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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.
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Second Revised Codition
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COPYRIGHT BY
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1901.
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PRESS OF
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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. 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. 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
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aN a 9
ra M i i
sosscusal any if
ass ‘5 We
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Ssnnee S,
yA
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. wee. .
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a ee beatae)
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‘ :
poke ) ehh ste
os tke RSS A
° e S5 fe et 8 qquets
o es e
“= A foe ie Waaoak % *e
abe, .
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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 *+ *
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. ), 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.
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