and deodorization of the surface of masses of or- 
ganic material in privy vaults, etc. : 
Chloride of lime in powder. 


FOR CLOTHING, BEDDING, ETC. 


(a) Soiled underclothing, bed linen, ete. : 
1. Destruction by fire, if of little value. 
2. Boiling for at least half an hour. 
3. Immersion in a solution of mercuric chloride of the strength of 
1:2,000 for four hours. 
4, Immersion in a two-per-cent solution of carbolic acid for four hours. 
(6) Outer garments of wool or silk, and similar articles, which would be 
injured by immersion in boiling water or in a disinfecting solution: 
1. Exposure in a suitable apparatus to a current of steam for ten min- 
utes. 
2. Herons to dry heat ata temperature of 110°C. (230° F.) for two 
ours. 
(2) Mattresses and blankets soiled by the discharges of the sick: 
1. Destruction by fire. 
2. Exposure to superheated steam, 105° C. (221° F.), for ten minutes. 
(Mattresses to have the cover removed or freely opened.) 
8. Immersion in boiling water for half an hour. 


FURNITURE AND ARTICLES OF WOOD, LEATHER, AND PORCELAIN. 


Washing, several times repeated, with— 
1. Solution of carbolic acid, two per cent. 


FOR THE PERSON. 


The hands and general surface of the body of attendants of the sick, and 
of convalescents, should be washed with— 
1. Solution of chlorinated soda diluted with nine parts of water, 1:10. 
2. Carbolic acid, two-per-cent solution. 
8. Mercurie chloride, 1: 1,000. 


FOR THE DEAD. 


Envelop the body in a sheet thoroughly saturated with— 
1. Chloride of lime in solution, four per cent. 
2. Mercurie chloride in solution, 1: 500. 
3. Carbolic acid in solution, five per cent. 


FOR THE SICK-ROOM AND HOSPITAL WARDS. 


(a) While occupied, wach all surfaces with— 
1. Mercuric chloride in solution, 1: 1,000. 
2. Carbolic acid in solution, two per cent. 

(6) When vacated, fumigate with sulphur dioxide for twelve hours, burn- 
ing at least three pounds of sulphur for every thousand cubic feet of air 
space in the room; then wash all surfaces with one of the above-mentioned 
disinfecting solutions, and afterward with soap and hot water; finally throw 
open doors and windows, and ventilate freely. 


1The addition of an equal quantity of potassium permanganate asa deodorant, 
and to give color to the solution, is tobe recommended. [The writer no longer in- 
dorses this recommendation. See his paper on ‘“‘ The Disinfection of Excreta,” ap- 
pended. ] 


214 PRACTICAL DIRECTIONS FOR DISINFECTION. 


FOR MERCHANDISE AND THE MAILS. 


The disinfection of merchandise and of the mails will only be required 
under exceptional circumstances; free aération will usually be sufficient. 
disinfection seems necessary, fumigation with sulphur dioxide will be the 
only practicable method of accomplishing it without injury. 


RAGS. 


(a) Rags which have been usea for wiping away infectious discharges 
should at once be burned. 
(b) Rags collected for the paper-makers during the prevalence of an epi- 
demic should be disinfected, before they are compressed in bales, by— 
1. Exposure to superheated steam of 105° C. (221° F.) for ten minutes. 
2. Immersion in boiling water for half an hour. 


SHIPS. 


(a) Infected ships at sea should be washed in every accessible place, and 

especially the localities occupied by the sick, with— 
1. Solution of mercuric chloride, 1: 1,000. 
2. Solution of carbolic acid, two per cent. 

The bilge should be disinfected by the liberal use of a strong solution of 
mercuric chloride. 

(b) Upon arrival at a quarantine station, an infected ship should at 
once be fumigated with sulphurous acid gas, using three pounds of sulphur 
for every thousand cubic feet of air space; the cargo should then be dis- 
charged on lighters; a liberal supply of the concentrated solution of mercuric 
chloride (four ounces to the gallon) should be thrown into the bilge, and at 
the end of twenty-four hours the bilge wate: should be pumped out and re- 
placed with pure sea water; this should be repeated. A second fumigation, 
after the removal of the cargo, is recommended; all accessible surfaces should 
be washed with one of the disinfecting solutions heretofore recommended, 
and subsequently with soap and hot water. 


FOR RAILWAY CARS. 


The directions given for the disinfection of dwellings, hospital wards, and 
ships apply as well to infected railway cars. The treatment of excreta with 
a disinfectant, before they are scattered along the tracks, seems desirable at 
all times in view of the fact that they may contain infectious germs. Dur- 
ing the prevalence of an epidemic of cholera this is imperative. For this 
purpose the standard solution of chloride of lime is recommended. 


DISINFECTION BY STEAM. 


The Committee on Disinfectants, in the above-quoted ‘‘ Conclu- 
sions,” recommends the use of ‘steam under pressure, 105° C. (221° 
F.), for ten minutes” for the destruction of spore-containing infec- 
tious material. The spores of all known pathogenic bacteria are de- 
stroyed by a temperature of 100° C. maintained for five minutes, and 
in view of this fact the temperature fixed by the committee is ample, 
and to exact a higher temperature or longer exposure would be un- 
reasonable. But in practical disinfection the temperature required 
to destroy infectious material is not the only question to be considered. 
Economy in the construction and operation of the steam disinfecting 
apparatus must have due attention, and an important point relates 


PRACTICAL DIRECTIONS FOR DISINFECTION. 215 


to the penetration of porous, non-conducting articles, such as rolls of 
blankets, clothing, etc. These points have been the subject of nu- 
merous experimental investigations, and the principles involved 
have been elucidated, especially by the investigations of Esmarch 
(1887), of Budde (1889), and of Teuschner (1890). 

It has been shown that streaming steam is more effective than 
confined steam at the same temperature, because it penetrates porous 
objects more quickly. Also that superheated, “‘ dry” steam is not as 
effective as flowing steam at 100° C.; on the other hand, it corre- 
sponds in effectiveness with dry air, and the temperature must be 
raised to 140° to 150° C. in order to quickly destroy the spores of 
bacilli. 

Esmarch’s investigations show that streaming steam penetrates 
porous objects, like rolled blankets, more readily than confined 
steam; but the later researches of Budde and of Teuschner show 
that a temperature of 100° C. is more rapidly reached in the interior 
of such rolls when the flowing steam is under pressure. With the 
same pressure (fifteen pounds) a temperature of 100° C. was reached 
in two and one-half minutes when the steam was flowing, and in 
eleven minutes by steam at rest (Budde). Intermittent pressure 
was not found by Budde to present any advantages over continuously 
flowing steam ; on the contrary, the time of penetration was longer. 

Teuschner, whose investigations are the most recent, arrives at 
the following conclusions : 


1. Strongly superheated steam is not to be recommended for practical 
disinfection. On the contrary, a slight superheating of the steam, such as 
occurs in the apparatus of Schimmel, is nof objectionable. 

2. Those forms of apparatus in which the steam enters from above are 
much safer and quicker in their disinfecting action than those in which this 
is not the case. In the construction of such apparatus care must be taken, 
in order to secure penetration of the objects, that the air and steam have a 
free escape below. 

3. Disinfection is hastened by previously warming the apparatus. 

4. The most rapid disinfecting action is secured by the use of streaming 
steam in a state of tension (under pressure). 

5. Objects which have been in contact with fatty or oily substances 
require a longer time for disinfection than those which have not. 

6. To accomplish disinfection it is necessary to expel, as completely as 
possible, all air from the objects to be disinfected, and also to secure a suffi- 
cient condensation of the steam. 

7. The condensation of the steam advances in a sharply defined line 
from the periphery to the centre of porous objects. 

8. The temperature necessary for disinfection is only found in the zone 
where condensation has already taken place. 

9. Only a few centimetres from the zone in which the temperature is 
100° C.—when disinfection is incomplete—there may be places in which 
the temperature is 40° C, or more below the boiling point. 


216 PRACTICAL DIRECTIONS FOR DISINFECTION. 


DISINFECTION BY FORMALDEHYDE GAS. 


Recent experiments have demonstrated the valuable germicidal 
properties of formaldehyde gas. Owing to its superior germicidal 
value and non-toxic properties, it has to a considerable extent taken 
the place of sulphur dioxide as a gaseous disinfectant. In making 
practical use of this agent a suitable apparatus will be required. For 
the disinfection of a room with its contents, freely exposed for sur- 
face disinfection, one pound of formalin should be volatilized for 
each thousand cubic feet of air space—the time of exposure to the 
disinfecting action of the gas being not less than twelve hours. 
When paraform is used the amount required will be sixty grammes 
to one thousand cubic feet (Novy). In the absence of any appa- 
ratus satisfactory results have been obtained by the Department of 
Health of the city of Chicago, as follows: 

‘‘Ordinary bed sheets were employed to secure an adequate evaporatory 
surface, and these, suspended in the room, were simply sprayed with a forty- 
per-cent solution of formalin through a common watering-pot rose-head. A 
sheet of the usual size and quality will carry from one hundred and fifty to 
one hundred and eighty cubic centimetres of the solution without dripping, 
and this quantity has been found sufficient for the disinfection of one thousand 
«ubic feet of space. Of course, the sheets may be modified to any necessary 
number. . . . Surface disinfection was thorough, while a much greater de- 
gree of penetration was shown than that secured by any other method.” 

Formalin may also be used in the disinfection of rooms and their 
contents by spraying all exposed surfaces. 

Experiments made by Kinyoun and others show that formalde- 
hyde gas does not injure the color or textile strength of fabrics of 
wool, silk, cotton, or linen, and that it has no injurious action upon 
furs, leather, copper, brass, nickel, zinc, polished steel or gilt work. 
Iron and unpolished steel are attacked by the gas. 


DISINFECTION OF THE HANDS. 


The importance of a reliable method of disinfecting the hands of 
surgeons, obstetricians, and nurses after they have been in contact 
with infectious material from wounds, puerperal discharges, etc., is 
now fully recognized, and some surgeons consider it necessary to 
completely sterilize the hands before undertaking any surgical opera- 
tion which will bring them in contact with the freshly-cut tissues. 
The numerous experiments which have been made with a view to 
ascertaining the best method of accomplishing such sterilization of 
the hands show that it is by no means a simple matter to effect it, 
and especially to insure the destruction of microdrganisms con- 
cealed beneath the finger nails. Fiirbringer, in an extended series 
of experiments (1888), found that a preliminary cleansing with soap 


PRACTICAL DIRECTIONS FOR DISINFECTION. 217 


and a brush was even more important than the degree of potency of 
the disinfecting wash subsequently applied. He recommends the 
following procedure : 

1. Remove all visible dirt from beneath and around the nails. 

2. Brush the spaces beneath the nails with soap and hot water 
for a minute. 

3. Wash for a minute in alcohol (not below eighty per cent), and, 
before this evaporates, in the following solution : 

4, Wash thoroughly for a minute in a solution containing 1 :500 
of mercuric chloride or three per cent of carbolic acid. 

Roux and Reynés tested the above method of Fiirbringer, and 
found that it gave better results than others previously proposed, al- 
though not always entirely successful in securing complete steriliza- 
tion. 

Boll has recently (1890) reported favorable results from the fol- 
lowing method : 


1. Cleanse the finger nails from visible dirt with knife or nail scissors. 

2. Brush the hands for three minutes with hot water and potash soap. 

3. Wash for half a minute in a three-per-cent solution of carbolic acid, 
and subsequently in a 1 : 2,000 solution of mercuric chloride. 

4. Rub the spaces beneath the nails and around their margins with iodo- 
form gauze wet in a five-per-cent solution of carbolic acid. 


Welch, asa result of extended experiments made at the Johns 
Hopkins Hospital, recommends the following procedure : 


1. The nails are kept short and clean. 

2. The hands are washed thoroughly for several minutes with soap and 
water, the water being as warm as can be comfortably borne, and being fre- 
quently changed. A brush sterilized by steam is used. The excess of soap 
is washed off with water. 

3. The hands are immersed for one or two minutes in a warm saturated 
solution of permanganate of potash and are rubbed over thoroughly with a 
sterilized swab. 

4. They are then placed in a warm saturated solution of oxalic acid, 
where they remain until complete decolorization of the permanganate 
occurs. 

5. They are then washed off with sterilized salt solution or water. 

6. They are immersed for two minutes in sublimate solution, 1 : 500. 

The bacteriological examination of the skin thus treated yields almost 
uniformly negative results, the material for the cultures being taken from 
underneath and around the nails. This is the procedure now employed in 
the gynecological and surgical wards of the hospital. 


THE DISINFECTION OF EXCRETA. 


The contents of privy vaults and cesspools should never be allowed 
to accumulate unduly or to become offensive. By frequent removal, 
and by the liberal use of antiseptics, such necessary receptacles of 
filth should be kept in a sanitary condition. The absorbent deodo- 


218 PRACTICAL DIRECTIONS FOR DISINFECTION. 


rants, such as dry earth or pounded charcoal, or the chemical de- 
odorants and antiseptics, such as chloride of zinc, sulphate of iron, 
etc., will, under ordinary circumstances, prevent such places from 
becoming offensive. Disinfection will be required only when it is 
known or suspected that infectious material, such as the dejections 
of patients with cholora, yellow fever, or typhoid fever, has been 
thrown into the receptacles. 

In the Manual for the Medical Department of the United States 
Army the following directions are given: 

92. When accumulations of organic material undergoing decomposition 
cannot be removed or buried, they may be treated with an antiseptic solu- 
tion, or with freshly burned quicklime. Quicklime is also a valuable disin- 
fectant, and may be substituted for the more expensive chloride of lime for 
disinfection of typhoid and cholera excreta, etc. For this purpose freshly 
prepared milk of lime should be used, containing about one part, by weight, 
of hydrate of lime, to eight of water. 

93. During the prevalence of an epidemic, or when there is reason to 
believe that infectious material has been introduced from any source, latrines 
and cesspools may be treated with milk of lime, in the proportion of five 
parts to one hundred parts of the contents of the vault, and the daily addi- 
tion of ten parts for one hundred parts of daily increment of feeces. 

According to Behring, lime has about the same germicidal value 
as the other caustic alkalies, and destroys the cholera spirillum and 
the bacillus of typhoid fever, of diphtheria, and of glanders after 
several hours’ exposure, in the proportion of fifty cubic centimetres 
normal-lauge per litre. Wood ashes or lye of the same alkaline 
strength may therefore be substituted for quicklime. 

Finally, it must not be forgotten that we have a ready means of 
disinfecting excreta in the sick-room or its vicinity by the application 
of heat. Exact experiments, made by the writer and others, show 
that the thermal death-point of the following pathogenic bacteria, 
and of the kinds of virus mentioned, is below 60° C. (140° F.): 
Spirillum of cholera, bacillus of anthrax, bacillus of typhoid fever, 
bacillus of diphtheria, bacillus of glanders, diplococcus of pneu- 
monia (Micrococcus Pasteuri), streptococcus of erysipelas, staphylo- 
cocci of pus, micrococcus of gonorrhcea, vaccine virus, sheep-pox 
virus, hydrophobia virus. Ten minutes’ exposure to the tempera- 
ture mentioned may be relied upon for the disinfection of material 
containing any of these pathogenic organisms, except the anthrax 
bacillus when in the stage of spore formation. The use, therefore, 
of boiling water in the proportion of three or four parts to one 
part of the material to be disinfected may be safely recommended 
for such material. Or, better still, a ten-per-cent solution of sulphate 
of iron or of chloride of zinc at the boiling-point may be used in the 


same way (three parts to one). 


PART THIRD. 


PATHOGENIC BACTERIA. 


I. Moves or Action. II. CHannets oF InFeEcTION. III. SuscEPTIBILITY AND 
Inuuniry. IV. Protective Inocunatrons. V. Pyrocentc Bacteria. VI. 
BacTERIA IN Croupous Pxreumonia. VII. PatHocrentc Micrococcr Not 
DESCRIBED IN Sections Y. anp VI. VIIL Tue Bacriuus or AN- 
THRAX. IX. Tue Bacinuus or Typnorp FEvErR. X. Bac- 
TERIA IN DrpHruEeRIA. XI. Bacriits or INFLuENZA. XII. 
BAcILLI IN CHronic INFEctrious Diseases. XIII. Bacrutr 
WHICH PRODUCE SEPTICZMIA IN SUSCEPTIBLE ANI- 

MALS. XIV. ParHocGentc AEROBIC BACILLI NOT 
DESCRIBED IN Previous SEcTIOoNS. XV. 

BacTERIA OF PiLant DisEaAsEs. XVI. 

PatHocEntic ANAEROBIC BAcrILit. 

XVII. PatHogEentc SPIRILua. 


PAB. TELED, 


PATHOGENIC BACTERIA. 


L 


MODES OF ACTION. 


Many of the saprophytic bacteria are pathogenic for man, or for 
one or more species of the lower animals, when by accident or ex- 
perimental inoculation they obtain access to the body ; these may be 
designated facultative parasites. Other species which, for a time 
at least, are able to lead a saprophytic mode of life have their nor- 
mal habitat in the bodies of infected animals, in which they produce 
specific infectious diseases. To this class belong the cholera spirillum, 
the anthrax bacillus, the bacillus of typhoid fever, and various other 
microérganisms which are the cause of specific infectious diseases in 
some of the lower animals. These we may speak of as parasites 
and facultative saprophytes. Still others are strict parasites and 
do not find the conditions for their development outside of the bodies 
of the animals which they infest, except under the special conditions 
in which bacteriologists have succeeded in cultivating some of them. 
The best known strict parasites are the tubercle bacillus, the bacillus 
of leprosy, the spirillum of relapsing fever, and the micrococcus of 
gonorrhcea, 

There can be but little doubt that even the strict parasites, at some 
time in the past, were also saprophytes, and that the adaptation to a 
parasitic mode of life was gradually effected under the laws of natural 
selection. In a previous chapter (Section III., Part Second) we have 
referred to the modifications in biological characters which may 
occur as a result of special conditions of environment. Thus we may 
obtain non-chromogenic varieties of species which usually produce 
pigment, or non-pathogenic varieties of bacteria which are usually 
pathogenic. There is also evidence that the tubercle bacillus, a strict 


222 MODES OF ACTION. 


parasite, may be so modified, by cultivation for successive genera- 
tions in a culture medium containing glycerin, that it will finally 
grow in ordinary beef infusion, thus showing a tendency to adapt 
itself to a saprophytic mode of life. 

Some of the saprophytic bacteria are indirectly pathogenic by 
reason of their power to multiply in articles of food, such as milk, 
cheese, fish, sausage, etc., and there produce poisonous ptomaines 
which, when these articles are ingested, give rise to various morbid 
symptoms, such as vomiting, gastric and intestinal irritation, fever, 
ete. Or similar symptoms may result from the multiplication of 
bacteria producing toxic ptomaines in the alimentary canal. No 
doubt gastric and intestinal disorders are largely due to this cause, 
and may be induced by a variety of saprophytic bacteria when these 
establish themselves in undue numbers in any portion of the ali- 
mentary tract. In Asiatic cholera the same thing occurs, but with 
more fatal results from the introduction of the East Indian cholera 
germ discovered by Koch. This is pathogenic for man, because it is 
able to multiply rapidly in the human intestine, and there produces a 
toxic substance which, being absorbed, gives rise to the morbid pheno- 
mena of the disease. The spirillum itself does not enter the blood or 
invade the tissues, except to a limited extent in the mucous coat of 
the intestine, and the true explanation of its pathogenic power is no 
doubt that which has been given. 

Other microérganisms invade the tissues and multiply in cer- 
tain favorable localities, but have not the power of developing in the 
blood, in which they are only found occasionally and in very small 
numbers or not at all. Thus the typhoid bacillus locates itself in the 
intestinal glands, in the spleen, and in the liver, forming colonies of 
limited extent, and evidently not finding the conditions extremely 
favorable for its growth, inasmuch as it does not take complete pos- 
session of these organs. The symptoms which result from its pre- 
sence are doubtless partly due to local irritation, disturbance of func- 
tion, and, in the case of the intestinal glands, necrotic changes 
induced by it. But in addition to this its pathogenic action depends 
upon the production of a poisonous ptomaine which has been isolated 
and studied by the German chemist Brieger (typhotoxin), 

Certain saprophytic bacteria, when injected beneath the skin of a 
susceptible animal, multiply at the point of inoculation and invade 
the surrounding tissues, giving rise in some instances to the forma- 
tion of a local abscess, in others to an infiltration of the tissues with 
bloody serum, and in others to extensive necrotic changes. These 
local changes are due not simply to the mechanical presence of the 
microorganisms which induce them, but to chemical products evolved 
during the growth of these pathogenic bacteria. Indeed, their patho- 


MODES OF ACTION. 223 


genic power evidently depends, in some instances at least, upon these 
toxic products of their growth, by which the vital resisting power of 
the tissues is overcome. 

Among the bacteria which in this way produce extensive local 
inflammatory and necrotic changes are certain anaérobic species 
found in the soil and in putrefying material, such as the bacillus of 
malignant cedema and the writer’s Bacillus cadaveris. The bacillus 
of symptomatic anthrax, an infectious disease of cattle, acts in the 
same way. All of these produce toxic substances which have a very 
pronounced local action upon the tissues invaded by them. Other 
bacteria, while they develop chiefly in the vicinity of the point of 
entrance—by accident or by inoculation—produce a potent toxic sub- 
stance which gives rise to general symptoms of a serious character, 
such as tetanic convulsions (bacillus of tetanus) or intense fever and 
nervous phenomena (micrococcus of erysipelas). Again, the local 
irritation resulting from the presence of parasitic bacteria may pri- 
marily give rise to the formation of new growths having alow grade 
of vitality, which later may undergo necrotic changes, as in tubercu- 
losis, glanders, and leprosy. In this case constitutional symptoms 
are not present, or are of a mild character during the development 
of these new formations, which apparently result from the local ac- 
tion of substances eliminated during the growth of the parasite, 
rather than from its simple presence. This is an inference based 
upon the fact that non-living particles, or even living parasites, as in 
trichinosis, do not produce similar new growths composed of cells, 
but become encysted in a fibrous capsule. 

In pneumonia we have a local process in which one or more lobes 
of the lung are invaded by a pathogenic micrococcus (Micrococcus 
pheumoniz croupos) which induces a fibrinous exudation that com- 
pletely fills the air cells. How far the symptoms of the disease are 
due to the local inflammation and disturbance of function, and to 
what extent they may be due to the absorption of a soluble toxic 
substance evolved as a result of the growth of the micrococcus, has 
not been determined. But the mild character of the general symp- 
toms when a limited area of lung tissue is involved leads to the in- 
ference that the pathogenic power of this particular pathogenic 
microérganism is chiefly exercised locally. 

The pus cocci and various other saprophytic bacteria, when intro- 
duced beneath the skin, give rise to the formation of abscesses, un- 
attended by any very considerable general disturbance ; and also to 
secondary purulent accumulations—metastatic abscesses. 

That this is not due simply to their mechanical presence is shown 
by the fact that powdered glass and other inert substances, when 
thoroughly sterilized, do not give rise to pus formation when intro- 


224 MODES OF ACTION. 


duced beneath the skin or injected into the cavity of the abdomen. 
On the other hand, it has been demonstrated by the experiments of 
Grawitz, De Bary, and others that certain chemical substances 
which act as local irritants when brought in contact with the tissues 
may induce pus formation quite independently of microérganisms : 
nitrate of silver, oil of turpentine, and strong liquor ammonie have 
been shown to possess this power. And it has been demonstrated by 
the recent experiments of Buchner that sterilized cultures of a long 
list of different bacteria—seventeen species tested—give rise to sup- 
puration when introduced into the subcutaneous tissues. 

Buchner has further shown that this property of inducing pus for- 
mation resides in the dead bacterial cells and not in soluble products 
present in the cultures. For, the clear fluid obtained by passing 
these sterilized cultures through a porcelain filter gave a negative re- 
sult, while the bacteria retained by the filter, although no longer 
capable of development, having been killed by heat, invariably 
caused suppuration. 

Individuals suffering from malnutrition are more susceptible to 
invasion by specific disease germs or by the common pus cocci 
than are those in vigorous health. Thus the sufferers from starva- 
tion, from crowd poisoning, sewer-gas poisoning, etc., are not only 
liable to be early victims during the prevalence of an epidemic dis- 
ease, but are very subject to abscesses, boils, ulcers, etc. A slight 
abrasion in such an individual, inoculated by the ever-present pus 
cocci, may give rise to an obstinate ulcer or a phlegmonous inflam- 
mation. 

In the same way some of the ordinary saprophytes, which usually 
have no pathogenic power, may be pathogenic for an animal whose 
strength is reduced by disease or injury. Thus necrotic changes 
may occur in injured tissues, or in those which havea deficient blood 
supply—from occlusion of an artery, for example—due to the presence 
of putrefactive bacteria which are incapable of development in the 
circulation of a healthy animal or in healthy tissues. We may also 
have a progressive gangrene, due to infection of wounds by bacteria 
which are able to invade healthy tissues. This is seen in the so- 
called hospital gangrene, which is undoubtedly due to microérgan- 
isms, although the species concerned in its production has not been 
determined, owing to the fact that modern bacteriologists have had 
few, if any, opportunities for studying it. The history of the disease, 
its rapid extension in infected surgical wards, the extensive slough- 
ing which occurs within a few hours in previously healthy wounds, 
and the effect of deep cauterization by the hotiron, nitric acid, or 
bromine in arresting the progress of the disease, all support this view 
of its etiology. Whether itis due toa specific pathogenic micro- 


MODES OF ACTION. 225 


organism, or to exceptional pathogenic power acquired by some one 
of the common bacteria which infest suppurating wounds, cannot be 
determined in the absence of exact experiments by modern methods. 
But the latter view has seemed to the writer the most probably cor- 
rect. There are many facts which go to show that pathogenic viru- 
lence may be increased by cultivation in animal fluids, and where 
wounded men are brought together under unfavorable sanitary con- 
ditions, as has been the case where hospital gangrene has made its 
appearance, it may be that some common saprophyte acquires the 
power of invading the exposed tissues instead of simply feeding upon 
the secretions which bathe its surface. 

Koch has described a progressive tissue necrosis in mice, due to a 
streptococcus, which he first obtained by inoculating a mouse in the 
ear with putrid material. The morbid process is entirely local and 
rapidly progressive, causing a fatal termination in about three days, 
without invasion of the blood. 

In diphtheritic inflammations of mucous membranes we have 
a local invasion of the tissues and a characteristic plastic exudation. 
In true diphtheria the local inflammation and necrotic changes in 
the invaded tissues are not sufficient to account for the serious gen- 
eral symptoms, and we now have experimental evidence that the 
diphtheria bacillus produces a very potent toxic substance to which 
these symptoms are no doubt largely due. The diphtheria bacillus 
of Léffler appears to be the cause of the fatal malady which goes 
by this name, but undoubtedly other microérganisms may be con- 
cerned in the formation of diphtheritic false membranes. In cer- 
tain forms of diphtheria, and especially when it occurs as a com- 
plication of scarlet fever, measles, and other diseases, the Klebs- 
Léffler bacillus is absent, and a streptococcus, which appears to be 
identical with Streptococcus pyogenes, is found in considerable num- 
bers and is probably the cause of the diphtheritic inflammation. 
An epidemic of diphtheria occurring among calves was studied by 
Loffler, and is ascribed by him to his Bacillus diphtheriz vitulo- 
yum. The same bacteriologist has shown that the diphtheria of 
chickens and of pigeons is due to a specific bacillus which differs 
from that found in human diphtheria, and which he calls Bacillus 
diphtheria columbrarum. 

Prof. Welch has studied the histological lesions produced by 
filtered cultures of the diphtheria bacillus. Cultures in glycerin- 
bouillon, several weeks old, were filtered through porcelain, and the 
sterile filtrate was injected beneath the skin of guinea-pigs. One 
cubic centimetre of this filtrate was injected into a guinea-pig on 
the 10th of December, and two cubic centimetres more on the 
14th of the same month. The animal succumbed at the end of 

15 


226 MODES OF ACTION. 


three weeks and five days after the first inoculation. At the autopsy 
‘“‘the lymphatic glands of the inguinal and axillary regions were 
found to be enlarged and reddened; the cervical glands were swollen 
and the thyroid gland was greatly congested. There was a consider- 
able excess of clear fluid in the peritoneal cavity. Both layers of the 
peritoneum were reddened, the vessels of the visceral layer being es- 
pecially injected. The spleen was enlarged to double the average 
size; it was mottled, and the white follicles were distinctly outlined 
against the red ground. The liver was dark in color and contained 
much blood. . . . The kidneys were congested and the cut surface 
was cloudy. . . . The pericardial sac was distended with clear se- 
rum. Under the epicardium were many ecchymotic spots. The 
lungs exhibited areas of intense congestion or actual haemorrhage 
into the tissues. . . . The histological lesions in this case are identi- 
cal with those observed by us in connection with the inoculation of 
the living organisms.” 

To what extent non-specific catarrhal inflammations of mucous 
membranes are caused by the local action of microédrganisms has 
not been determined, but in gonorrhcea the proof is now considered 
satisfactory that the ‘‘ gonococcus” of Neisser is the cause of the 
intense local inflammation and purulent discharge. In this disease 
the action of the pathogenic microérganism seems to be limited to 
the tissues invaded by it, as there is no general systemic disturbance 
indicating the absorption of a toxic ptomaine. 

Chronic catarrhal inflammations appear, in some cases at least, 
to be kept up by the presence of microédrganisms, which are always 
found in the discharges from inflamed mucous surfaces. 

The influence of microédrganisms, and especially of the pus cocci, 
in preventing the prompt healing of wounds, is now well established. 
An extensive suppurating wound or collection of pus, especially if 
putrefactive bacteria are present, causes fever and nervous symp 
toms, due to the absorption of toxic products. More intense general 
symptoms result from the presence of the streptococcus of pus than 
from the less pathogenic staphylococci ; this is seen in erysipelatous 
inflammations and in puerperal metritis due to the presence of this 
micrococcus. Like the other pus cocci, the Streptococcus pyogenes 
does not usually invade the blood, but when introduced into the sub- 
cutaneous tissues it induces a local inflammatory process, with a ten- 
dency to pus formation, and it invades the neighboring lymph chan- 
nels, in which the conditions appear to be especially favorable for its 
multiplication. 

Finally, certain pathogenic bacteria, when introduced into the 
bodies of susceptible animals, quickly invade the blood and multiply 
in it. Inso doing they necessarily interfere with its physiological 


MODES OF ACTION. 227 


functions by appropriating for their own use material required for 
the nutrition of the tissues ; and at the same time toxic substances 
are formed which play an important part in the production of the 
morbid phenomena, which in this class of diseases very commonly 
lead to a fatal result. The pathogenic bacteria which invade the 
blood may also, in certain cases, give rise to local necrosis and dis- 
turbance of function in various organs in a mechanical way by 
blocking up the capillaries. 

The invasion of the blood which occurs in anthrax and in vari- 
ous forms of septicemia in the lower animals, induced by subcuta- 
neous inoculation with pure cultures of certain pathogenic bacteria, 
does not generally immediately follow the inoculation. Usually a 
considerable local development first occurs, which gives rise to more 
or less inflammation of the invaded tissues, and very commonly to 
an effusion of bloody serum in which the pathogenic microédrganism 
is found in great numbers. Even in susceptible animals the blood 
seems to offer a certain resistance to invasion, which is overcome 
after a time by the vast number of the parasitic host located in the 
vicinity of the point of inoculation, aided probably by the toxic sub- 
stances developed as a result of their vital activity. 

The experiments of Cheyne (1886) seem to show that in the case 
of very pathogenic species, like the anthrax bacillus or Koch’s bacil- 
lus of mouse septiceemia, a single bacillus introduced subcutaneously 
may produce a fatal result in the most susceptible animals, while 
greater numbers are required in those which are less susceptible. 
Thus a guinea-pig succumbed to general infection after being inocu- 
lated subcutaneously with anthrax blood diluted to such an extent 
that, by estimation, only one bacillus was present in the fluid in- 
jected ; and a similar result in mice was obtained with Bacillus 
murisepticus. In the case of the microbe of fowl cholera (Bacillus 
septiceemize heemorrhagicee) Cheyne found that for rabbits the fatal 
dose is 300,000 or more, that from 10,000 to 300,000 cause a local 
abscess, and that less than 10,000 produce no appreciable effect. 
The common saprophyte Proteus vulgaris was found to be patho- 
genic for rabbits when injected into the dorsal muscles in sufficient 
numbers. But, according to the estimates made, 225,009,000 were 
required to cause death, while with doses of from 9,000,000 to 112,- 
000,000 a local abscess was produced, and less than 9,000,000 gave 
an entirely negative result. 

Secondary infections occurring in the course of specific infec- 
tious diseases are of common occurrence. Thus a pneumonia may 
be developed in the course of an attack of measles or of typhoid 
fever ; or infection by the common pus cocci in the course of scarlet 
fever, typhoid fever, mumps, etc., may give rise to local abscesses, 


228 MUDES OF ACTION. 


to endocarditis, ete. Again, méved infection may be induced by 
injecting simultaneously into susceptible animals two species of path- 
ogenic bacteria. 

Bumm, Bockhart, and others have reported cases of mixed gonor- 
rhceal infection in which the pyogenic micrococci gave rise to ab- 
scesses in the glands of Bartholin, to cystitis, parametritis, or to 
“ vonorrheeal inflammation ” of the knee joint. Babes gives numer- 
ous examples of mixed infection in scarlet fever and in other diseases 
of childhood. Anton and Fiitterer have studied the question of 
secondary infection in typhoid fever. Karlinski has reported a case 
of secondary infection with anthrax in a case of typhoid fever, infec- 
tion occurring by way of the intestine. Many other examples of 
secondary or mixed infection are recorded in the recent literature of 
bacteriology and clinical medicine, but enough has been said to call 
attention to the importance of the subject. 

The researches of Rémer, Kanthack (1892), and others show that 
the injection of the filtered products of certain bacteria (Bacillus 
pyocyaneus, Vibrio Metchnikovi, etc.) produces a decided leucocy- 
tosis in the animals experimented upon. And a similar result, prob- 
ably from a like cause, has been shown by recent experiments to 
occur in pneumonia (Billings) and other infectious diseases. 

Certain bacterial products have been shown by experiment to pro- 
duce fever when injected into the circulation or beneath the skin of 
lower animals; others produce rapid respiration, dilatation of pupils, 
diarrhoea, and paralysis or convulsions (typhotoxin of Brieger, 
methyl-guanidin, etc.) ; the toxic effects of some are immediate and 
of others more or less remote (toxalbumin of diphtheria) ; others have 
a primary toxic effect which is followed after a time by toxic symp- 
toms of a different order (Pneumobacillus liquefaciens bovis). 


IL. 
CHANNELS OF INFECTION. 


WE have abundant evidence that susceptible animals may be in- 
fected by the injection of various pathogenic bacteria beneath the 
skin, and'accidental infection through an open wound or abrasion 
of the skin is the common mode of infection in tetanus, erysipelas, 
hospital gangrene, and the “ traumatic infectious diseases” generally. 
Other infectious diseases, like anthrax and glanders, are frequently 
transmitted in the same way. We have also satisfactory evidence 
that tuberculosis may be transmitted to man by the accidental inocu- 
lation of an open wound; and in view of the fact that susceptible 
animals are readily infected in this way, it would be strange if it 
were otherwise. 

The question whether infection may occur through the unbroken 
skin has been studied by several bacteriologists and an affirmative 
result obtained. Thus Schimmelbusch produced pustules upon the 
thigh in two young persons suffering from pyzemia by rubbing upon 
the surface a pure culture of Staphylococcus pyogenes aureus which 
he had obtained from the pus of a furuncle. The same author aiso 
succeeded in infecting rabbits and guinea-pigs with anthrax, and 
rabbits with rabbit septicemia, by rubbing pure cultures upon 
the uninjured skin. Similar results had previously been reported 
by Roth, who also showed that infection might occur through 
the uninjured mucous membrane of the nose. Machnoff also suc- 
ceeded in infecting guinea-pigs with anthrax through the unin- 
jured skin of the back, and, as a result of subsequent microscop 
ical examination of stained sections, arrived at the conclusion that 
the principal channel through which infection was accomplished was 
the hair follicles. Braunschweig, in a series of experiments in which 
he introduced various pathogenic bacteria into the conjunctival sac 
of mice, rabbits, and guinea-pigs, obtained a negative result with the 
anthrax bacillus, the bacillus of mouse septicaemia, the bacillus of 
chicken cholera, and Micrococcus tetragenus; but the bacillus ob- 
tained by Ribbert from the intestinal diphtheria of rabbits gave a 
positive result in five mice, two guinea-pigs, and a rabbit. 


230 CHANNELS OF INFECTION, 


Infection through the mucous membrane of the intestine no 
doubt occurs in certain diseases. This is believed to be a common 
mode of the infection of sheep and cattle with anthrax, and probably 
also in the infectious disease of swine known as hog cholera. The 
anthrax bacillus would be destroyed by the acid secretions of the 
stomach, but if spores are present in food ingested they will reach 
the intestine. The experiments of Korkunoff do not, however, sup- 
port the view that infection is likely to occur in this way. In a series 
of experiments upon white mice fed with bread containing a quantity 
of anthrax spores the result was uniformly negative, but exception- 
ally infection occurred in rabbits. The same author obtained posi- 
tive results in rabbits fed with food to which a pure culture of the 
bacillus of chicken cholera had been added. 

Buchner, in experiments upon mice and guinea-pigs fed with 
material containing anthrax spores, obtained a positive result in four 
out of thirty-three animals. This is no doubt the usual mode of in- 
fection in typhoid fever in man. 

Infection may also occur through the mucous membrane of the 
respiratory organs. This has been demonstrated by several bac- 
teriologists, and especially by the experiments of Buchner, who 
mixed dried anthrax spores with lycopodium powder or pulverized 
charcoal, and caused mice and guinea-pigs to respire an atmosphere 
containing this powder in suspension. In a series of sixty-six experi- 
ments fifty animals died of anthrax, nine of pneumonia, and seven 
survived. That infection did not occur through the mucous mem- 
brane of the alimentary canal was proved by comparative experi- 
ments in which animals were fed with double the quantity of spores 
used in the inhalation experiments. Out of thirty-three animals fed 
in this way but four contracted anthrax. That infection occurred 
through the lungs was also demonstrated by the microscopical ex- 
amination of sections and by culture experiments, which showed that 
the lungs were extensively invaded, while in many cases the spleen 
contained no bacilli. Positive results were also obtained with cul- 
tures of the anthrax bacillus not containing spores, which the ani- 
mals were made to inhale in the form of spray. But in this case a 
considerable quantity was required, and a sero-fibrinous pneumonia 
was usually produced as well as general infection; the inhalation of 
small quantities gave no result. Positive results in rabbits were also 
obtained by causing them to inhale considerable quantities of a spray 
containing the bacillus of chicken cholera, 

The fact that large quantities of a liquid culture of these virulent 
bacilli were required to infect very susceptible animals by way of 
the pulmonary mucous membrane, and that Buchner failed to cause 
the infection of these animals with small quantities of a pure culture 


CHANNELS OF INFECTION. 231 


inhaled in the form of spray, indicates that this is not a common 
mode of infection in the absence of spores. This view receives 
further support from the experiments of Hildebrandt, who made 
tracheal fistula in three rabbits, and, after the wound had entirely 
healed, injected into the trachea of each a pure culture of the anthrax 
bacillus, which was proved to be virulent by inoculation in mice or 
guinea-pigs. All of the animals remained in good health. On the 
other hand, three rabbits which received in the same way a pure cul- 
ture of the bacillus of rabbit septicaemia died as a result of general 
infection. 

That man may be infected with anthrax by way of the respira- 
tory organs seems to be well established. In England the disease 
known as “‘ wool-sorter’s disease” results from infection in this way 
among workmen engaged in sorting wool, which is liable to contain 
the spores of the anthrax bacillus when obtained from the skin of an 
animal which has fallen a victim to this disease. That infection 
occurs through the lungs is shown by the fact that these organs are 
first involved, the disease being, in fact, a pulmonic anthrax. 

While these experiments prove the possibility of infection through 
the respiratory mucous membrane, other experiments made by Hil- 
debrandt show that under ordinary circumstances bacteria suspended 
in the air do not reach the trachea in rabbits, but are deposited upon 
the mucous membrane of the mouth, nares, and fauces. In healthy 
rabbits the tracheal mucus was, as a rule, found to be free from bac- 
teria, while they were very numerous in mucus obtained from the 
mouth or nares. But when a rabbit was made to inhale for half an 
hour an atmosphere charged with the spores of Aspergillus fumigatus 
their presence in the lungs was demonstrated by cultivation, the ani- 
mal being killed for the purpose half an hour after the inhalation 
experiment. 

The rapidity with which infection may occur is shown by the 
experiments of Nissen, Pfuhl, and others. In mice inoculated with 
anthrax bacilli at the tip of the tail fatal anthrax has resulted, 
although the tail was amputated ten minutes after the inoculation. 
Schimmelbusch inoculated fresh wounds with anthrax cultures (in 
mice) and immediately after treated the wounds with strong anti- 
septic solutions, but the animals succumbed to infection. Cultures 
of the anthrax bacillus have been obtained from the liver, spleen, and 
kidneys half an hour after the infection of an open wound on the 
surface of the body (Schimmelbusch and Ricker). The experiments 
of Sherrington and others show that pathogenic bacteria may escape 
by way of the kidneys into the bladder, or through the liver into the 
gall bladder. But his experiments indicate that such escape does not 
occur through healthy organs. Non-pathogenic bacteria injected 


232 CHANNELS OF INFECTION. 


into the circulation were not found in the urine, and when a consid- 
erable quantity of a pathogenic species was injected into a vein there 
was no immediate appearance of bacteria in the urine, but they were 
found later, probably as a result of lesions in the secreting organ due 
to their local action or to that of their toxic products. In man the 
presence of pathogenic bacteria in the urine has been frequently veri- 
fied, especially in typhoid fever, pneumonia, and streptococcus in- 
fection. When, asa result of the establishment of foci of infection 
in the liver, localized necrosis of tissue occurs, the pathogenic bac- 
teria to which the infection is due escape with the bile and enter 
the intestine. It is probable that escape through the walls of the 
intestine does not occur unless there is a local lesion of some kind, as 
in typhoid fever. 

The presence of tubercle baciili in the milk of cows has been 
repeatedly demonstrated, and in a certain proportion of the cases 
they have been found in the milk of cows whose udders gave 
no evidence of being the seat of a tubercular process. Usually, how- 
ever, when tubercle bacilli are found in the milk the cow’s udder 
is already involved in the disease. The milk of women with puer- 
peral fever has been found to contain streptococci; and in mastitis 
from a localized infection by pyogenic cocci these are found in the 
milk. It must be remembered, however, that both Staphylococcus 
albus and aureus have been found in the milk of healthy women. 
The micrococcus of pneumonia has been found in the milk of women 
suffering from croupous pneumonia (Hoa, and Bordoni-Uffreduzzi). 
Various observers (Brunner, Tizzoni, von Hiselsberg) have reported 
the presence of pus cocci in the sweat of patients suffering from sep- 
ticeemia, and the experiments of Brunner indicate that they may have 
escaped through the sweat glands. This, however, does not appear 
to be definitely established. 


Jil. 


SUSCEPTIBILITY AND IMMUNITY. 


No questions in general biology are more interesting, or more 
important from a practical point of view, than those which relate to 
the susceptibility of certain animals to the pathogenic action of cer- 
tain species of bacteria, and the immunity, natural or acquired, from 
such pathogenic action which is possessed by other animals. It has 
long been known that certain infectious diseases, now demonstrated 
to be of bacterial origin, prevail only or principally among animals 
of a single species. Thus typhoid fever, cholera, and relapsing 
fever are diseases of man, and the lower animals do not suffer from 
them when they are prevailing as an epidemic. On the other hand, 
man has a natural immunity from many of the infectious diseases of 
the lower animals, and diseases of this class which prevail among 
animals are frequently limited to a single species. Again, several 
species, including man, may be susceptible to a disease, while other 
animals have a natural immunity from it. Thus tuberculosis is 
common to man, to cattle, to apes, and to the small herbivorous ani- 
mals, while the carnivora are, as arule, Immune; anthrax may be 
communicated by inoculation to man, to cattle, to sheep, to guinea- 
pigs, rabbits, and mice, but the rat, the dog, carnivorous animals, and 
birds are generally immune ; glanders, which is essentially a disease 
of the equine genus, may be communicated to man, to the guinea- 
pig, and to field mice, while house mice, rabbits, cattle, and swine 
are to a great extent immune. 

In addition to this general race immunity or susceptibility we 
have individual differences in susceptibility or resistance to the ac- 
tion of pathogenic bacteria, which may be either natural or acquired. 
Asarule, young animals are more susceptible than older ones. 
Thus in man the young are especially susceptible to scarlet fever, 
whooping cough, and other ‘‘children’s diseases,” and after forty 
years of age the susceptibility to tubercular infection is very much 
diminished. Among the lower animals it is a matter of common 
laboratory experience that the very young of a susceptible species 
may be infected when inoculated with an ‘‘attenuated culture” 
which older animals of the same species are able to resist. 


234 SUSCEPTIBILITY AMD IMMUNITY. 


Considerable differences as to susceptibility may also exist among 
adults of the same species. In man these differences in individual 
susceptibility to infectious diseases are frequently manifested. Of a 
number of persons exposed to infection in the same way, some may 
escape entirely while others have attacks differing in severity and 
duration. In our experiments upon the lower animals we constantly 
meet with similar results, some individuals proving to be exception- 
ally resistant. Exceptional susceptibility or immunity may be to 
some extent a family characteristic or one of race. Thus the negro 
race is decidedly less subject to yellow fever than the white race, 
and this disease is more fatal among the fair-skinned races of the 
north of Kurope than among the Latin races living in tropical or sub- 
tropical regions. On the other hand, small-pox appears to be excep- 
tionally fatal among negroes and dark-skinned races generally. 

A very remarkable instance of race immunity is that of Algerian 
sheep against anthrax, a disease which is very fatal to other sheep. 

In the instances mentioned race immunity is probably an ac- 
quired tolerance due to natural selection and inheritance. If, for 
example, a susceptible population is exposed to the ravages of small- 
pox, the least susceptible individuals will survive and may be the pa- 
rents of children who will be likely to inherit the special bodily char- 
acters upon which this comparative immunity depends. The ten- 
dency of continuous or repeated exposure to the same pathogenic 
agent will evidently be to establish a race tolerance ; and there is 
reason to believe that such has been the effect in the case of some 
of the more common infectious diseases of man, which have been 
noticed to prevail with especial severity when first introduced among 
a virgin population, as in the islands of the Pacific, etc. 

In the same way we may explain the immunity which carnivor- 
ous animals have for anthrax and various forms of septicemia to 
which the herbivora are very susceptible when the pathogenic germ 
is introduced into their bodies by inoculation. From time immemo- 
rial the carnivora have been in the habit of fighting over the dead 
bodies of herbivorous animals, some of which may have fallen a prey 
to these infectious germ diseases, and in their fighting they receive 
wounds, inoculated with the infectious material from these bodies, 
which would be fatal to a susceptible animal. If at any time in the 
past a similar susceptibility existed among the carnivora, with indi- 
vidual differences as to resisting power, it is evident that there would 
be a constant tendency for the most susceptible individuals to perish 
and for the least susceptible to survive. 

But if we admit this to be a probable explanation of the immu- 
nity of carnivorous animals from septic infection, we have not yet 
explained the precise reason for the immunity enjoyed by the 


SUSCEPTIBILITY AND IMMUNITY. 235 


selected individuals and their progeny. The essential difference be- 
tween a susceptible and immune animal depends upon the fact that 
in one the pathogenic germ, when introduced by accident or ex- 
perimental inoculation, multiplies and invades the tissues or the 
blood, where, by reason of its nutritive requirements and toxic pro- 
ducts, it produces changes in the tissues and fluids of the body incon- 
sistent with the vital requirements of the infected animal; while in 
the immune animal multiplication does not occur or is restricted to a 
local invasion of limited extent, and in which after a time the re- 
sources of nature suffice to destroy the parasitic invader. 

Now the question is, upon what does this essential difference de- 
pend ? Evidently upon conditions favorable or unfavorable to the 
development of the pathogenic germ; or upon its destruction by 
some active agent present in the tissues or fluids of the body of the 
immune animal; or upon a neutralization of its toxic products by some 
substance present in the body of the animal which survives infec- 
tion. 

What, then, are the unfavorable conditions which may be supposed 
to prevent development in immune animals? In the first place, the 
temperature of the body may not be favorable. Certain pathogenic 
bacteria are only able to develop within very narrow temperature lim- 
its, and, if all other conditions were favorable, could not be expected 
to multiply in the bodies of cold-blooded animals. Or the temperature 
of warm-blooded animals, and especially of fowls, may be above the 
point favorable for their development. This is the explanation 
offered by Pasteur of the immunity of fowls, which are usually re- 
fractory against anthrax ; and in support of this view he showed by 
experiment that when chickens are refrigerated after inoculation, by 
being partly immersed in cold water, they are liable to become in- 
fected and to perish. But, as pointed out by Koch, the sparrow, 
which has a temperature as high as that of the chicken, may con- 
tract anthrax without being refrigerated. We must not, therefore, 
too hastily conclude that the success in Pasteur’s experiment de- 
pended alone upon a reduction of the body heat. Gibier has shown 
that the anthrax bacillus may multiply in the bodies of frogs or 
fish, if these are kept in water having a temperature of 35° C. 
But the anthrax bacillus grows within comparatively wide tempera- 
ture limits, while other pathogenic bacteria are known to have a 
more restricted temperature range and would be more decidedly 
influenced by this factor—e.g., the tubercle bacillus. 

The composition of the body fluids, and especially their reaction, 
is probably a determining factor in some instances. Thus Behring 
has ascribed the failure of the anthrax bacillus to develop in the 
white rat, which possesses a remarkable immunity against anthrax, 


236 SUSCEPTIBILITY AND IMMUNITY. 


to the highly alkaline reaction of the blood and tissue juices of this 
animal. Behring claims to have obtained experimental proof of the 
truth of this explanation by feeding white rats on an exclusively 
vegetable diet or by adding acid phosphate of lime to their food, by 
which means this excessive alkalinity of the blood is diminished. 
Rats so treated are said to lose their natural immunity, and to die as 
a result of inoculation with virulent cultures of the anthrax bacillus. 

The experiments of Nuttall, Behring, Buchner, and others have 
established the fact that recently drawn blood of various animals 
possesses decided germicidal power, and Buchner has shown that 
this property belongs to the fluid part of the blood and not to its 
cellular elements. It has also been shown that aqueous humor, the 
fluid of ascites, and lymph from the dorsal lymph sac of a frog 
possess the same power. This power to kill bacteria is destroyed by 
heat, and is lost when the blood has been kept for a considerable 
time, but it is not neutralized by freezing. Further, this power to 
destroy bacteria differs greatly for different species, being very de- 
cided in the case of certain pathogenic bacteria, less so for others, 
and absent in the case of certain common saprophytes. Behring 
has also shown that the blood of different animals differs consider- 
ably in this regard, and that the blood of the rat and of the frog, 
which animals have a natural immunity against anthrax, is espe- 
cially fatal to the anthrax bacillus. The experiments made show 
that this germicidal power is very prompt in its action, but that it is 
limited as to the number of bacteria which can be destroyed by a 
given quantity of blood serum. When the number is excessive, de- 
velopment occurs after an interval during which a limited destruc- 
tion has taken place. It would appear that the element in the blood 
to which this germicidal action is due is neutralized in exercising 
this power ; and as, independently of this, blood serum is an excel- 
lent culture medium for bacteria, an abundant development takes 
place when the destruction has been incomplete. 

Buchner (1889) first proved by experiment that the germicidal 
power of the blood of dogs and rabbits does not depend upon the 
presence of the cellular elements, but is present in clear serum which 
has been allowed to separate from the clot in a cool place. Exposure 
for an hour to a temperature of 55° C. destroys the germicidal action 
of serum as well as of blood. 

The researches of Buchner, of Hankin, and others, show that this 
germicidal power of fresh blood serum depends upon the presence of 
proteids, to which the first-named bacteriologist has given the name 
of “alexins.” Hankin, in his paper upon the origin of these “defen- 
sive proteids” in the animal body (1892), arrives at the conclusion 


ae 


SUSCEPTIBILITY AND IMMUNITY. 237 


that while they are present in the cell-free serum they are the prod- 

_uct of certain leucocytes—Ehrlich’s eosinophile cells. He believes 
that the eosinophile granules become dissolved in the serum and con- 
stitute the germicidal proteid which is shown to be present by ex- 
periments upon bacteria. According to Hankin the separation of 
these granules can be witnessed under the microscope. They first 
accumulate upon one side of the cell and then gradually disappear, 
and as this occurs a considerable increase in the bactericidal power 
of the serum can be demonstrated. The germicidal power of the 
blood serum is also said to be increased when the number of leuco- 
cytes is considerably augmented, as occurs when a sterilized culture 
of Vibrio Metschnikovi is injected subcutaneously. Also by treat- 
ment which favors a separation of the alexin from the leucocytes, 
2.e., a solution of the eosinophile granules. This may be accom- 
plished by the injection of an extract of the thymus gland of the 
calf, or by simply allowing the drawn blood to stand for several hours 
at a temperature of 38° to 40° C. 

Buchner’s latest communication upon the subject shows that he 
also attributes the origin of the germicidal proteid in fresh blood 
serum to the leucocytes. In his paper on “Immunity,” read at the 
Eighth International Congress on Hygiene and Demography (Buda- 
pest, 1894), he calls attention in the first place to the fact that a 
clearly marked distinction must be made between natural immunity 
and acquired immunity, inasmuch as the “ alexins” and “ antitoxins” 
have very different properties. The first-mentioned proteids are de- 
stroyed by a comparatively low temperature (55° to 60° C.), while the 
antitoxins resist a considerably higher temperature, and, unlike the 
alexins, have no bactericidal or globulicidal action. A very remark- 
able fact developed in Buchner’s experiments is that the blood serum 
from the dog and from the rabbit, when mixed, neutralize each other 
so far as their germicidal power is concerned. 

By injecting sterilized emulsions of wheat-flour paste in the 
pleural cavity of rabbits and dogs Buchner succeeded in obtaining an 
exudate which had more decided germicidal power than the blood 
or serum of the same animal. This was evidently due to the large 
number of leucocytes present, but not to their phagocytic action, as 
was shown by experiment. By freezing the exudate the leucocytes 
were killed, but the germicidal action of the fluid was rather in- 
creased than diminished by freezing. While freezing had no effect 
upon the germicidal action of the pleural exudate, this was always 
neutralized by exposure to a temperature of 55° C. 

Emmerich, Tsuboi, Steinmetz, and Léw (1892), as a result of ex- 
tended experiments, arrived at the conclusion that the germicidal 
action of blood serum “depends upon a specific property of the alkali 


238 SUSCEPTIBILITY AND IMMUNITY. 


serumalbumin, and that it is a purely chemical process.” They 
state that when the germicidal power is neutralized by heat it may 
be restored by the addition of an alkali. Buchner repeated the ex- 
periments of Emmerich and his associates and obtained similar re- 
sults, but interprets them differently. According to him the serum 
does not regain its germicidal power, but after the addition of an 
alkali and subsequent dialyzing the nutritive value of the serum is sO 
diminished that the bacteria do not develop in it. 

Pane (4892) has made experiments which give additional weight 
to the assumption that the alkalinity of the blood is an important 
factor in accounting for immunity. He states that carbonate of 
soda, dissolved in water, in the proportion of 1:3,000, has a de- 
cided germicidal action upon the anthrax bacillus, equal to that of 
the blood serum of the rabbit. And that when rabbit serum is com- 
pletely neutralized it no longer has any injurious action on anthrax 
bacilli. 

Zagari and Innocente (1892) also arrived at the conclusion that 
the diminished resistance to anthrax infection resulting from curare 
poisoning in frogs, and from chloral or alcohol in dogs (Platania), in 
fowls as a result of starvation (Canalis and Morpurgo), in white 
mice asa result of fatigue (Charin and Roger), is, in fact, due to 
diminished alkalinity of the blood, which they found to correspond 
with the increased susceptibility resulting from the causes men- 
tioned. 

Buchner (1892) states that several of the ammonium salts, and 
especially ammonium sulphate, cause an increase in the germicidal 
action of blood serum, and also increase its resistance to the neutral- 
izing effects of heat. The experiments of Pansini and Calabrese 
(1894) show, on the contrary, that the addition of uric acid to blood 
serum diminishes its bactericidal activity, as does also the presence 
of glucose. That certain infectious diseases are especially virulent 
in persons suffering from diabetes is a frequently repeated clinical 
observation. 

Van Fodor has shown by experiment that the injection of an 
alkali into the circulation of a rabbit increases its resistance to 
anthrax infection and the germicidal activity of its blood serum. 
The same bacteriologist has found that when a rabbit is infected 
with anthrax, the alkalinity of its blood is notably increased during 
the first twenty-four hours, when we may suppose that the powers 
of nature are brought to bear to resist the invading parasite, and that 
after this time it rapidly diminishes. Ten hours after infection (by 
subcutaneous inoculation?) the alkalinity of the blood had increased 
21.5 per cent. Shortly before the death of the animal a diminution 
of 26.3 per cent was noted. This diminution was observed in thirty- 


a4 


SUSCEPTIBILITY AND IMMUNITY. 239 


four out of thirty-nine animals experimented upon, and these ani- 
mals succumbed to the anthrax infection in a shorter time than did 
the other five in which there was no such diminution. 

It seems probable that the germicidal property of freshly drawn 
blood serum is not due to its alkalinity, per se, but to the fact that 
the germicidal constituent is only soluble in an alkaline fluid. The 
researches of Vaughn, McClintock, and Novy indicate that this ger- 
micidal constituent is a nuclein. Dr. Vaughn, in his last published 
paper upon “Nucleins and Nuclein Therapy,” says: “Kossel, of 
Berlin, has confirmed our statements concerning the germicidal 
action of the nucleins. Dr. McClintock and I have also demon- 
strated that the germicidal constituent of blood serum is a nuclein. 
This nuclein is undoubtedly furnished by the polynuclear white 
corpuscles.” Denys has (1894) reported the results of experi- 
ments made in his laboratory by Van der Velde, which give sup- 
port to the conclusion reached by Vaughn. In these experiments a 
sterilized culture of staphylococci was injected into the pleural cavity 
of rabbits in order to obtain an exudate. At intervals of two hours 
this exudate was obtained by killing one of the animals in the series 
experimented upon, and at the same time blood from the animal was 
secured. Both the exudate and the blood were placed in a centrifugal 
machine, in order to obtain a serum free from corpuscular elements. 
The germicidal activity of the serum was then tested. The general 
result of the experiments was to show that the longer the interval 
after the injection into the pleural cavity the more potent the ger- 
micidal activity of the exudate became, and that there was no corre- 
sponding increase in the activity of the blood serum obtained from 
the circulation. At the end of ten or twelve hours, the serum from 
the exudate killed all of the staphylococci in a bouillon culture twenty 
times as great in quantity as the germicidal serum used in the ex- 
periment. The absence of any increase in germicidal power in the 
blood serum taken from the general circulation shows that the nota- 
ble increase manifested by the exudate was due to local causes; and 
as a matter of fact it corresponded with an increase in the number of 
leucocytes as found in the pleural exudate. 

Thus it will be seen that the independent researches of Hankin, 
of Buchner, of Vaughn, and of other competent bacteriologists, have 
led them to the same ultimate result so far as the origin of the ger- 
micidal constituent of the blood is concerned, and that the leucocytes 
appear to play an important réle in the protection of the animal body 
from invasion by bacteria (natural immunity). 

It has been shown by several investigators that the number of 
leucocytes increases in certain infectious diseases, and this increase, 
together with an increased alkalinity of the blood, which has here- 


240 SUSCEPTIBILITY AND IMMUNITY. 


tofore been referred to, appears to be a provision of nature for over- 
coming the infection which has already occurred. 

It has been demonstrated by experiment that naturally immune 
animals may be infected by the addition of certain substances to cul- 
tures of pathogenic bacteria. Thus Arloing was able to induce symp- 
tomatic anthrax in animals naturally immune for this disease by 
mixing with his cultures various chemical substances, such as car- 
bolic acid, pyrogallic acid, and especially lactic acid (twenty per 
cent). Leo has shown that white mice, which are not subject to 
the pathogenic action of the glanders bacillus, may be rendered sus- 
ceptible by feeding them for some time upon phloridzin, which gives 
rise to an artificial diabetes, and causes the tissues to become im- 
pregnated with sugar. 

Bouchard has found that very small doses of a pure culture of 
Bacillus pyocyaneus are fatal to rabbits when at the same time a 
considerable quantity of a filtered culture of the same bacillus is in- 
jected into a vein. The animal could have withstood the filtered 
culture alone, or the bacillus injected beneath its skin; but its resist- 
ing power—natural immunity—is overcome by the combined action 
of the living bacilli and the toxic substances contained in the filtered 
culture. The same result may be obtained by injecting sterilized 
cultures of adifferent microérganism. Thus Roger has shown that 
the rabbit, which has a natural immunity against symptomatic 
anthrax, succumbs to infection when inoculated with a culture of the 
bacillus of this disease, if at the same time it receives an injection of 
a sterilized or non-sterilized culture of Bacillus prodigiosus. Monti 
has succeeded in killing animals with old and attenuated cultures of 
Streptococcus pyogenes, or of Staphylococcus pyogenes aureus, by in- 
jecting at the same time a culture of Proteus vulgaris. In a similar 
way, it seems probable, the normal resistance of man to infection by 
certain pathogenic bacteria may be overcome. Thus when water 
contaminated by the presence of the typhoid bacillus is used for 
drinking by the residents of a certain town or district, not all of 
those who in this way are exposed to infection contract typhoid 
fever; and among those who do, there is good reason to believe that, 
in certain cases at least, the result depends upon an additional factor 
of the kind suggested by the above-mentioned experiments—e.g., the 
consumption of food containing putrefactive products, or the respi- 
ration of an atmosphere containing volatile products of putrefaction. 

The natural immunity of healthy animals may also be neutralized 
by other agencies which have a depressing effect upon the vital re- 
sisting power. Thus Nocard and Roux found by experiment that an 
attenuated culture of the anthrax bacillus, which was not fatal to 
guinea-pigs, killed these animals when injected into the muscles of 


SUSCEPTIBILITY AND IMMUNITY. 241 


the thigh after they had been bruised by mechanical violence. 
Abarrin and Roger found that white rats, which are not susceptible 
to anthrax, became infected and frequently died if they were ex- 
hausted, previous to inoculation, by being compelled to turn a revolv- 
ing wheel for a considerable time. Pasteur found that fowls, which 
have a natural immunity against anthrax, become infected and 
perish if they are subjected to artificial refrigeration after inocula- 
tion. This has been confirmed by the more recent experiments of 
Wagner (1891). According to Canalis and Morpurgo, pigeons 
which are enfeebled by inanition eaily contract anthrax as a result 
of inoculation. Arloing states that sheep which have been freely 
bled contract anthrax more easily than others; and Serafini found 
that when dogs were freely bled the bacillus of Friedlander, injected 
into the trachea or the pleural cavity, entered, and apparently mul- 
tiplied to some extent in the blood, whereas without such previous 
bleeding they were not to be found in the circulating fluid. Certain 
anesthetic agents have been shown also to produce a similar result. 
Platania communicated anthrax to immune animals—dogs, frogs, 
pigeons—by bringing them under the influence of curare, chloral, or 
alcohol; and Wagner obtained similar results in his experiments 
upon pigeons to which he had administered chloral. In man, clini- 
cal experience shows that those who are addicted to the excessive use 
of alcohol are especially liable to contract certain infectious diseases 
—pneumonia, erysipelas, yellow fever, etc. 

The micrococcus of pneumonia is habitually present in the sali- 
vary secretions of many healthy individuals, and it is evident that 
an attack of pneumonia does not depend alone upon the presence of 
this micrococcus, which has, nevertheless, been*conclusively shown 
to be the usual infectious agent in cases of croupous pneumonia. No 
doubt the introduction of the pathogenic micrococcus to the vulner- * 
able point—the lungs—is an essential factor in the development of a 
case of pneumonia, but there is reason to believe that there are other 
factors equally essential. Thus it is well known that an attack of 
pneumonia often results from exposure to cold, which may act as an 
exciting cause; and, also, that a recent attack of an acute febrile 
disease—especially measles—constitutes a predisposing cause. It is 
generally recognized that malnutrition, want of exercise, insanitary 
surroundings, and continued respiration of an atmosphere loaded 
with dust, as in cotton mills, or a recent attack of pneumonia, con- 
stitute predisposing causes to tubercular infection by way of the 
lungs. 

While natural immunity may be overcome by the various depress- 
ing agencies referred to, it is also true that it has only a relative 


value in the absence of these predisposing causes, and may be over- 
16 


242 SUSCEPTIBILITY AND IMMUNITY. 


come by unusual virulence of the pathogenic infectious agent, or by 
the introduction into the body of an excessive amount of a pure cul- 
ture of the same. 

The pathogenic potency of known disease germs varies as widely 
as does the susceptibility of individuals to their specific action. In 
general it may be said that the more recently the germ comes from 
a developed case of the disease to which it gives rise the more viru- 
lent it is, and the longer it has been cultivated outside of the animal 
body the more attenuated is its pathogenic power. Thus when the 
discharges of a typhoid fever patient find their way directly to a 
water-supply of limited amount a large proportion of those who- 
drink the water are likely to be attacked; but when a considerable 
interval of time has elapsed since the contamination occurred, 
although the germs may still be present, the liability to attack is 
much less on account of diminished pathogenic virulence. 

The development of an attack also depends, to some extent, upon 
the number of germs introduced into a susceptible individual at one 
time. The resources of nature may be sufficient to dispose of a few 
bacilli, while a large number may overwhelm the resisting power of 
the individual. 

The experiments of Cheyne (1886) show that in the case of very 
pathogenic species a single bacillus, or at least a very small number, 
introduced beneath the skin, may produce fatal infection in a very 
susceptible animal, while greater numbers are required in those less 
susceptible. Thus a guinea-pig succumbed to general infection after 
being inoculated subcutaneously with anthrax blood diluted to such 
an extent that, by estimation, only one bacillus was present in the 
fluid injected; and wsimilar result was obtained in mice with Bacillus 
murisepticus. In the case of the microbe of fowl cholera (Bacillus 
‘septiceemia hemorrhagice), Cheyne found that for rabbits the fatal 
dose was 300,000 or more, that from 100,000 to 30,000 cause a local 
abscess, and that less than 10,000 produce no appreciable effect. The 
common saprophyte, Proteus vulgaris, was found to be pathogenic 
for rabbits when injected into the dorsal muscles in sufficient num- 
bers. But, according to the estimates made, 225,000,000 were re- 
quired to cause death, while doses of from 9,000,000 to 112,000,000 
produced a local abscess, and Jess than 9,000,000 gave an entirely 
negative result. 


ACQUIRED IMMUNITY. 


It has long been known that, in a considerable number of infec- 
tious diseases, a single attack, however mild, affords protection 
against subsequent attacks of the same disease; that in some cases 
this protéction appears to be permanent, lasting during the life of the 


SUSCEPTIBILITY AND IMMUNITY. 243 


individual; that in others it is more or less temporary, as shown by 
the occurrence of a subsequent attack. 

The protection afforded by a single attack not only differs in dif- 
ferent diseases, but in the same disease varies greatly in different 
individuals. Thus certain individuals have been known to suffer 
several attacks of small-pox'or of scarlet fever, although, as a rule, a 
single attack is protective. Exceptional susceptibility or insuscepti- 
bility may be not only an individual but a family characteristic, or 
it may belong to a particular race. 

In those diseases in which second attacks are not infrequent, as, 
for example, in pneumonia, in influenza, or in Asiatic cholera, it is 
difficult to judge from clinical experience whether a first attack exerts 
any protective influence. But from experiments upon the lower ani- 
mals we are led to believe that a certain degree of immunity, lasting 
for a longer or shorter time, is afforded by an attack of pneumonia 
or of cholera, and probably of all infectious diseases due to bacterial 
parasites. In the malarial fevers, which are due to a parasite of a 
different class, one attack affords no protection, but rather predis- 
poses to a subsequent attack. 

In those diseases in which a single attack is generally recognized 
as being protective, exceptional cases occur in which subsequent 
attacks are developed as a result of unusual susceptibility or expo- 
sure under circumstances especially favorable to infection. Maiselis 
(1894) has gone through the literature accessible to him for the 
purpose of determining the frequency with which second attacks 
occur in the various diseases below mentioned. The result is as 
follows: 

Second Third Fourth 


Attacks. Attacks. Attacks. Total. 
Small-pox............. ... 505 9 0 514 
Scarlet fever wpa 29 4 0 33 
Measles........... a 86 1 0 37 
Typhoid fever............... 202 5 1 208 
Choleray ss .is..054 neuesacen ce 29 3 2 34 


These figures support the view generally entertained by physi- 
cians that second attacks of scarlet fever and of measles are compar- 
atively rare, while second attacks of small-pox are not infrequently 
observed. Considering the very large number of cases of typhoid 
fever which occur annually in all parts of Europe and America, the 
number of second attacks. collected does not bear a very large propor- 
tion to the total number taken sick, although the recorded cases, of 
course, fall far short of the total number of second attacks of this 
and the other diseases mentioned. 

The second attacks of cholera recorded are not numerous, and, no 
doubt, a carefullly conducted investigation made in the areas of en- 


a244 SUSCEPTIBILITY AND IMMUNITY. 


demic prevalence of this disease would show that second attacks are 
more common than is indicated by these figures. 

That immunity may result from a comparatively mild attack as 
well as from a severe one is a matter of common observation in the 
case of small-pox, scarlet fever, yellow fever, etc.; and since the dis- 
covery of Jenner we have in vaccination a simple method of produc- 
ing immunity in the first-mentioned disease. Theacquired immunity 
resulting from vaccination is not, however, as complete or as per- 
manent as that which results from an attack of the disease. 

These general facts relating to acquired immunity from infectious 
diseases constituted the principal portion of our knowledge with re- 
ference to this important matter up to the time that Pasteur (1880) 
demonstrated that in the disease of fowls known as chicken cholera, 
which he had proved to be due to a specific microédrganism, a mild 
attack followed by immunity may be induced by inoculation with an 
“* attenuated virus *’—?.e., by inoculation with a culture of the patho- 
genic microérganism the virulence of which had been so modified 
that it gave rise toa comparatively mild attack of the disease in 
question. Pasteur’s original method of obtaining an attenuated virus 
consisted in exposing his cultures for a considerable time to the ac- 
tion of atmospheric oxygen. It has since been ascertained that the 
same result is obtained with greater certainty by exposing cultures 
for a given time to a temperature slightly below that which would 
destroy the vitality of the pathogenic microérganism, and also by ex- 
posure to the action of certain chemical agents. 

Pasteur at once comprehended the importance of his discovery, 
and inferred that what was true of one infectious germ disease was 
likely to be true of others. Subsequent researches, by this savant 
and by other bacteriologists, have justified this anticipation, and the 
demonstration has already been made for a considerable number of 
similar diseases—anthrax, symptomatic anthrax, rouget. 

A virus which has been attenuated artificially—by heat, for ex- 
ample—may be cultivated through successive generations without re- 
gaining its original virulence. As this virulence depends, to a con- 
siderable extent at least, upon the formation of toxic products during 
the development of the pathogenic microorganism, we naturally infer 
that diminished virulence is due to a diminished production of these 
toxic substances. 

There is reason to believe that a natural attenuation of virulence 
may occur in pathogenic bacteria which are able to lead a sapro- 
phytic existence during their multiplication external to the bodies of 
living animals, and the comparatively mild character of some epi- 
demics is probably due to this fact. 


SUSCEPTIBILITY AND IMMUNITY. 245 


Again, cultivation within the body of a living animal may, in 
certain cases, cause a diminution in the virulence of a pathogenic 
microérganism. Thus Pasteur and Thuiller have shown that the 
microbe of rouget when inoculated into a rabbit kills the animal, but 
that its pathogenic virulence is nevertheless so modified that a cul- 
ture made from the blood of a rabbit killed by it is a suitable “‘ vac- 
cine” for the pig. 

On the other hand, we have experimental evidence that the viru- 
lence of attenuated cultures may be reéstablished by passing them 
through the bodies of susceptible animals. Thus a culture of the 
bacillus of rouget, attenuated by having been passed through the 
body of a rabbit, is restored to its original virulence by passing it 
through the bodies of pigeons. Anda culture of the anthrax bacillus 
which will not kill an adult guinea-pig may be fatal to a very young 
animal of the same species or to a mouse, and the bacillus cultivated 
from the blood of such an animal will be found to have greatly in- 
creased virulence. 

In Pasteur’s inoculations against anthrax “ attenuated ” cultures 
are employed which contain the living pathogenic germ as well as 
the toxic products developed during its growth. Usually two inocu- 
lations are made with cultures of different degrees of attenuation— 
that is to say, with cultures in which the toxic products are formed 
in less amount than in virus of full power. The most attenuated 
virus is first injected, and after some time the second vaccine, which 
if injected first might have caused a considerable mortality. The 
animal is thus protected from the pathogenic action of the most 
virulent cultures. 

Now, it has been shown by recent experiments that a similar im- 
munity may result from the injection into a susceptible animal of the 
toxic products contained in a virulent culture, independently of the 
living bacteria to which they owe their origin. Chauveau, in 1880, 
ascertained that if pregnant ewes are protected against anthrax by 
inoculation with an attenuated virus, their lambs, when born, also 
give evidence of having acquired an immunity from the disease. As 
the investigations of Davaine seemed to show that the anthrax 
bacillus cannot pass through the placenta from the mother to the 
foetus, the inference seemed justified that the acquired immunity of 
the latter was due to some soluble substance which could pass the 
placental barrier. More recent researches by Strauss and Chamber- 
lain, Malvoz and Jacquet, and others, show that the placenta is not 
such an impassable barrier for bacteria as was generally believed at 
the time of Chauveau’s experiments, so that these cannot be accepted 
as establishing the inference referred to. But, as stated, we have 
more recent experimental evidence which shows that immunity may 


246 SUSCEPTIBILITY AND IMMUNITY. 


result from the introduction into the bodies of susceptible animals of 
the toxic substances produced by certain pathogenic bacteria. The 
first satisfactory experimental evidence of this important fact was 
obtained by Salmon and Smith in 1886, who succeeded in making 
pigeons immune from the pathogenic effects of cultures of the bacil- 
lus of hog cholera by inoculating them with sterilized cultures of 
this bacillus. In 1888 Roux reported similar results obtained by in- 
jecting into susceptible animals sterilized cultures of the anthrax 
bacillus. Behring and Kitasato, in 1890, reported their success in 
establishing immunity against virulent cultures ‘of the’ bacillus of 
tetanus and the diphtheria bacillus by inoculating susceptible ani- 
mals with filtered, germ-free cultures of these pathogenic bacteria. 

In 1892 Behring, Kitasato, and Wassermann published the re- 
sults of interesting experiments with a bouillon made from the 
thymus gland of the calf. They found that the tetanus bacillus cul- 
tivated in this bouillon did not form spores and had comparatively 
little virulence. Mice or rabbits inoculated with it in small doses— 
0.001 to 0.2 cubic centimetre for a mouse—proved to be subsequently 
immune. And the blood serum of an immune rabbit injected into 
the peritoneal cavity of a mouse—0.1 to 0.5 cubic centimetre—was 
found to give it immunity from the pathogenic action of a virulent 
culture of the tetanus bacillus. Similar results were obtained with 
several other pathogenic bacteria cultivated in the thymus bouillon— 
spirillum of cholera, bacillus of diphtheria, typhoid bacillus. We 
give here the directions for preparing the thymus bouillon as used by 
the authors named: 


Two or three thymus glands are chopped into small pices immediately 
after they are taken from the animal. An equal part of distilled water is 
added to the mass and stirred for some time ; it isthen placed in an ice chest 
for twelve hours. The juices are now expressed through gauze by means of 
a flesh press. A clouded, slimy fluid is obtained, which constitutes a stock 
solution. This is diluted with water, and a certain quantity of carbonate of 
soda is added to the solution before sterilization. By this means coagulation 
and precipitation of the active substance from the thymus gland are avoided. 
The exact amount of water and of sodium carbonate required to prevent pre- 
cipitation must be determined by experiment, asit differs for different glands. 
Usually an equal portion of water and sufficient soda solution to turn litmus 
paper feebly blue will give the desired result. The liquid is now heated in 
a large flask, which is left for fifteen minutes in the steam sterilizer, The 
liquid is allowed to cool and then filtered through fine linen to remove any 
suspended coagula ; the filtrate has a milky opalescence. It is now placed 
in test tubes and again stérilized. The active principle is precipitated by the 
addition of a few drops of acetic acid. 


In Pasteur’s inoculations against hydrophobia, made subsequently 
to infection by the bite of a rabid animal, an attenuated virus is in- 


SUSCEPTIBILITY AND IMMUNITY. 247 


troduced upon the surface of the brain, and immunity is established 
during the interval—so-called period of incubation—which usually 
occurs between the date of infection and the development of the 
disease. That the immunity in this case also depends upon the 
introduction of a chemical substance present in the desiccated spinal 
eord of rabbits which have succumbed to rabies, which is used in 
these inoculations, is extremely probable. But, as the germ of rabies 
has not been isolated or cultivated artificially, this has not yet been 
demonstrated. Wooldridge claims to have made susceptible animals 
immune against anthrax by inoculating them with an aqueous ex- 
tract of the testicle or of the thymus gland of healthy animals. 

We may mention also the interesting results obtained by Em- 
merich, Freudenreich, and others, who have shown that an anthrax 
infection in a susceptible animal inoculated with a virulent culture 
may be made to take a modified and non-fatal course by the simul- 
taneous or subsequent inoculation of certain other non-pathogenic 
bacteria—streptococcus of erysipelas, Bacillus pyocyaneus. 

In a series of experiments made by the writer some years ago 
evidence was obtained that, under certain circumstances, immunity 
from the effects of one pathogenic bacillus may be obtained by the 
previous injection of a pure culture of a different species. In the 
experiments referred to injections into the cavity of the abdomen of 
a culture of Bacillus pyocyaneus protected rabbits from the lethal 
effects of Bacillus cuniculicida Havaniensis, when subsequently in- 
jected into the cavity of the abdomen in such amount (one cubic 
centimetre of a bouillon culture) as invariably proved fatal in rabbits 
not protected by such injections. 

Before considering the theories which have been offered in expla- 
nation of acquired immunity it is desirable to call attention to certain 
observations which have been made during the past few years relat- 
ing to “‘ chemiotaxis.” 

The term chemotaxis was first used by Pfeffer to designate the 
property, observed by himself and others, which certain living cells 
exhibit with reference to non-living organic material, and by virtue 
of which they approach or recede from certain substances. The 
chemiotaxis is said to- be positive when the living cell approaches, and 
negative when it recedes from, a chemical substance. As examples 
of this we may mention the approach of motile bacteria to nutrient 
material or to the surface of a liquid medium where they find the 
oxygen required for their vital activities ; and of leucocytes to cer- 
tain substances when these are introduced beneath the skin of warm- 
or cold-blooded animals. This subject has recently received much 


248 SUSCEPTIBILITY AND IMMUNITY. 


attention and has been studied especially by Ali-Cohen, Massart and 
Bordet, Gabritchevski, and others. 

According to Gabritchevski, the following substances have a neg- 
ative chemiotaxis for the leucocytes : Sodium chloride in ten-per-cent 
solution, alcohol in ten-per-cent solution, quinine, lactic acid, gly- 
cerin, chloroform, bile. On the other hand, a positive chemiotaxis 
is excited by sterilized or non-sterilized cultures of various bacteria. 
This is shown by the fact that when a small capillary tube, closed at 
one end, which contains the substance to be tested, is introduced be- 
neath the skin of an animal, the leucocytes are repelled from the tube 
by certain substances, while those which incite positive chemiotaxis 
cause them to enter the tube in great numbers. The experiments of 
Buchner seem to show that the positive chemiotaxis induced by 
sterilized cultures of bacteria introduced beneath the skin of an 
animal, is due to the proteid contents of the cells rather than to the 
chemical products elaborated as a result of their vital activity. But 
that such chemical products may, in some instances at least, produce 
a positive chemiotaxis independently of the bacteria is shown by 
the experiments of Gabritchevski with filtered cultures of Bacillus 
pyocyaneus—confirmed by Massart and Bordet. 

An important observation made by Bouchard, and confirmed by 
Massart and Bordet, is the following: When a tube containing a cul- 
ture of Bacillus pyocyaneus is introduced beneath the skin of a rabbit 
it is found, at the end of afew hours, to contain a great number of 
leucocytes. Butif immediately after its introduction ten cubic centi- 
metres of a sterilized culture of the same bacillus are injected into the 
circulation through a vein, very few leucocytes enter the tube intro- 
duced beneath the skin—that is, the chemiotaxis of the leucocytes 
for the bacilli contained in the tube has been neutralized by injecting 
a considerable quantity of the soluble products of the same bacillus 
into the circulation. 

Buchner, having shown that the bacterial cells contain a proteid 
substance which attracts the leucocytes, experimented with various 
other proteids and found that gluten, casein from wheat, and legumin 
from peas had a similar effect. Starch has no effect, but a mass of 
flour, made from wheat or from peas, introduced beneath the skin of 
a rabbit or of a guinea-pig, with antiseptic precautions, in the course 
of a day or two is enveloped and penetrated by immense numbers of 
leucocytes. If, instead of introducing these substances which induce 
positive chemiotaxis beneath the skin, they are injected into the cir- 
culation, Buchner has shown that a great increase in the number of 
leucocytes occurs. 


SUSCEPTIBILITY AND IMMUNITY. 249 


. THEORIES OF IMMUNITY. 


Exhaustion Theory.—F¥or atime Pasteur supported the view 
that during an attack of an infectious disease the pathogenic micro- 
érganism, in its multiplication in the body of a susceptible animal, 
exhausts the supply of some substance necessary for its development, 
that this substance is not subsequently reproduced, and that conse- 
quently the same pathogenic germ cannot again multiply in the body 
of the protected animal. This view is sustained in a memoir pub- 
lished in the Comptes Rendus of the French Academy in 1880, in 
which Pasteur says : 


‘Tt is the life of a parasite in the interior of the body which produces the 
malady commonly called ‘choléra des poules,’ and which causes death. 
From the moment when this culture (7.¢., the multiplication of the parasite) 
is no longer possible in the fowl the sickness cannot appear. The fowls are 
then in the constitutional state of fowls not subject to be attacked by the 
disease. These last are as if vaccinated from birth for this malady, because 
the foetal evolution has not introduced into their bodies the material neces- 
sary to support the life of the microbe, or these nutritive materials have 
disappeared at an eatly age. 

‘Certainly one should not be surprised that there may be constitutions 
sometimes susceptible and sometimes rebellious to inoculation—that is to 
say, to the cultivation of a certain virus—when, as I have announced in my 
first note, one sees a preparation of beer yeast made, exactly like one from 
the muscles of fowls (bouillon), to show itself absolutely unsuited for the cul- 
tivation of the parasite of fowl cholera, while it is admirably adapted to the 
cultivation of a multitude of microscopic species, notably to the bactéride 
charbonneuse (Bacillus anthracis). 

“The explanation to which these facts conduct us, as well of the consti- 
tutional resistance of some individuals as of the immunity produced by 
protective inoculations, is only natural when we consider that every culture, 
in general, modifies the medium in which it is effected—a modification of 
the soil when it relates to ordinary plants; a modification of plants and ani- 
mals when it relates to their parasites ; a modification of our culture liquids 
when it relates to mucédines, vibrioniens, or ferments. 

“‘These modifications are manifested and characterized by the circum- 
stance that new cultivations of the same species in these media become 
promptly difficult or impossible. If we sow chicken bouillon with the mi- 
crobe of fowl cholera, and, after three or four days, filter the liquid in order 
to remove all trace of the microbe, and subsequently sow anew in the fil- 
tered liquid this parasite, it will be found quite powerless to resume the most 
feeble development. The liquid, which is perfectly limpid after being fil- 
tered, retains its limpidity indefinitely. 

“How can we fail to believe that by cultivation in the fowl of the atten- 
uated virus we place its body in the state of this filtered liquid which can 
no longer cultivate the microbe? The comparison can be pushed still 
further; for if we filter the boudllon containing the microbe in full develop- 
ment, not on the fourth day of culture, but on the second, the filtered liquid 
will still be able to support the development of the microbe, although with 
less energy than at the outset. We comprehend, then, that after a cultiva- 
tion of the modified (attenwé) microbe in the body of the fowl we may not 
have removed from all parts of its body the aliment of the microbe. That 
which remains will permit, then, a new culture, but in a more restricted 


measure. ; ; 
‘“‘This is the effect of a first inoculation ; subsequent inoculations will 


250 SUSCEPTIBILITY AND IMMUNITY. 


remove progressively all the material necessary for the development of ‘the 
parasite.” 


In discussing this theory, in a paper published in the American 
Journal of the Medical Sciences (April, 1881), the writer says: 


‘Let us see where this hypothesis leads us. In the first place, we must 
have a material of small-pox, and a material of measles, and a material of 
scarlet fever, etc., etc. Then we must admit that each of these different 
materials has been formed in the system and stored up for these emergencies 
—attacks of the diseases in question—for we can scarcely conceive that they 
were all packed away in the germ cell of the mother and the sperm cell of 
the father of each susceptible individual. If, then, these peculiar materials 
have been formed and stored up during the development of the individual, 
how are we to account for the fact that no new production takes place after 


an attack of any one of the diseases in question ? 

‘‘ Again, how shall we account for the fact that the amount of material 
which would nourish the small-pox germ, to the extent of producing a case 
of confluent small-pox, may be exhausted by the action of the attenuated 
virus«(germ) introduced by vaccination? Pasteur’s comparison of a fowl 
protected by inoculation with the microbe of fowl cholera, with a culture 
fluid in which the growth of a particular organism has exhausted the pabu- 
lum necessary for the development of additional organisms of the same kind, 
does not seem to me to be a just one, as in the latter case we have a limited 
supply: of nutriment, while in the former we have new supplies constantly 
provided of the material—food—from which the whole body, including the 
hypothetical substance essential to the development of the disease germ, was 
built up prior to the attack. Besides this we have a constant provision for 
the elimination of effete and useless products. 

‘This hypothesis, then, requires the formation in the human body, and 
the retention up to a certain time, of a variety of materials which, so far as 
we can see, serve no purpose except to nau the germs of various specific 
diseases, and which, having served this purpose, are not again formed in the 
same system, subjected to similar external conditions, and supplied with the 
same kind of nutriment.” 


It is unnecessary to discuss this hypothesis any further, inasmuch 
as it is no longer sustained by Pasteur or his pupils, and is evidently 
untenable. 

The Retention Theory, proposed by Chauveau (1880), is subject to 
similar objections. According to this view, certain products formed 
during the development of a pathogenic microérganism in the body 
of a susceptible animal accumulate during the attack and are subse- 
quently retained, and, being prejudicial to the growth of the particu- 
lar microédrganism which produced them, a second infection cannot 
occur. Support for this theory has been found by its advocates in 
the fact that various processes of fermentation are arrested after a 
time by the formation of substances which restrain the development 
of the microérganisms to which they are due. But in the case of a 
living animal the conditions are very different, and it is hard to con- 
ceive that adventitious products of this kind could be retained for 
years, when in the normal processes of nutrition and excretion the 
tissues and fluids of the body are constantly undergoing change. 
Certainly the substances which arrest ordinary processes of fermen- 


SUSCEPTIBILITY AND IMMUNITY. 251 


tation by their accumulation in the fermenting liquid, such as alco- 
‘ hol, lactic acid, phenol, etc., would not be so retained. But we can- 
not speak so positively with reference to the toxic albuminous 
substances which recent researches have demonstrated to be present 
in cultures of some of the best known pathogenic bacteria. It is 
difficult, however, to believe that an individual who has passed 
through attacks of half a dozen different infectious diseases carries 
about with him a store of as many different chemical substances pro- 
duced during these attacks, and sufficient in quantity to prevent the 
development of the several germs of these diseases. Nor does the 
experimental evidence relating to the action of germicide and germ- 
restraining agents justify the view that a substance capable of 
preventing the development of one microérganism should be with- 
out effect upon others of the same class; but if we accept the re- 
tention hypothesis we must admit that the inhibiting substance 
produced by each particular pathogenic germ is effective only in 
restraining the development of the microbe which produced it in the 
first instance. 
Pasteur discusses this hypothesis in his paper from which we 
have already quoted, as follows : 


‘“We may admit the possibility that the development of the microbe, in 
place of removing or destroying certain mattersin the bodies of the fowls, 
adds, on the contrary, something which is an obstacle to the future develop- 
ment of this microbe. The history of the life of inferior beings authorizes 
such a supposition. The excretions resulting from vital processes may arrest 
vital processes of the same nature. In certain fermentations we see anti- 
septic products make their appearance during, and as a result of, the fer- 
mentation, which put an end to the active life of the ferments and arrest 
the fermentations long before they are completed. In the cultivation of our 
microbe, products may have been formed the presence of which, possibly, 
may explain the protection following inoculation. 

“Our artificial cultures permit us to test the truth of this hypothesis. 
Let us prepare an artificial culture of the microbe, and after having evapo- 
rated it, in vacuo, without heat, let us bring it back to its original volume 
by means of fresh chicken bouillon. If the extract contains a poison for 
the life of the microbe, and if this is the cause of its failure to multiply in the 
filtered liquid, the new liquid should remain sterile. Now, thisis not the case. 
We cannot, then, believe that during the life of the parasite certain substances 
are.produced which are capable of arresting its ulterior development.” 


This experiment of Pasteur appears to be conclusive so far as the 
particular pathogenic microérganism referred to is concerned ; and 
we may say, in brief, that more recent inves‘igations do not sustain 
the view that acquired immunity is due to the retention of products 
such as are formed by pathogenic bacteria in artificial culture media, 
and which act by destroying these bacteria or restraining their devel- 
opment when they are introduced into the bodies of immune animals. 

Moreover, if we suppose that the toxic substances which give 
pathogenic power to a particular microérganism are retained in the 


252 SUSCEPTIBILITY AND IMMUNITY. 


body of an immune animal, we must admit that the animal has ac- 
quired a tolerance to the pathogenic action of these toxic substances, © 
for their presence no longer gives rise to any morbid phenomena. 
And this being the case, we are not restricted to the explanation 
that immunity depends upon a restraining influence exercised upon 
the microbe when subsequently introduced. 

The Vital Resistance Theory.—Another explanation offers itself, 
viz., that immunity depends upon an acquired tolerance to the 
toxic products of pathogenic bacteria. This is a view which the 
writer has advocated in various published papers since 1881. Ina 
paper contributed to the -Lmerican Journal of the Medical Sci- 
ences in April, 1881, it is presented in the following language: 


‘‘The view that I am endeavoring to elucidate is that, during a non- 
fatal attack of one of the specific diseases, the cellular elements implicated 
which do not succumb to the destructive influence of the poison acquire a 
tolerance to this poison which is transmissible to their progeny, and which 
is the reason of the exemption which the individual enjoys from future 
attacks of the same disease.” ' 


In my chapter on “‘ Bacteria in Infectious Diseases,” in ‘‘ Bac- 
teria,” published in the spring of 1884, but placed in the hands of the 
publishers in 1883, I say: 


“Tt may be that the true explanation of the immunity afforded by a mild 
attack of an infectious germ disease is to be found in an acquired tolerance to 
the action of a chemical poison produced by the microdrganism, and conse- 
quent ability to bring the resources of nature to bear to restrict invasion by 
the parasite.” 


The ‘‘ resources of nature” are referred to in the same chapter as 
follows : 


‘‘The hypothesis of Pasteur would account for the fact that one individual 
suffers a severe attack and another a mild attack of an infectious disease, 
after being subjected to the influence of the poison under identical cireum- 
stances, by the supposition that the pabulum required for the development: 
of this particular poison is more abundant in the body of one individual 
than in the other. The explanation which stems to us more satisfactory is 
that the vital resistance offered by the cellular elements in the bodies of 
these two individuals was not the same for this poison. It is well known 
that in conditions of lowered vitality resulting from starvation, profuse 
discharges, or any other cause, the power to resist disease poisons is greatly 
diminished, and, consequently, that the susceptibility of the same individual 
differs at different times. 

‘From our point of view, the blood, as it is found within the vessels of a 
living animal, is not simply a culture fluid maintained at a fixed tempera- 
ture, but under these circumstances is a tissue, the histological elements of 
which present a certain vital resistance to pathogenic organisms which may 
be introduced into the circulation. 

“Tf we add a small quantity of a culture fluid containing the bacteria of 
putrefaction to the blood of an animal, withdrawn from the circulation into 
a proper receptacle and maintained in a culture oven at blood heat, we will 
find that these bacteria multiply abundantly, and evidence of putrefactive 


1«« What is the Explanation of the Protection from Subsequent Attacks, result- 
ing from an Attack of Certain Diseases, ete?” American Journal of the Medical 
Sciences, April, 1881, p. 376. 


SUSCEPTIBILITY AND IMMUNITY, 253 


decomposition will soon be perceived. But if we inject a like quantity of 
the culture fluid with its contained bacteria into the circulation of a living 
animal, not only does no increase and no putrefactive change occur, but the 
bacteria introduced quickly disappear, and at the end of an hour or two the 
most careful microscopical examination will not reveal the presence of a 
single bacterium. This difference we ascribe to the vital properties of the 
fluid as contained in the vessels of a living animal; and it seems probable 
that the little masses of protoplasm known as white blood corpuscles are the 
essential histological elements of the blood, so far as any manifestation of 
vitality is concerned. The writer has elsewhere (1881) suggested that the 
disappearance of the bacteria from the circulation, in the experiment 
referred to, may be effected by the white corpuscles, which, it is well known, 
pick up, after the manner of amcebe, any particles, organic or inorganic, 
which come in their way. And it requires no great stretch of credulity to 
believe that they may, like an amoeba, digest and assimilate the protoplasm 
of the captured bacterium, thus putting an end to the possibility of its do- 
ing any harm. 

‘In the case of a pathogenic organism we may imagine that, when cap- 
tured in this way, it may share a like fate if the captor is not paralyzed by 
some potent poison evolved by it, or overwhelmed by its superior vigor and 
rapid multiplication. In the latter event the active career of our conserva- 
tive white corpuscle would be quickly terminated and its protoplasm would 
serve as food for the enemy. It is evident that in a contest of this kind the 
balance of power would depend upon circumstances relating to the inherited 
vital characteristics of the invading parasite and of the invaded leucocyte.” 


In the same chapter the writer quotes from his paper on acquired 
immunity, published in 1881, as follows : 


‘‘ The difficulties into which this hypothésis [the exhaustion theory of Pas- 
teur] leads us certainly justify us in looking further for an explanation of the 
phenomena in question. This explanation is, I believe, to be found in the 
peculiar properties of the protoplasm, which is the essential framework of 
every living organism. The properties referred to are the tolerance which 
living protoplasm may acquire to certain agents which, in the first instance, 
have an injurious or even fatal influence upon its vital activity ; and the 
property which it possesses of transmitting its peculiar qualities, inherent or 
acquired, through numerous generations, to its offshoots or progeny. 

‘*Protoplasm is the essential living portion of the cellular elements of ani- 
mal and vegetable tissues; but as our microscopical analysis of the tissues has 
not gone beyond the cells of which they are composed, and is not likely to 
reveal to us the complicated molecular structure of the protoplasm, upon 
which, possibly, the properties under consideration depend, it will be best, 
for the present, to limit ourselves to a consideration of the living cells of the 
body. ‘These cells are the direct descendants of the pre-existent cells, and 
may all be traced back to the sperm cell and the germ cell of the parents. 
Now, the view which I am endeavoring to elucidate is that, during a non- 
fatal attack of one of the specific diseases, the cellular elements implicated, 
which do not succumb to the destructive influence of the poison, acquire a 
tolerance to this poison which is transmissible to their progeny, and which 
is the reason of the exemption which the individual enjoys from future 
attacks of the same disease. 

‘“The known facts in regard to the hereditary transmission by cells of ac- 
quired properties make it easy to believe in the transmission of such a 
tolerance as we imagine to be acquired during the attack; and if it is shown 
by analogy that there is nothing improbable in the hypothesis that such a 
tolerance is acquired, we shall have a rational explanation, not of heredity 
and of the mysterious properties of protoplasm, but of the particular result 
under consideration. The transmission of acquired properties is shown in 
the budding and grafting of choice fruits and flowers, produced by cultiva- 


254 SUSCEPTIBILITY AND IMMUNITY. 


tion, upon the wild stock from which they originated. The acquired proper- 
tics are transmitted indefinitely; and the same sap which on one twig nour- 
ishes a sour crab apple, on another one of the same branch is elaborated into 
a delicious pippin. 

‘The tolerance to narcoties—opium and tobacco—and to corrosive poisons 

—arsenic—which results from a gradual increase of dose, may be cited as an 
example of acquired tolerance by living protoplasm to poisons which at the 
outset would have been fatal in much smaller doses. 
_ ‘The immunity which an individual enjoys from any particular disease 
must be looked upon as a power of resistance possessed by the cellular ele- 
ments of those tissues of his body which would yield to the poison in the 
case of an unprotected person.” . 


This theory of immunity, advanced by the author in 1881, has 
received considerable support from investigations made since that 
date, and especially from the experimental demonstration by Sal-. 
mon, Roux, and others that, as suggested in the paper from which I 
have quoted, immunity may result from the introduction into the 
body of a susceptible animal of the soluble products of bacterial 
growth—filtered cultures. 

The theory of vital resistance to the toxic products evolved by 
pathogenic bacteria is also supported by numerous experiments 
which show that natural or acquired immunity may be overcome 
when these toxic products are introduced in excess, or when the vital 
resisting power of the animal has been reduced by various’ agencies. 

More direct experimental evidence in favor of the view under con- 
sideration is that obtained by Beumer in his experiments with steril- 
ized cultures of the typhoid bacillus. He found that after the re- 
peated injection of non-lethal doses mice were able to resist an 
amount of this toxine which was fatal to animals of the same spe- 
cies not so treated. But, on the other hand, Gamaléia found, in his 
experiments upon guinea-pigs which had been made immune against 
the pathogenic action of a spirillum, called by him Vibrio Metschni- 
kovi, that these animals have no increased tolerance for the toxic 
products of this microédrganism. Although immune against infec- 
tion by the living microbe, they were killed by the same quantity of 
a sterilized culture as was fatal to guinea-pigs which had not been 
rendered immune. 

Charrin has obtained similar results in experiments with filtered 
cultures of Bacillus pyocyaneus. Rabbits which had an artificial im- 
munity against the pathogenic action of thé bacillus were killed by 
doses of a sterilized culture such as were fatal to other rabbits of the 
same size not immune. In subsequent experiments by Charrin and 
Gameléia “vaccinated” rabbits were found to be even more suscepti- 
ble to the toxic action of filtered cultures than were those not vacci- 
nated. Metschnikoff (1891) has followed up this line of experiment, 
and has shown that when considerable amounts of filtered cultures 
of Bacillus pyocyaneus are injected subcutaneously in rabbits a cer- 


SUSCEPTIBILITY AND IMMUNITY. 255 


tain tolerance to the toxic action of the same cultures is established 
in some instances. But his results do not give any substantial sup- 
port to the view that immunity depends upon an acquired tolerance 
to the toxic action of the chemical products contained in cultures of 
the pathogenic bacteria with which he experimented—Bacillus pyo- 
cyaneus and Vibrio Meiscbnikovi. 

In view of the results of experimental researches above recorded, 
and of other recent experiments which show that, in certain cases at 
least, acquired immunity depends upon the formation of an anti- 
toxine in the body of the immune animal, we are convinced that the 
theory of immunity under discussion, first proposed by the writer in 
1881, cannot be accepted as a sufficient explanation of the facts in 
general. At the same time we are inclined to attribute considerable 
importance to acquired. tolerance to the toxic products of pathogenic 
bacteria as one of the factors by which recovery from an infectious 
disease is made possible and subsequent immunity established. 

The “ vital-resistance theory ” of the present writer, as set forth 
in the above-quoted extracts from his published papers, is essentially 
the same as that advocated by Buchner at a later date (1883). Buch- 
ner supposes that during the primary infection, when an animal re- 
covers, a “‘ reactive change” has been produced in the cells of the 
body which enables it to protect itself from the pathogenic action 
of the same microérganism when subsequently introduced. 

Of course when we ascribe immunity to the “‘ vital resistance” of 
the cellular elements of the body, we have not explained the 
modus operandi of this vital resistance or “‘ reactive change,” but 
have simply affirmed that the phenomenon in question depends upon 
some acquired property residing in the living cellular elements of 
the body. We have suggested that that which has been acquired 
is a tolerance to the action of the toxic products produced by patho- 
genic bacteria. But, as already stated, in the light of recent experi- 
ments this theory now appears to us to be untenable as a general 
explanation of acquired immunity. 

The Theory of Phagocytosts.—The fact that in certain infectious 
diseases due to bacteria the parasitic invaders, at the point of inocu- 
lation or in the general blood current, are picked up by the leuco- 
cytes and in properly stained preparations may be seen in their in- 
terior, has been known for some years. In mouse septiceemia—an 
infectious disease described by Koch in his work on ‘‘ Traumatic 
Infectious Diseases,” published in 1878—the slender bacilli which are 
the cause of the disease are found in large numbers in the interior of 
the leucocytes. Koch says, in the work referred to: “Their rela- 
tion to the white blood corpuscles is peculiar ; they penetrate these 
and multiply in their interior. One often finds that there is 


256 SUSCEPTIBILITY AND IMMUNITY. 


hardly a single white corpuscle in the interior of which bacilli can- 
not be seen. Many corpuscles contain isolated bacilli only; others 
have thick masses in their interior, the nucleus being still recog- 
nizable ; while in others the nucleus can be no longer distinguished ; 
and, finally, the corpuscle may become a cluster of bacilli, breaking 
up at the margin—the origin of which one could not have explained 
had there been no opportunity of seeing all the intermediate steps 
between the intact white corpuscle and these masses” (Fig. 78). It 
will be noted that in the above quotation Koch affirms that the 
bacilli penetrate the leucocytes and multiply in their interior. Now, 
the theory of phagocytosis assumes that the bacilli are picked up by 
the leucocytes and destroyed in their interior, and that immunity de- 
pends largely upon the power of these “‘ phagocytes” to capture and 
destroy living pathogenic bacilli. 

The writer suggested this as an hypothesis as long ago as 1881, 
in a paper read before the American Association for the Advance- 
ment of Science, in the following language: 

“It has occurred to me that possibly the white corpuscles may 
have the office of picking up and digesting bacterial organisms which 


Fia. 78.—Bacillus of mouse septicemia in leucocytes from blood of mouse (Koch), 


by any means find their way into the blood. The propensity exhib- 
ited by the leucocytes for picking up inorganic granules is well 
known, and that they may be able not only to pick up but to assimi- 
late, and so dispose of, the bacteria which come in their way, does 
not seern to me very improbable, in view of the fact that amoeba, 
which resemble them so closely, feed upon bacteria and similar or- 
ganisms.” ? 

At a later date (1884) Metschnikoff offered experimental evi- 
dence in favor of this view, and the explanation suggested in the 
above quotation is commonly spoken of as the Metschnikoff theory. 


1“ A Contribution to the Study of Bacterial Organisms commonly found upon 
Exposed Mucous Surfaces and in the Alimentary Canal of Healthy Individuals.” II- 
lustrated by photomicrographs. Proceedings of the American Association for Ad- 
vancement of Science, 1881, Salem, 1882, xxx., 838-94. Also in Studies from the 
Biological Laboratory, Johns Hopkins University, Baltimore, vol. ii., No. 2, 1882. 


SUSCEPTIBILITY AND IMMUNITY. 257 


The observations which first led Metschnikoff to adopt this view 
were made upon a species of daphnia which is subject to fatal infec- 
tion by a torula resembling the yeast fungus. Entering with the 
food, this fungus penetrates the walls of the intestine and invades the 
tissues. In certain cases the infection does not prove fatal, owing, as 
Metschnikoff asserts, to the fact that the fungus cells are seized upon 
by the leucocytes, which appear to accumulate around the invading 
parasite (chemiotaxis) for this special purpose. If they are success- 
ful in overpowering and destroying the parasite the animal recovers ; 
if not, it succumbs to the general infection which results. In a simi- 
lar manner, Metschnikoff supposes, pathogenic bacteria are destroyed 
when introduced into the body of an immune animal. The colorless 
blood corpuscles, which he designates phagocytes, accumulate at the 
point of invasion and pick up the living bacteria, as they are known 
to pick up inorganic particles injected into the circulation. So far 
there can be no doubt that Metschnikoff is right. The presence of 
bacteria in the leucocytes in considerable numbers, both at the point 
of inoculation and in the general circulation, has been repeatedly 
demonstrated in animals inoculated with various pathogenic bacteria. 
The writer observed this in his experiments, made in 1881, in which 
rabbits were inoculated with cultures of his Micrococcus Pasteuri ; 
and it was this observation which led him to suggest the theory 
which has since been so vigorously supported by Metschnikoff. But 
the presence of a certain number of bacteria within the leucocytes 
does not prove the destructive power of these cells for living patho- 
genic organisms. As urged by Weigert, Baumgarten, and others, 
it may be that the bacteria were already dead when they were picked 
up, having been destroyed by some agency outside of the blood cells, 
As heretofore stated, we have now experimental evidence that blood 
serum, quite independently of the cellular elements contained in it 
in the circulation, has decided germicidal power for certain patho- 

. genic bacteria, and that the blood serum of the rat and other animals 
which have a natural immunity against anthrax is especially fatal 
to the anthrax bacillus. 

Numerous experiments have been made with a view to deter- 
mining whether pathogenic bacteria are, in fact, destroyed within 
the leucocytes after being picked up, and different experimenters 
have arrived at different conclusions. In the case of mouse septi- 
czmia, already alluded to, and in gonorrhcea, one would be disposed 
to decide, from the appearance and arrangement of the pathogenic 
bacteria in the leucocytes, that they are not destroyed, but that, 
on the other hand, they multiply in the interior of these cells, which 
in the end succumb to this parasitic invasion. In both of the dis- 


eases mentioned we find leucocytes so completely filled with the 
17 


258 SUSCEPTIBILITY AND IMMUNITY. 


pathogenic microérganisms that it is difficult to believe that they 
have all been picked up by a voracious phagocyte, which has 
stuffed itself to repletion, while numerous other leucocytes from 
the same source and in the same microscopic field of view have 
failed to capture a single bacillus or micrococcus. Moreover, the 
staining of the parasitic invaders, and the characteristic arrange- 
ment of the “gonococcus” in stained preparations of gonorrhceal 
pus, indicate that their vitality has not been destroyed in the interior 
of the leucocytes or pus cells, and we can scarcely doubt that the 
large number found in certain cells is due to multiplication in situ 
rather than to an unusual activity of these particular cells. But in 
certain infectious diseases, and especially in anthrax, the bacilli in- 
cluded within the leucocytes often give evidence of degenerative 
changes, which would support the view that they are destroyed by 
the leucocytes, unless these changes occurred before they were picked 
up, as is maintained by Nuttall and others. We cannot consider 
this question as definitely settled. 

Going back to the demonstrated fact that susceptible animals may 
be made immune by inoculating them with the toxic products pro- 
duced during the growth of certain pathogenic bacteria, we may 
suppose either that immunity results from the continued presence of 
these toxic products in the body of the inoculated animal, or from a 
tolerance acquired at the time of the inoculation and subsequently 
retained—by transmission from cell to cell, as heretofore suggested. 
Under the first hypothesis—retention theory—immunity may be ex- 
plained as due to a continued tolerance on the part of the cellular ele- 
ments of the body to the toxic substances introduced and retained ; 
or to the effect of these retained toxic products in destroying the 
pathogenic bacteria, or in neutralizing their products when these are 
subsequently introduced into the body of the immune animal. We 
eannot understand how toxic substances introduced in the first in- 
stance can neutralize substances of the same kind introduced at a 
later date. There is something in the blood of the rat which, accord- 
ing to Behring, neutralizes the toxic substances present in a filtered 
culture of the tetanus bacillus ; but whatever this substance may be, 
it is evidently different from the toxic substance which it destroys, 
and there is nothing in chemistry to justify the supposition last 
made. Is it, then, by destroying the pathogenic microérganism 
that these inoculated and retained toxic products preserve the animal 
from future infection ? Opposed to this supposition is the fact that 
the blood of an animal made immune in this way, when removed 
from the body, does not prove to haveincreased germicidal power as 
compared with that of a susceptible animal of the same species. 
Again, these same toxic substances in cultures of the anthrax bacillus, 


SUSCEPTIBILITY AND IMMUNITY. 259 


the tetanus bacillus, the diphtheria bacillus, etc., do not destroy the 
pathogenic germ after weeks or months of exposure. And when we 
inoculate a susceptible animal with a virulent culture of one of these 
microérganisms, the toxic substances present do not prevent the rapid 
development of the bacillus ; indeed, instead of proving a germicide, 
they favor its development, which is more abundant and rapid than 
when attenuated cultures containing less of the toxic material are 
used for the inoculation. In view of these facts we are unable to 
adopt the view that acquired immunity results from the direct action 
of the products of bacterial growth, introduced and retained in the 
body of the immune animal, upon the pathogenic microédrganism 
when subsequently introduced or upon its toxic products. 

But there is another explanation which, although it may appear 
a priort to be quite improbable, has the support of recent experimen- 
tal evidence. This is the supposition that some substance is formed 
in the body of the immune animal which neutralizes the toxic 
products of the pathogenic microdrgantsm. How the presence of 
these toxic products in the first. instance brings about the formation 
of an “antitoxin” by which they are neutralized is still a mystery; 
but that such a substance is formed appears to be proved by the ex- 
periments of Ogata, Behring and Kitasato, Tizzoni and Cattani, G. 
and F. Klemperer, and others. 

Ogata and Jasuhara, in a series of experiments made in the Hy- 
gienic Institute at Tokio (1890), discovered the important fact that 
the blood of an animal immune against anthrax contains some sub- 
stance which neutralizes the toxic products of the anthrax bacillus. 
When cultures were made in the blood of dogs, frogs, or of white 
rats, which animals have a natural immunity against anthrax, they 
were found not to kill mice inoculated with them. Further experi- 
ments showed that mice inoculated with virulent anthrax cultures 
did not succumb to anthrax septicemia if they received at the same 
time a subcutaneous injection of a small quantity of the blood of an 
immune animal. So small a dose as one drop of frog’s blood or one- 
half drop of dog’s blood proved to be sufficient to protect a mouse 
from the fatal effect of an anthrax inoculation. And the protective 
inoculation was effective when made as long as seventy-two hours 
before or five hours after infection with an anthrax culture. Fur- 
ther, it was found that mice which had survived anthrax infection as 
a result of this treatment were immune at a later date (after several 
weeks) when inoculated with a virulent culture of the anthrax 
bacillus. 

Behring and Kitasato have obtained similar results in their ex- 
periments upon tetanus and diphtheria, and have shown that the 
blood of an immune animal, added to virulent cultures before in- 


260 SUSCEPTIBILITY AND IMMUNITY. 


oculation into susceptible animals, neutralizes the pathogenic power 
of these cultures. 

They have shown by experiment that the blood of a rabbit which 
has an acquired immunity against tetanus, mixed with the virulent 
filtrate from a culture of the tetanus bacillus, neutralizes its toxic 
power. One cubic centimetre of this filtrate was mixed with five 
cubic centimetres of serum from the blood of an immune rabbit and 
allowed to stand for twenty-four hours ; 0.2 cubic centimetre of this 
injected into a mouse was without effect, while 0.0001 cubic centi- 
metre of the filtrate without such admixture was infallibly fatal to 
mice. The mice inoculated with this mixture remained immune for 
forty to fifty days, after which they gradually lost their immunity. 
The blood or serum from an immune rabbit, when preserved in a 
dark, cool place, retained its power of neutralizing the tetanus tox- 
albumin for about a week, after which time it gradually lost this 
power. The blood of chickens, which have a natural immunity 
against tetanus, was found not to havea similar power. Behring 
and Kitasato have also shown that the serum of a diphtheria-immune 
rabbit destroys the potent toxalbumin in diphtheria cultures. It 
does not, however, possess any germicidal power against the diph- 
theria bacillus. 

Ogata, in 1891, reported that he had succeeded in isolating from the 
blood of dogs and of chickensa substance to which he ascribes the nat- 
ural immunity of these animals from certain infectious diseases, and 
the power of their blood to protect susceptible animals from the same 
diseases. This substance is soluble in water and in glycerin, but in- 
soluble in alcohol or ether, by which it is precipitated without being 
destroyed. Its activity is neutralized by acids, but not by weak 
alkaline solutions. Ogata supposes the substance isolated by him to 
be the active agent in blood serum by which certain pathogenic bac- 
teria are destroyed, as shown by the experiments of Nuttall, Buchner, 
and others. Hankin had previously isolated an albuminoid sub- 
stance from the spleen and blood of the rat, to which he ascribed the 
immunity of this animal from anthrax. This substance, according 
to the author named, is a globulin; it is insoluble in alcohol and in 
distilled water, and does not dialyze. 

Tizzoni and Cattani ascribe the protection of animals which have 
acquired an immunity against tetanus to the présence of an albumi- 
nous substance which they call the tetanus-antitoxin. This they 
have isolated from the blood of immune animals. They arrive at 
the conclusion that it is a globulin, or a substance which is carried 
down with the globulin precipitate, and that it is different from the 
globulin, above referred to, obtained by Hankin from animals im- 
mune against anthrax. 


SUSCEPTIBILITY AND IMMUNITY. 261 


G. and F. Klemperer, in 1891, published an important memoir in 
which they gave an account of their researches relating to the ques- 
tion of immunity, etc., in animals subject to the form of septicaemia 
produced by the Micrococcus pneumonie croupose. They were able 
to produce immunity in susceptible animals by introducing into their 
bodies filtered cultures of this micrococcus, and proved by experiment 
that this immunity had a duration of at least six months. They 
arrived at the conclusion that the immunity induced by injecting fil- 
tered cultures is not directly due to the toxic substances present in 
these cultures, but that they cause the production in the tissues of an 
antitoxin which has the power of neutralizing their pathogenic 
action. The toxic substance present in cultures of the “ diplococcus 
of pneumonia” they call “pneumotoxin”; the substance produced in 
the body of an artificially immune animal, by which this pneumo- 
toxin is destroyed if subsequently introduced, they call “ anti-pneumo- 
toxin.” 

Emmerich, in a communication made at the meeting of the In- 
ternational Congress for Hygiene and Demography, in London, re- 
ported results which correspond with those of G. and F. Klemperer 
so far as the production of immunity is concerned, and also gave an 
account of experiments made by Donissen in which the injection of 
twenty to twenty-five cubic centimetres of blood or expressed tissue 
juices, filtered through porcelain, from an immune rabbit into an 
unprotected rabbit, subsequently to infection with a bouillon culture 
of “diplococcus pneumoniz,” prevented the development of fatal 
septicemia. Even when the injection was made twelve to fifteen 
hours after infection, by inhalation, the animal recovered. 

Emmerich and Mastraum had previously reported similar results 
in experiments made upon mice with the Bacillus erysipelatos suis 
(rothlauf bacillus). White mice are very susceptible to the patho- 
genic action of this bacillus. But mice which, subsequently to in- 
fection, were injected with the expressed and filtered tissue juices of 
an immune rabbit, recovered, while the control animals succumbed. 
According to Emmerich, the result in these experiments was due to 
a destruction of the pathogenic bacilli in the bodies of the infected 
animals ; and the statement is made that at the end of eight hours 
after the injection of the expressed tissue juices all bacilli in the body 
of the infected animal were dead. The same liquid did not, however, 
kill the bacilli when added to cultures external to the body of an 
animal. The inference, therefore, seems justified that the result de- 
pends, not upon a substance present in the expressed juices of an 
immune animal, but upon a substance formed in the body of the 
animal into which these juices are injected. 

We have, however, an example of induced immunity in which 


262 SUSCEPTIBILITY AND IMMUNITY. 


the result appears to depend directly upon the destruction of the 
pathogenic microérganism in the body of the immune animal. In 
guinea-pigs which have an acquired immunity against Vibrio Metsch- 
nikovi the blood serum has been proved to possess decided germicidal 
power for this ‘‘ vibrio,” whereas it multiplies readily in the blood 
serum of non-immune guinea-pigs (Behring and Nissen). 

There is experimental evidence that animals may acquire an arti- 
ficial immunity against the toxic action of certain toxalbumins from 
other sources than bacterial cultures. Thus Sewell (1887) has shown 
that a certain degree of tolerance to the action of rattlesnake venom 
may be established by inoculating susceptible animals with small 
doses of the “‘hemialbumose” to which it owes its toxic potency. 
These results have been confirmed by the more recent experiments of 
Calmette (1894) and of Fraser (1895). In his paper detailing the 
results of his experiments the first-named author says: 

‘‘Animals may be immunized against the venom of serpents either by 
means of repeated injections of doses at first feeble and progressively stronger, 
or by means of successive injections of venom mixed with certain chemical 
substances, among which I mention especially chloride of gold and the hypo- 
chlorites of lime or of soda. 

“The serum of animals thus treated is at the same time preventive, anti- 


toxic, and therapeutic, exactly as is that of animals immunized against 
diphtheria or tetanus. 

“Tf we inoculate a certain number of rabbits, under the skin of the 
thigh, with the same dose, one milligramme of cobra venom for example, 
and if we treat all of these animals with the exception of some for control, 
by subcutaneous or intraperitoneal injections of the serum of rabbits im- 
munized against four milligrammes of the same venom, all of the control 
animals not treated will die within three or four hours, while all of the 
animals will recover which receive five cubic centimetres of the therapeutic 
serum within an hour after receiving the venom.” 


In this connection we may remark that there is some evidence to 
show that persons who are repeatedly stung by certain poisonous in- 
sects—-mosquitoes, bees—acquire a greater or less degree of immu- 
nity from the distressing local effects of their stings. 

Ehrlich, of Berlin, in 1891, reported his success in establishing 
immunity in guinea-pigs against two toxalbumins of vegetable 
origin: one—ricin—from the castor-oil bean (Ricinus communis), 
the other—abrin—from the jequirity bean. The toxic potency 
of ricin is somewhat greater than that of abrin, and it is esti- 
mated by Ehrlich that one gramme of this substance would suffice 
to kill one and a half millions of guinea-pigs. When injected be- 
neath the skin, in dilute solution, it produces intense local inflamma- 
tion, resulting in necrosis of the tissues. Mice are less susceptible 
than guinea-pigs and are more easily made immune. This is most 
readily effected by giving them small and gradually increasing doses 
with their food. As a result of this treatment the animal resists 


SUSCEPTIBILITY AND IMMUNITY. 263 


subcutaneous injections of two hundred to four hundred times the 
fatal dose for animals not having this artificial immunity. The fatal 
dose of abrin is about double that of ricin. When injected into mice 
in the proportion of one cubic centimetre to twenty grammes of body 
weight a solution of one part in one hundred thousand of water 
proved to be a fatal dose. The local effects are also less pronounced 
when solutions of abrin are used ; they consist principally in an ex- 
tensive induration of the tissues around the point of injection and a 
subsequent falling off of the hair over this indurated area. When 
introduced into the conjunctival sac, however, abrin produces a 
local inflammation in smaller amounts than ricin, a solution of 1:800 
being sufficient to cause a decided but temporary conjunctivitis. 
Solutions of 1:50 or 1:100 of either of these toxalbumins, introduced 
into the eye of a mouse, give rise to a panophthalmitis which com- 
monly results in destruction of the eye. But in mice which have 
been rendered immune by feeding them for several weeks with food 
containing one of these toxalbumins, no reaction follows the intro- 
duction into the eye of the strongest possible solution, or of a paste 
made by adding abrin to a little ten-per-cent salt solution. Ehrlich 
gives the following explanation of the remarkable degree of im- 
munity established in his experiments by the method mentioned: 

** All of these phenomena depend, as may be easily shown, upon 
the fact that the blood contains a body—antiabrin—which completely 
neutralizes the action of the abrin, probably by destroying this body.” 

In a more recent paper Ehrlich has given an account of subse- 
quent experiments which show that the young of mice which have 
an acquired immunity for these vegetable toxalbumins may acquire 
immunity from the ingestion of the mother’s milk; and also that 
immunity against tetanus may be acquired in a very brief time by 
young mice through their mother’s milk. In his tetanus experi- 
ments Ehrlich used blood serum from an immune horse to give im- 
munity to the mother mouse when her young were already seven- 
teen days old. Of this blood serum two cubic centimetres were 
injected at a time on two successive days. The day after the first 
injection one of the sucklings received a tetanus inoculation by 
means of asplinter of wood to which spores were attached. The 
animal remained in good health, while a much larger control mouse 
inoculated in the same way died of tetanus at the end of twenty-six 
hours. Other sucklings, inoculated at the end of forty-eight and of 
seventy-two hours after the mother had received the injection of 
blood serum, likewise remained in good health, while other control 
mice died. 

The possibility of conferring immunity by means of the milk of 
an immune animal is further shown by the experiments of Brieger 


264 SUSCEPTIBILITY AND IMMUNITY. 


and Ehrlich (1892). A female goat was immunized against tetanus 
by the daily injection of “thymus-tetanus bouillon.” The dose was 
gradually increased from 0.2 cubic centimetre to 10 cubic centimetres. 
At the end of thirty-seven days a mouse, which received 0.1 cubic 
centimetre of the milk of this goat in the cavity of the abdomen, 
proved to be immune against tetanus. Further experiments gave a 
similar result, even when the milk of the goat was not injected into 
the peritoneal cavity of the mouse until several hours after inocu- 
lation with a virulent culture of the tetanus bacillus. 

When the casein was separated the milk retained its full im- 
munizing activity, and by concentration 7m vacuo a thick milk 
was obtained which had a very high immunization value—0.2 cubic 
centimetre of this milk protected a mouse against forty-eight times 
the lethal dose of a tetanus culture. 

In a subsequent communication (1893) Brieger and Ehrlich de- 
scribe their method of obtaining the antitoxin of tetanus from milk 
in a more concentrated form. They found by experiment that it was 
precipitated by ammonium sulphate and magnesium sulphate. From 
twenty-seven to thirty per cent of ammonium sulphate added to milk 
caused a precipitation of the greater part of the antitoxin. This pre- 
cipitate was dissolved in water, dialyzed in running water, then 
filtered and evaporated in shallow dishes at 35° C. in a vacuum. 
One litre of milk from an immune goat gave about one gramme of a 
transparent, yellowish-white precipitate, which contained fourteen 
per cent of ammonium sulphate. This precipitate had from four 
hundred to six hundred times the potency of the milk from which 
it was obtained in neutralizing the tetanus toxin. 

In a still later communication (1893) Brieger and Cohn give an 
improved method of separating the antitoxin from the precipitate 
thrown down with ammonium sulphate. The finely pulverized pre- 
cipitate is shaken up with pure chloroform, and when this is allowed 
to stand the antitoxin rises to the surface while the ammonium salt 
sinks to the bottom. By filling the vessel to the margin with chloro- 
form, the antitoxin floating on the surface can be skimmed off, after 
which it quickly dries. By this method the considerable loss which 
occurred in the dialyzer, used in the previously described method, is 
avoided. 

A most interesting question presents itself in connection with the 
discovery of the antitoxins. Does the animal which is immune 
from the toxic action of any particular toxalbumin also have an im- 
munity for other toxic proteids of the same class? The experimental 
evidence on record indicates that it does not. In Ehrlich’s experi- 
ments with ricin and abrin he ascertained that an animal which had 
been made immune against one of these subtances was quite as sus- 


SUSCEPTIBILITY AND IMMUNITY. 265 


ceptible to the toxic action of the other as if it did not possess this 
immunity, 7.e., the antitoxin of ricin does not destroy abrin, and 
vice versa. Asan illustration of the fact, he states that in one ex- 
periment a rabbit was made immune for ricin to such an extent, that 
the introduction into its eye of this substance in powder produced no 
inflammatory reaction; but the subsequent introduction of a solution 
of abrin, of 1 to 10,000, caused a violent inflammation. 

Evidently these facts are of the same order as those relating to 
immunity from infectious diseases, and, taken in connection with the 
experimental data previously referred to, give strong support to the 
view that the morbid phenomena ‘in all diseases of this class are due 
to the specific toxic action of substances resembling the toxalbumins 
already discovered ; and that acquired immunity from any one of 
these diseases results from the formation of an antitoxin in the body 
of the immune animal. 

Hankin calls these substances produced in the bodies of immune 
animals ‘‘ defensive proteids,” and proposes to classify them as fol- 
lows: First, those occurring naturally in normal animals, which he 
calls sozins ; second, those occurring in animals that have acquired 
an artificial immunity—these he calls phylaxins. Each of these 
classes of defensive proteids is further subdivided into those which 
act upon the pathogenic microédrganism itself and those which act 
upon its toxic products. These subclasses are distinguished by the 
prefixes myco and toxo attached to the class name. 

In accordance with this classification a mycosozin is a defensive 
proteid, found in the body of a normal animal, which has the power 
of destroying bacteria. 

A toxosozin is a defensive proteid, found in the body of a normal 
animal, which has the power of destroying the toxic products of bac- 
terial growth. 

A mycophylaxin is a defensive proteid produced in the body of 
an animal which has an acquired immunity for a given infectious 
disease, which has the power of destroying the pathogenic bacteria 
to which the disease is due. 

A toxophylaxin is a defensive proteid produced in the body of 
an animal which has an acquired immunity for a given infectious 
disease, which has the power of destroying the toxic products of the 
pathogenic bacteria to which the disease is due. 

Buchner had previously proposed the name “alexins” for these 
defensive proteids. 

The importance of the experimental evidence above referred to in 
explaining the phenomena of natural and acquired immunity is ap- 
parent. The facts stated also suggest a rational explanation of re- 


266 SUSCEPTIBILITY AND IMMUNITY. 


covery from an attack of an acute infectious disease. But the idea 
that during such an attack an antidote to the disease poison is de- 
veloped in the tissues is yet so novel, and the experimental evidence 
in support of this view is of such recent date, that it would be pre- 
mature to accept this explanation as applying to immunity in gene- 
ral, It seems difficult to believe that an individual who has passed 
through attacks of measles, mumps, whooping cough, scarlet fever, 
small-pox, etc., has in his blood or tissues a store of the antitoxin of 
each of these diseases, formed during the attack and retained during 
the remainder of his life, or continuously produced so long as the 
immunity lasts. Moreover, in those diseases to which the experi- 
mental evidence above recorded relates—diphtheria, tetanus, pneu- 
monia—as they occur in man, no lasting immunity has been shown 
to result from a single attack, and in this regard they do not come 
into the same class with the eruptive fevers and other diseases in 
which a single attack usually protects during the lifetime of the in- 
dividual. 

In those instances in which acquired immunity has been shown 
to be due to the production in the body of the immune animal of an 
antitoxin, it is still uncertain whether there is a continuous produc- 
tion of the protective proteid, or whether that formed during the 
attack remains in the body during the subsequent immunity. The 
latter supposition appears at first thought improbable ; but when we 
remember that the protective proteids which have been isolated by 
Hankin from the blood and spleen of rats, and by Tizzoni and Cat- 
tani from the blood of animals made immune against tetanus, do 
not dialyze, it does not seem impossible that these substances might 
be retained indefinitely within the blood-vessels. On the other hand, 
the passage of the tetanus antitoxin into the mother’s milk, as 
shown by Ehrlich’s experiments upon mice, indicates a continuous 
supply, otherwise the immunity of the mother would soon be lost. 

The writer has obtained (May, 1892) experimental evidence that 
the blood of vaccinated, and consequently immune, calves contains 
something which neutralizes the specific virulence of vaccine virus, 
both bovine and humanized. Four drops of blood serum from a calf 
which had been vaccinated two weeks previously, mixed with one 
drop of liquid lymph recently collected in a capillary tube, after con- 
tact for one hour was used to vaccinate a calf; the same animal was 
also vaccinated with lymph, preserved on three quills, which was 
mixed with four drops of serum from the immune calf and left for 
one hour. The result of these vaccinations was entirely negative, 
while vaccinations upon the same calf made with virus from the 
same source, and mixed with the same amount of blood serum from 
a non-immune calf, gave a completely successful and typical result. 


SUSCEPTIBILITY AND IMMUNITY. 267 


The experimental evidence detailed shows that in certain dis- 
eases acquired tmmunity depends upon the formation of anti- 
toxins in the bodies of immune animals. As secondary fac- 
tors it is probable that tolerance to the toxic products of pathogenic 
bacteria and phagocytosis have considerable importance, but it is 
evident that the principal réle cannot be assigned to these agencies. 

As arule the antitoxins have no bactericidal action; but it has 
been shown by the experiments of Gamaléia, Pfeiffer, and others, 
that in animals which have an acquired immunity against the spiril- 
lum of Asiatic cholera and against spirillum Metschnikovi, there is a 
decided increase in the bactericidal power of the blood serum, and 
that immunity probably depends upon this fact. 

The researches of Metschnikoff upon hog cholera, of Issaef upon 
pneumonia, and of Sanarelli upon typhoid fever indicate that the 
immunity conferred upon susceptible animals by protective inocula- 
tions is not due to an antitoxin but to a substance present in the 
blood of immune individuals which acts directly upon the pathogenic 
microérganism, as is the case in cholera-immune animals. The ani- 
mals immunized are said to be quite as sensitive to the action of the 
bacterial poisons as are those which have not received protective 
inoculations. “Their serum does not protect against the toxin, but 
against the microbe” (Roux). 

According to Buchner (1894) the antitoxins are to be regarded not 
as reactive products developed in the body of the immune animal, 
but as modified, changed, and “entgiftete” products of the specific 
bacterial cells. He insists that they do not neutralize the toxins by 
direct contact, but only through the medium of the living organism. 
This explanation scarcely appears tenable in view of the experimental 
evidence, and the fact that the antitoxin of tetanus escapes in con- 
siderable quantity with the milk of an immune goat without, ap- 
parently, diminishing the immunity ofthe animal. In the immunity 
against the toxic action of the vegetable toxalbumins—ricin and 
abrin—as shown by Ehrlich’s experiments, there are no “ products of 
bacterial cells” introduced with the pure toxalbumin from the castor 
bean or the jequirity bean; and we have sufficiently numerous ex- 
periments to show that immunity, with the presence of antitoxins in 
the blood, may be induced by precipitated and purified toxalbumins 
from filtered cultures. Several of the experimenters, also, have re- 
ported that the toxins from bacterial cultures are neutralized in vitro 
by blood-serum from an immune animal, or by the precipitated anti- 
toxin from such serum after contact for a certain number of hours. 
If they are correct in the statement that a certain time is required 
after the antitoxin has been brought in contact with the toxin, in 
order that the latter may be neutralized, as shown by injection of the 


268 SUSCEPTIBILITY AND IMMUNITY. 


mixture into a susceptible animal, then we must admit that this 
neutralizing effect occurs outside of the body of the animal, as has 
been generally assumed. 

The experiments of Vaillard are also opposed to Buchner’s view. 
He reports that in a rabbit immunized against tetanus, “a volume of 
blood equal to the total amount which circulates in its body may be 
withdrawn without diminishing, in an appreciable manner, the anti- 
toxic power of its serum. Therefore the antitoxin must be repro- 
duced as fast as it is withdrawn.” The author from whom we have 
just quoted (Roux) also reports the results of experiments which 
show that the antitoxic value of the serum of a rabbit immunized 
against tetanus does not bear a direct relation to the quantity of the 
tetanus toxin introduced, but depends also upon the method adopted. 
When a few large doses are given the result is far less favorable than 
that obtained by giving the same amount in repeated small doses. 
The serum of an animal immunized by thirty-three small doses was 
found to neutralize, in vitro, 150 parts of toxin, while that of an 
animal which received the same amount in nine doses only neutral- 
ized 25 parts of the same toxin. On the other hand we have experi- 
ments which indicate that the supposed neutralization of a toxin by 
an antitoxin in vittro is not really a chemical neutralization. Thus 
Buchner found in his experiments with the tetanus toxin and anti- 
toxin, in a dry powder, that when mixed in a certain proportion and 
injected into white mice no tetanicsymptoms were induced. Butthe 
same mixture gave rise to distinct tetanic symptoms in guinea-pigs, 
showing that the inference that the toxin had been neutralized in 
vitro, based upon the experiment on mice, would have been a mis- 
take. And certain observations made by Roux and Vaillard seem 
to give support to the view that neutralization does not occur im 
vitro, but that the result depends upon some physiological reaction 
induced by the antitoxin within the body of the livinganimal. These 
bacteriologists found that when the antitoxin was apparently in ex- 
cess, tetanic symptoms could be induced in susceptible animals if 
they had been in any way exhausted prior to the injection of the 
mixture of toxin and antitoxin; and that the same result followed 
when their resisting power had been reduced by injecting into them 
at the same time filtered cultures of other bacteria. 

In this connection the results reported by Calmette, Phisalix, 
and Bertrand are of interest. These investigators found that when 
the antitoxin of snake-poison was mixed with this venom in a pro- 
portion which neutralized its toxic properties, as shown by experi- 
mental inoculations, and the mixture then heated to 70° C., by which 


SUSCEPTIBILITY AND IMMUNITY. 269 


temperature the antitoxin is destroyed, subsequent inoculations 
showed that the toxin was still active. 

The experiments of Stern (1894) show that the typhoid bacil- 
lus not only grows in blood-serum from a typhoid convalescent, which 
has been proved to neutralize its pathogenic effects when injected 
into a susceptible animal, but also that its toxic products are de- 
veloped in this culture medium. From this Stern concludes that the 
serum must in some way act upon the infected animal, causing 
changes which enable it to resist infection, rather than upon the 
bacillus or upon its toxic products directly. It has also been shown 
by Behring (1890) for the diphtheria bacillus, by Vaillard for the 
tetanus bacillus (1892), and by Issaeff (1893) for the micrococcus of 
pneumonia, that these several pathogenic microdrganisms may be 
cultivated in the blood-serum of animals immunized for the diseases 
which they produce. 

In a paper published in 1897, Ehrlich advanced his “ side-chain ” 
(seitenkette) theory. He considers the individual cells of the body to 
be analogous, in a certain sense, to complex organic substances, and 
that they consist essentially of a central nucleus to which secondary 
atom-groups having distinct physiological functions are attached by 
“side chains ”—such as chemists represent in their attempts to illus- 
trate the reactions which occur in the building up or pulling down of 
complex organic compounds. The cell-equilibrium is supposed to be 
disturbed by injury to any of its physiological atom-groups—as by a 
toxin—and this disturbance results in an effort at compensatory repair 
during which plastic material in excess of the amount required is 
generated and finds its way into the blood. This Ehrlich regards as 
the antitoxin, which is capable of neutralizing the particular toxin 
to which it owes its origin, if this is subsequently introduced into the 
blood. In this theory a specific combining relation is assumed to 
exist between various toxic substances and the secondary atom- 
groups of certain cellular elements of the body. The atom-groups 
which, in accordance with this theory, combine with the toxin of any 
particular disease germ, Ehrlich calls the “toxophoric side chain.” 
Immunity, according to Ehrlich, is either “active” or “passive.” 
Passive immunity resalts from the introduction of the immunizing 
substance from an immunized animal into the circulation of a non- 
immune animal, e.g., the use of diphtheria antitoxin as a prophy- 
lactic. This passive immunity is more transient than the active 
immunity which results from an attack of an infectious disease, from 
inoculations with living vaccines, or from repeated injections of in- 
creasing doses of the toxins of pathogenic bacteria. Hhrlich’s ex- 


270 SUSCEPTIBILITY AND IMMUNITY. 


planation of immunity, however probable it may appear, can hardly 
be said to rest upon a substantial experimental foundation, and we 
must admit that the exact source and method of production of the 
antitoxins in the animal body, and their mode of action, are still 
undetermined; and, for the present, we must be satisfied with the 
knowledge that in some way these so-called antitoxins, which have 
been proved to be present in the blood-serum of immune animals, 
protect these animals from infection by pathogenic bacteria. And 
that when transferred to susceptible animals they confer upon them 
a temporary immunity; or if introduced after infection, may neutral- 
ize the pathogenic action of the toxins produced by specific “disease 
germs.” 

Finally, there is experimental evidence to show that immunity 
from the pathogenic action of certain bacteria may be produced by 
previous injections of cultures of other bacteria (sterilized or other- 
wise), and even by the injection of the blood-serum of normal indi- 
viduals or of other substances. 

Pasteur, in 1880, communicated to the French Academy of Sciences 
the results of experiments which led him to the conclusion that fowls 
which had an acquired immunity against chicken cholera also had 
an immunity against anthrax. Roux has reported that the blood- 
serum of a horse which has been immunized against tetanus neutral- 
izes the toxic power of cobra poison. But the contrary effect is not 
produced—7.e., the bleod-serum of an animal immunized against the 
cobra poison does not neutralize the tetanus toxalbumin. The state- 
ment is also made that the blood-serum of a rabbit which has been 
made immune against hydrophobia will protect a susceptible animal 
against the cobra venom in doses four or five times as large as the 
usually lethal dose. iso that rabbits which have been immunized 
against snake-poison are less susceptible to the toxic effects of abrin, 
and the reverse—?.e., antiabrin neutralizes, to some extent at least, 
the toxic action of snake-poison. 

The writer, in his “Report on the Etiology and Prevention of 
Yellow Fever ” (1890), gives, on pp. 196 and 197, experimental evi- 
dence which shows that the injection into the peritoneal cavity of 
rabbits of cultures of Bacillus pyocyaneus or of Bacillus gracilis pro- 
tected the animals from the fatal results of subsequent injections of 
my bacillus X, which was extremely fatal to rabbits when injected 
into the cavity of the abdomen in doses of 1 or 2¢.c. In referring 
to these experiments I say: “ The evidence favors the view that death 
results from peritonitis (and toxemia?) induced by intra-peritoneal 
injections, and that «a tolerance on the part of the peritoneum may 


PLATE IV. 


Fies. 1, 2, and 3.—Leucocytes from the spleen of an inoculated 
monkey, containing Spirillum Obermeieri. (Soudakewitch.) 

Fires. 4 and 5.—Leucocytes (“macrophages”) from a preparation of 
muscle from a pigeon which succumbed to an anthrax inoculation. In 
Fig. 4 the bacilli are deeply stained; in Fig. 5 they are pale. (Metsch- 
nikoff.) 

Fic. 6.—Leucocyte from a frog seventy-two hours after the injection 
of anthrax spores. (Trapeznikoff.) 

Fies. 7 and 8.—Leucocytes from a chicken four hours after the in- 
jection of anthrax spores. (Trapeznikoff.) 


Plate JV 


STERNBERG'S BACTERIOLOGY. 


2 


Fig. 2. 


Fig. 1. 


Fig. 5. 


Fig. 6. 


PHAGOCYTES. 


SUSCEPTIBILITY AND IMMUNITY. QTL 


be established by the injection of certain other bacilli, or of ster- 
alized cultures of bacillus X.” 

This corresponds with facts subsequently developed by Issaeff 
(1894) in his experiments with reference to immunity in guinea- 
pigs against cholera cultures injected into the cavity of the abdomen. 
He found that a certain degree of immunity was established by the 
previous injection of blood-serum from normal individuals, and also 
of various acids, alkalies, and neutral liquids. The immunity pro- 
duced in this way was, however, feeble and temporary, and could 
not properly be considered as identical with that produced by inocula- 
tions with attenuated cultures which give rise toa mild attack of a 
specific disease. : 

Cesaris-Demel and Orlandi have reported (1894) their success in 
immunizing animals against infection by the typhoid bacillus by 
means of sterilized cultures of Bacillus coli communis, and the 
reverse. 

While this chapter relates especially to acquired immunity from 
infectious diseases, and this immunity has been shown to depend, in 
a number of these diseases at least, upon the development of anti- 
toxins in the body of the immune animal, it may be worth while to 
refer briefly, before closing, to some examples of acquired immunity 
of a different order. We refer to the tolerance to extremes of heat 
and cold which may be established by habitual exposure, and, more 
especially, to the tolerance to narcotics and irritant poisons, which is 
very remarkable and has never been explained in a satisfactory 
manner. Samuel (1892) has presented experimental evidence which 
shows that the local inflammation which results from the application 
of croton-oil to the ear of a rabbit does not occur when a second ap- 
plication is made to the same ear after recovery from the effects of 
the first. That a tolerance may be acquired to comparatively large 
doses of arsenic is well known, and the tolerance which the victims 
of drug habits acquire to enormous doses of narcotics is a matter of 
daily observation. In the writer’s paper on acquired immunity, pub- 
lished in 1881, an attempt was made to account.for acquired im- 
munity in infectious diseases as analogous to the immunity to drugs 
just referred to; but the experimental evidence presented in the pres- 
ent chapter shows that the analogy has no scientific foundation in 
the absence of any evidence that there is an antitoxin of morphia, of 
cocaine, of narcotin, etc., in the blood of the habitués of these drugs. 


IV. 
PROTECTIVE INOCULATIONS. 


ANTHRAX. 


THE discovery of the anthrax bacillus by Davaine (1863), and the 
demonstration of its etiological relation to the disease with which it is 
associated, by the researches of Pasteur, Toussaint, Koch (1878-1881) 
and other pioneers in this field of investigation, constitute the foun- 
dation of our present knowledge of bacteriology and of the practical 
results attained in protective inoculations and serum-therapy. Anda 
review of the literature relating to the anthrax bacillus would show, 
in a most interesting manner, the successive steps by which we have 
arrived at the important results which have gone so far toward estab- 
lishing medicine upon a scientific basis. In the present volume, 
however, we must confine our attention to those investigations which 
relate directly to the subject in hand. 

Toussaint, a pioneer in researches relating to protective inocula- 
tions, has a short paper in the Comptes-Rendus of the French Academy 
of Sciences of July 12th, 1880, entitled “Immunity from Anthrax 
(“charbon”) Acquired as a Result of Protective Inoculations.” 

Tn this paper he announces his discovery of the important fact that 
the anthrax bacillus does not form spores in the tissues or liquids of 
the body of an infected animal, but multiplies alone by binary divi- 
sion—“sa multiplication se fait toujours par une division du mycélium.” 

In the same communication he reports his success in conferring 
immunity upon five sheep by means of protective inoculations, and 
also upon four young dogs. We must therefore accord him the prior- 
ity in the publication of experimental data demonstrating the practi- 
cability of accomplishing this result. 

Toussaint does not give his method in the communication above 
referred to, but the following quotation from a communication made 
to the Academy of Sciences on March 19th, 1881, by Pasteur, shows 
the method, and at the same time demonstrates the fact that Tous- 
saint was the first to produce immunity by the use of sterilized cul- 
tures. Pasteur says: 


‘‘By inoculating sheep either with defibrinated blood from an animal 
dead of anthrax, after filtration through several thicknesses of paper, or 


PROTECTIVE INOCULATIONS. 273 


with the same blood defibrinated and subjected to 55° C. for ten minutes, 
according to Toussaint, these sheep subsequently resist inoculations with 
anthrax blood. . . . The bacillus, according to Toussaint, deposits in the 
blood of animals in which it multiplies-a substance which may become its 
own vaccine. By filtration while cold in one case, by a temperature of 55°C, 
in the other, the bacillus is said to be removed or killed; so that the inocula- 
tion of filtered or heated blood introduces into the animal inoculated vac- 
cinal matter deprived of bacteria.” 


After thus stating Toussaint’s method and explanation Pasteur 
proceeds to raise objections against this method, the principal of 
which are that the anthrax bacillus is not killed by exposure to a tem- 
perature of 55° C. for ten minutes, and that inoculation with a virus 
prepared in this way would result in a considerable mortality among 
the animals inoculated, although those surviving the inoculation would 
be protected. . 

In a communication made to the French Academy of Sciences, 

September 27th, 1880, Pasteur gave an account of an experiment made 
July 14th, 1879, upon two cows, which in connection with a subsequent 
experiment, made August 6th, 1880, upon four cows, led him to the 
conclusion that a single attack of anthrax protects from subsequent 
attacks. He says in the paper referred to: 


‘On the 15th of September, 1880, two cows, A and C, which had been 
very ill as a result of the first inoculation, made August 6th, were reinoculated 
on the left side, that is to say, on the side opposite the first inoculation. We 
used five drops of culture of the bacillus of anthrax (‘bactéridies du char- 
bon’). The following days there was no perceptible cedema and no elevation 
of temperature in either cow. The question is then resolved : a single attack 
protects (‘le charbon ne récidive pas’).” 


The next important steps in the line of experimental research 
leading to protective inoculations in the disease under consideration 
were reported by Pasteur in his communication to the French Acad- 
emy made at the séance of February 28th, 1881 (with the collaboration 
of Chamberland and Roux), entitled “De l’atténuation des virus et 
de leur retour 3 la virulence.” In this connection Pasteur announces 
his discovery of the fact that when cultivated at a temperature of 42° 
to 48° C., the anthrax bacillus no longer forms spores and rapidly 
loses its virulence. He says: 


‘*As regards its virulence, the extraordinary fact has been ascertained that 
the bacillus is no longer virulent after it has been kept for eight days at a 
temperature of 42° to 43° C.; at least its cultures are inoffensive for the 
guinea-pig, the rabbit, and the sheep, three species of animals which are 
very susceptible to anthrax. We are able, then, not only to attenuate viru- ° 
lence, but to effect its complete extinction, by a simple method of cultivation. 

‘‘Before the extinction of its virulence the microbe of charbon passes 
through the intermediate degrees of attenuation, and, on the other hand, as 
happens also with the microbe of fowl cholera, each of these degrees of vir- 

1 


4 
274 PROTECTIVE INOCULATIONS. 


ulence may be reproduced by cultivation. Finally, as shown in one of our 
recent communications, since one attack of anthrax protects, each one of our 
attenuated microbes of charbon constitutes a vaccine for the microbe of 
superior virulence ; that is to say, a virus suitable to produce a more benign 
malady. What, then, is more easy than to find among these a virus suitable 
to give anthrax to sheep, cows, or horses, without causing them to perish, 
and capable of preserving them from a subsequent fatal attack? We have 
already practised this operation upon sheep with great success.” 


At the end of this important communication Pasteur says: 


“‘T concluded my communication of October 26th by remarking that the 
attenuation of virus by the influence of the air is probably one of the factors 
in the extinction of great epidemics. The facts just recorded, in their turn, 
may serve to explain the so-called spontaneous appearance of these scourges. 
An epidemic which has been terminated by the attenuation of its virus may 
be relighted by the reinforcement of this virus under certain influences. The 
accounts which I have read of the spontaneous appearance of the plague ap- 
pear to me to offer examples of this. The plague is a virulent malady which 
prevails in certain countries. In all of these countries its attenuated virus 
probably exists, ready to take its active form when the necessary conditions 
as to climate, famine, and distress again prevail. There are other virulent 
maladies which appear spontaneously in all countries, such as camp typhus. 
Without doubt the germs of the microbes which cause these.diseases are 
everywhere distributed. Man carries them about him, or in his intestine, 
without great damage, but ready, nevertheless, to become dangerous when, 
as a result of certain conditions or of successive development upon the sur- 
face of wounds, in bodies enfeebled or otherwise, their virulence is progres- 
sively reinforced: And from this point of view virulence appears to us 
under a new light which is somewhat disquieting for humanity, unless na- 
ture, in the evolution which has occurred during the past centuries, has al- 
ready encountered all possible occasions for the production of virulent or 
contagious diseases, an assumption which seems very improbable. 

‘What is an inoffensive microscopic organism for man or for a given 
animal? It is an organism which cannot develop in our body or in that of 
the animal; but nothing proves that if this microscopic organism should 
penetrate into some other of the thousands of species of the creation, it could 
not invade it and cause it to become sick. Its virulence, then, reinforced by 
passing through a series of individuals of this species, might become such 
that it could invade man or one of the domestic animals. By this means new 
contagions may be created. I am disposed to believe that it is in this way 
that, in the course of ages, have appeared small-pox, syphilis, the plague, 
yellow fever, etc.” 


This broad induction has received considerable support from more 
recent researches, which show that the typhoid bacillus, the cholera 
spirillum, and other important pathogenic bacteria become attenuated 
when they lead a saprophytic existence for some time, and regain their 
virulence when they are propagated within the bodies of susceptible 
animals. 

In a later communication (March 21st, 1881) Pasteur says that he 
has found by experiment that when attenuated varieties of the anthrax 
bacillus form spores, these again reproduce the same pathogenic va- 
riety, so that cultures of each degree of attenuation can be maintained . 
indefinitely. 


PROTECTIVE INOCULATIONS. 275 


On June 13th, 1881, Pasteur communicated the results of his famous 
experiment at Pouilly-le-Fort, near Melun. He says: 


‘On the 5th of May, 1881, we inoculated, by means of a Pravez syringe, 
twenty-four sheep, one goat, and six cows, each animal with five drops of 
an attenuated culture of the anthrax bacillus. On the 17th of May we rein- 
oculated these animals with a second virus, also attenuated, but more virulent 
than the first. 

‘On the 31st of May we proceeded to make a very virulent inoculation in 
order to test the efficacy of the preventive inoculations made on the 5th and 
17th of May. For this experiment we inoculated the vaccinated animals, 
and also twenty-four sheep, one goat, and four cows which had not received 
any previous treatment. 

‘“‘The very virulent virus used on the 31st of May was obtained from 
spores preserved in my laboratory since the 21st of March, 1877. 

‘‘In order to make the experiments more comparable we inoculated alter- 
nately a vaccinated and a non-vaccinated animal. When the operation was 
finished all of those present were invited to reassemble on June 2d, 7.e., 
forty-eight hours after the virulent inoculation was made. 

‘‘Upon the arrival of the visitors on June 2d, all were astonished at the 
result. The twenty-four sheep, the goat, and the six cows which had received 
the attenuated virus, all presented the appearance of health. On the con: 
trary, twenty of the sheep, and the goat, which had not been vaccinated, were 
already dead of anthrax ; two more of the non-vaccinated sheep died before 
the eyes of the spectators, and the last of the series expired before the end of 
the day. The non-vaccinated cows were not dead. We had previously 
proved that cows are less subject than sheep to die of anthrax. But all had 
an extensive cedema at the point of inoculation, behind the shoulder. Cer- 
tain of these cedematous swellings increased during the following days to such 
dimensions that they contained several litres of liquid, deforming the animal. 
One of them even nearly touched the earth. The temperature of these cows 
was elevated 3° C. The vaccinated cows did not experience any elevation of 
temperature, or tumefaction, or the slightest loss of appetite. The success, 
therefore, was as complete for the cows as for the sheep.” 


The facts that infection depends to some extent upon the number 
of bacilli introduced, and that animals which have a certain degree of 
immunity, like the Algerian race of sheep, may succumb when they 
are inoculated with a certain quantity of virus, although they resist 
asmaller amount, were announced by Chauveau in his communication 
to the French Academy at the séance of June 28th, 1880. He says: 


“The facts which I have just presented show that the anthrax bacillus 
behaves in the organism of Algerian sheep, not as if it were deprived of the 
principles necessary for its development, but rather as if it were in a medium 
rendered unsuitable for its growth by the presence of substances injurious to 
it. In avery small number the bacilli are arrested in their development by 
the inhibitory influence of these substances. When they are very numerous, 
on the contrary, they surmount more easily this obstacle to their prolifera- 
tion.” 


This quotation shows that Chauveau had at this early date arrived 
at an explanation of immunity very nearly in accord with that which 
is now generally accepted. 

The fact that infection is influenced by the quantity of the infec- 


276 PROTECTIVE INOCULATIONS. 


tious material introduced had previously been insisted upon by Da- 
vaine in his paper entitled “ Recherches sur quelques unes des conditions 
qui favorisent ou qui empéchent le dévelopment cde la septicémie,” pub- 
lished in the Bulletin of the Academy of Medicine, séance of February 
18th, 1879. 

Davaine says: 

“‘A third condition relates to the quantity of bacteria introduced into the 
tissues. This question of quantity has been made manifest in our experi- 


ments. Not only doesit differ in different species of animals, the rabbit and 
the dog, for example, but it varies in different animals of the same species.” 


In his communication to the Academy of Sciences, made on April 
4th, 1881, Chauveau gives the results of his experiments in producing 
immunity by inoculations with very small quantities of virus. After 
some preliminary experiments with a larger number, five sheep were 
inoculated with diluted anthrax blood estimated to contain two hun- 
dred and fifty bacilli for each. Atl of the animals survived the inocu- 
lation after having manifested some slight febrile reaction. Six weeks 
later all were reinoculated with a dose which should have been fatal 
to an unprotected animal. One of the animals died of anthrax, the 
other four resisted perfectly. 

On June 26th, 1882, Chauveau reported to the Academy of Sciences 
the results of his experiments relating to the protection of animals 
from anthrax infection by the method of Toussaint. By carefully 
conducted experiments Chauveau found that nine or ten minutes’ ex- 
posure to a temperature of 54° C. killed all of the bacilli in anthrax 
blood, and the same result was obtained by sixteen minutes’ exposure 
to 52° C., while at 50° C. the time required is twenty minutes. An 
attenuated virus suitable for protective inoculations is obtained by 
exposure for a somewhat shorter time, and as a result of his experi- 
ments Chauveau was led to the conclusion that for a first inoculation 
anthrax blood heated to 50° C. for fifteen minutes afforded a good at- 
tenuated virus. This was to be followed after an interval of ten to 
fifteen days by a second inoculation with a stronger virus, obtained 
by exposing anthrax blood to the same temperature (50° C.) for nine 
or ten minutes. These inoculations sufficed to protect the animals 
when they were subsequently inoculated with virus of full strength— 
blood from an animal which had recently succumbed to the disease. 
Chauveau says with reference to this method: 


‘‘In one hour, with a single guinea-pig [dead of anthrax], it is easy to 
prepare the quantity of vaccine required to inoculate more than five hundred 
sheep. The inoculation is made with the point of a lancet, charged, by the 
method in use in my: laboratory, with a very small quantity of virus. Two 


PROTECTIVE INOCULATIONS. QV? 


or three large punctures under the skin, upon the internal surface of the ear, 
suffice for a successful inoculation. ; 

‘The vaccine prepared in this way should be used at once, or.at least 
not later than tl:e day after it has been prepared. Experience has shown me 
that it is then quite as harmless and quite as efficacious as Pasteur’s vaccine.”’ 


In the preparation of an attenuated virus by this method Chauveau 
insists upon attention to the following points: 


‘‘The first rule to follow, and the principal one, is to practise the heating 
in such a manner that all parts of the anthrax blood are raised to the re- 
quired temperature and withdrawn from it at the same instant. When the 
quantity of blood to be transformed to a vaccine is too great, all parts are 
not uniformly acted upon by the very.short exposure to heat; the virulent 
agents in the deeper layers may, in that case, preserve all of their activity, 
and cause a fatal infection. To avoid this it is best to enclose the blood in 
little cylindrical pipettes, 1 mm. in diameter. The extremity of these 
pipettes is sealed, and the portion which contains the blood is immersed in a 
considerable quantity of water maintained at the proper temperature. At 
the end of the proper time they are taken from the hot bath and plunged into 
cold water. 

‘* Another rule should be rigorously observed if one wishes to be sure of 
success. The blood should be collected under conditions which make it sure 
that the virulent agents introduced into the tubes all have the same vitality, 
the same activity, and that they are impressed in the same degree by the 
heating. Thisis the case when we take the blood froma guinea-pig just 
dead, afler having survived from thirty-six to forty-eight hours an inocula- 
tion with very active virus. Before introducing the blood into the pipettes . 
it should be allowed to coagulate, and the coagula should be broken and 
crushed in order to obtain a defibrinated blood, which is always very rich in 
virulent bacilli.” 


In a subsequent communication (February 26th, 1883), Chauveau 
admits that the application of this method is somewhat difficult and 
delicate when blood is employed, and states that it is far more satis- 
factory to use pure cultures, which may be attenuated in the same 
way. He prefers to cultivate the bacillus in a bouillon made from 
the flesh of a chicken, and to start his culture by adding to this bouil- 
lon a drop of blood from an animal just dead from anthrax. The cul- 
ture is left for twenty hours in an incubating oven at a temperature of 
43° C. During this time there is an abundant development of the 
bacillus, and the culture is ready to be subjected to the attenuating 
action of a higher temperature. This is accomplished by exposure 
to a temperature of 47° C. for a period of one, two, three, or four 
hours, according to the degree of attenuation desired. After three 
hours’ exposure the attenuated culture no longer kills guinea-pigs. 
In a later communication (March 5th, 1883) Chauveau states that he 
has ascertained by experiment that the degree of attenuation produced 
by this method is maintained in subsequuent cultures made at 48° C., 
from the attenuated culture thus obtained. 

Another method of attenuating the virulence of anthrax cultures is 


278 PROTECTIVE INOCULATIONS. 


that described by Chauveau, in 1885. This consists in cultivating the 
bacillus at a temperature of 38° to 39° C., under a pressure of eight 
atmospheres. Cultures treated in this way killed guinea-pigs, but 
did not kill sheep, cattle, or horses, and constituted a suitable atten- 
uated virus for protective inoculations in these animals. One drop 
was used for a sheep, and two drops for a cow or a horse, and the 
immunity was proved to last for a vear. 

Kitt, in experiments made in 1884 and 1885, found that au atten- 
uation of the virulence of anthrax bacilli may be effected by passing 
them through birds, which have but little susceptibility to anthrax 
infection; but the results obtained were not uniform, and the method 
was not thought to have any great practical value. In the same paper 
Kitt gives an account of his experiments with Pasteur’s vaccine, No. 
1 and No. 2, which he obtained from the agent in Paris. These ex- 
periments led him to the conclusion that the attenuated cultures used 
by Pasteur are too weak. But by passing them through guinea-pigs 
their virulence was increased so that they served to protect cattle and 
sheep, although not without danger for the last-mentioned animals. 

During the vear 1882 Pasteur’s method was extensively practised 
in the department of Eure-et-Loir, where anthrax was very prevalent 
and had been the cause of extensive losses. The results of these pro- 
tective inoculations were reported to the Academy of Sciences (séance 
of December 18th, 1882) by Pasteur, who submitted, with some re- 
marks, a report prepared by M. Boutet, from which we quote as 
follows: 

“The number of sheep vaccinated during the year has been 79,392; 
among’ these flocks the average annual loss during the past ten years was 
7,237—9.01 percent. Since the vaccinations but 518 animals have died—0.65 
per cent. We must observe that this year, probably on account of the great 
humidity, the mortality in Eure-et-Loir has only been three per cent. The 
losses should therefore have been 2,382, instead of 518, without the vaccina- 
tions. In the flocks which were only partly vaccinated we had 2,308 vacci- 
nated and 1,659 not vaccinated ; the loss among the first was 8, or 0.4 per 
cent.; among the second the loss was 60, or 3.9 per cent. We call attention 
to the fact that in these flocks, in different cantons of the department, the 
sheep vaccinated and not vaccinated were subjected to the same conditions 
of soil, of lodging, of food, of temperature, and that consequently they were 
exposed to identical influences. 

‘The veterinary surgeons in Eure-et-Loir have vaccinated 4,562 animals 
of the bovine species. Out of this number the annual loss had been 322. 
Since the vaccinations only 11 cows have died. That is, the annual mortality 
has been reduced from 7.03 per cent. to 0.24 per cent. 

“Some engorgements, generally not serious, having occurred after vacci- 
nating horses, and the mortality not being great in this species, the veterina- 


rians have thought it prudent not to vaccinate horses on a large scale. Only 
524 were vaccinated ; three of these died after the first vaccination.” 


Notwithstanding this favorable report some bacteriologists, and 


PROTECTIVE INOCULATIONS. 279 


notably Koch, were not disposed to admit the practical value of Pas- 
teur’s anthrax inoculations. At the conclusion of an elaborate me- 
moir published in the second volume of the “Mittheilungen” of the 
Imperial Board of Health of Germany (1884), Koch and his collab- 
orators (Gaffky and Loffler) say: 


‘‘As now acertain immunity against inoculated anthrax cannot be ob- 
tained by the method of Pasteur, as we have seen, without considerable 
losses, and as the immunity secured at the expense of considerable loss is 
only an imperfect protection against contracting anthrax in the ordinary 
way, we must consider the protective inoculations heretofore practised as of 
doubtful utility, especially when we remember that the second inoculation 
with a yet stronger virus causes the death of more animals which may serve 
to further spread the disease.” 


The attenuating influence of light on the anthrax bacillus and the 
fact that cultures attenuated in this way may be used for protective 
inoculations was first ascertained by Arloing (1886). Roux subse- 
quently (1887) showed that the presence of oxygen is a necessary fac- 
tor in the sterilization of cultures by exposure to sunlight. Behring, 
who has since been so active in the field of research to which the 
present volume relates, published an article in the Centralblatt fir 
klinische Medicin in 1888 (September 22d) in which he attempted to 
explain the natural immunity of white rats against anthrax infection. 
His conclusions are given as follows: 


“4. The blood-serum of white ratsis nota favorable medium for the 


anthrax bacillus.” ; 
“2. The blood-serum of rats differs from that of animals susceptible to 


infection by its greater alkalinity.” 

‘«8. By the addition of an acid to the blood-serum of rats this becomes a 
favorable medium for the growth of the anthrax bacillus.” 

‘4, The blood-serum of rats which are treated, during life, in such a way 
as to reduce the alkalinity of the blood becomes a suitable medium for the 
development of the anthrax bacillus.” 


As we have pointed out in the chapter on Natural Immunity, the 
true explanation of the facts ascertained in Behring’s experiments is 
probably to be found, not in the germicidal power of the compar- 
-. atively small amount of alkali present in the rat’s serum, but in the 

. fact that the germicidal proteid produced by the leucocytes is only 
soluble in an alkaline medium. Ina paper published in the Annals 
of the Pasteur Institute (August, 1888), Roux and Chamberland have 
given an account of experiments made by them which establish the 
fact that immunity against anthrax may be established by inoculating 
susceptible animals with blood from an animal dead from anthrax, in 
which the anthrax bacilli had been killed by heat or removed by fil- 
tration (Sur ’immunité contre le charbon conférée par des substances 


280 PROTECTIVE INOCULATIONS. 


chimiques). These experiments were commenced in 1881. The 
authors named say : 


‘‘In repeating the experiments of Toussaint upon-anthrax blood which 
had been heated, we made several observations which convinced us that it is 
possible to confer immunity against anthrax upon sheep by injecting under 
their skin anthrax blood which does not contain any living bacilli.” 


While immunity was produced in this way, Roux and Chamber- 
land remark that the sheep which had received a comparatively large 
dose were quite sick when subsequently inoculated with a virulent 
culture, and the immunity acquired was less reliable than that ob- 
‘tained by Pasteur’s method with two vaccines of different degrees of 
attenuation. 

In an investigation made by Hankin, in the laboratory of Professor 
Koch at the Hygienic Institute of Berlin, the results of which are given 
in a preliminary account published in the British Medical Journal 
(October 12th, 1889), the important fact was ascertained that immunity 
may be produced in susceptible animals by inoculating them with an 
“albumose” isolated from anthrax cultures. Hankin gives the fol- 
lowing account of his method of obtaining this immunizing proteid 
from anthrax cultures: 


‘In the course of my process of preparation it is precipitated from its so- 
lution by the addition of a large bulk of absolute alcohol, and well washed 
in this liquid to free it from ptomaines; it is well known that all such sub- 
stances are soluble in alcohol. It is then filtered off and dried ; then it is re- 
dissolved and filtered through a Chamberland filter. A rough estimate of 
the percentage of albumose present in the clear solution thus obtained is 
made colorimetrically by means of the biuret reaction and a peptone solution 
of known strength.” 

“‘In one experiment four rabbits (Nos. 23 to 26) were inoculated subcuta- 
neously with virulent anthrax spores. No. 26 served as a control and died 
in about forty hours. The other three rabbits had the albumose solution in- 
jected into the ear-vein at the same time. Nos. 24 and 25 each had about 
the five-millionth of their body-weight, while No. 23 had only the ten-mil- 
lionth of its body-weight of albumose. No. 25 died in less than forty-eight 
hours, but Nos. 23 and 24 survived. Ten days later Professor Koch kindly 
reinoculated these two rabbits for me with very virulent anthrax from an 
agar-agar culture. Their temperature has remained normal since then, and 
they are now alive and well afortnight after this operation. I have also suc- 
ceeded in producing immunity in mice against attenuated anthrax.” 


In a paper published in the Proceedings of the Royal Society in 
1890, Dr. Sidney Martin has given an account of his researches relat- 
“ing to “The Chemical Products of the Growth of Bacillus Anthracis, 
and their Physiological Action.” In his experiments che cultures 
were maintained for from ten to fifteen days, and the bacilli were then 
‘ removed by filtering through a Chamberland filter. The filtrate was 
found to contain: 


PROTECTIVE INOCULATIONS. 281 


“1. Proto-albumose, deutero-albumose, and a trace of peptone, all with 
the same chemical reactions as the similar bodies formed in peptic digestion. 
2. An alkaloid. 3. Small quantities of leucin or tyrosin. The chief char- 
acteristic of the proto- and deutero-albumose obtained from anthrax cultures 
was found to be their strong alkalinity in solution. This was not removed 
by prolonged dialysis or by washing in alcohol, chloroform, benzene, or ether. 
These proteids are precipitated in an alkaline condition by saturation with 
NaCl (proto-albumose) or (NH,)2S0,.” 


The alkaloid found was soluble in water or in absolute alcohol, 
was strongly alkaline in solution, and readily formed salts with acids. 
Tt was slightly volatile and lost its poisonous properties to a great ex- 
tent when exposed to the air for some time. A mixture of the two 
albumoses was toxic, and when injected into mice in small amounts 
caused a local subcutaneous cedema ending in recovery. Larger 
doses caused more extensive cedema and death. A fatal dose for a 
mouse weighing twenty-two grammes was 0.3 gramme. Boiling for a 
short time diminished the toxicity of these proteids without com- 
pletely destroying it. The alkaloid produced similar symptoms 
when injected into mice, but more promptly and in a smaller dose—0.1 
to 0.15 gramme killed a mouse weighing twenty-two grammes in two 
or three hours. Hankin and Westbrook have more recently (1892) 
made researches with reference to the proteids present in anthrax 
cultures. To obtain an immunizing albumose they cultivated the 
bacillus at 20° C. in flesh-extract solution (1:1,000) to which fibrin 
was added. At the end of eight days a considerable precipitate was 
obtained by means of ammonium sulphate. This was placed in a 
dialyzer in running water at 42° to 45° C.; then precipitated by alco- 
hol and dissolved in a small quantity of water (thirty cubic centime- 
tres)—five hundred cubic centimetres of flesh extract treated in this 
way gave only 0.44 gramme of albumose. Experiments on mice - 
gave some evidence of the immunizing action of this albumose, but 
the results were apparently not so definite as those previously re- 
ported by Hankin. Nor are the experiments of Petermann, who 
followed Hankin’s method (1892), more satisfactory. Arloing ob- 
tained more favorable results by using culture liquids from which 

“the bacilli had been removed by sedimentation. A considerable 
precipitate was obtained when alcohol was added to the culture 
liquid, but it was found that this precipitate had no immunizing effect. 
On the contrary, there remained in solution an immunizing sub- 
stance. This was obtained in a concentrated form by evaporating at 
50° C. in a partial vacuum. Experiments upon lambs showed the 
protective power of this extract, and of the culture liquids before 
‘treatment when injected in considerable quantity. 


282 PROTECTIVE INOCULATIONS. 


In a paper published in the Fortschritte der Medicin, Wysokowicz 
gives a résumé of the results obtained in Russia in protective inocula- 
tions made up to date of publication (January, 1889). According to 
the author named, Professor Cenkowski, who had made himself 
familiar with Pasteur’s method while on a visit to Paris, was the first 
to employ it in Russia (1883). But he found its application to be 
attended with some difficulties. The cultures attenuated as directed 
by Pasteur at 42° to 43° C. “showed a very different degree of viru- 
lence in different experiments, and their virulence was also changed 
by keeping.” Experiments were therefore made with a view to secur- 
ing a more satisfactory vaccine. In an experiment made in 1885, 
1,333 sheep were inoculated; of these 21 died from the first inocula- 
tion and 4 from the second (1.86 per cent). Subsequently better 
results were obtained, and up to the end of 1888, 20,310 sheep had 
been inoculated, with an average mortality of 0.87 per cent as a re- 
sult of the inoculations. 

Professor Cenkowski found that greater losses occurred when the 
inoculations were made in midsummer or midwinter than when they 
were made in the spring or autumn. The losses from anthrax dimin- 
ished among the flocks in which the protective inoculations were prac- 
tised in proportion to the number of sheep inoculated, falling from 
8.3 per cent in 1884, the year before the inoculations were com- 
menced, to 0.13 per cent in 1888. The author of the paper states 
that in some parts of Russia the annual loss among the sheep from 
anthrax is as high as 33 per cent. 

The reliability of the protective inoculations was tested by a com- 
mission, to which Wysokowicz belonged. Fifty sheep which had been 
inoculated from two to four months previously were infected with viru- 
lent anthrax material. Of these only one died. Later, twenty sheep 
which had been inoculated thirteen months before were inoculated with 
virulent material. Of these two died. These favorable results are 
ascribed by Wysokowicz to the improved method of attenuating 
anthrax virus adopted by Professor Cenkowski. As a first vaccine 
he employed a culture which was stronger than that of Pasteur, and , 
which killed mice and caused the death of one-third of the Ziesel- 
mause (Spermophilus citillus) inoculated. He used as a vaccine an 
attenuated culture which had been carried through a series of the 
animals last mentioned. His vaccine, consisting of a bouillon cul- 
ture from a drop of blood of the animal, was preserved by the addi- 
tion of two parts of a thirty-per-cent solution of pure glycerin to 
one part of the culture. 

For inoculating a sheep of average size he used 0.1 to 0.2 cubic 


Shag se see oe 


PROTECTIVE INOCULATIONS. 283 


centimetre of this first vaccine; for a larger animal, from 0.3 to 0.5 
cubic centimetre. The second inoculation was made twelve days 
after the first, with a virus which killed three-fourths of the Ziesel- 
mause and from one-third to one-half of the rabbits inoculated with it. 
Numerous experiments convinced Cenkowski that no change occurred 
in the virulence of his different vaccines when they were carried 
through a series of mice or of earless marmots (Zieselmause). 

Hess reports that the anthrax inoculations made by Chauveau’s 
method in the Canton Bern, during the years 1886, 1887, and 1888, 
were not attended with any losses either from the inoculations or from 
subsequent attacks of anthrax among the inoculated animals (cattle?). 
Tn all, two hundred and fifty-three animals were inoculated during the 
three years specified. 

Hutyra (1890) has reported upon anthrax inoculations by Pasteur’s 
method, as carried out under the regulations of the Government in 
1889. The number of horses inoculated was 130, 2 of which died of 
anthrax at a later date—not as aresult of the inoculation. This gives 
a percentage of loss of 1.38, which is much below the usual rate with- 
out protective inoculations. Three thousand two hundred and sev- 
enty-nine cattle, belonging to 32 different estates, were inoculated. 
Of these 11 died from anthrax, and 2 of these as a result of the first 
inoculation. Deducting these 2 the loss was 0.27 per cent, whereas 
in former years the losses in the same herds had been from 6 to 
12 per cent. Twenty-two thousand seven hundred and sixty- 
seven sheep were inoculated on 23 different estates. One hundred 
and sixty-two of these died from the first inoculation and 59 within 
twelve days after the second inoculation. In the course of the year 
432 of the inoculated animals died from anthrax—in all a loss of 
9.18 per cent. In the absence of protective inoculations the annual 
loss in these flocks had been about 10 per cent. It was found that - 
lambs four months old could be inoculated with the same dose as the 
older sheep, and without any greater loss as a result of the operation. 

The result of anthrax inoculations made in France by Pasteur’s 
method during the twelve years ending in 1894 have been sum- 
marized by Chamberland. The veterinarians who made the inocula- 
tions were each year called upon to answer the following questions: 
1. Number of animals inoculated. 2. Number of deaths from first 
inoculation. 3. Number of animals dying within twelve days after 
second inoculation. 4. Number of animals dying of anthrax within 
a year after protective ‘inoculations. 5. The yearly average loss 
before inoculations were practised. The total number of animals 
inoculated during the period to which this report refers was 1,788, - 


"284 PROTECTIVE INOCULATIONS. 


677 sheep and 200,962 cattle. The average annual loss before 
these protective inoculations were practised is said to have beén 
about ten per cent for sheep and five per cent for cattle. The total 
mortality from this disease among inoculated animals, including that 
resulting from the inoculations, was 0.94 per cent for sheep and 0.34 
per cent for cattle. Chamberland estimates that the total saving as a 
result of the inoculations practised has been 5,000,000 francs for 
sheep and 2,000,000 frances for cattle. 

Podmolinoff gives the following summary of results obtained in 
1892 and 1893 in the “government of Cherson” (Austria): Number 
of sheep inoculated, 67,176; loss, 294 = 0.43 per cent. Number of 
horses inoculated, 1,452; loss, 8. Number of cattle inoculated, 
3,652; loss, 2. The conclusion is reached that Pasteur’s method of 
inoculation affords an immunity against infection with virulent an- 
thrax bacilli in greater amounts than could ever occur under natu- 
ral conditions. 


BUBONIC PLAGUE. 


A number of prominent bacteriologists have been engaged in re- 
searches relating to the prevention and cure of bubonic plague by 
means of an antitoxic serum, obtained by the same method and in 
accordance with the same fundamental scientific principle as in the 
case of the antitoxic serum which is now so successfully employed in 
the treatment of diphtheria.. The experiments thus far made have 
apparently been attended with a considerable degree of success. Pro- 
fessor Calmette reports that the serum of Yersin prepared at the Pas- 
teur Institute in Paris proved to be curative in a considerable propor- 
tion of the cases treated during the recent outbreak at Oporto, and that 
protective inoculation conferred a temporary immunity, which, how- 
ever, did not last longer than twenty days. The mortality in cases 
not treated by Yersin’s serum was 70 per cent, in those treated with 
it 18 per cent. 

The inoculations made by Haffkine in Bombay appear to have been 
quite successful. In his first experiment 8,142 persons were inocu- 
lated. Of these 18 subsequently contracted the disease and 2 died. 
Among 4,926 persons inoculated a single time at Dharwan, 45 were 
subsequently attacked and 15 died; while among 3,387 persons in 
whom a second inoculation was made, only 2 were attacked. Haff- 
kine uses in his inoculations a sterilized culture of the plague bacil- 
lus. The inoculation is followed by slight fever and enlargement of 
the nearest lymphatic glands. All symptoms disappear at the end 
of two or three days. 


PROTECTIVE INOCULATIONS. 285 


The duration of the immunity resulting from these inoculations 
has not been definitely determined, although in a majority of those 
inoculated it appears to have afforded protection for at least five or 
six months. Haffkine’s method of preparing his material for protec- 
tive inoculations isas follows: A kilogramme of finely chopped goat’s 
flesh is macerated in diluted hydrochloric acid, and then placed in an 
autoclave and heated for six hours under a pressure of three atmos- 
pheres. ‘This is filtered, neutralized’ with KOH, and diluted up to 
three litres. The plague bacillus is grown in this medium. Accord- 
ing to Haffkine, when the bacillus is planted upon the surface of this 
medium, a characteristic growth results. If undisturbed for five or 
six days delicate thread-like processes are seen hanging in the culture 
medium resembling stalactites suspended from the roof of a cavern. 
This growth is said to be peculiar to the plague bacillus. To make 
the prophylactic the bacillus is grown in a darkened room in large 
flasks. In India it is unnecessary to use a thermostat. Five or six 
crops of the stalactites are grown and shaken to the bottom of the 
flasks. This takes about six weeks. The culture is then sterilized 
in a water bath at 70° C., the time required being about three hours. 
A little carbolic acid or thymol is then added, and the material, after 
shaking to distribute the bacteria, is decanted into small bottles. It 
is now ready for use, and is usually injected into the subcutaneous 
connective tissue in doses of two cubic centimetres. A second inocu- 
lation in from fourteen to twenty days is recommended by Leumann, 
and after this the blood of the inoculated individual usually gives the 
Widal reaction. a 


CHICKEN CHOLERA. 


Pasteur’s researches with reference to the etiology of the disease 
known in France as choléra des poules first led him to the discovery 
that a virulent culture of a pathogenic bacterium may become “ atten- 
uated” by certain agencies, and that immunity may be conferred upon 
susceptible animals by inoculating them with such attenuated culture. 
We now know that his microbe of fowl cholera is a widely distributed 
bacillus, which is frequently encountered in putrefying material, and 
that it is also extremely fatal to pigeons, pheasants, sparrows, rabbits, 
and mice. Also that the same or nearly allied species may produce 
an infectious disease of swine (Schwetineseuche), of cattle (Rinder- 
seuche), and of deer ( Wildseuche). 

Subcutaneous injection of a minute quantity of a virulent culture 
usually kills chickens within forty-eight hours. Some time before 
death the fowl falls into a somnolent condition, and, with drooping 


286 PROTECTIVE INOCULATIONS. 


“wings and ruffled feathers, remains standing in one place until it dies. 
Infection may also occur from the ingestion of food moistened with a 
culture of the bacillus or soiled with the discharges from the bowels 
of other infected fowls. At the autopsy the mucous membrane of the 
small intestine is found to be inflamed and studded with small hem- 
orrhagic foci, as are also the serous membranes; the spleen is notably 
enlarged. The bacilli are found in great numbers in the blood, in the 
various organs, and in the contents of the intestine. In rabbits death 
commonly occurs in from sixteen to twenty hours, and is often pre- 
ceded by convulsions. The temperature is elevated at first, but 
shortly before death it is reduced below the normal. The post-mor- 
tem appearances are: swelling of the spleen and lymphatic. glands; 
ecchymoses or diffuse ‘hemorrhagic infiltrations of the mucous mem- 
branes of the digestive and respiratory passages, and in the 
muscles; and at the point of inoculation a slight amount of inflamma- 
tory edema. The bacilli are found in considerable numbers in the 
blood within the vessels, or in that which has escaped into the tissues 
by the rupture of small veins. They are not, however, so numerous 
as in some other forms of septicemia—e.g., anthrax, mouse septi- 
cemia—when an examination is madeimmediately after death; later, 
the number may be greatly increased as a result of post-mortem mul- 
tiplication within the vessels. The rabbit is so extremely susceptible 
to infection by this bacillus that inoculation in the cornea by a slight 
superficial wound usually gives rise to general infection and death. 
‘This animal may also be infected by the ingestion of food contami- 
nated with a culture of the bacillus. It is by this means that Pasteur , 
proposed to destroy the rabbits in Australia, which have multiplied 
in that country to such an extent as to constitute a veritable pest. 
Both in fowls and in rabbits the disease may, under certain circum- 
stances, run a more protracted course—e.g., when they are inoculated 
with a small quantity of un attenuated culture. In less susceptible 
animals—guinea-pigs, sheep, dogs, horses—a local abscess, without 
general infection, may result from the subcutaneous injection of the 
bacillus; but these animals are not entirely immune. In the infec- 
tious maladies of swine, cattle, deer, and other large animals, to 
which reference has been made, and which are believed to be due to 
the same bacillus, the symptoms and pathological appearances do not 
entirely correspond with those in the rabbit or the fowl; but the ba- 
cillus as obtained from the blood of such animals corresponds in its 
morphological and biological characters with Pasteur’s microbe of 
fowl cholera, and Koch’s bacillus of rabbit septicemia, and pure 
cultures from the various sources mentioned are equally fatal to rab- 


PROTECTIVE INOCULATIONS. 287 


bits and to fowls. In the larger animals pulmonary and intestinal 
lesions are developed, and in swine a diffused red color of the skin, 
similar to that observed in the disease known in Germany as Schiein- 
erothlauf (Fr. rouget) is sometimes seen. 

According to Baumgarten, bacilli from Wildseuche or from Rinder- 
seuche inoculated into swine give rise to fatal Schiweineseuche, and 
bacilli from any of these forms of disease, when inoculated into pig- 
eons, produce characteristic fowl cholera; but the bacillus as obtained 
from Schweinesuche or TVildseuche is not fatal to chickens, and the ba- 
cillus from Schweineseuche is fatal to guinea-pigs, which have but 
slight susceptibility to the bacillus of rabbit septicemia. Notwith- 
standing these differences, he agrees with Hueppe in the view that the 
bacilli from the various sources mentioned are specifically identical; 
although evidently, if this view is adopted, we must admit that varie- 
ties exist which differ somewhat in their pathogenic power. 

In this volume this bacillus is described under the name Bac.7lus 
septicemice hemorrhagice, first proposed for it by Hueppe. In the 
present chapter we shall give an account of the experimental evidence 
relating to protective inoculations in various animals, with the differ- 
ent varieties of the bacillus in question which have been encountered. 

It seems probable that the same bacillus was the cause of the fatal 
form of septiceemia studied by Davaine, which resulted from the in- 
oculation of susceptible animals with putrefying blood. These ex- 
periments by the distinguished French physician constitute an im- 
portant part of the pioneer work in this field of research. They were 
commenced in 1868, and are published in the Budletin of the Academy 
of Medicine (séance of Feburary 18th, 1879). 

Davaine, in the paper referred to, calls attention to the fact, devel- 
oped by his experiments, that there is a great difference in the resist- 
ing power of different animals to the form of septicemia which lad 

“been the subject of his investigations. Thus the rabbit succumbed 
when inoculated with a millionth part of a drop of blood, while guinea- 
pigs and dogs remained unaffected by such small doses. With refer- 
ence to the specific cause of the form of septicemia studied by him, 
Davaine says: 

‘The virus is one of the bacteria of putrefaction. I say ‘one of the bac- 
teria,’ because there is reason to believe that there are among these minute 


organisms numerous species which do not all develop at the same time when 
they are present in various media.” 


Davaine also discovered the fact that infection depends, within 
certain limits, upon the quantity of bacteria introduced into the tis- 
sues. He says: 


288 PROTECTIVE INOCULATIONS. 


_.This question of quantity was manifest in our experiments. Not only 
did it vary in different species, the rabbit and the dog, for example, but it 
may vary in the same species.” 


The identity of “ Davaine’s septicemia” with Pasteur’s choléra des 
poules is made still more probable by the experimental evidence 
offered by Toussaint in a communication to the French Academy of 


Sciences, made by M. Bouley at the séance of July 25th, 1881. In 
this communication Toussaint says: 


‘“‘Three years ago, July 8th, 1878, I had the honor to present to the 
Academy an account of a malady due to microbes, which I identified with 
that studied by Davaine in 1864 and 1865, and which he differentiated from 
anthrax, for which it had been mistaken by Leplat and Jaillard. 

‘‘In the month of December, 1878, I made acquaintance with fowl 
cholera, and already, in my thoughts, I identified this disease with that 
which I had observed in my experiments made early in the year. The mi- 
crobes of the two diseases resembled each other perfectly and behaved the 
same when inoculated in rabbits. I had, even in 1879, sent to M. Bouley 
two notes, in which I called attention to the analogies which exist between 
the parasites of the two diseases and the lesions which they determine, not 
only in the rabbit but also in pigeons and fowls. 

‘‘The experiments of the same kind made at the end of 1879 and in 1880 
caused me to insert the note published on page 301, vol. xci., of the Comptes- 
rendus, under the title of - ‘Identity of Acute Experimental Septiczemia 
and Fowl Cholera.’ I gavea résumé in this note of five series of experi- 
ments which had demonstrated to me that inoculations of the microbe of sep- 
ticzemia give rise to the manifestations of fowl cholera. These results have 
recently been confirmed by additional facts.” 


Toussaint closes his paper by some remarks upon the origin of 
epidemics of fowl cholera, which we quote because we believe that the 
additions made to our knowledge of the microbe which causes this 
disease give support to the views advanced by him in 1881: 


““The causes which determine epidemics of fowl cholera are yet unknown. 
It has been supposed that putrefactive substances may give rise to them, and 
this has led to the recommendation of cleanliness and disinfection for their 
prevention. The microbe which kills the first fowl in an epidemic certainly 
came from some anterior generation which had killed others. But how was 
it perpetuated? Do not the facts which demonstrate the development of sep- 
ticeemia from material undergoing putrefaction throwsome light on the ques- 
tion of etiology? Isit not probable: that the fowls find the conditions of 
infection with cholera in the presence of organic matter undergoing putrefac- 
tion, which may serve as a culture medium for the germs of septicemia 
herr oo in suspension in the air togéther with the ordinary germs of putre- 

action : 


Pasteur’s first communication relating to the etiology of fowl 
cholera was made to the French Academy at the séance of February 
9th, 1880. In‘this communication he calls attention to the fact that 
when fowls are fed with bread or meat’ soiled with a small quantity 
of a culture of the microbe of fowl cholera they become infected and 
their discharges contain the bacillus in large numbers, a fact which 


PROTECTIVE INOCULATIONS. 289 


readily accounts for the spread of the disease in a poultry yard when 
a case occurs. 

In the same communication Pasteur records his observation that 
“by a certain change in the method of cultivation the infectious mi- 
crobe may be caused to have a diminished virulence.” Also the fact 
that fowls inoculated with this “attenuated” virus recover and are 
subsequently immune against infection by the most virulent microbes. 
In concluding this communication Pasteur says: 

“Tt appears to be superfluous to point out the principal result of the facts 
which I have had the honor to present to the Academy. There are two, how- 
ever, which it may be useful to mention. These are, first, the hope of ob- 
taining artificial cultures of all kinds of virus; second, the idea of seeking 
for virus vaccines of the virulent maladies which have devastated so often, 


and still devastate, the human race, and are such a scourge to that branch of 
agriculture which relates to the breeding of domestic animals.” 


In his communication of October 26th, 1880, Pasteur gives his rea- 
sons for concluding that attenuation of virulence is due to the action 
upon the microbe of atmospheric oxygen. He infers this from the 
fact, demonstrated by experiment, that when cultures are placed in 
hermetically sealed tubes, from which the oxygen present is soon ex- 
hausted by the growth of the microbe, they do not become attenuated 
in virulence; whereas cultures which are freely exposed to the air 
gradually become attenuated. Pasteur sees in this an important fact 
bearing upon the explanation of the natural extinction of epidemics. 
He says: 


‘‘May we not suppose, then, that it is to this influence that we must at- 
tribute, in the present as in the past, the limitation of great epidemics ?” 

In his communication to the French Academy, made on February 
28th, 1881, Pasteur treats of the attenuation of virulence by the method 
above referred to and by the method of Toussaint, and also of the re- 
establishment of the virulence of attenuated cultures. He says: 


‘¢The secret of the return to virulence rests solely, at present, upon suc- 
cessive cultures in the bodies of certain animals.” 


Thus he had found by experiment that the anthrax bacillus might 
be so attenuated that it was harmless for grown guinea-pigs, or even 
for guinea-pigs a month or a week old, but it would still kill guinea- 
pigs just born—a day old. By inoculating an older pig with the blood 
of this one, and so on, the virulence was gradually augmented, until 
finally a virus might be obtained which would kill adult animals, and 
even sheep. In the same way the attenuated microbe of fowl cholera 
could be restored to virulence by first inoculating small birds, such as 


sparrows or canaries. 
19 


290 PROTECTIVE INOCULATIONS. 


Applying these facts, demonstrated by his experiments, to the ex- 
planation of the origin of epidemics, Pasteur says: . 


‘‘T finished my communication on October 26th by calling attention to the 
attenuation of viruses by exposure to the air as being probably one of the fac- 
tors in the extinction of great epidemics. The facts presented in this paper, 
in their turn, may serve to explain the so-called ‘spontaneous development’ 
of these scourges. 

‘* An epidemic which has been extinguished by the attenuation of its virus 
may be reborn by the reinforcement of this virus under certain influences. 
The accounts which [ have read of the spontaneous appearance of the plague 
appear to me to offer examples of this; for example, the plague at Benghazi, 
in 1856-58, the outbreak of which could not be traced. The plague is a viru- 
lent malady which belongs to certain countries. In all of these countries its 
attenuated virus ought to exist, ready to resume its active form when condi- 
tions as to climate, famine, and distress again occur. There are other viru- 
lent maladies which appear ‘spontaneously’ in all countries ; such as camp 
typhoid. Without doubt the germs of the microbes which cause these last- 
mentioned maladies are everywhere distributed. Man carries them upon him 
or in his intestinal canal without great damage, but ready to become danger- 
ous, when, owing to constipation or to successive development upon the sur- 
face of wounds, in_ bodies enfeebled or otherwise, their virulency is pro- 
gressively reinforced.” 


We believe that the more complete our knowledge relating to the 
origin and extinction of epidemics, of the kind referred to by Pasteur, 
becomes, the more apparent will be the value of his inductions and 
the clearness of his scientific foresight. 

Toussaint, on July 25th, 1881, reported the results of his experi- 
ments upon protecting fowls by a “new method of vaccination.” This 
consisted in inoculating them with the blood of a rabbit which had re- 
cently died from septicaemia produced by the same microbe. Asa 
result of such inoculations the fowls had slight local lesions at the 
point of inoculation, and soon recovered. They were subsequently 
found to beimmune. Cultures from the blood of a septicaemic rabbit 
were found to act in the same way. When the culture had been passed 
through a pigeon, and had then killed a fowl, according to Toussaint, 
it preserved its virulence when subsequently passed through the 
rabbit. : 

Salmon, in the “Report of the Commissioner of Agriculture” for 
1881 and 1882, gives an account of his experiments in producing im- 
munity by the use of a diluted virus. He says: 

‘“The experiments of Chauveau, taken with my own, indicate that this 
method is capable of generalization to the same extent as that discovered by 
Pasteur: while the ease and quickness with which the vaccine is prepared, 
the certainty of effects, the economy of material, and the more perfect pro- 
tection are points which would appear to make it decidedly superior. 
Wherever the cholera of fowls is raging a standard cultivation may be made 
and the vaccine obtained within twenty-four hours ; a single drop of such a 


cultivation will vaccinate ten, twenty, or even forty thousand fowls, and 
within three weeks of the commencement of work the most susceptible of our 


PROTECTIVE INOCULATIONS. 291 


fowls are insusceptible to inoculation with the strongest virus. And this, 
without any sickness, or even local necroses, which Pasteur describes as fol- 
lowing vaccinations with his attenuated virus.” 


In discussing the practical value of this method Salmon estimates 
the cost as trifling—-“ not more than half a day’s time of one man for 
one hundred fowls, even if three inoculations were made.” 

In a paper on protective inoculations against fowl cholera, by Kitt, 
in the Deutsche Zeitschrift fiir Thiermedicin (December 20th, 1886), the 
conclusion is reached that these inoculations undoubtedly protect the 
fowls from infection either in the natural way or by inoculations with 
virulent material. But Kitt doubts the practical utility of the method 
for the arrest of epidemics of this disease in the poultry yard; and, 
as we think with justice, prefers to depend upon cleanliness, disin- 
fection, and prompt removal of infected fowls. As he points out, a 
considerable time is required to produce complete immunity, and two 
inoculations are often insufficient. Pasteur had previously reported 
that a third inoculation is usually required. But the infection 
spreads so rapidly when an epidemic is developed in a poultry yard 
that a large proportion of the fowls would be likely to perish before 
the protective inoculations could be carried out. Another objection 
is that when inoculated in the breast muscle the value of the fowl for 
the table is reduced, and when inoculated in the wing an unpleasant- 
looking scab is left at the point of inoculation. The cost in material 
and time required to carry out the three successive inoculations is 
also an objection to the practical application of the method. More- 
over, the excreta of the inoculated fowls contain the pathogenic mi- 
crobe, and it would evidently be unwise to practise inoculations in 
poultry yards not already infected. Kitt states also that he has 
always succeeded in stamping out the disease very promptly by the 
other measures referred to—disinfection, cleanliness, separation of 
all fowls which show any indications of being infected. 

In a more recent paper (1893) Kitt reports his success in confer- 
ying immunity upon fowls by a new method, which is, however, 
rather of scientific interest than of practical value. He first experi- 
mented to see whether the blood serum or tissue juices of immune 
fowls would give immunity against cholera to other fowls, and ob- 
tained a successful result. He was not, however, able to produce im- 
munity in pigeons or in rabbits by thesame method. He next under- 
took to determine whether the immunizing substance was present in 
the eggs of fowls which had an immunity as a result of protective in- 
oculations. The albumen and yolk of the egg, in doses of five to ten 
cubic centimetres, was injected into the breast of fowls, and at the end 


292 PROTECTIVE INOCULATIONS. 


of ten days a second inoculation of the same kind was made. Six days 
after the second inoculation the fowls (five) and a control hen were 
inoculated with virulent blood from a pigeon, and at the same time fed 
with the chopped-up flesh and liver of a pigeon just dead from fowl 
cholera. The control hen died on the following day from typical 
cholera, the others remained in perfect health. 


CHOLERA. 


The spirillum discovered by Koch in 1884 is now generally recog- 
nized as the specific cause of Asiatic cholera. But recent researches 
indicate that there are numerous pathogenic varieties of this spirillum, 
and show that either an attenuated cholera spirillum or a closely allied 
saprophyte is not infrequently found in the water of rivers in various 
parts of Europe. As this spirillum is found in the intestine of cholera 
patients, and not in the blood, it is evident that its pathogenic action 
depends upon the chemical products developed during its growth, 
and this inference is fully justified by the results of experiments upon 
the lower animals. These chemical products have been studied by 
Brieger, Pfeiffer, Scholl, Gamaleia, Westbrook, and others. 

Brieger (1887) succeeded in isolating several toxic ptomaines from 
cultures of the cholera spirillum, some of which had previously been 
obtained from other sources—cadaverin, putrescin, creatinin, methyl- 
guanidin. In addition tothese he obtained two toxic substances not 
previously known. One of these is a diamine, resembling trimethyl- 
diamine; it gave rise to cramps and muscular tremor in inoculated 
animals. The other poison reduced the frequency of the heart’s 
action and the temperature of the body in the animals subjected to 
experiment. In more recent researches made by Brieger and Frankel 
(1890), a toxalbumin was obtained from cholera cultures which, when 
injected subcutaneously into guinea-pigs, caused their death in two or 
three days, but had no effect upon rabbits. 

Pfeiffer has more recently (1892) published his extended researches 
relating to the cholera poison. He finds that recent aérobic cultures 
of the cholera spirillum contain a specific toxic substance which is. 
fatal to guinea-pigs in extremely small doses. This substance stands 
in close relation to the bacterial cells, and is perhaps an integral part 
of the same. The spirilla may be killed by chloroform, thymol, or 
by desiccation, without apparent injury to the toxic potency of this 
substance. It is destroyed, however, by absolute alcohol, by concen- 
trated solutions of neutral salts, and by the boiling temperature, and 
secondary toxic products are formed which have a similar pathogenic 


PROTECTIVE INOCULATIONS. 293 


action but are from ten to twenty times less potent. Similar toxic 
products were obtained by Pfeiffer from cultures of the Finkler-Prior 
spirillum and from Spirillum Metchnikovi. 

Scholl (1890) took advantage of the fact, previously demonstrated 
by Hueppe, that cultures of the cholera spirillum in egg albumen, in 
the absence of oxygen, are more toxic than ordinary bouillon cultures. 
Cultures were made by Hueppe’s method in hen’s eggs. No poison- 
ous ptomaines were found, but two toxic albuminous substances were 
obtained. The albuminous liquid from the egg cultures was dropped 
into ten times its volume of absolute alcohol, which caused a white 
precipitate, a portion of which sank to the bottom while another por- 
tion floated on the surface. The portion which floated was easily 
dissolved in a very dilute solution of potash and could be precipitated 
from this solution by the careful addition of acetic acid, but dissolved 
in an excess of this acid. It dissolved also in a seven-per-cent salt 
solution, but was precipitated by a saturated solution. It gave the 
biuret and xanthoprotein reaction. This substance proved to be very 
poisonous. It killed guinea-pigs within twenty minutes when a few 
cubic centimetres of the alkaline solution—potash—were injected into 
the cavity of the abdomen. Scholl calls this substance cholera-toxo- 
globulin. The precipitate which fell to the bottom of the receptacle 
was washed with alcohol, then digested with water for twenty minutes 
at 40° C. Very little was apparently dissolved out by this procedure, 
but this little proved to be very toxic. In from one to three minutes 
after the injection of a few cubic centimetres of the solution into the 
peritoneal cavity of a guinea-pig the animal died. This aqueous 
solution gave the biuret and xanthoprotein reaction; it was precipi- 
tated by mercuric chlorid, nitrate of mercury, and tannin, but not by 
a saturated solution of ammonium sulphate or acetic acid. This sub- 
stance Scholl calls cholera-toxo-pepton. The toxic action of these 
substances is destroyed by a temperature of 100° C., maintained for 
half an hour, or by 40° to 45° C., maintained for twenty-four hours. 
But at ordinary temperatures they retain their toxic action for several 
weeks. 

Gruber (1892) has also obtained a toxic albuminous precipitate by 
allowing egg cultures to fall into alcohol, drying the precipitate, and 
then extracting it with water. 

Gamaleia (1893) has obtained a toxin which produces the typical 
phenomena of cholera, which, according to him, is closely associated 
with the bacteria cells, but can be extracted by a soda solution or 
by heating to 55° to 60° C. The conclusion is reached that it isa 
nucleo-albumin analogous to the toxalbumins of tetanus and of 


294 PROTECTIVE INOCULATIONS. 


diphtheria. It is precipitated by alcohol, acids, and by magnesium 
sulphate. 

Finally, Westbrook, in a still more recent research (1894), arrives 
at the conclusion that the cholera spirillum produces various toxic’ 
proteids which in small amounts produce immunity in susceptible 
animals, and the production of which depends to a certain extent 
upon the culture medium; or that its toxin is a substance of constant 
chemical composition which is mixed with various albuminous sub- 
stances, either contained in the culture medium or developed in the 
culture. Duclaux is of the opinien that the last supposition is cor- 
rect, and that the so-called toxalbumins are not bodies of definite 
chemical composition, but mixtures of toxins and albuminous sub- 
stances. 

Experiments made upon the lower animals show that the intro- 
duction of these cholera toxins into the body of a susceptible animal, 
either with or without the living cholera spirillum, results in the 
establishing ci a certain degree of immunity against the toxic action 
of cholera cultures. And there is good reason to believe that a non- 
fatal attack of cholera in man gives the individual a relative immunity 
from subsequent attacks, for sometime at least. This has led to ex- 
tended experiments with reference to the possibility of producing a 
similar immunity in man by means of protective inoculations. The 
experiments bearing upon this point which have been made upon the 
lower animais will first engage our attention. 

Hueppe (1887) first demonstrated the fact that injection of a small 
amount of a cholera culture into the peritoneal cavity of a guinea-pig 
is fatal to these animals. 

In the following year (1888) Gamaleia reported his success in in- 
fecting guinea-pigs by subcutaneous injections of blood from an in- 
fected pigeon. He found that by successive inoculations in pigeons 
a considerable increase in virulence is established; and that while 
guinea-pigs were not fatally infected by subcutaneous inoculations 
with ordinary cultures, they invariably died when inoculated with the 
more virulent culture in the blood of an infected pigeon. Also, that 
when guinea-pigs were inoculated with ordinary cultures, or with 
cultures sterilized by heat, they were subsequently immune, and re- 
sisted inoculations with the most virulent material. In the same 
year the author referred to announced the discovery of a spirillum 
which closely resembles the cholera spirillum—his “ Vibrio Metch- 
nikovi.” This was obtained from the intestinal contents of fowls 
suffering from a fatal infectious ‘malady (in Odessa). According to 
Gamaleia, chickens and pigeons which have survived an inoculation 


PROTECTIVE INOCULATIONS. 295 


with a culture of this spirillum are subsequently immune against the 
pathogenic action of the cholera spirillum, and vice versa. In subse- 
quent communications Gamaleia reported that sterilized cultures of 
his “Vibrio Metchnikovi” (sterilized by heat at 120° C.) were very 
pathogenic for rabbits, fowls, pigeons, and even for dogs and sheep. 
The rabbit proved to be the most susceptible animal, and succumbed 
to doses of four cubic centimetres in from twelve to twenty hours. 
Doses of one cubic centimetre per one hundred grammes of body 
weight caused a temporary indisposition followed by immunity. 
Pigeons were made immune by larger doses. 

The researches of Pfeiffer (1889) confirmed those of Gamaleia as 
to the fact that pigeons and guinea-pigs could be made immune 
against Vibrio Metchnikovi by the injection of sterilized cultures. 
But guinea-pigs which had been immunized against this pathogenic 
spirillum succumbed to cholera infection; and, on the other hand, 
animals which had been treated in various ways with a cholera cul- 
ture died without exception when infected with Vibrio Metchnikovi. 
The conclusion is therefore reached that the two pathogenic spirilla 
are distinct species, although very similar in many respects. 

Brieger and Wassermann (1892) have reported the results of ex- 
periments with the cholera spirillum cultivated in thymus bouillon. 
After twenty-four hours’ development in this medium the cultures 
were sterilized by heat (55° C. for fifteen minutes) and placed in an 
ice-chest for twenty-four hours. Four cubic centimetres of this fluid 
injected daily for four days into the peritoneal cavity of a guinea-pig 
made it immune to the cholera spirillum in doses three times as large 
as were required to kill an animal not so treated. This immunity 
lasted for two months. Fedoroff (1892) obtained similar results by 
the subcutaneous injection of sterilized cultures in doses of one cubic 
centimetre, in guinea-pigs. His cultures in thymus bouillon were 
kept for from seven to ten days at 37° C., then sterilized by heating 
for fifteen minutes at 65° C., then allowed to stand in a dark room for 
twenty-four hours, and finally mixed with an equal volume of glycerin. 

‘Ketscher (1892) has obtained evidence that the immunizing sub- 
stance in animals which have received protective inoculations is con- 
tained in the milk of females thus treated. Three goats received 
subcutaneous inoculations of virulent cholera cultures, and also injec- 
tions into a vein and into the peritoneal cavity. The milk of these 
goats was injected into the peritoneal cavity of rabbits; these proved 
to be immune when subsequently lethal doses of a virulent cholera 
culture were injected into the peritoneal cavity. 

According to Gamaleia (1892), dogs are very susceptible to infec- 


296 PROTECTIVE INOCULATIONS. 


tion with cholera spirilla, and present symptoms closely resembling 
those of cholerain man. They may also be easily immunized against 
the pathogenic action of cholera cultures. 

Gruber and Wiener (1892) have also found that susceptible ani- 
mals are easily immunized against cholera infection either by inocu- 
lation with small doses, with attenuated cultures, or with larger quan- 
tities of sterilized cultures. Haffkine (1892) also reports his success 
in immunizing guinea-pigs and pigeons. 

Pawlowsky (1893) claims to have obtained from the blood of ani- 
mals having an acquired immunity against cholera an antitoxin in the 
form of an amorphous powder; and Lazarus (1892) reports that the 
blood of man, after recovery from an attack of cholera, has the prop- 
erty of protecting guinea-pigs from fatal infection when injected, in 
very small amount, into the peritoneal cavity. Issaeff (1894) in an 
extended series of experiments was not able entirely to confirm the 
results reported by Lazarus. In a summary of results obtained in 
his own experiments he says: 


“1. The intraperitoneal or subcutaneous injection of blood serum from 
normal individuals [that is, persons who have not suffered an attack of 
cholera], and also of various acids, alkalies, and neutral liquids, gives to 
guinea-pigs a certain resistance against intraperitoneal cholera infection. 
This resistance, however, is feeble and temporary, aud cannot be considered 
as identical with the true immunity which results from vaccination with the 
products of the cholera bacteria. 

‘*2. Guinea-pigs vaccinated against cholera have no immunity against 
the toxins of the cholera vibrio, notwithstanding their high degree of insus- 
ceptibility to infection with cultures containing the living vibrio. The blood 
of immunized guinea-pigs does not possess antitoxic properties. The maxi- 
mum dose of cholera toxins which immune guinea-pigs can withstand is not 
greater than that which control animals withstand. : 

‘3. The blood of guinea-pigs carefully immunized against cholera pos- 
sesses specific and very pronounced immunizing, and, in a certain sense, 
curative powers. 

‘‘4, The blood of cholera convalescents possesses similar specific and 
curative powers. This property is first developed about the end of the third 
week oo the attack, and disappears completely at the end of two or three 
months.” 


In a series of experiments made by Pfeiffer and Issaeff the results 
obtained, as stated by Pfeiffer in a subsequent communication, were 
as follows: 


‘‘In my research with Issaeff ‘upon the explanation of cholera immu- 
nity’ I proved that the serum of animals which have an active acquired im- 
munity against cholera only has a specific action upon this particular species 
of vibrio, and as regards other species of bacteria does not differ in its action 
from the blood serum of normal animals. We also showed that this specific 
influence in respect to the intraperitoneal cholera infection of guinea-pigs 
was due exclusively to bactericidal processes which in some way were in- 
duced by the serum of immune animals.” 


PROTECTIVE INOCULATIONS. 297 


The view of Pfeiffer, founded upon his experimental results, is 
that the destruction of the living cholera spirilla, which quickly takes 
place in the peritoneal cavity of the guinea-pig, when at the same 
time a minute quantity of serum from an immune animal is intro- 
duced, is not directly due to the bactericidal action of this serum, but 
that in some way it gives rise to a specific bactericidal action in the 
exudate which is found in the peritoneal cavity as a result of such in- 
jections. His experiments also lead him to the conclusion that this 
is accomplished quite independently of phagocytosis. 

The brief review of experimental researches relating to cholera 
immunity which we have made shows that, while there is a general 
agreement as to the possibility of producing immunity in susceptible 
animals, there is considerable difference of opinion as to the true ex- 
planation of this immunity. The supposition that it is due to an 
antitoxin which has the power of neutralizing the toxic products of 
the cholera spirillum does not receive any support from the most re- 
cent investigations—those of Pfeiffer and Issaeff—which, on the con- 
trary, seem to establish the fact that this immunity depends upon an 
increased bactericidal activity of the blood serum of immune animals. 
A very curious fact developed by the researches of the bacteriologists 
last named is that— 

‘‘The cholera serum which in the peritoneal cavity of guinea-pigs acted 
only upon the cholera bacteria, and behaved toward other vibrios exactly like 
the serum of normal animals, in a test tube killed all four species of vibrios 
with equal rapidity.” 

Unfortunately the evidence relating to the value of protective in- 
oculations in man, although supported by the evidence already re- 
ferred to as regards the lower animals, is, to a considerable extent, 
unsatisfactory, owing to the difficulty of applying scientific methods 
to experiments of this kind. The evidence, however, is in favor of 
the view that a certain degree of protection is afforded by the subcu- 
taneous injection of cholera cultures. Such protective inoculations 
could not be expected to confer an absolute immunity, inasmuch as 
the immunity resulting from a single attack has only a relative value, 
and is probably not of long duration. 

We quote from Shakespeare’s “Report on Cholera in Europe and 
India, 1890,” the following paragraphs relating to immunity as a 
result of an attack of cholera: 


“IMMUNITY AFTER AN ATTACK OF CHOLERA—EXPERIENCES IN 
FRANCE, 1884. 


“The Academy of Medicine of Paris directed a circular letter of questions 
concerning cholera to the physicians of the localities infected by that disease 


298 PROTECTIVE INOCULATIONS. 


in 1884, and in group L of general observations in that questonario is found 
the following: ‘Have there been observed recurrences among the people 
attacked, either in a former epidemic or in the present one? Give the results 
of this:recurrence.’ In response to their questions the Academy received 184 
communications, but the committee appointed to analyze them eliminated 
79; for various reasons given only 104 were used for analysis. Of this num- 
ber only 8 bore upon the particular question above mentioned, and it is 
reasonable to assume that the other 96 observers said nothing concerning this 
point because they had observed nothing bearing upon it. The results of 
this analysis may be stated as follows : 

“From Castelnaudary, with a population of 10,000, we learn that there 
were 54 cases and 18 deaths from cholera, among which there was 1 recur- 
rence; from Aix, with 20,257, number of cases unknown, deaths, 117, among 
these 2 recurrences were observed, at intervals of ten and forty days; from 
Beseges, with 11,400 inhabitants, we learn of 124 cases and 40 deaths, among 
which were 2 recurrences; from Cette, with 35,000, the number of cases is 
not mentioned, but we learn that there were 92 deaths and 1 recurrence; 
from Nantes, with 124,300 inhabitants we learn of 251 cases and 112 deaths, 
with 1 recurrence ; from Perpignan, with 25,000 inhabitants, we hear of 325 
cases and 225 deaths, and receive the indefinite statement that there were 
some fatal recurrences ; from Pignans, population not stated, we learn of 22 
attacks and 12 deaths, with 1 recurrence ; from Cadenet, with a population 
of 26,000, we are not informed of the number of cases, but learned that there 
were 20 deaths and 2 recurrences.” 


“IMMUNITY AFTER AN ATTACK OF CHOLERA—EXPERIENCE IN 
SPAIN, 1885. 


“While examining cholera in Spain, the writer prepared a circular con- 
taining a series of twenty-five questions relating especially to the nature, eti- 
ology, and prophylaxis of cholera, one of which requested the physician to 
state whether or not, in his own personal experience, he had observed a sec- 
ond or a third attack of cholera during the same epidemic, and in case of a 
positive reply to detail the symptoms and all the circumstances surrounding 
it. This circular-letter was addressed to some twenty-five hundred Spanish 
physicians, located in the various cities, towns, and villages in that kingdom 
which had suffered from the epidemic. Among the large number of replies 
there were only eight in which a second attack was reported, and from an ex- 
amination of the details of these there was no doubt left in our mind that six 
were not genuine second attacks after a complete recovery, but were in 
reality relapses due to imprudences of diet or otherwise before convalescence 
and complete recovery had been established. Two of the eight cases, from the 
details of the reports given, may have been genuine recurrent attacks of 
Asiatic cholera, or may have been simply seizures of cholera morbus (cholera 
nostras). It is well known that after an attack of Asiatic cholera the di- 
gestive apparatus is left ina damaged condition, and disorders of the in- 
testines continue for a long time. The habits of life and the imprudences so 
common to theclass of people most frequently suffering from Asiatic cholera 
in that country are such as to render them more than usually liable to suffer 
attacks of cholera nostras. As having an important bearing upon this sug- 
gestion, the writer made an analysis of the vital statistics of Spain, covering 
the five years previous to 1885, for the purpose of learning the extent of 
prevalence of cholera nostras among that population, and the result of the in- 
quiry shows that the number of deaths attributed to that disease averaged 
per year sixteen per every million inhabitants.” 


Dr. Ferrén, who practised inoculations on an extensive scale dur- 
ing the epidemic of 1885, in Spain, gives the following account of his 
method of performing these inoculations: 


PROTECTIVE INOCULATIONS. 299 


‘‘1. The cholera vaccine is nothing more than a pure culture, in bouillon, 
of the comma bacillus. Its easy and long preservation (four to five days) 
allows of its transportability to great distances, taking care always to keep 
the flask which contains the material upright. 

‘*2. Heat and cold do not interfere with its preservation if the vaccine is 
to be used in a short time. It should not, however, be kept out of doors dur- 
ing the warm season. 

‘*3. The vaccine should be kept in flasks of the model of Ferran, with a 
flat bottom and a short neck. The stopper, which is of rubber, fits perfectly, 
and is penetrated by two glasstubes. One} straight and short, which does not 
extend below the inferior surface of the stopper, and which does not project 
above more than some two centimetres, is plugged with a small quantity of 
sterilized cotton and a superficial covering of wax. The other glass tube is 
longer, and extends on the lower side as far as the bottom of the flask, while 
its superior end is curved, and terminates in a capillary extremity, the tip of 
which is closed with wax. 

‘*4, When the vaccine is to be used 1t is necessary to make two principal 
preparations for the operation. A smallsyringe for the hypodermic injection, 
anda small vessel into which it is necessary to empty the fluid from the flask, 
are required. The syringe should have metallic pistons and mountings, 
without mastic of any kind and without rubber. Its capacity shou!d be one 
cubic centimetre, its needle thicker and shorter than that of ordinary use. 
Before beginning the vaccination the syringe must be filled two or three 
times with boiling water, which is aspirated and expelled through the needle. 
This is called sterilizing the instrument, and by this means the extraneous 
germs are destroyed which might be contained in it, in order to avoid the 
production of phlegmons and abscesses. The trouble in taking this precaution 
will be little. Acting thus, one may perform thousands of injections without 
fear of any accident. It is suggested that it is a bad custom to pass the nee- 
dle through a flame in order to sterilize it, because this mode of procedure 
draws the temper. Another precaution that must be taken relates to the 
examination of the syringe before using it, in order to be well assured that the 
piston acts perfectly and that not a single drop of the liquid escapes by a leak 
in thecannula. This latterdefect is sufficient to rejecttheinstrument. If the 
syringe aspires air because the leather washer, which is placed at the end of 
the glass tube in order to facilitate its adaptation, is dry, or the piston is in the 
same condition, it is necessary to delay a little while in order to take the 
syringe apart and soak it in warm water. It is convenient to keep several 
syringes for use, with a sufficient number of needles, when many inocula- 
tions are to be performed. 

“5. The small receptacle into which the vaccine is poured in order that 
the syringe may be filled readily is a capsule, a cup, or some similar vessel. 
Before use, it should be washed and dried with extreme care, and imme- 
diately before using passed through an alcohol or Bunsen flame, in order to 
sterilize it. 

‘6. All these preparations having been made, the drop of wax which 
closes the capillary extremity of the long tube of the flask is removed, and at 
the same time also the wax covering of the cotton stopper of the short tube, 
but by no means must this cotton stopper be removed ; a rubber tube, or the 
extremity of a small Richardson spray apparatus, is adjusted to the short 
tube. The capillary extremity of the long tube is now slightly warmed in 
order to soften somewhat the wax which may have been drawn into its 
lumen by capillarity, and air is forced into the flask, either by blowing into 
the rubber tube or by working the Richardson atomizer ; the air injected by 
pressure upon the vaccine fluid forces the latter out through the long tube 
with the capillary extremity, and it is collected in the cup or small sterilized 
vessel. This latter is then covered with white paper, which has been 
scorched in the flame, or with a sterilized glass plate ; as often as the syringe 
is filled this cover will be removed and again immediately afterward replaced. 


300 PROTECTIVE INOCULATIONS. 


“7, Never should the rubber stopper which closes the flask, or the cotton 
which plugs the short straight tube, be removed, because otherwise the 
germs of the external air might enter and contaminate the culture, and in 
this way give place to local and general accidents among the inoculated. 
Whenever, through the movements of transportation, the cotton plug in the 
short glass tube has become so wet as to impede the passage of the air which 
is to be forced into the flask in the act of expelling the vaccine from it, it 
may be removed with the point of a needle and rapidly substituted by an- 
other plug of surgical cotton which has been carbonized or salicylized. If 
this proceeds with cleanness and promptness, there is no danger in doing it. 
When the cotton, although wet, does not impede the injection of the air, it is. 
better not to change it. ' 

‘‘8. After terminating the vaccination, again the capillary extremity of 
the curved tubeis passed through the flame until the small quantity of liquid 
remaining in it is evaporated ; it is then stopped a second time with a small 
drop of wax; and from the other glass tube the rubber tube which has been 
employed for forcing in the air is removed and another thin layer of wax is 
placed over the cotton plug. 

‘*9, If in the smaller vessel or cup any of the vaccine fluid remains after 
the vaccination of all persons present, it is boiled, and in this manner the 
culture is killed, for it should not be used in another operation, because at- 
mospheric germs might become mixed with it. 

‘10. The technique for the practice of the inoculation is the same as for all 
hypodermic injections. The most convenient region is that of the brachial 
triceps. 

tT, The dose is one cubie centimetre—or the contents of a syringe—into- 
each arm, for individuals of all ages and conditions. 

‘12. Five days having elapsed, revaccinations may be performed by fol-. 
lowing the same instructions.” 


Shakespeare, who was sent by the United States Government to: 
Spain to investigate the results of these inoculations, reports as fol- 
lows: 


‘*And now with respect to the human inoculations: The most of these 
inoculations were performed in villages in the province of Valencia. The 
number of persons inoculated considerably exceeds thirty thousand. Much 
has been both said and written in Spain, France, and England concerning 
the results of these inoculations. The results which have been published 
have appeared to very strongly back up the claim of Dr. Ferran that chol- 
eraic inoculation has the power of protecting the individual against an at- 
tack of cholera, and that the extensive practice of this inoculation among 
villages already invaded by the epidemic is a powerful and at the same time 
harmless means of bringing the epidemic to an end. This being the case, 
for those who were unwilling to accept the deductions to be made from the 
published statistics the only way of escaping their force seemed to be by 
an attack upon their validity. 

‘‘The statistics of the anti-choleraic inoculations have been widely at- 
tacked. The first public onslaught upon these statistics of which the world, 
outside of Spain, had much knowledge was made in the report of the French 
Commission, with Dr. Brouardel at its head, which was presented to the 
Minister of Commerce after the return of that Commission from Spain in the 
summer of 1885. It is charged in that report that the results of the statistics 
therein reproduced are assailable on account of having been collected by 
physicians who were partisan supporters of Dr. Ferran, and that they neither 
possessed any adequate official character nor did they possess sufficient de- 
tails. As far as I can learn, the general impression entertained throughout. 
the world of the value of inoculation statistics is based, in the main, upon 
this report of the French Commission. 


PROTECTIVE INOCULATIONS. 301 


‘‘The statement of that Commission that the statistics which they had 
been able to obtain of the preventive inoculations of Ferran were to a con- 
siderable degree void of any official character may be true, and perhaps it is 
also true that they emanated from the partisan friends of Ferrdn ; but it must 
be distinctly remembered that at that day there were practically no official 
statistics of this kind in the hands of any one. The official statistics collected 
under the orders of the Spanish Government were gotten together at a far 
later date. 

“Upon the appointment of the Government at Madrid of the second offi- 
cial Spanish Commission to investigate the Ferrén question in the provinces 
where the inoculations were being practised, it was ordered that official sta- 
tistics of inoculation should be collected in the usual manner; that is to 
say, by the customary statistical officers of the Government. This second 
medical Commission was also accompanied by an independent statistical 
commission who were charged with the duty of forming statistics of those 
inoculations which were expected to be witnessed by the Medical Commission 
in their tour of investigation, and the report to the Spanish Government of 
this statistical commission is based exclusively upon the official statistics 
which they themselves collected. 

‘In estimating the value of the official character and the authority of the 
official statistics, which have since the visit of the French Commission to 
Spain been collected and published, the following circumstances should be 
taken into account: The provincial governments of Spain are somewhat 
peculiar, in that the civil governors change with the changes which take 
place in the Government at Madrid, so that the political constitution of the 
provincial governments is always a reflex of that of the central government 
at Madrid. Moreover, the political sentiment of the provincial government 
is also more or less perfectly reflected by the local governments of the towns 
of the province. 

‘‘The hostility of the Minister of the Interior at Madrid to Dr. Ferran, and 
his attempts at the prevention of cholera by inoculation, is a well-known fact 
now generally admitted ; and the hostility which Dr. Ferran met with from 
the civil governor of the province of Valencia was even greater than that 
manifested by the Minister of the Interior himself. 

“The official statistics of the Ferr4n inoculations are in the first place 
signed by the physicians of the locality ; and in the next place by the judge 
of the municipal court, and sometimes also by the president judge of the 
judicial district, by the parochial priest, and by the mayor of the municipality, 
whose signatures and seals are attested by an authorized notary public. 

“Tt must, therefore, be obvious that the charge made by the French Com- 
mission, which has been so constantly reiterated everywhere, that the public 
statistics of the anti-choleraic inoculations are void of official character and 
are to be regarded as ex-parte testimony of the partisans of Ferran, cannot 
apply to official statistics which were collected under the supervision of the 
municipal authorities of the villages wherein the inoculations were per- 
formed, and attested not only by the local judicial officers and the parochial 
priests, but also by the political officers—that is to say, the secretaries and 
the mayors of the municipalities ; for it must be admitted that neither the po- 
litical officers of the municipalities nor of the provincial governments, any 
more than the parochial priest, can reasonably be charged with being the 
partisans or friends of Ferran—the Minister of the Interior continuing dur- 
ing the time of collection of these official statistics to be hostile to the claims 
of Ferran. It therefore follows that the attack upon the statistics of the in- 
oculations made by the French Commission, and so widely accepted by the 
medical world as conclusive, does not apply to the official statistics of which 
we are speaking. And, in view of this fact, the evidence as to the efficiency 
and harmlessness of the anti-choleraic inoculations should be re-examined. 
As I have already said, the results of the preventive inoculations of Ferran 
as set forth in the official statistics appear to very strongly support his claim 


802 PROTECTIVE INOCULATIONS. 


of the protective value of the inoculations. In view of the great importance 
of this whole subject, I have determined to place these statistics in this report 
for the benefit of the readers of the English language, in order that they may 
judge for themselves of the facts as they appear to be recorded. 

‘‘From the Government statistics of cholera throughout the province of 
Valencia, it appears that among the villages invaded there were 62 attacks 
per one thousand of the population, and 31 deaths per thousand, which gives 
a mortality of 50 per cent of those attacked. It appears from analysis of 
the published official statistics of cholera in 22 towns where inocula- 
tion was performed the inhabitants were divided as follows: 104,561 not 
inoculated ; 30,491 inoculated. Of the latter there were 387 attacks of cholera, 
or 12 per thousand, and 104 deaths, or 3 per thousand ; the mortality of those 
attacked being 25 per cent. Of the former there were 8,406 attacks, 
or 77 per thousand, and 3,512 deaths, or 33 per thousand, being a mortality 
of those attacked of 43 per cent. It appears, therefore, that among 
the population of villages wherein anti-choleraic inoculations had been more 
or less extensively performed the liability of the inoculated to attacks of 
cholera was 6.06 times less than that of the non-inoculated, whilst the liability 
of the inoculated to death by cholera was 9.87 times less than that of the non- 
inoculated. These figures are based exclusively upon the data furnished by 
inoculations, the reinoculations being left out of consideration, because they 
are much less numerous, although from the records of the inoculations it 
would seem that the liability of attack, and especially of death by cholera, is 
many times less among them than among those inoculated a single time. 

‘*The charge has also been made with respect to the published records of 
the inoculations that the hygienic and physical condition of the subjects of 
inoculation have not been sufficiently indicated in the records, and that the 
vast majority of those profiting by the opportunity to receive the anti- 
choleraic inoculations were of the middle and upper classes, and therefore 
not of that class of inhabitants who are notoriously most liable to attack and 
death from cholera. This criticism may have some justness as respects some, 
perhaps many, of the villages where inoculations were performed ; but there 
are certainly many of the villages wherein the results of the inoculation 
seemed to be most positively in favor of the claim of Ferrdn where this 
criticism cannot hold. I refer to villages wherein three-fourths or four- 
fifths of the inhabitants were inoculated, leaving only the fraction of the 
population non-inoculated. Even in the absence of any special notes indi- 
cating the social conditions and hygienic surroundings of the inoculated in 
these villages, it is ridiculous to assume that the vast majority of these were 
people of the middle and upper classes, and were therefore but little liable to 
attack and death by cholera. Any one acquainted with the character of the 
Spanish population as it exists in the rural villages will admit at once that. 
the vast majority of this population consists of the wretched and the poor, 
who live under the most unhygienic and unsalubrious conditions, and there- 
fore are of that class most liable to suffer from cholera. 

‘‘There is still another result of the preventive inoculations of Ferran 
apparently shown by these statistics. I refer to the apparent marked short- 
ening of the course of the epidemic after a large percentage of the inhabitants 
had become inoculated. It would seem, therefore, from analysis of the 
official statistics, that the practice of the anti-choleraic inoculation after the 
method of Ferran, besides giving the subject inoculated a considerable im- 
munity from attack and death by cholera, furnishes a means of bringing an 
epidemic rapidly to an end.” 


With reference to Haffkine’s method of inoculation we cannot do 
better than to quote from a lecture which he gave in London, in 1893: 


‘In the research that I have done at the Pasteur Institute on vaccination 
against Asiatic cholera I have chosen for my starting-point the inoculation 


PROTECTIVE INOCULATIONS. 303 


of the animal into the peritoneal cavity. Starting from this point I have 
worked out a method which permits the culture of the microbe in the animal 
organism in a state of purity during indefinite generations, the exaltation of 
it to a well-determined maximum of strength, and keeping it at the same 
degree of virulence for an unlimited period of time. 

‘This method is illustrated by three series of experiments which were the 
subject of our publications in the Comptes rendus de la Société de Biologie 
of Paris, and which are: 

“1, Giving the first animal a dose larger than the fatal dose, and killing 
this animal in a sufficiently short space of time to be able to find the more 
resisting microbes. 

‘*2. To expose the exudation taken from the peritoneal cavity to the air 
for several hours. 

‘*3. Then to transfer this exudation to the next animal, of large or small 
size, according to the concentration of the exudation. : 

‘‘In the hands of a number of other experimenters this method has given 
the same results and showed a pertect consistency. 

‘‘The properties of the virus which is obtained in this manner of cultiva- 
tion are as follows: Upon intraperitoneal inoculation it kills guinea-pigs 
regularly in the space of about eight hours, and the fatal dose for this animal 
is reduced to about twenty times less than that which it would have been 
necessary to take for the microbe with which I started. The same inocula- * 
tion kills rabbits and pigeons with a dose which would have been perfectly 
harmless at the beginning of the experiments. It kills guinea-pigs by intra- 
muscular inoculation. 

‘‘The subcutaneous inoculation brings about the formation of a large 
cedema, which tends toward sequestration of a whole part of the cutaneous 
tissues and to the formation of a wide open wound, which is cured in from 
two to three weeks. 

‘‘The basis of anticholeraic vaccination is founded on the virus obtained 
in the manner we have just described. 

“‘This virus, injected under the skin of a healthy animal, gives it, after 
several days, immunity from all choleraic contamination, in whatever man- 
ner this may arise; that is to say, if an animal that has been thus treated be 
taken, and an attempt made to infect it either by the digestive canal, by 
neutralization of the gastric juice and the injection of opium into the peri- 
toneum, or by the introduction of the microbe into the intestines by the 
method of Nicati and Rietsch, or by intramuscular inoculation, or finally, 
by intraperitoneal injection, the most terrible of all, it resists, whilst the con- 
trol animals succumb. 

“‘ Anticholeraic vaccination of animals in this manner is then definitely 
established. But the operation described cannot be, such as it is, applied to 
man. The wound following on the subcutaneous inoculation is terrible to 
look at, and, in all probability, extremely painful. Besides, although it 
does not in itself present any danger to the health of the individual, it exposes 
him to all the complications inseparable from an open wound. 

“This power of producing necrosis of the cutaneous tissues has been 
removed from the exalted vaccine by cultivating it at a temperature of 39° 
C., and in an atmosphere constantly aérated. nder these conditions the 
first generations of the cholera microbe would die rapidly, in an interval of 
two to three days, and therefore care must be taken to sow them again in 
new media immediately before death, and after a series of generations of this 
kind a culture is obtained which, if injected under the skin of animals, even 
in exaggerated doses, produces only a passing cedema, and prepares the 
organism in such a, manner that the injection of exalted virus, the definite 
vaccine, only produces a local reaction of the slightest description. 


304 PROTECTIVE INOCULATIONS. 


“VACCINATION BY FIXED VACCINE. 


‘“The method of vaccination thus worked out comprises, then, two vac- 
cines—a mild vaccine, obtained by weakening the fixed virus; and a 
strengthened vaccine, which is presented by the virus itself. It is easy to 
understand why, to obtain the weakened vaccine, we do not use an ordinary 
virus, but a virus the nature of which has been previously fixed in the labor- 
atory. It is because the virus, such as is found in the natural state, especially 
when it has a saprophytic phase of development, presents such pathogenic 
differences that there is no certainty in its application. Respecting this we 
need only recall the story of variolization, and the great danger that an indi- 
vidual incurred when the infectious substance from a slightly attacked sub- 
ject was transferred to him. The mildness or the gravity of an infection does 
not depend only on the veritable strength of the contagious substance, but 
upon the resistance of the individual from whom it is taken. Thus it hap- 
pened that in taking vaccine lymph from a subject lightly affected, a very 
weak substance was sometimes produced, which was incapable of producing 
a protective action ; and sometimes a lymph of such strength that it killed 
less resistant individuals. The great benefit of Jenner’s discovery lay in 
that it precisely indicated a substance fixed by passages through animals, and 
of a virulence below that which is fatal to the human organism. Another 
example is given in the method of Toussaint of vaccination against anthrax, 
the first of its kind, which has been obliged to make way for the method of 
M. Pasteur, for the sole reason that the latter, based upon virus of a fixed 
nature, presented an absolute certainty in its results which was wanting in 
the other. Finally, in the history of cholera itself I may recall the attempt 
made in 1885 by Dr. Ferran, of Barcelona, who, with the object of preserv- 
ing the population of the Peninsula from the epidemic of cholera, made 
injections in his patients of the ordinary virus taken from dead bodies and 
cultivated in the laboratory. The statistics of the results obtained by this 
means showed such uncertainty that no one dared to recommend this opera- 
tion to his country in spite of the very numerous trials made in Spain. 

‘“The possibility of treating the animal organism by vaccines of an abso- 
lutely fixed nature, prepared by means of special operations, constitutes, on 
the contrary, the basis of the Pasteurian method, and here lies the whole 
secret and the sole guarantee of the success of its application. 


“APPLICATION OF THE METHOD TO MAN. 


‘‘The method of anticholeraic vaccination, worked out by experiments 
on guinea-pigs, was tried upon rabbits and pigeons before it was applied to 
man. These animals were chosen in order to have subjects very differently 
organized, and in order to be able to generalize the conclusions, and to be 
able to extend them to the human organism. 

‘‘The result obtained on all these animals being absolutely the same, it 
was decided to apply the operation to man. 

‘“The symptoms produced by this operation have been described in several 
scientific magazines. The method has been tried at Paris, Cherbourg, and 
at Moscow, on about fifty persons of both sexes, between the ages of nineteen 
and sixty-eight, of French, Swiss, Russian, English, and American nation- 
ality. 

‘Tn every case the method has shown itself absolutely harmless to health, 
and the symptoms that it evoked were a rise in temperature, a local sensitive- 
ness at the place of inoculation, and the formation of a transitory cedema at 
the same place. The first sensations are felt about two or three hours after 
‘inoculation ; fever and general indisposition disappear after twenty-four to 
thirty-six hours ; the sensitiveness and cedema last, gradually dying away in 


PROTECTIVE INOCULATIONS. 805 


from three to four days. The symptoms following the second inoculation 
were generally rather more marked, but of shorter duration. The whole 
recalls the sensation of a bad cold in the head, lasting about one or two days. 

‘‘The microbes introduced under the skin do not propagate, but after a 
certain time they die and disappear. Itis the substances which they contain, 
and which are set free when they die, that act upon the animal organism and 
confer immunity upon it. It is found that the same result can be obtained if 
the microbe be killed before inoculation, and if their dead bodies only be in- 
jected. Thus I have been enabled to prepare vaccines preserved in weak 
solutions of carbolic acid. In this the microbes die at the end of several 
hours, and the vaccine so prepared has been found still efficacious six 
months after its preparation. It is evident that there is much advantage in 
this state of preservation of the microbes. They can be used by persons 
having no bacteriological training, and the absence of every living organism 
makes them perfectly safe. The carbolic acid that they contain preserves 
them against any invasion of other microbes. Finally, as they can be kept 
for several months, their preparation can be entrusted toa central laboratory, 
whence the vaccine ampoules can be sent out to operators. But it may be 
presumed that immunity given by these preserved vaccines will not equal in 
persistency that produced by living ones, and as the method is not yet backed 
up by established statistics, it is better that vaccinations should be done as 
much as possible with living virus, so as to obtain the most conclusive 
results. 

“As to the length of time that immunity produced by living vaccine 
lasts, we have not yet at the laboratory animals that have been inoculated at 
avery distant date; those upon which we experimented dated from, at most, 
four months and a half. At the end of this time their immunity was found 
to be still perfect, and we do not despair of its lasting much longer yet. 


“HARMLESSNESS OF THE METHOD. 


“The inoculations upon man, added to the hundreds of experiments that 
we have made upon animals, testify to the perfect harmlessness of these 
operations, and there is no difficulty in proving their efficacy by experiment, 
no matter on what species of animal. We have taken twelve guinea-pigs, 
and vaccinated six of them with vaccines preserved in carbolic acid since 
September 8th last. Yesterday, at five o’clock, six days after the first vacci- 
nation, we injected into the peritoneal cavity of all the non-vaccinated ani- 
mals a fatal dose of virus, and into the vaccinated animals we injected a 
double dose. The six vaccinated animals are perfectly well, while of the 
others two have already died of choleraic poisoning, two are very ill, and the 
others will certainly soon become so. But it is evident that I cannot perform 
a like experiment on man (but, however, this would be the only means of 
being able to give a definite experimental demonstration).” 


Further details as to the method are given by Woodhead in the 
“Edinburgh Hospital Reports,” as follows: 


‘‘TIn order to be absolutely certain that the virus is pure, M. Haffkine 
makes cultivations before each inoculation of the human subject, by Roux 
and Yersin’s method, one devised for the separation of the diphtheria bacillus. 
A small drop of the virus exalté is taken on a spatula-shaped needle, and 
streak after streak is made with the flat of this needle on the surface of the 
agar in the tubes, a couple of tubes being used, so that twelve streaks per- 
haps, in all, are made without the needle being recharged; in the earlier 
streaks, of course, the seed bacilli are so close together that a continuous line 
of colonies makes its appearance ; but along the course of the later streaks, 
colonies, with distinct intervals between them, are developed ; part of one of 
these is examined under the microscope, in order to determine that it is made 


20 


306 PROTECTIVE INOCULATIONS. 


up only of comma bacilli, and then the other part is used for seed material 
for a tube culture preparatory to inoculation. 

‘‘The inoculation itself is an exceedingly simple process; the needle and 
the syringe are boiled; the tube containing the material to be used for inocu- 
lation receives a syringeful or pipetteful of sterilized beef broth, then with a 
platinum needle the culture is thoroughly mixed with this broth, so that a 
kind of emulsion is prepared; this emulsion is drawn up in a sterilized 
pipette, and is then passed into a sterilized conical glass covered with steril- 
ized paper. Ifasixth of the culture is to be introduced, two more syringe- 
fuls or pipettefuls of broth are to be added, so that we now have three in all ; 
if an eighth, three are added, and so on; the whole is mixed, and then half a 
syringeful is taken for use for each patient. In inoculating, the skin, just 
shave the crest of the ilium, is thoroughly cleansed with five-per-cent solu- 
tion of carbolic acid, the attenuated virus is inoculated on the left side, and 
then after an interval of four or five days the second vaccine, or the more 
virulent form, is inoculated on the right side. After inoculation everything 
that has been used is thoroughly boiled, the skin of the patient is again 
washed with five-per-cent carbolic acid, and the table is washed down with 
the same solution.” 


Haffkine commenced his experiments on man by inoculating him- 
self, and has repeated the inoculation three times. He next inoculated 
about fifty individuals in Paris, Cherbourg, and Moscow, and demon- 
strated in a satisfactory way that the inoculations are without danger. 

A first inoculation in an unprotected person is said to give rise to 
some malaise and febrile reaction, to pain and tumefaction at the 
point of inoculation, and swelling of the neighboring glands. The 
second inoculation with a strong virus, made after an interval of six 
days, causes also some elevation of temperature, but no swelling at 
the point of inoculation. This slight reaction from a strong virus is 
supposed to be satisfactory evidence of a certain degree of immunity 
as a result of the first inoculation. 

The results of the protective inoculations by Haffkine’s method, 
which have been practised in India, indicate that these inoculations 
have a real value, but that immunity is not immediately established, 
and consequently that during an epidemic a certain number of fatal 
cases may be expected among the inoculated as well as among the 
non-inoculated. This is illustrated by the results of inoculations 
made among the prisoners in Gaya jail (1894), reported by Surgeon- 
Major Macrae, I.M.S., from whose report we quote as follows: 

‘*Cholera broke out in Gaya ul on the 9th of July, and from that date 
until 2d August 34 cases occurred, with 20 deaths, there being on date of first 
attack 422 prisoners in jail. The disease was clearly traceable to importa- 
tion, but its diffusion among the prisoners was a question of much greater 
difficulty. The sanitary condition of the jail is excellent; it was built quite 
recently, on the latest plans, and is generally considered a model jail. The 
water supply, which is from a well, is of excellent quality and protected 
from pollution, and it is believed that the spread of the disease was largely 
due to the agency of flies finding access to food and milk after being in con- 


tact with cholera poison, and contaminating them. From the 9th to the 17th 
July six cases occurred, with five deaths. 


PROTECTIVE INOCULATIONS. 307 


‘‘Many of the prisoners on being told about preventive inoculation wished 
to be inoculated, and M. Haffkine, who had previously been communicated 
with, and whose zeal and enthusiasm in the cause that he so well advocates 
are beyond praise, arrived here on the 18th July, and in the presence of Sur- 
geon-Colonel Harvey, who kindly assisted, and myself, inoculated 147 
prisoners, and on the 19th 68, making a total of 215 out of 433 present in the 
jail on that date. 

‘‘Being purely voluntary, no selection of prisoners was possible ; but all 
classes in the jail were represented, male and female, old and young, habit- 
uals and less frequent offenders, strong and weakly, convalescent and even 
hospital patients sent their representatives. No difference of any kind was 
made between inoculated and non-inoculated prisoners; they were under 
absolutely identical conditions as regards food, water, accommodation, etc., 
in short, in every possible respect. 

‘As, owing to the progress of the epidemic, a large number of prisoners 
were removed from the jail into camp, it will be found convenient to con- 
sider the effect produced by the anticholera inoculation under three head- 
ings: : 

‘*(a) The first will include the period from the 18th July, the date of first 
inoculations, to the 24th July, the date on which final reinoculations were 
made, and refers to all the prisoners. ; 

‘*(b) The second concerns the prisoners who remained in jail after the 
majority were removed into camp, and comprises the period from 25th July 
to 2d August, on which date the final case occurred among this body of 
prisoners. 

‘‘(ce) The third refers to the body of prisoners who were moved into camp 
on 25th July, and includes the period between that date and ist August, 
when the final case occurred among this body. ‘ 


Percentage Percentage Percentage 
A Chol- eee 
Plena | Sea an | eee eee | Pade te 
* No. 1 
Tnoculated............. 211.2 5 2.37 4 1.89 80.0 
Not inoculated......... 209.0 7 en 5 2.39 71.42 
No. 
{noculated............. 32.5 a “3.07 vil, Nil. Nil. 
Not inoculated......... 48.55 7 1if42 / 3 6,18 42.86 
‘s No. Ill 
Inoculated............. 171.42 2 _ 116 1 0.58 50.0 
Not inoculated......... 146.5 6 4.09 2 1.36 33.33 


‘*The conclusions to be drawn from the results above recorded appear to 
me to be that for the first few days the inoculations have scarcely any protec- 
tive influence ; then their effect seems to gradually increase. M. Haffkine in 
his publications has laid stress on the fact that he anticipates a period of ten 
days would elapse from date of first inoculations before the full effect would 
be obtained. 


DURING THE FIRST FIVE || Frrsv THREE DAYS AFTER : 
DAYS AFTER FIRST INOC- SECOND INOCULATION, LAST SIX DAYS. 
ULATION. ; 
Cases. Deaths. Cases. | Deaths. Cases. | Deaths. 
Inoculated...... 5 4 3 1 Nil. Nil. 
Not inoculated... 7 5 5 3 8 2 


308 PROTECTIVE INOCULATIONS. 


‘‘ Further observations are necessary to prove whether the inoculations as 
now practised will prove of lasting benefit ; the results obtained in Gaya jail 
seem to me to justify the conclusion that their temporary beneficial effect is 
undoubted. 

‘‘T have been informed by M. Haffkine that he proposes to introduce a 
certain modification of his method, with the object of affording protection to 
patients during the ten days necessary for the action of his vaccines. I think 
there is every reason to believe that better results would have been obtained 
here had the inoculations been performed at an earlier period instead of dur- 
ing the epidemic.” 


In a paper published in the British Medical Journal (January 26th, 


1895), Haffkine gives the following summary of his inoculations in 
India: 


“TABLE SHOWING THE TOTAL NUMBER OF PERSONS ON WHOM OB- 
SERVATIONS HAVE BEEN MADE IN CALCUTTA, GAYA, CAWNPORE, 
AND LUCKNOW. 


Percentage Percentage 
Number. Cases. of Cases Deaths. of Deaths 
to Strength. to Strength. 
Non-inoculated ............. 1,735 174 10.63 118 6.51 
Inoculated. ...... 0... 000.0 e. 500 21 4.20 19 3.80 
TO wivcaswascans genes 2,235 195 132 


Other methods of producing immunity in man have been proposed, 
and experiments indicate that this may be accomplished through the 
digestive tract by the ingestion of considerable quantities of steril- 
ized cultures. Thus Klemperer (1892) has obtained results which 
seem to show that immunity in man may be induced, not only by the 
subcutaneous injection of virulent cultures, but also by the subcuta- 
neous injection of the milk of immunized goats and by the ingestion of 
cultures sterilized by heat. The degree of immunity, as determined 
by the activity of the blood serum of the immune individual for the 
protection of guinea-pigs, is considerably less, however, than when 
repeated injections of virulent cultures have been made. The blood 
serum of individuals made immune by the last-mentioned method is 
said by Klemperer to protect guinea-pigs when injected into the cav- 
ity of the abdomen in the dose of 0.005 cubic centimetre. And the 
injection of five cubic centimetres of milk from an immunized goat is 
said to confer such an immunity that 0.25 cubic centimetre of blood 
serum from the immune individual is sufficient to protect a guinea-pig 
from cholera cultures. 

Sawtschenko and Sabolotny (1893), as a result of a series of ex- 
periments made upon themselves and laboratory assistants, arrive at 
the following conclusions: 


PROTECTIVE INOCULATIONS. 309 


“1. After the ingestion of sterilized (by heat) and subsequently carbolized 
agar cultures of cholera bacteria the serum of man acquires an immunizing 
property as regards the cholera vibrio. 

_ 2 Asa result of the ingestion of sterilized agar cultures the individual 
is protected from infection with virulent cultures of the cholera vibrio by way 
of the intestine. 


‘*3. The discharges of individuals immune against cholera, and to all out- 
ward appearance in perfect health, may contain a great number of cholera 
vibrios (in case they are in any way introduced into the intestine) and may 
thus serve to propagate the malady.” 


DIPHTHERIA. 


According to Roux and Yersin, “attenuated varieties ” of the diph- 
theria bacillus may be obtained by cultivating it at a temperature of 
39.5° to 40° C. in a current of air; and these authors suggest that a 
similar attenuation of pathogenic power may occur in the fauces of 
convalescents from the disease, and that possibly the similar non- 
pathogenic bacilli which have been described by various investigators 
have originated in this way from the true diphtheria bacillus. These 
authors further state, in favor of this view, that from diphtheritic 
false membrane, preserved by them in a desiccated condition for five 
months, they obtained numerous colonies of the bacillus in question, 
but that the cultures. were destitute of pathogenic virulence. They 
say: 

‘Tt is then possible, by commencing with a virulent bacillus of diphtheria, 
to obtain artificially a bacillus without virulence, quite similar to the attenu- 
ated bacilli which may be obtained from a benign diphtheritic angina, or even 
from the mouth of certain persons in good health. This microbe, obtained 
artificially, resembles completely the pseudo-diphther:tic bacillus ; like it, it 


grows more abundantly at a low temperature ; it renders bouillon more rap- 
idly alkaline ; it grows with difficulty in the absence of oxygen.” 


Subcutaneous inoculations in guinea-pigs of a small quantity of a 
pure culture of the bacillus (0.1 to 0.5 cubic centimetre of a bouillon 
culture) cause death in from one to four or five days. The usual 
changes observed at the autopsy are— 


‘* An extensive local cedema, with more or less hyperzemia and ecchymo- 
sis at the site of inoculation, frequently swollen and reddened lymphatic 
glands, increased serous fluid in the peritoneum, pleura, and pericardium, 
enlarged and hemorrhagic suprarenal capsules, occasionally slightly swollen 
spleen, sometimes fatty degenerations in the liver, kidney, and myocardium. 
We have always found the Léffler bacilli at the seat of moculation most 
abundant in a grayish-white, fibrino-purulent exudate present at the point of 
inoculation, and becoming fewer at a distance from this, so that the more re- 
mote parts of the cedematous fluid do not contain any bacilli” (Welch and 
Abbott). 


The authors quoted agree with Loffler and others in stating that 
the bacillus is found only at the point of inoculation. In all cases 


310 PROTECTIVE INOCULATIONS. 


their cultures from the blood and from the various organs gave a 
negative result. 

Rabbits are not so susceptible, and may recover after the subcu- 
taneous inoculation of very small doses, but usually die in from four 
to twenty days when two to four cubic centimetres of a bouillon cul- 
ture have been introduced beneath the skin. In these animals, also, 
there is an extensive local cedema, an-enlargement of the neighboring 
lymphatic glands, and a fatty degeneration of the liver. Roux and 
Yersin have shown that in rabbits, when death does not ensue too 
quickly, paralysis of the posterior extremities frequently occurs, thus 
completing the experimental proof of the specific pathogenic power 
of pure cultures of this bacillus. 

Similar symptoms are produced in pigeons by the subeutaneous 
inoculation of 0.5 cubic centimetre or more, but they commonly re- 
cover when the quantity is reduced to 0.2 cubic centimetre (Roux and 
Yersin). 

The rat and the mouse have a remarkable immunity from the effects 
of this poison. Thus, according to Roux and Yersin, a dose of two 
cubic centimetres, which would kill in sixty hours a rabbit weighing 
three kilogrammes, is without effect upon a mouse which weighs only 
ten grammes. 

Old cultures are somewhat less virulent than fresh ones, but when 
replanted in a fresh culture medium they manifest their original viru- 
lence. Thus a culture upon blood serum which was five months old 
was found by Roux and Yersin to kill a guinea-pig in five days, but 
when replanted it killed a second animal of the same species in 
twenty-four hours. 

Evidently a microdrganism which destroys the life of a susceptible 
animal when injected beneath its skin in small quantity, and which 
nevertheless is only found in the vicinity of the point of inoculation, 
must owe its pathogenic power to the formation of some potent toxic 
substance, which, being absorbed, gives rise to toxemia and death. 
This inference in the case of the diphtheria bacillus is fully sustained 
by the results of experimental investigations. Roux and Yersin 
(1888) first demonstrated the pathogenic power of cultures which 
had been filtered through porous porcelain. Old cultures were found 
by these experimenters to contain more of the toxic substance than 
recent ones, and to cause the death of a guinea-pig in a dose of two 
cubic centimetres in less than twenty-four hours. The filtered cul- 
tures produced in these animals the same effects as those containing 
the bacilli—local cedema, hemorrhagic congestion of the organs, 
effusion into the pleural cavity. Somewhat larger doses were fatal 


PROTECTIVE INOCULATIONS. dll 


to rabbits, and a few drops injected subcutaneously sufficed to kill a 
small bird within a few hours. In their second paper (1889) the 
authors mentioned state that so long as the reaction of a culture in 
bouillon is acid its toxic power is comparatively slight, but that in 
old cultures the reaction is alkaline, and in these the toxic potency is 
greatly augmented. With such a culture, filtered after having been 
kept for thirty days, a dose of one-eighth of a cubic centimetre in- 
jected subcutaneously, sufficed to kill a guinea-pig; and in larger 
amounts it proved to be fatal to dogs when injected directly into the 
circulation through a vein. 

The same authors, in discussing the nature of the poison in their 
filtered cultures, infer that it is related to the diastases, and state that 
its toxic potency is very much reduced by exposure to a comparatively 
low temperature—58° C., for two hours—and completely destroyed by 
the boiling temperature—100° C., for twenty minutes. It was found 
to be insoluble in alcohol, and the precipitate obtained by adding al- 
cohol to an old culture proved to contain the toxic substance. ’Loffler 
also has obtained, by adding five volumes of alcohol to one of a pure 
culture, a white precipitate, soluble in water, which killed rabbits in 
the dose of 0.1 to 0.2 gramme when injected beneath the skin of these 
animals. It gave rise to a local cedema and necrosis of the skin in 
the vicinity of the point of inoculation, and to hyperemia of the in- 
ternal organs. This deadly toxin appears to be an albuminoid sub- 
stance, but its exact chemical composition has not yet been deter- 
mined. 

Brieger and Frankel (1891) obtained results corresponding with 
those previously reported by Roux and Yersin. Their researches 
showed that the toxic substance contained in diphtheria cultures is 
destroyed by a temperature of 60° C.; that it is soluble in water, and 
insoluble in alcohol; that it does not pass through a dialyzing mem- 
brane, and has not the chemical characters of the ptomaines or toxins, 
but is an albuminous body—a toxalbumin. It was obtained by the 
authors named by precipitation with slightly acidified (acetic acid) 
alcohol; the precipitate, after being washed in a dialyzer and dried 
in a vacuum ata temperature of 40° C., was a snow-white, amorphous, 
erumbling mass. 

Wassermann and Proskauer (1892) found that the alcoholic pre- 
cipitate from diphtheria cultures contains two different substances, 
which are distinguished by their different degrees of solubility in 
dilute and absolute alcohol; both, however, give the usual reactions 
of albuminous bodies, and pass very slowly through a dialyzing mem- 
brane. Only one of these substances possesses toxic properties. : 


312 PROTECTIVE INOCULATIONS. 


After the removal of peptone and globulin from the filtered cultures, 
these were evaporated and a precipitate obtained of one of the 
albuminous substances by means of sixty to seventy per cent al- 
cohol. The other substance remained in solution, and was sub- 
sequently obtained by precipitation with absolute alcohol. The 
substance first obtained by this method is toxic, and the other pre- 
cipitate is not. The authors named succeeded in killing rabbits with 
the toxalbumin obtained in this way, but were not able to produce 
immunity in these animals by the injection of non-fatal doses. Fréan- 
kel (1891) had previously reported his failure to immunize guinea-pigs 
by the injection of the dry precipitate, obtained in his experiments 
from diphtheria cultures; but when filtered cultures, or cultures 
sterilized by heat (55° C. for one hour), were injected into these ani- 
mals, they showed an increased resistance to the pathogenic action of 
virulent cultures. Still better results were obtained when ten cubic 
centimetres of a bouillon culture, heated to 100° C., were injected 
subcutaneously, but still this method was not entirely reliable. But 
true immunity was established by injecting into the peritoneal cavity 
ten to twenty ctbic centimetres of a bouillon culture heated to from 65° 
to 70° C. for one hour. The immunity was not fully established until 
about fourteen days after the protective inoculation. Frankel arrives 
at the conclusion that the cultures must contain an immunizing sub- 
stance as well as a toxic proteid, as the diphtheria toxalbumin is de- 
stroyed by the temperature (65° to 70° C.) used in the preparation of 
his cultures for producing immunity. 

Behring, in the same year (1891), commenced his experiments 
upon diphtheria immunity. Guinea-pigs were made immune by the 
use of sterilized cultures, and by inoculations with virulent cultures, 
four weeks old, to which iodine terchloride had been added in the 
proportion of 1:500—the mixture was allowed to stand for sixteen 
hours. Animals were also immunized by injecting beneath the skin 
a virulent culture of the bacillus, and then treating them with sub- 
cutaneous injections of iodine terchloride (two cubic centimetres), 
which was thrown under the skin for three days in succession in the 
vicinity of the point of inoculation. The guinea-pigs treated in this 
way remained sick for some time, but finally recovered and were 
subsequently immune. Still better results were obtained when rab- 
bits were subjected to the same treatment. The animals were im- 
mune against the toxic action of sterilized cultures, as well as against 
infection by virulent diphtheria bacilli. 

In subsequent experiments (1892) Behring and Wernicke used 
cultures which had been attenuated by contact with iodine terchloride 


PROTECTIVE INOCULATIONS. 313 


for from thirty-six to forty-eight hours, and proved that the method 
could be successfully employed in immunizing sheep; and the fact 
was ascertained that blood serum from an immune animal could be 
used with success in arresting diphtheritic infection in susceptible 
animals. To preserve the serum, which they obtained from immun- 
ized sheep, rabbits, and guinea-pigs, they added to it 0.5 per cent of 
pure carbolic acid. For producing immunity they found that a 
smaller amount of serum was required than was necessary for the 
cure of an animal already infected. If the injection was made imme- 
diately after infection, from one and a half to two times the amount 
was required; eight hours after infection the amount was three times 
as great, and twenty-four to thirty-six hours after infection the dose 
required was eight times the immunizing dose. 

The immunizing value of blood serum from different animals was 
estimated by finding the smallest dose which would protect an animal 
from fatal infection by the minimum lethal dose of a culture the toxic 
potency of which had been carefully determined. The value is ex- 
pressed in figures which give the proportion required compared with 
the body weight of the animal. Thus an immunizing value of one 
hundred would mean that one gramme of the serum is sufficient to 
protect an animal weighing one hundred grammes from the fatal 
effect which would be produced in a control animal of the same weight 
by infection with a virulent culture of the diphtheria bacillus in the 
minimum doses required to produce this result. The cultures em- 
ployed are made in bouillon containing one per cent of peptone; they 
are inoculated from agar cultures and are kept in the incubating oven 
for two days. Cultures prepared in this way were found to be quite 
uniform in their pathogenic virulence as tested upon guinea-pigs. But 
when cultures are kept for some time there is an increase in virulence. 
Thus a culture obtained from a fatal case of diphtheria which in 1890 
killed guinea-pigs in three to four days, when injected subcutaneously 
in the dose of 0.1 cubic centimetre (two-days-old bouillon culture), 
at the end of a year was fatal to these animals in the dose of 0.025 
cubic centimetre. This increase in virulence is ascribed to the fact 
that the cultures were renewed at long intervals. 

More recently (1894) Behring has fixed a standard for what he 
calls a normal therapeutic serum. This is a serum which when in- 
jected into guinea-pigs in the proportion of 1:5,000 of body weight 
saves the animal from the fatal effects of ten times the minimum dose 
of a culture in bouillon, two days old, which would kill a control ani- 


mal not treated. 
In a subsequent communication (November, 1894) Behring states 


314 PROTECTIVE INOCULATIONS. 


his conclusion that for producing immunity in man, one hundred and 
fifty normal antitoxin units should be given, instead of sixty as he 
had previously recommended. 

The serum manufactured under his direction is said (September, 
1894) to be of two kinds—one, obtained from the horse, has a value 
of sixty normal antitoxin units; the other has a value of one hundred 
and forty units. Of the weaker serum Behring says experience has 
demonstrated that for children under ten years of age ten cubic cen- 
timetres is sufficient to arrest the progress of the disease and effect a 
cure if given within two or three days from the outset of the attack. 
For producing immunity in children subject to infection, one-tenth 
of this amount (one cubic centimetre) is said to be sufficient. Of 
the stronger serum one cubic centimetre is sufficient to arrest the dis- 
ease during the incubation period; and, according to Behring, out of 
one hundred cases treated during the first forty-eight hours with a 
single therapeutic dose (ten cubic centimetres of serum having a value 
of sixty, equals six hundred normal units), not five will die. The 
later the treatment is commenced the larger will be the dose required. 
Behring further states that the diphtheria antitoxin has no injurious 
effect upon animals in the largest doses that have been employed, 
and that aside from its antitoxic power its properties are entirely 
negative so far as living animals are concerned. 

Aronson (1893), in experiments on dogs, succeeded in producing 
immunity by the use of attenuated cultures, or of cultures to which 
formaldehyde had been added; also by feeding the animal large quan- 
tities of diphtheria bouillon; and, finally, by injection of the blood of 
naturally immune animals (white rats) into which large quantities (ten 
cubic centimetres) of a virulent culture had been injected. Two months 
after receiving several such injections it was found that 0.2 gramme 
of blood-serum from the rat sufficed to save a guinea-pig from fatal 
infection. In experiments on dogs an immunity was established in 
six weeks by the injection of a large amount of a virulent culture. Its 
serum had a value of 1: 30,000, 7.e., 0.01 cubic centimetre of this serum 
sufficed to protect a guinea-pig weighing three hundred grammes. 
From one hundred grammes of this serum Aronson claims to have ob- 
tained 0.8 gramme of a substance which had a value of 1:500,000, as 
tested in the treatment of an animal which had received ten times 
the minimum fatal dose of a two-days’ bouillon culture. A ten-per- 
cent solution of this substance had, therefore, ten times the value of 
Behring’s “normal serum.” The precipitated antitoxin was soluble in 
water, and more readily so in a slightly alkaline solution, and gave 
all the reactions of an albuminous body. When dried in vacuo at 


PROTECTIVE INOCULATIONS. 315 


40° C., and then heated to 102° C., it still retained its antitoxic 
potency. 

Ehrlich, Kossel, and Wassermann (1894) have made experiments 
upon goats, which they found very susceptible to the action of the 
diphtheria poison. Sterilized cultures were first injected in gradually 
increasing amounts, and later virulent cultures. In this way they ob- 
tained a serum which has a value sixty times that of Behring’s “nor- 
mal serum.” In a subsequent communication (1894) Wassermann 
gives an account of his experiments with the milk of immunized 
goats, which contains the antitoxin in considerable quantity, and 
from which it was obtained in a concentrated form by the following 
method: The milk is obtained in sterilized vessels and twenty cubic 
centimetres of normal hydrochloric acid are added to each litre; a 
sufficient quantity of rennet is then added to coagulate the casein, 
and this is separated from the liquid, which is then shaken up with 
chloroform for some time. The liquid is now allowed to stand in 
order that the butter, which has been dissolved by the chloroform, 
may sink to the bottom. The clear liquid is then decanted and the 
antitoxin precipitated from it by means of ammonium sulphate (thirty 
to thirty-three per cent). The precipitate is rapidly dried upon 
porous porcelain plates, in vacuo, and then dissolved in water in the 
proportion of ten parts for one hundred of milk first employed—a 
concentration to one-tenth. Of this solution 0.125 cubic centimetre 
was found to neutralize 0.9 cubic centimetre of a toxin which killed 
guinea-pigs weighing five hundred grammes in the dose of 0.1 cubic 
centimetre. This toxin was an old bouillon culture of the diphtheria 
bacillus to which 0.5 per cent of carbolic acid had been added to 
preserve it. In a communieation of the same date Ehrlich and Was- 
sermann report that they have for some time had a cow immunized 
to such a degree that one cubic centimetre of its milk protects guinea- 
pigs from the fatal effects of 0.9 cubic centimetre of the above-men- 
tioned toxin. The antitoxic value of the milk of an immunized cow 
or goat, as compared with that of its blood, is estimated by Ehrlich 
and Wassermann as from 1:15 to 1:30—usually about 1:20. 

Aronson, in testing his antitoxin, uses a bouillon culture of the 
diphtheria bacillus two and one-half months old, which he preserves 
by the addition of 0.3 per cent of trikresol. He finds that the im- 
munity which results from injections of the antitoxin is established 
at once; that it is not accompanied by any reaction or symptom of 
sickness; and that it is of comparatively short duration. 

As a result of extended experiments made at the Pasteur Institute 
in Paris, Roux has perfected the following method for the production 


316 PROTECTIVE INOCULATIONS. 


of an antitoxin suitable for use in the treatment of diphtheria in man. 
The horse has been found the most suitable animal for this purpose, 
on account of his slight susceptibility and the ease with which a high 
degree of immunity can be established; and because of the large 
amount of blood that may be drawn without injury to the animal. 
Roux prepares his toxin by cultivating the diphtheria bacillus in a 
slightly alkaline bouillon made from beef and containing two per 
cent of peptone and 0.5 per cent of sodium chloride. This medium 
is placed in flat-bottomed flasks, and should not be more than balf an 
inch in depth. Two glass tubes pass into the flask, which serve for 
inlet and outlet tubes to be used in passing a current of air over the 
cultures. This is commenced when the growth is fairly started, at 
the end of twenty-four hours, and the air should be moist to prevent 
the evaporation of the culture. In Roux’s laboratory a flask is used 
which has a tube attached to one side, about an inch from the bottom, 
and which is known as a Fernback flask. A flocculent deposit falls 
to the bottom and gradually accumulates for about a month. This 
consists of bacilli which have for the most part lost their vitality and 
are undergoing degeneration. At the end of thirty days, during 
which time they are kept in an incubating oven at a temperature of 
37° C., the cultures are passed through a Pasteur-Chamberland filter, 
and 0.5 per cent of carbolic acid may be added in order to preserve 
them. This filtrate is so toxic that a dose of 0.1 cubic centimetre 
will kill a guinea-pig weighing five hundred grammes in less than 
forty-eight hours. A healthy horse is selected and receives at first a 
dose of 0.5 cubic centimetre of the filtered culture (or of the clear 
‘fluid obtained from a culture by decantation, and containing 0.5 per 
cent of carbolic acid). The dose is gradually increased at intervals 
of a few days, and is followed each time by some febrile reaction and 
tumefaction at the point of inoculation. When the reaction is exces- 
sive, a little Gram’s solution is added to the following dose. The 
usual plan of treatment is stated by Kinyoun as follows: 


‘‘First day, 1 to 2 ¢.c. of pure toxins, of which 1 to 10 c.c. fatal to a 500- 
gm. guinea-pig ; eighth day, 1c¢.c.; fourteenth day, 1.5 c.c. ;: twentieth day, 
2c.c.; twenty-eighth day, 3 c.c. ; thirty-third day, 5 ¢.c. ; thirty-eighth day, 
8 c.c.; forty-third day, 10 c.c. ; forty-seventh day, 20 ¢.c.; fifty-first day, 30 
c.c.; fifty-sixth day, 50 c.c.; sixty-second day, 50 c.c. ; sixty-eighth day, 60 
c.c. ; seventy-fourth day, 100 c.c. ; eightieth day, 250c.c.; eighty-eighth day, 
250 c.c. 

‘‘ When the first injections are given there is quite a marked local and gen- 
eral reaction to the poison; there is an cedema at the point of the injection, 
which is followed by a distinct inflammatory process—hard in the centre and 
soft and cedematous at its periphery. The general reaction is manifested by 
a rise in the temperature, 1° to 2° C., loss of appetite, and occasionally cramps. 
The reaction must be taken as the guide in the future dosage, and a sufficient 


PROTECTIVE INOCULATIONS. 317 


time must be allowed to elapse between the injections for the complete recov- 
ery from the general and local effects. As the quantity of the toxins is in- 
creased the general effects generally decréase, perhaps a rise of a degree for 
twenty-four hours. The local effect partakes more of an cedema, and has the 
character of an inflammation. 

‘At a certain stage, usually after two months’ treatment, when fifty to 
sixty cubic centimetres can be injected without harm, there is no general re- 
action, but a large cedema at the site of the injection, which disappears 
within from twenty-four to forty-eight hours. Toward the last, even when 
two hundred to three hundred cubic centimetres are given, there is only an 
enormous cedema, which disappears within from twelve to eighteen hours. 
When these inordinately large quantities can be given with only a local re- 
action being manifest, the horse has come well under the influence, and the 
blood will be found to be rich in the antitoxin. 

‘‘There is a curious fact well worth noting: At the end of the second 
month of treatment, when the horse can bear as much as fifty to sixty cubic 
centimetres of the toxins without discomfort, the blood will be found to con- 
tain but little of the antitoxin. The antitoxin only appears after repeated 
stimulation of the cells (?) by the large and frequent doses of the toxins.” 


The subcutaneous injections do not yield a serum as rich in the 
antitoxins as when the toxins are injected directly into the blood cur- 
rent. When it is desired to do this, toward the last of the treatment 
the toxins are injected directly into the jugular vein. The process is 
tedious and requires a longer time, and for practical purposes has not 
been found so satisfactory as the simple subcutaneous injection. The 
strength of the serum is tested by using young guinea-pigs of five 
hundred grammes weight. One gramme of the serum usually will 
protect fifty thousand grammes of guinea-pig against a fresh virulent 
culture of the Bacillus diphtherwe. This is the strength that is used 
in the hospitals. By the intravenous injections a serum of the pro- 
tective strength of 1:100,000 can be obtained. 

When fully immunized from six to eight litres of blood may be 
taken from a horse at one time, but as a rule it is better not to take 
more than three. The blood is drawn from the jugular vein, by means 
of a small trocar and cannula, into wide-mouthed bottles having a ca- 
pacity of 2.5 litres; these are placed in an ice chest for twenty-four 
hours to give time for the separation of the serum, which is then 
transferred to smaller receptacles for preservation. 

The dose of serum prepared in this way, when used to protect from 
diphtheria infection, is five cubic centimetres for a child under ten 
years of age, and ten cubic centimetres for older children. This does 
not afford an absolute protection, but is believed to be generally 
effective, and in case of failure the attack is said to be of a mild 
character. The curative dose of Roux’s serum is twenty cubic centi- 
metres for children, and thirty to forty cubic centimetres for patients 
over fifteen years of age. The larger dose is divided and given, at 
the same time, by subcutaneous injection in two places. Antiseptic 


318 PROTECTIVE INOCULATIONS. 


precautions are taken in giving these injections, and a little absorbent 
cotton is placed over the puncture. 


FOOT-AND-MOUTH DISEASE. 


This is an infectious disease of cattle, sheep, goats, and swine, the 
etiology of which, so far as the specific infectious agent is concerned, 
has not been determined. 

The extent to which the disease in question prevails in some parts 
of Europe is shown by the statistics for 1891 of the prevalence of this 
disease in Germany. According to the Reichsseuchenbericht it pre- 
vailed most extensively in the southern portion of Germany. The 
total number of infected farms was 47,865; the total number of in- 
fected cattle was 394,640; of sheep, 240,904; of goats, 3,378; of swine, 
182,208. Behla (1892) has made inoculation experiments with the 
filtered saliva of infected cattle to which he added one to two per cent 
of carbolic acid, and claims to have produced immunity in young pigs 
and lambs. The duration is not, however, very long even in animals 
which have recovered from an attack of the disease—said to be from 
six months to three years—and a practical method of restricting the 
disease by means of protective inoculations has not as yet been intro- 
duced. 

GLANDERS. 


_ The toxic substances produced in cultures of the glanders bacillus 
when concentrated in the form of a glycerin extract constitute the so- 
called mallein, which has been extensively used in the diagnosis of 
glanders in horses. As is the case when animals infected with tuber- 
culosis are inoculated with tuberculin, animals infected with glanders 
have a decided rise of temperature after receiving a sufficient dose of 
mallein beneath the skin. 

Babes (1892) reports that the toxic substance in cultures of the 
glanders bacillus may be obtained by precipitation with alcohol; and 
that mallein obtained from filtered cultures to which glycerin has 
been added, or the alcoholic precipitate, may be successfully used 
for protecting susceptible animals against glanders infection or for 
curing the disease after infection. He has demonstrated the thera- 
peutic value upon guinea-pigs and upon two horses which are said to 
have been cured of chronic glanders. When large and repeated doses 
are injected into healthy animals they produce nephritis and general 
marasmus. The action upon horses infected with glanders is very 
marked and small doses may even cause death. 

Kresling (1892) recommends potato cultures as preferable to bouil- 


PROTECTIVE INOCULATIONS. 319 


lon cultures for the preparation of mallein. The potatoes are to be 
washed, before sterilization, in a five-per-cent bicarbonate of soda 
solution, “until the wash-water remains clear.” They are then 
cooked for an hour and twenty minutes. After planting upon the 
surface glanders bacilli from a previous culture they are placed in an 
incubator at 36° to 36.5° C., with provision to prevent them from be- 
coming dry. At the end of two weeks the growth is removed with a 
platinum spatula and added to nine parts of water, in which it is well 
mixed by rubbing. It is then allowed to stand for twenty-four hours, 
after which it is sterilized for fifteen minutes at 110° C. (a lower tem- 
perature would no doubt answer quite as well). After cooling it is 
passed through a Chamberlain filter by means of a pressure of six 
atmospheres. The filtrate is then carefully evaporated over a water- 
bath to one-fourth its volume, and to this concentrated extract gly- 
cerin is added in the proportion of one part to two. The mixture is 
again sterilized in the autoclave at 110° C. When injected into 
healthy horses in the dose of two cubic centimetres this mallein does 
not cause an elevation of temperature exceeding 0.5° to 0.8°. But 
one cubic centimetre injected into a horse having glanders causes its 
temperature to mount to 40° C., and at the point of inoculation a con- 
siderable swelling is developed which lasts from four to six days— 
in healthy horses a swelling the size of a man’s fist is developed at 
the point of inoculation, which disappears within twenty-four hours. 

In Pasteur’s laboratory, according to Nocard (1892), mallein is 
prepared as follows: The glanders bacillus is first made so virulent 
by successive inoculations in susceptible animals that it will kill a 
rabbit or a white mouse in a fewhours. This virulent bacillus is cul- 
tivated in glycerin-peptone-flesh-infusion (five per cent of glycerin and 
five per cent of peptone). The cultures are kept in the incubating 
oven for four weeks at a temperature of 31° C., and then sterilized in 
the autoclave at 110° C. They are then filtered through paper and 
evaporated, in vacuo, over sulphuric acid, at-a low temperature, to one- 
tenth of the original volume. The result is a syrup-like, dark-brown; 
strong-smelling liquid, which is about one-half glycerin. This can 
be preserved in a cool and dark place for a long time. When it is to 
be used nine parts of a 0.5-per-cent solution of carbolic acid are 
added to one part of the glycerin extract. The concentrated extract, 
when injected into a healthy horse in the dose of one-half to one cubic 
centimetre, causes a local swelling which disappears after two or three 
days. The temperature of the body is elevated from 1.5° to 2° C. as 
a result of the injection, and there are chilliness, loss of appetite, and 
debility. When the diluted mallein is injected in healthy animals in 


320 PROTECTIVE INOCULATIONS. 


the dose of 2.5 cubic centimetres no reaction occurs. On the other 
hand, this dose causes an intense febrile reaction in horses with glan- 
ders. There is a chill followed by an elevation of temperature amount- 
ing to 2° to 3° C., accompanied by dyspnoea and great debility; in 
some cases the animal dies as a result of the inoculation. 

For the preparation of the active substance ina dry condition, 
Foth gives the following directions: The cultures are evaporated at 
a temperature not exceeding 80° C. to one-tenth of their volume, and 
filtered. The clear and thick, dark-brown liquid is then slowly 
dropped into absolute alcohol (twenty-five to thirty parts) with con- 
stant stirring. A flaky, white precipitate is thrown down, and ac- 
cumulates as a pale yellow mass upon the sides and bottom of the 
vessel. After standing for twenty-four hours the alcohol is carefully 
drawn off and the precipitate washed with absolute alcohol. This 
is to be carefully done, and to avoid loss will require several days. 
The precipitate is then placed upon a thick paper filter and thor- 
oughly washed by drawing alcohol through it by means of an exhaus- 
tion apparatus, after which the purified precipitate is collected and 
dried with care at a low temperature—best in a vacuum over sul- 
phuric acid. A spongy, crumbling mass is thus obtained, which is 
easily crushed to form an extremely light white powder. This is 
readily soluble in water. It is not at all hygroscopic, and can be 
preserved in a dry condition without difficulty. The dose for a horse 
is 0.1 gramme. ; 

De Schweinitz and Kilborne, in a paper published in November, 
1892, state that in December, 1890, they 


‘extracted from culture liquids of the Bacillus malleus an albumose which 
appeared to be the active principle in these cultures. At that time a pre- 
liminary experiment was conducted to see if this substance could be used 
to make guinea-pigs immune to the disease—glanders. The result was that 
out of a set of five, three vaccinated and two checks, only one, a vaccinated 
animal, recovered froin an inoculation of a glanders culture. This experi- 
ment has since been repeategl with sets of ten and twelve guinea-pigs each, 
with, at present writing, only negative results. A note of this work was 
published in the ‘Annual Report of the Department of Agriculture for 1891.’ 
The albumose was best obtained from the cultures, after the removal of the 
germ, by means of a Pasteur filter, by precipitation with absolute alcohol, 
resolution in water, and reprecipitation.” 


Babes (1892) claims to have succeeded in immunizing guinea-pigs 
against glanders by means of the toxic substances contained in cul- 
tures of the bacillus. 

Foth (1894) has reported the results of extended experiments 
which have been made with his “Malleinum siccum” in Austro-Hun- 
gary. These results are stated as follows: 


PROTECTIVE INOCULATIONS. 321 


The experiments were for the most part made by Professor Schin- 
delka, of Vienna. The tests were made with doses ranging from 0.1 
gramme to 0.2 gramme. The number of horses treated, for diagnos- 
tic purposes, was four hundred and fifty-five; of these one hundred 
and forty-seven were examined post mortem. In general the infected 
horses reacted and the others did not. A reaction of 2° C. and up- 
ward, running a typical course, was evidence that the animal was in- 
fected, and such animals were killed and carefully examined by au- 
topsy. 

A reaction of 1.3° to 1.9° C., running a typical course, was taken 
as evidence that the animal was probably infected, and called for its 
isolation and a subsequent inoculation after an interval of four weeks. 

A reaction of less than 1.2° C., or an atypical course of the febrile 
reaction, was taken as evidence of nou-infection. 

The typical febrile reaction consisted in a rapid or gradual eleva- 
tion, according to the dose, then a fall of some tenths of a degree, a 
subsequent elevation to the highest previously reached point or above, 
and a gradual fall to the normal. The atypical reaction, which some- 
times occurs in healthy animals, consists in an early and rapid eleva- 
tion followed by an equally rapid fall to the normal. To properly 
distinguish the typical temperature curve, upon which the diagnosis 
depends, hourly observations are considered necessary. 

Schutz (1894), as a result of his experiments on fifty-four horses, 
arrives at the conclusion that mallein may give rise to the so-called 
“typical reaction” in horses which are not infected with glanders. 

Hutyra and Preiz (1894), as a result of their extended researches, 
arrive at the conclusion that the use of mallein constitutes the most 
important means for the early diagnosis of glanders in horses. They 
conclude that a temperature of 39.4° C. may be accepted as a safe 
positive mallein reaction. According to them the reaction commences 
from four to six hours after the injection, and reaches its maximum 
in from eight to fourteen hours—rarely in sixteen to twenty hours. 
The return to the normal occurs in from twenty-four to thirty-six 
hours. The authors last named give the following directions for the 
preparation of mallein: The virulence of the glanders bacillus is first 
increased by passing it through a series of guinea-pigs. Cultures 
are then made upon sterilized potato. When the culture and potato 
have become quite dry and dark colored they are collected in a glass 
dish and covered with a liquid consisting of equal parts of glycerin 
and distilled water, containing three to five parts per thousand of 
mercuric chloride. After standing for from ten to fourteen days in 


an incubating oven at 37.5° C., the liquid is filtered through paper 
al 


B22 PROTECTIVE INOCULATIONS. 

and sterilized for an hour in a steam sterilizer. This liquid remains 
sterile on account of the presence of mercuric chloride, and may be 
preserved a long time without losing its activity. The dose is from 
0.3 to 0.5 cubic centimetre, which is diluted to three cubic centimetres 
with carbolic acid water (0.5-per-cent solution). The diluted solu- 
tion may also be kept a long time without losing its activity. 

Bonome and Vivaldi (1892) have tested the action of mallein ob- 
tained by precipitation with alcohol upon various animals. Guinea- 
pigs were found to resist comparatively large doses (ten to fifteen 
milligrammes), while rabbits and cats were more sensitive to the 
toxic action. In guinea-pigs and rabbits infected with glanders ba- 
cilli very small doses had a favorable influence upon the progress of 
the infection, and in healthy guinea-pigs a certain degree of immunity 
was induced by the repeated injection of small doses. 

In a subsequent paper (1894) Bonome reports that he has had 
favorable results in the treatment of chronic glanders in man by doses 
of ;'5 to 1; cubic centimetre. The first dose is said to have caused 
an elevation of temperature, headache, polyuria, etc., but upon re- 
peating the dose after two or three days a decided improvement of 
the general symptoms followed. 

Chenot and Picq (1892) claim to have cured glanders in guinea-pigs 
by injections of blood serum from the ox, which animal has an im- 
munity from the disease. They also state that the blood serum of . 
the ox is germicidal for the glanders bacillus. Guinea-pigs treated 
with ox serum, either before or after infection, recovered in seven 
cases out of ten. When inoculated with very virulent cultures, which 
usually killed these animals in five days, the animals are said to have 
survived from twenty-one to forty-two days. 

Bonome (1894) reports his success in curing infected guinea-pigs 
by means of filtered cultures made in the blood serum of the ox. He 
was not, however, successful in accomplishing this result with mallein 
made in the usual way. 


HOG CHOLERA. 


The experiments thus far made with reference to protective inocu- 
lations against hog cholera have not given very satisfactory results. 
Selander and Metchnikoff have reported success in immunizing rab- 
bits, but according to Smith their experiments were made with the 
bacillus of swine plague, and not with that of hog cholera as they 
supposed. The following conclusions have been formulated by Smith 
as a result of his extended experiments: 


“1. It is possible to produce immunity toward hog-cholera and swine- 


PROTECTIVE INOCULATIONS. 323 


plague bacteria in the very susceptible rabbit and the less susceptible guinea- 
pig. In the rabbit the only promising method of immunization toward hog 
cholera is the use of gradually augmented doses of attenuated cultures. 

“2. Immunization toward swine plague is produced artificially with 
much greater ease than toward hog-cholera bacteria. 

‘*3. The blood serum of animals protected against hog cholera and swine 
plague is almost as efficacious in producing immunity soon after treatment 
as the bacterial products obtained from cultures. 

‘‘4, Different degrees of culture in both hog cholera and swine plague 
lead to different forms of the inoculation disease. The greater the immunity 
short of complete protection the more prolonged and chronic the disease in- 
duced subsequently by inoculation. 

‘5, Pathogenic bacteria may remain in the organs of inoculated animals 
some time after apparently full recovery. Their presence may or may not 
be associated with lesions recognizable by the intel eye. 

‘*6. The toxicity of sterilized cultures appears to be directly proportional 
to the number of bacteria in the injected fluid.” 


The experiments of Moore, reported in Bulletin No. 6 of the 
Bureau of Animal Industry, show that the bacillus of hog cholera 
does not become attenuated by being passed through rabbits, and that 
in the experiments of Metchnikoff, which led him to conclude that 
this is the case, the bacillus of swine plague, and not that of cholera, 
was used. 

De Schweinitz studied the chemical products of the hog-cholera 
bacillus in 1890, and obtained from the cultures cadaverin, methyl- 
amine, a ptomaine (“sucholotoxin”), and an albumose (“sucholoal- 
bumin”’), 

Novy (1890) has also obtained, by Brieger’s method, a basic toxic 
substance (“susotoxin”) which kills'rats in the dose of 0.125 to 0.25 
cubic centimetre. He also obtained from concentrated cultures, by 
precipitation with absolute alcohol, a toxalbumin which, when dried, 
killed rats in three or four hours in the dose of 0.05 to 0.01 gramme. 

De Schweinitz in a later publication (1699) reports that he has 
obtained, by the method of Brieger and Boer for the isolation of the 
diphtheria antitoxin, an ash-free white powder, which possesses the 
antitoxic properties of serum from an immune animal; ninety cubic 
centimetres of serum gave him 0.152 gramme of this powder. The 
method referred to consists in precipitation by the use of zine sul- 
phate, repeated solution in sodium hydrate and precipitation by 
CO,. In preparing serum for his experiments, cattle, horses, mules, 
and monkeys were employed. “The animals received injections 
of the filtered, sterile or live, cultures of the hog-cholera germ and 
swine-plague germ, respectively, or the solutions of their products, 
including cell contents, extracts, and secretions. These injections 
were made either subcutaneously, intravenously, or intra-abdomi- 
nally, or a combination of two or more of these methods, depending 


324 PROTECTIVE INOCULATIONS. 


upon the results obtained. The quantities given at first were small, 
but increased gradually until large amounts of the material used could 
be injected without bad results. This treatment of the animals must 
be carried out very carefully, and requires six to eight months’ time 
before the serum is sufticiently potent to be of any practical use. As 
the treatment continues, the power of the serum to check the motility 
of the hog-cholera germ increases with rapidity. The length of im- 
munity produced by the injection of serum is short, and more perma- 
nent immunity can apparently be secured by using in addition to 
serum the products of the germs.” 

The results of extensive inoculations (thirty-five thousand animals) 
which have been made by the Agricultural Department during the past 
two years have not yet been published, but it is understood that as a 
rule these results have been quite satisfactory. 


HOG ERYSIPELAS. 


Pasteur’s first studies relating to the etiology of “rouget” were 
made, in collaboration with Chamberland, Roux, and Thuillier, in 
1882. Pasteur found that the virulence of his cultures was increased 
by passing them through pigeons and diminished by passing them 
through rabbits. By a series of inoculations in rabbits he obtained 
an attenuated virus suitable for protective inoculations in swine. In 
practice he recommended the use of a mild virus first, and after an 
interval of twelve days of a stronger virus. These inoculations have 
been extensively practised in France, and the fact that immunity may 
be established in this way is well demonstrated. There has been some 
doubt, however, as to the practical value of the method, as its appli- 
cation has been attended with some loss, and there appears to be 
danger that the disease may be spread by the alvine discharges of 
inoculated animals. In a region where the annual losses from the 
disease are considerable, and where the soil is, perhaps, thoroughly 
infected with the bacilli, protective inoculations probably afford the 
best security against loss. But when it is practicable to stamp out 
the disease by quarantine of infected animals, disinfection of localities 
in which cases have occurred, and strict attention to cleanliness, this 
will probably be found the best method of combating the malady. 

Chamberland (1894) states that in the preceding seven years, dur- 
ing which time protective inoculations were practised in France on a 
large scale, the mortality from rouget has been reduced to 1.45 per 
cent, whereas before these inoculations were practised the mortality 
from this disease was about twenty per cent. Losses amounting in 


PROTECTIVE INOCULATIONS. 325 


some instances to as much as ten per cent have resulted from the in- 
oculations. These are ascribed by Chamberland to secondary infec- 
tion, through the inoculation wound, with other pathogenic bacteria. 

Jakobi (1888) reports the results of inoculations made in 1887 and 
1888 with “vaccines” obtained from Pasteur’s agent in Paris. His 
results agree with those previously reported by Lydtin in showing a 
smaller loss, as a result of the inoculations, among young pigs than 
among older ones—over sixteen weeks. The loss among young pigs 
was only 1.3 per cent. The animals which survived subsequently es- 
caped infection, while others not inoculated, associated with them, 
succumbed to the disease. 

Hutyra has given the following statistics of inoculations made in 
Hungary during the year 1889, with “vaccines” obtained from the 
Pasteur laboratory in Vienna: 48,637 pigs were inoculated on 117 
different farms. Of these 142 (0.29 per cent) died between the first 
and second inoculation. After the second inoculation 59 animals died 
(0.1 per cent). During the year following the inoculations, 1,082 in- 
oculated pigs died of Rothlaw. Before the inoculations the annual 
loss in the same localities is said to have been from 10 to 30 per 
cent. Upon one farm 220 pigs which had been inoculated were as- 
sociated with 1,500 not inoculated. The loss among the latter was 
50 per cent; among the former 2.27 per cent. 

In a later communication (1894) Jakobi gives the following results 
of inoculations made since by the same method: 1889, inoculated 
133, loss 5; 1890, inoculated 151, loss 2; 1891, inoculated 158, loss 
0; 1893, inoculated 223, loss 0; 1894, inoculated 145, loss 4. Total 
inoculated, 1,036; totalloss, 14. These inoculations were made upon 
19 different farms, and principally upon pigs less than four months 
old. The inoculated pigs were isolated to prevent the communication 
of the disease to other unprotected pigs. 

Inoculations with Blood Serum of Immune Animals.—The experi- 
ments of Lorenz, commenced in 1891, seem to establish the fact that 
there is an antitoxin in the blood of animals which have an acquired 
immunity against this disease which may be used for producing im- 
munity in other animals, or for the cure of the disease in animals 
already infected. In his latest communication (1894) Lorenz says: 


‘‘When I read in the journals of the discovery of Behring and Kitasato 
that the blood of animals immunized against tetanus, when injected beneath 
the skin of other animals, gave them an immunity against tetanus, I had in 
my possession rabbits which were immunized against Rothlauf. I took from 
one of these some blood from the ear vein, injected it under the skin of a 
mouse,, inoculated this latter with a Rothlauf culture, and made the dis- 
covery, in this and a series of subsequent experiments, that the blood of an 


326 PROTECTIVE INOCULATIONS. 


animal immune against Rothlauf contains an immunizing substance. I 
further ascertained that this substance is found only in the blood serum, and 
not in the solid portions of the body organs, etc., and with the exception of 
the blood was found only in the secretions of serous membranes. I also 
found that the immunizing substance is only to be found for a certain time 
after renewed infection in the immune animals, and that it gradually disap- 
pears, without the loss of immunity in the animal, however. Finally, I dis- 
covered that the animals into which one injects blood serum from immune 
animals do not acquire a lasting immunity, but are only immune for a rela- 
tively short time.” 

In experiments made in 1898 and 1894, with a view to producing 
immunizing serum for protective inoculations on a large scale, Lorenz 
met with some disappointments; but he proposes to renew his attempts 
and hopes to avoid the difficulties which have been brought to light 
by experience, one of which he states as follows: 

‘When an animal already immunized against Rothlauf receives an in- 
jection of a considerable quantity of a culture of the bacillus, in order to 
cause the production in its blood of a serum of high therapeutic value, the 
animal bears these injections without any notable reaction. But its blood 
serum contains during the following days, besides the immunizing substance, 
-also poisonous substances, and blood ‘which is taken too soon (twenty-four 
hours) after the injection has a toxic action upon animals which are already 
infected. If this poisonous serum is injected into a mouse which has been 
infected two days before with Rothlauf bacilli, in the dose of about 0.05 cubic 
centimetre, death occurs in a few hours, even when scarcely any evidence of 
sickness had been observed before the injection.” 


The fact that mice infected with this bacillus may be cured by in- 
jecting into them blood serum from an immunized rabbit has also 
been demonstrated by F. Klemperer (1892). In his experiments with 
the bacillus of mouse septicemia, and with Friedlander’s bacillus, he 
found that serum from an immune rabbit may be used to immunize 
mice and also to cure them after infection, while serum from a non- 
immune rabbit has no such action. The immunity produced in this 
way was found to be specific. That. is, animals immunized against 
the pathogenic action of one of these bacilli were not protected against 
infection by the other. The “ Heilserum” when added to cultures in 
vitro did not prove to have any special bactericidal action. 


HYDROPHOBIA. 


Notwithstanding the extended researches made, especially in Pas- 
teur’s laboratory, the etiology of hydrophohia still remains unsettled. 
It has been demonstrated by experiment that the virus of the disease 
is located in the brain, spinal marrow, and nerves of animals which 
have succumbed to the disease, as well as in the salivary secretions 
of rabid animals; and that the disease may be transmitted by intra- 
venous inoculation, or by introducing a small quantity of virus beneath 


PROTECTIVE INOCULATIONS. 827 


the dura mater, with greater certainty than by subcutaneous fnocula- 
tions. But the exact nature of this virus has not been determined. 
The fact that a considerable interval elapses after inoculation before 
the first symptoms are developed indicates that there is a multiplica- 
tion of the virus in the body of the infected animal; and this is further 
shown by the fact that after death the entire brain and spinal marrow 
of the animal have a virulence equal to that of the material with which 
it was inoculated in the first instance. The writer’s experiments 
(1887) show that this virulence is neutralized by a temperature of 60° 
C., maintained for ten minutes—a temperature which is fatal to all 
known pathogenic bacteria in the absence of spores. But recent ex- 
periments show that certain toxic products of bacterial growth are 
destroyed by the same temperature. We are, therefore, not justified 
in assuming that the morbid phenomena are directly due to the pres- 
ence of a living micro-organism; and, indeed, it seems probable, from 
what we already know, that the symptoms developed and the death of 
the animal are due to the action of a potent chemical poison of the 
class known as toxalbumins. But, if this is true, we have still to ac- 
count for the production of the toxic albuminoid substance, and, in 
the present state of knowledge, have no other way to explain its in- 
crease in the body of the infected animal than the supposition that a 
specific, living germ is present in the virulent material, the introduc- 
tion of which into the body of a susceptible animal gives rise to the 
morbid phenomena characterizing an attack of rabies. 

Pasteur and his associates have thus far failed to demonstrate the 
presence of microorganisms in the virulent tissues of animals which 
have succumbed to an attack of rabies. Babes has obtained micro- 
cocci in cultures from the brain and spinal cord of rabid animals, and 
states in his article on hydrophobia in “ Les Bacteries”” (second edi- 
tion, p. 791) that pure cultures of the second and third generation in- 
duced rabies in susceptible animals; but his own later researches do 
not appear to have established the etiological relation of this micro- 
coccus. 

Gibier (1884) has reported the presence of spherical refractive 
granules, resembling micrococci, in the brain of rabid animals, which 
he demonstrated by rubbing up a little.of the cerebral substance with 
distilled water. As these supposed micrococci did not stain with 
the usual aniline colors and were not cultivated, it appears very doubt- 
ful whether the refractive granules seen were really microorganisms. 

Fol (1835) claims to have demonstrated the presence of minute 
cocci, 0.2 » in diameter, in sections of spinal cord from rabid ani- 
mals, by Weigert’s method of staining. The cords were hardened in 


328 PROTECTIVE INOCULATIONS. 


a solution of bichromate of potash and sulphate of copper, colored 
with a solution of hematoxylon, and decolorized in a solution of fer- 
rocyanide of potash and borax. 

The writer (1887) has made similar preparations, carefully follow- 
ing the method as described by Fol, but was not able to demonstrate 
the presence of microdrganisms inthe numerous sections made. Nor 
have the observations of Fol been confirmed by the researches of other 
bacteriologists who have given their attention to the subject since the 
publication of his paper. 

Pasteur first announced his success in reproducing rabies in sus- 
ceptible animals by inoculations of material “from the medulla oblon- 
gata, the frontal lobes of the cerebral hemispheres, and the cerebro- 
spinal fluid” in a communication to the Academy of Sciences made on 
May 30th, 1881. At the same time he reported his success in the 
discovery of “a method for considerably shortening the period of in- 
cubation in rabies, and also of reproducing the disease with certainty.” 
This was by inoculations made after trephining, upon the surface of 
the brain with material obtained from the brain of a rabid animal. 
Dogs inoculated in this way developed rabies in the course of two 
weeks, and died before the end of the third week—sometimes of furi- 
ous rabies and sometimes of the paralytic form of the disease. Ina 
second communication (December 11th, 1882) Pasteur reports his 
success in communicating the disease by the intravenous injection of 
virus from the central nervous system; also the experimental demon- 
stration of the fact that all forms of rabies may be produced by the 
same virus; also that all portions of the spinal cord of rabid animals 
are virulent, as well as all parts of the brain; also that an animal 
(dog) which had recovered from a mild attack after inoculation proved 
to be subsequently immune, and that “this observation constitutes a 
first step toward the discovery of the prophylaxis of rabies.” On 
February 25th, 1884, many important facts are stated which had been 
developed during the continuous study of the disease, and among 
others the fact that by passing the virus through a series of animals 
of the same species a fixed degree of virulence is established for each 
susceptible species, as shown by a definite and uniform period of in- 
cubation. By this method a virus had been obtained which produced 
rabies in rabbits in seven or eight days, and another which caused 
the development of the disease in guinea-pigs in five or six days after 
inoculation. In a subsequent communication (May 19th, 1884) evi- 
dence is given to show that by successive inoculations in monkeys the 
period of incubation is prolonged, and that the attenuated virus ob- 
tained from a monkey, after several successive inoculations in this 


PROTECTIVE INOCULATIONS. 829 


animal, when inoculated into the dog, no longer produces fatal rabies; 
and that dogs so treated are subsequently immune. 

In his address before the International Medical Congress at Copen- 
hagen (August 11th, 1884), after a review of the facts developed during 
his experimental researches made during the preceding four years, 
Pasteur gives an account of the test made by a commission, appointed 
by the Minister of Public Instruction, to determine the efficacy of his 
method as applied to the protection of dogs. Hesays that he gave to 
the commission nineteen dogs which had been rendered refractory 
against rabies by preventive inoculations. These nineteen dogs and 
nineteen control animals, obtained from the pound without any selec- 
tion, were tested at the same time. The test was made upon some of 
the animals of both series by inoculation with virulent material upon 
the surface of the brain, and upon others by allowing them to be bit- 
ten by rabid dogs, and upon still others by intravenous inoculations. 

Not one of the protected animals developed rabies; on the other 
hand, three of the control dogs out of six bitten by a mad dog devel- 
oped the disease, five out of seven which received intravenous inocu- 
lations died of rabies, and five which were trephined and inoculated 
onthe surface of the brain died of the same disease. In a subsequent 
report the commission, of which M. Boulley was president, stated that 
twenty-three protected dogs which were bitten by ordinary mad dogs 
all remained in perfect health, while sixty-six per cent of the control 
animals, bitten in the same way, developed rabies within two months. 

In his communication of October 26th, 1885, Pasteur reports his 
discovery of the fact that the virulence of the spinal cord of a rabbit 
is gradually attenuated by hanging it in a dry atmosphere, and is 
finally entirely lost; also that he had been able to make a practical 
application of this discovery in the protection of dogs by means of 
successive inoculations beneath the skin of an emulsion of spinal mar- 
row attenuated in this way. The first inoculation was to be made 
with a portion of spinal cord which had been kept long enough to de- 
prive it of all virulence, and this was followed by daily inoculations 
with more virulent material, until finally material was used from a 
cord only a day or two old. 

With reference to his first inoculations in man, Pasteur says: 

‘Making use of this method, I have already made fifty dogs of various 
races and ages immune to rabies, and had not met with a single failure, 


when, on the 6th of July, quite unexpectedly, three persons, residents of 
- Alsace, presented themselves at my laboratory.” 


These persons were Theodore Vone, who had been bitten on the 
arm on July 4th; Joseph Meister, aged nine, bitten on the same day 


330 PROTECTIVE INOCULATIONS. 


by the same rabid dog; and the mother of Meister, who had not been 
bitten. The child had been thrown down by the dog and bitten upon 
the hand, the legs, and the thighs, in all in fourteen different places. 
Pasteur commenced the treatment on July 6th, by injecting beneath 
the skin of this child an emulsion of cord which had been kept for 
fourteen days; this was followed by twelve more inoculations made 
on successive days with cord of increasing degrees of virulence—the 
last with cord a day old. On March Ist, 1886, Pasteur reported to 
the Academy of Sciences the fact that the boy Meister remained in 
good health and gave detailed information with reference to a number 
of cases which had since been treated by the same method. 

With reference to the duration of the immunity resulting from 
these inoculations Pasteur says (1886) that out of fourteen dogs in- 
oculated with “ ordinary street virus,” by trephining, at the expiration 
of a year after the protective inoculations had been practised, eleven 
resisted; out of six tested in the same way at the end of two years 
two proved to be immune. 

In November, 1886, Pasteur communicated to the Academy of 
Sciences the results of his experiments with reference to a modification 
of his method as at first employed—the so-called intensive method. 
This modification consisted in making the inoculations with cords of 
increasing virulence in more rapid succession. 

The method followed at Odessa, as reported by Gamaleia (1887), 
is shown below, the day being given above and age of the cord 
below. 


1 2 e 8 8. oF 2 Be 
14138 12-11 10-9 87 65 3 2-10 86 £4 BF 


Since the adoption of this method and the use of larger quantities 
of virus, according to Gamaleia, there have been no deaths among 
those inoculated, numbering more than two hundred at the time the 
report was made. The author last referred to concludes from his ex- 
perience that “the mortality diminishes in direct relation to the quan- 
tity of the vaccine injected.” 

Bujwid (1889) reports a total of 670 inoculations, with 9 deaths, 
made at Varsovie during the years 1886, 1887, and 1888. His method 
is shown below. 


oa 
1210 


2 Z 
8-6 3 


plo 


4 5 
3 6 


woo 


The results of inoculations made at the Pasteur Institute in Paris 
during the years 1886 to 1890 are given in the following table: 


PROTECTIVE INOCULATIONS. 331 


‘Year. Number Treated. Died. Mortality. 
ASEE sis owteasteniieee one cca mamone 2,671 25 0.94 
VSB Y, varies eicisigea. adie soa-evn-waniarars 1,770 13 0.73 
TS8S8)) aecisuag@eceisaaa aways 1,622 9 0.55 
A889 socesurduounwGe irk wea age meee 1,830 6 0.38 
USQO ssaddrsvaad eevee wea AGa ase 1,540 5 0.32 
Otel ceive v's Gave egwee ena da 9,433 58 0.61 


In the following table, A includes all persons treated who had been 
bitten by an animal proved to be rabid; B, persons bitten by animals 
examined by veterinary surgeons and pronounced rabid; C, persons 
bitten by animals suspected of being rabid. The figures relate to the 
year 1890: 


| Number Treated. | Died. Mortality. 
Aas visxe ce wsmiaineeeags's Sistadie 416 0 dives 
Boece cash hele Gee o eee ues aaae 909 4 0.44 
C s.wweedemasibva se cae a wEtaneonde 215 at 0,46 


Bordoni-Uffreduzzi gives the following statistics with reference to 
the inoculations practised at the Pasteur Institute in Turin during 
the years 1886 to 1891: 81 persons were inoculated by the method 
first proposed by Pasteur, with a mortality of 2.46 per cent; 925 
persons were subsequently inoculated by the same method, but with 
larger doses of virus, with a mortality of 1.72 per cent. Finally, 338 
persons were inoculated with still larger doses, with a mortality of 
0.29 per cent. 

At the Pasteur Institute in Palermo the number of persons inocu- 
lated in the four years prior to 1891 was 662, with a mortality among 
the inoculated of 0.6 per cent. In Bologna (1890) 210 persons bitten 
by dogs undoubtedly mad were inoculated, with a mortality of 0.47 
per cent. 

Tn the Pasteur Institute at Naples 810 persons were treated during 
the years 1886 to 1892, with a mortality of 0.86 per cent. 

During the year 1891, 1,564 persons were inoculated at the Pas- 
teur Institute in Paris, with a total mortality of 0.57 per cent. In 
394 of these cases the animal which inflicted the bite was proved to 
be rabid by experimental inoculations. 

Horsely (1889) has made a comparison of the results obtained by 
the “intensive treatment” as compared with thcse by the treatment 


first employed, and says: 


332 PROTECTIVE INOCULATIONS. 


“Tt is evident that the intensive treatment is very successful in coping 
with the worst cases, and that, instead of being itself a source of death, as 
asserted by those who gain notoriety and subsistence by villifying and mis- 
representing scientific progress, it is a powerful agent in saving life.” 


The following table is given by Horsely “as showing the contrast 
between the old or simple treatment and the intensive treatment”: 


Simple Treatment, 1886. Intensive Treatment, 1888. 
Odessa... ......0e ee eee 3.39 per cent. 0.64 per cent. 
WATSAW <4 ais iite apenas 4.1 a 0.0* “ 
Moscow.... ...seeeeee 82f * 1.6 


* The figures include sixteen months’ work, and thirty individuals bitten in the 
face—four by wolves. 

+ This unusually high rate was found to be due to imperfections in the manner 
of preparing the cords for the inoculation material. 


Perdrix (1890), in an analysis of the results obtained at the Pasteur 
Institute in Paris, calls attention to the fact that the mortality among 
those treated has diminished each year and ascribes this to improve- 
ment in the method. He says: 


‘* At the outset it was difficult to know what formula to adopt for the 
treatment of each particular case. Upon consulting the accounts of the bites 
in persons who have died of hydrophobia notwithstanding the inoculations, 
we have arrived at a more precise determination as to the treatment suitable 
for each case, according to the gravity of the lesions. In the cases with seri- 
ous wounds we inject larger quantities of the emulsion of cord and repeat the 
inoculations with the most virulent material. For the bites upon the head, 
which are especially dangerous, however slight their apparent gravity may 
be, the treatment is more rapid, and, above all, more intensive—that is to 
‘say, the virulent cord is injected several times.” 


The statistics arranged with reference to the location of the bite 
are given by Perdrix as follows: 


Bitten upon the head, 684; died, 12 = 1.75 per cent. 
- = “hands, 4,396; “ §=— 92 ae 
re . “ limbs, 2,889; “ 5=017 “ 


Other methods of making susceptible animals immune against 
hydrophobia have been proposed and proved by experiment to be 
successful. Thus Galtier in 1880-1881 claimed that the sheep and 
the goat could be protected by intravenous injections of the virus of 
rabies, and more recent experiments fully confirm this. Protopopoff 
(1888) by injecting an emulsion of cord from a rabid animal into the 
circulation of dogs succeeded in protecting them from hydrophobia 
as a result of subsequent inoculation with virulent material upon the 
surface of the brain. He injected into a vein, at intervals of three 
days, one cubic centimetre of an emulsion of cord—first of six days, 
second of three days, third of one day. Roux had previously accom- 
plished the same result by a single intravenous injection of a larger 


PROTECTIVE INOCULATIONS. 333 


quantity (thirty-five cubic centimetres) of cord which had been kept 
for five or six days. In discussing his results Roux calls attention to 
the fact, which had been developed during his experiments, that the 
virulence of the spinal cord of rabid animals does not depend entirely 
upon the length of time it has been kept, but that large doses of cord 
kept as long as twelve days will sometimes produce hydrophobia when 
injected into the circulation of dogs, when smaller doses of cord kept 
five or six days prove to be inoffensive. He supposes that during 
desiccation the virus may not be equally acted upon throughout the 
cord, but that certain “islands” in the central portion may remain 
living and virulent when all the rest has been modified. A practical 
point with reference to the preservation of virulent material is referred 
to by Roux in a note published in the Annels of the Pasteur Insti- 
tute. This is the fact that when preserved in glycerin, portions of 
the central nervous system retain their virulence for considerable 
time. Other forms of virus, e.g., vaccine, may also be preserved in 
the same way. 

Centanni (1892) has succeeded in making rabbits immune by in- 
oculating them with an attenuated virus obtained by subjecting viru- 
lent material to the action of an artificial gastric juice. After digestion 
for less than twelve hours the virus still kills rabbits, when inoculated 
beneath the dura mater, but the period of incubation is considerably 
prolonged. After from twelve to twenty hours’ digestion it no longer 
kills rabbits, but causes an infection, from which they recover, and 
after which they are immune. 

Serum-therapy.—Tizzoni and Centanni (1892) have reported suc- 
cess in the treatment of infected rabbits by the use of blood serum 
from immune animals of the same species—immunized by the “Ital- 
jan method” above described. The animals experimented upon were 
inoculated with a “street virus” which produced paralytic rabies in 
rabbits and caused their death in from fourteen to eighteen days. 
The blood serum was obtained from rabbits which had been proved 
to be immune by resisting inoculations of virus of full strength on 
the surface of the brain. The blood serum, in doses of three to five 
cubic centimetres, was injected subcutaneously, or into the peritoneal 
cavity, or into the circulation. Injections were made into each animal 
(in all from eleven to twenty-six cubic centimetres) after the first symp- 
toms of paralytic rabies had appeared (on the seventh, the tenth, the 
eleventh, and the fourteenth day after infection). Four rabbits treated 
in this way fully recovered. In a subsequent experiment the bacteri- 
ologists named treated three rabbits with a dry antitoxin obtained by 
precipitation from the blood serum of immune rabbits. The precipi- 


334 PROTECTIVE INOCULATIONS. 


tate was obtained by adding one part of serum to ten parts of alcohol, 
and was dried in vacuo. This dried precipitate, in doses of 0.18 to 
0.25 gramme, was dissolved in sterilized water and injected as in the 
previous experiment. Commencing on the eighth day after infection 
five or six doses were given—in all 0.9 to1.3 gramme. Allof the ani- 
mals treated recovered, while all of the control animals died. Babes 
had previously (1889) reported successful results in conferring im- 
munity upon susceptible animals by injections of blood serum from 
immune animals. 

Tizzoni and Schwartz, in pursuing this line of investigation (1892), 
report that while the blood serum of immune rabbits neutralizes the 
“fixed virus” of rabies in vitro, after short contact (five hours), the 
blood serum of immune dogs has but slight antitoxic potency. The 
immunizing substance in the rabbit serum does not dialyze, is soluble 
in glycerin, is precipitated by alcohol, and in general behaves like a 
globulin. In subsequent experiments Tizzoni and Schwartz used 
blood serum from dogs and rabbits immunized by Pasteur’s method. 
The blood was drawn from the carotid of the immune animals, and 
the serum from the same, mixed with virulent spinal marrow in the 
form of a homogeneous emulsion, obtained by crushing and pressing 
through linen. These experiments corresponded with those pre- 
viously made as to the superior antitoxic power of rabbit serum, 
which, after five hours’ contact, neutralized the virulence of the emul- 
sion of cord. By the injection of serum from an immune rabbit, in 
doses of five cubic centimetres, into the circulation of other rabbits, 
they were, as a rule, made immune. The immunizing substance 
(antitoxin) was shown by other experiments to be present only in the 
blood. Extracts from the liver, spleen, kidney, or muscles gave a 
negative result. 

In a later communication (1894) Tizzoni and Centanni give an 
account of further experiments made principally upon sheep and dogs. 
By repeated inoculations they succeeded in obtaining from these ani- 
mals a serum having an immunizing value of 1:25,000 or more, and 
from this a precipitate was obtained estimated to have a value of 
1:300,000, and which in doses of 0.23 gramme (of the dried precipi- 
tate), dissolved in five times its weight of water, ought to be a sufficient 
dose to protect a man from the development of hydrophobia after 
being bitten by a rabid animal. 

The authors named believe that inoculations with this antitoxin 
would be reliable for man, and that they would possess decided ad- 
vantages over Pasteur’s method of inoculation. These advantages are 
specified as follows: 


PROTECTIVE INOCULATIONS. 335 


‘‘ Applicability at any time during the period of incubation up to the 
moment of the appearance of symptoms of rabies ; absolute absence of viru- 
lence and of any injurious action ; very rapid treatment by the injection of 
one or several small doses of material ; complete solubility and consequently 


prompt absorption of the material injected and its easy preservation in a dry 
condition.” 


INFLUENZA. 


The bacillus discovered by Pfeiffer, in 1892, is now well estab- 
lished as the specific cause of this disease. Bruschettini has recently 
(1893) reported the details of his experiments upon rabbits, for which 
animals this bacillus is pathogenic. As a result of these experiments 
he has reached the following conclusions: 


‘‘1. Rabbits may be vaccinated against the pathogenic action of cultures 
of the influenza bacillus without great difficulty. 

‘2, The best material for producing a high grade of immunity is blood 
cultures which have been filtered through the Berkenfeld filter. 

‘3. The blood serum of immunized animals has strong antitoxic proper- 
ties, but has no germicidal power. 

‘4, The serum of vaccinated animals has the power of conferring im- 
munity upon other animals, in comparatively small amounts—in the pro- 
portion of 1:42,000 of body weight, and perhaps still less. 

‘*5. This serum has also a decided curative action, and rescues rabbits 
from death even as late as forty-eight hours after infection by injection of a 
culture of the bacillus into the trachea.” 


These results lead the author to hope that serum-therapy may 
afford a method of curing this disease in man. For this purpose the 
blood of an immune rabbit would appear to be the most promising 
source from which to procure an antitoxic serum. 


INFLUENZA IN HORSES. 


Scuitirz (1887) has described a minute oval bacillus, usually asso- 
ciated in pairs, which appears to be the specific infectious agent in 
the disease known in Germany as Brustseuche. This bacillus is path- 
ogenic for mice, rabbits, pigeons, and guinea-pigs, but not for swine 
or chickens. By injection of cultures into the parenchyma of the 
lungs Schiitz reproduced the disease—confirmed in 1888 by Hell. 

Horses which have suffered an attack of infectious influenza are 
subsequently immune, and the experiments of Hell have shown that 
an immunity also follows the disease which results from inoculations 
with pure cultures of the Schiitz bacillus. 

The extended experiments made by the War Department of the 
German Government show that the disease is not produced by intra- 
venous injections or by the ingestion of the bacillus with the food. 
Infection occurs, however, when cultures are injected into the re- 


336 PROTECTIVE INOCULATIONS. 


spiratory passages. Subcutaneous injections cause a painful local 
tumefaction, often followed by an abscess, but without the general 
symptoms of influenza. 

Experiments have been made in’ Germany by Hell, Siedamgrotzki, 
and others, which indicate that the subcutaneous injection of blood 
serum from immune horses may confer immunity on other horses. 
Hell usually injected forty cubic centimetres at a time, and repeated 
this at intervals until two hundred to two hundred and forty cubic 
centimetres had been injected in the course of two or three weeks. 
He also reports the results of treatment by injections of blood serum 
into the trachea in horses already infected, and thinks these injections 
had a favorable influence on the course of the disease. Experiments 
made subsequently by Toepper have givena similar result, but others 
have not been so fortunate, and the immunizing value of blood-serum 
injections, as practised by the authors referred to, seems to be still a 
matter of some doubt. Toepper (1893) gives full directions for col- 
lecting the serum and a detailed account of results of experimental 
inoculations made by himself and others. He prefers to inject the 
serum into the breast over the ensiform cartilage. No reaction oc- 
curs after the injection. 


PLEURO-PNEUMONIA OF CATTLE. 


Protective inoculations against this disease have long been success- 
fully practised. For this purpose serum obtained from the lungs of 
an animal recently dead has been employed, this having been proved 
by experiment to be infectious material, although the exact nature of 
the infectious agent present in it was not determined. 

Willems, who was one of the first to advocate the use of protective 
inoculations in pleuro-pneumonia (1852), gave a lecture in 1894 in 
which he reviewed the evidence in favor of these inoculations in 
the disease under consideration. Various methods had been em- 
ployed. Thus Willems states that the natives of the banks of the 
Zambéze cause animals to swallow a certain quantity of the liquid 
from the pleural cavity of an animal recently dead, and thus give 
them immunity. The virus has been injected into the circulation by 
some experimenters, and others have proposed to attenuate it by heat. 
But the method which has been most extensively employed is that 
discovered by the Dutch settlers at the Cape of Good Hope (the 
Boers), and consists in inoculating animals in the tail with serum 
from the lungs of an animal recently dead; or with a virus obtained 
from the tumefaction produced by such an inoculation in the tail. 


FROTECTIVE INOCULATIONS. 337 


This secondary virus was very extensively used by Lenglen, a veter- 
inarian at Arras, who communicated his results to the Academy of 
Science at Paris, in April, 1863, and Willems says, in his last pub- 
lished communication, that this is the method which he prefers. It 
is also the method most extensively employed in Australia, into which 
country infectious pleuro-pneumonia was introduced in 1858. It 
quickly spread and has caused enormous losses. The killing of all 
animals, sick or suspected of being infected, was tried for several 
years; but this proved to be ineffectual for stamping out the disease, 
and the sacrifice was so great that this measure of prophylaxis was 
abandoned. 

According to Loir, attention in Australia was called to Willems’ 
method of protective inoculations, in 1861, by a letter from Cape 
Colony published in the journals of Sydney and in Melbourne. The 
method was at once applied both in Victoria and in New South Wales, 
and since that date many thousands of cattle have been inoculated. 
In order to obtain a sufficient supply of virus the method recom- 
mended by Pasteur in 1882 has been followed. This is described by 
Pasteur himself in the following words: 

“With a single lung we may procure sufficient virus to serve for numer- 
ous series of animals. And without having recourse to other lungs this pro- 
vision may be maintained in the folowing manner: It is sufficient before the 
supply of virus is exhausted to inoculate a young calf in the dewlap or in 
the shoulder. The animal dies very promptly, and all its tissues near the 


point of inoculation are infiltrated with serum, which is virulent, and may be 
collected and preserved in a state of purity.” 


Loir prefers to obtain the virus in this way from a calf six to twelve 
months old, during the second week after inoculation, when the tem- 
perature of the animal has gone up to 40° to 42° C., as the virus is 
then said to possess the maximum degree of intensity. This vaccine 
seems to become attenuated in passing through a series of animals by 
inoculation, so that when it has been passed through a series of five 
animals it no longer produces death even when inoculated in the most 
dangerous localities. Loir testifies to the protective value of inocula- 
tions with this virus made in the tail of the animal, and gives the fol- 
lowing example: A few months prior to the publication of his paper 
(1893), about two thousand cows were inoculated with a virus which 
had been passed through a series of five calves. At the moment of 
being driven away they were joined by nineteen other cows not vac- 
cinated. After being on the road for a distance of two thousand kilo- 
metres, the animals arrived at their destination. The two thousand 
vaccinated were in good condition, while eight of the non-vaccinated 
had died of pleuro-pneumonia. 


338 PROTECTIVE INOCULATIONS. 


In the Bulletin of the Central Society of Veterinary Medicine of May 
24th, 1894, M. Robcis reports the results of inoculations made with 
cultures of Arloing’s Pnewmobacillus liquefaciens bovis, and with in- 
jections of pulmonary serum. His statistics with reference to the 
last-mentioned “legal” inoculations he has obtained from official 
documents relating to the Department of the Seine. 

The total number of infected localities in this department during 
the years 1885 to 1891 was 1,253; total number of contaminated ani- 
mals, 18,356; total number inoculated, 18,359; total number of deaths 
prior to inoculation, 1,753; total number of deaths after inoculation, 
2,741; total number of deaths due to the inoculation, 94; total per- 
centage of mortality, 22.8 per cent. After discussing these and other 
statistics Robcis arrives at the conclusion that Arloing’s method of 
preventive inoculations with cultures of the Pnewmobacillus liquefaciens 
bovis gives better results than the legal method with serum from an 
infected animal, the total loss among animals exposed to contagion 
not being over twelve to fourteen per cent. 

Nocard (1892) says that serum from the lungs of an animal dead 
from pleuro-pneumonia preserves its virulence and usefulness as a 
vaccine, when mixed with half a volume of pure neutral glycerin and 
half a volume of a five-per-cent solution of carbolic acid. At the end 
of two and a half months this mixture preserved its full virulence. 


PNEUMONIA. 


The micrococcus of croupous pneumonia was discovered by the 
present writer in the blood of rabbits inoculated subcutaneously with 
his own saliva in September, 1880. In 1885 this micrococcus, which 
I had repeatedly obtained in pure cultures from the blood of rabbits 
inoculated, as in the first instance, with my own saliva, was identified 
with the micrococcus of the same form present in the rusty sputum 
of patients with pneumonia. In a paper read before the Pathological 
Society of Philadelphia, in April, 1885, and published in the Ameri- 
can Journal of Medical Sciences on July Ist of the same year, I say: 

‘Tt seems probable that this micrococcus is concerned in the etiology of 


croupous pneumonia, and that the infectious nature of the disease is due to 
its presence in the fibrinous exudate into the pulmonary alveoli.” 


This has since been fully established by the researches of Frankel, 
Weichselbaum, Netter, Gameleia, and many others. Frankel first 
discovered this micrococcus in his own salivary secretions in 1883, 
and his first paper relating to its presence in the exudate of croupous 
pneumonia was published on July 13th, 1885, 7.e., thirteen days after 


PROTECTIVE INOCULATIONS. 339 


the publication of the paper from which the above quotation is made. 
Under these circumstances the writer feels justified in again calling 
attention to his priority in the discovery of this important pathogenic 
micrococcus, and in objecting to its being described as “Frankel’s 
pneumococceus,” the “ diplococcus of Frankel,” ete. 

In my paper above referred to (July, 1885) I described this micro- 
coccus under the name of Micrococcus Pasteuri, but in my “Manual 
of Bacteriology ” (1892) it is described under the name of Jicrococeus 
pneumonice crowpose. 

This micrococcus is very pathogenic for mice and for rabbits, less 
so for guinea-pigs and for dogs. Like other pathogenic microérgan- 
isms of the same class, it varies greatly in virulence when obtained 
from different sources. In the saliva of healthy persons, which 
seems to be its normal habitat, it sometimes has comparatively little 
virulence. On the other hand, when contained in the blood or in an 
exudate from a serous cavity of an infected rabbit or mouse, it is very 
virulent. In one instance (1881) the writer has seen a fatal result in 
a dog from the subcutaneous injection of one cubic centimetre of bloody 
serum from the subcutaneous connective tissue of a rabbit recently 
dead. 

Pneumonia never results from subcutaneous injections into sus- 
ceptible animals, but injections through the thoracic walls into the 
lung may induce a typical fibrinous pneumonia. This was first de- 
monstrated by Talamon (1883), who injected the fibrinous exudate of 
croupous pneumonia, obtained after death, or drawn during life by 
means of a Pravaz syringe, from the hepatized portion of the lung, 
into the lungs of rabbits. Gameleia has also induced pneumonia in 
a large number of rabbits, and also in dogs and sheep, by injections 
directly into the pulmonary tissue. Sheep were found to survive sub- 
cutaneous inoculations, unless very large doses (five cubic centimetres) 
of a virulent culture were injected. But intrapulmonary inoculations 
are said to have invariably produced a typical fibrinous pneumonia 
which usually proved fatal. In dogs similar injections gave rise to 
a “frank, fibrinous pneumonia’ which rarely proved fatal, recovery 
usually occurring in from ten to fifteen days, after the animal had 
passed through the stages of red and gray hepatization characteristic 
of this affection in man.” 

Without doubt an attack of pneumonia is followed by a certain 
degree of immunity of longer or shorter duration. According to 
Ruge, who'has made a careful study of the subject, relapses are 
very infrequent—indicating a temporary immunity—but subsequent 
attacks are more likely to occur in those who have once suffered an 


840 PROTECTIVE INOCULATIONS. 


attack of the disease, and as many as four or five attacks have been 
known to occur in the same individual. 

In 1,100 cases collected by Wagner but 2 relapses occurred 
(=0.18 per cent). Ruge reports that in 440 cases treated at the 
Charité in Berlin there were but 2 relapses. The liability to sub- 
sequent attacks at a later period is shown by the following figures, 
which we copy from Ruge’s paper: In 280 cases reported by Stortz, 
26.4 per cent, had previously suffered an attack of the disease; in 
133 cases reported by Morhart the proportion of previous attacks was 
41.3 per cent; in 157 cases by Pohlmann, 34.4 per cent; in 166 cases 
by Schapira, 31.3 per cent; in 128 cases by Keller, 36.9 per cent; in 
175 cases by Grisolle, 80.9 per cent. 

The writer, in a series of experiments made during the winter of 
1880-81, obtained experimental evidence which showed that suscep- 
tible animals (rabbits) acquire immunity from the pathogenic action 
of this micrococcus as a result of inoculations with an attenuated 
virus. The experiments referred to had as their object the determi- 
nation of the comparative value of various germicidal agents, as tested 
upon this micrococcus; incidentally it was found “that a protective 
influence has been shown to result from the injection” (into rabbits) 
“of virus, the virulence of which has been modified, without being 
entirely destroyed, by the agent used as a disinfectant.” (Quoted 
from the writer’s report of the experiments referred to, “Studies 
from Biological Laboratory,” Johns Hopkins University, Balti- 
more, 1882.) 

In 1891 G. and F. Klemperer published an important memoir re- 
lating to the pathogenic action of this micrococcus and the production 
of immunity in susceptible animals by means of filtered cultures. In 
some cases this immunity was found to last as long as six months. A 
curious fact developed in their researches was that the potency of the 
substance contained in the filtered cultures was increased by subject- 
ing these to a temperature of 41° to 42° C. for three or four days, or 
to a higher temperature (60° C.) for an hour or two. When injected 
into a vein after being subjected to such a temperature, immunity was 
complete at the end of three or four days; but the same material, not 
so heated, required larger doses and a considerably longer time (four- 
teen days) to confer immunity upon a susceptible animal. The un- 
warmed material caused a considerable elevation of temperature, last- 
ing for some days. The authors mentioned conclude from their 
investigations that the toxic substance present in cultures of Micro- 
coccus pheumonie croupose is a proteid substance, which they propose 
to call pneumotoxin. The substance produced in the body of an im- 


PROTECTIVE INOCULATIONS., 341 


mune animal, as a result of protective inoculations, upon which the 
immunity of these animals depends, is also a proteid, which they call 
antipneumotoxin. This they isolated from the blood serum of im- 
mune animals. By experiment they were able to demonstrate that 
the blood serum containing this protective proteid, when injected 
into other animals, rendered them immune; and also that it arrested 
the progress of the infectious malady induced by inoculating suscep- 
tible animals with virulent cultures of the micrococcus. When in- 
jected into the circulation of an infected animal, its curative action 
was manifested by a considerable reduction of the body temperature. 
The toxalbumin was obtained from filtered bouillon cultures of a viru- 
lent variety of the micrococcus of pneumonia, in the form of an amor- 
phous, yellowish-white powder. This was thrown down from the 
filtered cultures by means of alcohol, and again dissolved in water 
and reprecipitated in order to purify it. 

Issaeff (1893) as a result of his experiments has found that the 
virulence of this micrococcus can be greatly increased by successive 
inoculations in the peritoneal cavity of rabbits, and that after a series 
of ten or twelve such inoculations the blood of the infected animal does 
not coagulate and becomes extremely toxic. In order to obtain the 
toxins from this blood, Issaeff collects the blood of three or four ani- 
mals just dead in a sterilized vessel, and adds to this an equal volume 
of sterilized water containing one per cent of glycerin, made alkaline 
by the addition of a few drops of a concentrated solution of bicarbo- 
nate of soda. The mixture is sterilized by passing it through a Cham- 
berland filter. This liquid sometimes kills rabbits when injected into 
the circulation in the proportion of one per cent of the weight of the 
animal. When heated to 70° C. its toxic power is considerably di- 
minished, and a temperature of 100° C. neutralizes it completely. 

Emmerich (1891) has succeeded in immunizing rabbits and mice 
by the intravenous injection of a very much diluted but virulent cul- 
ture of the micrococcus. Other rabbits and mice were rendered im- 
mune by injecting into them material obtained from rabbits immu- 
nized with diluted cultures. The flesh of these animals was rubbed up 
into a pulp, and the juices were obtained by pressure through a piece 
of sterilized cloth. The bloody juice, after standing for twelve hours 
at a temperature of 10° C., was passed through a Pasteur filter and 
then served to immunize the animals referred to. 

Belfanti (1892) has succeeded in immunizing rabbits against the 
pathogenic action of this micrococcus by injecting into the circulation 
a filtrate obtained from the sputa of pneumonia cases. The viscid 
sputa mixed with an equal part of distilled water was kept on ice for 


342 PROTECTIVE INOCULATIONS. 


twenty-four hours and then passed through a Chamberland filter. 
Ten cubic centimetres of this filtrate was injected into the ear vein of 
rabbits. Some of the animals so treated proved to be immune against 
general infection when inoculated with a virulent culture of the micro- 
coccus, but they had a localized inflammation and cedema about the 
point of inoculation. After recovering from this they proved to be 
entirely refractory against subsequent inoculations. 

Foa and Scabia (1892) have reported success in producing immu- 
nity with filtered cultures, and also with a glycerin extract from the 
blood of an infected rabbit. This, after filtration, was injected sub- 
cutaneously in doses of two cubic centimetres at intervals of five days. 
The authors named have also produced immunity in rabbits by the 
use of “pneumo-protein.” This is an extract from the bacterial cells 
obtained by first collecting these from the surface of a Chamberland 
filter through which the cultures have been passed; then digesting 
them for three hours at 55° C. in a five-per-cent solution of glycerin. 
According to Foa and Scabia immunity produced in this way is more 
decided and of longer duration than that resulting from the other 
methods tested by them. 

Mosny (1892) has also made numerous experiments which show 
that rabbits may be immunized by means of filtered cultures, or by 
the juices from the tissues of an immune animal obtained by macer- 
ation and filtration. When sterilized cultures were employed the best 
results were obtained by first heating very virulent cultures for three 
hours at 60° C. The dose employed was ten cubic centimetres, and 
immunity was not established immediately but required a period of 
at least four days for its development. 

The blood serum of immune rabbits was not found to have any 
bactericidal power, and the micrococcus of pneumonia preserved its 
vitality longer in the blood serum of immune rabbits than in that of 
other animals of the same species. 

G. and F. Klemperer had previously reported that the blood of 
immune rabbits does not destroy the micrococcus of pneumonia or re- 
strict its development. 

Issaeff (1893) also reports his success in immunizing rabbits by 
means of sterilized cultures or filtered blood from infected animals 
recently dead. A single intravenous injection of ten cubic centi- 
metres of filtered blood, prepared as heretofore indicated (p. 340), 
sufficed to confer immunity. To test immunity the animals were 
subsequently inoculated with two to four drops of virulent blood; and 
to maintain it the inoculations (0.5 cubic centimetre) were repeated 
every four weeks. Although immune against infection these animals 


PROTECTIVE INOCULATIONS. 343 


are said not to have acquired any immunity against the toxins of the 
micrococcus of pneumonia. Contrary to the conclusion reached by 
G. and F. Klemperer, Issaeff concludes from his experiments that 
“rabbits, although completely refractory against pneumonic infec- 
tion, remain highly sensitive to the toxins of this microbe. Even 
small doses of the toxins are not neutralized in the blood of vacci- 
nated animals. We are therefore brought to the conclusion that the 
existence of an antitoxic property of the blood of vaccinated animals 
cannot be admitted.” 

The serum of immunized rabbits was not found by Issaeff to pos- 
sess any bactericidal power for the micrococcus of pneumonia, and 
no attenuation of virulence occurred as a result of cultivation in this 
serum. But when introduced beneath the skin of an immune rabbit, 
the micrococcus quickly loses its virulence. At the end of eighteen 
hours it has completely lost its pathogenic power, and cultures made 
in bouillon no longer have any injurious effect upon rabbits. This 
attenuating effect produced in the body of an immune animal is 
ascribed by Issaeff to the action of phagocytes, which are said to be 
very numerous, and in the course of five or six hours to pick up all of 
the cocci in the vicinity of the point of inoculation. These are not, 
however, immediately destroyed in the interior of the phagocytes, but 
preserve their vitality for nearly forty-eight hours, and when intro- 
duced into bouillon give a culture which has no longér any patho- 
genic virulence. 

RINDERPEST. 


The disease of cattle known in Germany as rinderpest is due to a 
bacillus closely resembling the bacillus of fowl cholera and of swine 
plague (Bacillus septicceemice hemorrhagice). 

Professor Semmer, of St. Petersburg, has reported (1892) his suc- 
cess in immunizing cattle against this disease. The virulence of cul- 
tures was attenuated by passing them through guinea-pigs, or by 
exposure to heat, and this attenuated virus was used in protective 
inoculations. Semmer says: 


“By the subcutaneous injection of blood serum from immune animals 
their susceptibility to rinderpest was diminished, and such blood serum de- 
stroyed the ‘rinderpest contagium’ in one to twenty-four hours.” 


SWINE PLAGUE. 


As stated in the chapter on cholera in fowls, the bacillus of swine 
plague (Schweineseuche, Loffler and Schiitz) very closely resembles 
Pasteur’s microbe of fowl cholera and Koch’s bacillus of rabbit sep- 


O44 PROTECTIVE INOCULATIONS. 


ticeemia, and if not identical with these at least varies from them so 
slightly in its morphological and biological characters that recent 
authors do not feel justified in considering it a distinct species. 
Koch first obtained his bacillus of rabbit septicemia by inoculating 
rabbits with putrefying flesh infusion. Gaffky produced the same in- 
fectious disease in rabbits by inoculating them with impure river water. 
Davaine had previously obtained similar results by inoculating rabbits 
with putrefying blood. The writer in 1887 produced the same disease 
in rabbits, while in Cuba, by inoculating them with putrefying liver 
from a yellow-fever cadaver. A similar, and possibly identical, ba- 
cillus has been found in the blood of deer (Hueppe), of cattle (Kitt, 
and of buffalo (Oreste-Armanni) suffering from a fatal infectious dis- 
ease. And all of these allied species or varieties are included by 
Hueppe and by the present writer under the single specific name 
Bacillus septicemice hemorrhagice. The bacillus of the disease known 
in this country as swine plague, according to Smith, agrees in all par- . 
ticulars with that of the German swine plague (Schweineseuche) de- 
scribed by LitHer and Schiitz, except that the latter is more patho- 
genic for swine and for rabbits. 

In a publication by Smith and Moore (United States Department 
of Agriculture, Bureau of Animal Industry, Bulletin No. 6, 1894) they 
have given an account of their experiments relating to immunizing 
animals against the pathogenic action of this bacillus. The bacilli 
used in these experiments were sufficiently virulent to kill rabbits in 
twenty hours when injected beneath the skin of these animals in doses 
of 0.001 cubic centimetre of a fresh bouillon culture. The experiments 
were made upon young adult rabbits by various methods, viz.: with 
sterilized bouillon cultures; with sterilized suspensions of agar cul- 
tures; with the filtrate of agar suspensions; with defibrinated, steril- 
ized blood of infected rabbits ; with blood serum from immune animals. 

‘‘A greater or less degree of immunity was produced in rabbits by steril- 
ized bouillon cultures, sterilized agar suspensions, sterilized blood from 
infected rabbits, and blood serum from immunized rabbits. The sterilized 


blood of diseased rabbits was capable of producing immunity, while the 
blood serum of immune rabbits produced rather equivocal results.” 


The different degrees of immunity which may be acquired by 
rabbits, as shown by a subsequent inoculation with virulent material, 
are classified by Moore as follows: 


“1, No resistance—acute septicaemia. 

‘2. Slight resistance—peritonitis. 

‘*3. Increased resistance—pleuritis and pericarditis with or without sec- 
ondary pneumonia. 

‘4. Higher degree of resistance—pleuritis and peritonitis. 


PROTECTIVE INOCULATIONS. 845 


“5. Still greater resistance—irregular lesions in the form of abscesses, 
subcutaneous and subperitoneal. 


‘‘6. Nearly complete immunity—very slight reaction at the point of in- 
oculation.” 

Up to the year 1894 the bacteriological experts of the Depart- 
ment of Agriculture had not proposed to make a practical application 
of the facts developed in their experimental work in the way of pro- 
tecting herds of swine by means of inoculations with an attenuated 
virus, or with sterilized cultures. In the report on swine plague, 
made by the Bureau of Animal Industry published in 1891, the fol- 
lowing measures for arresting an epidemic are recommended: 

_ ‘“When the disease has actually appeared in a herd the question generally 
arises whether it is worth while to make any attempt to save a portion of the 
herd or to leave them to their fate. Asa rule it may be stated that it is best 
to slaughter both healthy and diseased at once, and give the surroundings 
sufficient time to rid themselves of the infection before fresh animals are 
brought into them. If this be not desirable, we should recommend the fol- 
lowing measures to be vigorously carried out : 

‘“‘a. Removal of still healthy animals to uninfected grounds or pens as 
soon as possible. 

‘*b, Destruction of all diseased animals. 

‘‘¢e. Careful burial or burning of carcasses. ? 

‘‘d. Repeated thorough disinfection of the infected premises. 

‘“‘e, Great cleanliness both as to surroundings and as regards food.” 

In the same report (1891) the following reference is made to pro- 
tective inoculations: 

‘‘ As regards swine plague the experiments which have thus far been car- 
ried out indicate that this disease may prove amenable to preventive inocula- 
tion. We have been able, by the injection of both living cultures and those 
sterilized at a low temperature (58° C.), to make the most susceptible animals 
—rabbits—insusceptible to the most virulent swine plague bacteria. By two 
subcutaneous injections of cultures of swine-plague bacteria, swine have 
been made insusceptible to doses injected into the circulation which proved 
fatal to control pigs within twenty-four hours.” 

According to Smith the experiments of Metchnikoff (1892), re- 
ported as made with the bacillus of hog cholera, were in fact made 
with the bacillus of swine plague; we therefore refer to them here. 
These experiments showed that rabbits could be easily immunized 
against the pathogenic action of virulent cultures by means of blood, 
from an infected animal, sterilized by heat. Doses of 1.5 cubic centi- 
metres, or more, were fatal to rabbits; but smaller doses, repeated 
several times, given either subcutaneously or by injection into the 


circulation, caused the animal to become immune. 


STREPTOCOCCUS INFECTION. 


It is now generally recognized by pathologists that erysipelas, 
puerperal fever, certain forms of diphtheritic inflammation of the 


346 PROTECTIVE INOCULATIONS. 


fauces, and certain acute abscesses are due to infection by a strepto- 
coccus described by recent authors under the name of Streptococcus 
pyogenes. This streptococcus, like other pathogenic microdrganisms 
of the same class, varies greatly in its pathogenic power as a result of 
conditions relating to the source of the particular variety under culti- 
vation. As obtained from a case of erysipelas or puerperal fever it is 
extremely virulent, but when it has led a saprophytic existence for 
some time, or has been cultivated for a considerable time in the 
usual artificial culture media, its pathogenic potency is greatly 
diminished. 

Mironoff (1893) has made a series of experiments with a view to 
determining whether rabbits can be immunized against the pathogenic 
action of this streptococcus, and has obtained successful results by the 
following method: 

Vigorous rabbits, weighing two kilogrammes, were inoculated 
subcutaneously with from three to six cubic centimetres of a sterilized 
bouillon culture of the streptococcus. Cultures three days old were 
employed, and these were sterilized for twenty minutes at 120° C.— 
the reason for using so high a temperature is not apparent, inasmuch 
as this streptococcus is destroyed in a few minutes by a temperature 
of 60° C. At the end of ten to fifteen days, ‘when the animal has 
fully recovered,” a second dose of from six to twelve cubic centimetres 
of a culture, sterilized in the same way, is injected beneath the skin. 
After another interval of ten to fifteen days two cubic centimetres of a 
virulent non-sterilized culture are injected subcutaneously, and this is 
repeated with gradually increasing doses (one to two cubic centime- 
tres more) at intervals of the same period. Finally the animals 
“support without reaction” a dose five times as great as would be re- 
quired to kill an animal of the same weight not immunized. But the 
author adds that more than half the animals thus treated died before 
the completion of the immunizing process. These deaths resulted 
from local infectious processes, such as peritonitis, pericarditis, men- 
ingitis, or abscesses formed at the point of inoculation. 

Further experiments showed that the blood serum of animals im- 
munized in this way when injected into susceptible animals (rabbits) 
in the dose of 1.5 cubic centimetres per kilogramme of body weight 
conferred upon them a certain degree of immunity against streptococ- 
cus infection, and with twice this amount (three cubic centimetres) a 
very decided immunity was produced. The blood serum of immune 
rabbits in doses of three to four cubic centimetres per kilogramme of 
body weight was found to exercise a curative power, and completely to 
arrest the acute septicemia resulting from inoculations with a virulent 


PROTECTIVE INOCULATIONS. 347 


culture of this streptococcus, or to cause the disease to run a chronic 
course, with formation of abscesses and final recovery. 

In this connection we may call attention to the experiments of 
Emmerich (1886), which show that the fatal course of anthrax infec- 
tion, in rabbits, may be arrested by the subcutaneous or intravenous 
injection of this streptococcus. Subsequent experiments by Emmerich 
and de Mattei (1887) showed that eleven hours after such an injection 
the anthrax bacilli were all dead and were already undergoing degen- 
erative changes. 

Emmerich and his associates (1894) have reported numerous addi- 
tional experiments which show that the blood serum of a rabbit which 
is suffering from streptococcus septiceemia (third day), when filtered 
through a Pasteur-Chamberland filter to remove all living cocci, may 
be used with success in arresting anthrax infection in rabbits. The 
filtered serum was given four hours after the anthrax infection in the 
dose of twenty-five cubic centimetres in the peritoneal cavity and fif- 
teen cubic centimetres subcutaneously. This was repeated the fol- 
lowing day at nine o’clock in the morning and five o’clock in the 
evening, and again on the third day inthe morning. Favorable re- 
sults were also obtained by using in the same way blood serum from 
a sheep infected with the streptococcus. 

Cobbett (1894) reports success in immunizing rabbits by means of 
attenuated varieties of the streptococcus or by filtered cultures. Also 
that cutaneous erysipelas, produced by inoculation, after recovery 
leaves the patient immune from a repetition of the local inflammatory 
process as a result of a subsequent inoculation, and also confers a gen- 
eral immunity against streptococcus infection. But this immunity is 
of short duration, not lasting longer than a few weeks. Inoculation 
in the ear of a rabbit, protected by a previous inoculation in the same 
locality, is followed by an inflammatory reaction; but this is of brief 
duration and has disappeared before the erysipelatous inflammation 
produced in a control is well under way. 


SYMPTOMATIC ANTHRAX. 


This disease of cattle is popularly known as “ black leg,” or “ quar- 
ter evil,” and is described by German authors under the name of 
Rauschbrand—French, “charbon symptomatique.” The disease pre- 
vails during the summer months in various parts of Europe, and to 
gome extent in the United States. It is characterized by the appear- 
ance of irregular, emphysematous swellings of the subcutaneous tis- 
sues and muscles, especially over the quarters. The muscles in the 


348 PROTECTIVE INOCULATIONS. 


affected areas have a dark color and contain a bloody serum in which 
the bacillus is found to which the disease is due. This is an an- 
aérobic bacillus which forms large oval spores. 

The etiology of the disease was first clearly established by the re- 
searches of Arloing, Cornevin, and Thomas (1880 to 1883), and sub- 
sequent researches have shown that immunity may be produced in 
susceptible animals by protective inoculations. 

The disease causes considerable losses among cattle in certain sec- 
tions. Horses do not contract it spontaneously, and when inoculated 
with a culture of the bacillus present only a limited local reaction. 
Swine, dogs, rabbits, fowls, and pigeons have but slight susceptibility. 
The researches of the authors above mentioned have shown that the 
virulence of a culture is greatly increased by adding to it twenty per 
cent of lactic acid. The guinea-pig is the most susceptible animal, 
and succumbs in from twenty-four to thirty-six hours when inoculated 
subcutaneously with a small quantity of a pure culture. According 
to Kitasato cultures in a bouillon made from the flesh of the guinea- 
pig soon lose their virulence, while cultures in solid media preserve 
their virulence for a long time. Cultures are readily attenuated by 
heat, according to the method of Toussaint and Chauveau—exposure 
to a temperature of 42° to 48° C. in the absence of spores. The 
spores are attenuated by exposure for several hours to a temperature 
of 80° to 100° C. Arloing, Cornevin, and Thomas recommend for the 
production of immunity in cattle inoculation with a dried powder of 
the muscles of animals recently dead from the disease. This is at- 
tenuated by heat. According to Kitt the muscles should first be 
dried at 32° to 35° C. and then powdered. Two “vaccines” are pre- 
pared from this powder—a strong vaccine by exposure to a temper- 
ature of 85° to 90° C. for six hours, and a weaker vaccine by exposure 
for the same time to a temperature of 100° to 104° C. (dry heat). An 
inoculation is first made with the weaker vaccine which gives rise to 
a local reaction of moderate intensity. Later a second inoculation is 
made with the stronger vaccine, after which the animal is immune 
from the pathogenic action of the most virulent material. Immunity 
may also be secured by intravenous injections; or, in guinea-pigs, by 
inoculations with cultures which have become attenuated by being kept 
a few days, or by exposure to a temperature of 42° to 483° C.; or by 
inoculation with a very small quantity of a pure culture; or by inocu- 
lations with filtered cultures (Roux and Chamberland) ; or with cultures 
sterilized by heat (Kitasato). A non-fatal and protective local infec- 
tion may also be produced in cattle by inoculations with virulent ma- 
terial made into the extremity of the tail. Roux has claimed that 


PROTECTIVE INOCULATIONS. 349 


animals which have un acquired immunity against symptomatic an- 
thrax are also immune against the pathogenic action of the bacillus of 
malignant oedema; but Kitasato was unable to confirm this. 

Strebel, in 1885, published the results of protective inoculations 
made in Switzerland in 1884. The inoculations were made in the end 
of the tail with two “vaccines,” with an interval between the two of 
from nine to fourteendays. The vaccines were prepared by exposure 
to heat, as above recommended by Arloing, Cornevin, and Thomas. 
The most favorable season for inoculations was found to be the spring, 
and the most favorable age of cattle for inoculation from five months 
to two years. 

Tn seven Swiss cantons 2,199 cattle were inoculated; 1,810 inocu- 
lations were made among animals which were exposed in dangerously 
infected pastures. Of these but 2 died, one two months and the 
other four months after the protective inoculations. Among 908 in- 
oculated cattle, which were pastured with 1,650 others not inoculated, 
the mortality was 0.22 per cent, while the loss among the latter was 
6.1 per cent. The following year (1885), according to Strebel, the 
number of inoculations, exclusive of those made in the canton of Bern, 
was 35,000. The losses among inoculated animals are reported as hav- 
ing been about five times less than among those not protected in this 
way. In the canton of Bern, in the same year, according to Hess, 
15,187 cattle were inoculated by thirty-eight veterinarians—12,190 of 
these were pastured in dangerously infected pastures. The results 
are said to have been favorable to the method, but the abstract at 
hand does not give the precise figures. 

In 1887 Kitt reported the results of his investigations, which were 
confirmatory of those previously published by Arloing, Cornevin, 
and Thomas, and also of a new method of inoculation, which pre- 
sented the advantage that a single inoculation was sufficient to confer 
immunity. This was made in the region of the shoulder with a vac- 
cine somewhat stronger than that employed by the French bacteriol- 
ogists, but which was found to be without danger for cattle. It 
produced only a slight local effect. His vaccine was prepared by 
heating the moistened flesh of an animal just dead from the disease 
to 85° to 90° GC. for six hours. This did not kill the spores present, 
but caused a sufficient attenuation in their virulence. 

Tn a later communication (1888) Kitt recommends that the flesh of 
the diseased animal be first dried and pulverized, and then subjected 
to a temperature of 100° C. in streaming steam for six hours, after 
which it is to be again dried and used for subcutaneous inoculations. 
The dose is from five to fifteen centigrammes. 


850 PROTECTIVE INOCULATIONS. 


Roux (1888) has shown by experiment that sterilized cultures of 
the bacillus, which have been exposed to a temperature of 115° C., 
when injected in doses of forty cubic centimetres, three times repeated, 
into the cavity of the abdomen of guinea-pigs, cause these animals to 
be completely immune against the most virulent material. Cultures 
from which the bacilli have been separated by filtration are still more 
active. And immunity could easily be conferred by the subcutaneous 
inoculation, in guinea-pigs, of one cubic centimetre of the filtrate from 
the serum obtained from the cedematous tissues of a diseased animal. 

Schuhanka (1888) has reported the results of inoculations made 
in the dukedom of Salzburg during the year 1887. In all 2,596 cattle 
were inoculated once, and 2,472 twice, with an attenuated virus, in 
forty-seven different parishes. Most of these were from six months 
to a year old. No losses occurred as a result of the inoculations. 
During the summer of 1887 the 2,472 cattle which had been twice in- 
oculated were associated in infected pastures with 3,561 unprotected 
cattle. The loss among the former was 8, = 0.32 per cent; among the 
latter it was 235, = 6.31 per cent. 

Strebel reports similar results, in 1887, in the canton Freiburg, 
where 1,725 cattle which had been inoculated suffered a loss of 0.23 
per cent, and 1,945 associated cattle a loss of 5.28 per cent. 

Lydtin (1892) reports the results of inoculations made in five dis- 
tricts (Amtsbezirken) in Baden during the years 1886-91: 2,797 cattle 
were inoculated with a loss of 3 only as a result of the inoculation. 
None of the inoculated cattle subsequently contracted the disease. 

In the Bulletin of the Central Society of Veterinary Medicine of 
France (1892), Guillod and Simon give the results of 3,500 inocula- 
tions made since 1884. The mortality among cattle in the region 
where these inoculations were practised had been from 10 to 20 per 
cent, but fell to 0.5 per cent among the inoculated animals. 

The authors last named prefer inoculations in the region of the 
shoulder to the plan first practised of inoculating in the end of the 
tail. Strebel also (1892) advocates this method, which is quickly car- 
ried out and attended with but little loss. According to Strebel the 
loss among 13,022 inoculated in this way only amounted to 5, while 
the loss among animals inoculated by the old method was twice as 
great. 


TETANUS. 
The experiments of Kitasato (1889) show that pure cultures of the 


tetanus bacillus injected into mice, rabbits, or guinea-pigs produce 
typical tetanic symptoms and death. As the presence of this bacillus 


PROTECTIVE INOCULATIONS. 351 


at the seat of injury, in cases of tetanus in man, has now been 
demonstrated by numerous observers, there is no longer any ques- 
tion that tetanus must be included among the traumatic infectious 
diseases, and that the bacillus of Nicolaier and of Kitasato is the 
specific infectious agent. Kitasato’s experiments (1890) show that 
cultures of the tetanus bacillus which have been sterilized by fil- 
tration through porcelain produce the same symptoms, and death, in 
the animals mentioned, as result from inoculation with cultures con- 
taining the bacilius. It is evident, therefore, that death results from 
the action of a toxic substance produced by the bacillus. This is 
further shown by the fact that the bacillus itself cannot be obtained in 
cultures from the blood or organs of an animal which has succumbed 
to an experimental inoculation with an unfiltered culture; but the 
blood of an animal killed by such an inoculation contains the tetanus 
poison, and when injected into a mouse causes its death with tetanic 
symptoms. 

When a platinum needle is dipped into a pure culture of the teta- 
nus bacillus, and a mouse is inoculated with it subcutaneously, the 
animal invariably falls sick within twenty-four hours and dies of 
typical tetanus in two or three days. Rats, guinea-pigs, and rabbits 
are killed in the same way by somewhat larger quantities—0.3 to 0.5 
cubic centimetre (Kitasato). Pigeons are very slightly susceptible. 
The tetanic symptoms are first developed in the vicinity of the point 
of inoculation: if the animal is inoculated in the posterior portion of 
the body, the hind legs first show tetanic contraction; if in the fore- 
part of the body, the muscles of the neck are first affected. At the 
autopsy there is a certain amount of hyperemia at the point of inocu- 
lation, but no pus is formed; in inoculations with garden earth, or 
accidental inoculations in man, pus is commonly found in the vicinity 
of the inoculation wound. The various organs are normal in appear- 
ance. Kitasato says that he has not been able to demonstrate the 
presence of the bacillus or of spores in the spinal marrow, the nerves, 
muscles, spleen, liver, lungs, kidneys, or blood from the heart; nor 
has he been able to obtain cultures from the various organs. In mice 
which were inoculated at the root of the tail Kitasato was able to de- 
monstrate the presence of the bacilli at the point of inoculation by the 
microscopical examination of an excised piece of the tissues for eight 
to ten hours after the inoculation; later than this they were not found. 
In pus from the inoculation wounds of men and animals accidentally 
infected the bacilli are present, but the formation of spores does not 
always occur. According to Kitasato, the sooner death has occurred 
after accidental inoculation the less likely are spores to be found in 


352 PROTECTIVE INOCULATIONS. 


the rods, but from pus in which no spores are seen cultures of the 
bacillus may be obtained in which spores will develop in the usual 
manner. 

Guinea-pigs are even more susceptible to the tetanus poison than 
mice, and rabbits less so. The amount of filtrate from a slightly 
alkaline bouillon culture required to kill a mouse is extremely minute 
—0.00001 cubic centimetre (Kitasato). The tetanic symptoms are 
developed within three days; if the animal is not affected within four 
days it escapes entirely. The tetanus poison is destroyed by a tem- 
perature of 65° C. maintained for five minutes, or 60° for twenty 
minutes, or 55° for an hour and a half; in the incubating oven at 37° 
C. it gradually loses its toxic potency’; in diffuse daylight, also, its 
toxic power is gradually lost; in a cool, dark place it retains its origi- 
nal potency indefinitely ; in direct sunlight it is completely destroyed 
in from fifteen to eighteen hours; it is not injured by being largely 
diluted with distilled water; it is destroyed in an hour by hydro- 
chloric acid in the proportion of 0.55 per cent; terchloride of iodine 
destroys it in the proportion of 0.5 per cent; cresol in one per cent— 
one hour’s exposure. In general it is destroyed by acids and by 
alkalies. Blood serum from cattle, horses, sheep, rabbits, rats, or 
guinea-pigs does not modify its toxic properties. 

Brieger (1886) first succeeded in obtaining from impure cultures 
of the tetanus bacillus a crystallizable toxic substance, called by him 
tetanin, which was found to kill small animals in very minute doses 
and with the characteristic symptoms of tetanus. More recently Kit- 
asato and Weyl have obtained the same substance, by following 
Brieger’s method, from a pure culture of this bacillus. From a bouil- 
lon made from one and one-fourth kilogrammes of lean beef, with the 
addition of twenty-five grammes of peptone, they obtained 1.7118 
grammes of hydrochlorate of tetanin. This proved fatal to white mice 
in six hours in the dose of 0.05 gramme, and a dose of 0.105 gramme 
caused characteristic tetanic convulsions and death within an hour. 
The bacteriologists last named also obtained from their cultures the 
tetanotoxin of Brieger. Two mice were inoculated subcutaneously 
with 0.003 gramme of this substance; one died at the end of five 
hours without the development of tetanic symptoms; the other sur- 
vived. In addition to these substances, indol, phenol, and buty- 
ric acid were demonstrated to be present in cultures of the tetanus 
bacillus. 

The more recent researches of Brieger and Frankel, and of Kita- 
gato, show that the toxic ptomain discovered by Brieger in 1886 is 
not the substance to which cultures of the tetanus bacillus owe their 


PROTECTIVE INOCULATIONS. 3853 


great and peculiar pathogenic power. The distinguished German 
chemist and his associate have succeeded in isolating from tetanus 
cultures a toxalbumin which is far more deadly than tetanin. 

Brieger and Cohn in more recent investigations (1898) relating to 
the toxic products of the tetanus bacillus have arrived at the following 
results: The cultures were made in veal bouillon containing one per 
cent of peptone and one-fifth per cent of chloride of sodium. Large 
quantities of the cultures in this medium were filtered through porce- 
lain filters. The active substance was precipitated from the filtrate 
by means of a saturated solution of ammonium sulphate. By adding 
this salt in excess the precipitate is made to rise to the surface and is 
skimmed off with a platinum spatula. The liquid is removed by plac- 
ing this upon porous porcelain plates and the crude toxin is dried in 
avacuum. It still contains 6.5 per cent of ammonium sulphate. The 
tetanus bouillon after filtration is said to be fatal to mice in the dose 
of 0.00005 cubic centimetre. A litre of this bouillon gave about one 
gramme of the dried precipitate, which produced characteristic te- 
tanic symptoms and death when injected into mice in the dose of 
0.0000001 gramme. Kitasato in his experiments had previously ob- 
tained a tetanus bouillon which was five times as toxic as that used by 
Brieger and Cohn in their experiments, and which killed mice in the 
dose of 0.00001 cubic centimetre. The dried precipitate obtained by 
Brieger and Cohn contained various impurities, including a certain 
amount of ammonium sulphate, but was found to kill susceptible ani- 
mals in the proportion of 0.0000066 gramme per kilogramme of body 
weight. 

It was purified without loss of toxic power by placing it in a dialyzer 
in running water for from twenty-four to forty-eight hours, after which 
it was dried in vacuo at 20° to 22° C. The purified toxin thus ob- 
tained had a slightly yellowish color, and was in the form of trans- 
parent scales, which were odorless, tasted like gum acacia, and were 
easily soluble in water. The chemical reactions of this purified toxin, 
according to Brieger and Cohn, show that it is not a true albuminous 
body. When injected beneath the skin of a mouse weighing fifteen 
grammes, in the dose of 0.00000005 gramme, it causes its death, and 
one-fifth of this amount gave rise to tetanic symptoms from which the 
animal recovered after a time. The lethal dose for a man weighing 
seventy kilogrammes is estimated by the authors named to be 0.00023 
gramme (0.23 milligramme). Comparing this with the most deadly 
vegetable alkaloids known it is nearly six hundred times as potent as 
atropine and one hundred and fifty times as potent as strychnine. 

Fermi and Pernossi (1894), as a result of an elaborate research, 

23 


354 PROTECTIVE INOCULATIONS. 


have determined many of the chemical characters of the tetanus toxin. 
When in solution it is destroyed by a comparatively low temperature 
(55° C. for one hour) and by exposure to direct sunlight, but the dry 
powder resists a temperature of 120° C. It has not the properties of 
an alkaloid, as it is not dissolved by any of the usual solvents of these 
bodies—the only solvent thus far discovered is said to be water. It 
resembles the albumins and peptones in its failure to pass through 
a dialyzing membrane. The authors last referred to conclude their 
summary of results as follows: 


‘‘The appended table shows that the tetanus poison, like that of diph- 
theria, in its behavior as regards the action of light, heat, chemical agents, 
and dialysis, as also its solvents, the agents which precipitate it, and its action 
upon living animals, closely resembles the poisons of serpents (Naja tripu- 
dians, Crotalus, etc.). As to the chemical nature of this group of substances, 
we can at present only say that they rather have the characters of collodial 
substances than otherwise, and more nearly resemble the albuminoid bodies 
than the bases. We do not, however, reject the very probable hypothesis 
that these toxins are acids or bases, or other very unstable, peculiar substances, 
which are closely united with colloidal substances, as is the case, for example, 
with the alkali and acid albumins and so many other albuminous bodies.” 


While the exact nature of the toxic substance contained in tetanus 
cultures has not been determined, we probably cannot, at present, do 
better than to continue to speak of it as a “toxalbumin.” 

Kitasato (1891) was not able to produce immunity in mice by in- 
oculations with minute doses of the poison, or with a filtrate which 
had been exposed to various degrees of temperature by which its 
activity was diminished or destroyed. But immunity lasting for 
about two months was produced in rabbits by inoculating them with 
the filtrate from a culture of the tetanus bacillus, and subsequently, 
in the same locality, with three cubic centimetres of a one-per-cent 
solution of terchloride of iodine; this last solution was injected sub- 
cutaneously in the same dose at intervals of twenty-four hours for five 
days. Of fifteen rabbits treated in this way six proved to be immune 
against large doses of a virulent culture of the tetanus bacillus. The 
same treatment was not successful in producing immunity in mice or 
guinea-pigs, but the important discovery was made that a small quan- 
tity of blood (0.2 cubic centimetre) from an immune rabbit, when in- 
jected into the abdominal cavity of a mouse, gave it immunity from 
the effects of inoculations with the tetanus bacillus. Moreover, mice 
which were first inoculated with a virulent culture of the bacillus, and, 
after tetanic symptoms had appeared, received in the cavity of the 
abdomen an injection of blood serum from an immune mouse, were 
preserved from death. The power of the blood of an immune animal 
to neutralize the tetanus poison was further shown by mixing the fil- 


PROTECTIVE INOCULATIONS. 355 


trate from a virulent culture with blood serum from an immune animal 
and allowing it to stand for twenty-four hours; a dose three hundred 
times greater than would have sufficed to kill a mouse proved to be 
without effect after such admixtures with blood serum; as before 
stated, the blood serum of animals which are not immune has no 
effect upon the poison. The duration of immunity induced in this 
way was from forty to fifty days. Blood serum from an immune rab- 
bit, preserved in a cool, dark room, retains its power of neutralizing 
the tetanus poison for about a week, after which time it gradually 
loses it. Having found that chickens have a natural immunity against 
tetanus, Kitasato made experiments to ascertain whether their blood 
serum would also neutralize the tetanus poison; the result was nega- 
tive. 

That the tetanus poison is present in the blood of individuals who 
die from tetanus has been proved by Kitasato by injecting a small 
quantity (0.2 to 0.3 cubic centimetre) of blood from the heart of a 
fresh cadaver into mice; the animals develop typical tetanic symp- 
toms and die in from twenty hours to three days. 

Tizzoni and Cattani have (1891) reported results similar to those 
obtain by Kitasato. By repeated inoculations with gradually in- 
creasing doses of the tetanus poison they succeeded in making a dog 
and two pigeons immune, and found that blood serum from this im- 
mune dog, in very small amount, completely destroyed the toxic 
power of a filtrate from cultures of the tetanus bacillus—one to two 
drops of serum neutralized 0.5 cubic centimetre of filtrate after fifteen 
to twenty minutes’ contact. They also ascertained that small amounts 
of blood serum from this immune dog injected into other dogs or 
white mice produced immunity in these animals; but they were not 
able to produce immunity in guinea-pigs or rabbits by the same 
method. 

In a later communication (May, 1891) Tizzoni and Cattani give 
an account of their experiments made with a view to determining 
the nature of the substance in the blood serum of an immune animal 
which has the power of destroying the toxalbumin of tetanus—“tet- 
anus antitoxin.” They found, in the first place, that this antitoxin 
in blood serum is destroyed in half an hour by a temperature of 68° 
C.; further, that it does not pass through a dialyzing membrane; that 
it ig destroyed by acids and alkalies. As a result of their researches 
they conclude that it is an albuminous substance having the nature of 
an enzyme. 

Vaillard has succeeded in producing immunity in rabbits by re- 
peated injections into the circulation of filtered cultures—in all twenty 


356 PROTECTIVE INOCULATIONS. 


cubic centimetres—which had been exposed for one hour to a temper- 
ature of 60° C. At a temperature of 65° C. both the toxic and the 
immunizing action is destroyed. 

Behring (1892) gives the following account of a method which he 
has successfully employed for producing immunity in large animals— 
especially in horses: A culture of the tetanus bacillus is made, in 
bouillon, of such toxic potency that 0.75 cubic centimetre will kill a 
rabbit in three or four days. To two hundred cubic centimetres of 
this culture he adds carbolic acid in the proportion of 0.5 per cent for 
the purpose of preserving it. The horse first receives a subcutaneous 
injection of ten cubic centimetres of this culture fluid to which ter- 
chloride of iodine (ICIl,) has been added in the proportion of 0.25 
per cent; at the end of eight days twenty cubic centimetres of the 
same mixture are given; again in eight days the dose is repeated; 
then, after an interval of three days, thirty cubic centimetres of the 
same mixture. Following this, at an interval of eight days, he gives 
two injections of thirty cubic centimetres each of a mixture containing 
one-half the quantity of ICI, (0.175 per cent). The proportion of 
the iodine terchloride is then reduced to 0.125 per cent, and two 
doses of twenty cubic centimetres each are given. Finally the culture 
fluid is administered in the dose of 0.5 cubic centimetre, and this dose 
is doubled every five days. Before giving the first dose of culture 
fluid without the addition of ICI,, the immunizing value of the blood 
serum of the horse is tested on mice, and if it falls below 1:100 a 
dose of 0.25 cubic centimetre is given instead of the larger dose (0.5 
cubic centimetre) above mentioned. 

Schitz (1892) has applied Behring’s method to a considerable 
number of horses and sheep, and arrives at the conclusion that it is a 
reliable method of protecting these animals against infection with liv- 
ing tetanus bacilli and against the toxic action of filtered cultures; 
that the degree of immunity and the antitoxic power of the blood 
serum increase as larger doses are gradually given. According to 
Behring the immunizing value of blood serum from a horse treated in 
this way is very high. As tested on mice it may be 1:200,000, or even 
more. According to his calculations a serum having a value of 1:100,- 
000, as tested on mice, should be given to a man weighing fifty kilo- 
grammes in the quantity of fifty cubic centimetres, given in the course 
of two days, in order to insure immunity. 

The same author in a subsequent paper (1892) gives details as to 
the method of estimating the therapeutic value of serum from an im- 
mune animal. He first calls attention to the fact that the only re- 
agent by which the antitoxic potency of this serum can be tested is 


PROTECTIVE INOCULATIONS. 357 


the body of a living animal. The test animal selected is the white 
mouse. When the statement is made that a serum has the value of 
1:1,000,000, he means that by an experimental test, made upon white 
mice, it has been ascertained that these animals are protected from 
fatal infection with the minimal lethal dose of a tetanus culture by the 
use of 0.00002 gramme of the serum for a mouse weighing twenty 
grammes. For the cure of tetanus in the mouse, after the first symp- 
toms of the disease have appeared, a dose at least one thousand times 
as great as the immunizing dose is required, and the more advanced 
the progress of the case the greater the dose must be. A serum of 
the strength above indicated, if used for the treatment of a case of 
tetanus in man, should, according to Behring, be employed in doses 
amounting altogether to at least one hundred cubic centimetres—given 
inside of twenty-four hours in doses of twenty cubic centimetres each. 
For persons sixteen years old he would give doses of ten cubic centi- 
metres, and for children under six, five cubic centimetres at a dose. 
The serum of this strength which he had prepared for testing its 
curative value on man was preserved by the addition of 0.5 per cent 
of carbolic acid. 

Rotter (1892) reports a case successfully treated by Behring’s 
serum. In all two hundred and fifty cubic centimetres was adminis- 
tered subcutaneously. The case was not, however, one of the most 
severe forms of the disease. 

Brieger and Ehrlich (1892) have succeeded in immunizing goats 
by means of gradually increasing doses of a culture of the tetanus 
bacillus in thymus bouillon. The amount given at first was 0.2 cubic 
centimetre, and this was gradually increased to ten cubic centimetres. 
At the end of thirty-seven days the animal was found to be immune 
against virulent cultures, and the important fact was demonstrated 
that the immunizing substance (antitoxin) was present in its milk. 
A mouse which received 0.1 cubic centimetre of the milk of this goat 
in the peritoneal cavity proved to be immune against infection as a 
result of inoculation with a tetanus culture. The immunizing value 
of the milk from this goat was found to be 1,600. That is, a dose of 
0.2 cubic centimetre, which was equal to 1:100 of the body weight of 
the animal, protected a mouse from sixteen times the fatal dose of a 
tetanus culture. After precipitation of the casein the milk still pre- 
served its antitoxic power unimpaired, and by concentrating it 7 vacuo 
a fluid was obtained which proved to have an immunizing value of 
5,000. 

Tn a later communication (1893) Brieger and Cohn give the results 
of additional experiments with the milk of immunized goats. Ani- 


858 PROTECTIVE INOCULATIONS. 


mals were chosen which were two or three years old and had given 
birth to young a few weeks before the inoculations were commenced. 
It having been previously shown by Ehrlich that the precipitated 
tetanus toxin from cultures could be successfully used to immunize 
guinea-pigs, the same substance was employed in these experiments. 
The treatment was commenced with a dose of 0.00001 gramme, which 
was carefully increased to 0.00007 gramme, the injections being made 
at intervals of four days. But this proved to be too much, and the 
animal died of typical tetanus after the last dose. In a subsequent 
experiment Brieger and Coln succeeded in immunizing a goat ina 
month and a half so that the animal finally withstood a dose of 0.06 
gramme, but this animal ceased to give milk, became anemic, and 
finally died. 

The authors therefore resorted to a different method which had 
previously been successfully employed by Ehrlich, Behring, and 
others. Cultures of the tetanus bacillus in bouillon were heated to 
65° C. for half an hour, and then used for immunizing two goats. 
After five weeks’ treatment the animals resisted doses of the precipi- 
tated toxin, which were gradually increased to ten grammes, at which 
time the treatment had been carried on for nearly six months and the 
antitoxic value of the milk was found to be 90,000 immunization units. 

The method of determining antitoxic values adopted by Brieger 
and Cohn is the following: They had found by carefully conducted 
experiments that their precipitated toxin (Rohgifte) killed a mouse 
weighing twenty grammes in the dose of 0.0000003 gramme, but 
failed to kill when injected in the dose of 0.0000002 gramme. The 
first-mentioned dose was therefore accepted as the minimum fatal dose 
for an animal weighing eighteen to twenty grammes, and the object 
in view was to find the minimum amount of milk required to prevent 
the toxic action of such a dose. 

The antitoxin was obtained from the goat’s milk by precipitation 
with ammonium sulphate, thirty-two per cent; the precipitate was 
again dissolved and treated with a solution of basic acetate of lead; 
this salt does not precipitate the antitoxin when the solution is slightly 
alkaline; the voluminous precipitate produced by the lead acetate is 
filtered out and repeatedly washed with water; the filtered fluid and 
wash water are again treated with ammonium sulphate, added to 
saturation, and the resulting precipitate is dissolved in a small quan- 
tity of water; a precipitate is again obtained by saturation with am- 
monium sulphate, and this is dried upon porcelain plates in a vac- 
uum. The ammonium sulphate remaining could not be removed by 
dialysis, as experiment showed that a considerable loss of the antitoxin 


PROTECTIVE INOCULATIONS. 359 


occurred in a dialyzer placed in running water. But by shaking up 
the dry powder in chloroform the heavy salt sank to the bottom and 
the purified antitoxin floated on the surface and could be recovered by 
skimming it off. The powder thus obtained consisted of a mixture of 
various substances, including the antitoxin, and when obtained from 
milk having an antitoxic value of 90,000 it was found to have a value 
of 25,000,000 immunization units. By further purification a still 
higher value was obtained (55,000,000). In experiments on mice a 
dose ten thousand times as great as was necessary to produce immu- 
nity proved to exercise a curative power—i.e., a dose of 0.02 gramme 
for a mouse weighing twenty grammes saved it from being killed by 
double the minimum fatal dose of the tetanus toxin, after tetanic 
symptoms had been developed. 

Reference has been made to the production of immunity by the 
use of cultures made in thymus bouillon. This was made known 
through the experiments of Brieger, Kitasato, and Wassermann 
(1892). The thymus bouillon is made from the thymus glands of 
calves, which are chopped fine in a hash machine and covered with 
an equal volume of distilled water. The mixture is stirred for some 
time and then placed in an ice chest for twelve hours; the liquid is 
then obtained by filtration through gauze with pressure—by means of 
a flesh-press machine. A turbid, slimy fluid is thus obtained, which 
is diluted with an equal volume of water and made slightly alkaline 
by the addition of soda solution. It is then sterilized at 100° CO. for 
fifteen minutes. As a result of this the liquid has a grayish-brown 
color, and some large flocculi in suspension, which are removed by 
passing it through fine linen. The fluid is then of a milky opal- 
escence. It is next placed in test tubes and again sterilized. The 
tetanus bacillus when cultivated in this medium does not form spores, 
and the toxic potency of the culture is very much reduced—1: 5,000 to 
1:3,000 of the toxic potency manifested by cultures of the same bacil- 
lus in ordinary media. Inoculations with cultures in thymus bouillon 
were found to kill mice in the dose of 0.5 cubic centimetre, while 
smaller amounts failed to kill and caused the animals to be immune. 
A culture in ordinary bouillon was fatal to mice in the dose of 0.001 
cubic centimetre. 

Experiments on rabbits (thirty-five) gave a uniformly successful 
result in immunizing these animals. Immunity was established in 
the course of two weeks, and the blood serum of these animals tested 
on mice showed an antitoxic value of 1,000. 

Reference has already been made to the earlier researches of the 
Italian investigators, Tizzoni and Cattani. These have been followed 


360 PROTECTIVE INOCULATIONS. 


by additional investigations, the results of which have been reported 
in numerous published papers. The authors named have ascertained 
that when kept in a cool place (15° to 25° ©.) the blood serum of 
immune rabbits retains its antitoxic power for several months, and 
the antitoxin, obtained by precipitation with alcohol, kept in a dry 
condition for more than ten months, was found to preserve its original 
activity. 

Having succeeded in their earlier experiments in immunizing rab- 
bits and dogs, Tizzoni and Cattani (in 1893) proceeded to experiment 
upon horses, and were equally successful with these animals. As a 
result of numerous injections with an attenuated virus, continued for 
a period of ninety-seven days, they established an immunity which 
was tested by inoculating the animal with ten cubic centimetres of a 
gelatin culture, of which one two-hundredth part of a drop killed a 
white mouse. The antitoxic value of the blood serum of this horse 
was 1:5,000,000—7.e., one gramme of this serum would immunize five 
million grammes of mice, or two hundred and fifty thousand mice 
weighing twenty grammes each. In a later communication (1894) the 
authors named report that after freely bleeding immunized horses, and 
allowing them to rest for one or two months, and then again treating 
them with small doses of tetanus cultures, the blood serum soon be- 
comes as active as before the bleeding. The greatest antitoxic power 
was manifested from twenty to twenty-three days after the completion 
of the protective inoculations, and a serum was obtained possessing a 
value of 1:10,000,000. According to the authors named the precipi- 
tated (by alcohol) and purified antitoxin from such a serum, judging 
from their experiments on lower animals, should cure a case of teta- 
nus in man in the dose of from forty to fifty centigrammes. 

The authors last mentioned have reported (1892) that the young of 
immune parents have a certain degree of inherited immunity. And 
the more recent experiments of Ehrlich and Hubener have confirmed 
this so far as the inheritance of immunity from the mother (in mice) 
is concerned; but their results did not show any immunity in the 
young when only the father had been rendered immune; and the im- 
munity inherited from the mother only lasted for two or three months 
after birth. 


TUBERCULOSIS. 


Metchnikoff states that when kept at a temperature of 42° C. for 
some time the tubercle bacillus undergoes a notable diminution in its 
pathogenic power, and that when kept at a temperature of 43° to 44° 
C. it after a time only induces a local abscess when injected subcu- 


PROTECTIVE INOCULATIONS. 361 


taneously into guinea-pigs. The experiments of Lite also indicate 
that an “attenuation of virulence” has occurred in the cultures pre- 
served in Koch’s laboratory, originating in 1882 from the lungs of a 
tuberculous ape. The author named made experiments with cultures 
from this source (ninetieth to ninety-fifth successive cultures), and at 
the same time with a culture obtained from Roux, of Pasteur’s labor- 
atory. Rabbits inoculated with cultures from the last-mentioned 
source developed a hectic fever at the end of two weeks, and died tu- 
berculous at the end of twenty-one to thirty-nine days. Twelve rab- 
bits were inoculated with the cultures from Koch’s laboratory; the 
injections were made either subcutaneously, or into a vein, or into the 
pleural cavity, or into the cavity of the abdomen. No elevation of 
temperature occurred in any of the animals, and they were found at 
the end of a month to have increased in weight. At the end of six 
weeks one of them was killed and tubercular nodules were found in 
various organs. The remaining animals were killed at the end of 
one hundred and forty-four to one hundred and forty-eight days. 
The two inoculated subcutaneously presented no sign of general tu- 
berculosis, but a small yellow nodule containing bacilli was found at 
the point of inoculation. Those inoculated by injection into a vein 
showed one or two nodules in the lungs containing a few bacilli. In 
Koch’s original experiments rabbits were killed by intravenous inocu- 
lation of his cultures in from thirteen to thirty-one days. That this 
attenuation of virulence depends upon a diminished production of 
toxic product to which the bacillus owes its pathogenic power appears 
to be very certain, in view of the fact that the late cultures in a series 
have a more vigorous and abundant development than the more patho- 
genic cultures obtained directly from the animal body. 

The discovery by Koch of a toxin in cultures of this bacillus, 
which is soluble in glycerin, and which in very minute doses pro- 
duces febrile reaction and other decided symptoms when injected 
subcutaneously into tuberculous animals, must rank as one of the first 
importance in scientific medicine, whatever the final verdict may be 
as to its therapeutic value in tuberculous diseases in man. 

The toxic substance contained in Koch’s glycerin extract from cul- 
tures of the tubercle bacillus, now generally known under the name of 
tuberculin, is soluble in water, insoluble in alcohol, and passes readily 
through dialyzing membranes. It is not destroyed by the boiling 
temperature. According to the chemical examination of Jolles, the 
“lymph” contains fifty per cent of water and does not contain alka- 
loids or cyanogen compounds. It contains albuminates, which are 
thrown down as a voluminous white precipitate by tannic acid, and 


362 PROTECTIVE INOCULATIONS. 


are redissolved by hot water containing sodium chloride and very 
dilute potash solution. The elementary analysis gave N 5.90 per 
cent, C 35.19 per cent, and H 7.02 per cent. The results obtained 
are believed to show that the active substance present in the lymph 
is a toxalbumin. In experiments made with Koch’s lymph in Pas- 
teur’s laboratory by Bardach, a very decided elevation of temperature 
was produced in tuberculous guinea-pigs by the subcutaneous injection 
of 0.1 gramme, and a fatal result by the injection of 0.2 to 0.5 gramme. 
In man a decided febrile reaction is produced in tuberculous patients 
by very much smaller doses—0.001 cubic centimetre. 

Hammerschlag, in his chemical researches, found that the tubercle 
bacillus yields a larger proportion of substances soluble in alcohol and 
ether than any other bacilli tested (twenty-seven per cent). The alco- 
holic extract contains fat, lecithin, and a toxic substance which pro- 
duces convulsions in rabbits and guinea-pigs. The portion insoluble 
in alcohol and ether contains cellulose and an albuminoid substance. 
No ptomains were found, but a toxalbumin was isolated, which 
caused an elevation of temperature in rabbits of 1° to 2° C., lasting 
for a day or two. 

Koch (1891) has given a full account of his method of preparing 
crude tuberculin, and also the process by which he obtains from this 
a tuberculin which appears to be pure, or nearly so. To obtain con- 
siderable quantities of the crude product the tubercle bacillus is culti- 
vated in an infusion of calves’ flesh, or of beef extract to which one 
per cent of peptone and four or five per cent of glycerin have been 
added. This culture liquid must be made slightly alkaline, and it 
is placed in flasks with a flat bottom, which should not be more than 
half filled—thirty to fifty cubic centimetres. The inoculation is made 
upon the surface with small masses from a culture upon blood serum 
or glycerin agar. By accident Koch discovered that these masses 
floating upon the surface give rise to an abundant development, and 
to the formation of a tolerably thick and dry white layer, which finally 
covers the entire surface. At the end of six to eight weeks develop- 
ment ceases, and the layer after a time sinks to the bottom, breaking 
up meanwhile into fragments. These cultures, after their purity has 
been tested by a microscopical examination, are poured into a suitable 
vessel and evaporated to one-tenth the original volume over a water 
bath. The liquid is then filtered through procelain. The crude tu- 
berculin obtained by this process contains from forty to fifty per cent 
of glycerin, and consequently is not a suitable medium for the develop- 
ment of saprophytic bacteria, if they should by accident be introduced 
into it. It keeps well and preserves its activity indefinitely. 


PROTECTIVE INOCULATIONS. 363 


From this crude tuberculin Koch has obtained a white precipitate 
with sixty-per-cent alcohol which has the active properties of the 
crude tuberculin as originally prepared. This is fatal to tuberculous 
guinea-pigs in doses of two to ten milligrammes. It is soluble in 
water and in glycerin, and has the chemical reactions of an albuminous 
body. In preparing it one volume and a half of absolute alcohol is 
added to one volume of the crude tuberculin, and, after stirring it to 
secure uniform admixture, this is put aside for twenty-four hours. 
At the end of this time a flocculent deposit will be seen at the bot- 
tom of the vessel. The fluid above this is carefully poured off; and 
an equal quantity of sixty-per-cent alcohol is poured into the vessel 
for the purpose of washing the precipitate. This is again allowed to 
settle, and the procedure is repeated three or four times, after which 
the precipitate is washed with absolute alcohol. It is then placed 
upon a filter and dried in a vacuum exsiccator. 

The “tuberculocidin ” of Klebs is a purified tuberculin obtained by 
precipitation with alcohol. The precipitate is washed in chloroform 
and then dissolved in a mixture of carbolic acid and glycerin. 

Bujwid (1894) prepares tuberculin as follows: He uses cultures on 
glycerin agar or in glycerin bouillon which have been kept at a suit- 
able temperature for five to eight weeks. The glycerin-agar cultures 
are treated with distilled water by which the tuberculin is extracted. 
After adding the water the test tubes are kept in a cool place for 
twenty-four hours, and this is repeated two or three times. The ex- 
tract from the agar cultures or the bouillon cultures is then sterilized 
by exposure for from five to ten minutes to a temperature of 100° C.; 
then filtered through a Chamberland filter; then evaporated at a low 
temperature to a syrup-like consistence. When this crude tuberculin 
is dropped into ten times its volume of strong alcohol a brown pre- 
cipitate is thrown down which contains the active principle. From 
the tubercle bacilli obtained by filtering his cultures Bujwid also 
obtained an active substance which in doses of two milligrammes 
caused an elevation of 2° C. in the temperature of an infected guinea- 
pig. This substance was obtained by digesting the bacilli for two 
months in glycerin and water (three per cent of glycerin), filtering 
and evaporating the extract, and precipitation in six volumes of 
ninety-five-per-cent alcohol. The precipitate when dried was in the 
form of a white powder. 

Helman (1894) obtains tuberculin from potato cultures. The sec- 
tions of potato are neutralized by leaving them for half an hour ina 
solution of one-half to one per cent of bicarbonate of soda, after which 
they are sterilized for twenty minutes in the autoclave at 120° C. The 


364 PROTECTIVE INOCULATIONS. 


best results were obtained when the potatoes were wet with a five-per- 
cent solution of glycerin. The sections of potato were placed in Petri’s 
dishes upon blotting paper wet with a sublimate solution, and the 
dishes containing the cultures were surrounded with cotton wet with 
the same solution. The cultures were subsequently treated with dis- 
tilled water, to extract the active principle, which was also obtained 
from the bacilli by mixing them with glycerin in the proportion of 
1:10. 

Numerous experiments have been made with dead tubercle bacilli, 
as well as with the toxic products developed in cultures. Héricourt 
and Richet (1890) found by experiment that old cultures heated to 80° 
C., several days in succession, when injected into a vein in rabbits, in 
the dose of ten to twenty cubic centimetres, caused the death of these 
animals. Smaller doses from which the animals recovered seemed to 
make them less susceptible to infection than control animals, but the 
number of experiments was too limited to establish this as a fact. In 
a subsequent (1891) communication the authors named claim to have 
succeeded in immunizing rabbits by injecting filtered and sterilized 
cultures of the tubercle bacillus, either subcutaneously (five to fifteen 
cubic centimetres) or into a vein (twenty to forty drops). The injections 
were repeated every second or third day for a period of fifteen days, 
after which the test inoculation was made with a culture, obtained 
from a tuberculous cow in one series, and from tuberculous fowls in 
another. Four vaccinated rabbits in the first series escaped general 
tuberculosis, while four out of eight control animals died tuberculous. 
In the second series five vaccinated animals resisted infection and 
three out of four control animals died tuberculous. 

De Schweinitz (1894) has reported the results of experiments with 
attenuated cultures of the tubercle bacillus, and has, apparently, suc- 
ceeded in conferring immunity upon guinea-pigs by inoculations 
with such cultures. 

Klebs (1891), in experiments on guinea-pigs and rabbits, convinced 
himself that the fatal result of an inoculation with tubercle bacilli (in 
the cavity of the abdomen or subcutaneously in guinea-pigs, and in 
the eye in rabbits) was greatly delayed by injections of Koch’s tuber- 
culin (0.3 to 0.5 cubie centimetre) either before or after infection. 

Baumgarten (1891), in experiments upon rabbits inoculated with 
tubercle bacilli in the anterior chamber of the eye, failed to obtain 
favorable results from treatment with Koch’s tuberculin given in con- 
siderable doses (0.5 to one gramme) either before or after infection. 

The results reported in the same year by Gramatschikoff, by 
Popoff, by Alexander, and by Gasparini and Mercanti, were also un- 


PROTECTIVE INOCULATIONS. 3865 


favorable as regards an immunizing or curative effect from inocula- 
tions of tuberculin in rabbits. Dénitz, on the contrary, arrives at the 
conclusion that when early treatment is instituted iris tuberculosis 
may be arrested and cured, and the more recent experiments of Tru- 
deau (1893) give support to this conclusion. Baumgarten, however, 
insists that the tuberculin treatment does not prevent metastasis to 
the lungs after inoculations in the anterior chamber of the eye. 

Pfuhl (1891) treated forty-seven infected guinea-pigs, and at the 
date of his report forty-four had died tuberculous, but the date of 
death was somewhat postponed by the treatment. The animals not 
treated succumbed at the end of eight weeks (average of all controls), 
and those treated with small doses of tuberculin lived, on the average, 
ten weeks. With larger doses still more favorable results were ob- 
tained—tour lived on an average twelve weeks, and three were still 
living, eleven, fifteen, and sixteen weeks after infection, at the date of 
publication. 

Kitasato (1892) also obtained favorable results in the treatment 
of infected guinea-pigs, and arrives at the conclusion that guinea-pigs 
which have been cured by the treatment are not susceptible to a sec- 
ond infection, for a certain time at least. 

Bujwid (1892), in experiments upon guinea-pigs, found that in- 
fected animals which received from 0.05 to 0.1 gramme of tuberculin 
within three hours showed an elevation of temperature of 1.5° to 2° C. 
Thirteen infected guinea-pigs treated with tuberculin lived from two 
and a half to eight months, while all of the control animals (eighteen) 
died in from six to nine weeks. The animal which survived eight 
months was found not to be tuberculous, but presented evidence of re- 
covery from a former tuberculous process. In two rabbits inoculated 
in the anterior chamber the iris tuberculosis was favorably influenced 
by the tuberculin treatment, but general infection occurred, and the 
animals died about the same time as the controls. Three apes were 
treated without any apparent result; they all died within two months 
after infection. 

The experiments of Gramatschikoff, Czaplewski, and Roloff, and 
of Yamagiva, published in 1892, show that the tuberculin treatment 
does not cure tuberculous infection in inoculated guinea-pigs and rab- 
bits, and that the bacilli retain their vitality in such animals in spite 
of the most persistent treatment. 

Héricourt and Richet (1892), in experiments made for the purpose 
of immunizing animals against tuberculous infection, failed to obtain 
positive results in the most susceptible species—guinea-pigs, rabbits, 
and apes—but claim to have succeeded in immunizing dogs by intra- 


366 PROTECTIVE INOCULATIONS. 


venous injections of cultures of the bacillus of tuberculosis in fowls. 
Animals which had been so treated after an interval of two to six 
months received an intravenous injection of one cubic centimetre of a 
culture of the bacillus tuberculosis from man. This was fatal to 
“non-vaccinated ” dogs, as a rule, in about three weeks, but the “ vac- 
cinated” animals survived the injection. 

The results obtained by Trudeau (1893) are of such interest that 
we shall quote in extenso what he says with reference to preventive 
inoculations : 


‘Antitubercular inoculation was first tried by Falk in 1883, and all 
attempts in this direction have resulted until recently in but an unbroken 
record of failures. In 1890 I added my name to the list of those who found 
it impossible to produce immunity in animals by this method. In 1890, 
Martin and Grancher, and Courmont and Dor, claimed to have produced in 
rabbits a certain degree of immunity by previous inoculation, after Pasteur’s 
hydrophobia method, of avian tubercle bacilli of graded and increasing viru- 
lence. These vaccinations were, however, frequently fatal to the animals, 
and the immunity obtained was but slight. Richet and Héricourt have since 
claimed to produce complete immunity in dogs by intravenous inoculations 
of bird tubercle bacilli. These experimenters found that though harmless to 
the dog when first derived from the chicken, bird bacilli, by long cultivation 
in liquid media, become pathogenic for this animal, and by thus grading the 
virulence of the injections complete immunity against any form of tubercu- 
lar infection was produced in the dog. As yet these striking results have not 
been confirmed. The animals which I now present to you illustrate an at- 
tempt I have made along the same line to produce immunity in the rabbit. 
Cultures grown directly from the chicken’s lesions in bouillon for, first, five 
weeks, then six months, were twice injected subcutaneously at intervals of 
twenty-one days in doses of 0.025 and 0.05, and a third injection of a still 
older culture was occasionally given. About one in four of the rabbits died 
within three months, profoundly emaciated, but without any visible tubercu- 
lar lesions. The remaining animals recovered aud were apparently in good 
health, when, together with an equal number of controls, they were inocu- 
lated in the anterior chamber of the eye with cultures of Koch’s bacillus 
derived from the tuberculous lesions of the rabbit, and cultivated about three 
months on glycerin agar. The results of these inoculations present many 
points of interest. In the controls, as is usually the case, if the operation 
has been done carefully and aseptically, and with a moderate amount of 
dilute virus, two days after the introduction of the virulent material in the 
eye little or no irritation is observed, and little is to be noticed for two weeks, 
when a steadily increasing vascularity manifests itself, small tubercles ap- 
pear on the iris, which gradually coalesce and become cheesy, intense iritis 
and general inflammation of the structures of the eye develop, the inocula- 
tion wound becomes cheesy, and in six to eight weeks the eye is more or less 
completely destroyed and the inflammation begins to subside. The disease, 
however, remains generally localized in the eye for many months, and even 
permanently. In the vaccinated animals, on the contrary, the introduction 
of the virulent bacilli at once gives rise to a marked degree of irritation. On 
the second day the vessels of the conjunctiva are tortuous and enlarged, 
whitish specks of fibrinous-looking exudation appear in the iris and in the 
anterior chamber, and more or less intense iritis supervenes; but at the end 
of the second to the third week, when the eyes of the controls begin to show 
progressive and steadily increasing evidence of inflammatory reaction, the 
irritation in those of the vaccinated animals begins slowly to subside and the 
eyes tomend. The vascularity is less, the whitish spots of fibrinous material 


PROTECTIVE INOCULATIONS. 367 


appear smaller, the structures of the eye become clearer, the inoculation 
wound is but a bluish fibrous scar, until in from six to twelve weeks, in suc- 
cessful cases, all irritation has disappeared and the eyes present, as in the 
animals I now show you, but fibrous evidence of the traumatism and the in- 
flammatory processes which have been set up by the inoculation. In all the 
controls, as you see, the inoculation wound is cheesy and the cornea and iris 
are more or less destroyed by tubercle and cheesy areas. 

‘Some of the protected animals slowly relapse, and the one I now show 
you has small tubercles growing on the iris; but even in such eyes the 
entire absence of caseation is noticeable, and the disease progresses almost 
imperceptibly. I have repeated this experiment on three sets of rabbits with 
about the same results each time. The vaccinations as practised are of them- 
selves, in some instances, fatal; but the fact remains that where recovery 
takes place a marked degree of immunity has been acquired. I do not lay 
any claim, therefore, to have produced a complete or permanent immunity 
by a safe method, but it seems to me that these eyes constitute a scientific 
demonstration of the fact that in rabbits preventive inoculation of bird- 
tubercle bacilli can retard, and even abort, an otherwise progressive localized 
tubercular process so completely as to prevent destruction of the tissues 
threatened, and that the future study of anti-tubercular inoculation may not 
be as entirely hopeless as it has until recently appeared.” 


TYPHOID FEVER. 


Brieger (1885) found in cultures of the typhoid bacillus small 
amounts of volatile fat acids, and when grape sugar has been added 
to the culture medium lactic acid. He also obtained a highly alka- 
line basic substance possessing toxic properties which he named 
typhotoxin (C,H,,NO,). This he supposes to be the specific product 
to which the pathogenic action of the bacillus is due. It produces in 
mice and guinea-pigs salivation, paralysis, dilated pupils, diarrhcea, 
and death. 

More recent experiments by Pfeiffer (1894) lead him to conclude 
that the specific poison of the typhoid bacillus is not present in fil- 
tered cultures, but is closely associated with the bacterial cells. Ac- 
cording to Pfeiffer the bacillus may be killed by a temperature of 54° C. 
without injury to this toxic substance. The fatal dose of the dead 
bacilli is from three to four milligrammes per one hundred grammes 
of body weight for guinea-pigs. Susceptible animals may be im- 
munized by means of this toxic substance, and their blood is found to 
contain an antitoxin which has a specific bactericidal action upon the 
typhoid bacillus. But, according to Pfeiffer, the blood serum of ani- 
mals immunized in this way does not differ from normal serum in 
its action on bacillus coli communis and other species of bacteria. 
These results are believed, by the author referred to, to settle the 
question of the specific character of the typhoid bacillus, and to dif- 
ferentiate it from nearly allied species. The presence of a typhoid 
antitoxin in the blood serum of individuals who have recently suffered 
an attack of typhoid fever has also been demonstrated by Pfeiffer. 


368 PROTECTIVE INOCULATIONS. 


Chantemesse and Widal (1888) first showed by experiment that 
susceptible animals could be made immune against the pathogenic 
action of this bacillus by the subcutaneous injection of sterilized cul- 
tures. Having found that four drops of a bouillon culture, three 
days old, injected into the peritoneal cavity of white mice caused the 
death of these animals within thirty-six hours, they proceeded to in- 
ject small quantities (one-half cubic centimetre) of a culture which 
had been sterilized by heat, and found that after several such protec- 
tive inoculations the mice no longer succumbed to infection by an 
unsterilized culture. 

In experiments made upon rabbits, Bitter (1892) arrived at the 
conclusion that the immunity which he produced in these animals by 
the intravenous injection of concentrated sterilized (by filtration) cul- 
tures was due to the presence of an antitoxin in the blood of the 
immune animals. Having found that control animals were killed by 
intravenous injections of one cubic centimetre of his concentrated 
solution of the products of the typhoid bacillus, he added to twice 
this amount of the toxic solution a certain quantity (?) of blood 
serum from an immune rabbit, and injected the mixture into the 
circulation of rabbits with a negative result. Control experiments 
in which the toxic solution was mixed with L.ood serum from non- 
immune animals showed that this had no antitoxic effect, and the ani- 
mals died. Bruschettini obtained (189%) similar results in his ex- 
periments upon rabbits with cultures sterilized by heat (60° C.). He 
concludes from his experiments that the blood serum of rabbits im- 
munized in this way not only possesses antitoxic properties, but that 
it has greater germicidal potency for the typhoid bacillus than the 
blood of normal rabbits. 

Stern (1892) has made experiments to determine whether the blood 
of recent convalescents from typhoid has greater germicidal power 
for the typhoid bacillus than that of other individuals. The result 
showed that the blood serum from persons who had recently recovered 
from typhoid fever had no increased germicidal power, but rather 
showed diminished potency for the destruction of typhoid bacilli. 
But blood from a man who had suffered an attack seventeen and a 
half years previously was found to have unusual bactericidal power, 
although it did not protect white mice from typhoid infection. On 
the other hand, blood from recent convalescents served to immunize 
white mice, thus indicating the presence of an antitoxin. This is 
also shown by the experiments of Chantemesse and Widal (1892), 
who report their success in immunizing susceptible animals by in- 
jecting the blood serum of other animals previously made immune by 


PROTECTIVE INOCULATIONS. 369 


repeated injections of sterilized (by heat) cultures. The authors last 
named have also tested the blood serum of typhoid-fever patients, of 
recent convalescents from the disease, and of persons who had sutf- 
fered an attack some years before the experiment was made. The ex- 
periments were made upon guinea-pigs. The authors conclude that 
‘in general the guinea-pig is immunized against the action of virulent 
typhoid cultures by the subcutaneous injection of a small quantity 
of serum of persons who have suffered an attack of the disease, no 
matter how remote.” But this immunity was shown to be of short 
duration, and quite different from that induced by the injection of 
sterilized cultures, which does not immediately follow the introduc- 
tion of the toxic substances, but requires a certain number of days for 
its development. The degree of immunity is said by the authors last 
named to depend to a considerable extent upon the dose given, and 
the animals treated in this way still resisted virulent cultures at the 
end of two months. On the other hand, injections of blood serum 
from immune individuals were effective in doses of a single cubic cen- 
timetre, within a few hours, and the immunity conferred had a com- 
paratively brief duration. 

Protective inoculations in man have been practised on quite a large 
scale by surgeons of the English army in India and in South Africa. 
The method of Wright has been followed in preparing sterile cultures 
for inoculation. Cultures in bouillon are made and kept in the incu- 
bator at 37° C. for two or three weeks. The cultures are then drawn 
into small glass tubes, which are sealed by heat. The tubes are 
placed in a vessel containing cold water, and the temperature is grad- 
ually raised to 60° C., where it is maintained for five minutes. Plant- 
ings in a culture medium are made from these tubes to make sure that 
sterilization is complete. As a further protection against the intro- 
duction of living bacteria, one-half per cent of lysol may be added to 
the sterilized culture. The amount used for protective inoculations 
in man has been fixed at two-fifths of the minimum amount, which 
would be fatal to a guinea-pig weighing two hundred and fifty 
grammes. The inoculation gives rise to a well-marked local reaction, 
which does not result in suppuration, and to more or less pronounced 
general disturbance. Usually this is slight, but sometimes rigors, 
nausea, and a tendency to syncope occur. That these inoculations 
are not without effect is shown by the fact that the blood serum of 
an inoculated individual exercises a marked agglutinating action 
upon the typhoid bacillus in a recent culture (Widal reaction). This 
is said to be equal to that resulting from an attack of typhoid fever. 
Cameron, after an inoculation practised upon himself, found that at 


“370 PROTECTIVE INOCULATIONS. 


the end of twenty days his blood serum exhibited an agglutinating 
power forty times greater than that of normal blood. In practice it 
has been found advisable to repeat the inoculation at the end of a 
week. Wright reports that among 11,295 British soldiers inoculated 
in India, the percentage of those who subsequently contracted ty phoid 
fever was 0.95, while 2.5 per cent of those not inoculated suffered an 
attack of this disease. According to Foulerton the soldiers in South 
Africa, during the Boer war, who have been inoculated have contracted 
typhoid fever in the proportion of six per thousand, while those not 
inoculated have suffered to the extent of nine per thousand. How 
much value should be attached to these statistics it is difficult to say, 
on account of the numerous factors which are likely to influence the 
result. Thus a command on the march in a sparsely inhabited coun- 
try would be much less liable to suffer from typhoid fever than another 
located in a town and remaining for a considerable time on the same 
camping ground. In a recent report (February, 1901) Professor 
Wright states that of 539 officers, men, and women connected with the 
Fifteenth Hussars at Meerut, India, 360 received protective inocula- 
tion in England against typhoid fever and 179 did not. Of the former 
2 (0.55 per cent) were admitted to the hospital, suffering from ty phoid 
fever, with 1 death (0.27 per cent); while of the latter 11 (6.14 per cent 
were attacked by the fever, with 6 deaths (8.35 per cent). 

It is evident that, while the results reported are encouraging, this 
method should not be relied upon as a substitute for those sanitary 
measures which must be our main reliance for the prevention of epi- 
demics of this disease, viz., sterilization of drinking-water, disinfec- 
tion of excreta, sanitary police of camps, etc. 


Vv. 
PYOGENIC BACTERIA. 


Tuer demonstration made by Ogston, Rosenbach, Passet, and 
others that micrococci are constantly present in the pus of acute 
abscesses, led to the inference that there can be no pus formation in 
the absence of microdrganisms of this class. But it is now well 
established, by the experiments of Grawitz, De Bary, Steinhaus, 
Scheurlen, Kaufmann, and others, that this inference was a mis- 
taken one, and that certain chemical substances introduced beneath 
the skin give rise to pus formation quite independently of bacteria. 
Among the substances tested which have given a positive result are 
nitrate of silver, oil of turpentine, strong liquor ammonia, cada- 
verin, etc. The demonstration has also been made by numerous in- 
vestigators that cultures of pus cocci, when sterilized by heat, still 
give rise to pus formation when injected subcutaneously. This was 
first established by Pasteur in 1878, who found that sterilized cul- 
tures of his “microbe générateur du pus” induced suppuration as 
well as cultures containing the living microbe. This fact has since 
been confirmed, as regards the pus staphylococci and various bacilli, 
by a number of bacteriologists. Wyssokowitsch produced abscesses 
containing sterile pus by injecting subcutaneously agar cultures of 
the anthrax bacillus sterilized by heat. Buchner obtained similar 
results in a series of forty experiments from the injection of steril- 
ized cultures of Friedlander’s bacillus (‘‘ pneumococcus ”), and has 
shown that the pus-forming property belongs to the bacterial cells 
and not to a soluble chemical substance produced by them. When 
cultures were filtered by means of a Chamberlain filter the clear 
fluid which passed through the porous porcelain was without effect, 
while the dead bacteria retained by the filter produced aseptic pus 
infiltration in the subcutaneous tissues within forty-eight hours 
after having been injected. Subsequent experiments gave similar 
results with seventeen different species tested, including Staphylo- 
coccus pyogenes aureus, Staphylococcus cereus flavus, Sarcina auran- 
tiaca, Bacillus prodigiosus, Bacillus Fitzianus, Bacillus subtilis, 
Bacillus coli communis, Bacillus acidi lactici, etc. From the experi- 


372 PYOGENIC BACTERIA. 


ments made to determine the exact cause of pus formation following 
the injection of sterilized cultures Buchner arrives at the conclusion 
that it is due to the albuminous contents of the bacterial cells. 

While it is demonstrated that a large number of microérganisms, 
either living or in sterilized cultures, may give rise to the formation 
of pus, the extended researches of Rosenbach, Passet, and other 
bacteriologists show that few species are usually concerned in the 
formation of acute abscesses, furuncles, etc., in man. Of these the 
two most important, by reason of their frequent occurrence and path- 
ogenic power, are Staphylococcus pyogenes aureus and Strepto- 
coccus pyogenes; next to these comes Staphylococcus pyogenes 
albus, and the following species are occasionally found : Staphylo- 
coccus pyogenes citreus, Staphylococcus cereus flavus, Staphylococcus 
cereus albus, Micrococcus tenuis, Bacillus pyogenes fcetidus, Micro- 
coccus tetragenus, Micrococcus pneumonis croupose. Two or more 
species are often found in the same abscess ; thus Passet, in thirty- 
three cases of acute abscess, found Staphylococcus aureus and albus 
associated in eleven, albus alone in four, albus and citreus in two, 
Streptococcus pyogenes alone in eight, albus and streptococcus in 
one, and albus, citreus, and streptococcus in one. Hoffa found, in 
twenty-two cases of inguinal bubo, aureus in ten, albus in nine, and 
citreus in three. Bumm, in ten cases of puerperal mastitis, found 
aureus in seven and Streptococcus pyogenes in three. Rosenbach 
found staphylococci alone sixteen times, Streptococcus pyogenes alone 
fifteen times, staphylococci and streptococci associated five times, 
and Micrococcus tenuis three times in thirty-nine acute abscesses and 
phlegmons examined by him. 

Robb and Ghrisky have shown that under the most rigid antisep- 
tic treatment microdrganisms are constantly found attached to su- 
tures when these are removed from wounds made by the surgeon, 
and that a skin abscess frequently results from the presence of the 
most common of these microédrganisms—Staphylococcus epidermidis 
albus. 

The authors named state their conclusions as follows : 


‘‘A wound, at some time of ‘its existence, always contains organisms. 
They occur either on the stitches or in the secretions. _ 

‘The number of bacteria is influenced by the constricting action of the 
ligatures or drainage tube, or anything interfering with the circulation of 
the tissues. fy 

‘The virulence of the organisms present will influence the progress of 
the wound. : : . 

‘The body temperature is invariably elevated if the bacteria are viru- 
lent; and, indeed, in cases where many of the less virulent organisms are 
found, almost without exception there is some rise of temperature.” 


The organism most frequently found—Staphylococcus epidermi- 


PYOGENIC BACTERIA. 373 


dis albus—has but slight virulence. Out of forty-five cases in which 
a bacteriological examination was made this micrococcus was ob- 
tained in pure cultures in thirty-three ; in five cases it was associated 
with Staphylococcus pyogenes aureus, in one case with Streptococ- 
cus pyogenes, in three cases Streptococcus pyogenes was obtained 
alone. 

In abscesses resulting from inflammation of the middle ear the 
micrococcus commonly known under the name of ‘“‘ diplococcus 
pneumoniz ”—Micrococcus pneumoniz crouposee—has been obtained 
in pure cultures in a considerable number of cases when the pus has 
been examined immediately after paracentesis of the tympanic mem- 
brane. We shall not, however, describe this among the pyogenic 
bacteria, but will give an account of it in the following section (Bac- 
teria in Croupous Pneumonia, etc.). Bacillus pyocyaneus, which is 
described by some authors among the pyogenic bacteria, is found 
only in the pus of open wounds, where its presence is evidently acci- 
dental. We shall describe it among the chromogenic saprophytes. 


STAPHYLOCOCCUS PYOGENES AUREUS. 


Synonym.—Micrococcus of infectious osteomyelitis (Becker). 

Observed by Ogston (1881) in the pus of acute abscesses, but not 
differentiated from the associated staphylococci and the streptococ- 
cus of pus. Obtained by Becker from the pus of osteomyelitis (1883). 
Isolated from the pus of acute abscesses and accurately described by 
Rosenbach (1884) and by Passet (1885). 

The Staphylococcus pyogenes aureus is a facultative parasite, and — 
is the most common pyogenic micrococcus found in suppurative pro- 
cesses generally. But it is also a common and widely distributed 
saprophyte, which finds the conditions necessary for its existence on 
the external surface of the human body and of moist mucous mem- 
branes. This is shown by the researches of numerous bacteriolo- 
gists. Thus Ullmann found it upon the skin and in the secretions of 
the mouth of healthy persons, and also in the dust of occupied apart- 
ments, in water, etc.; Bockhart obtained it in cultures from the 
surface of the body and from the dirt beneath the finger nails of 
healthy persons ; Biondi, Vignal, and others in the salivary secre- 
tions; B. Frankel in mucus from the pharynx; Von Besser and 
Wright in nasal mucus; Escherich in the alvine discharges of 
healthy infants ; C. Frankel in the air ; and Liibbert in the soil. Its 
presence in the air, in water, or in the soil is, however, quite excep- 
tional, and is probably to be considered the result of accident, its 
normal habitat as a saprophyte appearing to be rather upon the sur- 
face of the body and of mucous membranes. 


374 PYOGENIC BACTERIA. 


Morphology.—Spherical cells having a diameter of 0.7 « (Hade. 
lich) to 0.9 # (0.87 «4 Passet), solitary, in pairs, or in irregular 
groups, occasionally in chains of three or four elements or in groups 
of four. The dimensions vary somewhat in dif- 


eds Bis ferent culture media, being larger in a favorable 
e than in an unfavorable medium. The individual 
sigh? cells, as pointed out by Hadelich, consist of two 


hemispherical portions separated from each other 

Fig. 79.—Staphylococ- by a very narrow cleft, which is not visible when 

from. a drawing by the cells are deeply stained, but may be demon- 

Rosenbach. strated, with a high power, by staining for a short 
time (two minutes or less) in a solution of fuchsin in aniline water. 

This micrococcus stains quickly in aqueous solutions of the basic 
aniline colors, and may also be stained with acid carmine and hama- 
toxylin. It is not decolorized by iodine solution when stained with 
methyl violet-—Gram’s method. 

Biological Characters.—Staphylococcus pyogenes aureus grows 
either in the presence or absence of oxygen, and is consequently a 
facultative anaérobic. It multiplies rapidly at a temperature of 18° 
to 20° C. in milk, flesh infusions, and various other liquid media, 
and in nutrient gelatin or agar. It liquefies gelatin, and in stab 
cultures liquefaction occurs all along the line of puncture, forming a 
pouch which is largest above and at the end of three or four days has 
extended to the full capacity of the test tube at the surface. The 
liquefied gelatin in this pouch is at first opaque from the presence of 
little agglomerations of micrococci in suspension, but after a time 
these are deposited and the gelatin becomes transparent. During 
the period of active growth the cocci accumulate near the surface of 
the gelatin, and, in contact with the air, the characteristic golden-yel- 
low pigment is produced. By the subsidence of the colored masses 
of cocci from this superficial stratum a yellow deposit is gradually 
formed at the bottom of the pouch of liquefied gelatin (Fig. 80), This 
pigment, which is the principal character distinguishing the micro- 
coccus under consideration from certain other liquefying staphylo- 
cocci, is only formed in the presence of oxygen. Upon the surface 
of nutrient agar development occurs in the form of a moist, shining 
layer, with more or less wavy outlines, having at first a pale-yellow 
color, which soon deepens to an orange- or golden-yellow. The col- 
onies which develop upon agar plates are spherical and opaque, and 
usually acquire the golden-yellow color within afewdays. Colonies 
on gelatin plates or in Esmarch roll tubes first appear as small white 
dots, which later are more or less granular in appearance and present 
the yellow color, especially towards the centre ; but, owing to the 
extensive liquefaction of the gelatin caused by them, their develop- 


PYOGENIC BACTERIA. 375 


ment can only be followed for two or three days. Upon potato, ata 
temperature of 35° to 37° C., a rather thick, moist layer of consider- 
able extent forms at the end of twenty-four to forty-eight hours ; 
this is also at first of a pale-yellow, and later 
of an orange-yellow color. The temperature 
mentioned is most favorable for the rapid 
development of this micrococcus, although 
multiplication may occur at a comparatively 
low temperature and is tolerably abundant at 
the ordinary room temperature. 

Cultures of the ‘‘ golden staphylococcus,” 
and especially those upon potato, give off a 
peculiar odor which resembles that of sour 
paste. When cultivated in milk it gives rise 
to the formation of lactic and butyric acids 
and to coagulation of the casein. No poison- 
ous ptomaines or toxalbumins have been iso- 
lated from cultures of this micrococcus, but, 
like other liquefying bacteria, it forms a sol- 
uble peptonizing ferment, by which gelatin 
may be liquefied independently of the living 
microérganism. While the Staphylococcus 
aureus gives rise to the production of acids— yg ¢0.—Gelatin culture of 
principally lactic acid—in media containing Staphylococcus pyogenes aureus 
glucose or lactose, it has also been shown by @*uméa"e™- 

Brieger that ammonia is one of the products of its vital activity. 
Unlike some other pathogenic bacteria, it is able to grow in a medium 
having a distinctly acid reaction. A non-poisonous basic substance 
has been isolated by Brieger from old cultures in meat infusion which 
differs from any of the ptomaines obtained by him from other sources. 

The thermal death-point of this micrococcus, in recent cultures in 
flesh-peptone-gelatin, as determined by the writer, is between 56° and 
58° C., the time of exposure being ten minutes. When in a desic- 
cated condition a much higher temperature is required—0° to 100° C. 
—for its destruction ; and it retains its vitality for more than ten 
days when dried upon a cover glass (Passet). It retains its vitality 
for a long time in cultures in nutrient gelatin or agar, and may grow 
when transplanted from such cultures even at the end of a year. 

Very numerous experiments have been made to determine the 
proportion of various chemical agents required to destroy the vitality 
or to restrain the growth of this important pyogenic micrococcus. 
The extended researches of Liibbert (1886) with reference to the 
antiseptic power of agents added to a suitable culture medium—nu- 
trient gelatin—gave the following results: Development was _ pre- 


¢ 


376 PYOGENIC BACTERIA. 


vented by the agents named in the proportion given: Nitric acid, 
1:797; phosphoric acid, 1:50; boracic acid, 1:327; oxalic acid, 
1 :433 ; acetic acid, 1 : 720; citric acid, 1:433; lactic acid, 1 :350 ; 
benzoic acid, 1:400; salicylic acid, 1:655; iodine dissolved with 
potassium iodide, 1:1,100; arsenite of potash, 1: 733; mercuric 
chloride, 1:81,400; chloral hydrate, 1:133; carbolic acid, 1 : 814; 
thymol, 1:11,000; resorcin, 1:122; hydrochinon, 1:353; kairin, 
1:407 ; antipyrin, 1:26; muriate of quinine, 1:550; muriate of 
morphia, 1:60. For the destruction of vitality very much larger 
amounts are required. In Bolton’s experiments (1887) a one-per-cent 
solution of carbolic acid was successful after two hours’ exposure, 
but two per cent failed to completely destroy vitality in the same 
time ; one per cent of sulphate of copper was also successful, and but 
a single colony developed after exposure to a solution of 1:200. In 
the experiments of Gartner and Plagge the Staphylococcus aureus in 
bouillon cultures is said to have been killed in a few seconds (eight) 
by a solution of mercuric chloride of the proportion of 1: 1,000; Behr- 
ing found it was killed by the acid sublimate solution of La Place, 
in the proportion of 1:1,000, in ten minutes; Tarnier and Vignal 
found that a solution of 1:1,000 was successful in two minutes. 
Abbott (1891) has shown that in the same culture there may be a 
considerable difference in the resisting power of the cocci, and that 
while frequently all are destroyed in five minutes by a 1 :1,000 solu- 
tion, it occurs quite as frequently that some may survive after an ex- 
posure of ten, twenty, and even thirty minutes. 
Pathogenests.—Subcutaneous inoculation with a small quantity 
of a culture of Staphylococcus pyogenes aureus is without result in 
rabbits, guinea-pigs, or mice, but when a considerable quantity is 
injected beneath the skin of a rabbit or a guinea-pig an abscess is 
produced, which usually results in recovery, but may give rise to 
general infection and the death of the animal. Injection into a 
vein or into the cavity of the abdomen in the animals mentioned 
usually induces a fatal result within a few days. The most charac- 
teristic pathological changes are found in the kidneys, which con- 
tain numerous small collections of pus and under the microscope 
present the appearances resulting from embolic nephritis. Many of 
the capillaries and some of the smaller arteries of the cortex are 
plugged up with thrombi consisting of micrococci. Metastatic ab- 
scesses may also be found in the joints and muscles. The micro- 
cocci may be recovered in pure cultures from the blood and the 
various organs ; but they are not numerous in the blood, and a sim- 
ple microscopical examination will often fail to demonstrate their 


presence. 
Animals frequently survive the injection of a small quantity of 


PYOGENIC BACTERIA. : 377 


a pure culture made directly into the circulation, and there is evi- 
dence that the pathogenic potency of this micrococcus may vary 
considerably as a result of conditions relating to its origin and culti- 
vation in the animal body or in artificial media. When injected in 
considerable quantities it may be obtained in cultures from the 
urine, but not sooner than six or eight hours after the injection, and 
not until the formation of purulent foci in the kidneys has already 
occurred (Wyssokowitsch). 

The pyogenic properties of this micrococcus have been demon- 
strated upon man by the experiments of Garré, of Bockhart, and of 
Bumm. The first-named observer inoculated a small wound at the 
edge of one of his finger nails with a minute quantity of a pure cul- 
ture, and a subepidermal, purulent inflammation extending around 


Fia. 81.—Vertical section through a subcutaneous abscess caused by inoculation witb staphylo- 
cocci, in the rabbit, forty-eight hours after infection; margin towards the normal tissue. x 950. 
(Baumgarten.) 


the margin of the nail resulted from the inoculation. Staphylococ- 
cus aureus was recovered in cultures from the pus thus formed. A 
more extensive and extremely satisfactory experiment was subse- 
quently made by Garré, who applied a considerable quantity of a 
pure culture obtained from the above-mentioned source—third gene- 
ration—to the uninjured skin of his left forearm. At the end of 
four days a large carbuncle, surrounded by isolated furuncles, de- 
veloped at the point where the culture had been applied. This ran 
the usual course, and it was several weeks before it had completely 
healed. No less than seventeen scars remained to give evidence of 
the success of the experiment. 

In Bockhart’s experiments a similar but milder result was ob- 
tained, the conditions having been somewhat different. A small 


378 PYOGENIC BACTERIA. 


quantity of an agar culture was suspended in 0.5-per-cent salt solu. 
tion, and this was rubbed upon the uninjured skin of the left fore- 
arm. By gentle scratching with a disinfected finger nail the epithe- 
lium was removed in places over the area to which the micrococcus 
had been applied. As a result of this procedure numerous impe- 
tigo pustules and occasionally a genuine furuncle developed. Por- 
tions of the skin containing the smaller pustules were excised and 
examined microscopically. As a result of this examination Bock- 
hart concluded that the cocci penetrate by way of the hair follicles, 
the sebaceous and sudoriparous glands, or, where the epidermis had 
been removed by scratching, directly to the deeper layers of the skin. 

In Bumm’s experiments, made upon himself and several other 
persons, Staphylococcus aureus suspended in sterilized salt solution 
was injected beneath the skin. An abscess resulted in every case. 

The very extended researches made by bacteriologists during the 
past five or six years show that the golden staphylococcus is the 
most common pyogenic microérganism. Its presence has been de- 
monstrated not only in furuncles and carbuncles, but also in various 
pustular affections of the skin and mucous membranes—impetigo, 
sycosis, phlyctenular conjunctivitis ; in purulent conjunctivitis and 
inflammation of the lacrymal sac; in acute abscesses formed in the 
lymphatic glands, the parotid gland, the tonsils, the mamme, etc. ; 
in metastatic abscesses and purulent collections in the joints ; in em- 
pyema ; in infectious osteomyelitis ; and in ulcerative endocarditis. 
The evidence relating to its presence and etiological import in the 
last-mentioned affections demands special consideration. 

Infectious osteomyelitis appears from the researches of Becker, 
Rosenbach, Krause, Passet, and others, to be usually due to the pre- 
sence of Staphylococcus aureus, although Kraske has shown that in 
certain cases this is associated with other microédrganisms. Becker, 
who obtained this micrococcus from the pus of osteomyelitis in 1883, 
was the first to show by experiment that the same affection might be 
induced in rabbits by injecting cultures of the micrococcus into the 
circulation, after having crushed or fractured a bone in one of its 
legs. The animal usually died in from twelve to fourteen days and 
presented the usual appearances of osteomyelitis at the fractured 
point. The abundant yellowish-white pus contained the golden 
staphylococcus which was described by Becker, and subsequently 
known in the bacteriological laboratories of Germany as the ‘‘ mi- 
crococcus of infectious osteomyelitis.” Becker’s experimental re- 
sults have been confirmed by Krause and Rosenbach; and Rodet, by 
injecting smaller quantities of a culture into the circulation, has suc- 
ceeded in producing an osteomyelitis without previous injury to the 
bone. 


PYOGENIC BACTERIA, 379 


Ulcerative endocarditis has been shown by the researches of 
numerous bacteriologists to be occasionally accompanied by a mycotic 
invasion of the affected tissues by the golden staphylococcus ; in 
other cases Streptococcus pyogenes is present. The researches of 
Weichselbaum, and of E. Frankel and Sanger, also show that it is 
present in a certain proportion of the cases, at least, of endocarditis 
verrucosa, although in smaller numbers. That the diseased condi- 
tion of the cardiac valves in ulcerative endocarditis is due to mycotic 
invasion is now generally admitted and is supported by experimental 
evidence. Rosenbach first (1873) produced an endocarditis in lower 
animals by mechanical injury to the cardiac valves, effected by in- 
troducing a sound through the aorta. Following his method, Wys- 
sokowitsch (1885), after injuring the cardiac valves in rabbits, in- 
jected into the circulation pure cultures of various bacteria. He 
obtained positive results with Staphylococcus aureus and Strepto- 
coccus pyogenes only. When these micrococci were injected into 
the trachea or subcutaneously the result was negative, as was the 
case when very few cocci were injected into a vein, or when two 
days or more were allowed to elapse after injury to the cardiac 
valves. Subsequently Weichselbaum, Prudden, and Frankel and 
Sanger obtained confirmatory results, thus establishing the fact that 
when the valves are first injured mechanically (or chemically— 
Prudden) the injection into a vein of a pure culture of Staphylococcus 
aureus gives rise to a genuine ulcerative endocarditis. It has been 
further shown by Ribbert that the same result may be obtained with- 
out previous injury to the valves by injecting into a vein the staphy- 
lococcus from a potato culture suspended in water. In his experi- 
ments not only the micrococci from the surface but the superficial 
layer of the potato was scraped off with a sterilized knife and mixed 
with distilled water ; and the successful result is ascribed to the fact 
that the little agglomerations of micrococci and infected fragments 
of potato attach themselves to the margins of the valves more readily 
than isolated cocci would do. In these experiments the mitral and 
tricuspid valves were affected, while the semilunar valves remained 
intact. In ulcerative endocarditis it is evident that cocci detached 
from the diseased valves must find their way into the circula- 
tion, As a matter of fact, masses of micrococci are carried away by 
the blood stream and form emboli in various parts of the body, which 
become secondary foci of infection and give rise to local necrotic 
changes and accumulations of pus. While this undoubtedly occurs, 
it is generally admitted that the mycotic infection of the cardiac 

‘valves is usually a secondary affection, resulting from the transpor- 
tation of micrococci in the blood current from some other infected 
focus. But there is no general development of micrococci in the cir- 


380 PYOGENIC BACTERIA. 


culating fluid, and in man, as in animals infected experimentally, a 
microscopic examination of the blood for microérganisms usually 
gives a negative result. Culture experiments may, however, demon- 
strate their presence. Thus recent investigations by Netter, Hisel- 
berg, and others show that the pus cocci are usually present in the 
blood in small numbers, as demonstrated by culture experiments, in 
septic infection from wounds. 


STAPHYLOCOCCUS PYOGENES ALBUS. 


Isolated by Rosenbach (1884) from the pus of acute abscesses, in 
which it is sometimes the only microdrganism present, and some- 
times associated with other pus cocci. In thirty-three acute abscesses 
examined by Passet (1885) it was associated with Staphylococcus 
aureus in eleven, with Staphylococcus citreus in two, with Strepto- 
coccus pyogenes in one, with both Staphylococcus citreus and Strep- 
tococcus pyogenes in one, and was obtained alone from four. 

In its morphology this micrococcus is identical with the preced- 
ing, but it is distinguished from it by the absence of pigment and 
by being somewhat less pathogenic. Surface cultures upon nutrient 
agar or potato have a milk-white color. It liquefies gelatin in the 
same way as does the golden staphylococcus, but the deposit at the 
bottom of the liquefied gelatin is without color. In the temperature 
conditions favorable to its growth, and in its biological characters 
generally, with the exceptions noted, it is not to be distinguished 
from the species previously described. According to Fliigge, it is 
more common than aureus among many of the lower animals. 

Pathogenesis.—Fortunati has tested the comparative pathogenic 
power of Staphylococcus aureus and Staphylococcus albus by inocu- 
lations into the cornea of rabbits. A. purulent infiltration of the 
cornea and panophthalmitis resulted when Staphylococcus aureus 
was inoculated upon the surface of the cornea by scratching with an 
infected needle, but inoculations made in the same way with Staphy- 
lococcus albus healed spontaneously or gave rise to a perforating 
ulcer. After paracentesis of the cornea with an instrument infected 
with Staphylococcus aureus panophthalmitis developed in thirty hours; 
the same result occurred at the end of sixty to seventy-two hours 
when the instrument was infected with Staphylococcus albus. When 
a sterilized instrument was used the result was negative. In bacteri- 
ological researches made by Gallenga, in cases of panophthalmitis in 
man, Staphylococcus albus was found in ten cultures and Staphy- 
lococcus aureus in nine. 


Staphylococcus Epidermidis Albus (Welch). 


The researches of Welch show that a white staphylococcus, prob- 
ably identical with Staphylococcus pyogenes albus of Rosenbach, is 


PYOGENIC BACTERIA. 381 


the most common microorganism upon the surface of the body, and 
that “itis very often present in parts of the epidermis deeper than 
can be reached by any known means of cutaneous disinfection save 
the application of heat.” With reference to this coccus Welch 
says: 

‘So far as our observations extend—and already they amount to a 
large number—this coccus may be regarded as a nearly, if not quite, con- 
stant inhabitant of the epidermis. It is now clear why I have proposed to 
eall it the Staphylococcus epidermidis albus.’ It possesses such feeble pyo- 
genic capacity, ag is shown by its behavior in wounds as well as by experi- 
ments on rabbits, that the designation Staphylococcus pyogenes albus does 
not seem appropriate. Still, I am not inclined to insist too much upon this 
point, as very probably this coccus, which has hitherto been unquestionably 
identified by Bossowski and others with the ordinary Staphylococcus pyo- 
genes albus of Rosenbach, is an attenuated or modified form of the latter 
organism, although, as already mentioned, it presents some points of differ- 
ence from the classical description of the white pyogenic coccus.” 


According to Welch, this coccus differs from Staphylococcus pyo- 
genes aureus not only in color, but also in the fact that it liquefies 
gelatin more slowly, does not so quickly cause coagulation of milk, 
and is far less virulent when injected into the circulation of rabbits. 
It has been shown by the researches of Bossowski and of Welch 
that this coccus is very frequently present in aseptic wounds, and 
that usually it does not materially interfere with the healing of 
wounds, although sometimes it appears to cause suppuration along 
the drainage tube, and it is the usual cause of ‘stitch abscess.” 
Bossowski, in fifty cases of wounds treated antiseptically, obtained 
bacteria from the discharges in forty, and in twenty-six of these 
eases he found Staphylococcus pyogenes albus; Staphylococcus au- 
reus was found nine times, Streptococcus pyogenes in two, and vari- 
ous non-pathogenic bacteria in eight. In forty-five laparotomy 
wounds examined by Ghrisky and Robb, in which strict antiseptic 
precautions had been observed, bacteria were found in thirty-one, and 
in nineteen of this number Staphylococcus albus was present, 
Staphylococcus aureus in five, Bacillus coli communis in six, and 
Streptococcus pyogenes in three. 


STAPHYLOCOCCUS PYOGENES CITREUS. 


Isolated by Passet (1885) from the pus of acute abscesses. In thirty- 
three cases examined it was found associated with Staphylococcus albus in 
two and with Staphylococcus albus and Streptococcus pyogenes in one. 

In its morphology this coccus is identical with the two preceding species 
from which it is distinguished by the formation of a lemon-yellow pigment, 
instead of a pofen or orange-yellow as in Staphylococcus aureus. The 
pigment is only formed in the presence of oxygen. This coccus is said by 
Frankel to liquefy gelatin more slowly than the previously described species 
—Staphylococcus aureus and Staphylococcus albus. 

As to its pathogenic properties we have no definite information. It is 
included among the pyogenic bacteria because of its occasional presence in 


382 PYOGENIC BACTERIA. 


the pus of acute abscesses, although it has heretofore only been found in as- 
sociation with other microérganisms. 


MICROCOCCUS PYOGENES TENUIS. 


Obtained by Rosenbach (1884) from pus in three cases out of thirty-nine 
examined. 

Morphology.—Micrococci, somewhat irregular in size, but larger than 
Staphylococcus albus, and seldom associated in masses. Frequently the in- 
dividual cocci present the appearance of consisting of two deeply stained 
masses separated from each other by a paler interspace. Cultures upon the 
surface of nutrient agar form a very thin, transparent layer of about one 
millimetre in breadth along the line of inoculation; this resembles a thin 


layer of varnish. f 
Pathogenesis undetermined. (Micrococcus pneumoniz croupose ?) 


STREPTOCOCCUS PYOGENES. 


Synonyms.—Micrococcus of erysipelas (Fehleisen) ; Streptococcus 
erysipelatos ; Streptococcus of pus ; Streptococcus longus (Von Lin- 
gelsheim). 

Obtained by Fehleisen from the skin involved in cases of erysipe- 
las (1883), and by Rosenbach (1884) and Passet (1885) from the pus 
of acute abscesses. The characters of the ‘‘ streptococcus of erysipe- 
las” of Fehleisen and the ‘‘ Streptococcus pyogenes” of Rosenbach 
and Passet are generally admitted to be identical, although some 
bacteriologists still describe them separately and cultures from the 
two sources are still retained in bacteriological laboratories under the 
names originally given them. 

Rosenbach found Streptococcus pyogenes alone in fifteen cases, 
and associated with staphylococci in five cases, out of thirty-nine 
cases examined of acute pus formation. Passet, in thirty-three 
similar cases, obtained the streptococcus alone in eight and associated 
with staphylococci in two. Subsequent researches show that this 
micrococcus is frequently, if not constantly, present in puerperal 
metritis ; that it is the most frequent microédrganism associated with 
ulcerative endocarditis ; that it is frequently present in diphtheritic 
false membranes, and especially in those cases of diphtheritic inflam- 
mation which are secondary to scarlet fever and measles (Prudden). 
Numerous investigations made by bacteriologists during the past few 
years indicate that this isa very important and widely distributed 
pathogenic microérganism. It has also been frequently found upon 
exposed mucous surfaces—mouth, nose, vagina—of healthy in- 
dividuals. ° 

According to the researches (1891) of Von Lingelsheim, the Strep- 
tococcus pyogenes differs from Streptococcus erysipelatos in be- 
ing pathogenic both for mice and rabbits, while the latter is patho- 


PYOGENIC BACTERIA. 383 


genic for rabbits only. The author named, as a result of extended and 
carefully conducted comparative studies, arrives at the following 
conclusions: 


‘* According to my observations, there are two great groups among the 
streptococci. These cannot be distinguished one from the other in cultures 
in highly albuminous media (pus, blood serum), but present constant dif- 
ferences when cultivated in bouillon. The decisive characteristics in this 
medium are: macroscopic, the cloudiness of the medium ; microscopic, the 
length of thechains. The two groups are with difficulty distinguished in 
agar cultures; more easily in gelatin, in which the streptococcus which 
forms short chains causes a slight liquefaction, while the Streptococcus 
longus does not. Upon potato Eareptocoocus brevis alone shows a visible 
growth. . . . Wesee here a group of streptococci which we separate from 
the others, because of their microscopic and cultural differences, under the 
name of Streptococcus brevis, which is also distinguished by having no 
pathogenic action upon the animals usually experimented upon. We 
recognize, on the other hand, the streptococci which we have grouped to- 
gether as Streptococcus longus as all pathogenic and about in equal degree 
for a certain species of animal (rabbits); but by experiments upon other 
species (mice) we arrive at the conclusion that there must also be differences 
between these streptococci. It appears that the streptococci which are dis- 
tinguished by their high degree of pathogenic power upon mice are also 
those which are distinguished in bouillon cultures by the formation of con- 
glomerate masses. We find among these also one which is distinguished 
by especial virulence for mice, and that this one is distinguished in cultures 
by its scanty growth upon ox serum.” 


The more recent researches of Knorr (1893), and of Waldvogel 
(1894), indicate that the classification of the streptococci proposed by 
von Lingelsheim has no great value, and show that marked changes 
in biological characters and in pathogenic power may result from 
cultivation in special media, or from successive inoculations into 
animals. 

Morphology.—Spherical cocci, from 0.4 4 to 1 « in diameter, but 
varying considerably in dimensions in different cultures, and even 
in asingle chain. Multiply by binary division, 
in one direction only, forming chains, in which 
the elements are commonly associated in pairs. 
Under certain circumstances, instead of form- 
ing chains, a culture may contain only, or 
chiefly, diplococci ; but usually chains contain- 
ing from four to twenty or more elements are 
formed, and these are frequently associated 
in tangled masses. Occasionally one or more 
cells in a chain greatly exceed their fellows in 
size, and some bacteriologists suppose that Fie. 8—Pus containing 
these cells serve as reproductive spores—arthro- Hoa eee 

é ‘ igge.) 
spores—but this has not been definitely proven. 

Stains readily with the aniline colors and by Gram’s method. 


384 PYOGENIC BACTERIA. 


Biological Characters.—Grows readily in various liquid and 
solid culture media, including all of those usually employed in bac- 
teriological researches. The most favorable temperature for its de- 
velopment is from 30° to 37° C., but it multiplies freely at the ordi- 
nary room temperature—16° to 18° C. 

Streptococcus pyogenes is a facultative anaérobic, growing 
both in the presence and absence of oxygen. It 
does not liquefy gelatin, and in gelatin stab 
cultures it grows along the line of puncture, 
forming numerous small, spherical, translu- 
cent, whitish colonies, which are closely crowd- 
ed together at the upper portion of the line of 
growth, and often distinctly separated from 
each other below; upon the surface there is 
often no growth, or a scanty development may 
occur about the point of entrance of the inocu- 
lating needle. The minute colonies along the 
line of puncture are already visible at the end 
of twenty-four hours in cultures kept in the 
incubating oven at 30° to 35° C., and at the end 
of three or four days they have reached their 
full development, forming a semi-opaque, white, 
granular column, upon the margins of which 
the separate colonies are seen projecting into the 
gelatin. On gelatin plates very small, translu- 
\ p cent colonies are developed, which upon the sur- 
> face spread out to form a flat, transparent disc 

Fic. 83—Streptococeus of about one-half millimetre. Under a low mag- 

pelatin; stick culture at nifying power these colonies are seen to be slight- 

end of four days at 16-- ly granular and have a yellowish color. Ata 

ae ice i later date they become darker and less trans- 
parent, and the margin may show irregular projections made up of 
tangled masses of cocci in chains. The characters of growth in 
nutrient agar and in jellified blood serum are similar to those in gela- 
tin, and on agar plates colonies are formed similar to those above 
described, except that they are somewhat smaller and more trans- 
parent. Fehleisen and De Simone state that the erysipelas coccus' 
may develop upon the surface of cooked potato, but most authorities: 
—Fligge, C. Frankel, Passet, Baumgarten—agree that no growth 
occurs upon potato, Milk is a favorable medium for the growth of 
this micrococcus, and the casein is coagulated by it. A slightly acid 
reaction of the culture medium does not prevent its development. 
The thermal death-point, as determined by the writer, is between 
52° and 54° C., the time of exposure being ten minutes. According 


PYOGENIC BACTERIA. 385 


to De Simone, a temperature of 39.5° to 41° C. maintained for two 
days is fatal to this micrococcus. 

Manfredi and Traversa have injected filtered cultures into frogs, 
guinea-pigs, and rabbits for the purpose of ascertaining if any solu- 
ble toxic substance is produced during the growth of Streptococcus 
pyogenes. They report that in some cases convulsions and in others 
paralysis resulted from these injections. 

Von: Lingelsheim has (1891) reported the following results 
obtained in an extended series of experiments made to determine 
the germicidal power of various chemical agents as tested upon 
this microérganism—time of exposure two hours : Hydrochloric acid 
1: 250, sulphuric acid 1: 250, caustic soda 1:130, ammonia 1 : 25, 
mercuric chloride 1 :2,500, sulphate of copper 1: 200, chloride of 
iron 1: 500, terchloride of iodine 1 : 750, peroxide of hydrogen 1 : 50, 
carbolic acid 1 : 300, cresol 1 : 250, lysol 1 : 300, creolin 1 : 130, naph- 
thylamin 1 : 125, malachite green 1.:3,000, pyoktanin 1 : 700. 


Fic. 84.—Section from margin of an erysipelatous inflammation, showing streptococci in 
lymph spaces. From a photograph by Koch. x 900. 


Pathogenesis.—When inoculated into the cornea of rabbits 
Streptococcus pyogenes gives rise to keratitis. Inoculations into the 
ear of the same animal usually give rise to a localized erysipelatous 
inflammation accompanied by an elevation of temperature in the in- 
oculated ear; at the end of thirty-six to forty-eight hours the in- 
flamed area, which has well-defined margins and a bright-red color, 
extends from the point of inoculation along the course of the veins to 
the root of the ear. This appearance passes away in the course of a 
few days and the animal recovers. Subcutaneous injections into mice 
or rabbits are usually without result, and the last-named animal also 
withstands injections of considerable quantities into the general cir- 
culation through a vein. When, however, the animal has previously 
been weakened by the injection of toxic substances the streptococcus 
may multiply in its body and cause its death (Fligge). 

Fehleisen has inoculated cultures, obtained in the first instance 
from the skin of patients with erysipelas, into patients in hospital 
suffering from lupus and carcinoma, and has obtained positive re- 
sults, a typical erysipelatous inflammation having developed 

25 


386 PYOGENIC BACTERIA. 


around the point of inoculation after a period of incubation of from 
fifteen to sixty hours, This was attended with chilly sensations and 
an elevation of temperature. Persons who had recently recovered 
from an attack of erysipelas proved to be immune. 

Sections made from the ear of an inoculated rabbit, or of skin taken 
from the affected area in erysipelas in man, show the streptococci in 
considerable numbers in the lymph channels, but not in the blood 
vessels. They are more numerous, according to Koch and to Fehl- 
eisen, upon the margins of the erysipelatous area, and may even be 
seen in the lymph channels a little beyond the red margin which 
marks the line of progress of the infection. 

The researches of Weichselbaum and others show that Strepto- 
coccus pyogenes is the infecting microorganism in a certain propor- 
tion of the cases of ulcerative endocarditis. The author named 
found it in four cases out of fifteen examined, and in two cases of 
endocarditis verrucosa outof thirteen. In a previously reported series 
of sixteen cases (fourteen of ulcerative endocarditis and two of ver- 
rucosa) the streptococcus was found in six. 

In diphtheritic false membranes this streptococcus is very com- 
monly present, and in certain cases attended with a diphtheritic exu- 
dation, in which the Bacillus diphtheriz has not been found by com- 
petent bacteriologists, it seems probable that Streptococcus pyogenes 
is the pathogenic microérganism responsible for the local inflamma- 
tion and its results. Thus in a series of twenty-four cases studied by 
Prudden in 1889 the bacillus of Léffler was not found, “but a strep- 
tococcus apparently identical with Streptococcus pyogenes was found 
in twenty-two.” Chantemesse and Widal have also reported cases 
in which a fibrinous exudate resembling that of diphtheria was as- 
sociated with a streptococcus. ‘‘ These forms of so-called diphtheria 
are most commonly associated with scarlatina and measles, erysipe- 
las, and phlegmonous inflammation, or occur in individuals exposed 
to these diseases ; but whether exclusively under these conditions is 
not yet established ” (Prudden). 

Léffler has described under the name of Streptococcus articu- 
lorum a micrococcus obtained by him from the affected mucous 
membrane in cases of diphtheria, and which he believes to be acci- 
dentally present and without any etiological import in this disease. 
In its characters it closely resembles Streptococcus pyogenes and is 
perhaps a variety of this widely distributed species. Its characters 
are described by Fligge as follows : 


“Cultivated in nutrient gelatin, it forms at the end of three days small, 
transparent, light-gray drops, upon the margin of which, under the micro- 
scope, the cocci in twisted chains may be observed. As many as one hun- 


PYOGENIC BACTERIA. 387 


dred elements may be found in a single chain, and some of these are distin- 
guished by their size; occasionally whole chains are made up of these large 
cocci, and when closely observed some of these may present indications of 
division transversely to the axis of the chain. Subcutaneous inoculation of 
cultures into mice results in the death of a considerable number of these ani- 
mals—more than half; and the streptococci are found in the spleen and other 
organs. Inoculation into the ear of rabbits causes an erysipelatous inflam- 
mation. When injected into the circulation of these animals through a vein 
joint affections are developed in from four to six days, and a purulent ac- 
cumulation occurs in which the streptococci are found. In two rabbits in- 
oculated in the same way with a culture of the streptococcus of erysipelas, 
Loffler has observed a similar result.” 


Numerous researches indicate that infection by Streptococcus 
pyogenes through the endometrium is the usual cause of puerperal 
fever. Thus Clivio and Monti demonstrated its presence in five 
cases of puerperal peritonitis. Czerniewski found it in the lochia of 
a large number (thirty-five out of eighty-one) of women suffering 
from puerperal fever, but in the lochia of fifty-seven healthy puer- 
peral women he was only able to find it once. In ten fatal cases he 
found it in every instance, both in the lochial discharge during life 
and in the organs after death. Widal carefully studied a series of 
sixteen cases and arrived at the conclusion that this was the infect- 
ing microérganism in all. Bumm and other observers have given 
similar evidence. Hiselsberg and Emmerich have succeeded in de- 
monstrating the presence of the streptococcus in hospital wards con- 
taining cases of erysipelas. That puerperal fever may result from 
infection through the finger of the accoucheur, when he has previ- 
ously been in contact with cases of erysipelas, has long been taught, 
and, in view of the facts above recorded, is not difficult to under- 
stand. But in view of the fact that the streptococcus of pus has been 
found in vaginal mucus and in the buccal and nasal secretions of 
healthy persons, it may appear strange that cases of puerperal fever 
not traceable to infection from erysipelas or from preceding cases 
do not occur more frequently. This is probably largely due to an 
attenuation of the pathogenic power of the streptococcus when it 
leads a saprophytic existence. Widal asserts that, when cultivated 
in artificial media for a few weeks, the cultures no longer have their 
original virulence, and Bumm has made the same observation. On 
the other hand, in ‘‘ streptococcus-peritonitis ” occurring as a result 
of puerperal infection Bumm states that the thin, bright-yellow, 
odorless fluid contained in the cavity of the abdomen is extremely 
virulent ; a very slight trace, a fragment of a drop, injected into the 
abdominal cavity of a rabbit, is sufficient within twenty-four hours 
to cause a general septic inflammation with a bloody serous exuda- 
tion, quickly terminating in the death of the animal ; injected sub- 
cutaneously it gives rise to an enormous phlegmon which also 


388 PYOGENIC BACTERIA. 


quickly proves fatal. But cultures of Streptococcus pyogenes, after 
it has been carried through successive generations in artificial media, 
injected beneath the skin of a rabbit, usually produce no result, or 
at most an abscess of moderate dimensions. 

It seems probable that the micrococcus isolated by Fligge from 
necrotic foci in the spleen of a case of leucocythemia, and described 
by him under the name of Streptococcus pyogenes malignus, was 
simply a very pathogenic variety of the streptococcus of pus. He 
was not able to differentiate it from Streptococcus pyogenes by its 
morphology or growth in culture media, but it proved far more 
pathogenic when tested upon animals. Mice inoculated subcutane- 
ously with a minute quantity of a pure culture died, without excep- 
tion, in three to five days. A large abscess was formed at the point 
of inoculation, and the blood of the animal contained numerous cocci 
in pairs and chains. Rabbits inoculated in the ear showed at first 
the same local appearances as result from inoculations with strepto- 
coccus of pus and of erysipelas, but after two or three days symp- 
toms of general infection were developed, and death occurred at the 
end of three or four days. At the autopsy the cocci were found in 
the blood, and frequently there were purulent collections in the 
joints containing the same microérganism. Krause has also de- 
scribed a streptococcus which only differs from Streptococcus pyo- 
genes of Rosenbach and Passet by the greater virulence manifested 
by its cultures. 

The fact that pathogenic bacteria may attain an intensified de- 
gree of virulence by cultivation in the bodies of susceptible animals 
was demonstrated by Davaine many years ago, and is fully estab- 
. lished by the experiments of Pasteur and others. It is true of the 
anthrax bacillus, of the writer’s Micrococcus Pasteuri, and of other 
well-known pathogenic microédrganisms. The reverse of this—at- 
tenuation of virulence as a result of cultivation in artificial media— 
is also well established for several pathogenic species. Now it 
appears that the attenuated streptococcus is far less likely to give 
rise to erysipelas or to puerperal infection than is the same micro- 
organism as obtained from a case of one or the other of these infec- 
tious diseases. The same is probably true also of Staphylococcus 
aureus and other facultative parasites which are found as sapro- 
phytes upon the surface of the body and upon exposed mucous mem- 
branes in healthy persons. And it is not improbable that attenuated 
varieties of these micrococci which find their way into open wounds, 
or into the uterine cavity shortly after parturition, if they escape 
destruction by the sanguineous discharge, acquire increased patho- 
genic power from their multiplication in it, as a result of which they 
are able to invade the living tissues. But it appears probable that 


PYOGENIC BACTERIA. 389 


infection through open wounds does not depend alone upon the 
potency of the pathogenic micrococci present in them, but also upon 
the absorption of chemical poisons produced by septic (putrefactive) 
bacteria, which weaken the vital resisting power of the tissues. 
Gottstein, as a result of experiments made by him, is of the opinion 
that the resorption of broken-down red blood corpuscles favors infec- 
tion by pathogenic bacteria present in wounds; and he has shown 
that the injection into animals of certain toxic substances which de- 
stroy the red corpuscles in the circulation makes them susceptible to 
the pathogenic action of certain bacteria which are harmless for 
them under ordinary circumstances. Thus a guinea-pig, an animal 
which is immune against the bacillus of fowl cholera, succumbed to an 
inoculation made after first injecting subcutaneously 0.06 gramme of 
hydracetin dissolved in alcohol. At the autopsy hemorrhagic exu- 
dations were found in the serous cavities, hemorrhagic infarctions 
in the lungs, and quantities of the bacillus injected were found in 
the blood and in fluid from the cavity of the abdomen. 

In man the ever-present pus cocci are more likely to invade the 
tissues, forming furuncles, carbuncles, and pustular skin eruptions, 
or erysipelatous and phlegmonous inflammations, when the standard 
of health is reduced from any cause, and especially when by absorp- 
tion or retention various toxic organic products are present in the 
body in excess. It is thus that we would explain the liability to these 
localinfections, as complications or sequele of various specific infec- 
tious diseases, in the victims of chronic alcoholism, in those exposed 
to septic emanations from sewers, etc., and probably in many cases 
from the absorption of toxic products formed in the alimentary canal 
as a result of the ingestion of improper food, or of abnormal fermen- 
tative changes in the contents of the intestine, or from constipation. 

The Pus Cocct in Inflammations of Mucous Membranes.— 
To what extent the pus cocci are responsible for inducing and main- 
taining non-specific inflammations of mucous membranes has not 
been determined ; but having demonstrated the pyogenic properties 
of these cocci, their presence in the purulent discharges from inflamed 
mucous membranes can scarcely be considered as unimportant, not- 
withstanding the fact that they are also frequently found in secre- 
tions from healthy mucous surfaces. They are likewise found upon 
the skin of healthy persons, and yet we have unimpeachable experi- 
mental evidence that they may produce a local inflammation, at- 
tended with pus formation, when injected subcutaneously, or even 
when freely applied to the uninjured surface. 

In otitis media Levy and Schrader obtained Staphylococcus 
albus in pure cultures in three cases out of ten in which paracentesis 
was performed, and in two others it was present in association with 


390 PYOGENIC BACTERIA. 


other microérganisms. In eighteen cases of otitis media in young 
children Netter found Staphylococcus aureus six times and Strepto- 
coccus pyogenes thirteen times. Scheibe, in eleven cases in which 
perforation had not yet taken place, found Staphylococcus albus in 
two and various other microdrganisms in the remaining cases ; Sta- 
phylococcus aureus was not present in any. Habermann obtained 
aureus associated with other bacteria in a single case of purulent 
otitis media. In a series of eight cases occurring as a sequela of 
influenza Scheibe obtained Streptococcus pyogenes in two, “ diplo- 
coccus pneumonis” in two, Staphylococcus aureus in one, Strepto- 
coccus pyogenes and Staphylococcus albus together in two, and Strep- 
tococcus pyogenes in association with an undescribed micrococcus in 
one. In all of these cases a slender bacillus was also present, as 
shown by microscopical examination, which did not grow in any of 
the culture media employed. Bordoni-Uffreduzzi and Gradenigo 
have tabulated the results obtained by various bacteriologists who 
have examined pus obtained through the previously intact tympanic 
membrane. In thirty-two cases of this character the microérganism 
most frequently found was diplococcus pneumonize (Micrococcus 
pneumoniae croupose of the present writer), which was present in a 
pure culture in thirteen and associated with Staphylococcus aureus 
in one, with Staphylococcus albus in one, and with Streptococcus 
pyogenes in one. In the other sixteen cases the pyogenic cocci were 
present in all but two, in which bacilli were found—Bacillus tenuis 
in one, a non-liquefying bacillus in one. In twenty-seven cases in 
which the pus was withdrawn from one to thirty days after paracen- 
tesis or spontaneous rupture of the membrane, the pyogenic cocci 
were present in twenty and diplococcus pneumoniz in seven. 

In acute nasal catarrh Paulsen found Staphylococcus aureus in 
seven cases out of twenty-four examined, and HK. Frankel in two out of 
four ; but it must be remembered that Von Besser has shown that this 
micrococcus is frequently present in the secretions from the healthy 
nasal mucous membrane, and we have experimental evidence that 
the pus organisms, when introduced into the conjunctival sac of 
rabbits (Widmark), do not give rise to catarrhal inflammation. On 
the other hand, Widmark found that when inoculated into the cornea 
of rabbits an intense conjunctivitis resulted, together with keratitis 
and perforation of the cornea in fifteen per cent of the cases. The 
same author in his bacteriological researches obtained the pyogenic 
staphylococci from the circumscribed abscesses of blepharadenitis, 
while in inflammation of the lacrymal sac Streptococcus pyogenes 
was usually present. 

Shougolowicz,in the bacteriological examination of twenty-six cases 
of trachoma, found Staphylococcus albus in twelve, Staphylococcus 


PYOGENIC BACTERIA. 391 


aureus in nine, Staphylococcus citreus in three, and Staphylococcus 
cereus albus in three. These pus organisms were in a number of 
the cases associated with other well-known saprophytes, and in seven 
cases a short bacillus not previously described was found. That 
various bacilli are found in the conjunctival sac of healthy eyes 
and in different forms of conjunctivitis has been shown by Fick, 
whose results do not correspond in this respect with those of Gif- 
ford, who found almost exclusively micrococci. Whatever may be 
the final conclusion as to the réle of the pus cocci heretofore de- 
scribed in the etiology of acute or chronic conjunctivitis, there can be 
no doubt of the power of the “‘ gonococcus” to induce a virulent in- 
flammation of the conjunctive when introduced into healthy eyes. 


MICROCOCCUS GONORRH@A. 


Synonym.—Gonococcus (Neisser). 

Discovered by Neisser (1879) in gonorrheeal pus and described by 
him under the name of ‘‘ Gonococcus.” Cultivated by Bumm (1883), 
and infective virulence proved by inocula- 
tion into man. Constantly present in viru- 
lent gonorrhceal discharges, for the most 
part in the interior of the pus cells or at- ce See 
tached to the surface of epithelial cells. @ 

Morphology.—Micrococci, usually join- @ by 
ed in pairs or in groups of four, in which by 
the elements are flattened — “ biscuit- 
shaped.” The flattened surfaces face each a & ® @ @& 
other and are separated, in stained pre- ee” @ ww BWM OW 
parations, by an unstained interspace. Gite (i na, ae - 
The diameter of an associated pair of cells pure euture, % about 1,000: b. gon. 
varies from 0.8 to 1.6 HM in the long dia- cocci in pus cells and epithelial cell 

from case of gonorrhceal ophthzl- 
meter—average about 1.25 m—and from jnia; ¢, formand mode of division 
0.6 to 0.8 «4 in the line of the interspace of gonococci—schematic. (Bumm.) 
between the biscuit-shaped elements, which 
sometimes present a slight concavity of the flattened surfaces. Mul- 
tiplication occurs alternately in two planes, and as a result of this 
groups of four are frequently observed. But diplococci are more 
numerous and are considered as the characteristic mode of grouping. 
Single, spherical, undivided cells are rarely seen. 

It must be remembered that the morphology of this micrococcus 
as above described does not suffice to distinguish it, for Bumm has 
shown that ‘the biscuit form is not at all specific for the gonococcus, 
but is shared with it by a number of microérganisms, which consist 
of two hemispherical elements with the flattened surfaces facing each 


392 PYOGENIC BACTERIA. 


other and separated by a cleft, and some of these correspond in their 
morphology, in every detail, with the gonococcus.” 

Stains quickly with the basic aniline colors, especially with 
methyl violet, gentian violet, and fuchsin; not so quickly with 
methylene blue, which is, however, one of the most satisfactory 
staining agents for demonstrating its presence in pus. Beautiful 
double-stained preparations may be made from gonorrheal pus, 
spread upon a cover glass and “‘ fixed,” secundum artem, by the use 
of methylene blue and eosin. Does not stain by Gram’s method— 
2.€., the cocci are decolorized, after having been stained with an ani- 
line color, by being immersed in the iodine solution employed in 
Gram’s method of staining. But this character cannot be depended 
upon alone for establishing the diagnosis, for Bumm has shown that 


Fia. 86.—“* Gonococcus ” in gonorrheeal pus. From a photomicrograph by Frinkel and Pfeiffer 
x 1,000. 


other diplococci are occasionally found in gonorrhceal pus which do 
not stain by this method. Itserves to distinguish them, however, from 
the common pus cocci heretofore described—Staphylococcus aureus, 
Staphylococcus albus, Staphylococcus citreus—which retain their 
color when treated in the same way. A more trustworthy diagnostic 
character is that these biscuit-shaped diplococci are found within the 
pus cells, sometimes one or two pairs only, but more frequently in 
considerable numbers, and occasionally in such numbers as to com- 
pletely fill the cell. No similar picture is presented by pus from any 
other source, with the exception of that from a form of “ puerperal 
cystitis” which Bumm has described. But in this the diplococci 
contained in the pus cells were to be distinguished by the fact that 
they retained their color when treated by Gram’s method. Owing 


PYOGENIC BACTERIA. 393 


to the difficulty of cultivating this micrococcus, and the importance, 
under certain circumstances, of not making a mistake in its diag- 
nosis, these characters are of exceptional value. 

Biological Characters.—Bumm (1885) first succeeded in culti- 
vating the “gonococcus” upon human blood serum, obtained from 
the placenta of a recently delivered woman. He found that the cul- 
tures thrive best in a moist atmosphere at 30° to 34° C. The growth 
under the most favorable conditions is slow, and frequently no devel- 
opment occurs when pus containing numerous gonococci is placed 
upon blood sérum in an incubating oven; or after a slight multi- 
plication development ceases and the cocci undergo degenerative 
changes and quickly disappear. 

Cultures upon the surface of blood serum form a very thin, often 
scarcely visible layer, with a smooth, moist, shining surface, and 
by reflected light a grayish-yellow color. The growth at the end of 
twenty-four hours may extend for a distance of a millimetre along 
the line of inoculation, but at the end of two or three days no fur- 
ther development occurs and the cocci soon lose their vitality. This 
micrococcus, then, is aérobic. Whether it may also be a facultative 
anaérobic has not been definitely determined, but it doesnot grow 
along the line of puncture when stick cultures are made in blood se- 
rum. Its rapid and abundant multiplication in gonorrhceal infection 
of mucous membranes, and the difficulties attending its cultivation 
in artificial media, show that the gonococcus is a strict parasite, 

Lestikow and Léffler, prior to the publication of Bumm’s impor- 
tant monograph, had reported successful results in cultivating the 
gonococcus upon a mixture of blood serum and gelatin. Bockhart 
has since recommended a mixture of nutrient agar (two parts), lique- 
fied at a temperature of 50° C., with blood serum (two to three parts) 
at 20° C. By quickly mixing with this a little pus containing the 
gonococcus he was able to obtain colonies upon plate cultures, made 
by pouring the liquid medium upon sterile glass plates in the usual 
manner. 

Ghon and Schlagenhaufer in 1893 reported that they obtained 
good results by adding phosphate of soda to blood-serum agar, made 
according to the method of Wertheim—one part of human blood 
serum from the placenta to two or three parts of nutrient agar. Also 
that they were successful in cultivating the gonococcus in an acid 
medium made by adding one part of urine to two of nutrient agar 
(two per cent). Turro (1894) has since published the results of his 
experiments relating to the cultivation of this micrococcus in acid 
media. According to him it grows in normal urine, either with or 
without the addition of peptone (one per cent); also in acid gelatin, 
prepared in the usual way but without neutralization (?). 


394 PYOGENIC BACTERIA. 


Turro also claims to have produced specific urethritis in dogs by 
inoculation with his cultures. Heiman (1895) as a result of an ex- 
tended experimental research, arrives at the conclusion that “the 
diplococcus described by Turro in connection with his acid media is 
not the gonococcus.” His inoculation experiments in dogs, made 
with pure cultures of the gonococcus, gave an entirely negative result. 
For the cultivation of the gonococcus, Heiman recommends a medium 
made from “chest serum” obtained from a patient suffering with 
hydrothorax or acute pleurisy. This was found to be superior to 
placenta serum, sheep-blood serum, or peritoneum serum, because of 
the great amount of serum albumin which it contains. Two per 
cent of agar, one per cent of peptone, and one-half per cent of sodium 
chloride were added to the chest serum, and the medium was sterilized 
by “fractional sterilization.” 


Fie. 87.—Gonorrhoeal conjunctivitis, second day of sickness; section through the mucous mem- 
brane of upper eyelid; invasion of the epithelial layer by gonococci. (Bumm.) 


Schrétter and Winkler (1890) report their success in cultivating 
the gonococcus upon albumin from the egg of the pewit—“‘ Kibitz.” 
In the culture oven at 38° C. a thin, transparent, whitish layer was 
already visible at the end of six hours and rapidly extended ; the 
growth was less abundant at the end of three days, and had entirely 
ceased by the fifth day. Attempts to cultivate the same microér- 
ganism in albumin from hens’ eggs gave a negative result. 

Aufuso (1891) has cultivated the gonococcus in fluid obtained 
from the knee joint in a case of chronic synovitis, but failed to culti- 
vate it in the fluid of ascites. A culture of the twelfth generation 
made upon the culture medium mentioned, solidified by heat, was 
introduced into the urethra of a healthy man and gave rise to a 
characteristic attack of gonorrhea. 

Development does not occur below 25° or above 38° GC. The 
writer has shown that a temperature of 60° C. maintained for ten 
minutes destroys the infective virulence of gonorrhceal pus. 

Pathogenesis.—That the gonococcus is the cause of the specific 
inflammation and purulent discharge characteristic of gonorrhea is 
now generally admitted upon the experimental evidence obtained by 


PYOGENIC BACTERIA. 395 


Bumm. Having succeeded in obtaining it in pure cultures from 
gonorrhceal pus, he made successful inoculations in the healthy ure- 
thra in two cases—once with a third culture and once with one 
which had been transferred through twenty successive generations. 
In both cases a typical gonorrhcea developed as a result of the inocu- 
lation. 

The mucous membranes in man which are subject to gonorrhceal 
infection are those of the urethra, the conjunctiva, the cervix uteri, 
and the vagina in children—the vagina in adults is not involved. 
Inoculations of gonorrhceal pus into the vagina or conjunctival sac of 
the lower animals—dogs, rabbits, horses, apes—are without result. 

The very numerous researches which have been made by compe- 
tent bacteriologists show that the gonococcus is constantly present in 
gonorrhceal discharges, and in view of the facts above stated its etio- 
logical import appears to be fully established. Bumm has studied 
the development of blennorrhwa neonatorum, and has shown that 
soon after infection the presence of gonococci may be demonstrated 
in the superficial epithelial cells of the mucous membrane and be- 
tween them ; that they soon penetrate to the deeper layers, and that 
by the end of forty-eight hours the entire epithelial layer is invaded 
by the diplococci, which penetrate by way of the connecting mate- 
rial—‘* Kittsubstance ”—between the cells. They also multiply in 
the superficial layers of connective tissue and give rise to an inflam- 
matory reaction, which is shown by an abundant escape of leuco- 
cytes from the dilated capillary network. The penetration of the 
gonococci to the deeper layers of the mucous membrane of the ure- 
thra, and even to the corpus cavernosum, was observed by Bockhart 
in a case studied by him in which death occurred during an acute 
attack of gonorrhea. But Bumm concludes from his researches 
that this is not usual, and that the invasion is commonly limited to 
the superficial layers of the mucous membrane. 

Staphylococcus pyogenes aureus is not infrequently associated 
with the gonococcus in late gonorrhceal discharges, and the abscesses 
which occasionally develop as a complication of gonorrhcea, in the 
prostate, the inguinal glands, or around the urethra, are probably 
due to its presence, which has been demonstrated in the pus from 
such abscesses in a number of cases. The same is true of the joint 
affections and endocarditis which sometimes occur in the course of 
an attack of gonorrhea, Although some authors have claimed to 
find the gonococcus in these so-called metastatic gonorrhceal inflam- 
mations, the evidence is not satisfactory, and it seems probable that 
the Staphylococcus aureus is the usual microédrganism concerned in 
these affections. 


VI. 
BACTERIA IN CROUPOUS PNEUMONIA. 


BACILLUS OF FRIEDLANDER. 


Synonyms.—Pneumococeus (Friedlander); Bacillus pneumonia 
(Fligge). 

Obtained by Friedlander and Frobenius in pure cultures (1883) 
from the exudate into the pulmonary alveoli in cases of croup- 
ous pneumonia. Subsequent researches show that it is only present 
in asmall proportion of the cases—nine times in one hundred and 
twenty-nine cases examined by Weichselbaum, three times in seventy 
cases examined by Wolf. 

Morphology.—Short rods with rounded ends, often so short as 

; to resemble micrococci, especially in very 
OG recent cultures ; commonly united in pairs 


op 8. OO \@ or chains of four, and under certain cir- 
0 a STO) cumstances surrounded by a transparent 
2 Q capsule. The gelatinous envelope —so- 


Fic. 88.—Bacillusof Friedlinder; called capsule—is not seen in preparations 
a, from a culture; , from bloodof ~made from cultures in artificial media, but 
mouse, showingcapsule. (Fligge.) . . s a 

is very prominent in properly stained prepa- 
rations from the blood of an inoculated animal. It often has a diame- 
ter equal to or greater than that of the enclosed cell, and appears to 
consist of a substance resembling mucin, whichis soluble in water or 
dilute alcohol. Where several cells are united in a chain they may 
all be enclosed in a common envelope, or each may have its own cap- 
sule. This capsule is not peculiar to Friedlander’s bacillus, as he 
at first supposed, but is found in other bacilli and also in the writer’s 
Micrococcus Pasteuri. 

Friedlander’s bacillus stains readily with the aniline colors, but 
is decolorized by the iodine solution used in Gram’s method. In 
preparations from the blood of an inoculated animal, stained by an 
aniline color, the capsule appears as an unstained envelope surround- 
ing the stained cell, but by special treatment the capsule may also be 
stained. Friedlander’s method is as follows: The section or cover- 


BACTERIA IN CROUPOUS PNEUMONIA. 397 


glass preparation is placed for twenty-four hours in a solution of 
gentian violet and acetic acid, containing fifty parts of a concentrated 
alcoholic solution of gentian violet, one hundred parts of distilled 
water, and ten parts of acetic acid. The stained preparation is 
washed for a minute or two in a one-per-cent solution of acetic acid, 
dehydrated with alcohol, cleared up with oil of cloves or cedar, and 
mounted in balsam. The bacillus is quickly stained in dried cover- 
glass preparations by immersion in aniline-water-gentian-violet solu- 
tion (two or three minutes). The stained preparation should be de- 
colorized by placing it in absolute alcohol for half a minute, and then 
washed in distilled water. 

Biological Characters.—This bacillus does not, so far as is 
known, form reproductive spores; it is non-motile and does not 
liquefy gelatin. It is aérobic and a facultative anaérobic. In 
gelatin stab cultures it presents the ‘‘nail-shaped” growth first 
described by Friedlander, which is not, however, peculiar to this 
bacillus. The head of the nail is formed by the 
development around the point of entrance of the 
inoculating needle of a rounded, white mass hav- 
ing a smooth, shining surface, and its stem by the 
growth along the line of puncture. This consists 
of closely crowded, opaque, white, spherical colo- 
nies. Gas bubbles sometimes develop in gelatin 
cultures, and in old cultures the gelatin about the 
line of growth acquires a yellowish-brown color. 
The growth in nutrient agar resembles that in 
gelatin. Upon the surface of blood serum abun- 
dant grayish-white, viscid masses are developed. 
Upon potato the growth is abundant, quickly cov- 
ering the entire surface with a thick, yellowish- 
white, glistening layer which often contains gas 
bubbles when the temperature is favorable. Col- 
onies in gelatin plates appear at the end of twenty- 
four hours as small, white spheres, which increase 
rapidly in size, and upon the surface form round- 
ed, smooth, glistening, white masses of consider- 
able size. Under the microscope the colonies pre- — 
sent a somewhat irregular outline and a slightly a ee ee 
granular appearance. Growth occurs at compara- gelatin; end of four days 
tively low temperatures—16° to 20° C.—butis more # 16°18 C. Baumgar- 
rapid in the incubating oven, The thermal death- — 
point, as determined by the writer, is about 56° C. In the ordinary 
culture media it retains its vitality for a long time, and may grow 
when transplanted to fresh culture material after having been pre- 


398 BACTERIA IN CROUPOUS PNEUMONIA. 


served for a year or more. Ata temperature of 40° C. development 
ceases. 

Pathogenesis.—In Friedlinder’s experiments the bacillus from 
pure cultures, suspended in water, was injected through the thoracic 
wall into the right lung of dogs, rabbits, guinea-pigs, and mice. 
Rabbits proved to be immune; one dog out of five, six guinea-pigs 
out of eleven, and all of the mice (thirty-two) succumbed to the 
inoculation. At the autopsy the pleural cavities were found to con- 
tain a sero-purulent fluid ; the lungs were intensely congested, con- 
tained but little air, and in places showed limited areas of red infil- 
tration; the spleen was considerably enlarged; the bacillus was 
found in great numbers in the lungs, the fluid in the pleural cavi- 
ties, and in the blood obtained from the general circulation or from 
the various organs of the body. Similar appearances presented them- 
selves in the case of the guinea-pigs which succumbed to the inocu- 
lation. 

These results show that the bacillus under consideration is path- 
ogenic for mice and for guinea-pigs, but they are by no means 
sufficient to prove that it is capable of producing a genuine croupous 
pneumonia in man, and it is still uncertain whether its occasional 
presence in the exudate into the pulmonary alveoli in cases of this 
disease has any etiological importance. 


MICROCOCCUS PNEUMONIZ CROUPOSA, 


Synonyms.—Micrococcus Pasteuri (Sternberg) ; Micrococcus of 
sputum septicemia (Frankel) ; Diplococcus pneumoniz (Weichsel- 
baum); Bacillus septicus sputigenus (Fligge); Bacillus salivarius 
septicus (Biondi) ; Lancet-shaped micrococcus (Talamon) ; Strepto- 
coccus lanceolatus Pasteuri (Gameléia). 

Discovered by the present writer in the blood of rabbits inocu- 
lated subcutaneously with his own saliva in September, 1880; by 
Pasteur in the blood of rabbits inoculated with the saliva of a child 
which died of hydrophobia in one of the hospitals of Paris in De- 
cember, 1880 ; identified with the micrococcus in the rusty sputum of 
pneumonia, by comparative inoculation and culture experiments, by 
the writer in 1885 (paper published in the American Journal of the 
Medical Sciences, July 1st, 1885). Proved to be the cause of croup- 
ous pneumonia in man by the researches of Talamon, Salvioli, Stern- 
berg, Frankel, Weichselbaum, Netter, Gameléia, and others, 

The Presence of Micrococcus Pasteurt in the Salivary Secre- 
tions of Healthy Individuals.—In September, 1880, while engaged 
in investigations relating to the etiology of the malarial fevers, I in- 
jected a little of my own saliva beneath the skin of two rabbits as a 
control experiment. To my surprise the animals died, and I found 


BACTERIA IN CROUPOUS PNEUMONIA. 3uy 


in their blood a multitude of oval microérganisms, united for the 
most part in pairs, or in chains of three or four elements. These 
experiments are recorded in my paper entitled ‘‘ Experimental Inves- 
tigations Relating to the Etiology of the Malarial Fevers,” published 
in the Report of the National Board of Health for 1881, pp. 74, 75. 

Following up my experiments made in New Orleans (in Septem- 
ber, 1880), in Philadelphia (January, 1881), and in Baltimore (March, 
1881), I obtained the following results : 

‘‘ The saliva of four students, residents of Baltimore (in March), 
gave negative results ; eleven rabbits injected with the saliva of six 
individuals in Philadelphia (in January) gave eight deaths and three 
negative results; but in the fatal cases a less degree of virulence was 
shown in six by a more prolonged period between the date of injec- 
tion and the date of death. This was three days in one, four days 
in four, and seven days in one.” 

In a paper published in the Journal of the Royal Microscopical 
Society (June, 1886) I say : 

“* My own earlier experiments showed that there is a difference in 
the pathogenic potency of the saliva of different individuals, and I 
have since learned that the saliva of the same individual may differ 
in this respect at different times. Thus during the past three years 
injections of my own saliva have not infrequently failed to cause a 
fatal result, and in fatal cases death is apt to occur after a some- 
what longer interval, seventy-two hours or more; whereas in my 
earlier experiments the animals infallibly died within forty-eight 
hours.” 

The presence of my Micrococcus Pasteuri was demonstrated in 
the blood of the rabbits which succumbed to the inoculations. 

Claxton, in a series of experiments made in Philadelphia in 1882, 
injected the saliva of seven individuals into eighteen rabbits. Five 
of these died within five days, and nine at a later period. 

Frankel, whose first publication was made in 1885, discovered 
the presence of this micrococcus in his own salivary secretions in 1883, 
and has since made extended and important researches with refe- 
rence to it. The saliva of five healthy individuals and the sputa 
of patients suffering from other diseases than pneumonia, injected 
into eighteen rabbits, induced fatal ‘‘sputum septicemia” in three 
only. When he commenced his experiments his saliva was uni- 
formly fatal to rabbits, but a year later it was without effect. 

Wolf injected the saliva of twelve healthy individuals, and of 
three patients with catarrhal bronchitis, into rabbits, and induced 
“ sputum septicaemia ” in three. 

Netter examined the saliva of one hundred and sixty-five healthy 
persons, by inoculation experiments in rabbits, and demonstrated 
the presence of this micrococcus in fifteen per cent of the number. 


400 BACTERIA IN CROUPOUS PNEUMONIA. 


Vignal, in his recent elaborate paper upon the microorganisms 
of the mouth, says: 

‘* Last year I encountered this microbe continually in my mouth 
during a period of two months, then it disappeared, and I did not 
find it again until April of this year, and then only for fifteen days, 
when it again disappeared without appreciable cause.” 

T he Presence of Micrococcus Pneumonicee Croupose in Pneu- 
monic Sputum.—Talamon, in 1883, demonstrated the presence of this 
micrococcus in pneumonic sputum, described its morphological char- 
acters, and produced typical croupous pneumonia in rabbits by in- 
jecting material containing it into the lungs through the thoracic 
walls. 

Salvioli, in 1884, demonstrated its presence in pneumonic sputum 
by injections into rabbits. 

In 1885 the writer made a similar demonstration, and by compara- 
tive experiments showed that the micrococcus present in the bleod 
of rabbits inoculated with the rusty sputum of pneumonia was iden- 
tical with that which he had discovered in 1880 in rabbits inoculated 
with his own saliva. 

The same year (1885) A. Frankel made a similar demonstration, 
and published a paper containing valuable additions to our knowl- 
edge relating to the biological characters of this microérganism (first 
publication appeared July 13th, 1885). 

In 1886 Weichselbaum published the results of his extended re- 
searches relating to the presence of this micrococcus in the fibrinous 
exudate of croupous pneumonia. He obtained it in ninety-four cases 
(fifty-four times in cultures) out of one hundred and twenty-nine cases 
examined. 

Wolf (1887) found it in sixty-six cases out of seventy examined. 

Netter (1887) in seventy-five per cent of his cases, and in the sputum 
of convalescents from pneumonia in sixty per cent of the cases ex- 
amined, by inoculations into rabbits. 

Gameléia (1887) in twelve fatal cases of pneumonia in which he 
collected material from the lungs at the post-mortem examination. 

Goldenberg, whose researches were made in Gameléia’s labora- 
tory, foundit in pneumonic sputum in forty consecutive cases, by 
inoculations into rabbits and mice. 

The Presence of Micrococcus Pneumonice Croupose in Menin- 
gitts.—Numerous bacteriologists have reported finding diplococci in 
the pus of meningitis, and frequently the microérganisms have been 
fully identified as ‘‘ diplococcus pneumoniz.” Thus Netter (1889), in 
a résumé of the results of researches made by him in twenty-five 
cases of purulent meningitis, reports as follows : 


BACTERIA IN CROUPOUS PNEUMONIA. 401 


Thirteen cases were examined microscopically, by cultures, and 
by inoculations into susceptible animals ; six cases by microscopical 
examination and experiments on animals; and the remainder only by 
microscopical examination. Four of the cases were complicated 
with purulent otitis, six with pneumonia, three with ulcerative endo- 
carditis. The ‘‘pneumococcus” was found in sixteen of the twenty- 
five cases; in four Streptococcus pyogenes was present; in two 
Diplococcus intracellularis meningitidis of Weichselbaum ; in one 
Friedlander’s bacillus ; in one Newmann and Schiaffer’s motile ba- 
cillus ; in one a small curved bacillus. 

In forty-five cases collected from the literature of the subject by 


Fie. 90. Fia, 91. Fie. 92. 


Fia. 90.—Micrococcus pneumonie croupose from blood of rabbit inoculated with normal human 
saliva (Dr. 8.). x 1,000. 

Fie. 91.—Micrococcus pneumoniz croupose from blood of rabbit inoculated subcutaneously 
with fresh pneumonic sputum from a patient in the seventh day of the disease. > 1,000. 

Fig. 92.—Surface culture of Micrococcus pneumoniz croupose, on nutrient agar, showing the 
development of long chains. x 1,000.1 


Netter this micrococcus was present in twenty-seven, Streptococcus 
pyogenes in six, and the Diplococcus intracellularis meningitidis of 
Weichselbaum in ten. 

Monti (1889), in four cases of cerebro-spinal meningitis, demon- 
strated the presence of the same micrococcus. In three of his cases 
pneumonia was also present. In two Staphylococcus pyogenes aureus 
was associated with the ‘‘ diplococcus pneumoniz.” 

Micrococcus Pneumonice Croupose in Ulcerative Endocar- 
ditis.—Weichselbaum, in a series of twenty-nine cases examined 
(1888), found ‘‘ diplococcus pneumoniz ” in seven. 

Micrococcus Pneumonice Crouposce in Acute Abscesses.—In a 
case of parotitis occurring as a complication of croupous pneumonia 
this micrococcus was obtained from the pus in pure cultures by Testi 
(1889); and in another case in which, as a complication of pneumonia, 
there developed a purulent pleuritis, abscess of the parotid on both 
sides, and multiple subcutaneous abscesses, the pus from all of the 
sources named contained the “‘diplococcus” in great numbers, as 
shown not only by microscopical examination but by inoculation into 
rabbits. 

1The above figures are from Dr. Sternberg’s paper published in the American 


Journal of the Medical Sciences for July and October, 1885. 
26 


402 BACTERIA IN CROUPOUS PNEUMONIA. 


In a case of tonsillitis resulting in the formation of an abscess 
Gabbi (1889) obtained the same coccus in pure cultures. 

In otitis media this micrococcus has been found in a consider- 
able number of cases in the pus obtained by paracentesis of the 
tympanic membrane, and quite frequently in pure cultures—by Zau- 
fal (1889) in six cases; Levy and Schrader (1889) in three out of ten 
cases in which paracentesis was performed; by Netter (1889) in five 
out of eighteen cases occurring in children. 

Monti (1889) and Belfanti (1889) report cases of arthritis of the 
wrist joint, occurring as a complication of pneumonia, in which this 
micrococcus was obtained in pure cultures. Ortmann and Samter 
(1889), in a case of purulent inflammation of the shoulder joint fol- 
lowing pneumonia and pleurisy, obtained the “diplococcus pneu- 
moniz” in pure cultures. 

Morphology.—Spherical or oval cocci, usually united in pairs, or 
in chains consisting of three or four elements. Longer chains, con- 
taining ten or more elements, are frequently formed, especially in 
cultures upon the surface of nutrient agar, and in liquid media; it 
may therefore be regarded asa streptococcus. As observed in the 
blood of inoculated animals it is usually in pairs consisting of oval 
or lance-oval elements, which are surrounded by a transparent cap- 
sule. Owing to the elongated form of the cocci when in active 
growth, it has been regarded by some authors as a bacillus; but in 
cultures in liquid media, when development by binary division has 
ceased, the cells are spherical, or nearly so, and in cultures on the 
surface of nutrient agar the individual cells more nearly approach a 
spherical form than in the blood of an inoculated animal. The “lan- 
ceolate” form was first referred to by Tala- 
mon, who described it as having the form of 
a grain of wheat, or even still more elongated 
like a grain of barley, as seen in the fibrin- 
ous exudate of croupous pneumonia. The 
transparent material surrounding the cells— 
so-called capsule—is best seen in stained 

— preparations from the fibrinous exudate of 

Bi 9 a meray era croupous pneumonia or from the blood of an 
sule, attached to pus cells from inoculated animal. It appears as an un- 
ae avis stained marginal band surrounding the ellip- 
tical cells, and varies greatly as to its extent 

in different preparations. This capsule probably consists of a sub- 
stance resembling mucin, and, being soluble in water, its extent de- 
pends partly upon the methods employed in preparing specimens for 
microscopical examination. It is occasionally seen in stained prep- 
arations from the surface of cultures on blood serum; and in drop 


BACTERIA IN CROUPOUS PNEUMONIA. 403 


cultures examined under the microscope, by using a small diaphragm 
it may be seen to surround the cocci as a scarcely visible halo. 

This micrococcus stains readily with the aniline colors; and also 
by Gram’s method, which constitutes an important character for dis- 
tinguishing it from Friedlander’s bacillus. 

Biological Characters.—Grows in the presence of oxygen— 
aérobic—but is also a facultative anaérobic. Like other micro- 
cocci, it has no spontaneous movements. It grows in a variety of 
culture media when they have a slightly alkaline reaction, but will 
not develop in a medium which contains the slightest trace of free 
acid. Nor will it grow at the ordinary room temperature. Scanty 
development may occur at a temperature of 22° to 24° C., buta 
temperature of 35° to 87° C. is most favorable for its growth, which 
is very rapid in a suitable liquidmedium. In aninfusion made from 
the flesh of a chicken or a rabbit it multiplies, in the incubating 
oven, with remarkable rapidity ; at the end of six to twelve hours 
after inoculation the previously transparent fluid will be found to 
present a slight cloudiness and to be filled throughout with the cocci 
in pairs and short chains. It does not produce a milky opacity in 
liquid media, like the pus cocci, for example, but the fluid becomes 
slightly clouded ; multiplication ceases at the end of about forty- 
eight hours or less, and the liquid medium again becomes transpa- 
rent as a result of the subsidence of the cocci to the bottom of the 
receptacle. 

It may be cultivated in flesh-peptone-gelatin, containing fifteen 
per cent of gelatin, at a temperature of 24° C., or in liquefied gela- 
tin (ten per cent) in the incubating oven. 
In gelatin (fifteen ‘per cent) stab cultures 
small white colonies develop all along the 
line of puncture, and in gelatin plates 
small, spherical, slightly granular, whitish 
colonies are formed: the gelatin ts not 
liquefied. In agar plates extremely mi- 
nute colonies are developed in the course 
of forty-eight hours, which resemble little, 
transparent drops of fluid, and under the RSE: 
microscope some of these are observed to yng, 94 —gingle colony of Micro- 
have a compact, finely granular central coccus pneumonie croupose upon 
portion surrounded by a paler, transparent, ee ae 
finely granular marginal zone. Upon the 
surface of nutrient agar or coagulated blood serum development 
occurs in the form of minute, transparent, jelly-like drops, which 
form a thin layer along the line of inoculation in ‘‘ streak cultures” ; 
and in agar stick cultures the growth along the line of puncture is 


404 BACTERIA IN CROUPOUS PNEUMONIA. 


rather scanty, almost homogeneous, and semi-transparent. Upon 
potato no development occurs, even in the incubating oven. Milk is 
a favorable culture medium, and the casein is coagulated as a result 
of its presence. 

It ceases to grow on solid media at about 40° C., and in favorable 
liquid media at 42°C. Its thermal death-point, as determined by 
the writer, is 52° C., the time of exposure being ten minutes. It 
loses its vitality in cultures in a comparatively short time—four or 
five days on agar—and is very sensitive to the action of germicidal 
agents. Its pathogenic power also undergoes attenuation very 
quickly when it is cultivated in artificial media, but may be restored 
by passing it through the bodies of susceptible animals. Attenua- 
tion of virulence may also be effected by exposing bouillon cultures 
to a temperature of 42° C. for twenty-four hours, or by five days’ 
exposure to a temperature of 41° C. 

Emmerich reported in 1891 to the Congress of Hygiene and 
Demography in London the results of experiments made by him 
relating to immunity in rabbits and mice. Rabbits were rendered 
immune by the intravenous injection of a very much diluted but 
virulent culture of the micrococcus. The flesh of these immune 
rabbits was rubbed up into a fine paste, and the juices obtained by 
compressing it in a clean, sterilized cloth. This bloody juice was kept 
for twelve hours at a temperature of 10° C., and then sterilized by 
passing it through a Pasteur filter. Some of this juice was injected 
into a rabbit, which with twenty-five others was then made to re- 
spire an atmosphere charged with a spray of a bouillon culture of 
the micrococcus. As a result of this all of the rabbits died except the 
one which had previously been injected with the immunizing juice. 
In a similar experiment upon mice six of these animals, which had 
previously been injected with the immunizing juice, survived the in- 
jection of a full dose of a virulent culture, while a control mouse, 
not previously injected with the juice, promptly died after receiving 
the same quantity of the virulent culture. 

The writer in 1881, in experiments made to determine the value 
of various disinfectants, as tested upon this micrococcus, obtained 
experimental evidence that its virulence is attenuated by the action 
of certain antiseptic agents. Commenting upon the results of these 
experiments in my chapter on ‘‘ Attenuation of Virus,” in ‘‘ Bacte- 
ria” (1884), I say: 


‘‘Sodium hyposulphite and alcohol were the chemical reagents which 
produced the result noted in these experiments ; but it seems probable that 
a variety of antiseptic substances will be found to be equally effective when 
used in the proper proportion. Subsequent experiments have shown that 
neither of these agents is capable of destroying the vitality of this septic 
micrococcus in the proportion used (one per cent of sodium hyposulphite or 


BACTERIA IN CROUPOUS PNEUMONIA. 405 


one part of ninety-five-per-cent alcohol to three parts of virus), and that 
both have a restraining influence upon the development of this microdrgan- 
ism in culture fluids.” 


The following results were obtained by the writer in his experi- 
ments (1881 and 1883) to determine the germicidal and antiseptic 
value of the agents named, as tested upon this micrococcus. 

Alcohol.—A twenty-four-per-cent solution was effective upon 
bouillon cultures in two hours. 

Boric Acid.—A saturated solution failed to destroy vitality after 
two hours’ exposure, but | : 400 restrained development. 

Carbolic Acid.—A one-per-cent solution destroys vitality in two 
hours, and 1 : 500 restrains development. 

Cupric Sulphate destroys the virulence of the coccus in the 
blood of a rabbit in the proportion of 1 : 400 in half an hour. 

Ferric Sulphate failed to destroy vitality in a saturated solution, 
but restrained development in the proportion of 1 : 200. 

Hydrochloric Acid destroys the virulence of the blood of a rab- 
bit containing this micrococcus in the proportion of 1 : 200. 

Iodine, in aqueous solution with potassium iodide, destroys vital- 
ity in the proportion of 1: 1,000 and prevents development in 1: 4,000. 

Mercuric Chloride.—One part in forty thousand prevents the 
development of this micrococcus, and 1 : 20,000 was found to destroy 
vitality in two hours. 

Nitric Acid.—One part in four hundred destroyed the virulence 
of rabbit’s blood containing this micrococcus. 

Caustic Potash.—A two-per-cent solution destroyed vitality in 
two hours. 

Potassium Permanganate.—A two-per-cent solution,destroyed 
the virulence of rabbit’s blood containing this coccus. 

Salicylic Acid, dissolved by the addition of sodium biborate.— 
A solution of 1 : 400 prevented development. 

Sulphuric Acid.—One part in two hundred destroys vitality, and 
1: 800 prevents development. 

In a paper by Bordoni-Uffreduzzi relating to the resisting power 
of pneumonic virus for desiccation and light, the following results are 
given: Pneumonic sputum attached to cloths, when dried in the air 
and exposed to diffuse daylight, retained its virulence, as shown by 
injection in rabbits, for a period of nineteen days in one series of ex- 
periments and for fifty-five days in another. Exposed to direct sun- 
light the same material retained its virulence after twelve hours’ 
exposure. Cultures have far less resistance, and the protection 
afforded by the dried albuminous material in which the micrococci 
were embedded, in the experiments referred to, probably accounts 
for the virulence being retained so long a time. 


406 BACTERIA IN CROUPOUS PNEUMONIA. 


Kruse and Pansini (1892) have published an elaborate paper giv- 
ing an account of their researches relating to “diplococcus pneumo- 
nix” and allied streptococci. We give below a summary statement 
of their results: 


Many varieties were obtained by the observers named in their cultures 
from various sources—from the lungs of individuals dead from pneumonia, 
from pleuritic exudate, from pneumonic sputa, from bronchitic sputa, from 
the saliva of healthy persons, from the secretion in a case of subacute nasal 
catarrh, from the urine of a patient with nephritis. 

Pure cultures were obtained by the use of agar plates or by inoculations 
into rabbits. In all about thirty varieties were obtained and cultivated 
through many successive generations. As a rule, the different varieties, 
which at first were seen to have the form of diplococci, when cultivated for 
a length of time in artificial media presented the form of streptococci ; and 
the elements which at first were lancet-shaped showed a tendency to become 
spherical. 

‘ The more virulent varieties usually presented the form of diplococci 
with lancet-shaped elements, or of short chains. A variety which formed 
long chains could be pronounced, in advance of the experiments on animals, 
to possess comparatively little virulence. When by inoculations in animals 
the virulence of such a variety was restored, the tendency to form chains 
was less pronounced. 

Although, as a rule, no development occurs at 20° C., certain varieties 
were obtained which, after long cultivation in artificial media, showed a de- 
cided growth at 18° C. 

Decided differences were shown by the cultures from various sources as 
regards their growth in milk. Out of eighty-four cultures from various 
sources eleven did not produce coagulation. As arule, cultures which caused 
coagulation of milk were virulent for rabbits, and when such cultures lost 
their virulence they usually lost at the same time the power of coagulating 
milk. Virulent cultures die out sooner than those which have become at- 
tenuated by continuous cultivation in artificial media; the first, on the sur- 
face of agar, usually fail to grow at the end of a week, while the attenuated 
cultures may survive for three weeks or more, 


Pathogenesis.—This micrococcus is very pathogenic for mice and 
for rabbits, less so for guinea-pigs. The injection of a minute quan- 
tity—0.2 cubic centimetre or less—of a virulent culture beneath the 
skin of a rabbit or a mouse usually results in the death of the animal 
in from twenty-four to forty-eight hours. The following is from the 
writer’s first published paper (1881), and refers to the pathological 
appearances in rabbits : 


‘The course of the disease and the post-mortem appearances indicate that 
it is a form of septicemia. Immediately after the injection there is a rise of 
temperature, which in a few hours may reach 2° to 3° C. (8.6° to 5.4° F.); 
the temperature subsequently falls, and shortly before death is often several 
degrees below the normal. There is loss of appetite and marked debility 
after twenty-four hours, and the animal commouly dies during the second 
night or early in the morning of the second day after the injection. Death 
occurs still more quickly when the blood from a rabbit recently dead is in- 
jected. Not infrequently convulsions immediately precede death. 

‘‘The most marked pathological appearance is a diffuse inflammatory 
cedema or cellulitis, extending in all directions from the point of injection, 


BACTERIA IN CROUPOUS PNEUMONIA. 407 


but especially to the dependent portions of the body. Occasionally there is 
a little pus near the puncture, but usually death occurs before the cellulitis 
reaches the point of producing pus. The subcutaneous connective tissue 
contains a quantity of Vieods serum, which possesses virulent properties and 
which contains a multitude of micrococci. There is usu: lly more or less in- 
flammatory adhesion of the integument to the subjacent tissues. The liver 
is sometimes dark-colored and gorged with blood, but more frequently it is 
of a lighter color than normal and contains much fat. The spleen is either 
normal in appearance or enlarged and dark-colored. Changes in this organ 
are more marked in those cases which are of the longest duration. 
‘The blood commonly contains an immense number of micrococci, usually 
joined in pairs and having a diameter of about 0.5 “. These are found in 
lood drawn from superficial veins, from arteries, and from the cavities of 
the heart immediately after death, and in a few cases their presence has been 


Fig. 95.—Micrococcus pneumonie croupose in blood of rabbit inoculated with pneumonic spu- 
tum. x 1,000. 


verified during life. Observations thus far made, however, indicate that it 
is only during the last hours of life that these parasites multiply in the cir- 
culating fluid, and in a certain proportion of the cases a careful search has 
failed to reveal their presence in the blood in post-mortem examinations 
made immediately-after the death of the animal.” 


Tn animals which are not examined until some hours after death 
a considerable increase in the number of micrococci occurs post mor- 
tem. The fact that this micrococcus varies very much as to its 
“pathogenic power, as a result of conditions relating to the medium in 
which it develops, was insisted upon in my first published paper, and 
has been fully established by later researches (Frankel, Gameléia). 
Susceptible animals inoculated with attenuated cultures acquire an 
immunity against virulent cultures. 

In dogs subcutaneous injections usually give a negative result, 
or at most a small abscess forms at the point of inoculation. In a 


a ij 


408 BACTERIA IN CROUPOUS PNEUMONIA. 


single experiment, however, the writer has seen a fatal result in a 
dog from the injection of one cubic centimetre of bloody serum from 
the subcutaneous connective tissue of a rabbit recently dead. This 
shows the intense virulence of the micrococcus when cultivated in 
the body of this animal. Pneumonia never results from subcutane- 
ous injections into susceptible animals, but injections made through 
the thoracic walls into the substance of the lung may induce a typi- 
cal fibrinous pneumonia. This was first demonstrated by Talamon 
(1883), who injected the fibrinous exudate of croupous pneumonia, 
obtained after death, or drawn during life by means of a Pravaz 
syringe from the hepatized portions of thelung, into the lungs of 
rabbits. According to Sée, eight out of twenty animals experi- 
mented upon exhibited ‘‘a veritable lobar, fibrinous pneumonia, 
with pleurisy and pericarditis of the same nature.” Gameléia has 
also induced pneumonia in a large number of rabbits, and also in the 
dog and the sheep, by injections directly into the pulmonary tissue. 
Sheep were found to survive subcutaneous inoculations, unless very 
large doses (five cubic centimetres) of the most potent virus were in- 
jected. But intrapulmonary inoculations invariably induced a typi- 
cal fibrinous pneumonia which usually proved fatal. In dogs simi- 
lar injections gave rise to a ‘frank, fibrinous pneumonia which 
rarely proved fatal, recovery usually occurring in from ten to fifteen 
days, after the animal had passed through the stages of red and 
gray hepatization characteristic of this affection in man.” 

Monti claims to have produced typical pneumonia in rabbits by 
injecting cultures of this micrococcus into the trachea. 

From the evidence obtained in these experimental inoculations, 
and that recorded relating to the presence of this micrococcus in the 
fibrinous exudate of croupous pneumonia, we are justified in con- 
cluding that it is the usual cause of this disease, and consequently 
have described it under the name Micrococcous pneumonix crou- 
pose. We prefer this to the name commonly employed by German 
authors—“‘ diplococcus pneumonis ”—because this micrococcus, al- 
though commonly seen in pairs, forms numerous short chains of 
three or four elements in cultures in liquid media, and upon the sur- 
face of nutrient agar may grow out into long chains. It would, 
therefore, more properly be called a streptococcus than a diplococcus. 

While the micrococcus of pneumonia is not usually seen in the 
blood in cases of pneumonia it is probably present in small numbers, 
and secondary infection of the kidneys appears to be a common occur: 
rence. Thus Frankel and Reiche (1894) report that in twenty-two 
cases out of twenty-four in which they had an opportunity to exam- 
ine the kidneys, this micrococcus was present. It was found espe- 


1 


BACTERIA IN CROUPOUS PNEUMONIA. 409 


cially in the larger branches of the veins and arteries, but also in the 
intertubular vessels and the glomeruli. The kidneys gave evidence 
of degenerative changes, and it is probable that the “ pneumococcus” 
would have been found in the urine of some of these cases if a bac- 
teriological examination had been made during life. 


VII. 


PATHOGENIC MICROCOCCI NOT DESCRIBED IN 
SECTIONS V. AND VI. 


DIPLOCOCCUS INTRACELLULARIS MENINGITIDIS. 


DISCOVERED by Weichselbaum (1887) in the exudate of cerebro- 
spinal meningitis (six cases), for the most part within the cells. 

Morphology.—Micrococci, usually united in pairs, in groups of 
four, or in little masses ; sometimes solitary and larger (probably 
being upon the point of dividing). Distinguished by their presence 
in the interior of pus cells in the exudate, in this respect resembling 
the gonococcus. 

Stain best with Léffler’s alkaline solution of methylene blue. 
Do not retain their color when treated with iodine solution (Gram’s 
method). 

Biological Characters.—This micrococcus does not grow at the 
room temperature, but upon nutrient agar an abundant development 
occurs in the incubating oven. Upon the surface of agar a tolerably 
luxuriant, viscid growth, which by reflected light is gray and by 
transmitted light grayish-white ; along the line of puncture growth 
occurs only near the surface, indicating that this micrococcus will 
not grow in the absence of oxygen. Upon plates made from agar- 
agar (one per cent) and gelatin (two per cent) very small colonies are 
formed in the interior of the mass, and larger ones, of a grayish 
color, on the surface. The former, under the microscope, are seen to 
be round or slightly irregular, finely granular, and of a yellowish- 
brown color. The superficial colonies have a yellowish-brown nu- 
cleus, surrounded by a more transparent zone. The growth upon 
coagulated blood serum is very scanty, as is that in bouillon; no 
growth occurs upon potato. This micrococcus quickly loses its power 
of reproduction in artificial cultures—within six days—and should 
be transplanted to fresh material at short intervals—two days. 

Pathogenesis.—Mice are especially susceptible, and usually die 
within forty-eight hours after inoculation. Also pathogenic for 
guinea-pigs, rabbits, and dogs. 


NOT DESCRIBED IN SECTIONS V. AND VI. 411 


MICROCOCCUS TETRAGENUS. 


First described by Gaffky (Fligge). Obtained by Koch and 
Gaffky (1881) from a cavity in the lung in a case of pulmonary 
phthisis. Since found occasionally in normal saliva (three times in 
fifty persons examined by Biondi), and in the pus of acute abscesses 
(Steinhaus, Park, Vangel). Rather common in the sputum of phthi- 
sical cases. 

Morphology.—Micrococci, having a diameter of about one p, 
which divide in two directions, forming tetrads, which are enclosed 
in a transparent, jelly-like envelope—especially well developed as 
seen in the blood and tissues of inoculated animals. In cultures the 
cocci are seen in the various stages of division, as large single cells, 


Fia. 96.—Micrococcus tetragenus; section of lung of mouse. x 800. (Fligge.) 


pairs of oval elements, or groups of four resulting from the trans- 
verse division of these latter. 

Stains quickly with aniline colors, and in preparations from the 
blood of an inoculated animal the transparent envelope may also be 
feebly stained. Stains also by Gram’s method. 

Biological Characters.—This micrococcus grows, rather slowly, 
in nutrient gelatin at the ordinary room temperature, without lique- 
faction of the gelatin. Upon gelatin plates small white colonies are 
developed in from twenty-four to forty-eight hours, which under the 
microscope, with a low power, are seen to be spherical or lemon- 
shaped, finely granular, and with a mulberry-like surface. When 
they come to the surface they form white, elevated, and rather thick 
masses having a diameter of one to two millimetres. In gelatin 
stab cultures a broad and thick white mass forms upon the surface, 


412 PATHOGENIC MICROCOCCI 


and along the line of puncture a series of round, milk-white or yel- 
lowish masses form, which usually remain distinct, but may become 
confluent. Upon the surface of agar the growth is similar to that 
upon gelatin, or in streak inoculations may consist of a series of 
spherical, white colonies. Upon cooked potato a thick, viscous layer 
is formed of milk-white color ; the growth upon blood serum is also 
abundant, especially in the incubating oven. This micrococcus is a 
facultative anaérobic. 

Pathogenesis.—Subcutaneous inoculation of a culture of this 
micrococcus in minute quantity is fatal to white mice in from two to 
six days. The animals remain apparently well for the first day or 
two, then remain quiet and somnolent until death occurs. The cocci 
are found in comparatively small numbers in the blood of the heart, 
but are more numerous in the spleen, lungs, liver, and kidneys, from 
which organs beautiful stained preparations may be made show- 
ing the tetrads surrounded by their transparent capsule. Common 
house mice and field mice are, for the most part, immune, as are the 
rabbit and the dog. Guinea-pigs sometimes die from general infec- 
tion, and sometimes a local abscess is the only result of a subcutane- 
ous inoculation. 


MICROCOCCUS BOTRYOGENUS (Rabe). 


Synonyms.—Micrococeus of ‘‘ myko-desmoids ” of the horse; Mi- 
crococcus askoformans (Johne) ; Ascococcus Johnei (Cohn), 

First described by Bollinger (1870) ; morphological characters and 
location in the diseased tissues described by Johne (1884) ; biological 
characters determined by Rabe (1886). 

Is found in certain diffused or circumscribed growths in the con- 
nective tissue of horses—‘‘ myko-desmoids.” 

Morphology.—Micrococci, having a diameter of 1 to 1.5 yu, usu- 
ally united in pairs. 

In the tissues the cocci are united in colonies of fifty to one hun- 
dred y in diameter, and these are associated in mulberry-like masses 
visible to the naked eye. The separate colonies are enclosed in a 
homogeneous, transparent envelope—as in Ascococcus Billrothii. 
This is not the case, however, in cultures in artificial media. 

Stains with the aniline colors. 

Biological Characters.—In gelatin plate cultures spherical, 
sharply defined, silver-gray colonies are developed ; later these have 
a yellowish color and a metallic lustre, and the plate presents the ap- 
pearance of being powdered with grains of pollen. It gives off a 
peculiar fruit-like odor, reminding one of the odor of strawberries. 
In gelatin stab cultures growth occurs along the line of puncture as 
a pale grayish-white line, which later becomes milk-white; an air 


NOT DESCRIBED IN SECTIONS V. AND VI. 413 


bubble forms near the surface of the gelatin ; very slight liquefac- 
tion occurs in the immediate vicinity of the line of growth, and after 
atime the grayish-white thread sinks into an irregular mass, lying 
at the bottom of the puncture. Upon nutrient agar scarcely any de- 
velopment occurs. Upon potato the growth is abundant, in the form 
of a pale-yellow, circular layer, and the culture gives off the peculiar 
odor above described. 

Pathogenesis.—When inoculated into guinea-pigs general infec- 
tion and death result. In sheep and goats it produces a local in- 
flammatory cedema and sometimes necrosis of the tissues. In horses 
inoculated subcutaneously an inflammatory cedema first occurs, fol- 
lowed at the end of from four to six weeks by the development of new 
growths in the connective tissue, resembling the tumors found in 
cases of the disease in the animal from which the micrococcus in 
question was first cultivated. These tumors contain characteristic 
mulberry-like conglomerations of colonies made up of the coccus. 


MICROCOCCUS OF MANFREDI. 


Synonym.—Micrococcus of progressive granuloma formation. 

Obtained by Manfredi (1886) from the sputum of two cases of 
croupous pneumonia following measles. 

Morphology.—Oval micrococci, having a diameter of 0.6 to 1.0 
and from 1.0 to 1.5 in length ; usually associated in pairs, and oc- 
casionally in short chains containing three or four elements. 

Stains with the aniline colors and by Gram’s method. 

Biological Characters.—Aérobic; does not liquefy gelatin. 
Upon gelatin plates forms small, spherical colonies, at first grayish- 
white, which spread out upon the surface as thin, transparent plates, 
which by transmitted light have a bluish, by reflected light a pearl- 
gray color. Later these become. thicker and have a pearly lustre. 
Under the microscope (forty to fifty diameters) the colonies are seen 
to be slightly granular and the margins have an irregular outline. 
In gelatin stab cultures a scanty growth occurs along the line of 
puncture, and a rather thin and limited growth about the point of 
inoculation. Upon blood serum a thin, greenish-yellow layer, which 
has irregular margins and a slightly granular, shining surface, is 
developed. The growth upon potato, at 37° C., is scanty, and con- 
sists of a very thin, moist layer, which has a yellowish color and is 
slightly granular. Growth occurs in favorable media—bouillon, 
gelatin—at temperatures of 18° to 48° C., but ceases at a temperature 
of 48° to 50° C. 

Pathogenesis.—Pathogenic for dogs, rabbits, guinea-pigs, mice, 
and birds. In mammals the principal pathological appearance re- 
sulting from infection consists in the formation of ‘‘ granulation tu- 


414 PATHOGENIC MICROCOCCI 


mors ” in the parenchymatous organs. These vary in size from that 
of a millet seed to that of a pea, and undergo caseation. They con- 
tain the micrococcus and are infectious. Mammals die in from nine 
to fifteen days; birds in from one to three or four, and without the 
formation of the characteristic granuloma, but with general infec- 
tion of the blood. Cultures which have been kept for several months 
retain their pathogenic power. 


MICROCOCCUS OF BOVINE MASTITIS (Kitt). 


Obtained by Kitt (1885) from the udder of cows suffering from mastitis 
and giving milk mixed with pus. 

Morphology.—Micrococci, having a diameter of 0.2 to 0.5 yw, solitary, 
united in pairs, in irregular groups, and occasionally in chains. 

Stains with the aniline colors. 

Biological Characters.—Does not liquefy gelatin. Upon gelatin plates 
forms spherical, translucent, glistening colonies, the size of a hemp seed to 
that of a pin’s head; in gelatin stab cultures a nail-shaped growth occurs, 
the mass at the point of puncture being opaque and of a white color. Upon 
potato, colonies are quickly developed which have a grayish-white or dirty 
yellow color, and after a few days have a shining, wax-like appearance. 
Grows rapidly in milk, causing an acid reaction; in six hours in the incu- 
ce oven the milk is pervaded by the micrococcus, or in twelve hours at 
20° C. 

Pathogenesis.—Injection of pure cultures, suspended in distilled water, 
into the mammary ands of cows, produces typical, acute, purulent mas- 
titis (Kitt). The micrococcus produced the same result after having been 
cultivated in artificial media for a year. Subcutaneous inoculations in cows, 
pigs, guinea-pigs, rabbits, and mice were without result. Injections into 
the mammary gland of goats were also without effect. 


MICROCOCCUS OF BOVINE PNEUMONIA (?). 


Isolated by Poels and Nolen (1886) from the lungs of cattle suffering 
from ‘*Lungenseuche” (infectious pleuro-pneumonia of cattle). 

Morphology.—Micrococci, varying considerably in size—average dia- 
meter 0.9 4; solitary, in pairs, or in chains containing several elements; sur- 
rounded by a transparent capsule, which stains with difficulty. 

Stains with all the aniline colors, and with difficulty by Gram’s method. 

Biological Characters.—Does not liquefy gelatin, and grows like the ba- 
cillus of Friedlander in gelatin stab cultures (nail-shaped growth). In gela- 
tin plates the colonies are spherical, white, and have a very faint yellowish 
tinge. Grows more rapidly on agar in the incubating oven, and upon po- 
tato in the form of a very pale-yellowish layer. Is destroyed by a tempera- 
ture of 66° C. maintained for fifteen minutes. 

Pathogenesis —Pure cultures injected into the lungs of dogs, rabbits, 
and guinea-pigs are said to give rise to pneumonic inflammation, and simi- 
lar results were obtained by injection into the trachea of dogs and by in- 
halation experiments. Injection of a pure culture into the lungs of a cow 
caused extensive pneumonic changes; but these did not entirely correspond 
with the appearances found in the lungs of cattle suffering from infectious 
pneumonia, Cattle inoculated with a pure culture, by means of a sterilized 
lancet, did not fall sick, but are believed by Poels and Nolen to have been 
protected from the disease by such inoculations. 

The specific relation of the micrococcus above described to the disease 
with which it was associated, in the researches of the authors mentioned, has 
not been established by subsequent investigations. 


NOT DESCRIBED IN SECTIONS V. AND VI. 415 


STREPTOCOCCUS SEPTICUS (Fliigge). 


Found by Nicolaier and by Guarneri in unclean soil during researches 
made in Fliigge’s laboratory in Gottingen. 

Morphology.—Cannot be distinguished from Streptococcus pyogenes, but 

does not so constantly form chains, being found in the tissues of inoculated 
animals, for the most part in pairs. 
_ Biological Characters.—Grows more slowly than Streptococcus pyogenes ; 
in gelatin plates very minute colonies first appear at the end of three or four 
days, or along the line of puncture in gelatin stick cultures after five or six 
days. Does not liquefy gelatin. 

Pathogenesis.—Is very pathogenic for mice and for rabbits, causing death 
from general infection in two or three days. 


STREPTOCOCCUS BOMBYCIS. 


Synonym.—Microzyma bombycis (Béchamp). 

Found in the bodies of infected silkworms suffering from la flacherie 
(maladie des morts-plats). Etiological relation established by Pasteur. 

Morphology.—Oval cells, not exceeding 1.5 # in diameter, in pairs or in 
chains. 

Biological Characters.—Not determined with precision. 

Pathogenesis.—The infected silkworm ceases to eat, becomes weak, and 
dies. Its body is soft and diffluent, and at the end of twenty-four to forty- 
eight hours is filled with a dark-brown fluid and with gas. 


NOSEMA BOMBYCIS. 


Synonyms.—Micrococcus ovatus; Panhistophyton ovatum. 

Found in the blood and all of the organs of silkworms infected with 
pébrine (Fleckenkrankheit). 

First observed by Cornalia. Etiological relation established by Pasteur. 

Morphology.—Shining, oval cells, three to four « long and two / broad; 
solitary, in pairs, or in irregular groups. 

Biological Characters.—Not determined with precision. 

Puthogenesis.—Dark spots appear upon the skin of infected silkworms, 
which lose their appetite, become slender and feeble, and soon die. The 
oval corpuscles are found in all of the organs, and also in the eggs of 
butterflies hatched from infected larvee. Some authors are of the opinion 
that the oval corpuscles found in this disease do not belong to the bacte- 
ria, but to an entirely different class of microdrganisms—the Psorospermia 
(Metschnikoff). 


MICROCOCCUS OF HEYDENREICH. 


Synonyms.—Micrococcus of Biskra button—F'r. ‘‘clou de Biskra”; Ger. 
‘‘Pendesche Geschwur.” . ; 

Found by Heydenreich (1888) in pus and serous fluid obtained from the 
tumors and ulcers in the Oriental skin affection known as Biskra button. 

Morphology.—Diplococci, from 0.86 to 1 # in length, surrounded by a 
capsule; sometimes associated to form tetrads. 

Stains with the usual aniline colors. __ : i 

Biological Characters .--An aérobic, liquefying micrococcus. Grows in 
the usual culture media at the room temperature. In gelatin stick cultures, 
at 20° C., at the end of forty-eight hours growth occurs along the line of 
puncture in the form of small, crowded colonies, which produce a grayish- 
white line; upon the surface a thin, circular layer of a yellowish-white 
color is developed. At the end of three to four days liquefaction commences 
near the surface, where a funnel is formed which extends until about the 
fourteenth day, when the gelatin is completely liquefied. Upon the surface 


416 PATHOGENIC MICROCOCCI 


of agar, at 37° C., a grayish-white or yellowish layer is formed at the end of 
twenty-four hours, which has a varnish-like lustre. Upon potato, at 30° to 
aA C., at the end of forty-eight hours a white or yellow layer has de- 
veloped. : . 

Pathogenesis.—According to Heydenreich, inoculations in rabbits, dogs, 
chickens, horses, and sheep cause a skin affection which is identical with 
that which characterizes Biskra button in man. When rubbed into the 
healthy skin of man it also produces the development of abscesses. 


MICROCOCCUS ENDOCARDITIDIS RUGATUS (Weichselbaum). 


Obtained by Weichselbaum (1890) from the affected cardiac valves in a 
fatal case of ulcerative endocarditis. 

Morphology.—Micrococci, resembling the staphylococci of pus in dimen- 
sions and mode of grouping; solitary, in pairs, in groups of four, or in ir- 
regular masses. 

Biological Characters.—An aérobic micrococcus. Does not grow at the 
room temperature. Upon agar plates, at 37° C., at the end of three or four 
days the superficial colonies consist of a small, brown, central mass sur- 
rounded by a granular, semi-transparent, grayish marginal zone; gradually 
they attain a characteristic wrinkled appearance; the deep colonies, under a 
low power, are irregular, finely granular, and contain a large central, yel- 
lowish-brown nucleus surrounded by a narrow, grayish-brown peripheral 
zone. Inagar stab cultures small, spherical colonies are formed upon the 
surface, which become confluent, forming a grayish-white, wrinkled layer 
which has a stearin-like lustre and is very viscid; a scanty growth occurs 
along the line of puncture. Upon potato, at 37° C., a scanty development 
occurs in the form of a small, dry, pale-brown mass. Upon blood serum 
isolated or confluent, colorless colonies are formed the size of a poppy seed; 
these are closely adherent to the surface of the culture medium. 

Pathogenesis.—W hen injected subcutaneously into the ear of a rabbit it 
produces tumefaction and redness; in guinea-pigs, formation of pus. When 
injected into the circulation of dogs, after injury to the aortic valves, an en- 
docarditis is developed. 


MICROCOCCUS OF GANGRENOUS MASTITIS IN SHEEP. 


Obtained by Nocard (1887) from the milk of sheep suffering from gan- 
grenous mastitis (mal de pis or d’araignée), a fatal disease which attacks 
especially sheep which are being milked for the manufacture of cheese, at 
Roquefort and elsewhere in France. 

Morphology.—Micrococci, solitary, in pairs, or in irregular groups, resem- 
bling the staphylococci of pus in dimensions and arrangement. 

Stains with the usual aniline colors and also by Gram’s method. 

Biological Characters.—An aérobic and facultative anaérobic, liquefy- 
ing micrococcus. Grows at the room temperature in the usual culture me- 
dia. Upon gelatin plates, at the end of forty-eight hours, the colonies are 
spherical and white in color; under a low power the superficial colonies are 
circular in outline, homogeneous, and brown in color; they are surrounded 
by a semi-transparent aureole ; liquefaction around the superficial colonies 
occurs sooner than around those beneath the surface of the gelatin. In 
gelatin stick cultures, at 18° to 20° C., on the second day liquefaction of the 
gelatin commences near the surface ; by the fifth day a pouch of liquefied‘ 
gelatin has formed, which has the shape of an inverted cone; at the bottom 
of this an abundant deposit of micrococci is seen, while the liquefied gela- 
tin above is clouded throughout. In agar stick cultures development oc- 
curs upon the surface as a thick white layer, which gradually extends 
over the entire surface, and after a time acquires a yellowish tint; develop- 
ment also occurs along the line of puncture. Upon potato a thin, viscid, 
grayish layer is peice developed; the outline is irregular and the edges 
thicker than the central portion ; the central portion of this layer gradually 


NOT DESCRIBED IN SECTIONS V. AND VI. 417 


acquires a yelluw color, while the periphery remains of a dirty-white or 
grayish color. Blood serum is liquefied by this micrococcus. 

Pathogenesis.—A few drops of a pure culture injected subcutaneously or 
into the mammary gland of sheep cause an extensive inflammatory oedema 
and the death of the animal in from twenty-four to forty-eight hours. A 
cubic centimetre injected into the mammary gland of a goat produced no re- 
sult; the horse, the calf, the pig, the cat, chickens, and guinea-pigs also proved 
to beimmune. Subcutaneous injections in rabbits produce an extensive ab- 
scess at the point of inoculation, 


STREPTOCOCCUS OF MASTITIS IN COWS. 


Obtained by Nocard and Mollereau (1887) from the milk of cows suffering 
from a form of chronic mastitis (mammite contagieuse). 

Morphology.—Spherical or oval cocci, a itttle Tees than one in diameter, 
usually united in long chains. 

Stains with the usual aniline colors and also by Gram’s method. 

Biological Characters.—An aérobic and facultative anaérobic, non- 
liquefying streptococcus. Grows in the usual culture media at the room 
temperature. Develops rapidly in milk or in bouillon at a temperature of 
16° to 30°C. The milk of a cow suffering from the form of mastitis produced 
by this micrococcus, when drawn with proper precautions in sterilized test 
tubes, at the end of twenty-four hours is acid in reaction; the lower two- 
thirds of the tube is filled with an opaque, dirty-white, homogeneous deposit, 
and above this is an opalescent, serous fluid of a bluish or dirty-yellow or 
slightly reddish color, according to the age of the lesion. A drop of this 
milk examined under the microscope shows the presence of the streptococcus 
in great numbers. The addition of two to five per cent of glucose or of gly- 


Fic. 97.—Streptococcus of mastitis in cows (Nocard). 


eerin to bouillon makes it a more favorable culture medium; the reaction 
should be neutral or slightly alkaline, as this streptococcus does not grow 
readily in an acid medium, although it produces an acid reaction in media 
containing sugar, the acid formed bene ee In gelatin stab cultures the 
growth upon the surface is scanty, in the form of a thin pellicle around the 
point of puncture; along the line of inoculation minute, opaque, granular 
colonies are developed, which, being closely crowded, form a thick line with 
jagged margins. Sse 

nagar stab cultures the growth is similar but more abundant. Upon 
the surface of nutrient gelatin, agar, or blood serum a large number of mi- 


a4 


418 PATHOGENIC MICROCOCCI 


nute, spherical, semi-transparent colonies are developed among the impfstrich ; 
these have a bluish tint by reflected light; they may become confluent, form- 
ing a thin layer with well-defined margins. Upon gelatin plates, at 16° to 
18° C., colonies are first visible at the end of two or three days; they are 
spherical and slightly granular, at first transparent and later of a pale-yellow 
color by transmitted light, which gradually becomes brown. At the end of 
five or six weeks the colonies are still quite small, well defined, and opaque. 
Pathogenesis.—Pure cultures injected into the mammary gland of cows 
and goats gave rise to a mastitis resembling in its development that from 
which the streptococcus was obtained in the first instance. Injections into 
the cavity of the abdomen or into a vein, of one cubic centimetre of a pure 
culture, gave a negative result in dogs, cats, rabbits, and guinea-pigs. 


DIPLOCOCCUS OF PNEUMONIA IN HORSES. 


Obtained by Schiitz (1887) from the lungs of horses affected with pneu- 
monia. 

Morphology.—Oval cocci, usually in pairs. surrounded by a homogene- 
ous, transparent capsule. * 

Does not stain by Gram’s method. 

Biological Characters.—An aérobic, non-liquefying micrococcus. Grows 
at the room temperature. Upon gelatin plates forms small, spherical, white 
colonies. 

In gelatin stick cultures grows along the line of puncture 4s small, white, 
separate colonies, which grow larger without becoming confluent. Upon 
the surface of agar small transparent drops are developed along the impf- 
strich. 

~ Pathogenesis.—The injection of a pure culture into the lung of a horse 
produces pneumonia and causes its death in eight or nine days. Pathogenic 
for rabbits, guinea-pigs, and mice. 


STREPTOCOCCUS CORYZH CONTAGIOSZ EQUORUM. 


Obtained by Schiitz (1888) from pus from the lymphatic glands involved 
in horses sutfering from the disease known in Germany as Druse des 
Pferdes. 

Morphology.—Oval cocci, in pairs, in chains containing three or four 
elements, or in long chaplets. d 

Stains with the usual aniline colors—very intensely with Weigert’s or 
Ehbrlich’s solution. 

Biological Characters.—An aérobic and facultative anaérobic micrococ- 
cus. Grows slowly at the room temperature, more rapidly at 37° C. Upon 
gelatin plates at the end of three to five days minute colonies become visible; 
these never exceed the size of a pin’s head. In gelatin stab cultures growth 
upon the surface is scanty or absent; along the line of puncture minute 
colonies are developed in rows. Upon agar plates, at 37° C., at the end of 
twenty-four hours lentil-shaped colonies are developed the size of a pin’s 
head; under a low power the superficial colonies are seen to have a well-de- 
fined, opaque nucleus surrounded by a grayish, transparent marginal zone, 
which represents a half-fluid, slimy growth which does not extend after the 
third day and later disappears entirely; the deep colonies are at first well- 
defined, and later surrounded by wing-like outgrowths. Upon blood serum, 
at 37° C., pees transparent drops are first developed; these become con- 
fluent and form a viscid and tolerably thick layer; this later becomes dry 
and iridescent. 

Pathogenesis.--Pathogenic for horses and for mice, producing in these 
animals an abscess at the point of inoculation, and metastatic abscesses in 
the neighboring lymphatic glands, Not pathogenic for rabbits, guinea-pigs, 
or pigeons. 


NOT DESCRIBED IN SECTIONS V. AND VI. 419 


HZMATOCOCCUS BOVIS (Babes). 


Obtained by Babes (1889) from the blood and various organs of cattle 
which had died of an epidemic malady (in Roumania) characterized by haemv- 
globinuria. The cocci are found in the blood in great numbers, for the most 
part enclosed in the red corpuscles. 

Morphology.—Biscuit-shaped cocci united in pairs; sometimes oblong in 
form, isolated or united in groups; the free cocci are surrounded by a pale- 
yellowish, shining aureole of 0.5 to 1 w in diameter. 

Stains best with Léffler’s solution of methylene blue; does not stain by 
Gram’s method. 

Biological Characters.—An aérobic and facultative anaérobic, non- 
liquefying micrococcus. Grows very slowly at the room temperature—not 
below 20° C. In the incubating oven grows in the usual culture media. In 
gelatin stab cultures a scanty development of small, white colonies occurs 
along the line of puncture. Upon the surface of agar small, transparent 
drops are developed along the impfstrich. Upon potato, at 37° C.. a thin, 
broad, yellowish, shining layer is developed in the course of a few days— 
scarcely visible. Upon blood serum small, moist, transparent colonies are 
developed. 

Pathogenesis —Pathogenic for rabbits and rats, which die in from six to 
ten days after inoculation with a pure culture; the spleen is found to be en- 
larged, the lungs hypereemic, and a bloody serum is found in the cavity of 
the abdomen; the cocci are present in the blood in considerable numbers, 
but are rarely seen in the red corpuscles. Inoculations in oxen, horses, 
goats, sheep, guinea-pigs, and birds were without effect. 


STREPTOCOCCUS PERNICIOSUS PSITTACORUM. 


Micrococcus of gray pirrot disease. Eberth and Wolff have described 
an infectious disease of gray parrots, which is said to be extremely fatal 
among the imported birds. The disease is characterized by the formation of 
nodules upon the surface and in the interior of various organs, and especially 
in the liver. Micrococci of medium size are found in these nodules and in 
blood from the heart; these are sometimes in chains. Microscopic examina- 
tion of stained sections shows that these cocci are directly related to the tis- 
sue necrosis which characterizes the disease. But the micrococcus has not 
been cultivated and its biological characters are undetermined. 


STREPTOCOCCUS AGALACTIAZ CONTAGIOS AL. 


Obtained by Adametz (1894) from the milk of cows suffering from mas- 
titis (Gelben Galt). According to Adametz all of the streptococci which 
have been described by different investigators (Kitt, Nocard and Mollereau, 
Guillebeau, and others) are probably varieties of a single species. 
Morphology.—Spherical cocci in short chains—1 » in diameter. 
Biological Characters.—An aérobic and facultative anaérobic, non- 
liquefying streptococcus. : ; ; 

pon gelatin plates forms flat, transparent, white or bluish-white, 
slimy colonies, having a slight pearly lustre and an irregular outline. In 
nutrient gelatin containing five per cent of milk sugar the colonies, at the 
end of eight.days, have a diameter of 0.85 to 1 millimetre; they are milk- 
white and of a semi-fluid, slimy, consistence. 

Upon agar plates the deep colonies are punctiform and white in color— 
under a low power they are seen to have an irregular dentate contour and a 
brownish color; the superficial colonies gradually assume the appearance 
of transparent, flat drops having a diameter of 0.5 to 0.7 millimetre. In 
sterilized milk fermentation occurs, at 37° C., in from twenty to twenty-four 


420 PATHOGENIC MICROCOCCI 


hours ; some hours later the casein is precipitated, fine gas bubbles are seen 
in the lower part of the fluid and a foam upon the surface; the reaction is 
acid and the casein is not peptonized. The power of producing acid and gas 
is diminished or lost after a few successive cultures have been made. 

Streptococcus mastitis sporadice (Guillebeau) is said by Adametz to be 
distinguished from the streptococcus above described (No. 444) by being 
smaller—0.5 in diameter—and by the fact that the cultures do not lose the 
power of producing fermentation in milk. 


MICROCOCCUS MELITENSIS. 


Surgeon-Major Bruce, of the British army, in 1887 demonstrated 
the etiological relation of a micrococcus, now known as Aicrococcus 
melitensis, to the infectious disease known as Malta fever (syn- 
onyms: Mediterranean fever; Neapolitan fever; Rock fever of Gib- 
raltar, etc.). Subsequent researches show that this fever is not re- 
stricted to the Mediterranean region, and it will probably be found 
to have an extensive area of prevalence on both continents. Cases 
have been recognized in America and by medical officers of the army 
stationed in the Philippine Islands. Curry (Captain and Assistant 
Surgeon United States Volunteers), in arecent report to the Surgeon- 
General of the army, says: 


‘‘T had the honor to report to the Surgeon-General of the Army on Jan- 
uary 2d, 1900, four cases of Mediterranean or Malta fever, which came under 
my observation, while on duty as pathologist to the 1st Reserve Hospital in 
Manila, P. I., cases occurring among our troops and originating on the 
Island of Luzon. 

‘Later, in a report on the ‘ Diseases of the Philippine Islands,’ I reported 
twelve additional cases. In all these cases a positive serum reaction with 
the Micrococcus melitensis was obtained, and the clinical history of the cases 
corresponds with the descriptions of Malta fever as given by the English 
army surgeons Bruce, Hughes, Wright, Semple, and others, and that de- 
scribed by Manson. Included in these sixteen cases is one autopsy. 

‘‘In my report on the ‘ Diseases of the Philippine Islands,’ under the head- 
ing of ‘Fevers of the Philippines,’ I expressed the belief that ‘Malta fever 
is not an uncommon disease in the Philippine Islands,’ and that it appeared 
that ‘Malta fever is by no means as limited geographically as has been 
thought heretofore.’ 

‘“Our experience here in the Army and Navy General Hospital, Hot 
Springs, Ark., has convinced me that Malta fever is widespread in tropical 
and sub-tropical regions. We are but having a repetition of the experience 
of the English army surgeons at the Royal Victoria Hospital, Netley. 

‘“‘Among the soldiers and sailors, here in our wards, who have been 
returned from tropical stations, we have found already four to have Malta 
fever. These four cases came from widely separated stations. Two cases 
are in soldiers, one from the Philippines, and one from Cuba, and two are 
among sailors of the United States navy who were recently returned from 
South Atlantic stations. 

‘* All four cases entered this hospital with a diagnosis of rheumatism.” 


Morphology.—Micrococci, about 0.5 » in diameter, usually soli- 
tary or in pairs; occasionally short chains are seen in cultures. In 


NOT DESCRIBED IN SECTIONS V. AND VI. 421 


old cultures kept at the room temperature the cells may be oval or 
elongated. 

Biological Characters.—An aérobic, non-liquefying micro- 
coccus. Does not stain by Gram’s method. Grows best in nutrient 
agar. In stab cultures no growth is seen for several days. ‘‘ At 
length the growth appears as pearly-white spots scattered around the 
point of puncture and minute, round, white colonies are also seen 
along the course of the needle track”; these increase in size, and 
after some weeks a rosette-shaped growth is seen upon the surface, 
and the growth along the line of puncture has a yellowish-brown 
color. At the end of nine or ten days, at 37° C., some of the colonies 
on the surface of nutrient agar are as large as No. 4 shot; by trans- 
mitted light they have a yellowish color at the centre, and the per- 
iphery is bluish-white; by reflected light they have a milky-white 
color. At 25° C. colonies first become visible at the end of about 
seven days, at 37° C. in three to four days. Does not grow upon 
potato. Very scanty growth upon nutrient gelatin at 22° C. at the 
end of a month. 

This micrococcus has usually been described as non-motile, but 
Gordon has demonstrated that it has from one to four flagella, which 
are difficult to demonstrate by the usual staining methods. 

Pathogenests.—Pathogenic for monkeys, which suffer from fever 
as a result of subcutaneous inoculations and usually die in from thir- 
teen to twenty-one days. The spleen is found to be enlarged and 
contains the micrococcus. Not pathogenic for mice, guinea-pigs, 
or rabbits. 

In man the micrococcus is found in large numbers in the spleen, 
which is greatly enlarged. 

Widal Reaction.—The blood serum of patients suffering from 
Malta fever and of individuals who have recently recovered from the 
disease causes the agglutination of Micrococcus melitensis in recent 
cultures. According to Wright and Smith this reaction may be 
manifested a year after recovery. Dilution of 1:1000 will in ex- 
ceptional cases give a distinct agglutinating effect. 


VIII. 


THE BACILLUS OF ANTHRAX. 


[Fr., CHARBON; Ger., MILZBRAND. | 


ANTHRAX is a fatal infectious disease which prevails extensively 
among sheep and cattle in various parts of the world, causing heavy 
losses. In Siberia it constitutes a veritable scourge and is known 
there as the Siberian plague ; it also prevails to a considerable extent 
in portions of France, Hungary, Germany, Persia, and India, and 
local epidemics have occasionally occurred in England, where it is 
known under the name of splenic fever. It does not prevail in the 
United States. In infected districts the greatest losses are incurred 
during the summer season. 

In man accidental inoculation may occur among those who come 
in contact with infected animals, and especially during the removal of 
the skin and cutting up of dead animals, when there is any cut or 
abrasion upon the hands. A malignant pustule is developed as the 
result of such inoculation, but, as a rule, general infection does 
not occur, as is the case when inoculations are made into the more 
susceptible lower animals—rabbit, guinea-pig, mouse. Those who 
handle the hair, hides, or wool of infected animals are also liable to 
contract the disease by inoculation through open wounds, or by the 
inhalation of dust containing spores of the anthrax bacillus. Cases 
of pulmonic anthrax, known formerly in England as “ wool-sorters’ 
disease,” have been occasionally observed in England and in Ger- 
many, and are now recognized as being due to infection through the 
lungs in the manner indicated. 

The French physician Davaine, who had observed the anthrax 
bacillus in the blood of infected animals in 1850, communicated to 
the French Academy of Sciences the results of his inoculation experi- 
ments in 1863 and 1864, and asserted. the etiological relation of the 
bacillus to the disease with which his investigations showed it to be 
constantly associated. This conclusion was vigorously contested by 
conservative opponents, but has been fully established by subsequent 
investigations, which show that the bacillus, in pure cultures, induces 


THE BACILLUS OF ANTHRAX. 423 


anthrax in susceptible animals as certainly as does the blood of an 
animal recently dead from the disease. 

Owing to the fact that this was the first pathogenic bacillus cul- 
tivated in artificial media, and to the facility with which it grows ix 
various media, it has served more than any other microérganism for 
researches relating to a variety of questions in pathology, general 
biology, and public hygiene, some of which are discussed in other 
sections of this volume. 


BACILLUS ANTHRACIS. 


Synonyms.—Milzbrandbacillus, Ger. ; Bactéridie du charbon, F'r. 
First observed in the blood of infected animals by Pollender (1849) 
and by Davaine (1850). Etiological relation affirmed by Davaine 


tit 


es 
OE ee 
= ==. 


‘ , 
i Sy 
; ., 
——— AN 
pcan 
wm swe". 
wf % Ba . 
‘ a 
y --227=-tSs 
oN sre us 
, 4 . 4 
rae . OWE 
“sr” S/h ou 
527 4 
om “ Mao 
‘ 
aN a 9 
ra M i i 
sosscusal any if 
ass ‘5 We 
‘ aay 
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Fig. 98.—Bacillus anthracis, from a culture, showing development of long threads in convo- 
luted bundles. x 300. (Klein.) 


(1863), and established by the inoculation of pure cultures by Pasteur 
(1879) and by many other investigators. 

‘Morphology.—Rod-shaped bacteria having a breadth of 1 to 
1.25 p, and 5 to 20 » in length; or, in suitable culture media, growing 
out into long, flexible filaments, which are frequently united in 
twisted, cord-like bundles. These filaments in hanging-drop cul- 
tures, before the development of spores, appear to be homogeneous ; 
or the protoplasm is clouded and granular, but without distinct seg- 
mentation. But in stained preparations the filaments are seen to be 
made up of a series of rectangular, deeply stained segments. In 
hanging-drop cultures the ends of the rods appear ruunded, but in 
stained preparations from the blood of an infected animal they are 
seen to present a slight concavity, and a lenticular interspace is 
formed where two rods come together. The diameter of the rods 


424 THE BACILLUS OF ANTHRAX. 


varies considerably in different culture media; and in old cultures 
irregular forms are frequently seen—“ involution forms.” 

Under favorable conditions endogenous spores are developed in 
the long filaments which grow out in artificial culture media. 
These first appear as refractive granules distributed at regular inter- 
vals in the segments of the protoplasm, which gradually disappear 
as the spores are developed; and these are left as oval, highly re- 
fractive bodies, held together in a linear series by the cellular enve- 
lope, and subsequently set free by its dissolution. The germination 
of these reproductive bodies results in the formation of rods and 
spore-bearing filaments like those heretofore described. In this pro- 
cess the spore is first observed to 
lose its brilliancy, from the ab- 
sorption of moisture, a promi- 
nence occurs at one end of the 
oval body, and soon the external 
envelope — exosporium—is rup- 
tured, permitting the softened 
protoplasmic contents enclosed 
in the internal spore membrane 
—endosporium—to escape as a 
short rod, to which the empty 
exosporium sometimes remains 
attached. 

The anthrax bacillus stains 
readily with the aniline colors 
and also by Gram’s method, 
when not left too long in the 
, decolorizing iodine solution. 
; Fig. 99.—Bacillus anthracis, from a culture, show- ,offler’s solution of methylene 
ing formation of spores. X 1,000. (Klein.) ‘ 7 

blue is an especially good stain- 
ing fluid for this as well as for many other bacilli. Bismarck brown 
is well adapted for specimens which are to be photographed, and also 
for permanent preparations, as it is less liable to fade than the blue 
and some other aniline colors. 

Biological Characters.—The anthrax bacillus is aérobic, but 
not strictly so, as is shown by the fact that it grows to the bottom of 
the line of puncture in stab cultures in solid media. It is non-mo- 
tile, and is distinguished by this character from certain common 
bacilli resembling it in morphology—Bacillus subtilis—which were 
frequently confounded with it in the earlier days of bacteriological 
investigation. 

The anthrax bacillus grows in a variety of nutrient media at a 


THE BACILLUS OF ANTHRAX. 425 


temperature of 20° to 38°C. Development ceases at temperatures 
below 12° C. or above 45° C. 

This bacillus grows best in neutral or slightly alkaline media, and 
its development is arrested by a decidedly acid reaction of the cul- 
ture medium. It may be cultivated in infusions of flesh or of vari- 
ous vegetables, in diluted urine, in milk, etc. 

In gelatin plate cultures small, white, opaque colonies are devel- 
oped in from twenty-four to thirty-six hours, which under the micro- 
scope are seen to be somewhat irregular in outline and of a greenish 
tint ; later the colonies spread out upon the surface of the gelatin, 
and the darker central portion is surrounded by a brownish mass of 
wavy filaments, which are associated in tangled bundles. Mycelial- 
like outgrowths from the periphery of 
the colony may often be seen extending 
into the surrounding gelatin. At the 
end of two or three days liquefaction of 
the gelatin commences, and the colony 
is soon surrounded by the liquefied me- 
dium, upon the surface of which it floats 
as an irregular white pellicle. In gela- 
tin stab cultures growth occurs all 
along the line of puncture as a white cen- 
tral thread, from which lateral thread- 
like ramifications extend into the culture 
medium. At the end of two or three 
days liquefaction of the culture medium 
commences near the surface, where the 
development has been most abundant. 
At first a pasty, white mass is formed, 
but as liquefaction progresses the upper 
part of the liquefied gelatin becomes 
transparent from the subsidence of the ig 109 culture of Bacillus an- 
motionless bacilli, and these are seen thracis in nutrient gelatin: a, end 
upon the surface of the non-liquefied 0] [our Qaysi, > end of eight days. 
portion of the medium in the form of 
cloudy, white masses, while below the line of liquefaction the charac- 
teristic branching growth may still be seen along the line of puncture. 

In agar plate cultures, in the incubating oven at 35° to 37° C., 
colonies are developed within twenty-four hours, which under the. 
microscope are seen to be made up of interlaced filaments and are 
very characteristic and beautiful. Upon the surface of nutrient agar 
a grayish-white layer is formed, which may be removed in ribbon-like 
strips ; and in stick cultures in this medium a branching growth is 
seen, like that in gelatin, but without liquefaction. The addition of 


426 THE BACILLUS OF ANTHRAX. 


a small quantity of agar toa gelatin medium prevents liquefaction 
of the gelatin (Fligge). 

Upon blood serum a rather thick, white layer is formed and 
liquefaction slowly occurs. 

Upon potato the growth is abundant as a rather dry, grayish- 
white layer, of limited extent, having a somewhat rough surface and 
irregular margins. 

Spores are formed only in the free presence of oxygen, as in sur- 
face cultures upon potato or nutrient agar, or in shallow cultures in 
liquid media, and at a temperature of 20° to 35° C. They are not 
formed during the development of the bacilli in the bodies of living 


Fic. 101.—Colonies of Bacillus anthracis upon gelatin plates: a, at end of twenty-four hours; 
b, at end of forty-eight hours. x 80. (Fligge.) 


animals, but after the death of the animal the bacillus continues to 
multiply for a time, and spores may be formed where the fluids 
containing it come in contact with the air—as, for example, in 
bloody discharges from the nostrils or from the bowels of the dead 
animal. 

Varieties incapable of spore production have been produced arti- 
ficially, by several bacteriologists, by cultivating the bacillus under 
unfavorable conditions. Roux was able to produce a sporeless va- 
riety by successive cultivation in media containing a small quantity 
of carbolic acid—1 : 1,000. 

Varieties differing in their pathogenic power may also be pro- 
duced by cultivation under unfavorable conditions. Thus Pasteur 


THE BACILLUS OF ANTHRAX. 427 


produced an “ attenuated virus” by keeping his cultures for a con- 
siderable time before replanting them upon fresh soil, and supposed 
the effect was due to the action of atmospheric oxygen. It seems 
probable that it was rather due to the deleterious action of its own 
products of growth present in the culture media. It has been 
shown by Chamberlain and Roux that cultivation in the presence 
of certain chemical substances added to the culture medium—e.g., 
bichromate of potassium 0.01 per cent—causes an attenuation of 
virulence. The same result occurs when cultures are subjected to a 
temperature a little below that which is fatal to the bacillus—50° C. 
for eighteen minutes (Chauveau); 42.5° C. for two or three weeks 
(Koch). Attenuation of pathogenic virulence is also effected by cul- 
tivation in the body of a non-susceptible animal, like the frog (Lu- 
barsch, Petruschky); or in the blood of a rat (Behring); by exposure 
to sunlight (Arloing); and by compressed air (Chauveau). 

Anthrax spores may be preserved ina desiccated condition for 
years without losing their vitality or pathogenic virulence when in- 
oculated into susceptible animals. They also resist a comparatively 
high temperature. Thus Koch and Wolffhigel found that dry spores 
exposed in dry air required a temperature of 140° C., maintained for 
three hours, to insure their destruction. But spores suspended in a 
liquid are destroyed in four minutes by the boiling temperature, 
100° C. (writer’s determination). 

The bacilli, in the absence of spores, according to Chauveau, are 
destroyed in ten minutes by a temperature of 54° C. 

For the action of various antiseptic and germicidal agents upon 
this bacillus we must refer to the sections especially devoted to this 
subject (Part Second). 

Toussaint, by injecting filtered anthrax blood into animals, obtained 
evidence that it contained some toxic substance which in his experi- 
ments gave rise to local inflammation without any noticeable general 
symptoms. More recent investigations show that a poisonous sub- 
stance is formed during the growth uf the anthrax bacillus, and that 
cultures containing this toxin, from which the bacilli have been re- 
moved by filtration through porcelain, produce immunity when in- 
jected into susceptible animals, similar to that resulting from inocu- 
lations with an attenuated virus. It is probable that the pathogenic 
power of the anthrax bacillus depends largely upon the presence of 
this toxin, and that the essential difference between virulent and 
attenuated varieties depends upon the more abundant production of 
this toxic substance by the former. It has also been shown that 
virulent cultures produce a larger quantity of acid than those which 
have been attenuated by any of the agencies above mentioned 
(Behring). 


428 THE BACILLUS OF ANTHRAX. 


Pathogenesis.—The anthrax bacillus is pathogenic for cattle, 
sheep, horses, rabbits, guinea-pigs, and mice. White rats, dogs, and 
frogs are immune, as is also the Algerian race of sheep. The spar- 
row is susceptible to general infection, but chickens, under normal 
conditions, are not. Young animals are, as a rule, more susceptible 
than adults of the same species. Man does not belong among the 
most susceptible animals, but is subject to local infection as a result 
of accidental inoculation—malignant pustule—and to pulmonic an- 
thrax from breathing air, containing spores of the anthrax bacillus, 
during the sorting of wool or hair from infected animals. In animals 
which havea partial immunity, natural or acquired, as a result of 
inoculations with attenuated virus, the subcutaneous introduction of 
virulent cultures may give rise to a limited local inflammatory pro- 
cess, with effusion of bloody serum in which the bacillus is found in 
considerable numbers ; but the blood is not invaded, and the animal, 
after some slight symptoms of indisposition, recovers. In susceptible 


Fig. 102.—Bacillus anthracis in liver of mouse. x 700. (Fligge.) 


animals injections beneath the skin or into a vein give rise to general 
infection, and the bacilli multiply rapidly in the circulating fluid. 
Death occurs in mice within twenty-four hours, and in rabbits, as a. 
rule, in less than forty-eight hours. The blood of the heart and 
large vessels may be found, in an autopsy made immediately after 
death, to contain comparatively few bacilli; but in the capillaries of 
the various organs, and especially in the greatly enlarged spleen, in 
the liver, the kidneys, and the lungs, they will be found in great 
numbers, and well-stained sections of these organs will give an as- 
tonishing picture under the microscope, which the student should not 
fail to see in preparations made by himself. The capillaries in many 
places will be found stuffed full of bacilli; or they may even be rup- 


THE BACILLUS OF ANTHRAX. 429 


tured as a result of the distention, and the bacilli, together with 
escaped blood corpuscles, will be seen in the surrounding tissues. In 
the kidneys the glomeruli, especially, appear as if injected with col- 
ored threads, and by rupture these may find their way into the urini- 
ferous tubules. 

These appearances and the general symptoms indicate that the 
‘disease produced by the introduction of this bacillus into the bodies of 
susceptible animals is a genuine septicemia. As in other forms of 
septicaemia, the spleen is found to be greatly enlarged ; it has a dark 
color and is soft and friable. With this exception the organs pre- 
sent no notable changes, although the liver is apt to be somewhat 
enlarged. Inthe guinea-pig an extensive inflammatory cedema, ex- 
tending from the point of inoculation to the most dependent parts of 
the body, is developed ; the subcutaneous connective tissue is infil- 
trated with bloody serum and has a gelatinous appearance. This 
animal comes next to the mouse in susceptibility, and cultures which 


Fig. 103.—Bacillus anthracis in kidney of rabbit. x 400. (Baumgarten.) 


are attenuated to such an extent that they will not kill a rabbit or a 
sheep may still kill a guinea-pig ; or, if not, may killamouse. Pasteur 
has shown that the pathogenic power of the bacillus may be reéstab- 
lished by inoculations into susceptible animals, and that an attenu- 
ated culture which will not kill an adult guinea-pig may be fatal to 
a very young animal of this species, and that cultures from the blood 
of this will have an increased pathogenic virulence. 

Very minute quantities of a virulent culture are infallibly fatal to 
these most susceptible animals, but for rabbits and other less sus- 
ceptible animals the quantity injected influences the result, and re- 


430 THE BACILLUS OF ANTHRAX. 


covery may occur after subcutaneous or intravenous injection of a 
very small number of bacilli. 

Infection in cattle and sheep commonly results from the ingestion 
of spores while grazing in infected pastures. The bacillus itself, in 
the absence of spores, is destroyed in the stomach. While spores are 
not formed in the bodies of living animals, their discharges contain 
the bacillus, and this is able to multiply in them and to form spores 
upon the surface of the ground when temperature conditions are 
favorable. Itis probable that this is the usual way in which pastures 
become infected, and that the bloody discharges from the bladder 
and bowels of animals suffering from the disease furnish a nidus for 
the external development of these reproductive elements ; as also do 
the fluids escaping from the bodies of dead animals. And possibly, 
under specially favorable conditions, the bacillus may lead a sapro- 
phytic existence for a considerable time in the superficial layers of the 
soil. 

Buchner has shown by experiment that infection in animals may 
result from respiring air in which anthrax spores are in suspension 
in the form of dust ; and in man this mode of infection occurs in the 
so-called wool-sorters’ disease. 

The question of the passage of the anthrax bacillus from the 
mother to the foetus in pregnant females has received considerable 
attention. That this may occur is now generally admitted, and ap- 
pears to be established by the investigations of Strauss and Chamber- 
lain, Morisani, and others. That it does not always occur is shown, 
however, by the researches of other bacteriologists, and especially by 
those of Wolff. 

Sirena and Scagliosi (1894) report, as the result of extended experi- 
ments made by them, that anthrax spores may survive in distilled 
water for twenty months; in moist or dry earth for two years and 
nine months; in sea-water for one year and seven months; in sewage 
nearly sixteen months. 

Marmier (1895) has made an extended experimental research to 
determine the nature of the specific toxin of the anthrax bacillus. 
This he obtains from cultures, at a low temperature, in media con- 
taining peptone and glycerin. It has not the reactions of an albu- 
minoid body and is not destroyed by a temperature of 100°C. In 
comparatively large doses it kills animals susceptible to anthrax, and 
by the administration of smaller doses immunity may be established 
in such animals. This toxin is contained in the bacterial cells, and 
is obtained by subjecting these to the action of alcohol, or from the 
filtrate when cultures are made ata low temperature in a medium 
containing peptone. It has not, however, been obtained in a pure 
form, and its exact nature has not been determined. 


IX. 
THE BACILLUS OF TYPHOID FEVER. 


NUMEROUS researches support the view that the bacillus described 
by Eberth in 1880 bears an etiological relation to typhoid fever— 
typhus abdominalis of German authors; and pathologists have ac- 
cepted this bacillus as the veritable “germ” of typhoid fever, not- 
withstanding the fact that the final proof that such is the case is still 
wanting. 

This final proof would consist in the production in man or in one 
of the lower animals of the specific morbid phenomena which char- 
acterize the disease in question, by the introduction of pure cultures 
of the bacillus into the body of a healthy individual. Evidently it is 
impracticable to make the test upon man, and thus far we have no 
satisfactory evidence that any one of the lower animals is subject to 
the disease as it manifests itself in man. The experiments of 
Frankel and Simmonds show, however, that this bacillus is patho- 
genic for the mouse and the rabbit. We shall refer to the experi- 
ments of these authors later. 

Before the publication of Eberth’s first paper Koch had observed 
this bacillus in sections made from the spleen and liver of typhoid 
cases, and had made photomicrographs from these sections. His 
name is, therefore, frequently associated with that of Eberth as one 
of the discoverers of the typhoid bacillus. Other investigators had no 
doubt previously observed the same organism, but some of them had 
improperly described it as a micrococcus. Such a mistake is easily 
made when the examination is made with a low power; even with a 
moderately high power the closely crowded colonies look like masses 
of micrococci, and it is only by focussing carefully upon the scattered 
organisms on the outer margin of a colony that the oval or rod-like 
form can be recognized. 

Several observers had noted the presence of microédrganisms in 
the lesions of typhoid fever prior to the publication of Eberth’s pa- 
per, and Browicz in 1875, and Fischel in 1878, had recognized the 
presence of oval organisms in the spleen which were probably identi- 
cal with the bacillus of Eberth. 

The researches of Gaffky (1884) strongly support the view that 


432 THE BACILLUS OF TYPHOID FEVER. 


the bacillus under consideration bears a causal relation to typhoid 
fever. Eberth was only successful in finding the bacillus in the 
lymphatic glands or in the spleen in eighteen cases out of forty in 
which he searched for it. On the other hand, he failed to find it in 
eleven cases of various nature—partly infectious processes—and in 
thirteen cases of tuberculosis in which the lymphatic glands were 
involved, and in several of which there was ulceration of the mucous 
membrane of the intestine. 

‘Koch, independently of Eberth and before the publication of his 
first paper, had found the same bacillus in about half of the cases 
examined by him, and had pointed out the fact that they were lo- 
cated in the deeper parts of the intestinal mucous membrane, beyond 
the limits of necrotic changes, and also in the spleen, whereas the 
long, slender bacillus of Klebs was found only in the necrosed por- 
tions of the intestinal mucous membrane. 

The researches of W. Meyer (1881) gave a larger proportion of 
successful results. This author confined his attention chiefly to the 
swollen plaques of Peyer and follicles of the intestine which had not 
yet undergone ulceration. The short bacillus which had been de- 
scribed by Eberth and Koch was found in sixteen out of twenty cases 
examined. The observations of this author are in accord with those 
of Eberth as to the presence of the bacillus in greater abundance in 
cases of typhoid which had proved fatal at an early date. 

The fact that in these earlier researches the bacilli were not found 
in a considerable proportion of the cases examined is by no means 
fatal to the view that they bear an etiological relation to the disease. 
As Gaffky says in his paper referred to : ; 

“This circumstance admits of two explanations. Either in those 
cases in which the bacillus has been sought with negative results 
they may have perished collectively, before the disease process which 
thev had induced had run its course ; or the proof of the presence of 
bacilli was wanting only on account of the technical difficulties which 
attend the finding of isolated colonies.” 

Gaffky’s own researches indicate that the latter explanation is the 
correct one. 

In twenty-eight cases examined by this author characteristic 
colonies of the bacillus were found in all but two. In one of these, 
one hundred and forty-six sections from the spleen, liver, and kid- 
neys were examined without finding a single colony, and in the other 
a like result attended the examination of sixty-two sections from the 
spleen and twenty-one sections from the liver. In the first of these 
cases, however, numerous colonies were found in recent ulcers of the 
intestinal mucous membrane, deeply located in that portion of the 
tissue which was still intact. These recent ulcers were in the neigh- 


THE BACILLUS OF TYPHOID FEVER. 433 


borhood of old ulcers and are supposed to have indicated a relapse 
of the specific process. In the second case the negative result is 
thought by Gaffky to have been not at all surprising, as the patient 
died at the end of the fourth week of sickness, not directly from the 
typhoid process, but as a result of perforation of the intestine. 

Gaftky has further shown that in those cases in which colonies 
are not found in the spleen, or in which they are extremely rare, the 
presence of the bacillus may be demonstrated by cultivation ; and 
that, when proper precautions are taken, pure cultures of the bacil- 
lus may always be obtained from the spleen of a typhoid case. 
Hein has been able to demonstrate the presence of the bacillus and 
to start pure cultures from material drawn from the spleen of a living 
patient by means of a hypodermatic syringe. Philopowicz has re- 
ported his success in obtaining cultures of the bacillus by the same 
method. 

The fact that a failure to demonstrate the presence of microdér- 
ganisms by a microscopic examination cannot be taken as proof of 
their absence from an organ, is well illustrated by a case (No. 18) in 
which the bacillus was obtained by Gaffky from the spleen and also 
from the liver, in pure cultures ; whereas in cover-glass preparations 
made from the same spleen he failed to find a single rod, and more 
than one hundred sections of the spleen were examined before he 
found a colony. 

To obtain pure cultures from the spleen Gaffky first carefully 
washes the organ with a solution of mercuric chloride, 1:1,000, A 
long incision is then made through the capsule with a knife sterilized 
by heat. A second incision is made in this with a second sterilized 
knife, and a third knife is used to make a still deeper incision in the 
same track. By this means the danger of conveying organisms from 
the surface to the interior of the organ isavoided. From the bottom 
of this incision a little of the soft splenic tissue is taken up on a, ster- 
ilized platinum needle, and this is plunged into the solid culture 
medium, or drawn along the surface of the same, or added to lique- 
fied gelatin and poured upon a glass plate. The colonies develop, in 
an incubating oven, in the course of twenty-four to forty-eight hours. 

Gaffky has also shown that the bacillus is present in the liver, in 
the mesenteric glands, and, in a certain proportion of cases at least, 
in the kidneys, in which it was found in three cases out of seven. 

The appearance of the colonies in stained sections of the spleen 
is shown in Figs. 104 and 105. Two colonies are seen in Fig. 104 
(at a, a) as they appear under a low power—about sixty diameters. 
In Fig. 105 one of the colonies is seen more highly magnified—about 
five hundred diameters. 

Frankel and Simmonds have demonstrated that the bacilli multi- 

28 


434 THE BACILLUS OF TYPHOID FEVER. 


ply in the spleen after death, and that numerous colonies may be 
found in portions of the organ which have been kept for twenty- 
four to forty-eight hours before they were placed in alcohol, when 
other pieces from the same spleen placed in alcohol soon after the 
death of the patient show but few colonies or none at all. 

This observation does not in any way weaken the evidence as to 
the etiological réle of the bacillus, but simply shows that dead ani- 
mal matter is a suitable nidus for the typhoid germ—a fact which 
has been repeatedly demonstrated by epidemiologists and insisted 
upon by sanitarians. 

The authors last referred to confirm Gaffky as regards the con- 
stant presence of the bacillus in the spleen. In twenty-nine cases 
they obtained it by plate cultures twenty-five times, and remark 
that in the four cases attended with a negative result this result is 


Fig. 104. 


not at all surprising, inasmuch as the typhoid process had termi- 
nated and death resulted from complications. 

Gaffky did not succeed in obtaining cultures from the blood of 
typhoid-fever patients, and concludes from his researches that if the 
bacilli are present in the circulating fluid it must be in very small 
numbers. He remarks that possibly the result would be different if 
the blood were drawn directly from a vein instead of from the capil- 
laries of the skin. Frankel and Simmonds also report that gelatin, 
to which blood drawn from the forefinger of typical cases had been 
added, remained sterile when poured upon plates in the usual man- 
ner—Koch’s method. The blood was obtained from six different in- 
dividuals, all in an early stage of the disease—the second to the 
third week. A similar experiment made with blood obtained, post 
mortem, from the large veins or from the heart, also gave a negative 
result in every instance save one. In the exceptional case a single 


THE BACILLUS OF TYPHOID FEVER. 435 


colony developed upon the plate. In view of these results we are 
inclined to attribute the successful attempts reported by some of the 
earlier experimenters (Letzerich, Almquist, Maragliano) to accidental 
contamination and imperfect methods of research. The more recent 
work of Tayon does not inspire any greater confidence. This author ° 
obtained cultures in bouillon by inoculating it with blood drawn 
from a typhoid patient, and found that these were fatal, in a few 
hours, to guinea-pigs, when injected into the peritoneal cavity. The 
lesions observed are said to have resembled those of typhoid fever— 
congestion and tumefaction of Peyer’s plaques and of the mesenteric 
glands, congestion of the liver, the kidneys, etc. 

The presence of the bacillus of Eberth in the alvine evacuations of 
typhoid patients has been demonstrated by Pfeiffer and by Frankel 
and Simmonds. This demonstration is evidently not an easy mat- 
ter, for while the bacilli are probably always present in some portion 
of the intestine during the progress of the disease, it does not follow 
that they are present in every portion of the intestinal contents. As 
only a very small amount of material is used in making plate cul- 
tures, and as there are at all times a multitude of bacteria of various 
species in the smallest portion of fecal matter, it is not to be ex- 
pected that the typhoid bacillus will be found upon every plate. 
Frankel and Simmonds made eleven attempts to obtain the bacillus. 
by the plate method, using three plates each time, as is customary 
with those who adhere strictly to the directions of the master, and 
were successful in obtaining the bacillus in three instances—in two 
in great numbers and in the third in a very limited number of colo- 
nies. 

The numerous attempts which have been made to communicate 
typhoid fever to the lower animals have given a negative result in 
every instance. Murchison, in 1867, fed typhoid-fever discharges to 
swine, and Klein has made numerous experiments of the same kind 
upon apes, dogs, cats, guinea-pigs, rabbits, and white mice, without 
result. Birch-Hirschfeld, in 1874, by feeding large quantities of 
typhoid stools to rabbits, produced in some of them symptoms which 
in some respects resembled those of typhoid ; but these experiments 
were repeated by Bahrdt upon ten rabbits with an entirely negative 
result. Von Motschukoffsky met with no better success in his at- 
tempts to induce the disease by injecting blood from typhoid patients 
into apes, rabbits, dogs, and cats. Walder also experimented with 
fresh and with putrid discharges from typhoid patients, and with 
blood taken from the body after death, feeding this material to 
calves, dogs, cats, rabbits, and fowls, without obtaining any posi- 
tive results. Klebs has also made numerous experiments of a simi- 
lar nature, and in a single instance found in a rabbit, which died 


436 THE BACILLUS OF TYPHOID FEVER. 


forty-seven hours after receiving a subcutaneous injection of a cul- 
ture fluid containing his ‘typhoid bacillus,” pathological lesions re- 
sembling those of typhoid. 

Eberth and Gaffky very properly decline to attach any import- 
ance to this solitary case, in which, as the first-named writer re- 
marks, a different explanation is possible, and the possibility of an 
intestinal mycosis not typhoid in its nature must be considered. 

Gaffky has also made numerous attempts to induce typhoid 
symptoms in animals by means of pure cultures of Eberth’s bacillus, 
given with their food or injected into the peritoneal cavity or subcu- 
taneously. The first experiments were made upon five Java apes. 
For a considerable time these animals were fed daily with pure cul- 
tures containing spores. The temperature of the animals was taken 
twice daily. The result was entirely negative. No better success 
attended the experiments upon rabbits (16), guinea-pigs (13), white 
rats (7), house mice (11), field mice (4), pigeons (2), one hen and a calf. 

Cornil and Babes report a similar negative result from pure cul- 
tures of the typhoid bacillus injected into the peritoneal cavity and 
into the duodenum in rabbits and guinea-pigs. 

Frankel and Simmonds have made an extended series of experi- 
ments upon guinea-pigs, rabbits, and mice, and have shown that 
pure cultures of the bacillus of Eberth injected into the last-men- 
tioned animals—mice and rabbits—may induce death, and that the 
bacillus may again be obtained in pure cultures from their organs. 
It is not claimed that the animals suffer an attack of typhoid fever 
as the result of these injections, but that their death is due to the 
introduction into their bodies of the typhoid bacillus, and that this 
bacillus is thereby proved to be pathogenic. 


BACILLUS TYPHI ABDOMINALIS. 


Synonyms.—Bacillus typhosus ; Typhus bacillus. 

Eberth (1880 and 1881) demonstrated the presence of this bacillus 
in the spleen and diseased glands of the intestine in typhoid cada- 
vers. Gaffky (1884) first obtained it in pure cultures from the same 
source and determined its principal biological characters. 

It is found, in the form of small, scattered colonies, in the spleen, 
the liver, the glands of the mesentery, the diseased intestinal glands, 
and in smaller numbers in the kidneys, in fatal cases of typhoid fever; 
it has also been obtained, by puncture, from the spleen during life, 
from the alvine discharges of the sick, and rarely from the urine. 
It is not found in the blood of the general circulation, unless, pos- 
sibly, in rare cases and in small numbers. 

Morphology.—Bacilli, usually one tothree 4 inlength and about 


THE BACILLUS OF TYPHOID FEVER. 437 


0.5 to 0.8 » broad, with rounded ends; may also grow out into long 
threads, especially upon the surface of cooked potato. The dimen- 
sions of the rods differ considerably in different media. Spherical or 
oval refractive granules are often seen at the extremities of the rods, 
especially in potato cultures kept in the incubating oven; these are 
not reproductive spores, as was at first supposed. The bacilli have 
numerous flagella arranged around the periphery of the cells—usually 
from five to twenty, but many short rods have but a single 


SAS 
WN 


\\ 


WANS 

ANY AWRY 
UNG 

SS 


SHIZS 


Fie. 106, Fia. 107. 


Fic. 106.—Bacillus typhi abdominalis, from single gelatin colony. X 1,000. From a photo- 
micrograph. (Frinkel and Pfeiffer.) 

FiG. 107.—Bacillus typhi abdominalis, from single gelat‘n colony. X 1,000. From a photo- 
micrograph. (Sternberg.) 


terminal flagellum. These flagella are spiral in form, about 0.1 «in 
thickness, and from three to five times as long as the rods (Babes). 

In stained preparations unstained “‘ vacuoles” may often be seen 
at the margins of the rods, either along the sides or at the ends ; 
these appear to be due to a retraction of the protoplasm from the cell 
membrane. 

The typhoid bacillus stains with the aniline colors, but more 
slowly than many other bacteria, and easily parts with its color when 
treated with decolorizing agents—e.g., iodine solution as employed in 
Gram’s method. Léffler’s solution of methylene blue is an excellent 
staining agent for this bacillus, but permanent preparations fade out 
after a time ; fuchsin, gentian violet, or Bismarck brown, in aqueous 
solution, may also be used. The flagella may be demonstrated by 
Léffler’s method of staining (p. 32). 

To stain the bacillus in sections of the spleen, etc., itis best to 
leave these in Léffler’s methylene blue solution or in the carbol- 
fuchsin solution of Ziehl for twelve hours or more; or the aniline- 


438 THE BACILLUS OF TYPHOID FEVER. 


fuchsin solution may be used. The sections should be washed in 
distilled water only, when Ziehl’s solution is used, or with a very di- 
lute solution of acetic acid when Ehrlich’s tubercle stain is employed 


(Baumgarten). 


Fig. 108.—Bacillus typhi abdominalis. stained by Liffler’s method, showing flagella. x 1,000. 
From a photomicrograph by Frankel and Pfeiffer. 


Biological Characters.—The typhoid bacillus is a motile, aéro- 
bic, non-liquefying bacillus, which grows readily in a variety of 
culture media at the ‘‘room temperature.” Although it grows most 
abundantly in the presence of free oxygen, it may also develop in its 
absence, and is consequently a facultative anaérobic. 

In gelatin plate cultures small, white colonies are developed at 
the end of thirty-six to forty-eight hours, which under the microscope 


Fig. 109,—Single colony of Bacillus 
typhi abdominalig, jn nutrient gela- 
tin. (x?) From a photograph by 
Roux. 


are seen to be somewhat irregular in 
outline and of a spherical, oval, or long- 
oval form ; these have by transmitted 
light a slightly granular appearance and 
a yellowish-brown color. At the end of 
three or four days the colonies upon the 
surface of the gelatin form a grayish- 
white layer of one to two millimetres in 
diameter, with more or less irregular 
margins, and, when developed from deep 
colonies, with an opaque central nucleus. 
These colonies, by transmitted light, 
have a yellowish-brown color towards 
the centre, where they are thickest, 


while the margins are colorless and transparent ; the surface is com- 


THE BACILLUS OF TYPHOID FEVER. 439 


monly marked with a network of lines and furrows. Stab cultures in 
ten-per-cent gelatin, at 18° to 20° C., at the end of three days show 
upon the surface a whitish, semi-transparent layer, with sharply 
defined margins and irregular outline, which has a shining, pearly 
lustre; and along the line of puncture a gray- 
ish-white growth, made up of crowded colo- 
nies, which are larger and more distinct at the 
bottom of the line of growth. Upon nutrient 
agar, at a temperature of 35° to 37° C., the 
growth is more rapid and forms a whitish, 
semi-transparent layer. The cultures give off 
a faint putrefactive odor. The growth upon 
blood serum is rather scanty, in the form of 
transparent, shining patches along the line 
of inoculation. 

The typhoid bacillus develops abundantly 
in milk, in which fluid it produces an acid 
reaction; it also grows in various vegetable in- 
fusions and in bouillon. 

None of the above characters of growth are 
distinctive, as certain common bacilli found 
in normal faeces present a very similar appear- 
ance when cultivated in the same media. 

The growth of this bacillus upon potato is 
an important character, as was first pointed 
out by Gaffky. In the incubating oven at the 
end of forty-eight hours, or at the room tem- ®»dominalis; stick culture 

: in nutrient gelatin, eighth 
perature in three or four days, the surface of aay at 16°-20° C. (Baum- 
the potato has a moist, shining appearance, garten.) 
but there is no visible growth such as is produced by many other bac- 
teria upon this medium. <A simple inspection would lead to the belief 
that no growth had occurred; but if with a platinum needle a little ma- 
terial is scraped from any portion of the shining surface and a stained 
preparation is made from it, numerous bacilli will be seen, some of 
which are likely to be in the form of quite long threads, while others 
are short and have rounded extremities. This “‘invisible growth” 
has been shown by the researches of Buchner and others to be most 
characteristic upon potatoes having a decidedly acid reaction, as is 
usually the case. When cultivated upon potatoes having an alkaline 
reaction a thin, visible film of a yellowish-brown color and of limited 
extent may be developed. Inasmuch as several common and widely 
distributed bacteria closely resemble the typhoid bacillus in form and 
in their growth in nutrient gelatin, this character of invisible growth 


440 THE BACILLUS OF TYPHOID FEVER. 


upon potato is very important for its differentiation, especially as the 
common bacilli referred to—Bacillus coli communis, bacillus of Em- 
merich—produce a very distinct and rather thick, yellowish-white 
mass upon the surface of potato. But recent researches show that 
this invisible growth, although not a common character, does not 
belong exclusively to the typhoid bacillus (Babes). 

This bacillus in its development in culture media produces acids— 
according to Brieger small quantities of volatile fat acids, and, in 
presence of grape sugar, lactic acid. It also grows readily in a de- 
cidedly acid medium, and this character has been employed as a test 
for differentiating it from other similar bacilli; but some of these 
also grow in a decidedly acid medium, and too much reliance cannot 
be placed upon this test. 

Brieger has shown that indol is not produced in cultures of the 
typhoid bacillus, and Kitasato has proposed to use the indol test for 
differentiating this from other similar bacilli which are said, asa 
rule, to give the indol reaction. This test consists in the addition to 
ten cubic centimetres of a bouillon culture which has been in the in- 
cubating oven for twenty-four hours, of one cubic centimetre of a 
solution of sodium nitrite (0.02 gramme to one hundred cubic centi- 
metres of distilled water), together with a few drops of concentrated 
sulphuric acid. If indol is present a red color is developed. 

None of the above-mentioned tests are entirely reliable, but, taken 
together with the morphological and biological characters above de- 
scribed, they may enable the bacteriological expert to give a tolerably 
confident opinion as to the presence of this bacillus in a water supply 
suspected of contamination, ete. And when a bacillus having these 
characters is obtained in a pure culture from the spleen of a typhoid 
cadaver the student may be very sure that he has the typhoid bacillus. 
But in the presence of various similar bacilli, as in faeces, very careful 
comparative researches will be required to determine in a definite 
manner that a non-liquefying bacillus obtained in pure cultures by 
the plate method is really the one now under consideration—espe- 
cially so as the cultures of the typhoid bacillus in the same medium 
may differ considerably at different times, and a number of bacilli 
are known which resemble it so closely that it is still uncertain 
whether they are to be considered as varieties of the typhoid bacillus 
or as distinct species. Thus Babes, in an extended research, found in 
the organs of typhoid cases, associated with the true typhoid bacillus, 
other bacilli or varieties very closely resembling it. He has also 
described three varieties (?), obtained by him from other sources, 
which could only be differentiated from the true typhoid bacillus by 
very careful comparison of cultures made side by side in various 


media. 


THE BACILLUS OF TYPHOID FEVER. 441 


Cassedebat, also, in an extended examination of the river water 
at Marseilles with reference to the presence of the typhoid bacillus, 
found three species which very closely resembled it, but which by 
careful comparison were shown to present stight but constant dif- 
ferences in their biological characters. He was not able to find the 
true typhoid bacillus, and his researches, together with those of Babes 
and other recent investigators, make it appear probable that numerous 
mistakes have been made by bacteriologists who have reported the 
finding of the typhoid bacillus in river and well water, in faces, etc., 
and who have depended mainly upon the character of invisible 
growth upon potato in making their diagnosis. Cassedebat states 
that all three of his pseudo-typhoid bacilli corresponded in their 
growth upon potato with the bacillus of Eberth. They also corre- 
sponded in their growth on gelatin, agar-agar, and blood serum, 
which, as heretofore remarked, has no characteristic features. They 
all gave a negative indol reaction. Like the typhoid bacillus, they 
grew in milk without causing coagulation of the casein, but two of 
them produced an alkaline reaction in this fluid, while the third cor- 
responded with the typhoid bacillus in producing a decided acid re- 
action. Differences were also observed in bouillon cultures, and in 
bouillon and milk to which various aniline colors had been added, as 
recommended by Holz. 

Whether the typhoid bacillus, as obtained from the spleen of a 
typhoid cadaver, is in truth specifically distinct from these similar 
bacilli, or whether they are all varieties of the same species, result- 
ing from modifications in their biological characters acquired during 
their continuous development under different conditions, is an un- 
settled question. But, in view of the experimental evidence now 
available, there is nothing improbable in the supposition that they are 
simply varieties, and that, as the result of a saprophytic mode of 
life, this bacillus may undergo more or less permanent modifications. 

In the writer’s experiments (1887) the thermal death-point of the 
typhoid bacillus was found to be 56° C., the time-of exposure being 
ten minutes ; and potato cultures containing the refractive granules 
described by Gaffky as spores were found to be infallibly destroyed 
by a temperature of 60° C. This result has been confirmed by Buch- 
ner (1888) and by Janowsky (1890), and the inference seems justified 
that these granules are not reproductive bodies, as was at first be- 
lieved ; for spores are distinguished by their great resistance to heat 
and other destructive agencies. According to Buchner, the bacilli 
containing these refractive granules are even less resistant than fresh 
cultures in which they are not present, and he is disposed to look 
upon them as representing a degeneration of the protoplasm of the 
cells. They do not stain by the methods which are successful in 


442 THE BACILLUS OF TYPHOID FEVER. 


staining the spores of other bacilli, and, in short, present none of the 
characters which distinguish spores, except the form and high re- 
fractive power. 

The typhoid bacillus retains its vitality for many months in cul- 
tures; the writer has preserved bouillon cultures for more than a year 
in hermetically sealed tubes, and has found that development 
promptly occurred in nutrient gelatin inoculated from these. Dried 
upon a cover glass, it may grow in a suitable medium after having 
been preserved for eight to ten weeks (Pfuhl). When added to 
sterilized distilled water it may retain its vitality for more than four 
weeks (Bolton), (forty days Cassedebat), and in sterilized sea-water 
for ten days (De Giaxa). Added to putrefying faeces it may preserve 
its vitality for several months (Ufflemann), in typhoid stools for three 
months (Karlinski), and in earth upon which bouillon cultures had 
been poured for five and one-half months (Grancher and Deschamps). 

In hanging-drop cultures this bacillus may be seen to exhibit very 
active movements, the shorter rods rapidly crossing the field with a 
darting or to-and-fro, progressive motion, while longer filaments 
move in a serpentine manner. 

In addition to the volatile fat acids which, according to Brieger, 
are formed in small amounts in cultures of the typhoid bacillus, and 
to lactic acid formed in solutions containing grape sugar, a basic 
substance possessing toxic properties has been isolated by the chemist 
named—his typhotoxine (C,H,,NO,). Brieger supposes that other 
basic substances are likewise formed, but believes this to be the speci- 
fic product to which the pathogenic action of the bacillus is due. It 
is a strongly alkaline base, which produces in mice and guinea-pigs 
salivation, paralysis, dilated pupils, diarrhoea, and death. 

Numerous experiments have been made to determine the amounts 
of various germicidal agents required to destroy the vitality of this 
bacillus, and the action of antiseptics in restraining its development. 
For the results of these experiments the reader is referred to the 
sections in Part Second relating to the action of antiseptics and disin- 
fectants. 

Pathogenests.—The very numerous experiments which have been 
made on the lower animals have not been successful in producing in 
any one of them a typical typhoid process. Nor is this surprising, 
in view of the fact that, so far asis known, no one of them is liable to 
contract the disease, as man does, by the use of infected food or 
water. 

The experiments of Frankel and Simmonds show that when con- 
siderable quantities of a pure culture of this bacillus are injected into 
the circulation of rabbits through the ear vein, or into the peritoneal 
cavity of mice, a certain proportion of the inoculated animals die, 


THE BACILLUS OF TYPHOID FEVER.. 443 


usually within forty-eight hours, and that the bacillus may be re- 
covered from the various organs, although itis not present in the 
blood. But death does not always occur from intravenous injections, 
and subcutaneous or intraperitoneal injections in rabbits are usually 
without result. Subcutaneous injections in mice proved to be fatal in 
ten cases out of sixteen inoculated by A. Frankel. Seitz, by following 
Koch’s method—i.e., by rendering the contents of the stomach alka- 
line, and arresting intestinal peristalsis by the administration of 
opium—obtained a fatal result, in a majority of the guinea-pigs experi- 
mented upon, from the introduction of ten cubic centimetres of a 
bouillon culture into the stomach through a pharyngeal catheter. 
We may remark, with reference to these results, that while they show 
that cultures of the typhoid bacillus have a certain pathogenic power, 


Fic. 111.Section through wall of intestine, showing invasion by typhoid bacilli. x 950. 
(Baumgarten.) 


‘they also show that the animals experimented upon frequently re- 
covered after comparatively large doses, and that the typhoid bacil- 
lus is not pathogenic in the same sense as are those microérganisms 
which, when introduced into the body of a susceptible animal in very 
minute amount, give rise to general infection and death. On the 
other hand, a fatal result depends upon the quantity of the culture 
introduced in the first instance, rather than upon the multiplication 
of the bacillus in the body of the inoculated animal. This view is 
confirmed by the experiments of Sirotinin, which show not only that 
a fatal result depends upon the quantity injected, but also that a 
similar result follows the injection of cultures which have been ster- 
ilized by heat or filtration. The pathogenic action, then, depends 
upon the presence of toxic substances produced during the growth of 
the bacillus in artificial culture media. The researches of Brieger, 
heretofore referred-to, show the presence in such cultures of a toxic 
ptomaine—his typhotoxine—to which the pathozenic potency of these 


444 THE BACILLUS OF TYPHOID FEVER. 


cultures appear to bedue. White mice and guinea-pigs usually die 
in from twenty-four to forty-eight hours when inoculated in the 
cavity of the abdomen with a virulent culture of the typhoid bacillus 
—0.1 cubic centimetre to 0.5 cubic centimetre of a bouillon culture 
three days old. According to Kitasato, the virulence of cultures 
from different cases of typhoid fever varies considerably. 

Detection of the Typhoid Bacillus in Water.—The generally 
recognized fact that typhoid fever is usually contracted by drink- 
ing water contaminated by the typhoid bacillus has led to numer- 
ous researches having for their object the discovery of a reliable 
method of detecting this bacillus when present in water in compara- 
tively small numbers in association with the ordinary water bacilli. 
The use of Koch’s plate method, as commonly employed, will 
not suffice, because the water bacilli present grow more rapidly 
and cause liquefaction of the gelatin before visible colonies of the 
typhoid bacillus are formed ; and, owing to the relatively small 
number of typhoid bacilli, these are likely to escape detection. The 
aim of bacteriologists has, therefore, been to restrain the growth of 
these common water bacilli by some agent which does not at the 
same time prevent the development of the typhoid bacillus. Chan- 
temesse and Widal were the first to propose the use of carbolic acid 
for this purpose. They recommended the addition of 0.25 per cent 
of this agent to nutrient gelatin ; but, according to Kitasato, the de 
velopment of the typhoid bacillus is restrained by an amount exceed- 
ing 0.20 per cent. 

Holz prepares an acid medium by adding gelatin (ten per cent) to 
the juice of raw potatoes, and asserts that while the typhoid bacillus 
grows luxuriantly in this medium, many other bacilli fail to develop 
init. The test is said to be still more reliable if 0.05 per cent of car- 
bolic acid is added to the “‘ potato-gelatin.” According to Holz, the 
addition of more than 0.1 per cent of carbolic acid to nutrient gelatin 
prevents the free development of the typhoid bacillus. 

Thoinot has claimed to be able to obtain the typhoid bacillus from 
mixed cultures—as, for example, from feeces—by suspending a small 
amount of material containing it for several hours in a solution con- 
taining 0.25 per cent of carbolic acid. While other bacilli are 
destroyed, the typhoid bacillus is said to survive such exposure. 

The method of Partettt has been tested in a practical way by 
Kamen, and proved to be satisfactory for the detection of the typhoid 
bacillus in water which was supposed to be the source of a local 
epidemic of the disease. The following solution is used: 


Carbolic acid, ; - : . é : - 5 grammes, 
Hy drochloric acid (pure), : : . : 4 “ 
Distilled water, . 3 : ‘ : ‘ . 100 ae 


THE BACILLUS OF TYPHOID FEVER. 445 


Several test tubes, each of which contains ten cubic centimetres 
of neutral, sterilized bouillon, are used in the experiment. From 
three to nine drops of the acid solution are added to each of these, 
and the tubes are then placed in an incubating oven for twenty-four 
hours to ascertain whether they are still sterile after this addition. 
If the bouillon remains clear, from one to ten drops of the suspected 
water are added to each tube and they are returned to the incubating 
oven. If at the end of twenty-four hours the bouillon becomes 
clouded, this is due, according to Parietti, to the presence of the 
typhoid bacillus, which is then to be obtained in pure cultures by the 
plate method. 

The following method, suggested by Hazen and White, has been 
tested with favorable results by Foote. This method depends upon 
the fact that most of the common water bacilli do not grow at a tem- 
perature of 40° C., whereas this is a favorable temperature for the 
development of the typhoid bacillus. A small quantity of the sus- 
pected water is added to liquefied nutrient agar in test tubes, and 
plates are made. ‘These are placed in an incubating oven at 40° C., 
and the typhoid bacillus, if present, will develop colonies within two 
or threedays. At the ordinary room temperature the more numerous 
water bacilli would develop upon the same plates so abundantly that 
it would be difficult to recognize colonies of the typhoid bacillus. 

Theobald Smith (Centralb. f. Bakteriol., Bd. xii., page 367), 
has shown that the typhoid bacillus may be differentiated from other 
similar bacilli (Bacillus coli communis, bacillus of hog cholera, etc.) 
by the fact that it does not produce gas in culture media containing 
sugar—grape sugar, cane sugar, or milk sugar. The medium recom- 
mended by Smith for making this test is a peptone-bouillon contain- 
ing two per cent of grape sugar and made slightly alkaline with 
carbonate of soda. The liquid becomes clouded throughout at the 
end of twenty-four hours, but not a trace of gas is developed even 
after several days. On the other hand, the colon bacillus and other 
bacilli which closely resemble the typhoid bacillus cause an abundant 
development of gas in this medium. 

The method of Wurtz will be found useful in the detection of 
colonies of the typhoid bacillus in plate cultures from contaminated 
water, etc. This consists in the addition to the nutrient medium of 
lactose (two per cent) and a solution of litmus. When the colonies 
develop in plates made from this medium the typhoid colonies re- 
main blue, while colonies of the “colon bacillus” have a red color, on 
account of the development of lactic acid. 

Schild (1894) uses a bouillon containing formalin (1:7,000) and 
claims that the typhoid bacillus fails to grow in this medium, while 


446 THE BACILLUS OF TYPHOID FEVER. 


the bacilli of the colon group multiply in it and cause the me- 
dium to become clouded within twenty-four hours. Abel (1894), asa 
result of extended experiments, arrives at the conclusion that the 
formalin test cannot be relied upon for distinguishing the typhoid 
bacillus from certain similar bacilli, which also fail to grow in for- 
malin solution. But, on the other hand, a bacillus which grows in 
bouillon containing 1:7,000 of formalin can be definitely pronounced 
to be not the typhoid bacillus. 

Elsner (1895) recommends the following method for the detection 
of the typhoid bacillus in water or in feeces: To potato gelatin, pre- 
pared by the method of Holz, he added one per cent of potassium 
iodide. But few species of bacteria will grow in this medium, but 
Bacillus coli communis grows in it luxuriantly, forming fully de- 
veloped colonies at the end of twenty-four hours. The typhoid col- 
onies, on the contrary, are only just visible under a low power at the 
end of twenty-four hours, and at the end of forty-eight hours are 
seen as small, shining, drop-like, very finely granular colonies. At 
the same time the colonies of the colon bacillus are much larger, 
coarsely granular, and of a brownish color. By this method Elsner 
succeeded in obtaining pure cultures of the typhoid bacillus from 
the feeces in fifteen out of seventeen cases of typhoid fever, in various 
stages of the disease. Lazarus (1895) has tested this method and re- 
ports that he succeeded without any difficulty in obtaining pure cul- 
tures of the typhoid bacillus from the alvine discharges of typhoid 
patients. 

When the typhoid bacillus and the colon bacillus are planted to- 
gether, in the same liquid medium, the first-mentioned bacillus, even 
when in excess at the outset of the experiment, soon disappears and 
the Bacillus coli communis remains in full possession. According 
to Wathelet (1895) the colon bacillus will grow in bouillon which 
has served as a culture medium for the typhoid bacillus, or on the 
surface of an agar plate from which a typhoid culture has been re- 
moved; but the typhoid bacillus fails to develop in culture media 
which have served for the development of the colon bacillus. 

The various diagnostic tests which have been proposed, and the 
extensive literature of the subject, show that the recognition of the 
typhoid bacillus in water, feeces, etc., is attended with serious diffi- 
culties. This is chiefly due to the fact that bacilli have been ob- 
tained from various sources which resemble more or less closely the 
typical typhoid bacillus as obtained from the spleen of a typhoid 
patient (or cadaver) and the “colon bacillus” as found in the alimen- 
tary canal of healthy men and animals; and also from the fact that 
the bacillus, as obtained from typhoid cases, varies to some extent in 
its biological characters, and that varieties may be produced in the 


THE BACILLUS OF TYPHOID FEVER. 447 


bacillus as obtained, from a single colony, by special modes of culti- 
vation. From a consideration of these facts certain authors have 
been led to the conclusion that Bacillus typhi abdominalis and Bacillus 
coli communis are simply varieties of the same species. This view, 
however, is not generally acespted, and the characters which serve to 
differentiate the two bacilli are sufficiently well defined when typical 
cultures are compared. These characters, briefly stated, are: The 
invisible growth of the typhoid bacillus on potato; its failure to give 
the indol reaction; its failure to coagulate milk, or to produce a 
change of color in litmus milk; its failure to produce gas in culture 
media containing glucose or lactose; its failure to grow in formalin 
bouillon (1:7,000); and its active motility. Whether the closely re- 
lated bacilli which present some of the characters above indicated, 
without corresponding in all particulars with typical cultures of the 
typhoid bacillus, are varieties of this bacillus, which under favorable 
circumstances could give rise to typhoid infection, has not been defi- 
nitely determined, but appears to be quite probable. It may be that 
such varieties are developed when the typhoid bacillus in feeces finds 
its way into surface waters, under conditions which are favorable for 
its continued development as a saprophyte. On the other hand, it 
may be that one or more of the saprophytic bacilli, which are found 
in water and which closely resemble the typhoid bacillus, may give rise 
to the infectious disease which we know as typhoid fever when in- 
troduced into the alimentary canal of a particularly susceptible indi- 
vidual, and that the special conditions attending its development as 
a parasite give rise to certain modifications in its biological charac- 
ters of a more or less permanent kind. 

Frankland (1895), as aresult of extended experiments, has arrived 
at the conclusion that when the typhoid bacillus is cultivated for a 
long time in media which are more and more largely diluted with 
water, it acquires an increased ability to survive in river water. 

A predisposition to typhoid infection is established by various 
depressing agencies, such as inanition, overwork, mental worry, in- 
sanitary surroundings, etc. And there is considerable evidence in 
support of the supposition that exposure to the offensive gases 
given off from ill-ventilated sewers constitutes a predisposition to 
the disease. 

Experiments made by Alessi (1894), in the Hygienic Institute 
of the University of Rome, give support to this view. The ex- 
periments were made upon rats, guinea-pigs, and rabbits. The 
rats were confined in a close cage with perforated bottom, which was 
placed over the opening of a privy; the guinea-pigs and rabbits in 
similar cages having a receptacle below in which their own excreta 
was allowed to accumulate. The animals which breathed an atmo- 


448 THE BACILLUS OF TYPHOID FEVER. 


sphere vitiated in this way lost, after a time, their usual activity and 
became emaciated, although they continued to eat greedily. When 
these animals were inoculated with a small quantity of a culture of 
the typhoid bacillus (0.25 to 0.5 cubic centimetre) they died within 
from twelve to thirty-six hours. The same amount of the typhoid 
culture injected into control animals produced no injurious effect. In 
the animals which succumbed to typhoid infection there was found a 
hemorrhagic enteritis, increase in volume of Peyer’s glands and of the 
spleen, and typhoid bacilli in the blood, liver, and spleen. The char- 
acteristic appearances of typhoid infection were more pronounced in 
the rabbits and guinea-pigs than in rats. Similar experiments with 
Bacillus coli communis gave similar results. The time required to 
induce this predisposition for typhoid infection was from five to 
seventy-two days for the rats, seven to fifty-eight for the guinea- 
pigs, and three to eighteen for the rabbits. Alessi found that the 
susceptibility to infection diminished after a certain time, and sug- 
gests that in a similar way man may become habituated to breathing 
an atmosphere containing sewer gases. 

Pus-Production by Typhotd Bactlli._The literature relat- 
ing to the typhoid bacillus includes many observations as to its 
presence in accumulations of pus in various parts of the body—often 
ina pure culture. It has been found in a considerable number of 
cases of periostitis secondary to typhoid fever, in purulent syno- 
vitis, and in abscesses in various parts of the body. 

Dmochowski and Janowski (1895), as the result of a review of the 
literature and a painstaking experimental research, arrive at the con- 
clusion that even in abscesses, occurring in typhoid fever cases, in 
which only the pus cocci are found, it is probable that the typhoid 
bacillus originated the process resulting in abscess formation. They 
assert that the typhoid bacillus dies out in a comparatively short 
time in abscesses which are directly due to its presence, and that 
often it may be found in the abscess walls when its presence can no 
longer be demonstrated in the purulent contents of the abscess cavity. 


PLATE V. 


PATHOGENIC BACTERIA. 


Fie. 1.—Bacillus anthracis from cellular tissue of inoculated mouse. 
Stained with gentian violet. x 1,000. Photomicrograph by Frankel 
and Pfeiffer. 

Fre. 2.—Bacillus anthracis in section of liver of inoculated rabbit. 
Stained with Bismarck brown. x 250. Photomicrograph by Sternberg. 

Fie. 3.—Micrococeus gonorrhcee in gonorrheal pus. Stained with 
gentian violet. x 1,000. Photomicrograph by gaslight. (Sternberg.) 

Fre. 4.—Anthrax spores from a bouillon culture. Double-stained 
preparation—with carbol-fuchsin and methylene blue. x 1,000. Photo- 
micrograph by Frankel and Pfeiffer. 

Fie. 5.—Spirillum cholere Asiaticee from a culture upon starched 
linen at end of twenty-four hours. Stained with fuchsin. x 1,000. 
Photomicrograph by Frankel and Pfeiffer. 

Fic. 6.—Bacillus diphtheriz from colony upon an agar plate, twenty- 
four hours old. Stained with Léffler’s solution of methylene blue. x 
1,000. Photomicrograph by Frankel and Pfeiffer. 


PLATE ¥. 
STERNBERG’S BACTERIOLOGY. 


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Que ee SS 
"py: 
ay Aa ide 
a ee beatae) 
- oat 
‘ : 
poke ) ehh ste 
os tke RSS A 
° e S5 fe et 8 qquets 
o es e 
“= A foe ie Waaoak % *e 
abe, . 
. peers nto 
aes pee Mee ON 
5 Vie 
, "@ . 
e i . 
Fig. 4. 


PATHOGENIC BACTERIA. 


Xx. 
BACTERIA IN DIPHTHERIA. 


DIPHTHERIA is generally recognized by physicians as a specific 
infectious disease, and, owing to its wide prevalence and fatal char- 
acter, a precise knowledge of its etiology is of the greatest import- 
ance. Until, as a result of recent researches, this was determined, 
pathologists were in doubt as to whether diphtheria should be con- 
sidered as primarily a local infection, or whether the local manifesta- 
tions were secondary toa general systemic infection. But this question 
appears now to be definitely settled in favor of the former view. We 
have to-day a very precise knowledge of the specific infecting agent, 
and have evidence that it produces during its growth. a very potent 
toxic substance, the absorption of which from the seat of local infec- 
tion accounts in a satisfactory manner for the general symptoms of 
the disease, which are due to toxzemia and not to an invasion of the 
blood and tissues by the pathogenic microérganism producing it. 

Numerous researches by competent bacteriologists have failed to 
demonstrate the presence of bacteria in the blood of patients suffer- 
ing from diphtheria, but a variety of microdrganisms have been ob- 
tained in cultures from diphtheritic pseudo-membranes, and may be 
demonstrated by the microscopical examination of stained prepara- 
tions. Among these are the well-known pus organisms, and espe- 
cially the Streptococcus pyogenes, which appears to be very commonly 
present, and is perhaps the active agent in the production of certain 
forms of pseudo-diphtheria. But the malignant, specific diphtheria, 
so well known in this country and in Europe, has been demonstrated 
by the recent researches of bacteriologists to be due to a bacillus first 
recognized by Klebs in stained preparations of diphtheritic false 
membranes (1883), and cultivated and described by Léffler in 1884. 
In his first publication Léffler did not claim to have fully demon- 
strated the etiological relation of this bacillus, but this appears to be 
fully established by subsequent researches. 

In his first research Léffler studied twenty-five cases, and in the 
greater number of them found in stained preparations the bacil- 
lus previously described by Klebs. From six of these cases he 

29 


450 BACTERIA IN DIPHTHERIA. 


obtained it in pure cultures, and by inoculations in pigeons, chickens, 
rabbits, and guinea-pigs proved that it gave rise to a diphtheritic 
inflammation when inoculated into the mucous membrane of the 
trachea, conjunctiva, pharynx, or vagina. In a second communica- 
tion Léffler reported his success in finding the same bacillus in ten 
additional cases, and also that he had isolated from the same source 
a non-pathogenic bacillus which resembled it very closely. This 
pseudo-diphtheria bacillus has since been found by other bacteri- 
ologists (Von Hoffmann, Roux and Yersin), and it is uncertain 
whether it is to be considered a distinct species, or a non-pathogenic 
variety of the diphtheria bacillus as maintained by Roux and Yersin. 
But its occasional presence does not invalidate the very positive ex- 
perimental evidence relating to the specific pathogenic power of the 
true diphtheria bacillus. 

Léffler, in 1890, reviewed the evidence upon which this bacillus is 
now generally conceded by bacteriologists to be the specific infectious 
agent in true diphtheria. The following are the principal points in 
the demonstration : 

First.—It is found in all undoubted cases of diphtheria, In 
support of this we have the results of researches made by Léffler, 
Wyssokowitsch, D’Espine, Von Hoffmann, Ortmann, Roux and 
Yersin, Kolisko and Paltauf, Zarinko and Sérensen, who in nearly 
every case have demonstrated without difficulty the presence of this. 
bacillus. On the other hand, Prudden failed to find it in a series of 
twenty-four cases studied by him; but his own account of these 
cases indicates that they were not cases of true diphtheria. He says 
in a subsequent communication : 

“In view of the doubt existing among practitioners as. to whether all 
forms of pseudo-membranous inflammation should be called diphtheria or 
not, and with the purpose of making a wholly objective study, the writer 
distinctly stated at the outset of that paper that all the fatal cases of exten- 
sive pseudo-membranous laryngitis, as well as pharyngitis, should in his 
study be considered as cases of diphtheria. This left the question as to the 
propriety of establishing separate groups of pseudo-membranous inflamma- 
tion open and free from bias. It was distinctly stated, however, that six- 
teen out of the twenty-four cases occurred in a large asylum, in which 
measles and scarlet fever were prevalent during the period in which these 
studies were under way. Five other cases in another asylum were ex- 
posed to similar conditions.” 

In a subsequent series of “‘ twelve cases of fatal pseudo-mem- 
branous inflammation occurring in two children’s asylums, in which 
for many months there had been no scarlatina and no measles, and 
in which there was no complicating suppurative inflammation and 
no erysipelas,” Prudden (1890) obtained Léffler’s bacillus in cultures 
from eleven, and he says: 


‘‘We are now, it would seem, justified, as it did not appear to the writer 


BACTERIA IN DIPHTHERIA. 451 


that we were two years ago, owing to the large number of important re- 
searches which have been made in the interim, in saying that the name 
diphtheria, or at least primary diphtheria, should be applied, and exclusively 
applied, to that acute infectious disease, usually associated with a pseudo- 
membranous inflammation of the mucous membranes, which is primarily 
caused by the bacillus called Bacillus diphtheriz of Loffler.” 

With reference to the question as to how long after convalescence 
is estabiished the diphtheria bacillus may be present in the throat 
of an infected person, Léffler has made the following research (1890). 
In a typical case a bacteriological examination was made daily from 
the commencement until fourteen days after its termination. Fever 
disappeared on the fifth day, and the exudation had all disappeared 
on the sixteenth day. Up to this time the bacillus was daily ob- 
tained in cultures, and subsequently nearly every day up to the 
twenty-fifth—that is, for three weeks after the febrile symptoms had 
disappeared. Roux and Yersin have also obtained the bacillus in 
cultures from mucus scraped from the throats of convalescents sev- 
eral days after the disappearance of all evidence of the disease. 

Seconp. The Klebs-Léffler bacillus is found only in diph- 
theria.—In his earlier researches Léffler obtained the bacillus in a 
single instance from the mouth of a healthy child, and this fact led 
him to hesitate in announcing it as his conviction that it was the 
true cause of diphtheria. Butin extended researches made subse- 
quently he has not again succeeded in finding it, except in associa- 
tion with diphtheria, and admits now that he may have been mis- 
taken as to the identity of the bacillus found. This seems not 
improbable in view of the fact that very similar bacilli have been 
found by various bacteriologists. Thus Von Hoffmann obtained a 
very similar but non-pathogenic bacillus from the mucus of chronic 
nasal catarrh and from healthy mucous membranes; Babes from 
cases of trachoma, Neisser from ulcers, Zarinko from the surface of 
various mucous membranes. But all of these were shown to present 
certain differences in their biological characters by which they could 
be differentiated from the true diphtheria bacillus. 

Welch and Abbott in their comparative studies did not find the 
Léffler bacillus, “‘or any bacillus that an experienced bacteriologist 
would be likely to confound with it.” They examined mucus from 
the throats of healthy children, from those suffering from simple in- 
flammation of the tonsils and pharynx, and from four cases of so- 
called follicular tonsillitis. As a result of their investigations they 
agree with Léffler, and with Roux and Yersin, as to “the great prac- 
tical value, for diagnostic purposes, of a bacteriological examination 
of cover-glass specimens and by cultures” of cases in which there is 
any doubt of the true character of the disease. They say further : 


452 BACTERIA IN DIPHTHERIA. 


‘The only species of bacteria which we have found constantly in the 
cases of diphtheria has been the Léffler bacillus. Two other species have 
been present in many cases, viz., the well-known streptococcus, which grows 
in much smaller colonies and less rapidly than the Léffler bacillus, and a 
short, oval, often slightly pointed bacillus, growing in long chains running 
parallel to each other. ‘There are often marked irregularities in shape and 
especially in size of this bacillus, even of individuals in the same chain. 
The colonies of this bacillus are grayish-white, moist, larger than those of 
the streptococcus, but smaller than those of the Loffler bacillus.” 


THIRD. As shown by Liffler’s earlier researches, pure cultures 
of this bacillus induce characteristic diphtheritic inflammation 
when inoculated into the mucous membranes of certain lower ani- 
mals. Roux and Yersin have also shown that local paralysis is 
likely to occur in inoculated animals, as is the case in diphtheria in 
man. In speaking of their inoculations into the trachea in rabbits 
these investigators say : 


“‘The affection which is thus induced in the rabbit resembles croup in 
man. The difficulty which the animal experiences in breathing; the noise 
made by the air in passing through the obstructed trachea: the aspect of the 
trachea, which is congested and covered with false membranes; the cedema- 
tous swelling of the tissues and glands of the neck, make the resemblance 
absolutely remarkable.” 


Welch and Abbott give the following account of the results of 
inoculations into the trachea in kittens : 


‘‘A half-grown kitten is inoculated into the trachea with one platinum 
loop from a pure culture of the Loffler bacillus on glycerin-agar, eleven days 
old, derived from Case IV. For the inoculation a small median incision was 
made over the trachea, in which a hole just large enough to admit the plati- 
num loop was made. Theculture was rubbed over the mucosa of the trachea 
for an extent about three centimetresin length, and in this process sufficient 
force was used to abrade the mucous membrane. On the day following the 
inoculation no special alteration in the animal was observed, but on the 
morning of the second day it was found very weak. In the course of this 
day it became so weak as to lie completely motionless, apparently uncon- 
scious, with very feeble, shallow respiration ; several times it was thought to 
be dead, but on careful examination proved still to be breathing feebly. It 
was found dead on the morning of the third day. At the autopsy the wound 
was found gaping and covered with a grayish, adherent, necrotic, distinctly 
diphtheritic layer. For a considerable distance around the wound the sub- 
cutaneous tissues were very cedematous, the oedema extending from the 
lower jaw down over the sternum, and to the sides of the neck, and along 
the anterior extremities. Thelymphatic glands at the angle of the jaw were 
markedly swollen and reddened. The mucous membrane of the trachea, 
beginning at the larynx and extending down for six centimetres, was covered 
with a tolerably firm, grayish-white, loosely attached pseudo-membrane, in 
all respects identical with the croupous membranes observed in the same 
situation in cases of human diphtheria.” 


BACTERIA IN DIPHTHERIA. 453 


BACILLUS DIPHTHERI. 


First observed by Klebs (1883) in diphtheritic false membranes. 
Isolated in pure cultures and pathogenic power demonstrated by 
Léffler (1884). 

Found in diphtheritic pseudo-membranes, and especially in the 
deeper portions, intermingled with numerous cellular elements; while 
the superficial layers of the membrane commonly contain but few 
cells or bacilli, or are invaded by other species, especially by Strep- 
tococeus pyogenes. The bacilli are not found in the affected mucous 
membrane, or in sections from the internal organs in fatal cases of 
this disease. 

Morphology.—Rods, straight or slightly curved, with rounded 
ends, having a diameter of 0.5 to 0.8 
mM, and from 2 to 3 y/ in length. Iv- 
regular forms are very common, and, 
indeed, are characteristic of this bacil- 
lus. In the same culture, and especially 
in an unfavorable culture medium, very 
great differences in form and dimen- 
sions may be observed ; one or both ends 
may appear swollen, or the central por- 
tion may be notably thicker than the 
extremities, or the rod may be made up ane: 
of irregular spherical or oval segments. Fig. 112, — Bacillus diphtheria, 
Multiplication occurs by fission only, ‘om * sulture ‘upon blood serum. 
and the bacilli do not grow out into fila- eae ste 
ments. 

In unstained preparations certain portions of the rod, and espe- 
cially the extremities, are observed to be more highly refractive than 
the remaining portion ; and in stained preparations these portions 
are seen to be most deeply colored. The diphtheria bacillus may be 
stained by the use of Léffler’s alkaline solution of methylene blue, 
but is not so readily stained with some of the other aniline colors 
commonly employed. It stains also by Gram’s method. For the 
demonstration of the bacillus in sections of diphtheritic membrane 
“nothing can surpass in brilliancy and sharp differentiation sections 
stained doubly by the modified Weigert’s fibrin stain and picro-car- 
mine” (Welch and Abbott). 

Biological Characters.—The diphtheria bacillus is aérobic, non- 
motile, and non-liquefying; it does not form spores. It grows most 
freely in the presence of oxygen, butis also a facultative anaérobie. 

Development occurs in various culture media at a temperature of 
from 20° to 42° C., the most favorable temperature being about 35° C, 


454 BACTERIA IN DIPHTHERIA. 


It grows readily in nutrient gelatin having a slightly alkaline reac- 
tion, in nutrient agar, glycerin-agar, or in alkaline bouillon, but the 
most favorable medium appears to be that first recommended by 
Léffler—viz., a mixture of three 
parts of blood serum with one part 
of bouillon, containing one per cent 
of peptone, one per cent of grape 
sugar, and 0.5 per cent of sodium 
chloride. This mixture is steril- 
ized and solidified at a low tem- 
perature, as is usual with blood 
serum. Upon this the develop- 
Fic. 113,—Colonies of Bacillus diphtherz ment is so rapid in the incubating 
ty nutrient agar ond of emirfour POE. oven that, at the end of twenty- 
four hours, the large, round, ele- 
vated colonies, of a grayish-white color and moist appearance, may 
be easily recognized, while other associated bacteria will, as a rule, 
not yet have developed colonies large enough to interfere with the 
recognition of these. 

Upon nutrient agar plates the deep-lying colonies, when magni- 
fied about eighty diameters, appear as round or oval, coarsely granu- 
lar discs, with rather ill-defined margins, or, when several colonies 
are in juxtaposition, as figures of irregular form. The superficial col- 
onies are grayish-yellow in color, have an irregular, not well-defined 
outline and a rough, almost reticulated surface. The growth upon 
glycerin-agar is very similar. The first inoculations in a plain nu- 
trient agar tube often give a comparatively feeble growth, which be- 
comes more abundant in subsequent inoculations in the same medium. 
In stick cultures in glycerin—or plain—agar, growth occurs to the 
bottom of the line of inoculation, and also upon the surface, but is 
not at all characteristic. The same may be said with reference to 
cultures in nutrient gelatin. Plate cultures in this medium contain- 
ing fifteen per cent of gelatin, at 24° C., give rather small colonies, 
which are white by reflected light and under the microscope are seen 
as yellowish-brown, opaque discs, having a more or less irregular 
outline and a granular structure. In alkaline bouzllon the growth is 
sometimes in the form of small, whitish masses along the sides and 
bottom of the tube, but at others a diffusely clouded growth occurs 
in this medium ; after standing for some time in the incubating oven 
a thin, white pellicle may form upon the surface of the bouillon. 
The reaction of the bouillon becomes at first acid, but later it has an 
alkaline reaction (Welch). With reference to the growth on potato, 
authors have differed, probably because the growth is scarcely vis- 
ible ; upon this point we quote from Welch and Abbott : 


BACTERIA IN DIPHTHERIA. 455 


‘‘ Our experience has been that the Bacillus diphtherize grows on ordinary 
steamed potato without any preliminary treatment, but that the growth is 
usually entirely invisible or is indicated by a dry, thin glaze after several 
days. Doubtless the invisible character of the growth has led most observers 
into the error of supposing that no growth existed, whereas the microscopi- 
cal examination reveals a tolerably abundant growth, which on the first po- 
tato is often feebler than on succeeding ones. Irregular forms are par- 
ticularly numerous in potato cultures, and in general the rods are thicker 
than on other media. In twenty-four hours, at a temperature of 35° C., 
microscopical examination shows distinct growth. We have cultivated the 
bacillus for many generations on potato.” 


Milk is a favorable medium for the growth of this bacillus, and, 
as it grows at a comparatively low temperature (20° C.), it is evi- 
dent that this fluid may become a medium for conveying the bacillus 
from an infected source to the throats of previously healthy children. 

Cultures of the diphtheria bacillus may retain their vitality for 
several months, and when dried upon silk threads for several weeks 
colonies are still developed in a suitable medium—in the room from 
three to four weeks, in an exsiccator five to ten, and in one instance 
fourteen weeks. In dried diphtheritic membrane, preserved in small 
fragments, the bacillus retained its vitality for nine weeks, and in 
larger fragments for twelve to fourteen weeks. 

The thermal death-point, as determined by Welch and Abbott, is 
58° C., the time of exposure being ten minutes. Léffler had previ- 
ously found that it did not survive exposure for half an hour to 60° 
C. With reference to the action of germicidal and antiseptic agents, 
we refer to the sections in Part Second relating to this subject. 

Pathogenesis.—In view of the evidence heretofore recorded, it 
may be considered as demonstrated that this bacillus gives rise to 
the morbid phenomena which characterize the fatal disease in man 
known as diphtheria. 

We have already referred to the effects of inoculations into the 
trachea in rabbits and cats, which give rise to a characteristic diph- 
theritic inflammation, with general toxeemia and death from the 
absorption of soluble toxic products formed at the seat of local in- 
fection. This inference as to the cause of death seems justified by 
the fact that the pathogenic bacillus does not invade the blood and 
tissues, and is supported by additional experimental evidence (see 
pages 309-317). 


PSEUDO-DIPHTHERITIC BACILLUS. 


Léffler, Von Hoffmann, and others have reported finding bacilli 
which closely resemble the Bacillus diphtheriae, but which differ 
from it chiefly in being non-pathogenic. The following account we 


456 BACTERIA IN DIPHTHERIA. 


take from a paper upon the subject by Roux and Yersin (troisiéme 
mémoire, 1890). 

Found by Roux and Yersin in mucus from the pharynx and ton- 
sils of children—from forty-five children in Paris hospitals, suffering 
from various affections, not diphtheritic, fifteen times; from fifty- 
nine healthy children in a village school on the seaboard, twenty-six 
times. Of six children with a simple angina but two furnished cul- 
tures of this bacillus, while it was obtained in five out of seven cases 
of measles. 

Its characters are given as follows: 


‘* The colonies of the pseudo-diphtheritie bacillus, cultivated upon blood 
serum, are identical with the true diphtheria bacillus Ata temperature of 
33° to 85° multiplication is rapid, and it continues at the ordinary tempera- 
ture, although slowly. Under the microscope the appearance of the bacillus 
which forms these colonies is the same as that of Bacillus diphtheria. It 
stains readily with Loffler’s solution of methylene blue, and intensely by 
Gram’s method. Sometimes it colors uniformly, at others it appears granu- 
lar. It grows in alkaline bouillon, giving a deposit upon the walls of the 
vessel containing the culture, and in this medium often presents the inflated 
forms, pear-shaped, or club-shaped. It is destroyed in aliquid medium bya 
temperature of 58° C. maintained for ten minutes. All of these characters 
are common to the pseudo-diphtheritic bacillus and the true Bacillus diphthe- 
riz. Asa difference between them we may note that the pseudo diphtheritic 
bacillus is often shorter in colonies grown upon blood serum; thatitscultures 
in bouillon are more abundant; that they continue at a temperature of 20° to 
22°, at which the true bacillus grows very slowly. When we make a com- 
parison of cultures in bouillon they become acid and then alkaline, but the 
change occurs much sooner in the case of the pseudo-diphtheritic bacillus. 
Like the true bacillus, the pseudo diphtheritic grows in a vacuum, but less 
abundantly than the other. 

‘*Tnoculations into animals of cultures of this bacillus have never caused 
their death; but we may remark that in some experiments a notable edema 
has been produced in guinea-pigs at the point of inoculation, while in others 
there has been no local lesion. The most marked cedema resulted from cul- 
tures obtained from cases of measles. ; 

“ Do the facts which we have reported explain the question which occupies 
us? Can we conclude that there is a relation between the two bacilli? On 
the one side, the presence of the pseudo diphtheritic bacillusin the mouths of 
healthy persons, and of those who have anginas manifestly not diphtheritic, 
seems to be opposed to the idea of a relationship between them. On the 
other hand, when we consider that the non-virulent bacillus is very rare in 
fatal diphtheria, that itis more abundant in benign diphtheria, that it be- 
comes more common in severe cases as they progress towards recovery, and, 
finally, that they are more numerous in persons who have recently had 
diphtheria than in healthy persons, it is difficult to accept the idea that the 
two microbes are entirely distinct. The morphological differences which 
have been referred to are so slight that they prove nothing. The twomicro- 
organisms can only be distinguished by their action upon animals, but the 
difference of virulence does not at all correspond with the ditference of ori- 
gin. Asregards the form and the aspect of cultures, the true and false 
diphtheria bacilli differ less than virulent anthrax differs from avery attenu- 
ated anthrax bacillus, which, however, originate from the same source, 
Besides, the sharp distinction which we make between the virulent and non- 
virulent bacilli is arbitrary; it depends upon the susceptibility of guinea- 
pigs. If we inoculate animals still more susceptible, there are pseudo diph- 
theritic bacilli which we must class as virulent; and if, on the contrary, we 
substitute rabbits for guinea pigs in_our experiments, there are diphtheritic 
bacilli which we must call pseudo-diphtheritic. In our experiments we do 


BACTERIA IN DIPHTHERIA. 457 


not simply encounter bacilli which are very virulent and_bacilli which are 
non-virulent; between these two extremes there are bacilli of every degree 
of virulence.” 


Abbott, in 1891, published the result of his researches with 
reference to the presence of the pseudo-diphtheritic bacillus in 
benign throat affections. He made a bacteriological study of fifty- 
three patients, nine of whom were suffering from acute pharyngitis, 
fourteen from acute follicular tonsillitis, eight from ordinary post- 
nasal catarrh, two from simple enlarged tonsils, fifteen from chronic 
pharyngitis, one from subacute laryngitis, one from chronic laryngi- 
tis, one from rhinitis, and two from an affection of the tonsils and 
pharynx. In forty-nine cases nothing of particular interest was ob- 
served. A variety of microérganisms were isolated, and of these 
the pyogenic micrococci were the most common. 

In four cases microérganisms were found which resembled the 
Bacillus diphtheriz of Léffler in their morphology and growth in cul- 
ture media, but which proved not to be pathogenic. Abbott says : 
“The single point of distinction that can be made out between the 
organisms obtained from Cases I., III., and IV. and the true bacil- 
lus of diphtheria is in the absence of pathogenic properties from the 
former, whereas in addition to this point of distinction the organism 
from Case II. gives, as has been stated, a decided and distinct 
growth upon the surface of sterilized potato.” 

Recent authors are generally inclined to the opinion that bacilli 
which resemble the diphtheria bacilli in every respect except that 
they are non-pathogenic should be regarded as attenuated varieties 
of the diphtheria bacillus rather than as belonging to a distinct 
species—the so-called “ pseudo-diphtheria” bacillus. However, there 
are bacilli which closely resemble the bacillus of diphtheria and yet 
may be differentiated from it otherwise than by the test upon sus- 
ceptible animals. Neisser has given us a staining method which is 
especially useful in making this differential diagnosis. The culture 
of the bacillus to be tested is grown upon Loffler’s blood-serum mix- 
ture. This is solidified at a temperature of 100° C., and grown in 
an incubator at a temperature between 34° and 36°C. The staining 
of a cover-glass preparation from such a culture is effected by the 
following method: Methylene blue, one gramme; alcohol (96°), two 
cubic centimetres; dissolve and add distilled water, nine hundred and 
fifty cubic centimetres, and acetic acid, fifty cubic centimetres. From 
one to three seconds only will be required to stain the cover-glass 
preparation with this solution; it should then be carefully washed in 
water and stained in a solution made by adding two grammes of 
vesuvin to one litre of boiling water. This solution is allowed to 
cool before using, and from three to five seconds will be sufficient 


458 BACTERIA IN DIPHTHERIA. 


time for the action of the stain, after which the cover glass is again 
washed and is then ready for examination. The diphtheria bacillus 
appears in such a preparation as faintly stained brown rods, in the 
interior of which one to three Gark-blue granules may be seen. These 
are oval in form and are found at the extremities of the bacterial 
cells. Neisser and others who have made use of this method agree 
that bacilli which do not stain in this way are not diphtheria bacilli. 


BACILLUS DIPHTHERIZ COLUMBARUM. 


Described by Léffler (1884), who obtained it from diphtheritic pseudo-mem- 
branes in the mouths of pigeons dead from an infectious form of diphtheria 
which prevails in some parts of Germany among these birds and among 
chickens. 

Reddened patches first appear upon the mucous membrane of the mouth 
and fauces, and these are covered later with a rather thick, yellowish layer 
of fibrinous exudaie. In pigeons the back part of the tongue, the fauces, 
and the corners of the mouth are especially affected; in chickens the tongue, 
the gums, the nares, the larynx, and the conjunctival mucous membrane. 
The disease is especially fatal among chickens, the young fowls and those of 
choice varieties being most susceptible. It is attended at the outset by fever, 
and usually proves fatal within two or three weeks, but may last for several 
months. 

Morphology.—Short bacilli with rounded ends, usually associated in ir- 
regular masses, and resembling the bacilli of rabbit septicemia (fowl 
cholera), but a little longer and not quite so broad. In sections from the 
liver they are seen in irregular groups in the interior of the vessels. 

Biological Characters —An aérobic, non-motile, non-liquefying bacillus. 

Grows in nutrient gelatin in the form of spherical, white colonies along 
the line of puncture, and upon the surface as a whitish layer. Under the 
microscope the colonies in gelatin plates have a yellowish-brown color and 
a slightly granular surface. Upon blood serum the growth consists of a 
semi-transparent, grayish-white layer. Upon potato a thin layer is formed 
having a grayish tint. 

Pathogenesis.—Pigeons inoculated with a pure culture in the mucous 
membrane of the mouth are affected exactly as are those which acquire the 
disease naturally. Subcutaneous inoculations in pigeons give rise to an in- 
flammation resulting in local necrotic changes. Pathogenic for rabbits and 
for mice. Subcutaneous injections in mice give rise toa fatal result in about 
five days. The bacillus is found in the blood and in the various organs, in 
the interior of the vessels, and sometimes in the interior of the leucocytes; 
they are especially numerous in the liver. The lungs are dotted with red 
spots, the spleen is greatly enlarged, and the liver has a marbled appearance 
from the presence of numerous irregular white masses scattered through the 
pale-red parenchyma of the organ. These white masses are seen, in sec- 
tions, to consist of necrotic liver tissue, In the centre of which the bacilli 
are found in great numbers, in the interior of the vessels. This appearance 
is so characteristic that Ldffler considers inoculations in mice to be the most 
reliable method of establishing the identity of the bacillus. Not pathogenic 
for chickens, guinea-pigs, rats, or dogs. 

There seems to be some doubt whether the form of diphtheria which pre- 
vails among pigeons, and which Loffler has shown to be due to the bacillus 
above described, is identical with the diphtheria of chickens. Diphtheria in 
man has been supposed by some authors to be identical with that which 
prevails among fowls, and_possibly this may be the case under certain cir- 
cumstances. But the evidence seems to be convincing that there is an 


BACTERIA IN DIPHTHERIA. 459 


infectious diphtheria of fowls which is peculiar to them, and which, under 
ordinary circumstances, is not communicated to man. 


BACILLUS DIPHTHERIZ VITULORUM. 


Described by Léffler (1884) and obtained by him from the pseudo-mem- 
branous exudation in the mouths of calves suffering from an infectious form 
of diphtheria. The disease is characterized by the appearance of yellow 
patches upon the mucous membrane of the cheeks, the gums, the tongue, 
and sometimes of the larynx and nares of infected animals. There is a yel- 
lowish discharge from the nose, an abundant flow of saliva, occasional at- 
tacks of coughing, and diarrhoea. Death may occur at the end of four or 
five days, but usually the animal survives for several weeks. Diphtheritic 
patches similar to those in the mouth are also found in the large intestine, 
and scattered abscesses in the lungs. 

Léffler, in a series of seven cases examined, obtained from the deeper por- 
tions of the pseudo-membranous deposit a long bacillus which appears to be 
the cause of the disease. 

Morphology.—Bacilli, five to six times as long as broad, usually united in 
long filaments. The diameter of the rods is about half that of the bacillus 
of malignant cedema. 

Biological Characters.— Attempts to cultivate this bacillus in nutrient 
gelatin, blood serum from sheep, and various other media were unsuccessful. 
But when fragments of tissue containing the bacillus were placed in blood 
serum from the calf a whitish border, consisting of the long bacilli, was de- 
veloped. These could not, however, be made to grow when transferred to 
fresh blood serum. 

Pathogenesis.— Mice inoculated subcutaneously with the fresh diph- 
theritic exudation died in from seven to thirty days. The autopsy disclosed 
an extensive infiltration of the entire walls of the abdomen, which often pene- 
trated the peritoneal cavity and enveloped the liver, the kidneys, and the 
intestine*in a yellowish exudate. The bacillus was found in this exudate, 
and by inoculating a little of it into another animal of the same species a 
similar result was obtained. Not pathogenic for rabbits or guinea-pigs. 


BACILLUS OF INTESTINAL DIPHTHERIA IN RABBITS. 


Described by Ribbert (1887) and obtained by him from the organs of rab- 
bits which succumbed to an affection characterized by a diphtheritic inflam- 
mation of the mucous membrane of the intestine. The autopsy revealed also 
swelling of the mesenteric glands and minute necrotic foci in the liver and 
spleen. 

e Morphology.—Bacilli with slightly rounded ends, from three to four /# 
long and 1 to 1.4 in diameter; often united in pairs or in filaments con- 
taining several elements. 

Stains with the aniline colors, but not so readily in sections as some 
other microdrganisms. Ribbert recommends staining with aniline-water- 
fuchsin solution, washing in water, then placing the sections in methylene 
blue solution, and decolorizing in alcohol. Does not stain by Gram’s 
method. 

Biological Characters.—An aérobic, non-liquefying (non-motile ?) ba- 
cillus. Upon gelatin plates semi-transparent, grayish colonies are formed 
which later have a brownish color; the surface of these is finely granular 
and of a pearly lustre. In stick cultures in nutrient gelatin the growth 
along the line of puncture is very scanty. On potato a flat, whitish layer is 
formed, which extends slowly over the surface. Grows best at a temperature 
of 30° to 35° C. 

Pathogenesis.—Pure cultures injected into the peritoneal cavity or sub- 
cutaneously in rabbits caused the death of these animals in from three to 
fourteen days, according to the quantity injected. At the autopsy necrotic 


460 BACTERIA IN DIPHTHERIA. 


foci are found in the liver and spleen, and the mesenteric glands are en- 
larged, but the intestine presents a healthy appearance. But when cultures 
ave introduced into the alimentary canal the characteristic diphtheritic in- 
flammation of the mucous membrane of the intestine is induced. This re- 
sult was obtained both by direct injection into the lumen of the intestine 
and by injecting cultures into the mouth. 


Additional Notes upon Diphtheria and the Diphtheria Bacil- 
lus.—C. Frankel (1895) reports that he has repeatedly observed 
branching forms of the diphtheria bacillus in cultures upon Léf- 
fler’s blood-serum medium, and that these branching forms are seen 
more constantly and in greater numbers in cultures made upon the 
surface of hard-cooked albumen from hen’s eggs. 

The continued presence of virulent diphtheria bacilli in the fauces 
of patients who have recovered from the disease, either after the use 
of the antitoxin or under other treatment, has been demonstrated by 
several bacteriologists. Silverschmidt (1895), in forty-five cases 
treated by Behring’s antitoxic serum, found that the number of ba- 
cilli usually diminished some days after the treatment was com- 
menced, but that in cases in which complete recovery had taken 
place not infrequently virulent bacilli could be obtained many days 
(in one case thirty-one days) after convalescence was established. 

Escherich (1893) opposes the view that the pseudo-diphtheria bacil- 
lus is simply a non-virulent variety of the diphtheria bacillus. He 
found this pseudo-diphtheria bacillus in the throats of thirteen out 
of three hundred and twenty individuals examined. According to 
him there is no evidence that this completely non-virulent pseudo- 
diphtheria bacillus ever acquires pathogenic virulence, while attenu- 
ated varieties of the true diphtheria bacillus readily recover their 
power to produce the toxic products upon which virulence depends. 

Sevestre (1895), as a result of researches made by himself and 
several other bacteriologists who have made similar investigations, 
arrives at the conclusion that: 

“First. In a certain number of cases the bacillus of Léffler disap- 
pears about the same time as the false membranes; or it may persist 
for some time, but ceases to be virulent—in this case it seems to have 
undergone modifications and presents the form of short bacilli. . . . 

“Second. In another series of cases, less numerous but neverthe- 
less considerable, the bacillus persists in a virulent condition for a 
longer or shorter time after the apparent cure of the malady. . 

“Third. The observations collected up to the present time do not 
enable us to fix precisely the limits of persistence, but it is not far 
out of the way if we place it at several weeks to a month for the 
throat. In the nasal fossee the bacillus often persists for a still 
longer time, and its presence commonly coincides with a more or less 
abundant discharge from the nose.” 


BACTERIA IN DIPHTHERIA. 461 


Park and Beebe (1894), in an extended research made for the pur- 
pose of determining the persistence of the diphtheria bacillus in the 
throats of convalescents (2,566, cultures made), found that in 304 out 
of 605 consecutive cases the bacillus disappeared within 3 days after 
the disappearance of the exudate; in 176 cases it persisted for 7 days; 
in 64 cases for 12 days; in 36 cases for 15 days; in 12 cases for 3 
weeks; in 4 cases for 4 weeks; in 2 cases for 9 weeks. Park and 
Beebe arrive at the following conclusion with reference to pseudo- 
diphtheria bacilli: 

“The name pseudo-diphtheria bacillus should be regarded as ap- 
plying to those bacilli found in the throat which, though resembling 
the diphtheria bacilli in many respects, yet differ in others equally im- 
portant. These bacilli are rather short, and more uniform in size 
and shape than the typical Léffler bacillus. They stain equally 
throughout with the alkaline ‘methyl-blue solution, and produce 
alkali in their growths in bouillon. They are found in about one 
per cent of the healthy throats in New York City, and seem to have 
no connection with diphtheria. They are never virulent.” 

Park (1894) has shown that virulent diphtheria bacilli are fre- 
quently found in the throats of persons who have been associated 
with diphtheria patients, although no manifestations of the disease 
were visible. It is therefore apparent that infection requires not 
only the presence of virulent bacilli, but also of a predisposition to 
the disease. This corresponds with the facts relating to other in- 
fectious diseases—e.g., tuberculosis, typhoid fever—and among’ the 
probable predisposing causes we may mention “sewer-gas poisoning,” 
catarrhal inflammations of the mucous membranes most commonly 
involved, inanition, “crowd poisoning,” and depressing agencies 
generally. 

Bacteriologists have given much attention to the question of mixed 
infection in diphtheria. Funck (189+) accepts the generally received 
view that mixed infections with the diphtheria bacillus and Strepto- 
coccus pyogenes are more serious than an uncomplicated diphtheria, 
and in an experimental research has attempted to determine whether 
this is due to an increased production of the diphtheria bacillus or to 
the presence of the streptococcus. His experiments on guinea-pigs 
showed that when infected with streptococci these animals did not 
prove to be more sensitive to the action of the diphtheria poison 
(without living bacilli), and he concludes that the unfavorable influ- 
ence of the streptococcus in mixed infections is due to increased patho- 
genic activity on the part of the diphtheria bacillus. Bernheim 
(1894) found, in his experiments on guinea-pigs, that they suc- 
cumbed more rapidly to diphtheria infection when they previously 


462 BACTERIA IN DIPHTHERIA. 


or simultaneously received an injection of a streptococcus culture— 
filtered or unfiltered. 

Results of Treatment with the Antitoxin.—While questions re- 
lating to therapeutics are not considered in this manual, a brief note 
upon the results of treatment by the serum of immunized animals 
may not be out of place. A collective investigation (1895) un- 
‘dertaken by the Deutsche medicinische Wochenschrift gave the 
following results: The number of cases collected was 10,312; all of 
these occurred between the 1st of October, 1894, and the Ist of April, 
1895; 5,883 of these cases were treated with the antitoxin and 4,479 
without it. In the first group the mortality was 9.6 per cent, and in 
the second group 14.7 per cent. Two thousand five hundred and fifty 
six children treated with the antitoxin were between two and ten 
years of age; among these the mortality was 4 per cent, while 
among children of the same age not treated with the antitoxin the 
mortality was 15.2 per cent. Six hundred and ninety-six patients 
above ten years of age were treated with a mortality of 1 per cent. 

Monod (1895), at a meeting of the Paris Academy of Medicine, 
presented the following statistics demonstrating the influence upon 
the mortality from diphtheria in France exerted by the antitoxin 
since its employment from November, 1894. The following figures 
represent the number of deaths from diphtheria during the first six 
months in eight years in 108 French cities having a population of 


more than 20,000: 
1888-94, 1895. 


Average. Average. 
SABUALY sec te sso 4G. Hise Wee Lea eter eed on Se as 469 205 
Februar yiiis: ccs. ca viwseieay 1220405 50s Mee ewe we see es 466 187 
Mareh:. $5.00 civ cnkve So PS a08 MA OHRSR AE owe eoN ES oe 155 
SPT ivat deep snesei pod 8ctek soaks euwtoine Wiedaspoe © aigus lage ay woes 44 160 
AMER Yiscde< cosshsrie ca patcvavavanecaiaan nsavaunronse sedgcan boo: exbsora te cousebusteonisesce ata teasss A17 1138 
UG a sesork jak a diacsnaava wai aavaveseesaia a3 Cah ava satrap Racal. 333 84 

2,656 904 


It will be seen from the above statement that during the first six 
months in the year 1895 after the introduction of the antitoxin treat- 
ment, the number of deaths from diphtheria in the 108 French cities 
referred to was 1,552 less than the average for the preceding ten 
years, and we are justified in concluding that a considerable propor- 
tion of this saving at least is due to this method of treatment. 


XI. 


BACILLUS OF INFLUENZA. 


DISCOVERED by Pfeiffer (1892) in the purulent bronchial secretion, 
and by Canon in the blood of patients suffering from epidemic in- 
fluenza. Pfeiffer found the bacillus in thirty-one cases examined by 
him, and in uncomplicated cases it was present in the purulent bron- 
chial secretion in immense numbers and in a pure culture. Canon, 
whose independent observations were published at the same time, 
examined the blood of twenty influenza patients in stained prepara- 
tions, and found the same bacillus in nearly all of them. His method 
of demonstrating it is as follows: 

The blood is spread upon clean glass covers in the usual way. 
After the preparations are thoroughly dry they are placed in abso- 
lute alcohol for five minutes. They are then transferred to the fol- 
lowing staining solution (Czenzynke’s): concentrated aqueous solu- 
tion of methylene blue, forty grammes ; one-half-per-cent solution of 
eosin (dissolved in seventy-per-cent alcohol), twenty grammes ; dis- 
tilled water, forty grammes. The cover glasses immersed in this 
staining solution are placed in an incubating oven at 37° C. for from 
three to six hours, after which they are washed with water, dried, 
and mounted in balsam. In successful preparations the red blood 
corpuscles are stained red by the eosin, and the leucocytes blue. The 
bacillusis seen in these as ashort rod, often resembling a diplococcus. 
It is sometimes seen in large numbers, but usually only a few rods 
are seen after a long search—four to twenty in a single preparation. 
In six cases it was found in numerous aggregations containing from 
five to fifty bacillieach. In these cases the blood was drawn during 
a fall of temperature or shortly after. 

Morphology.—Very small bacilli, having about the same diameter 
as the bacillus of mouse septicaemia, but only half as long. Solitary 
or united in chains of three or four elements. 

Stains with difficulty with the basic aniline dyes—best with 
dilute Ziehl’s solution, or Léffler’s methylene blue solution, with heat. 
The two ends of the bacilli are most deeply stained, causing them to 
resemble diplococci. Pfeiffer says: ‘‘I am inclined to believe that 
some of the earlier observers also saw the bacilli described by me, 
but that, misled by their peculiar behavior with regard to staining 
agents, they described them as diplococci or streptococci.” Do not 
stain by Gram’s method. 


464 BACILLUS OF INFLUENZA. 


Biological Characters.—An aérobic, non-motile bacillus. Does 
not grow in nutrient gelatin at the room temperature. Spore forma- 
tion not observed. Upon thesurface of glycerin-agar in the incubat- 
ing oven very small, transparent, drop-like colonies are developed at 
the end of twenty-four hours. These can only be recognized by the 
aid of alens. ‘‘A remarkable point about them is that the colonies 
always remain separate from each other, and do not, as all other 
species known to me do, join together and form a continuous row. 
This feature is so characteristic that the influenza bacilli can be 
thereby with certainty distinguished from other bacteria ” (Kitasato). 
On 1.5 per cent sugar-agar the colonies appear as extremely small 
droplets, clear as water, often only recognizable with a lens 
(Pfeiffer). 

In bourlion a scanty development occurs, and at the end of twen- 
ty-four hours small, white particles are seen upon the surface, which 
subsequently sink to the bottom, forming a white, woolly deposit, 
while the bouillon above remains transparent. This bacillus does 
not grow at temperatures below 28° C, 

Canon has obtained colonies, resembling those described by Kita- 
sato, in cultures from the blood of influenza patients. His cultures 
were made upon glycerin-agar in Petri’s dishes. Tenor twelve drops 
of blood from a puncture made in the finger of the patient, after 
sterilization of the surface, were allowed to fall upon the agar medium, 
and this was placed in the incubating oven. As the number of ba- 
cilli in the blood is small, a considerable quantity is used. The 
colonies are visible at the end of twenty-four to forty-eight hours. 

The influenza bacillus is quickly destroyed by desiccation; a 
pure culture diluted with water and dried is destroyed with cer- 
tainty in twenty hours; in dried sputum the vitality is retained 
somewhat longer, but no growth occurs after forty hours. The 
thermal death-point is 60° C. with five minutes’ exposure (Pfeiffer 
and Beck). 

Pathogenesis.—Pfeiffer infers that this is the specific cause of 
influenza in man for the following reasons : 

1. They were found in all uncomplicated cases of influenza ex: 
amined, in the characteristic purulent bronchial secretion, often in 
absolutely pure cultures. They were frequently situated in the pro- 
toplasm of the pus corpuscles ; in fatal cases they were found to 
have penetrated from the bronchial tubes into the peribronchitic tis- 
sue, and even to the surface of the pleura, where in two cases they 
were found in pure cultures in the purulent exudation. 

2. They were only found in cases of influenza. Numerous con- 
trol experiments proved their absence in ordinary bronchial ca 


tarrh, etc. 


PLATE VI. 


PATHOGENIC BACTERIA. 


Fig. 1.—Bacillus of influenza in bronchial mucus. x 1,000. Photo- 
micrograph by Frankel. 

Fig. 2.—Bacillus of influenza in bronchial mucus, after the termina- 
tion of the febrile period. The bacilli are for the most part in pus cells. 
x 1,000. Photomicrograph by Frankel. 

Fie. 3.—Bacillus tetani from an agar culture. x 1,000. Photo- 
micrograph by Frankel and Pfeiffer. 

Fic. 4.—Micrococcus pneumonize croupose in sputum of a patient 
with pneumonia. x 1,000. Stained by Gram’s method. Photomicro- 
graph by Frankel and Pfeiffer. 

Fie. 5.—Mierococeus pneumonie croupose in blood of rabbit. x 
1,000. Photomicrograph made at the Army Medical Museum, Washing- 
ton, by Gray. 

Fic. 6.—Bacillus of hog cholera, showing flagella. Stained by 
Loffler’s method. x 1,000. Photomicrograph made at the Army Medi- 
cal Museum, Washington, by Gray. 


PLATE VI. 
STERNBERG’S BACTERIOLOGY, 


PATHOGENIC BACTERIA. 


BACILLUS OF INFLUENZA. 465: 


3. The presence of the bacilli corresponded with the course of the 
disease, and they disappeared with the cessation of the purulent 
bronchial secretion. 

In his preliminary report of his investigations Pfeiffer says : 

“Numerous inoculation experiments were made on apes, rabbits, 
guinea-pigs, rats, pigeons, and mice. Only in apes and rabbits 
could positive results be obtained. The other species of animals 
showed themselves refractory to influenza.” 

Kruse (1894) reports that he found the bacillus of Pfeiffer in 
eighteen influenza patients examined by him in the hospital at Bonn. 
On the other hand, he failed to find it in a considerable number of pa- 
tients suffering from other diseases of the respiratory passages. His 
evidence is the more valuable as he had previously (1890) reported 
his failure to find the bacillus in typical cases of influenza. He now 
ascribes his failure at that time to imperfect technique. 

Huber (1893), Richter (1894), Borchardt (1894), and other com- 
petent bacteriologists, have also confirmed the results reported by 
Pfeiffer as regards the presence of this bacillus in the bronchial 
secretions of persons suffering from epidemic influenza, and as to 
its biological characters. Bujwid (1893) recognizes the bacillus of: 
Pfeiffer as identical with a bacillus which he cultivated from the 
spleen of an influenza patient in 1890. 

The researches of Weichselbaum, Kowalski, Friedrich, Kruse, 
Bouchard, and others have given a negative result as regards the 
presence of the influenza bacillus in the blood. They were not able 
to demonstrate its presence either in stained preparations or by cul- 
ture methods. Pfeiffer, also, during the last epidemic, has made 
special researches upon this point and has never succeeded in finding 
the bacillus. Day after day, both in mild and severe cases, he placed 
from ten to twenty drops of blood from influenza patients on blood- 
agar—a most favorable medium—but his cultures always remained 
sterile. 

In his experiments upon rabbits, Pfeiffer (1893) found that the 
intravenous injection of a small quantity of culture on blood-agar, 
twenty-four hours old, suspended in one cubic centimetre of bouillon, 
caused a characteristic pathogenic effect. The first symptoms were 
developed within one and a half to two hours after the injection. 
The animals became extremely feeble, lying flat upon the floor with 
their limbs extended, and suffered from extreme dyspncea. The tem- 
perature mounted to 41°C. or above. At the end of five or six hours 
they were able to sit upon their haunches again, and in twenty-four 
hours had nearly recovered from all indications of ill-health. Larger 
doses caused the death of the inoculated animals. These results are 
due to toxic products present in the cultures, and Pfeiffer has never 

30 


466 BACILLUS OF INFLUENZA. 


observed a septiceemic infection as a result of his inoculation ex- 
periments. 

Pfeiffer has found in three cases of bronchopneumonia a pseudo- 
influenza bacillus which closely resembles the bacillus previously de- 
scribed by him as peculiar to that disease. This pseudo-influenza 
bacillus resembles the genuine one in its growth in culture media, 
but is larger and shows a decided inclination to grow out into long 
threads. By these morphological characters, which are said to be 
constant, it may, according to Pteiffer, be readily distinguished. 


XII. 


BACILLI IN CHRONIC INFECTIOUS DISEASES. 


In tuberculosis, leprosy, glanders, and syphilis we have a group 
of infectious diseases which present many points of resemblance. 
Allrun a chronic course; all may be communicated to susceptible 
animals by inoculation ; in all, the lymphatic glands in the vicinity 
of the point of inoculation become enlarged, and new growths, con- 
sisting of various cellular elements of a low grade of vitality, are de- 
veloped in the tissues which are the point of predilection for each ; 
in all, these new growths show a tendency to degenerative changes, 
as a result of which abscesses, caseous masses, or open ulcers are 
formed. 

In two of the diseases in this group—tuberculosis and glan- 
ders—the infectious agent has been obtained in pure cultures and its 
specific pathogenic power demonstrated by inoculations in susceptible 
animals; in one—leprosy—there is but little doubt that the bacillus con- 
stantly found in the new growths characteristic of the disease bears 
an etiological relation to it, although this has not been demonstrated, 
the bacillus not having as yet been cultivated in artificial media. 
The evidence with reference to the parasitic nature of the fourth dis- 
ease mentioned as belonging to this group—syphilis—is still unsatis- 
factory, but there is every reason to believe that it will also eventu- 
ally be proved to be due to a parasitic microérganism. 

The announcement of the discovery of the tubercle bacillus was 
made by Koch, in March, 1882, at a meeting of the Physiological 
Society of Berlin. At the same time satisfactory experimental evi- 
dence was presented as to its etiological relation to tuberculosis in 
man and in the susceptible lower animals, and its principal biologi- 
cal characters were given. 

Baumgarten independently demonstrated the presence of the tu- 
bercle bacillus in tuberculous tissues and published the fact soon 
after the appearance of Koch’s first paper. The previous demonstra- 
tion by Villemin (1865)—confirmed by Cohnheim (1877) and others— 
that tuberculosis might be induced in healthy animals by inocula- 
tions of tuberculous material, had paved the way for his discovery, 


468 BACILLI IN CHRONIC INFECTIOUS DISEASES. 


and advanced pathologists were quite prepared to accept it. The 
more conservative have since been obliged to yield to the experi- 
mental evidence, which has received confirmation in all parts of the 
world. To-day it is generally recognized that tuberculosis is a spe- 
cific infectious disease due to the tubercle bacillus. 

As evidence of the thorough nature of Koch’s personal researches 
in advance of his first public announcement, we give the following 
résumé of his investigations : 

In nineteen cases of miliary tuberculosis the bacilli were found in 
the tubercular nodules in every instance ; also in twenty-nine cases 
of pulmonary phthisis, in the sputum, in fresh cheesy masses, and in 
the interior of recently formed cavities; in tuberculous ulcers of the 
tongue, tuberculosis of the uterus, testicles, etc. ; in twenty-one cases 
of tuberculous—scrofulous—lymphatic glands ; in thirteen cases of 
tuberculous joints ; in ten cases of tubercular bone affections ; in four 
cases of lupus; in seventeen cases of Perlsucht in cattle. His ex- 
perimental inoculations were made upon two hundred and seventy- 
three guinea-pigs, one hundred and five rabbits, forty-four field 
mice, twenty-eight white mice, nineteen rats, thirteen cats, and upon 
dogs, pigeons, chickens, etc. Very extensive comparative researches 
were also made, which convinced him that the bacillus which he had 
been able to demonstrate in tuberculous sputum and tissues by a spe- 
cial mode of staining was not to be found in the sputa of healthy 
persons, or of those suffering from non-tubercular pulmonary affec- 
tions, or in organs and tissues involved in morbid processes of a 
different nature. 


BACILLUS TUBERCULOSIS. 


Discovered by Koch (first public announcement of discovery 
March 24th, 1882). The bacilli are found in the sputum of persons 
suffering from pulmonary or laryngeal tuberculosis, either free or in 
the interior of pus cells; ey miliary tubercles and fresh caseous 
masses, in the lungs or elgewhere ; in recent tuberculous cavities in 
the lungs ; in tuberculous glands, joints, bones, and skin affections 
(lupus) ; in the lungs of cattle suffering from pulmonary tubercu- 
losis—Perlsucht ; and in tubercular nodules generally in animals 
which are infected naturally or by experimental inoculations. 

In the giant cells of tubercular growths they have a peculiar and 
characteristic position, being found, as a rule, upon the side of the 
cell opposite to the nuclei, which are crowded together in a crescentic 
arrangement at the opposite pole of the cell. Sometimes a single 
bacillus will be found in this position, or there may be several. 
Again, numerous bacilli may be found in giant cells in which the 
nuclei are distributed around the periphery. They are more numer- 


BACILLI IN CHRONIC INFECTIOUS DISEASES. 469 


ous in tuberculous growths of recent origin, and often cannot be 
demonstrated, by microscopical examination, in caseous material 
from the centre of older nodules. But such material, when inocu- 
lated into susceptible animals, gives rise to tuberculosis, and the 
usual inference is that it contains spores of the tubercle bacillus. 

Morphology.—The tubercle bacilli are rods with rounded ends, 
of from 1.5 to 3.5 win length, and are commonly slightly curved or 
bent at an angle; the diameter is 
about 0.2 4. In stained preparations 
unstained portions are frequently 
seen, which are generally believed to 
be spores, but this is by no means 
certain. From two to six of these 
unstained spaces may often be seen 
in a single rod, and owing to this al- 
ternation of stained and unstained 
portions the bacilli may, under a low 
power, be mistaken for chains of mi- 
crococci The reds are usually soli- 
vary » but wud be united ee gina se Fie. 114. — Bacillus tuberculosis. 
in short chainscontaining three or four x 1,000. From a photomicrograph. 
elements. In old cultures irregular 
forms may be observed, the rods being sometimes swollen at one 
extremity, or presenting the appearance of having a lateral bud-like 
projection—involution forms. 

The staining charaeters of this bacillus are extremely important 
for its differentiation and recognition in preparations of sputum, etc. 
Unlike most microérganisms of the same class, it does not readily 
take up the aniline colors, and when stained it is not easily decolorized, 
even by the use of strong acids. The failure to observe it in tuber- 
culous material, prior to Koch’s discovery, was no doubt due to the 
fact that it does not stain in the usual aqueous solutions of the aniline 
dyes. Koch first recognized it in preparations placed in a staining 
fluid to which an alkali had been added—solution of methylene blue 
with caustic potash ; but this method was not very satisfactory, and 
he promptly adopted the method devised by Ehrlich, which consists 
essentially in the use of a solution of an aniline color—fuchsin or 
methyl violet—in a saturated aqueous solution of aniline oil, and de- 
colorization with a solution of a mineral acid—nitric acid one part to 
three parts of water. 

The original method of Ehrlich gives very satisfactory results, 
but various modifications have since been proposed, some of which 
are advantageous. The carbol-fuchsin solution of Ziehl is now 
largely employed ; it has the advantage of prompt action and of 


470 BACILLI IN CHRONIC INFECTIOUS DISEASES. 


keeping well. The staining is effected more quickly if heat is ap- 
plied. The tubercle bacilli stain by Gram’s method, but this is not 
to be recommended for general use, owing to the fact that the pro- 
toplasm of the rods is frequently contracted into a series of spheri- 
cal, stained bodies, which might easily be mistaken for micrococci. 

The examination of sputum for the presence of the tubercle ba- 
cillus is recognized as a most important procedure for the early diag- 
nosis of pulmonary tuberculosis. It is at- 
tended with no special difficulties, and every 
physician should be acquainted with the 
technique. 

The patient should be directed to expec- 
torate into a clean, wide-mouthed bottle or 
glass-covered jar the material coughed up 
from the lungs, and especially, in recent 
cases, that which is coughed up upon first 
rising in the morning. This should be 
placed in the physician’s hands as promptly 
as possible ; although a delay of some days 
does not vitiate the result, and the tubercle 
bacilli may still be demonstrated after the sputum has undergone pu- 
trefaction. Itis well to pour the specimen into a clean, shallow vessel 
having a blackened bottom—a Petri’s dish placed upon a piece of dead- 
black paper will answer very well. In tuberculous sputum small, len- 
ticular masses of a yellowish color may usually be observed, and one 
of these should be selected for microscopical examination, by picking 
it up with a platinum needle and freeing it as far as possible from 
the tenacious mucus in which it is embedded. If such masses are 
not recognized take any purulent-looking material present in the 
specimen, whether it be in small specks distributed through the mu- 
cus, or in larger masses. A little of the selected material should be 
placed in the centre of a clean cover glass and another thin glass 
cover placed over it. By pressure and a to-and-fro motion the mate- 
rial is crushed and distributed as evenly as possible; the glasses are 
then separated by a sliding motion. The film is permitted to dry by 
exposure in the air. When dry the cover glass, held in forceps, is 
passed three times through the flame of an alcohol lamp or Bunsen 
burner to fix the albuminous coating. Too much heat causes the film 
to turn brown and ruins the preparation. The staining fluid (Ziehl’s 
carbol-fuchsin) may then be poured upon the cover glass, or this may 
be floated upon the surface of the fluid contained in a shallow watch 
glass. Heat is now applied by bringing the cover glass over a 
flame and holding it there until steam begins to be given off from 
the surface of the staining fluid; it is then withdrawn and again 


sis in sputum, x 1,000. (Baum- 
garten.) 


BACILLI IN CHRONIC INFECTIOUS DISEASES. 471 


gently heated at intervals for a minute or two. The cover glass is 
then washed in water, and the film will be seen to have a uniform 
deep-red color. The next step consists in decolorization in the acid 
solution (twenty-five-per-cent solution of nitric or of sulphuric acid). 
The cover glass is gently moved about in this solution for a few 
seconds, and the color will be seen to quickly fade to a greenish 
tint. The object is to remove all color from the cells and the al- 
buminous background, so that the bacilli, which retain their color in 
presence of the acid, may be clearly seen. The preparation is next 
washed in dilute alcohol (sixty per cent) to remove the fuchsin 
which has been set free by the acid. If decolorization was not car- 
ried far enough the film will be seen to still have a red color, espe- 
cially in places where it is thickest, when it is removed from the 
dilute alcohol and washed out in water. In this case it will be 
necessary to return it to the acid solution and again wash it in the 
dilute alcohol and in water. It may now be placed in a solution 
of methylene blue or of vesuvin for a contrast stain. The tubercle 
bacilli are distinguished by the fact that they retain the red color 
imparted to them in the fuchsin solution, while other bacteria pre- 
sent, having been decolorized in the acid solution, take the contrast 
stain and appear blue or brown, according to the color used. The 
double-stained preparation, after a final washing in water, may be 
examined at once, or dried and mounted in balsam for permanent 
preservation. 

Of the various other methods which have been proposed, that of 
Frankel, as modified by Gabbett, appears to be the most useful. This 
consists in staining as above directed with Ziehl’s carbol-fuchsin solu- 
tion, and in then placing the cover glass directly in a second solution 
which contains both the acid for decolorizing and the contrast stain. 
This second solution contains twenty parts of nitric acid, thirty parts 
of alcohol, fifty parts of water, and sufficient methylene blue to make 
a saturated solution (one to two parts in one hundred). After re- 
maining in this solution for a minute or two the cover glass is washed 
in water, and upon microscopical examination the tubercle bacilli, if 
present, will be seen as red rods which strongly contrast with the 
blue background. 

The methods recommended for cover-glass preparations may also 
be used for staining the tubercle bacillus in thin sections of tuber- 
culous tissues, except that itis best not to employ heat. The sec- 
tions may be left for an hour in the carbol-fuchsin solution, or for 
twelve hours in the Ehrlich-Weigert tubercle stain—eleven cubic 
centimetres of saturated alcoholic solution of methyl violet, ten cubic 
centimetres of absolute alcohol, one hundred cubic centimetres of ani- 
line water. They should then be decolorized by placing them for 


472 BACILLI IN CHRONIC INFECTIOUS DISEASES. 


about half a minute in dilute nitric acid (ten per cent); then wash 
out color in sixty-per-cent alcohol ; counter-stain for two or three 
minutesin a saturated aqueous solution of methylene blue ; dehydrate 
with absolute alcuhol or with aniline oil; clear up in oil of cedar, 
and mount in xylol balsam. If the aniline-water-methyl-violet solu- 
tion has been used for staining the bacilli a saturated solution of 
vesuvin may be used as a contrast stain. : 

Biological Characters.—A parasitic, aérobic, non-motile ba- 
cillus, which grows only at a temperature of about 37° C. Is also a 
facultative anaérobic (Frankel). 

The question as to spore formation has not been definitely deter- 
mined. It has been generally assumed that the unstained spaces 
which are frequently seen in the bacilli are spores ; and the fact that 


Fic. 116.—Section through a tuberculous nodule in the lung of a cow, showing two giant cells 
containing tubercle bacilli. x 950, (Baumgarten.) 


caseous material in which a microscopical examination has failed to 
demonstrate the presence of bacilli may produce tuberculosis, with 
bacilli, when inoculated into guinea-pigs, has been explained upon the 
supposition that this material contained spores. But a few bacilli 
present in such caseous material might easily escape detection. As 
pointed out by Frankel, the oval spaces in stained specimens have 
not the sharply defined outlines of spores. Moreover, the bacilli, when 
examined in unstained preparations, do not contain corresponding re- 
fractive bodies, recognizable as spores. And when the bacilli are 
stained by Gram’s method the protoplasm is often contracted in the 
form of little, spherical stained masses, while the unstained spaces 
are larger and no longer have the oval form presented in rods stained 
by Ehrlich’s method. The great resisting power of the bacillus to 
heat and to desiccation has been supposed to be due to the presence 


‘BACILLI IN CHRONIC INFECTIOUS DISEASES. 473 


of spores. But, so far as resistance to heat is concerned, this is not 
so great as was at one time believed. Schill and Fischer (1884), as- 
suming that the tubercle bacillus forms spores, made quite a number 
of experiments to determine its thermal death-point. They sub- 
jected sputum containing the bacillus to a temperature of 100° C., and 
tested the destruction of vitality by inoculations into guinea-pigs. 
Exposure to steam at a temperature of 100°C. for two to five min- 
utes was effective in every experiment, with one exception. One 
guinea-pig died tuberculous after having been inoculated with 
sputum exposed to this temperature for two minutes. This result 
was assumed to show that the bacillus would survive lower tempera- 
tures, but it is evident that additional experiments were required to 
establish this fact. In 1887 the writer made a few similar experi- 
ments at alower temperature, and guinea-pigs inoculated with tuber- 
culous sputum exposed for ten minutes to a temperature of 90°, 80°, 
and 60° C. failed to become tuberculous, while another guinea-pig, 
inoculated with the same material after exposure to a temperature of 
50° C. for ten minutes, died tuberculous. These results correspond 
with those subsequently (1888) reported by Yersin, who tested the 
thermal death-point of this bacillus by the culture method. This 
author assumes that the bacilli form spores, but states as a result of 
his experiments that “‘at the end of ten days bacilli heated for ten 
minutes at 55° C. gave a culture in glycerin-bouillon ; those heated 
to 60°, at the end of twenty-two days; while those heated to 70° and 
above failed to grow in every instance. This experiment, repeated a 
great number of times, always gave the same result. The tubercle 
bacilli then resist a temperature of 60° C. for ten minutes, and it is 
to be remarked that the resistance of spores to heat appears to be no 
greater than that of the bacilli themselves.” Yersin remarks in a 
footnote that ‘‘the spores which served for these experiments did 
not appear as more or less irregular granules taking the coloring 
matter strongly, but as veritable spores with sharply defined outlines, 
to the number of one or two ina bacillus, or three at the outside. 
These spores are particularly clear in cultures upon glycerin-agar 
several weeks old.” 

It may be that bacteriologists have been mistaken in the infer- 
ence that all spores possess a greater resisting power for heat than 
that exhibited by bacilli in the absence of spores. That this is true 
as regards anthrax spores ard many others, the thermal death-point 
of which has been determined by exact experiments, does not prove 

that itis true for all. Anditis known that there are wide differ- 
ences in the resisting power both of the spores of different species 
and in the vegetating cells. To admit that the tubercle bacillus or 
the typhoid bacillus, etc., may form spores which have no greater 


474 BACILLI IN CHRONIC INFECTIOUS DISEASES. 


resisting power against heat than the bacilli themselves, would there- 
fore simply be an admission that soma bacteriologists had made a 
mistaken inference based upon incomplete data. In view of the 
facts stated we can simply repeat what was said at the outset, viz., 
the question as to spore formation has not been definitely deter- 
mined. 

The tubercle bacillus is a strict parasite, and its biological char- 
acters are such that it could scarcely find natural conditions, outside 
of the bodies of living animals, favorable for its multiplication. It 
therefore does not grow as a saprophyte under ordinary circum- 
stances. But it has been noted by Roux and Nocard that when it 
has been cultivated for a time in artificial media containing glycerin 
it may grow in a plain bouillon of veal or chicken, in which media it 
fails to develop when introduced directly from a culture originating 
from the body of an infected animal. This would indicate the pos- 
sibility of its acquiring the ability to grow as a saprophyte ; and we 
can scarcely doubt that at some time in the past it was a true sapro- 
phyte. The experiments of Nuttall indicate that the bacillus may 
multiply, under favorable temperature conditions, in tuberculous 
sputum outside of the body. And it is extremely probable that mul- 
tiplication occurs in the muco-purulent secretion which accumulates 
in pulmonary cavities in phthisical patients. In these cavities its de- 
velopment may, in a certain sense, be regarded as saprophytic, as it 
feeds upon non-living organic material. 

Koch first succeeded in cultivating this bacillus upon coagulated 
blood serum, prepared as directed in Section VIII., Part First, of the 
present volume. Roux and Nocard have since shown (1888) that it 
grows very well on nutrient agar to which glycerin has been added 
(six to eight per cent), and also in veal broth containing five per cent 
of glycerin. It is difficult to obtain pure cultures from tuberculous 
sputum, on account of the presence of other bacteria which grow 
much more rapidly and take full possession of the medium before the 
tubercle bacillus has had time to form visible colonies. For this rea- 
son it is best to first inoculate a guinea-pig with the tuberculous spu- 
tum and to obtain cultures from it after tuberculous infection has 
fully developed. The inoculated animals usually die at the end of 
three or four weeks. It is best to kill one which gives evidence of 
being tuberculous, and to remove one or more nodules from the 
lungs through an opening made in the chest walls. The greatest 
care will be required to prevent contamination by other common 
microérganisms. The instruments used must be sterilized by heat, 
and the skin over the anterior thoracic wall carefully turned back ; 
then, after again sterilizing knives and scissors, cut an opening into 
the chest cavity, draw out the root of the lung, and take up with 


BACILLI IN CHRONIC INFECTIOUS DISEASES. 475 


slender sterilized forceps, or with a strong platinum loop, one or 
more well-defined tubercular nodules. These may be conveyed di- 
rectly to the surface of the solid culture medium and then broken 
up and rubbed over the surface as thoroughly as possible ; or they 
may first be crushed between two sterilized glass slides, and then 
transferred with the platinum loop and thoroughly rubbed into the 
surface of the culture medium. 

This breaking-up of the tuberculous nodules and distribution of 
the bacilli upon the surface of the culture medium is essential for 
the success of the experiment. Instead of using the tubercular 
nodules in the lungs, an enlarged lymphatic gland from the axilla or 
elsewhere may be used, as first recommended by Koch. This is to 
be crushed in the same way ; and it will be best to inoculate a num- 
ber of tubes at the same time, as accidental contamination or failure 
to develop is very liable to occur in a certain number. Owing to the 
liability of the blood serum to become too dry for the development of 
the bacillus, it is best to keep the cultures in a moist atmosphere, or 
to prevent evaporation by applying a rubber cap over the open end 
of the test tube. This should be sterilized in a solution of mercuric 
‘chloride (1 :1,000) ; and the end of the cotton plug should be burned 
off just before applying it, for the purpose of destroying the spores 
of mould fungi, which in a dry atmosphere would be harmless, but 
under the rubber cap are likely to sprout and to send their mycelium 
through the cotton plug to the interior of the tube, thus destroying 
the culture. 

Upon coagulated blood serum the growth first becomes visible at 
the end of ten to fourteen days (at 37° C.), and at the end of three 
weeks a very distinct and characteristic develop- 
ment has occurred. The first appearance is that of 
dry-looking, grayish-white points and scales, which 
are without lustre, and are sometimes united to 
form a thin, irregular, membranous-looking layer. 
Under the microscope, with an amplification of 
eighty diameters, the early, thin surface growth 
upon blood serum presents a characteristic appear- 
ance. The bacilli, arranged in parallel rows, form 
variously curved figures, of which we may obtain 
impressions by carefully applying a dry cover glass, BY. 17. Mubercle 
to the surface. Upon staining the preparation in culture upon blood se- 
the usual way the same arrangement of the bacilli "™™. x 500. och.) 
which adhered to the thin glass cover will be pre- 
served. The growth is more abundant in subsequent cultures, 
which have been kept up in Koch’s laboratory from his original 
pure cultures up to the present time ; in these the bacillus still pre- 


476 BACILLI IN CHRONIC INFECTIOUS DISEASES. 


serves its characters of form and growth, and its specific pathogenic 
power. 

Pastor (1892) has succeeded in obtaining pure cultures of the 
tubercle bacillus from sputum by the following ingenious method : 
After proving by microscopic examination that the sputum of a 
tuberculous individual contains numerous bacilli, he has the patient 
cleanse his mouth as thoroughly as possible with sterilized water, 
and then expectorate some material, coughed up from the lungs, into 
a sterilized test tube. By shaking with sterilized water a fine emul- 
sion is made, and this is filtered through fine gauze. The filtrate, 
which is nearly transparent, contains numerous tubercle bacilli. A 
few drops of the emulsion are now added to liquefied gelatin in a test 
tube, and a plate is made in the usual way. This is kept for three 
or four days at the room temperature, during which time the com- 
mon mouth bacteria capable of growth form visible colonies. By 
means of a hand lens a place is now selected in which no colonies are 
seen, and a bit of gelatin is excised with a sterilized knife. This 
piece is transferred to the surface of blood serum or glycerin-agar, 
and placed in the incubating oven, where in due time colonies of 
the tubercle bacillus will usually be foand to develop. 

Another method of accomplishing the same result has been 
described by Kitasato. This isa method devised by Koch some time 
since and successfully employed in his laboratory. The morning 
expectoration of a tuberculous patient, raised from the lungs by 
coughing, is received in a Petri’s dish. A bit of sputum, such as 
comes from the tuberculous cavity in the lungs of such a patient, is 
now isolated with sterilized instruments and carefully washed in at 
least ten successive portions of sterilized water. By this procedure 
the bacteria accidentally attached to the viscid mass of sputum dur- 
ing its passage through the mouth are washed away. In the last 
bath the mass is torn apart and a small portion from the interior is 
used to make a microscopic preparation, the examination of which 
shows whether only tubercle bacilli are present. If this be the case 
cultures upon glycerin-agar are started from material obtained from 
the interior of the same mass. The colonies obtained in this way 
appear in about two weeks as round, white, opaque, moist, and shin- 
ing masses. Kitasato’s researches show that the greater portion of 
the tubercle bacilli in sputum obtained in this way, and in the con- 
tents of lung cavities, are incapable of development, although this 
fact cannot be recognized by a microscopic examination of stained 
specimens. 

On account of the greater facility of preparing and sterilizing 
glycerin-agar, and the more rapid and abundant development upon 
this medium, it is now usually employed in preference to blood 


BACILLI IN CHRONIC INFECTIOUS DISEASES. 477 


serum. The growth at the end of fourteen days is more abundant than 
upon blood serum at the end of several weeks. When numerous 
bacilli have been distributed over the surface of the culture medium 
a rather uniform, thick, white layer, which subsequently acquires a 
yellowish tint, is developed ; when the bacilli 
are few in number or are associated in scattered 
groups separate colonies are developed, which 
acquire considerable thickness and have more 
or less irregular outlines; they are white at 
first, then yellowish-white. Frankel describes 
the tubercle bacillus as a facultative anaérobic, 
and it would appear that it must be able to grow 
in situations where it can obtain very little oxy- 
gen from its development in the interior of tu- 
berculous nodules, lymphatic glands, etc. But 
in stick cultures in glycerin-agar development 
only occurs near the surface, and not at all in 
the deeper portion of the medium. In view of 
its abundant growth on the surface it is diffi- 
cult to understand this failure to grow along 
the line of puncture, if it is in truth a faculta- 
tive anaérobic. 7 

In peptonized veal broth containing five per 
cent of glycerin the bacillus develops at first in 
the form of little floceuli, which accumulate at 
the bottom of the flask and which by agitation 
are easily broken up. At the end of two or 
three weeks the bottom of the flask is covered 
with similar flocculi, which form an abundant 
deposit. 

Pawlowski and others report success in cul- 
tivating the tubercle bacillus upon the surface 
of cooked potato enclosed in a test tube after 5, 18 culture of tu. 
the method of Bolton and Roux. The open end __ vercle bacillus upon glyce- 
of the tube is hermetically sealed in a flame che Photograph by 
after the bacilli have been planted upon the 
obliquely-cut surface of the potato; this prevents drying. Ac- 
cording to Pawlowski, better results are obtained if the surface of 
the potato is moistened with a five-per-cent solution of glycerin. The 
growth is said to be seen at the end of about twelve days as grayish, 
dry-looking flakes ; at the end of three or four weeks it forms a dry, 
smooth, whitish layer, and no further development occurs. 

The range of temperature at which this bacillus will grow is 
very restricted ; 37° C. is usually given as the most favorable point, 


478 BACILLI IN CHRONIC INFECTIOUS DISEASES, 


but Roux and Nocard say that the most favorable temperature ap- 
pears to be 39°, and that development is slower at 37°. 

The experiments of Koch, Schill and Fischer, and others show 
that the bacilli retain their vitality in desiccated sputum for several 
months (nine to ten months—De Toma); but they are said to undergo 
a gradual diminution in pathogenic virulence, which is more rapid 
when the desiccated material is kept at a temperature of 30° to 40° C. 
In the experiments of Cadéac and Malet portions of the lung from 
a tuberculous cow, dried and pulverized, produced tuberculosis in 
guinea-pigs at the end of one hundred and two days. They retain 
their vitality for a considerable time in putrefying material (forty- 
three days—Schill and Fischer ; one hundred and twenty days—Ca- 
déac and Malet). The resisting power of this bacillus against ger- 
micidal agents is also greater than that of certain other pathogenic 
microérganisms, but not so great as to justify the inference that it 
forms spores. It is not destroyed by the gastric juice in the sto- 
mach, as is shown by successful infection experiments in suscep- 
tible animals, by mixing cultures of the bacillus with their food 
(Baumgarten, Fischer), and also by experiments with an artificially 
prepared gastric juice (Falk). They are destroyed, in sputum, in 
twenty hours by a three-per-cent solution of carbolic acid, even 
when they present the appearance usually ascribed to the presence 
of spores (Cavagnis) ; also by absolute alcohol, a saturated aqueous 
solution of salicylic acid, saturated aniline water, etc. (Schill and 
Fischer). The more recent experiments of Yersin upon pure cul- 
tures of the bacillus gave the following results: ‘‘ Tubercle bacilli, 
containing spores, were killed by a five-per-cent solution of carbolic 
acid in thirty seconds, by one-per-cent in one minute ; absolute alco- 
hol, five minutes ; iodoform-ether, one per cent, five minutes ; ether, 
ten minutes; mercuric chloride, 1:1,000 solution, ten minutes ; 
thymol, three hours ; salicylic acid, 2.5 per cent, six hours. 

The tubercle bacillus appears to be especially susceptible to the 
action of light. In his address before the Tenth International Medi- 
cal Congress (Berlin, 1890) Koch says that when exposed to direct 
sunlight the tubercle bacillus is killed in from a few minutes to sev- 
eral hours, according to the thickness of the layer; it is also de- 
stroyed by diffuse daylight in from five to seven days when placed 
near a window. This fact has an important hygienic bearing, espe- 
cially in view of the fact that the tubercle bacillus is not readily 
killed by desiccation, putrefaction of the material containing it, etc. 
Tuberculous sputum expectorated upon sidewalks, etc., being ex- 
posed to the action of direct sunlight, will in many cases be disin- 
fected by this agent by the time complete desiccation has occurred— 
7.e., before it is in a condition to be carried into the air as dust. 


BACILLI IN CHRONIC INFECTIOUS DISEASES. 479 


Sawizky in 1891 made a series of experiments to determine 
the length of time during which dried tuberculous sputum retains 
its virulence. He arrived at the conclusion that virulence is not sud- 
denly but gradually lost, and that in an ordinary dwelling room 
dried sputum retains its specific infectious power for two and one- 
half months. 

Tizzoni and Cattani (1892) have presented some experimental evi- 
dence which indicates that injections of Koch’s tuberculin into 
guinea-pigs may produce in these animals a certain degree of im- 
munity against tuberculosis; and that this immunity depends upon 
the presence of an anti-tuberculin formed in the body of the partially 
immune animal. 

Numerous experiments made by veterinary surgeons upon tuber- 
culous cows show that the injection of Koch’s tuberculin in these 
animals, in doses of thirty to forty centigrammes, produces a rise of 
temperature of from 1° to 3° C. The febrile reaction usually occurs 
in from twelve to fifteen hours after the injection. Its duration and 
intensity do not depend upon the extent of the tuberculous lesions, 
but is even more marked when these are slight than in advanced 
cases. In non-tuberculous animals no reaction occurs, and the ex- 
periments made justify the suspicion that tuberculosis exists if an 
elevation in temperature of a degree or more occurs as a result of 
the subcutaneous injection of the dose mentioned. 

When the number of tubercle bacilli in sputum is comparatively 
small they may easily escape observation. Methods have therefore 
been suggested for finding them under these circumstances. Ribbert 
(1886) proposed the addition to the sputum of a two-per-cent solution 
of caustic potash, and boiling the mixture. The tenacious mucus is 
dissolved, and when the mixture is placed in a conical glass vessel 
the bacilli are deposited at the bottom and may easily be found in 
the sediment after removing the supernatant fluid. The same object 
is accomplished by Stroschein (1889) by the addition to sputum of 
three times its volume of a saturated solution of borax and boracic 
acid in water. 

A method of estimating the number of baciili in sputum has 
been proposed by Nuttall, which appears to give sufficiently ac- 
curate results and to be useful in judging of the progress of a 
case or of the results of treatment. For the details of this method 
we must refer to the author’s paper (Johns Hopkins Hospital Bulle- 
tin, vol. xi., No. 13, 1891). It consists essentially in first making 
the sputum fluid by the addition of a solution of caustic potash; in 
then shaking it thoroughly in a bottle containing sterilized gravel 
or pounded glass ; in carefully measuring the total quantity of fluid, 
and in dropping upon glass slides uniform drops by means of a grad- 


480 BACILLI IN CHRONIC INFECTIOUS DISEASES. 


uated pipette; in spreading these uniformly by means of a platinum 
needle and a turn table; in covering the dried film with a film of 
blood serum, and coagulating this by heat; and, finally, in staining 
and counting the bacilli in a series of slides from the same specimen, 
and from the average number found in a single drop estimating the 
total number in the sputum for twenty-four hours. 
Pathogenesis.—Man, cattle, and monkeys are most subject to 
contract the disease naturally, and it may be communicated by in- 
oculation to many of the lower animals—guinea-pigs, field mice, rab- 


Fic. 119.—Limited epithelioid celled tubercle of theiris. x 950. (Baumgarten) 


bits, and cats are among the most susceptible animals ; and in larger 
doses dogs, rats, white mice, and fowls may also be infected. 

When tuberculous sputum is introduced beneath the skin of a 
guinea-pig the nearest lymphatic glands are found to be swollen at 
the end of two or three weeks, at the same time there is a thickening 
of the tissues about the point of inoculation ; later a dry crust forms 
over the local tuberculous tumefaction, and beneath this is a flattened 
ulcer covered with cheesy material. The animals become emaciated 
and show difficulty in breathing, and usually succumb to general 
tuberctlosis, especially involving the lungs, within four to eight 
weeks. Injections of tuberculous sputum, or of pure cultures of the 
bacillus, into the peritoneal cavity give rise to extensive tuberculo- 
sis of the liver, spleen, and lungs, and to death, asa rule, within 
three or four weeks. Rabbits are less susceptible to subcutaneous 


BACILLI IN CHRONIC INFECTIOUS DISEASES. 481 


injections, but die within seventeen to twenty days when virulent— 
recent—cultures are injected into the circulation. As a result of 
such an inoculation the animal rapidly loses flesh and has a decided 
elevation of temperature, commencing at the end of the first week 
and increasing considerably during the last days of life. At the 
autopsy the spleen and liver are found to be greatly enlarged, but 
they do not contain any tubercles that can be recognized by the naked 
eye (Yersin). They contain, however, great numbers of tubercle 
bacilli, both free and in the cells. Injections of a small quantity of 
a pure culture into the anterior chamber of the rabbit’s eye cause 
first iris-tuberculosis, followed by swelling and caseation of the near- 
est lymph glands, and finally general infection and death ; when 
larger quantities are injected general tuberculosis is quickly devel- 
oped. The influence of quantity—number of bacilli—is also shown 
in subcutaneous, intravenous, or intraperitoneal injections into guinea- 
pigs and rabbits (Hirschberger, Gebhardt, Wyssokowitsch). Thus 
rabbits which received less than one hundred and fifty bacilli, in 
sputum, in the experiments of Wyssokowitsch, did not develop tuber- 
culosis ; and in guinea-pigs the smaller the number injected the more 
protracted the course of the disease was found to be. 

Tuberculosis in man no doubt results, in a large proportion of the 
cases, from the respiration, by a susceptible individual, of air con- 
taining the tubercle bacillus in suspension in a desiccated condition. 
As already stated, it has been demonstrated by experiment that the 
bacillus retains its vitality in desiccated sputum for several months. 
The experiments of Cornet have demonstrated that in the dust of 
apartments occupied by tuberculous patients tubercle bacilli are very 
commonly present in sufficient numbers to induce tuberculosis in 
guinea-pigs inoculated in the peritoneal cavity with such dust, while 
negative results were obtained from inoculations with dust from 
other localities. In view of these facts the usual mode of infection 
is apparent. Infection may also occur through an open wound or 
abrasion of the skin, as in the small, circumscribed tumors which 
sometimes develop upon the hands of pathologists as a result of 
handling tuberculous tissues. A few instances of accidental inocu- 
lation through wounds made by glass or earthen vessels containing 
tuberculous sputum have also been recorded. A more common mode 
of infection, especially in children, is probably by way of the intesti- 
nal glands, from the ingestion of the milk of tuberculous cows. That 
infection may occur by way of the intestine has been proved by ex- 
periments upon rabbits, which develop tuberculosis when fed upon 
tuberculous sputum. And that the tubercle bacillus is frequently, if 
not usually, present in the milk of tuberculous cows has been proved 
by the experiments of Bollinger, Hirschberger, Ernst, and others. 


31 


482 BACILLI IN CHRONIC INFECTIOUS DISEASES. 


In Hirschberger’s investigations milk from tuberculous cows in- 
duced tuberculosis in guinea-pigs, when injected subcutaneously or 
into the peritoneal cavity, in fifty-five per cent of the cases studied 
(twenty). The conclusion is reached that the milk may contain tu- 
bercle bacilli even when the udder of the cow is not involved. Ernst 
also, from an examination of the milk from thirty-six tuberculous 
cows in which the udder was apparently not involved, found the 
tubercle bacillus by microscopical examination in five per cent of the 
samples examined (one hundred and fourteen). 

The prevalence of tuberculosis among cattle is shown by numer- 
ous investigations, and especially by the official inspections of 
slaughtered animals made in Germany. Thus in Saxony, in the 
year 1889, of 611,511 cattle examined 6,135 were found to be tubercu- 
lous (about one per cent); in Berlin, 1887-1888, out of 130,733 ani- 
mals slaughtered 4,300 were found to be tuberculous (3.2 per cent). 
In view of the facts stated the great mortality from tubercular dis- 
eases among children, many of whom are removed from other prob- 
able sources of infection, is not difficult to understand, and the 
practical and simple method of preventing infection in this way, af- 
forded by the sterilization (by heat) of milk used as food for infants, 
must commend itself to all. 


BACILLUS TUBERCULOSIS GALLINARUM. 


The researches of Maffucci (1889) and of Cadiot, Gilbert, and 
Roger (1890) show that the bacillus obtained from spontaneous tu- 
berculosis in chickens, although closely resembling the bacillus of 
human tuberculosis, is not identical with it, varying especially in its 
pathogenic power. This view is sustained by the observations of 
Koch, who says in his address before the Tenth International Medi- 
cal Congress (Berlin, 1890): 


‘The care which it is necessary to exercise in judging of the characters 
which serve to differentiate bacteria, even those which are well known, I 
have learned in the case of the tuberclebacillus This species is so definitely 
characterized by its staining reactions, its growth in pure cultures, and its 
pathogenic qualities, and indeed by each of these characters, that it seems 
impossible to confound it with other species. Nevertheless in this case also 
one should not rely upon a single one of the characters mentioned for de- 
termining the species, but should follow the safe rule that all available 
characters should be considered, and the identity of a certain bacterium 
should only be regarded as demonstrated when it has been shown to corre- 
spond in all of these particulars. When I made my first researches with 
reference to the tubercle bacillus I was controlled by this rule, and tested 
tubercle bacilli from various sources, not only with reference to their stain- 
ing reactions, but also with reference to their growth in culture media and 
pathogenic characters. Only in the tuberculosis of chickens I was not able 
to apply this rule, as at that time it was not possible for me to obtain fresh 
material from which to make pure cultures. As, however, all other forms 


BACILLI IN CHRONIC INFECTIOUS DISEASES. 483 


of tuberculosis had given identical bacilli, and the bacilli of chicken tuber- 
culosis in their cand and. behavior towards the aniline colors entirely 
corresponded with these, I believed myself justified in assuming their iden- 
tity, notwithstanding the incompleteness of the research. Later I received 
pure cultures from various sources, which apparently originated from tuber- 
cle bacilli, but in several regards differed from these; especially in the fact 
that inoculation experiments, made by experienced and reliable investigators, 
led to dissimilar results, which it was necessary to regard as unexplained con- 
tradictions. At first I believed that these differences depended upon changes 
suchas are frequently observed in pathogenic bacteria, when these are culti- 
vated in pure cultures outside of the body fora long time under more or less 
unfavorable conditions. In order to solve the riddle I attempted by various 
influences to change the common tubercle bacilli into the presumed variety 
referred to. They were cultivated for several months at so high a tempera- 
ture that only a scanty growth was obtained; in other experiments still 
higher temperatures were allowed to act repeatedly for so long a time that 
the cultures were brought as nearly as possible to the point of killing the 
bacilli. In asimilar way I subjected the cultures to the action of chemical 
agents, of light, or absence of moisture; they were cultivated for many gen- 
erations in association with other bacteria; inoculated successively in ani- 
mals having but a slight susceptibility. But, in spite of all these attempts, 
only slight variations were obtained in their characters—far less than other 
pathogenic bacteria undergo under similar circumstances. Itappears, there- 
fore, that the tubercle bacilli retain their characters with special obstinacy ; 
this is in accord with the fact that pure cultures which have now been cul- 
tivated by me in test tubes for more than nine years, without in the mean- 
time having been in a living body, are still entirely unchanged with the ex- 
ception of a slight diminution of virulence. . .. It happened about a year 
ago that I received a living chicken which wassuffering from tuberculosis, 
and I used this opportunity to make cultures directly from the diseased or- 
gans of this animal, which previously I had not been able todo. "When the 
cultures grew I.saw to my surprise that they had precisely the appearance 
and all of the characters possessed by the enigmatical cultures resembling 
those of the genuine tubercle bacillus. Later I learned that these also ori- 
ginated from tuberculosis in fowls, but, upon the assumption that all forms 
of tuberculosis are identical, had been considered genuine tubercle bacilli. 
A verification of my observations I find in the recently published researches 
of Prof. Maffucci with reference to tuberculosis of fowls.” 


According to Maffucci, adult chickens are refractory against the 
action of the Bacillus tuberculosis from man, and there are slight 
morphological and biological differences in the bacilli from the two 
sources. 

Cadiot, Gilbert, and Roger (1891) have made a series of experi- 
ments with the bacillus of tuberculosis in fowls. They found 
the bacilli to be very numerous in the livers of chickens suffering 
from spontaneous tuberculosis, and inoculated with material from 
this source six chickens, five rabbits, and twelve guinea-pigs. The 
chickens, when inoculated in the cavity of the abdomen or by injec. 
tion into a vein, died in from forty-one to ninety-three days from 
general tuberculosis. Four of the rabbits died of general tuberculosis, 
presenting the same appearance as that following inoculation with 
bacilli from human tuberculosis. Of the guinea-pigs, which were 
inoculated in the cavity of the abdomen, eleven remained in good 


484 BACILLI IN CHRONIC INFECTIOUS DISEASES. 


health and one only died of general tuberculosis. These experi- 
ments show a decided difference in the pathogenic properties of 
tubercle bacilli from the two sources, for the guinea-pig is especially 
susceptible to tuberculosis as a result of similar inoculations with 
bacilli from human tuberculosis. We must therefore conclude that 
the bacillus found in spontaneous tuberculosis in fowls is a distinct 
variety of Bacillus tuberculosis. Whether this variety would cause 
tuberculosis in man, if introduced into susceptible subjects, has not 
been determined ; and, as pointed out by Koch, this question can 
only be answered in the affirmative if it should be obtained in pure 
cultures from cases of human tuberculosis. 

Since the above was written Maffucci has published (1892) an 
elaborate memoir upon tuberculosis of fowls. His conclusions are 
stated as follows : 


‘The bacillus cf tuberculosis in fowls is distinguished from that of tuber- 
culosis in mammals by the following points of difference: 

‘‘1. It does not induce tuberculosis in guinea-pigs, and seldom causes 
general tuberculosis in rabbits. 

‘©2. The cultures in various media havea different appearance from those 
of the Bacillus tuberculosis of mammals, 

' 3, The temperature at which it develops varies between 35° and 45° C., 
and the thermal death-point is 70° C. 

“4, At 45° to 50° C. the cultures show long, thick, and branched forms. 

‘“5. The bacillus retains its vegetative and pathogenic power at the end 
of two years. 

‘6, This bacillus produces a substance which is toxic for guinea-pigs and 
is but slightly toxic for grown fowls. 

‘“7, The tuberculosis produced in fowls by this bacillus is without giant 
cells.” 

Additional Notes upon the Tubercle Bacillus (1895).—Several 
authors (Metschnikoff, Czaplewski, Fischel) have described branch- 
ing forms of the tubercle bacillus, and Lubinsky (1895) reports that 
in certain media it grows out into long threads, which, however, he 
has never observed to be branched. The media used by him are said 
to give a more abundant growth than occurs upon glycerin-agar; 
the most favorable being made of flesh-peptone agar, or flesh-peptone 
bouillon, containing four per cent of glycerin and mashed _ potato, 
one kilo of finely chopped and washed potato to fifteen hundred cubic 
centimetres of water; this is cooked for three or four hours and filtered 
—to the filtrate is added four per cent of glycerin; one and a half 
per cent of agar is now added and the mixture is again cooked and 
filtered. 

Jones (1895) has observed the branching forms previously de- 
scribed by several authors, and states that they are only found upon 
the surface of culture media where there is free access of oxygen. 


He concludes that the tubercle bacillus does not form endogenous 


BACILLI IN CHRONIC INFECTIOUS DISEASES. 485 


spores, such as are found in various other bacilii, but that in the rods 
and branched filaments certain objects are seen which are probably 
reproductive elements, and which closely resemble similar bodies 
(“ Kolben ”) seen in the actinomyces fungus, to which Jones believes 
the tubercle bacillus is closely related. 

Prudden and Hodenpyl (1891) have shown that the injection of 
dead tubercle bacilli in rabbits gives rise to the development of nod- 
ules in the lung containing epithelioid and giant cells, but that these 
never undergo caseation. This fact is supposed to justify the infer- 
ence that caseation is due to the products elaborated during the 
growth of living tubercle bacilli. The results reported by Vissmann 
(1892) correspond with those reported by Prudden and Hodenpyl. 
Gamaléia (1892) has also obtained nodules with epithelioid and 
giant cells from the injection of dead tubercle bacilli, but in his ex- 
periments he also found caseation of the nodules. Baumgarten sug- 
gests that this was probably due to the fact that there were some liv- 
ing tubercle bacilli remaining in the cultures which he injected. 

Loomis (1890) and Pizzini (1892) have shown that living tubercle 
bacilli are not infrequently found in the bronchial glands of individ- 
uals who present no evidence of tubercular disease of the lungs or else- 
where. The author last mentioned inoculated thirty guinea-pigs 
with the bronchial, mesenteric, and cervical glands of thirty in- 
dividuals in whom death was due to accident or acute disease, and 
who were free from tuberculosis. Twelve of these thirty guinea- 
pigs developed tuberculosis as a result of the inoculation. 

Straus (1894) has found tubercle bacilli in the nasal cavities o 
healthy individuals. ; 

Ernst (1895), as the result of extended researches made under the 
auspices of the Massachusetts Society for Promoting Agriculture, 
has arrived at the following conclusions with reference to the pres- 
ence of the tubercle bacillus in the milk of tuberculous cows: 

“The possibility of milk from tuberculous udders containing the 
infectious element is undeniable. 

“ With the evidence here presented, it is equally undeniable that 
milk from diseased cows with no appreciable lesion of the udder may, 
and not infrequently does, contain the bacillus of the disease.” 

De Schweinitz (1894) has found that by continued cultivation in 
an artificial medium the tubercle bacillus becomes attenuated, so that 
when inoculated into guinea-pigs these animals give no evidence of 
tubercular infection for six months or more. And his experiments 
indicate that animals which have survived an inoculation with the 
attenuated tubercle bacillus acquire an immunity against the patho- 
genic action of virulent cultures. 


486 BACILLI IN CHRONIC INFECTIOUS DISEASES. 


Amann (1895) has given in the Centralblatt fiir Bakteriologie 
(Bd. xvii., page 513) a detailed account of his method for demon- 
strating the presence of tubercle bacilli in sputum by sedimentation. 
He mixes the sputum with two to four volumes of cold distilled 
water, in a glass cylinder which should not be more than half full. 
He adds one cubic centimetre of chloroform and a small quantity 
of shot; the glass cylinder is then closed with a rubber cork and vio- 
lently shaken for some minutes. From four to six volumes of dis- 


Fig. 120.—Section of a recent lepra nodule of theskin, x 950. (Baumgarten.) 


tilled water are then added and the mixture is placed in a V-formed 
glass tube for sedimentation; two cubic centimetres of carbol-fuchsin 
solution are added and distributed by gentle agitation of the tube. 
At the end of two days the sedimentation is complete and the stained 
bacilli, cells, connective-tissue fibres, etc., are taken up with a pipette 
for examination under the microscope. 


BACILLUS LEPR. 


Discovered by Hansen (1879), chiefly in the interior of the peculiar 
round or oval cells found in leprous tubercles. Discovery confirmed 
by Neisser (1879) and by many subsequent observers. 

While found chiefly in the leprous tubercles of the skin and mucous 
membranes, the bacilli have also been foundin the lymphatic glands, 
the liver, the spleen, the testicles, and, in the anzesthetic form of the 
disease, in the thickened portions of nerves involved in the leprous 
process. Some observers have also reported finding them in the 
blood, but this appears to be quite exceptional. In the leprous cells 
they are commonly found in great numbers, and they may also be 
seen in the lymph spaces outside of these cells. They are not found 
in the epidermal layer of the skin, but, according to Babes, they may 
penetrate the hair follicles. 

Morphology.—The bacillus of leprosy resembles the tubercle ba- 
cillus in form, but is of more uniform length and not so frequently 


BACILLI IN CHRONIC INFECTIOUS DISEASES, 487 


bent or curved. The rods have pointed ends; and in stained pre- 
parations unstained spaces, similar to those observed in the tubercle 
bacillus and generally assumed to be spores, are to be seen, although 
not quite so distinctly as in the latter. The bacilliare said by Fliigge 
to be from four to six “in length and less than one “in width— 
probably considerably less, for the same author states that the tubercle 
bacillus has about the diameter of the bacillus of mouse septiceemia, 
and this is given as 0.2 ». 

This bacillus stains readily with the aniline colors and also 
by Gram’s method. Although it differs from the tubercle bacillus 
in the ease with which it takes up the ordinary aniline colors, it re- 
sembles it in retaining its color when subsequently treated with 
strong solutions of the mineral acids. Double-stained prepara- 
tions are therefore easily made by first staining sections or cover- 
glass preparations in Ziehl’s carbol-fuchsin solution or in an aqueous 
solution of methyl violet, decolorizing in acid, washing in alcohol, 
and counter-staining with methylene blue—or, if methyl violet was 
used in the first instance, with vesuvin. 

Biological Characters.—The earlier attempts to cultivate this 
bacillus were without success, but recently Bordoni-Uffreduzzi has 
obtained from the marrow of the bones of a leper a bacillus which 
he believes to be the leprosy bacillus, and which he was able to culti- 
vate upon blood serum to which a certain amount of peptone and of 
glycerin had been added. At first this bacillus only grew with diffi- 
culty and in the incubating oven ; but after it had been cultivated 
artificially through a number of generations it is said to have grown 
upon ordinary nutrient gelatin at the room temperature. The bacillus 
obtained in this way is said to have retained its color when treated 
with acids, after having been stained with aniline-fuchsin, correspond- 
ing in this respect with the bacillus of leprosy and the tubercle ba- 
cillus. But it differed considerably in its morphology from the Ba- 
cillus lepree as seen in the tissues of lepers, being considerably thicker, 
and it was not so promptly stained by the aniline colors as is the 
bacillus found in the tissues. Moreover, attempts to cultivate thc 
same bacillus from leprous tubercles of the skin were unsuccessful, 
as were also inoculation experiments into the anterior chamber of the 
eye inrabbits. It is therefore a matter of doubt as to whether the 
bacillus obtained by Bordoni-Uffreduzzi is identical with that present 
in such numbers in the cells of the leprous tubercles, to which the 
name Bacillus lepre has been given. 

Some of the earlier observers described the bacillus of leprosy as 
motile, but this assertion seems to have been based upon some error 
of observation, and it is now generally agreed that, like the tubercle 
bacillus, it is without proper movements. The question of spore for- 


* 488 BACILLI IN CHRONIC INFECTIOUS DISEASES. 


mation has not been definitely settled. As before remarked, un- 
stained portions, occurring at regular intervals, are seen in the rods in 
stained preparations ; but no satisfactory evidence has been presented 
to show that these are truly reproductive spores. 

Pathogenesis.—The inference that the bacillus above described 
bears an etiological relation to the disease with which it is associated 
is based upon the demonstration of its constant presence in leprous 
tissues—which has now been repeatedly made in various and distant 
parts of the world—and of its absence from the same tissues involved 
in different morbid processes. As it has not been obtained in pure 
cultures, the final proof of such etiological relation is still wanting. 
We have, however, experimental evidence to show that leprous tis- 
sues containing this bacillus are infectious and may reproduce the 
disease. The experiment has been made upon man by Arning, who 
inoculated a condemned criminal subcutaneously with fresh leprous 
tubercles. The experiment was made in the Sandwich Islands, and 
the man was under observation until his death occurred from leprosy 
at the end of about five years. The first manifestations of the disease 
became visible in the vicinity of the point of inoculation several 
months after the experimental introduction of the infectious material. 

Positive results have also been reported in the lower animals by 
Damsch, by Vossius, and by Melcher and Ortmann. The last-named 
investigators inoculated rabbits in the anterior chamber of the eye 
with portions of leprous tubercles excised for the purpose from a 
leper. The animals died from general infection at the end of several 
months, and the characteristic tubercles containing the bacillus were 
distributed through the various organs. 

Wolters (1893) who has made numerous inoculation experiments 
and has made a critical review of all the recorded experimental evi- 
dence, arrives at the conclusion that the comparatively small number 
of successful results reported cannot be accepted as evidence that 
leprosy can be transmitted to the lower animals by inoculation. He 
believes that in some cases the tubercle bacillus has been present in 
the material inoculated and that the infectious process following the 
inoculation was tuberculous and not Jeprous. In inoculations into 
the anterior chamber, in the eyes of rabbits, the considerable number 
of bacilli introduced with the leprous tissue remain and retain their 
staining properties, so that the bacilli originally introduced are found 
in the leucocytes of the inflammatory exudate or granulation tissue 
formed as a result of the introduction of foreign material. Wolters 
also doubts whether the few successful results reported in the culti- 
vation of the lepra bacillus are trustworthy. He has never succeeded 
in his efforts to cultivate the bacillus. 


BACILLI IN CHRONIC INFECTIOUS DISEASES. 489 


BACILLUS MALLEI. 


Synonyms.—The bacillus of glanders; Der Rotzbacillus, Ger.; 
Bacille de la morve, Fr. 

Discovered by Léffler and Schiitz (1882), and proved to be the 
cause of glanders by the successful inoculation of pure cultures. 
Found especially in the recent nodules in animals infected with 
glanders ; also in the same after ulceration, and in the discharge 
from the nostrils, pus from the specific ulcers, etc.; sometimes in the 
blood of infected animals (Weichselbaum). 

Morphology.—Bacilli with rounded ends, straight or slightly 
curved, rather shorter and decidedly thicker than the tubercle bacil- 
lus ; usually solitary, but occasionally united in 
pairs, or in filaments containing several elements ees 
(in potato cultures). In stained preparations Oey Wi 4 
unstained or feebly stained spaces are seen in GS ¥ 
the rods, alternating with the deeply stained & ef (ad 

: ak Ww @? 
protoplasm of the cell. As in the tubercle bacil- & 
lus, which presents a similar appearance, these ay 4 Lg& ~* 
spaces have been supposed by some bacteriolo- Fic. 121.—Bacillus mal. 
gists to represent spores; but Léffler believes i _X 1,000. From a pho- 
them to represent rather a degeneration of the {vapremey 
protoplasm. Baumgarten and Rosenthal claim 
to have demonstrated the presence of spores by the use of Neisser’s 
method of staining, but they do not consider it established that the 
unstained spaces in the rods referred to are of this nature. 

The glanders bacillus may be stained with aqueous solutions of 
the aniline colors, but the staining is more intense when the solution 


Fig. 122.—Section of aglanders nodule. x 700. (Fligge.) 
v 


is made feebly alkaline. Add to three cubic centimetres of a 1: 10,000 
solution of caustic potash, in a watch glass, one cubic centimetre of 
a saturated alcoholic solution of an aniline color (methylene blue, 


490 BACILLI IN CHRONIC INFECTIOUS DISEASES. 


gentian violet or fuchsin); or the aniline-water-fuchsin, or methyl 
violet solution of Ehrlich may be used, with the addition just be- 
fore use of an equal quantity of 1: 10,000 solution of caustic potash. 
Loffler recommends that cover-glass preparations be placed in Ehr- 
lich’s solution and heated for five minutes; then decolorized in a one- 
per-cent solution of acetic acid to which sufficient tropeolin has 
been added to give it the yellow color of Rhine wine; then quickly 
washed in distilled water. This bacillus presents the peculiarity of 
losing very quickly in decolorizing solutions the color imparted to it 
by the aniline staining solutions. For this reason the staining of the 
bacillus in sections is attended with some difficulty. Léffler recom- 
mends his alkaline methylene-blue solution for staining sections ; and 
for decolorizing, a mixture containing ten cubic centimetres of distilled 
water, two drops of strong sulphuric acid, and one drop of a five- 
per-cent solution of oxalic acid. Thin sections should be left in this 
acid solution about five seconds. The method more recently recom- 
mended by Kiihne also gives good results in skilful hands (see p. 35). 

Biological Characters.—An aérobic, non-motile, parasitic 
bacillus, which may be cultivated in various artificial media at a 
temperature of 37° C. The lowest temperature at which develop- 
ment occurs (22° ©.—Léffler) is a little above that at which nutrient 
gelatin is liquefied ; the highest limit is 43°C. According to Frankel, 
the glanders bacillus is a facultative anaérobic. Baumgarten and 
Rosenthal claim to have demonstrated the presence of spores by 
Neisser’s method of staining. Léffler was led to doubt the forma- 
tion of spores from the results of his experiments upon the thermal 
death-point of this bacillus, and its comparatively slight resistance 
to desiccation and destructive chemical agents. He found that ex- 
posure for ten minutes to a temperature of 55° C., or for five minutes 
to a three- to five-per-cent solution of carbolic acid, or for two min- 
utes to a 1:5,000 solution of mercuric chloride, was effectual in de- 
stroying its vitality. As a rule, the bacilli do not grow after having 
been preserved in a desiccated condition for a few weeks ; and in a 
moist condition the cultures cannot be preserved longer than three 
or four months—usually not so long as this (Léffler). The bacillus 
does not grow in infusions of hay, straw, or horse manure, and it is 
doubtful whether it finds conditions in nature favorable for its sap- 
rophytic existence. It grows, in the incubating oven, in neutral 
bouillon, in nutrient gelatin, or in nutrient agar, and still better in 
glycerin-agar. Upon the last-mentioned medium it grows, even at 
the room temperature (Kranzfeld), but better still in the incubating 
oven, as a pale-white, transparent streak along the line of inocula- 
tion, which at the end of six or seven days may have a width of 
seven to eight millimetres. According to Raskina, nutrient agar 


— 


BACILLI IN CHRONIC INFECTIOUS DISEASES. 491 


made with milk forms an extremely favorable medium, upon which 
a thick, pale-white layer develops in two or three days, which on the 
third or fourth day acquires an amber-yellow color, and the deeper 
layers acquire a brownish-red tint. 

The growth upon solidified blood serwm, in the course of three or 
four days at 37° C., consists of yellowish, transparent drops, which 
later coalesce into a viscid layer, which has a milky appearance from 
the presence of numerous small crystals (Baumgarten). The growth 
upon cooked potato is especially characteristic. In the incubating 
oven, at the end of two or three days, a rather thin, yellowish, trans- 
parent layer develops, which resembles a thin layer of honey. Later 
this ceases to be transparent, and the amber color changes, at the 
end of six to eight days, to a reddish-brown color ; and outside of 
the reddish-brown layer, with more or less irregular outlines, the 
potato for a short distance acquires a greenish-yellow tint. 

Pathogenesis.—Glanders occurs principally among horses and 
asses, but may- be contracted by man from contact with infected 
animals ; it has also been communicated, in one instance with a fatal 
result, by subcutaneous inoculation, resulting accidentally from the 
use of an imperfectly sterilized hypodermic syringe which had pre- 
viously been used for injecting cultures of the bacillus into guinea- 
pigs. The field mouse and the guinea-pig are especially susceptible 
to infection by experimental inoculations ; the cat and the goat may 
be infected in the same way. Lions and tigers in menageries are 
said to have contracted glanders from being fed upon the flesh of in- 
fected animals (Baumgarten). » Rabbits have but slight susceptibility, 
and the same is true of sheep and dogs; swine, cattle, white mice, 
and common house mice are immune. 

The etiological relation of the bacillus is fully established by the 
experiments of Léffler and Schiitz, confirmed by other bacteriologists, 
which show that pure cultures injected into horses, asses, and other 
susceptible animals, produce genuine glanders. The disease is char- 
acterized in the equine genus by the formation of ulcers upon the 
nasal mucous membrane, which have irregular, thickened margins 
and secrete a thin, virulent mucus; the submaxillary lymphatic 
glands become enlarged and form a tumor which is often lobulated ; 
other lymphatic glands become inflamed, and some of them suppurate 
and open externally, leaving deep, open ulcers; the lungs are also 
involved and the breathing becomes hurried and irregular. In farcy, 
which is a more chronic form of the same disease, circumscribed 
swellings, varying in size from a pea to a hazelnut, appear on differ- 
ent parts of the body, especially where the skin is thinnest ; these 
suppurate and leave angry-looking ulcers with ragged edges, from 
which there is an abundant purulent discharge. The specific bacillus 


492 BACILLI IN CHRONIC INFECTIOUS DISEASES. 


can easily be obtained in pure cultures from the interior of suppurat- 
ing nodules and glands which have not yet opened to the surface, 
and the same material will give successful results when inoculated 
into susceptible animals. But the discharge from the nostrils or from 
an open ulcer contains comparatively few bacilli; and as these are 
associated with various other bacteria which grow more readily in 
our culture media, it is not easy to obtain pure cultures, by the plate 
method, from such material. 

In the guinea-pig subcutaneous inoculation is followed in four or 
five days by tumefaction at the point of inoculation, and after a time 
a prominent tumor with caseous contents is developed ; ulceration of 
the skin follows, anda chronic, purulent ulcer with irregular, indu- 
rated margins results; after a time this may cicatrize. Meanwhile 
the lymphatic glands become involved, and the symptoms of general 


Fig. 123.—Section through a glanders nodule in liver of field mouse. Tissue 250. Bacilli 
x 500. (Baumgarten.) 


infection are developed at the end of four or five weeks ; the glands 
suppurate, and in males the testicles are also involved ; finally a dif- 
fuse inflammation of the joints occurs, and death results from ex- 
haustion. In the guinea-pig the specific ulcers upon the nasal mu- 


BACILLI IN CHRONIC INFECTIOUS DISEASES. 493 


cous membrane, which characterize the disease in the horse, are rarely 
developed to any great extent. 

In field mice general infection occurs at once asa result of the 
subcutaneous injection of a small quantity of a pure culture, and the 
animal dies at the end of three or four days. Upon post-mortem 
examination the principal changes are found in the liver and in the 
greatly enlarged spleen. Scattered through these organs are minute 
gray points which are scarcely visible to the naked eye. In the 
guinea-pig, which succumbs at a later date, these nodules are larger 
and closely resemble miliary tubercles, both macroscopically and 
under the microscope, in stained sections of the tissues. Similar 
nodules are also found in the kidneys and in the lungs; they have a 
decided tendency to undergo purulent degeneration. The bacilli are 
found principally in these nodules, of recent formation, and are com- 
monly associated in groups, as if they had been enclosed in the inte- 
rior of a cell the membranous envelope of which had undergone 
degeneration and disappeared. 

As before remarked, it is not an easy matter to demonstrate the 
bacillus in sections of the tissues containing these nodules, owing to 
the facility with which they lose their color in alcohol and other de- 
colorizing agents. For this reason it will be best to dehydrate sec- 
tions by the use of aniline oil (Weigert’s method) or to resort to 
Kiihne’s method of staining. 

It is also difficult to demonstrate the presence of the bacillus in 
nodules which have undergone purulent degeneration, in the secre- 
tions from the nostrils of horses suffering from glanders, or in the 
pus from the specific ulcers and suppurating glands ; for they are 
present in comparatively small numbers. But the virulent nature of 
these discharges is shown by inoculations into guinea-pigs or mice, 
and it is easier to obtain a pure culture from such virulent material 
by first inoculating a susceptible animal than directly by the plate 
method; for the small number of bacilli present, and their associa- 
tion with other bacteria which develop more rapidly in our culture 
media, make this a very uncertain procedure. For establishing the 
diagnosis of glanders, therefore, Liffler recommends the inoculation 
of guinea-pigs with pus from a suppurating gland or ulcer, or the 
nasal discharge from a suspected animal, rather than a direct attempt 
to demonstrate the presence of the bacillus by staining and culture 
methods. 

The method proposed by Strauss gives more prompt results. 
This consists in the intraperitoneal injection of cultures or of the 
suspected products into the cavity of the abdomen of male guinea- 
pigs. If the glanders bacillus is present the diagnosis may be made 
within three or four days from the infectious process established in 


494 BACILLI IN CHRONIC INFECTIOUS DISEASES. 


the testicles. At the end of this time the scrotum is red and shining, 
the epidermis desquamates, and suppuration occurs, the pus some- 
times perforating the integument, This pus is found to contain the 
glanders bacillus. The animal usually dies in the course of twelve 
to fifteen days. When the animals are killed three or four days 
after the inoculation, the two layers of the tunica vaginalis testis 
are found to be covered with a purulent exudate containing the 
glanders bacillus and to be more or less adherent. Even as early 
as the second day the tunica vaginalis is seen to be covered with 
granulations. 

An attenuation of virulence occurs in cultures which have been 
kept for some time, and inoculations with such cultures may give a 
negative result ; or, when considerable quantities are injected, may 
produce a fatal result at a later date than is usual when small 
amounts of a recent culture are injected into susceptible animals. 

Kalning, Preusse, and Pearson have obtained from cultures of 
the glanders bacillus a glycerin extract similar to the crude tubercu- 
lin of Koch—mallein. This, when injected into animals suffering 
from glanders, gives rise to a considerable elevation of temperature, 
and it is used as a means of diagnosis in cases of suspected infection in 
animals in which the usual symptoms have not yet manifested them- 
selves. The value of the test has been demonstrated by numerous 


experiments. 
Bonome (1894), as a result of extended researches, arrives at the 
following conclusions: : 


“1. The bacillus is found not only in the diseased tissues and 
purulent discharges, but also in the urine and milk of infected ani- 
mals. 

“2. The bacillus is found in the foetus of infected animals even 
when the placenta is free from any pathological change. 

“3. The glanders bacillus is very sensitive to desiccation and will 
not grow after being preserved for ten days at 25° C. 

“4, In distilled water the bacillus dies out in six days. 

“5. On the contrary, when protected from desiccation it resists 
a comparatively high temperature—70° C. for six hours; a temper- 
ature of 90° to 100° C. destroys it in three minutes.” 


‘ 


BACILLUS OF LUSTGARTEN. 


Synonym.—Syphilis bacillus. 

_, Found by Lustgarten (1884) in syphilitic lesions and secretions of syphi- 
litic ulcers, and believed by him to be the specific infectious agent in this 
disease. No satisfactory experimental evidence that this is the case has yet 
been obtained. 

Morphology.—Straight or curved bacilli, which bear considerable resem- 
blance to tubercle bacilli, but differ from them in the staining reactions. 

They are usually more or less curved, or bent at a sharp angle, or S-shaped ; 


BACILLI IN CHRONIC INFECTIOUS DISEASES. 495 


the ends often present slight knob-like swellings; the length is from three 
and one-half / to four and one-half “, and the diameter is from 0.25 to 0.3 u. 
With a high power the contour is seen to be not quite regular, but wavy in 
outline, and bright shining spaces in the deeply stained rods may be ob- 
served ; these, from two to four in a single rod, are believed by Lustgarten 
to be spores. The bacilli are not found free in the tissues, but are enclosed 
in cells of a round-oval or polygonal form, which are said to be about double 
the size of a white blood corpuscle. The bacilli are not numerous, and very 
commonly only one or two are found in a single cell, but groups of six or 
eight may sometimes be seen, especially upon the margins of a syphilitic 
lesion, and in the tissues in the immediate vicinity of the infiltration, which 
show but little change or are apparently healthy (Lustgarten). 

The presence of these bacilli in syphilitic lesions was demonstrated by 
Lustgarten by the following staining method: The thin sections are placed 
in the Ehrlich-Weigert gentian-violet solution (one hundred parts aniline 
water, eleven parts saturated alcoholic solution of gentian violet) for from 
twelve to twenty-four hours at the room temperature, and two hours in the 
incubating oven at 40°C. The sections are then thoroughly washed in alco- 
hol and placed for ten seconds in a 1.5-per-cent solution of potassium per- 
manganate; in this solution a precipitate of peroxide of manganese is 


Fic, 124. Fic. 125. 


Fic. 124.—Migrating cell containing syphilis bacilli. CLustgarten.) 


Fic. 125,—Pus from hard chancre containing syphilis bacilli (Lustgarten.) ‘ 


formed, which adheres to the section ; this is dissolved and washed off in a 
dilute aqueous solution of pure sulphuric acid; the sections are then washed 
in water, and, if not completely decolorized, are returned for a few seconds to 
the permanganate solution and again washed .off in the acid; it may be 
necessary to repeat this operation three or four times. Finally the sections 
are dehydrated and mounted in balsam in the usual manner. Cover-glass 
preparations are made in the same way, except that, after being taken from 
the staining solution, they are washed off in water instead of in alcohol. — 

Another method of staining, reeommended by De Giacoma, consists in 
placing the sections for twenty-four hours in aniline-water-fuchsin solution 
(cover-glass preparations may be stained in the same solution, hot, in a few 
minutes), then washing them in water, and decolorizing in a solution of per- 
chloride of iron—first in a dilute and then in a saturated solution. 

The method of staining employed by Lustgarten serves to differentiate 
his bacillus from many other microdrganisms, but not from the tubercle ba- 
cillus and the bacillus of leprosy, which, as he pointed out, may be stained 
in the same way. And it has since been shown by Alvarez and Tavel, and 
by Matterstock, that in smegma from the prepuce or the vulva, bacilli are 
found which have the same staining reaction and are similar in their mor- 
phology to the bacillus of Lustgarten. This by no means proves that the 


496 BACILLI IN CHRONIC INFECTIOUS DISEASES. 


smegma bacilli found under the prepuce of healthy persons are identical 
‘with the bacilli found by Lustgarten and others mm sections of tissues involved 
in syphilomata. In the absence of pure cultures and inoculation experiments 
it is impossible to establish identity, however similar may be the characters 
referred to. Several well-known pathogenic bacilli resemble quite as closely 
in these particulars other bacilli which have, nevertheless, been differentiated 
from them by culture and inoculation experiments. We may mention 
especially in this connection the bacillus of diphtheria, as obtained from the 
pseudo-membranous exudation in a genuine case of this disease, and the 
pseudo-diphtheria bacilli found by Roux and Yersin in the fauces of healthy 
children. On the other hand, since it has been shown that similar bacilli 
are common in preputial smegma, we cannot attach great importance to the 
finding of Lustgarten’s bacillus in primary syphilitic sores; and it has not 
been found in sufficient numbers, or with sufficient constancy, by those who 
have searched for it subsequently to the publication of Lustgarten’s inves- 
tigations, to give strong support to the view that it is the specific infectious 
agent in syphilis. Baumgarten, who has searched in vain for Lustgarten’s 
bacillus in uncomplicated visceral syphilomata, suggests that the bacilli 
found occasionally in such lesions were perhaps tubercle bacilli and repre- 
sented a mixed infection. As the bacillus under consideration has not been 
obtained in cultures, we have no information as to its biological characters 
and pathogenesis. 


BACILLUS OF RHINOSCLEROMA. 


First observed by Von Frisch (1882) in the newly formed tubercles of 
rhinoscleroma. Cultivated by Paltauf and Von Hiselberg (1880). 

Rhinoscleroma is a chronic affection of the skin, and. especially of the 
mucous membrane of the nares, which is characterized by the formation of 
tubercular thickenings of the skin and tumefaction of the nasal mucous 
membrane, followed sometimes by ulceration. It prevails in Italy, Austria, 
and to aslight extent in some parts of Germany. Pathologists generally 
regard it as an infectious process, although this has not been proved. 

The bacilli, first described by Von Frisch, appear to be constantly present 
in the newly formed tubercles. They are commonly found in certain large 


Fic. 126.—Bacillus of rhinoscleroma in lymphatic vessels of the superficial part of tumor. 
* 1,200. (Cornil aud Babes ) 


hyaline cells peculiar to the disease, and may also be observed in the lym- 

phatic vessels or scattered about in the involved tissues. 
Morphology.—Short bacilli with rounded ends, usually united in pairs, 

and surrounded bya gelatinous capsule resembling that of Friedlinder’s 


BACILLI IN CHRONIC INFECTIOUS DISEASES. 497 


bacillus. According to Eisenberg, the bacilli are two to three times as long 
as broad, and may grow out into filaments. 

These bacilli stain readily with the aniline colors and by Gram’s method. 
The capsule may be demonstrated by the methods usually employed in stain- 
ing Friedlander’s bacillus, or by the following method which is especially 
recommended by Alvarez: The excised portions of tissue involved in the dis- 
ease are placed for twenty-four hours in a one-per-cent solution of osmic 
acid and then in absolute alcohol. When properly hardened thin sections 
are made; these are stained in a hot solution of aniline-water-methyl-violet 
for a few minutes, and then decolorized, by Gram’s method, in iodine so- 
lution. 

Biological Characters.—An aérobic, non-motile, non-liquefying bacillus 
(facultative anaérobic ?). 

In gelatin stab cultures the growth resembles that of Friedlander’s ba- 
cillus—z.e., a nail-like growth, consisting of densely crowded, opaque colonies 
along the line of puncture, and a heaped-up, white, glistening mass upon the 
surface, hemispherical in form and viscous in consistence. Upon gelatin 
plates yellowish-white, spherical colonies are developed within two or three 
days, which under the microscope are seen to be granular. Upon potato a 
cream-like growth occurs along the line of inoculation, which is white or 
yellowish-white in color, and in which gas bubbles may be developed. De- 
velopment is most rapid at a temperature of 35° to 38°, but also occurs at the 
room temperature. 

Pathogenesis.—The etiological relation of this bacillus to the disease with 
which it is associated has not been established. It is pathogenic for mice 
and for guinea-pigs, less so for rabbits; in this regard, as in its morphology 
and growth in various culture media, it bears a close resemblance to Fried- 
lander’s bacillus, which is also found not infrequently in the nasal secretions 
of healthy persons and in those suffering from chronic nasal catarrh or ozzena. 

The principal points of difference, as pointed out by Baumgarten, are as 
follows: The bacillus of rhinoscleroma is usually more decidedly rod-shaped 
than Friedlander’s bacillus, although both may be of so short an oval as to 
resemble micrococci. The first-mentioned bacillus constantly presents the 
appearance of being surrounded by a transparent capsule, even in the cul- 
tures in artificial media, while Friedlander’s bacillus in such media does not 
usually present this appearance, unless as a result of special treatment. 
Finally, the bacillus of rhinoscleroma may retain its color, in part at least, 
when treated by Gram’s method, while Friedlander’s bacillus is completely 
decolorized when placed in the iodine solution employed in this method. 

Notwithstanding these points of difference, Baumgarten is not entirely 
satisfied that’ this bacillus is a distinct species, and several bacteriologists 
have maintained that it is identical with the bacillus of Friedlander. 


32 


XIII. 


BACILLI WHICH PRODUCE SEPTICAIMIA IN 
SUSCEPTIBLE ANIMALS. 


WHEN, as a result of accidental (natural) or experimental inocula- 
tion, a microdrganism is introduced into the body of a susceptible. 
animal which is able to multiply in its blood, producing a general in- 
fection, we speak of this general blood infection as a septiccemia. 
When pathogenic microérganisms which are unable to multiply in 
the blood establish themselves in some particular locality in the ani- 
mal body which is favorable for their growth, and by the formation 
of toxic products, which are absorbed, give rise to general symptoms 
of poisoning, we designate the affection toxemia. As examples of 
this mode of pathogenic action we may mention diphtheria and 
tetanus. As a rule, the various forms of septicemia are quickly 
fatal, and, as the microdrganisms to which they are due multiply in 
the blood of the infected animal, this fluid possesses infectious pro- 
perties, and, when inoculated in the smallest quantity into another 
susceptible animal, reproduces the same morbid phenomena. A typi- 
cal example of this class of diseases is found in anthrax, to which 
disease a special section has already been devoted (VIII.). But in 
this and other forms of septicemia subcutaneous inoculations do not, 
as a rule, result in the immediate invasion of the blood by the para- 
sitic microérganism. Often a local inflammatory process of consider- 
able extent is first induced ; and in some cases general infection only 
occurs a short time before the death of the animal, depending, per- 
haps, upon a previous toxemia from the absorption of toxic products 
developed at the seat of localinfection. The pathogenic action, then, 
in acute forms of septicaemia appears to result, not alone from the 
presence and multiplication of the pathogenic microdrganism in the 
blood, but also from the toxic action of products evolved during its 
growth. 

Some of the pathogenic bacilli of this class now known to bac- 
teriologists have been discovered by studying the infectious diseases 
induced by them in lower animals among which these diseases pre- 
vail naturally—?.e., independently of human interference. Many 


BACILLI WHICH PRODUCE SEPTICAMIA. 499 


more are known to us from experiments made in pathological labora- 
tories, in testing by inoculations into animals bacteria obtained from 
various sources, with reference to their pathogenic power. We in- 
clude in this group only those bacilli which induce fatal septicemia 
‘in susceptible animals when injected into the circulation or sub- 
cutaneously in a comparatively small quantity—e.g., less than half 
a cubic centimetre of a bouillon culture. 


BACILLUS SEPTICHMIA HAMORRHAGICA, 


Synonyms.—Bacillus of fowl cholera; Microbe du choléra des 
poules (Pasteur); Bacillus cholere gallinarum (Fligge); Bacillus der 
Hiihnercholera ; Bacillus of rabbit septicemia; Bacillus cuniculi- 
cida (Fliigge) ; Bacillus der Kaninchenseptikimie (Koch) ; Bacillus 
der Rinderseuche (Kitt) ; Bacillus der Schweineseuche (Léffler and 
Schiitz) ; Bacillus der Wildseuche (Hueppe) ; Bacillus der Biiffel- 
seuche (Oreste-Armanni) ; (Bacterium of Davaine’s septiczemia ?) 

It is now generally admitted by bacteriologists that Koch’s ba- 
cillus of rabbit septicemia (1881) is identical with the bacillus 
(‘‘micrococcus”) of fowl cholera previously described by Pasteur 
(1880). The similar bacilli found in the blood of animals dead from 
the infectious diseases known in Germany as Wildseuche (Hueppe), 
Rinderseuche (Kitt), Schweineseuche (Schtitz), and Biiffelseuche 
(Oreste-Armanni) appear also to be identical with the bacillus of 
rabbit septicemia and fowl cholera. This view is sustained by 
Hueppe and by Baumgarten, and by the comparative researches of 
Caneva (1891) and of Bunzl-Federn (1891). 

This is evidently a widely distributed pathogenic bacillus ; it was 
obtained by Koch from rabbits inoculated with pu- 
trefying flesh infusion, by Gaffky from impure river . | Oe: 
water, and by Pasteur from the blood of fowls suffer- O)% iC me) 
ing from the infectious disease known in France as *+ * <t wee. 
choléra des poules. It is not infrequently found in °°~() «=D < 
putrefying blood, and its presence in the salivary 20 ter an oe 
secretions of man has occasionally been demonstrated 4,4. 497 _ Bacillus 
(Baumgarten). septicaemia hemor- 

With reference to the American swine plague ee ea 
described by Salmon and Smith, we are informed by (aumgarten.) 
Smith, in his most recent publication upon the subject 
(Zeitschrift fiir Hygvene, Band x., page 493), that cultures of the 
German Schweineseuche bacillus, received from the Berlin Hygienic 
Institute, compared with his cultures from infected swine in this 
country, agreed in all particulars, except that the former were de- 
cidedly more pathogenic for swine and for rabbits. 

It appears extremely probable that the form of septicemia studied 


500 BACILLI WHICH PRODUCE SEPTICEMIA 


by Davaine (1872), which he induced in the first instance by inject- 
ing putrid ox blood into rabbits, was due to the same pathogenic ba- 
cillus. The writer obtained this bacillus (1887) in Cuba from the 
blood of rabbits inoculated with liver tissue taken from a yellow- 
fever cadaver and kept for forty-eight hours in an antiseptic wrap- 
ping. Thename which we have adopted is that proposed by Hueppe 
for the form of septicemia to which it gives rise—‘‘Septikaémia 
haémorrhagica.” 

Morphology.—Short bacilli with rounded ends, from 0.6 to 0.7 
/ in diameter and about 1.4 4 long; sometimes united in pairs, or 
in chains of three or fourelements. In stained preparations the ex- 
tremities are usually stained, while the central portion of the rod 
remains unstained. This ‘‘ end staining” causes the rods to present 
the appearance of diplococci when examined with a comparatively 
low power, and some of the earlier observers described the microér- 
ganism under consideration as a micrococcus. It is quickly stained 
by the aniline colors usually employed, but loses its color when 
treated by Gram’s method. 

Biological Characters.—A. non-motile, aérobic, non-liquefy- 
ing bacillus. Does notform spores. Grows in various culture media 
at the room temperature, but more rapidly at 35° to 37° C.—the 
lowest temperature at which development occurs is about 13° C. 
Although this is an aérobic bacillus and a certain amount of oxygen 
is necessary for its development, it appears to grow better when the 
amount is somewhat restricted than it does on the surface of nutrient 
media. 

Upon gelatin plates, at the end of two or three days, small, 
white colonies are developed upon or near the surface; these are 
finely granular and spherical, with a more or less irregular outline, 
and by transmitted light have a yellowish color ; later the central 
portion of the colonies is of a yellowish-brown color and is sur- 
rounded by a transparent peripheral zone. The superficial colonies 
are commonly smaller than those which develop a little below the 
surface of the gelatin. In stab cultures in nutrient gelatin the 
growth upon the surface consists of a thin, whitish layer in the 
vicinity of the point of puncture, having an irregular, jagged out- 
line—sometimes there is no development upon the surface; along 
the line of puncture the growth consists of rather transparent, dis- 
crete. or confluent colonies. In streak cultures upon nutrient agar, 
or gelatin, or blood serum the growth ‘is limited to the immediate 
vicinity of the line of inoculation, and consists of finely granular, 
semi-transparent colonies, which form a thin, grayish-white layer 
with irregular, somewhat thickened margins. Upon potato no de- 
velopment occurs, as a rule, at the room temperature, but in the in- 


IN SUSCEPTIBLE ANIMALS. 501 


cubating oven a rather thin, transparent, grayish-white or yellowish, 
waxy eee is developed in the course of a few days. According to 
Bunzl-Federn, the bacillus of fowl cholera and that 
of rabbit septicemia grow upon potato, while the 
bacillus of Wildseuche, Schweineseuche, and Biif- 
felseuche do not. According to 
Caneva, none of the bacilli of this 
group grow upon potato. The 
same author states thatthe growth 
in milk is scanty and does not 
produce coagulation, while Bunzl- 
Federn finds that the bacillus of 
fowl cholera and of rabbit septi- 
cemia produce coagulation and 
the others do not. These differ- 
ences are not, however, consid- 
ered by the author last named as 
sufficient to establish the specific 
difference of the bacilli from these 
different sources. He looks upon 
them rather as varieties of the 
same species. Bunzl-Federn has 
also ascertained that when cul- 
tivated in a peptone solution all 
of the bacilli of this group, with 


Fig. 128, — Bacflius 


septiceemize hemor- 
rhagice; stick culture 
in nutrient gelatin, 
end of four days at 16°- 
18°C. (Baumgarten ) 


Fie. 129,—Bacillus 
of Schweineseuche ; 
old stick culture 
in nutrient gela- 
tin. (Schitz,) 


the exception of that obtained 
from the so-called Biiffelseuche; 
give the reaction for phenol and 


for indol—the bacillus of Biiffel- 
seuche gives the indol reactiononly. Development in bouillon is rapid 
and causes a uniform turbidity of the fluid. Cultures of this bacillus 
may retain their vitality for three months or 
more when kept in a moist condition; but 
the bacillus usually fails to grow after having 
been kept for a few days in a desiccated con- 
dition ; according to Hueppe, it may resist 
desiccation for fourteen days. The thermal 
death-point, as determined by Salmon for 
the bacillus of fowl cholera, is 56° C., the time 
of exposure being ten minutes (55° C. with 
fifteen minutes’ exposure—Baumgarten). It 
isnotreadily destroyed by putrefaction (Kitt). 
A solution of mercuric chloride of 1 :5,000 
destroys it in one minute, and a three-per-cent solution of varbolic 


Fic. 130.— Bacillus of swine 
plague; colonies on gelatin 
plate, end of scvea days, 
x 60. (Snuith.) 


502 BACILLI WHICH PRODUCE SEPTICZMIA 


acid in six hours (Hueppe). Pasteur (1880) has shown that when 
cultures of this bacillus (microbe of fowl cholera) in bouillon are 
kept for some time they gradually lose their pathogenic virulence, 
and he has ascribed this ‘‘ attenuation of virulence” to the action of 
atmospheric oxygen. He also ascertained that the particular degree 
of virulence manifested by the mother culture after a certain interval 
could be maintained in successive cultures made at short intervals. 
He was thus able to cultivate different pathogenic varieties, and to 
use these in making protective inoculations, by which susceptible ani- 


Fie. 131.—Bacillus of Schweineseuche, in blood of rabbit. (Schiitz.) 


mals were preserved from the effects of virulent cultures injected 
subsequently. 

Attenuated cultures recover their virulence when inoculated into 
very susceptible animals. Thus a culture which would produce a 
non-fatal and protective attack in a chicken may, according to Pas- 
 teur, kill asmall bird, like a sparrow; and by successive inoculations 
from one sparrow to another the original degree of virulence may be 
restored, so that a minute quantity of a pure culture would be fatal 
to a chicken. 

Pathogenesis.—Pathogenic for chickens, pigeons, pheasants, 
sparrows, and other small birds, for rabbits and mice, also for swine 
(Schweineseuche), for cattle (Rinderseuche), and for deer (Wil]. 
seuche). (See supra, pp. 285-287.) 

The researches of Smith and of Moore show that “an attenuated 
variety of bacteria, belonging to the group of swine-plague bacteria 
and not distinguishable from them, inhabit the mouth and upper air 


IN SUSCEPTIBLE ANIMALS. 503 


passages of such domesticated animals as cattle, dogs, and cats” 
(Smith), 
BACILLUS OF CHOLERA IN DUCKS. 


Obtained by Cornil and Toupet (1888) from the blood of ducks, in the 
Jardin d’Acclimation at Paris, which had died of an epidemic disease charac- 
terized by diarrhoea, feebleness, aud muscular tremors, and which resulted 
fatally in two or three days. 

Morphology.—Does not differ in its morphology from the bacillus of 
fowl cholera (Bacillus septicaemia hemorrhagice) ; short rods with rounded 
ends, from 1 to 1.5 w in length and 0.5 u broad. 

Stains with the usual aniline colors, but not by Gram’s method; the ends 
stain more deeply than the central portion. 

Biological Characters.—An aérobic, non-liquefying, non-motile bacillus. 
Does not form spores. Grows in the usual culture media at the room tem- 
perature. Initsgrowth in various media, as wellasin its morphology, Cornil 
and Toupet found this bacillus to correspond with the bacillus of fowl 
cholera. In gelatin stab cultures the growth upon the surface consists of a 
thin, grayish layer, and along the line of puncture as small, semi-transpa- 
rent, Hiontly yellowish, spherical colonies. Upon agar, in the incubating 
oven, at the end of twelve hours small, lentilsshaped, waxy colonies are 
formed, which later may have a diameter of three to four millimetres. 
Upon potato circular, yellowish colonies are formed, which become con- 
fluent and form a somewhat depressed, pale-yellow layer. 

Pathogenesis.—According to Cornil and Toupet, this bacillus is patho- 

enic for ducks, but not for chickens or pigeons, and only kills rabbits when 
Injected in considerable quantity. Ducks die in from one to three days 
from subcutaneous injections, or by the ingestion of food to which the bacil- 
lus has been added. 


BACILLUS OF HOG CHOLERA (Salmon and Smith). 


Synonyms.—Bacillus of swine plague (Billings); Bacillus of swine- 
pest (Selander). 

According to Smith, this bacillus was first described by Klein 
(1884) ; it was first obtained in pure cultures and its principal char- 
acters determined by Salmon and Smith (1885), and has since been 
studied in cultures and by experimental inoculations by Selander, 
Billings, Frosch, Welch, Caneva, Bunzl-Federn, and cthers. 

The bacillus is found in the blood and various organs of hogs 
which have succumbed to theinfectious disease known in this country 
as hog cholera; and also in the contents of the intestine, from which 
it may be obtained by inoculations into rabbits, but is not easily iso- 
lated by the plate method owing to the large number of other bac- 
teria present (Smith). 

Morphology.—Short bacilli with rounded ends, 1.2 to 1.5 «in 
length and 0.6 to 0.7 in breadth ; usually united in pairs. 

This bacillus is easily stained by the aniline colors usually em- 
ployed, but does not retain its color when treated by Gram’s method. 
When the staining agent is allowed to act for a very short time the 
ends of the rods may be stained while the central portion remains 
unstained. 


504 BACILLI WHICH PRODUCE SEPTICAIMIA 


Biological Characters.—An aérobic (facultative anaérobic), non- 
liquefying, actively motile bacillus. 1n many of its characters this 
bacillus closely resembles the one last described (Bacillus septiceemiz 
hemorrhagice), but it is distinguished from it by its active move- 
ments, which, according to Smith, may be still observed in cultures 
which have been kept for weeks or months. Does not form spores, 
Grows readily in various culture media at the room temperature— 
more rapidly in the incubating oven. Upon gelatin plates colonies 
are developed in from twenty-four to forty-eight hours. The deep colo- 
nies are spherical and homogeneous, and have a brownish color by 
transmitted light; they seldom exceed one-half millimetre in diameter. 


Fie. 132.—Bacillus of hog cholera; stained by Liffler’s method to show flagella. x 1,090. From 
a photomicrograph mace at the Army Medical Museum, (Gray.) 


The superficial colonies may attain a diameter of two millimetres ; 
they present no distinctive characters. Upon agar plates the colonies 
may have adiameter of four millimetres ; they have a grayish, trans- 
parent appearance and a shining surface. In gelatin stab cultures 
small, yellowish-white colonies are developed along the line of in- 
oculation, which may become confluent; upon the surface a thin, 
pearly layer is developed about the point of inoculation, which may 
have a diameter of six millimetres or more. Upon potato a straw- 
yellow layer is developed, which later acquires a darker color. In 
slightly alkaline boucllon a slight cloudiness may be observed at the 
end of twenty-four hours, and at the end of one or two weeks, if 
not disturbed, a deposit is seen at the bottom of the tube and a thin, 
broken film may form upon the surface. The development of this 
bacillus in milk produces a direct solution of the casein without pre- 
vious coagulation ; when a solution of litmus has been added to milk 


IN SUSCEPTIBLE ANIMALS. 505 


it retains its blue color in presence of this bacillus, while the bacillus 
previously described causes it to change to red. Neither phenol 
nor indol is produced in solutions containing peptone (Bunzl-Federn) 
—another distinguishing character from the Bacillus septiceemize 
hemorrhagice. This bacillus may be cultivated in slightly acid 
media, which after a time acquire an alkaline reaction. 

In Smith’s experiments this bacillus was found to resist desicca- 
tion from nine days to several months, according to the thickness of 
the layer dried upon the cover glass; bacilli from an agar culture in 
some experiments failed to grow after seventeen days, and in others 
still gave cultures after four months. Bouillon cultures are steril- 
ized in four minutes by a temperature of 70° C., in fifteen minutes 
by 58° C., and in one hour by 54° C. (Smith). Novy has isolated 
from cultures of the hog-cholera bacillus a toxic basic substance 
which he calls susotoxin, This was obtained by Brieger’s method ; 
it is a yellowish-brown, syrup-like liquid, which, when injected into 
rats in doses of 0.125 to 0.25 cubic centimetre, causes their death in 
less than thirty-six hours. He also obtained by precipitation with 
absolute alcohol, from cultures concentrated in a vacuum at 36° C., 
a toxalbumin which when dried was in the form of a white powder 
easily soluble in water. Rats died in three or four hours after re- 
ceiving subcutaneously a dose of 0.1 to 0.5 gramme. 

Pathogenesis.—Pathogenic for swine, rabbits, guinea-pigs, mice, 
and pigeons. 

Tn certain parts of the United States the disease known as “‘ hog 
cholera” frequently prevails among swine asa fatal epidemic. It 
may occur as an acute and quickly fatal septiczemia, or in a more 
chronic form lasting from two to four weeks or even longer. In 
the acute form death may occur within twenty-four hours, and hem- 
orrhagic extravasations are found upon the mucous and serous 
membranes and in the parenchyma of the lungs, kidneys, and lym- 
phatic glands. The spleen is greatly enlarged, soft, and dark in 
color. In the chronic form of the disease the most notable changes 
are found in the alimentary canal. These are most constant and 
characteristic in the cecum and colon, which may be studded with 
spherical, hard, necrotic masses or extensive diphtheritic patches. 
According to Smith, the hemorrhagic and necrotic form of the dis- 
ease may exist at the same time in different animals of the same 
herd. The bacilli are found in all of the organs, and especially in 
the spleen, where they are associated in irregular colonies similar 
to those of the typhoid bacillus. Smith has demonstrated their pre- 
sence in urine taken from the bladder immediately after the death 
of the animal, and states that the kidneys are almost always in- 


506 BACILLI WHICH PRODUCE SEPTICAMIA 


volved, as shown by the presence of albumin and tube casts in the 
urine. 

An extremely minute quantity of a bouillon culture injected be- 
neath the skin of a rabbit causes its death in from seven to twelve 
days; a larger quantity may produce a fatal result in five days ; in- 
travenous injections of very small amounts may be fatal within 
forty-eight hours. After a subcutaneous injection the animal re- 
mains in apparent good health for three or four days, after which it 
loses its appetite and is indisposed to move ; several days before 
death the temperature is suddenly elevated from 2° to 3° C., and it 
remains high until the fatal termination. At the autopsy the spleen 
is found to be enlarged and of a dark-red color ; the liver is studded 
with small, yellowish-white, necrotic foci; the kidneys have undez- 
gone parenchymatous changes ; the heart is fatty ; and the intestinal 
mucous membrane is more or less marked with hemorrhagic extra- 
vasations. The bacilli are found in all of the organs. In house 
mice the results of experimental inoculations are similar to those in 
rabbits. Guinea-pigs succumb when inoculated subcutaneously with 
one-tenth cubic centimetre; pigeons require a still larger dose— 
about three-quarters of a cubic centimetre. Swine are killed by the 
intravenous injection of one to two cubic centimetres of a recent 
bouillon culture, but, as a rule, do not succumb to subcutaneous 
injections. Cultures recently obtained from diseased animals are 
more virulent than those which have been propagated for a consider- 
able time in artificial media. 

Smith has described a variety of the hog-cholera bazillus obtained during 
an epidemic in which the disease was of longer duration—about four weeks 
—than is usual, and in which there was commonly found at the autopsy a 
diphtheritic inflammation of the mucous membrane of the stomach. This 
bacillus differed from the typical form by being somewhat larger and in 
forming considerably larger colonies in gelatin plates—two or three times 
as large. It also produced a greater opacity in peptonized bouillon, and in 
general showed a more vigorous growth in various nutrient media. It dif- 
fered also in its pathogenic power, as tested upon rabbits, causing death at a 
later date or not at all; and in fatal cases the swelling of the spleen and 
necrotic foci in the liver, produced by the first-described species, were absent. 

Bang (1892) has obtained ‘a bacillus from infected swine in Denmark 
which corresponds with the American hog-cholera bacillus. In chronic 
forms of the disease pneumonia and an extensive diphtheritic process in the 
intestines occurred as a complication. This was found to be due to another 
bacillus, called by Bang ‘‘ vacuole-bacillus.” This produced a fatal pleuro- 
pneumonia when injected into the lungs in pigs. According to Bang, his 
‘‘vacuole-bacillus” is without doubt identical with the swine-plague bacillus 
of Salmon and Smith, and the disease of swine studied by him was a mixed 
infection. The necrotic changes in the intestine, found in cases running a 
chronic course, are believed by Bang to be due to still another bacillus—his 
‘* necrosis-bacillus.” Affanassieff (1892) confirms the results previously ob- 
tained by several independent observers as to the identity of the swine-plague 


bacillus of Salmon and Smith with the Loffler-Schiitz bacillus. The only 
difference observed was a difference in pathogenic virulence—the bacillus 


IN SUSCEPTIBLE ANIMALS. 507 


from America corresponding with a somewhat attenuated variety of that 
from Germany. 
_ Welch (1894), as a result of his extended researches, arrives at the follow- 
ing conclusion : 

‘‘Our own conclusion as to the bacteria of Schweineseuche and of swine 
plague is that no difference exists between them as regards morphology, 
culture behavior, and pathogenic effects on rabbits, mice, and other labora- 
tory animals. Cultures of each occur which are also indistinguishable by 
inoculation of pigs. The only difference by laboratory experiment which 
has thus far been brought out is that there oceur Schweineseuche bacilli of 
higher degree of virulence as tested on pigs than any swine-plague bac- 
teria which have hitherto been isolated from pigs in this country. Another 
point to be considered in this connection is that Schweineseuche occurs as 
an independent disease in Germany without association with hog cholera, 
whereas swine plague has not been shown to prevail with the same inde- 
pendence as an epizootic in this country.” 

Silberschmidt (1895) arrives at a different conclusion from that reached 
by Smith, Welch, Bang, and others, He believes that the diseases of swine 
known as hog cholera, swine plague, and infectious pneumo-enteritis are all 
due to one and the same bacillus, which, however, varies considerably both 
in its morphological characters and its pathogenic power. In view of the 
results previously reached by equally competent bacteriologists, and especially 
by Smith and by Welch in this country, we are not disposed to accept the 
view maintained by Silberschmidt. 

Smith has described several varieties of the hog-cholera bacillus, and_in 
his account of the ‘‘ hog-cholera group of bacteria” shows that the Bacillus 
enteriditis of Girtner and the Bacillus typhi murium of Loffler belong to 
this group. The characters of the different varieties (or species?) belonging 
to the group are given by Smith in detail (United States Department of Agri- 
culture, Bureau of Animal Industry, Bulletin No. 6, 1894), and the follow- 
ing general statement is made: ; ; 

“Tf we attempt to sum up those characters which are to circumscribe the 
hog-cholera group of bacteria we are at once confronted by the scarcity of 
common characters. Pathogenesis, though of great importance from the 
standpoint of pathology, is probably the last character acquired and 
evidently the most variable and most readily lost. If we base the unity 
of this group on morphological and biological characters, we are like- 
wise met by variations in size, absence of motility, variations in the ap- 
pearance of the colonies. There are, however, certain underlying char- 
acters, as expressed by the behavior of these bacteria in bouillon con- 
taining dextrose, saccharose, and lactose, which I think will serve as a very 
important group character, differentiating such groups sharply from the 
colon group. I would therefore suggest that for the present all bacteria 
whose size approximates that of this group, which do not liquefy gelatin, and 
whose fermentative properties are the same as those described for this group, 
should be ranged under it. Future investigations into the biochemical char- 
acters of these varieties or sub-species may reveal other differential charac- 
ters, but the time has not yet come when such laborious work will be under- 
taken oma sufficiently extensive scale to be of any service in differentiating 
varieties and sub-species.” : 

Selander in 1890, and Metschnikoff in 1892, have reported a rapid increase 
in virulence of the bacillus of hog cholera by successive inoculations in 
rabbits or pigeons. Moore (1894) has shown that this is a mistake, and that 
the bacteriologists named probably did not experiment with cultures of the 
hog-cholera bacillus, as they supposed, but that their experiments were 
made with the bacillus of swine plague—Bacillus septiceemize hemorrhagi- 
cze—which when passed through a series of rabbits attains a notable increase 
in pathogenic virulence. eich : 

In a recent article, Klein, of London (1895) says: ‘‘ The bacillus of 
English swine plague, which I described in 1884, in Virchow’s Archiv, as 


é 


508 BACILLI WHICH PRODUCE SEPTIC ZMIA 


shown by Smith and Welch, is identical with the bacillus of American hog 
cholera.” 


BACILLUS OF BELFANTI AND PASCAROLA. 


Synonym.—Impftetanusbacillus. ; . 

Obtained by Belfanti and Pascarola (1888) from the pus of wounds in an 
individual who succumbed to tetanus. 

Morphology.—Bacilli with rounded ends, sometimes so short as to resemble 
micrococci; resemble the Bacillus septiczeemize haeemorrhagice (fowl cholera). 

Stains with the usual aniline colors and also by Gram’s method. The 
ends are commonly more deeply stained than the central portion. | 

Biological Characters.—An aérobic and facultative anaérobic, non- 
liquefying, non-motile bacillus. Spore formation not observed. Grows in 
the usual culture media at the room temperature. Upon gelatin plates yel- 
lowish-gray, finely granular, spherical colonies with smooth outlines are 
developed. In gelatin stab cultures, at 18° to 25° C., at the end of twenty- 
four hours small, spherical colonies are developed along the line of punc- 
ture, which are isolated or closely crowded; upon the surface a rather thin, 
shining, grayish-white, iridescent, circular layer is formed; gas is given off 
which has not a disagreeable odor. Upon the surface of agar elevated, 
shining, gray colonies develop along the impfstrich, or a gray, shining band 
is formed which increases in thickness but not in breadth—usually less than 
one-half centimetre broad. Old cultures give off an acidodor. Upon blood 
serum a thin, white layer is developed along the line of inoculation. Upon 
potato a thin, white, varnish-like layer is formed. 

Pathogenesis.—Very pathogenic for rabbits, guinea-pigs, white mice, and 
sparrows. Not pathogenic for chickens, pigeons, or geese. 


BACILLUS OF SWINE PLAGUE, MARSEILLES. 


Synonyms.—Bacillus der Schweineseuche, Marseilles (Rietsch 
and Jobert) ; Bacillus der Frettchenseuche—ferret disease (Eberth 
and Schimmelbusch); Bacillus der Amerikanischen Rinderseuche 
(Caneva); Bacillus of spontaneous rabbit septicemia (Eberth). 

The researches of Caneva and of Bunzl-Federn agree as to the 
identity of the bacillus obtained by Rietsch and Jobert (1887) from 
swine attacked with a fatal epidemic disease in Marseilles, and the 
bacillus found by Eberth and Schimmelbusch (1889) in the blood of 
ferrets suffering from a fatal form of septicaemia studied by them. 
The first-named bacteriologist also identifies a bacillus supposed 
by Billings to be the cause of ‘‘ Texas fever” in cattle (‘‘ Ameri- 
kanische Rinderseuche”) and the bacillus of swine plague (Billings) 
with the above. Bunzl-Federn obtained cultures of Billings’ swine- 
plague bacillus at two different times. He identifies the one first re- 
ceived with the bacillus now under consideration, and the other with 
the bacillus of hog cholera (Salmon).! 

1The author named says: ‘ With reference to the bacillus of swine plague 


(Billings), I obtained, as did Caneva, a decided production of acid in the cultures 
first sent by Billings; but upon testing later cultures received directly from Bil- 


IN SUSCEPTIBLE ANIMALS. 509 


Morphology.—Bacilli with rounded ends, about twice as long as 
broad, and one-third smaller than the bacillus of typhoid fever 
(Eberth and Schimmelbusch). The bacillus of hog cholerais shorter 
and more slender than the Marseilles bacillus, and the bacillus of 
Léffler and Schiitz is still smaller (Rietsch and Jobert). 

In stained preparations the extremities of the rods are usually 
deeply stained, while the central portion remains unstained—“ polar 
staining.” By Léffler’s method of staining the presence of flagella 
may be demonstrated (Frosch). 

Stains readily with the aniline dyes usually employed, but does 
not retain its color when treated by Gram’s method. 

_Biological Characters.—An aérobic (facultative anaérobic), 
non-liquefying, actively motile bacillus. Grows readily at the 
room temperature, and is distinguished from the bacillus of septi- 
cemia hemorrhagica by its active movements and more rapid and 
abundant development in the various culture media usually em- 
ployed. It is distinguished from the bacillus of hog cholera by pro- 
ducing phenol and indol in solutions containing peptone, by causing 
coagulation of milk, and by producing an acid reaction in this fluid. 
Grows in culture media having an acid reaction. 

Rietsch and Jobert give the following account of the characters 
of growth in various culture media, as compared with the bacillus of 
hog cholera and the bacillus of Schweineseuche (Léffler, Schutz) : 

Gelatin streak cultures. At the end of twenty-four hours this 
bacillus had developed considerably, while the growth of the hog- 
cholera bacillus was scarcely to be discerned with the naked eye, and 
the bacillus of Schweineseuche did not form a visible growth until 
the end of forty-eight hours. After several days the bacillus of 
swine plague (Marseilles) formed an opaque, yellowish-white streak, 
which, when examined with a low-power lens, had a brown color by 
transmitted light and a bluish-white color by reflected light. The 
streak of the Léffler-Schiitz bacillus was not so thick and not so 
opaque, and was made up of small, nearly transparent colonies ; the 
hog-cholera bacillus came between the other two. Upon blood 
serum, agar, and glycerin-agar the Marseilles bacillus grew more 
rapidly than the other two, forming a layer which was opaque and 
of a white color, with bluish and reddish reflections. Upon potato 
it formed a thick, opaque, yellowish layer, while the growth of the 
hog-cholera bacillus was much thinner and that of the Léffler-Schiitz 
bacillus scarcely to be seen. In bouzllon the Léffler-Schiitz bacillus, 
at the end of three days at 37° C., had not produced any perceptible 


lings and from other sources, the result was exactly the opposite—viz., a decided 
production of alkali in milk and identity with the hog-cholera bacillus of Salmon.” 


510 BACILLI WHICH PRODUCE SEPTICAMIA 


cloudiness, while the Marseilles bacillus at the end of twenty-four 
hours had caused the fluid to be clouded, a film of bacteria had 
formed upon the surface anda deposit at the bottom of the tube ; the 
hog-cholera bacillus produced a less degree of opacity in the bouillon. 

Pathogenesis.—This bacillus is pathogenic for sparrows and 
other small birds when injected beneath the skin in small amounts, 
and also for pigeons in a longer time—five to fourteen days. Frosch 
reports a negative result from subcutaneous injections into rabbits, 
guinea-pigs, mice, and pigeons, but his cultures appear to have be- 
come attenuated, as the recent cultures of Eberth and Schimmelbusch 
were fatal to pigeons in four out of five experiments. Two rabbits 
were inoculated subcutaneously by Rietsch and Jobert with half a 
Pravaz syringeful of a pure culture of the Marseilles bacillus ; one of 
these died on the sixth day and the other survived. 

In sparrows, which succumb in from twenty-four to thirty-six 
hours after receiving a small amount of a pure culture in the breast 
muscle, the bacillus is present in the blood in large numbers, and a 
purulent pleuritis and pericarditis is found at the autopsy. In the 
ferrets from which Eberth and Schimmelbusch obtained their cultures 
the bacillus was not present in the blood in sufficient numbers to be 
readily demonstrated by microscopical examination, but it was ob- 
tained in pure cultures from the liver, spleen, and lungs. The prin- 
cipal pathological appearances noted were enlargement of the spleen 
and pneumonia. Caneva reports that the Marseilles bacillus injected 
into white mice gives rise to an extensive abscess at the point of in- 
oculation, but does not kill adult animals. In a young mouse which 
succumbed to such an injection the bacilli were not generally dis- 
tributed in the tissues, but were found as emboli in the smaller capil- 
laries. This bacillus, then, is distinguished from the similar bacilli 
previously described by its comparatively slight pathogenic power, 
as well as by its more vigorous growth in culture media, and the 
other characters heretofore mentioned. 


BACILLUS SEPTICUS AGRIGENUS. 


Obtained by Nicolaier from soil which had been manured. 

Morphology.—Resembles the bacillus of fowl cholera and of rabbit sep- 
ticeemia, of which it is perhaps a variety, but is usually somewhat longer. 
It also sometimes shows the end-staining characteristic of Bacillus septice- 
mize hemorrhagic, but not so constantly and not so sharply defined. 

Biological Characters.—An aérobic, (non-liquefying 2%), non-motile ba- 
cillus. Does not form spores. 

In gelatin plate cultures spherical, finely granular colonies are developed 
having a yellowish-brown central portion, which is separated by a dark 
ring from a grayish-brown marginal zone; later this difference in color dis- 
appears and thecolonies become more decidedly granular. In stick cultures 
the growth consists of a thin layer which is not at all characteristic. 

Pathogenesis.—Small quantities of a pure culture injected into the ear 


®| & 


IN SUSCEPTIBLE ANIMALS. 511 


vein of a rabbit cause its death in from twenty-four to thirty-six hours; 
pathogenic also for house miceand for field mice. Atthe autopsy no notable 
pathological changes are observed. The bacilli are found in blood from the 
heart and in the capillaries of the various organs, but are not so numerous 
as in rabbit septiceemia; they show a special inclination to adhere to the 
margins of the red blood corpuscles. 


BACILLUS ERYSIPELATOS SUIS. 


Synonyms.—Bacillus of hog erysipelas; Bacillus des Schweine- 
rothlauf (Léffler, Schiitz) ; Bacille du rouget du pore (Pasteur) ; Ba- 
cillus of mouse septicemia; Bacillus murisepticus (Fliigge) ; Bacil- 
lus des Mauseseptikamie (Koch). 

The bacillus of mouse septiceemia, first described by Koch (1878), 
resembles so closely in its morphology, characters of growth, and 
pathogenic power the bacillus of Schweinerothlauf of Léffler and 
Schiitz (1885) that they can scarcely be considered as distinct spe- 
cies, although, from slight differences which have been observed, they 
are perhaps entitled to separate consideration as varieties of the 
same species. Fliigge, Eisenberg, Frankel, and other authors, while 
recognizing the fact that the bacilli from the two sources closely re- 
semble each other, apparently do not consider 
them identical, and describe them separately. 
Baumgarten, on the other hand, describes them 
under one heading and considers it highly prob- 
able that they are identical, although he also 
admits slight differences in the morphological 
characters and growth in culture media. These 
differences are, however, no greater than we 
have in artificially produced varieties of other 5, 195 Bacitius of 
well-known microorganisms, and we think it mouse septicemia in leu- 
best to follow Baumgarten in describing them ‘Svs ra rer 
under a single heading. 

Koch first obtained this bacillus by injecting putrefying blood or 
flesh infusion, during the first days of putrefactive change, beneath 
the skin of mice. A certain proportion of the animals experimented 
upon contracted a fatal form of septicemia, and the bacillus under 
consideration was found in their blood. The bacillus of Schweine- 
rothlauf was obtained by Léffler and by Schitz from the blood and 
various organs of swine which had succumbed to the infectious 
malady known in Germany as rothlauf and in France as rouget. 

Morphology.—Extremely minute bacilli, about 1 # in length and 
0.2 » in diameter. The Schweinerothlauf bacilli are described as 
somewhat thicker and longer by Fliigge, by Frankel, and by Ei- 
senberg, but Baumgarten states that they are somewhat more 


512 BACILLI WHICH PRODUCE SEPTICAMIA 


slender and on the average shorter than the bacillus of mouse septi- 
cemia. The bacilli are solitary, or in pairs the elements of which 
are often united at an angle ; occasionally a chain of three or four 
elements may be observed, and in old cultures the bacilli may 
grow out into short threads which are straight or more or less 
curved and twisted. Small refractive bodies may sometimes be dis- 
tinguished in the rods, and these have been supposed by some authors 
to be spores, but this has not been demonstrated. 

This bacillus stains readily with the ordinary aniline staining 
agents and also by Gram’s method. 

Biological Characters.—A facultative anaérobic, non-liquefy- 
ing bacillus. According to Schottelius, the rothlauf bacilli are some- 


Fig. 134.—Bacillus of rouget, from a pure culture. x 1,000. Froma photomicrograph. (Roux.) 


times motile, but Fligge states that other observers have not seen 
them in active motion. Frankel says they have the power of volun- 
tary motion. Eisenberg says that the bacillus of mouse septiceemia 
is motionless, and Frankel says they “seem to be incapable of volun- 
tary motion.” Baumgarten remarks: “ Whether the bacilli exhibit 
voluntary movements has not been determined.” Although this 
bacillus is not strictly anaérobic, it grows better in the absence of 
oxygen than in its presence. Development occurs in various culture 
media at the room temperature, but is more rapid in the culture 
oven. In gelatin stab cultures no development occurs upon the 
surface, but the growth along the line of puncture is very character- 
istic; this consists of a delicate cloud-like, radiating growth, which 
extends, in the course of a few days, almost to the walls of the test 
tube. The rothlauf bacillus does not extend so rapidly through the 


iN SUSCEPTIBLE ANIMALS. 


513 


gelatin, and the branching, cloud-like growth is not as delicate; 
Fliigge compares it to the brush of bristles used for cleansing test 


tubes. In old cultures in nutrient gelatin a 
slight softening of the gelatin occurs along the 
line of growth, and as a result of evaporation 
and desiccation a funnel-shaped cavity is formed 
in the culture medium in the course of two or 
three weeks. In gelatin plates colonies are 
developed in the course of two or three days in 
the deeper layers of the gelatin, but not upon 
the surface; these are nebulous, grayish-blue, 
radiating masses, which are so delicate as to be 
scarcely visible without the aid of a lens or a 
dark background. Under a low power they 
appear as branching feathery masses, which 
have been compared by Fligge to the radiating 


growth of ‘‘bone corpuscles.” In older cultures , 


they coalesce and cause a nebulous opacity of 
the whole plate, which has a bluish-gray lustre. 

Upon the surface of nutrient agar or blood 
serum a very scanty development occurs along 
the line of inoculation. No growth occurs upon 
potato. In bouillon the bacilli cause a slight 
cloudiness at the outset, and later a scanty gray- 
ish-white deposit upon the bottom of the test 
tube; no film is formed upon the surface. 


Fic. 135.— Bacillus of 
mouse septicemia; 
culture in nutrient gela- 
tin, end of four days at 
18°C. (Baumgarten.) 


The thermal death-point of this bacillus, as determined by the 
writer (1887), is 58° C., the time of exposure being ten minutes. In 
the experiments of Bolton it was destroyed in two hours by mercuric 
chloride solution in the proportion of 1:10,000; by carbolic acid and 


Fic. 136.—Bacillus of mouse septicaemia; single colony in nutrient gelatin. x 80. (Fliigge.) 


by sulphate of copper in one-per-cent solution. 


These results are 


opposed to the view that the minute refractive granules which may 
sometimes be seen in the interior of the rods are reproductive spores, 


33 


514 BACILLI WHICH PRODUCE SEPTICAMIA 


for all known spores have a much greater resisting power to heat 
and the chemical agents named. 

Pathogenesis.—Pathogenic for swine, rabbits, white mice, house 
mice, pigeons, and sparrows. Field mice, guinea-pigs, and chickens 
are immune. 

Swine may be infected by the ingestion of food containing the 
rothlauf bacillus, as has been demonstrated by allowing them to eat 
the intestine of an animal which had recently succumbed to the dis- 
ease, and also by the subcutaneous injection of pure cultures. The 
disease usually terminates fatally within three or four days, and 
sometimes in less than twenty-four hours. It is characterized by 


Fie. 137.—Section of diaphragm of a mouse dead from mouse septicemia, showing bacilli in a 
capillary blood vessel. (Baumgarten.) 


fever, debility, loss of appetite, and by the appearance upon the sur- 
face of the body of red patches, which gradually extend and become 
confluent, producing after a time a uniform dark-red or brown color 
of the entire surface. The discharges from the bowels frequently 
contain bloody mucus. At the autopsy, in acute cases, the spleen is 
notably enlarged, and the liver and kidneys are likely to be more or 
less swollen, as are also the lymphatic glands, especially those of 
the mesentery; the gastric and intestinal mucous membranes are 
usually inflamed and spotted with hemorrhagic extravasations ; the 
serous membranes also may be inflamed, and the cavities of the 
pleure, pericardium, and peritoneum usually contain more or less 
fluid. The bacilli are found in the blood vessels throughout the 
body and are especially numerous in the interior of the leucocytes. 


IN SUSCEPTIBLE ANIMALS. 515 


Cornevin and Kitt have shown that the contents of the intestine 
also contain the bacilli in large numbers, and the disease appears to 
be propagated among swine principally by the contamination of their 
food with the alvine discharges of diseased animals. 

Pigeons are very susceptible to the pathogenic action of this ba- 
cillus, and usually die within three or four days after inoculation 
with a pure culture. Rabbits are not so susceptible, although a 
certain proportion die from general infection after being inoculated 
in theear. The first effect of such an inoculation is to produce an 
erysipelatous inflammation. When the animal recovers it is subse- 
quently immune. 

White mice and house mice are extremely susceptible, but field 
mice are immune. This remarkable fact was first ascertained by 
Koch by experiments with his bacillus of mouse septicemia. House 
mice which have been inoculated with a minute quantity of a pure 
culture of the rothlauf, or mouse septiceemia, bacillus, die in from 
forty to sixty hours. The animal is usually found dead in a sitting 
position, with its back strongly curved, and for many hours before 
death it remains quietly sitting in the same position ; the eyes are 
glued together by a sticky secretion from the conjunctival mucous 
membrane. At the autopsy the spleen is found to be very much en- 
larged, and there may be a slight amount of cedema at the point of 
inoculation. 

The bacilli are found in the blood vessels generally, and are very 
numerous in the interior of the leucocytes, which are sometimes com- 
pletely filled with them. 


BACILLUS COPROGENES PARVUS. 


Synonym. —Mauseseptikaimiedhnlicher Bacillus (Hisenberg). 

Obtained by Bienstock from human faces. 

Morphology.—A very minute bacillus, which is but little longer than it 
is broad, and might easily be mistaken for a micrococcus. 

Biological Characters.—Grows very slowly on nutrient gelatin, forming 
a scarcely visible film along the line of inoculation, which at the end of 
several weeks is scarcely one millimetre wide. Is not motile. 

Pathogenesis.—In white mice an extensive cedema is developed at the 
point of inoculation at the end of ten or twelve hours, and the animal dies 
within thirty-six hours. The bacilli are found in great numbers in the 
effused serum at the point of inoculation and in comparatively small num- 
bers in the blood. A rabbit inoculated with a pure culture obtained from a 
mouse died at the end of eight days. The inoculation, which was made in 
the ear, gave rise to a local erysipelatous inflammation. 


516 BACILLI WHICH PRODUCE SEPTICAMIA 


BACILLUS CAVICIDA. 


Synonym.—Brieger’s bacillus. Probably a pathogenic variety of Bac- 
terium coli commune of Escherich. 

Obtained by Brieger (1884) from human feces. . ; 

Morphology.—Small bacilli, about twice as long as broad, which closely 
resemble the colon bacillus of Escherich (Bacterium coli commune). 

Biological Characters.—An aérobic (facultative anaérobic), non-liquefy- 
ing bacillus. ar we 

The growth in gelatin plate cultures is said to be very characteristic, the 
colonies being ‘‘in the form of very beautifully grouped, whitish, concentric 
rings, which are arranged like the scales upon the back of a turtle” (Hisen- 
berg). The writer has studied cultures of this bacillus brought from the 
bacteriological laboratories of Germany, side by side with cultures of the 
Bacterium coli commune of Escherich, and has found no appreciable differ- 
ences in the colonies in gelatin plates, or in the growth in various culture 
media. Upon potato it grows rapidly in the incubating oven, forming a 
dirty-yellow, moist layer. 

Pathogenesis.—This bacillus, as first obtained by Brieger, was character- 
ized by being very pathogenic for guinea-pigs, which were invariably killed, 
within seventy-two hours, by the subcutaneous injection of a minute quan- 
tity of a pure culture. The bacillus was found in great numbers in the 
blood of animals which succumbed to an experimental inoculation, The 
writer’s experiments with this bacillus, made in 1889, indicate that its patho- 
genic power had become attenuated, inasmuch as considerable quantities of 
a pure culture injected into guinea-pigs did not cause the death of the ani- 
mals—culture used came originally from Germany. Not pathogenic for 
rabbits or for mice. 


BACILLUS CAVICIDA HAVANIENSIS. 


This bacillus was obtained by the writer from the contents of the intestine 

of a yellow-fever cadaver, in Havana, 1889, through inoculated guinea-pigs. 

7 Morphology.—A bacillus with rounded ends, 

from two to three » long and about 0.7 » broad, 
frequently united in pairs. 

Stains readily with the ordinary aniline colors, 

Biological Characters.—-An aérobic and fac- 
ultative anaérobic, non-liquefying, actively mo- 
tile bacillus. 

In gelatin stab cultures the growth upon the 
surface is very scanty and thin, not extending far 
from the point of puncture ; along the line of 

uncture are developed small, translucent, pearl- 
ike, spherical colonies, which later become opaque 
s and sometimes granular. In gelatin roll tubes, 
Fic. 138.—Bacillus cavicida at the end of twenty-four hours at 22° C., 
Havaniensis; from « potato the deep colonies are very small spheres, of a pale 
culture. x 1,000. From apho- straw color; later they becomeopaque, light-brown 
tomicrograph. (Sternberg.) spheres, or may have a dark central mass sur- 
rounded by a transparent zone. The superficial 
colonies at the end of five days are small, translucent masses of a pale straw 
color towards the centre, with thin and irregular margins, sometimes with 
acentral light-brown nucleus; at the end of ten days the deep colonies are 
still quite small, of a brown color, and opaque. 

In glycerin-agar roll tubes, at the end of twenty-four hours, the deep colo- 
nies are in the form of a biconvex lens, and appear spherical when viewed 
in face and biconvex when seen from the side; they have a straw color 


IN SUSCEPTIBLE ANIMALS. 517 


by transmitted light and are bluish-white by reflected light; the superficial 
colonies are translucent, with a bluish-white lustre. 2 

On potato, at 22° C., at the end of forty eight hours there is a thin, dirty- 
yellow growth of limited extent; at the end of ten days there is a thin, 
gamboge yellow layer and little masses of the same color; the growth is 
quite thin, with irregular outlines, and is confined to the vicinity of the 
impfstrich. 

Grows in nutrient agar containing 0.2 per cent of hydrochloric acid. 
Thermal death point 55° C. Grows in agua coco without forming gas, and 
causes this liquid and bouillon to become slightly translucent—not milky. 

_ Pathogenesis.—Pathogenic for guinea-pigs, less so for rabbits. Guinea- 
pigs inoculated subcutaneously with a few drops of a pure culture die in ten 
or twelve bours from general infection. There is usually a considerable 
effusion of bloody serum in the vicinity of the point of inoculation, and the 
spleen is more or less enlarged. 


BACILLUS CRASSUS SPUTIGENUS. 


Obtained by Kreibohm (1886) from the sputum of two individuals, and 
once in scrapings from the tongue. 

Morphology.—Short, thick bacilli, of oblong form, with rounded corners, 
often bent or twisted—‘‘sausage-shaped.” Immediately after division the 
bacilli are about one-half longer than they are broad, but before dividing 


Fie. 139.—Bacillus crassus sputigenus, from blood of mouse. x 700. (Fligge.) 


again they may <ttain a length of three to four times the breadth. Irregular 
forms with swollen ends or uneven contour are frequently seen. 

This bacillus is quickly stained by the ordinary aniline colors and also 
by Gram’s method. 

Biotogical Characters.—An aérobic, non-liquefying (non-motile % ba- 
cillus. Grows in various culture media at the room temperature—more 
navily in the incubating oven. ‘‘Appears to form spores at 35° C.” 
(Fligge). 

Taaeris plates, at the end of thirty-six hours, grayish-white colonies are 
developed, which soon reach the surface of the gelatin and spread out as 
round, viscid, grayish white drops, which project considerably above the 
surface of the culture medium. Undera low magnifying power recent colo- 


518 BACILLI WHICH PRODUCE SEPTICEMIA 


nies appear as spherical, grayish-brown discs, the surface of which is marked 
with dark points or lines. The superficial colonies are more transparent, 
have irregular outlines, and the surface, especially near the margins, 1s 
coarsely granular. The development in stab cultures is very rapid and re- 
sembles that of Friedlander’s bacillus—‘“‘ nail-shaped” growth. Upon potato 
the growth is also similar to that of Friedlainder’s bacillus, and consists of a 
thick, grayish-white, moist, and shining layer. 

Pathogenesis.—Mice inoculated with a small quantity of a pure culture 
die from acute septicaemia in about forty-eight hours. The bacilli are found 
in blood from the heart and from the various organs—most numerous in 
the liver. Rabbits are killed within forty-eight hours by intravenous injec- 
tion of a small quantity, and the blood contains the bacillus in great num- 
bers. Larger amounts injected into the circulation of rabbits or dogs cause 
death in a few hours (three to ten), preceded by diarrhcea, and in some in- 
stances bloody discharges from the bowels. At the autopsy an acute gastro- 
enteritis is found, 


BACILLUS PYOGENES FCETIDUS. 


Obtained by Passet (1885) from an abscess of the anus. 

Morphology.—Short bacilli with rounded ends, 1.45 mu long and 0.58 u 
broad; usually associated in pairs or in short chains. 

Biological Characters.—An aérobic, non-liquefying, motile bacillus. 
Grows rapidly in the usual culture media at the room temperature. In the 
interior of the rods, in stained preparations, one or two unstained, spherical 
places may sometimes be seen, which have been supposed to be spores (?). 
The independent motion exhibited by this bacillus is not very active. In 
gelatin plates white colonies are developed at the end of twenty-four hours, 
which upon the surface spread out as grayish-white plaques, having a dia- 
meter sometimes of one centimetre; these are thickest in the centre and of 
a whitish color; the colonies may become confluent. In gelatin stab cul- 
tures the growth upon the surface, at the end of twenty-four hours, consists 
of a thin, grayish white layer with rather thick, irregular margins; along the 
line of puncture more or Aes crowded colonies. Upon potato the bacillus 
forms an abundant, shining, pale-brown layer. The cultures give off a dis- 
agreeable putrefactive odor. 

According to Hisenberg, mice and guinea-pigs are killed in twenty-four 
hours by injections beneath the skin or into the cavity of the abdomen, and 
numerous bacilli are found in the blood. 


PROTEUS HOMINIS CAPSULATUS. 


Obtained by Bordoni-Uffreduzzi (i887) from two cadavers presenting the 
pathological appearances of the so-called ‘‘ Hadernkrankheit.” 

Morphology.—Bacilli, varying considerably in dimensions; somewhat 
thicker than the anthrax bacillus; often swollen in the middle or at the ex- 
tremities; more or less curved; isolated, united in pairs or in long filaments; 
in stained preparations from agar cultures or from blood the bacilli are sur- 
rounded by a ‘‘ capsule.” 

Stains with the usual aniline colors and also by Gram’s method. 

Biological Characters.—An aérobic (facultative anaérobic 2), non-lique- 
Fying, non-motile bacillus. Formation of spores not observed. Grows in 
the usual culture media at the room temperature. A+ a temperature of 15° to 
17° C. long filaments are formed, in which the bacilli are surrounded with a 
capsule; at 22° to 24° C. the bacilli are for the most part isolated, but few fila- 
ments being formed ; at 32° to 37° C. the bacilli are so short as to resemble 
Teerences development ceases at a temperature of 8° and is very slow at 


IN SUSCEPTIBLE ANIMALS. 519 


This bacillus grows as well in an acid medium as in one which is slightly 
alkaline. In gelatin plates, at the end of eighteen to twenty-four hours, 
colonies are formed which under a low power are seen to be spherical and 
to contain a quantity of shining granules; the following day, at a tempera- 
ture of 15° to 17° C., the colonies may be as large asa pin’s head and still 
remain spnerical or slightly oval, but 
the outline is no longer so uniform, 
and between the shining points in the 
interior a confused network may be 
seen; as the colony becomes larger it 

_is raised above the surface of the gela- 
tin, becomes opaque, and has a pearly 
lustre like that of Friedlander’s bacil- 
lus. In gelatin stab cultures the 
growth resembles that of Friedlin- 
der’s bacillus—‘‘ nail-shaped growth.” 
Upon the surface of nutrient agar a 
rapidly extending, semi-transparent 
layer is formed. Spon potato, at 15° 
to 17° C., at the end of twenty-four 
hours transparent drops are seen in 
the vicinity of the point of inocula- 
nee ‘aie Tier a ane shining, color- ie 

ess layer, of tough consistence, is ra 
formed, which gradually extends over on pci ae ato sacs ag 
the surface. The growth upon blood  guzzi) E seeeds 

serum resembles that upon nutrient : ee . 
agar, and the blood serum is not liquefied. In liquid blood serum or in 
bouillon the bacilli are isolated—not in filaments; they cause a clouding of 
the liquid, and an abundant deposit accumulates at the bottom of the tube, 
while a film of bacilli forms upon the surface. The cultures never give off 
a putrefactive odor. : f 

Pathogenesis.—Pathogenic for dogs and for mice, less so for rabbits and 
for guinea-pigs. Agar cultures grown in the incubating oven at 32° to 37 
C. are more pathogenic than cultures in gelatin at the room temperature. 
A small quantity of a recent culture injected subcutaneously in mice causes 
their death in from one to four days, according to the quantity and age of 
the culture; the recent cultures are most virulent. When the animal lives 
more than twenty-four hours it has a mucous diarrhea. At the autopsy the 
spleen is found to be much enlarged and dark in color ; the lymp atic 
glands are also swollen and hemorrhagic, the liver and kidneys hyperemic; 
in the vicinity of the point of inoculation is a subcutaneous cedema of jelly- 
like appearance and numerous punctiform hemorrhages are seen. The ba- 
cillus is found in great numbers in the effused serum from the subcutaneous 
tissues, in the blood, the contents of the intestine, and in the parenchyma of 
the various organs. When examined at once the bacilli in the subcutaneous 
cedema and in the lymphatic glands are usually quite short, and even spherical, 
while in the blood they are somewhat longer and may appear as short fila- 
ments with swollen ends, surrounded by a capsule. When the examination 
is made some time after the death of the animal longer filaments are quite 
numerous. Rabbits and guinea-pigs are killed by the intravenous injection 
of comparatively small amounts of a recent culture, but quite large doses 
are required to producea fatal result when the injection is made beneath 
the skin. From two to three cubic centimetres of a recent culture injected 
into the circulation of a dog give rise to symptoms of toxzemia, and the ani- 
mai usually dies on the second day. At the autopsy the abdominal organs 
are found to be hypereemic, the mucous membrane of the intestine swollen, 
red in color, and covered with bloody mucus. The bacillus is found in the 
blood and in the various organs. When smaller doses are injected into a 
vein (a few drops) the animal, after a few hours, has a mucous diarrhoea and 


520 BACILLI WHICH PRODUCE SEPTICMIA 


vomiting, or efforts to vomit. Death usually occurs at the end of two or 
three days. At the autopsy the spleen is found to be normal, the other or- 
gans slightly hyperzemic, and the intestinal mucous membrane in a state of 
éatarrhal inflammation. The bacilli are found in the blood and in the vari- 


ous organs in considerable numbers. 


PROTEUS CAPSULATUS SEPTICUS. 


Obtained by Banti (1888) from a case of ‘t acute hemorrhagic infection.” 

According to Banti, this is possibly identical with the preceding species— 
Proteus hominis capsulatus—but in some respects more nearly resembles 
Friedlinder’s bacillus. 


BACILLUS ENTERITIDIS. 


Obtained by Giirtner (1888) from the tissues of a cow which was killed in 
consequence of an attack characte1ized by a mucous diarrhoea, and also from 
the spleen of a man who died twelve hours after eating the flesh of this 
animal. 

Morphology.—Short bacilli, about twice as long as broad, frequently united 
in pairs; chains of four to six elements are sometimes seen. 

Stains with the usual aniline colors, and presents the peculiarity of 
staining deeply at one end while the remainder of the rod is but slightly 
stained. When two bacilli are united the deeply stained ends are in apposi- 
tion. 

Biological Characters.—An aérobic, non-liquefying, motile bacillus. 
Spore formation not determined. Grows in the usual culture media at the 
room temperature. Upon gelatin plates pale-gray, superficial colonies are 
formed at the end of twenty-four hours; under a low power these are seen 
to be coarsely granular and transparent; the central portion usually pre- 
sentsa greenish color ; deep colonies are spherical, indistinctly granular, 
and of a brownish color ; in older colonies a marginal transparent zone is 
seen which appears to be made up of minute fragments of glass of a pale- 
brown color. In gelatin stab cultures but slight development occurs along 
the line of puncture ; upon the surface a thick, grayish-white layer is 
formed, which after a time becomes very much wrinkled. Upon the surface 
of agar, at 37° C., at the end of eighteen to twenty hours a grayish-yellow 
layer has formed. Upon potato a moist, shining, yellowish-gray layer is 
developed. The growth upon blood serum is rapid in the form of a gray 
layer along the line of inoculation. 

Pathogenesis.—White mice and house mice usually die in from one to 
three days when fed with a pure culture of this bacillus. Rabbits and gui- 
nea-pigs die in from two to five days from subcutaneous injections—less 
pathogenic for pigeons and canary birds. Dogs, cats, chickens, and sparrows 
areimmune. A goat died in twenty hours after receiving an intravenous 
injection of two cubic centimetres of a culture in blood serum. The princi- 
pal pathological appearance consists in an intense inflammation of the in- 
testinal mucous membrane. The bacilli are found in blood from the heart 
and also in the contents of the stomach. 


BACILLUS OF GROUSE DISEASE. 


Obtained by Klein (1889) from the lungs and liver of grouse which had 
succumbed to an epidemic disease. 

Morphology.—Bacilli with rounded ends, from 0.8 to 1.6 «long; may 
also be seen as spherical or oval cells 0.6 4 long and 0.4 « thick; solitary, in 
pairs, or in chains of three to four elements. 

Stains best with Weigert’s solution of methylene blue in aniline water. 

Biological Characters.—An aérobic, non-liquefying, non-motile bacillus. 
Spore formation not observed. Grows in the usual culture media at the 


IN SUSCEPTIBLE ANIMALS. 621 


room temperature—better in the incubating oven. Upon gelatin plates, at 
20° C., at the end of twenty-four hours small, angular, transparent scales 
may be seen upon the surface with a low-power lens; at the end of three or 
four days these form flat, more or less irregular, shining, gray colonies, with 
tin and often dentate margins; these colonies may become confluent and 
form adry, scaly layer which by reflected light has a peculiar, fatty lustre 
In gelatin stab cultures the superficial growth is in the form of a trans- 
parent, dry, grayish layer with dentate margins, not more than three to five 
millimetres in diameter. Upon agar, at 36° to 37° C., a thin, whitish-gray, 
dry layer is formed. 

Pathogenesis.—Pathogenic for mice, for guinea-pigs, for linnets, and for 
green-finches; less so for sparrows. Chickens, pigeons, and rabbits, accord- 
ing to Klein, areimmune. Of eight mice inoculated subcutaneously with 
one or two drops of a bouillon culture, six died within forty-eight hours 
and two recovered. Out of eight guinea-pigs inoculated in the same way 
four died in forty-eight hours and two recovered. At the autopsy the 
lungs and liver were found to be hyperemic, the spleen not enlarged. The 
peu? were present in large numbers in blood from the heart and in the 

ungs. 


BACILLUS GALLINARUM. 


Obtained by Klein (1889) from the blood of chickens which succumbed 
to an epidemic disease resembling ‘‘ fowl cholera.” The bacillus is believed 
by Klein not to be identical with Pasteur’s bacillus of fowl cholera, and is 
said not to be pathogenic for rabbits, which would seem to differentiate it 
from this bacillus (Bacillus septicaemize haeemorrhagicz). 

Morphology.—Bacilii with rounded ends, from 0.8 to 2 # long and 
0.3 to 0.4.4 thick; often in pairs. 

Stains with the usual aniline colors. 

Biological Characters.—An aérobic, non-liquefying, non-motile bacillus. 
Does not form spores. Grows in the usual culture media at the room tem- 
perature—better in the incubating oven. Upon gelatin plates forms grayish- 
white, superficial colonies, which later present the appearance of fiat, homo- 
geneous, whitish discs with thin edges and irregular margins, and by 
transmitted light have a brownish color. The deep colonies are smfll and 
spherical, and have a brownish color by transmitted light. In gelatin stab 
cultures a thin, gray layer with irregular margins and of limited extent 
forms upon the surface, and a scanty growth occurs along the line of punc- 
ture in the form of a grayish-white line. Upon the surface of agar, at 
87° C., a thin, gray layer with irregular margins has developed at the end of 
twenty-four hours; later this extends over the entire surface as a thin, gray- 
ish-white layer. No growth occurs upon potato at 37°C. In bouillon, at 37° 
C., development occurs, with clouding of the bouillon, within twenty-four 
hours; later a deposit consisting of bacilli is seen at the bottom of the tube, 
but no film forms upon the surface. 

Pathogenesis. —Chickens inoculated subcutaneously with a pure culture 
die in from twenty-four hours to eight or nine days. Pigeons and rabbits 
are immune. 


BACILLUS CAPSULATUS. 


Obtained by Pfeiffer (1889) from the blood of a guinea-pig which died 
spontaneously. : 

Morphology.—Thick bacilli with rounded ends, usually two or three 
times as long as broad; often united in chains of two or three elements; may 
grow out into homogeneous filaments. Stained preparations show the ba- 
cilli to be enveloped in an oval capsule which may be considerably broader 
than the bacilli themselves—two to five times as broad; where several ba- 
cilli are united they are surrounded by a single capsular envelope. 

Stains with the usual aniline colors, but not by Gram’s method. In pre- 
parations which are deeply stained with hot fuchsin or gentian violet solu- 


§22 BACILLI WHICH PRODUCE SEPTICEMIA 


tion the capsule is so deeply stained that the bacillus is hidden; by careful 
treatment with a weak solution of acetic acid the capsule may be differen- 
tiated as a pale-red or violet envelope surrounding the deeply stained bacilli. 

Brologicat Characters.—An aer- 
obic aud facultative anaérobic, 
non -liquefying, non-motile bacillus. 
Spore formation not observed. 
Grows in the usual culture media 
at the room temperature. The cul- 
tures in agar or upon potato are very 
viscid and draw out into long 
threads when touched with the pla- 
tinum needle; the blood of an ani- 
mal killed by inoculation with this 
bacillus has the same viscid charac- 
ter. Upon gelatin plates minute 
colonies are first visible at the end 
of twenty-four to thirty-six hours; 
later the deep colonies are white, 
oval masses the size of a pin’s head; 
the superficial colonies attain the 
size of a lentil, and are flattened, 

hemispherical masses with a porce- 
Fie 141.—Bacillus capsulatus, from peritoneal Jain-white color. In gelatin stab 
exudate of an inoculated guinea-pig. x 1,000. exyltures growth occurs to the bot- 
From a photomicrograph. (Ffeiffer.) tom of the line of puncture, and on 
the surface a shining white, circular, 
arched mass forms around the point of puncture, resembling the growth of 
Friedlander’s bacillus. Upon the surface of agar, at 37° C, at the end of 
twenty-four hours a thick, soft layer of a pure white color is formed, which 
is very viscid and resembles the growth of Micrococcus tetragenus upon the 
same medium. Upon potato an abundant and viscid, shining, yellowish- 
white layer is quickly developed. 

Pathogenesis.—Pathogenic for white mice and for house mice, which die 
at the end of two or three days after being inoculated at the root of the tail 
with a small quantity of a pure culture. Inoculation from mouse to mouse 
increases the virulence of the cultures. At the autopsy the superficial veins 
are distended with blood, the inguinal glands enlarged, the spleen consid- 
erably enlarged, the liver and kidneys hyperemic, the intestine pale, the 
heart distended with blood, which usually is very viscid and is drawn out 
into threads when touched with the platinum needle. The bacilli are found 
in the blood and in all of the organs, in the contents of the peritoneum and 
pleuree, and in the exudate in the vicinity of the point of inoculation. 
Pathogenic also for guinea pigs and for pigeons; guinea-pigs are infallibly 
killed within thirty-six hours by the injection of a single drop of a bouillon 
culture, twenty-four hours old, into the cavity of the abdomen; the blood 
contains the bacillus in enormous numbers, as does the viscid fluid found in 
the peritoneal cavity. Rabbits do not succumb to intraperitoneal or subcu- 
taneous inoculations, but are killed by the intravenous injection of one 
cubic centimetre of a recent bouillon culture. Putrefactive changes occur 
very quickly in animals killed by inoculation with this bacillus. 


BACILLUS HYDROPHILUS FUSCUS. 


Obtained by Sanarelli (1891) from the lymph of frogs suffering from a 
fatal infectious disease. 

Mor phology.—Bacilli with rounded ends, usually from 1 to 3 « in length; 
often short oval; may grow out into filaments of 12 to 20 # in length. 
_ Biological Characters.—An aérobic, liquefying, motile bacillus. Grows 
in the usual culture media at the room temperature. In gelatin stab cul- 


IN SUSCEPTIBLE ANIMALS. 523 


tures, at 18° to 20° C., liquefaction has already commenced along the line of 
puncture at the end of twelve hours, and at the end of thirty-six to forty- 
eight hours half of the gelatin is liquefied in funnel shape; on the third or 


fourth day the gelatin is completely lique- 
fied, and a thick, white, flocculent deposit 
is seen at bottom of the tube. In glycerin- 
agar, at 87° C., a slight, bluish, diffuse 
fluorescence is seen upon the surface at the 
end of twelve hours, and soon after a luxu- 
riant growth, which soon covers the entire 
surface, is developed; at the end of twenty- 
four to thirty-six hours large gas bubbles 
begin to form in the agar; gradually the 
fluorescence disappears, the surface growth 
becomes thicker and hasa dirty-gray color 
which changes later to brownish. “Blood 
serum is a favorable medium and is rapidly 
liquefied by this bacillus. Upon potato the 
growth is most characteristic. At the end 
of twelve hours a thin, straw-yellow layer 
is developed along the impfstrich; this 
gradually becomes yellow, and at the end 


Fie. 142.—Bacillus hydrophilus fus- 
cus, in blood of triton. (Sanarelli.) 


of four to five days hasa brown color, resembling that of the glanders bacil- 


lus upon potato. 


Pathogenesis.—Pathogenic for frogs, toads, lizards, and oth ‘“‘cold- 


blooded” animals; also for guinea-pigs, rabbits, dogs, 
cats, mice, chickens, and pigeons. When a few drops of 


Fig. 143.—Bacillus 
hydrophilus fuscus; 
culture in nutrieut 
gelatin, end of six- 
teenhours. (Sana- 
relli.) 


a bouillon culture are injected into the muscles of the 
thigh, swelling and redness at the point of inoculation 
are quickly developed, and death usually occurs in eight 
to ten hours. The bacilli are found in great numbers in 
the blood and in all of the organs. Guinea pigs die from 
general infection within twelve hours after receiving a 
subcutaneous injection of a small amount of a pure cul- 
ture; the spleen is enlarged and the liver and spleen hy- 
pereemic; an extensive inflammatory cedema in the vicin- 
ity of the inoculation wound is frequently observed; the 
bacilli are very numerous in the blood and in all the or- 
gans. Rabbits die in five to six hours from an intravenous 
injection. Adult dogs are immune, but new-born dogs 
(three to four days old) die infallibly, after receiving a 
subcutaneous injection of a small quantity of a pure cul- 
ture, in twelve to thirty-six hours. Young cats also suc- 
cumb to similar inoculations. Chickens and pigeons die 
within five to seven hours after receiving an intravenous 
injection, but resist subcutaneous injections. 


BACILLUS TENUIS SPUTIGENUS. 


Obtained by Pansini (1890) from sputum. 

Morphology. Short bacilli, usually in pairs and sur- 
rounded by a capsule. 

Stains by Gram’s method. 

Biological Characters.—An aérobic, non-liquefying, 
non-motie bacillus. Grows in nutrient gelatin at the 
room temperature. Develops abundantly on potato. 
Coagulates milk and produces an acid reaction in this 
medium. 


Pathogenesis.— Pathogenic for rabbits and white rats; not for guinea- 
pigs or for white mice (in small doses). 


524 BACILLI WHICH PRODUCE SEPTICZMIA 


BACILLUS OF LASER. 


Obtained by Laser (1892) from mice which succumbed to an epidemic dis 
ease in Frinkel’s laboratory at Kénigsberg. : . 

In its characters this bacillus closely resembles the bacillus of swine 
plague, and is perhaps identical with it. ’ 

Morphology.—A small bacillus, with rounded ends, about twice as long 
as broad. Has flagella both at the extremicies and sides. 

Stains by the usual aniline colors and also by Gram’s method. 

Biological Characters.—An_ aérobie and facultative anaérobic, non- 
liquefying, actively motile bacillus. Spore formation not observed. Grows 
either in the incubating oven or at the room temperature, Thermal death- 
point 65° to 70° C.—ten minutes’ exposure. Upon gelatin plates, at the end 
of two days, the deep colonies are spherical, finely granular, and brownish 
in color; the superficial are transparent, finely granular, and leaf-like. 
In gelatin stab cultures growth occurs along the entire line of puncture as 
well as upon the surface. At the end of three days a considerable evolution 
of gas is usually observed. In agar an abundant development is seen at the 
oni of twenty-four hours in the incubating oven; upon the surface a gray- 
ish-white, shining layer with dentate margins is formed along the track of 
the needle. In bouillon, at 37° C., development is abundant and rapid; a 
thin film is formed on the surface at the end of thesecond day. Upon potato 
a brownish layer is formed at the end of twenty-four hours. In milk an 
acid reaction is produced. 

Pathogenesis.—Pathogenic for field mice, guinea-pigs, rabbits, and 
pigeons. The bacillus is found in the blood and various organs of infected 
mice, The spleen is found to be greatly enlarged. 


BACILLUS TYPHI MURIUM (LOffler). 


Obtained bv Léffler (1889) from mice which died in his laboratory from 
an epidemic disease due to this bacillus, 

Morphology.—Short bacilii, resembling the bacillus of diphtheria in 
pigeons, aa varying considerably in dimensions—like the bacillus of 
typhoid fever; grows out into flexible filaments. 

‘i Stains with the aniline colors—best with Loffler’s solution of methylene 
ue. 

Biological Characters.—An aérobic and facultative anaérobic, non- 
liquefying, motile bacillus. Spore formation not determined. Has flagella 
around the periphery of the cells, like those of the typhoid bacillus, and ex- 
hibits similar active movements. In gelatin stab cultures, at the room 
temperature, growth occurs upon the surface, at the end of forty-eight hours, 
in the form of a flat, grayish-white, round, semi-transparent mass the size of 
a pin’s head; later the surface colony increases in extent and has more or 
less irregular margins. In gelatin plate cultures the deep colonies are at 
first round. slightly granular, transparent, and grayish; later they are of a 
yellowish-brown color and decidedly granular. The superficial colonies are 
very granular and marked by delicate lines—similar to colonies of the 
typnoid bacillus. Upon agar a grayish-white layer is developed which is 
not at all characteristic. Upon potato arather thin, whitish layer isformed, 
and around. this the potato acquires a dirty bluish-gray color. In milk an 
abundant development occurs, and a decidedly acid reaction is produced 
without causing any perceptible change in the appearance of the fluid. 

Pathogenesis.—Pathogenic for white mice, which die in from one to two 
weeks after infection ; also to field mice, which succumb to subcutaneous in- 
jections of a pure culture, and also, in from eight to twelve days, when fed 
upon potato cultures or bread moistened with a small quantity of a bouillon 
culture. L6ffler believes that this bacillus may be used for the destruction 
of field mice in grain fields, inasmuch as they invariably die after ingesting 
food which has been contaminated with it, and also from eating the bodies 


IN SUSCEPTIBLE ANIMALS. 525 


of other mice which have died as a result of infection. House mice are also 
susceptible. Rabbits, guinea pigs, pigeons, and chickens were found by 
Léffler not to be susceptible to infection by feeding. 


BACILLUS OF CAZAL AND VAILLARD. 


Obtained by Cazal and Vaillard (1891) from cheesy nodules upon the 
peritoneum and in the pancreas of an individual who died in the hospital 
at Val de Grace. 

Morphology.—Bacilli with rounded ends, but little longer than they are 
broad; solitary, in pairs, or in chains of ten to fifteen or more elements. 

Stains with the usual aniline colors, but not by Gram’s method; the 
extremities of the rods are more deeply stained than the central portion— 
‘‘ polar staining.” 

Biological Characters.—An aérobic and facultative anaérobic, liquefy- 
ing, motile bacillus. Doesnotform spores. Grows in the usual culture media 
at the room temperature—more rapidly in the incubating oven at 37° C. In 
gelatin stab cultures, at the end of twenty-four nours, a series of puncti- 
form, white colonies is developed along the line of puncture; upon the sur- 
face development is more abundant, and at the end of forty-eight hours 
liquefaction commences ; this progresses slowly from above downward, 
and a white, flocculent deposit accumulates at the bottom of the liquefied 
eee Upon the surface of agar, at the end of twenty-four hours at 37° 

-, &@ moist, transparent, opalescent layer is developed, which rapidly ex- 
tends over the entire surface ; later this layer becomes somewhat thicker, 
whitish, and cream-like in consistence, without losing its transparency. 
Upon potato a thick, prominent, moist, and slightly viscid layer is devel- 
oped, which at first has a pale-yellow and later a yellowish-brown color. 
In bouillon development is abundant, producing a milky opacity of the 
liquid; a thick, flocculent deposit accumulates at the bottom of the tube ; 
the reaction of the culture liquid becomes very alkaline. All of the cultures 
give off a peculiar odor, slightly ammoniacal and resembling that of putrid 
urine. The cultures retain their vitality for several months—in a closed 
tube for more than a year. The thermal death-point is 60° C. with fifteen 
minutes’ exposure. 

Pathogenesis.—Pathogenic for rabbits and mice, but not for guinea-pigs. 
In mice death occurs from general infection, at the end of forty-eight to 
sixty hours, from the subcutaneous injection of one eighth cubic centimetre 
of a recent bouillon culture. In rabbits injection of one cubic centimetre 
into the circulation causes the death of the animal in thirty-six to fifty 
hours. The symptoms induced are a foetid diarrhcea and paralysis of the 
extremities. When smaller doses are injected (0.5 cubic centimetre) a 
chronic malady is developed, characterized at the outset by diarrhcea and 
emaciation, then by the development of tumors which resemble those found 
in the man from whom the cultures were first obtained. These tumors are 
for the most part located in the subcutaneous connective tissue; after a time 
they attain the size of a chestnut and ulcerate, allowing the escape of a 
semi-fluid, purulent material. The animalsusually recover. Similar tumors 
are developed as a result of subcutaneous injections of one to three cubic 
centimetres of a recent bouillon culture. 


BACILLUS OF BABES AND OPRESCU. 


Obtained by Babes and Oprescu (1891) from a case of septiceemia hamor- 
rhagica presenting some resemblance to exanthematic typhus. 

Morphology.— In agar cultures the bacilli are from 0.4 to 0.5 # thick, and 
are frequently united in pairs; associated with these rod-shaped bacteria are 
forms which are of a short oval. In gelatin cultures oval forms are more 
numerous ; they have a diameter of 0.3 to 0.4 uw, and often appear to be 
surrounded by a capsule. In fresh cultures the bacilli are often in form of 


526 BACILLI WHICH PRODUCE SEPTIC4MIA 


a figure 8, and are only stained at the point of contact of the two segments. 
In potato cultures they are sometimes elongated and swollen at one ex- 
tremity. 

Stains with the usual aniline colors and by Gram’s method. | ; 

Biological Characters.—An aérobic and facultative anaérobic, non-lique- 
fying, actively motile bacillus. Spore formation not observed. Grows in the 
usual culture media at the room temperature—more rapidly at 37° C. In 
gelatin stab cultures yellowish-white colonies are developed along the line 
of puncture; at the bottom these may have a diameter of one to two millime- 
tres, and they havea brown color. Upon the surface an irregular, lobulated, 
whitish, translucent, paraffin like layer is developed. At the end of eight 
days the surface growth consists of large, confluent, transparent plaques, 
with irregular outlines and crenated, elevated margins ; along the line of 
puncture large, separate, lenticular or spherical cvlonies are seen ; these 
have a brownish-white color. At the end of two months the surface growth 
is concentric and still more transparent, while the colonies near the surface 
have become almost brown. Upon the surface of agar, at 87° C., a narrow 
band is developed along the line of inoculation; above, this is composed of 
transparent, shining, flat, round colonies having a diameter of one milli- 
metre or more; below, the colonies are confluent and form a transparent, 
whitish layer. In glycerin-agar development is still more abundant, and 
may already be perceived at the end of twelve hours. Crystals are seen 
below the surface in agar cultures and about the superficial colonies in gela- 
tin. Upon potato a uniform, thin, grayish, very transparent layer is de- 
veloped, which sometimes has a brownish-gray tint. At the end of a few 
days the potato acquires a brownish color, In bouillon cloudiness of the 
medium is apparent at the end of ten hours ; twenty-four hours later a 
whitish precipitate is seen at the bottom of the tube, which is more abun- 
dant when the culture medium contains glucose; later a thin pellicle is 
seen upon the surface and the bouillon acquires a yellowish color. 

Pathogenesis.—Recent cultures are pathogenic for rabbits, guinea-pigs, 
pigeons, and mice, which die from general infection in from two to four 
days. Old cultures are less virulent. 


BACILLUS OF LUCET. 


Obtained by Lucet (1891) from chickens and turkeys suffering from an 
infectious form of septiczemia characterized by dysenteric discharges—'‘‘ Dy- 
senterie epizootique des poules et des dindes.” 

Resembles Bacillus gallinarum of Klein, and is perhaps identical with 
this microdrganism. ; 

Morphology.—Short bacilli, from 1.2 to 1.8 # long, usually in pairs. 

Stains with the usual aniline colors, but not by Gram’s method. 

Biological Characters.—An aérobic and facultative anaérobic, non-lique- 
fying, non-motile bacillus. Spore formation not observed. Grows slowly in 
the usual culture media at the room temperature—more rapidly at 37° C. 

In gelatin plates small, shining, moist, white, circular colonies are devel- 
oped, which look like little drops of wax; later these increase in size, and 
especially in thickness, forming hemispherical masses. In gelatin stab cul- 
tures grayish, punctiform colonies are developed along the line of puncture, 
and upon the surface a circular, prominent, whitish plaque. Streak cultures 
upon the surface of gelatin are in the form of a dirty-white or grayish-white, 
moist streak, with regular margins, limited to the line of inoculation, but 
increasing in thickness until it breaks loose and slips down the oblique sur- 
face of the culture medium. _ The deposit which collects in this way acquires, 
as it becomes old, in the deepest portion a reddish color. Upon agar it forms 
a thick, yellowish-white, mucus-like layer with straight or slightly dentate 
margins. In bouillon it produces a decided clouding of the liquid, and an 
abundant grayish, pulverulent sediment accumulates at the bottom of the 
tube; the bouillon after a time becomes transparent above this sediment and 


PLATE VII. 


BACILLUS OF GLANDERS. 


Fig. 1.—Bacillus mallei from the liver of a field mouse, cover-glass 
preparation. (Loftler.) 

Fie. 2.—Bacillus mallei from a recent culture upon blood serum. 
(Loffler. ) 

Fig. 3.—Bacillus mallei in section of spleen of a field mouse dead 
from glanders. (Léoffler.) 

Fie. 4.—Culture of glanders bacillus upon cooked potato. (Loffler.) 


STERNBERG'S BACTERIOLOGY. * Plate VU. 


Fig.+. 


BACILLUS OF GLANDERS (LOEFFLER ) 


IN SUSCEPTIBLE ANIMALS. 527 


is viscid, drawing out into threads. In the absence of oxygen the characters 
of growth are the same as in its presence. The cultures acquire an alkaline 
reaction; they are sterilized by exposure for ten minutes to a temperature of 
60° C. Does not grow upon potato. 

_ Pathogenesis.—Pathogenic for chickensand turkeys. Not pathogenic for 
pigeons, pune ries, or rabbits when injected subcutaneously or into the 
peritoneal cavity, but kills rabbits when injected into a vein. In the in- 
fected fowls the bacilli are found in small numbers in the blood, more nu- 
merous in the kidneys and liver, still more numerous in the spleen, and in 
enormous numbers in the intestinal mucus, where in acute cases it is found 
almost in a pure culture. Fowls do not contract the disease as a result of 
the ingestion of grains soiled with cultures of the bacillus, but become in- 
fected when fed with animal food to which a pure culture has been added. 


XIV. 


PATHOGENIC AKROBIC BACILLI NOT DESCRIBED IN 
PREVIOUS SECTIONS. 


A CONSIDERABLE number of saprophytic bacilli are pathogenic for 
small animals when injected into the circulation, or subcutaneously, 
or into a serous cavity in considerable quantity—one to five cubic 
centimetres or more—but fail to produce any appreciable effect 
when introduced into the bodies of these animals in minute doses, 
and do not multiply in the blood to any considerable extent, al- 
though in fatal cases they may usually be recovered in cultures from 
the blood and tissues. These bacilli are pathogenic by reason of the 
toxic ptomaines produced by them, or because of local inflammatory 
processes which they induce, or for both of these reasons combined. 
Some of them may also, under certain circumstances, multiply in 
the blood and thus give rise to septiceemia as well as to toxeemia ; 
this is the case, for example, with the “‘ colon bacillus” of Escher- 
ich. When injected in considerable quantity into the circulation 
of a guinea-pig it causes the death of the animal within twenty-four 
hours, and the bacillus is found in the blood in great numbers ; but 
minute amounts injected into a vein, or larger amounts injected 
subcutaneously, do not usually produce general infection. It is, 
therefore, not included among the ‘‘ bacilli which produce septi- 
ceemia in susceptible animals.” There is reason to believe, however, 
that under certain circumstances this bacillus may have sufficient 
pathogenic potency to produce a genuine septicemia in guinea-pigs. 
Thus the original cultures of Brieger’s bacillus, which appears to be 
a variety of the colon bacillus, are reported to have produced fatal 
septicemia in guinea-pigs when injected subcutaneousiy in small 
amounts. <A strict division into pathogenic bacilli which produce 
general blood infection—septicemia—and those which produce a 
fatal result owing to the production of toxic chemical substances is 
not possible; for many pathogenic bacteria produce general infection 
when injected in comparatively large doses, and at the same time 
give rise to symptoms of toxemia; or general infection may occur 
in animals of one species, and fatal toxsmia without septicemia in 


PATHOGENIC AEROBIC BACILLI NOT BEFORE DESCRIBED. 529 


those of another species. Many of the bacilli described in the pre- 
sent section are common saprophytes, which have been shown by 
laboratory experiments to be pathogenic for certain animals when 
introduced into their bodies in a certain amount, which differs greatly 
for different bacteria and for different species of animals. The ex- 
periments of Cheyne and others show how largely the pathogenic 
power of saprophytic bacteria depends upon the quantity of a cul- 
ture which is injected, as well as upon the age of the culture and 
the seat of the inoculation—in the blood, the abdominal cavity, the 
subcutaneous tissues, or the muscles. And the bacteriologist named 
has also shown that pathogenic power depends, in some instances at 
least, upon the combined action of the toxic substances introduced 
in the first instance and of the living bacteria. Thus Cheyne found 
that one-tenth of a cubic centimetre of a bouillon culture of Proteus 
vulgaris injected into the dorsal muscles of a rabbit infallibly caused 
its death within forty-eight hours, but when the dose was reduced 
to one-fortieth cubic centimetre the animal recovered. But if to 
this amount (one-fortieth cubic centimetre) he added one cubic cen- 
timetre of a sterilized (by heat) culture of the same bacillus instead 
of diluting with distilled water, and injected the mixture into the 
dorsal muscles of a rabbit, death occurred in every experiment 
within forty-eight hours. The sterilized culture injected by itself 
produced no effect in this dose (one cubic centimetre), and Cheyne 
believes that the fatal result in these experiments was due to the 
fact that the toxic products present in the sterilized culture over- 
came the natural resisting powers of the tissues and enabled the 
bacillus to multiply over a larger area than would otherwise have 
been the case. As a result of this, toxic substances were produced in 
the body of the animal in sufficient quantity to cause general toxe- 
mia and death ; whereas the bacilli alone, in the dose mentioned, 
were not able to invade the tissues in the vicinity of the point of 
inoculation, and gave rise to a local abscess only. The same ex- 
planation is probably true for very many of the saprophytic bacteria 
which have been shown to possess pathogenic power ; and it is prob- 
able that many of those which are now classed by bacteriologists as 
non-pathogenic would prove to be pathogenic in the same way if 
thoroughly tested upon various species of animals, although it might 
be necessary to use unusually large doses to accomplish the same 
result. 


BACILLUS COLI COMMUNIS. 


Synonyms.—Bacterium coli commune (Escherich); Colon bacillus 
of Escherich ; Emmerich’s bacillus (Bacillus Neapolitanus). Prob: 
ably identical with Bacillus cavicida (Brieger’s bacillus), 

84 


530 PATHOGENIC AEROBIC BACILLI 


Obtained by Emmerich (1885) from the blood, various organs, and 
the alvine discharges of cholera patients at Naples; by Weisser 
(1886) from normal and abnormal human feces, from the air, and 
from putrefying infusions; by Escherich (1886) from the fasces of 
healthy children ; since shown to be commonly present in the alvine 
discharges of healthy men, and probably of many of the lower ani- 
mals. Found by the writer in the blood and various organs of yellow- 
fever cadavers, in Havana (1888 and 1889). 

Numerous varieties have been cultivated by different bacteriolo- 
gists, which vary in pathogenic power and to some extent in their 
growth in various culture media; but the differences described are 
not sufficiently characteristic or constant to justify us in considering 
them as distinct species. 

Morphology.—Differs considerably in its morphology as obtained 
from different sources and in various culture media. The typical 
form is that of short rods with rounded ends, from two to three pu in 
length and 0.4 to 0.6 4 broad; but under certain cir- 
cumstances the length does not exceed the breadth— 


e% 
Rie! , about 0.5 u—and it might be mistaken for a micrococ- 
Vays? © cus; again the prevailing form in a culture is a short 
® oval ; filaments of five “or more in length are often 


Fie. 144.—Ba- observed in cultures, associated with short rods or oval 
wrong nw ton cells. The bacilli are frequently united in pairs. The 
(scherich.) presence of spores has not been demonstrated. In un- 

favorable culture media the bacilli, in stained prepara- 
tions, may present unstained places, which are supposed by Escherich 
to be due to degenerative changes in the protoplasm. Under certain 
circumstances some of the rods in a pure culture have been observed 
by Escherich to present spherical, unstained portions at one or both 
extremities, which closely resemble spores, but which he was not able 
to stain by the methods usually employed for staining spores, and 
which he isinclined toregard as “‘ involution forms.” 

This bacillus stains readily with the aniline colors usually em- 
ployed by bacteriologists, but quickly parts with its color when 
treated with iodine solution—Gram’s method—or with diluted al- 
cohol. 

Biological Characters.—An aérobic and facultative anaérobic, 
non-liquefying bacillus. Sometimes exhibits independent move- 
ments, which are not very active. One rod of a pair, in a hanging- 
drop culture, may advance slowly with a to-and-fro movement, 
while the other follows as if attached to it by an invisible band 
(Escherich), The writer’s personal observations lead him to believe 
that, as a rule, this bacillus does not exhibit independent movements. 
Does not form spores. Grows in various culture media at the room 


NOT DESCRIBED IN PREVIOUS SECTIONS. 531 


temperature—more rapidly in the incubating oven. Grows in a de- 
cidedly acid medium. 

In gelatin plates colonies are developed in from twenty-four to 
forty-eight hours, which vary considerably in their appearance ac- 
cording to their age, and in different cultures in the same medium. 
The deep colonies are usually spherical and at first are transparent, 
homogeneous, and of a pale-straw or amber color by transmitted 
light ; later they frequently have a dark-brown, opaque central por- 
tion surrounded by a more transparent peripheral zone ; or they may 
be coarsely granular and opaque; sometimes they have a long-oval 
or ‘‘whetstone” form. The superficial colonies differ still more in 
appearance ; very young colonies by transmitted light often resemble 
little drops of water or fragments of broken glass ; when they have 
sufficient space for their development they quickly increase in size, 
and may attain a diameter of three to four centimetres ; the central 
portion is thickest, and is often marked by aspherical nucleus of a 
dark-brown color when the colony has started below the surface of 
the gelatin; the margins are thin and transparent, the thickness 
gradually increasing towards the centre, as doesalso the color, which 
by transmitted light varies from light straw color or amber to adark 
brown. The outlines of superficial colonies are more or less irregular, 
and the surface may be marked by ridges, fissures, or concentric 
rings, or may be granular. The writer has observed colonies re- 
sembling a rosette, or a daisy with expanded petals. Escherich 
speaks of colonies which present star-shaped figures surrounded by 
concentric rings. 

In gelatin stab cultures the growth upon the surface is rather 
dry, and may be quite thin, extending over the entire surface of the 
gelatin, or it may be thicker with irregular, leaf-like outlines and 
with superficial incrustations or concentric annular markings. An 
abundant development occurs all along the line of puncture, which 
in the deeper portion of the gelatin is made up of more or less closely 
crowded colonies ; these are white by reflected light, and of an am- 
ber or light-brown color by transmitted light ; later they may become 
granular and opaque. Frequently a diffused cloudy appearance is 
observed near the surface of the gelatin, and under certain circum- 
stances branching, moss-like tufts develop at intervals along the line 
of growth. One or more gas bubbles may often be seen in recent 
stick cultures in gelatin. 

Upon nutrient agar and blood serum, in the incubating oven, an 
abundant, soft, white layer is quickly developed. Upon potato an 
abundant, soft, shining layer of a brownish-yellow color is developed. 
The growth upon potato differs considerably, according to the age of 
the potato. According to Escherich, upon old potatoes there may 


532 PATHOGENIC AEROBIC BACILLI 


be no growth, or it may be scanty and of a white color. In milk, at 
37° C., an acid reaction and coagulation of the casein are produced at 
the end of eight or ten days, In the absence of oxygen this bacillus 
is able to grow in solutions containing grape sugar (Escherich). In 
bouillon it grows rapidly, producing a milky opacity of the culture 
liquid. The thermal death-point of Emmerich’s bacillus, and of the 
colon bacillus from feces, was found by Weisser to be 60° C., the 
time of exposure being ten minutes. The writer has obtained corre- 
sponding results. Weisser found that when the bacilli from a bouil- 
lon culture were dried upon thin glass covers they failed to grow 


Fia. 145. Fig. 146. 


Fia. 145.—Bacillus coli communis in nutrient gelatin containing twenty per cent of gelatin, end 
of two weeks, showing moss-like tufts along the line of growth. (Sternberg.) 

Fie. 146.—A portion of the growth shown in Fig 147, at a, magnified about six diameters. 
From a photograph. (Sternberg.> 


after twenty-four hours. These results give confirmation to the 
view that the bacillus under consideration does not form spores. 
This view receives further support from the experiments of Wal- 
liczek (1894), who found that when dried upon pieces of sterile filter 
paper the bacillus failed to grow at the end of eighteen hours. 
Pathogenesis.—Comparatively small amounts of a pure culture 
of the colon bacillus injected into the circulation of a guinea-pig 
usually cause the death of the animal in from one to three days, and 
the bacillus is found in considerable numbers in its blood. But when 


NOT DESCRIBED IN PREVIOUS SECTIONS. 533 


injected subcutaneously or into the peritoneal cavity of rabbits or 
guinea-pigs, a fatal termination depends largely on the quantity in- 
jected; and although the bacillus may be obtained in cultures from 
the blood and the parenchyma of the various organs, it is not present 
inlarge numbers, and death appears to be due to toxzemia rather than 
to septicaemia. Mice are not susceptible toinfection by subcutaneous 
injections. Small quantities injected beneath the skin of guinea-pigs 
usually produce a local abscess only ; larger amounts—two to five 
cubic centimetres—frequently produce a fatal result, with symptoms 
and pathological appearances corresponding with those resulting 
from intravenous injection. These are fever, developed soon after 
the injection, diarrhoea, and symptoms of collapse appearing shortly 
before death. At the autopsy the liver and spleen appear normal, or 
nearly so; the kidneys are congested and may present scattered 
punctiform ecchymoses (Weisser). According to Escherich, the 
spleen is often somewhat enlarged. The small intestine is hyper- 
zemic, especially in its upper portion, and the peritoneal layer pre- 
sents a rosy color; the mucous membrane gives evidence of more 
or less intense catarrhal inflammation, and contains mucus, often 
slightly mixed with blood. In rabbits death occurs at a somewhat 
later date, and diarrhcea is a common symptom. In dogs the subcu- 
taneous injection of a considerable quantity of a pure culture may 
give rise to an extensive local abscess. 

In human pathology the colon bacillus plays an important réle. . 
It is concerned in the etiology of a considerable proportion of the 
cases of cystitis and of pyelonephritis, and peritonitis resulting from 
perforation. It appears to be the cause of certain affections of the 
anal region (Hartmann and Lieffring). It has been obtained in pure 
culture from abscesses in various parts of the body, from the valves 
of the heart in endocarditis, from the pleural cavity in empyema, etc. 
It has also been found in the blood, as a result of general infection 
following cystitis and pyelonephritis (Sittmann and Barnow). 

Varieties.—Booker, in his extended studies relating to the bac- 
teria present in the faeces of infants suffering from summer diarrhea, 
has isolated seven varieties “ which closely resemble Bacterium coli 
commune in morphology and growth in agar, neutral gelatin, and 
potato, but by means of other tests a distinction can be made between 
them.” 

Some of the pathogenic bacteria heretofore described are also 
closely allied to the “colon bacillus” and by some bacteriologists are 
supposed to belong to the same group—7.e., to be varieties of the 
same species rather than independent species with fixed characters. 
Whatever may be the remote relationship, the typhoid group, the hog- 


534 PATHOGENIC AEROBIC BACILLI 


cholera group, the Bacillus typhi murium of Léffler, the bacillus of 
Laser, the Bacillus enteritidis of Gartner, and other similar bacilli 
appear to be differentiated from one another by characters which 
justify their description under separate names. Still it is difficult to 
fix upon any one of these characters to which specific value can be 
attached; and, in view of the many varieties found in nature or pro- 
duced artificially in laboratory experiments, we are not justified in 
asserting that our classification of these low organisms has any sub- 
stantial scientific foundation. The difficulties attending an attempt 
to establish specific characters are well illustrated by the extensive 
literature relating to the differentiation of bacilli belonging to the 
typhoid group from those belonging to the colon group. The main 
points upon which the distinction must depend have been referred to 
in the section devoted to the typhoid bacillus. 

Fremlin (1893) has made a comparative study of the colon bacil- 
lus from various sources. He finds the common characters of gas 
production in media containing sugar and coagulation of milk. Cul- 
tivated from different animals the morphology is the same, but there 
are differences as regards motility. The most active movements are 
said to be exhibited in the bacillus from man, while the variety ob- 
tained from the intestines of rabbits showed scarcely any movements. 
The different varieties displayed considerable differences in their 
growth upon potato. 

Dreyfuss (1894) finds decided differences in the pathogenic viru- 
lence of the colon bacillus from healthy individuals and from those 
suffering from various intestinal disorders. A culture from the dis- 
charges of a fatal case of cholera nostras proved to be exceptionally 
virulent—tested by intraperitoneal injections in guinea-pigs. Gilbert 
(1895), as a result of his extended researches, concludes that there are 
five principal types among the bacilli most nearly related to the colon 
bacillus: 1st. Bacilli which differ from the colon bacillus by their 
being non-motile. This type includes two varieties: one gives thick 
yellowish colonies upon gelatin plates and numerous gas bubbles on 
potato—this is the bacille lactique of Pasteur and the Bacillus lactis 
aérogenes of Hscherich; the other gives thin, bluish-white colonies 
and includes the bacille de l’éndocardite of Gilbert and Lion. 2d. 
Bacilli which differ from the colon bacillus by the fact that cultures 
do not give the indol reaction. 3d. Bacilli which do not cause the 
fermentation of lactose. 4th. Bacilli which are not motile and 
do not ferment lactose. 5th. Bacilli which are not motile, do not 
give the indol reaction, and do not ferment lactose. 

Theobald Smith (1895) gives the following account of his method 
of detecting bacilli of the ‘‘colon group ’’ in water : 


NOT DESCRIBED IN PREVIOUS SECTIONS. 535 


‘The method followed by the writer in the general bacteriological exam- 
ination of water consists, first, in the preparation of gelatin plates for the 
usual enumeration ; and, second, in the addition to every one of ten fermen- 
tation tubes, containing a one-per-cent dextrose bouillon, a certain quantity 
of water. This is added most easily by first diluting the water, so that one or 
two cubic centimetres are equivalent to the quantity which it is desired to add 
toeach tube. Pipettes graduated by drops are convenient, but not so accurate. 
In case of ground water it is well to prepare in addition a flask containing 
fifty to one hundred cubic centimetres of the water, and an equal, or greater, 
quantity of bouillon, to which sugar is not added. Plates may be prepared 
from this flask after sixteen to twenty-four hours. When gas begins to ap- 
pear in the fermentation tubes, the amount accumulated at the end of eac 
twenty-four hours should be marked with a glass pencil on the tube. From 
these tubes, which contain fifty to sixty per cent of gas on the third day, and 
are very strongly acid, plates may be prepared to confirm the indications of 
Bacillus coli. This, however, is not essential, for the writer has found as 
yet no species having these fermentative characters which is not one of the 
following : Bacillus coli, Bacillus lactis aérogenes, Bacillus enteriditis, Bacil- 
lus typhi murium, Bacillus cholere suis. The three last-mentioned species 
are probably as rare in water as Bacillus typhosus itself. 

“ My own experience coincides with that of Matthews when he states that 
ninety-two per cent of all bacteria in ground water are suppressed in the 
thermostat. While the addition of 0.5 cubic centimetre, or even more, of 
such water may fail to produce cloudiness in any of the series of fermenta- 
pen sae the same quantity, or less, of surface water never fails to infect 
the tubes.” 


Bacillus Coli Communis in Peritonitis.—The researches of A. 
Frankel show that Bacillus coli communis may be obtained in pure 
cultures from the exudate into the peritoneal cavity in a considerable 
proportion of the cases of peritonitis, and there is good reason for 
believing that in these cases it was the cause of the inflammatory 
process. Thirty-one cases were examined by Frankel, with the fol- 
lowing result: Pure cultures of Bacillus coli communis were obtained 
in nine cases; of Streptococcus (pyogenes ?) in seven; of Bacillus 
lactis aérogenes in two; of ‘‘diplococcus pneumoniz” in one ; of 
Staphylococcus pyogenes aureus inone. Of the remaining eleven 
cases, seven gave mixed cultures, and in three of these Bacillus coli 
communis was the most abundant species. The author referred to 
has also shown that pure cultures of Bacillus coli communis injected 
into the cavity of the abdomen of rabbits cause a typical peritonitis. 
The present writer has frequently obtained the same result in experi- 
ments made with this bacillus. It would appear, therefore, that the 
peritonitis which so constantly results from wounds of the intestine 
is probably due, to a considerable extent, to the introduction of this 
microérganism from the lumen of the intestine, where it is con- 
stantly found, into the peritoneal cavity, where the conditions are 
favorable for its rapid development. 


536 PATHOGENIC ABROBIC BACILLI 


BACILLUS LACTIS AEROGENES. 


Obtained by Escherich (1886) from the contents of the small intestine of 
children and animals fed upon milk ; in smaller numbers from the faeces of 
milk-fed children, and in one instance from uncooked cow’s milk. 

Morphology.—Short rods with rounded ends, from 1 to 
2 in length and from 0.1 to 0.5 # broad; short oval and 


oft, spherical forms are also frequently observed, and, under 
68 certain circumstances, longer rods —3 “—may be developed: 
3° £é usually united in pairs, and occasionally in chains contain- 


ing several elements. In some of the larger cells Escherich 
. has observed unstained spaces, but was not able to obtain 
Fic. 147,—Bacil- any evidence that these represent spores. 


om gee 8 This bacillus stains readily with the ordinary aniline 
aoe colors, but does not retain its color when treated by Gram’s. 
oe method 


' Biological Characters. —An aérobic (facultative anaérobic), non-liquefy- 


ing, non motile bacillus. Does not form spores. Grows in various culture 
media at the room temperature—more rapidly in the incubating oven. 
Upon gelatin plates, at the end of twenty-four hours, small white colonies. 
are developed. Upon the surface these form hemispherical, soft, shining 
masses which, examined under the microscope, are found to be homogeneous. 
and opaque, with a whitish lustre by reflected light. The deep colonies are 
spherical and opaque and attain a considerable size. In gelatin stab cul- 
tures the growth resembles that of Friedlinder’s bacillus—i.e.. an abundant. 
growth along the line of puncture and a rounded mass upon the surface, 
forming a ‘‘nail-shaped” growth. In old cultures the upper portion of the 
gelatin is sometimes clouded. and numerous gas bubbles may form in the 
gelatin. Upon the surface of nutrient agar an abundant, soft, white layer 
is developed. Upon old potatoes, in the incubating oven, at the end of 
twenty-four hours a yellowish-white layer, several millimetres thick, is 
developed, which is of paste-like consistence and contains about the peri- 
phery a considerable number of small gas bubbles; this layer increases in 
dimensions, has an irregular outline, and larger and more numerous gas 
bubbles are developed about the periphery, some the sizé of a pea; later the 
whole surface of the potato is covered with a creamy, semi-fluid mass filled 
with gas bubbles. On young potatoes the development is different; a rather 
luxuriant, thick, white or pale-yellow layer is formed, which is tolerably 
dry and has irregular margins; the surface is smooth and shining, and a 
few minute gas bubbles only are formed after several days. 

Pathogenesis.—Injections of a considerable quantity of a pure culture 
into the circulation of rabbits and of guinea-pigs give rise to a fatal result 
within forty-eight hours. 

In his first publication relating to ‘‘ the bacteria found in the dejecta of 
infants afflicted with summer diarrhoea,” Booker has described a bacillus 
which he designates by the letter B, which closely resembles Bacillus lactis 
aérogenes and is probably identical with it. He says: 

“Summary of Bacillus B.—Found nearly constantly in cholera infan- 
tum and catarrhal enteritis, and generally the predominating form. It 
appeared in larger quantities in the more serious cases. It was not found 
in the dysenteric or healthy feces. It resembles the description of the Ba- 
cillus lactis aérogenes, but the resemblance does not appear sufficient to con- 
stitute an identity, and, in the absence of aculture of the latter for com- 
parison, it is considered a distinct variety for the following reasons: Bacillus 
B is uniformly larger, its ends are not so sharply rounded, and in all culture 
media long, thick filaments are seen, and many of the bacilli have the pro- 
toplasm gathered in the centre, leaving the poles clear. There is some 


ae 


NOT DESCRIBED IN PREVIOUS SECTIONS. 537 


difference in their colony growth on gelatin, and in gelatin stab cultures 
bacillus B does not show the nail-form growth with marked end swelling in 
the depth. In potato cultures the Bacillus lactis aérogenes shows a differ- 
ence between old and new potatoes, while bacillus B does not show any 
difference. 

‘* Bacillus B possesses decided pathogenic properties, which was shown 
both by hypodermic injections and feeding with milk cultures.” 


BACILLUS ACIDIFORMANS. 


Obtained by the writer (1888) from a fragment of yellow-fever liver pre- 
served for forty-eight hours in an antiseptic wrapping; since obtained from 


Fig. 148. Fig, 149, 


Fie. 148.—Bacillus acidiformans, from a potato culture. x 1,000. From wu photomicrograph 
(Sternberg ) 

Fia. 149.—Culture of Bacillus acidiformans in nutrient gelatin, end of four days at 22° C. 
From a photograph. (Sternberg.) 


liver preserved in the same way from two comparative autopsies—i.e., not 
cases of yellow fever. 

Morphology.—A. short bacillus with rounded corners, sometimes short. 
oval in form; from 14 to 3 # in length and about 1.2 u in breadth; may grow 
out into filaments of 5 to 10 ~, or more, in length; in some cultures the short. 
oval form predominates. 

Stains readily with the aniline colors usually employed, and by Gram’s 
method. 

Biological Characters.—An aérobic and facultative anaérobic, non- 
liquefying, non-motile bacillus. Does not form spores. Grows rapidly at 
the room temperature in the usual culture media. Grows in decidedly acid 
media; in culture media containing glycerin or glucose it produces an abun- 
dant evolution of carbon dioxide, and a volatile acid is formed. 

It does not liquefy gelatin, and in stab cultures grows abundantly both 
on the surface and along the line of puncture. At the end of twenty-four 
hours, at 22°C., a moniled white mass is formed upon the surface, resembling 
the growth of Friedlander’s bacillus; at the bottom of the line of puncture 
the separate colonies are spherical, opaque, and pearl-like by reflected light. 
Gas bubbles are formed in the gelatin. At the end of a week thesurface is 
covered with a thick, white, semi-fluid mass. _ 

In gelatin roll tubes the superticial colonies are translucent or opaque, 
and circular or somewhat irregular in outline; by reflected light they are 


538 PATHOGENIC AEROBIC BACILLI 


slightly iridescent; the deep colonies are spherical, opaque, and homo- 
eneous. 

. The growth upon the surface of nutrient agar is abundant and rapid, of 

ashining milk-white color, and cream-like in consistence. An abundant 

development forms along the line of puncture and the culture medium is 

split up by gas bubbles. In glycerin-agar the evolution of gas is very abun- 

dant and the culture medium acquires an intensely acid reaction. 

On potato the growth is abundant and rapid at a temperature of 20° to 
30° C., forming a thick, semi-fluid mass of a milk-white color. 

I have not obtained any evidence that this bacillus forms spores; the 
cultures are sterilized by ten minutes’ exposure to a temperature of 160° F. 

When cultivated in bouillon to which five per cent of glycerin has been 
added the culture medium acquires a milky opacity, and there is a copious 
precipitate, of a viscid consistence, consisting of bacilli; during the period 
of active development the surface is covered with gas bubbles, as in a sac- 
charine liquid undergoing alcoholic fermentation, and the liquid has a de- 
cidedly acid reaction. 

Pathogenesis.—Pathbogenic for rabbits and for guinea pigs when injected 
into the cavity of the abdomen—one to two cubic centimetres of a culturein 
bouillon. The animal usually dies in less than twenty-four hours. The 
bacilli are found in the blood in rather small numbers, and are frequently 
seen in the interior of the leucocytes. The spleen is enlarged, the liver 
normal, the intestine usually hyperzemic. 


BACILLUS CUNICULICIDA HAVANIENSIS. 


Obtained by the writer (1889) from the contents of the intestine of yellow- 
fever cadavers, and also from fragments of yellow-fever liver preserved for 
forty-eight hours in an antiseptic wrap- 
ping—my bacillus x, Havana, 1889. 

Morphology.—This bacillusresembles 
the colon bacillus in form, but is some- 
what larger, from 2 to 4 “ in length and 
from 0.8 tol “ in diameter ; sometimes 
associated in pairs; may grow out into 
short filaments—not common. The ends 
of the rods are rounded, and under cer- 
tain circumstances vacuoles are seen at 
the extremities, especially in potato cul- 
tures. 

Stains quickly with the aniline colors 
usually employed, and also by Gram’s 
method. 

Biological Characters.—An aérobic 
and facultative anaérobic, non-lique- 

Fig. 150.—Bacillus cuniculicida Havani- fying bacillus. Under certain circum- 
‘ensis, from a sinzle colony in nutrient gela- stances may exhibit active movements, 
tin. x 1,000. From a photomicrograph, yt is usually motionless. 

Sternberg.) A very curious thing with reference 
: : to this bacillus is that it presented ac- 
tive movements in my cultures made directly from yellow-fever cadavers, 
but that these movements were not constant, and that since my return to 
Baltimore I have not, as a rule, observed active movements in cultures from 
the same stock, which, however, preserved their pathogenic power and other 
characters. In Havana these movements were usually not observed in all 
the bacilli in a field under observation, but one and another would start from 
a quiescent condition on an active and erratic course; sometimes spinning 
actively upon its axis, and again shooting across the field as if propelled by 
a flagellum, 


NOT DESCRIBED IN PREVIOUS SECTIONS. 539 


My notes indicate that cultures passed through the guinea-pig are more 
apt to be motile. 

In gelatin stab cultures the growth of bacillus w resembles that of the 
colon bacillus, but the colonies at the bottom of the line of puncture are 
more opaque and not of a clear amber color like that of colonies of the colon 
bacillus. Upon the surface the growth is thicker than that of the colon 
bacillus, and forms a milk-white, soft mass. 

_ The colonies in gelatin Esmarch roll tubes vary considerably at different 
times. Deep colonies are usually spherical, homogeneous, light brown in 
color, and more opaque than the similar colonies of the colon bacillus. At 
the end of a few days the deep colonies become quite opaque, and may be 
lobate, like a mulberry, or coarsely granular; sometimes the deep colonies 
have an opaque central portion surrounded by a transparent marginal zone. 

In old gelatin roll tubes these deep colonies form opaque white hemi- 


Fie. 151. Fie. 152. 


Fig. 151.—Bacillus cuniculicida Havaniensis; colonies in gelatin roll tube, third day at 20° C, 
x6. From aphotograph. (Sternberg.) 

Fie. 152.—Bacillus cuniculicida Havaniensis ; colonies in gelatin roll tube, end of forty-eight 
hours. X10. Froma photograph. (Sternberg.) 


spheres projecting from the surface of the dried culture medium, and little 
tufts of acicular crystals are sometimes observed to project from the side of 
such old colonies. 

The superficial colonies are circular or irregular in outline, with trans- 
parent margins and an opaque central portion, sometimes corrugated. They 
are finely granular and iridescent by reflected light, and of a milk-white 
color; by transmitted light they have a brownish color. Young colonies 
closely resemble those of the colon bacillus. This bacillus grows well at a 
temperature of 20° C. (68° F.), but more rapidly and luxuriantly at a higher 
temperature—30° to 35° C. 

It grows well in agar cultures, and especially in glycerin-agar, in which 
it produces some gas and an acid reaction. The growth on the surface 
of glycerin-agar cultures is white, cream-like in consistence, and quite abun- 
dant. 

It grows well in an agar or gelatin medium made acid by the addition of 
0.2 per cent (1: 500) of hydrochloric acid. 

n cocoanut water it multiplies rapidly, producing a milky opacity of the 
Breviauehy transparent fluid, an acid reaction, and an evolution of carbon 
dioxide. 

On potato it produces a thick layer, which may. cover the entire surface 
in three or four days, and which has a dirty-white, cream-white, or pinkish- 


540 PATHOGENIC AEROBIC BACILLI 


white color and cream-like consistence. The growth upon potato varies at 
different times, evidently owing to ditferences in the potato. 

When stained preparations are examined with the full light of the Abbe 
condenser the ends of some of the rods appear to be cut away, leaving a con- 
cave extremity; but by using a small diaphragm to obtain definition it will 
be seen that the cell wall extends beyond the stained portion of the rod and 
includes what appears to bea vacuole. There is no reason to believe that 
this appearance is due to the presence of an end spore, for the supposed 
vacuole is not refractive, as a spore would be, and my experiments on the 
thermal death-point of this bacillus indicate that it does not form spores. 
Cultures are sterilized by exposure for ten minutes to a temperature of 160° 
F. (71.2° C.). 

Pathogenesis.—Very pathogenic for rabbits when injected into the cavity 
of the abdomen. Injections of asmall quantity of a pure culture into the 
ear vein or subcutaneously generally give a negative result. Injections of 
from one to five cubic centimetres of a culture in bouillon, blood serum, or 
agua coco, into the cavity of the abdomen, frequently prove fatal to rabbits 
in a few hours—two to six. 

The negative results obtained in injecting cultures beneath the skin or 
into the ear vein of rabbits show that this bacillus does not induce a fatal 
septicemia in these animals, and the fatal result when injections are made 
into the peritoneal cavity does not appear to be due to an invasion of the 
blood, but rather to the local effect upon the peritoneum, together with the 
toxic action of the chemical products resulting from its growth. 

It is true that I have always been able to recover the bacillus from the 
liver, or from blood obtained from one of the cavities of the heart, even in 
animals which succumb within a few hours to an injection made into the 
cavity of the abdomen. But the direct examination of the blood shows that 
the bacilli are present in very small numbers, and leads me to believe that 
the bacillus does not multiply, to any considerable extent at least, in the 
circulating fluid. 

The spleen is not enlarged, as is the case in anthrax, rabbit septicemia, 
and other diseases in which the pathogenic microdrganism multiplies abun- 
dantly in the blood. 

On the other hand, there is evidence of local inflammation in the peri- 
toneal cavity. When death occurs within a few hours the peritoneum is: 
more or less hyperzemic and there is a considerable quantity of straw-colored. 
fluid in the cavity of the abdomen. When the animal lives for twenty 
hours or more there is a decided peritonitis with a fibrinous exudation upon 
the surface of the liver and intestine. Usually the liver, in animals which: 
die within twenty-four hours, is full of blood, rather soft, and dark in color. 
ae a single instance I found the liver to be of a light color and loaded with 

at. 

The rapidly fatal effect in those cases in which I have injected two or 
more cubic centimetres of a culture into the cavity of the abdomen has led 
me to suppose that death results from the toxic effects of a ptomaine con- 
tained in the culture at the time of injection. The symptoms also give sup- 
port to this supposition. The animal quickly becomes feeble and indisposed 
to move, and some time before death lies helpless upon its side, breathing 
regularly, but is too feeble to get up on its feet when disturbed. Death some- 
times occurs in convulsions, but more frequently without—apparently from 
heart failure. 

Pathogenic also for guinea-pigs when injected into the cavity of the 
abdomen, but death does not occur in so short a time—eighteen to twenty 
hours. The comparative researches of Reed and Carroll indicate that this is: 
a pathogenic variety of the colon bacillus. 


NOT DESCRIBED IN PREVIOUS SECTIONS. 541 


BACILLUS LEPORIS LETHALIS. 


Obtained by Dr, Paul Gibier (1888) from the contents of the intestine of 
yellow-fever patients; also by the writer from the same source (1888, 1889) 
in exceptional cases and in comparatively small numbers. Named and de- 
scribed by present writer. 

Morphology.—Bacilli with rounded ends, from 1 to 3 win length and 
about 0.5 “in breadth. The length may vary in the same culture froma 
short oval to rods which are two or three times as long as broad, or it may 
grow out into flexible filaments of considerable length. In recent cultures 
the bacilli are frequently united in pairs. 

Stains readily with the aniline colors usually employed. In cultures 
which are several days old, or in recent cultures when the stained prepara- 
tion is washed in alcohol, the ends of the rods are commonly more deeply 
stained than the central portion—‘‘end staining”; and in old cultures some 
of the bacilli are very faintly stained. 

Biological Characters.—An aérobic, liquefying, actively motile bacillus. 
Does not form spores. 

In gelatin stab cultures, at the end of twenty-four hours at a tempe- 
rature of 20° to 22° C., there isan abundant development along the line of 
puncture and commencing liquefaction at the surface. Later the liquefaction 
is funnel-shaped, and there is an opaque white central core along the line 
of puncture, with liquefied gelatin around it. Liquefaction progresses most 
rapidly at the surface, and in the course of three or four days the upper por- 
tion of the gelatin for a distance of half an inch or more is completely lique- 
fied, and an opaque white mass, composed of bacilli, rests upon the surface 
of the unliquefied portion. 

In gelatin roll tubes the young colonies upon the surface are transparent 
and resemble somewhat small fragments of broken glass; later liquefaction 
occurs rapidly. Deep colonies in gelatin roll tubes, or at the bottom of stick 
cultures, are spherical, translucent, and of a pale straw color. 

Upon the surface of nutrient agar it grows rapidly, forming a rather thin, 
translucent, shining, white layer, which covers the entire surface at the end 
of two or three days at a temperature of 20° C. 

Upon potato the growth is rapid and thin, covering the entire surface, 
and is of a pale-yellow color. 

This bacillus grows at a comparatively low temperature, and its vitality 
is not destroyed by exposure for an hour and a half in a freezing mixture at 
15° C. below zero (5° F.). 

Decided growth occurred in a stick culture in gelatin exposed in Balti- 
more during the month of January in an atticroom. During the twenty- 
two days of exposure the highest temperature, taken at 9 a.M. each day, 
was 11° C., and the lowest 2° C. Ata temperature of 16° to 20° C. develop- 
ment in a favorable culture medium is rapid. 

There is no evidence that this bacillus forms spores; cultures are sterilized 
by exposure to a temperature of 60° C. for ten minutes. 

Coagulated blood serum is liquefied by this bacillus. It retains its vitality 
for along time in old cultures, having grown freely when replanted at the 
end of a year from a hermetically sealed tube containing a pure culture in 
blood serum. : ; 

Pathogenesis.—This bacillus is very pathogenic for rabbits when injected 
into the cavity of the abdomen in quantities of one cubic centimetre or more; 
it is less pathogenic for guinea-pigs, and is not pathogenic for white rats 
when injected subcutaneously. Gelatin cultures seem to possess more in- 
tense pathogenic power than bouillon cultures, and cultures from the blood 
of an animal recently dead as the result of an inoculation are more potent 
than those from my original stock which had not been passed through a 
susceptible animal. 


542 PATHOGENIC AEROBIC BACILLI 


The mode of death in rabbits is quite characteristic. A couple of hours 
after receiving in the cavity of the abdomen two or three cubic centimetres. 
of a liquefied gelatin culture the animal becomes quiet and indisposed to eat 
or move about. Soon after it becomes somnolent, the head drooping for- 
ward and after a time resting between the front legs, with the nose on the 
floor of its cage. Itcan be roused from this condition, and raises its head in 
an indifferent and stupid way when pushed or shaken, but soon drops off 
again into a profound sleep. Frequently the animals die in a sitting posi- 
tion, with their nose resting upon the floor of the cage between the front 
legs. I have not seen this lethargic condition produced by inoculations with 
ae oe microédrganism. Convulsions sometimes occur at the moment of 

eath, 

The time of death depends upon the potency of the culture and its quan- 
tity as compared with the size of the animal. From three to four cubie 
centimetres of a liquefied gelatin culture usually kill a rabbit in from three 
to seven hours. : 

The rapidity with which death occurs when a considerable quantity of a 
liquefied gelatin culture is injected into the cavity of the abdomen, and the 
somnolence which precedes death, give rise to the supposition that the lethal 
effect is due to the presence of a toxic chemical substance rather than toa 
multiplication of the bacillus in the body of the animal. And this view is 
supported by the fact that animals frequently recover when the dose admin- 
seipred is comparatively small and especially when it is injected subcuta- 
neously. 

In all cases in which death occurs, even when but a few hours have 
elapsed since the inoculation was made, I have recovered the bacillus in 
cultures made from blood obtained from the heart or the interior of the 
liver, and, as stated, these cultures appear to have a greater virulence than 
those not passed through tke rabbit. 

In sections of the liver and kidney stained with Léffler’s solution of 
methylene blue the bacilli are seen, and are often in rather long-jointed fil- 
aments, 


BACILLUS PYOCYANEUS. 


Synonyms.—Bacillus of green pus ; Microbe du pus bleu; Bacil- 
len des griinblauen Eiters; Bacterium aéruginosum. 
Obtained by Gessard (1882) from pus having a green or blue 
color, and since carefully studied by Gessard, Charrin, and others. 
This bacillus appears to be a widely distributed 
®, (gy saprophyte, which is found occasionally in the 
purulent discharges from open wounds, and some- 
times in perspiration and serous wound secretions 
(Gessard). The writer obtained it, in oneinstance, 
Fie. 153,—Bacillus in cultures from the liver of a yellow-fever cada- 
triers) * 700. ver (Havana, 1888), 

Morphology.—A slender bacillus with rounded 
ends, somewhat thicker than the Bacillus murisepticus and of about 
the same length (Fligge); frequently united in pairs, or chains of four 
to six elements; occasionally grows out into filaments, 

Biological Characters.—An aérobic, liquefying, motile bacil- 
lus. Grows readily in various culture media at the room tempera- 
ture—more rapidly in the incubating oven. Is a facultative anaé- 


ne 


- 


NOT DESCRIBED IN PREVIOUS SECTIONS. 543. 


robic (Frankel). Does not form spores. The thermal death-point, 
as determined by the writer, is 56° C., the time of exposure being ten 
minutes. In gelatin plate cultures colonies are quickly developed, 
which give to the medium a fluorescent green color; at the end of 
two or three days liquefaction commences around each colony, and 
usually the gelatin is completely liquefied by the fifth day. Before 
liquefaction commences the deep colonies, under a low power, appear 
as spherical, granular masses, with a serrated margin, and have a. 
yellowish-green color; the superficial colonies are quite thin and 
finely granular ; at the centre, where they are thickest, they have a. 
greenish color, which fades out towards the periphery. 

In stab cultures in nutrient gelatin development is most abun- 
dant near the surface, and causes at first liquefaction in the form 
of a shallow funnel; later the liquefied gelatin is separated from 
that which is not liquefied by a horizontal plane, and a viscid, yel- 
lowish-white mass, composed of bacilli, accumulates upon this sur- 
face, which gradually has a lower level as liquefaction progresses ;. 
the transparent, liquefied gelatin above is covered with a delicate, 
yellowish-green film, and the entire medium has a fluorescent green 
color. Upon nutrientagar a rather abundant, moist, greenish-white 
layer is developed, and the medium acquires a bright green-color, 
which subsequently changes to olive green. Upon potato a viscid 
or rather dry, yellowish-green or brown layer is formed, and the 
potato beneath and immediately around the growth has a green color: 
when freely exposed to the air or to the vapors of ammonia. In milk 
the casein is first precipitated and then gradually dissolved, while at 
the same time ammonia is developed. The green pigment is formed 
only in the presence of oxygen; it is soluble in chloroform and may 
be obtained from a pure solution in long, blue needles ; acids change 
the blue color to red, and reducing substances to yellow. According 
to Ledderhose, it is an aromatic compound resembling anthracene, 
and isnot toxic. According to Gessard’s latest researches (1890), two. 
different pigments are produced by this bacillus, one of a fluorescent. 
green and the other—pyocyanin—of a blue color. Cultivated in egg 
albumin the fluorescent green pigment, which changes to brown. 
with time, is alone produced. In bouillon and in media containing 
peptone or gelatin both pigments are formed, and the pyocyanin 
may be obtained separately by dissolving it in chloroform. In an 
alkaline solution of peptone (two per cent) to which five per cent of 
glycerin has been added the blue pigment alone is formed. 

Pathogenesis.—The experiments of Ledderhose, Bouchard, and: 
others show that this bacillus is pathogenic for guinea-pigs and rab-. 
bits. Subcutaneous or intraperitoneal injections of recent cultures— 


544 PATHOGENIC AEROBIC BACILLI 


one cubic centimetre or more of a culture in bouillon—usually cause 
the death of the animal in from twelve to thirty-six hours. An ex- 
tensive inflammatory cedema and purulent infiltration of the tissues 
result from subcutaneous inoculations, and a sero-fibrinous or puru- 
lent peritonitis is induced by the introduction of the bacillus into the 
peritoneal cavity. The bacillus is found in the serous or purulent 
fluid in the subcutaneous tissues or abdominal cavity, and also in the 
blood and various organs, from which it can be recovered in pure 
cultures, although not present in great numbers, as is the case in 
the various forms of septiczemia heretofore described. When smaller 
amounts are injected subcutaneously the animal usually recovers 
after the formation of a local abscess, and it is subsequently immune 
when inoculated with doses which would be fatal to an unprotected 
animal. Immunity may also be secured by the injection of a con- 
siderable quantity of a sterilized culture. Bouchard has also pro- 
duced immunity in rabbits by injecting into them the filtered urine 
of other rabbits which had been inoculated with a virulent culture of 
the bacillus. It has been shown by Bouchard, and by Charrin and 
Guignard, that in rabbits which have been inoculated with a culture 
of the anthrax bacillus a fatal result may be prevented by soon after 
inoculating the same animals with a pure culture of the Bacillus 
pyocyaneus. The experiments of Woodhead and Wood indicate that 
the antidotal effect is due to chemical products of the growth of the 
bacillus, and not to an antagonism of the living bacterial cells. They 
were able to obtain similar results by the injection of sterilized cul- 
tures of Bacillus pyocyaneus, made soon after infection with the 
anthrax bacillus. 

Schimmelbusch (1894) reports that in researches made by Mih- 
sam this bacillus was found in the axilla, the anal region, or the in- 
guinal fold in fifty per cent of the healthy individuals examined. 
Its presence in wounds greatly delays the process of repair and may 
give rise to a general depression of the vital powers from the ab- 
sorption of its toxic products. Schimmelbush states that a physician 
injected 0.5 cubic centimetre of sterilized (by heat) culture into his 
forearm. That as a result of this injection, after a few hours he had 
a slight chill, followed by fever, which at the end of twelve hours 
reached 38.8° ; an erysipelatous-like swelling of the forearm oc- 
curred, and the glands in the axilla were swollen and painful. Re- 
covery occurred without the formation of an abscess. Buchner has 
related a similar case. 

Krannhals (1894) refers to seven cases in which a general pyocy- 
aneus infection in man was found, and adds an eighth from his own 
experience. In this the Bacillus pyocyaneus was obtained, post mor- 


NOT DESCRIBED IN PREVIOUS SECTIONS. 545 


tem, from green pus in the pleural cavity, from serum in the peri- 
cardial sac, and from the spleen, in pure culture. 

Martha, Gruber, Maggiora, Gradenigo, Kossel, and Rohrer have 
reported cases in which the Bacillus pyocyaneus has been obtained in 
pure cultures from pus obtained from the tympanic cavity in middle- 
ear disease. Kossel (1894) relates several cases in his own experience 
which led him to the conclusion that, in children, the Bacillus pyocy- 
aneus, through general blood infection or indirectly through the 
absorption of its toxic products, may be the cause of death. 

The following varieties of this bacillus have been described by 
bacteriologists: 


BACILLUS PYOCYANEUS  (P. Ernst). 


Found in pus from bandages colored green. 

Morphology.—Slender bacilli from 2 to 4ulong—occeasionally 5 to 6 u— 
and from 0.5 to 0.75broad ; sometimes united in pairs, or chains of three 
elements. 

Biological Characters.—An aérobic, liquefying, actively motile, chro- 
mogenic bacillus. Produces a yellowish-green pigment; when old cul- 
tures are shaken up with chloroform and this is allowed to stand, three 
layers are formed—an upper, clouded, dirty-yellow layer ; below this is a 
milky, pale-green layer ; and at the bottom a transparent, azure-blue layer. 
Spore formation has not been demonstrated. Grows in the usual culture 
media at the room temperature—more rapidly at 35°C. Upon gelatin plates 
colonies are formed resembling those of the well-known Bacillus pyocyaneus, 
but liquefaction is more rapid. In gelatin stick cultures funnel-shaped 
liquefaction occurs at the upper part of the line of puncture by the third 
day, and progresses more rapidly than is the case with Bacillus pyocyaneus 
under,.the same circumstances ; on the fifth day a bluish-green color is de- 
veloped; by the twelfth day liquefaction has obliterated the entire line of 
growth and extends to the margins of the tube; the liquefied gelatin for a 
depth of about one centimetre hasa dark emerald-green color, and a film 
consisting of bacilli isseen upon the surface. Upon the surface of agar a 
flat, greenish-white, dry layer is formed along the line of inoculation, and 
the agar around, at the end of a week, acquires a bluish-green color. Upon 
potato, at the end of three days, an abundant dry layer of a fawn-brown 
color has developed ; this is surrounded by a pale-green coloration of the 
potato, and at points where the surface is fissured, an intense dark-green 
color is developed; the growth on potato has a more or less wrinkled appear- 
ance ; when one of the fawn-colored colonies 1s touched with the platinum 
needle, the point touched, at the end of two to five minutes, acquires an in- 
tense dark leaf-green color, which reaches its maximum intensity in about 
ten minutes, and has faded out again at the end of half an hour. Ernst con- 
siders this ‘‘chameleon phenomenon” the most characteristic distinction 
between the bacillus under consideration and Bacillus pyocyaneus. In milk 
a green color is developed at the surface, the casein is precipitated and sub- 
sequently peptonized. 


Bacillus pyocyaneus pericarditidis. Found by H. C. Ernst in 
fluid obtained by tapping the pericardial sac of a man aged forty- 
seven years. Fluid was drawn from the pericardial sac on four dif- 
ferent occasions. The man subsequently “eloped.” Ernst gives the 
following description of this bacillus: 

35 


546 PATHOGENIC AEROBIC BACILLI 


ORIGIN.—Pericardial fluid, containing also bacilli of tuberculosis. —- 

FoRM AND ARRANGEMENT,—Small straight bacilli, with rounded ends, 
three or four times as long as broad, and on most media slightly larger than 
the Bacillus pyocyaneus of Gessard, occurring within the cells in the origi- 
nal fluid, aa sometimes showing two or three end to end, but never observed 
in long chains. 

Moriniry.—Actively motile in hanging-drop culture. No cilia or flagel- 
la have been demonstrated. 

GrowTH—Gelatin: Plates.—Colonies appear at the end of thirty-six to 
forty-eight hours as fine white points in the interior, and upon the surface of 
the medium; edges are sharply defined ; soon there appears a circular zone 
of liquefaction, finally passing through the stratum of the medium with 
the colony at the bottom. Under a low power the centre of the colony may 
be of a brownish color. On the second day a greenish tinge may be seen 
about the individual colonies on the surface which spreads through the 
entire medium. The plates may always be distinguished from those of the 
Bacillus pyocyaneus of Gessard by the bluish-green when contrasted with 
the yellowish-green color of this latter. 

Gelatin: Needle Cultures.—At the end of twenty-four hours a small, 
saucer-shaped depression of liquefaction at upper end of needle track, which 
gradually spreads and deepens until the liquefaction extends straight across 
the tube, and about half-way down the needle track. <A bluish-green fluores- 
cence appears about the liquefied portion at the very upper part of the gela- 
tin, later changing into a yellowish green. The colony is deposited as a. 
yellowish, heavy sediment at the bottom of the liquefied portion, the upper 
part of which is clear. A small, whitish growth occurs along the remainder 
of the needle track. Old cultures, in which a certain amount of evapora- 
tion has occurred, assume a very dark greenish-black color. 

Agar-agar.—Along the needle track appears a flat, dry colony of a dirty 
grayish-white color spreading out upon each side of the needle track and 
growing at first upon the surface of the water of condensation, later depos- 
iting a white sediment at the bottom. From the first there may be detected, 
by reflected light, a metallic lustre on the surface of the colony in places, 
which metallic sheen later spreads over the whole colony and furnishes a 
marked differentiating point. In addition to this, within twenty-four to 
forty-eight hours at 37° C., there appears a green fluorescence throughout 
the whole of the medium, which increases slowly to a marked bluish-green 
color, and never assumes the nut-brown of the Bacillus pyocyaneus of 
Gessard upon the same medium. The colony is not especially viscid. 

Potato.—There appears a reddish-brown colony along the needle track, 
elevated and moist, confined to the line of the needle. It presents no change 
of color upon touching with the needle, but certain specimens (as do some of 
the Bacillus pyocyaneus) develop later a heavy green color extending over 
the whole surface of the potato, which later changes almost to black. 

Bouillon.—Twenty-four hours at 37° C. gives a growth, especially on the 
surface, which is a wrinkled scum; no cloudiness of the bouillon, and avery 
faint greenish fluorescence one centimetre below the surface. At this time 
it differs from the Bacillus pyocyaneus of Gessard, in that the latter shows 
cloudiness of the medium all through. Later the same cloudiness appears in 
bouillon cultures of this new bacillus, together with a whitish sediment de- 
posited at the bottom of the tube, and then the cultures are indistinguishable 
ieee each other. The same changes, but slower, occur at room tempera- 

re. 

Peptone.—One, 3.5, and six-per-cent solution. Twenty-four hours at 37° 
C. gives a faint bluish tinge at upper edge of medium with very faint cloudi- 
ness; later (in one or two weeks) there forms a marked scum upon the sur- 
face that is difficult to break up by shaking, and the whole medium assumes 
a grass-green color of more or less intensity, and not seen on other similar 
bacilli. The shape and size of the organism, under the microscope, differ 


NOT DESCRIBED IN PREVIOUS SECTIONS, 547 


very markedly in this medium from any other bacilli examined. The same 
changes are to be seen at room temperature, but more slowly. 

Egg-Albumin: Plain.—Twenty-four hours at 37° C., ‘yellowish-white, 
very profuse growth all along the needle track ; yellowish-green spreading 
out from it almost to sides of tube, and in the condensation water as well. 
The growth has no especial distinguishing characteristics. Irregular lique- 
faction occurs, but the growth at no time differsin any marked way from 
other varieties of the Bacillus pyocyaneus. 

_ Blood Serum.—Twenty-four hours at 37° C. shows flat, moist colony 
with bluish-green fluorescence in its neighborhood. Liquefaction begins 
early and goes on slowly until complete in from one to two weeks, with an 
increasing intensity of color which becomes markedly blue, and eventually 
almost black. 

Milk.—Behaves as do the other bacteria. 

BEHAVIOR TO TEMPERATURE.—Grows at 15°-25° C. slowly; much more 
freely at 35°-38° C., when it produces the color more quickly. 

RaPIDITY OF GROWTH.—Moderate. 

SPORE-PRODUCTION.—Not observed. 

NEED oF AIR.—Does not grow undermica. Facultatively anaérobic, but 
does not produce color except with free access of oxygen. 

Gas-PRODUCTION.—Produces faint foul odor. 

BEHAVIOR TO GELATIN.—Liquefies gelatin slowly. 

CoLor-PRropuction.—Produces a bluish-green color which in old cul- 
tures changes almost to a black. Upon the addition of acids (both vegetable 
and mineral) to cultures the color changes to red, and upon the addition of 
alkalies abright grass-green appears. This reaction is best seen in bouillon 
and gelatin cultures, but occurs in other media as well, notably blood-serum. 

BEHAVIOR To ANILINE DyEs.—Stains easily and well with any of the 
aniline dyes usually employed, and by Gram’s method. 

Microscopic APPEARANCE IN DIFFERENT Mep1a.—Under the micro- 
scope, its general appearance on various media is of a rod larger than the 
Bacillus pyocyaneus. In peptone cultures this difference is verv marked. 
In this case, the Bacillus pyocyaneus tested appeared as very short, oval, 
bacilli, almost like micrococci, while the new bacillus showed as a long, 
fine rod, from four to six times as long as broad—length about one-half the 
diameter of a red-blood corpuscle—and arranged sometimes two or three 
end to end. These same cultures transferred to gelatin became indistin- 
guishable from each other in size. 

PaTHOGENESIS.—Injections of small quantities (0.5 centimetre) of a bouil- 
lon culture twenty-four hours old into the abdominal cavity of rabbits and 
guinea-pigs, killed fifty per cent in from twenty-four to thirty-six hours. 
Autopsy showed general congestion of abdominal viscera, slight effusion into. 
the peritoneal cavity, and cover-glass preparations and cultures showed the 
bacilli in the effusion in the abdominal cavity, as well as in the blood from. 
the heart and various organs. 


BACILLUS OF FIOCCA. 


Found by Fiocca in the saliva of cats and dogs. 

Closely resembles the influenza bacillus of Pfeiffer and of Canon, 

Morphology.—Resembles the bacillus of rabbit septicaemia, but is only 
half as large—from 0.2 to 0.33 # in breadth. The length is but little greater 
than the breadth. Usually seen in pairs, closely resembling diplococci. 
When cultivated on potato it appears to be a micrococcus, but in the blood 
of infected animals and in bouillon cultures it is seen to be a short bacillus. 

Stains with difficulty with the usual aniline colors, but is readily stained 
by Ehrlich’s method or with Ziehl’s solution. 

Biological Characters.—An aérobic and facultative anaérobic, non- 
liquefying, non-motile bacillus. Spore formation not observed. Grows best 


548 PATHOGENIC AEROBIC BACILLI 


at 37° C. and does not develop at temperatures below 15° C. _ In agar plates, 
at 37° C., small, punctiform colonies are developed at the end of twenty-four 
hours; these do not increase in size later; under the microscope the deep 
colonies are seen to be spherical, granular, and dark yellow in color; the 
superficial colonies are more or less round, with irregular outlines, trans- 
arent, slightly granular, and often have a shining nucleus at the centre. 
on gelatin plates the colonies have a similar appearance, but are not vis- 
ible in less than four or five days. In streak cultures upon the surface of 
agar small, punctiform colonies are seen along the track of the needle at the 
end of twenty-four hours, resembling fine dewdrops; the following day 
these colonies are a little larger and less transparent; they remain distinct, 
especially along the margins of the line of growth. Upon potato a very 
thin, transparent layer is developed, which does not change the appearance 
of the surface of the potato, but slightly increases its resistance to the plati- 
num needle. In bouillon small flocculi, suspended in the clear liquid, are 
developed within twenty-four hours; these subsequently sink to the bottom. 

Milk is not coagulated by this bacillus, and no gas is produced in media 
containing sugar. 

Pathogenesis.—Pathogenic for rabbits, guinea-pigs, young rats, and mice, 
in which animals it produces general infection, and death—in rabbits—at 
the ee of twenty-four hours. The bacillus is found in the blood in great 
numbers. 


PROTEUS VULGARIS. 


Obtained by Hauser (1885) from putrefying animal substances, 
and since shown to be one of the most common and widely distrib- 
uted putrefactive bacteria. This and the other species of Proteus 


Fia. 154.—Proteus vulgaris; ‘‘ swarming islands” from a gelatin culture. x 285. (Hauser.) 


described by the same bacteriologist (Proteus mirabilis, Proteus Zen- 
keri) have no doubt frequently been encountered by previous observ- 
ers, and areamong the species formerly included under the name 
“* Bacterium termo,” which was applied to any minute motile bacilli 
found in putrefying infusions. 

Morphology.—Bacilli with rounded ends, about 0.6 4 broad, and 


NOT DESCRIBED IN PREVIOUS SECTIONS. 549 


varying greatly in length, being sometimes short oval, and at others 
from 1.25 to 3.75 jv in length ; also grow out into flexible filaments, 
which may be more or less wavy or spiral in form. The short rods 
are commonly seen in pairs ; they have terminal flagella ; involution 
forms are frequently seen, the most common being spherical bodies 
about 1.6 in diameter. In old cultures in bouillon, or in cultures 
made in meat infusion in the incubating oven, the short oval forms 
greatly predominate, but in recent cultures in nutrient gelatin fila- 
ments of considerable length are encountered in association with 
shorter rods. 

Stains readily with fuchsin or gentian violet—not so well with 
the brown aniline colors ; does not stain by Gram’s method (Cheyne). 

Biological Characters.—An aérobic and facultative anaérobic, 
liquefying, motile bacillus. Grows rapidly in. the usual culture 
media at the room temperature. 

The growth upon gelatin plates (five per cent of gelatin) at the 
room temperature is very characteristic ; at the end of six or eight 
hours small depressions in the gelatin are observed, which contain 
liquefied gelatin and grayish-white masses of bacilli. Under alow 
power these depressions are seen to be surrounded by a marginal 
zone consisting of two or three layers, outside of which is a zone of a 
single layer, from which amceba-like processes extend upon the sur- 
face of the gelatin. These processes are constantly undergoing 
changes in their form and position, and may become separated from 
the mother colony, or remain temporarily attached to it by a narrow 
thread consisting of bacilli; after a time the entire surface of the 
gelatin is covered with wandering, amceba-like colonies; these 
rapidly cause liquefaction, which by the end of twenty-four to forty- 
eight hours has reached a depth of one millimetre or more over the 
entire surface. The deep colonies also are surrounded by processes 
projecting into the gelatin, which may be observed to suddenly ad- 
vance and again to be retracted towards the central zodgloea-like 
mass. Liquefaction around the colony rapidly progresses, and 
actively motile rods and spiral filaments may be seen about the peri- 
phery of this liquefied gelatin, while about it is a radiating crown of 
irregular processes, some of which may be screw-like or corkscrew- 
formed. In ten-per-cent gelatin the migration of surface colonies, 
above described, is not observed. In gelatin stab culturcs liquefac- 
tion occurs along the entire line of puncture, and soon the contents 
of the tube are completely liquefied ; near the surface of the liquefied 
gelatin the growing bacilli form a grayish-white cloudiness, and at 
the bottom of the tube an abundant flocculent deposit is formed. 
Upon the surface of nutrient agar a rapidly extending, moist, thin, 
grayish-white layer is formed. Upon potato this bacillus produces a 


550 PATHOGENIC AEROBIC BACILLI 


dirty-white, moist layer. The cultures in media containing albumin 
or gelatin have a putrefactive odor and acquire a strongly alkaline 
reaction. A temperature of 20° to 24° C. is most favorable for the 
growth of this bacillus. It isa facultative anaérobie and grows in 
an atmosphere of hydrogen or of carbon dioxide, although not so 
rapidly as in the presence of oxygen. The movements are often ex- 
tremely active and difficult to follow under the microscope ; again 
they may be quite deliberate, or the bacilli may remain motionless 
for a time and again dart off in active motion. The long terminal 
flagella may sometimes be discerned by means of a good objective 
and careful manipulation of the light. 

Pathogenesis.—Pathogenic for rabbits and for guinea-pigs when 
injected into the circulation, into the cavity of the abdomen, or sub- 
cutaneously in considerable quantity. Cultures in nutrient gelatin 
are said by Cheyne to be more pathogenic (toxic) than those in bouil- 
lon. When injected into the muscles of rabbits a much smaller 
dose produces a fatal result than when injected subcutaneously. 
In Cheyne’s experiments, made in London (1886), one-tenth cubic 
centimetre of a liquefied gelatin culture, injected into the dorsal 
muscles, was invariably fatal in from twenty-four to thirty-six hours; 
a dose of one-twentieth cubic centimetre, injected in the same way, 
usually caused death; while one-fortieth cubic centimetre gave rise to 
an extensive local abscess, and the animals died at the end of six or 
eight weeks. Dosesof less than one-five-hundredth cubic centimetre 
produced no effect. Cheyne estimates that one cubic centimetre ofa 
culture in nutrient gelatin contains 4,500,000,000 bacilli, and, conse- 
quently, that a smaller number than 9,000,000 produced no effect when 
injected into the muscular tissue of rabbits. Injections into the sub- 
cutaneous connective tissues of a dose twice as large as that which in- 
variably proved fatal when injected into the muscles usually caused 
an extensive abscess, but did not kill the animal; and, after re- 
covery from the effects of such an injection, the rabbit was found to 
be immune against a similar dose injected into the muscles. Foa 
and Bonome have succeeded in producing immunity against the 
etrects of virulent cultures of this bacillus by inoculating rabbits with 
filtered cultures, and also by injecting beneath the skin of these ani- 
mals a solution of neurin, which they believe to be the principal 
toxic product present in the cultures. 

Proteus Vulgaris in Cholera Infantum.—The extended re- 
searches of Booker have led him to the conclusion that this bacillus 
plays an important part in the production of the morbid symptoms 
which characterize cholera infantum. Proteus vulgaris was found 
in the alvine discharges in a considerable proportion of the cases ex- 
amined, but was not found in the feeces of healthy infants. ‘‘ The 


NOT DESCRIBED IN PREVIOUS SECTIONS. 551 


prominent symptoms in the cases of cholera infantum in which the 
proteus bacteria were found were drowsiness, stupor, emaciation 
and great reduction in flesh, more or less collapse, frequent vomiting 
and purging, with watery and generally offensive stools.” 

The researches of Krogius, Schnitzler, Schmidt and Aschoff, and 
others, show that in cases of cystitis and of pyelonephritis this bacil- 
lus is frequently found in pure cultures, or associated with other bac- 
teria. The authors last named state that in sixty cases of cystitis 
reported by various authors the colon bacillus was found in pure cul- 
tures, and in thirteen cases the proteus of Hauser. Next to Bacillus 
coli communis Proteus vulgaris appears to be the microérganism 
most frequently concerned in the etiology of pyelonephritis. 

Levy (1895) isolated from sour yeast a bacillus, which he identified 
as “Proteus Hauseri,” and made numerous experiments on dogs to 
test its pathogenic power. From five to ten cubic centimetres of a 
liquefied gelatin culture injected into the circulation, through a vein, 
caused the typical symptoms of “sepsin poisoning,” as formerly de- 
scribed by Bergmann and Schmeideberg (1868). In two dogs which 
died at the end of forty-eight hours the intestinal tract was found in 
a condition of intense hemorrhagic infiltration. The spleen and 
glands of the mesentery were much enlarged. But a bacteriological 
examination gave an entirely negative result, showing that death 
resulted from toxeemia and not from septicemia. Further experi- 
ments showed that the dried precipitate obtained from liquefied gela- 
tin cultures, by the addition of alcohol, had the same pathogenic 
action on dogs, rabbits, and mice as cultures containing the living 
-bacilli. That a similar pathogenic effect is produced in man by the 
products of growth of this bacillus was shown by the following facts: 
While conducting his experiments Levy had an opportunity to make 
a bacteriological examination in the case of a man who died after a 
brief attack of cholera morbus. From the vomited material and the 
stools he obtained a pure culture of proteus; but the blood, collected 
at the autopsy, was sterile. In the mean time seventeen other per- 
sons who had eaten at the same restaurant were taken sick in the same 
way. Upon an examination at the restaurant it was found that the 
bottom of the ice chest in which the proprietor kept his meats was 
covered with a slimy, brown layer, which gave off a disagreeable 
odor. Cultures from this gave the proteus as the principal micro- 
organism present. Levy concludes from his own investigations and 
those of other bacteriologists that in so-called “flesh-poisoning” bac- 
teria of this group are chiefly at fault, and that the pathogenic effects 
are due to toxic products evolved during their development. 


552 PATHOGENIC AEROBIC BACILLI 


PROTEUS OF KARLINSKI. 


_. Synonym.—Bacillus murisepticus pleomorphus (Karlinski). Probably 
identical with Proteus vulgaris of Hauser. 

Obtained by Karlinski (1889) from a fibro-purulent uterine discharge, and 
from abscesses in the uterus and its appendages in a puerperal woman. 

Morphology.—Resembles Proteus vulgaris of Hauser in its morphology, 
and presents various forms under ditferent circumstances relating to the 
culture medium, the temperature, age of culture, etc.—sometimes as spheri- 
cal or short oval cells, at others as longer or shorter-rods or spiral filaments; 
usually as bacilli with round ends two anda half times as long as thick, 
often united in pairs. 

Stains with the usual aniline colors, but not by Gram’s method. 

Biological Characters.—An aérobic and facultative anaérobic, liquefy- 
ing, motile bacillus. Spore formation not observed. Grows rapidly in the 
usual culture media at the room temperature. In gelatin plate cultures, at 
the end of ten hours, small colonies are developed which have well-defined 
outlines, are oval or whetstone-shaped, of a light-brown color by transmitted 
light and white by reflected light, with a somewhat darker margin and a 
smooth surface, sometimes marked by shallow clefts; at the end of twenty 
hours the colonies commence to have irregular margins, and the surface of 
the gelatin above them is marked by concentric rings. At the end of thirty 
hours the colonies have formed a bulb-shaped liquefaction of the gelatin, 
and delicate, ray-like offshoots are seen around the margin. At the end of 
two days the built ous cavities are about one and a half millimetres in diameter 
and contain a cloudy, grayish-white liquid; they are surrounded by a moist- 
looking, gray, irregular marginal zone. In gelatin stab cultures, at the end 
of twenty-four hours, a funnel-shaped.liquefaction of the gelatin occurs near 
the surface, and a grayish-white, cloudy mass is developed along the line of 
puncture; at the end of forty-eight hours a sac-like pouch of liquefied gela- 
tin has formed, and in the course of four or five days the gelatin is entirely 
liquefied. Upon agar plates the colonies are at first oval in form and white 
by reflected light, or pale brown by transmitted light ; at the end of thirty 
hours the surface becomes wrinkled or folded and is surrounded by radiat- 
ing, delicately twisted offshoots. Upon the surface of agar a white layer 
is developed. Upon potato a whitish-gray, soft, homogeneous layer, which 
after standing along time hasa darker color. Upon blood serum a thin, 
grayish-white layer is formed and the serum is rapidly liquefied. Gelatin 
cultures acquire a strongly alkaline reaction and give off a disagreeable 
odor resembling that of butyric acid. 

Pathogenesis.—White mice inoculated at the root of the tail die in from 
twenty-two to twenty-four hours ; the spleen is greatly enlarged; the bacilli 
are found in blood from the various organs—less numerous in blood from 
the heart. Field mice and house mice are less susceptible. Subcutaneous 
injections in rabbits may give rise to local inflammation and also to general 
infection. In white rats and guinea-pigs a local abscess may result from a 
subcutaneous inoculation, 


PROTEUS MIRABILIS. 


Obtained by Hauser (1885) from putrefying animal substances. 

Morphology.—Bacilli resembling very closely the preceding species (Pro- 
teus vulgaris), but presenting more numerous involution forms, which may 
be spherical, pear-shaped, or spermatozoa-like, etc. The bacilli are about 
0.6 « in diameter and vary greatly in length, being sometimes nearly spheri- 
cal, or forming rods of 2 to 8.75 w in length, or long filaments. 

Biological Characters.—An aérobic and facultative anaérobic, liquefy- 
ing, motile bacillus. Spore formation has not been observed. Grows in the 
usual culture media at the room temperature. Does not liquefy gelatin as 


NOT DESCRIBED IN PREVIOUS SECTIONS. 553 


rapidly as Proteus vulgaris. Upon gelatin plates, at the end of twelve 

ours, superficial colonies of two to three millimetres in diameter are formed; 
under a low power these appear finely granular and brownish in color, and 
have an irregular outline; outgrowths from the margin extend in various 
directions and form new colonies, which may be attached for a time by a 
long and slender thread consisting of bacilli. The movement of these new 
colonies is not as pronounced as in the case of the preceding species, and 


Fie. 155.—‘‘ Swarming islands *’ of Proteus mirabilis, from a gelatinculture. x 285. (Hauser.) 


they are characterized by the presence of numerous distorted bacilli—invo- 
lution forms. The deep colonies form spiral zodglcea masses. 

In gelatin stab cultures the whole surface is first covered with threads 
and islands of bacilli, which after atime form an anastomosing network, and 
finally a confluent layer which at the end of forty-eight hours is rather thick, 


Fia. 156.—Spiral zodglosa from a culture of Proteus mirabilis. x 95. (Hauser.) 


with a moist, shining surface and grayish color, and appears to be perforated 
with numerous small, sieve-like openings. These thinner and transparent. 
places disappear after a time, and at the end of two or three days liquefac- 
tion of the gelatin commences; complete liquefaction does not occur until 
the fifth or sixth day, or even later. Along the lineof puncture finely gran- 
ular colonies are first formed, from which long threads are given off, which 
form after a short time a tolerably broad zone of threads and spiral zodgloea 


masses. 


554 PATHOGENIC AEROBIC BACILLI 


Pathogenesis.—In Hauser’s experiments filtered cultures (two to six cubic 
centimetres), injected into the circulation or into the cavity of the abdomen 
in rabbits, caused fatal toxeemia. 


PROTEUS ZENKERI. 


Obtained by Hauser (1885) from putrefying animal substances. 

Morphology.—Bacilli which vary greatly in length—average about 1.65 », 
and about 0.4 u broad. 

Biological Characters.—An aérobie and facultative anaérobic, non- 
liquefying, motile bacillus. Spore formation not observed. Grows in the 
usual culture media at the room temperature. Upon the surface of nutrient 
gelatin a laminated mass forms about the point of puncture, from the peri- 
phery of which offshoots are given off, at the extremities of which colonies 
are formed, as in the case of Proteus mirabilis. Gradually a rather thick, 
grayish-white, opaque layer is formed, which covers the entire surface of the 
gelatin and is easily detached from it. This species is distinguished from 
the two preceding by the fact that it does not liquefy gelatin or blood serum 
eee does not give off a decided putrefactive odor when cultivated in these 
media. 

Pathogenesis.—Considerable quantities injected into small animals give 
rise to local abscesses and to symptoms of toxemia. 


PROTEUS SEPTICUS. 


Obtained by Babes (1889) from the mucous membrane of the intestine and 
‘the various organs of a boy who died of septiczemia. 

Morphology.—Bacilli about 0 4 4 broad and varying greatly in length; 
‘slightly curved rods or flexible filaments, often associated in loose chains. 

Stains by the usual aniline colors and by Gram’s method. 

Biological Characters.—An aérobic, liquefying, motile bacillus. Spore 
formation not observed. Grows in the usual culture media at the room 
‘temperature. In gelatin plates centres of liquefaction are quickly formed 
and rapidly extend. The spherical, liquefied places have at first a wavy or 
-dentate outline, and are surrounded by a branching, transparent, granular 
margin which rapidly extends in advance of the liquefaction. In stab cul- 
tures in nutrient gelatin liquefaction of the entire contents of the tube may 
take place within twenty-four hours, or a broad, liquefied sac is formed 
along the line of puncture. Gelatin cultures give off a very disagreeable 
odor. Upon the surface of nutrient agar, at 37° C., a peculiar, thick net- 
work extends over the surface in the course of a few hours. Upon potato an 
elevated, brownish-white, shining layer is formed. Blood serum is lique- 
fied by this bacillus. 

Pathogenesis.—Pathogenic for mice, less so for rabbits. In mice death 
-occurs in from one to three days after the subcutaneous injection of a small 
ee of a pure culture ; the bacilli are present in the blood in small’ 
numbers. 


PROTEUS LETHALIS. 


Synonym.—Proteus bei Lungengangran des Menschen (Babes). 

Obtained by Babes (1889) from the spleen and gangrenous portions of the’ 
lung of aman who died of septicaemia. 

Morphology.—Short rods with round ends, from 0.8 to 1.5 u thick ; often’ 
‘swollen in the middle, like a lemon ora flask ; forms short, flexible filaments 
which also present similar swellings. 

Stains with the usual aniline colors and also by Gram’s method. 

Biological Characters.—An aérobie’ and facultative anaérobic, non- 
liquefying, motile bacillus. Net observed to form spores. Grows in the 
usual culture media at the room temperature. In gelatin plates forms hemi- 
spherical, elevated, whitish, translucent colonies, which later send out 


NOT DESCRIBED IN PREVIOUS SECTIONS. 555 


coarse branches which ramify over the surface of the gelatin. A similar 
growth is observed upon the surface of gelatin stab cultures, and an abun- 
dant development takes place along the line of puncture. Upon nutrient 
agar a thick, opaque, slightly yellowish layer is formed. Upon potato a 
moist, shining, brownish layer is developed, and the potato acquires a 
brownish color. Upon blood serum the growth is less abundant than on 
agar; the blood serum is not liquefied. This bacillus grows rapidly at the 
room temperature; it is destroyed by a temperature of 80° C., and presum- 
ably does not form spores. 

Pathogenesis.—Recent cultures are very pathogenic for mice and for 
rabbits, less so for guinea-pigs. The subcutaneous injection of a small 
quantity of a pure culture kills susceptible animals in two or three days. 
More or less cedema is found at the point of inoculation. Injections into the 
rectum of rabbits gave rise to hemorrhagic enteritis, peritonitis, and death 
at the end of four days. 


BACILLUS A OF BOOKER. 


Obtained by Booker (1889) from the alvine discharges of children suffer- 
ing from cholera infantum. 

Morphology.—Bacilli with round ends, varying greatly in length, usually 
three to four 4 long and 0.7 u broad (in recent agarcultures). In older cul- 
tures the bacilli are shorter and smaller. 

Biological Characters.—An aérobic and facultative anaérobic, lique- 
JSying, motile bacillus. Grows at the room temperature in the usual culture 
media. In gelatin plates colonies are visible at the end of twenty-four 
hours; under the microscope these are nearly colorless, and liquefaction 
soon occurs around them. a gelatin stab cultures complete liquefaction 
occurs in three or four days. Upon agar a colorless layer covering the entire 
surface is developed in three or four days, and an abundant development 
occurs along the line of puncture. Agar colonies have a bluish look, and 
are surrounded by an indistinct halo which shades off gradually into the 
surrounding agar ; under a Jow power the colonies are light-brown and the 
borders indistinct; the surface has a delicate, wavy appearance. Upon po- 
tato the growth is luxuriant and of a dirty-brown color. Blood serum is 
liquefied by this bacillus. 2 : : 

Milk is coagulated into a gelatinous mass having an alkaline reaction; 
later the coagulum is dissolved. ee Ee 

Pathogenesis.—Mice and guinea-pigs fed with cultures in milk die in from 
one to eight days. 


BACILLUS ENDOCARDITIDIS GRISEUS. 


Obtained by Weichselbaum (1888) from the affected valves in a case of 
endocarditis recurrens ulcerosa. : 

Morphology.—Short rods with rounded or somewhat pointed ends, about 
two to three times as long as broad—of about the same dimensions as the 
bacillus of typhoid fever. 

Stains with the usual aniline colors and also by Gram’s method; the 
longer rods from old cultures are irregularly stained. _ : 

Biological Characters.—An aérobic, non-liquefying, motile bacillus. 
Refractive bodies may be seen in some of the rods, which resemble spores and 
are stained by the method of Ernst, but they do not show the resistance of 
known spores to physical and chemical agents. Grows well in the usual 
culture media at the room temperature. Upon gelatin plates colonies are 
formed which resemble those of Friedlinder’s bacillus, but which gradually 
acquire a gray or grayish-white color. The prominent, convex, superficial 
colonies under a low power are finely granular and grayish-brown in color; 
the deep colonies are yellowish-brown in color, have slightly notched_mar- 
gins, and the surface is covered with minute projections. In stab cultures 


556 PATHOGENIC AEROBIC BACILLI. 


a rather thin, circular layer forms about the point of puncture; this has the 
appearance of stearin; later it becomes grayish-white and the margins are 
marked by radiating lines. Upon the surface of nutrient agar a similar 

rowth occurs which has a pale-brown or reddish-gray color. Upon potato 
in the incubating oven an abundant development occurs, forming a dry- 
looking layer of a grayish-brown color and having irregularly notched mar- 
gins. Upon blood serum an abundant, grayish-white growth of cream-like 
consistence forms along the impfstrich; later this has a reddish gray color. 
This bacillus grows to the bottom of the line of puncture in stick cultures, 
and is no doubt a facultative anaérobic. 

Pathogenesis.—Pathogenic for white mice and for guinea-pigs. 


BACILLUS ENDOCARDITIDIS CAPSULATUS. 


Obtained by Weichselbaum (1888) from thrombi and embolic infarctions 
in the spleen and kidneys of a man who died from endocarditis with forma- 
tion of thrombi. 

Morphology.—Resembles Friedlainder’s bacillus, and is frequently sur- 
rounded by a capsule, which may be stained; also forms long, curved fila- 
ments, in the protoplasm of which vacuoles may be observed in stained pre- 
parations. 

Stains with the usual aniline colors, but not by Gram’s method; by 
staining with fuchsin and carefully decolorizing with diluted alcohol the 
presence of a capsule may be demonstrated. 

Biological Characters.— An aérobic, non-liquefying bacillus. Grows in 
the usual culture media at the room temperature. 

In gelatin stab cultures development occurs along the line of puncture, 
and on the surface asa rather thin, white, dry layer which resembles stearin. 
In agar plates the superficial colonies are thin, about two millimetres in 
diameter and gray in color; under a low power the margins are trans- 
parent and colorless, and the centre resembles the deep colonies; these are 
very small and grayish-white in color ; under a low power the surface is 
seen to be covered with tooth-like, projecting masses, the margin is dentate 
and has a pale-yellow color, while the centre is yellowish-brown. 

_ Pathogenes?s.—Rabbits are killed by the injection of a considerable quan- 
tity of a pure culture into the cavity of the abdomen or subcutaneously. 


BACILLUS ALVEI. 


Synonym.—Bacillus of foul brood (of bees). 

_ Obtained by Cheshire and Cheyne (1885) from the larvae in hives infected 
with ‘‘ foul brood.” The larve in the interior of cells in the comb die and 
become almost fluid as a result of parasitic invasion by this bacillus. 

Morphology.—Bacilli with rounded ends, from 2.5 to 5 4 in length (aver- 
age about 3.6 ~) and 0.8 u in diameter. Grow out into filaments and form 
large oval spores which have a greater diameter than the rods in which they 
are developed—1.07 u. 

es readily with the aniline colors usually employed, also by Gram’s 
method. 

_ Biological Characters.—An aérobic and facultative anaérobic, liquefy- 
ing, motile bacillus. Forms endogenous spores. Grows readily in the usual 
culture media at the room temperature. 

In gelatin plates small, round or oval colonies are formed, which later 
become pear-shaped; a branching outgrowth occurs about the margins of the 
colonies, and especially from the small end of the pear-shaped mass. In 
streak cultures upon the surface of gelatin growth occurs first along the impf- 
strich, and from this an outgrowth occurs consisting of bacilli in a single 
row or in several parallel rows, and forming irregular or circular figures, 


NOT DESCRIBED IN PREVIOUS SECTIONS. 557 


from which other similar outgrowths occur ; the branching outgrowths may 
anastomose. The gelatin is liquefied in the vicinity of these lines of growth, 
forming a network of channels. A similar growth is seen upon the surface 
of gelatin stab cultures, and along the line of puncture white, irregular 
masses are formed, from which rather coarse branches are given off which 
often have a club-shaped extremity. In older cultures the finer branches 
disappear, so that the secondary centres of growth are disconnected from the 
original colonies; complete liquefaction of the gelatin occurs in about two 
weeks; the liquefied gelatin has a yellowish color and peculiar odor. Upon 
the surface of nutrient agar, at 37° C., a white layer isformed. Upon potato 
the development is slow and results in the formation of a dry, yellowish 
layer. In milk coagulation first occurs, and the coagulum is subsequently 
dissolved; a slightly acid reaction is produced. This bacillus grows best in 
the incubating oven at 37°, and does not develop at temperatures below 16° 
C. The spores require for their destruction a temperature of 100° C. main- 
tained for four minutes (determined by the writer, 1887). 

Pathogenesis. —The introduction of pure cultures of this bacillus into 
hives occupied by healthy swarms causes them to become infected with foul 
brood; grown bees also become infected when given food containing the ba- 
cillus (Cheshire). Mice injected subcutaneously with a considerable quan- 
tity die within Liskin ae hours, guinea-pigs in six days (Hisenberg). 
Smell amounts injected beneath the skin of mice or rabbits produce no appa- 
rent result. 


BACILLUS OF ACNE CONTAGIOSA OF HORSES. 


Obtained by Dieckerhoff and Grawitz (1885) from pus and dried scales 
from the pustules of ‘‘ acne contagiosa ” of horses. 

Morphology.—Short rods, straight or slightly bent, 0.2 ~ in diameter. 

Stains best with an aqueous solution of fuchsin, and also by Gram’s 
method; does not stain well with Léffler’s alkaline solution of methylene 
blue. 

Biological Characters.—An aérobic, non-liquefying bacillus. In gelatin 
stab cultures a very scanty growth occurs along the line of puncture; upon 
the surface a white mass forms about the point of puncture. Upon blood 
serum and nutrient agar an abundant growth at the end of twenty-four 
hours at 37° C., consisting of white colonies along the impfstrich, which 
later have a yellowish-gray color. The growth is more abundant and rapid 
upon blood serum than upon other media. — . ; ; 

Pathogenesis. —Pure cultures of the bacillus described are said by Diecker- 
hoff and Grawitz to produce typical acne pustules when rubbed into theskin 
of horses, calves, sheep, and dogs. When rubbed into the intact skin of 
guinea-pigs a phlegmonous erysipelatous inflammation was produced, and 
the animal died at the end of forty-eight hours with symptoms of toxsemia. 
Subcutaneous injections in guinea-pigs caused toxzemia and death at theend 
of twenty-four hours. At the autopsy a hemorrhagic infiltration of the in- 
testinal mucous membrane was observed; the bacilli were not found in the 
internal organs. In rabbits pure cultures rubbed into the intact skin caused 
a development of pustules and a severe inflammation of the subcutaneous 
connective tissue, from which the animal usually recovered. Subcutaneous 
injections in rabbits sometimes caused a fatal toxemia. House mice, field 
mice, and white mice were not affected by the application of cultures, by 
rubbing, to the uninjured skin, but succumbed to subcutaneous injections in 
twenty-four hours or between the fifth and tenth days. Those which died’ 
at a late date presented the pathological appearances which characterize 
pyzmia. 


558 PATHOGENIC AEROBIC BACILLI 


BACILLUS OF PURPURA HAZ MORRHAGICA OF TIZZONI AND GIO- 
VANNINI. 


Obtained by Tizzoni and Giovannini (1889) from the blood of two children. 
who died of purpura hemorrhagica following impetigo. 

Morphology.—Bacilli with round ends, from 0.75 to 1.3 # long and 0.2 
to 0.4 x broad; often seen in pairs or in groups like streptococci. 

Stains with the usual aniline colors, but not by Gram’s method. 

Biological Characters.—An aérobie and facultative anaérobic, non- 
liquefying, non-motile bacillus. Spore formation not observed. Grows in 
the usual culture media at theroom temperature. Upon gelatin plates the 
colonies at first resemble those of Streptococcus pyogenes. Upon the surface 
small, opaque points are seen at the end of forty-eight hours, which at the 
end of four to five days develop into spherical, yellowish-gray colonies with 
irregular margins, surrounded by a growth resembling tufts of curly hair. 
Upon agar the growth is similar, but more rapid and of a pale color, often 
with a central nucleus surrounded by a. net-like marginal zone. Upon 
blood serum the growth is similar to that upon agar. Upon potato, at 37° 
C., a limited development occurs about the point of inoculation, which has 
a dark-yellow color. The cultures give off a very penetrating odor. 

Pathogenesis.—Pathogenic for dogs, rabbits, and guinea-pigs when in- 
jected subcutaneously. Not pathogenic for white mice or pigeons. The 
symptoms resulting from a subcutaneous injection are said to be fever, al- 
buminuria and, in some cases, anuria, hemorrhagic spots upon. the skin, 
convulsions ; death occurs in from one to three days. Atthe autopsy there 
are found cedema about the point of inoculation, hemorrhages in the skin and 
muscles, and sometimes in the internal organs and in serous cavities; the 
blood does not coagulate. The bacilli are found in the subcutaneous con- 
nective tissue, but not in the blood or in the various organs. Sections show 
coagulation necrosis of the liver cells and of the renal epithelium. 


BACILLUS OF PURPURA HMMORRHAGICA OF BABES. 


Obtained by Babes (1890) from the spleen and lungs of an individual who- 
died from purpura hzmorrhagica with symptoms of septicemia. Resembles 
the bacillus previously described by Tizzoni and Giovannini, and still more 
that of Kolb; but, according to Babes, differs in some respects from both of 
these, although they all belong evidently to the same group. 

Morphology.—Bacilli with rounded ends, oval or pear-shaped, about 0.3 
thick, surrounded by a narrow capsule. 

Stains with the aniline colors, but not deeply, and still less intensely by 
Gram’s method. 

Biological Characters.—An aérobic and facultative anaérobic, non-- 
liquefying, non-motile bacillus. Does not form spores. Grows in the usual 
culture media at the room temperature. In gelatin stick cultures, at the 
end of three days, a thin, transparent, irregular layer has developed upon 
the surface, and a whitish, punctate stripe along the line of inoculation. In 
agar stick cultures an abundant development occurs along the line of punc- 
ture, and at the end of three days the growth upon the surface consists of 
small, moist, transparent drops; later of larger, flat, shining, yellowish- 
white plaques which have ill-defined margins. Upon blood serum the de- 
velopment is somewhat more abundant in the form of small, white, moist 
colonies one to two millimetres broad. Upon potato, at the end of three 
days, moist, whitish drops with ill-defined margins. 

Pathogenesis.—Inoculations in the conjunctive of rabbits produce ecchy- 
moses of the conjunctiva. At the autopsy numerous hemorrhagic extrava- 
sations are found in all the organs, especially in the lungs and liver; the 
spleen is enlarged; the bacilli can be recovered in pure cultures from the 
various organs. Old cultures proved to have lost their virulence. Patho- 
genic for mice, which die from general infection in the course of a few days; 


NOT DESCRIBED IN PREVIOUS SECTIONS. 559 


the spleen is enlarged, and hemorrhages in the serous membranes are usually 
seen, 


BACILLUS OF PURPURA HAMORRHAGICA OF KOLB. 


Obtained by Kolb (1891) from the various organs of three individuals: 
who died in from two to four days from attacks characterized by suddenly 
developed fever, purpura, and albuminous urine. 

Morphology.—Oval bacilli, usually in pairs, 0.8 to 1.5 long and 0.8 4 
broad, surrounded by a narrow capsule, which is only seen distinctly in 
preparations from the organs. 

Stains with the aniline colors, but not deeply, and still more feebly by 
Gram’s method. 

Biological Characters.—An aérobic and facultative anaérobic, non- 
liquefying non-motile bacillus. Does not form spores. Grows in the usual 
culture media at the room temperature. In gelatin. stick cultures, at the end 
of four days, a very small, thin, hyaline growth is seen about the point of 
inoculation. The development is more abundant along the line of puncture. 
Upon the surface of agar a thin layer is formed with smooth margins. 
Upon potato, at the end of three to four days, a whitish, moist, shining stripe 
is seen along the impfstrich which is about three millimetres broad. 

Pathogenesis.—Injections of 0.5 to 1 cubic centimetre of a bouillon 
culture into the abdominal eavity of rabbits cause symptoms of general in- 


4 
2 


7 ' " 
are ry 7, 
Af S68 NS oo * 

Net Nt ‘ 


me iy 
¢ 
“. 
cs 


Fie. 157. Fia. 158. 
Fig. 157,—Bacillus gracilis cadaveris, from a gelatin culture. x 1,000.. From a photomicro- 


ph, (Sternberg.) ; 
Ee, 158.—Bacillus gracilis; colonies in gelatin roll tube, end of forty-eight hours. X 12. From 


a photograph. (Sternberg ) 


fection in the course of a few days, and not infrequently hemorrhagic ex- 
travasations are seen in the ear muscles. More than one cubic centimetre 
may cause death in from one to three days. At the autopsy hemorrhagic 
extravasations are found in the subcutaneous tissues and in the serous and 
mucous membranes. The blood has little disposition to coagulate; the 
bacillus may be recovered in pure cultures from the various organs. In 
guinea-pigs local ecchymoses are sometimes produced, otherwise not patho- 
genic for this animal. Pathogenic for mice, which die from general infec- 
tion, after being inoculated with a small quantity of a pure culture, in from 
two to three days; spleen enlarged; lymphatic glands often hemorrhagic. 
Not fatal to dogs, but animals which were inoculated with one cubic centi- 
metre of a bouillon culture and subsequently killed proved to have hemor- 
rhagic extravasations in the various organs. 


560 PATHOGENIC AEROBIC BACILLI 


BACILLUS GRACILIS CADAVERIS (Sternberg). 


Obtained (1889) from a fragment of liver, of man, kept for forty-eight 
hours in an antiseptic wrapping. ; : 

Morphology.—Bacilli about 1 « broad and 2 “ long, associated in long 
chains. 

Biological Characters.—An aérobie and facultative anaérobic, non- 
motile, non-liquefying bacillus. Spore formation not observed. In gelatin 
roll-tubes the deep colonies are opaque and spherical; superficial colonies 
circular or slightly irregular in outline, white in color, and opaque or slightly 
translucent. In gelatin stab cultures, at 22° C., at the end of five days a 
rather thick, white mass at the point of puncture, covering one-third of the 
surface, and closely crowded, opaque colonies at bottom of line of puncture, 
with slender, branching outgrowth above. In nutrient agar, at the end of 
five days at 22° C.,a milk-white growth upon the surface and opaque 
growth to bottom of line of puncture. On potato, at end of five days at 
22° C., rather thick, cream-white growth with irregular margins along the 
impfstrich. Cultures in bouillon have a milky opacity and a very disagree- 
able odor. Grows in agua coco without formation of gas.- 

Pathogenic for rabbits when injected into the cavity of the abdomen. 


CAPSULE BACILLUS OF NICOLAIER. 


Obtained by Nicolaier (1894) from pus contained in an abscess of the kid- 
ney—obtained post-morten.. 

Morphology.—Thick bacilli, with rounded ends, usually four times as 
long as thick, and frequently presenting irregular outlines ; often united in 
pairs, and sometimes growing out into filaments; cocci-like forms also occur. 
Often surrounded by a capsule which vemains unstained in stained prepara- 
tions. Does not stain by Gram’s method. 

Biological Characters.—An aérobic, and facultative anaérobic, non- 
liquefying, non-motile bacillus. Does not form spores, Grows at the room 
temperature and more rapidly at 37° C. Upon gelatin plates at 20° C., at 
the end of twenty-four to thirty-six hours punctiform colonies are devel- 
oped, which under a low power appear finely granular, and grayish-yellow 
spheres. At the end of forty-eight to sixty hours the superficial colonies ap- 
pear as round or slightly irregular, grayish-white discs, which project but lit- 
tle above the surface of the gelatin, and have thin, transparent margins. 
The deep colonies have a sharply defined contour, with dark-brown centre 
and a purely granular pale-brown marginal zone. In gelatin stab cultures 
aslightly elevated, moist-looking, sticky layer with more or less transparent 
margins is developed. In slanting cultures this growth gradually slips down 
to the lowest part of the exposed surface, leaving a thin, gray, transparent 
layer over the gelatin ; along the line of puncture a ribbon-like, grayish- 
white growth with irregular margins is developed. In media containin 
glucose some gas bubbles are developed. The growth is much more rapi 
in the incubating oven at 37° C., and there is an abundant development of 
gas in agar tubes. Upon potato a grayish-white, slimy mass with a shining 
surface is quickly developed. In bouillon, at the end of twenty-four hours, 
at 37° C., the medium is clouded throughout, and a grayish-white deposit ac- 
aes at the bottom of the tube. Development occurs also in acid 
media. 

Pathogenesis.—Pathogenic for house mice, white mice, and for rats—not 
for rabbits or guinea-pigs—by subcutaneous injections. As Nicolaier has 
made a careful comparison of the characters of the various ‘ capsule bacilli” 
described, we quote from him as follows : 

‘Our bacillus in its morphology and growth in various media closely re- 
sembles that of Fasching and of Abel, both of which were obtained in patho- 


NOT DESCRIBED IN PREVIOUS SECTIONS. 561 


logical products from man. It is distinguished from them by its pathogenic 
action upon mice. White and gray mice when infected with our bacillus 
die from septicaemia and show, in addition to a serous exudation at the point 
of inoculation, constant pathological changes in the kidneys, which may usu- 
ally be recognized by a macroscopic examination. Also by the spleen, which 
is not always enlarged, and the liver, which only in a few cases showed any 
microscopic changes. In mice inoculated with the bacillus of Fasching, or 
that of Abel, which died of septicae:mia, there was constantly seen an en- 
largement of the spleen (Fasching, Abel) and of the liver (Abel), and a 
cloudy swelling of the liver and kidneys (Abel) which our mice failed to 
show. The macroscopic and microscopic changes which we found in the 
kidneys in mice, and also in some cases in the liver and spleen, were not ob- 
served by Fasching or by Abel. Recently Paulsen has described a capsule ba- 
cillus from atrophic rhinitis, and Marchand a capsule bacillus—not further 
described—which he obtained in great numbers from the exudate in a case 
of lobar pneumonia. Both appear to be very similar to Fasching’s bacillus. 
They are pathogenic for mice, but do not cause the changes in the kidneys 
which we have described. These capsule bacilli are therefore not identical 
with ours. Marchand’s bacillus is further distinguished by the fact that it is 
pathogenic for guinea-pigs. . . . The bacillus of Kockel is distinguished 
from ours by the following characters: It forms upon the surface of gelatin, 
as well as in stick cultures, highly elevated, button-like colonies, while our 
bacillus grows more in flat and broad layers. It also lacks the semi-fluid 
character of growth upon slanting agar, which distinguishes our bacillus, 
and as a result of which the growth slips down to the lowest point on the 
slanting surface ; further it forms upon potato a yellowish layer, while ours 
is grayish-white ; and it does not grow in acid media. Finally, it is patho- 
genic for rabbits by intravenous injection, while ours is not.” 


BACILLUS MUCOSUS OZANAL. 


Obtained by Abel (1893) from cases of ozeena simplex (rhinitis atrophicans 
foetida). As this bacillus appears to correspond in its morphological and bio- 
logical characters with the capsule bacillus above described we shall not 
repeat this description, but quote from Abel, as follows: 

“This bacillus. found in the secretion from cases of ozzena, as the de- 
scription we have given shows, closely resembles Friedlinder’s pneumo- 
bacillus. It is distinguished from it by certain constant characters. The 
ozeena bacillus forms in cultures a more fluid mass than Friedlander’s. As 
aresult of this it does not form the characteristic nail-head culture, but 
spreads out over the surface of the gelatin. Upon slanting gelatin cultures 
the growth slips down to the lowest point. In old cultures it never shows a 
brown coloring of the culture medium. It never forms gas on potato, and 
in agar and gelatin cultures but little gas is developed. Mice always suc- 
cumb to subcutaneous inoculations, while Friedlander’s bacillus does not 
kill mice. Intraperitoneal infection of guinea-pigs with the ozeena bacillus 
always causes their death. Friedlandev’s bacillus only killed about half the 
guinea-pigs inoculated in the cavity of the abdomen. Finally, Friedlander’s 
bacillus has a greater tendency to cocci-like forms. The resemblance to 
Pfeiffer’s capsule bacillus is closer. But the tenacious layer described by 
Pfeiffer as found upon the intestinal coils and the lungs in mice, and the 
sticky condition of the blood and tissue juices (fadenziehende) are want- 
ing. The reaction at the point of inoculation in mice is also much more 
pronounced with my bacillus.” ; 

It seems extremely probable that this bacillus,the Bacillus capsulatus mu- 
cosus of Fasching, and the above-described capsule bacillus of Nicolaier 
are simply pathogenic varieties of one and the same bacillus. 


36 


562 PATHOGENIC AEROBIC BACILLI 


CAPSULE BACILLUS OF VON DUNGERN. 


Obtained by von Dungern (1893), post mortem, from a new-born child 
which died of hemorrhagic septicemia—infection through umbilicus. 

Morphology.—A short, thick bacillus, from 1 to 2 » long and half as 
broad, surrounded by a capsule which is slightly stained by gentian violet— 
best seen in the body of infected mice; sometimes seen in pairs or in chains 
of four elements; also grows out into filaments, especially in bouillon. 
Upon potato usually only small spherical elements, resembling micrococci, 
are seen. Does not stain by Gram’s method. 

Biological Characters.—An aérobic and facultative anaérobic, non- 
motile, non-liquefying bacillus. Does not form spores. Coagulates milk, 
in which it causes an abundant development of gas at 38°C. Has feeble 
indol reaction. Grows well at room temperature, more rapidly in incubator. 
Upon gelatin plates the deep colonies at end of twelve hours are the size of a 
pin’s head, finely granular, spherical, and sharply defined. Upon the sur- 
face, porcelain-like, elevated, white colonies are developed, which in two or 
three days attain the size of lentils. In gelatin stab cultures development 
occurs all along the line of puncture, frequently with formation of gas bub- 
bles. Upon agar a thick, softlayer of a white color is developed. In bouil- 
lon, at 38° C., there is considerable development of gas. Upon potato the 
growth is very abundant, of a pale yellowish-white color, thick, soft, some- 
what sticky, and filled with gas bubbles. A great portion of the surface is 
covered by this growth at the end of twenty-four hours, even at the room 
temperature. These cultures give off a peculiar odor, sometimes aromatic- 
feetid and sometimes recalling that of fresh bread. Some of the cultures on 
potato soon become cream-like in consistence. At first they have an alkaline 
and later an acid reaction, when they have the odor of acetic acid. 

Pathogenesis.—Very pathogenic for white mice. The bacilli are found 
in the blood and in all the organs in enormous numbers. At the point of 
inoculation there is frequently a hemorrhagic cedema. The spleen is greatly 
enlarged. Also pathogenic for guinea-pigs when injected into the cavity of 
the abdomen—less pathogenic for rabbits. 

According to von Dungern, this bacillus can not be distinguished by its 
morphological and biological characters from Friedlander’s bacillus, Bacil- 
lus capsulatus of Pfeiffer, or Bacillus canalis capsulatus of Mori. But it is 
distinguished from these by greater virulence, especially for rabbits, and by 
the fact thatit frequently gives rise to hemorrhagic extravasations in inocu- 
lated animals. ln our opinion the characters given do not justify the view 
that this bacillus is a distinct species from the bacilli above mentioned. 


BACILLUS PESTIS (Kitasato and Yersin). 


Discovered by Kitasato (1894) in the blood of living patients, and 
in the buboes, blood, and organs of those who had recently died from 
the infectious malady known as bubonic plague. Kitasato was sent 
to Hong-Kong by the Japanese Government for the purpose of inves- 
tigating this disease. According to Lowson the bacilli are found in 
the faeces, in the contents of the buboes, and in the blood. 

Morphology.—In his preliminary note, Kitasato described the 
plague bacilli as “rods with rounded ends,” which are readily 
stained by the ordinary aniline dyes, the poles being stained darker 
than the middle part, especially in blood preparations, and present- 
ing a capsule sometimes well marked, sometimes indistinct. 


NOT DESCRIBED IN PREVIOUS SECTIONS. 563 


Yersin, who was sent by the French Government to study the 
bubonic plague at Hong-Kong, arrived in that city on the 15th of 
June, 1894. He describes the bacillus found in the contents of the 
buboes as being short and thick, with rounded ends, staining easily 
with the aniline colors, butnot by Gram’s method. “The extremities 
stain more intensely than the centre, so that they often present a 
clear space in the middle. Sometimes the bacilli appear to be sur- 
rounded by a capsule. . . . In bouillon the bacillus has a very char- 
acteristic appearance, resembling the cultures of the streptococcus of 
erysipelas—a clear liquid with grumous deposits on the walls and at 
the bottom of the tube. These cultures examined under the micro- 
scope show veritable chains of short bacilli, presenting in places a 
considerable spherical enlargement.” 

This bacillus is sometimes seen to be motile, and it has flagella, 
which, however, are difficult to stain (Gordon). 

In agar cultures, in the incubator at 37° C., involution forms soon 
appear. These may be spherical, oval, pyriform, etc., and are often 
many times larger than the typical bacillus. 

Biological Characters.—We quote from Kitasato’s preliminary 
report as follows: 


The bacilli show very little movement, and those grown in the incubator, 
in beef-tea, make the medium somewhat cloudy. The growth of the bacilli is 
strongest on blood serum at the normal temperature of the human body 
(34° C.); under these conditions they develop luxuriantly and form a col- 
ony moist in consistence and of a yellowish-gray color; they do not liquefy 
the serum. On agar-agar jelly (the best is good glycerin agar) they also 
grow freely. The different colonies are of a whitish-gray color and by re- 
flected light have a bluish appearance ; under the microscope they appear 
moist and in rounded patches with uneven edges ; at first they appear every- 
where as if piled up with ‘‘glass-wool,” later as if having dense, large cen- 
tres. If acover-glass preparation is made from a cultivation on agar-agar, 
and, after having been stained, is observed under the microscope, long 
threads of bacilli are seen, which might, by careless inspection, be mistaken 
for a coccus chain, but are recognized with certainty as ‘‘ threads of bacilli” 
under closer observation. The growth on agar-gelatin is similar to that on 
agar-agar ; in a puncture cultivation at the ordinary temperature after a few 
days they are found growing as a fine dust in little points alongside the 
puncture, but with very little growth on the surface. Whether these ba- 
cilli are able to liquefy ordinary gelatin or not Iam at present unable to de- 
cide, as the temperature of Hong-Kong ranges so high that the employment 
of simple nutritive gelatin is out of the question. I shall give further infor- 
mation on this question later. On potatoes at a temperature of from 28° 
to 30° C., there was no growth after ten days’ observation, but at a tempera- 
ture of 37° C. the bacilli developed sparingly after a few days; the growth 
was whitish-gray in color and exsiccated. As mentioned before, the bacilli 
grow best at a temperature of from 38° to 39° C.; at how low a temperature 
growth is possible Iam unable at present to state. So far 1 have been un- 
able to observe the formation of spores. 

Experiments on Animals.—Mice, rats, guinea-pigs, and rabbits are sus- 
ceptible to inoculation. If these animals are inoculated with pure culti- 
vations, or with the blood of a plague patient in which the bacilli have been 


564 PATHOGENIC AEROBIC BACILLI 


observed, or with the contents of a bubo, or with pieces of internal organs, 
or even with the contents of the intestine, they begin to become ill in from 
one to two days, according to the size of the animal. Their eyes become wa- 
tery, they begin to show disinclination for any effort, later they avoid their 
food, and hide quietly in a corner of the cage. The temperature rises to 
41.5° C., and with convulsive symptoms they die in from two to five days. I 
must observe that in Hong-Kong I could only obtain small guinea-pigs 
{weight from one hundred to one hundred and fifty grammes) and small 
rabbits (from two hundred to two hundred and fifty grammes). If I could 
have experimented upon larger animals it is possible that life would have 
been prolonged somewhat beyond the periods mentioned above. The parts 
around the point of inoculation are infiltrated with a reddish gelatinous 
exudation, the spleen is enlarged, sometimes there is a swelling of the lym- 
phatic glands, and in all the organs the bacilli are found. _ The results found 
after death in animals are very similar to those found in anthrax and in 
oedema malignum. Pigeons do not appear to be susceptible to the influence 
of the bacilli. I made experiments by feeding some mice and guinea-pigs 
with pure cultivations of the bacillus and with small pieces of the internal 
organs: the result was, such animals perished in a few days under the same 
symptoms as those which had been inoculated. In all the internal organs 
of animals so destroyed I found the bacilli. With the dust of dwelling- 
houses from which the plague-stricken had been removed, I made sev- 
eral experiments upon animals. Some of the animals died from tetanus. 
In one case only a guinea-pig died with plague symptoms, and in this ani- 
mal the same bacilli were found in the internal organs as in those of 
plague patients who had succumbed. These experiments with the dust from 
infected houses I shall certainly continue. Many rats and mice at present 
die spontaneously in Hong-Kong. I examined some of them. In the inter- 
nal organs of a mouse I discovered the same bacilli. : 

Experiments with Desiccation,—The contents of a bubo in which the 
bacilli were present in great numbers were wiped over cover glasses (per- 
fectly cleansed by heat and alcohol), and some of these cover-glasses were 
dried in the air of a room at a temperature ranging from 28° to 30°C. Oth- 
ers I exposed directly to the sun’s rays, and from among them, after an expo- 
sure of from one, two, and three hours up to six days, I removed some parts, 
putting such portions in beef-tea and placing them in the incubator. Those 
which had been standing in the room from one to thirty-six hours showed a 
pretty good growth in the incubator, but those which had been in the room 
for more than four days were unable to show any growth even after one 
week’sincubation. Those exposed directly to the sun were all destroyed after 
from three to four hours. Further cultivations on serum were treated 
exactly like the contents of the bubo with very similar results. 

Experiments with Heat.—Beef-tea cultivations which had been heated 
for thirty minutes ma water bath up to 80° C. were destroyed; at 100° C., in 
the vapor apparatus they were destroyed in a few minutes. 


Yersin reports that when fragments of the spleen or liver of 
animals which have died of the plague are fed to rats and mice they 
usually become infected and die, and the bacillus is found in their 
organs, lymphatic glands, and blood. He also demonstrated the pres- 
ence of the bacilli in dead rats found in the houses or streets of 
Hong-Kong. 

Without doubt rats play an important part in the propagation of 
the disease. Monkeys are also very susceptible to infection, and it is 
said that the disease has been known to occur as an epidemic among 
these animals. There is also good reason to believe that fleas have 
some influence in the propagation of the disease, by transferring the 


NOT DESCRIBED IN PREVIOUS SECTIONS. 565 


bacillus from infected rats to man, or from one individual to another. 
Infection in man occurs by inoculation through lesions of the skin 
and also by the respiratory passages (pulmonic form). 


BACILLUS PISCICIDUS AGILIS (Sieber). 


Discovered by Sieber (1895) in infected fish, which died of an epidemic 
disease in the laboratory of Professor Nencki, at St. Petersburg. 

Morphology.—Short bacilli, often united in pairs. 

Biological Characters.—An aérobic and facultative anaérobic, motile, 
liquefying bacillus. In old cultures in bouillon spores are developed. 
Grows at temperatures of from 12° to 37.5° C. Thermal death point, 60° to 
65° C. On gelatin and agar plates forms granular, grayish, or yellowish 
colonies, which appear to be made up of three concentric rings—the outer one 
having a jagged outline. Gasis developed during the growth of the bacillus 
—carbon dioxide and methy] merecaptan in small amount. Upon potato it 
forms yellowish-brown, pearl-like colonies. Causes coagulation of milk. 
Retains its vitality and virulence for months in well or river water. 

Pathogenesis.—Pathogenic for fish, frogs, guinea-pigs, rabbits, mice, 
and dogs (not for birds). Old cultures are more pathogenic than recent 
ones, and gelatin cultures are the most active. Frogs are killed in half an 
hour by 0.1 cubic centimetre of a bouillon culture six days old. Filtered 
cultures are as toxic as those containing the living bacillus; they give with 
iron chloride a characteristic color reaction—an intense red color. Sieber 
has obtained from his cultures an extremely toxic alkaloid in the form of a 
hydrochlorate. Two litres of filtered culture gave 0.1 gramme of this salt. 
An aqueous solution of this killed a frog in fifteen minutes in the dose of 
0.0035 gramme. 


BACILLUS OF MERFSHKOWSKY. 


Obtained by Mereshkowsky (1894) from infected animals (Spermophilus 
musicus) which died from an epidemic malady developed in his laboratory. 

Morphology.—Closely resembles Loffler’s Bacillus typhi murium. 

Biological Characters.—An aérobic, motile, uon-liquefying bacillus. 
Spore formation not observed. Grows in the usual culture media at the 
room temperature —best at 37.5° C. In bouillon, at the end of twenty-four 
hours, the medium is clouded aud a white pellicle is seen upon the surface, 
which breaks up into small flocculi and falls to the bottom when the tube 
is slightly shaken. On gelatin plates minute, slightly granular, pale-brown 
colonies may be seen, under a low power at the end of twenty-four hours; 
on the second day these are visible as white spheres, which under the micro- 
scope have a pale-brown color and a more or less transparent, peripheral 
zone. In media containing glucose no gas is developed. The growth upon 
agar and potato presents nothing characteristic. ; : 

Pathogenesis.—Pathogenic for Zieselmausen (Spermophilus musicus), 
for Spermophilus guttatus, for squirrels (Sciurus vulgaris) for house mice, 
for field mice (Arvicola arvalis). Not pathogenic for man or forthe domes- 
tic animals tested, horse, swine, sheep, fowls. Mereshkowsky proposes to 
use cultures of this bacillus for the extermination of field mice, which die in 
from one to ten days after being fed upon biscuit wet with a bouillon cul- 
ture. 


BACILLUS OF EMMERICH AND WEIBEL. 


Obtained by Emmerich and Weibel (1894) from infected trout in ponds 
belonging to an establishment for raising these fish. The disease appeared 
as a superficial ‘‘furunculosis with secondary development of abscesses con- 
taining bloody pus.” Death occurred in from twelve to twenty days. The 
pustules and secondary abscesses and blood from the heart and various or- 
gans contained bacilli, which proved to be the cause of the infectious malady. 


566 PATHOGENIC AEROBIC BACILLI 


Morphology.—Bacilli about as long as the typhoid bacillus, but not so 
thick, very frequently united in pairs ; occasionally grows out into filaments. 
Biological Characters.—An aérobic and facultative anaérobice, lique- 
fying, non-motile bacillus. Does not form spores. Thermal death point, 60° 
. Stains with the usual aniline colors but not by Gram’s method. Grows 
best at 10° to 15° C. The growth in gelatin is quite characteristic. At the 
end of two or three days, in gelatin plates, at the room temperature, small 
white colonies are developed ; in four or five days small gas bubbles or ex- 
cavations are seen, at the bottom of which lie the scale-like or rosetta-formed 
colonies. The margin of the colonies is irregular and later jagged. At 
first the colonies are grayish-white or yellowish, later brownish. The 
superficial colonies have a peculiar lustre. In gelatin stab cultures, colo- 
nies develop along the line of puncture, which at first resemble the growth 
of Streptococcus pyogenes, and no development is seen on the surface. At 
the end of five to seven days in place of the line of colonies is seen a channel 
filled with air, or gas developed by the separate colonies, the bubbles from 
which coalesce. The funnel formed in this way is somewhat larger above, 
and at the bottom contains a whitish sediment consisting of bacteria con- 
tained in a few drops of liquefied gelatin. Along the sides of the funnel 
bubble-like cavities may frequently be seen, at the bottom of which the bac- 
teria have accumulated. In bouillon a slight cloudiness is seen near the 
surface, on the walls of the test tube; when slightly shaken this falls to the 
bottom, leaving the bouillon entirely clear. In agar-agar tubes, a veil- 
like stripe develops along the line of puncture, and a grayish-yellow, moist 
layer, with irregular outlines upon the surface. After some weeks this 
acquires a brown color. No growth occurs upon potato. No development 
occurs in the incubating oven at 37° C. 

Pathogenesis.—Trout became infected and died through direct infection, 
subcutaneous or intramuscular inoculations, or through the addition of cul- 
tures to the water in which they were kept, or by placing infected fish in the 
same tank with healthy ones. 


BACILLUS OF BECK. 


Synonym.—Der Bacillus der Brustseuche beim Kaninchen. 

Obtained by Beck (1892) from rabbits which died of an infectious malady 
in the Institut fiir Infectionskrankheiten, in Berlin. 

Morphology.—Very small and slender bacilli, about twice as long and 
twice as thick as the influenza bacillus ; somewhat pointed at the extremities ; 
show a tendency to grow out into filaments. 

Biological Characters.—An aérobic (strict) non-liquefying, non-motile 
bacillus. Spore formation not observed. Grows at the room-temperature 
and more vigorously at 88° C. Does not stain by Grain’s method. Thermal 
death point, 50° C. (five minutes). Resists desiccation, at the room tempera- 
ture, for seventeen days, at 87° C. for three days. 

On gelatin plates, at tle end of forty-eight hours, small, finely granular, 
glass-like colonies are developed ; older colonies have a pale-brown appear- 
ance. In gelatin stab cultures a granular growth of a white color is seen 
along the line of puncture. Upon agar, at 37° C., an abundant development 
occurs in twenty-four hours. The line of puncture seen from above is gray- 
ish-white, by transmitted light bluish and porcelain-like with a brownish 
tint. On agar plates the colonies have a yellowish-gray appearance; the 
margin of the finely granular colonies is sharply defined. In agar cultures 
several days old the colonies are sticky and may be picked up as a compact 
mass, or drawn out into threads. In bouillon, at 37° C., there is a slight 
cloudiness at the end of twenty-four hours ; later the bouillon is clear and a 
white sediment is seen at the bottom of the tube. In bouillon cultures 
especially, the bacillus grows out into long filaments. 


NOT DESCRIBED IN PREVIOUS SECTIONS. 567 


_ _ Pathogenesis.—From 0.28 to 1 cubic centimetre of a bouillon culture 
injected into the pleural cavity of a rabbit caused a development of all of 
the symptoms of influenza (Brustseuche)—viz., elevation of temperature at 
the end of five or six hours, cough, nasal discharge, dyspnoea, and death— 
usually in from three to five days. The autopsy showed a distinct pleuro- 
pneumonia and a general blood infection by the bacillus in question. 
Injections into the circulation also give rise to the symptoms of influenza, 
including pneumonia, and to death at the end of from ten to fourteen days. 
Subcutaneous injections resulted in the development of an abscess and of ex- 
tensive necrosis of the tissues, but did not cause a general blood infection. 
Guinea-pigs were somewhat less susceptible than rabbits, but injections into 
the pleural cavity produced similar symptoms and death at a later date. 
White mice and house mice, as a result of intraperitoneal injections, died 
within two or three days from general blood infection. 


BACILLUS PISCICIDUS (Fischel and Enoch). 


Obtained by Fischel and Enoch (1892) from an infected carp. 

Morphology.—Bacilli solitary or in chains of four to five elements, 1.2 
to 3 long and 0.25 thick. Stains by the usual aniline colors and by 
Gram’s method. 

Biological Characters.—An aérobic and facultative anaérobic, non- 
motile, liquefying bacillus. Forms spores. In gelatin plates forms round 
colonies of a pale yellowish-brown color, having a slightly toothed border 
anda granular surface. At the end of twenty-four hours a narrow zone of 
liquefaction can be discerned around the colonies, and at the end of about 
ten days the gelatin is entirely liquefied. In gelatin stab cultures a scanty 
growth is seen along the line of inoculation at the end of twelve hours; the 
growth upon the surface is rapid, and liquefaction commences at the end of 
twenty-four hours. Upon agar, at 37° C. at the end of eighteen hours, a thin 
granular layer is seen, which consists of small, pale-gray colonies. In agar 
stick cultures a scanty growth occurs along the line of puncture, which does 
not increase after thirty-six hours. Upon the surface the growthis abundant, 
forming, at the end of five days a tolerably thick grayish-white layer. No 
growth occurs upon potato at the room temperature, but at 87° C. a tolera- 
bly thick, sticky layer of a grayish-white color is developed in three or four 
days. In bouillon, at 37° C., the medium is clouded at the end of twelve 
hours, and a thin pellicle is seen upon the surface at the end of thirty-six 
hours; this falls to the bottom when the tube is slightly agitated. At the 
end of four days development has ceased, and the bouillon is again transpar- 
ent, while a flocculent deposit is seen at the bottomof thetube. The bouillon 
gives off a penetrating odor, like that of burnt milk. Thesame odor is given 
off from cultures in milk, which is peptonized by the action of the bacillus. 
At the end of twenty days, at 37° C., the entire contents of the tube have be- 
come transparent. 

Pathogenesis.—Produces a fatal infectious disease in fish (‘gold carp”) 
when inoculated beneath the skin ; also pathogenic for mice and for guinea- 


pigs. 
BACILLUS PYOGENES FILIFORMIS (Flexner). 


Obtained by Flexner (1895) from the interior of the uterus and from an 
exudate in the pericardial and pleural cavities, of a rabbit which died on the 
fifth day after parturition. ; oe 

Morphology.—Pleomorphous cocci-like forms, short or long bacilli, and 
long threads are seen in cover slips prepared from the exudate. ‘‘Very few 
of the bacilli stain regularly ; for the most part brightly stained spots appear 
between stained areas. An outer membrane always stains, enclosing the 


568 PATHOGENIC AEROBIC BACILLI 


stained dots in a colorless ground. The threads, as a rule, present delicate, 
sinuous, and wavy outlines; the short forms are straight with rounded ends.” 

Biological Characters.—All attempts to cultivate this bacillus in the 
usual media, either in the presence of oxygen or in an atmosphere of hydro- 
gen, proved unsuccessful. Butsuccessive cultures were made by inoculations 
in the pleural cavity of rabbits—a bit of pleural exudate suspended in bouil- 
lon was used for this purpose. The bacillus was also propagated upon the 
lungs, heart, uterus, and kidney of healthy rabbits. The organs were re- 
moved with great care to prevent contamination and placed in sterilized test 
tubes. Transplantations from these cultures were only successful for one or 
two generations. Better results were obtained Ly cultivating the bacillus 
upon the one-third to one-half grown foetuses of rabbits. 

Pathogenesis.—‘' Considerable variations were observed according as the 
inoculations were made into the pleural cavity, the peritoneal cavity, the sub- 
cutaneous tissue, beneath the dura mater, or directly into the circulation. 
The inoculations gave positive results in all cases except a few, in which they 
were made subcutaneously. The death of the animal occurred soonest when 
inoculation was made beneath the dura mater. A small portion of the skull 
was trephined, care being taken to exclude extraneous microdrganisms, and 
a drop of the pleural fluid or a speck of the fibrinous exudate was introduced 
beneath the membranes, care being taken not to injure the brain. These 
animals, which quickly recovered from the effects of the operation, died on 
an average about twelve hours after the inoculation. . . . 

‘The pleural inoculations were followed by death, as before stated, in ev- 
ery instance, the death of the animal occurring upon the third or fourth day. 
The appearances presented at the autopsy were for the most part an exact 
reproduction of those observed in the animal which had succumbed to the 
natural disease. Upon the side of inoculation a thick, grayish-yellow, shaggy 
membrane covered the pleural surfaces, being at times four or five millime- 
tres in thickness. The pleural cavity contained several cubic centimetres of 
a clear heemoglobin-colored fluid, the lung for the most part being com- 
pressed. At times smaller or larger areas of lobular pneumonia would be 
present ; and, as a rule, the inflammation was not limited to the serous mem- 
brane of the side of inoculation, but extended into the opposite pleural cavity 
and into the pericardial sac. However, in these situations the process was, 
as a rule, less intense, the solid exudate being less considerable, and in the 
case of the opposite pleural cavity sometimes entirely wanting. The super- 
ficial vessels, however, were injected and the serous surface of the affected 
membrane covered with a slimy, clear fluid. In addition to this the oppo- 
site pleural cavity always contained a similar pink serum to that described 
upon the side of inoculation. 

‘The study of the exudate upon the side of inoculation as well as the 
fluid contained in the opposite pleural cavity and in the pericardium showed 
the same organisms as had been introduced.” 


BACILLUS DYSENTERIA. 


The researches of Shiga, of Flexner, and of the board of medical 
officers of the army engaged in the study of tropical diseases in the 
Philippine Islands (1890) give support to the belief that there is a 
form of acute dysentery which is due to infection by the bacillus of 
Shiga, which Flexner describes as follows:’ 


‘‘Bacillus of the average size of B. coli communis. There is variation in 
length: almost none in thickness. The individuals are usually separate; 
g Pp 


1 Johns Hopkins Hospital Bulletin, vol. xi., No. 115. 


NOT DESCRIBED IN PREVIOUS SECTIONS. 569 


Sometimes they are united in pairs, but only very rarely do they occur as 
filaments, The ends are slightly rounded. The bacillus shows moderate 
motility ; Gram’s stain is negative. 

Growth takes place upon all culture media at the room temperature, 
but better in the thermostat. Gelatin is not liquefied. The colonies resemble 
those of B. typhosus, being more nearly like them when first isolated from 
the dejecta than after a period of cultivation outside the body. After many 
months of such saprophytic growth the colonies become thicker, exhibit a 
moist surface, and are less translucent. The strokes upon agar slants show a 
similar alteration. At first the growth extends but little lateral] y, but later 
on it becomes two to three millimetres in width, and generally shows distinct 
indentations at the edges. Upon gelatin the colonies are more delicate ; the 
stab extends along the line of puncture only, spreading very little at the sur- 
face of the medium. 

‘On potato, growth takes place along the line of inoculation and spreads 
beyond. After some days it is a little elevated and of a pale-brown tint. On 
unfavorable potatoes the growth is slight, moist, and membranous, resem- 
hee rent for the greater amount of moisture, that of B. typhosus when 

ypical. 
'‘“ Sugars—glucose, lactose, and saccharose—are not fermented gaseously. 
Iu glucose media a moderate acid production takes place. . 

‘* Bouillon is clouded diffusely and a sediment forms. There is no pro- 
duction of a pellicle. 

‘‘ Litmus milk assumes, after twenty-four to seventy-two hours, a faint 
lilac tinge. After the lapse of from six to eight days alkali begins to be pro- 
duced, which increases in amount until the litmus is rendered deep blue in 
color. No coagulation of the milk ensues. 

‘‘Indol is not always formed. Even in sugar-free bouillon it may fail to 
appear, or it may be produced in small quantities only. 

‘‘Suitable cultures of this organism, when tested for the agglutination re- 
action with the blood serum of persons suffering from dysentery—the host of 
another individual—give, in many cases, a positive result. 

‘‘The bacillus is pathogenic for the ordinary laboratory animals. It is 
abundant in the acute cases in which it may be the predominating organism ; 
it becomes more difficult to find as the cases progress toward recovery or 
chronicity. In the ordinary chronic dysentery of Manila, in which amcebse 
are commonly encountered, it was not found. It can be cultivated from the 
dejecta during life, and the intestinal contents, mucous membrane, and 
mesenteric glands in fatal cases. 

‘Since the publication of Shiga’s studies, Escherich and Celli have both 
attempted to show that the organisms obtained from their respective epidemics 
of dysentery are identical with the B. dysenteriz. In both cases they have 
proceeded upon the false assumption that Shiga’s microérganism was a 
variety of B. coli communis, whereas, in point of fact, it is much more nearly 
related in its cultural and physiological properties to B. typhosus. 

“The question naturally arises, In what ways does it differ from B. 
typhosus? Comparison of the Eberth-Gaffky and Shiga bacilli show the 
criteria of difference to be by no means numerous. The main features, how- 
ever, are as follows: The latter shows less marked motility when first iso- 
lated and a tendency to lose motility rapidly in artificial cultivations; it 
displays a more uniform generation of indol; after a brief preliminary acid 
production in milk it gives rise to a gradually increasing alkalinization ; it is 
inactive to blood serum from typhoid cases; but reacts with serum from 
dysenteric cases to which B. typhosus does not respond. . . . : 

‘‘Bearing directly upon these considerations are the results of Lieutenant 
Strong’s studies, continued after our departure from Manila. He writes: 
‘After you left we had a large number of acute cases of dysentery. It seems 
certain that this form, which we have begun to speak of as acute infectious 
dysentery, is independent of amcebe. I have now records of fourteen cases 


570 PATHOGENIC AEROBIC BACILLI 


(not all were fatal) which I studied bacteriologically. From the stools in all 
of these, there has been obtained a bacillus which agrees with the organism 
obtained by you. I have also obtained the organisms from the mesenteric 
glands in three fatal cases. In one case of acute dysentery with secondary 
acute fibrinous peritonitis I obtained it from the exudate. The agglutina- 
tion reaction is not invariable. Amoebze were never demonstrable in any of 
these fourteen cases. On the other hand, in every case with certain anatomi- 
cal lesions we always find the amcebze. In some cases of dysentery in which 
the amcebee were absent and the bacilli present, that have lasted four to five 
weeks (one case lasted nearly two months) and then resulted fatally, we see 
a continuation of the same process that is observed in the acute fatal cases. 
The lesions are those of necroses of the mucous membrane and induration of 
the gut.’” 


XV. 
BACTERIA OF PLANT DISEASES. 


I SHALL not attempt to give a full account of the bacteria which 
have been described as bearing an etiological relation to various in- 
fectious diseases of plants, but a “text-book of bacteriology” would 
be incomplete without some reference to the best known of these 
bacteria. In the following descriptions of species I have preferred 
to quote largely from the published papers of Dr. Erwin F. Smith, 
of the Department of Agriculturé, United States, a recognized au- 
thority in the investigation of plant diseases, rather than to rewrite 
his descriptions. 


BACILLUS SOLANACEARUM (Sinith). 


Causes a bacterial disease of the tomato, egg plant, and Irish 
potato. 


‘* Morphology.—A medium-sized bacillus, with rounded ends; often in 
pairs, with a plain constriction ; elliptical, but of variable size, depending on 
age of culture or the length of time the tissues of the plant have been occu- 
pied ; usually one and one-half to three times as long as broad. On cover- 
glass preparations made from peptone beef bouillon cultures forty-eight 
hours old and stained with a watery solution of methyl violet, many are 1.5 
by 0.5 x, but these measurements must not be taken too literally, since the size 
depends not only on the age of the culture but also on the kind of stain em- 
ployed, 7.e., on whether or not the cell wall stains. Organism motile, often 
only sluggishly so, especially when taken from the plant, but sometimes very 
actively motile, especially in young cultures. Flagella much longer than 
the rod; several—exact number and place of attachment not made out 
clearly, owing to imperfect preparations (Van Ermengem’s method), but ap- 
parently arising from any part of the rod. An attempt to stain them by 
Loéffler’s method was unsuccessful. No spores observed either in the plant 
or in culture media, but the search has not been continued long enough to 
warrant any opinion as to their existence. Zodgloea are formed almost from 
the start in fluid culture media. 

“ Symptoms Produced in the Plant.—The first indication of this disease, 
or at least the first one to attract the farmer’s attention, is the sudden wilting 
of the foliage. This may occur first on a single shoot, but finally it affects 
the whole plant. Subsequently, and especially if the plant is young or not 
very woody, the stem shrivels, first changing to a yellowish-green or to a 
muddy green, and finally to brown or black. The vascular bundles become 
brown long before the shrivelling takes place, and in the potato often show 
through the outer green parts of the stem as long, dark streaks, or the bac- 
teria run out on the petioles, after the manner of pear blight, forming nar- 
row, black lines. The vessels of such bundles are filled with the bacilli, 


572 BACTERIA OF PLANT DISEASES. 


which ooze out when the stem is cut across. The foliage may wilt with or 
without a preliminary yellowing. If the bacteria are sharia in the ves- 
sels of the stem, the wilt is often very sudden and the foliage has no time to 
become yellow. The progress of the disease seems to be more rapid in young 
than in old plants and in hot than in cold weather. 

‘In the case of the potato the tubers are also finally attacked and de- 
stroyed, the organism reaching them by way of the vascular bundles of the 
stem, A brown or black rot ensues, beginning in the stem end of the tuber 
in the vascular ring and extending in all directions therefrom. Al] stages 
of this rot of the tubers (both in 1895 and in 1896) were obtained repeatedly 
from pure cultures of the bacillus pricked into the stem several feet above 
ground. 

‘*Boutllon and Peptone Cultiures.—This organism grows well at room 
temperatures of 20° to 30° C., in beef broth peptonized (Witte’s peptonum 
siccum). It seemed to make little difference whether the bouillon was left 
acid or rendered slightly alkaline with carbonate of soda. The gathering of 
the zodglcea in the upper layers of the fluid is very distinct, especially if the 
tubes are left undisturbed in an upright position for some days. On shaking 
the turbidity becomes uniform. The organism produces a copious, dirty 
white precipitate (much more precipitate than B. tracheiphilus). 

‘‘The inoculated tubes of Iitinws milk developed no acid—i.e., showed 
no trace of reddening. After two or three days the litmus became perceptibly 
bluer than in the control tubes, and this bluing increased from day to day, 
indicating a progressing alkalinity. This change took place at room tem- 
peratures of 20° to 30 C., and also in the thermostat at 37° C. The casein 
was not precipitated. 

‘* Gelatin.—In plate cultures of nutrient gelatin the buried colonies are 
circular in outline (globose), yellowish or brownish, granular (under Zeiss 
sixteen millimetres objective and 12 compensating ocular), and with well- 
defined margins. No oblong or spindle-shaped colonies could be found. 
The circular outline and regularity and distinctness of the margin of the 
colony were especially noteworthy. Whether these features will be found 
constant with all gelatins is a question yet to be determined. Occasionally, 
aftei™ few days, a narrow, clear zone appeared around the margin of many of 
these colonies as if liquefaction had setin. This, however, did not progress, 
or increased but very slowly, and was clearly visible only under the compound 
microscope. The buried colonies remained small, as if requiring more 
oxygen than they were able to get. The surface colonies were circular, thin, 
thin-edged, smooth, white, and wet-shining. They did not spread over the 
plate rapidly or cause any liquefaction (fifteen per cent gelatin, temperature 
20° to 27° C.). 

‘“‘The organism grew best in a gelatin of the following composition: 
Lean minced beef, five hundred cubic centimetres ; distilled water, one thou- 
sand cubic centimetres; mixed and set twenty-four hours in a cool place ; 
filtered and added ten grammes of Witte’s peptonum siccum and one hundred 
and fifty grammes of L. and F. gelatin. This gelatin was clarified with ege 
and rendered alkaline with sodium hydrate, titrating with phenolphthalein. 
The degree of alkalinity was between twelve and fourteen of Mr. Fuller's 
scale. 

“‘ Agar.—In poured plates of nutrient agar the buried colonies differed 
considerably from those in gelatin. Instead of being circular with a very 
smooth margin, they were irregularly round or even oblong, with a decidedly 
irregular granular margin. These colonies were brown or yellowish-brown 
under sixteen millimetres objective and 12 ocular. After some weeks the 
whole body of the agar became decidedly brown. No spindle-shaped colonies 
were to be seen The surface colonies grew rather slowly. They were dirty- 
white, smooth, wet-shining, and did not spread widely over the agar. 

‘‘The behavior on potato is very characteristic. In twenty-four to forty- 
eight hours (temperature 27° to 32° C.) the fluid became turbid and the pro- 


BACTERIA OF PLANT DISEASES. 573 


Jecting part of the cylinder was covered with a copious, wet-shining growth. 
-\t first this growth was white or dirty white, but after some days (three to 
ten) it became brown, and finally, in places, nearly or quite black (smoke 
brown is perhaps the proper term). The growth on potato was not wrinkled. 
The substratum and the fluid in the bottom of the tube also became brown. 
The rapidity and the degree of pigmentation seem to depend on the slightly 
varying composition of the potato, apparently on the amount of glucose 
present. No gas was formed in any of the many potato cultures. No acid 
was detected in any stage of the growth of the cultures, not even when tested 
at the end of the first twenty-four hours. The potato cultures, which were 
slightly acid on the start (mormal acidity of the tuber), soon became strongly 
alkaline to litmus paper. With Nessler’s solution the alkaline potato cultures 
gave an immediate, copious, orange-yellow reaction, indicating ammonia. 
These cultures developed a peculiar odor, often noticed in rotting potatoes, 
but not specially disagreeable. This odor was likened by one person to the 
smell of sour bran. Its chemical nature has, not been determined. The 
Sets did not fall into pieces, but retained their shape for several 
weeks. 

‘Gas Production.—No gas appeared in any of the many cultures. The 
organism is not a gas producer. 

‘* Relation to Oxygen.—This bacillus appears to be strictly aérobic. If 
aes uate anaérobic, itis not so with any of the carbohydrates yet 
tested. 

‘* Acids—No acid reaction could be detected in any stage of any of the 
cultures. Potato cultures only twenty-four hours old and which were acid 
on the start (normal acidity of the tuber) gave a decided alkaline reaction to 
litmus paper. If any acid whatever is formed it is masked by the presence 
of alkali and is not butyric acid. 

‘* Alkalies.—This organism is a very vigorous alkali producer. On 
warming the cultures over a gas flame or on placing the blued strips of 
litmus paper on a warm glass plate the alkaline reaction quickly disappears. 
On adding a few drops of Nessler’s reagent, as already stated, a copious 
orange-yellow precipitate is at once developed. This would indicate that at 
least a part of the alkali is due to ammonia. Probably amine bases are-also 

resent. 
. “‘The bacillus grows well in the thermostat at 87° C.—possibly a trifle 
better than outside at summer temperatures ranging from 25° to 32° C. 
Under either condition it grows rapidly. It still grew readily from bouillon 
cultures after several weeks’ exposure to 37°C. (three weeks’ exposure in 
one case, seven weeks’ exposure in another). 

‘* Pigments.—A brown pigment is formed in course of a few days in the 
host plants (potato, tomato, etc.), and in culture media containing grape, 
fruit, or cane sugar (nutrient agar, steamed potato, fermentation tubes). 
This pigment is soluble in water and glycerin. It is insoluble in ethyl alco- 
hol, ether, chloroform, xylol, and carbon bisulphide.” 


“BACILLUS HYACINTHI (Wakker). 


‘« Pseudomonas hyacinthi (Wakker).—A yellow, rod-shaped organism, 
multiplying by fission ; ends rounded ; single, in pairs, or fours, more rarely 
in the form of chains or filaments; motile by means of one polar flagellum. 
In the host plant, when the bundles are crowded full of the yellow slime and 
broken down, it is generally 0.8 to 1.2 by 0.4 to 0.6. In alkaline beef 
broth or on agar it usually measures 1 to 2 by 0.4 to 0.6%, In old cultures 
rich in sugar it often grows out into long, slender chains, or into filaments 
(50 to 100“ long) in which there are no distinct septa. Non-sporiferous. Color 
distinctly yellow, but somewhat variable. Chrome yellow to pale cadmium 
in the host plant, 7.e., bright yellow (Ridgway’s nomenclature of colors). 
On culture media, when not interfered with by the brown pigment, generally 


574 BACTERIA OF PLANT DISEASES. 


gamboge, chrome yellow, or canary yellow, but sometimes paler. Old 
cultures on some media darken from the production of a soluble, pale-brown 
pigment. This feeble brown stain is best developed in hyacinth broth, in 
potato broth with peptone, on turnips, on radishes, and on banana rinds. It 
was not observed in acid or alkaline beef broth, on cocoanut flesh, on sugar 
beets, in nutrient starch jelly, in agar, or in gelatin, with or without sugar. 
This organism grows readily on potato cylinders standing in distilled water, 
but it never becomes copious or fills the water with a solid yellow slime, 
owing to its feeble diastatic action. Potatoes on which it has grown, even for 
several months, always give a strong starch reaction with iodine. It behaves 
the same on nutrient starch jelly free from assimilable sugars. It liquefies 
nutrient gelatin and Léffler’s blood serum, but does so slowly, and will not 
liquefy gelatin at all if ten per cent cane sugar is added. Growth on 
nutrient agar or nutrient starch jelly is inhibited (unless the inoculation be 
from a solid culture and very copious) by the addition of ten per cent 
glycerol, and is greatly retarded by five-per-cent glycerol; even two anda 
half per cent of glycerol retarded growth. Growth in beef broth was much 
retarded by the addition of 1.5-per-cent sodium chloride. Organisms ex- 
tremely sensitive to plant acids, including those of the hyacinth. Aérobic; 
doubtfully, if ever, facultative anaérobic; not a gas producer. Does not 
redden litmus milk, but makes it bluer, and slowly separates the casein from 
the whey by means of a lab ferment. Produces under some circumstances, 
and slowly, asmall amount of non-volatile acid (slime acid?) with various 
sugars (grape, cane, etc.), which acid is frequently obscured by the moderate 
production of alkali. In the presence of air produces an organic acid 
(probably acetic) from ethyl alcohol dissolved in milk or bouillon.. Inverts 
cane sugar, but apparently without the intervention of any enzyme. Will 
not grow on thirty-per-cent grape-sugar agar. Resists dry air very well, z.e., 
more than forty-eight days when spread on cover glasses in thin layers. 

‘*In Dunham’s solution with methylene blue the color is reduced in a 
few days, but re-oxidizes quickly on shaking ; final color (fifty-six days) bright 
blue. In Dunham’s solution with indigo carmine the color changes to a 
bright blue, which persists for a long time; final color yellowish. In Dun- 
ham’s solution with rosolic acid and enough HCl to render the fluid yellow- 
ish, Ps. hyacinthi did not redden the fluid, but made it colorless, the bac- 
terial precipitate becoming rosy or salmon-colored. Produces indol slowly 
in peptonized beef broth and in peptonized Uschinsky’s solution ; does not 
produce nitrites in these solutions. Does not reduce potassium nitrate to 
nitrite in peptonized beef bouillon. Not a strong-smelling germ. Not 
readily mayen by its own decomposition products except in media contain- 
ing alcohol. 

~ “Will not grow in the thermostat at 37° C., and grows very feebly on 
some media and not at all on others at 34° to 35° C. Optimum temperature 
28° to 30° C., or thereabouts. Minimum temperature approximately 4° C. 
Thermal death point (ten minutes’ exposure), 47.50’ C.; nearly all the rods 
are killed at 47° and a great many at 46.50° C. Did not grow at room tem- 
perature after six days’ exposure in alkaline beef broth in the thermostat at 
35° to 36.35°. Does not grow well in Uschinsky’s solution. Grows much 
better in Uschinsky’s solution when peptone is added to it. ..Grows well 
with a bright yellow color on cylinders of steamed cocoanut flesh, standing 
with one end in distilled water. 

‘*Pathogenic to hyacinths. Enters the plant through wounds, through 
the blossoms, etc., and multiplies in the vascular system, filling the vessels, 
especially those of the bulb, with a bright yellow slime consisting of bacteria. | 
The walls of the vessels are destroyed and extensive cavities are formed in 
the bundles. The parenchyma around the bundles is also involved, but only 
very slowly, the organism being a feeble destroyer of cell walls. The host 
plant is not rapidly destroyed, a year or more being necessary. The cells 
are first separated by solution of the middle lamella, but the wall itself seems 


BACTERIA OF PLANT DISEASES. 575 


finally to disappear. The cavities contain innumerable bacteria mingled 
with fragments of the dissolved bundles and of the surrounding parenchyma. 

First described by Dr. J. H. Wakker from the Netherlands, where it 
often causes serious losses in the hyacinth gardens. Not known to occur in 
any other part of the world” (E. F. Smith). 


BACILLUS CAMPESTRIS (Pammel). 


The cause of brown rot in Cruciferous plants. 


_ ‘Pseudomonas canrpestris (Pammel).— Yellow, rod-shaped, motile 
micro-organism. Size and color varying according to substratum, food 
supply, ete. Generally 0.7 to 3.0 by 0.4 to 0.5 # Co.or dull wax yellow or 
canary yellow. Occasionally as bright as light cadmium or as pale as prim- 
rose yellow (Ridgway’s color scale). One polar flagellum. Non-sporiferous, 
so far as known. Pathogenic for various Cruciferous plants, entering and 
dwarfing or destroying the host plant through the vascular system, which 
becomes decidedly brown. Aérobic but, so far as known, not a gas or acid 
producer, 7.e., not facultative anaérobic. Forms cavities around the bundles 
but seems to be only feebly destructive to cellulose. Produces a brown pig- 
ment in the host plant and on steamed Cruciferous substrata, especially the 
turnip. Grows very rapidly on steamed potato cylinders at room tempera- 
tures, but without odor or the formation of any brown pigment. Liquefies 
gelatin. Grows feebly at 7° C., better at 10° C., but still feebly ; grows well 
at 17° to 19° C. ; grows luxuriantly at 21° to 26° C. ; grows very feebly at 37° 
to 38° C. ; will not grow at 40° C.; and is killed by ten minutes’ exposure to 
51°C. Organism closely related to Wakker’s Bacterium hyacinthi, from 
which it differs, so far as I have been able to observe, chiefly in its patho- 
genic properties, its duller yellow color and its higher thermal death point” 
(E. F. Smith). 


PSEUDOMONAS STEWARTI (Smith). 


‘A medium-sized rod rounded at the end and motile by means of one 
polar flagellum, size 0.5 to 0.9 by 1 to 2, no spores observed ; found in enor- 
mous numbers in the vascular bundles of corn (Zea mays) associated with a 
destructive disease of which it is probably the cause; color in the host plant 
and in culture media yellow (buff to chrome or ochre, occasionally a pale, 
dirty yellow) ; aérobic and facultative anaérobic ; grows in all ordinary cul- 
ture media; bears alkali well (soda) and plant acids extremely well; grows 
luxuriantly in Uschinsky’s solution ; growth enormously stimulated by cane 
sugar, grape sugar, and galactose; growth not favored by five-per-cent doses 
of lactose, maltose, dextrin, mannite, or glycerin in nutrient starch jelly ; 
diastatic action feeble, z.e., able to obtain food from starch only with much 
difficulty ; produces alkalies in all sorts of media and acids in the presence of 
grape and cane sugar; reduces litmus slowly; does not liquefy gelatin 
(Stewart); does not liquefy Loffler’s blood serum; grows well at summer 
temperatures of 25° to 30° C. ; does not die out quickly in culture media ; does 
not produce gas; sensitive to light (Stewart); occurs in New York and 
Michigan and may be looked for in all parts of the United States” (EK. F. 
Smith). 

BACILLUS AMYLOVORUS (Burrill). 


Described by Burrill (1880) as the cause of pear blight. Etiologi- 
eal relation to this disease confirmed by Arthur (1884 to 1887) and 
by Waite (1891 to 1895). 


‘Beginning in the spring the germs on the new growth of the season first 
appear on the negative discs of the blossoms. The bacilli live and multiply 
in the nectar and are able to enter the nectar glands without a puncture or 


576 BACTERIA OF PLANT DISEASES. 


injury, and thus normally get inside their hosts. The distribution from 
flower to flower and tree to tree is through the agency of insects, mainly 
flower-visiting source. Infection also occurs on the young shoots and less 
frequently on the fleshy bark through injuries. Insects and birds are agents 
of distribution and inoculation in these cases. No evidence could be found 
that the germs are carried by the wind. The blight germs usually die out in 
the twigs which are blighted and dead, but in certain cases the germs manage 
to keep alive through the summer by making slow progress in the fleshy 
bark. Such cases may succeed in living over winter. Winter weather is 
favorable to the longevity on account of the moisture and low temperature. 
The cases of ‘‘hold over” blights start off again in spring and exude quanti- 
ties of gummy matter full of the bacilli. This is visited by insects, especially 
flies and wasps, and carried on to the newly opened flowers, thus completing 
the life cycle. 

‘* An oval rod-like bacillus 0.6-0.8“ by 1 to6#long. Constant in diameter 
but varying greatly in length. Occurs singly or in young cultures in pairs, 
chains, or masses. Stains readily with the ordinary aniline dies either watery 
or alcoholic solutions. Has no capsule, but is supplied with several flagella 
scattered over the surface. Itis actively motile. Does not produce spores. 
On nutrient beef and potato broth produces first a strong turbidity and a 
slight granular pellicle on the surface, which breaks up and settles to the 
bottom. The color of the mass is milky white on all solid media. 

‘*On agar plates the outside colonies at ordinary temperature (18° to 20° C.) 
reach a diameter of about one millimetre in forty-eight hours, and at the end 
of a week become five to six millimetres across. A temperature of 36° to 
87° C. starts the growth more promptly, but results in a feebler ultimate de- 
velopment. 

“‘The addition of malic or citric acid in small amounts so as to acidify 
the agar, increases the vigor of growth, while an excess of alkali diminishes 
it. On gelatin made from the commercial brands the opposite effect is pro- 
duced. Gelatin should be neutral to phenolphthalein to insure vigorous 
development. There is moderate liquetaction in good gelatin culture. A 
moderate growth is made on sterile potato cylinders. 

‘In the fermentation tube it decomposes sugar without the formation of 
gas. It is most vigorous on maltose, the cultures becoming strongly acid, 
and is slightly less so on cane sugar, dextrose, and levulose. It is aérobic and 
facultative anaérobic. It produces no pigment or coloring matter of any sort, 
and no odor. It does not decompose starch. Its principal food consists of 
nitrogenous matter, sugars, and probably, to some extent, certain organic 
acids, the very substances which occur in vigorous, young, growing tissues 
of the host. Certain statements formerly made are now known to be erro- 
neous. 

‘The germ mass is said to be yellowish-white on potato. This could 
only come from an impure culture, as the true pear-blight germ is always 
white. Gas, in some places COs, is said to be formed. This never oc- 
curs. Butyric acid is said to be one of the products of its decomposition. 
The germ produces acid but never butyric. Starch is said to be decomposed 
and used as a food, but so far we have never been able to demonstrate this. 
The germ is said to live over winter in the soil. In our search we have 
failed to find itin such places, and its life cycle is complete without it” (Waite). 


BACILLUS TRACHEIPHILUS (Smith). 
The cause of ‘‘ wilt ’’ in various species of Cucurbitacee—cucum- 


bers and melons. 


‘‘Bacilli, often two or three times as long as broad, of medium size; soli- 
tary or in pairs, occasionally in chains of four. The dimensions vary greatly 
in the infected plant; many rods are 1.2 to 2.5 » long by 0.5 to 0.7 » broad 


BACTERIA OF PLANT DISEASES. 577 


In cultures the dimensions vary still more. In recent cultures the bacilli 
exhibit active movements, which are soon lost. The bacilli are often asso- 
ciated in viscous masses, forming milk-white drops, which when touched 
with a platinum needle may be drawn out into long threads. This viscosity 
appears to be due to a swollen and partially liquefied capsule, which may be 
demonstrated under the microscope in stained or unstained preparations. 
Does not form spores. Grows in bouillon, Dunham’s solution, ete. Does 
not form a surface film ora deposit at the bottom of the test tube, but the 
culture medium is slightly clouded. Grows very slowly or not at all in gela- 
tin and does not liquefy. Upon agar-agar it grows as a thin, smooth, milk- 
‘white, sticky layer, which extends only a short distance from the point of 
inoculation. In stab cultures it grows all along the line of puncture, form- 
ing, after a time, finger-like projections, which under a lens are seen to be 
finely granular. Upon potato it forms a thin, smooth, white, moist-looking 
layer, which only extends a short distance from the line of inoculation. 
The color of the growth resembles that of the potato, and is much whiter 
than that of most bacteria. It produces no pigment and causes no change 
in the color of the potato. In culture solutions containing dextrose, saccha- 
rose, lactose, or maltose no gas is developed. It does not cause coagulation 
of milk. It grows best in alkaline media. It is destroyed by a temperature 
of 43° C. maintained for ten minutes. Cultures in liquid media or on potato 
usually die out within three weeks. It stains best with carbol-fuchsin solu- 
tion. In properly stained preparations it is seen to have a capsule and 
flagella—in some bacilli one flagellum at each extremity of the rod, while in 
others there are more” (Smith). 


37 


XVI. 


PATHOGENIC ANAEROBIC BACILLI. 


STRICTLY anaérobic bacilli are not able to multiply in the blood 
of living animals; but some of them may multiply in the subcuta- 
neous connective tissue or in the muscles, when introduced by in- 
oculation, and are pathogenic because of the local inflammatory or 
necrotic processes to which they give rise, or because they produce 
soluble toxic substances which are absorbed and cause death by 
their special action upon the nervous system or by general toxemia, 


BACILLUS TETANI. 


Synonyms.—The bacillus of tetanus ; Tetanusbacillus, Ger. 

Nicolaier (1884) produced tetanus in mice and rabbits by intro- 
ducing garden earth beneath their skin, and showed that the disease 
might be transmitted to other animals by inoculations with pus or 
cultures in blood serum containing the tetanus bacillus, which, how- 
ever, he did not succeed in obtaining in pure cultures. Carle and 
Rattone (1884) showed that tetanus is an infectious disease, which 
may be transmitted by inoculation from man to lower animals—a 
fact which has since been verified by the experiments of Rosenbach 
and others. Obtained in pure cultures by Kitasato (1889). 

The writer produced tetanus in a rabbit in 1880 by injecting be- 
neath its skin a little mud from the street gutters in New Orleans. 
The tetanus bacillus appears to be a widely distributed microérgan- 
ism in the superficial layers of the soil in temperate and especially in 
tropical regions. In Nicolaier’s experiments it was not found in soil 
from forests or in the deeper layers of garden earth. 

Morphology.—Slender, straight bacilli, with rounded ends, 
which may grow out into long filaments. Spores are developed at 
one extremity of the bacilli, which are spherical in form and consid- 
erably greater in diameter than the rods themselves, giving the 
spore-bearing bacilli the shape of a pin. 

Stains with the usual aniline colors and also by Gram’s method. 
The method of Ziehl may be employed for double-staining bacilli and 
spores, 


PATHOGENIC ANABROBIC BACILLI, 579 


Biological Characters.—An anaérobic, liquefying, motile 
bacillus. Forms spores. Grows at the room temperature, in the 
absence of oxygen, in the usual culture media. Grows best at a 
temperature of 36° to 38° C.; in nutrient gelatin, at 20° to 25° C., 
development is first seen at the end of three or four days ; does not 
grow at a temperature below 14° C. Spores are formed in cultures 
kept in the incubating oven at 36° C., at the end of thirty hours ; 
in gelatin cultures at 20° to 25° C., at the end of a week (Kitasato). 
The bacilli exhibit voluntary movements which are not very active ; 
those containing spores are not motile. It may be cultivated in an 
atmosphere of hydrogen, but does not grow in the presence of oxy- 
gen—strictly anaérobic—or in an atmosphere of carbon dioxide. 
The addition of one and one-half to two per cent of grape sugar to 
nutrient agar or gelatin causes the development to be more rapid 


Fie. 159. Fie. 160, 


Fie. 159.—Tetanus bacillus, from a gelatin culture. x 1,000. From a photomicrograph by 
Pfeiffer. 

Fria. 160.—Tetanusbacillus, from an agar culture ; spore-bearing rods. x 1,000. From a photo- 
micrograph by Pfeiffer. 


and abundant. The culture medium should have a feebly alkaline 
reaction. 

Colonies in gelatin plates, in an atmosphere of hydrogen, re- 
semble somewhat colonies of Bacillus subtilis, the opaque central 
portion being surrounded by a circle of diverging rays ; liquefaction 
is, however, much slower, and the resemblance is lost after a short 
time. Older colonies resemble the colonies of certain microscopic 
fungi, being made up of diverging rays. In long gelatin stab cul- 
tures development occurs along the line of puncture, at a consid- 
erable distance below the surface, in the form of a radiate out- 
growth ; the gelatin is slowly liquefied, and asmall amount of gas is 
at the same time formed. In peptonized bouillon having a slightly 
alkaline reaction, under hydrogen gas, the development is abundant 


580 PATHOGENIC ANAKROBIC BACILLI. 


and the cultures give off a characteristic odor— brenzlichen Ge- 
ruch ” (Kitasato). 

According to Kitasato, blood serum is not a very favorable me- 
dium for the growth of the tetanus bacillus, and—contrary to the 
statement of Kitt, Tizzoni, and others— 
it does not cause liquefaction of this 
medium. 

The spores of the tetanus bacillus re- 
tain their vitality for months in a desic- 
cated condition, and are not destroyed in 
two and one-half months when present 
in putrefying material (Turco). They 
withstand a temperature of 80° C. main- 
tained for an hour, but are killed by 
five minutes’ exposure to steam at 100° C. 
They are not destroyed in ten hours by 
a five-per-cent solution of carbolic acid, 
but did not grow after fifteen hours’ ex- 
posure in the same solution. A five- 
per-cent solution of carbolic acid, to 
which 0.5 per cent of hydrochloric acid 
has been added, destroys them in two 
hours ; in sublimate solution containing 
1:1,000 of mercuric chloride they are 
destroyed at the end of three hours, or 
in thirty minutes when 0.5 per cent of 
hydrochloric acid is added to the solu- 
tion. Kitasato succeeded in obtaining 
pure cultures from the pus formed in 

Fie. 161.—Culture of Bacillus tetani the vicinity of inoculation wounds, by 
in nutrient gelatin. (Kitasato.) destroying the associated bacilli after 
the tetanus bacilli had formed spores. 
This was effected by heating cultures from this source for about an 
hour at a temperature of 80°C. The spores of the tetanus bacillus 
survived this exposure, and colonies were obtained from them. in flat 
flasks especially devised for anaérobic cultures ; from these colonies 
pure cultures in nutrient agar or gelatin—long stick cultures—or in 
peptonized bouillon were easily obtained. 


BACILLUS CEDEMATIS MALIGNI. 


Synonyms.—Bacillus of malignant oedema; Vibrion septique 
(Pasteur). 
Discovered by Pasteur (1877); carefully studied by Koch (1881). 


PATHOGENIC ANAEROBIC BACILLI. 581 


This bacillus is widely distributed, being found in the superficial 
layers of the soil, in dust, in putrefying substances, in the blood of 
animals which have been suffocated (by invasion from the intestine), 
in foul water, etc. 

It may usually be obtained by introducing beneath the skin of a 
rabbit or a guinea-pig a small quantity of garden earth. The animal 
dies within a day or two, and this bacillus is found in the bloody 
serum effused in the subcutaneous connective tissue for a consider- 
able distance about the point of inoculation. 

Morphology.—Bacilli from 3 to 3.5 # long and 1 to 1.1 « broad; 


Fie. 162.—Bacillus cedematis maligni, from subcutaneous connective tissue of inoculated 
guinea-pig. x 950. (Baumgarten.) 


frequently united in pairs, or chains of three elements ; may grow 
out into long filaments 15 to 40 « long—these are straight, or bent 
at an angle, or more or less curved. They resemble the bacillus of 
anthrax, but are not quite as broad, have 

rounded ends, and in stained preparations 

\ = on the long filaments are not segmented as is 
X ( the case with the anthrax bacillus. By 

00 § Léffler’s method of staining they are seen to 
KS have flagella arranged around the periphery 
of the cells. Large, oval spores may be de- 

my veloped in the bacilli (not in the long fila- 

fia. tie eas elem ments), which are of greater diameter than 
tis maligni, from an agarcul. the rods, and produce a terminal or central 


ture, showing spores. 1,000 gwelling of the same, according to the loca- 
From a _ photomicrograph, 


(Frinkel and Pfeiffer.) tion of the spore. 
Stains readily by the aniline colors usu- 


ally employed, but is decolorized when treated by Gram’s method. 


C= 


o 
4 


582 PATHOGENIC ANAEROBIC BACILLI. 


In stained preparations the long filaments may present a somewhat 
granular appearance from unequal action of the staining agent. 

Biological Characters.—A strictly anaérobic, liquefying, mo- 
tule bacillus. Forms spores. Grows in the usual culture media 
when oxygen is excluded—in an atmosphere of hydrogen. Grows 
at the room temperature—better in the incubating oven at 37° C. 
The spores are formed most abundantly in cultures kept in the in- 
cubating oven, but may also be formed at a temperature of 20° C. 
In the bodies of animals which succumb to an experimental inocula- 
tion no spores are found immediately 
after death, but the bacilli multiply rap- 
idly in the cadaver, and form spores 
when the temperature is favorable. 

The malignant-cedema bacillus may 
be cultivated in ordinary nutrient gela- 
tin, but its development is more abun- 
dant when one to two per cent of grape 
sugar has been added to the culture 
medium. In deep stab cultures in this 
medium development occurs at first only 
near the bottom of the line of puncture ; 
the gelatin is liquefied and has a grayish- 
white, clouded appearance ; an abundant 
development of gas occurs, and as this 

Fre. 164.—Bacillus cedematis ma. @ccumulates the growth and liquefaction 
ligni, culturesin nutrient gelatin; a, of the gelatin extend upward. <A very 
tae Leapilian naire pinnae bot- characteristic appearance is obtained 

when the bacilli are mixed in a test 
tube with gelatin which has been liquefied by heat, and which is then 
allowed to solidify. Spherical colonies are developed, in the course 
of two or three days, in the lower portion of the gelatin ; these are 
filled with liquefied gelatin of a grayish-white color, and when ex- 
amined with a low power are seen to be permeated with a network 
of filaments, while the periphery presents a radiate appearance. In 
nutrient agar growth also occurs at the bottom of a deep punc- 
ture ; it has an irregular, jagged outline and a granular appearance; 
the considerable development at. the deepest portion and gradual 
thinning out above give the growth a club shape ; in the incubating 
oven there is an abundant development of gas, which often splits up 
the agar medium and forces the upper portion against the cotton 
stopper. An abundant development of gas also occurs in cultures 
in blood serum, and the medium is rapidly liquefied ; at a tempera- 
ture of 37° it is changed in a few days toa yellowish fluid, at the 
bottom of which some irregular, corroded fragments of the solidified 


PATHOGENIC ANABROBIC BACILLI. 583 


serum may be seen. In agar plates, placed in a close receptacle 
from which oxygen is excluded, cloudy, dull-white colonies are 
formed which have irregular outlines and under the microscope 
are seen to be made up of branching and interlaced filaments radi- 
ating from the centre. Cultures of the malignant-cedema bacillus 
give off a peculiar, disagreeable odor, which cannot, however, be 
designated as ‘‘ putrefactive.” 

Pathogenesis.—Pathogenic for mice, guinea-pigs, rabbits, and, 
according to Kitt, for horses, dogs, goats, sheep, calves, pigs, chick- 
ens, and pigeons. According to Arloing and to Chauveau, cattle are 
immune. The disease is rarely developed except as a result of ex- 
perimental inoculations, but horses occasionally have malignant 
cedema from accidental inoculation, and cases have been reported 
in man—‘‘ gangréne gazeuse.” A small quantity of a pure cul- 
ture injected beneath the skin of a susceptible animal gives rise to 
an extensive inflammatory cedema of the subcutaneous connective 
tissue and of the superficial muscles, which extends from the point 
of inoculation, especially towards the more dependent portions of 
the body. The bloody serum effused is without odor and contains 
little if any gas. But when malignant edema results from the in- 
troduction of a little garden earth beneath the skin of a guinea-pig or 
other susceptible animal, the effused serum is frothy and has a pu- 
trefactive odor, no doubt from the presence of associated bacteria. 
Injections into the circulation do not give rise to malignant cedema, 
unless at the same time some bacilli are thrown into the connective 
tissue. While small animals usually die from an experimental in- 
oculation with a moderately small quantity of a pure culture, larger 
ones (dogs, sheep) frequently recover. At the autopsy, if made at 
once, the bacilli are found in great numbers in the effused serum, 
but not in blood from the heart or in preparations made from the 
parenchyma of the various organs; later they may be found in all 
parts of the body as a result of post-mortem multiplication. This 
applies to rabbits and to guinea-pigs, but not to mice ; in these little 
animals the bacilli may find their way into the blood during the last 
hours of life, and their presence may be demonstrated in smear prepa- 
rations of blood from the heart or from the parenchyma of the spleen 
or liver. In mice the spleen is considerably enlarged, dark in color, 
and softened ; in rabbits and guinea-pigs less so. With this excep- 
tion the internal organs present no very notable pathological changes. 

Animals which recover from malignant cedema are said to be 
subsequently immune (Arloing and Chauveai). Roux and Cham- 
berlain have shown that immunity may be induced in guinea-pigs by 
injecting filtered cultures of the malignant-cedema bacillus (about 
one hundred cubic centimetres of a bouillon culture in three doses) 


584 PATHOGENIC ANAEROBIC BACILLI. 


into the abdominal cavity; or, better still, by the injection of fil- 
tered serum from animals which have recently succumbed to an ex- 
perimental inoculation (one cubic centimetre repeated daily for seven 
or eight days). 

BACILLUS CADAVERIS. 


Obtained by the writer (1839) from pieces of liver and kidney, from yel- 
low-fever cadavers, which had been preserved for forty-eight hours in an 
antiseptic wrapping, at the summer temperature of Havana; also in two 


Fia. 165.—Bacillus cadaveris; smear preparation from liver of yellow-fever cadaver, kept 
twenty-four hours in an antiseptic wrapping. x 1,000, Froma photomicrograph. (Sternberg.) 


cases from pieces of yellow-fever liver immediately after the autopsy; also 
from liver preserved in an antiseptic wrapping from comparative autopsies 
made in Baltimore. 

Morphology.—Large bacilli with 
square or slightly rounded corners, 
from 1.5 to 4“ in length and about 
1.2 » broad; frequently associated in 
pairs; may grow out into straight or 
slightly curved filaments of from 5 
to 15 » in length. 

Biological Characters.—An an- 
aérobic, non-motile bacillus; not 
cultivated in nutrient gelatin; not 
observed to form spores. 

Bacillus cadaveris is a strict anaé- 
robic and is difficult to cultivate. I 
have succeeded best with nutrient 
agar containing five per cent of 
glycerin, removing the oxygen 
thoroughly by passing a stream of 
hydrogen through the liquefied me- 
dium. The colonies in a glycerin- 
Fre. 166,—Bacillus cadaveris, from ananaé- agar roll tube (containing hydrogen 
robic culturein glycerin-agar. 1,000, From and hermetically sealed) are opaque, 
a photomicrograph. (Sternberg.) irregular in outline, granular, and of 


PATHOGENIC ANAEROBIC BACILLI. 585 


a white color by reflected light. The culture medium acquires an acid re- 
action as a result of the development of the bacillus. ; ‘ 

Liver tissue containing this bacillus, after having been kept in an anti- 
septic wrapping for forty-eight hours, has a fresh appearance, a very acid re- 
action, and is without any putrefactive odor. 

Pathogenesis.—Liver tissue containing this bacillus is very pathogenic 
for guinea-pigs when injected subcutaneously, and causes an extensive in- 
flammatory edema extending from the point of inoculation. Pure cul- 
tures of the bacillus are less pathogenic, and the few experiments which I 
made in Havana gave a somewhat contradictory result, recovery having 
occurred in one guinea-pig which received a subcutaneous injection of ten 
minims of liquid from an anaérobic culture in glycerin-agar, while another 
died at the end of twenty hours from a subcutaneous injection of three 
apse ta extensive inflammatory cedema in the vicinity of the point of 
inoculation. 


BACILLUS OF SYMPTOMATIC ANTHRAX. 


Synonyms.—Rauschbrandbacillus, Ger. ; Bacille du charbon 
symptomatique, Fr. 

First described by Bollinger and Feser (1878); carefully studied 
and its principal characters determined by Arloing, Cornevin, and 
Thomas (1880-83). 


Fie. 167. Fic. 168. 


Fia. 167.—Bacillus of symptomatic anthrax, from an agar culture. x 1,000. From a photomi- 
erograph. (Frankel and Pfeiffer.) 

Fig. 168.—Bacillus of symptomatic anthrax, from muscles of inoculated guinea-pig. From a 
photomicrograph. (Roux.) 


Found in the affected tissues of animals—principally cattle—suf- 
fering from ‘‘ black leg,” “‘ quarter evil,” or symptomatic anthrax (Fr., 
‘‘charbon symptomatique”; Ger., ‘‘ Rauschbrand”). The disease 


586 PATHOGENIC ANAEROBIC BACILLI. 


prevails during the summer months in various parts of Europe, and 
is characterized by the appearance of irregular, emphysematous 
swellings of the subcutaneous tissue and muscles, especially over the 
quarters, hence the name “quarter evil.” The muscles in the 
affected areas have adark color and contain a bloody serum in 
which the bacillus is found. 

Morphology.—Bacilli with rounded ends, from three to five “ 
long and 0.5 to 0.6 4 broad ; sometimes united in pairs, but do not 
grow out into filaments. The spores are oval, somewhat flattened on 
one side, thicker than the bacilli, and lie near the middle of the rods, 
but a little nearer to one extremity. The bacilli containing spores 
are somewhat spindle-formed (Kitasato). ‘‘Involution forms” are 
quite common in old cultures or in unfavorable 
media ; in such cultures variously distorted and 
often greatly enlarged bacilli may be seen, some 
being greatly swollen in the middle — spindle- 
shaped. When properly stained, by Léffler’s 
method, a number of flagella are seen around the 
periphery of the cells. 

Stains with the aniline colors usually em- 
ployed, but not by Gram’s method. Spore-bear- 
ing bacilli may be double-stained by first stain- 
ing the spores by Ziehl’s method, and then the 
bacilli with a solution of methylene blue. 

Biological Characters.—An anaérobic, lig- 
uefying, motile bacillus. Forms spores. Grows 
at the room temperature in the usual culture media, 
in the absence of oxygen, in an atmosphere of hy- 
drogen, but not in carbon dioxide. This bacillus 
grows more rapidly and abundantly in nutrient 
agar or gelatin to which 1.5 to 2 per cent of 
grape sugar or five per cent of glycerin has been 
added. Colonies in gelatin, in an atmosphere of 
hydrogen, are at first spherical, with irregular out- 
lines and a wart-like surface ; later the gelatin is 

Fic. 160, Bacillus /iquefied around them, and radiating filaments 
‘of symptomatic an- grow out into the gelatin, so that by transmitted 
eee a ie light they present the appearance of an opaque 
tin, ten days at 18°- central mass with an irregular surface surrounded 
20°C, Kitasato.) by rays. In stab cultures in nutrient gelatin, at 

20° to 25° C., at the end of two or three days 
development occurs at the bottom of the line of puncture to within 
about two fingers’ breadth of the surface; the gelatin is slowly 
liquefied and considerable gas is formed. In old cultures the 


PATHOGENIC ANABROBIC BACILLI. 587 


growth and liquefaction of the gelatin extend nearly to the sur- 
face. In agar stab cultures, in the incubating oven, develop- 
ment begins within a day or two and extends to within one 
finger’s breadth of the surface; considerable gas is evolved, and 
the cultures have a peculiar, acid, penetrating odor. Development 
is most rapid at 36° to 38° C., but may occur at a temperature of 16° 
to 18° C.—not lower than 14°. Spores are quickly formed in cul- 
tures kept in the incubating oven—not so quickly at the room tem- 
perature. These withstand a temperature of 80° C. maintained for 
an hour, but are killed in five minutes by a temperature of 100° C. 
(in steam). In the bodies of infected animals spores are not formed 
until after the death of the animal, at the end of twenty-four to forty- 
eight hours (Kitasato). 

The spores are destroyed by a five-per-cent solution of carbolic 
acid in ten hours, and the bacilli, in the absence of spores, in five 
minutes ; a 1:1,000 solution of mercuric chloride destroys the spores 
in two hours (Kitasato). According to Kitasato, certain shining 
bodies of irregular form, which stain readily with the aniline colors, 
are to be seen in the rods as they are found in the bloody serum from 
an animal recently dead ; but these are not spores, as some bacterio- 
logists have supposed. 

Pathogenests.—Cattle, which are immune against malignant 
cedema, are most subject to infection by the bacillus of symptomatic 
anthrax, and the disease produced by this anaérobic bacillus prevails 
almost entirely among them ; horses are not attacked spontaneously 
—1.e., by accidental infection—and when inoculated with a culture of 
this bacillus present only a limited local reaction. Swine, dogs, rab- 
bits, fowls, and pigeons have but slight susceptibility, but the re- 
searches of Arloing, Cornevin, and Thomas, and of Roger show that 
by the addition of a twenty-per-cent solution of lactic acid to a cul- 
ture its virulence is greatly increased, and animals which have but 
little susceptibility, like the rabbit or the mouse, succumb to such in- 
jections ; similar results were obtained by Roger by the simultaneous 
injection of sterilized or non-sterilized cultures of Bacillus prodigiosus 
or of Proteus vulgaris. 

Klein (1894) has obtained from the spleen of sheep a bacillus 
which corresponds with the bacillus of malignant cedema in every 
respect, except that it proved to be without pathogenic power—“a 
non-virulent variety of the Rauschbrand bacillus” (Klein), 


BACILLUS C2DEMATIS MALIGNI NO. 1 (Novy). 


Obtained by Novy (1894) from the subcutaneous cedema in guinea-pigs 
which were inoculated with a solution of milk-nuclein, which had been pre- 
pared from fresh casein. _ 

Morphology.—Bacilli with rounded ends, usually solitary, from 2.5 to 


588 PATHOGENIC ANAEROBIC BACILLI. 


5 # long and from 0.8 to 0.9 u broad. Occasionally short and straight fila- 
ments, 8 to 14 » long, are seen—very rarely these reach a length of 22 to 35 y. 
Long and slender spiral filaments are found in pure cultures which are be- 
lieved to be gigantic flagella. These are seen in preparations stained with 
gentian violet as unstained spiral filaments, usually from 17 to 25 « long ; 
some are of uniform thickness and others spindle-formed, having a thickness 
of 1.7 to 2.6 “ in the middle, and tapering to a scarcely visible line at the ex- 
tremities. These flagella are readily stained by Léffler’s method. They are 
attached to the periphery of the rods, asin the typhoid bacillus. In artifi- 
cial cultures they are usually from 40 to 50 » long. With reference to the 
peculiar spindle-formed bodies found in the cultures Novy says: ‘‘ As to the 
character of these gigantic flagella little can be said. Ld6ffler, who, so far as 
I know, was the first to observe these singular forms, regarded them as bun- 
dles or collections of flagella.” 

Although at first inclined to doubt this, Novy says, in a postscript to his 
paper, that an examination of photo-micrographs, which had been made to 
accompany it, convinces him that Loffler’s explanation is probably correct. 

Biological Characters.—An anaérobic, motile bacillus. The motions are 
not active, but consist in a very moderate to-and-fro swinging motion. Does 
not form spores. Does not grow at the room temperature. Grows at tem- 
peratures of 24° to 38° C. The best media for its development are slightly 
alkaline bouillon, gelatin, or agar, containing two per cent of glucose. May 
be cultivated in a vacuum or in an atmosphere of hydrogen, carbon dioxide, 
or illuminating gas. Also in long stick cultures in agar. In glucose-agar 
plates colonies develop in fifteen hours at 38°C. These appear as small, 
white masses the size of a pin’s head, which, under the microscope, appear 
to be made up of thickly felted threads. The smaller colonies appear asa 
network of branching lires, very similar to the colonies of the tetanus ba- 
cillus ; larger colonies have a dark centre, withan irregular, fringed margin, 
and are surrounded by delicate filaments. In glucose-agar stab cultures 
growth occurs along the line of puncture to within one cubic centimetre of 
the surface, but is not as abundaat as the growth of the bacillus of malig- 
nant oedema or of symptomatic anthrax. At38°C. development occurs within 
twelve to sixteen hours, and has reached its maximum at the end of twenty- 
four hours. An abundant development of gas occurs, which splits up the 
agar and forces the upper portion towards the top of the tube. The develop- 
ment of gas is most abundant in alkaline media, being almost absent in media 
having a neutral or acid reaction. The most favorable medium is a fresh al- 
kaline bouillon containing two per cent of gelatin, of glucose, and of pep- 
tone. 

Pathogenesis.—Pathogenic for rabbits, guinea-pigs, white mice, white rats, 
pigeons, and cats. Death usually results in from twelve to thirty-six hours 
after the subcutaneous injection of one-tenth to one-fourth cubic centimetre 
of a pureculture. At the autopsy an extensive subcutaneous cedema is found 
extending from the point of inoculation. The fluid in the brawny connective 
tissue is usually colorless, sometimes of a pale-red color. A small amount of 
gas is commonly present. The pleural cavities contain an enormous amount. 
of serous exudate, which at first is fluid, but when the autopsy is delayed be- 
comes gelatinous. In rabbits and guinea-pigs the amount of this serum ob- 
tained from the pleural cavities may be from fifty to sixty cubic centimetres. 
The bacilli are usually not very numerous in this serum from the subcuta- 
neous tissues and pleural cavity. 

Kerry (1894) has described a ‘‘ new pathogenic anaérobic bacillus” which 
resembles that of Novy in several particulars. It does not grow at the room 
temperature, does not form spores, and is pathogenic for mice, rats, rabbits, 
and guinea-pigs ; it forms ‘‘ very long and thick flagella, which may be spir- 
alig gechlingelt.” This bacillus was obtained from a guinea-pig inoculated 
with dried blood (suspended in water containing lactic acid and glucose) 
which had been obtained from a cow that was supposed to have died of 
Rauschbrand. 


PATHOGENIC ANAEROBIC BACILLI. 589 


BACILLUS AEROGENES CAPSULATUS. 


Found by Welch in the blood vessels of a patient with thoracic aneurism 
opening externally; autopsy made in cool weather eight hours after death— 
the vessels found full of gas bubbles. 

Morphology.—Straight or slightly curved bacilli with slightly rounded 
or sometimes square-cut ends; a little thicker than Bacillus anthracis, and 
varying in length—average length 8 to 6 w; long threads and chains are oc- 
casionally seen. The bacilli, both from cultures and in the animal body, are 
enclosed in a transparent capsule. 

Biological Characters.—An anaérobic, non-motile, non-liquefying ba- 
cillus. Does not form spores. Grows in the usual culture media, in the ab- 
sence of oxygen, at the room temperature, and produces an abundant de- 
velopment of gas inall.. In nutrient gelatin there is no marked liquefaction, 
but the gelatin is slightly peptonized. In agar, colonies are developed which 
are usually one to two millimetres in diameter, but may attain a diameter of 
one centimetre; they are grayish-white in color and in the form of flattened 
spheres, ovals, or irregular masses, beset with little projections or hair-like 
processes. Bouillon is rendered diffusely cloudy, with an abundant white 
sediment. Milk is coagulated in one or two days. The culturesin agar and 
bouillon have a faint odor, comparable to that of stale glue. Upon potatoa 
pale grayish-white layer is developed; growth occurs at 18° to 20° C., but is 
much more rapid at 80° to 87° C. Bouillon cultures are sterilized by ex- 
posure to a temperature of 58° C. for ten minutes. : 

Pathogenesis.—‘' Quantities up to 2.5 cubic centimetres of fresh bouillon 
cultures were injected into the circulation of rabbits without any apparent 
effect, except in one instance in which a pregnant rabbit was killed, by the 
injection of one cubic centimetre, in twenty-one hours. If the animal is 
killed shortly after the injection the bacilli develop rapidly after death, with 
an abundant formation of gas in the blood vessels and organs, especially the 
liver. At temperatures of 18° to 20° C. the vessels, organs, and serous cavi- 
ties may be full of gas in eighteen to twenty-four hours, and at tempera- 
tures of 30° to 32° C. in four to six hours, when one cubic centimetre of a 
bouillon culture has been injected into the circulation shortly before death.” 

It is suggested by Welch and Nuttall that in some of the cases in 
‘which death has been attributed to the entrance of air into the veins, the gas 
found at the autopsy may not have been atmospheric air, but may have been 
produced by this or some similar microdrganisiu entering the circulation and 
developing after death. 


In a paper published in the Bulletin of the Johns Hopkins Hos- 
pital (September, 1900) Professor Welch says: “Our further studies 
of the gas bacillus obtained from different sources have shown a 
moderate range of variation in some of its properties. This is true 
especially of spore formation, rapidity of liquefaction of gelatin, 
presence of capsules, and virulence.” 

This bacillus has been shown by recent researches to be widely 
distributed in nature, its natural habitat being the intestinal canal of 
man and lower animals and the soil. It has considerable importance 
in human pathology, having been found in various localized infec- 
tious processes in the subcutaneous tissues, the uterus, the urinary 
tract, the liver, the lungs, and the pleural cavities. 


XVII. 
PATHOGENIC SPIRILLA. 


SPIRILLUM OBERMEIERI. 


Synonyms.—Spirochete Obermeieri; Spirillum of relapsing fe- 
ver ; Die Recurrensspirochate. 

Discovered by Obermeier (1873) in the blood of persons suffering 
from relapsing fever. 

This spirillum is present, in very great numbers, in the blood of 
relapsing-fever patients during the febrile paroxysms. It has not 
been found under any other circumstances, and its etiological rela- 
tion to the disease with which it is associated is generally admitted. 

Morphology.—Very slender, flexible, spiral or wavy filaments, 
with pointed ends; from sixteen to forty “in length and consider- 
ably thinner than the cholera spirillum—about 0.1 4. Koch has 
demonstrated the presence of flagella (Eisenberg). 

Stains readily with the aniline colors, especially with fuchsin, 
Bismarck brown, and in Léffler’s solution of methylene blue. 

Biological Characters.—An aérobic, motile spirillum which 
has not been cultivated in artificial media. This spirillum appears to 
be a strict parasite, whose habitat is the blood of man. The disap- 
pearance of the parasite from the blood soon after the termination 
of a febrile paroxysm, and its reappearance during subsequent par- 
oxysms, have led to the inference that it must form spores, but this 
has not been demonstrated. In fresh preparations from the blood 
the spirillum exhibits active progressive movements, accompanied 
by very rapid rotation in the long axis of the spiral filaments, or by 
undulatory movements. The movements are so vigorous that the 
comparatively large red blood corpuscles are seen, under the micro- 
scope, to be thrown about by the slender spiral filaments, which it is 
difficult to see in unstained preparations. When preserved in a one- 
half-per-cent salt solution they continue to exhibit active movements 
for a considerable time. Efforts to cultivate this spirillum in artificial 
media have thus far been unsuccessful, although Koch has observed 
an increase in the length of the spirilla and the formation of a 
tangled mass of filaments. 


ERG'S BACTERIOLOGY. 


% £ es 
Sipe 


ey Sew sere 
HENS ON CK 
Seo Sesh 


PATHOGENIC SPIRILLA. 591 


In experiments made by Heydenreich the spirillum was found to 
preserve its vitality (motility) for fourteen days at a temperature of. 


Fie. 170.—Spirillum Obermeieri in blood of maa. x 1,000. From a pbotomicrograph.. 
(Frankel and Pfeiffer. ) 


16° to 22° C., for twenty hours at 37°, and at 42.5° for two or three 


hours only. 
Pathogenesis.—Causes in man the disease known as relapsing 


fever. Miinch and Moczutkowsky have produced typical relapsing: 


Fic. 171.—Spirillum Obermeieri in blood of an inoculated ape. x 700. (Koch. 


fever in healthy persons by inoculating them with blood containing 
the spirillum of Obermeier. The spirilla are found in the blood dur- 
ing the febrile paroxysm, and for a day or two, at the outside, after 


592 PATHOGENIC SPIRILLA. 


its termination ; sometimes they are present in great numbers, and 
at others can only be found by searching several microscopic fields; 
they are not present in the various secretions—urine, sweat, saliva, 
etc. In fatal cases the principal pathological changes are found in 
the spleen, which is greatly enlarged, and in the liver and marrow 
of the bones, which contain inflammatory and necrotic foci. Koch 
and Carter have succeeded in transmitting the disease to monkeys - 
by subcutaneous inoculations with small amounts of defibrinated 
blood containing the spirillum. After an incubation period of seve- 
ral days typical febrile paroxysms were developed, during which 
the actively motile spirilla were found in the blood in large numbers. 
Blood from one animal, taken during the attack, induced a similar 
febrile paroxysm when inoculated into another of the same species— 
relapses, such as characterize the disease in man, were not observed. 
One attack did not preserve the animals experimented upon from a 
similar attack when they were again inoculated after an interval of 
a few days. Soudakewitch (1891) has made successful inoculation 
experiments in monkeys, and has shown thatin monkeys from which 
the spleen has previously been removed the spirilla continue to 
multiply very abundantly in the blood and the disease has a fatal 
termination, whereas in monkeys from which the spleen has not been 
removed the spirilla disappear from the blood within a few days 
after the access of the febrile paroxysm and the animal recovers. 


SPIRILLUM ANSERUM. 


Synonym.—Spirocheta anserina (Sakharoff). 

Obtained by Sakharoff (1890) from the blood of geese affected by a fatal 
form of septicemia due to this spirillum. This disease prevails among geese 
in Caucasia, especially in swampy regions, appearing annually and destroy- 
ing a large number of the domestic geese. 

Morphology.—Resembles the spirillum of relapsing fever. The long and 
flexible spiral filaments, when the disease is at its height, are often seen in 
interlaced masses, around the margins of which radiate single filaments 
which by their movements cause the whole mass to change its place, as if it 
were a Single organism. These masses are sometimes so large that a single 
‘one occupies the entire field of the microscope. 

Stains with the usual aniline colors. 

Biological Characters.—An aérobic, motile spirillum. Not cultivated 
in artificial media. The movements-are very active, resembling those of 
Spirillum Obermeieri, but cease in an hour or two in preparations made from 
the blood of geese containing it. 

Pathogenesis.—A small quantity of blood from an infected goose inocu- 
lated into a healthy animal of the same species induces the disease after a 
period of incubation of four to five days. The infected goose ceases to eat, 
‘becomes apathetic, remaining in one a Ae and usually he at the end of a 
week ; the temperature is increased, and in some cases there is diarrhea. 
The spirilla are found in the blood at the outset of the malady, but after 
death they are not seen either in the blood orin the various organs. The 
heart and the liver are found to have undergone a fatty degeneration, and 
yellowish, cheesy granules the size of a millet seed are seen upon the surface 
of these organs. The spleen is soft and easily broken up by the fingers, 


PATHOGENIC SPIRILLA. 593 


Inoculations into chickens and pigeons were without result ;,in one 
chicken the spirilla were found in the blood on the fourth day after inocula- 
tion, but the fowl recovered. 


SPIRILLUM CHOLERA ASIATIC A. 


Synonyms.—Spirillum (“bacillus”) of cholera ; Comma bacillus 
of Koch; Kommabacillus der Cholera Asiatica; Bacille-virgule 
cholerigéne. 

Discovered by Koch (1884) in the excreta of cholera patients and 
in the contents of the intestine of recent cadavers. , 

The researches of Koch, made in Egypt and in India (1884), and 
subsequent researches by bacteriologists in various parts of the 
world, show that this spirillum—so-called ‘‘ comma bacillus ”—is con- 
stantly present in the contents of the intestine of cholera patients 
during the height of the disease, and that it is not found in the con- 
tents of the intestine of healthy persons or of those suffering from 


® 
i e »@ oS 
6.3 5 
Fic. 172. Fie. 173, 


Fie. 172.—Spirillum cholerze Asiatic. % 1,000. From a photomicrograph. (Koch.) 
Fig. 173.—Spirillum cholere Asiaticz, involuticaforms. x 700. (Van Ermengem.) 


other diseases than cholera. The etiological relation of this spiril- 
lum to Asiatic cholera is now generally admitted by bacteriologists. 
Jforphology.—Slightly curved rods with rounded ends, from 0.8 
to 2 in length and about 0.3 to 0.4 in breadth. The rods are 
usually but slightly curved, like a comma, but are occasionally in 
the form of a half-circle, or two united rods curved in opposite 
directions may form an S-shaped figure. Under certain circum- 
stances the curved rods grow out into long, spiral filaments, which 
may consist of numerous spiral turns, and in hanging-drop cultures 
the S-shaped figures may also be seen to form the commencement 
_of a spiral; in stained preparations the spiral character of the long 
filaments is often obliterated, or nearly so. When development is 
very rapid the short, curved rods or S-shaped spirals only are seen ; 
but in hanging-drop cultures, or in media in which the develop. 
38 


594 PATHOGENIC SPIRILLA. 


ment is retarded by an unfavorable temperature, the presence of a 
little alcohol, etc., the long, spiral filaments are quite numerous, and 
bacteriologists generally agree that the so-called ‘‘ comma bacillus ” 
is really only afragment of a true spirillum. By Léffler’s method 
of staining the rods may be seen to have a single terminal flagel- 
lum. In old cultures the bacilli frequently lose their characteristic 
form and become variously swollen and distorted—involution forms. 
Hueppe has described the appearance of spherical bodies in the 
course of the spiral filaments, which he believes to be reproductive 
elements—so-called arthrospores. 

Stains with the aniline colors usually employed, but not as quick- 
ly as many other bacteria; an aqueous solution of fuchsin is the 


Fie. 174, Fie. 175. 
Fig. 174.—Spirillum choleree Asiatic; colonies upon gelatin plate, end of thirty hours. x 160. 
Photograph by Frankel and Pfeiffer. 
Fia. 175.—Spirillum cholerz Asiatic, from a gelatinculture. x 1,000. From a photomicro- 
graph. (Frankel and Pfeiffer.) 


most reliable staining agent; is decolorized by iodine solution— 
Gram’s method. Sections may be stained with Léffler’s solution. 

Biological Characters.—An aérobic (facultative anaérobic), 
liquefying, motile spirillum. Grows in the usual culture media at 
the room temperature—more rapidly in the incubating oven. Does 
not grow at a temperature above 42° or below 14° C. Does not form 
endogenous spores (forms arthrospores, according to Hueppe ?). 

In gelatin plate cultures, at 22° C., at the end of twenty-four 
hours small, white colonies may be perceived in the depths of the 
gelatin ; these grow towards the surface and cause liquefaction of 
the gelatin in the form of a funnel which gradually increases in 


PATHOGENIC SPIRILLA. 595 


depth, and at the bottom of which is seen the colony in the form of 
asmall, white mass; asa result of this the plates on the second or 
third day appear to be perforated with numerous small holes ; later 
the gelatin is entirely liquefied. Under 
a low power the young colonies, before 
liquefaction has commenced, present a 
rather characteristic appearance ; they 
are of a white or pale-yellow color, and 
Fic. 176.—Colonies of the cholera have a more or less irregular outline, 
ee a ane margins being rough and uneven; 
end of thirty hours; c, end of forty- . 
eight hours; d, after liquefaction of the texture is coarsely granular, and the 
chee ie neese surface looks as if it were covered with 
, little fragments of broken glass, while 

the colony has a shining appearance ; when liquefaction commences an 
ill-defined halo is first seen to surround the granular colony, which 
by transmitted light has a peculiar roseate hue. In stab cultures in 
nutrient gelatin development occurs all along the line of inoculation, 


a 6 c d e il 


Fig. 177.—Spirillum cholere Asiatic; a, one day old; b, three days old; c, four days old; d, five 
days old, e, seven days old; f,10days old. From photographs by Koch. 


but liquefaction of the gelatin first occurs only near the surface ; on 
the second day, at 22° C., a short funnel is formed which has a 
comparatively narrow mouth, and the upper portion of which con- 
tains air, while just below this is a whitish, viscid mass ; later the 
funnel increases in depth and diameter, and at the end of from four 
to six days may reach the edge of the test tube; in from eight to 
fourteen days the upper two-thirds of the gelatin is completely lique- 
fied. Owing to the slight liquefaction which occurs along the line of 
growth during the first three or four days, the central mass which 


596 PATHOGENIC SPIRILLA. 


had formed along the line of inoculation settles down as a curled 
or irregularly bent, yellowish-white thread in the lower part of a 
slender tube filled with liquefied gelatin, the upper part of which 
widens out and is continuous with the funnel above. Upon the sur- 
face of nutrient agar a moist, shining, white layer is formed along 
the line of inoculation—impfstrich. Blood serum is slowly liquefied 
by this spirillum. Upon the surface of cooked potato, in the incu- 
bating oven, a rather thin and semi-transparent brown or grayish- 
brown layer is developed. In bouillon the development is rapid and 
ly slightly 


Fia. 178.—Cultures in nutrient gelatin, at the room temperature (16° to 18° C.), at the com- 
mencement of the fourth dey; a, Spirillum choleree Asiatice; b, Spirillum tyrogenum; ¢, Spirillum 
of Finkler and Prior. (Baumgarten.) 
clouded, but the spirilla accumulate at the surface, forming a wrin- 
kled membranous layer. Sterilized milk is also a favorable culture 
medium. In general this spirillum grows in any liquid containing a 
small quantity of organic pabulum and having a slightly alkaline 
reaction. An acid reaction of the culture medium prevents its de- 
velopment, as a rule, but it has the power of gradually aceommo- 
dating itself to the presence of vegetable acids, and grows upon 
potatoes—in the incubator only—which have a slightly acid reaction. 
Abundant development occurs in bouillon which has been diluted 
with eight or ten parts of water, and the experiments of Wolffhiigel 


PATHOGENIC SPIRILLA. 597 


and Riedel show that it also multiplies to some extent in sterilized 
river or well water, and that it preserves its vitality in such water 
for several months. But in milk or water which contains other bac- 
teria it dies out in a few days. Gruber and Schottelius have shown, 
however, that in bouillon which is greatly diluted the cholera spiril- 
lum may take the precedence of the common saprophytic bacteria, 
and that they form upon the surface of such a medium the charac- 
teristic wrinkled film. Koch found in his early investigations that 
rapid multiplication may occur upon the surfce of moist linen, and 
also demonstrated the presence of this spirillum in the foul water of 
a “tank” in India which was used by the natives for drinking 
purposes. In the experiments of Bolton (1886) the cholera spirilluam 
was found to multiply abundantly in distilled water to which 
bouillon was added in the proportion of fifteen to twenty-five parts 
in one thousand. 

The thermal death-point of the cholera spirillum in recent cul- 
tures in flesh-peptone-gelatin, as determined by the writer (1887), is 
52° C., the time of exposure being four minutes ; a few colonies only 
developed after exposure to a temperature of 50° for ten minutes. 
In Kitasato’s experiments (1889) ten or even fifteen minutes’ expo- 
sure to a temperature of 55° C. was not always successful in destroy- 
ing the vitality of the spirillum, although in certain cultures exposure 
to 50° for fifteen minutes was successful. He was not, however, 
able to find any difference between old and recent cultures as regards 
resistance to heat or to desiccation. In a moist condition this spiril- 
lum retains its vitality for months—as much as nine months in agar 
and about two months in liquefied gelatin. Itis quickly destroyed 
by desiccation, as first determined by Koch, who found that it did 
not grow after two or three hours when dried in a thin film on a 
‘glass cover. In Kitasato’s experiments (1889) the duration of vital- 
ity was found to vary from a few hours tv thirteen days, the differ- 
ence depending largely upon the thickness of the film. When dried 
upon silk threads they may retain their vitality for a considerably 
longer time (Kitasato). Very numerous experiments have been 
made to determine the amount of various disinfecting agents re- 
quired to destroy the vitality of this microérganism. We give be- 
low the results recently reported by Boer (1890), whose experiments 
were made in Koch’s laboratory. Experiments upon a culture in 
bouillon kept for twenty-four hours in the incubating oven, time of 
exposure two hours: hydrochloric acid, 1:1,350; sulphuric acid, 
1:1,300; caustic soda, 1:150; ammonia, 1:350; mercuric cyanide, 
1 :60,000 ; gold and sodium chloride, 1 : 1,000; silver nitrate, 1: 4,000; 
arsenite of soda, 1:400; malachite green, 1:5,000; methyl violet, 
1:1,000; carbolic acid, 1: 400; creolin, 1:3,000; lysol, 1:500. In 


598 PATHOGENIC SPIRILLA. 


Bolton’s experiments (1887) mercuric chloride was effective in two 
hours in the proportion of 1:10,000 ; sulphate of copper, 1 : 500. 

The low thermal death-point and comparatively slight resisting 
power for desiccation and chemical agents indicate that this spiril- 
lum does not form spores, and most bacteriologists agree that this 
is the case. Hueppe, however, has described a mode of spore for- 
mation which is different from that which occurs among the bacilli, 
viz., the formation of so-called arthrospores ; these are said to be 
developed in the course of the spiral threads, not as endogenous re- 
fractive spores, but as spherical bodies which have a somewhat 
greater diameter than the filament and are somewhat more refrac- 
tive. This mode of spore formation has not been observed by Kita- 
sato and other bacteriologists who have given attention to the ques- 
tion, and cannot be considered as established. In competition with 
the ordinary putrefactive bacteria the cholera spirillum soon disap- 
pears, and, as determined by Neffelman and by Kitasato, they only 
survive for a few days when mixed with normal feces. 

A test for the presence of the cholera spirillum has been found 
by Bujwid and by Dunham in the reddish-violet color produced in 
bouillon cultures containing peptone, or in cultures in nutrient gela- 
tin, when a small quantity of sulphuric acid is added to the culture. 
According to Frankel, this test serves to distinguish it from the ordi- 
nary bacteria of the intestine and from the Finkler-Prior spirillum, 
but not from Metschnikoff’s spirillum (‘‘ vibrio”). The reaction is 
shown by bouillon cultures which have been in the incubating oven 
for ten or twelve hours, and by gelatin cultures in which liquefac- 
tion has occurred. The sulphuric acid used should be quite pure ; 
the color quickly appears and is reddish-violet or purplish-red. Ac- 
cording to Salkowski, the red color is due to the well-known indol 
reaction, which in cultures of the cholera spirillum is exceptionally 
intense and rapid in its development. A test which is said to dis- 
tinguish cultures of the cholera spirillum from the spirillum of De- 
neke and that of Finkler-Prior, has been proposed by Cahen. This 
consists in adding a solution of litmus to the bouillon and in making 
the culture at 37°C. The cholera cultures show on the following 
day a decoloration which does not occur at this temperature with the 
other spirilla named. 

For determining as promptly as possible whether certain suspected 
excreta contain cholera spirilla, a little of the material may be used 
to inoculate greatly diluted bouillon, gelatin plates being made at 
the same time. At the end of ten or twelve hours the cholera spiril- 
lum, if present, will already have formed a characteristic wrinkled 
film upon the surface ; a little of this should be used to start a new 
culture in diluted bouillon, and a series of gelatin plates made from 


PATHOGENIC SPIRILLA. 599 


it, after which the color test may be applied. The result of this, in 
connection with the morphology of the microédrganisms forming the 
film and the character of growth in the gelatin plates, will estab- 
lish the diagnosis if the cholera spirillum is present in considerable 
numbers. If but few are present in the original material it may be 
necessary to make two or more series of plates and bouillon cultures 
before a pure culture can be obtained and a positive diagnosis made. 


aN 7 
ee 


we 
Gee, ve 


Fie. 179.—Section through mucous membrane of intestine from cholera cadaver; a tubular 
gland (a) is cut obliquely; in the interior of this (>), and between the epithelial and basement 
membrane. are numerous spirilla, x 600. (Pligge.) 


The spirillum is not found in the blood or in the various organs of 
individuals who have succumbed to an attack of cholera, but it is 
constantly found in the alvine discharges during life and in the con- 
tents of the intestine examined immediately after death; frequently in 
almost a pure culture in the colorless “rice-water” discharges. It is 
evident, therefore, that if we accept it as the etiological agent in this 
disease, the morbid phenomena must be ascribed to the absorption of 
toxic substances formed during its multiplication in the intestine. In 
cases which terminated fatally after a very brief sickness Koch found 
but slight changes in the mucous membrane of the intestine, which 
was slightly swollen and reddened; but in more protracted cases the 
follicles and Peyer’s patches were reddened around their margins, and 
an invasion of the mucous membrane by the “comma bacilli” was 
observed in properly, stained sections; they penetrated especially 
the follicles of Lieberktihn, and in some cases were seen between the 
epithelium and basement membrane. As a rule, the spirillum is not 


600 PATHOGENIC SPIRILLA. 


present in vomited matters, but Koch found it in small numbers in 
two cases and Nicati and Rietsch in three. In about one hundred 
cases in which Koch examined the excreta, or the contents of the in- 
testine of recent cadavers, during his stay in Egypt, in India, and in 
Toulon, his ‘‘ comma bacillus” was constantly found, and other ob- 
servers have fully confirmed him in this particular—Nicati and 
Rietsch in thirty-one cases examined at Marseilles ; Pfeiffer, twelve 
_ cases in Paris; Schottelius in cases examined in Turin; Ceci in 
Genoa, etc. On the other hand, very numerous control experiments 
made by Koch and others show that it is not present in the alvine 
discharges of healthy persons or in the contents of the intestine of 
those who die from other diseases. In the writer’s extended bacte- 
riological studies of the excreta, and contents of the intestine of ca- 
davers, in’ yellow fever, he has not once encountered any microér- 
ganism resembling the cholera.spirillum. 

As none of the lower animals are liable to contract cholera during 
the prevalence of an epidemic, or as a result of the ingestion of food 
contaminated with choleraic excreta, we have no reason to expect 
that pure cultures of the spirillum introduced by subcutaneous inocu- 
lation or by the mouth will give rise in them to a typical attack of 
cholera. Moreover, it has been shown by experiment that this spi- 
rillum is very sensitive to the action of acids, and is quickly de- 
stroyed by the acid secretions of the stomach, of man or the lower 
animals, when the functions of this organ are normally performed. 
By a special method of procedure, however, Nicati and Rietsch, and 
Koch, have succeeded in producing in guinea-pigs choleraic symp- 
toms and death. The first-named investigators injected cultures of 
the spirillum into the duodenum, after first ligating the biliary duct; 
the animals experimented upon died, and the intestinal contents con- 
tained the spirillum in large numbers. The fact that this procedure 
involves a serious operation which alone might be fatal, detracts 
from the value of the results obtained. Koch’s experiments on 
guinea-pigs are more satisfactory, and, having been fully controlled 
by comparative experiments, show that the ‘‘comma bacillus” is 
pathogenic for these animals when introduced in a living condition 
into the intestine. This was accomplished by first neutralizing the 
contents of the stomach with a solution of carbonate of toda—five 
cubic centimetres of a five-per-cent solution, injected into the stomach 
through a pharyngeal catheter. For the purpose of restraining in- 
testinal peristalsis the animal also receives, in the cavity of the abdo- 
men, a tolerably large dose of laudanum—one gramme tincture of 
opium to two hundred grammes of body weight. The animals are 
completely narcotized by this dose for about half an hour, but re- 
cover from it without showing any ill effects. Soon after the ad- 


PATHOGENIC SPIRILLA. 601 


ministration of the opium a bouillon culture of the cholera spirillum 
is injected into the stomach through a pharyngeal catheter. Asa 
result of this procedure.the animal shows an indisposition to eat and 
other signs of sickness, its posterior extremities become weak and 
apparently paralyzed, and, as a rule, death occurs within forty-eight 
hours. At the autopsy the small intestine is found to be congested 
and is filled with a watery fluid containing the spirillum in great 
numbers. Comparatively large quantities of a pure culture injected 
into the abdominal cavity of rabbits or of mice often produce a fatal 
result within two or three hours; and Nicati and Rietsch have ob- 
tained experimental evidence of the pathogenic power of filtered cul- 
tures not less than eight days old. The most satisfactory evidence 
that this spirillum is able to produce cholera in man is afforded by an 
accidental infection which occurred in Berlin (1884), in the case of a 
young man who was one of the attendants at the Imperial Board of 
Health when cholera cultures were being made for the instruction of 
students. Through some neglect the spirillum appears to have been 
introduced into his intestine, for he suffered a typical attack of 
cholera, attended by thirst, frequent watery discharges, cramps in 
the extremities, and partial suppression of urine. Fortunately he 
recovered ; but the genuine nature of the attack was shown by the 
symptoms and by the abundant presence of the ‘‘ comma bacillus” 
in the colorless, watery discharges from his bowels. Nicati and 
Rietsch observed a certain degree of attenuation in the pathogenic 
power of the spirillum after it had been cultivated for a considerable 
time at 20° to 25° C. ; and the observation has since been made that 
cultures which have been kept up from Koch’s original stock have 
no longer the primitive pathogenic potency. 

Cunningham, as a result of researches made in Calcutta (1891), 
arrives at the conclusion that Koch’s “comma bacillus” cannot 
be accepted as the specific etiological agent in this disease. This 
conclusion is based upon the results of his own bacteriological 
studies, which may be summed up as follows: First, in many un- 
doubted cases of cholera he has failed to find comma bacilli. Sec- 
ond, in one case he found three different species. Third, in one case 
the reaction with acids could not be obtained. From sixteen cases 
in which Cunningham made cultures he obtained ten different vari- 
eties of comma bacilli, the characters of which he gives in his pub- 
lished report. It may be that in India, which appears to be the 
permanent habitat of the cholera spirillum, many varieties of this 
microorganism exist ; but extended researches made in the laborato- 
ries of Europe show that Cunningham is mistaken in supposing that 
spirilla resembling Koch’s “ comma, bacillus” are commonly present 
in the intestine of healthy persons. The view advocated is that 


602 PATHOGENIC SPIRILLA. 


during the attack these spirilla are found in increased numbers be- 
cause conditions are more favorable for their development, but that 
they have no etiological import. The writer would remark that, in 
very extended researches made in the United States and in Cuba, he 
has never found any microdrganism resembling Koch’s cholera spi- 
rillum in the feeces of patients with yellow fever or of healthy indi- 
viduals, or in the intestinal contents of yellow-fever cadavers. 


SPIRILLUM OF FINKLER AND PRIOR. 


Synonym.— Vibrio proteus. ; . 
Obtained by Finkler and Prior (1884) from the feeces of patients with 
cholera nostras, after allowing the dejecta to stand for some days. Subse- 


Fic, 181. Fia. 182. 
Fic. 180,—Spirillum of Finkler and Prior, from 2 gelatin culture. X 1,000. From a photomicro- 
graph. (Frinkel and Pfeiffer.) 
Fig. 181.—Spirillum of Finkler an1 Prior; colonies ujzon gelatin plate; a, end of sixteen hours; 
b, end of twenty-four hours; c, end of thirty-six hours. x 80. (Fligge) 
Fig. 182.—Spirillum of Finkler and Prior; culture in nutrient gelatin; c, two days old; d, four 
days old. (Fligge.) 


quent researches have not sustained the view that this spirillum is the speci- 
fic cause of cholera morbus. 

Morphology.—Resembles the spirillum of Asiatic cholera, but the curved 
segments (‘‘ bacilli” ) are somewhat longer and thicker and not so uniform 
in diameter, the central portion being usually thicker than the somewhat 
pointed ends; forms spiral filaments, which are not as numerous, and are 
usually shorter than those formed by the cholera spirillum. In unfavorable 
media involution forms are common—large oval, spherical, or spindle- 
shaped cells, etc. Has a single flagellum at one end of the curved segments, 
which is from one to one and one-half times as long as these. 

Stains with the usual aniline colors—best with an aqueous solution of 
fuchsin. 


° 


PATHOGENIC SPIRILLA. 603 


Biological Characters.—An aérobic and facultative anaérobic, liquefy- 
ing, motile spirillum. Spore formation not demonstrated. Grows in the 
usual culture media at the room temperature. Upon gelatin plates small, 
white, punctiform colonies are developed at the end of twenty four hours, 
which under the microscope are seen to be finely granular and yellowish or 
yellowish-brown in color; liquefaction of the gelatin around these colonies 
progresses rapidly, and at the end of forty-eight hours is usually complete in 
plates where they are numerous. Isolated colonies on the second day form 
saucer-shaped depressions in the gelatin the size of lentils, having a sharply 
defined border. In gelatin stab cultures liquefaction progresses much more 
rapidly than in similar cultures of the cholera spirillum, and a stocking- 
shaped pouch of liquefied gelatin is already seen on th second day, which 
rapidly increases in dimensions, so that by the end of a week the gelatin is 
usually completely liquefied; upon the surface of the liquefied medium a 
whitish film is seen. Upon agara moist, slimy layer, covering the entire 
surface, is quickly developed. The growth in blood serum is rapid and 
causes liquefaction of the medium. Upon potato this spiriilum grows at the 
room temperature and produces a slimy, grayish-yellow, glistening layer, 
which soon extends over the entire surface. The cholera spirillum does not 
grow upon potato at the room temperature. The cultures of the Finkler- 
Prior spirillum give off a tolerably strong putrefactive odor, and, according 
to Buchner, in media containing sugar an acid reaction is produced as a re- 
sult of their development. They lhuvea greater resistance tu desiccation than 
the cholera spirillum. 

Pathogenesis.—Pathogenic for guinea-pigs when injected into the 
stomach by Koch’s method, after previous injection of a solution of car- 
bonate of soda, but a smaller proportion of the animals die from such injec- 
tions (Koch). At the autopsy the intestine is pale, and its watery contents, 
oa contain the spirilla in great numbers, have a penetrating, putrefactive 
odor. 

SPIRILLUM TYROGENUM. 


Synonyms —Spirillum of Deneke; Kasespirillen. 

Obtained by Deneke (1885) from old cheese. 

Morphology.—Curved rods and long, spiral filaments resembling the 
spirilla of Asiatic cholera. The diameter of the curved segments is some- 
what less than that of the cholera spirillum, and the turns in the spiral fila- 
ments are lower and closer together. The diame- 


ter of the “‘commas” is uniform throughout, so = - . 

that this spirillum more closely resembles the s* _.  --> 

cholera spirillum than does that of Finklerand = _- at, Pe =; 

Prior. =) Weve 
Stains with the usual aniline colors—best SS ar ge a 3 

with an aqueous solution of fuchsin. aaa 9 = = hh 
Biological Characters.—An aérobic and fac- ‘ m~tw 

ultative anaérobie, iquefying, motile spirillum. Pcie 


Spore formation not demoustrated. Grows in 

the usual culture media at the room temperature Fie. 183.—Spirillum tyroge- 
—more rapidly than the cholera spirillum and num. x 700. (Fligge. 

less so than that of Finkler and Prior. Upon 

gelatin plates small, punctiform colonies are developed, which on the second 
day are about the size of a pin’s head and have a yellowish color; under 
the microscope they are seen to be coarsely granular, of a yellowish-green 
color in the centre and paler towards the margins. The outlines of the colo- 
nies are sharply defined at first, but later, when liquefaction has commenced, 
the sharp contour is no longer seen. At first liquefaction of the gelatin 
causes funnel-shaped cavities resembling those formed by the cholera spiril- 
lum, but liquefaction is more rapid. In gelatin stab cultures liquefaction 
occurs all along the line of puncture, and the spirilla sink to the bottom of 


604 PATHOGENIC SPIRILLA. 


the liquefied gelatin in the form of a coiled mass, while a thin, yeflowish 
layer forms upon the surface; complete liquefaction usually occurs in 
about two weeks. Upon the surface of agar a thin, yellowish layer forms 


a b c 


Fig. 184.—Spirillum tyrogenum; colonies in gelatin plate; a,end of sixteen hours; b, end of 
twenty-four hours; c, end of thirty-six hours. Xx 80. (Fliigge.) 


along the impfstrich. Upon potato, at a temperature of 37° C., a thin, yel- 
low layer is usually developed (not always—Hisenberg) ; this contains, as a 
rule, beautifully formed, long, spiral filaments. 

Pathogenesis.—Pathogenic for guinea-pigs when introduced into the 
stomach by Koch’s method ; three out of fifteen animals treated in this way 
succumbed. 


SPIRILLUM METSCHNIKOVI. 


Synonym.—Vibrio Metschnikovi (Gameléia). 
Obtained by Gameléia (1888)-from the intestinal contents of chickens 
dying of an infectious disease which prevails in certain parts of Russia dur- 
: ing the summer months, and which in some respects re- 
sembles fowl cholera. The experiments of Gameléia show 
’ that the spirillum under consideration is the cause of the 
disease referred to, which he calls gastro-enteritis cholerica. 

Morphology.—Curved rods with rounded ends, and spi- 
ral filaments ; the curved segments are usually somewhat 
shorter, thicker, and more decidedly curved than the 
“*comma bacillus” of Koch. The size differs very, consid- 
erably in the blood of inoculated pigeons, the diameter 
being sometimes twice as great as that of the cholera spiril- 
lum, and at others about the same. A single, long, undu- 
lating flagellum may be seen at one extremity of the spiral 
filaments or curved rods in properly stained preparations. 

Stains with the usual aniline colors, but not by Gram’s 
method. 

Biological Characters.—An aérobic (facultative an- 
aérobic?), liquefying, mvtile spirillum. According to 
Gamaléia, endogenous spores are formed by this spirillum ; 
but Pfeiffer does not confirm this observation, and it must 
be considered extremely doubtful in view of the slight 
resistance to heat—killed in five minutes by a temperature 
ee 50° | ae in me usual Rossa media at the room 

epi, temperature. Upon gelatin plates small, white, puncti- 

ringer ris A mee form colonies are developed at the end of inalee to SiX- 
culture in nutrient t€€0 hours; these rapidly increase in size and cause lique- 
gelnditi,eudartorty: faction of the gelatin, which is, however, munch more rapid 
eight hours. From a with some than with others. At the end of three days 
photograph. (Fran- large, saucer-like areas of liquefaction may be seen resem- 
kel and Pfeiffer.) bling that produced by the Finkler-Prior spirillum and the 
contents of which are turbid, while other colonies have 

produced small, funnel-shaped cavities filled with transparent, tiquefied gel- 
atin and resembling colonies of the cholera spirillum of the same age. Under 


PATHOGENIC SPIRILLA. 605 


the microscope the larger liquefied areas are seen to contain yellowish-brown 
granular masses which are in active movement, and the margins are sur- 
rounded by a border of radiating filaments. In gelatin stab cultures the 
growth resembles that of the cholera spirillum, but the development is more 
rapid. Upon agar, at 37° C., a yellowish layer resembling that formed by 
the cholera spirillum is quickly developed. Upon potato no growth occurs 
at the room temperature, but at 37° C. a yellowish-brown or chocolate-col- 
ored layer is formed, which closely resembles that produced by the cholera 
spirillum under the same circumstances. In bouillon, at 37° C., develop- 
ment is extremely rapid, and the liquid becomes clouded and opaque, having 
a grayish-white color, while a thin, wrinkled film forms upon the surface. 
When muriatic or sulphuric acid is added to a culture in peptonized bouillon 
ared color is produced similar to that produced in cultures of the cholera 
spirillum, andeven more pronounced. In milk, at 35° C., rapid development 
occurs, and the milk is coagulated at the end of a week ; the precipitated 
casein accumulates at the bottom of the tube in irregular masses and is not 
redissolved. The milk acquires a strongly acid reaction and the spirilla 
quickly perish. 

Pathogenesis.—Pathogenic for chickens, pigeons, and guinea-pigs; rab- 
bits and mice are refractory except for very large doses. Chickens suffering 
from the infectious disease caused by this spirillum remain quiet and somno- 
lent, with ruffled feathers; they have diarrhoea; the temperature is not ele- 
vated above the normal, as is the case in chicken cholera. At the autops. 
the most constant appearance is hyperzemia of the entire alimentary canal. 
A_grayish-yellow liquid, more or less mixed with blood, is found in con- 
siderable quantity in the small intestine; the spleen is not enlarged and the 
organs generally are normal in appearance. In adult chickens the spirillum 
is not found in the blood, but in young ones its presence may be verified by 
the culture method and by inoculation into pigeons, which die in from 
twelve to twenty hours after being inoculated with two to four cubic cen- 
timetres. The pathological appearances in pigeons correspond with those 
found in chickens, but usually the spirillum is found in great numbers in 
blood taken from the heart. A few drops of a pure culture inoculated sub- 
cutaneously in pigeons or injected into the muscles cause their death in 
eight td twelve hours. Gameléia claims that the virulence of cultures is 
greatly increased by successive inoculations in pigeons, but Pfeiffer has 
shown that very minute doses are fatal to pigeons and that no decided in- 
crease of virulence occurs as a result of successive inoculations. Accordin 
to Gameléia, chickens may be infected by giving them food sf hear | 
with the cultures of the spirillum, but pigeons resist infection in this way. 
Guinea-pigs usually die in from twenty to twenty-four hours after receiving 
a subcutaneous inoculation ; at the autopsy an extensive subcutaneous 
cedema is found in the vicinity of the point of inoculation, and a superficial 
necrosis may be observed ; the blood and the organs generally contain the 
‘‘vibrio” in great numbers, showing that the animals die from general in- 
fection—acute septicemia. When infection occurs in these animals by way 
of the stomach the intestine will be found highly inflamed and its liquid con- 
tents will contain numerous spirilla. 

Gameléia has shown that plepons and guinea-pigs may be made immune 
by inoculating them with sterilized cultures of the spirillum—sterilized by 
heat at 100° OC. Old cultures contain more of the toxic substance than those 
of recent date. Thus two to three cubic centimetres of a culture twenty days 
old will kill a guinea-pig when injected subcutaneously, while five cubic 
centimetres of a culture five days old usually fail to do so. According to 
Pfeiffer, old cultures have a decidedly alkaline reaction, and their toxic power 
is neutralized by the addition of sulphuric acid. 

Gameléia has claimed that by passing the cholera spirillum of Koch 
through a series ef pigeons, by successive inoculation, its pathogenic power 


606 PATHOGENIC SPIRILLA. 


is greatly increased, and that when sterilized cultures of this virulent vari- 
ety of the ‘‘ comma bacillus” are injected into pigeons they become Immune 
against the pathogenic action of the ‘‘ vibrio Metschnikoff,” and the reverse. 
Pfeiffer (1889), in an extended and carefully conducted research, was not 
able to obtain any evidence in support of this claim, 


NOTES RELATING TO THE PATHOGENIC SPIRILLA. 


Quite a number of spirilla have been obtained from various sources 
which resemble more or less closely the spirillum of Asiatic cholera. 
It appears probable that some of these are in fact varieties of Koch’s 
“eomma bacillus” which have undergone various modifications as a 
result of the conditions under which they have maintained their ex- 
istence as saprophytes. Others are evidently essentially different, 
and have no very near relationship to the cholera spirillum. The 
principal points of difference between these recently described spirilla 
and Spirillum cholere Asiatice are given in the following résumé, 
for which we are indebted to Dieudonné (1894). 


‘Since the outbreak of cholera in 1892, various vibrios have been de- 
scribed which resemble more or less closely the cholera vibrio. When these 
are tested as to their morphological characters, growth in peptone solutions, 
in gelatin and agar plates, cholera-red reaction, and pathogenic power, they 
may be divided, at the outset, into two groups: viz., such vibrios as show 
only a remote resemblance to the cholera vibrio, and therefore are easily dif- 
ferentiated from it, and such as present only minor differences or none at 
all that have been demonstrated. 

‘“To the first group belongs the spirillum isolated by Russell from sea 
water—Spirilum marinum—which rapidly liquefies gelatin and does not 
grow at the body temperature. Rénon isolated from water, obtained at Bil- 
Jancourt, a vibrio which likewise quickly liquefies gelatin, but is not patho- 
ae for guinea-pigs, either by subcutaneous or intraperitoneal inoculation. 

linther, in examining the Spree water, found a vibrio which, upon gelatin 
plates, formed circular colonies with smooth margins, very finely granular 
and of a brown color. This vibrio did not give the indol reaction, and all 
infection experiments gave a negative result. Giinther named this sapro- 
phyte Vibrio aquatilis. About the same time (1892) Kiessling obtained from 
water, from Blankenese, a vibrio which presented similar characters and 
probably is identical with that of Gtinther. Weibel obtained from well-water 
a vibrio which liquefies gelatin more rapidly than the cholera vibrio ; its 
pathogenic action was not tested. Bujwid (1893) isolated from Weichsel 
water a vibrio which at low temperatures (12° C.) grew almost the same as 
the cholera vibrio, but at higher temperatures was easily distinguished from 
it. Bujwid’s assistant, Orlowski, found in a well at Lubin a very similar 
vibrio. Lo6ffler (1893) obtained from the Peene water a vibrio which at 37° 
C. grows rapidly and liquefies gelatin very rapidly, like the Finkler-Prior 
spirllum. Fokker (1893), from water of the harbor at Groningen, obtained 
a vibrio which rapidly liquefied gelatin and occasionally gave the indol re- 
action. Injections into the peritoneal cavity of mice and guinea-pigs gave 
a negative result. Fokker supposes that this is an attenuated cholera bacil- 
lus, because it forms the same ensyme as cholera bacteria, and when culti- 
vated for three months its characters, especially its peptonizing power, had 
changed. Fischer (1893) found in the stools of a woman suffering from diar- 
rhoea a vibrio which in gelatin cultures resembled that of Finkler and 
Prior. In bouillon and peptone solution it caused clouding and formation of 


PATHOGENIC SPIRILLA. 607 


a pellicle, but only gave a slight indol reaction. A portion of the mice in- 
oculated subcutaneously had after a time abscesses, from the contents of 
which Fischer was able to cultivate his vibrio, which he named Vibrio helco- 
genes. Vogler (1893), in an extended series of examinations of faeces, found 
a vibrio which showed many points of resemblance to the cholera vibrio in 
its growth in gelatin. But it constantly gave a negative indol reaction, and 
was not pathogenic for guinea-pigs when injected into the peritoneal cavity. 
Bleisch obtained from the dejecta of a man who died with choleraic symptoms 
abacterium which upon gelatin plates grew at first like the cholera bacillus, but 
was distinguished from it by many points of difference in other respects : 
short rods, sometimes bent, but never showing spiral forms. It gave the 
cholera-red reaction. Wolf (1883) obtained from cervical secretion, from a 
woman suffering from chronic endometritis, acomma-formed bacillus, which 
in its growth on gelatin plates resembled the cholera vibrio. The liquefac- 
tion was, however, much more rapid, a culture a day old being as far ad- 
vanced as a cholera culture of three to four days. The addition of sulphuric 
acid to a bouillon culture caused a faint rose-red color, which upon standing 
changed tobrown. The addition of sulphuric acid and potassium iodide paste 
did not cause a blue color, so there was no formation of nitrites. Bonhoff 
(1893), in water from Stolpe, in Pommerania, discovered two vibrios, one of 
which in the first twenty-four hours grew like the cholera vibrio, but did not 

ive the cholera-red reaction. Out of four guinea-pigs inoculated one only 

ied with cholera-like symptoms. The other vibrio gave the cholera-red reac- 
tion, but did not liquefy gelatin and was very inconstant as regards its patho- 
genic power. Zorkendorfer (1893) isolated a vibrio from the stools of a 
woman who died with choleraic symptoms, which at first grew upon gelatin 
plates like the cholera vibrio, but after the second day liquefied the gelatin 
very rapidly, so that it could no longer be taken for the same. The indol 
reaction was constantly absent, and it was not pathogenic for guinea-pigs, 
rabbits, or pigeons. Blackstein (1853) obtained from the water of the Seine 
a comma bacillus which resembled the cholera vibrio in many particulars, but 
was distinguished by the finer granulation and more opaque appearance of 
its colonies. Sanarelli (1893), by the use of special media, isolated from the 
water of the Seine and of the Marne no less than thirty-two vibrios, four of 
which resembled the cholera vibrio in Came the indol reaction. Three 
others gave the indol reaction after eight days ; the remainder did not give it 
at ail, or only very faintly. The vibrios which upon a first inoculation gave 
no results or only very slight evidence of pathogenic power, when carried 
through a series of animals caused a fatal infection. When a sterilized cul- 
ture of the colon bacillus was injected at the same time death always oc- 
curred. Sanarelli believes that these vibrios must have had a common ori- 
gin—from the dejecta of cholera patients. Fischer (1894) has described a 
number of vibrios from sea-water which are distinguished from the cholera 
vibrio especially by a preference for media containing sea-water. Finally, 
the vibrios found in water, referred to by Koch (‘ Ueber den augenblicklichen 
Stand der Cholera-diagnose,’ Zeitschr. fiir Hygiene, Bd. xiv., page 319), 
belong here. 

Oui te different from these is a second group of vibrios which in their in- 
vestigation offered great and often almost insuperable difficulties for the 
differential diagnosis. Here, first of all, is the Vibrio Berolinensis, found by 
Neisser in August, 1893, and described by Rubner, Neisser, and Giinther. 
This was isolated from water which had previously contained cholera vibrios, 
for which reason Dunbar considers it not impossible that this is a genuine 
cholera vibrio, somewhat changed perhaps by long-continued development 
in water. Neither in its morphology nor in its behavior in gelatin stick eul- 
tures, in milk and other media, could it be distinguished from the genuine 
comma, bacillus; the indol reaction and pathogenic action upon guinea-pigs 
were the same; oni the contrary, a differentiation was easily made in gelatin 
plate cultures. Atthe end of twenty-four hours it formed small, spherical, 


608 PATHOGENIC SPIRILLA. 


finely granular colonies, which at the end of forty-eight hours were not yet 
visible to the naked eye. Heider (1893) isolated from the water of the Donau 
canal a vibrio which he called Vibrio Danubicus. This resembles the chol- 
era vibrio fully in its morphology. As a distinguishing character it was 
found that this vibrio, in thinly planted plates, forms flat, superficial colo- 
nies having irregularly rounded margins and other slight differences; also 
the pathogenic action upon mice inoculatedsubcutaneously, and the ease with 
which guinea-pigs are infected by way of the respiratory passages. It is 
worthy of note that the day after the sample was taken a man was taken sick 
with cholera who had worked on the Donau the day before—on the principal 
stream at a place far below the junction of the canal. Dunbar (1893) found 
vibrios in the Elbe, in the Rhine, in the Pegnitz, and in the Amstel at Amster- 
dam. These presented no decided characters by which he was able to differ- 
entiate them from the cholera vibrio. The most careful comparative investi- 
gations did not lead to the discovery of any points of difference which had 
not already been observed in genuine cholera cultures. Everything, there- 
fore, indicated that these were genuine cholera bacilli, especially as these 
vibrios disappeared from the rivers when cholera ceased to prevail. It was 
first possible through an observation of Kutscher’s to differentiate a portion 
of these water bacteria, and certain vibrios isolated from the discharges of 

ersons suspected of having cholera from cultures of the cholera spirillum. 

n the presence of oxygen, at asuitable temperature, they give off a greenish- 
white phosphorescence. 

‘As phosphorescence has never been observed in undoubted cholera cul- 
tures, we can assert with tolerable certainty that such phosphorescent vibrios 
are not genuine cholera bacteria. But as this phosphorescent property was 
inconstant in thirty-eight out of sixty-eight cultures, Dunbar believes that 
some reserve must be exercised in accepting this as evidence that these are 
not genuine cholera vibrios. Maassen (1894) givesas a further distinguishing 
character of these phosphorescent vibrios the fact that they form a strong, 
usually wrinkled pellicle in bouillon, of proper alkalinity, containing gly- 
cerin or carbohydrates (cane sugar, lactose); also that in such media the 
formation of indol and a subsequent return to an alkaline reaction may be 
observed. 

‘‘ As already stated, Sanarelli isolated from Seine water a considerable 
number of vibrios, and among them four—viz.: one from St. Cloud, Point- 
du-Jour, Gennevilliers No. 5, and Versailles (Seine), which after twenty-four 
hours gave a distinct indol reaction and were more or less pathogenic for 
guinea-pigs (the one from St. Cloud was also pathogenic for pigeons). Ivan- 
off (1893) Geaeribes a vibrio which he isolated from the feeces of a patient with 
typhoid fever. Butas the discharges had been mixed with Berlin hydrant 
water, Ivdnoff admits the possibility that his vibrio came from this water. 
It closely resembles the cholera vibrio, but is distinguished by its colonies in 
gelatin plates, which, at the end of twenty-four to thirty-six hours, in place 
of the usual coarse granulation of cholera colonies shows a distinct formation 
of filaments. Morphologically the vibrio is distinguished by a decided ten- 
dency to preserve the spiral form, and especially by its size. Celli and San- 
tori (1893) describe a Vibrio romanus, which they isolated from twelve 
undoubted cases of cholera. This does not give the indol reaction, is not 
pathogenic for animals, and does not grow in bouillon or agar at 37° C. 
This is considered by the authors named an atypical variety of the cholera 
vibrio, especially as the distinguishing characters did not prove to be perma- 
nent. After eight months’ cultivation the cultures gave the indol reaction, but 
the pathogenic power was still almost absent. Recently Chantemesse (1894) 
has described a vibrio which he found in the spring of 1894 during the chol- 
era epidemic at Lisbon. This differed in many particulars from the genuine 
cholera vibrio, resembling more closely the vibrio of Finkler-Prior. As in 
the Lisbon epidemic, with a large number taken sick, only one death occurred, 
and in view of the results of the bacteriological examination, Chantemesse 


PATHOGENIC SPIRILLA. 609 


supposes this to have been an epidemic of cholera nostras. Finally, Pfuhl 
(1894) found a vibrio in the north harbor of Berlin which from its growth in 
gelatin and pathogenesis for pigeons he believes to be identical with Vibrio 
Metschnikovi.” 


To the list of vibrios above referred to as resembling more or less 
closely the cholera spirillum we must add those described by Cun- 
ningham (1894) and obtained by him from the discharges of cholera 
patients. He has described “thirteen distinct forms obtained from 
cases of cholera and one of non-choleraic origin.” 

Pfeiffer and Issaeff (1894) report that they have found a sen- 
sitive test for the differentiation of these vibrios in the specific 
character of cholera immunity. They found that guinea-pigs which 
were immunized against cholera infection have a lasting immunity, 
and that the serum of such immunized animals has a specific ac- 
tion in protecting against infection by genuine cholera vibrios 
only, while for other species it has no action different from that 
of the blood serum of normal animals. In all cases where the cholera 
serum acted specifically the vibrios were promptly destroyed, while 
in cases where this specific action was absent the injected vibrios 
multiplied rapidly and caused the death of the animal. By means of 
this method the vibrios isolated from water—the phosphorescent 
vibrios of Dunbar, Vibrio Danubicus, Cholera Massanah—are shrwn 
to be distinct species, while the vibrio of Ivanoff behaves like the 
genuine cholera vibrio. In a subsequent paper Pfeiffer reports the 
interesting fact that a trace of highly active cholera serum, added to 
a culture of the cholera spirillum, when injected into the peritoneal 
cavity of a guinea-pig, within a surprisingly brief time causes the 
destruction of the cholera vibrios; whereas no such effect is produced 
upon other species. A similar destruction occurs when cholera vib- 
rios are injected into the abdominal cavity of immunized guinea- 
pigs. The researches of Dunbar (1894) indicate that Pfeiffer’s test 
is not so reliable as he supposed; and also that phosphorescence can- 
not be relied upon for distinguishing similar water bacteria from 
genuine cholera vibrios. Rumpel has reported the fact that two un- 
doubted cultures of the cholera spirillum, from different sources, after 
being passed through pigeons and cultivated for some time in arti- 
ficial media, showed phosphorescence. One of these cultures was ob- 
tained originally from the discharges of Dr. Oergel, who was a vic- 
tim to cholera from laboratory infection (case reported by Reincke, in 
the Deutsche medicinische Wochenschrift, No. 41, 1894). Anvther 
case of supposed laboratory infection, in which recovery occurred, is 
reported by Lazarus, in the Berliner medicinische Wochenschrift, 
1893, page 1,241. 

That cholera vibrios may be present in the alimentary canal of 

39 


610 PATHOGENIC SPIRILLA. 


healthy individuals without giving rise to any symptoms of ill-health 
appears to be demonstrated. In support of this conclusion we quote 
as follows from a recent paper by Abel and Claussen: 

“In Wehlau (East Prussia), in the autumn of 1894, seven cases of 
cholera, occurred about the same time. The members of the family 
were at once isolated and their feces examined almost daily. Of 
especial interest were seventeen individuals who belonged to families 
in which three fatal cases occurred. Of these seventeen persons, who 
were not sick at all or only had for a brief time a diarrhcea, thirteen 
had cholera vibrios in their discharges for a considerable time. As 
the table shows, many of these comma bacilli were not found in dis- 
charges every day, but were obtained again after being absent” (in 
the cultures) “for a day or two.” 

Abel and Claussen (1895), as a result of very extended experi- 
ments, arrive at the conclusion that cholera vibrios in feeces as a rule 
do not survive longer than twenty days, and often cannot be ob- 
tained after two or three days; exceptionally they were obtained in 
cultures at the end of thirty days—Karlinsky and Dunbar have re- 
ported finding them at the end of fifty-two days and four months. 
Karlinsky (1895) has also reported that upon woollen and linen goods, 
cotton batting and wool, which were soaked in the discharges of 
cholera patients and preserved from drying by being wrapped in 
waxed paper, the cholera vibrio retained its vitality for from twelve 
to two hundred and seventeen days. 

The researches of Kasansky (1895) show that the cholera spiril- 
lum is not destroyed by alow temperature (—30 C.) and that it 
even resists repeated freezing and thawing—three or four times. 

Behring and Ransom (1895) as a result of an extended experi- 
mental research, arrive at the conclusion that cholera cultures from 
which the bacteria have been removed have specific toxic properties, 
and cause symptoms similar to those which result from the intro- 
duction into guinea-pigs of the living bacteria; that from these fil- 
tered cultures a solid substance can be obtained having the same 
toxic properties, and that from susceptible animals which have been 
treated with this toxic substance a serum can be obtained which is 
active not only against the cholera poison, but against the cholera 
vibrio. These results support those previously reached by other 
bacteriologists and lead to the hope that a specific treatment of the 
disease may be successfully employed. The results obtained by 
Haffkine in India are favorable to the view that his method of prophy- 
laxis, by the subcutaneous injection of virulent cholera cultures, has 
a real value. 


PLATE IX. 


Fie. 1.—Bacillus diphtheriz (Klebs-Léfiler) from culture on. blood 
serum. Stained with Léffler’s solution of methylene blue. x 1,000. 
Photomicrograph by oil lamp. (Borden.) 

Fig. 2.—Micrococcus gonorrhee in urethral pus. Stained with 
Léffler’s solution of methylene blue. x 1,000. Photomicrograph by oil 
lamp. (Borden.) 

Fig. 3.—Bacillus tuberculosis in sputum. x 1,000. Photomicro- 
graph by oil lamp. (Borden.) 

Fig. 4.—Bacillus typhi abdominalis, from agar culture. x 1,000. 
Photomicrograph by oil lamp. (Borden.) 

Fie. 5.—Streptococcus pyogenes (longus). x 1,000. Photomicro- 
graph made at the Army Medical Museum by sunlight. (Gray.) 

Fic. 6.—Bacillus mallei. x 1,000. Photomicrograph made at the 
Army Medical Museum by sunlight. (Gray.) 


PLATEIX. 
STERNBERG'S BACTERIOLOGY. 


Fig. 2. 


Fig. 


Fig. 3. 


Fig. 6. 


PATHOGENIC BACTERIA. 


PART FOURTH. 


SAPROPHYTES. 


I. Bacteria IN THE AIR. II. BacTERIA IN Water. III. Bacteria IN 
THE Sort. IV. BacTERIA ON THE SURFACE OF THE BODY AND OF EX- 
POSED Mucous MEMBRANES. V. BACTERIA OF THE STOMACH AND 
INTESTINE. VI. BACTERIA OF CADAVERS AND OF PUTREFYING 
MaTERIAL FROM VARIOUS SouRcES. VII. Bacteria 
IN ARTICLES OF Foon. 


I. 


BACTERIA IN THE ATR. 


THE saprophytic bacteria are found wherever the organic material 
which serves as their pabulum is exposed to the air under conditions 
favorable to their growth. The essential conditions are presence of 
moisture and a suitable temperature. The organic material may be 
in solution in water or in the form of moist masses of animal or 
vegetable origin, and the temperature may vary within considerable 
limits—0° to 70° C. But the species which takes the precedence will 
depend largely upon special conditions. Thus certain species multi- 
ply abundantly in water which contains comparatively little organic 
pabulum, and others require a culture medium rich in albuminous 
material or in carbohydrates ; some grow at a comparatively low or 
high temperature, while others thrive only at a temperature of 20° to 
40° C. or have a still more limited range; some require an abun- 
dant supply of oxygen, and others will not grow in the presence of 
this gas. Our statement that saprophytic bacteria are found wherever 
the organic material which serves as their pabulum is exposed to the 
air—under suitable conditions—relates to the fact that it is through 
the air that these bacteria are distributed and brought in contact 
with exposed material. It is a matter of common laboratory experi- 
ence that sterilized organic liquids quickly undergo putrefactive de- 
composition when freely exposed to the air, and may be preserved in- 
definitely when protected from the germs suspended in the air by 
means of a cotton air filter. But the organic pabulum required for 
the nourishment of these bacteria is not found in the air in any con- 
siderable amount, and if they ever multiply in the atmosphere it 
must be under very exceptional conditions. Their presence is due to 
the fact that they are wafted from surfaces where they exist in a 
desiccated condition, and, owing to their levity, are carried by the 
wind to distant localities. But, under the law of gravitation, when 
not exposed to the action of currents of air they constantly fall 
again upon exposed surfaces, which, if moist, retain them, or from 
which, if dry, they are again wafted by the next current of air. 
Under these circumstances it is easy to understand why, as deter- 


614 BACTERIA IN THE AIR. 


mined by investigation, more bacteria are found near the surface of 
the earth than at some distance above the surface, more over the 
land than over the ocean, more in cities with their dust-covered 
streets than in the country with its grass-covered fields. 

Careful experiments have shown that bacteria do not find their 
way into the atmosphere from the surface of liquids, unless portions 
of the liquid containing them are projected into the air by some 
mechanical means, such as the bursting of bubbles of gas. Cultures 
of pathogenic bacteria freely exposed to the air in laboratories do not 
endanger the health of those who work over them; but if such a cul- 
ture is spilled upon the floor and allowed to remain without disin- 
fection, when it is desiccated the bacteria 
contained in it will form part of the dust of 
the room and might be dangerous to its 
occupants. Bacteria do not escape into the 
air from the surface of the fluid contents of 
sewers and cesspools, but changes of level 
may cause a deposit upon surfaces, which 
is rich in bacteria, and when dried this ma- 
terial is easily carried into the atmosphere 
by currents of air. 

Tyndall’s experiments (1869) show that 
in a closed receptacle in which the air is 
perfectly still allsuspended particles are af- 
ter a time deposited on the floor of the closed 
air chamber. And common experience de- 
monstrates the fact that the dust of the at- 
mosphere is carried by the wind from ex- 
posed surfaces and again deposited when the 
air is at rest. This dust as deposited, for 
example, in our dwellings contains innu- 
merable bacteria in a desiccated condition, 
and the smallest quantity of it introduced 
into a sterile organic liquid will cause it to 
undergo putrefactive decomposition, and 
by bacteriological methods it will be found 
tocontain various species of bacteria. Such 

Fic. 186.—Penicilium. glau- dust also contains the spores of various 
cum; m, mycelium, from which ould fungi which are present in the atmo- 
is given off a branching pedicle “ 
bearing spores. x 150. sphere, usually in greater numbers than the 

bacteria. The mould fungi are air plants 
which vegetate upon the surface of moist organic material and form 
innumerable spores, which are easily wafted into the air, both on 
account of their low specific gravity and minute size, and because they 


BACTERIA IN THE AIR. 61d 


are borne upon projecting pedicles by which they are removed from 
the moist material upon which and in which the mycelium develops 
(Fig. 186), and, being dry, are easily carried away by currents of air. 

Bacteriologists have given much attention to the study of the mi- 
croérganisms suspended in the atmosphere, with especial reference to 
hygienic questions. The methods and results of these investigations 
will be considered in the present section. 

Pasteur (1860) demonstrated the presence of living bacteria in the 
atmosphere by aspirating a considerable quantity of air through a 
filter of gun-cotton or of asbestos contained in a glass tube. By dis- 
solving the gun-cotton in alcohol and ether he was able to demon- 
strate the presence of various microédrganisms by a microscopical ex- 
amination of the sediment, and by placing the asbestos filters in 
sterilized culture media he proved that living germs had been filtered 
out of the air passed through them. 


: 


tl 


| 


Fie. 187. 


A method employed by several of the earlier investigators con- 
sisted in the collection of atmospheric moisture precipitated as dew 
upon a surfaco cooled by a freezing mixture. This was found to con- 
tain living bacteria of various forms. The examination of rain water, 
which in falling washes the suspended particles from the atmosphere, 
gave similar results. 

The first systematic attempts to study the microédrganisms of the 
air were made by Maddox (1870) and by Cunningham (1873), who 
used an aéroscope which was a modification of one previously de- 
scribed by Pouchet. In the earlier researches of Miquel a similar 
aéroscope was used. This is shown in Fig. 187, The opening to the 
cylindrical tube A is kept facing the wind by means of a wind vane, 
and when the wind is blowing a current passes through a small aper- 
ture in a funnel-shaped partition which is properly placed in the 
cylindrical tube. A glass slide, upon the lower surface of which a 


616 BACTERIA IN THE AIR. 


mixture of glycerin and glucose has been placed, is adjusted near the 
opening of the funnel, at a distance of about three millimetres, so 
that the air escaping through the small orifice is projected against it. 
By this arrangement a considerable number of the microérganisms 
present in the air, as well as suspended particles of all kinds, are ar- 
rested upon the surface of the slide and can be examined under the 
microscope or studied by bacteriological methods. But an aéroscope 
of this kind gives no precise information as to the number of living 
germs contained in a definite quantity of air. The microscopical ex- 
amination also fails to differentiate the bacteria from particles of 
various kinds which resemble them in shape, and the microérgan- 
isms seen are for the most part spores of various fungi mingled with 
pollen grains, vegetable fibres, plant hairs, starch granules, and 
amorphous granular material. 

Another method, which has been employed by Cohn, Pasteur, 
Miquel, and others, consists in the aspiration of a definite quantity of 
air through a culture liquid, which is then placed in an incubating 
oven for the development of microédrganisms washed out of the air 
which has been passed through it. This method shows that bacteria 
of different species are present, but gives no information as to their 
relative number, and requires further researches by the plate method 
to determine the characters of the several species in pure cultures. 

A far simpler method consists in the exposure of a solid culture 
medium, which has been carefully sterilized and allowed to cool on a 
glass plate or in a Petvri’s dish, for a short time in the air to be ex- 
amined. Bacteria and mould fungi deposited from the air adhere to 
the surface of the moist culture medium, and form colonies when the 
plate, enclosed in a covered glass dish, is placed in the incubating oven. 
The number of these colonies which develop after exposure in the 
air for a given time enables us to estimate in a rough way the num- 
ber of microérganisms present in the air of the locality where the 
exposure was made ; and the variety of species is determined by ex- 
amining the separate colonies,each of which is, as a rule, developed 
from a single germ. By exposing a number of plates at different 
times this method enables us to determine what species are most 
abundant in a given locality and the comparative number in dif- 
ferent localities, as determined by counting the colonies after ex- 
posure for a definite time—e.g., ten minutes. Of course we will only 
obtain evidence of the presence of such aérobic bacteria as will 
grow in our culturemedium. The anaérobic bacteria may be studied 
by placing plates exposed in a similar way in an atmosphere of hydro- 
gen. Bacteria which grow slowly and only under special conditions, 
like the tubercle bacillus, would be likely to escape observation, as 
the mould fungi and common saprophytes would take complete pos- 


BACTERIA IN THE AIR. 617 


session of the surface of the culture medium before the others had 
formed visible colonies. Students will do well to employ this simple 
and satisfactory method for the purpose of making themselves familiar 
with the more common atmospheric organisms, and they will find 
the shallow glass dishes with a cover, known as Petri’s dishes, very 
convenient for the purpose. These dishes should be sterilized in the 
hot-air oven and sufficient sterile nutrient gelatin or agar poured 
into them to cover the bottom. After the culture medium has be- 
come solid by cooling, the exposure may be made by simply remov- 
ing the cover and replacing it at the end of the time fixed upon. 


Or gs 
i 
WF 


Poa 


aoe 


Fie. 188, 


To determine in a more exact way the number of microérganisms 
contained in a given quantity of air will require other methods. But 
we may say, en passant, that such a determination is usually not of 
great scientific importance. The number is subject to constant fluc- 
tuations in the same locality, depending upon the force and direction 
of the wind. If we have on one side of our laboratory a dusty 
street and on the other a green field, more bacteria will naturally be 
found when the wind blows from the direction of the street than 
when it comes from the opposite direction ; or, if the air is filled with 
dust from recently sweeping the room, we may expect to find very 


618 BACTERIA IN THE AIR. 


many more than when the room has been undisturbed for some time. 
The painstaking researches which have already been made have es- 
tablished in a general way the most important facts relating to the 
distribution of atmospheric bacteria, but have failed to show any de- 
finite relation between the number of atmospheric bacteria and the 
prevalence of epidemic diseases. In the apparatus of Hesse, Fig. 
188, a glass tube, having a diameter of four to five centimetres and a 
length of half a metre toa metre, isemployed. In use this is sup- 
ported upon a tripod, as shown in the figure, and air is drawn 
through it by a water aspirator consisting of two flasks, also shown. 
The upper flask being filled with water, this flows into the lower 
flask by siphon action, and upon reversing the position of the flasks 
number one is again filled. By repeating this operation as many 
times as desired a quantity of air corresponding with the amount of 
water passed from the upper to the lower flask is slowly aspirated 
through the horizontal glass tube. The microérganisms present are 
deposited upon nutrient gelatin previously allowed to cool upon the 
lower portion of the large glass tube. The air enters through a small 
opening in a piece of sheet rubber which is tied over the extremity 
of the horizontal tube, and before the aspiration is commenced this 
opening is covered by another piece of sheet rubber tied over the 
first. Experience shows that when the air is slowly aspirated most 
of the germs contained in it are deposited near the end of the tube 
through which it enters. The colonies which develop upon the nu- 
trient gelatin show the number and character of living microérgan- 
isms contained in the measured quantity of air aspirated through the 
apparatus. The method with a soluble filter of pulverized sugar, to 
be described hereafter, is preferable when exact results are desired; 
and for the purpose of determining the relative abundance and the 
variety of microédrganisms present in the atmosphere of a given lo- 
cality the exposure of nutrient gelatin in Petri’s dishes is far simpler, 
and, as a rule, will furnish all the information that is of real 
value. 

In his extended researches made at the laboratory of Montsouri, 
in Paris, Miquel has used various forms of apparatus and has ob- 
tained interesting results ; but his method of ensemencements frac- 
tronnés requires a great expenditure of time and patience, and the 
more recent method with soluble filters is to be preferred. 

In his latest modification of the method referred to Miquel used a 
flask like that shown in Fig. 189. From twenty to forty cubic cen- 
timetres of distilled water are introduced into this flask. The cap A 
contains a cotton air filter and is fitted to the neck of the flask by a 
ground joint. This is removed during the experiment. The tube C 
is connected with an aspirator. It contains two cotton or asbestos 


BACTERIA IN THE AIR. : 619 


filters, cand b. The cap being removed and the aspirator attached, 
the air is drawn through the water, by which suspended germs are 
arrested ; or if not they are caught by the inner cotton plug b. The 
sealed point of the tube B is now broken off, and the contents of the 
flask equally divided in thirty to forty tubes containing bouillon, 
which are placed in the incubating oven. 
Twenty-five cubic centimetres of bouillon 
are also introduced into the flask, and the 
cotton plug 0 is pushed into it so that any 
bacteria arrested by it may develop. If 
one-fourth or one-fifth of the bouillon tubes 
show a development of bacteria it is in- 
ferred that each culture originated from 
a single germ, and the number present in 
the amount of air drawn through the flask = 
is estimated from the number of tubes in Fie. 189, 

which development occurs. 

The method adopted by Straus and Wiirtz is more convenient and 
more reliable in its results. This consists in passing the air by means 
of an aspirator through liquefied nutrient gelatin or agar. The ap- 
paratus shown in Fig. 190 is used for this purpose. Two cotton 
plugs are placed in the tube B, to which the aspirator is attached, 
and afterthe determined quantity of air has been passed through the 
liquefied medium the inner plug is pushed down with a sterilized 
platinum needle so as to wash out in the culture 
medium any germs arrested by it. Finally the 
gelatin or agar is solidified upon the walls of 
the tube A by rotating it upon a block of ice or 
under a stream of cold water. It is now put 
aside for the development of colonies, which are 
counted to determine the number of germs pre- 
sent in the quantity of air passed through the 
liquefied culture medium. The main difficulty 
with this apparatus is found in the fact that the 
nutrient gelatin foams when air is bubbled 
through it; for this reason an agar medium is 
to be preferred. In using this it will be neces- 
sary to place the liquefied agar in a bath main- 
tained at 40° C. Foaming of the gelatin is pre- 
vented by adding a drop of olive oil before ster- 
ilization in the steam sterilizer. But this inter- 
feres with the transparency of the medium. 

In the earlier experiments upon atmospheric organisms Pasteur 
used a filter of asbestos, which was subsequently washed out in a 


Fie. 190. 


620 BACTERIA‘ IN THE AIR. 


culture liquid. A filter of this kind washed out in liquefied gelatin 
or nutrient agar would give more satisfactory results, as the culture 
medium could be poured upon plates or spread upon the walls of a 
test tube and the colonies counted in the usual way. Petri prefers 
to use a filter of sand, which he finds by experiment arrests the mi- 
croérganisms suspended in the atmosphere, and which is subsequently 
distributed through the culture medium. The sand used is such as 
has been passed through a wire sieve having 
openings of 0.5 millimetre indiameter. This is 
sterilized by heat, and is supported in a cylin- 
drical glass tube by small wire-net baskets. The 
complete arrangement is shown in Fig. 191. 
Two sand filters, c, and c,, are used, the lower 
one of which serves as a control to prove that 
all microérganisms present in the air have been. 
arrested by the upper one. The upper filter is 
protected, until the aspirator attached to the 
tube fh is put in operation, by a sterile cotton 
plug, not shown in the figure which represents 
the filter in use. Petri uses a hand air pump as 
an aspirator, and passes one hundred litres of 
air through the sand in from ten to twenty 
minutes. The sand from the two filters is then 
distributed in shallow glass dishes and liquefied 
gelatin is poured over it ; this is allowed to sol- 
idify and is put aside for the development of 
colonies. The principal objection to this method 
is the presence of the opaque particles of sand 
in the culture medium. This objection has been 
overcome by the use of soluble filters, a method 
first employed by Pasteur and since perfected 
by Sedgwick and by Miquel. The most useful 
material for the purpose appears to be cane 
sugar, which can be sterilized in the hot-air oven 
at 150° C. without undergoing any change in 
C its physical characters. Loaf sugar is pulver- 
Fic. 191. ized in a mortar and passed through two sieves 
in order to remove the coarser grains and the 
very fine powder, leaving for use a powder having grains of about 
one-half millimetre in diameter. This powdered sugar is placed 
in a glass tube provided with a cap having a ground joint and a cot- 
ton plug to serve as an air filter (A, Fig. 192), or in a tube such as is 
shown at B, having the end drawn outand hermetically sealed. Two 
cotton plugs are placed at the lower portion of the tube, at a and at b. 


BACTERIA IN THE AIR. 621 


Glass tubing having a diameter of about five millimetres is used in 
making these tubes, and from one to two grammes of powdered sugar 
ix a suitable quantity to use asa filter. The whole apparatus is steril- 
ized for an hour at 150° C. in a hot-air oven after the pulverized 
sugar has been introduced. Before using it will be necessary to 
pack the sugar against the supporting plug a by gently striking the 
lower end of the tube, held in a vertical position, upon some horizon- 
tal surface; and during aspiration 
the tube must remain in a vertical 
position, or nearly so, in order that 
the sugar may properly fill its entire 
calibre. The aspirator is attached to 
the lower end of the tube by a piece 
of rubber tubing. When the tube B 
is used the sealed extremity is broken 
off at the moment that the aspirator 
is set in action, and it is again sealed 
in a flame after the desired amount 
of air has been passed through the 
filter. The next step consists in dis- 
solving the sugar in distilled water 
or in liquefied gelatin. To insure 
the removal of all the sugar the cot- 
ton plug a may be pushed out with a 
sterilized giass rod, after removing:b 
with forceps. From fifty to five hun- 
dred cubic centimetres of distilled 
water, contained in an Erlenmeyer 
flask and carefully sterilized, may be 
used, the amount required depending 


upon circumstances relating to the Y ss 

conditions of the experiment. By a 

adding five or ten cubic centimetres Sib 

of this water, containing the sugar es me site 36h 


and microérganisms arrested by it, 
to nutrient gelatin or agar liquefied by heat, and then making Es- 
march roll tubes, the number of germs in the entire quantity is easily 
estimated by counting the colonies which develop in the roll tubes. 
Sedgwick and Tucker, in a communication made to the Boston 
Society of Arts, January 12th, 1888, were the first to propose the use 
of a soluble filter of granulated sugar for collecting atmospheric 
germs. Their complete apparatus consists of an exhausted receiver, 
from which a given quantity of air is withdrawn by means of an air 
pump. A vacuum gauge is attached to the receiver, which is coupled 


622 BACTERIA IN THE AIR. 


with the glass tube containing the granulated-sugar filter by a piece 
of rubber tubing. Instead of transferring the soluble filter to gela- 
tin in test tubes, they use a large glass cylinder having a slender 
stem, in which the sugar is placed (Fig. 193). After the aspiration 
liquefied gelatin is introduced into the large glass cylinder, which is 
held in a horizontal position ; the sterilized cotton plug is then re- 
placed in the mouth of the cylinder, the sugar is pushed into the 
liquefied gelatin and dissolved, and by rotating the cylinder upon a 
block of ice the gelatin is spread upon its walls as in an Esmarch roll 
tube. For convenience in counting the colonies lines are drawn upon 
the surface of the cylinder, dividing it into squares of uniform di- 
mensions. 


GENERAL RESULTS OF RESEARCHES MADE. 


As already stated, the presence of bacteria in the atmosphere de- 
pends upon their being wafted by currents of air from surfaces where 
they are present in a desiccated condition. That they are not carried 
away from moist surfaces is shown by the fact that expired air from 
the human lungs does hot contain microdrganisms, although the in- 
spired air may have contained considerable numbers, and there are 
always a vast number present in the salivary secretions. The moist 
mucous membrane of the respiratory passages constitutes a germ 
trap which is much more efficient than the glass slide smeared with 
glycerin used in some of the aéroscopes heretofore described, for it 
is a far more extended surface. As a matter of fact, most of the sus- 
pended particles in inspired air are deposited before the current of 
air passes through the larynx. 

Air which passes over large bodies of water is also purified of its 
germs and other suspended particles. The researches of Fischer 
show that ata considerable distance from the land no germs are 
found in the atmosphere over the ocean, and that it is only upon ap- 
proaching land that their presence is manifested by the development 
of colonies upon properly exposed gelatin plates. 

Uffelmann found, in his researches, that in the open fields the 
number of living germsin a cubic metre of air averaged two hundred 
and fifty, on the sea coast the average was one hundred, in the court- 
yard of the University of Rostock four hundred and fifty. Thenum- 
ber was materially reduced after a rainfall and increased when a 
dry land wind prevailed. 

Frankland found that fewer germs were present in the air in 
winter than in summer, and that when the earth was covered with 
snow the number was greatly reduced, as also during a light fall of 
snow ; the air of towns was found to be more rich in germs than the 


BACTERIA IN THE AIR. 623 


air of the country; the lower strata of the atmosphere contained 
more than the air of elevated localities. 

Von Freudenreich also found that the air of the country contained 
fewer germs than that of the city. Thus in the city of Berne acubic 
metre of air often contained as many as two thousand four hundred 
germs, while the maximum in country air was three hundred. His re- 
sults corresponded with those of Miquel in showing that the number 
of atmospheric organisms is greater in the morning and the evenins, 
between the hours of 6 and 8, than during the rest of theday. Neu- 
mann, whose researches were made in the Moabite Hospital, found 
the greatest number of bacteria in the air in the morning after the 
patients able to sit up had left their beds and the wards had been 
swept. The number of germs was then from eighty to one hundred 
and forty in ten litres of air, while in the evening the number fell to 
four to ten germs in ten litres. 

Miquel has given the following summary of results obtained in 
his extended experiments, made in Paris during the years 1881, 1882, 
and 1883 : 


Number of Germs in a Cubic Metre of Air. 


Air of Laboratory, Air of Park, Mont 
Montsouri. souri. 
Average for 1880.......00c0005 seeeeee cee 215 71 
hig” ETO S Vis woot cten dedi ad ian 348 62 
ae pr ee 550 51 


Rue de Rivoli, average for one year, 750; summit of Pantheon, 28 ; 
Hotel-Dieu, 1880, average for four months, male ward 6,300, female 
ward 5,120; La Piété Hospital, average of fifteen months, 11,100. 

It must be remembered that the figures given relate both to bac- 
teria and to the spores of mould fungi, and that the latter are com- 
monly the most numerous when the experiment is made in the open 
air. Petri has shown that when gelatin plates are exposed in the air 
the relative number of spores of mould fungi deposited upon then: is 
less than is obtained in aspiration experiments. 

The number of colonies which develop on exposed plates does not 
represent the full number of bacteria deposited, for these colonies 
very frequently have their origin in a dust particle to which several 
bacteria are attached, or in a little mass of organic material contain- 
ing a considerable number. 

It is generally conceded that sea air and country air are more 
wholesome than the air of cities, and especially of crowded apart- 
ments, in which the number of bacteria has been shown to be very 
much greater. But it would be a mistake to ascribe the sanitary 
value of sea, country, and mountain air to the relatively small num- 


624 BACTERIA IN THE AIR. 


ber of bacteria present in such air. There are other important fac- 
tors to be considered, and we have no satisfactory evidence that the 
number of saprophytic bacteria present in the air has an important 
bearing upon the health of those who respire it. We do know that 
the confined air of crowded apartments, and especially of factories 
in which a large quantity of dust is suspended in the air, predisposes 
those breathing such air to pulmonary diseases and lowers the gen- 
eral standard of health. But it has not been proved that this is due 
to the presence of bacteria. Infectious diseases may, under certain 
circumstances, be communicated by way of the respiratory passages 
as a result of breathing air containing in suspension pathogenic bac- 
teria; but there is reason to believe that this occurs less frequently 
than is generally supposed. 

Kriiger has shown that the dust of a hospital ward in which pa- 
tients with pulmonary consumption expectorated occasionally upon 
the floor contained tubercle bacilli. This was proved by wiping up 
the dust on a sterilized sponge, washing this out in. bouillon, and in- 
jecting this into the cavity of the abdomen of guinea-pigs. Two 
animals out of sixteen injected became tuberculous. In pulmonic 
anthrax, which occasionally occurs in persons engaged in sorting 
wool— wool-sorters’ disease ”—infection occurs as a result of the 
respiration of air containing the spores of the anthrax bacillus. 

Among the non-pathogenic saprophytes found in the air certain 
aérobic micrococci appear to be the most abundant, and, as a rule, 
bacilli are not found in great numbers or variety. In some localities 
various species of sarcinz are especially abundant. The following 
is a partial list of the species which have been shown by the researches 
of various bacteriologists to be occasionally present in the air. But, 
as heretofore remarked, their presence is to be regarded as acci- 
dental, and so far as we know there is no bacterial flora properly be- 
longing to the atmosphere : 


Micrococcus ures (Pasteur), Diplococcus roseus (Bumm), Diplococcus 
citreus conglomeratus (Bumm), Micrococcus radiatus (Fliigge), Micrococcus 
flavus desidens (Hltigge), Micrococcus flavus liquefaciens (Fliigge), Micro- 
coccus tetragenus versatilis (Sternberg), Micrococcus pyogenes aureus (Rosen- 
bach), Micrococcus pyogenes citreus (Passet), Micrococcus cinnabareus 
(Fliigge), Micrococcus flavus tardigradus (Fliigge), Micrococcus versicolor 
(Fliigge), Micrococcus viticulosus (Katz), Micrococcus candidans (Fliigge), 
Pediococcus cerevisize (Balcke), Sarcina lutea (Schréter), Sarcina rosea 
(Schréter), Sarcina aurantiaca, Sarcina alba, Sarcina candida (Reinke), 
Bacillus tumescens (Zopf), Bacillus subtilis (Ehrenberg), Bacillus multipedi- 
culosus (Fliigge), Bacillus mesentericus fuscus (Fliigge), Bacillus mesenteri- 
cus ruber (Globig), Bacillus inflatus (A. Koch), Bacillus mesentericus vul- 
gatus, Bacillus prodigiosus, Bacillus aérophilus (Liborius), Bacillus pestifer 
(Frankland), Spirillum aureum (Weibel), Spirillum flavescens (Weibel), Spi- 
rillum flavum (Weibel), Bacillus Havaniensis (Sternberg). 

In the researches of Welz, made in the vicinity of Freiburg, twenty- 
three different micrococci and twenty-two bacilli were obiaiued from the 
air. 


BACTERIA IN THE AIR. 625 


ADDITIONAL NOTES UPON BACTERIA IN THE AIR. 


Ruete and Enoch (1895) have examined the air of closed schoolrooms 
with the following results. Eighteen different species were obtained, only 
one of which proved to be pathogenic for mice, guinea-pigs, and rabbits. The 
number of bacteria per cubic metre varied from 1,500 to 3,000,000, the aver- 
age pene about 268,000. The observations were made during the winter 
mouths. 

Marpmann (1893), in his examination of dust collected in the streets of Leip- 
zig for tubercle bacilli, obtained positive results from a considerable pro- 
portion of the specimens examined. Evidently these bacilli in dust from the 
streets are liable to be blown into the air and deposited upon the mucous 
membrane of the respiratory passages of those breathing this air. Christiani 
(1893) has shown that, as a rule, no bacteria are present in the air at an alti- 
tude of one thousand metres or more above the soil (air collected during 
balloon ascensions). 

Dyar (1895) has made a careful study of the microdrganisms found in the 
air in the city of New York. He has described numerous species of micro- 
cocci and bacilli found chiefly in the air of the hallway of the College of 
Physicians and Surgeons. Some of these are new and some have been 
identified as previously described species. 


40 


II. 


‘BACTERIA IN WATER. 


THE water of the ocean, of lakes, ponds, and running streams 
necessarily contains bacteria, as they are constantly being carried 
into it by currents of air passing over the neighboring land surfaces, 
and by rain water which washes suspended microérganisms from 
the atmosphere ; and, as such water contains more or less organic 
material in solution, many of the saprophytic bacteria multiply in it 
abundantly. It is only in the water of springs and wells which 
comes from the deeper strata of the soil that they are absent. The 
number and variety of species present in water from any given 
source will depend upon conditions relating to the amount of organic 
pabulum, the temperature, the depth of the water, the fact of its 
being in motion or at rest, its pollution from various sources, etc. 
The comparatively pure water of lakes and running streams contains 
a considerable number of bacteria which find their normal habitat 
in such waters and which multiply abundantly in them, notwith- 
standing the small quantity of organic matter and salts which they 
contain. The water of stagnant, shallow pools, and of sluggish 
streams into which sewage is discharged, contains a far greater 
number and a greater variety of species. 

The study of these bacteria in water has received much attention 
on account of the sanitary questions involved, relating to the use of 
water from various sources for drinking purposes. In the present 
section we shall first give an account of the methods of bacteriologi- 
cal water analysis, and then a condensed statement of results ob- 
tained in the very numerous investigations which have been made. 

A very important point to be kept in view is the fact that a great 
increase in the number of bacteria present, in samples of water col- 
lected for investigation, is likely to occur if these samples are kept 
for some time. A water which, for example, contains only two 
hundred to three hundred bacteria per cubic centimetre when the ex- 
amination is made at once, may contain several thousand at the end 
of twenty-four hours, and at the end of the second or third day 
twenty thousand or more may be present in the same quantity. 


aw 


BACTERIA IN WATER. 627 


Later, on account of the exhaustion of organic pabulum, the num- 
ber is again reduced as the bacteria present gradually lose their 
vitality. Under these circumstances-it is evident that an estimate of 
the number of bacteria present in water from a given source can 
have no value, unless a sample is tested by bacteriological methods 
within a short time after it has been collected. Not more than an 
hour or two should be allowed to elapse, especially in warm weather. 
By placing the water upon ice the time may be extended somewhat, 
but Wolffhiigel has shown that the number of germs is gradually 
diminished when water is preserved in this way, and it will be safest 
to make an immediate examination when this is practicable. 

The collection may be made in a sterilized Erlenmeyer flask pro- 
vided with a cotton air filter, or in a bottle having a ground-glass 
stopper which has been wrapped in tissue paper and sterilized for an 
hour or more at 150° C. in the hot-air oven. Or the small flasks with 
a long neck may be used, as first recommended by Pasteur. These 
are prepared as follows: The bulb is first gently heated, and the ex- 
tremity of the tube dipped into distilled water, which mounts into 


Fie, 194, 
a7 


the bulb as it cools; the water is then made to boil, and when al] 
but a drop or two has escaped and the bulb is filled with steam the 
extremity of the tube is hermeticallyesealed. When the steam has 
condensed by the cooling of the bulb a partial vacuum is formed, 
and the tube is ready for use at any time. It is filled with water by 
breaking off the sealed extremity under the surface of the water of 
which a sample is desired. This is done with sterilized forceps, and 
care must be taken that the exterior of the tube is properly sterilized 
before the collection is made. The end is immediately sealed in the 
flame of a lamp. A difficulty with these vacuum ‘tubes is that they 
are so completely filled with water that this cannot be readily drawn 
from them again in small quantities. The writer therefore prefers 
to make the collection in a tube shaped as shown in Fig. 194, in which 
a partial vacuum is formed just before the collection by heating the 
air in the bulb. The water mounts into the tube as the air in the 
bulb cools, and is readily forced out again for making cultures by 
applying gentle heat to the bulb, Asa lamp is needed to seal the end 
of the tube in either case, there is no special advantage in having a 
vacuum formed in advance, and, as stated, the vacuum tubes are so 


628 BACTERIA IN WATER. 


nearly filled with water that it is not so simple a matter to obtain the 
contents for our culture experiments without undue exposure to at- 
mospheric germs. In practice small glass bottles with ground-glass 
stoppers will be found most convenient, and, when properly steril- 
ized, are unobjectionable. They should be filled at a little distance 
below the surface, as there is often a deposit of dust upon the surface 
- of standing water, and sometimes a 
: delicate film made up of aérobic bac- 
‘ teria. When water is to be obtained 
: from a pump or a hydrant it should 


x be allowed to flow for some time before 
the collection is made. To collect 
water at various depths the apparatus 


shown in Fig. 195 isrecommended by 
Lepsius. An iron frame supports an 
inverted flask, A, filled with sterilized 
mercury and containing about three 
hundred cubic centimetres. The flask 
B is intended to receive the mercury 
when, at the desired depth, it is al- 
c al lowed to flow through the capillary 
tube 6. Thisis sealed at the extremity 
i) and bent as shown in the figure. By 
ON pulling upon the cord ¢ this tube is 

an broken, and as the mercury flows from 
the flask this is filled with water 
through the tube a. The extremity 
of the broken tube b is closed by the 
mercury in the flask B when A is full 
of water, and the apparatus can be 
brought to the surface with only such 
water as was collected at the depth 
from which a sample was desired. 

The bacteriological analysis is 
made by adding a definite quantity 
of the water under investigation to 
liquefied gelatin or agar-gelatin, and 
making a plate or Esmarch roll tube, which is put aside for the devel- 
opment of colonies. Miquel and others have preferred to use liquid 
cultures and the method of fractional cultivation described in the 
previous section, The use of a solid culture medium has, however, 
such obvious advantages that we do not consider it necessary to do 
more than refer to the other method as one which, when applied 
with skill and patience, may give sufficiently accurate results. 


ae aN 
fi Oy 


Lh 


i 


BACTERIA IN WATER. 629 


The amount of water which should be added to the usual quan- 
tity of liquefied flesh-peptone-gelatin in a test tube, in order that the 
colonies which develop may be well separated from each other and 
easily counted, can only be determined by experiment. If the water 
is from an impure source a single drop may be too much, and it will 
be necessary to dilute it with distilled water recently sterilized. But 
for ordinary potable water it will usually be best, in a first experi- 
ment, to make two trials, one with one cubic centimetre and one 
with one-half cubic centimetre added to the liquefied nutrient gelatin. 
The water in the collecting bottle should be shaken, to distribute the 
bacteria which may have settled to the bottom, before drawing off by 
means of a sterilized pipette the amount used for the experiment, and 
the germs present in it are to be distributed through the liquefied 
gelatin by gently moving the tube to and fro. 

Koch’s method of preparing a gelatin plate is illustrated in Fig. 
196, A glass dish, containing ice water and covered with a large 


Fig. 196, 


plate of glass, is supported upon a levelling tripod. By means of a 
spirit level this is adjusted to a horizontal position, so that when the 
liquefied gelatin is poured upon the smaller sterilized glass plate, seen 
in the centre of the large plate of-glass, it will not flow, but may be 
evenly distributed over the surface by means of a sterilized glass rod. 
The glass cover resting against the side of the apparatus is placed 
over the gelatin plate while it is cooling, to protect it from atmo- 
spheric germs, and when the gelatin is hard the plate is transferred 
to a shallow glass dish, which is kept ata temperature of about 
20° C. for several days for the development of colonies. Itis difficult 
to count colonies when more than five thousand develop upon a plate 
of the usual size, and for this reason it will be best to repeat the ex- 
periment with a smaller quantity of water from the same source, if 
this is at hand, rather than to attempt to count an overcrowded 
plate. Before pouring the gelatin upon the plate the lip of the test 
tube containing it should be sterilized by passing it through a flame. 
The liquefied gelatin should be carefully distributed to cover a rect: 


650 BACTERIA IN WATER. 


angular surface and leaving a margin of about one centimetre around 
the edge of the plate. The Koch’s dish in which the gelatin plate is 
placed for the development of colonies should be carefully sterilized 
by heat or by washing it out with a sublimate solution. A circular 
piece of filtering paper, saturated with sublimate solution or distilled 
water, is placed at the bottom of the lower dish to keep the air in a 
moist condition and prevent drying of the gelatin. Usually two or 
three plates made at the same time are placed one above the other on 
glass supports made for this purpose. If many liquefying organisms 
are present it will be necessary to count the colonies before these run 
together—usually on the second day ; but in the absence of liquefy- 
ing colonies it is best to wait until the third, or even the fifth day, as 
the number of visible colonies and the ease of counting them will be 
greater than at an earlier date. The development of afew scattered 
liquefying colonies which threaten to spoil the plate may be arrested 
by taking up the liquefied gelatin from each with a bit of filtering 
paper, and then, by means of a camel’s-hair brush, applying a solu- 
tion of potassium permanganate to the margin of the colony. The 
growth of colonies of mould fungi, which have developed from spores 
from the atmosphere falling upon the plate while it is exposed, can 
be checked by the application of collodion containing bichloride of 
mercury. 

Counting of the colonies is a simple matter when they are few 
in number; when they are numerous it is customary to place the 
plate over a dark background, and to place above it a glass plate 
divided into square centimetres by lines ruled with a diamond. By 
means of a lens of low power the colonies in a certain number of 
squares are counted and the average taken. This multiplied by the 
number of square centimetres in the gelatin-covered surface gives 
approximately the entire number of colonies which have developed 
from the amount of water used in the experiment. 

Instead of using Koch’s original plate method, as above described, 
the shallow, covered glass dishes recommended by Petri may be 
employed. These are from one to one and one-half centimetres high 
and from ten to fifteen centimetres in diameter. The liquefied gel- 
atin is poured into the lower dish and the cover at once placed over 
it. The gelatin does not dry out very soon, but, if necessary, several 
.of these Petri’s dishes may be placed in a larger jar, which serves as 
a moist chamber. 

The roll tubes of Esmarch may also be used, and have the ad- 
vantage that accidental colonies from air-borne germs are excluded. 
The counting of colonies is not quite as easy, but by the use of a 
mounted lens especially designed for the purpose it is attended with 
no great difficulty. The surface of the tube is divided into squares 


BACTERIA IN WATER. 631 


by colored lines, and the number of colonies in several squares is 
counted in order to obtain an average and estimate the entire 
number, 

Water which contains numerous liquefying bacteria had better 
be examined by the use of nutrient agar instead of gelatin; and in 
very warm weather it will be necessary to use an agar medium, as 
ten-per-cent gelatin is likely to melt if the temperature goes above 
22°C. A difficulty in the use of agar for plates consists in the lia- 
bility of the film to slip from the glass. This may be remedied to 
some extent by adding a few drops of a concentrated solution of gum 
acacia to the liquefied agar medium. Petri’s dishes are well adapted 
for the use of the agar medium, as the objection referred to does not 
apply to them. The gelatin-agar medium, containing 5 per cent 
of gelatin and 0.75 per cent of agar, may also be used with advan- 
tage in the bacteriological analysis of water. Much stress was at 
one time laid upon the enumeration of liquefying colonies, upon 
the supposition that the liquefying bacteria were especially harmful 
as compared with the non-liquefying, and that a water containing 
many liquefying colonies was to be looked upon with suspicion. We 
now know, however, that there are many common and harmless 
saprophytes which cause the liquefaction of gelatin, and that some 
of the most dangerous pathogenic bacteria do not liquefy gelatin. 
This distinction has therefore no special value, and the question for 
bacteriologists to-day is not how large is the comparative number of 
liquefying colonies, but what species are represented by the colonies 
present, liquafying and non-liquefying, and what are the special 
pathogenic properties of each. The answer to these questions, in 
the case of any particular water supply, calls for special knowledge 
and great patience and care in the isolation in pure cultures, and 
careful study of the various species present. 

It is now generally recognized that a mere enumeration of the 
number of colonies which develop from a water under investigation 
is not a sufficient indication upon which to found an opinion as to its 
potability. An excessive number of bacteria is an indication that 
the water contains a large amount of the organic material which 
serves as pabulum for these microédrganisms. But the chemists are 

able to determine the amount of organic matter present in water 
with greater precision ; and, as we have seen, the number of bacteria 
may increase many-fold in water which is kept standing in the labo- 
ratory for two or three days in a well-corked bottle. As a matter of 
fact, the enumeration of bacteria in water, although it has given us 
results of scientific interest, has not materially added to the methods 
previously applied for estimating the sanitary value of water ob- 
tained from various sources for drinking purposes. But the bacte- 


632 BACTERIA IN WATER, 


riological examination may prove to be of great value if it succeeds 
in demonstrating the presence of certain pathogenic bacteria and in 
thus preventing the use of a dangerous water. We do not mean to 
say, however, that an enumeration of the bacteria present in drink- 
ing water has no practical value. An excessive number indicates an 
excessive amount of organic pabulum, which may have come from 
a dangerous source; and the dangerous pathogenic bacteria are not 
only more likely to be present in such water, but they can more 
readily multiply in it, while ina pure water they would fail to in- 
crease in number, and, as has been shown by experiment, would die 
out within a short time. 

The number of bacteria present in rain water, or in snow which 
has recently fallen, varies greatly at different times. Naturally the 
number is greater when the surface of the earth is dry and the at- 
mosphere loaded with dust by currents of wind passing over it, and 
less when the surface is moist and the atmosphere has been purified 
by recent rains. 

In snow from the surface of a glacier in Norway, Schmelck found 
two bacteria and two spores of mould fungi per cubic centimetre of 
water from the melted snow. Ganowski, in experiments made with 
freshly fallen snow collected in the vicinity of Kiew, obtained the fol- 
lowing results: February 2d, 1888: temperature of the air, 7.2°C.; 
snowfall, 0.1 millimetre ; number of bacteria in 1 cubic centimetre 
of water from melted snow, 34 in one sample and 38 in another. 
February 20th, 1888: temperature, 11.1° C.; snowfall, 1.1 milli- 
metres ; number of bacteria in one sample, 203, in another 384. 

Miquel obtained from rain water collected at Montsouri during a 
rainy season 4.3 germs per cubic centimetre ; in rain water collected 
in the centre of the city of Paris, 19 per cubic centimetre. 

Hazithas also been shown to contain bacteria in considerable num- 
bers. Bujwid found in hailstones which fell at Warsaw 21,000 
bacteria in 1 cubic centimetre ; but this is exceptional, and is supposed 
to be due to the fact that surface water had been carried into the air 
by the storm and frozen. Fontin examined hail which fell in St. 
Petersburg, and obtained an average of 729 bacteria per cubic centi- 
metre of water from the melted hail. 

River water has been carefully examined by numerous bacterio- 
logists in various localities and at different seasons of the year. We 
give below some of the results reported : 

Water of the Seine at Choisy, before reaching Paris, 300; at 
Bercy, 1,200; at Saint-Denis, after receiving the sewer water from 
the city, 200,000 germs per cubic centimetre (Miquel). 

Water of the Spree beyond Képenick, 82,000 ; two hundred steps 
below the mouth of the Wuhle, 118,000; in Berlin above the mouth 


BACTERIA IN WATER, 633 


of the Panke, 940,000; below the mouth of the Panke, 1,800,000 
(Koch). 

Water of the Main above the city of Wurzburg, in the month of 
February, 520; below the city, 15,500 (Rosenberg). 

Water of the Potomac, at Washington, in 1886 : January, 3,774; 
February, 2,536; March, 1,210; April, 1,521; May, 1,064; June, 
348; July, 255; August, 254; September, 178; October, 75; No- 
vember, 116 ; December, 967 (Theobald Smith). 

The Thames, in the autumn of 1885, in the vicinity of London 
Bridge two hours after high water, contained 45,000 germs per cubic 
centimetre ; the water of the Lea at Lea Bridge, 4,200,000 (Bisch- 
off). 

The Neva inside the city of St. Petersburg, in September, 1883, 
contained 1,500 in one sample and 1,040 in another ; in November 
(20th), 6,500 (Poehl). 

The water of the Oder, collected within the limits of the city of 
Stettin, was found by Link to contain from 5,240 to 15,000 bacteria 
per cubic centimetre ; that of the Limmat, at Zurich, 346 in one 
specimen and 508 in another (Cramer). 

Lake water, as a rule, contains fewer bacteria than river water. 

Wolffhiigel, in researches extending from July, 1884, to July, 
1885, obtained from the water of the Tegeler Lake an average of 396 
bacteria per cubic centimetre. Cramer obtained an average of 168 
per cubic centimetre during the months of October, December, and 
January, 1884, from the water of Lake Zurich ; in June of the same 
year the average of 42 examinations gave 71 per cubic centimetre. 
In Lake Geneva, Fol and Dunant obtained from water collected some 
distance from the shore an average of 38 bacteria per cubic centi- 
metre. 

Ice which is usually collected from lakes and rivers contains a 
greater or less number of bacteria, according to the depth and purity 
of the water. The ice used in Berlin, collected from the surface of 
lakes and rivers in the vicinity of the city, contains from a few hun- 
dred to 25,000 bacteria to the cubic centimetre (Frankel). In the ex- 
periments of Heyroth samples of ice from the same source gave less 
than 100 per cubic centimetre in three, from 100 to 500 in eight, from 
500 to 1,000 in six, from 1,000 to 5,000 in seven, and 14,400 in one. 

Prudden obtained from Hudson River ice, put up six miles below 
the city of Albany, an average of 398 bacteria per cubic centimetre 
from transparent ice, and in the superficial “‘ snow ice” 9,187. Ice 
collected lower down the river contained an average of 189 in the 
transparent and 3,693 in the snow ice. 

Ice from the Dora at Turin was found by Bordoni-Uffreduzzi to 
contain from 120 to 3,546 bacteria per cubic centimetre. 


634 BACTERIA IN WATER. 


Hydrant water, as supplied to cities, has received the attention 
of numerous investigators. The water supply of Berlin was ex- 
amined by Plagge and Proskauer at intervals of a week from June, 
1885, to April, 1886. Their tabulated results show considerable 
variations. We give the figures for a single day, June 30th, 1885: 
Stralauer works, water of the Spree, unfiltered 4,400, filtered 53 ; 
Tegeler works, water of the lake, unfiltered 880, filtered 44; high re- 
servoir at Charlottenberg, 71; 75 W. Wilhelmstrasse, 121 ; Fried- 
richstrasse, 41-42 8. W., 160; Schmidstrasse, 165 E., 51 ; Friedrich- 
strasse, 126 N., 151; Weinmeisterstrasse, 15 C., 63. 

Wells which are supplied by water from deep strata contain few 
bacteria, unless contaminated by surface water in which they are 
usually very abundant. Roth examined the water of sixteen surface 
wells in Belgard, which has a very porous subsoil, and found from 
4,500 to 5,000 bacteria in three, from 7,800 to 15,000 in six, from 
18,000 to 35,000 in six, and 130,000 per cubic centimetre in one. 

Forty-seven wells in Stettin, the water of which was examined by 
Link, gave the following results : Less than 100 in six, 100 to 500 in 
twenty-one, and in the remainder (sixteen) from 1,000 to 18,000. 

Sixty-four wells in Mainz examined by Egger, and 53 in Gotha 
by Becker, gave more favorable results ; the number of wells in the 
former city, in which less than 100 colonies developed from 1 cubic 
centimetre, was 34, and in the latter the same (34). Bolton examined 
the water of 13 wells in Gittingen, and found but 1 in which the 
number of colonies from 1 cubic centimetre was less than 100 ; in 12 
the number varied from 180 to 4,940. 

The water of deep wells and springs may be entirely free from 
bacteria, or nearly so. Egger found in the water of an artesian well 
at Mainz 4 bacteria per cubic centimetre, and the same number was 
found by Hueppe in the deep well at the Wiesbaden slaughter-house. 
The artesian well at the gasworks of Kiel was found by Brennig to 
contain from 6 to 30 bacteria per cubic centimetre. In a spring at 
Batiolettes, Fol and Dunant found 57 bacteria per cubic centimetre. 
Fiirbringer obtained from springs at Jena 156 from one, 51 from 
another, 32 from another, and 109 fromanother. The water supplied 
to Danzig from the Prangenaur Spring was found in several experi- 
ments to be free from bacteria (Freimuth). 

In a summary of results obtained in various German cities Tie- 
mann and Gartner find that ‘sixty-nine per cent of the wells from 
which samples of water were examined contained less than 500 bac- 
teria per cubic centimetre. 

The water of sewers is naturally rich in bacteria. Miquel found 
that at Clichy the sewer water contained 6,000,000 bacteria per cubic 
centimetre. Bischoff found in water from London sewers 7,500,000, 


BACTERIA IN WATER. 635 


and numerous observations show that the number of bacteria in river 
water is greatly increased in the vicinity of and below the mouths 
of city sewers. 

We conclude from the experimental data recorded that water 
containing less than 100 bacteria to the cubic centimetre is presum- 
ably from a deep source and uncontaminated by surface drainage, 
and that it will usually be safe to recommend such water for drink- 
ing purposes, unless it contains injurious mineral substances. 
Water that contains more than 500 bacteria to the cubic centimetre, 
although it may in many cases be harmless, is to be looked upon 
with some suspicion, and water containing 1,000 or more bacteria is 
presumably contaminated by sewage or surface drainage and should 
be rejected or filtered before it is used for drinking purposes. But, 
as heretofore stated, the danger does not depend directly upon the 
number of bacteria present, but upon contamination with pathogenic 
species which are liable to be present in surface water and sewage. 
In swallowing a glassful of pure spring water a number of bacteria 
from the buccal cavity are washed away and carried into the stomach, 
which, if enumerated, would doubtless far exceed in numbers those 
found in the most impure river water. 

The number of bacteria does not depend alone upon the amount 
of organic pabulum contained in a water, and cannot be depended 
upon in forming an estimate of this; for, as has been shown by 
Bolton, certain water bacteria multiply abundantly in water con- 
taining comparatively little organic matter, while other species fail 
to grow unless the quantity is greater. In a water containing con- 
siderable nutrient material the water bacteria may be restrained in 
their development by other species present until the amount of pabu- 
lum is reduced so that these no longer thrive, when the common 
water bacteria will take the precedence, and an enumeration may 
show a greater number of colonies than at first. But, in general, 
water rich in organic material contains a greater number of bacteria 
and a greater variety of species than that which is comparatively 
pure. 

That certain bacteria may multiply in water which has been 
carefully distilled has been shown by Bolton and others. Two com- 
mon water bacteria—Micrococcus aquatilis and Bacillus erythrospo- 
rus—multiplied abundantly in doubly distilled water, and when 
this water was again sterilized and re-inoculated with one of these 
species the same abundant increase occurred. This was repeated six 
times with the same result (Bolton). Computing the number of 
these water bacteria in ten cubic centimetres of distilled water at 
twenty millions, and estimating their specific gravity at one, and the 
diameter of the individual cells at one s, the total weight of the entire 


636 BACTERIA IN WATER. 


number, according to Bolton, would be less than one-hundredth 
of a milligramme, and at least three-fourths of this must consist of 
water. Theorganic material represented by this number of bacteria 
would therefore be so minute that it might be supplied by dust par- 
ticles accidentally falling into the distilled water. 

Rosenberg has shown that while many of the species which he 
obtained in pure cultures from the water of the river Main multiplied 
in sterilized distilled water, other species quickly died out in such 
water. The growth of certain bacteria depends not only upon the 
quantity of nutritive material present, but upon its quality, the con- 
ditions in this regard being widely different for different species. 

In view of the facts heretofore stated bacteriologists are now giv- 
ing more attention to a careful study of the kinds of bacteria pre- 
sent in their examinations of water. Rosenberg, in his examinations 
of the water of the Main in the vicinity of Wtrzburg (1886), found 
that before the river reached the city the water contained more 
micrococci than bacilli, but that after receiving the sewage of the 
city the number of bacilli was greatly in excess. 

Adametz (1888) has described eighty-seven ‘species obtained by 
him from water in the vicinity of Vienna; Maschek found fifty-five 
different species in the drinking water used at Leitmeritz; and Tils 
(1890) has described fifty-nine species obtained by him from the city 
water supply at Freiburg. 

Among the pathogenic bacteria which are liable to find their 
way into water used for drinking purposes, the most important, from 
a sanitary point of view, are the bacillus of typhoid fever and the 
spirillum of Asiatic cholera. Both of these microdrganisms are pre- 
sent in great numbers in the excreta of persons suffering from the 
specific forms of disease to which they give rise, and are consequently 
liable to contaminate wells and streams which receive surface water, 
when such excreta are thrown upon the surface or into sewers, etc. 
Epidemics of these diseases have frequently been traced to the use 
of such contaminated water, and in a few instances the presence of 
these specific disease germs in water has been demonstrated by bac- 
teriological methods. Laboratory experiments indicate, however, 
that an increase of these pathogenic bacteria in drinking water is not 
likely to occur, except under special conditions, and that they die 
out after a time, being ata disadvantage in the struggle for exist- 
ence constantly going on among the numerous species which have 
their normal habitat in water. 

Bolton, Frankland, and others have shown that the anthrax ba- 
cillus, not containing spores, dies out in hydrant water within five or 
six days. In the experiments of Kraus the anthrax bacillus added 
to well water, not sterilized, ata temperature of 10.5° C., was still 


BACTERIA IN WATER. 637 


present in a living condition on the second day, but no colonies de- 
veloped after the third day ; the typhoid bacillus died out between 
the fifth and seventh days ; the cholera spirillum was no longer found 
on the second day. In the meantime the common water bacteria 
had increased in numbers enormously. Similar results have been 
reported by Hochstetter and others. Hueppe, in ten experiments in 
which the typhoid bacillus was added to well water of a bad quality, 
found that in two no development of this bacillus occurred after the 
fifth day, while a few colonies developed in the other experiments as 
late as the tenth day. In these experiments the temperature was 
comparatively low (10.5° C.). At a higher temperature the experi- 
ments of Wolffhiigel and Riedel show that an increase may take 
place. At the room temperature (about 20° C.) the typhoid bacillus 
added to distilled water, to well water, and to Berlin hydrant water 
was still present, in some instances, at the end of thirty-two days. 
And it was found that in some cases a decrease in the number 
occurred, then a notable increase, and finally a second diminution. 

Koch found the cholera spirillum in a water tank at Calcutta 
during a period of fourteen days, and in his experiments showed that 
it preserved its vitality in well water for thirty days, in Berlin sewer 
water for six to seven days, and in the same mixed with feces for 
twenty-seven hours only. In the experiments of Nicati and Rietsch 
the cholera spirillum preserved its vitality in distilled water for 
twenty days, in sewer water (of Marseilles) thirty-eight days, in 
water of the harbor for eighty-one days. The numerous experiments 
recorded by the observers named, and by Bolton, Hueppe, Hoch- 
stetter, Maschek, Kraus, and others, show that while the cholera 
spirillum may sometimes quickly die out in distilled water, in other 
experiments it preserves its vitality for several weeks (Maschek), and 
that it lives still longer in water of bad quality, such as is found in 
sewers, harbors, etc. Bolton found that for its multiplication a 
water should contain at least 40 parts in 100,000 of organic material, 
while the typhoid bacillus grew when the proportion was considerably 
less than this—6.7 parts in 100,000. 

Russell (1891) has studied the bacterial flora of the Gulf of 
Naples, and of the mud at the bottom of this gulf, collected at 
various depths up to eleven hundred metres. His investigations 
show that sea water does not contain as many bacteria as an 
equal volume of fresh water; that bacteria are found in about 
equal numbers in water from the surface and in that from various 
depths ; that the mud at the bottom constantly contains large num- 
bers of bacteria; that some of the species isolated grow best in a 
culture medium containing sea water. 

At a depth of 50 metres the water contained 121 bacteria per cubic 


638 BACTERIA 1N WATER. 


centimetre, and the mud from the bottom 245,000 ; at 100 metres the 
water contained 10 and the mud 200,000 per cubic centimetre ; at 
500 metres the water contained 22 and the mud 12,500 per -cubic 
centimetre ; at 1,100 metres the mud contained 24,000. 

The following new species were obtained by Russell from the 
source mentioned: Bacillus thalassophilus, Cladothrix intricata, 
Bacillus granulosus, Bacillus limosus, Spirillum marinum, Bacillus 
litoralis, Bacillus halophilus. 

The bacterial flora of fresh and sea water is very extensive, as 
will be seen by the following list of species which have been described 
by various bacteriologists who have given their attention to its 


study : 
NON-PATHOGENIC MICROCOCCI. 


Micrococcus aurantiacus (Cohn), Micrococcus luteus (Cohn), Micrococcus 
violaceus (Cohn), Micrococcus flavus liquefaciens (Fltigge), Micrococcus fla- 
vus desidens (Fliigge), Micrococcus radiatus (Fliigge), Micrococcus cinnaba- 
reus (Fliigge), Micrococcus flavus tardigradus (Fliigge), Micrococcus versi- 
color (Fliigge), Micrococcus agilis (Ali-Cohen), Micrococcus fuscus (Maschek), 
Diplococcus luteus (Adametz), Pediococeus albus (Lindner), Micrococcus 
cerasinus siccus (List), Micrococcus citreus (List), Micrococcus aquatilis 
(Bolton), Micrococcus fervidosus (Adametz), Micrococcus plumosus (Brauti- 
gam), Micrococcus viticulosus (Katz), Micrococcus cremoides (Zimmermann), 
Micrococcus carneus (Zimmermann), Micrococcus concentricus (Zimmer- 
mann), Micrococcus rosettaceus (Zimmermann), Micrococcus ureze (Pasteur), 
Weisser Streptococcus (Maschek), Wurmformiger Streptococcus (Maschek), 
Micrococcus aérogenes (Miller), Sarcina alba, Sarcina candida (Reinke), 
Sarcina lutea. 

PATHOGENIC MICROCOCCI. 


Staphylococcus pyogenes aureus (Rosenbach), Micrococcus of Heyden- 
reich—‘‘ Micrococcus Biskra.” 


NON-PATHOGENIC BACILLI. 


Bacillus arborescens (Frankland), Bacillus viscosus (Frankland), Bacil- 
lus aquatilis (Frankland), Bacillus liquidus (Frankland), Bacillus nubilis 
(Frankland), Bacillus vermicularis (Frankland), Bacillus aurantiacus 
(Frankland), Bacillus eceruleus (Smith), Bacillus glaucus (Maschek), Bacil- 
lus albus putidus (Maschek), Bacillus fluorescens liquefaciens, Bacillus fluo- 
rescens nivalis (Schmolck), Bacillus lividus (Plagge and Proskauer), Bacil- 
lus rubidus (Eisenberg), Bacillus sulfureum_(Holschewnikoff), Bacillus 
violaceus, Bacillus gasoformans (Hisenberg), Bacillus liquefaciens (Hisen- 
berg), Bacillus phosphorescens indicus (Fischer), Bacillus phosphorescens 
indigenus (Fischer), Bacillus phosphorescens gelidus (Katz), Bacillus sma- 
ragdino-phosphoresceus (Katz), Bacillus argenteo-phosphorescens Nos. I., 
II., and III. (Katz), Bacillus cyaneo-phosphorescens (Katz), Bacillus ar- 
genteo-phosphorescens liquefaciens (Katz), Bacillus ramosus, Bacillus sub- 
tilis (Ehrenberg), Proteus sulfureus (Lindenborn), Bacillus aureus (Ada- 
metz), Bacillus brunneus (Adametz), Bacillus flavocoriaceus (Adametz), 
Bacillus fluorescens non-liquefaciens, Bacillus latericeus (Adametz), Bacillus 
stolonatus (Adametz), Bacillus berolinensis indicus (Classen), Bacillus ery- 
throsporus (Eidam), Bacillus luteus (List), Bacillus aquatilis suleatus Nos. 
1, 2, 8, 4, and 5 (Weichselbaum), Bacillus albus (Eisenberg), Bacillus multi- 
pediculosus(Fliigge), Bacillus Ziirnianum (List), Bacillus fulvus (Zimmer- 
mann), Bacillus helvolus (Zimmermann), Bacillus ochraceus (Zimmer- 


9 


BACTERIA IN WATER. 639 


mann), Bacillus plicatus, Bacillus devorans (Zimmermann), Bacillus gracilis 
(Ziemormanin), Bacillin guttatus (Zimmermann), Bacillus implexus (Zim- 
mermann), Bacillus punctatus (Zimmermann), Bacillus radiatus aquatilis 
(Zimmermann), Bailie vermiculosus (Zimmermann), Bacillus constrictus 
(Zimmermann), Bacillus fluorescens aureus (Zimmermann), Bacillus fluo- 
rescens longus (Zimmermann), Bacillus fluorescens tenuis (Zimmermann), 
Bacillus fuscus (Zimmermann), Bacillus rubefaciens (Zimmermann), Bacil- 
lus subflavus (Zimmermann), Bacillus janthinus (Zopf), Bacillus mycoides 
(Fligge), Bacillus tremelloides (Tils), Bacillus cuticularis (Tils), Bacillus 
filiformis (Tils), Bacillus ubiquitus (Jordan), Bacillus circulans (Jordan), 
Bacillus superficialis (Jordan), Bacillus reticularis (Jordan), Bacillus ru- 
bescens (Jordan), Bacillus hyalinus (Jordan), Bacillus cloacee (Jordan), 
Bacillus delicatulus (Jordan), Bacillus violaceus laurentius (Jordan). 


PATHOGENIC BACILLI. 


Bacillus typhi abdominalis (Kberth, Gaftky), Bacillus erysipelatos suis 
(‘‘ Bacillus murisepticus,” Koch), Bacillus septicemiz hemorrhagice 
(‘‘ Bacillus cuniculicida,” Koch), Proteus vulgaris (Hauser), Proteus mira- 
bilis (Hauser), Bacillus canalis capsulatus (Mori), Bacillus canalis parvus 
(Mori), Spirillum cholerse Asiaticae (‘‘ Comma bacillus,” Koch), Bacillus coli 
communis (Escherich), Bacillus hydrophilus fuscus (Sanarelli), Bacillus 
venenosus (Vaughan), Bacillus venenosus brevis (Vaughan), Bacillus vene- 
nosus invisibilis (Vaughan), Bacillus venenosus liquefaciens (Vaughan). 

The following additional species are described by Zimmermann (1894) in 
his second publication (‘‘ Die Bakterien unserer ‘rink- und Nutzwasser”). 
Micrococcus candidus, Micrococcus coralloides, Streptococcus cinereus, Mi- 
crococcus sulphureus, Micrococcus galbanatus, Micrococcus erythromyxa, 
Sarcina flavea, Sarcina aurantiaca, Sarcina rosea. Bacillus ruber, Bacillus 
miniaceus, Bacillus mesentericus roseus, Bacillus carnosus, Bacillus chryso- 

loia, Bacillus multipediculus flavus, Bacillus villosus, Bacillus radiatus, 
Bacillus fluorescens albus, Bacillus viridans, Bacillus turcosa, Bacillus halans, 
Bacillus nacreaceus, Bacillus mirabilis, Bacillus umbilicatus, Bacillus lactis 
viscosus, Bacillus synxanthus, Bacillus sericeus, Bacillus minutus, Bacillus 
stellatus, Bacillus radicosus, Bacillus vernicosus, Bacillus mucosus, Bacillus 
centralis, Bacillus spumosus, Bacillus annulatus, Bacillus liquefaciens, Bacil- 
lus disciformans. 

The following spirilla and ‘‘vibrios” have also been found in water— 
chiefly in river water : : a ae 

Spirillum volutans, Spirillum sanguineum, Spirillum serpens, Vibrio ru- 
gula, Spirillum plicatile, Spirillum marinum (Russell). Spirillum cholerze 
Asiaticee, Spirillum of Rénon, Vibrio aquatilis (Gunther), Vibrio of Weibel, 
Vibrios of Bujwid (Bacillus choleroides a and 6), Vibrio of Litfler, Vibrios 
of Bonhoff, Vibrio of Blackstein, Vibrios of Sanarelli, Vibrios of Fischer, 
Vibrio Berolinensis, Vibrio Danubicus, Vibrio of Pfuhl (v. Metchnikovi 2). 
Several of the ‘‘ vibrios” in this list which have recently been obtained from 
river water in various parts of Europe are probably varieties of the cholera 


spirillum, 
ADDITIONAL NOTES UPON BACTERIA IN WATER. 


It is now generally recognized by bacteriologists that the potability of 
water is to be determined by an investigation relating to the presence or ab- 
sence of known pathogenic bacteria, rather than by an estimate of the num- 
ber of bacteria present in each cubic centimetre of the water under exami- 
nation. From asanitary point of view the most important of these pathogenic 
bacteria are the cholera spirillum and allied ‘‘ vibrios,” the bacilli of the ** ty- 
phoid group” (Bacillus typhi abdominalis and allied forms), the bacilli of 
the ‘‘colon group” (Bacillus coli communis with its varieties and similar 
bacilli of fecal origin). When one of these pathogenic bacilli is present in a 


640 BACTERIA IN WATER. 


water-supply in small numbers as compared with the number of saprophytic 
bacteria, it is not an easy matter to demonstrate the fact by the ordinary 

late method, especially in the case of non-liquefying species like the typhoid 
Pacillne, If we have, for example, one typhoid bacillus to one thousand ba- 
cilli of other species it is evident that in a series of three plates, made in the 
usual way for the purpose of obtaining isolated colonies, there would be but a 
small chance of obtaining a colony of the typhoid bacillus in plate No. 3, 
and a plate containing one thousand colonies or more would be so crowded 
that the detection of the single typhoid colony would be very difficult. For 
this reason, it is necessary to resort to special methods by which the more 
numerous saprophytic bacteria will be excluded, or their numbers greatly 
reduced. Some of the methods which have been successfully employed for 
the detection of the typhoid bacillus and of the cholera spirillum are given 
in the sections devoted to these microdrganisms. We give below some de- 
tails relating to the methods employed by bacteriologists of recognized com- 
petence in recent investigations : 

Marpmann (1895) considers all water which contains fecal bacteria as 
dangerous as a supply for drinking purposes. For the detection of patho- 
genic bacteria he recommends the following procedure : 

The pathogenic bacteria are divided into two groups by cultivation in nu- 
trient agar containing 0.2 percent of citric acid, and in the same medium con- 
taining two per cent of sodium carbonate. The bacilli of the typhoid group 
are said to grow in the acid medium but not in that containing two per cent 
of sodium carbonate. On the other hand, cholera vibrios develop in the al- 
kaline medium but not in that containing 0.2 per cent of citric acid. The ba- 
cilli of the colon group also (‘‘cloaca-bacilli”) do not grow in the medium 
containing citric acid. Bouillon containing the same amounts of acid and 
alkaliis also employed. The water to be examined is first mixed with an 
equal portion of acid and of alkaline bouillon in two test tubes, and these are 
kept at a temperature of 30° C. for twenty-four hours, during which time 
the pathogenic bacteria, if present, will multiply and cause a clouding of the 
culture media. Inoculations are now made into the acid and alkaline agar 
and gelatin. Growth in alkaline gelatin at the room temperature (10° to 18° 
C.) is due to ‘‘ cloaca-bacteria” ; growth in acid gelatin at 20° to 23° C. is due 
to bacilli of the typhoid group. Plates should also be made from the clouded 
bouillon, acid and alkaline ; and the colonies resembling those of the typhoid 
or of the colon group should be tested in nutrient gelatin containing sugar 
to ascertain whether there is development of gas, in which case the bacilli are 
of the colon group. 

When typhoid and colon bacilli are associated in water the last-mentioned 
bacillus takes the precedence, and the typhoid bacillus has a tendency to dis- 
appear. Thisisshown by the experiments of Gimbert (1894), who introduced, 
at the same time, colon bacilli and typhoid bacilli into water, and found that 
at the end of forty-eight hours he was no longer able to isolate the typhoid 
bacillus from plates. In view of this fact failure to find the typhoid bacillus 
does not relieve the water from the suspicion of being dangerous if the colon 
bacillus is present. But, on the other hand, this bacillus is so common that 
it is perhaps the exception when it is not present in surface waters. As 
pointed out by von Freudenreich (1895) it may, however, escape detection 
unless a considerable quantity of water is used in making the test. When 
the quantity is from one hundred to five hundred cubic centimetres, in- 
stead of from one to five cubic centimetres, as wasformerly the usual amount 
Stoned it is found not infrequently even in spring water (von Freuden- 
reich). 

The author last mentioned says that when present in small numbers it 
saay be demonstrated by the method of Vincent, as follows: Mix of the water 
ninety cubic centimetres with ten cubic centimetres of a twenty-per-ceut 
solution of peptone, and one cubic centimetre of a seven-per-cent solution of 
carbolic acid; place in the incubating oven at 42°C. If development '1- 


BACTERIA IN WATER. 641 


curs it will probably be due to the colon bacillus, but it will be necessary to 
make plates and pure cultures from single colonies in order to determine 
this with certainty. The demonstration may be made more quickly, accord- 
ing to von Freudenreich, by using a medium containing milk sugar (five per 
cent) and cultivating at 35° C. If the colon bacillus is present there will be 
an abundant development of gas in from twelve to twenty-four hours, and 
the bacillus may then be readily isolated by the plate method. The colon 
bacillus has been found by Moissan and Gimbert in mineral waters bottled in 
France. Poncet (1895) has made a careful study of the bacteria found in the 
various springs at Vichy. The species described are all harmless water bac- 
teria and have little interest from a sanitary point of view. 

Kruse (1894), as a result of his extended researches and of a critical con- 
sideration of the experimental data available, arrives at the conclusion that a 
sanitary inspection of the sources of supply is more important, in determin- 
ing the safety of the supply from a sanitary point of view, than a chemical 
or bacteriological examination. The writer has for some years past enter- 
tained the same opinion. Kruse says, however, that for the control of fil- 
tering plants bacteriological ‘‘counting-methods” are indispensable. He 
also ascribes a ‘‘high scientific value” to investigations relating to the pres- 
ence of the more important pathogenic bacteria; but says that, notwith- 
standing the improvements in methods of research, we cannot wait for a 
demonstration of the presence of the cholera or typhoid bacteria before con- 
demning a water as probably unsafe, if sources of contamination are dis- 
covered—or, we would add, if cases of cholera or typhoid fever can be traced 
with a fair degree of certainty to the use of water from a given source. 

Fischer (1894), in his account of the researches made during the Plankton 
expedition, has given a summary of the experimental evidence relating to the 
presence of bacteria in the waters of the ocean. The species found were for 
the most part different from those found in lakes and rivers, and at some 
distance from the shore none of the previously known species of micrococci 
and bacilli were encountered. The number of bacteria in samples from the 
surface ata distance from the shore was comparatively small (usually less 
than five hundred per cubic centimetre), but in the vicinity of land very 
large numbers were sometimes found. At adistance of ten metres below the 
surface the number found was greatly in excess of the number at the surface 
—the difference being probably due to the germicidal action of sunlight. At 
depths of four hundred metres bacteria were constantly found in great num- 
bers, and water from a depth of eleven hundred metres was still found to 
contain them. 


41 


Til. 
BACTERIA IN THE SOIL. 


SuRFACE soil, and especially that which is rich in organic matter, 
contains very numerous bacteria of many different species. Some of 
these are of special interest on account of their pathogenic power. 
Thus the bacillus of malignant cedema and the bacillus of tetanus 
have been shown to be widely distributed species, which have been 
obtained by investigators in various parts of the world by inoculating 
susceptible animals—guinea-pigs or mice—with a little rich surface 
soil. Other species are interesting because of their action in nitrifi- 
cation and in the destructive decomposition of organic material by 
which it is fitted for assimilation by the higher plants. Many of the 
bacteria present in the soil dre strictly anaérobic, and in attempts to 
estimate the number and kind of microérganisms present in a given 
sample this fact must be kept in view. 

The simplest method of studying the bacteria in the soil consists 
in introducing a small quantity into liquefied gelatin in test tubes, 
and, after carefully crushing it with a sterilized glass rod and thor- 
oughly mixing it with the gelatin, making roll tubes in the usual 
way. Some of these should be put up for anaérobic cultures—z.e., 
the tube should be filled with an atmosphere of hydrogen according 
to Frankel’s method. If the object in view is to estimate the num- 
ber of bacteria in a given sample of soil the difficulty is encountered 
that, however finely crushed, the little masses of earth are likely to 
contain numerous bacteria, and we cannot safely assume that each 
colony originates from a single germ. Thoroughly washing a small 
quantity of soil, by agitation, in a considerable quantity of distilled 
water, and then adding a definite quantity of the water to nutrient 
gelatin and making roll tubes or plates, as in water analysis, sug- 
gests itself as a simple method ; but Frankel has shown that it is far 
from being reliable when the object is to estimate the number of 
bacteria. He obtained more uniform and accurate results by intro- 
ducing the earth at once into liquefied gelatin and crushing it as 
thoroughly as possible with a strong platinum wire, after which as 
thorough a mixture as possible was effected by tilting the tube up 


BACTERIA IN THE SOIL. 643 


and down. But for the purpose of obtaining pure cultures from sin- 
gle colonies of the various species present, we should prefer to wash 
the earth in distilled water and to allow the sediment to settle before 
taking a portion of the water to add to the nutrient medium. 

In some experiments made in 1881 Koch ascertained that in soil 
which had not been disturbed but few bacteria were to be found at 
the depth of a metre; and this fact has since been established by the 
extended researches of Frankel, who devised a special boring instru- 
ment for obtaining samples of earth from different depths. Miquel, 
in 1879, estimated the number of bacteria in one gramme of earth 
collected in the park of Montsouri, Paris, ata depth of twenty centi- 
metres, at 700,000; and in a cultivated field which had been treated 
with manure,.at 900,000. The following results were obtained by 
Adametz: One gramme of earth from a sandy soil contained at the 
surface 880,000, at a depth of twenty to twenty-five centimetres 
400,000 ; the same quantity of clayey soil contained at the surface 
500,000, at a depth of twenty to twenty-five centimetres 460,000. 

In experiments made by Beumer (1886) and by Maggiora (1887) 
considerably greater numbers were found, but the last-named ob- 
server, in some instances at least, kept the earth for some time after 
collecting it, which may have materially influenced the result. 
Beumer obtained from a specimen of sandy humus taken from a 
depth of three metres 45,000,000 to the gramme; at four metres, 
10,000,000; at five metres, 8.000,000; at six metres, 5,000,000. 
These specimens were obtained from the vicinity of hospitals at 
Greifswald. In a churchyard, at a depth of four metres, the num- 
ber in one experiment was 1,152,000, and in another 1,278,000. 

Frankel has given special attention to the examination of undis- 
turbed soil not in the immediate vicinity of dwellings. In samples 
from a fruit orchard near Potsdam he found that the superficial 
layers contained from 50,000 to 350,000 germs per cubic centimetre. 
The greatest number was not immediatel; upon the surface, but at 
from one-quarter to one-half metre below the surface. The num- 
ber was found to be greater in summer than in winter, the maximum 
being in July and August. Ata depth of three-quarters of a metre 
to a metre and a half there wasavery great and abrupt diminutionin 
thenumber of germs, From 200,000 at one-half metre the number fell 
to 2,000 at a depth of a metre, from 250,000 at three-quarters of a 
metre to 200 at one metre, etc., and at a depth of one and one-half 
metres, in some instances, no more living germs were obtained. In 
other experiments a few colonies developed from earth obtained ata 
depth of three or four metres, but these were slow in making their 
appearance, and often several days, or even weeks, elapsed before 
they became visible in Esmarch roll tubes. In experiments with sur- 


644 BACTERIA IN THE SOIL. 


face soil, on the contrary, a multitude of colonies developed within 
twenty-four to forty-eight hours, and, as many liquefying bacteria 
were present, it was necessary to make the enumeration on the first 
or second day, at which time, no doubt, many of the bacteria present 
had not yet formed visible colonies. The results obtained have, 
therefore, only a relative value. 

The most important fact developed by Frankel’s researches is that 
in virgin soil there is a dividing line at a depth of from three-quarters 
to one and one-half metres, below which very few bacteria are found, 
and that, consequently, the ‘‘ ground-water region ” is free from micro- 
érganisms, or nearly so, notwithstanding the immense numbers pre- 
sent in the superficial layers. 

The extended researches of Maggiora, made in the vicinity of 
Turin, led him to the following conclusions : 


1. The number of germs in desert and forest soils is much smaller, other 
conditions being equal, than in cultivated lands, and in these it is less than 
in inhabited localities. 

2. In desert soils the number of germs bears a relation (a) to the geologi- 
cal epoch to which the lands belong, and, within certain limits, to the height 
above the level of the sea—the older the soil and the greater the altitude, 
other things being equal, the fewer the germs ; (6) to the compactness and 
aération of the soil—the more compact and impermeable to air the smaller 
the number of germs capable of developing in gelatin ; (c) to the nature of 
ie soil—sandy soils contain fewer germs than soils rich in clay and in 

umus. 

3. In cultivated lands the number of germs augments with the activity 
of cultivation and the strength of the fertilizers used. 

4, In inhabited localities the number of germs in the superficial layers is 
very great. In the deep layers it usually diminishes rapidly, as is the case 
in all other soils. 


As to the kinds of bacteria present, and their biological characters 
and functions in preparing organic material for assimilation by the 
plants whose roots penetrate the soil, we have yet much to learn. 
Frankel remarks that the species most frequently encountered in the 
deeper strata of the soil were three bacilli which also abound in the 
superficial layers—viz., the ‘‘ hay bacillus,” the ‘‘ wurzel bacillus,” 
and the “‘hirnbacillus.” In all eleven bacilli were isolated and cul- 
tivated. Micrococci were only found four times, and spirilla not at 
all. Mould fungi were more abundant, and especially one previously 
obtained from the air by Hesse and called by him “‘ brauner Schim- 
melpilz.” Anaérobic bacilli, contrary to expectation, were not ob- 
tained in Frankel’s researches, and no pathogenic species were found 
in the deeper layers of the soil. We have already referred to the 
fact that the bacillus of malignant cedema and the bacillus of tetanus, 
two pathogenic, anaérobic species, are common in rich surface soil in 
various parts of the world. 


BACTERIA IN THE SOIL. 645 


The results obtained in the researches referred to, in which nutri- 
ent gelatin was used as a culture medium, are no doubt very in- 
complete, not only on account of the liquefaction of the gelatin by 
common liquefying bacilli before other species present have formed 
visible colonies, but also because this is not a favorable culture me- 
dium for some of the species present in thesoil. Thus Frankland has 
succeeded in isolating a nitrifying ferment which he calls ‘‘ Bacillo- 
coccus,” which grows abundantly in bouillon, but fails to grow in 
nutrient gelatin. Winogradski has also obtained in pure cultures a 
nitrifying ferment from the soil in the vicinity of Zurich, which he 
has called ‘‘ Nitromonas.” 

Comparatively few micrococci are found in the soil, while in the 
air they are usually found to be more abundant than bacilli. This 
is perhaps due to the fact that the bacilli are more promptly destroyed 
by desiccation and the action of sunlight. 

Several bacteriologists have made investigations relating to the 
duration of vitality of pathogenic bacteria in the soil. Frankel found 
that in Berlin the bacillus of anthrax, in Esmarch roll tubes, when 
buried in the soil ata depth of two metres, only occasionally gave 
evidence of growth, and at three metres no development occurred. 
The comparatively low temperature at this depth was no doubt an 
important factor in influencing the result. The cholera spirillum in 
the months of August, September, and October grew at a depth of 
three metres, but in the remaining months of the year failed to grow 
at two, while growth occurred at one and one-half metres. The 
bacillus of typhoid fever grew at three metres during the greater 
portion of the year. 

Giaxa has made extended and interesting experiments with the 
cholera spirillum, cultures of which he added to different kinds of 
soil (garden earth, clay, sand) and placed at different depths below 
the surface—one-quarter, one-half, and one metre. Some of theearth 
was sterilized and some was not. In the unsterilized earth he found 
the cholera spirillum in considerable numbers at the end of twenty- 
four hours at the greatest depth tested (one metre), but at the end of 
forty eight hours it had disappeared in five experiments out of seven 
—the lowest temperature at this depth was 20° C. In the sterilized 
soil the result was different ; the cholera spirillum was present in 
enormous numbers at the end of four days ata depth of a metre, 
and was still found in smaller numbers at the end of twelve days, but 
had disappeared at the end of twenty-onedays. These resultsindicate 
that the presence of common saprophytes in the soil is prejudicial to 
the development of the cholera spirillum, and that under ordinary 
circumstances it succumbs in the struggle for existence with these 
more hardy microérganisms. 


646 BACTERIA IN THE SOIL. 


The researches of Proskauer (1891) confirm those of Frankel and 
others as to the rapid diminution in the number of bacteria in the 
deeper layers of the soil. They also agree with those of Gartner in 
showing that in the soil of churchyards the number of bacteria 
diminishes greatly in the soil beneath the layer containing coffins. 
In general the influence of dead bodies upon the bacteria in the soil 
in the vicinity of coffins was very slight; in the subsoil of the grave- 
yard there were not many more bacteria than in similar soil outside 
of this. Reimers had previously shown that samples of earth from 
two graves, in one of which the body had been buried for thirty-five 
years and in the other for one and one-half years, gave similar re- 
sults when examined by bacteriological methods. 

Manfredi in 1892 published the results of his extended investiga- 
tions relating to the dust in the streets of Naples. The number of 
bacteria varied greatly in different parts of the city. In streets 
where the traffic was least and hygienic conditions the best the 
average number was 10,000,000 per gramme. In dirty and busy 
thoroughfares the average was 1,000,000,000, and in certain locali- 
ties the number was even five times as great as this. Injections into 
guinea-pigs gave a positive result in seventy-three per cent of the 
animals experimented upon. Among the known pathogenic bacte- 
ria obtained in this way were the pus cocci (in eight), Bacillus tuber- 
eulosis (in three), the bacillus of malignant cedema, and the tetanus 
bacillus. 

In the memoir of Fiilles (1891) the following species are described 
as having been found by him in the soil at Freiburg, Germany: 


MICROCOCCI. 


(a) Non-liquefying.—Micrococcus aurantiacus (Cohn), Micrococcus can- 
didus (Cohn), Micrococcus luteus (Cohn), Micrococcus candicans (Fliigge), 
Micrococcus versicolor (Fliigge), Micrococcus cinnabareus (Fliigge), Micro- 
coccus cereus albus (Passet), Micrococcus fervitosus (Adametz), Rother coc- 
cus (Maschek). 

(b) Liquefying.—Micrococcus flavus liquefaciens (Fliigge), Micrococcus 
flavus desidens (Fliigge), Diplococcus luteus (Adametz), Sarcina lutea. 


NON-PATHOGENIC BACILLI. 


(a) Non-liquefying.—Bacillus fluorescens putidus (Fliigge), Bacillus mus- 
ecoides (Liborius), Bacillus scissus (Frankland , Bacillus candicans, Bacillus 
diffusus (Frankland), Bacillus filiformis (Tils), Bacillus luteus (Fliigge), 
Fluorescent water bacillus (Eisenberg), Bacillus viridis pallescens (Frick), 
Bluish-green fluorescent bacillus (Adametz), Bacillus stolonatus (Adametz), 
Bacillus Ziirnianum (List), Bacillus aérogenes (Miller), Bacillus No. 1 and 
Bacillus No. 2 (Fiilles). 

(b) Liquefying.—Bacillus ramosus liquefaciens (Fliigge), Bacillus liqui- 
dus (Frankland), Bacillus ramosus—‘‘ wurzel bacillus,” Bacillus subtilis 


BACTERIA IN THE SOIL. 647 


(Ehrenberg), Bacillus mesentericus fuscus (Fliigge), Bacillus mesentericus 
vulgatus Giggs), Bacillus fluorescens liquefaciens (Fliigge), Lemon-yellow 
bacillus (Maschek), Green yellow bacillus (Hisenberg), Gas-forming bacillus 
(Eisenberg), Gray bacillus (Maschek), Bacillus prodigiosus (Ehrenberg), 
Proteus mirabilis (Hauser), Proteus vulgaris (Hauser), Bacillus mesentericus 
vulgatus, Bacillus cuticularis (Tils), ‘‘ Weisser bacillus ” (Hisenberg). 

(c) Pathogenic.—Bacillus cedematis maligni (Koch). 

In addition to the above the following species have been described by 
other authors: Bacillus liquefaciens magnus (Liideritz), Bacillus radiatus 
(Liideritz), Bacillus solidus (Liideritz), Bacillus mycoides roseus (Scholl), 
Bacillus viscosus (Frankland), Bacillus candicans (Frankland), Bacillus 
poliformis (Liborius), Clostridium foetidum (Liborius). 

Pathogenic species.—Staphylococcus pyogenes aureus (Rosenbach), Ba- 
cillus tetani (Nicolaier), Streptococcus septicus (Nicolaier), Pseudo-cedema ba- 
cillus (Liborius), Bacillus septicus agrigenus (Nicolaier), Bacillus of Utpadel. 


IV. 


BACTERIA OF THE SURFACE OF THE BODY AND OF 
EXPOSED MUCOUS MEMBRANKES. 


GREAT numbers of bacteria of various species multiply upon the 
surface of the human body, where they find the necessary pabulum 
in the excretions from the skin and the exfoliated epithelium. Evi- 
dently the number will be largely influenced by the clothing worn, 
the atmospheric conditions as to heat and moisture, personal habits, 
etc. The writer has frequently inoculated culture media with a drop 
of sterilized fluid which had been placed upon the surface of the body 
of patients in hospitals and of healthy persons. By friction with a 
platinum needle at the point where the drop of fluid is applied the 
surface is washed and a little epithelium detached. Cultures may , 
always be obtained by inoculating nutrient media from a drop of fluid 
applied in this way. Micrococci of various species, including the pus 
cocci, are very commonly encountered ; sarcinze and various bacilli 
are also frequently met with. Even the hands, which by reason of 
their exposure and frequent ablutions are freer from exfoliated epi- 
thelium than portions of the body covered with clothing, have con- 
stantly attached to their surface a considerable number of bacteria. 
This is shown by the experiments of Ktimmel and Forster, of Fiir- 
bringer and others, with reference to the disinfection of the hands. 
Forster found that after the most careful cleaning of the hands with 
soap, water, and a brush, contact of the fingers with nutrient gelatin 
always resulted in the development of a greater or less number of 
colonies. 

Bordoni-Uffreduzzi, in his researches relating to the bacteria of 
the skin, obtained in pure cultures five different species of micrococci 
and two bacilli. Pure cultures of his Bacterium graveolens, which 
was usually found between the toes, gave off a disagreeable odor like 
that observed from this locality in certain individuals. In his re- 
searches made in Havana the writer frequently encountered in cul- 
tures from the surface, associated with various micrococci, his Micro- 
coccus tetragenus versatilis. 

Fiirbringer found quite frequently in the spaces beneath the fin- 


BACTERIA OF THE SURFACE OF THE BODY. 649 


ger nails Staphylococcus pyogenes aureus associated with various 
other microérganisms. A similar result had previously been reported 
by Bockhart. 

In his examinations of water from various sources Miquel found 
that ‘‘wash-water” from the floating laundries on the Seine con- 
tained more bacteria than water from any other source, even than 
the water of the Paris sewers. His enumeration gave twenty-six 
million germs per cubic centimetre. 

Hohein has enumerated the colonies developing from undercloth- 
ing worn for various lengths of time and made of different kinds of 
material. A piece of the goods to be tested was sewed fast to the 
underclothing, so as to come in immediate contact with the body ; at 
the end of a given time a fragment one-quarter of a centimetre square 
was cut up as fine as possible and distributed in nutrient gelatin. 
Plates were made and the colonies counted at the end of five or six 
days. 

In an experiment in which sterilized woven goods were worn next 
to the skin of the upper arm the following results were obtained : 
Linen goods, at the end of’ one day 28, two days 4,180 colonies ; cot- 
ton goods, end of one day 105, end of two days 1,870 ; woollen goods, 
end of one day 606, end of two days 6,799. When the material had 
been in contact with the skin for four days the colonies which devel- 
oped were so numerous that they could not be counted. 

Maggiora isolated twenty-two species of bacteria from his cultures 
inoculated with epidermis from the foot. None of these proved to 
be pathogenic for mice, rabbits, or guinea-pigs. Several gave off a 
strong odor of trimethylamin, similar to that of sweating feet. 

The following species have been found upon the surface of the 
body : 

Non-pathogenic.—Diplococcus albicans tardus (Unna and Tommasoli), 
Diplococcus citreus liquefaciens (Unna and Tommasoli), Diplococcus flavus 
liquefaciens tardus (Unna and Tommasoli), Staphylococcus viridis flaves- 
cens (Guttmann), Bacillus graveolens (Bordoni-Uffreduzzi), Bacillus epider- 
midis (Bordoni), Ascobacillus citreus (Unna and Tommasoli), Bacillus fluo- 
rescens liquefaciens minutissimus (Unna and Tommasoli), Bacillus aureus 
(Unna and Tommasoli), Bacillus ovatus minutissimus (Unna and Tomma- 
soli), Bacillus albicans pateriformis (Unna and Tommasoli), Bacillus spini- 
ferus (Unna and Tommasoli), Bacillus of Scheurlen, Micrococcus tetragenus 
versatilis (Sternberg), Bacillus Havaniensis liquefaciens (Sternberg). 

Pathogenic. —Staphylococeus pyogenes albus, Staphylococcus pyogenes 
aureus, Streptococcus pyogenes, Diplococcus of Demme, Bacillus of | emme, 


Bacillus of Schimmelbusch, Bacillus of Tommasoli, Bacillus saprogenes IT. 
(Rosenbach), Bacillus parvus ovatus (LOffler). 


SURFACE OF MUCOUS MEMBRANES. 


Cultures made from the conjunctive of healthy persons usually 
show the presence of various micrococci, and sometimes of bacilli. 


650 BACTERIA OF THE SURFACE OF THE BODY 


McFarland (1895) says that in his researches the microdrganisms 
found were for the most part “those already described by others and 
of common occurrence in the air.” He encountered, however, sev- 
eral bacilli not previously described (“Bacillus hirsutus, Bacillus 
coerulefaciens, Bacillus circumscriptus, Bacillus succinacius, Bacillus 
violaceus flavus”). Lachowicz (1895) failed to obtain any bacteria 
in his cultures from the conjunctival sac in sixty-nine per cent of the 
healthy eyes examined by him (sixty-three eyes in all). He con- 
cludes that the microédrganisms, which at times are found in the 
healthy conjunctival sac, come principally from the air; that they 
are present in small numbers and probably remain only for a short 
time. His experiments show that most species when artificially 
introduced rapidly diminish in numbers and soon disappear entirely. 
Cultures of Streptococcus pyogenes and of Bacillus xerosis conjunc- 
tives introduced into healthy eyes did not cause the slightest irrita- 
tion. In this connection we may remark that the same is true as 
regards pathogenic bacteria introduced into the bladder, but that 
when there is some cause of local irritation or injury a chronic 
cystitis is likely to be developed. In like manner, we believe, chronic 
conjunctivitis may be developed as the result of local irritation in 
connection with the presence of pathogenic bacteria and especially of 
the pyogenic micrococci. 

The extended researches of Bach (1894) gave results corresponding 
with those of previous investigators, and not with those reported by 
Lachowicz, who, as stated above, failed to obtain cultures from sixty- 
nine per cent of the healthy eyes examined. Bach says: “In a large 
percentage of the cases the presence of bacteria may be demonstrated, 
even when the conjunctiva presents a perfectly normal appearance; 
the conjunctival sac must therefore be regarded as constantly in- 
fected.” Bach describes twenty-seven different microdrganisms ob- 
tained by him in pure cultures from this source, of these eighteen 
are micrococci. Herecognizes the fact that most of them come from 
the air, while others are introduced by the hands in rubbing the 
eyes, etc. In diseased conditions these are more numerous than in 
health, but the pus cocci are not infrequently found in healthy eyes. 

As bacteria are constantly present in the air, they are necessarily 
deposited upon the moist mucous membrane of the nose during in- 
spiration. Indeed, it would appear as if an important function of 
this extended mucous membrane is to purify the air from suspended 
particles, and it has been shown by experiment that expired air is 
practically free from bacteria. The greater number of those con- 
tained in inspired air are deposited upon the mucous membrane of 
the anterior nares. In culture experiments made by Von Besser, 
Wright, and others the aasal mucus was found to contain a great 


AND OF EXPOSED MUCOUS MEMBRANES. 651 


variety of bacteria; among others the pus cocci were frequently 
found by both of the observers mentioned. In eighty one cases Von 
Besser found the “diplococcus pneumoniz” fourteen times, Staphy- 
lococcus pyogenes aureus fourteen times, Streptococcus pyogenes 
seven times, and Friedlander’s bacillus twice. Twenty-eight of the 
cases examined were convalescents in hospital; among these the 
pathogenic species mentioned were found less frequently than in other 
individuals. The following non-pathogenic species were isolated: 
Micrococcus liquefaciens albus in twenty-two cases, Micrococcus al- 
bus in nine cases, Micrococcus cumulatus tenuis in fourteen cases, 
Micrococcus flavus liquefaciens in three cases, Bacillus striatus albus 
in ten cases, ete. 

Paulsen (1890) made thirty-one cultures in nutrient gelatin from 
sixteen persons and thirty-three in nutrient agar from twenty-two 
persons, with the following result: Eleven remained sterile, nineteen 
showed not more than ten colonies, sixteen less than one hundred, 
twelve more than one hundred, and in six the number was so great 
that they could not be counted. Micrococci were more numerous 
than bacilli; of these a “sulphur-yellow coccus” in tetrads was found 
in eight individuals. Various species of liquefying cocci, resem- 
bling the pus cocci, were isolated, but the conclusion was reached 
that none of these were identical with the staphylococci of pus, 
which Von Besser and Wright both found in a considerable propor- 
tion of the culture experiments made by them. 

Thomson and Hewlett (1895) have recently reported results which 
differ to some extent from those previously reported. While they 
found numerous bacteria in the vestibulum naris, cultures made from . 
mucus obtained from the interior of the nose usually gave a negative 
result—sixty-four out of seventy-six remained absolutely sterile, 
while in seven there was a scanty growth only. They conclude that 
while microédrganisms are occasionally found upon the Schneider- 
ian membrane they are not numerous and are often entirely absent; 
and that they are rarely found upon the pituitary membrane. Straus 
(1895) has examined the nasal secretions of persons associated with 
tubercular patients for the purpose of ascertaining if the tubercle ba- 
cillus was present. The presence of this bacillus was demonstrated, 
by inoculation into guinea-pigs, in nine healthy individuals out of 
twenty-nine examined; two of these were physicians and six were 
nurses. 

Very extended researches have been made with reference to the 
bacteria present in the human mouth, which show that numerous 
species are constantly present in the buccal secretions and upon the 
surface of the moist mucous membrane. Some of these are occa- 
sional and accidental, while others appear to have their normal habi- 


652 BACTERIA OF THE SURFACE OF THE BODY 


tat in the mouth, where the conditions as to temperature, moisture, 
and presence of organic pabulum are extremely favorable for their 
development. A minute drop of saliva spread upon a glass slide, 
dried, and stained with one of the aniline colors, will always be 
found to contain an immense number of bacteria of various forms. 
Some of these are attached to epithelial cells and some scattered about 
singly or in groups. Among those seen in a single specimen we will 
usually find cocci in tetrads, in chains, and in irregular groups, 
bacilli of various dimensions, and occasionally spirilla. According 
to Prof. Miller, of Berlin, the following species almost invariably 
occur inevery mouth: Leptothrix innominata, Bacillus buccalis max- 
imus, Leptothrix buccalis maxima, Iodococcus vaginatus, Spirillum 
sputigenum, Spirochete dentium. All of these fail to grow in ordi- 
nary culture media. Miller has made extended attempts to obtain 
cultures by varying the medium used and attempting to imitate as 
nearly as possible the natural medium in which they are found; but 
his attempts have been unsuccessful, or nearly so—“ only line cultures 
afforded a limited growth, but the colonies never developed more 
than fifteen to twenty cells, aud a transference to a second plate 
proved futile, no further growth taking place.” 

Up to the year 1885 Miller had isolated twenty-two different 
species of bacteria from the human mouth. Ten of these were cocci, 
five short bacilli, six long bacilli, and one a spirillum. Later the 
same author cultivated eight additional species. Vignal has isolated 
and described seventeen species obtained by him in pure cultures 
from the healthy human mouth; most of these are bacilli, and Miller, 
who found micrococci to be more numerous, supposes the difference 
in results to be due to the fact that many of the cocci do not grow in 
nutrient gelatin, which was the medium employed by Vignal. In 
the researches of the last-named author the following species were 
obtained most frequently, in the order given: 1. Bacterium termo. 
2. Bacilluse (Bacillus ulna ?). 3. Potatobacillus. 4. Coccusa. 5. 
Bacillusb. 6. Bacillusd. 7%. Bacillus c (Bacillusalvei ?). 8. Bacil- 
lus subtilis. 9. Staphylococcus pyogenesalbus. 10. Staphylococcus 
pyogenes aureus. 

Among the species above enumerated we find two of the most 
common pus cocci, Staphylococcus albus and aureus, but no mention 
is made of another important pathogenic micrococcus which is fre- 
quently found in the healthy human mouth, viz., the micrococcus of 
sputum septicemia, first named by the writer Micrococcus Pasteuri. 
This does not grow at ordinary temperatures, and consequently 
would not be obtained in gelatin plate cultures. Very different re- 
sults have been reported by different observers as to the frequency 
with which the pathogenic cocci are found in the buccal cavity. 


AND OF EXPOSED MUCOUS MEMBRANES. 653 


Black found in the saliva of ten healthy individuals the Staphy- 
lococeus pyogenes aureus seven times, Staphylococcus pyogenes al- 
bus four times, and Streptococcus pyogenes three times. On the 
other hand, Netter found Staphylococcus aureus only seven times in 
one hundred and twenty-seven individuals examined. Miller also 
has rarely found the pus cocci in the mouths of healthy persons. 
Streptococcus pyogenes was not found by Vignal in his extended 
researches. The experiments of the writer, of Vulpian, Frankel, 
Netter, Claxton, and others show that the micrococcus which in 
1885 I named Micrococcus Pasteuri, and which is identical with the 
‘ diplococcus pneumoniz ” of German authors, is frequently present 
in the healthy human mouth—now called Micrococcus pneumonize 
croupose. Netter examined the saliva of one hundred and sixty-five 
healthy individuals and obtained it in fifteen per cent of the number 
examined, 

Another pathogenic micrococcus which is frequently present in 
the mouths of healthy persons is the Micrococcus tetragenus of Koch. 
The following pathogenic bacteria have also been isolated and de- 
scribed : Bacillus crassus sputigenus (Kreibohm), Bacillus salivarius 
septicus (Biondi). The Streptococcus septo-pyzemicus of Biondi is 
described as having characters identical with those of the Strepto- 
coccus pyogenes of Rosenbach. Two other pathogenic species de- 
scribed by Biondi were each found in a single case only. Miller 
has described the following pathogenic species isolated and studied 
by him: Micrococcus gingives pyogenes, Bacterium gingive pyo- 
genes, Bacillus dentalis viridans, Bacillus pulpz pyogenes. 

Rosenthal (1893) examined the secretions from the mouths of 
fourteen individuals and obtained twenty-eight different bacteria; of 
these twenty-one had been previously described. Five species be- 
lieved to be new are described in detail by Rosenthal, viz.: Sarcina 
viridis flavescens, Micrococcus Reessii, Micrococcus ochraceus, Dip- 
lococcus Hauseri, Bacterium cerasinum. 

Vignal has tested a considerable number of microérganisms, 
obtained by him in his cultures from the healthy human mouth, with 
reference to their peptonizing action upon various kinds of food, with 
the idea that some of them may have an important physiological 
function of this kind. Out of nineteen species he found ten which, 
after a longer or shorter time, dissolved fibrin, nine which dissolved 
gluten, ten which dissolved casein, and five which dissolved albumin; 
nine changed lactose into lactic acid, seven inverted cane sugar, seven 
caused the fermentation of glucose, and seven coagulated milk. 

Sanarelli (1891) has shown that normal saliva has the power 
of destroying the vitality of a limited number of certain patho- 
genic bacteria, including the following species: Staphylococcus 
pyogenes aureus, Streptococcus pyogenes, Micrococcus tetragenus 


654 BACTERIA OF THE SURFACE OF THE BODY 


Bacillus typhi abdominalis, Spirillum cholere Asiatice. When to 
ten cubic centimetres of saliva, sterilized by filtration through porce- 
lain, the above-mentioned pathogenic bacteria were added in small 
numbers by means of a platinum needle carried over from a pure 
culture, no development occurred, and at the end of twenty-four 
hours the bacteria introduced were incapable of growth in a suitable 
medium. But when this amount of filtered saliva was inoculated 
with a large platinum loop—an dse—a certain number of the bacteria 
survived, and at the end of three or four days an abundant develop- 
opment occurred. At first, however, the number of living cells was 
considerably diminished. In saliva to which one ése of a culture of 
Staphylococcus aureus was added thirteen thousand eight hundred 
and forty colonies developed in a plate made immediately after inocu- 
lation, while a plate made at the end of twenty-four hours contained 
but one hundred and thirty-two colonies, and one at the end of forty- 
eight hours had but eight colonies. Subsequently multiplication 
occurred, and a plate made on the ninth day after inoculation con- 
tained so many colonies that they could not be counted. 

The diphtheria bacillus was not destroyed in filtered saliva, but 
did not multiply in it. On the other hand, it proved to be a very 
favorable medium for the development of Micrococcus pneumoniz 
croupose. 

Mucus from the surface of the meatus urinarius of man and 
woman, or from the vagina, will always be found to contain various 
bacteria ; but the bladder, the uterus, and Fallopian tubes in healthy 
individuals are free from microérganisms. 

Winter has isolated twenty-seven different species from vaginal 
and cervical mucus, and reports that he found Staphylococcus pyo- 
genes albus in one-half of the cases examined. <A streptococcus was 
also encountered which resembled Streptococcus pyogenes, although 
not positively identified with it. Samschin, on the other hand, failed 
to obtain the pus cocci in vaginal mucus from healthy women. 

Dénderlein, Von Ott, and others have carefully examined the 
lochial discharge with reference to the presence of bacteria. The 
first-named author found thatin healthy women the lochial discharge 
obtained from the uterus was free from germs, but when collected 
from the vagina various microdrganisms were obtained. In one case 
in which some fever existed Staphylococcus pyogenes aureus was 
found in the vagina, while the discharge from the uterus was free 
from germs. In five cases of puerperal fever Streptococcus pyogenes 
was obtained in the lochial discharge from the uterus. The results 
of Von Ott correspond with those of Dénderlein. Czerniewski, in 
the lochia of fifty-seven healthy women, found the Streptococcus 
pyogenes but once, while in the lochial discharge of fatal cases of 
puerperal fever it was always present. 


AND OF EXPOSED MUCOUS MEMBRANES. 655 


Steffeck (1892) has examined the vaginal secretion of twenty-nine 
pregnant females who had not been subjected to digital examina- 
tion, and found Staphylococcus pyogenes albus in nine, Staphylo- 
coccus pyogenes aureus in three, and Streptococcus pyogenes in one. 
These results indicate that puerperal septicemia from self-infection 
may occur in exceptional cases. In seventeen of the twenty-nine 
cases examined none of these pyogenic micrococci were found. 

Hofmeister (1894) has shown that bacteria are found not only 
upon the mucous membrane of the meatus urinarius in man, but 
that they may usually be obtained from the urethral canal at a depth 
of eight centimetres or more, although the number rapidly diminishes 
in the deeper portion of the urethra. 

Walthard (1895) arrives at the conclusion that while in pregnant 
females bacteria are constantly found in the vagina and the lower . 
portion of the cervical canal, they are absent from the upper part of 
the cervical canal, the uterus, and the tubes; and that during the 
puerperal condition the uterine cavity is preserved from spontaneous 
infection per vias naturalis by the plug of mucus in the cervical 
canal. In the vaginal secretions of one hundred pregnant women, 
. who had not been subjected to a digital examination, streptococci 
were obtained twenty-seven times in cultures. These were not viru- 
lent, but, according to Walthard, these saprophytic streptococci be- 
come virulent when, owing to a diminished resisting power, they are 
enabled to invade the tissues as parasites. 

Krénig (1894) concludes from his investigations that the vaginal 
secretions of pregnant women are usually so acid that Streptococcus 
pyogenes could not multiply in them; also that when the secretion is 
normal it is almost always sterile. 

Déderlein (1894) insists that the failure of Krénig to obtain micro- 
organisms in his cultures was due to the fact that suitable media 
were not used; also that certain bacilli are constantly found in nor- 
mal, acid vaginal secretions, and that in the pathological secretions 
which are feebly acid, neutral, or in some cases slightly alkaline a 
great variety of bacteria are found, including Streptococcus pyo- 
genes, as demonstrated by himself and other investigators. In a 
later paper (1894) Kroénig reports his success in obtaining cultures 
from normal, acid vaginal secretions by using acid media and by 
cultivating under anaérobic conditions. He reports also that patho- 
genic bacteria (streptococci, staphylococci, and Bacillus pyocyaneus) 
introduced into the vagine of pregnant women lose their power of 
reproduction in from six to forty-eight hours (streptococci did not 
grow after six hours). Ina still later communication (1894) Kronig 
reports that the bacteria present in the vaginal secretions of pregnant 


656 BACTERIA OF THE SURFACE OF THE BODY 


women are for the most part strictly anaérobic species, and that 
among these he found two non-pathogenic streptococci. 

Menge (1894) has examined the vaginal secretions in fifty non- 
pregnant women who had been in bed for at least fourteen days— 
after laparotomy. Microscopical examination showed the presence 
of bacteria in all cases, but in only six cases was a development of 
colonies obtained—upon agar plates; in one case Streptococcus pyo- 
genes was present. Menge concludes from his investigations that 
spontaneous infection during childbirth cannot occur, and that with 
the exception of the gonococcus the known pathogenic bacteria can- 
not multiply in the cervical canal. 

Gawronsky (1894) has examined the secretions from the healthy 
urethra in sixty-two women, most of whom were under treatment 
for uterine disease or displacement. The material for his cultures 
was obtained by means of a platinum loop, introduced through a 
glass cylinder, at a distance of one or one and one-half centimetres 
from the external orifice of the urethra. In fifteen out of the sixty- 
two cases examined a positive result was obtained, as follows: In 
three cases Streptococcus pyogenes, in eight Staphylococcus pyogenes 
aureus, in one Staphylococcus pyogenes albus, in two Bacillus coli 
communis, in one Bacterium tholoideum of Gessner. 

The following species have been obtained from the nasal and 


buccal secretions : 
FROM THE NOSE. 


Non-pathogenic.—Micrococcus nasalis (Hajek), Diplococcus coryze 
(Hajek). Micrococcus albus liquefaciens (Von Besser), Micrococcus cumu- 
latus tenuis (Von Besser), Micrococcus tetragenus subflavus (Von Besser), 
Diplococcus fluorescens foetidus (Klamann), Micrococcus feetidus (Klamann), 
Vibrio nasalis (Weibel), Bacillus striatus flavus (Von Besser), Bacillus 
striatus albus (Von Besser). 

Pathogenic.—Staphylococcus pyogenes aureus, Staphylococcus pyogenes 
albus, Streptococcus pyogenes, Bacillus of Friedlander, Bacillus of rhino- 
scleroma (?), Bacillus foetidus ozeenze (Hajek), Bacillus mallei (L6ffler), Ba- 
cillus smaragdinus feetidus (Reimann). 


FROM THE MOUTH. 


Non-pathogenic.—Micrococcus roseus (Hisenberg), Micrococcus A, B, CO, 
D, E of Podbielskij, Sarcina pulmonum (Hauser), Sarcina lutea, Micrococcus 
candicans (Fligge), Bacillus of Miller, Bacillus virescens (Frick), Vibrio 
rugula, Vibrio lingualis (Weibel), Pseudo-diphtheria bacillus (Von Hoft- 
mann), Bacillus mesentericus vulgatus, Bacillus subtilis, Bacillus a, ), ¢, d, 
e, f, g, h, i, andj of Vignal, Bacillus subtilis similis, Bacillus radiciformis 
(Hisenberg’), Bacillus luteus, Bacillus fluorescens non-liquefaciens, Bacillus 
ruber, Bacillus viridiflavus, Proteus Zenkeri, Bacillus G, H, I, J, K, L, M, 
N, and Vibrio O and P of Podbielskij, Vibrio viridans (Miller), Micrococcus 
nexifer (Miller), Iodococcus magnus (Miller), Ascococcus buccalis (Miller), 
Bacillus fuscans (Miller). 

Pathogenic.—Staphylococcus pyogenes albus, Staphylococcus pyogenes 
aureus, Staphylococcus salivarius septicus (Biondi), Streptococcus pyogenes, 
Micrococcus salivarius septicus (Biondi), Micrococcus tetragenus (Gaftky), 


AND OF EXPOSED MUCOUS MEMBRANES. 657 


Micrococcus gingivee pyogenes (Miller), Streptococcus septo-pyzemicus (Bi- 
ondi), Streptococcus articulorum (Léffler), Micrococcus of Manfredi, Micro- 
coccus pneumoniz croupossee—‘‘ Micrococcus Pasteuri” (Sternberg); Bacillus 
diphtheriz (Loffler), Bacillus tuberculosis (Koch), Bacillus of Friedlander, 
Bacillus bronchitidz: putridee (Lumnitzer), Bacillus septicaemize hzemorrha- 
gice, Bacillus gingivee pyogenes (Miller), Bacillus pulps pyogenes (Miller), 
Bacillus dentalis viridans (Miller), Bacillus crassus sputigenus (Kreibohm), 
Bacillus saprogenes No. 1 (Rosenbach), Bacillus pneumoniz agilis (Schou), 
Bacillus pneumoniz of Klein, Bacillus pneumosepticus (Babes). 


42 


Vv, 


BACTERIA OF THE STOMACH AND INTESTINE. 


As the secretions of the mouth contain numerous bacteria, these 
must constantly find their way to the stomach, but conditions are 
not favorable for their development when the stomach is in a healthy 
state and its secretions normal. Under certain circumstances, how- 
ever, there may be an abundant development in the stomach of spe- 
cies which give rise to various fermentations, and no doubt dyspep- 
tic symptoms are frequently due to this cause. In the present 
section we are, however, only concerned with the bacteria of the 
healthy stomach. Most of these, we think, are to be considered as 
only temporarily and accidentally present in this viscus as the result 
of the swallowing of the buccal secretions and of food and drink con- 
taining them. 

The experiments of Straus and Wirtz and of others show that 
normal gastric juice possesses decided germicidal power, which is 
due to the free hydrochloric acid contained in it. Hamburger (1890) 
found that gastric juice containing free acid is almost always free 
from living microérganisms, and that it quickly kills the cholera 
spirillum and the typhoid bacillus, but has no effect upon anthrax 
spores. Straus and Wiirtz found that the cholera spirillum is killed 
by two hours’ exposure in gastric juice obtained from dogs, the 
typhoid bacillus in two to three hours, anthrax bacilli in fifteen to 
twenty minutes, and the tubercle bacillus in from eighteen to thirty- 
six hours. The experiments of Kurlow and Wagner, made with 
gastric juice obtained from the stomach of healthy men by means of 
a stomach sound, gave the following results: Anthrax bacilli with- 
out spores failed to grow after exposure to the action of human gas- 
tric juice for half an hour, but spores were not destroyed in twenty- 
four hours; the typhoid bacillus was killed in one hour; the 
cholera spirillum, the bacillus of glanders, and Bacillus pyocyanus 
were all destroyed at the end of half an hour ; the pus cocci showed 
greater resisting power. Certain bacteria have a greater resisting 
power for acids than any of those above mentioned, and some of them 
may consequently pass through the healthy stomach to the intestine 


BACTERIA OF THE STOMACH AND INTESTINE. 659° 


in a living condition, but there is good reason to believe that the 
spirillum of cholera or the bacillus of anthrax would not. On the 
other hand, the tubercle bacillus and the spores of other bacilli can, 
no doubt, pass through the stomach to the intestine without losing 
their vitality. 

Of nineteen species isolated by Vignal in his cultures from the 
healthy human mouth, the greater number resisted the action of the 
gastric juice for more than an hour, and six species which did not 
form spores were found to retain their vitality in gastric juice for 
more than twenty-four hours. 

In making a bacteriological analysis of the contents of the healthy 
stomach the more resistant microérganisms and those which form 
spores will naturally be found in greater or less numbers, inasmuch 
as some of them are likely to be present in food and water ingested. 

Van Puteren (1888) obtained a variety of microérganisms in very 
considerable numbers from the stomachs of infants fed upon un- 
sterilized cow’s milk, but in healthy nursing infants the number was 
much smaller, especially when the mouth was washed out with dis- 
tilled water immediately before and after nursing. In 18 per cent 
of the cases no microédrganisms were found under these circum- 
stances, and in 41 per cent the number fell below one thousand per 
cubic centimetre. Among the nursing infants examined (eighty- 
five) the following species were most numerous: Monilia candida, 
Bacillus lactis aérogenes, a non-liquefying coccus, Staphylococcus 
pyogenes aureus, Bacillus subtilis. In infants fed upon cow’s milk 
(eleven) Bacillus lactis aérogenes was present in 45.4 per cent of 
the cases, and Staphylococcus pyogenes aureus in 27.2 per cent, non- 
liquefying cocci in 54.4 per cent, liquefying cocci in 72.7 per cent, 
Bacillus subtilis in 36.3 per cent, and Bacillus butyricus (Hueppe) 
in all of the cases; next to these Bacillus flavescens liquefaciens 
was the most abundant. The author named reaches the conclusion 
that no species is constant and that the presence of those found de- 
pends upon accidental circumstances. 

Abelous (1889) found in his own stomach, washed out while fast- 
ing, a considerable number of species of bacteria, viz.: Sarcina 
ventriculi, Bacillus pyocyaneus, Bacillus lactis aérogenes, Bacillus 
subtilis, Bacillus mycoides, Bacillus amylobacter, Vibrio rugula, 
and eight other undescribed bacilli and one coccus. All of these 
microérganisms were able to resist the action of hydrochloric acid 
in the proportion of 1.7 grammes in 1,000 grammes of water. 
Several were found to be facultative anaérobics. 

The action of the bacteria isolated by him was tested by Abelous 
upon various alimentary substances. The time required to effect 
changes, such as the digestion of fibrin, the changing of starch 


660 BACTERIA OF THE STOMACH AND INTESTINE. 


into glucose, etc., was found to be so long that there was no reason 
to suppose that any one of the microérganisms tested was con- 
cerned in ordinary stomach digestion. 

In the zntestine conditions are favorable for the development of 
many species of saprophytic bacteria, and the smallest quantity of 
excrementitious material from the bowels, spread upon a glass slide 
and stained with one of the aniline colors, will be found to contain 
a multitude of microédrganisms of this class, of various forms. 
Among these are certain species which have their normal habitat in 
the intestine, and which may always be obtained in cultures from 
this source, while others, having been present in food or water in- 
gested, and having escaped destruction in the acid juices of the 
stomach, are accidentally and temporarily present. These latter 
may or may not increase in the organic pabulum which abounds in 
the intestine, according as the conditions are favorable or otherwise. 
The strictly aérobic bacteria could not multiply because of the ab- 
sence of oxygen, and the species encountered are for the most part 
anaérobics or facultative anaérobics. The Bacillus coli communis 
of Escherich, which is the most constant and abundant species found 
in the intestine of man and of certain of the lower animals, is a facul- 
tative anaérobic, which grows readily in the ordinary culture media, 
either in the presence of oxygen or in an atmosphere of hydrogen. 
But certain other bacteria of the intestine are strictly anaérobic and 
do not grow readily in the media commonly employed by bacteri- 
ologists. 

Escherich has shown that in new-born infants the meconium is 
free from bacteria. At the end of twelve to eighteen hours after 
birth bacteria appear in the alvine discharges, and the number is 
already considerable at the expiration of the first twenty-four hours 
of independent existence. The species first found are cocci and yeast 
cells which no doubt come from the atmosphere, having been de- 
posited upon the moist mucous membrane of the mouth and swal- 
lowed with the buccal secretions. When the meconium is replaced 
by “‘milk feeces” these contain in large numbers the Bacillus coli 
communis, heretofore spoken of as the most common species found in 
the intestine of adults. Another species associated with this, but 
not so abundant, is the Bacillus lactis aérogenes of Escherich. 
Other bacilli and cocci are found occasionally in smaller numbers. 
These bacilli do not liquefy gelatin, and, as a rule, the microér- 
ganisms found in the alvine discharges of healthy persons are non- 
liquefying bacteria. Escherich’s researches led him to the conclu- 
sion that the Bacillus lactis aérogenes is constantly present in the 
small intestine of milk-fed children as the most prominent species, 
and that its multiplication there is favored by the presence of milk 


BACTERIA OF THE STOMACH AND INTESTINE. 661 


sugar, and that Bacillus coli communis finds the most favorable 
conditions for its growth in the large intestine. 

Brieger, in 1884, isolated from faces and carefully studied two 
bacilli, one of which has since been called by hisname. This is a 
non-liquefying bacillus which is very pathogenic for guinea-pigs, 
and which in its morphology and characters of growth closely re- 
sembles the Bacillus coli communis of Escherich. Indeed, a num- 
ber of non-liquefying bacilli, differing but slightly in their morpho- 
logical and biological characters, have been obtained by various 
investigators from the alimentary canal of man and the lower ani- 
mals, and it is still a question whether they are to be regarded as 
distinct species or as varieties of the ‘‘colon bacillus ” of Escherich. 
The bacillus obtained by Emmerich from cholera cadavers in Na- 
ples belongs to this group, and, if not identical with the colon bacil- 
lus, resembles it so closely that its differentiation is extremely diffi- 
cult. Brieger’s bacillus forms propionic acid in solutions containing 
grape sugar. A second bacillus obtained by him from the same 
source resembles the ‘‘ pneumococcus” of Friedlander ; this causes 
the fermentation of saccharine solutions, with production of ethyl 
alcohol. 

Bienstock (1883) isolated four species of bacilli from normal feces, 
two of which are comparatively large and resemble Bacillus sub- 
tilis in their morphology and in the formation of spores. A third 
species is described as an.extremely slender pathogenic bacillus, re- 
sembling the bacillus of mouse septicemia. The fourth species is an 
actively motile bacillus which forms end spores, causing the rods to 
have the form of a drumstick. This is said to cause the decomposi- 
tion of albumin, with production of ammonia and carbon dioxide. 
Later researches do not sustain Bienstock’s conclusion that the ba- 
cilli described by him are the principal forms found in normal feces. 

Among the species encountered by Escherich, in addition to those 
mentioned above (Bacillus coli communis and Bacillus lactis aéro- 
genes), are the following: Proteus vulgaris, found three times in 
meconium, and constantly in the faeces of dogs fed upon flesh ; Strep- 
tococcus coli gracilis, found in meconium, but not during the period 
of nursing, is constantly present in the intestine when a flesh diet is 
employed. 

The intestine of carnivorous and omnivorous animals contains a 
greater number of bacteria than that of the herbivora, and in the 
large intestine they are far more numerous than in the small intes- 
tine (De Giaxa). Sucksdorf has enumerated the colonies developing 
from one milligramme of feces from individuals on mixed diet. He 
obtained an average of 380,000 from a series of observations in which 
the maximum was 2,300,000 and the minimum 25,000. 


662 BACTERIA OF THE STOMACH AND INTESTINE. 


The constant presence of certain species of bacteria in the intes- 
tine of man and the lower animals has led to the supposition that 
they may serve a useful purpose, or perhaps even have an essential 
physiological réle in connection with intestinal digestion. While 
this question has not been definitely settled, the experiments of 
Vallin, Abelous, and others have thrown some light upon it, and a 
recent experiment by Nuttall and Thierfelder (1895) has considerable 
importance as bearing upon its solution. The experiment consisted 
in removing a foetus from a pregnant guinea-pig by Ceesarean sec- 
tion, placing it under conditions which protected it from the micro- 
organisms present in the atmosphere, and feeding it upon sterilized 
milk. Great technical skill was shown in carrying out this experi- 
ment for a period of eight days, during which time the little animal 
was kept in a sterilized atmosphere and was fed every hour day and 
night. At the end of this time it had consumed over three hundred 
and thirty cubic centimetres of sterilized milk, and was as active and 
healthy as other guinea-pigs of the same age. It was now killed, and 
a careful bacteriological examination showed that the discharges 
from the bowels and the contents of the intestine were entirely sterile. 


ADDITIONAL NOTES UPON BACTERIA OF THE STOMACH AND 
INTESTINE. 


Oppler (1894) has examined material, obtained in the early morning, from 
the stomach of persons suffering from indigestion, and found nearly always 
numerous masses of sarcine. Five different species were obtained from this 
source, which were distinguished by the following characters: No. 1, colo- 
nies sulphur yellow; No. 2, colonies greenish yellow ; No. 3, colonies white ; 
No. 4, colonies white, does not liquefy gelatin ; No. 5, colonies orange yel- 
low. Nos. 1 and 3 were most frequently encountered. 

Kauffmann (1895)in a carefully studied case of chronic dyspepsia obtained 
from the contents of the stomach in the morning before breakfast, and after 
atest meal, the following bacteria: Yellow sarcina, Micrococcusaurantiacus, 
Staphylococcus cereus albus, Bacillus subtilis, Bacillus ramosus, ‘‘ a large 
thick bacillus,” ‘‘a short bacillus resembling Bacillus coli communis.” The 
last-mentioned bacillus was found in large numbers, and Kauffmann suggests 
that it may have been the cause of the fermentation in the digestive tract 
which caused the unpleasant symptoms in the case under investigation. 

Macfayden (1887) and Gillespie (1893) have also obtained a bacillus from 
the stomach which appears to be identical with Bacillus coli communis. In 
the researches of Gillespie it was obtained from a patient with dilatation of 
the stomach who suffered from flatulence, etc. In all, twenty-four different 
microdrganisms were obtained by Gillespie from the contents of the stomach 
of different individuals. This number includes three species of saccha- 
romyces and a mucor. Among the conclusions reached by Gillespie are the 
foilowing : 

‘14, Although bacteria are of no aid to peptic digestion, and a hindrance 
to the pancreatic ferment if in quantity in the duodenum, they still are of 
great use in the smal] intestine, where they control putrefaction. This seems 
paradoxical : microdrganisms obstructing microdrganisms but assisting diges- 
tion. It seems, however, to be true. The organisms which most easily 
pass the searching examination of the stomach are those which give rise by 


BACTERIA OF THE STOMACH AND INTESTINE, 663 


their growth to the fatty acids, as they are the most resistant to the action of 
acids. Their products in the small intestine are sufficient to keep the con- 
tents of that viscus acid, and they thereby prevent or control putrefaction. 
In the large intestine the secretion is so alkaline that the putrefactive organ- 
isms reassert themselves. 

“*15. Increased putrefaction in the intestinal canal may therefore be due, 
in some cases, either to insufficient mortality among the putrefactive organ- 
isms in the stomach, or to too great mortality among the acid-forming bac- 
teria and yeasts. 

‘‘16. The lactic acid which appears during the first stages of digestion is 
due to the action of organisms. 

‘17, The lactic, acetic, butyric, and succinic acids found in gastroectasis 
are due also to organisms which luxuriate in the too stationary contents. 
The marsh gas, the Brennender-gas of the Germans, is probably due to the 
same cause ; in the only case of this character with edileh I have had the 
good fortune to meet no material for examination could be obtained.” 


The following species have been isolated from feeces and the con- 
tents of the intestine of cadavers : 


Non-pathogenic.-—Streptococcus coli gracilis (Escherich), Micrococcus 
aérogenes (Miller), Micrococcus tetragenus versatilis (Sternberg), Micrococ- 
cus ovalis (Escherich), ‘' Yellow liquefying staphylococcus ” (Escherich), 
‘*Porzellancoccus” (Escherich), Bacillus subtilis, Bacillus aérogenes (Miller), 
Bacterium aérogenes (Miller), Bacillus lactis erythrogenes (Hueppe), Clostri- 
dium foetidum (Liborius), Bacillus muscoides (Liborius), Bacillus putrificus 
coli (Bienstock), Bacillus subtilis similis I. and II. (Bienstock), Bacillus 
Zopfii, Bacillus liquefaciens communis (Sternberg), Bacillus intestinus lique- 
faciens (Sternberg), Bacillus intestinus motilis (Sternberg), Bacillus fluores- 
cens liquefaciens (Fliigge), ‘‘ Colorless fluorescent liquefying bacillus” 
(Escherich), ‘‘ Yellow liquefying bacillus” (Escherich), Bacillus mesenteri- 
cus vulgatus, Bacilli of Booker, A to T, first series; a to s, second series; 
Bacilli of Jeffries A to Z, and a, #. 

Pathogenic.—Staphylococcus pyogenes aureus, Bacillus typhi abdo- 
minalis, Bacillus septiceemiee heemorrhagice, Bacillus of Belfanti and Pas- 
carola, Bacillus enteritidis (Gartner), Bacillus of Lesage, Bacillus pseudo- 
murisepticus (Bienstock), Bacillus coli communis (Escherich), Bacillus lactis 
aérogenes (Escherich), Bacillus cavicida (Brieger), Bacillus of Emmerich, 
Bacillus coprogenes foetidus (Schottelius), Bacillus of Utpadel, Bacillus leporis 
lethalis (Sternberg), Bacillus acidiformans (Sternberg), Bacillus cuniculicida 
Havaniensis (Sternberg), Bacillus cadaveris (Sternberg), Bacillus cavicida 
Havaniensis (Sternberg), Proteus vulgaris (Hauser), Bacillus tuberculosis, 
Spirillum choleree Asiaticee Spirillum of Finkler and Prior. 


VI. 


BACTERIA OF CADAVERS AND OF PUTREFYING 
MATERIAL FROM VARIOUS SOURCES. 


THE putrefactive changes which occur so promptly in cadavers, 
when temperature conditions are favorable, result chiefly from post- 
mortem invasion of the tissues by bacteria contained in the alimen- 
tary canal. But it is probable that under certain circumstances 
microérganisms from the intestine may find their way into the cir- 
culation during the last hours of life, and that the very prompt putre- 
factive changes in certain infectious diseases in which the intestine 
is more or less involved are due to this fact. The writer has made 
numerous experiments in which a portion of liver or kidney re- 
moved from the cadaver at an autopsy made soon after death—one 
to six hours—has been enveloped in an antiseptic wrapping and kept 
for forty-eight hours at a temperature of 25° to 30° C. In every in- 
stance there has been an abundant development of bacteria, although 
as a rule none were obtained from the same material immediately after 
the removal of the organ from the body. This shows that a few 
scattered bacteria were present. The same result was obtained in 
cases of sudden death from accident, as from portions of liver or 
kidney removed from the bodies of persons dying of yellow fever, 
tuberculosis, and other diseases. 

Numerous researches show that the blood of healthy men and 
animals is free from bacteria, and that saprophytic bacteria injected 
into a vein soon disappear from the circulation; and recent experi- 
ments show that blood serum has decided germicidal power. Butin 
spite of this fact the experiments of Wyssokowitsch show that cer- 
tain bacteria injected into the circulation may be deposited in the 
liver, the spleen, and the marrow of the bones, and there retain their 
vitality for a considerable time. The spores of Bacillus subtilis were 
found by the observer named to preserve their vitality in the liver or 
spleen of animals into which they had been injected, for a period of 
two or three months. In the writer’s experiments the microérgan- 
isms which first developed in fragments of liver preserved in an an- 
tiseptic wrapping were certain large anaérobic bacilli, and especially 


BACTERIA OF CADAVERS AND OF PUTREFYING MATERIAL. 665 


my Bacillus cadaveris, together with the Bacillus coli communis 
of Escherich, my Bacillus hepaticus fortuitas, and other non-lique- 
fying bacilli of the ‘‘colon group.” 

These bacteria did not give rise to a putrefactive odor, and the 
fragment of liver when cut into had a fresh appearance and a very 
acid reaction, Later, putrefactive changes occurred and Proteus 


Fig. 197.—Smear preparation from liver of yellow-fever cadaver, kept forty-eight hours in an 
antiseptic wrapping. x 1,000. Fromaphotomicrograph. (Sternberg. 


vulgaris and other putrefactive bacteria obtained the precedence. 
Evidently all of these species must have been present in the liver at 
the time it was removed from the cadaver, although in such small 
numbers that they were rarely seen in smear preparations or ob- 
tained in cultures from the fresh liver tissue. The appearance of a 
smear preparation from the interior of a fragment preserved for 
forty-eight hours in an antiseptic wrapping is shown in Fig. 197. 
The horribly offensive gases which are given off from dead ani- 
mals in a state of putrefaction appear to be due to certain large an- 
aérobic bacilli which are found in such material, 
and which have not yet been thoroughly studied \ 
owing to the difficulty of cultivating them in arti- 
ficial media ; among them is a large bacillus with XY o 
round ends which forms an oval spore at one ex- _ 
tremity of the rather long rod. This the writer oC 14 
has described under the name of Bacillus cada- NX 
veris grandis, Fig. 198. ite 
Tn the interior of a putrefying mass of this kind 
only those bacteria are found which are able to grow in the absence 
of oxygen, but aérobic saprophytes may multiply upon the surface of 


666 BACTERIA OF CADAVERS AND OF PUTREFYING MATERIAL. 


such a mass, or in organic liquids to which the air has free access. 
Among the most common putrefactive bacteria are the Proteus vul- 
garis, Proteus mirabilis, and Proteus Zenkeri of Hauser. Formerly 
the minute motile bacteria found in putrefying animal infusions, etc., 
were commonly spoken of as belonging to the species ‘‘ Bacterium 
termo,” but recent researches show that several different species were 
included under this name by those whose researches were made be- 
fore the introduction of Koch’s method for isolating and differentiat- 
ing microérganisms of this class by the use of solid culture media. 
The different species of Proteus are all facultative anaérobics. They 
are more or less pathogenic, and according to Hauser produce a chem- 
ical poison which, when injected into small animals, causes death with 
all of the symptoms of putrid intoxication. The bacillus of mouse 
septicemia, which was first obtained by Koch from a putrefying meat 
infusion, is also pathogenic, as are the writer’s Bacillus cadaveris 
and various other anaérobic bacteria found in putrefying material. 

Some account of the various products of putrefaction and the 
microérganisms concerned in their production will be found in Sec- 
tion IV., Part Second, of the present volume. 


VIL. 


BACTERIA IN ARTICLES OF FOOD. 


Milk always contains bacteria, unless drawn with special precau- 
tions into a sterilized flask. In the healthy udder of the cow it is 
sterile, but in tuberculous cows, when the milk glands are involved, 
tubercle bacilli may find their way into the milk in considerable 
numbers. Ag ordinarily obtained and preserved, milk is greatly ex- 
posed to bacterial contamination from various sources ; desquamated 
cuticle from the external surface of the udder and from the hands of 
the milker, and floating particles from the air of the stable, fall into it 
at the very moment it is drawn, and it is subsequently contaminated 
by bacteria from the air, and from water used in washing the recep- 
tacles in which it is placed or added to it by the thrifty milkman. 
As it furnishes an excellent nutrient medium for many of the bacteria 
which are thus introduced into it, under favorable conditions of tem- 
perature it quickly undergoes changes due to the multiplication in it 
of one or more of these microédrganisms. The acid fermentation and 
coagulation of the casein which so constantly occurs is completely 
prevented by sterilizing fresh milk in flasks provided with a close- 
fitting cork or cotton air filter. Numerous researches have been 
made with reference to the microdrganisms found in milk and the 
various fermentations to which they give rise. Naturally a great 
variety of species will be found in an extended research, but all are 
accidentally present, and only those demand special attention which 
produce the various fermentations of this fluid commonly encoun- 
tered, or which have special pathogenic properties. 

Several different bacteria produce an acid fermentation and con- 
sequent coagulation of milk, but the usual agent in producing this 
fermentation is the Bacillus acidi lactici, which is identical with the 
“ferment lactique” of Pasteur. Whena pure culture of this bacillus 
is introduced into sterilized milk kept at a temperature of 25° to 30°C., 
coagulation occurs in from fifteen to twenty-four hours. A uniform, 
gelatinous mass is produced which does not subsequently become 
dissolved (Adametz). Various other bacteria produce a similar 
change, including a number of common water bacteria, several spe- 


0 


668 BACTERIA IN ARTICLES OF FOOD. 


cies of sarcina, Staphylococcus pyogenes aureus, and other pus cocci. 
Usually coagulation is due to the combined action of several bacteria, 
among which Bacillus acidi lactici is apt to be the most prominent. 

Other bacteria produce coagulation without the lactic acid fer- 
mentation. This appears to be due to the formation of a soluble 
ferment which acts like rennet, causing the coagulation of milk 
which has a neutral or slightly alkaline reaction. The coagu- 
lated casein in this case is subsequently redissolved. The bacteria 
which produce this change for the most part form spores, while the 
lactic acid ferments do not. If, therefore, milk is heated nearly to the 
boiling point the acid-forming bacteria will be destroyed and the 
spores of the other species surviving will give rise to coagulation 
without the production of lactic acid. Among the more common 
microérganisms of this group are the Bacillus butyricus (Hueppe), 
Bacillus mesentericus vulgatus, Léffler’s ‘‘ white milk-bacillus,” and 
the bacilli described by Duclaux under the generic name of Tyrothrix. 

Other fermentations are produced by certain chromogenic bacteria, 
and these, as a rule, are not as harmless from a sanitary point of view 
as those above referred to. Blue milk is produced by the presence of 
Bacillus cyanogenus, yellow milk by Bacillus synxanthus (Schréter) 
and by a species obtained by List from the feces of a sheep and 
another found by Adametz in cheese. The well-known Bacillus 
prodigiosus produces its characteristic red pigment when present in 
milk, and a bluish-red color is caused by Bacterium lactis erythrogenes 
(Hueppe). 

Viscous fermentation in milk is produced by several different bac- 
teria, among others by a micrococcus studied by Schmidt-Mthlheim, 
and a short bacillus isolated by Adametz—Bacillus lactis viscosus. 
Milk which has undergone this change is unwholesome as food ; it 
is recognized by the long filaments which are produced when it is 
touched with any object and this is slowly withdrawn. 

The Caucasian milk ferment, Bacillus Caucasicus, produces a 
special fermentation, which has been referred to in Section IV., Part 
Second (page 139). 

Various pathogenic bacteria have occasionally been found in milk 
in addition to the tubercle bacillus already referred to. Thus Adametz 
found Staphylococcus pyogenes aureus in two samples which had 
been submitted to him for examination, one of which had given rise 
to vomiting and diarrhoea. Wyssokowitsch cultivated from milk 
which had been standing some time a pathogenic bacillus, named by 
him Bacillus oxytocus perniciosus. 

The special microérganism which produces the poisonous pto- 
maine called by Vaughan tyrotoxicon has not yet been isolated ; nor 
do we know the exact cause of scarlet fever, although there is evi- 


BACTERIA IN ARTICLES OF FOOD. 669 


dence that this disease uas been spread by the use of contaminated 
milk, as have also diphtheria and typhoid fever, which diseases are 
due to bacilli now well known. As the cholera spirillum grows 
readily in milk, this disease could no doubt also be transmitted in 
the same way. 

Sedgwick and Batchelder (1892) have examined a large number 
of specimens of milk obtained in Boston and vicinity, for the purpose 
of determining the number of bacteria present. They found, as an 
average of several trials, that milk obtained in a clean stable, from 
a well-kept cow, by milking in the usual way into a sterilized bottle, 
contained 530 bacteria per cubic centimetre. “When, however, the 
milkman used the ordinary milk pail of flaring form, seated himself 
with more or less disturbance of the bedding, and vigorously shook 
the udder over the pail during the usual process of milking,” the 
numbers were very much higher—on an average 30,500 per cubic 
centimetre immediately after milking. The average of fifteen sam- 
ples taken from the tables of persons living in the suburbs of Boston 
was 69,143 per cubic centimetre. The average of fifty-seven sam- 
ples of Boston milk, obtained directly from the milk wagons and 
plated at once, was 2,355,500 per cubic centimetre. The average of 
sixteen samples from groceries in the city of Boston was 4,577,000 
per cubic centimetre. 

Prof. Renk found in the milk supply of Halle from 6,000,000 to 
30,000,000 bacteria per cubic centimetre—a number considerably ex- 
ceeding that usually found in the sewage of American cities (Sedg- 
wick). 

oe and Neumann (1891) have shown that the milk of healthy 
women frequently contains bacteria, and that Staphylococcus pyo- 
genes albus is the species most frequently found. This has been 
confirmed by the researches of Palleske (1892), Ringel (1893) and 
others. The last-mentioned author examined the milk of 25 women 
recently confined, “12 of whom were healthy and 13 sick.” In 38 
cases only was the milk sterile; in 17 cases Staphylococcus pyogenes 
albus was found; in 2 cases Staphylococcus pyogenes aureus; in 1 
case both albus and aureus; in 2cases Staphylococcus pyogenes albus 
and Streptococcus pyogenes. The streptococci were found in a case 
of mild puerperal fever and in a case of phlebitis. 

The researches of Hirshberger (1889), of Ernst (1895), and of 
others show that the milk of tuberculous cows may contain tubercle 
bacilli even when the udder of the animal presents no evidence of a 
localized tubercular infection. In 121 samples of milk examined by 
Ernst from 36 different cows, 19 gave a positive result; all from the 
milk of 12 cows in which no evidence of tuberculosis of the udder 
was found in a carefully made post-mortem examination. Among 


670 BACTERIA IN ARTICLES OF FOOD. 


the bacteria which produce unwholesome chauges in milk are several 
which cause it to become viscous or soapy. Among these we may 
mention Micrococcus lactis viscosus of Conn, Micrococcus Freuden- 
reichi of Guillebeau, Bacillus mesentericus vulgatus, and Bacillus 
lactis saponacei of Weighmann and Zirn. A considerable number 
of bacilli are known which give rise to the production of butyric 
acid fermentation in milk and its products. Some of these are an- 
aérobic and some aérobic. The list includes the following: Bacillus 
butyricus of Prazmowski, Bacillus of Liborius, Bacillus of Botkin, 
Bacilli of Kadrowski. 

The bitter taste which milk and cheese sometimes acquire is due 
to the presence of special bacterial ferments; among these the best 
known are an aérobic, liquefying micrococcus described by Conn, a 
bacillus described by Weighmann, Micrococcus casei amari and Ba- 
cillus liquefaciens lactis amari of De Freudenreich (1895). 

In fresh butter of good quality comparatively few microérganisms 
are found, but the researches of Conn show that the characteristic 
and agreeable flavor of fresh butter is due to, or at least may be imi- 
tated by, a bacillus which is concerned in the ripening of cream 
under normal conditions. Cultures of this bacillus (Bacillus 41 of 
Conn) have already been used in a practical way by butter makers 
to improve the flavor of their product. 

Kreuger (1890) obtained from “cheesy butter,” having a disa- 
greeable odor, various bacteria. Among these the most numer- 
ous were an oval micrococcus (Micrococcus acidi lactici, Kreuger), a 
slender bacillus resembling Bacillus fluorescens, and Bacillus acidi 
lactici of Hueppe. 

Klecki (1894) has isolated from rancid butter several bacteria not 
previously described, one or more of which are no doubt concerned , 
in the production of the rancid taste and odor. These are described 
under the following names: Bacillus butyri, Diplococcus butyri, a 
bacillus resembling Iodococcus vaginatus of Miller, Tetracoccus 
butyri, Bacillus butyri No. 2. 

Duclaux (1887) has isolated from different kinds of cheese no less 
than eleven different species of bacteria, which he believes are con- 
cerned in the “ripening process.” Seven of these are aérobie and 
four anaérobic species. Adametz (1889) has also isolated and studied 
a number of species to which he attributes the ripening of cheese. 

More recently Henrici (1895) has studied the bacterial flora of 
cheese, and Marchal (1895) has shown that the ripening of certain 
kinds of cheese (fromages mous) is probably due to Oidium lactis. 

Meats, even when salted and smoked, may contain living patho- 
genic bacteria which were present prior to the death of the animal, 
and, when not properly preserved, are of course liable to be invaded 
by putrefactive bacteria. 


BACTERIA IN ARTICLES OF FOOD. 671 


The researches of Foster (1889) show that the typhoid bacillus, 
the pus cocci, the tubercle bacillus, and the bacillus of swine plague 
resist the action of a saturated solution of salt for weeks and even for 
months; and the same observer found that the ordinary processes of 
salting and smoking did not destroy the tubercle bacillus in the flesh 
of a cow which had succumbed to tuberculosis. Beu has made cul- 
tures from a large number of specimens of fresh, salted, and smoked 
meats and fish, with the general result that the fresh and salted meats 
were found to contain a limited number of bacteria of various species, 
and that smoking for several days did not insure the destruction of 
these microdrganisms. In specimens of sausage six days’ smoking 
did not destroy a liquefying bacillus which was present, but at the 
end of six weeks’ exposure to smoke this bacillus no longer grew, 
while a non-liquefying bacillus present in the same specimen had not 
been destroyed. Fourteen days’ smoking sufficed to destroy all the 
microérganisms in a specimen of bacon, but this was not sufficient 
for the interior portions of a ham. Among the bacteria obtained by 
Beu from smoked meats he mentions the following: Staphylococcus 
cereus albus, Proteus vulgaris, Staphylococcus pyogenes aureus, Ba- 
cillus liquefaciens viridis, etc. The number of colonies which de- 
veloped from a fragment, the size of a mustard seed to that of a flax- 
seed, taken from the interior of the meats examined, was usually 
small; and the presence of a few scattered bacteria of these common 
species has no significance from a sanitary point of view, except as 
showing that pathogenic bacteria may survive in infected meats after 
they have been exposed to the usual processes of salting and smoking. 

Petri, in experiments upon the bacillus of swine plague (Schweine- 
rothlauf), arrived at the following results : 

The flesh of swine which died of this disease preserved its infec- 
tious properties after having been preserved in brine for several 
months, and the same flesh salted or pickled for a month and then 
smoked for fourteen days contained the rothlauf bacillus in a living 
and unattenuated condition. At the end of three months virulent 
rothlauf bacilli were still obtained from a smoked ham, but they were 
no longer found at the end of six months. 

Schrank (1888) has made cultures from both the albumin and the 
yolk of fresh eggs, and finds that they are free from bacteria. He 
thinks that, as a rule, putrefactive bacteria obtain access to the inte- 
rior through injured places in the shell, although exceptionally the 
egg may be infected with them in the oviduct of the fowl. The usual 
bacteria concerned in the putrefactive changes in eggs are, according 
to the author mentioned, a variety of Proteus vulgaris and Bacillus 
fluorescens putidus. 

Zérkendorfer (1893) has cultivated from rotten eggs sixteen dif- 


672 BACTERIA IN ARTICLES OF FOOD. 


ferent bacilli, all of which are described in detail and none of which 
were found to correspond with previously described species as given 
in Hisenberg’s Bacteriological Diagnosis. 

Peters (1889) has studied the flora of the “sauerteig” used in 
Germany as yeast for leavening bread. In addition to the numerous 
cells of three species of Saccharomyces, he finds that bacilli are present 
in great numbers, as shown by direct microscopical examination and 
culture experiments. He describes five species, designated Bacillus 
A, B, C, D, and E, which are commonly present, and to which the 
acid fermentation of the dough is ascribed. 

In Graham bread which: had undergone changes making it unfit 
to eat, Kratschmer and Niemilowicz have found the Bacillus mes- 
entericus vulgatus, which appears to have been the cause of the 
fermentation, which was produced in bread having a slightly alka- 
line reaction by inoculating it with a pure culture of this bacillus. 
The infected bread has a brownish color, a peculiar odor, and be- 
comes sticky and viscid. 

Uffelmann (1890) has also studied the bacteria in spoiled rye 
bread, and obtained, in addition to common mould fungi, Bacillus 
mesentericus vulgatus and Bacillus liodermus. 

Waldo (1894) has shown that baking does not sterilize bread. 
This was to have been expected in the case of the spores of bacilli, 
but it is somewhat surprising to find that two species of Sarcina and 
two micrococci survived the baking process. In all Waldo obtained 
thirteen species of bacteria from the interior of sixty-two loaves 
examined. Bacillus subtilis and allied spore-forming bacilli were 
most frequently found, and the statement is made that a loaf “from 
a low-class, dirty bakery will almost invariably contain more living 
bacteria (or their spores) than one from a good, clean bakery.”’ 

Lehmann (1894) under the name Bacillus levans has described a 
microérganism which closely resembles Bacillus colicommunis. This 
was obtained from sour dough, and was believed to be the cause of 
the acid fermentation which so often interferes with success in ob- 
taining sweet and wholesome bread. When a culture of this bacil- 
lus was added to flour and water, without the addition of yeast, an 
active fermentation occurred and the dough became acid. 


INDEX. 


Axpotr’s study of pseudo-diphtheritic 
bacilli, 456 
Abel’s capsule bacillus, 561 
Abrin, 262, 265, 267 
Abscesses, formation of, 223 
acute, micrococci in, 14 
micrococcus pneumonie crou- 
pose in, 401 
Absinthe, antiseptic power of, 203 
Acetic acid, action of, on spores, 182 
as a decolorizer in staining, 28 
Aceton-celluloidin solution, 36 
Acetone, germicidal power of, 197 
Acetylene gas in photomicrography, 110 
Acid media, bacterial growth in, 166 
Acids, action of, on bacteria, 180 
produced by bacteria, 137 
in culture media, 46 
prejudicial to the vitality of the 
cholera spirillum, 600 
diluted, as decolorizers in staining, 
28 
Acne contagiosa of horses, bacillus of, 557 
Aérobic bacteria, 16, 172 
in stab cultures, 70 
Agar as a medium of growth for: 
bacillus A of Booker, 555 
aérogenes capsulatus, 589 
of Babes and Oprescu, 526 
Beck, 566 
of Belfonti and Pascarola, 508 
capsulatus, 522 
capsule, of Nicolaier, 560 
of Cazal and Vaillard, 525 
of cholera in ducks, 503 
dysenteriz, 569 
enteritidis, 520 
of Fiocca, 548 
gallinarum, 521 
of grouse disease, 521 


43 


Agar as a medium of growth for: 
bacillus of hog cholera, 504, 509 
of Laser, 524 
of Lucet, 526 
piscicidus, 567 
piscicidus agilis, 565 
of purpura heemorrhagica, Kolb, 
559 
of purpura hemorrhagica, Tiz- 
zoni and Giovannini, 558 
pyocyaneus 8, 545 
pyocyaneus pericarditidis, 546 
cedematis maligni. No. II., 588 
solanacearum, 572 
of symptomatic anthrax, 586, 587 
ot swine plague, Marseilles, 509 
typhi murium, 524 
mnicrococcus tetragenus, 412 
proteus of Karlinski, 552 
spirillum of Finkler and Prior, 603 
Metschnikovi, 605 
tyrogenum, 604 
Agar-agar as a solid culture medium, 43 
characters of growth in stab cultures 
in, 72 
bacillus of Emmerich and Weibel in, 
566 
bacillus pestis in, 563 
Agar-gelatin, 44 
growth of bacillus pestis in, 563 
bacillus acidiformans, 538 
Agar, nutrient, Abbott’s method of pre- 
paring, 46 
in analysis of water, 631 
as a culture medium for: 
bacillus of acne contagiosa of 
horses, 557 
bacillus alvei, 557 
bacillus coli communis, 531 
bacillus diphtheriz, 454 


674 


Agar as a culture medium for: 
bacillus endocarditidis griseus, 
556 
bacillus erysipelatos suis, 513 
bacillus gracilis cadaveris, 560 
bacillus leporis lethalis, 541 
bacillus cedematis maligni, 582 
bacillus pyocyaneus, 543 
bacillus typhi abdominalis, 489 
micrococcus botryogenus, 413 
micrococcus melitensis, 421 
proteus hominis capsulatus, 519 
proteus lethalis, 555 
proteus septicus, 554 
proteus vulgaris, 549 
nutrient, Shultz’s method of prepar- 
ing, 46 
spirillum cholere asiatice in, 
596 
staphylococcus pyogenes aureus 
in, 374 
streptococcus pyogenes in, 384 
plates, bacillus anthracis on, 425 
bacillus endocarditidis capsula- 
tus on, 556 
bacillus cedematis maligni on, 
583 
micrococcus pneumoniz croupo- 
se, 403 
solutions, filtration of, 44 
stick cultures, bacillus of purpura 
hemorrhagica, Babes, 558 
Age, as influencing susceptibility to dis- 
ease, 233 
Agitation, influence of, 
growth, 163 
Agua coco as a culture medium, 39 
analysis of, 40 
bacillus cavicida havaniensis 
in, 517 
bacillus cuniculicida havanien- 
sis in, 588 
Air, demonstrations of micro-organisms 
in, 615 
list of saprophytes found in, 624 
optically pure, 5 
of city streets, bacteria in, 625 
of closed schoolrooms, bacteriology 
of, 625 
of hospitals, pathogenic germs in,624 


on bacterial 


INDEX. 


Albuminous fluids, germicidal powers of, 
210 
Alcohol as a decolorizer in staining, 28 
action of, on bacterial growth, 197 
on micrococcus pneumonie crou- 
pose, 405 
absolute, action of, on bacillus tuber- 
culosis, 478 
Alessi’s experiments on the pathogenic 
action of typhoid bacilli, 447 
Alexins (Buchner), 236, 265 
Algerian sheep, insusceptibility of, to 
anthrax, 234 
Alkalies, action of, on bacteria, 180 
Alkalinity of blood as affecting immu- 
nity, 236 
Alkaloid in anthrax cultures, 428 
Almonds, bitter, antiseptic power of, 
208 


. Alpha-naphthol, antiseptic power of, 206 


Alum, antiseptic value of, 188 
Aluminum acetate, antiseptic value of, 
188 
chloride, antiseptic value of, 188 
Amann’s method of separating tubercle 
bacilli from sputum, 486 
Ammonia, action of, on bacterial growth, 
184 
as a nutrient of bacteria, 126 
oxidation of, by bacteria, 144 
Ammonium carbonate, antiseptic value 
of, 188 
fluosilicate, antiseptic value of, 188 
sulphate, antiseptic value of, 188 
Amplification of photomicrographs, 106 
Anaérobic bacteria, 16 
cultivation of, 80 
in stab cultures, 71 
isolation of, 83 
concerned in putrefaction, 143 
Anesthetics in experiments on animals, 
99 
Analysis, chemical, of Friedlinder’s ba- 
cillus, 124 
of putrefactive 
Nencki, 124 
of purified tuberculin, 362 
Angelica, antiseptic power of, 203 
Aniline blue solution, Loffler’s, 29 
colors as stains, 25 


bacteria by 


INDEX. 


Aniline colors for staining the anthrax 
bacillus, 424 
in culture media, 46, 64 
dyes, germicidal power of, 197 
Aniline-gentian-violet solution, 
lich’s, 29 
Aniline-methyl-violet solution, Ehrlich- 
Weigert, 29 
Aniline oil, germicidal power of, 198 
water for staining solutions, 29 
Animals, experiments upon, 96 
inoculations into susceptible, 169 
post-mortem examinations of inocu- 
lated, 101 
Anise, antiseptic power of, 203 
Annales de l'Institut Pasteur, a reference 


Ehr- 


to, 8 
de Micrographie, a reference to, 
9 
Anthrax, causation of, discovered by 


Davaine, 6 
animals immune to, 233, 234 
antidotal effect of bacillus pyocya- 
neus in, 544 
susceptibility of herbivora to, 234 
bacillus, 422 
general infection by, 428 
pathogenesis of, 428 
specific toxin of, 430 
sporeless varieties of, 426 
staining of, 424 
thermal death-point of, 157 
vitality of, in various waters, 
637 
cultures, attenuation of, 277 
chemical products in, 428 
toxalbumin of, 150 
protein, 124 
spores, formation of, 424, 426 
influence of chlorine on, 177 
influence of sunlight on, 161 
survival of, in various media, 
430 
symptomatic, 347 
bacillus of, 585 
Antiabrin, 263 
Anticholeraic inoculations, Haffkine’s, 
802 
harmlessness of, 305 
Antipneumatoxin, 261, 341 


675 


Antiseptic agents, attenuation of viru- 
lence by, 131 
power, 164 
determination of, 165 
values, Miquel’s tables of, 186 
Antiseptics, definition of, 164 
Antitetanic blood serum, therapeutic 
value of, 356 
Antitoxic values, Brieger and Cohn’s 
method of determining, 358 
Antitoxin, diphtheria, 314 
from goat’s milk, 358 
tetanus, curative power of, 359 
typhoid, present in the blood serum 
of persons who have recently suf- 
fered from the fever, 367 
Antitoxins, 237 
bactericidal value of, 267 
discovery of, 259 
immunity dependent on, 267 
Apparatus, sterilization of, 53 
for coagulating blood serum, 58 
for filtering nutrient agar-agar, 45 
for hydrogen in cultivating anaé- 
robic bacteria, 86 
incubation, of d’Arsonval, 94 
for photomicrography, 106 
for sterilizing by filtration, 59, 60 
Aqueous humor, germicidal power of, 236 
Arloing’s experiments on spore forma- 
tion, 160 
Aromatic products of decomposition, 
germicidal power of, 198 
Aronson’s diphtheria antitoxin, 314 
Arsenious acid, action of, on bacteria, 
183 
Arthritis of the wrist, micrococcus pneu- 
moniz croupose in, 402 
Arthrobacterium of Hueppe, 128 
Arthrospores, 16, 19, 123 
of the comma bacillus, 554, 598 
Artificial culture media, 40 
Asbestos, filtration of air through, 619 
Ascitic fluid, germicidal power of, 286 
Ascococcus, general characters, 17 
grouping of, 21 
Johnei (Cohn), 412 
Aseptol, germicidal power of, 198 
Atmosphere, the, relatively free from 
bacteria, 613, 622 


676 INDEX. 


Attenuated virus, Pasteur’s method of 
obtaining, 244 
restoration of virulence to, 245 
of anthrax, 427 
cultures of the anthrax bacillus, 429 
Attenuation of virulence, 130 
by cultivation in the blood of an 
immune animal, 132 
of the bacillus of diphtheria, 457, 
458 
of the bacillus of tuberculosis. 360 
of cultures of bacillus mallei, 494 
of the microbe of fowl cholera, 502 


Bazes, on the pigments of bacillus pyo- 
_eyaneus, 134 
Babes’ bacillus of purpura hemorrhagica, 
558 
Bacille du charbon symptomatique, 585 
de la morve (B. mallei), 489 
du rouget du pore (Pasteur), 511 
virgule cholérigéne, 595 
Bacillen des griinblanen Liters, 542 
Bacilli, general characters, 18 
morphology, 22 
anaérobic (pathogenic), 578 
in chronic infectious diseases, 467 
movements, 120 
of Baumgarten, 12 
non-pathogenic, list of species found 
in the soil (Fiilles), 646 
list of species found in water, 
638 
pathogenic (anaérobic), 578 
list of species found in soils 
(Filles), 646 
list of species found in water, 
639 
pseudo-typhoid, 441 
which produce septicemia in sus- 
ceptible animals, 498 
Bacillus A of Booker (from cholera in- 
fantum), 555 
aceticus, acid produced by, 137 
acidiformans (Sternberg), 139,537 
pathogenesis of, 538 
acidi lactici, 667 
thermal death-point of, 155 
of acne contagiosa of horses, 557 
aérogenes capsulatus (Welch), 587 


Bacillus alvei, 556 


pathogenesis, 557 
amylovorus (Burrill), 575 
anthracis, 422 
biological characters, 424 
destroyed by gastric juice, 658 
morphology of, 423 
pathogenesis, 428 
spore formation, 426 
resistance of, to high tempera- 
tures, 154 : 
thermal death-point of, 156 
vitality of, in waters of various 
characters, 637 
vitality of, when buried in the 
soil, 645 
of anthrax, symptomatic, 585 
biological characters, 586 
methods by which virulence is 
increased, 587 
morphology, 586 
pathogenesis of, 587 
vital resistance of its spores, 586 
B of (Booker), 536 
of Babes and Oprescu, 525 
of Beck, from infected rabbits, 566 
of Belfanti and Pascarola, 508 
der Brustseuche beim Kaninchen, 
566 
der Biiffelseuche (Oreste-Armanni), 
499 
butyricus, acid produced by, 138 
cadaveris (Sternberg) from yellow- 
fever cadavers, 584 
grandis (Sternberg) of putrefac- 
tion, 665 
campestris (Pammel), 575 
capsulatus, 521 
mucosus (Fasching), 561 
cavicida, 516 , 
havaniensis (Sternberg), 516 
thermal death-point of, 155 
vitality of, in moist atmos- 
pheres, 159 
of Cazal and Vaillard, 525 
of cholera (see Spirillum), 593 
of cholera in ducks (Cornil and 
Toupet), 503 
cholere gallinarum (Fliigge), 499 
coli communis, 529 


INDEX. 


Bacillus coli communis, probably identi- 


cal with bacillus cavicida, 529 
comniunis, discovery of, 53! 
communis, biology and mor- 

phology of, 530 
communis, pathogenesis of, 532 
communis, varieties of, 533 

coprogenes parvus, 515 
crassus sputigenus, 517 
sputigenus, thermal death-point 
of, 155 
cuniculicida (Fltigge), 499 
havaniensis (Sternberg), 538 
havaniensis pathogenesis, 540 
cyanogenus, thermal death-point of, 
155 
diphtheriz, 449, 453 
biological and morphological 

characters, 453 
columbarum, 458 
columbarum, pathogenesis, 455 
columbarum, thermal  death- 

point of, 455 
vitulorum, 459 

dysenteriz (Shiga), 568 
distinguished from bacillus ty- 

phosus, 569 

of Eberth, 431 
of Emmerich and Weibel from in- 
fected trout, 565 
endocarditidis capsulatus, 556 
griseus, 555 
enteritidis (Gartner), 520 
erysipelatos suis, 511 
suis, biological characters, 512 
suis, morphology, 511 
suis, pathogenesis, 514 
suis, action of germicides on, 513 
of Fiocca, from saliva of cats and 
dogs, 547 
fluorescens, thermal death-point of, 

155 
putridus, influence of light on, 

161 

of foul brood (of bees), 556 
of fowl cholera, 499 
thermal death-point of, 501 
action of antisepties on, 501 
der Frettchensenche (Eberth and 
Schimmelbusch), 508 


GIT 


Bacillus of Friedlander, 396 


gallinarum (Klein) not identical with 
Pasteur’s bacillus of fowl chol- 
era, 521 
thermal death-point of, 156 
gracilis cadaveris (Sternberg) from 
iver of man, 560 
of green pus, 542 
of grouse disease (Klein), 520 
of hog cholera (Salmon and Smith), 
503 
pathogenesis, 505 
der Hiihnercholera, 499 
hyacinthi (Walker), 573 
hydrophilus fuscus (Sanarelli), 522 
indicus, thermal death-point of, 155 
of influenza (Pfeiffer), 463 
toxin of, 465 
der Kaninchenseptikimie (Koch), 499 
lactis aérogenes (Escherich), 536 
of Laser, perhaps identical with the 
bacillus of swine plague, Mar- 
seilles, 524 
leporis lethalis (Sternberg, Gibier), 
541 
leprae, 486 
pathogenesis, 488 
of Lucet, perhaps identical with ba- 
cillus gallinarum of Klein, 526 
mallei, 489 
pathogenesis, 491 
thermal death-point of, 156 
des Mauseseptikaimie (Koch), 511 
of Mereshkowski, 565 
of mouse septicemia, 511 
action of chlorine on, 177 
mucosus ozene (Abel), 561 
murisepticus (Fltigge), 511 
thermal death-point of, 155 
pleomorphus (Karlinski), 552 
neapolitanus, thermal death-pvint 
of, 155 
ceedematis maligni, 580 
maligni, biology, 582 
maligni, morphology, 581 
maligni, pathogenesis, 583 
maligni, No. II. (Novy). 587 
pestis (Kitasato and Yersin), 562 
biological characters as given by 
Kitasato. 563 


678 


Bacillus pestis, influence of desiccation 
on, 564 
piscicidus (Fischel and Enoch), 567 
agilis (Sieber), 565 
pneumoniz (Fliigge) (see Micrococ- 
cus), 596 
thermal death-point of, 155 
of preputial smegma similar mor- 
phologically to the syphilis bacil- 
lus, 495 
prodigiosus, pigment produced by, 135 
influence of electric currents on, 
163 
influence of light on, 161 
influence on the virulence of 
{ anthrax cultures, 587 
thermal death-point of, 155 
vitality of, in moist atmospheres, 
159 
of purpura hemorrhagica of Babes, 
558 
hemorrhagica of Kolb, 559 
hemorrhagica of Tizzoni and 
Giovanini, 558 
pyocyaneus, 542 
antidotal effect of, in inoculated 
anthrax, 544 
butyric acid produced by, 139 
green pigment of, 134, 543 
general infection by, 544 
pathogenesis of, 543 
thermal death-point of, 155 
varieties of, 545 
pyocyaneus 3 (P. Ernst), 545 
distinguished from 
pyocyaneus, 545 
pyocyaneus pericarditidis (H. C. 
Ernst), 545 
pyogenes filiformis (Flexner), 567 
foetidus (Passet), 518 
of rabbit septicemia, 499 
of rhinoscleroma, 496 
similarity of, to Friedlander’s 
bacillus, 497 
der Rinderseuche (Kitt), 499 
amerikanischen (Caneva), 508 
salivarius septicus (Biondi), 398 
septicemize hemorrhagice, 499 
hemorrhagice, biology and mor- 
phology of, 500 


bacillus 


INDEX. 


Bacillus septicemize hemorrhagice, va-~ 
rieties of, 344 
septicus agrigenus (Nicolaier), 510 
sputigenus (Fliigge), 398 
des Schweinerothlauf (Loéffler and 
Schtitz), 511 
thermal death-point of, 155 
der Schweineseuche (Loéffler and 
Schiitz), 499 
Marseilles (Rietsch—Jobert), 508 
solanacearum (Smith), 571 
of spontaneous rabbit septicemia 
(Eberth), 508 
of swine pest (Selander), 503 
plague (Billings), 503 
plague, Marseilles, 508 
post-mortem appearances in 
death from, 510 
subtilis, resistance of, to high tem- 
perature, 154 f 
tenuis sputigenus (Pansini), 523 
tetani, 578 
tracheiphilus (Smith), 576 
tuberculosis, 468 
attenuation of, 485 
biological characters, 472 
branching forms of, 484 
chlorine, action of, on, 177 
cultures, difficulties of obtaining 
pure, 474 
cultures, methods of obtaining, 
476 
gallinarum, not a variation of 
bacillus tuberculosis, 482 
germicides, action of, on, 478 
light, action of, on, 161 
morphology, 469 
in nasal cavity, healthy, 485 
pathogenesis, 480 
reproductive elements of, 485 
resistance of, to freezing, 153 
not ordinarily saprophytic, 474 
spore formation undetermined, 
472 
sputum, examination of, for, 470 
sputum, estimation of number 
of, in, 479 
staining methods for, 30 
thermal death-point of, 158 
vitality of, 478 


INDEX. 


Bacillus, typhi abdominalis (see Typhoid 
Bacillus), 486 
murium, 524 
typhosus, 431, 436 
ures, 140 
der Wildseuche (Hueppe), 499 
X (Sternberg) from yellow-fever ca- 
davers, 538 é 
Bacteria, a distinct class of vegetable 
organisms, 11 
action of acids on, 180 
of alkalies, 183 
of coal-tar products, 197 
of gases, 172 
of the haloid elements, 172 
of salts, 186 
chemical composition of, 124 
classification of, 10 
conditions of growth of, 125 
culture media for, 37 
dependent on pabulum, 153 
dimensions of, 20 
influence of pressure on growth of, 
163 
of physical agents, 153 
of one species on the growth of 
another, 128 
mistaken for infusoria, 3, 10 
modes of grouping, 20 
modifications of biological charac- 
ters, 129 
morphology of, 20 
motions of, 117 
the photographing of, 103 
probably seen by Leeuwenhoeck, 3, 10 
products of vital activity, 133 
acids, 137 
butyric acid, 138 
fermentation, 136 
fermentation, alkaline in urine 
141 
phosphorescence, 145 
a possible nucleus in, 117 
reproduction of, 117 
rapidity of reproduction, 120 
structure of, 117 
thermal death-point of, 155 
varieties of, 117 
vitality of, in moist atmospheres, 
159 


679 


Bacteria in the air, 613 
number of relatively unimpor- 
tant, 624 
in articles of food, 667 
in croupous pneumonia, 396 
in diphtheria, 449 
in the dust of city streets, 646 
in the soil, methods of studying, 642 
action of, on buried pathogenic 
bacteria, 645 
number of, in various localities 
and at various depths, 643 
of graveyards, 643, 646 
in water, 626 
species very numerous, 636 
distilled, 125 
of cadavers and of putrefying ma- 
terial from various sources, 664 
of plant diseases, 571 
of the skin and exposed mucous 
membranes, 648 
species found on, 649 
of the stomach and intestine, 658 
nitrifying, 144 
non-pathogenic, thermal death-point 
of, 157 
parasitic, 127 
pathogenic, fatal dose of, 242 
modes of action of, 219 
Bacteriacee of Zopf, 12 
Bacterial diseases, immunity afforded by 
an attack, 243 
Bactéridie du charbon, 423 
Bacteriology, literature of, & 
early progress of, 6 
Bacterium aéruginosum, 542 
coli commune (Escherich), 516, 529 
of Davaine’s septicemia, 499, 500 
of Ehrenberg, 3, 10 
termo, 548 
Barium chloride, antiseptic value of, 188 
Baumgarten, bacilli of, 12 
classification of bacteria, 12 
on the influence of Koch’s tubercu- 
lin, 364 
Bedding, disinfectants for, 213 
Beef extracts as culture media, 41 
Beggiatoa, sulphur in, 24 
Behring’s discovery of the antitoxins, 8 
antitoxin of diphtheria, 314 


680 


Behring’s discovery of the immunization 
of large animals against tetanus, 356 
serum for tetanus, strength and doses 
of, 357 
standard of diphtheritic antitoxin, 
313 
Belfonti’s experiments on micrococcus 
pneumoniz croupose, 341 
Benzene, germicidal power of, 198 
Benzoic acid, antiseptic value of, 183 
Benzo-naphthol, no germicidal power, 
206 
Beta-naphthol, antiseptic power of, 206 
Binary division of bacteria, 120 
Biological classification of bacteria, 13 
Biskra button, micrococcus of, 415 
Bismarck brown as a stain, 25 
for staining anthrax bacilli, 424 
Blackleg, 347, 585 
Bladder, urinary, free from bacteria in 
health, 654 
Blood, alkalinity of, as affecting immu- 
nity, 236 
free from bacteria in health, 664 
germicidal power of, 286 
of typhoid patients does not yield 
cultures, 434 
serum as a culture medium, 37 
germicidal powers of, 208, 257 
immunizing value of, 313 
mixture, Léffler’s, 48 
solidification of, 57 
solidified, streak cultures on, 77 
sterilization of, 57 
Sternberg’s method of collect- 
ing, 38 
Blood serum as a medium of growth for: 
bacillus A of Booker, 555 
bacillus of acne contagiosa of 
horses, 557 
bacillus anthracis, 426 
bacillus coli communis, 531 
bacillus endocarditidis griseus, 
556 
bacillus enteritidis, 520 
bacillus erysipelatos suis, 513 
bacillus of Friedlander, 397 
bacillus hydrophilus fuscus, 523 
bacillus mallei, 491 
bacillus cedematis maligni, 582 


INDEX. 


Blood serum as a medium of growth for: 
bacillus pestis, 563 
bacillus of purpura hemorrhag- 
ica (Baves), 558 
bacillus of purpura hemorrhag- 
ica (Tizzoni and Giovannini), 
558 
bacillus of swine plague, Mar- 
seilles, 509 
bacillus tetani, 580 
bacillus tuberculosis, 474, 475 
bacillus typhi abdominalis, 439 
micrococcus gonorrhee, 393 
micrococcus Manfredi, 413 
proteus hominis capsulatus, 519 
proteus of Karlinski, 552 
proteus lethalis, 555 
proteus septicus, 554 
spirillum cholere asiatice, 596 
spirillum of Finkler and Prior, 
603 
Blucher’s apparatus for an atmosphere of 
hydrogen, 87 
Blue milk, 668 
rays of spectrum, germicidal power 
of, 161 
Bohn's thermo-regulator, 90 
Boiling in water, as a disinfectant, 212, 
213 
Booker’s bacillus B, probably identical 
with bacillus lactis aérogenes, 536 
Boracic acid, action of, on bacteria, 182 
Borden’s method of photographing by oil 
light, 111 
Bordoni-Uffreduzzi on the bacteria of the 
human skin, 648 
bacillus of, from the marrow of 
the bones of a leper, probably 
not identical with bacillus 
lepree, 487 
Boric acid, action of, on micrococcus 
pneumonie croupose, 405 
Botkin’s apparatus for an atmosphere of 
hydrogen, 87 
bacillus butyricus, 189 
Bougies of the Chamberland filter, 59, 
60 
Bouillon as a culture medium, 41 
for bacillus aérogenes capsula- 
tus, 589 


INDEX. 


Bouillon as a culture medium for bacil- 
lus of Babes and Oprescu, 526 
for bacillus of Beck, 566 
for bacillus of Cazal and Vail- 
lard, 525 
for bacillus 
532 
for bacillus diphtherie, 454 
for bacillus dysenteriz, 569 
for bacillus of Emmerich and 
Weibel, 566 
for bacillus erysipelatos suis, 
513 
for bacillus of Fiocca, 548 
for bacillus gallinarum, 521 
for bacillus gracilis cadaveris, 
560 
for bacillus of hog cholera, 504, 
509 
for bacillus of influenza, 464 
for bacillus of Laser, 524 
for bacillus of Lucet, 526 
for bacillus of Mereshkowsky, 
565 
for bacillus of Nicolaier (cap- 
sule), 560 
for bacillus cedematis maligni, 
No. II., 588 
for bacillus piscicidus, 567 
for bacillus pyocyaneus pericar- 
ditidis, 546 
for bacillus septicemiz hem- 
orrhagice, 501, 509 
for bacillus solanacearum, 572 
for bacillus of swine plague, 
Marseilles, 509 
for bacillus tetani, 579 
for bacillus of von Dungern 
(capsule), 562 
for micrococcus Manfredi, 413 
for proteus hominis capsulatus, 
519 
for spirillum cholere asiatice, 
596 
for spirillum of Metschnikovi, 
605 
Bovine mastitis, micrococcus of (Kitt), 
414 
pneumonia, micrococcus of, 414 
Branching forms of bacilli, 123 


coli communis, 


681 


Bread, not sterilized by the heat of bak- 
ing, 672 
paste as a culture medium, 49 
Brieger’s bacillus, 516 
toxic ptomains from cholera cultures, 
292 
typhotoxin, 367 
Bromine, influence of, 
growth, 178 
Bronchial glands, living tubercle bacilli 
found in, 485 
Broncho-pneumonia, a pseudo-influenza 
bacillus in, 466 
Brownian movements, 119 
Brustseuche, 335 
Bubonic plague, 562 
discovery of the bacillus of, 8 
infection in man by inoculation 
through cutaneous lesions and 
by the respiratory passages, 
565 
rats and fleas as propagators of, 
564 
Buchner’s method of cultivating anaéro- 
bic bacteria, 85 
theory of immunity, 255 
Bujwid’s test for the presence of the 
cholera spirillum, 598 
experience with tuberculin, 365 
method of preparing tuberculin, 363 
Bulbs, Sternberg’s, 38, 66 
Bureau of Animal Industry, report of 
measures for arresting swine plague, 
345 
Butter, bacteria in, 670 
Butyric acid, action of, on bacteria, 183 
produced by bacteria, 138 


on bacterial 


CapaveERIn, 146 
Cajuput, antiseptic power of, 203 
Calamus, antiseptic power of, 205 
Calcium chloride, antiseptic value of, 188 
hydroxide, action of, on bacterial 
growth, 184 
hypochlorite, 
188 
light in photomicrography, 107 
Camphor, germicidal power of, 199, 203 
Canada balsam for mounting bacterial 
slides, 28 


antiseptic value of, 


682 


Canon’s method of demonstrating the ba- 
cillus of influenza, 463 
Capsule bacilli, Nicolaier’s comparison 
of the characters of, 560 
bacillus of Nicolaier, 560 
of von Dungern, 562 
Caraway, antiseptic value of, 203 
Carbolic acid in culture media, 47 
as a disinfectant, 212, 213 
action of, on anthrax spores, 


587 

action on bacillus tuberculosis, 
478 

action on micrococcus pneu- 


monie croupose, 405 
for restraining growth of water 
bacteria, in searching for the 
typhoid bacillus, 444 
Carbol-fuchsin solution, Ziehl’s, 29 
Carbon for bacterial growth, 126 
dioxide, intluence of, on bacterial 
growth, 174 
prevents growth of the anaérobic 
bacillus of symptomatic an- 
thrax, 586 
does not prevent growth of the 
anaérobic bacillus cedematis 
maligni No. II., 588 
monoxide, influence of, on bacterial 
growth, 174 
Carnivora immune to tuberculosis, 233 
Carrot, antiseptic power of, 2038 
Cars, railway, disinfection of, 214 
Cascarilla, antiseptic power of, 203 
Cattle, prevalence of tuberculosis among, 
482 
Caucasian milk ferment, 139 
Caustic potash, action of, on micrococcus 
pneumoniz croupose, 405 
Cedar, antiseptic power of, 203 
Celery, antiseptic power of, 203 
Cell-globulin, germicidal power of, 210 
Cells, vital activity of, 133 
Cellular structure of bacteria, 117 
Cellulose, fermentation of, 141 
“Centralblatt fiir Bakteriologie,” 9 
Cerebrospinal meningitis, diplococcus 
of, 410 
Cesspools, disinfection of, 217 
Chamomile, antiseptic power of, 203 


INDEX. 


Chantemesse and Widal’s immunization 
against the typhoid bacillus, 
368 
method of searching for the 
typhoid bacillus in water sup- 
plies, 444 
Chaplets, grouping of bacteria in, 22 
Charbon, bacilius of, 422 
symptomatique, 347 
Chauveau’s retention theory of immu- 
nity, 250 
Cheese, bacteria in, 670 
toxic principle in, 148 
Chemical agents, influence of, on cultures 
of the cholera spirillum, 597 
analysis of water, advantages of, 631 
Chemiotaxis, 247, 257 
Cherry laurel, antiseptic power of, 203 
Cheyne’s experiments on the toxicity of 
cultures of proteus vulgaris, 550 
observations on the pathogenic power 
of saprophytes, 529 
Chloral hydrate, antiseptic value of, 189 
Chloride of lime asa disinfectant, 212, 213 
Chlorinated soda as a disinfectant, 213 
Chlorine, influence of, on bacterial 
growth, 177 
Chloroform, action of, on bacteria, 177 
Cholera, a disease of man, 233 
cultures, toxalbumin of, 150 
experimental evidence of its produc- 
tion in man by the comma bacil- 
lus, 601 
immunity after an attack of, 297 
infection, heat as a disinfectant for, 
154 
ptomains, 149 
second attacks of, 243 
spirillum, influence of light on, 162 
vaccine (Ferran), 299 
(Haffkine), 302 
Cholera infantum, bacillus A of Booker 
in, 555 
proteus vulgaris in, 550 
Cholera morbus not caused by the Fink- 
ler-Prior spirillum, 602 
proteus Hauseri in, 551 
Cholin, 147 
Chromic acid, action of, on putrefactive 
bacteria, 181 


INDEX, 


Chromogenic organisms, 14, 135 
Cinnamon, antiseptic power of, 203 
Cities, bacteriology of the air of, 625 
Citric acid, action of, on pathogenic bac- 
teria, 182 
Citron, antiseptic power of, 203 
Cladothrix, apparent branching in, 24 
Cladotrichee (Zépf), general characters, 
12, 19 
Classification of bacteria, 10 
Clostridium, general characters of, 18 
spore formation in, 122 
of bacilli, 23 
Clothing, disinfection of, 213 
Clou de Biskra, micrococcus of, 415 
Cloves, antiseptic power of, 203 
Cocci of Baumgarten, 12 
Coccocee (Zopf), 12 
Cocoanut water analysis of, 40 
as a culture medium, 39 
for bacillus cavicida havanien- 
sis in, 517 
for bacillus cuniculicida hava- 
niensis in, 539 
Coffee infusion, antiseptic power of, 200 
Cohn’s classification of bacteria, 11 
solution, 40, 126 
Cohnheim, production of tuberculosis by 
inoculation of tuberculous material, 467 
Cold, influence of, on bacterial vitality, 
153 
Colon bacilli, difficulty of distinguishing 
from allied groups, 554 
and typhoid bacilli, varieties of 
the same species? 447 
frequency of, in water supplies, 
640 
methods for their detection in 
water supplies, 640 
Smith's method for their detec- 
tion, 535 
pacillus of Escherich, 529 
its important réle in human 
pathology, 538 
Colonies on plates of solid media, 72 
Comma bacillus of Koch, 593 
Conjunctiva, bacterial flora of, 649 
Contact preparations, 27 
Copaiba, antiseptic power of, 208 
Copper sulphate as a disinfectant, 212 


683 


Coriander, antiseptic power of, 203 
Corn, Indian, bacterial diseases of, 575 
Cotton, first use of, as a filter for micro- 
organisms, 4 
plugs for test tubes, 62 
for tubes containing aérobic bac- 
teria, 54 } 
in growing tubercle bacilli, 475 
Cover-glass preparations, 25 
Cows, cocci of hemoglobinuria of, 419 
streptococcus of mastitis of, 419 
Creolin, germicidal power of, 201 
Creosote, germicidal power of, 201 
Cresol, germicidal power of, 201 
Cruciferous plants, brown rot in, 575 
Cuba, cases of Malta fever from, 420 
Cucumbers, bacterial diseases of, 576 
Cucurbitaces, bacterial diseases of, 576 
Culture media, 37 
artificial, 40 
for bacillus mallei, 490 
for bacillus tuberculosis, 474 
liquid, 40 
natural, 37 
neutralization of, 49 
solid, 41 
sterilization of, 52 
Cultures of anthrax, chemical products 
in, 428 
of bacillus diphtheriz, vitality of, 455 
of typhoid bacilli obtained from the 
spleen in typhoid fever cases, 433, 
436 
drop, 64 
in liquid media, 62, 67 
in solid media, 69 
stab, 69 
streak, 77 
Cumin, antiseptic power of, 203 
Cunningham’s objections to accepting 
Koch’s bacillus as the specific 
cause of cholera, 601 
vibrios resembling those of cholera, 
609 
Cupric chloride, antiseptic value of, 189 
sulphate, antiseptic value of, 189 
action of, on micrococcus pneu- 
moniz croupose, 405 
Curry on Malta fever in the Philippine 
Islands, 420 


684 


Cystitis, proteus vulgaris in, 551 
Czenzynke’s staining solution for the 
bacillus of influenza, 468 


D’ArsonvaL’s incubating apparatus, 94 
Davaine, causation of anthrax discovered 
by, 6, 422 
classification of bacteria, 10 
Dead, disinfection of the, 213 
Death-point, thermal, of bacteria, 155 
De Bary’s classification of bacteria, 12 
Decolorization in staining, 28 
Defensive proteids of Haffkine, 265 
De Giacoma’s method of staining syph- 
ilis bacilli, 495 
Deneké’s spirillum, 603 
De Schweinitz, immunity from tubercu- 
losis conferred by attenuated cultures, 
364 
Desiccation, influence of, on bacteria, 
159 
resistance of anthrax spores to, 427 
influence of, on bacillus pestis, 564 
on the comma bacillus, 597 
Developers in photomicrography, 113 
Diaphtherin, germicidal power of, 202 
Dieudonné on the vibrios resembling the 
cholera spirillum, 606 
Dimethylamine, 147 
Diphtheria antitoxin, 314 
results of treatment with, 462 
bacillus of, found in throat for weeks 
after subsidence of fever, 451, 
460 
attenuated varieties, 309 
branching forms of (C, Frankel), 
460 
bacteria in, 449 
cultures, toxalbumin of, 312 
evidence on which the Klebs-Liffler 
bacillus is regarded as the cause 
of, 450 
heat as a disinfectant for, 154 
intestinal in rabbits, bacillus of, 459 
a local infection, 449 
mixed infections in, 461 
post-mortem appearances of, 226 
toxalbumin of, 149 
Diphtheritic false membranes, strepto- 
coccus pyogenes in, 386 


INDEX. 


Dipbtheritic 
225 
Diplococeus, general characters, 17 
of Frankel, 339 
grouping of, 21 
intracellularis meningitidis, 410 
pneumoniz, discovery of, 7 
pneumoniz (Weichselbaum), 398 
in meningitis, 400 
in otitis media, 373, 390 
Discontinuous heating in sterilizing, 53, 
56, 57 
Disease, race immunity to, 234 
susceptibility to, 233 
Diseases of plants, bacteria of, 571 
Disinfectant, importance of sunlight as 
a, 162 
Disinfectants, action of, 164 
influence of, on cholera cultures, 597 
Disinfection, practical directions for, 212 
Disinfektol, germicidal power of, 202 
Distilled water, bacteria in, 125, 635 
Double staining of bacterial preparations, 
28 
of spore preparations, 31 
Drop cultures, 64 
Druse des Pferdes, streptococcus of, 418 
Ducks, bacillus of cholera in (Cornil and 
Toupet), 503 
Duclaux’s solution for butyric-acid fer- 
ment, 138 
Dunham’s solution, 151 
peptone solution, 41 
test for the presence of the cholera 
spirillum, 598 
Dust, atmospheric, bacterial constitution 
of, 614 


inflammation, cause of, 


Expertu’s bacillus, 481 
Egg albumen as a culture medium for 
bacillus pyocyaneus pericarditidis, 547 
Eggs, bacteriology of, 671 
as a culture medium, 40 
Ehrenberg’s vibrioniens, 3, 10 
Ehrlich’s aniline-gentian-violet solution, 
29 
solution in staining bacillus mallei, 
490 
method of staining bacillus tubercu- 
losis, 469 


INDEX. 


Ehrlich’s side chain theory, 269 
Ehrlich-Weigert’s aniline-methyl-violet 
solution, 29, 471 
method of staining the tubercle ba- 
cillus, 30 
stain for syphilis bacilli, 495 
Electric light in photomicrography, 107 
Electricity, influence of, on bacterial 
growth, 162 
Elsner’s method for detecting the ty- 
phoid bacillus in water and faces, 
446 
Emmerich’s bacillus (B. neapolitanus), 
529 
thermal death-point of, 155 
experiments on micrococcus pneu- 
moniz croupose, 341 
Endocarditis, ulcerative, micrococcus 
pneumonie croupose in, 401 
Endospores, 16 
Enzymes produced by liquefying bac- 
teria, 136 
Eosin as a double stain, 28 
collodion in photographing bacteria, 
103 
Eosinophile cells,. 237 
Epidemics, Pasteur on the origin of, 290 
Erlenmeyer flasks, 60, 63 
Escherich’s view that pseudo-diphther- 
itic bacilli are distinct from the true 
bacillus, 460 
Esmarch’s roll tubes, 76 
tubes in water analysis, 628, 
630 
method of cultivating anaérobic bac- 
teria, 84 
Essential oils, antiseptic power of, 202 
Ether, action of, on bacillus tuberculosis, 
478 
antiseptic power of, 202 
in experiments on animals, 99 
Eugenol, antiseptic power of, 203 
Euphorin, feeble germicidal activity of, 
204 
Eucalyptol, antiseptic power of, 203 
Eucalyptus, antiseptic power of, 203 
Excreta, disinfection of, 212, 217 
method of testing for the cholera 
spirillum, 598 
Exhaustion theory of immunity, 249 


685 


Expired air free from micro-organisms, 
622 
Eye, inoculations into, 99 


FacuLtaTIvE anaérobics, 16, 18 
in stab cultures, 70 
parasites, 15, 127, 221 
saprophytes, 221 
Feces, list of species of pathogenic bac- 
teria found in, 663 
Fallopian tubes free from bacteria in 
health, 654 
Farcy, symptoms of, 491 
Fasching’s capsule bacillus, 561 
Feet, the, bacteria on, 649 
Fennel, antiseptic power of, 203 
Ferment lactique of Pasteur, 667 
Fermentation by bacteria, 136 
of grape sugar in water sup- 
plies, 535 
in liquid media, 68 
putrefactive, 141 
test for colon and typhoid bacilli in 
water, 445 
tubes, 68 
of urea, 140 
viscous, 141 
Ferments, soluble, produced by bacteria, 
143 
Fermi and Pernossi on the properties of 
tetanus poison, 354 
Ferran’s anticholeraic inoculations, 301 
Ferret disease, bacillus of, 508 
Ferric chloride, antiseptic value of, 
189 
sulphate, action of, on micrococcus 
pneumoniz croupose, 405 
antiseptic power of, 189 
Filters, soluble, in analysis, 620 
Filtration as a means of sterilizing, 58 
Finger-nails, bacteria found under, 648 
Finkler and Prior, spirillum of, 602 
Fiocca’s bacillus, 547 
method of staining spores, 32 
Fire as a disinfectant, 212, 213 
Fish, boiled, as a culture medium, 40 
Flagella of bacteria, 24, 119 
methods of staining, 32 
of bacillus amylovorus, 576 
campestris, 575 


686 


Flagella of bacillus of hog cholera 
(Loffler’s staining), 504 
hyacinthi, 573 
cdematis maligni, 581 
cdematis maligni, No. II., 588 
solanacearum, 571 
of swine plague, 509 
of symptomatic anthrax, 586 
typhi abdominalis, 437 
typhi murium, 524 
proteus vulgaris, 550 
pseudomonas Stewarti, 575 
spirillum of Finkler and Prior, 602 
Metschnikovi, 604 
Obermeieri, 590 
Flasks, Erlenmeyer’s, for liquid media, 
60, 63 
Pasteur’s, 63 
Sternberg’s, 38, 66 
Fleas, propagators of bubonic plague, 564 
Fleckenkrankheit, 415 
Flesh-peptone solution as a culture me- 
dium, 41 
gelatin, preparation of, 41 
Koch’s, use of, 69 
Flesh water as a culture medium, 40 
Flexner’s description of bacillus dysen- 
terie, 568 
Fliigge’s micrococcus septicus, 415 
directions for preparing fermented 
milk, 139 
Foa and Scabia’s experiments on micro- 
coccus pheumonis croupose, 342 
Foot-and-mouth disease, protective inoc- 
ulations in, 318 
Formaldehyde as a disinfectant, 204, 216 
Formalin, disinfectant power of, 204 
use of, as a test for typhoid bacilli, 
446 
Formic acid as a germicide, 183 
Formol, disinfectant power of, 204 
Foth’s directions for preparing mallein, 
320 
Fowl cholera, causes of, 288 
bacillus of, action of chlorine 
on, 177 
bacillus of, action of germicides 
on, 501 
bacillus of, thermal death-point 
of, 156 


INDEX. 


Frankel’s method of cultivating anaé- 
robic bacteria, 82 
pneumococcus, 339 
researches on the bacteria of the soil, 
643 
Frankel-Gabbett method of staining ba- 
cillus tuberculosis, 471 
Frankel and Simmonds, multiplication 
of typhoid bacilli in the spleen after 
death, 483 
Freezing, influence of, on bacterial vi- 
tality, 153 
French commission, report of, on Fer- 
rd4n’s anticholeraic inoculations, 301 
Freudenreich’s modification of Koch’s 
plate method, 79 
Friedlander’s bacillus, analysis of, 124 
thermal death-point of, 155 
pheumococcus, 396 
method of staining the tubercle ba- 
cillus, 80 
Fuchsin as a stain, 25, 27 
Fuller’s method of determining the re- 
action of nutrient media, 50 
Furniture, disinfectants for, 213 


GaBBeTT’s method of staining bacillus 
tuberculosis, 30 
Gaffky’s explanation of the failure to 
find Eberth’s bacillus in every case of 
typhoid fever, 482 
Galbanum, antiseptic power of, 203 
Gallic acid, action of, on bacteria, 183 
Gas developed by the bacillus of Em- 
merich and Weibel, 566 
by bacillus cedematis maligni, 
No. IL, 588 
by bacillus piscicidus agilis, 565. 
by bacillus of von Dungern (cap- 
- sule), 562 
bacillus (the) in its relations to hu- 
man pathology, 589 
Gases, action of, on bacteria, 172 
Gaslight, photomicrography by, 109 
Gastric juice, germicidal power of, 658 
protective by its acidity against 
attacks of cholera, 600 
Gastro-enteritis cholerica of chickens, 
symptoms and post-mortem appear- 
ances, 605 


INDEX. 


Gelatin as a culture medium, 41 


liquefaction of, by bacteria, 135 
growth of bacillus aérogenes capsu- 
latus on, 589 
of bacillus dysenteriz on, 569 
of bacillus cdematis maligni 
No. II. on, 588 
of symptomatic anthrax on, 586 
of bacillus solanacearum on, 572 
nutrient, as a culture medium, 42 
for bacillus diphtheria, 454 
for bacillus cedematis maligni, 
582 
plates, counting the colonies on, 6380 
plates in the cultivation of: 
bacillus A of Booker, 555 
bacillus acidiformans, 537 
bacillus alvei, 556 
bacillus anthracis, 425 
bacillus of Beck, 566 
bacillus of Belfonti and Pasca- 
rola, 508 
bacillus capsulatus, 522 
bacillus cavicida, 516 
bacillus coli communis, 531 
bacillus crassus sputigenus, 517 
bacillus of Emmerich and Wei- 
bel, 566 
bacillus endocarditidis griseus, 
555 
bacillus enteritidis, 520 
bacillus erysipelatos suis, 512, 
513 
bacillus of Fiocca, 548 
bacillus of Friedlander, 397 
bacillus gallinarum, 521 
bacillus of grouse disease, 521 
bacillus of hog cholera, 504, 509 
bacillus lactis aérogenes, 536 
bacillus of Laser, 524 
bacillus of Lucet, 526 
bacillus of Mereshkowsky, 565 
bacillus of Nicolaier (capsule), 
560 
bacillus piscicidus, 567 
bacillus piscicidus agilis, 565 
bacillus of purpura hemorrhagi- 
ca, Tizzoni and Giovannini, 
558 
bacillus pyocyaneus, 543 


687 


Gelatin plates in the cultivation of: 


bacillus pyocyaneus , 545 

bacillus pyeeyaneus pericardi- 
tidis, 546 

bacillus pyogenes foetidus, 518 

bacillus septicemiz hemor- 
rhagice, 500, 509 

bacillus tetani, 579 

bacillus typhi abdominalis, 488 

bacillus typhi murium, 524 

bacillus of von Dungern (cap- 
sule, 562 

micrococcus bothryogenus, 412 

micrococcus Manfredi, 413 

micrococcus tetragenus, 411 

proteus hominis capsulatus, 519 

proteus of Karlinski, 552 

proteus lethalis, 554 

proteus mirabilis, 553 

proteus septicus, 554 

proteus vulgaris, 549 

proteus Zenkeri, 554 

spirillum cholere asiatice, 594 

spirillum of Finkler and Prior, 
603 

spirillum Metschnikovi, 604 

spirillum tyrogenum, 603 

staphylococcus pyogenes aureus, 
3874 

streptococcus pyogenes, 384 

in water analysis, 628 

stab cultures of: 

bacillus A of Booker, 555 

bacillus acidiformans, 537 

bacillus acne contagiosa of 
horses, 557 

bacillus alvei, 557 

bacillus anthracis, 425 

bacillus of Babes and Oprescu, 
526 

bacillus of Beck, 566 

bacillus of Belfonti and Pasca- 
rola, 508 

bacillus capsulatus, 522 

bacillus cavicida havaniensis, 
516 

bacillus of Cazal and Vaillard, 
525 

bacillus of cholera in ducks, 503 

bacillus coli communis, 531 


688 


Gelatin, stab cultures of : 

bacillus crassus sputigenus, 518 

bacillus cuniculicida havanien- 
sis, 539 

bacillus of Emmerich and Wei- 
bel, 566 

bacillus endocarditidis capsula- 
tus, 556 

bacillus endocarditidis griseus, 
555 

bacillus enteritidis, 520 

bacillus erysipelatos suis, 512 

bacillus of Friedlander, 397 

bacillus gallinarum, 521 

bacillus gracilis cadaveris, 560 

bacillus of grouse disease, 521 

bacillus of hog cholera, 504 

bacillus hydrophilus fuscus, 522 

bacillus lactis aérogenes, 536 

bacillus of Laser, 524 

bacillus leporis lethalis, 541 

bacillus of Lucet, 526 

bacillus of Nicolaier (capsule), 
560 

bacillus cedematis maligni, 582 

bacillus piscicidus, 567 

bacillus of purpura hemorrha- 
gica (Babes), 558 

bacillus of purpura hemorrha- 
gica (Kolb), 559 

bacillus pyocyaneus, 543 

bacillus pyocyaneus f, 545 

bacillus pyocyaneus pericardi- 
tidis, 546 

bacillus pyogenes foetidus, 518 

bacillus of symptomatic anthrax, 
586 

bacillus tetani, 579 

bacillus typhi abdominalis, 439 

bacillus typhi murium, 524 

bacillus of von Dungern, 562 

micrococcus bothryogenes, 412 

micrococcus Manfredi, 413 

micrococcus tetragenus, 411 

proteus hominis capsulatus, 519 

proteus of Karlinski, 552 

proteus lethalis, 555 

proteus mirabilis, 553 

proteus septicus, 554 

proteus vulgaris, 549 


INDEX. 


Gelatin stab cultures of : ; 
spirillum cholere asiatice, 595 
spirillus of Finkler and Prior, 

603 
spirillum Metschnikovi, 605 
spirillum tyrogenum, 603 
staphylococcus pyogenes aureus, 
374 
streptococcus pyogenes, 384 
roll-tubes for: 
bacillus acidiformans, 537 
bacillus cavicida havaniensis, 
516 
bacillus cuniculicida havanien- 
sis, 539 
bacillus grandis cadaveris, 560 
bacillus leporis lethalis, 541 
stick cultures, staining for, 36 
streak culture of bacillus of Lucet, 
526 
culture of bacillus of swine 
plague, Marseilles, 509 

Gentian violet as a stain, 25, 27 

Geranium, antiseptic power of, 203 

Gernicides, definition of, 164 

methods of determining value of, 166 

Germs in air, number of, 623 

Gessard on the pigments produced by 

bacillus pyocyaneus, 134 
researches on bacillus pyocyaneus, 
548 
Giant cells in tuberculous nodules,468,472 
in lungs from injection of dead 
tubercle bacilli, 485 
Gleogenes of Cohn, 11 
Glanders, animals affected by, 233 
bacillus of (B. mallei), 489 
methods by which infection is com- 
municated, 491 
test for, by mallein, 152, 321, 494 
by obtaining pure cultures by 
inoculation, 493 
Glucose in culture media, 46 
influence of, on bacterial growth, 128 
Glycerin, action of, on bacteria, 204 
Glycerin-agar, preparation of, 45 
bacillus acidiformans, growth of, on, 
538 
of Babes and Oprescu, 626 
cuniculicida havaniensis, 539 


INDEX. 


Glycerin-agar, bacillus hydrophilus fus- 
cus, 523 
mallei, 490 
pestis, 563 
of swine plague, Marseilles, 509 
tuberculosis, 476 
roll-tube cultures of bacillus cavi- 
cida havaniensis, 517 
of bacillus cadaveris in hydro- 
gen atmospheres, 584 
Goat’s milk immunized, antitoxin of, 357 
precipitation of antitoxin from, 
358 
Gold chloride as an antiseptic, 190 
Gonococcus of Neisser, 226, 391 
biological characters of, 393 
discovery of, 7 
multiplication of, in leucocytes, 
258 
Graham bread, bacteriology of, 672 
Gram’s method of staining, 29, 34 * 
solution for decolorizing in staining, 
28 
Grape sugar in liquid culture media, 68 
growth of bacillus coli com- 
munis in solutions of, 532 
Graveyards, bacteria in the soil of, 643, 
646 ; 
Gray-parrot disease, micrococcus of, 419 
Ground-water region of the soil, free from 
bacteria, 644 
Guaiacol, antiseptic power of, 204 
Guinea-pigs, effects of injection of tuber- 
culous matter into, 480 


Hamarococcus bovis (Babes), 419 

Hemoglobinuria of cows, cocci of, 419 

Haffkine’s anticholeraic inoculations, 
302 

Hail, number of bacteria in, 682 

Haloid elements, action of, on bacteria, 
172 

Hands, disinfection of the, 216, 648 

Hanging-drop cultures, 64 

of typhoid bacilli, appearance 
of, 442 

Hansen’s discovery of the bacillus of 
leprosy, 7 

Hazen and White’s method of separating 
typhoid bacilli in water supplies, 445 


44 


689 


Heat, action of, on anthrax spores, 587 
attenuation of virulence by, 131 
influence of, on bacterial growth, 

153 
on bacillus pestis, 564 
isolation of spores by, 68 
as a sterilizer of culture media, 52 
dry, as a disinfectant, 212, 213 
influence of, on bacterial growth, 
154 
non-penetration of, 154 
as a sterilizing agency, 53 
moist, influence of, on bacterial vi- 
tality, 154 
Heating, discontinuous, as a sterilizing 
measure, 53 
Heliostat in photomicrography, 107 
Helman’s tuberculin from potato cul- 
tures, 363 
Helmholtz’s experiments opposed to spon- 
taneous generation, 4 
Hemialbumose, 262 . 
Herbivora, susceptible to bacterial injec- 
tion, 96 
to anthrax, 234 
Héricourt and Richet, experiments of, on 
bacillus tuberculosis, 364, 365 
Hesse’s apparatus for the bacteriological 
analysis of air, 618 
Heterogenesis or spontaneous genera- 
tion, 5 
Heydenreich’s micrococcus, 415 
Hoffman’s experiments on the preserva- 
tion of putrescible liquids, 5 
Hog cholera, bacillus of (Salmon and 
Smith), 503 
prevalence, symptoms and post- 
mortem appearances of, 505, 
506 
protective inoculations against, 
322 
group of bacteria, varieties or 
species ? 506, 507 : 
erysipelas, bacillus of, 511 
protective inoculations against, 
824 
symptoms and post-mortem ap- 
pearances of, 514 
Holz’s method of searching for the ty- 
phoid bacillus in water supplies, 444 


690 INDEX. 


Horse, symptoms of glanders in the, 491 
immunization of, against tetanus, 
360 
Horses, bacillus of acne contagiosa of, 
557 
diplococcus of pneumonia in, 418 
streptococcus coryze contagiose of, 
418 
Hot-air oven for sterilizing, 53 
Hot Springs, Ark., Malta fever from the 
tropics at, 420 
Hueppe’s apparatus for sterilizing and 
coagulating blood serum, 58 
classification of bacteria, 12 
Hyacinth, bacterial disease of the, 574 
Hydrant water, number of bacteria in, 
634 
Hydrocele fluid as a culture medium, 40 
Hydrochloric acid, action of, on micro- 
coccus pneumoniz croupose, 
405 
as a decolorizer in staining, 28 
influence of, on bacterial growth, 
181 
Hydrofluoric acid, influence of, on the 
tubercle bacillus, 179 
Hydrogen generator, 86 
in the cultivation of anaérobic bac- 
teria, 82 
influence of, on bacterial growth, 173 
peroxide as a disinfectant, 173 
Hydronaphthol, antiseptic power of, 205 
Hydrophobia, influence of temperature 
on the infectious material of, 156 
protective inoculations against, 332 
toxalbumin of, 327 
Hydrosulpburic acid, a bacterial produc- 
tion, 141 
influence of, on bacterial growth, 
175 
Hydroxylamin, antiseptic power of, 204 
Hypodermic injection of cholera vaccine, 
299 
syringe for inoculations, 97 


Ice, number of bacteria in, 633 
cream, toxic principle in, 148 
Ichthyol, antiseptic power of, 204 
Illicium, antiseptic power of, 203 
Immunity from disease, 233 


Immunity, acquired, 242 
alkalinity of the blood as affecting, 
236 
dependent on antitoxins, 267 
influence of animal temperature on, 
235 
natural agencies by which neutral- 
ized, 240 
theories of, 249 
to anthrax by filtered cultures of the 
bacillus, 428 
by anthrax toxin, 427 
to cholera by a previous attack, 297 
to serpent venom, 262 
to tetanus by cultures in thymus 
bouillon, 359 
inherited, 360 
to tuberculosis by attenuated cul- 
tures, 485 
Immunization of swine against rouget, 
method and disadvantages of, 515 
Impftetanus bacillus, 508 
Incubating ovens, 88 
Indigestion, sarcinz in stomach of per- 
sons suffering from, 662 
Indol, antiseptic power of, 205 
production of, 151 
by the cholera spirillum, 598 
by vibrios resembling the chol- 
era spirillum, 607 
by bacillus septicemiz hzmor- 
rhagicze in peptone solution, 
501 
by bacillus of swine plague, 
Marseilles, 509 
by capsule bacillus of von Dun- 
gern, 562 
not produced by the bacillus of hog 
cholera, 505 
test for the typhoid bacillus, 440 
Infection, rapidity of, 231 
by inhalation, 102 
Infections, mixed, 228 
secondary, 227 
Infectious diseases, chronic, bacilli in, 
467 
Influenza, protective inoculations against, 
335 
bacillus, discovery of, 8 
morphology, 463 


INDEX. 


Influenza, bacillus, pathogenesis, 464 
stains for, 463 
toxin of, 465 
_ Vitality of, 464 2 
in horses, protective inoculations 
against, 335 
Inhalation, infection of animals by, 102 
Inoculation experiments with bacillus 
pestis, 563 
of liquid media, 63 
Inoculations, protective, in cholera, 294 
Insects, transmission of plant diseases 
by, 576 
Intestinal bacteria, not concerned in the 
physiology of digestion, 662 
mucousmembrane, infection through, 
230 
Intestine, bacterial injections into, 99 
of man, bacteria found in, 660, 663 
Involution forms of bacilli, 23 
of bacillus of symptomatic an- 
thrax, 586 
Iodine, action of, on micrococcus pneu- 
moniz croupose, 405 
influence of, on bacterial growth, 178 
trichloride as a disinfectant, 178 
Iodoform as an antiseptic, 178 
Iodoform-ether, action of, on bacillus 
tuberculosis, 179, 478 
Iodol without germicidal power, 179 
Issaeff’s experiments on the micrococcus 
pneumoniz crouposee, 342 
Izal, disinfectant power of, 205 


JAHRESBERICHT (the) of Baumgarten, 9 

Jequirity solution as a culture medium, 47 

“Journal of Hygiene,” 9 

“Journal of Pathology and Bacteri- 
ology,” 9 

Juniper, antiseptic power of, 203 


KAESESPIRILLEN, 603 
Kidneys, cultures of typhoid bacilli ob- 
tained from the, 483, 436 
specially affected by staphylococcus 
pyogenes aureus, 376 
Kinyoun’s treatment of the horse for 
diphtheria antitoxin, 316 
Kitasato, bacteriological work of, 8 
distinguishing biological characters 
of the bacillus of influenza, 464 


691 


Kitasato, experiments on the bacillus of 
tetanus, 351 
results with tuberculin, 365 
Kitt’s micrococcus of bovine mastitis, 
414 
Klebs on the influence of Koch’s tuber- 
culin, 364 
method of obtaining pure cultures, 
67 
Klebs-Léffler bacillus of diphtheria, 449 
value of, for diagnostic purposes, 
451 
Klemperer’s experiments on cholera im- 
munization, 308 
Koch’s apparatus for coagulating blood 
serum, 58 
bacteriological researches, 7 
decolorizing solution, 28 
differentiation of species, 482 
discovery of the tubercle bacillus, 
467, 468 
experiment showing that the comma 
bacillus will infect when intro- 
duced in a living condition into 
the intestine, 600 
experiments on the thermal death- 
point of bacteria, 154 
flesh-peptone gelatin, use of, 69 
inoculation syringe, 97 
method of determining the germi- 
cidal power of chemical agents on 
anthrax spores, 168 
method of staining flagella, 32 
plate method, 74 
method, modifications of, 79 
method, in water analysis, 629 
statement of the action of sunlight 
on the tubercle bacillus, 478 
steam sterilizer, 55 
tuberculin, 151, 361 
production of, 362 
experiments favorable to, 479 
typhoid bacillus, 432 
vibrios remotely resembling the chol- 
era spirillum, 607 
views of the interior structure of the 
bacterial cell, 118 
Kockel’s capsule bacillus, 561 
Kolb’s bacillus of purpura hemorrhagica, 
559 


692 


Kommabacillus der cholera asiatica, 593 
Kreibohm’s bacillus, 517 
Kruse’s modification of Koch’s plate 
method, 79 
Ktihne’s method of staining bacteria in 
tissues, 35 
silicate jelly, 47 
staining method for bacillus mallei, 
490 


Lactic acid, action of, on bacteria, 182 
Lactose-litmus-agar, 47 
Lactose and litmus for detecting typhoid 
bacilli, 445 
Lake water, number of bacteria in, 633 
Lancet-shaped micrococcus (Talamon), 
398 
Lanolin, antiseptic power of, 205 
Laundries, bacteria in wash-water from, 
649 
Lavender, antiseptic power of, 203 
Lead chloride as an antiseptic, 190 
nitrate as an antiseptic, 190 
Leprosy, bacillus of, 487 
Leptotrichez of Zopf, general characters, 
12, 19 
Leucocytes, protective against bacterial 
invasion, 239 
multiplication of gonococci in, 258 
Leuconostoc, general characters of, 17 
Liborius’ method of cultivating anaéro- 
bic bacteria, 85 
Light, attenuating influence of, 279 
influence of, on bacterial growth, 159 
action of, on bacillus tuberculosis, 
478 
for photomicrography, 107 
Linen underclothing, bacteria on, 649 
Liquefying bacteria, 16 
in stab cultures, 71 
Liquid media, cultures of anaérobic bac- 
teria in, 81 
Liquids, non-evolution of bacteria from 
the surface of, 614 
Lithium chloride as an antiseptic, 190 
Litmus, in culture media, 46, 64 
action of bacillus of hog cholera on, 
504, 509 
Liver, cultures of typhoid bacilli ob- 
tained from the, 483, 43 


INDEX. 


Lochial discharge, presence of bacteria 
in, 654 
Loffler’s aniline-blue solution, 29 
plood-serum mixture, 48 
culture medium for bacillus diph- 
therice, 454 
method of staining bacteria in tis- 
sues, 34 
of staining flagella, 32 
solution for staining anthrax bacilli, 
424 
for staining the typhoid bacil- 
lus, 437 
Loretin, disinfectant power of, 205 
Liibert’s researches on the action of anti- 
septic agents on staphylococcus pyo- 
genes aureus, 375 
Lustgarten’s bacillus, 494 
Lydton’s report on protective inocula- 
tions in Baden, 350 
Lymph, germicidal power of, 236 
Lysol, disinfectant power of, 205 


Mace, antiseptic power of, 203 
Maffucci’s conclusions on the tuberculo- 
sis of fowls, 484 
Maggiora’s researches on the bacteria of 
the soil, 644 
Magnesium light, in photomicrography, 
107 
Mail matter, disinfection of, 214 
Mal d’araignée, micrococcus of, 416 
Mal de pis, micrococcus of, 416 
Malic acid as a germicide, 183 
Malignant cedema, bacillus of, 580 
pustule in man, 422, 428 
Mallein, a glycerin extract from cultures 
of the bacillus of glanders, 318, 494 
as a test for glanders in horses, 321 
preparation of, in Pasteur’s labora- 
tory, 319 
Foth’s method of preparing, 320 
Malnutrition, influence of, on develop- 
ment of pus cocci, 224 
Malta fever, micrococcus of, 420 
Widal reaction in, 421 
Mammite contagieuse, micrococcus of, 
417 
Manganese protochlorite as an antiseptic, 
190 
eee 


INDEX. 


Marchand’s capsule bacillus, 561 
Marjoram, antiseptic power of, 203 
Marmier’s experiments on the toxin of 
anthrax, 430 
Marpmann’s method for the detection of 
pathogenic bacteria in water, 640 
Marsh gas, a bacterial product, 141 
Mastitis in cows, micrococcus of, 417 
streptococci of, varieties of a 
single species, 419 
gangrenous, in sheep, micrococcus 
of, 416 
Matico, antiseptic power of, 203 
Mauseseptikimiedhnlicher bacillus (Bis- 
enberg), 515 
Measles, second attacks of, 243- 
Meat, infusions as culture media, 40 
Meats, bacteria in, 670 
Meatus urinarius of man, bacteria in, 
654 
Meconium, free from bacteria, 660 
Media, liquid, cultures in, 62 
characters of growth in, 64 
solid, cultures in, 69 
Mediterranean fever, micrococcus of, 420 
Melons, bacterial diseases of, 576 
Merchandise, disinfection of, 214 
Mercuric chloride action of, on anthrax 
spores, 587 
action of, on bacillus tubercu- 
losis, 478 
action of, on micrococcus pneu- 
moniz croupose, 405 
as a disinfectant, 212, 213 
as an antiseptic, 190 
cyanide as a disinfectant, 192 
iodide as an antiseptic, 192 
Merismopedia, general characters of, 17 
grouping of, in tetrads, 22 
Mesenteric glands, cultures of typhoid 
bacilli obtained from, 433, 436 
Metachromatic granules, 118 
Metacresol, germicidal power of, 202 
Methane, influence of, on bacterial 
growth, 175 
Methylamine, 147, 323 
Methylene blue as a stain, 25, 27 
Methyl-guanidin, 148 
Metschnikoff’s theory of phagocytosis, 
208, 256 


693 


Mice, subcutaneous injection of, 99 
field, immune to mouse septicemia, 
515 ; 

Mereshkowsky’s suggested meth- 
od of destroying, 567 
Microbe du cholera des poules (Pasteur), 
499 
du pus bleu, 542 
Micrococci, general characters, 17 
infrequent in the soil, 645 
list of species found in the soil 
(Fulles), 646 
non-pathogenic, list of species found 
in water, 638 
pathogenic, list of species found in 
water, 638 
Micrococcus askoformans (Johne), 412 
of Biskra button, 415 
bothryogenus (Rabe), 412 
of bovine mastitis, 414 
pneumonia, 414 
endocarditidis rugatus (Weichsel- 
baum), 416 
of erysipelas (Fehleisen), 382 
of fowl cholera, thermal death-point 
of, 156 
of gangrenous mastitis in sheep, 416 
gonorrheeal, 391 
growth of, in acid media, 393 
thermal death-point of, 156 
of Heydenreich, 415 
of Manfredi, 413 
melitensis, 420 
of myko-dermoids of the horse, 412 
ovatus, 415 
Pasteuri (Sternberg), 339, 398 
discovery of, 7, 339, 398 
found in the mouth, 652 
thermal death-point of, 155 
pneumonie croupose, 339, 398 
croupose, biological characters 
of, 403 , 
croupose, capsule of, 402 
croupose, discovery of, 7 
croupose, morphology of, 402 
croupose, pathogenesis, 400 
croupose, thermal death-point 
of, 404 
croupose, Belfonti’s 
ments on, 341 


experi- 


694 


Micrococcus pneumoniz croupose, Em- 
merich’s experiments on, 341 
croupose, Foa and Scabia’s ex- 
periments on, 342 
croupose, Issaeff’s experiments 
on, 341 
croupose, Klemperer’s experi- 
ments on, 342 
croupose in endocarditis, ulcer- 
ative, 401 
croupose in meningitis, 400 
croupose in otitis media, 390, 
402 
- of progressive granuloma formation, 
413 
pyogenes tenuis, 382 
of sputum septicemia (Frinkel), 398 
tetragenus, 411 
action of chlorine on, 177 
thermal death-point of, 155 
ure, 140 
liquefaciens, 140 
Micromillimetre (the), 20 
Microzyma bombycis, 415 
Middle-ear disease, bacillus pyocyaneus 
in, 545 
Milk, bacteria in, 232, 663 
number of present, 669 
fermented, 139 
immunization by, 263 
of immunized goats, antitoxic, 357 
toxic principle in, 148 
of healthy women, bacteria in, 669 
of cows affected with chronic masti- 
tis, 417 
of tuberculous cows, infective, 482, 
485, 669 
as a culture medium, 39 
for bacillus A of Booker, 555 
for bacillus aérogenes capsulatus, 
589 
for bacillus alvei, 557 
for bacillus coli communis, 532 
for bacillus diphtheria, 455 
for bacillus dysenterie, 569 
for bacillus of Fiocca, 548 
for bacillus of hog cholera, 504, 
509 
for bacillus of Laser, 524 
for bacillus piscicidus, 567 


INDEX. 


Milk as a culture medium for bacillus 
piscicidus agilis, 565 
for bacillus pyocyaneus, 543 
for bacillus pyocyaneus pericar- 
ditidis, 547 
for bacillus solanacearum, 572 
for bacillus tenuis sputigenus, 
523 
for bacillus typhi abdominalis, 
439 
for bacillus typhi murium, 524 
for bacillus of von Dungern (cap- 
sule), 562 
for micrococcus 
croupose, 404 
for spirillum cholere asiatica, 
596 
for spirillum Metschnikovi, 605 
Milzbrand, bacillus of, 422 
Mint, antiseptic power of, 203 
Miquel’s aéroscope, 615 
later methods of air analysis, 618 
tables of antiseptic values, 186 
Mironoff’s experiments against strepto- 
coccic infection, 346 
Mitylotoxin, 148 
Mixed infections, 228 
Moist chamber for drop cultures, 65 
Moitessier’s gas-pressure regulator, 88 
MOller’s method of staining spore prepa- 
rations, 32 
Monkeys, susceptible to the infection of 
bubonic plague, 564 
Monochlorophenol as a disinfectant, 179 
Morphia hydrochlorate as an antiseptic, 
193 
Morphological classification of bacteria, 
12 
Morphology of bacteria, general account, 


pneumonia 


20 
insufficient to differentiate species, 

20 
Mosny, experiments on micrococcus 


pneumoniz croupose, 342 

Motile bacteria, 16 

Moulds, spores of, in atmospheric dust, 
614 

Mouse septicemia, symptoms and post- 
mortem appearances of, 515 

Mouth, bacteria found in the, 651, 656 


INDEX. 


Mucorini of Nigeli, 11 
Mucus, germicidal power of, 210 
Miincke’s thermo-regulator, 92 
Muscarini, 147 
Mushrooms, toxic principle of, 147 
Mussels, toxic principle in, 148 
Mustard, antiseptic power of, 203 
oil of, antiseptic power of, 206 
Mycelium of moulds, 614 
Mycoderma, 64 
acetic acid produced by, 137 
Mycophylaxin, 265 
Mykoprotein, 124 
Myrtle, antiseptic value of, 203 


NAGeEtt’s classification of bacteria, 11 
Nails, finger, surgical care of, 217 
Nail-shaped growth of stab cultures, 397 
Naphthol, disinfectant power of, 205 
Nasal catarrh, pus cocci in, 390 
Natural culture media, 37 
Neapolitan fever, micrococcus of, 420 
Negro, insusceptibility of, to yellow 
tever, 234 
Neisser’s gonococcus, 226 
discovery of, 7 
stain for bacillus diphtherie, 457 
double staining of spore prepara- 
tions, 31 
Nematogenes of Cohn, 11 
Neuridin, 146 
Neurin, 147 
Nickel sulphate as an antiseptic, 193 
Nicolaier’s capsule bacillus, 560 
discovery of the tetanus bacillus, 8 
Nitrates, reduction of, 144 
Nitric acid as a decolorizer in staining, 
28 
influence of, on bacterial growth, 
181 
action of, on micrococcus pneu- 
moniz croupose, 405 
Nitrification produced by bacteria, 144 
Nitrogen necessary for bacterial growth, 
126 
dioxide, influence of, on bacterial 
growth, 175 
Nitrous acid as a test for indol, 151 
influence of, on bacterial growth, 
181 


695 


Nitrous oxide, influence of, on bacterial _ 
growth, 175 
Non-liquefying bacteria, 16 
characters of growth in stab 
cultures, 70 ‘ 
Non-motile bacteria, 16 
Non-pathogenic bacteria from the mouth, 
656 
from the nose, 656 
list of species from faces, 663 
Nose, bacteria found in the, 650, 656 
Nosema bombycis, 415 
Nosophen, 179 
Nuclein, the germicidal constituent of 
blood serum, 239 
Nucleins, germicidal power of, 210 
Nutmeg, antiseptic power of, 203 
Nutrient agar (see Agar, nutrient) 
gelatin (see Gelatin, nutrient) 
Nuttall’s method of estimating bacilli in 
tuberculous sputum, 479 


O11 light, Borden’s method of photograph- 
ing by, 111 

Oleic acid, influence of, on bacterial 
growth, 183 

Olive oil, no influence on anthrax spores, 
206 

Onion, antiseptic power of, 203 

Opopanax, antiseptic power of, 203 

Orange of Portugal, antiseptic power of, 
203 

Origanum, antiseptic power of, 203 

Orthochromatic photographic plates, 105, 
113 

Orthocresol, germicidal power of, 202 

Orthophenol, germicidal power of, 197 

Ose, 74 

Osmic acid, fatal to anthrax spores, 
181 

Osteomyelitis, micrococcus of, 373 

thermal death-point of, 375, 378 
Otitis media, micrococcus pneumonia 
croupose in, 402 
pus cocci present in, 3889 

Oxalic acid, action of, on bacteria, 182 

Oxychinaseptol, antiseptic power of, 202 

Oxycyanide of mercury as a disinfectant, 
192 

Oxygen, as a disinfecting agent, 172 


696 


Oxygen, essential for certain species of 


bacteria, 172 
influence of, on pigment production, 
133 


on spore formation, 123 
nascent, as a germicidal agent, 172 


PANHISTOPHYTON ovatum, 415 
Paracresol, germicidal power of, 202 
Paralysis, local, caused by diphtheritic 
inoculations, 452 
Parasites, strict, 221 
Parasitic bacteria, 15 
Parietti’s method of separating typhoid 
bacilli from water supplies, 444 
Park and Beebe’s observations on the 
disappearance of the Loffler bacillus in 
cases of diphtheria, 461 
Parrot disease, micrococcus of, 419 
Pasteur’s bacteriological researches, 7 
cholera des poules, 288 
demonstration of living organisms in 
the atmosphere, 615 
discovery of the causation of pé- 
brine, 6 
experiments on the attenuation of 
virus, 150 
on the attenuation of the mi- 
crobe of fowl cholera, 502 
on the etiology of rouget, 515 
on the preservation of putresci- 
ble liquids, 5 
flasks for liquid media, 63 
inoculations of rabbits with hydro- 
phobic virus, 99 
solution, 40, 126 
tubes for the collection of water 
samples, 627 
views on the origin of epidemics, 290 
on rabies, 328 
on the influence of temperature 
on the destruction of organ- 
isms, 5 
Pasteur-Chamberland filter, 60 
Pasteur Institute, production of diph- 
theria antitoxin at, 316 
of Naples, statistics of, 331 
of Palermo, statistics of, 831 
of Paris, statistics of, 331 
of Turin, statistics of, 331 


INDEX. 


Patchouly, antiseptic power of, 203 
Pathogenic anaérobic bacteria, 80 - 
bacteria, list of species found in 
feces, 663 
in milk, 668 
in the mouth, 652, 656 
in the nose, 656 
in the soil, duration of vitality 
of, 645 
in water, 636 
in water, detection of, 640 
organisms, 14 
power of saprophytes dependent on 
quantity and age of cultures and 
site of the inoculation, 529 
spirilla, 590 
Paulsen’s capsule bacillus, 561 
Pear blight, 575 
Pébrine, silkworms infected with, 415 
causation of, discovered by Pasteur, 
6 
not a bacterial disease ? 415 
Pendesche Geschwur, 415 
Pepper, antiseptic power of, 203 
Peptone, a product of bacterial action, 
142 
Peptotoxin, 148 
Peritoneum, injections into, 98 
Peritonitis, bacillus coli communis in, 
535 
Person, disinfection of the, 213 
Petermann’s experiments with filtered 
anthrax cultures, 428 
Petri dishes, 75 
use of, in air analyses, 617 
use of, in water analyses, 630, 
631 
Petri’s sand filter for air analyses, 620 
Petroleum lamps for incubating ovens, 
95 
Pfeiffer’s capsule bacillus, 561 
evidence of his bacillus being the 
specific cause of influenza, 464 
researches into the toxins of cholera 
cultures, 599 
views on the toxin of the typhoid 
bacillus, 367 
Pfeiffer and Issaeff’s method of distin- 
guishing the cholera spirillum from 
other vibrios, 609 


INDEX. 


Pfuhl’s experiments with tuberculin, 365 
Phagocytosis, 255, 267 
Metschnikoff’s theory of, 208, 256 
Phenol, produced by bacillus septicz- 
mize hemorrhagice, 501 
by bacillus of swine plague, 
Marseilles, 509 
not produced by bacillus of hog 
cholera, 505 
Phenolphthalein, to determine the reac- 
tion of media, 49 
Philippine Islands, Malta fever in the, 
420 
Phosphorescence by bacteria, 145 
an indication that vibrios are not 
the cholera spirillum, 608, 609 
Phosphoric acid, influence of, on the ty- 
phoid bacillus, 181 
Photomicrographs of bacteria 103 
amplification of, 106 
Photomicrography, apparatus for, 106 
Physicians, interest of, in bacteriological 
work, 6 
Pigment, production of, by bacteria, 133, 
135 
by staphylococcus pyogenes au- 
reus 374 
Pitfield-Muir method of staining flagella, 
33 
Plant diseases bacteria of, 571 
Platinum bichloride as an antiseptic, 193 
loop for inoculations, 63 
needle for inoculations, 65, 69 
Plectridium, spore formation in, 122 
Pleuropneumonia of cattle, protective 
inoculations against. 336 
Pneumonia, causation of, 241 
the micrococcus of, 339 
percentages of relapses in, 340 
protective inoculations against, 338 
Pneumotoxin, 261, 340 
Polar granules, 118 
Porcelain, unglazed, as a filtering me- 
dium, 59 
Post-mortem examination of inoculated 
animals, 101 
Potash soap, 
growth, 185 
Potassium hydroxide, influence of, on 
bacteria, 183 


action of, on _ bacterial 


697 


Potassium nitrate in culture media, 46 
permanganate as an antiseptic, 194 
action of, on micrococcus pneu- 
moni croupose. 405 
salts as antiseptics 193 
Potato as a culture medium, 40, 48, 78 
for bacillus A of Booker, 555 
for bacillus acidiformans, 538 
for bacillus aérogenes capsulatus, 
589 
+ for bacillus alvei, 557 
for bacillus anthracis. 426 
for bacillus of Babes and Opres- 
cu, 526 
for bacillus capsulatus, 522 
for bacillus cavicida havanien- 
sis, 516 
for bacillus of Cazal and Vail- 
lard, 525 
for bacillus of cholera in ducks, 
503 
for bacillus coli communis 531 
for bacillus crassus sputigenus, 
518 
for bacillus cuniculicida havani- 
ensis, 539 
for bacillus diphtheriz, 454 
for bacillus dysenteriz. 569 
for bacillus of Emmerich and 
Weibel, 566 
for bacillus endocarditidis gri- 
seus, 556 
for bacillus enteritidis, 520 
for bacillus of Fiocca, 548 
for bacillus of Friedlander, 397 
for bacillus gallinarum, 521 
for bacillus gracilis cadaveris, 
554 
for bacillus of hog cholera 504, 
509 
for bacillus hydrophilus fuscus, 
523 
for bacillus lactis aérogenes 536 
for bacillus of Laser, 524 
for bacillus leporis lethalis, 541 
for bacillus of Lucet, 527 
for bacillus mallei, 491 
for bacillus of Nicolaier (cap- 
sule), 560 
for bacillus pestis, 563 


698 


Potato as a culture medium for bacillus 

piscicidus, 567 

for bacillus piscicidus agilis, 565 

for bacillus of purpura hemor- 
rhagica (Babes), 558 

for bacillus of purpura hemor- 
rhagica (Kolb), 559 

for bacillus of purpura hamor- 
rhagica (Tizzoni and Giovan- 
nani), 558 

for bacillus pyocyaneus, 543 

for bacillus pyocyaneus 8, 545. 

for bacillus pyocyaneus pericar- 
ditidis, 546 

for bacillus pyogenes fcetidus, 
518 

for bacillus septicemiz hzmor- 
rhagice, 500, 509 

for bacillus solanacearum, 572 

for bacillus of swine plague, 
Marseilles, 509 

for bacillus tenuis sputigenus, 
523 

for bacillus tuberculosis, 477 

for bacillus typhi abdominalis, 
439 

for bacillus typhi murium, 524 

for bacillus of von Dungern, 562 

for micrococcus bothryogenus, 

. 413 

for micrococcus Manfredi, 413 

for micrococcus pneumonie 
croupose, 403 

for micrococcus tetragenus, 412 

for proteus hominis capsulatus, 
518 

for proteus of Karlinski, 552 

for proteus lethalis, 555 

for proteus septicus, 554 

for proteus vulgaris, 549 

for spirillum cholere asiatice, 


596 

for spirillum of Finkler and 
Prior, 603 

for spirillum of Metschnikovi, 
605 


for spirillum tyrogenum, 604 
for staphylococcus pyogenes au- 
reus, 375 
rot, symptoms of, 571 


INDEX. 


Pregl’s method of staining tubercle ba- 
cilli in tissues, 36 
Pressure, influence of, on.growth of bac- 
teria, 163 
Privy vaults, disinfection of, 217 
Protection (standard of) in inoculation 
of antitetanic serum, 356 
Protective inoculations in tuberculosis, 
360 
against typhoid fever in the 
British army, 369 
Proteus capsulatus septicus (Banti), 520 
Hauseri (Levy) from sour yeast, 
561 
of Karlinski, probably identical 
with proteus vulgaris (Hauser), 552 
lethalis, 554 
bei Lungengangrin des Menschen 
(Babes), 554 
mirabilis, swarming islands of, 552, 
653 
septicus, 554 
vulgaris (Hauser), 548 
in cholera infantum, 550 
influence of, on the virulence of 
anthrax cultures, 587 
instanced as illustrating varia- 
tions in the pathogenic power 
of saprophytes, 529 
Zenkeri, 554 
Prudden’s failure to find the Klebs- 
Loffler bacillus in pseudo-membranous 
inflammations, 450 
Pseudo-diphtheria bacilli, 461 
non pathogenic. 450, 455 
attenuations of bacillus diph- 
theria, 260 
Pseudo-influenza bacillus in broncho- 
pneumonia, 466 
Pseudomonas campestris, 575 
hyacinthi (Wakker), 573 
Stewarti (Smith), 575 
Ptomains, 133, 146 
Puerperal fever caused by streptococcus 
pyogenes, 387, 654 
self-infection possible, 655, 656 
Pulmonary mucous membrane, infection 
through, 230 
Pulmonic anthrax, 422, 428 
Pure cultures on gelatin plates, 72 


INDEX. 


Purpura hemorrhagica, bacillus of Babes, 
558 
bacillus of Kolb, 559 
bacillus of Tizzoni and Giovan- 
nini, 558 
Pus, formation of, 223 
independent of bacteria, 371 
of acute abscess micrococci of, 14 
production of, by typhoid bacilli 
448 
cocci found in healthy eyes, 650 
found in the mouth, 652 
present in diphtheritic mem. 
branes, 449 
on inflamed mucous membranes, 
389 
sterilized by heat give rise to 
pus formation, 371 
Putrefaction, 141, 665 
anaérobic bacteria active in, 142 
products of, 142 
in small intestine restrained by 
bacteria, 662 
Putrescin 147 
Pyelonephritis, proteus vulgaris in, 551 
Pyocyanin, 134, 548 
Pyogenic bacteria, 371 
Pyoktanin, germicidal power of, 197 
Pyrogallic acid in the cultivation of an- 
aérobic bacteria, 85 


Quarter evil, 347, 586 
Quinine, salts of, as antiseptics, 194 


Rassits, bacillus of intestinal diphtheria 
in, 459 
inoculations of with the diphtheria 
bacillus, 452, 455 
with hydrophobic virus, 99 
with tuberculous matter, 481 
Race immunity to disease. 234 
Rags, disinfection of, 214 
Railway cars, disinfection of, 214 
Rain-water. number of bacteria in, 682 
Ranvier’s moist chamber, 65 
Rats as propagators of bubonic plague, 
564 
Rauschbrand, 347 
Rauschbrandbacillus. 585 
anon-virulent variety of (Klein), 587 


699 


Reaction of culture media, 127 
Recurrens spirochaete, 590 
Red rays of spectrum non- germicidal, 161 
Reichert’s thermo-regulator, 90 
Relapsing fever, a disease of man, 233 
spirillum of, 590 
transmission of, by inoculation 
with infected blood, 591 
Retention theory of immunity, 250 
Rhinoscleroma, bacillus of, 496 
Rhorbeck’s thermo-regulator. 88 
Ricin, immunity against, 262, 265, 267 
Rinderpest, influence of temperature on 
the infectious material, 156 
protective inoculations against, 343 
Rinderseuche, 287 
River water, number of bacteria in, 632 
Rose, antiseptic power of, 203 
Rosemary, antiseptic power of, 203 
Rotzbacillus (see Bacillus mallei), 489 
Rouget, 287 
bacillus of, 511 
Koch’s experiments on the etiology 
of, 515 
protective inoculations against, 324 
Roux’s attenuated varieties of diphtheria 
bacillus 309 
method of producing diphtheria anti- 
toxin, 316 
incubator and thermo-regulator, 95 - 
Roux and Yersin, inoculation of the ba- 
cillus of diphtheria into rabbits, 
452 
study of the pseudo-diphtheritic ba- 
cilli, 456 


SaccuaromycetTes of Nageli, 11 
Sach’s classification of bacteria 11 
Sage, antiseptic power of, 203 
Salicylic acid, action of. on bacillus tu- 
berculosis, 478 
germicidal action of, 182 
action of, on micrococcus pneu- 
moniz croupose, 405 
Saliva, peptonizing action of, 653 
prejudicial to the action of certain 
pathogenic organisms 653 
Salmon’s immunizing experiments, 290 
Salmonson’s culture method, 79 
tube, 82 


700 


Salt meat, bacteria in, 671° 
saturated solution of, inefficient as a 
germicide, 573 
Salts, antiseptic and germicidal value of, 
158 
Sanarelli’s water vibrios resembling the 
cholera spirillum, 607 
Sandalwood, antiseptic power of, 203 
Sand filtration in air analysis, 620 
Saprin, 147 
Saprol, disinfectant power of 206 
Saprophytes, 611 
conditions favorable to their growth, 
613 
in air, list of, 624 
Saprophytic bacilli which are patho- 
genic, 528 
bacteria, 15 
Sarcina, general characters of, 17 
grouping of, 22 
in stomach of persons suffering from 
indigestion, 662 
aurantiaca, thermal death-point of, 
155 ; 
lutea, thermal death-point of, 155 
Sassafras, antiseptic power of, 203 
Savin, antiseptic power of, 203 
Sawizky on the vitality of dried tuber- 
culous sputum, 479 
Scarlet fever, second attacks of, 243 
Scheinfaden, 120 
Schizomycetes of Nageli, 11 
Schoolrooms, bacteriology of the air of, 
625 
Schréder and Van Dusch, use of cotton 
as a filter by, 4 
Schuhanka, protective inoculations in 
Salzburg 350 
Schulze’s (Franz) experiments opposed to 
spontaneous generation 4 
Schiitz’s diplococcus of pneumonia in 
horses, 418 
Schwann’s experiments opposed to spon- 
taneous generation, 4 
Schweinerothlauf, 287 
Schweineseuche, 287, 344 
(Léffler and Schiitz), 343 
Sea water. number of bacteria in, 637, 
641 
Secondary infections, 227 


INDEX. 


Septicemia, definition and general char- 
acters of, 498 
produced by saprophytic bacteria, 
528, 529 
Septicemic bacteria, attenuated varie- 
ties in the mouth and air passages of 
certain animals, 503 
Serpent venom, immunization against, 
262 
Serum-albumin, germicidal power of, 210, 
238 
Serum of blood, germicidal action of, 
236, 257 
immunizing value of, 313 
Sevestre’s observations on the disappear- 
ance of the Léffler bacillus after diph- 
theria, 460 
Sewer water, number of bacteria in, 634 
Shakespeare’s report on anticholeraic in- 
oculations, 300 
Sheep, micrococcus of gangrenous masti- 
tis in, 416 
Sheep-pox, influence of temperature on 
the infectious material of, 156 
Ships, disinfection of, 214 
Siberian plague, 422 
Silicate jelly as a culture medium, 47 
Silkworms infected with “la flacherie,” 
415 
with pébrine, 415 
Silver chloride as an antiseptic, 194 
nitrate as an antiseptic, 194 
Sjobring’s method of demonstrating the 
structure of bacterial cells, 117 
Skatol, no germicidal power, 206 
Skin, abscesses caused by staphylococcus 
epidermidis albus, 372 
human, bacterial flora of, 648 
list of species found on the, 649 
infection through the, 229 
Sleskin’s silicate jelly, 47 
Smallpox, heat as a disinfectant for, 154 
second attacks of, 243 
Smear preparations, 26 
Smith (Erwin F.) quoted on bacterial 
diseases of plants, 571 
Smith (Theobald), fermentation tube of, 
68 
views on the hog cholera group of 
bacteria, 506 


INDEX. 


Smith (Theobald), detection of colon ba- 
cilli in waters, 535 
identification of colon and typhoid 
bacilli in water supplies, 445 
Smoke, antiseptic power of, 206 
tobacco, antiseptic power of, 207 
Snow, number of bacteria in, 632 
Sodium borate as an antiseptic, 195 
carbonate as an antiseptic, 195 
chloride as an antiseptic, 195 
in culture media, 46 
influence of, on bacterial growth, 
128 
hydroxide, influence of, on bacterial 
growth, 184 
hyposulphite as an antiseptic, 195 
sulphite as an antiseptic, 195 
Soil bacteria, 642 
Solid culture media, 41 
Solidification of blood serum, 57 
Sour dough, bacteria of, 672 
Sozoiodol acid as a disinfectant, 179 
Spallanzani’s sterilization of putrescible 
liquids, 4 
Species not differentiated by morpholog- 
ical characters alone, 13 
Sperulina(Hueppe), general characters of, 
18 
Spirilla, general characters of, 18 
of Baumgarten, 13 
list of species found in water, 639 
movements of, 119 
pathogenic, 590 
resembling the spirillum of Asiatic 
cholera, 606 
Spirillum anserum, from blood of af- 
fected geese, 592° 
cholere asiatice, 292, 593 
- asiaticze, attenuation of, by con- 
tinuous cultivation, 601 
asiatice, biological characters, 
594 
asiaticz, influence of disinfect- 
ing agents on, 597 
asiatice influence of gastric 
juice on, 658 
asiaticze, influence of light on, 
162 
asiaticz, influence of tempera- 
ture on, 610 


701 


Spirillum cholerz asiatice occasionally 

present in healthy persons, 610 

asiaticee, phosphorescence occa- 
sionally found in, 609 

asiatice, thermal death-point of, 


155 

asiatice, vitality of, in feces, 
610 

asiatice, vitality of, when 


buried in the soil, 645 
asiatice, vitality of, in waters 
of various characters, 637 
of Ehrenberg, 3, 10 
of Finkler and Prior, 602 
pathogenesis, 603 
thermal death-point of, 155 
Metschnikovi, 604 
pathogenesis, 605 
murinum (Russell) remotely resem- 
bling the spirillum of cholera, 606 
Obermeieri, in the blood during the 
febrile paroxysms of relapsing 
fever, 590 
pathogenesis, 591 
tyrogenum (Deneke), 603 
thermal death-point of, 155 
Spirochaete, general characters, 18 
anserina (Sakharoff), 592 
of Ehrenberg, 3, 10 
Obermeieri, 590 
discovery of, 7 
Spleen, influence of, on the spirillum of 
relapsing fever, 592 
pure cultures of the typhoid bacillus 
from the, 483 
Splenic fever, 422 
Spontaneous generation proved errone- 
ous, 4 
Spores, 16 
action of acetic acid on, 182 
endogenous, 120 
germination of, 122 
influence of oxygen and temperature 
on, 123 
isolation of, by heat, 68 
resisting powers of, 5, 157 
restraining influence of temperature 
on, 166 
staining of, 31 
thermal death-point of, 58, 473 


702 


Spores, vitality of, 121, 122 
Spores formed by : 
bacillus alvei, 556 
bacillus anthracis, 424, 426 
bacillus anthracis, multiplica- 
tion of, 429 
bacillus anthracis, survival of, 
in various media, 430 
bacillus anthracis, exceptions 
to, 426 
bacillus crassus sputigenus, 517 
bacillus cedematis maligni, 582 
bacillus piscicidus agilis, 565 
bacillus of symptomatic anthrax, 
586, 587 
bacillus tetani, 578 
bacillus tetani, influence of heat 
and germicides on, 580 
Spores not formed by or not observed in: 
bacillus acidi formans, 538 
bacillus aérogenes capsulatus, 
589 
bacillus amylovorus, 576 
bacillus of Babes and Oprescu, 
526 
bacilus of Beck, 566 
bacillus coli communis, 530 
bacillus cuniculicida havanien- 
sis, 540 
bacillus of Emmerich and Wei- 
bel, 566 
bacillus erysipelatos suis, 513 
bacillus of Fiocca, 547 
bacillus gracilis cadaveris, 560 
bacillus hyacinthi, 573 
bacillus lactis aérogenes, 536 
bacillus of Laser, 524 
bacillus lepre, 488 
bacillus mallei, 489, 490 
bacillus of Mereshkowsky, 565 
bacillus of Nicolaier (capsule), 
560 
bacillus cedematis maligni No. 
II., 588 
bacillus pestis, 563 
bacillus piscicidus, 567 
bacillus of purpura bemor- 
rhagica (Babes), 558 
bacillus of purpura hemor- 
rhagica (Kolb), 559 


INDEX. 


Spores not formed by or not observed in: 
bacillus pyocyaneus, 543. 
bacillus pyocyaneus pericardi- 

tidis, 547 
bacillus solanacearum, 571 
bacillus tracheiphilus, 577 
bacillus tuberculosis, 472, 478 
bacillus of von Dungern (cap- 
sule), 562 
proteus lethalis, 554 
proteus Zenkeri, 554 
spirillum cholere asiatice, 594 
spirillum of Finkler and Prior, 
603 
spirillum of Metschnikovi, 604 
spirillum tyrogenum, 603 
Spores of moulds and fungi in atmos- 
pheric dust, 614 
of non-pathogenic bacteria, resist- 
ance of, to heat, 158 
Spring water, number of bacteria in, 634 
Sputum, examination of, for bacillus 
tuberculosis, 470 
micrococcus tetragenus in, 411 
separation of bacilli from, by sedi- 
mentation, 486 
tuberculous, estimation of the num- 
ber of bacilli in, 479 

Stab cultures, 69 
character of growth in, 70 
of anaérobic bacteria, 81 
from roll-tube colonies, 77 

Stained preparations, the photographing 

of, 104 

Staining methods in bacteriology, 25 
of a dried bacterial film, 27 
sections of gelatin stab cultures, 
36 
bacteria in tissues, 34 
sections of tuberculous tissues, 471 
of spores, 31 
Staining materials and methods for: 
bacillus acidiformans, 537 
bacillus acne contagiosa of 
horses, 557 

bacillus alvei, 556 

bacillus anthracis, 424 

bacillus of Babes and Oprescu, 
526 

bacillus of Beck, 566 


INDEX. 


Staining materials and methods for: 


bacillus of Belfonti and Pasca- 
rola, 508 

bacillus capsulatus, 521 

bacillus cavicida havaniensis, 
516 

bacillus of Cazal and Vaillard, 
525 

bacillus of cholera in ducks, 
503 

bacillus coli communis, 530 

bacillus crassus sputigenus, 517 

bacillus cuniculicida havanien- 
sis, 538, 540 

bacillus diphtherie, 453 

bacillus dysenteriz, 569 

bacillus of Emmerich and Wei- 
bel, 566 

bacillus endocarditidis capsula- 
tus, 556 

bacillus endocarditidis griseus, 
555 

bacillus enteritidis, 520 

bacillus erysipelatos suis, 512 

bacillus of Fiocca, 547 

bacillus of Friedlander, 396 

bacillus of grouse disease, 520 

bacillus of hog cholera, 504 

bacillus of influenza, 463 

bacillus of intestinal diphtheria 
in rabbits, Ribbert’s method, 
459 

bacillus lactis aérogenes, 536 

bacillus leporis lethalis, 541 

bacllius lepre, 487 

bacillus of Lucet, 526 

bacillus mallei, 489 


703 


Staining materials and methods for: 


bacillus pyocyaneus pericardi- 
tidis, 547 

bacillus pyogenes filiformis, 567 

bacillus cf rhinoscleroma, 497 

bacillus of septicemia hzmor- 
rhagica, 500 

bacillus solanacearum, 571 

bacillis of swine plague, Mar- 
seilles, 509 

bacillus of syphilis, 495 

bacillus of symptomatic anthrax, 
586 

bacillus of tetani, 578 

bacillus tuberculosis, 30, 469 

bacillus typhi abdominalis, 437 

bacillus typhi murium, 524 

bacillus of von Dungern, 562 

diplococcus intracellularis men- 
ingitidis, 410 

micrococcus bothryogenus, 412 

micrococcus gonorrheea, 392 

micrococcus of Manfredi, 413 

micrococcus tetragenus, 411 

proteus hominis capsulatus, 518 

proteus lethalis, 554 

proteus septicus, 554 

proteus vulgaris, 549 

spirillum cholere asiatice, 594 

spirillum of Finkler and Prior, 
602 

spirillum of Metschnikovi, 604 

spirillum of Obermeieri, 590 


spirillum tyrogenum, 603 


staphylococcus pyogenes aureus, 
374 
streptococcus pyogenes, 383 


bacillus of Nicolaier (capsule), Staphylococcus, grouping of, 21 
560 epidermidis albus (Welch), 380 
bacillus cedematis maligni, 581 pyogenes albus, 380 


bacillus cedematis maligni No. 
II., 588 

bacillus piscicidus, 567 

bacillus of purpura hemor- 
rhagica (Babes), 558 

bacillus of purpura hemor- 
rhagica (Kolb), 559 

bacillus of purpura hemor- 
rhagica (Tizzoni and Giovan- 
nintl), 558 


albus, thermal death-point of, 
155 

aureus, 373 

aureus, resistance of, to freez- 
ing, 153 

aureus, thermal death-point of, 
155 

aureus toxalbumins .from cul- 
tures of, 150 

citreus, 381 


704 


Staphylococcus pyogenes citreus, thermal 
death-point of, 155 
Starvation, influence of, on development 
of pus cocci, 224 
Steam under pressure as a disinfectant, 
159, 212, 213 
compared with streaming steam, 
215 * 
as a sterilizer, 56 
sterilizers, 55 
Sterilization of media, etc., 37, 52 
by filtration, 58 
imperfect, errors arising from, 62 
of a piston syringe, 97 
of a platinum loop for inoculation, 
63 
Sternberg’s bacillus X, 538 
bulbs, 38, 66 
discovery of bacillus coli communis 
in the blood and organs of yellow- 
fever cadavers, 580 
experiments on attenuation of virus 
by antiseptic agents, 131 
on the thermal death-point of 
bacteria, 154 
method of collecting blood serum, 38 
liquid culture method, 65 
method of cultivating anaérobic bac- 
teria, 83 
of determining the germicidal 
power of chemical agents on 
anthrax spores, 168 
syringe for inoculation, 97 
theory of immunity, 252 
thermo-regulator, 92 
tubes for the collection of 
samples, 627 
Stomach, bacteria of, not concerned in 
digestive action, 660 
of nursing infants, bacteria in, 659 
Strauss’ method of diagnosticating glan- 
ders, 493 
Streak cultures of bacillus septicemize 
hemorrhagice, 500, 509 
Strebel’s protective inoculations, 348, 349 
Streptococci, 17, 129 
grouping of, 22 
infection of, 347 
Streptococcus agalactiz contagiose, 419 
articulorum (Léffler), 386 


water 


INDEX. 


Streptococcus bombycis, 415 
coryze contagiose equorum, 418 
erysipelatos, 382 
action of chlorine on, 177 
lanceolatus Pasteuri (Gameléia), 398 
longus (von Lingelsheim), 382 
germicidal power of chemicals 
on, 385 
of mastitis in cows, 417 
mastitis sporadiz, 420 
perniciosus psittacorum, 419 
pyogenes, 346, 382 
in diphtheria, 449, 452, 453, 461 
thermal death-point of, 155, 384 
malignus (Fliigge), 388 
septicus (Fliigge), 415 
Strict anaérobics, 16 
parasites, 15, 221 
Sucholoalbumin, 323 
Sucholotoxin, 323 
Sugar as a filter in air analysis, 620 
Sulpho-carbolic acid, germicidal power 
of, 197 
Sulphur dioxide, influence of, on bac- 
terial growth, 176 
as a disinfectant, 212, 213, 214 
Sulphuretted hydrogen, a bacterial pro- 
duction, 141 
Sulphuric acid as a decolorizer in stain- 
ing, 28 
action of, on bacterial growth,180 
action of, on micrococcus pneu- 
moni croupose, 405 
Sulphurous acid, action of, on bacterial 
growth, 180 
Sunlight, importance of, as a disinfect- 
ant, 162 
Susceptibility to disease, 233 
Susotoxin, 323 
of Novy from cultures of bacillus of 
hog cholera, 505 
Swine plague, 343 
(American) bacillus of (Salmon 
and Smith), identical with 
bacillus septicemize hemor- 
rhagice, 499 
practical measures for arresting, 
845 
protective inoculations against, 
322 


INDEX. 


Symptomatic anthrax, 347 
bacillus of, 585 

Syphilis, Lustgarten’s bacillus of, 494 
not obtained in cultures, 496 


Tannic acid as a disinfectant, 183 
Tartaric acid as a disinfectant, 183 
Temperature, influence of, on bacterial 
growth, 126, 153 
on spore formation, 123 
on the destruction of organ- 
isms, 5 
resistance of anthrax spores to a 
high, 427 
animal, influence of, on immunity, 
235 
Test tubes, use of, for cultures, 62 
sterilization of, 53 
Tetanin, 149 
Brieger’s method of obtaining, 352 
Tetanotoxin of Brieger, 149, 352 
Tetanus, a traumatic infection, 350 
antitoxin, 260 
curative power of, 359 
Tizzoni and Cattani’s experi- 
ments on, 855 
bacillus, 578 
cultures, toxalbumin of, 150, 151 
poison of, 352 
neutralized by the blood of an 
immune animal, 354 
not neutralized by the blood of 
chickens, 355 
Tetrads, 22, 120 
Tetraiodphenolphthalein, antiseptic val- 
ue of, 179 
Texas fever in cattle, bacillus of, 508 
Thallophytes of Sachs, 11 
Therapeutic value of antitetanic blood 
serum, 356 
Thermal death-point of bacteria, 52, 155 
of bacillus of Beck, 566 
of bacillus cavicida havaniensis, 
517 
of bacillus of Cazal and Vaillard, 
525 
of bacillus coli communis, 532 
of bacillis of Emmerich and 
Weibel, 566 
of bacillus erysipelatos suis, 518 


45 


705 


Thermal death-point of bacillus of Laser, 
524 
of bacillus piscicidus agilis, 565 
of bacillus pyocyaneus, 548 
of bacillus septicemize hemor- 
rhagice, 501 
of bacillus typhi abdominalis, 
441 
of spirillum cholere asiatice, 
597 
Thermo-regulators, 88 
Thoinot’s method of isolating the typhoid 
bacillus, 444 
Thyme, antiseptic power of, 203 
Thymic acid, action of, on bacteria, 183 
Thymol, antiseptic power of, 207 
action of, on bacillus tuberculosis, 
478 
Thymus bouillon, 246, 357 
preparation of, 359 
immunization to tetanus by cul- 
tures in, 359 
Tin chloride as an antiseptic, 195 
Tizzoni and Cattani, on the preservation 
of tetanus antitoxin, 359 
experiments favorable to Koch's 
tuberculin, 479 
Tizzoni and Giovannini’s bacillus of 
purpura hemorrhagica, 558 
Tobacco smoke, antiseptic power of, 207 
Torula chains, 22 
Toussaint on fowl cholera, 288 


-Toxemia, definition of, 498 


caused by anaérobic bacteria, 578 
by saprophytic bacilli, 528, 529 
in diphtheria, 449 
Toxalbumin of diphtheria cultures, 312 
of hog cholera, 505 
of hydrophobia, 327 
of tetanus cultures, 353 
of typhoid culture, 150 
Toxalbumins, 146, 149 
Toxic power of the culture products of 
bacillus tetani, 353 
of virulent cultures neutralized 
by the blood of immune ani- 
mals, 259 
Toxin of anthrax, 430 
immunity produced by, 427 
of cholera cultures, 610 


706 


Toxin of bacillus of hog cholera, 505 
of influenza, 465 
piscicidus agilis, 565 
tuberculosis, 152, 361 
Toxins, 146, 151 
Toxophylaxin, 265 
Trachoma, pus cocci in, 390 
Tribromophenol as a disinfectant, 179 
Trikresol, germicidal power of, 202 
Trimethylamine, a non-toxic ptomain, 
147 
Trudeau’s successful antitubercular inoc- 
ulations, 366 
Tryptic enzymes, 135 
Tubercle bacilli, method of staining in 
tissues, 36 
in milk of cows, 232 
bacillus (see Bacillus tuberculosis) 
Tuberculin, action of, on tuberculous ani- 
mals, 479 
Bujwid’s method of preparing, 363 
Helman’s method of preparing, 363 
Koch’s, 151, 361 
use of, by various experimenters, 
364, 365 
Tuberculocidin of Klebs, 363 
Tuberculosis, carnivora immune to, 233 
among cattle, prevalence of, 482 
usual methods of infection, 481 
protective inoculations in, 360 
Tuberose, antiseptic power of, 203 
Turpentine, antiseptic power of, 208, 
206 
Tympanic cavity, bacillus pyocyaneus 
present in inflammation of, 545 
Tyndall’s optically pure air, 5 
Typhoid bacilli and colon bacilli, varie- 
ties of the same species? 447 
bacillus, 431, 486 
attenuation of, 242 
biological characters of, 488 
culture medium for, 47 
dead animal matter a suitable 
nidus for, 434 
destroyed by gastric juice, 658 
difficulty of differentiating, 440 
influence of light on the growth 
of, 161 
morphology, 486 
pathogenesis of, 442 


INDEX. 


Typhoid bacillus, pathogenic to certain 
of the lower animals, but does 
not convey typhoid fever, 436 
pus production by, 448 
resistance of, to freezing, 153 
thermal death-point of, 155, 441 
vitality of, in various media, 
442 
vitality of, in moist atmospheres. 
159 
vitality of, when buried in the 
soil, 645 
colonies in the spleen, post-mortem 
multiplication of, 434 
cultures, toxalbumin of, 150 
fever, a disease of man, 233 
failure to communicate it to ex- 
perimental animals, 4385, 442 
heat as a disinfectant for, 154 
protective inoculations in. 367 
second attacks of, 243 
Typhotoxin, 148, 222 
of Brieger, 442 
Tyrotoxicon, 148 


ULcEeRATIVE endocarditis, 379, 386 

Underclothing, worn, bacteria on, 649 

Urea, fermentation of, 140 

Uric acid, influence of, on the bacterici- 
dal activity of blood serum, 2388 

Urine as a culture medium, 39 

germicidal power of, 211 
Uterus, free from bacteria in health, 654 


Vaccine virus, influence of temperature 
on, 156 

Vagina, bacteria of, 654 

Vaillard, immunity to tetanus produced. 
in rabbits, 355 

Valerian, antiseptic power of, 203 

Valerianic acid as a disinfectant, 183 

Van Ermengem’s method of staining 

flagella, 33 

Veal broth peptonized as a culture me- 
dium for bacillus tuberculosis, 477 

Vegetable infusions as culture media, 41 

Veins, injections into, 98 

Venom, serpent, immunization against, 
262 

Verbena, antiseptic power of, 203 


INDEX. 


Vesuvin as a stain, 25 
as a double stain in bacterial prepa- 
rations, 28 
Vibrio, general characters, 18 
aquatilis (Giinther), remotely resem- 
bling the spirillum of cholera, 606 
Berolinensis (Neisser), its differenti- 
ation from the cholera spirillum, 
607 
Danubicus (Heider), its differentia- 
tion from the cholera spirillum, 
608 
of Ehrenberg, 3, 10 
helcogenes (Fischer), remotely resem- 
bling the cholera spirillum, 607 
Metschnikovi (Gameléia), 604 
Vibrion septique (Pasteur), 580 
Vibrioniens of Ehrenberg, 3, 10 
Vibrio-proteus, 602 
Vibrios of Celli and Santori, 608 
resembling the spirillum of cholera, 
606 
list of species found in water, 639 
phosphorescent, not genuine cholera 
germs, 608, 609 
Vincent’s method of detecting colon ba- 
cilli in water supplies, 640 
Violet rays of spectrum, germicidal 
power of, 161 
Virulence, attenuation of, in cultures, 
130, 388 
intensification of, 132, 388 
recovery of, 132 
Viscous fermentation, 141 
Vital resistance theory of immunity, 252 
Vitality of bacillus of hog cholera, 505 
of the typhoid bacillus in cultures, 
442 
Von Dungern’s capsule bacillus distin- 
guished from others of similar mor- 
phology, 562 


Water, action of, on the cholera spiril- 
lum, 597 
bacteria in, 626 
list of species found in, 638 
number not dependent on the 
amount of organic matter pres- 
ent, 635 
distilled, growth of bacteria in, 685 


107 


Water, pathogenic bacteria that may be 
present in, 636 
bacteria, detection of, 640 
bacteriological analysis of, 628 
collection of, for analysis, 627 
importance of immediate exam- 
ination, 627 
chemical analysis of, advantages of, 
631 
filtration, efficiency determined by 
counting colonies, 641 
supplies, detection of colon bacilli 
in, 535 
difficulty of finding typhoid 
bacilli in, 441, 444 
sanitary inspection of source 
more important than bacterio- 
logical or chemical analysis, 
641 
vibrios, remotely resembling the 
spirillum of cholera, 606 
Waters, surface, frequency of the colon 
bacilli in, 640 
Weichselbaum’s diplococcus intracellu- 
laris meningitidis, 410 
micrococcus endocarditidis rugatus, 
416 
Weigert’s method of staining bacteria in 
tissues, 35 
of staining used in sections of 
nodules of glanders, 493 
Welch and Abbott’s cultivation of the 
bacillus of diphtheria on po- 
tato, 455 
inoculations of kittens with the 
diphtheria bacillus, 452 
Well water, number of bacteria in, 634 
Widal reaction given by blood serum of 
those subjected to antityphoid 
inoculation, 369 
in Malta fever, 421 
Wildseuche, 287, 499 
Winogradsky’s silicate jelly, 47 
Wintergreen, antiseptic power of, 203 
Wolffhiigel’s experiments on the thermal 
death-point of bacteria, 154 
Wolter’s opinion that positive results in 
attempts to inoculate leprosy were due 
to associated tubercle bacilli, 488 
Woollen underclothing bacteria on, 649 


a. 
708 e 
Wool-sorter’s disease, 422, 430 
a pulmonary affection, 231 
Woven underclothing, bacteria on, 649 
Wurtz’s method of identifying typhoid 
bacilli in water supplies, 445 


XyLou for solution of Canada balsam, 28 


Ye Ltow fever, insusceptibility of negro 

to, 234 
cadavers, bacillus coli communis 
in the blood and organs of, 530 

milk, 668 
Yersin’s attenuated varieties of diph- 
theria bacillus, 309 

description of the bacillus pestis, 563 
aie 


ZEA mays, bacterial diseases of, 575 


INDEX. 


Zedvary, antiseptic power of, 203 


-“ Zeitschrift fir Hygiene,” 8 


Ziehl’s carbol-fuchsin solution, 29 
for bacillus lepree, 487 
for bacillus tuberculosis, 469- 
471 
for the typhoid bacillus, 488 
Ziehl-Nelson method of staining the tu- 
bercle bacillus, 30, 36 
Zinc chloride as an antiseptic and disin- 
fectant, 195, 212 
sulphate as a germicide, 196 
Zooglea, 21 
formed by bacillus solanacearum, 571 
proteus mirabilis, 5538 
Zopf, classification of, 12 
Zymogenic organisms, 14 


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