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MICRO-ORGANISMS AND DISEASE

INTRODUCTION TO THE STUDY OF
SPECIFIC MICRO-ORGANISMS

E. KLEIN, M.D., F.R.S.

Lecturer on General Anatomy and Physiology in the Medical School oj

IL o n ti o it

MACMILLAN AND CO., Ltd.

new YORK : MACMILLAN & CO.

1896

'I he Right oj Translation and Reproduction is Resettled.

P.ichard Clay and Sons, Limited,

LONDON AND BUNGAY.

First Edition , 1884. Reprinted , with additions
and alterations, 1885. Second Edition, 1886. Third
Edition, 1896.

SIR JOHN SIMON, K.C.B., D.C.L., LL.D., F.R.S.

THIS EOOK

3s iRcspcctfullv? BefctcateD

THE AUTHOR

PREFACE TO THE NEW EDITION

Ten years have passed since the last (third) Edition of
this book appeared. Great changes have taken place since
that time. The phenomenal extension of the study of
Bacteriology, its application to the study of Public Health,
and to Medical and Surgical Practice ; the remarkable
extension of our knowledge of the chemical activity of
Micro-organisms ; the results achieved in the battle against
infectious diseases by means of accurate bacterioscopic
analysis and diagnosis, and by the brilliant application of
serum therapeutics, are matters obvious to the Biologist, the
Chemist, the Sanitary Expert, the Physician and Surgeon.
To get an idea how much the study of micro-organisms in
their relation to disease has extended we may state that when
Baumgarten’s Jahresbericht — an annual review giving a
tolerably complete account of the work done in pathological
mycology — first appeared (Vol. I.) in 1886 its size was 185
pages; in 1892 (Vol. VII.) it had reached the size of 862
pages, and it has been steadily increasing since. Since 1886
several most important periodicals have come into existence

PREFACE

viii

which are almost entirely devoted to the publication of
original work in bacteriology : Zeitschrift fiir Hygiene ,
Ann ales de P Inst i tut Pasteur, the Journal of Pathology and
Bacteriology ; in addition the Hygienische Rundschau , the
Fortschritte der Medicin, the Archiv fiir Hygiene , the Pro-
ceedings of the Royal Society, various medical and veterinary
periodicals in this country and abroad, continue, as before,
to publish occasional papers on bacteriological subjects. It
must be obvious that it is an impossibility to give, even in
its essential outlines, an account of all this enormous
progress in a small handbook like the present volume, but
I have striven to add the more important results of the
work of the last decade, principally so far as they refer to
the relation of micro-organisms to disease. For this purpose
it was found necessary to revise and to rewrite some, and to
materially alter others, of the chapters of the old edition.

A considerable number of illustrations taken from photo-
grams have been added ; these have been, for the most part,
prepared from my own preparations, unless otherwise stated,
by Messrs. A. Pringle and E. Bousfield.

E. KLEIN.

March, 1896

CONTENTS

1*AGE

INTRODUCTION i

CHAPTER I

MICROSCOPIC EXAMINATION 7

CHAPTER II

PREPARATION OF CULTURE MATERIAL 24

CHAPTER III

VESSELS AND INSTRUMENTS USED IN CULTIVATIONS 38

CHAPTER IV

PREPARATION OF CULTURE-MEDIA FOR INOCULATION . .

CONTENTS

CHAPTER V

PAGE

METHODS OF INOCULATION . . 53

CHAPTER VI

GENERAL CHARACTERS OF BACTERIA 88

CHAPTER VII

CHEMISTRY OF BACTERIA 122

CHAPTER VIII

MICROCOCCI J3S

CHAPTER IX

bacillus ( Desmobacterium , coiln) 164

CHAPTER X

BACILLI : SPECIAL *7$

CHAPTER XI

BACILLI SPECIFICALLY PATHOGENIC TO MAN OR ANIMALS . . 204

CONTENTS

CHAPTER XII

PATHOGENIC BACILLI : GROUP C

CHAPTER XIII

THE MICROBES OF MALIGNANT ANTHRAX* OF DIPHTHERIA,
AND OF GLANDERS

CHAPTER XIV

BACILLUS TUBERCULOSIS AND BACILLUS LEPR/E

CPIAPTER XV

ANAEROBIC BACILLI

CHAPTER XVI

VIBRIO AND SPIRILLUM .

CHAPTER XVII

YEAST FUNGI : TORULACE/E, SACCHAROMYCES .

CHAPTER XVIII

MOULD-FUNGI

HYPLIOMYCETES OR MYCELIAL FUNGI

PAGE

248

271

333

36S

404

47i

477

CONTENTS

xii

CHAPTER XIX

PROTOZOA CAUSING DISEASE

PAGE

498

CHAPTER XX

ANTAGONISM AMONGST BACTERIA . . . .

527

CHAPTER XXI

THE RELATION OF SAPROPHYTIC TO PATHOGENIC ORGANISMS . 534

INDEX

585

MICRO-ORGANISMS AND DISEASE

INTRODUCTION

The relation of micro-organisms to disease is admitted
to be very intimate ; with special regard to infectious
diseases there exists now no doubt that specific microbes
are the causa causans, but also in a number of diseases not
infectious in the ordinary term — i.e. not communicable from
individual to individual — an important relation between a
specific microbe and the disease itself has been proved to
exist. Amongst the infectious or communicable diseases
there are still some in which the satisfactory demonstration
of specific microbes has not been achieved yet, as in hydro-
phobia, variola, syphilis, measles, whooping-cough, &c. ; but
in a large number of maladies which affect man and animals,
and which from the ascertained etiological data are of the
nature of communicable diseases, the demonstration of the
specific microbes is an established fact. Amongst the
diseases which do not strictly belong to the communicable
diseases, there are some in which it has been either proved
or made highly probable that microbes have an important

MICRO-ORGANISMS AND DISEASE [INTRO.

bearing on their production in particular individuals specially
predisposed.

To mention two series only : it has been shown that there
occur in some individuals a variety of localised inflam-
matory and suppurative foci, which are intimately associated
not necessarily with pus-forming cocci — the microbes of typi-
cal suppuration — but with one or the other kind of microbes
— e.g. proteus vulgaris, bacillus coli, pneumococcus, and other
microbes. These organisms have in these particular cases
only caused disease ; under healthy conditions their presence
in the various tissues is insufficient to do so. Or, to take
another series of disorders : fatal summer diarrhoeas in
children and in adults. Bacillus coli or proteus vulgaris
are under ordinary normal conditions found in the intestine,
in the large intestine and in the lower ileum, as also in
many other conditions associated with putrid proteid
decomposition. Under certain abnormal conditions of the
intestine caused, in the first instance, by fermentative
changes, such as the lactic or acetic fermentation or others,
the intestinal tract, specially the small intestine, is rendered
highly favourable for the multiplication of those microbes,
so that it practically contains a pure culture of them.
Bacillus coli and proteus vulgaris, being both endowed
with the power of intensive proteid decomposition, would
under these favourable conditions of multiplication produce
copiously poisonous alkaloids, ptomaines or allied toxins,
which, absorbed into the circulation, might produce fatal
results.

In order to pass in review all the ascertained facts and
observations in this vast and constantly growing field of
pathology, and to appreciate and to assign their true value
to the many observations bearing on this relation of micro-
organisms to disease, it is necessary that the reader, and still

INTRO.] MICRO-ORGANISMS AND DISEASE

more the worker in this field, should be enabled to criticise
the observations and facts brought forward by the numerous
writers on this subject, for otherwise he would probably
take as proved what has really not passed beyond the stage
of possibility. And it is this point which requires the most
careful attention — viz., to be able to see at a glance that,
owing to the imperfect or faulty methods employed, or that,
owing to certain inferences incompatible with the general
laws and general tendency of the well-founded and ex-
perimentally proved facts, the statements set forth in a
particular observation or series of observations are not to be
accepted.

In all investigations of the relation of micro-organisms to
disease it is necessary to bear in mind that, as Koch1 has
pointed out, no observation can be said to be complete, or,
one should rather say, in no instance can it be said to have
been satisfactorily proved , that a particular morbid process
is due to a particular micro-organism if any one of the
following conditions remains unfulfilled : — (i) It is absolutely
necessary that the micro-organism in question is present
either in the blood or the diseased tissues of man or of an
animal suffering or dead from the disease. In this respect
great differences exist, for in some infectious diseases the
micro-organisms, although present in the diseased tissues, are
not present in the blood ; while in others they are present
in large numbers in the blood only or in the lymphatics only.
rl hese points will be considered hereafter in the special
cases. (2) It is necessary to take these micro organisms
from their nidus, from the blood or the tissues as the case
may be, to cultivate them artificially in suitable media — i.e.
outside the animal body, but by such methods as to exclude
the accidental introduction into these media of other micro-
1 Die Mi hire nd- im pf ung, Cassel and Berlin, 1S83.

n 2

MICRO-ORGANISMS AND DISEASE [intro.

organisms ; to go on cultivating them from one cultivation
to another for several successive generations, in order to
obtain them free of every kind of matter derived from the
animal body from which they have been taken in the first
instance. (3) After having thus cultivated the micro-
organisms for several successive generations it is necessary
to re-introduce them into the body of a healthy animal
susceptible to the disease, and in this way to show that this
animal becomes affected with the same disease as the one
from which the organisms were originally derived. (4) And,
finally, it is necessary that in this so affected new animal the
same micro-organisms should again be found. A particular
micro-organism may probably be the cause of a particular
disease, but that really and unmistakably it is so can only be
inferred with certainty when every one of these desiderata
has been satisfied.

Now, at the time when Koch laid down these principles,
which being of the nature of exactness were accepted by
all, there still existed amongst medical men a considerable
number who doubted that microbes have any primary
relation to disease, the doctrine of contagium vivum was
still to them an unproven view, although the practice in
sanitary science had long accepted the correctness of that
view. And it may be said to have been one of the most
complete and exact achievements of Koch to have been the
first who by exact methods conclusively demonstrated the
correctness of the view in the case of malignant anthrax.
The same principles led him to the demonstration of the
causative relation between certain microbes and septi-
cemic infections in animals, and he may be said to have
crowned the edifice by his brilliant discovery of the tubercle
bacilli. In all these instances it was possible for him to
absolutely and by exact methods and without cavil to

intro.] MICRO-ORGANISMS ANI) DISEASE

establish the correctness of the theory of contagium vivirn.
Many facts have since come to light which necessitate a
relaxation of these rigid rules. As then so also now these
principles hold good, if the true causative relation between
a particular microbe and a particular disease is to claim
unequivocal acceptance, but there are a variety of conditions
under which such rigorous proof is impossible. Koch
himself has seen this in the case of cholera Asiatica. As
he himself so also others before and after him have
accepted as correct the everyday experience that Asiatic
cholera is a disease of the human subject only, that
domestic animals under natural conditions, in localities
where cholera is endemic or in localities where epidemics
prevail, have never been known to have been subject to
this disease. It is therefore obvious that since no animal
can be said to be susceptible to cholera in the sense in
which man is, the third and fourth points above stated, in
furnishing proof positive, cannot be fulfilled. True, under
certain modes of experimentation with cultures of the
cholera vibrio (see the Chapter on Cholera), guinea-pigs are
capable of developing an acute fatal disease ; but under the
same methods of experimentation other microbes, not
connected with cholera or any other disease, produce the
identical results or results differing only in degree.

Again, take the case of typhoid fever : the microbe
which is found in the tissue of the spleen and mesenteric
glands in large numbers in cases of typhoid fever only is,
from its constant occurrence and its special biological
characters, justly considered to be the microbe of that
disease, but since animals are not susceptible to typhoid
fever, the two conditions mentioned sub 3 and 4 cannot
be verified. The pathogenic action which this microbe
is capable of exerting on guinea-pigs when injected sub-

MICRO-ORGANISMS AND DISEASE [intro.

cutaneously or intraperitoneally, is not of a specific nature
and is not of the nature of typhoid fever in man. As a
last example we may mention leprosy. No one doubts that
the bacilli so peculiar in their morphological and biological
characters and in their distribution, which are found
crowding the cells and tissues of the leprosy nodules,
are the real microbe of leprosy, but no one has succeeded
as yet in producing leprosy in an animal.

It will be my aim in the following pages, first to describe
the methods that may be employed with success in inves-
tigations bearing on the relation of micro-organisms to
disease ; secondly, to describe in conformity with reliable
observations the morphology and physiology of the micro-
organisms that bear any relation to disease ; and thirdly, to
enumerate the principal observations that have been made
in recent years to prove the existence of such an intimate
relation. Last, but not least, we shall consider the precise
relation of the principal micro-organisms and their chemical
products to the causation of disease.

CHAPTER I

MICROSCOPIC EXAMINATION

For the examination of micro-organisms good high powers
are essential, at the least a power magnifying 300 to 400
linear diameters. Zeiss’ D or E and Zeiss’ or Leitz’s or
Reichert’s oil immersion i-i2th or i-i6th inch (2 mm.) will
be found sufficient for all purposes. In the case of tissues
stained with aniline dyes a good substage-condenser such as
Abbe’s or Powell and Lealand’s, is invaluable. I use Zeiss’
or Leitz’s stand with Abbe’s condenser, open diaphragm,
and plane mirror. As Koch 1 pointed out, and what is now
universally acted upon, stained specimens mounted in
Canada-balsam solution or Dammar varnish, when exa-
mined over an Abbe’s condenser, show the micro-organisms
with extreme clearness and sharpness.

The examination of the morphological characters of an
organism is carried out on fresh unstained, as well as on
fresh stained, microscopic specimens. Although the latter
method is, for reasons hereafter to be mentioned, by far the
most perfect and reliable one, it is nevertheless important to

1 Die Aetiologie d. Wundinfectiomkrankheiten, p. 34, Leipzig, 1879.
Iranslated as Traumatic Infective Diseases (New Syd. Soc.), London,

1880.

MICRO-ORGANISMS AND DISEASE [chap.

ascertain as far as possible the motility, chemical reactions,
and general morphology of living fresh specimens. Blood,
juices, tissues, and fluids in which the micro organisms are
present, are subjected directly, without any previous prepa-
ration, to microscopic examination. In the case of artificial
media in which micro-organisms have been growing, the
examination of fresh specimens is of great importance, for
the reason that the organisms can be easily identified and
their size and general morphological characters be more
correctly ascertained than after drying, hardening, and
staining. Besides, the chemical reactions can be satisfac-
torily studied in fresh specimens only. All one has to do
is to draw up with a capillary pipette or to take up with the
point of a platinum needle a drop or particle of the mate-
rial, to place it on an object-glass, and to cover it up with a
thin cover-glass. Where one has to deal with liquids, such
as artificial nourishing fluids, blood serum, tissue-juices,
secretions, transudations and exudations, no addition is re-
quired. In the case of more solid material, such as solid
artificial nourishing material, bits of tissue, &c., the addition
of a drop of neutral previously well-boiled saline solution
(of o-6 to 075 per cent.) is advantageous although not
absolutely necessary, since by pressing down the cover-glass
a layer of the material sufficiently thin for examination can
be obtained. In some instances a bit of tissue can be
teased out into fine particles by means of two clean
needles. Where it is a question of micro-organisms suffi-
ciently conspicuous by their shape, size, and general appear-
ance, their identification in the fresh condition is not
difficult ; this is the case with bacilli, vibrios, actinomyces,
and mycelia, but in the case of micrococci, especially when
isolated or in couples, and lying in blood, juices, or tissues,
their recognition is often extremely difficult. When in large

MICROSCOPIC EXAMINATION

clumps, such as larger or smaller masses of zoogloea, or
when in the shape of chains, the identification is not diffi-
cult; but in the more isolated state they are not easily
recognised, owing, as a rule, to the presence of granules or
particles of various kinds, from which morphologically their
distinction is well-nigh impossible. In such cases there are
certain rules of thumb, if I may say so, which assist, although
they do not absolutely insure, the diagnosis. These are
the uniform size and shape and micro- chemical reactions.
The addition of liquor potassse leaves micro-organisms quite
unaltered, whereas fatty and most albuminous granules alter
or altogether disappear by it. Acetic acid from 5 to 10 per
cent, strong does not affect micro-organisms, but albuminous
and other granules become in most instances altered. These
two re-agents, I think, are as reliable as any others ; if they
fail, then others like alcohol, chloroform, sulphuric ether,
&c., are not of any greater help, but the latter re agents may
be used, for instance, when it is a question between fat-
granules and micrococci, or crystals and bacilli.

Micro-organisms have a great affinity for certain dyes,
especially aniline dyes, and therefore these are used with
great success to demonstrate their presence, and to
differentiate in many instances morphological details which
in the unstained condition are not discernible. The
staining is effected on fresh unaltered organisms, or after
they have been dried. In the first instance the process is
carried out thus : — A microscopic specimen is made, and to
it is added afterwards drop after drop of the dye, passing
it through the specimen in the usual way of applying fluids
to a microscopic specimen — i.e. by adding with a capillary
pipette the dye at one margin of the cover-glass and sucking
it up with a strip of filter-paper applied to the opposite
margin of the cover-glass. When the staining has taken

IO MICRO-ORGANISMS AND DISEASE [chap.

place the excess of the dye is washed away with salt
solution, water or alcohol, or both, as the case may be (see
below). Unless the organisms are embedded in continuous
masses of solids, this method gives good results. In the
latter case, say if they are embedded in a microscopic lump
of tissue, or in a particular spot of a fine section of a fresh
tissue, it is necessary, after having placed the lump or
section on an object-glass, to drop the dye on to this previous
to putting on the cover-glass. After some minutes the dye
is allowed to run off by inclining the object-glass, and then
the washing is proceeded with till all the excess of the dye
is removed ; the mounting is then done by placing a drop
of water or salt solution on the specimen and covering
it with a cover-glass. In the case of sections through
fresh and hardened tissues containing micro-organisms,
the method of staining and of permanently mounting
them as a whole is more complicated, and will be detailed
presently.

When one has to deal with coherent masses of micro-
organisms, present either in natural media (i.e. animal tissue)
or artificial cultivations, such as zooglea and pellicles of
micrococcus or bacillus , these can be bodily transferred to
a watch-glass, stained, washed, and mounted without much
difficulty, either for immediate or permanent use. The
permanent specimens are made in this way : — Place the
section or pellicle in a watch-glass containing the dye, leave
it there till deeply tinted, take out with a needle, section
lifter, or the like, wash in water, then in alcohol, leave
here for sufficient time till most of the excess of the
colouring-matter is removed, then lift it on to an object-
glass, spread well out, place on it a drop of xylol or clove-
oil, and after a minute or two drain off, add a drop of
Canada-balsam solution (in chloroform or xylol), and cover

I] MICROSCOPIC EXAMINATION u

with a cover-glass. In some special instances, such as the
bacilli of leprosy and tuberculosis, double staining is
required. With other organisms, such as the bacilli of
glanders or tuberculosis, the washing is carried out, not
with water but with acid (acetic acid and nitric acid
respectively). All the details will be stated when dealing
with these special organisms.

The method extensively and successfully used for the
demonstration and preservation of microscopic specimens
of micro-organisms in fluids, in blood, pus, mucus, and
juices, is that of Weigert and Koch, which consists in
spreading out on a glass slide or cover-glass a very thin
film — the thinner the better — of the fluid (artificial or
natural culture medium), blood, pus, or juice, and drying
it rapidly by holding it for ten to twenty seconds over the
flame of a spirit-lamp or gas-burner. The most successful
preparations are obtained when the heating is carried on for
such a time that the film, having become opaque at first,
rapidly turns transparent. Several drops of the aniline dye
to be used are then poured over the specimen, or the film
is placed over the dye contained in a watch-glass, and
after remaining in contact from half to thirty minutes or
more, according to the nature of the microbes and the dye,
the specimen is removed.

The cover-glass specimen is then well rinsed with distilled
water, dried over the flame, and mounted in Canada-balsam
solution or Dammar varnish — of course always bearing in
mind on which surface of the cover-glass the film has been
spread. If the film has been well heated in the first
instance washing in water is quite sufficient, but if the
drying has been insufficient a good deal of diffuse staining
of the ground substance has taken place, and then the
cover-glass specimen must be also washed in alcohol

MICRO-ORGANISMS AND DISEASE [chap.

sufficiently long to remove this undesirable staining, then
washed in water, dried and mounted. In some instances,
washing with alcohol removes also the dye from the
bacteria, but as a rule it is better to first over-stain the
cover-glass specimen, then wash well in alcohol so as to
remove the dye from all except the bacteria, but do not
wash with alcohol too long, then rinse in distilled water, dry
and mount.

A method extensively used and yielding the best speci-
mens is the one known as the method of making impression
specimens — Klatschprceparate of the Germans. This method
aims at representing in stained films the impression of bac-
teria in the actual position on a solid culture medium. Be
the bacteria growing in a streak or in isolated colonies on
the surface of gelatine or agar in a plate cultivation (see
below), by pressing a clean cover-glass on to the surface of
the growth an impression is obtained of the growth, the
cover-glass is heated, and stained and treated as before.
When it is successful the bacteria are seen in the exact
position which they occupied in the culture, be that in a
streak or in separate colonies ; the manner in which they
arrange themselves and the manner in which the growth
proceeds at the margin is well shown. Care must be taken
to make impression specimens of young growths, for if late
the impression is too thick ; but even in such cases the
second or third impression of the same colony gives the
desired result.

In the case of liquefying bacteria impression preparations
must be made from gelatine growths at an early stage
before liquefaction commences ( vide Fig. 44) of a young
colony of anthrax bacilli on gelatine.

The most useful dyes in the examination of animal tissues
for bacteria are those aniline dyes that are soluble in water ;

l] MICROSCOPIC EXAMINATION 13

these are preferable to those soluble in alcohol only. They
have all great affinity for cell nuclei (Hermann) and belong
to the group of neutral or basic aniline colours. Methyl-
blue, methyl-violet, vesuvin, Bismarck-brown, magenta,
fuchsin, gentian-violet, Spiller’s purple, rosaniline, Hum-
boldt’s red (purple), are the dyes most commendable.

For staining of cover-glass specimens, as well as for sections
made of fresh tissues, the above dyes can be advantageously
used in the following manner : 2 to 5 grammes of the solid
dye are rubbed up in a mortar with 10 ccm. of absolute
alcohol ; add then gradually, while mixing, warm distilled
water, to bring up the total to 100 ccm.; filter and keep in
stoppered bottle. For use, filter a little of the dye into a
watch-glass. For staining film preparations or sections of
hardened tissues, the above dyes prepared with aniline
oil are preferable ; they are prepared thus : ( a ) Make a
saturated watery solution of pure aniline (aniline oil) by
mixing in a bottle one part of aniline oil with three parts
of distilled water; shake well every half hour for four to
six hours, decant the water as the oil settles to the bottom.
The decanted fluid is the saturated watery solution of aniline.
Of this take 100 ccm. Add to this (i) a saturated alcoholic
solution of either fuchsin, gentian-violet, Humboldt’s red,
methyl-blue or methyl-violet, 1 1 ccm. ; mix well, filter
into stoppered bottle. The sections are left in this dye for
from a few minutes to several hours (Humboldt’s red
requires only a few seconds). Different bacteria require
different periods to stain. As a rule warming the dye
facilitates the staining of the bacteria ; occasionally, also,
the addition of a few drops of liquor potassae. All sections,
after having been sufficiently stained, are transferred to and
washed in water, then methylated spirit, then in absolute
alcohol, then clarified in xylol or clove-oil, and finally

MICRO-ORGANISMS AND DISEASE [char

mounted in Canada- balsam (dissolved in chloroform, or
better still in xylol) or in Dammar varnish.

After a very extensive experience in staining film speci-
mens and sections, carried on for nearly eighteen years, I have
come to the conclusion that for all purposes of bacteriological
work the following stock of dyes is sufficient : (a) methyl-blue,
and (b) gentian-violet, both these prepared with a saturated
watery solution of aniline oil as described on the previous
page ; for staining of cover-glass film specimens use this
gentian-violet aniline water, and absolute alcohol in equal
volumes in a watch-glass, and allow the specimen to remain
in this mixture for a few seconds to half a minute ; afterwards
wash well in water, dry and mount in balsam ; (c) carbol-
fusin prepared after Ziehl 1 ; and (d) Loffler’s methyl-blue :
of a 2 per cent, watery solution of methyl-blue a little is
mixed with equal volume of potassic hydrate i in 10,000 ;
the staining must be of a prolonged character ; after
staining, wash well in water acidulated with acetic acid.
( e ) Alcoholic solution of eosin per cent, j (/) watery
solution of rubin 2 per cent. ; (g) watery solution of Bis-
marck-brown 2 per cent.

In order to bring out by the dye more conspicuously the
bacteria present in fluids or tissues various methods are used,
all of which are based on the principle that the bacteria have
an affinity to the dye which is greater than that of the tissue-
elements. Hence after staining, the tissue-elements may
be decolourised without abstracting the colour from the
bacteria. Cover-glass specimens or sections, after having
been well stained with a dye, are subjected to various
decolourising re-agents, whereby the tissue-elements become
deprived of the dye, but the bacteria retain it. Although in

1 1 ‘5 grammes fuchsin, 10 ec. absolute alcohol, 100 cc. of a 5 per cent,
watery phenol solution.

MICROSCOPIC EXAMINATION

1 5

some instances this is not easy of achievement, since by
such decolourising processes also the bacteria are liable to
lose the stain, it nevertheless is possible in the majority of
instances. In many cases prolonged washing in alcohol
absolutus and in clove-oil is sufficient to abstract the dye
from the tissue-elements, but in some special cases, owing to
peculiar chemical properties possessed by certain bacteria,
the decolourising process requires special methods. Of these
the following are the most useful : —

i. In some instances the specimens (cover-glass specimens
and particularly sections) are stained in one dye, then
washed in alcohol till quite pale, then transferred to a
contrast dye. As contrast dyes are to be regarded blue
and red, or red and brown, or blue and brown, or violet
and brown. In some cases only the bacteria retain the first
dye, the tissue-elements become stained by the second dye.
A similar result is often obtained by mixing the two dyes,
and then using them like a single dye ; hereby occasionally
the bacteria are found to take one colour, while the tissue-
elements take the contrast dye.

For double-staining of film specimens or sections the
following methods will be found most practicable for
general purposes : — ( a ) As a first stain methyl-blue aniline
water is used ; after well staining the specimen it is well
washed in water and then placed in -J- per cent, alcoholic
solution of eosin for from half to one minute, then washed
in water and prepared for mounting in balsam as usual ; (b)
a 2 per cent, watery solution of rubin is used as first dye,
then well washed in water, then placed in methyl-blue
aniline water for half to one minute, washed in water and
proceeded in the usual manner for balsam mounting.

The number of methods for successfully and differentially
double and treble staining normal and pathological tissues
is legion, and those who consult the excellent books by

16 MICRO-ORGANISMS AND DISEASE [chap.

Behrens, and by Kanthack and Drysdale, will find'all they
require not only with reference how to prepare the dyes and
how to apply them, but particularly in what cases and for
what tissues they were first employed and found most
useYul. Without wishing in the slightest degree to convey
that those engaged in pathological work should not avail
themselves to the full of every method that is recommended
and that has been found useful, I venture to say here tha^
the methods of staining, mentioned in this book, which
after many years’ experience have been successfully em-
ployed in my laboratory, have been found quite sufficient in
bacteriological work. '

2. One of the most useful methods for staining bacteria
in sections of hardened tissues or in films is Gram’s method.
Film specimens or sections are kept for five to ten minutes
in absolute alcohol, are then placed in any of the above
mixtures of aniline water and dye (fuchsin, magenta, Hum-
boldt’s red or gentian-violet, methyl-blue or methyl-violet),
and kept there for from two to five minutes or more ; they
are then washed in alcohol for from one to three minutes,
and are then transferred into the following solution: one
part of iodine, two parts of iodide of potash, 300 parts of
distilled water ; they are kept here till their colour com-
pletely changes (as a rule into dark purple), they are then
transferred into alcohol till all colour has apparently gone.
If successful, such sections when examined under the micro-
scope, show only the bacteria stained, while the tissue-
elements are quite colourless. To bring out these latter
more strikingly the sections are stained in a contrast dye,
vesuvin or Bismarck-brown, if red, violet, or blue has been
used as the first dye.

This method is of great diagnostic value, inasmuch as it
represents an important distinction between species which
otherwise may be difficult to distinguish ; one kind becom-

MICROSCOPIC EXAMINATION

ing decolourised by the iodine, while another retains the
first dye after passing through the iodine.

3. Ehrlich's method, used specially for demonstrating
tubercle-bacilli and leprosy-bacilli.— The specimens, after
having been well stained with carbol fuchsin (by heating
in a watchglass till the fluid begins to bubble), are trans-
ferred for 10-30 seconds into 30 per cent, watery solution
of nitric acid ; according to Friedlander a mixture of three
parts of nitric acid in 100 parts of alcohol is equally good.
A to per cent, watery solution of nitric acid is quite
strong enough. All bacteria except the tubercle-bacilli and
leprosy-bacilli lose the dye by this treatment. The prepara-
tions are then stained for contrast in methyl-blue vesuvin
or Bismarck-brown.

4. Koch’s method. — According to this the sections, after
having been stained, are transferred to a saturated solution
of carbonate of potash to which previously an equal volume
of water has been added. The preparations remain here
for from five to ten minutes, are then washed in water,
alcohol, clove-oil, and finally mounted in Canada-balsam
solution or Dammar varnish.

5. Lustgarten’s 1 method, used for the demonstration of
the syphilis-bacilli. — The sections are stained for from twelve
to twenty-four hours at ordinary temperature, and then for
an additional two hours at 40° C. in aniline water gentian-
violet ; they are then washed for a few minutes in absolute
alcohol, and then transferred to a 1 '5 per cent, solution of
permanganate of potash for ten seconds, then for the same
period into a watery solution of pure sulphurous acid ; wash
in distilled water, repeat the above process of placing the
sections first into the permanganate of potash solution, then
into the sulphurous acid water till they become apparently

1 Lustgarten, Med. Jahrbiichev der K.K. Gcs. d. Aerz/c, Vienna, 1 SS5-

i8 MICRO-ORGANISMS AND DISEASE [chap.

quite colourless. Only the syphilis-bacilli, tubercle-bacilli,
and leprosy-bacilli, are able to retain the dye ; other
bacteria lose it by being subjected to the permanganate.

De Giacomi 1 has improved this method of decolourising
by oxidation. Cover-glass specimens made of syphilis material
are stained with warm fuchsin for a few minutes, are then
washed in water to which a few drops of solution of iron
perchloride have been added, then placed into concentrated
solution of iron perchloride till the preparations have lost
all colour ; they are then stained for contrast in vesuvin or
Bismarck-brown.

A. Gottstein 2 places sections of syphilis material for
twenty-four hours in fuchsin or aniline water genetian-violet ;
wash with distilled water, then place them for a few seconds
into a pure or dilute solution of liquor ferri, then wash in
alcohol, clarify in cloveoil, mount in Canada-balsam.

It may not be unnecessary to point out, that if sections are kept for
many hours in the staining fluid, there may be found in them micro-
organisms (particularly bacilli) which have been accidentally introduced
into them by the solutions of aniline dye. Many of these, particularly
when used alkaline, contain organisms, and if the sections are kept
in them for many hours, notably in warm weather, bacteria will be
found to have not only invaded the tissue but to have multiplied
therein.

In examining fresh or hardened tissues for micro-organisms
it is necessary to make thin sections, which can be easily
done with the aid of any of the microtomes in common use,
amongst which Williams’s microtome for ice or ether freezing,
Cathcart’s for simple ether freezing, Minot’s microtome and
the Cambridge rocker with ordinary razor for cutting riband
sections from paraffin-embedded hardened materials, are
easiest to manipulate. As a matter of fact we now use

1 De Giacomi, Schrweizer Correspondezblatt , xv. 12.

■ A. Gottstein, Fortschritte d. Mcdizin, Berlin, 1SS5, No. 16, p. 545.

MICROSCOPIC EXAMINATION

either Williams’s or Cathcart’s microtome, and above all for
hardened materials, the Cambridge rocker. By this latter
the most exquisite and uniformly thin sections in a riband
are obtained.

As regards hardened material, it is necessary to remember
that the hardening must be carried out properly, small bits
—about a half to one cubic inch — of tissue being placed in
alcohol, or better, in Muller’s fluid, and kept there ; in the
first instance, for two to five days ; in the second for from
one to three weeks or more. Then small bits are cut out,
of which it is desired to make sections. Those hardened
in spirit must be soaked well in water to enable the material
to freeze, then superficially dried with blotting-paper, and
then used for cutting sections with the microtome. Those
hardened in Muller’s fluid are at once superficially dried
with blotting-paper and cut. When making sections with
Williams’s freezing microtome it is necessary to soak the
material first in gum mucilage and then to freeze and to cut.
Fresh tissues are at once cut with the freezing microtome,
the sections placed in a o-6 per cent, saline solution, floated
out and well spread out, and then stained by transferring
them in this condition' — well spread out, into a watch-
glass containing the dye. The sections of hardened tissues
are floated out in water, well spread out, and then trans-
ferred to the dye or dyes as the case may be.

It is necessary to prevent too much shrinking of the
sections, especially those of fresh tissues ; for this reason it
is advisable to float the sections in the saline solution or
water, as the case may be, on a broad lifter or spatula, to
spread them well out upon it, and to transfer them carefully
into the dye, then into the dish with water used for washing
off the excess of the dye, to transfer them, well spread out
on the lifter, to alcohol, then after several minutes to oil of

c 2

MICRO-ORGANISMS AND DISEASE [chap.

cloves, and finally on to a glass slide, on which they are
mounted in the usual manner with Canada-balsam solution,
the excess of clove-oil being previously drained off.

It is advisable, although not absolutely essential, to keep
the sections in a well-spread-out condition for a few seconds
in alcohol before placing them into the dye.

For the preparation of mounted stained specimens by the
rocking microtome the following method is used in my
laboratory :

Small pieces of tissue are put in Muller’s fluid 1 and
changed on the second and third day, are then allowed to
stand for seven days, then put in two-thirds methylated
spirit and one-third water, change after twenty-four hours to
absolute alcohol, and allow to properly harden.

Embedding in Paraffin.

Selected piece of tissue is then treated as follows :

1. Absolute alcohol, twenty-four hours.

2. Absolute alcohol and cedar wood oil, equal parts,
twelve hours.

3. Cedar wood oil, twelve hours.

4. Paraffin liquid, 50 to 520 C., bath No. 1, twelve hours.

5. Paraffin liquid, 50 to 520 C., bath No. 2, twelve hours.

6. Pour paraffin in a small box of brass or lead and place
tissue in it, when set take the paraffin block out of the box,
trim and fix on rocking microtome. Cut number of sec-
tions and float into warm water at 30° C. Fix sections on
cover-glasses, and allow to dry in incubator at 370 C.

7. Xylol, five minutes.

1 Glanders tissues are best placed.at once in absolute alcohol.

MICROSCOPIC EXAMINATION

8. Absolute alcohol, five minutes.

9. Methylated spirit, five minutes.

10. Stain specimens in selected dye.

11. Wash in water.

12. Wash in methylated spirit.

13. Absolute alcohol.

14. Oil of cloves.

15. Xylol.

16. Mount in xylol Canada-balsam.

I Demonstration of flagella. — Loffler ( Central l. f. Baht, und

Parasitenkunde, Bd. VI. Nos. 8, 9) has shown by a new
method that flagella of bacteria can be stained. Although
motile bacteria have been known or supposed to owe this
motility to the presence of flagella, these have in most cases
eluded demonstration, till Loffler by using a mordant of
tannin and ferrosulphate solution, previous to the stain,
showed with extreme clearness the actual presence of flagella
even in the weakest motile bacteria. Moreover, he showed
the quite unexpected and remarkable fact that while in
some only one flagellum at one end is present there are
others in which there are several such flagella, and even the
body of the bacteria may be completely invested in flagella
— e.g. in the case of the typhoid bacilli. Loffler’s beautiful
photographs created deservedly great sensation amongst
bacteriologists, and a host of workers have devoted at once
careful attention to the subject, hence resulted several useful
modifications of the composition, reaction, and duration of
action of the mordant. We shall limit ourselves to the
single statement that by the flagella staining alone a
diagnostic differentiation between bacillus coli and bacillus
typhosus has become possible, the former possessing two to
eight, at any rate a limited number of flagella, whereas the
latter possesses quite a mass of wavy spirilla-like flagella ex-

MICRO-ORGANISMS AND DISEASE [chap.

tending over the whole body at each end and at right angles
from the cylindrical body.

The method of flagella staining which both in Dr. Kan-
thack’s laboratory, and owing to his initiative also in my
laboratory, is used with facility and with unequivocal success,
is that described by van Ermengem, which I copy from Dr.
Kanthack’s manual. It ought to be stated that I have seen
specimens, prepared by beginners, of culture of bacillus coli
and of typhoid bacilli, which showed the flagella in a manner
and quantity that can without exaggeration be described as
striking. But in all these cases it must be added that they
were prepared without deviating in any essential point from
van Ermengem’s prescription. The flagella appear not as
prolongations of the protoplasm of the bacteria as generally
supposed, but seem to be part or outgrowths of the sheath
itself.

The method is this 1 : —

Staining of Flagella (Van Ermengem).

Prepare the following solutions :

(a) Osmic acid (2 per cent, solution) 1 part.

Tannin (10 to 25 per cent, solution) 2 parts.

To each 100 cc. of the tannin solution add four or five drops of
glacial acetic acid.

(/3) Nitrate of silver ('25 to '5 per cent solution).

(7) Gallic acid 5 grammes.

Tannin 3 grammes.

Fused acetate of soda 10 grammes.

Distilled water 350 cc.

Boil the cover-slips to be used in the following solution : —

Potassium bichromate 60 grammes.

Concentrated sulphuric acid 60 grammes.

Water . . . 1000 cc.

1 From Practical Pathology, Kanthack and Drysdale, pp. 3S and 39.

i] MICROSCOPIC EXAMINATION 23

Then wash them repeatedly in water. Keep them in absolute
alcohol and before use allow them to dry, without wiping, by placing
them in a vertical position, protected from dust.

Bacillus of Typhoid Fever and Vibrio Cholera Asiatic*:.

Carefully suspend one or two loops of an Agar-Agar culture (ten to
eighteen hours old) in a watch-glassful of distilled water.

(a) With a single loopful of this “suspension” prepare a cover-glass
film and allow it to dry in the air.

W Fix it by passing it three times through the flame, holding the
specimen in the fingers, so as to avoid over heating.

(r) Pour a few drops of solution (a) on the film and allow them to
act for half an hour.1

(rf) Wash very carefully in a large excess of distilled water, and
then in alcohol.

(e) Now keep it for three to five seconds in solution (0).

(/) Without washing, pass quickly through solution (7).

(g) Wash again in a fresh quantity of solution (0), moving the
specimen about gently and withdrawing it when the solution begins to
turn black.

(h) Wash it thoroughly in several changes of distilled water.

( i ) Dry it carefully between blotting-paper.

Mount it first in water and examine it with TV in. oil immersion, and
if the specimen be satisfactory, mount it permanently in xylol balsam.

If the flagella are not sufficiently stained, float the cover-slip off the
slide and begin again at (/).

Care must be taken to change the nitrate of silver solution as soon as
any precipitation shows itself.

This is an easy and very trustworthy method.

Mr Mervyn Gordon has succeeded in producing a uniform and
perfect dark staining of the flagella of the typhoid bacilli — far more
exquisite than I have seen it produced previously, by introducing in
the above method the following alterations :

(d) The cover-glass is left for five minutes in alcohol.

( e ) It is kept for two minutes in solution (0).

(fi) It is drained on blotting-paper, and left for one and a hatfi to two
minutes in solution (7). [The last half minute determines the degree of
staining.]

1 At a temperature of 605 C. five minutes is sufficient.

CHAPTER II

PREPARATION OF CULTURE MATERIAL

Artificial cultivations of micro-organisms in suitable
nourishing media in the incubator (Figs, i and 2) at tem-

Fig. 1.— Incubator, with Pace’s Regulator.

A. Page's Regulator. — This consists of a tube filled with mercury, and immersed
in the water surrounding the chamber of the incubator. In the upper part of the
tube, above the mercurial column, is a fine open glass tube, having near the lower

CH. n] PREPARATION OF CULTURE MATERIAL 25

end a fine hole. When the temperature of the water rises, the mercurial column
rises, and at a certain temperature rises above the lower open end of the small inner
glass tube just mentioned. If this point is reached, then the burner at C receives
only the amount of gas that passes through the fine lateral hole of that inner glass
tube. If the temperature of the water falls, the mercury falls, and the lower end of
the inner glass tube becomes again free, and now the burner at C receives a much
greater supply of gas. If so, the temperature of the water again rises, the mercury
rises, obstructs the lower end of the inner glass tube, the supply of gas is reduced to
what can pass through the fine lateral hole, and consequently the temperature again
falls, and so on. To adjust the regulator it is necessary when the thermometer in-
dicates the required degree of temperature to push the outer large glass tube, and
with it the inner tube, of the regulator so far down that the top of the mercurial
column just obstructs the free end of the inner glass. The temperature then regulates
itself for the reasons stated previously These regulators are sufficient for all
practical purposes when it is not a question of small differences in temperature, since
they are tolerably constant within one or two centigrades. The trouble one ex-
periences in the working of these and other similar regulators arises from the incon-
stancy of the main gas supply, this, as is well known, varying within wide limits.
The stopcock, £, obviates this to a limited extent ; when this is put at an angle of
45° only a limited amount of gas passes from the main supply tube to the regulator,
and therefore the variations in pressure of the gas are not felt to their full extent.
A Sugg’s regulator interposed between E and the main supply tap is very useful.

B. Thermometer to indicate the temperature in the chamber.

C. Gas burner.

D. Chamber of incubator. The front and back of the incubator is either a movable
tin plate or glass covered with black paper.

E. Stopcock to regulate, when required, the supply of gas.

F. Main supply. — The upper, lower, right and left walls of the incubator are
made of a double layer of tin ; between the two is water. The front and back of
the chamber are closed by a movable plate.

An excellent incubator for constant temperature is made by the Cambridge
Scientific Instrument Company. It has a double gas supply : one small permanent
flame, and a second one subject to the regulator.

peratures varying between 20° and 38° C., are necessary in
order to study more accurately the life-history of the septic
as well as the pathogenic organisms. Moreover, large
numbers of them become available in a short time, and
their relation to disease can be tested more conveniently.
For if it should be found that, having carried on outside the
animal body successive cultivations of a particular organism,
the re-introduction of this cultivated organism into the
animal body is again productive of the same disordei as
before, then the conclusion becomes inevitable that this
organism is intimately related to the causation of the
disease. It must be conceded that after several successive
cultivations in fluids any hypothetical substance supposed
to be the maleries tnorbi, and introduced at first from the
blood or tissues, being in a very diluted condition in the

MICRO-ORGANISMS AND DISEASE [chap.

first cultivation, would after several cultivations be practically
lost. But if this last cultivation should be found to act in
the same manner pathogenically — i.e. if a small quantity of
it, charged with the new brood of the organism, nevertheless

Fig. 2. — Heakson’s Incubator.

An excellent incubator for higher temperatures, as it possesses a very sensitive

regulator.

possesses full pathogenic power, then it is logical to say
that this pathogenic property rests with the organism. For
this and other reasons it is of essential importance to be

Ii] PREPARATION OF CULTURE MATERIAL 27

able to carry on successive cultivations of one and the
same organism without any accidental contamination or
admixture — i.e. it is necessary to carry on ptire cultivations.

ARTIFICIAL CULTIVATION MEDIA.

A. — Fluids.

As fluid nourishing material the following are used with
preference : —

1. Broth made from Meat— pork, beef, rabbit, chicken. — The
connective tissue and fat are first cut out from the fresh meat
— in the case of rabbit or chicken the whole animal without
head or viscera is used — and then placed in water and boiled.
Generally for each pound half an hour’s good boiling is
allowed. With regard to the quantity of water, each pound
of meat ought to yield ultimately at least one pint of broth.
When boiled, the broth is allowed to stand, the fat is skimmed
off, and the broth well neutralised, or even made faintly
alkaline by adding liquor potassse, or, better still, carbonate
of sodium.

The fresher the meat the less acid (sarcolactic acid) is in
the broth before neutralisation. The broth is then filtered
through a filter 1 into flasks previously sterilised (see below).
As a rule beef broth is clear, but if not it is filtered again.
If not clear then, it is allowed to stand for several hours.
A fine sediment is found at the bottom of the vessel, and
from this the clear supernatant fluid is decanted into a
sterilised vessel. The broth, if not clear after the first
filtering, can be cleared by boiling it with the broken shell

1 Unless otherwise stated all filtration is carried out by means of
folded Swedish filter paper.

MICRO-ORGANISMS AND DISEASE [chap.

and white of egg. The now clear fluid is filtered again.
The flasks which receive the broth are well plugged with
sterilised cotton-wool (see below). In this state the flask is
placed over a Bunsen burner on a wire netting, and boiled
for half an hour or more ; during the boiling the cotton-
wool plug is lifted out for half its length. The flask ought
not to contain more broth than about one-half or one-third
of its volume, to prevent the broth from rising too much
and wetting the plug. When turning off the flame the plug

is pushed down so as fully to plug the neck and mouth of
the flask ; a beaker with sterile cotton-wool cap is placed
over the mouth of the flask, and this is allowed to stand for
one night. Next day the boiling is repeated for half an
hour or more in the same manner as before. If the meat
has been fresh and the vessels and cotton-wool have been
sterile, twice boiling is found sufficient to destroy every
impurity. But to make sure, the broth is placed in the
incubator and kept there for twenty-four hours at a tern-

II] PREPARATION OF CULTURE MATERIAL

perature of 320 to 38° C., and then boiled on the next day
for half an hour in the usual way. The supposition is
made, that if by any chance after twice boiling the broth it
should contain unchanged spores of bacilli — the only
organisms that will resist boiling, although they do not resist
boiling for more than several minutes — the spores would
germinate into bacilli when kept for twenty-four hours in
the incubator at 320 to 38°, and these would then be killed
by the third boiling. As a matter of fact I have not as a
rule found any contaminating germs survive the second
boiling. It is of course to be borne in mind that during
the first as well as second and subsequent boiling the cotton-
wool plug is not removed from the mouth of the flask, but
is only raised out half its length from the neck. The
cotton wool and the cotton-wool cap and beaker are re-
placed immediately or simultaneously with the turning off
of the burner.

2. Peptone and Salt Solution. — Beef peptone (Savory and
Moore’s) is dissolved in distilled water, over a burner, to
the amount of 1 per cent. ; to the solution is added common
salt to the amount of o-5 per cent. ; so that every 100 ccm.
of the fluid contains one gramme of peptone and £ gramme
of salt. When dissolved it is made faintly alkaline, and
then filtered (the vessels being of course also in this, as in
all other cases, sterilised by heat).

A 10 per cent, peptone, 5 per cent, salt solution, repre-
sents a useful stock, because it can be kept as a smaller bulk,
and used by dilution if large quantities of 1 percent, peptone
be required.

For general use where broth is required as culture fluid,
the above stock-broth, plus 1 per cent, peptone and o-5 per
cent, salt, represents an excellent fluid, the nutrient broth ; it
is of course made faintly alkaline after the peptone is added

3° MICRO-ORGANISMS AND DISEASE [ciIAP.

and dissolved, then boiled, and either kept in the flask as
stock or decanted into sterile test-tubes to the amount of
5-10 cc., plugged with sterile cotton-wool. These are
steamed (see below) on two successive days, each time for
twenty minutes.

This nutrient broth, with 5-8 per cent, pure glycerin
represents glycerin broth.

3. Buchner's Fluid. — 10 parts of Liebig’s extract, and 8
parts of peptone, in 1,000 parts of water.

4. Blydrocele Fluid (Koch). — A new or well sterilised
trocar and cannula are used for the tapping ; to the cannula
is fixed an india-rubber tube that has been soaking in strong
carbolic acid solution for forty-eight hours. The distal end
of the tube is introduced carefully and rapidly into the neck
of a sterilised flask plugged with sterile cotton-wool, and the
fluid thus allowed to flow into the flask to about two-thirds
of its volume. This fluid is then decanted into sterile test-
tubes (plugged with sterile cotton-wool), each tube receiving
about 5 to 10 ccm. The tubes are then exposed in the incu-
bator to a temperature of from 550 to 60° C. for two to
three hours on two or three consecutive days.

Ascites fluid is obtained in the same way.

5. Blood Serum (Koch). — A glass cannula and india-
rubber tubing are soaked for forty-eight hours in strong
carbolic acid ; the cannula is tied into the carotid artery of
a healthy horse, and the arterial blood, after opening the
clip at the proximal end of the artery, is allowed to flow
into sterile flasks, or cylinders with stoppers. After letting
the blood stand for 24 to 48 hours in a refrigerator or in an
ice-box, the serum is taken off by means of large sterile
glass pipettes and introduced into sterile test-tubes, each
receiving about 5 to 10 ccm. The test-tubes, plugged with
sterile cotton-wool, are then exposed in the incubator to a

n] PREPARATION OF CULTURE MATERIAL

temperature of 58° to 62° C. in the same manner and for
the same time as the hydrocele fluid was.

Blood of ox or sheep obtained in the slaughter-house is
the blood from which generally “ serum ” is obtained ; it is
received into sterile glass vessels, and treated in the same
way as just described.

6. Urine is neutralised and sterilised by boiling for 20 to
30 minutes like broth.

7. Milk (pure or better separated) is sterilised by gentle
and careful steaming for 20 to 30 minutes on three succes-
sive days.

8. Whey is now also used as such, or better as an admix-
ture to gelatine or agar ; in either case it can be easily
sterilised by steaming.

Of less common use are : —

9. Pasteur's Fluid. — In 100 parts of distilled water are
dissolved 10 parts of pure cane-sugar, 1 part of ammonium
tartrate, and the ash of 1 part of yeast.

10. Cohn's Fluid. — 100 ccm. of distilled water, x gramme
of ammonium tartrate, no sugar, and instead of the ash of
yeast are substituted (A. Mayer) 0-5 gramme of potassium
phosphate, or o-5 gramme of crystallised magnesium sul-
phate, o‘o5 gramme of (tribasic) calcium phosphate. These
two fluids are sterilised in the same manner as the broth
and peptone solutions. Pathogenic organisms do not thrive
in either of these two fluids.

B. — Solids.

The solid media have the great advantage over the fluids
that in the former artificial cultures can be carried out more
easily ; as, owing to the resistance the solid basis offers to

32 MICRO-ORGANISMS AND DISEASE [chap.

the growth of the organisms, they remain more limited
to the spot or spots on which they are sown, and therefore
can be watched more easily; besides, an accidental con.
tamination — i.e. a growth appearing at a spot at which no
sowing was made, can be recognised at once. These advan-
tages are perhaps of the greatest use when it is intended to
grow the organisms on a surface exposed to the influence
of air — of course protected from contamination with other
organisms.

These advantages of solid media have been very minutely
pointed out by Koch in his researches on pathogenic
bacteria.1

As solid media are used : —

1. Slices of Boiled Potato or Boiled White of. Egg or Paste
(Fokker, Schroter, Cohn, Wernich). — A boiled potato or a
boiled unshelled egg is cut in half with sterile scalpel, and
the cut surface is inoculated. Immediately after, it is placed
on a clean glass plate and covered with a bell-glass, the
edges of the latter being fixed on the former by vaseline or
grease, the chamber is kept moist by a piece of wet blotting-
paper being placed inside the bell-glass, or a glass capsule
covered with another, both sterile, receive the potato. The
progress of growth of a particular organism or of different
organisms sown at a particular spot or line on the surface
of these substances can be easily watched with the unaided
eye.

Blocks of potato cut with a sterile cork-borer from a clean-
cut potato are placed into test-tubes over a cushion of sterile
cotton-wool, the test-tubes are then plugged and steamed on
two successive occasions for 20 minutes each time.

2. Gelatine (Brefeld, Grawitz, Koch). — This is used ad-
vantageously as a mixture with broth, peptone, beef-extract,

1 Mitthcilungen d. k. Gesundheitsamtes, i. iS8t.

li] PREPARATION OF CULTURE MATERIAL 33

blood serum, or hydrocele fluid. Koch, who introduced
this mixture, used it for the cultivation of bacteria on solids,
to be exposed to the air; the proportion of gelatine in the
mixture was 2 to 3 per cent. But this mixture, although
solid at ordinary temperature, does not keep solid in the
incubator, not even at 20° C. I have found that at least
7 ‘5 per cent, of gelatine must be contained in the mixture
to keep it solid at 20° to 250 C. Above this last tempera-
ture not even 1 1 per cent, gelatine will keep solid.

Nutrient Gelatine , most useful for the growth of all kinds
of bacteria, is prepared in this way : —

One pound of lean beef is cut up, to it is added one pint
of water, and is kept boiling in the digester or any other
vessel for from half to three-quarters of an hour. After
having been strained through fine calico it is filtered through
paper into a beaker ; bring up by adding water to 600 ccm. ;
add to this 60 grams of the finest gold label gelatine cut up
in small pieces, 6 grams of peptone, and 6 grains of common
salt. Dissolve on waterbath, but do not let the water boil ;
neutralise with carbonate of soda or, better, liquor potassae
till faintly alkaline ; steam for half an hour, filter by hot filter
(see Fig. 6) into a sterile flask plugged with sterile cotton-
wool, and bring it up to boiling point, at which it is kept for
a few minutes. This can be kept as stock gelatine, or can
be decanted at once into sterile test-tubes plugged with
sterile cotton-wool. These are steamed on two successive
days for 20 minutes each time. Keeps solid up to about
25° C.

Prepared in this manner the nutrient gelatine passes easily
and comparatively rapidly through filter paper on hot filter.

The same 10 per cent, nutrient gelatine can be of course
obtained if broth is already made — e.g. broth in a stock
flask, by adding the above-named quantities of gelatine,

MICRO-ORGANISMS AND DISEASE [chap.

peptone, and salt to 600 can. of the broth ; further process
is as above.

It is this gelatine which I generally use as “nutrient
gelatine,” not Koch’s meat infusion gelatine, for I find that
beef decoction gelatine as prepared above is conspicuously
a better nutritive medium than the meat infusion gelatine
prepared after Koch’s method.

3. If it is necessary to expose the cultivation to higher
temperatures than 25° C., the nutrient gelatine cannot be
used as a solid medium. Solidified blood serum, or solidi-
fied hydrocele fluid, or solidified ascites fluid, or solid
Agar-Agar mixture (Koch) must then be employed.

The first — i.e. the serum of blood, the hydrocele fluid,
and ascites fluid — can be made solid by heating them in
tubes (see page 51) gradually up to 68°, 70° or 710 C.
When this temperature is reached the material soon turns
solid, losing slightly its limpidity, but when solidified with
slanting surface (see serum inspissator) is sufficiently trans-
parent for all practical purposes. By heating it rapidly, or
heating it above 720, it becomes solid, granular, and opaque.
Of course, once thus made solid it cannot be liquefied
again, and therefore must be already contained in the
vessels (test-tubes and small flasks) in which the growth of
organisms is to be carried on.

4. Lofflers serum , very useful for cultivation of the
diphtheria bacillus on which this microbe grows with pre-
dilection, is composed of two parts of blood serum and
one part of faintly alkaline beef broth ; the fluid contained
in sterile plugged test-tubes is sterilised and solidified with
slanting surface in serum inspissator just like ordinary blood
serum.

Serum solidified with slanting surface always shows on
cooling a small amount of “ condensation water ; but this

Ii] PREPARATION OF CULTURE MATERIAL

can be easily driven off by placing the test-tubes in a
slanting position in the hot incubator (370) and leaving
them here for a few days. Owing to the large surface of
evaporation the condensation water is soon got rid of.

5. Kanthack's serum is a mixture of solidified ascites
fluid and Agar, which far surpasses all other media for the
isolation of the diphtheria bacilli, even when in a given
material these latter are almost swamped by other microbes :
the cultivation of this material rubbed over the slanting
surface of this serum (solidified) will at twenty-four to forty-
eight hours’ incubation show and pick out in a remarkable
manner the colonies of the diphtheria bacilli in almost pure
culture. It is prepared thus : Ascites fluid is received by
sterile trocar and tubing into a sterile flask plugged with
sterile cotton-wool. For each 100 cc. of ascitic fluid to be
treated, take 2 grams of Agar-Agar and treat as follows :
2 to 3 cc. glacial acetic acid in 500 cc. of distilled water;
put Agar in this solution and allow to soak for thirty
minutes, wash in several lots of tap-water and finish with
distilled water, thoroughly drain. For each 100 cc. of
ascitic fluid add 2 cc. of a 10 per cent, solution of caustic
potash, and very thoroughly mix. Add this to the Agar,
now add 6 per cent, of glycerine, and place in steamer and
steam at ioo° C. for one and a half hours; filter through
Chardin’s filter paper, decant into tubes and steam at
ioo° C. on three successive days for thirty minutes.

6. Nutrient Agar-Agar. — “Nutrient Agar” is Japan
isinglass. This was first used for preparation of solid
culture medium by Koch. The best and quickest mode of
preparing nutrient Agar is the following modification of
Tischutkin’s method described in Schenk’s Elements of
Bacteriology, English translation, p. 44. It is the nutrient
Agar which I now use, being easily and quickly pre-

Li 2

36 MICRO-ORGANISMS AND DISEASE [chap.

pared and of considerable transparency. The method is
this : —

Twenty grams of Agar s/rips are placed in 500 cc. of
distilled water in a flask to which are added 2 cc. glacial
acetic acid ; in this the Agar is allowed to soak and swell
up for fifteen to twenty minutes ; then, after pouring off,
it is well washed in tap-water, and finally distilled water;
the fluid is well drained off. After this process the Agar
is easily soluble ; to it (in the flask) are now added of
(a) the ordinary beef broth (above stock broth) 600 cc. ;
this is boiled for thirty minutes, in which time all the Agar
has dissolved. To this solution is added the following
mixture (b) consisting of 400 cc. of broth, 10 grams of
solid peptone and 10 grams of solid salt ; the whole is now
made slightly alkaline with liquor potassae and clarified
with white of egg. After mixing well up the flask is put
into the autoclave and kept therein at 120° C. for fifteen
minutes; it is then filtered through (folded) Chardin filter
on the “hot filter.” This Agar broth mixture is beautifully
clear and limpid and filters rapidly — one liter per hour, a
great advantage over other Agar preparations. This
nutrient Agar contains then 2 per cent. Agar, 1 per cent,
peptone, 1 per cent, salt all dissolved in beef broth. It is
decanted into sterile test tubes, steamed on two successive
days each time for twenty minutes; when allowed to cool
becomes solid. It can be solidified with slanting surface
by being placed on the special tray. When quite solid
with slanting surface a small amount of “ condensation
water ” accumulates at the bottom of the tube.

7. Grape Sugar Gelatine and Grape Sugar Agar are the
media best suited as solid media for the growth of anaerobic
microbes. The first is the nutritive gelatine as above
described, sub 2, but containing 2 per cent, of grape sugar.

ii] PREPARATION OF CULTURE MATERIAL 37

In order to avoid the brown colour, which the nutritive
gelatine assumes when the grape sugar is added, and after
heating it, it is necessary to prepare a watery solution of the
required amount of grape sugar separately, to add this to
the solution of gelatine, peptone, and salt in beef broth,
then to make the whole alkaline and steam. The grape
sugar Agar differs from nutrient Agar described sub 6, in
containing 2 per cent, of grape sugar added in substance to
the Agar mixture before putting into the autoclave.

8. Glycerin Agar, first used by Roux and Nocard for
the cultivation of the tubercle bacillus. It is, however, not
only useful for this, but also for other microbes. It is the
nutrient Agar mixture, sub 6, to which 5 to 8 per cent,
pure glycerin has been added before putting the mixture
into the autoclave.

CHAPTER III

VESSELS AND INSTRUMENTS USED IN CULTIVATIONS

All instruments, vessels (flasks, test-tubes, beakers, cotton-
wool, filters, calico) to be used are first thoroughly sterilised
by heating. In the case of flasks and test-tubes, this can be
done by exposing them thoroughly in all parts to the open
flame of a large Fletcher’s burner ; while thoroughly heated
the mouth is plugged with a good long plug (i to 2 inches)
of sterile cotton wool, this being pushed in by means of
sterile forceps. The plug in all cases must not be loose,
but also not too firm — an error in the latter direction being
of course preferable to one in the former. Or the flasks and
test-tubes, instruments, cotton-wool, &c., are placed in an
air-chamber (see Fig. 4) heated by a large Fletcher's burner
for several hours, up to between 130° and 150° C. In the
case of small flasks, test-tubes, and cotton-wool this process
is of course much more convenient, since a large number
can be heated simultaneously. Beakers and glass filters to
be used merely for a temporary operation are placed over a
wire net on a tripod and heated by the flame of a Bunsen’s
burner. In the case of test-tubes which are to receive
cultivation-fluids, I generally expose them, after having been
cleaned with strong acid, washed out with water and dried

CH. in] VESSELS, ETC., USED IN CULTIVATIONS 39

in the air-chamber for several hours (three to six) to a tem-
perature of from 130° to 150° C. : while hot they are taken
out seriatim , plugged with the sterile cotton-wool, and re-
placed in the air-chamber, and heated again for several
hours. All this, and other operations to be described below,

Fig. 4. — Hot Air-chamber for Sterilising Test-tubes, Cotton-wool, &c.

An iron chamber with double wall, the inner chamber having separate folding
doors. In the inner chamber are placed the test-tubes, glasses, &c., and the
cotton-wool, the latter in a loose condition. Both sets of doors are closed, and
the apparatus heated by a large Fletcher’s burner. A thermometer passing from
the inner chamber through the upper wall indicates the temperature of the chamber.
The hot-air apparatus can, according to the requirements of the laboratory, be
constructed of larger size than the one here depicted. I use one that is made four or
five times this size and is divided into several compartments.

may appear to some rather tedious and unnecessarily com-
plicated, but it cannot be too strongly insisted on that in
these matters one cannot be too scrupulous. A slight re-
laxation may, and occasionally is, followed by disastrous
consequences in the shape of accidental contamination, and
consequent loss of materials prepared at the cost of much

MICRO-ORGANISMS AND DISEASE [CHAP.

labour and time. Long experience in these matters has
taught me that, although in some instances less scrupulous
care has not been followed by bad results, still I have seen
occasionally unpleasant failures owing to slight laxity in
these matters.

Several weeks’ work may be annihilated by a single omis-
sion. Sometimes one is perhaps in a slight hurry, and does
not think the want of an additional heating of the test-tube
or cotton-wool or an additional boiling of the fluid will be
followed by any bad consequences. But, alas, nature does
not take into account our convenience, and failure is our
reward. If in any kind of experiments “ overdoing” is an
error in the right direction, it is in these very experiments in
the cultivation of micro-organisms.

The cotton-wool used for plugging flasks • and test-tubes is
prepared by pulling up loosely a quantity of good cotton-
wool and exposing it in a loose state in the air-chamber to a
temperature of 130° to 150° C .for several hours on tzoo suc-
cessive days. The cotton-wool ought to be just slightly
brownish — i.e. just faintly singed. Too much singeing makes
it brittle, and it is then difficult to make of it a satisfactory
plug. The plug used should not be too firm and not too
loose : in the former case it is not easy to lift it up quickly,
and in the latter it does not close sufficiently well. Cotton-
wool that has been kept in the air-chamber for an hour or
two is not absolutely sterile ; nor is cotton-wool that has been
kept in a compressed state in the air-chamber for several
days. The central portions remain under these conditions
quite white and are not sterile. No cotton-wool that is
not just brown — i.e. just faintly singed — is safe from risk of
impurity. No cotton-wool steeped in absolute alcohol,
strong carbolic acid, or any other disinfecting fluid, for ever
so many days or weeks, can be absolutely relied on.

Ill] VESSELS, ETC., USED IN CULTIVATIONS

As stated above, a plug of sterile cotton-wool tolerably
firm, of about one to two inches, is used for the plugging of
the flasks and test-tubes. An assertion such as that made
by Dr. Williams at the British Association (Bio-
logical Section, September 1883), that cotton-
wool plugs are not reliable, because they do
not protect the fluids in the vessels plugged with
them from accidental air-contamination, is to
be accepted only as applying to very loose
plugs and to cotton-wool not properly sterilised.

To good firm plugs of sterile cotton-wool it
evidently cannot apply, since all the results of
all workers in this field (Pasteur, Sanderson,

Cohn, Koch, Klebs, Buchner, and many others)
are against it.

Instruments , such as the points of needles,
and forceps, used in the processes of cultiva-
tion, lifting up cotton-wool plugs, making cotton-
wool plugs, inoculations, &c., must be heated
in the open flame of a Bunsen burner, if they
are to be absolutely relied on for cleanliness.

Scissors and knives used for cutting tissues
which are intended for inoculation, ought to be
likewise scrupulously clean. One ought to keep
a special set of instruments in a metal box, the
whole capable of being sterilised in the hot
air-chamber.

Syringes used for cutaneous, subcutaneous,
or other inoculations, ought to be capable ol
being sterilised by heat. The ordinary Pravaz
syringe of vulcanite not being capable of undergoing this
process, Koch has devised a glass syringe similar to the
Pravaz syringe. I do not use any syringe for inoculation

H .
2 t/i
- td

? *■
< o

w H
id '

42 MICRO-ORGANISMS AND DISEASE [chap.

of small quantities, but prefer using each time a fresh
capillary glass pipette made just before the inoculation.
Into this pipette I draw the small quantity to be used
for inoculation, and having made a very small puncture

Fig. 6.— Hot-water

Filter for Filtering Nutritive Gelatine or
Agar-Agar Mixture.

through the skin, the pointed end of the pipethej pushed
through it into the subcutaneous tissue or * ‘

inch or o!re inch and then the fluid is blown out rn.o he
tissue. In this way I am always absolutely safe horn « >
contamination with a previously used virus, wuci m.g

Hi] VESSELS, ETC., USED IN CULTIVATIONS 43

possibly adhere to one or other part of a syringe not
thoroughly sterilised.

The fine point of capillary pipettes (Fig. 5), used for in-

Kic. 7.— Steamer for Sterilising Culture Material contained in

Test-tubes.

1 Wire-net lo hold the test-tubes; 2. Tin vessel; 3. Wire diaphragm to hold 1;
underneath it is water ; 4. Lid ; 5. Gas-burner.

oculation of animals, or for drawing out a drop of fluid of a
cultivation in a flask or test-tube, or for inoculating material
contained in a test-tube or flask, are thus made : while one

MICRO-ORGANISMS AND DISEASE [CH. Ill

hand holds the bulb of the pipette, the other holds one end,
and putting at some distance from this end the tube into an
ordinary flame and quickly drawing it out, a point of extreme
fineness can be made. The same is done with the other end.
Such a pipette can be considered as practically closed at
both ends.

Amongst the apparatus useful in bacteriological work the
autoclave 1 deserves a place : it is a cylindrical metal chamber
heated by gas flame, and containing a small amount of
water ; the lid can be hermetically screwed down ; the tem-
perature of the steam developed inside is under pressure
easily raised beyond the boiling point of water, as when any
fluid culture medium (e.g. nutrient Agar as described above)
is to be heated to say no-1150 C. ; one atmosphere pres-
sure corresponds to this temperature of 110-115° C.

Platinum needles, platinum loops, or platinum lancets two
to three inches long are fastened (melted) either in glass rod
handles or in wooden handles by means of a long metal
cylinder; in the latter case the sterilising by heat of the near
end of the needle can be just as easily carried out as of the
glass rod, though a cracking and breaking of this latter is
avoided. Copper ovens of various sizes are used for the
heating (melting) of paraffin, &c., where a constant definite
temperature is to be maintained.

The serum inspissator for the solidification of serum which
is most useful is the one of Hueppe’s design as shown in
Fig. 10.

Trays of wood or tin are useful for obtaining gelatine, or
Agar tubes with large slanting surface during cooling (setting)
are shown in Fig. 9.

1 Sold by Wiesnegg in Palis.

CHAPTER IV

PREPARATION OF CULTURE-MEDIA FOR INOCULATION

We have on a former page described the methods to obtain
sterile stock of nourishing media suitable for artificial cul-
tivations. Those media which are to be used in a solid
state must, before solidification, be contained in test-tubes
and small flasks, sterilised and solidified in the manner
above described, so as to be ready for establishing cultures —
i.e. for inoculation. The Agar-Agar mixture however can,
like broth, peptone mixture, beef extract solution, and
gelatine mixtures, be kept as stock in large flasks. When
thus sterile these latter can be decanted when required into
a number of test-tubes or small flasks, in which the cultiva-
tion is to be carried out. Gelatine mixtures (gelatine and
broth, gelatine and peptone, gelatine and beef extract) and
the Agar-Agar mixtures, must of course be liquefied over a
flame before being ready for decanting. The test-tubes
most suitable are about six inches long, and should not be
less than about $ to f inch broad ; the flasks are of the
capacity of one, two or more ounces, and ought to have a
neck of compararively good width. The test-tubes receive
the fluids for about one and a half to two and a half inches
in depth — more (up to four inches) for anaerobic cultures »

46 MICRO-ORGANISMS AND DISEASE [chap,

the flasks for about one-fourth to one-third of their bulk.
All these test-tubes and flasks with their cotton-wool plugs,
before receiving the material, should be thoroughly sterilised
by heating. As I mentioned in the previous chapter, this
ought to be well borne in mind, for starting with a sterile
nourishing fluid — i.c. one that has been kept in the stock
flask for several days to several weeks in the incubator at a
temperature of from 320 to 38° C. and that has remained
perfectly clear and limpid— and working with thoroughly
sterilised test-tubes and cotton-wool plugs— very little care
is required to obtain sterile material ready for inoculation.
To start with a stock of nourishing material, however well
sterilised, and to decant it into test-tubes with cotton-wool
plugs not absolutely sterile must lead to failure. I have
seen this happen over and over again, and all the material
decanted became consequently contaminated and thereby
useless for inoculations. The test-tubes, glass dishes, and
flasks must be well cleaned with strong acid, then well
washed with water, then dried, placed in the hot air-chamber,
and kept there exposed for several hours to a temperature
of from 1 30° to T500 C., or they may be thoroughly heated
in all parts over the open flame of a gas-burner. The test-
tubes and flasks are plugged by means of sterile forceps with
the cotton-wool which is just faintly brown, and then replaced
in the air-chamber and again heated up to a temperature of
130° to 150° C. To decant sterile stock fluid into these
test-tubes and flasks I proceed thus : A clean beaker with
spout, covered with a clean glass plate, is placed inverted on
a net on a tripod over the flame of a Bunsen burner, and
thoroughly heated for half an hour or so ; then it is allowed
to cool, and when cool the plug of the stock flask is lifted
with forceps, and some of the sterile fluid quickly poured
from the flask into the beaker. The plug is replaced in the

iv] PREPARATION OF CULTURE-MEDIA

neck of the stock flask and the beaker covered with the
glass plate. Of course the quantity poured into the beaker
should be large enough to supply the required number of
test-tubes or small flasks. The stock flask containing still
some fluid, having been opened for however short a time,
has of course been exposed to air-contamination, and there-
fore must be treated accordingly, if the fluid left in it is to
serve as sterile nourishing material on a future occasion.
Consequently it is subjected to boiling for from fifteen to
thirty minutes.

Next, the fluid that has been poured into the beaker
(covered with the glass plate) is poured as quickly as pos-
sible into the test-tubes, one after the other, by lifting with
sterile forceps the plug and pouring in the fluid to a depth
of one and a half to two and a half inches, and the plug
replaced.

During this procedure contamination with air-organisms,
if there be any about, becomes inevitable. To lessen this
chance as much as possible, it is necessary to lift the plug
with sterile forceps, to pour the fluid as rapidly as is prac-
ticable into the test-tube or flask, and to replace immediately
the cotton-wool plug. Further, it is necessary to bear in
mind that the atmosphere is not at all times and everywhere
equally contaminated (see Prof. Tyndall’s observations). I
generally avoid undertaking this process on windy days, and
when I do it, I generally close windows and doors and keep
the air in the room as still as possible. I do not do it in a
room in which recently (say an hour or two previously) the
floor, walls, or tables have been swept.

1 have opened under these conditions the plugs of test-
tubes containing sterile material, and purposely exposed them
for a time varying from one to ten seconds, and I have not
seen more than from i to 2 per cent, contaminated.

MICRO-ORGANISMS AND DISEASE [chap.

Now, having filled the required number of test-tubes and
flasks with the required quantity of fluid, 1 subject these
seriatim to boiling. By means of an ordinary test-tube

Fig. 8.— A Beaker containing a Number of Culture-tubes Plugged
with Cotton-wool.

holder I hold them above a very small flame until the fluid
boils, and keep it so boiling for from half to one minute.
During this process of boiling the cotton-wool is only slightly
pulled up, and immediately before ceasing to boil the plug

iv] PREPARATION OF CULTURE-MEDIA

is again replaced, and pushed down with sterile forceps.
Then the test-tube is placed (of course upright) in a beaker
at the bottom of which a layer of cotton-wool — a sort of
cushion— has been placed. When finished, the test-tubes in
the beaker are all transferred to the incubator and kept
there for from one to three days, and all those in which the
fluid has remained limpid and clear are considered sterile
and ready for use. As a rule, starting with sterile stock
fluid, and using thoroughly sterile test-tubes and cotton-wool
plugs, after once or twice boiling after decanting there ought
to be no loss of tubes through accidental contamination with
air-organisms (during decanting). Sometimes, however, I
have had loss to the amount of 5 per cent, or more, but
then there was always a hitch of some kind traceable. To
decant under carbolic acid spray is not necessary or practic-
able, and possesses many unpleasant drawbacks, besides, in
some instances when I used it there was really a greater
percentage of contaminated tubes than without it.

A simple method and now generally used is to subject
the whole number of test-tubes or flasks into which the
nutritive material had been decanted (broth, peptone broth,
potato, milk, alkaline serum agar, nutritive gelatine, Agar-
Agar mixture) to a steamer (see Fig. 7). The test-tubes are
placed into the wire net (see figure), the top of the group of
test-tubes is covered with tinfoil, so as to protect the plugs
from becoming wet, and then the wire net is placed into
the steamer — the water at the bottom of which has been
previously heated to boiling, — the lid is put on and the
steaming is kept up for from fifteen to twenty minutes.
This is repeated on one or two successive days. I have not
seen any tubes go bad, after they have thus been steamed
on three successive days each time for twenty minutes.
Placed in the incubator and kept at a temperature of 35

MICRO-ORGANISMS AND DISEASE [chap.

to 38° C., from some days to one or two weeks, they
remain free of any growth.

Test-tubes containing solid nourishing material are for
the purpose of large culture surface, kept sufficiently

Fig 9. — Tray of Tin used for solidifying Gelatines or Agar with
Slanting Surface.

inclined during solidification of the material to allow the
material to spread into a layer of large area.

When test-tubes with sterile fluid blood-serum are to be
subjected to the process of solidification, it is advisable to

iv] PREPARATION OF CULTURE-MEDIA

keep the tubes in a slanting position, so as to allow the
serum to spread out into a layer which is sufficiently

Fig. 10. — HuEPPi&'s Serum Inshssatok. (Made by Lautenschlager in Berlin.)

transparent even after solidification, l'or this purpose
Hueppe’s serum inspissator is generally used.

In order to keep and protect cultures in tubes or flasks

e 2

MICRO-ORGANISMS AND DISEASE [ch. iv

from drying up, it is necessary to seal them up or to cap
them. The well-known indiarubber caps fulfil this purpose ;
unfortunately they are relatively expensive. Pouring a
layer of melted paraffin over the cotton-wool plug of the
mouth of the tube or flask is also used, but the process is
not a clean one, and this is specially felt when it is intended
to re-open the culture. I have for some years used, instead,
a method of sealing up which is not only reliable, but is
clean and very cheap, and easily renewed. I use gutta-
percha paper, of which a whole sheet only costs, a few pence,
and is sufficient for several dozens of tubes ; circular pieces
are cut out of the sheet sufficiently large to cover the mouth
and neck of the culture-tubes ; the mouth and neck, after
burning in the flame the upper part of the plug, are slightly
warmed, the circular piece is neatly placed over it and the
outer part of the piece pressed on to the glass of the neck,
if necessary warmed so as to stick well, with the pressure of
the finger a complete air-tight closure can be effected. It
can again, when it is required to re-open the culture, be
easily pulled off and then replaced by a new gutta-percha
cap. As stated above this mode of closure is easy, cheap,
and neat.

CHAPTER V

METHODS OF INOCULATION

Having now in test-tubes and small flasks sterile material
ready for inoculation, it is necessary to describe the mode
of inoculating the same.

i. Inoculations from Artificial Cultures. — The first and
simplest is the case where it is required to inoculate a new
tube or flask with a definite organism that has been growing
previously in a culture tube; that is to say, where it is
required to establish from an artificial cultivation a new
subculture. Take a freshly drawn-out capillary pipette,
with a fine point, as described in a former chapter ; draw
up with sterile forceps slightly the top part of the cotton-
wool plug of the old tube or flask, push carefully and gently
one of the pointed ends of the capillary pipette — the other
can be broken off blunt — through the remaining part of the
cotton wool plug, and push it downwards till it emerges
into the culture-fluid, or, if this be solid material, till it
reaches the spot or place where the organism is growing ;
allow a small droplet to ascend into the capillary pipette,
which it readily does by capillarity ; or if a larger quantity
is required draw it up by gently sucking at the outer end of
the capillary pipette. Then draw the capillary pipette

MICRO-ORGANISMS AND DISEASE [chap.

altogether out of the tube and cotton-wool plug, and push
this latter down with the forceps into its former position.
Immediately after this proceed to inoculate the new culture-
tube by doing exactly the same as before — viz., draw up
slightly with the forceps the top part of its cotton- wool plug,
push through the remainder of this plug the pointed end of
the capillary pipette, i.e. the one containing the droplet of
the material to be sown, and push it into the material at the
bottom of the test-tube or flask. A trace of the sowing
material flows out by itself, or, if a large quantity is required,
it is carefully blown from the pipette, but, of course, not
so that the tube is emptied by the blowing. If the sowing
is to be carried out on the surface of solid material, the
seed is deposited on the surface ; if in the depth, the end of
the pipette is pushed down into the depth of the material
and the seed there deposited. The pipette is then altogether
withdrawn and the plug replaced as before. The new tube
is then placed in a beaker on a cushion of cotton-wool, and
exposed to the required temperature in the incubator.

The simple and now universally adopted method, par-
ticularly in a place kept ordinarily clean, is this : the culture-
tube or flask is held slantingly, the plug is withdrawn with
forceps, and with a sterile platinum needle or loop a trace of
the old culture is transferred to culture-medium in a new
tube or flask, also kept slantingly, and of which the plug has
also been withdrawn. After the transference, both the old
culture and new subculture are plugged, both plugs before
insertion having been passed through the flame. If the
new subculture is made on solid medium, the inoculation is
made either as stab-culture — i.e., by dipping the end of the
sterile platinum needle into the old culture material, then
stabbing (piercing) about the central part the new solid
medium ; streak-culture , by drawing the charged needle or

METHODS OF INOCULATION

loop in one, two, three or more lines along the slanting
surface of the solid medium, or rubbing it all over this
surface.

If we have, however, a culture -fluid or any material that
contains, as the microscopical examination proves, various
species of organisms, which we wish to isolate, then the
method of Klebs of “ fractional cultivation,” or the method
of Lister and v. Nageli of “ dilution,” or better still, the
now universally-adopted method of Koch’s “ plate-culti-
vation,” is resorted to.

The “ fractional cultivation ” consists in the attempt to
isolate by successive cultivations the different organisms
that have been growing previously in the same culture. If
we take up by means of a capillary pipette or the point of a
platinum needle a trace of the culture-material, and inocu-
late with it in the manner above described a successive
series of new culture-tubes containing various nourishing
materials, and expose these tubes in the incubator to a definite
temperature, say 370 C., then the chances are that in the
first twelve or twenty-four hours not all the different species
of organisms sown out will have increased equally in num-
bers in all tubes ; most probably only one or two species in
each tube — i.e., the ones that grow best in this particular
medium and at this particular temperature — will be found to
have increased to an enormous extent, while the others have
made little or no progress as yet. The nourishing fluid
appears turbid, and filled chiefly with the one or two kinds
of organisms. Now take out with a fresh capillary pipette
or a platinum needle a minute droplet of this new culture
and inoculate with a trace of it a new culture-tube. I he
chances are that you inoculate only one kind, that is, the
one which is most abundant or perhaps is solely present.
After twelve or twenty-four hours’ incubation this new tube

MICRO-ORGANISMS AND DISEASE [chap.

contains now probably only one kind of organism. To
make it quite certain, inoculate from this a new culture-
tube in the same manner, and now you probably have
sown only a single species. In this manner by continued
transference it is possible to obtain cultures of only one
species of organisms. Many conditions, such as naked-eye
appearances of a particular kind, coloration of the culture-
medium, formation of a pellicle, the quantity of growth in a
given time, soon indicate whether we have the desired single
species ; in some instances it is, however, extremely difficult
to isolate after this method.

The method of “ dilution ” means diluting the material
containing the mixture of the various species to a very
large extent with some sterile indifferent fluid, such as well-
boiled saline solution of o‘6 per cent., and then inoculating
new tubes with a droplet of this greatly diluted material.
For this purpose take up with a platinum needle or loop a
droplet of the mixture, then transfer it in to a test-tube or flask
containing well-boiled saline solution, so as to greatly dilute
(iooo-fold or more) the particle or droplet of old culture-
material, and from this dilution inoculate then a series of new
culture-tubes containing different nourishing material, using
always only a trace for inoculation. In this way it is prob-
able that, owing to the great dilution, the trace of a droplet
of this mixture used for the new inoculation contains only
one species. Using a series of new culture-tubes and inocu-
lating them thus, after twenty-four hours of incubation it
will be found that some tubes have not received any seed,
others only one species. If it be required to dilute the
original fluid greatly, say if it teems with and is turbid by
different organisms, then a droplet of this is placed into a
large flask containing the well-boiled saline solution, so
that a dilution of i in t, 000,000 or more can be effected.

METHODS OF INOCULATION

The two methods— i.e.y that of fractional culture and of
dilution — may be successfully combined in this way : from
the first or second new culture, established after the method
of fractional cultivation, in which after twenty-four or thirty-
six hours one species greatly predominates, draw out with a
large capillary pipette a droplet, and dilute this to a great
extent with the saline solution, as described above, and now
inoculate with a trace of this mixture a new culture-tube.
Or, if after twenty-four hours’ incubation the microscope
reveals in this further culture more than one species, continue
the process of dilution and inoculation for a further genera-
tion. Thus it is possible to obtain cultures of only one
species, although the original fluid contained several species
of organisms.

One of the best and universally adopted methods for
isolation is that of the plate-cultivation introduced by Koch
in connection with the isolation of the choleraic comma
bacilli. A test-tube containing sterile nutritive gelatine as
above prepared is liquefied by gentle heat, best by being
kept in water of about 40° C., then the plug is lifted with
sterile forceps and the gelatine inoculated with a mere trace
of the bacterial mixture, either by means of the point of the
capillary pipette or of the heated and cooled point of a
platinum needle ; the plug is replaced and the gelatine
shaken so as to distribute uniformly the bacteria that had
been introduced. A shallow glass dish with flat bottom and
ground edge, and covered with a similar but slightly larger
dish,1 has previously been sterilised in the oven and then
allowed to cool ; the liquefied nutrient gelatine inoculated
with the trace of the bacterial material is then poured out

1 These plate-dishes are known as Petri’s dishes, but it might to lie
stated that several years before Petri I have described and figured this
dish — viz., in fig. 9 (present fig. 11) of the third edition of this work.

58 MICRO-ORGANISMS AND DISEASE [CHAP.

into the lower dish so as to form a thin layer at its bottom ;
the lifting off of the dish-cover, the pouring in of the gelatine,
and the replacing of the cover, ought to occupy only a
moment. In order to allow the gelatine to set rapidly the
dish is placed on moist blotting-paper; in hot weather a few
bits of ice are placed on the paper.

Fic. xi.— Plate-Cultivation.

Glass plate.

Bell-glass.

S.S EEning ,1.. plate-cultivation on a thin layer oh -ttiti.e
This glass dish is covered by a second glass dish-

This plate is then placed in the incubator as such or on a
olass plate, to which by means of greased edge, a bell-,,
can be fixed, on the interior of which , a piece of w
blotting-paper. In this way a closed moist chambe
established. But this is only of use if the plate is o
kept in the incubator for a long time; for ordinary work the
p'aie is Placed in the incubator without further add, t, on.

METHODS OF INOCULATION

The whole is then put into an incubator, the temperature of
which does not reach above 210 or 220 C. (or the tempera-
ture of the room in the warm months), in order to insure
the gelatine setting and remaining so. If a trace of material
containing various species of bacteria is thus distributed into
several cc. of gelatine, each microbe fixed by the gelatine on
setting will start a separate colony after a few days’ growth,
and the individual colonies, if different, will be appparent
by different characters, according to shape, colour, size of
the colonies, and according to whether they liquefy the
gelatine or not during their growth. In order to insure

Fig. 12. — Two Gf.latinf. Plate-cultures containing growing Colonies
on the Surface of Gelatine.

success, it is necessary to infect the original gelatine in the
test-tubes with only a trace of the bacterial mixture ; if too
many bacteria are introduced, their colonies sprouting up
are too numerous and soon become confluent. But if the
experiment is successful, the colonies are well separated from
one another, and from the individual and separate colonies
it is then easy by re-inoculation of gelatine tubes, or othei
nutritive material, to start pure subcultures of the different
species. It must be borne in mind that not all bacteria can
be isolated by this method, for some species of pathogenic
organisms require for their growth a higher temperature

MICRO-ORGANISMS AND DISEASE [chap.

than the one at which the nutrient gelatine remains solid,
while others refuse altogether to grow in gelatine, or grow
only too slow. In the latter case no success can be looked
for, if those bacteria which form their colonies much faster
and are present in large numbers crowd out the others that
require a long time to come up.

In such cases, particularly when one has to deal with
bacteria that do not grow in gelatine at the temperature at
which this latter remains solid, the same method of plate-
cultivation can be used, but substituting the gelatine by the
Agar-Agar peptone mixture above mentioned, previously
liquefied by heating, care must be taken not to proceed with
the inoculation of the Agar-Agar mixture before the tem-
perature has fallen to about 420 to 50° C. All other mani-
pulations remain the same.

It is perhaps not unnecessary to state that if the Agar
mixture is of recent date — contains, therefore, while in the
tube condensation fluid, after pouring it out in a plate dish,
cooling this, letting the Agar set, and placing the cultivation
in the incubator at 370 C., in all probability there will again
appear condensation water in the plate ; as the colonies
begin to develop on the surface they will be swamped by
that water and the whole surface will become covered with
an indiscriminate film of growth. It is therefore advisable
to keep the plate-dish in the incubator inverted and in
slanting position. If, however, the Agar mixture used for
the plate-culture is of some standing this is not necessary.
But I keep also gelatine plates in an inverted condition in
the incubator for the first day, in order to avoid too great
a loss of water by evaporation : care must of course be taken
that as soon as liquefying colonies appear and begin to
spread the plate-cultivation must again be placed upright.

A method which I have found very useful for making

METHODS OF INOCULATION

permanent plate-cultivations on gelatine or Agar, and which
I first described in the Reports of the Medical Officer of the
Local Government Board for 1886-1887, is the test-tube
plate-cultivation. It is this : in ordinary plate-cultivations
such as were described above it is obvious that only a
certain proportion of the colonies appearing on subsequent
incubation are situated on the surface, another proportion are
in the depth, and these latter are either not characteristic
for differential purposes or cannot easily be used for further
operations. It is therefore advantageous to have all colonies
appearing on the surface. This can be done in two ways :
(a) By pouring out into the plate-dish the gelatine or Agar
before inoculation, letting it set, and then smear or rub
over the surface of the set gelatine or Agar by means of
the platinum loop the infective material (see Fig. 12),
or ( b ) by rubbing it over the surface of gelatine or Agar,
set with slanting surface in test-tubes. In both cases all
colonies make their appearance on the surface only. The
latter method allows of the test-tube plate-cultivation to be
preserved uncontaminated, as the test-tube on opening for
the object of making subcultures can be held mouth down-
wards and safe against accidental contamination. When we
are dealing with microbes liquefying gelatine the test-tube
cultivations in gelatine are not practicable. The test-tube
gelatine-cultivations yield excellent impression-preparations
(see Chapter I.) ; for this purpose the interior of the test-
tube can be easily cast out on to a glass plate by dipping
the lower part of the test-tube for a few seconds in hot
water; too long exposure would melt the gelatine and spoil
the gelatine block for further operations.

2. Inoculations with Blood , Juices , and Tissues. Jo
establish a cultivation from blood of a dead animal, cut open
the thorax by removing the sternum with clean scissors, cut

MICRO-ORGANISMS AND DISEASE [chap.

open the pericardial sac, pierce with the pointed end of a
fresh capillary pipette the wall of the right ventricle or right
auricle, and allow a drop or two of blood to ascend into the
pipette, or if a larger quantity is required suck it up.- With-
draw the pipette and inoculate new culture-tubes as above.
Or, if blood of a large vein is required, separate the vessel
with sterile instruments, and make a small incision with sterile
scissors and push the pointed end of the capillary pipette
well forward. If juice of a lymphatic gland, or spleen, or
other parenchymatous organ be required, pierce the organ
after having washed its surface with strong solution of per-
chloride of mercury (Koch), with the pointed end of a
capillary pipette, then push it into the part required for a
little distance, and squeezing the organ press a drop or two
of the juice into it. The same procedure is adopted when
the pus of an abscess is required, the wall of which can be
pierced with the pointed end of the capillary pipette. If
not, a slight incision is made and the pipette introduced
through this into the abscess. If blood of a living animal is
required, expose a vessel with sterile instruments, make a
small incision with sterile scissors, push through this incision
the pointed end of the capillary pipette well forward, and
allow the blood to rise into the capillary tube. If blood of
a living human being is required, clean well with soap and
water and then with strong carbolic acid or perchloride of
mercury solution the tip of a finger, make a venous conges-
tion in the last phalanx by compressing it with a corner of a
handkerchief, prick the volar skin of the phalanx with a
clean (heated and cooled) needle, and plunging the pointed
end of the pipette into the drop of blood, allow a droplet
to ascend into the capillary tube of the pipette. But
all these inoculations can also be practised by means
of the platinum loop, only in this case contamination

v] METHODS OF INOCULATION 63

with extraneous organisms is more possible than by the
other method.

If solid tissues or parts of tissues are required — e.g. the
base of an ulcer, a tubercle of the liver, spleen, or lung— it
is possible to squeeze into the capillary tube of a pipette,
after pushing its pointed end into the part, a small droplet
of juice of the part required; but if this be not practicable
— i.e. if a solid particle be required — or if it be preferred
because simpler, then follow Koch’s method, now generally
used. This is as follows : Cut with clean sterile scissors or
scalpel into the part, take up rapidly with the point of a
needle or platinum wire previously heated in the flame of
a burner a small particle, a drop of blood,, pus, juice,
or solid material, and quickly introduce this into the
culture-tube to the place required — e.g. surface or depth of a
solid or fluid nourishing material. Of course in this case
the cotton-wool must be altogether lifted, and therefore
contamination with air-organisms is possible. But inocu-
lating several tubes at once and performing the operation
quickly, and working in an ordinarily clean place, one
always succeeds in getting most of the tubes without any
air-contamination. I have made numerous inoculations
with solid particles of different morbid tissues and products
in this manner, and, like Koch and others, have seen only
a very small percentage of tubes becoming contaminated
with air-organisms, chiefly moulds.

The same plan — i.e. of using the clean point of a sterile
needle or platinum wire for taking up the material to be
used for inoculation — is resorted to if one has to deal with
the culture in solid nourishing material, on or in which the
organisms are growing that we want to transplant either for
inoculation of a new tube or of an animal. A useful
method, which does not require the lifting out of the plug

64 MICRO-ORGANISMS AND DISEASE [chap.

at all, and which can easily be employed in the last case, is
this ; deposit from the pointed end of a capillary pipette a
droplet of some sterile fluid (broth or thoroughly-boiled
saline solution) on the spot of the solid medium on which
the organisms are growing, then scratch this spot with the
end of the capillary pipette in order to get the organisms off
from the solid basis and mixed with the drop of fluid
deposited there, then let this drop again ascend into the
end of the capillary pipette, and withdraw this altogether.
All this can be done without lifting out the cotton-wool
plug of the test tube or flask in which the growth is
proceeding.

If one has to use a particle of tissue the surrounding
portions of which are probably contaminated by putrefactive
organisms — e.g. a tubercle in the lung or a tubercle in the
spleen — it is well to follow Koch, and to disinfect the
surrounding parts by just washing them with a dilute solution
of corrosive sublimate, and then to remove these parts with
clean scissors so as to obtain the central particle which one
wishes to use for inoculation : of course one must not steep
the organ too long in sublimate solution, since this would
naturally destroy all organisms.

All these methods can be easily modified according to the
requirements of the special cases, and it is not necessary
here to give more than what has already been described in
the preceding.1

3. Fixing of cultures. — In connection with this a method
must be mentioned for the permanent fixing of plate- and
tube-cultures. The growth in these can be at any moment
arrested, and all further contamination and growth in

1 Compare also Koch, Untersuchungcn iibcr pathogens Baita ten, in
Berichte aus dem k. Gesunitheitsamte, Berlin, 188 r : and Die Act iologie
d. Tuberculose , Berlin, klin. Wochenschrift , No. 15, 1882.

METHODS OK INOCULATION

them prevented, by devitalising the microbes, and by sterilis-
ing the mecTium on and in which the growth has been taking
place. This is done by the fumes of formalin (i- strength);
commercial formalin is a 40 per cent, solution of formal-
dehide. A tube, or a number of culture-tubes, in which the
further growth of the microbe is to be arrested, are placed
best without their cotton-wool plugs into a wide-mouthed
bottle or glass cylinder, into which a small quantity of for-
malin (^ strength) has been poured, then close the cylinder
air-tight and let it stand. The vapours of formalin penetrat-
ing the tubes even through the wool plugs do their work in a
day or two. If a plate- cultivation is to be fixed, a few drops
of formalin are placed on the middle of the cover-dish, the
plate-dish is now inverted, and allowed to stand for from some
hours to a day or two. The formalin vapours fix thereby
permanently and kill the colonies, and no further growth
either of the colonies already formed or of new contaminating
colonies occurs.

4. Hanging drop cultures. — In order to study microbes
in the living state as for motility, growth, multiplication,
and spore formation, the methods used are practically those
known as “ hanging drop preparations ” first used by Koch.
An object-glass slide, possessing a shallow circular pit, is
covered, over the pit, with a cover-glass in the centre of which
a drop of the fluid suspension of bacteria, or of serum, blood,
&c., is deposited, the drop facing the pit; the edge of the
cover-glass can be fixed around the pit by paraffin or oil or
cement, and the observation can be carried out either at
the temperature of the laboratory or by placing the object on
a warm stage at any desired temperature. The droplet being
small the examination can be carried out even with high
powers as easy as an ordinary fresh preparation. Motility,
the elongation of bacilli, their division, germination of spores,

66 MICRO-ORGANISMS AND DISEASE [chap.

of bacilli, and of fungi, and other life processes can be
watched and noted. I have made very extensive observations
on the growth and division of bacilli and of mycelial fungi,
extending for hours, and noting the progress from day to
day, by distributing a limited number of the microbes (by
the aid of the point of a thin platinum needle) in a droplet
of melted nutrient gelatine or Agar deposited in the centre
of the cover-glass,1 and then flattening the droplet by the
platinum needle out into a film, of course limited to the
centre of the cover-glass, and finally fixing this latter by
means of sweet oil to the glass side film downwards. The
gelatine, as also the Agar, sets at the ordinary temperature
of the laboratory, and by a power up to 700 the bacteria
or other microbes can be easily focussed and kept under
observation, they being fixed in the set gelatine or Agar.

The glass cell (see Fig. 13) which I use is based on the
same principle ; it has the advantage of allowing the ob-
servation to be extended over longer periods as it is easier
to keep the chamber of the cell moist by depositing a droplet
of water on its floor.

These methods of watching and studying bacteria and
fungi in the living state with high powers in a gelatine film or
Agar film cannot be too strongly recommended ; it can be
carried out and extended over hours and days. A direct
insight is obtained into the phenomena of growth, germina-
tion, and division, as also of spore formation which as a
rule is only indirectly deduced. It is one of the most in-
teresting experiments to make such a preparation from the
blood of an animal dead of anthrax (care being taken to
introduce only a limited number of bacilli), and to watch

1 If a culture is used it is best first to make a distribution in sterile
salt, or water, or broth, by shaking in it a small particle of the growth
transferred by a platinum needle, and to inoculate the gelatine or Agar
drop from this dilution.

V1 METHODS OF INOCULATION 67

the growth oCjhe individual bacilli fixed in the set gelatine
into threads and the formation of the characteristic colonies
made up of curved and convoluted threads. Equally in-
teresting is it to watch the formation of colonies by the
proteus vulgaris or proteus Zenkeri, the “ swarming ” of them,
and the manifold sprouting of threadlike outgrowths; or
the gradual formation of bright globules and their enlarge-
ment into the characteristic oval spores in the threads of
bacillus anthracis or of hay bacillus, and their ultimate

Fig. 13.— A Glass Cell, for Observing under the Microscoi'e the
Progress of Growth of Micro-organisms.

The upper figure shows the cell in perspective ; the lower figure in profile or cross

section.

A. Glass slide.

B. Cover-glass.

C. Glass ring forming the wall of the chamber.

P. Drop of nourishing material in which the micro-organisms grow.

discharge and disintegration of the bacilli themselves. All
these points can be directly studied by intermittent obser-
vation in the above preparations extending over several
days.

5. Bacterioscopic Examination of Water. — Most waters
contain bacteria of some kind, sometimes in great numbers
without altering the limpidity of the water, at any rate not
for the unaided eye or the ordinary tests of transparency.

In order to directly demonstrate the bacteria, the water

f 2

MICRO-ORGANISMS AND DISEASE [chap.

is allowed to stand ; from the bottom layer a small quantity
is withdrawn and of this a drop or two are deposited in the
centre of a clean cover-glass and evaporated by heating.
This represents a film specimen, which is then stained,
washed, and mounted in the usual way. On microscopic
examination, according to the source from which the water
is derived, there will be found particles of amorphous debris,
cotton-wool threads, spores, and bits of mycelial threads and
stained bacteria in small or great numbers according to the
amount of pollution that the water has been exposed to.
In a so prepared specimen of the water that the different
London water companies, drawing their water from the
Thames and Lea, distribute to their consumers, as a rule
besides numerous bacteria, cotton fibres and amorphous
debris, there will be found various infusoria (see below). In
order to accurately study the number and character of the
bacteria, present in water, cultivations must be made. These
are of two kinds :

A. — To Determine the Numrer and General
Character of the Bacteria.

Plate-cultivations are used for this purpose, generally
gelatine plate-cultivations. But it must be remembered
that by determining the number of microbes in a given
small quantity of the water, added to the gelatine, by means
of gelatine plate-cultivation, we are determining only the
relative number of bacteria, that is to say, only those that
do and can grow at the temperature at which the gelatine
keeps solid, but there may be and sometimes are some
species present which only grow well at higher temperatures ;
in such cases their numbers must be determined by Agar
plates. But as a general rule in practice it is sufficient to

METHODS OF INOCULATION

determine tl>o. number of bacteria by means of gelatine
plates. For this purpose a definite— i.e. measured— small
quantity,1 -Ja, tV to 1 cc. (according to the turbidity) of
the water after shaking, is added to a gelatine tube ; this is
melted in warm water and then poured out into a sterile
plate-dish. This gelatine is allowed to set, and after
incubation at 20° C. for three or four days the number of
colonies that have sprung up are counted, and according to
the quantity of the water that has been added to the
gelatine for plate-cultivation the number is calculated per
1 cc. Several points have to be remembered in making
an estimate that is to be approximately correct. (1) There
ought to be always two plates made, and the number ought
to be determined by the average. (2) The sample of water
to be tested ought to be well shaken up before withdrawing
the required quantity for the plates, in order to make as
uniform a distribution of the bacteria in the water as
possible. But notwithstanding this sometimes enormous
differences will be found in two plates made from different
portions ; this is probably due to the fact that in some

1 For measuring definite small quantities of water or any other fluids,
I use a series of glass pipettes on the plan of those added to a hremo-
cytometer : 5 cmm. (^5), 10 cmm. (tW. 20 cmm. (-55), 5° cmm. (J5),
100 cmm. (tV), and 250 cmm. (\), \ cc. and 1 cc.; each of these
pipettes can be fitted with an india-rubber tube with porcelain or glass
mouthpiece ; these latter receive a plug of sterile cotton wool. 1 he
pipettes are sterilised, and when ready for use the tube is fitted on ; of
the fluid a little is poured into a sterile watchglass, and of this the re-
quired quantity is withdrawn and blown out into the test-tube containing
the culture-medium. If a quantity smaller than Att cc. is required a
dilution is previously made with a definite quantity of sterile distilled
water ; for instance, if ttiVtt cc. °f a given fluid is required, I take 5 cmm.
— i.e. of the fluid — and add this to 5 cc. of sterile distilled water,

each 1 cc. of this would contain 1 cmm. or tAt; cc.

The pipettes can be sterilised either in the hot air-chamber, which is
best, or by letting them lie for an hour or so in disinfecting fluid, then
empty them, and wash them thoroughly out twice or more times in
distilled water.

70 MICRO-ORGANISMS AND DISEASE [chap.

waters — e.g. the London waters— there are suspended in the
water microscopic masses of organic debris loaded with
bacteria. If such a mass happens to be in the particu-
lar quantity of the water that is added to the gelatine, and
after shaking this up the bacterial mass is broken up the
resulting number of colonies in the plate may be greatly
in excess.

(3) It ought to be remembered that the number of
bacteria in water is liable to considerably increase as time
goes on, for some bacteria — special water bacteria — are
capable of living and multiplying in water; they are capable
of thriving even on very small amounts of organic matter
present in the water. It is therefore necessary to make the
plate-cultivations as early as possible after the water is taken
from its stock. It is not necessary to do this on the spot,
if the place of work is within a comparatively short distance
say if the water can be delivered in the laboratory within a
few hours after ; but if water is sent from long distances,
when it has to travel many hours by rail in the summer
months, then the actual number of bacteria present at
starting is not to be measured by that found in plates made
say twenty-four hours after. In the cold weather and if the
sample of water sent is kept in a cool place, the multiplica-
tion in twenty-four hours is not very great. To obviate these
errors when water is sent from long distances, it is advisable
to keep the sample packed in ice or in cotton wool in a
cool place. It is obvious that the bottles in which the water
is received and sent should be sterile ; glass stoppered,
narrow necked, about two- to four-ounce bottles, sterilised in
the hot chamber, are best for this purpose — it does not
require to fill the water in vacuum tubes. From a large
experience I found that glass-stoppered bottles first well
washed out with nitric acid or methylated spirit, then twice

METHODS OF INOCULATION

successively withvthe water with which they are to be filled,
are quite satisfactory.

(4) In order to determine the number of bacteria present
per 1 cc. in a given sample of water by means of gelatine
plates, a sufficient quantity of the water ought to be used to
yield a fair number of colonies, such as can be counted
fairly accurately.1 If the water has a large number of
bacteria — this can be easily determined in a few minutes by
making a film preparation — i.e. by depositing one or two
drops of the water, after shaking, in the centre of the cover-
glass, drying and heating, staining and mounting and sub-
jecting it to microscopic examination ; after some practice
it is quite possible to say from such examination whether the
water has comparatively few or many bacteria, and accord-
ingly to make the plate-cultivation with a small quantity,
say or less or more up to 1 cc.

It ought further to be remembered that the rapidity with
which the colonies appear in the plates depends in the first
place on the temperature at which the plates are kept. If
the plates are kept in a cool place — e.g. in a cupboard at the
temperature of the room in the cold months — the growth is
extremely slow, and the colonies appear only after many
days. I have made comparisons in this respect. I have
seen it stated by the water analysts of the London water
companies that during particular months of the cold weather
the number of bacteria in London waters as determined by
gelatine plates kept in a dark cupboard at the temperature
of the laboratory, which was certainly under i7°C., and the
counting being done forty-eight hours after, was between 7
and 70 per 1 cc. ; whereas in my experiments under similar
conditions the plates were counted not after forty-eight hours

1 What is here stated of water refers to ail other fluids of which the
number of microbes are to be determined by pla' e-cultivation.

MICRO-ORGANISMS AND DISEASE [chap.

only but after seven days, and the number of colonics in the
plates was over twelve hundred per i cc. The plates ought
to be kept in the incubator always at a temperature of 20 to
21° C, if counted after two days it will be found that even
under these conditions not all colonies have sufficiently
developed as yet, and for these reasons the counting ought
to be repeated after three or four days. It seems to me that
the low figures of bacteria present in London waters as
published by the public analyst of the Local Government
Board as also by the analysts of the London water com-
panies are not to be accepted without hesitation, for the
reason that in neither case were the conditions for obtaining
a full and correct number of the bacteria fulfilled : the plates
were not kept at the most favourable temperature, or the
counting was done too early.

Koch has devised a plate in equal squares of known area
which enables one to count the number of colonies easily.
But it is just as easy and just as accurate to count the
colonies in a plate-cultivation, by drawing in ink lines on
the outside of the plate- dish (not the cover) by which the
area is divided in two, four, eight, sixteen, and so on, and to
count under a magnifying glass on a black ground the actual
number of colonies in each division. If the number of.
colonies in the plate-cultivation is small or moderately large
counting is easily and soon done, but if the number is ex-
cessively large, say several thousand, then only an approxi-
mate estimate can be made, by selecting two, three, or four
small subdivisions, say Txg- or -£■%, representing a fair average
distribution of the colonies, to count them patiently in these
divisions, and then by calculation give the total.

Messrs. Washbourne and Fakes have designed a print
which embodies the same principle, only is much simpler ;
it is a printed circular area in black of the size of a plate

METHODS OF INOCULATION

dish ; in this circuit area arc white radii subdividing the area
into sectores Tlff each, and cross circles subdividing these
sectores into three.

Under these circumstances the estimate can only be an
approximate one, but as stated above if the number of
colonies is not excessively large, the counting can be done
accurately.

A question that is constantly being asked is as to the
number of bacteria that ought not to be exceeded in water
if this is to be regarded as of good quality. It is quite clear
that water in which there is an average amount of vegetable
matter ought, and as a rule does, contain large numbers of
bacteria — eg. moorland water, lake water supplied from
moorland, water in lakes and ponds in meadows surrounded
by reeds, Szc. — yet this number of bacteria need not in the
least interfere with or deteriorate the good quality of the water,
whereas water even if taken from deep wells, in the chalk or
other formations, may contain a small number of bacteria
yet be wholly unfit for drinking purposes if at any point
percolation of sewage into this water takes place. Koch’s
standard which is accepted now generally is : that wherever
pollution of water with animal refuse potentially or actually
takes place, the number of bacteria should not exceed ioo
per i cc. But this, for the above-named reasons, does not
apply to waters which are not and cannot be so polluted.
Taking, for instance, the water which the London water
companies distribute to the London inhabitants, we find
that with the exception of the Kent Company — which
nominally, at any rate, draws its whole stock from the chalk
— all other companies draw their raw water from the
Thames or Lea, that is to say, from sources which are
notoriously open to pollution, and as a matter of fact are
constantly actually polluted with animal refuse — human ex-

MICRO-ORGANISMS AND DISEASE [chap.

crements included— such water should, on Koch’s standard,
not contain above ioo bacteria per x cubic centimetre.
I had the opportunity of examining these waters (eight
companies) for eight consecutive weeks, and found that out
of sixty-four samples thus examined only in eight were the
number one hundred or below, in the others above, in a
majority as numerous as in the unfiltered raw water.

The plate-cultivations thus made for ascertaining the
number of bacteria can be used for a superficial estimation
and the study of the character of the microbes, but it must
be understood that having used for each plate only a very
small quantity of the water, a fraction, say, of one to two
cubic centimetres, only those microbes will be met with in
these plates which occur in large numbers ; as to those that are
distributed in the water as a whole in limited numbers, there
is little chance of meeting them in a couple of plates inocu-
lated with only 1-2 cc. of the water. The bacteria almost
constantly present in small quantities, leaving out yeast and
fungi, are : — ( a ) bacillus fluorescens liquescens, easily recog-
nised by the rapid liquefaction and greenish tint of the
liquefied parts ; (71) bacillus sulcatus, in several varieties, not
liquefying, white, rounded, flat moist colonies ; (<r) micrococ-
cus, liquefying and non-liquefying ; ( d ) not infrequently one
or the other variety of bacillus mesentericus, motile large
bacilli, liquefying slower than fluorescens. The most im-
portant part of the examination refers to the detection of
microbes which are present in putrefying animal matter,
notably in sewage or animal excrements, or are derived from
the diseased bowels of man. Amongst these are bacillus coli,
proteus vulgaris, proteus Zenkeri, or a variety of it, and
above all bacillus of typhoid and vibrio of cholera.

METHODS OF INOCULATION

/?. — Detection of Special Microbes by
Special Methods.

Bacillus coli, being chiefly derived from the intestinal con-
tents of man and animals, would in the nature of things
occur in all matters : dust, earth, food-stuffs, mucous mem-
branes which have been exposed to pollution with matter
tainted with dejecta. Thus in large towns almost everything
is liable to become so polluted owing to the almost ubiqui-
tous presence of dust tainted with animal dejecta. The
same applies to any place and any material to which such
dejecta find access. This bacillus coli can, therefore, under
particular conditions of locality, be regarded as almost ubi-
quitous. The same applies to proteus vulgaris, but in a
somewhat more limited degree, since this organism, although
present in the alimentary canal, is nevertheless not so common
in this ; but being the chief organism producing the putrid
decomposition of albuminous substances, it will be found
wherever such substances undergo this change. These two
organisms or either, notably bacillus coli, if present in large
numbers in any water, would indicate that that water had been
subject to excremental pollution or that there exist in the
water putrid animal matter. A very limited number of bacillus
coli need not be and is not sufficient to condemn such water,
because the accession to it of a little dust, carried there by
air currents, originally impregnated with animal excreta,
would produce such a result, but in this case the bacillus
coli would in a large bulk of water be very scantily dis-
tributed. It is different if the bacillus coli or proteus vul-
garis, particularly the former, be present in large numbers,
for then pollution with excremental matter has probably
taken place. Take, for instance, water derived from deep

MICRO-ORGANISMS AND DISEASE [chap.

wells ; if there be no soakage of sewage, practically no
bacillus coli will be found in it, but if there be such soakage
bacillus coli is easily found in moderate numbers. It must
be obvious that where sewage pollution does take place, the
facility of discovering the bacillus coli in the water will
depend cceteris paribus on the relative amount of pollu-
tion and water. If, for instance, it is a case of a small
water-course to which sewage has continually access, num-
bers of colonies of bacillus coli would be found in a gelatine
plate made with even small quantities of the water, say
^ — i or 2 cc. But if it is a question of a water-course like
the river Thames, even after a moderate sewage pollution, the
volume of water is so great that bacillus coli can be demon-
strated only by subjecting large quantities of the water to the
culture test. To expect to find the bacillus coli in a few
drops or even a few cc. of water taken from the Thames at
Hampton, above the intake of the water companies’ water,
is very strange, and stranger still to say that not finding it
in so small a quantity, it is absent from the unfiltered
Thames water at Hampton ; such statements are liable to
throw doubt on bacteriological examinations in the eyes
of sanitarians, for it is notorious that apart from the surface
flushing of streets in many places on the upper Thames,
there is obvious pollution of the Thames with human excre-
ment?, along the shore, from barges, house-boats, &c. And
the same applies to the examination of water for typhoid
bacillus or cholera vibrio— viz., it is essential that large
volumes of water should be subjected to bacterioscopic
examination, and even then a negative result should be put
forward for what it is worth. Statements such as one
occasionally sees— viz., a few drops or a few cc. of the water
had been examined and no bacillus coli, or no bacillus of
typhoid, or no cholera vibrio was found, therefore such

METHODS OF INOCULATION

and such water does ribt contain any of these organisms, are
absurd; the latter statement would be unjustified even if large
quantities of the water had been subjected to examination.

For the detection of bacillus coli, bacillus of typhoid, and
a certain variety of proteus Zenkeri, a normal inhabitant of
sewage, the following method1 will be found to answer well :
Through a Berkefeld or Pasteur pressure filter a large vol-
ume of the water is pumped. The filter I generally use for
the purpose is a Berkefeld large bougie, which by a screw
can be well and tightly fastened into one end of a cylindrical
glass; the metal tube projecting from the candle is fixed
through an indiarubber stopper into a large bottle holding
about 1,000 — 1,200 cc.; this bottle has at the neck a lateral
glass tube, which by means of a stout indiarubber tube is
connected with an exhaustion pump, a good size hand-
pump. Before using the filter the candle, screw, indiarubber
stopper, glass cylinder, and glass bottle are all sterilised, the
glass and screw in the hot air-chamber, the candle and
indiarubber stopper in boiling water, in which the candle is
kept from half to one hour.

The water to be examined is poured by means of a sterile
glass beaker into the glass cylinder, and exhausting the air
in the bottle the water filters easily and rapidly; 1,200—1,500
or 2,000 cc. are thus easily filtered in a moderate space of
time; 1,200 cc. pass through in about 15-20 minutes.
Then the candle is unscrewed carefully, taken out with clean
hands, and 10 cc. of sterile water (or of the filtered water
from the bottle) are measured into a sterile glass dish ; the
whole surface of the candle is well brushed into these 10 cc.

1 I have- practised this method since 1892 ; when with Dr. Theodore
Thomson the outbreak of typhoid fever in Worthing, 1893, was
investigated, it was this method by which the typhoid bacillus in the
suspected water was demonstrated.

78 MICRO-ORGANISMS AND DISEASE [chap.-

of sterile water by means of a thoroughly clean nail-brush."
In this way the whole or practically the whole of the par-
ticulate matter of the bulk of water that had been filtered
is distributed in the io cc. of sterile water. By brushing
the surface some of the soft filter material is also brushed
off, but since the bulk of this easily settles down it is of
no material consequence. This distribution is used for
cultivation ; every cubic centimetre of it contains the amount
of the particulate matter of a definite bulk of the original
water. If, for instance, 1,200 cc. had been driven through
the filter, each 1 cc. would contain the particulate matter
of 120 cc. of the original water; if 2,000 cc., each cc.
of the distribution contains the particulate matter of
200 cc. of the original water. There is no difficulty in
subjecting to analysis if necessary the whole 10 cc. — i.e.
the whole particulate matter of the whole of the original
2,000 cc. of the water.

The cultivations of the distribution are made after Pari-
etti’s method in phenolated gelatine, or in phenolated broth,
or in both. Parietti first pointed out that by adding a solu-
tion of phenol to the broth or the nutrient gelatine previously
melted, these media while remaining favourable for the
growth and development of the bacillus coli and typhoid,
are not so well suited to that of the ordinary water bacteria,
some of the latter being either altogether suppressed while
others thrive only slowly, and therefore bacillus coli or
typhoid have in the meantime an opportunity to develop.
Of a 5 per cent, solution of absolute phenol o‘i cc. (or
100 cmm.) are added to 10 cc. of broth or gelatine; this
represents the phenolated gelatine or phenolated broth
respectively. The addition of hydrochloric acid, as
recommended by some, is according to my experience not
required.

METHODS OF INOCULATION

Of the above distribution then, to each phenol gelatine
or phenol broth tube or } cc. is added, the gelatine is
shaken and poured out into a plate, and after setting kept at
20° C., the phenol broth is incubated at 370 C. From the
phenol broth incubated for twenty-four hours, plates in
phenolated gelatine are then made. If in the original water
the bacillus coli or the sewage variety of proteus Zenkeri or
the typhoid bacillus be present, the phenol broth-cultivation
will be found uniformly turbid after twenty-four hours’ in-
cubation ; by placing a droplet of this culture into 10 cc. of
sterile salt solution and making with a platinum loop of this
dilution a ph&nol gelatine plate, this after incubation for
two to three days will show either of the above organisms in
numerous colonies.

The phenol gelatine plates when ready —after two to
four days’ incubation at 20° C. — must be carefully examined,
and all the surface colonies which resemble in aspect the
above organisms have to be tested by fresh preparation, by
flagella staining, and by subcultures in different media.
Gf this more when we come to deal with the differential
characters of the bacillus coli and typhoid bacillus. The
sewage variety of the proteus Zenkeri, as will be also de-
scribed later, is in its surface colonies so characteristic and
conspicuous that this and the microscopic examination are
sufficient for diagnosis.

Another method is this : mix in a sterile flask equal
■ volumes— 50, 100 or 200 cc. — of the water and broth,
having added to the latter the required quantity of phenol,
then incubate the flask at 370 C. for twenty-four hours,
and make phenol gelatine plates as before.

By these methods I was enabled to demonstrate the pres-
ence of an abundance of the typical bacillus coli not only
in Thames water above Hampton, that is the intake of the

MICRO-ORGANISMS AND DISEASE [chap.

London water companies, but also on several occasions
during the examination of samples of the water taken once
a week during January and February, 1895, in the filtered
waters distributed by the London water companies. This
is shown in the following table : —

and

week.

3rd

week.

4th

week.

5th

week.

6th

week.

7th

week.

8th

week.

Southwark ....

East London . . .

—

- i

Kent Water . . .

—

Lambeth

—

New River ....

—

West Middlesex .

—

Chelsea

—

•

Grand Junction . .

—

+ = Bacillus coli in London waters.

The third test to which drinking waters ought to be sub-
jected, is the microscopic examination of its suspended
matter, particularly as to the presence of protozoa.

The water remaining after the quantity required for
above filtration had been withdrawn is put away and allowed
to stand in a cool place for twenty to twenty-four hours.

By means of a pipette drawn out into a long capillary
tube 5-8 cc. or more are drawn up from the bottom layer,
the end of the capillary tube is sealed and the pipette fixed
in an upright position so as to allow the suspended matter
to settle in the capillary tube. When this has taken place,
the capillary tube is broken off and its contents subjected
to microscopic examination

I will give here a table showing what kind of living ani-
malculi were found by me in examining the “ filtered ” water
as distributed by the various London water companies
during six weeks of examination, and from this it will be

f=«

'Z!

METHODS OF INOCULATION

iQ -o
£ -
2 rt

3 <3"S

a is

8th week.

I 1 1

irt

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Very nt
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cercomonas.

MICRO-ORGANISMS AND DISEASE [chap.

seen that the London waters must have been considerably
polluted, and at the same time imperfectly filtered.

For the detection of the cholera vibrio in water the
peptone salt method is the simplest and best. A stock
solution of xo per cent, best peptone and 5 per cent,
common salt in distilled water is made ; this is made
faintly alkaline and sterilised by boiling. To each 90 cc.
of the water to be examined, contained in a sterile flask, 10
cc. of the above peptone solution are added, so as to
make the mixture in reality a 1 per cent, peptone 0*5 per
cent, salt solution. The flask is then incubated at 370 C. for
twelve to twenty-four hours.

The cholera vibrio grows well and rapidly in a 1 per cent,
peptone, 4 per cent, salt solution (Dunham), and is un-
doubtedly for this reason the best means of detecting the
vibrio. Such a peptone solution shows already, provided
cholera vibrios had been present, after twelve hours’ distinct
turbidity, and if of the top layer a droplet is removed and
examined fresh, briskly moving (revolving) comma bacilli
will be found ; they are easily recognised as commas if
a drop of the top layer of the cultivation fluid is deposited
in the centre of the cover-glass and without spreading it
out is dried, stained, and mounted. After twenty-four hours
the turbidity is much more pronounced, and cholera vibrios,
whether in pure or impure condition — i.e., without or with
admixture of other microbes, notably bacillus coli or proteus
vulgaris— can be isolated by gelatine or Agar plates in the
usual way, and then subjected to the various tests for
cholera vibrios (see cholera).

The two microbes which next to the cholera vibrio grow
fairly well in the above peptone-salt solution, are the bacillus
coli and the proteus vulgaris; for the detection of the latter in
water the peptone method is excellent, since a large quantity

METHODS OF INOCULATION

of water can hereby be subjected to examination, otherwise
ordinary gelatine plate-cultivations must be relied upon.
Hut since for these only small quantities of the water can be
used, the former method is far preferable, as by the ordinary
gelatine plate method the proteus could be detected only if
present very freely.

Examination of Air. — Miquel, Hesse, P. Frankland,
Carnelly, Robertson, and others have investigated the
number of microbes present in various samples of air,
under various conditions (town air, country air, mountain
air, air of schools-, dwelling rooms, hospitals, &c.). The
method is always in principle this : by means of an ordin-
ary gas clock or gasometer a measured quantity of air is
drawn at a moderate rate by means of an aspirator — fall
of water or mercury — through a cylindrical tube (Hesse),
or through a flask (Frankland, Carnelly) containing a
thin layer of solidified nutrient gelatine. Hesse’s tubes
are cylindrical, in which nutrient gelatine while still liquid
has, by slightly rolling the tubes, set at one side in a
thin film ; they are plugged at each end with a sterile india-
rubber stopper containing a sterile glass tube ; to each is
fixed a sterile tube, one is connected during the experiment
with the gas clock, the other with the aspirator ; the time
during, or the rapidity with which the air is drawn through
and the amount of air so drawn through are accurately
noticed ; after the experiment the glass tubes are plugged
with cotton wool so as to serve as a filter against the
entrance of further microbes. In Dr. P. Frankland’s ex-
periments, the air is passed through a sterile plug (asbestos
or glass wool) contained in a tube or flask, and this having
retained all microbes is then thrown into the gelatine ; this
is liquefied and well shaken so as to wash out of the plug
all microbes and to uniformly distribute them in the gelatine.

c. 2

84 MICRO-ORGANISMS AND DISEASE [chap.

The gelatine is then used for making plate-cultivations in
the ordinary plates, or is set as a thin film on the inside of
the flask. The writer uses a glass tube four to six inches
long, half an inch wide, containing in the middle a cotton-
wool or glass-wool plug about one and a half to two inches
long; at each end the tube is plugged with a small cotton-
wool plug. One end is drawn out in the shape of a large

Fig. 14.— Plate-cultivation in which the surface of the Gelatine set in
a Plate-dish had been exposed for Three Minutes to Air in Oxford
Street. Natural size of the colonies.

canula ; the whole is sterilised. When used the plugs of
the ends are removed, the canula end is joined to an as-
pirator and air is drawn through ; at the end of the experi-
ment the ends are again plugged. In order to use it after-
wards for plate-cultivations, the plugs of the ends are
removed, the central plug is then pushed out by means
of a thin glass rod, placed in liquefied nutrient gelatine

v] METHODS OF INOCULATION 85

or Agar, well shaken, and then plate-cultivations are
made.

In Carnelly’s experiments the nutrient gelatine is allowed
to set at the bottom of a large sterile flask, the mouth of
which is closed by a sterile indiarubber stopper, through
which two glass tubes are passed — one long one through
which the air passes from the gas meter, the other a short
one connected with the aspirator : as the air passes into
and out of the flask the microbes are deposited on the
surface of the set gelatine.

Examination of Ice. — A piece of ice is dug out from a
block, the surface of this having been previously well washed
with sterile water ; the piece is placed into a sterile test-
tube and after it has melted is treated like water.

Milk, for the detection of the number and general
character of bacteria, is treated like water — viz., a small
quantity, ^ to 1 cc., is used for ordinary plate-culture. If
bacillus coli, bacillus of typhoid, sewage bacillus, or cholera
vibrio are searched for, the best method is this : —

The whole or half of the quantity of milk sent for ex-
amination is put into a sterile flask or flasks, then of a 5 per
cent, phenol solution is added, so as to make the whole
contain o-o5 per cent, phenol ; then it is incubated at 37s C.
Next day phenol gelatine plates are made as in the case of
water. For cholera vibrios the same method is used as for
water.

Examination of soil , mud, earth, food-stuffs, or any other
solid material. Here, as in the examination of water, the deter-
mination is (a) of the number and {/>) of the character of the
organisms, and it is followed on exactly the lines described
of water examination.

(a) To determine the number : a definite weighed amount
of the solid material is distributed in a definite quantity of

MICRO-ORGANISMS AND DISEASE [char

sterile fluid, salt solution, or distilled water, and then plate-
cultivations in gelatine or Agar are made with definite
quantities of the distribution.

(b) To determine the general character of the microbes,
the colonies in the gelatine or Agar plates are subjected to a
close study in microscopic specimens and in subculture.
For determining the presence of bacillus coli, bacillus of
typhoid, the sewage variety of proteus Zenkeri, particles of
the solid matter are inoculated into melted phenol gelatine
or. phenol broth and then proceeded with as in the case
of water. For detection of the diphtheria bacilli see the
chapter on diagnosis of diphtheria.

For the diagnosis of the cholera vibrio, particles of the
material are inoculated into tubes containing i per cent, of
peptone and ^ per cent, salt, incubated at 370 C. for twelve
to twenty-four hours and examined in the- same way as
mentioned of water.

Methods of Anaerobic Cultivation. — If it is required to grow
and isolate bacteria which cannot grow or only very slowly and
feebly in free air, it is necessary to make anaerobic cultures.
Various methods and modifications have been designed for
this purpose, but I have found in actual practice that all those
species which have hitherto been described can be grown in
the depth of grape sugar gelatine (at 2o°C.) or in grape sugar
broth or grrfpe sugar Agar (at 370 C.), without any difficult)-.
A test tube containing to two-thirds its height the solid sugar
gelatine or solid sugar Agar, or fluid sugar broth, is easily in-
oculated in the deeper parts — i.e., that nearer the bottom than
the surface of the tube — by means of a capillary pipette con-
taining the bacteria in fluid suspension, the pipette being
well pushed down into the medium and a droplet pressed
out by blowing. The tube is then sealed up with tissue
paper or paraffin or indiarubber.

v] METHODS OF INOCULATION 87

Buchner places the culture-tubes, after inoculation,
plugged simply with cotton wool but not sealed, into a glass
bottle, and then adds into this carefully and liberally
pyrogallic acid and liquor potassse (for each gramme of
pyrogallic acid 1 cc. of liquor potassse) and hermetically
closes the bottle. I have not found any advantage in
using other methods over the two just described, and I
use the second or Buchner’s method to grow anaerobic
microbes on the slanting surface of solid media.

CHAPTER VI

GENERAL CHARACTERS OF BACTERIA

Bacteria are minute organisms not containing chlorophyll,
and multiplying by fision — hence the term schizomycetes (v-
Nageli). They are composed of a kind of protoplasm, the
mycoprotein of Nencki, and are invested with a membrane,
which is composed chiefly of cellulose and a certain amount
of mycoprotein (Nencki).

Their contents are transparent and clear, but sometimes
contain minute bright granules of sulphur (Beggiatoa).
Owing to the cellulose membrane they resist the action of
acids and alkalies. Under favourable conditions of growth
bacteria are able by rapid multiplication to form colonies ;
the individuals are then embedded in a hyaline gelatinous
matrix produced by them ; this is also mycoprotein. Some
species are possessed of one, two or more straight or wavy
or spiral cilia or flagella, and thereby they are capable of
locomotion ; some darting through or spinning round in the
fluid in which they are suspended. Such is the case with
some kinds of bacilli and spirilla as will be described later.

Bacteria grow best when left undisturbed in the dark :
movement of the vessel in which they grow is not advan-
tageous. Light and electricity do not appear to have a

CH. vi] GENERAL CHARACTERS OF BACTERIA 89

decided influence on some bacteria^ since they grow well in
the light, while on others diffuse daylight, and still more
decidedly direct sunlight has a strongly deleterious effect.
According to Cohn and Mendelssohn,1 strong electric
currents have a noxious influence on the growth of micro-
cocci.

Engelmann 2 describes a bacterium photometricum, the
motility of which directly depends on light ; it ceases in
the dark. Duclaux found that exposure to direct sunlight
injures the life and growth of some bacteria, both septic and
pathogenic.

The powerful inhibitory influence which insolation has on
the 'growth and life of aerobic bacteria has been first in-
vestigated by Duclaux, then by Downes and Lunt, and
more recently by Buchner, Marshall Ward and others.
This latter observer was the first to demonstrate the
important differences of action that exist in the red and
blue end of the spectrum, the latter acting more decidedly
bactericidal than the former. Dr. Westbrooke made the
important contribution to this subject by showing that this
germicidal action of sunlight depends on, or rather comes
into play during free supply of oxygen, that this action is
absent when oxygen is absent (or for instance in the case of
anaerobic microbes which grow only in absence of oxygen) ;
he further suggests that in the case of aerobic bacteria the
germicidal influence of light may be due to oxydising or
ozonising influences.

Bacteria may be roughly divided after Pasteur into two
great groups, according to whether they grow under, and
require free access of oxygen — aerobic , or whether they
can do and grow better without it — anaerobic. On more

1 Cohn’s Beitr. z. Biol. J. P/I. Bd. iii. 1.

- (Jitters, aus. d. physiol. Labor. Utrecht, 1882.

yo MICRO-ORGANISMS AND DISEASE [chap.

careful examination, however, it is found that while some
bacteria cannot at all or only very feebly grow in air (or
oxygen), there are others which cannot at all or only very
feebly grow without it : the first are obligatory anaerobic,
the second obligatory aerobic. Further it is found that
some bacteria can grow fairly well without oxygen, but grow
very much faster and more copiously under free access of
oxygen (air) ; these are facultative anaerobic ; while other
bacteria can grow fairly well under free access of air but
grow much better without it ; these are facultative aerobic.

Thus the bacillus of malignant oedema, quarter evil or
symptomatic anthrax, of tetanus Clostridium butyricum are
obligatory anaerobic, though also these are capable of
becoming more or less accustomed by subcultures to grow
on the surface under access of air.

The bacillus of anthrax, bacillus mesentericus, bacillus
prodigiosus, bacillus tuberculosis, bacillus coli and typhosus,
bacillus diphtheriae, vibrio of cholera and many others grow
best aerobically and show either no growth or only very
feebly so in the absence of free oxygen.

For a large number of bacteria it is difficult to assign a
correct place amongst the facultative anaerobic or faculta-
tive aerobic bacteria, because the boundary line between
these is somewhat ill defined.

The growth and multiplication of bacteria is cceteris
paribus principally influenced by the nature of the nutritive
medium. Since the substance of bacteria contains
proteid, all bacteria obviously require for their growth and
multiplication nitrogenous matter which in most instances
of pathogenic bacteria must be of the nature of albumin.
While there are bacteria which can exist on extremely
simple nitrogenous matter — e.g. ammonium carbonate — as is
the case with the nitrifying microbes, there are others

vi] GENERAL CHARACTERS ^OF BACTERIA

which can obtain this nitrogen from air and from nitrates,

e,g' bacillus radicicola — a bacterium which forms part of the

substance of the nodules on the roots of leguminosae, other
bacteria can exist on organic nitrogen in low composition
—e.g. ammonium tartrate (in Pasteur’s and Cohn’s fluid) ;
or urea (micrococcus ureae and other bacteria that thiive

Fig. 15. — Gelatine Plate-cultivation of Bacillus Radicicola, the
Colonies are liquefying.

Natural Size.

in urine). Most bacteria thrive well in media like the usual
culture media containing albuminous substances. But also
in this latter case great differences exist ; while for instance,
the bacteria occurring in water (bacillusfluorescens liquescens,
bacillus sulcatus and others) can even when the water contains
only traces of albuminous matter, well thrive therein and under

92 MICRO-ORGANISMS AND DISEASE [chap.

favourable conditions of temperature can rapidly and
strikingly multiply, there are bacteria which under ordinary
conditions live on rich albuminous food and for their
multiplication require a comparatively large amount of
albuminous matter. Of this nature are most pathogenic
bacteria, for their natural breeding-ground are the animal

Fig. 16. — Stab-cultivation in Gelatine of the Liquefying Bacillus

Radicicola.

Natural Size.

tissues, and the preparation of all our culture media
previously described is based on this fact.

All nutritive media must contain salts (sodium or po-
tassium salts), in some cases the addition of particular salts
(nitrates, phosphates) enhances the growth. Some bacteria
require other special additions — e.g. grape sugar in the case
of bacillus of Koch’s malignant oedema, bacillus of

vi] GENERAL CHARACTERS ^)F BACTERIA

symptomatic anthrax, bacillus of tetanus ; asparagin and
sodium salt in the case of phosphorescent bacteria (Bey-
rinck), milk sugar in the case of bacterium lactis.

The nature of the nutritive medium has in many cases an
important effect not only on the morphology but also on the
physiological action of bacteria in general, and of pathogenic

Fig. 17. — Impression of a very young Growth of Bacillus
Radicicola.

X 1000

bacteria especially. Thus, for instance, the addition of
excess of salt to nutritive gelatine affects considerably the
morphology of a series of bacteria — e.g. bacillus coli and its
varieties. This in ordinary gelatine are mostly short oval
bacteria, some are cylindrical, and few even threadlike, but
if an excess of chloride of sodium is added most of these
bacteria grow out into threads, some of great length. Or

MICRO-ORGANISMS ANI) DISEASE [chap.

take bacillus anthracis, this bacillus in nutritive gelatine
(beef broth, peptone, gelatine) often forms already during the
first twenty-four to forty-eight hours toruia-like chains and
filaments, of which the elements are spindle-shaped or pear-
shaped. Bacillus diphtheriae grown on nutritive Agar,
forms already after forty-eight hours longish jointed filaments,
whereas in gelatine most of the bacilli are short cylindrical.
The cholera vibrios forms in fluid media in a few days longish
spirals, on solid media it sometimes takes weeks. I have
isolated from the human tonsils a microbe which, grown on
blood serum or Agar forms exquisite commas, semicircular
forms, and spiral and wavy threads, grown on gelatine
the microbes are rod-shaped with just a faint indication of
curvature, transferred back on to serum or Agar they
promptly yield commas, semicircles, and spirals. Some species
of streptococci — eg. streptococcus pyogenes and erysipelas
form long and exquisite chains in fluid, very short chains
and diplococci on solid media, &c. And the same applies
to physiological action ; thus the tubercle bacillus grown on
glycerine Agar after a series of transfers on glycerine Agar
loses considerably in virulence when tested on the guinea-
pig, while when grown on serum formally generations retains
its full virulence. When it is kept growing on glycerine Agar
for a considerable series of generations it loses almost
entirely its virulence, but when so weakened it is replanted
in glycerine serum it soon regains it.

Many instances can be mentioned when similar alterations
in physiological functions of bacteria take place differing with
the medium in which they are grown.

The temperatures at which bacteria best grow show con-
siderable differences : ( a ) while some grow best at tempera-
tures at or below 20 or 210 C., and do not grow at all, or
only very feebly, above these— eg. bacillus prodigiosus, vibrio

Vi] GENERAL CHARACTERS qF BACTERIA 95

Finkler, certain water bacteria (bacillus sulcatus) ; (/>) the ma-
jority grow well not only at low temperatures but grow best at
temperatures above 22° C. ; and a still further group (c) com-
prises bacteria which do not grow at all or only very feebly
at temperatures below 22° C. To the latter class belong the
pneumococcus of Fraenkel, the bacillus tuberculosis. Amongst
groups b and c the optimum temperatures lie between 28
and 38° C. Fligher temperatures than 38° C. have on these
two groups a retarding influence, which is feeble one or two
degrees above this figure, but becomes in many cases pro-
nounced above 40° C. But this is not the case with all,
since, for instance, bacillus anthracis and bacillus tuber-
culosis at 42 -5 or even 430 C. show still copious growth.

Miquel described one species of spore-forming bacillus,
which, strange to say, has its natural habitat in soil, the
bacillus termophilus ; it grows well at temperatures at
which other bacteria are prevented from growing, injured and
even killed; this bacillus termophilus grows well at 65° C.,
and forms spores at 70-75° C.

In all cases of bacteria that do not form spores — and the
majority of species are of this kind — an exposure to a tem-
perature of 60-70° C. for ten to thirty minutes devitalises
them, but there are slight differences to be noticed — e.g.
whereas the bacillus coli is not killed by exposure for five
minutes to a temperature of 62° C., the typhoid bacillus is
killed under these conditions. All non-sporing bacteria
(also those that are capable of forming spores but do not
contain them, or had not formed them yet in particular
cultures) are invariably killed when exposed for five to ten
minutes to a temperature of 70° C.

Spores of bacilli are not killed even by an exposure to
98" C. for a minute or two; there exist differences in this,
for while the spores of bacillus anthracis arc killed at ioo° C.

96 MICRO-ORGANISMS AND DISEASE [chap.

in half a minute, those of bacillus subtilis (hay bacillus) are
not killed at this temperature in less than five to seven
minutes, and some spores (in some species of bacillus mes-
entericus) require ten and even fifteen minutes’ exposure.

Owing to the great resistance of spores to heat it is possible
and is easy in a mixture of non-sporing and sporing bacilli
to separate the former from the latter, by subjecting the fluid
containing the mixture to a temperature of 70° C. for five or
ten minutes, here by all non-sporing forms are killed whereas
the spores remain unharmed, and cultures made from the so
heated mixture produce growths of the spores only.

I will take a case in point. If a guinea-pig be injected
subcutaneously in the groin with a fair quantity of recently
manured garden earth, it dies as a rule in twenty-four to
thirty-six hours from Koch’s malignant oedema ; the mal-
odorous sanguineous fluid in the subcutaneous tissue of the
groin, thigh, abdomen, and chest contains large numbers of
motile and non-motile bacilli, some of these latter contain-
ing bright oval spores ; the fluid is collected in a sterile
test-tube and this is kept exposed for ten minutes to a tem-
perature of 70° C., then anaerobic cultures are made in
grape sugar gelatine by deep inoculation. The tubes are
then sealed and incubated at 20° C. The growth that
makes its appearance now is a pure growth of the anaerobic,
liquefying bacillus of Koch’s malignant oedema. But if
a culture be made with the subcutaneous fluid not heated,
generally only a copious growth of a bacillus similar to the
bacillus coli is produced, at best an abundance of the latter
and a very scarce growth of the former (Koch’s malignant
oedema) is produced. And the same method of separating
the spores of any other microbe : anthrax, symptomatic
anthrax, tetanus bacillus, butyricus from a non-sporing
microbe that happened to be coexistent in a given material:

Vi] GENERAL CHARACTERS OF BACTERIA 97

solids and fluids, as exudations, water, cultures, tissues, &c.,
can be successfully employed.

Growth and Division } — The rapidity with which bacteria
grow and multiply is subject to very great variations, and
Cceteris paribus constitutes definite and characteristic pecu-
liarities ; that is to say, some species Under the same
conditions of soil, temperature, &c., show a more rapid
growth and multiplication than others, these bearing no
relation to any known condition. Thus of the staphy-
lococcus. aureus and the streptococcus pyogenes, growing
under exactly the same conditions, and on good nutritive
media, the former shows incomparably greater rapidity in
multiplication, and produces much more copious growth in
a given time than the latter ; or if the bacillus of swine
erysipelas and the bacillus of swine fever be taken, the
latter is found to grow much more rapidly than the former ;
and, again, bacillus subtilis and Tinkler's spirillum grow
very much faster than bacillus anthracis and cholera spirillum
respectively.

Comparative experiments which the writer has made with
a number of microbes as to the rapidity of multiplication,
by way of observing them directly under the microscope in
a drop of solidified nutrient gelatine at 22° C. (“ suspended
solid drop ”) show as the average of several observations —

(a) The streptococcus pyogenes. Complete division of
the cocci took place in thirty minutes.

(b) The staphylococcus aureus liquescens in twenty

minutes. ; ;

{0) I he streptococcus of erysipelas in forty-five minutes.

(d) An orange coloured non-liquefying micrococcus in
forty minutes.

From Klein s article “Infectious Diseases ” in Stevenson and
Murphy’s Treatise on Hygiene , &c., vol. ii., pp. 19-23.

98 MICRO-ORGANISMS AND DISEASE [chap.

(e) The bacillus anthracis in thirty minutes.

(/) The bacillus subtilis of hay infusion in twenty
minutes. ^

(, g ) A filamentous bacillus liquefying gelatine, not mobile
and isolated from sewage, in eighteen minutes.

(h) A mobile bacillus (bacillus fluorescens liquescens),
rapidly liquefying gelatine and common in ordinary London
drinking water, in eighteen minutes.

(t) A bacillus, non-mobile, non-liquefying, rapidly form-
ing spores, and slightly filamentous, isolated from London
sewage, in forty minutes.

(/) The bacillus of the Middlesbrough pneumonia in
eighteen minutes.

{k) The bacillus of fowl enteritis in twenty-four minutes.

(/) The bacillus of typhoid fever in thirty minutes.

(;«) The bacillus diptherise in forty-five minutes.

In all these instances a single organism lying isolated was
focussed and watched, and, after a distinct division had
been noticed, the time was marked, and the interval it took
for one of these to again completely divide was taken as the
time for a division. In these observations, which do not
claim more than approximate accuracy, it was remarked
that the division of the two members of the dumb-bell cocci
or dumb-bell rods does not proceed at the same rate, the
difference being as much as a quarter to a third of the whole
time. The above numbers indicate the average of three
successive divisions, and therefore they only represent
approximately the main periods that these several microbes
require for dividing under the above conditions. Buchner
( Cent r alb 1. fur Bad. und Parasit. II. No. 1) calculated
the time required for the cholera vibrio for a division at
370 C., and found it to amount to twenty minutes on
an average.

Vi] GENERAL CHARACTERS OE BACTERIA 99

Observations were made on the common staphylococcus
pyogenes aureus, the bacillus of swine fever, the bacillus of
grouse disease, the bacillus of fowl enteritis, and the bacillus
of diphtheria, as to the amount of multiplication these
several microbes undergo when a definite number of them
is introduced into faintly alkaline beef broth (eight to ten
cubic centimetres), and kept in the incubator at about
370 C. All these different organisms grow with great
rapidity, and after twenty-four hours the broth is uniformly
turbid, provided the number introduced at starting be com-
paratively large. By making gelatine plate-cultivations with
a given small quantity of the broth previously diluted to a
definite degree, and then counting the number of colonies
that make their appearance on incubation, it is easy to
calculate the number of microbes present per cubic centi-
metre in the broth. In some experiments made with the
staphylococcus pyogenes aureus it was found that on intro-
ducing 248 microbes per cubic centimetre, they increased
in the first twenty-four hours to 20,000,000 per cubic centi-
metre; in another experiment 640,000 per cubic centimetre
were counted after the first twenty-four hours’ growth,

248.000. 000 per cubic centimetre after the second twenty-
four hours — i.e. after forty-eight hours’ incubation, and

1.184.000. 000 per cubic centimetre after the third twenty-
four hours — i.e. after seventy-two hours’ incubation. From
a number of experiments it was calculated that for each
microbe introduced, the multiplication during the first
twenty-four hours is 80,000 fold, during the second twenty-
four hours 400-fold, and during the third twenty-four hours
5-fold.

The rapidity of the growth and multiplication of the
bacillus of fowl cholera in the living blood was ascertained
in an experiment made on a rabbit. Of the microbes

h 2

too MICRO-ORGANISMS AND DISEASE [chap.

20,000 were subcutaneously injected into a rabbit. The
animal died in about twenty hours. The bacilli in the
heart’s blood were then counted by the ordinary methoA of
gelatine plate-cultivation, and it was found that their
number per cubic centimetre of heart’s blood amounted to

14.150.000. The weight of the rabbit was 1,250 grammes,
and taking eighty-three grammes (^5) as the amount of
blood present in the animal’s body, and assuming that the
bacilli were more or less uniformly distributed through the
blood, it follows that the total blood contained about

1.200.000. 000 of the bacilli. This would mean that each
one of the 20,000 bacilli injected had given origin to a host
of 60,000 bacilli in twenty hours.

The manner in which the individuals of the same species
divide varies considerably ; thus in the streptococcus scar-
latinae and str. pyogenes the writer has observed that in
gelatine some of the elements of a colony increase rapidly
to five, six, and more times the size of a typical coccus,
grow, in fact, into a ball of great size, then a cleft appears
by which the organism splits up into two demilunes, then
each of these again divides under a right angle to the
former line of division, so that the original ball is divided
into four quarters, each of which separates gradually from
its neighbour and becomes more or less spherical, and a
further division into two, and even into four, cocci of the
average size takes place. But the above mode of division
does not take place everywhere in the preparation, for many
of the typical cocci only slightly enlarge and then divide
into two, thus forming a diplococcus ; each of these divides
again transversely, and thus a chain of four minute cocci is
the result.

In broth cultures the writer has observed, as a rule, the
latter mode of division, though also here occasionally an

VI] GENERAL CHARACTERS OF BACTERIA ioi

element is noticed in a chain which is much larger than the
rest, and this larger element divides into two and four cocci
successively. So also those large elements described above
as occurring in the chains of streptococci show the suc-
cessive fission into two and four cocci. And it is this which
prompts him to say that these large elements found oc-
casionally in the chains or in the diplococci are not in-
volution forms, but are active elements which before
successively dividing grow up to large size. In staphylo-
coccus aureus liquescens, growing in gelatine, he has also
observed some of these large elements, though on the
whole they are not so numerous as in the streptococcus
growing in the same kind of medium. The normal mode
of division of a coccus is then (i) a slight enlargement
and division into two by transverse fissure, or (2) a coccus
enlarged to considerable size (four to six and more times)
and then successively divided into two and four and further
eight cocci of the normal size.

As regards bacilli all observations hitherto recorded agree
that a rod before dividing elongates sometimes more some-
times less, and then a transverse indentation appears about
midway, which ultimately becomes a fissure by which the
originally single rod divides into two ; according as the rod
was short or long, the resulting offsprings are more coccus-
like or more cylindrical. Now, in the observations which
the writer has carried out as to the time of the division of
the different microbes mentioned above the writer has
repeatedly noticed that a single cylindrical bacillus not
infrequently divides almost simultaneously into three and
even four short rods. The writer has observed cylindrical
bacilli in preparations of bacillus anthracis, made directly
from the blood of guinea-pigs, which were uniform, and
there was no indication in the fresh specimen that they

102 MICRO-ORGANISMS AND DISEASE [chap.

were other than single elements. These elements he has
seen to give origin almost simultaneously to as many as
four short slightly rod-shaped elements ; and these same
elements were, on continued observation, seen to elongate
and the terminals within several minutes seen to have in-
creased almost to twice their length, then each of them
again to have divided, one into three, the other into two
distinct rods.

Observations were carried out on a filamentous bacillus
isolated from sewage liquefying gelatine as a clear fluid ;
it was non-motile, rapidly growing into threads, and in the
filaments copious spore formation took place ; this bacillus
resembled morphologically the bacillus antbracis, but it
grows on the surface of gelatine more as a continuous
membrane of threads arranged parallel and coming off" at
right angle from a central stalk ; it grows much more
rapidly than the bacillus antbracis.

Now, directly observing under the microscope the growth
and multiplication of this bacillus in solidified gelatine,
threads of bacilli are seen shooting out with considerable
rapidity from a short cylindrical bacillus measuring 0-5 /x,
to iyu,, a thread more or less wavy is formed in the course of
two hours and a half, which extends across the whole field
of the microscope under a magnifying power of 500. On
such a growing thread the simultaneous division after elonga-
tion of cylindrical elements into three and four rods is also
distinctly and repeatedly noticed.

Also on the rods of the bacillus of diphtheria the same
simultaneous fission of elementary cylindrical cells into two,
three, and four elements was noticed. We conclude then
that in the division of bacilli the elements increase in length
and then by transverse fission divide into two, three, or four
elements, and according to the length of the cell before

VI] GENERAL CHARACTERS OF BACTERIA 103

division the elements resulting from the division differ in
length.

Spores. — One of the most important and interesting
phenomena in the life-history of bacteria is the power of
some species to form permanent seeds or spores , by which the
species can preserve itself and can withstand a variety of
adverse circumstances. Various conditions in nature
are often at play, in consequence of which weaker
species are less liable to survive in the severe struggle for
existence. There is first the adverse circumstance of com-
petition, such as constantly obtains under the general con-
ditions of growth in soil, in water, and in various organic
materials exposed to contamination from air, water, and soil.
Here numerous species find access and multiply, some
more, others less easily, till all the available nutriment is
exhausted. Some species, capable of forming spores, when
this stage of the exhaustion of the nutriment has been
reached, remain as spores and, till they are transferred by
some means or other to new material, or till new nutriment
is added, retain their power of again germinating and giving
rise to a new crop of the same species, and this survival
occurs even under severe adverse circumstances — e.g., the
presence of various noxious chemicals, cold, heat, drying,
&c. ; but those species that do not form spores retain life
only under exceptionally favourable conditions : as a rule,
owing to the presence of acids or other chemicals, e.g.,
products of the growth of bacteria, and owing to drying, &c.,
they are easily deprived of life. This question of the
formation of spores for the above reasons plays a most
prominent role as regards infectious diseases. A few
illustrations will easily show this. Take, for instance, the
bacillus anthracis. This organism, although present in
enormous numbers in the blood and blood-vessels of

104

MICRO-ORGANISMS AND DISEASE [chap.

animals dead of the disease, does not at any time form
spores when kept away from the air — i.e., from a supply of
oxygen ; consequently in such an animal when left unopened
all the bacilli, after having gone on increasing in numbers
after death for some time, gradually degenerate and dis-
appear, so that sometimes after five to eight days in the case
of small animals like mice and guinea-pigs, living anthrax
bacilli are no longer to be found in the tissues, they having
been suppressed by putrefactive organisms. The spleen of
such an animal after this distance of time produces no in-
fection with anthrax after inoculating it in comparatively
large doses into a fresh guinea-pig or mouse ; whereas if a
trace of a droplet of the splenic blood of an animal dead
of anthrax is used for inoculation of a guinea-pig or mouse,
say within three days after death, virulent anthrax follows.
Or if the blood and tissues of an animal dead of virulent
anthrax are by some means or other thoroughly dried, such
blood loses all virulent power, since by thorough drying the
bacilli anthracis are killed. But let either the blood or the
nasal or other discharges of an animal dead from anthrax
be exposed to air for a sufficient time to allow the
bacilli to form spores, then neither putrefaction, nor drying,
nor chemical agencies such as acids and alkalies, will affect
the power of these spores to germinate again into bacilli and
to produce virulent anthrax when finding access to a suitable
animal body. This is actually the case when cattle and sheep
are sojourning on and feeding in a field, where months or
even years previously an animal having died from anthrax,
the blood and discharges of such an animal found access
to the surface of the soil, that is where the bacilli anthracis
find opportunity to multiply and to form spores. It is
these spores which afterwards are picked up by the animals
grazing in such a field. The same thing occurs in wool-

VI] GENERAL CHARACTERS OF BACTERIA 105

sorters’ and hide-sorters’ disease, which is virulent anthrax
in the human beings engaged in the sorting of wool or the
handling of hides derived from animals — sheep, goats, and
cattle respectively— which had succumbed to fatal anthrax.
In these cases it is always spores of the bacillus anthracis
which are the cause of infection of the human beings
handling these articles.

Observing bacilli, which do form spores (e.g. bacillus
subtilis, various species of “ potato bacillus,” bacillus
mesentericus, bacillus anthracis, and the bacillus fila-
mentosus above mentioned), it is noticed that the first sign
of the appearances of spores is indicated by the presence
of a bright, glistening globule in the protoplasm of the
bacillus ; at the same time the bacillus is distinctly broader
and paler in its substance as compared with the other
bacilli. This globule gradually enlarges in diameter,
becoming at the same time slightly oval ; this continues till
the thickness of the globule often exceeds the breadth of
the bacillus, this latter being now markedly pale and trans-
parent. The writer has watched in bacillus anthracis and
bacillus filamentosus the spores from their first appearance
as bright globules till they had reached their full thickness
and length ; this took about three hours, and he has also
noticed that after sowing on the surface of solidified agar
the blood of the heart or spleen of a guinea-pig dead of
anthrax and keeping it under observation at the tempera-
ture of 200 C. spores would be noticed in a few of the
bacillary filaments after twelve hours ; in the case of the
bacillus subtilis, various potato bacilli, and bacillus fila-
mentosus growing in broth, copious spore formation was
noticed in a superficial pellicle after sixteen hours. Koch
first observed that spore formation in bacillus anthracis
occurred after six hours. But not all bright granules that

io6

MICRO-ORGANISMS AND DISEASE [chap.

make their appearance in bacilli arc spores ; thus in the
typhoid bacillus growing on potato, and in other species of
bacilli growing on potato, there appear bright granules,
either terminally or centrally, which are not spores ; they
do not show the reaction of spores, either to dyes, or on
drying, or heating. Nor are all the bright granules that
make their appearance in bacilli capable of forming spores
to be at once taken as spores, since under certain con-
ditions such granules do occur, but never reach the size of
full spores ; this is observed occasionally in anthrax bacilli
when growing under conditions unfavourable for the forma-
tion and development of spores. The appearance of real
spores in all bacillary species is very characteristic : the spores
are of a bright, glistening aspect, are oval in shape, and gener-
ally thicker than the typical bacilli. The substance of these
latter is at the same time pale and transparent, and broader
than the bacilli not containing spores; the threads of the
bacilli appear beaded by the spores, the beads being the
glistening oval thick spores, while the rest of the thread is
pale and appears thinner. In the single bacilli the spores
are placed either centrally or terminally ; in the latter case,
if the bacillus is of some length, it looks not unlike a
spermatozoon, the spore corresponding to the head, the
bacillus to the tail. Sometimes in motile bacilli short
chains are noticed, in which, in one terminal element, a
spore has already made its appearance, while the other
bacillus is still possessed of motility ; and here, on account
of the motility, the resemblance to a spermatozoon is still
more striking. Under the most favourable conditions
almost every element constituting a bacillary thread or
chain forms a spore, in other threads only here and there
a cell contains a spore ; in the first case the thread is
regularly and densely beaded, in the latter the beads are

Vi] GENERAL CHARACTERS OF BACTERIA 107

relatively few and far between. The last phase is reached
when the bacillus itself swells up into a gelatinous capsule
enveloping the spore and ultimately altogether disappears ;
then the spore is free and has reached its full size and
development. Examining in stained specimens spore-
hearing bacilli, the spores appear unstained, whereas the
rest of the bacillary substance takes readily the dye ; under
these conditions the spore looks like an oval clear space,
not unlike the vacuoles above mentioned ; but the spore
has a sharp outline of its own, the vacuole has not. It is,
however, not easy to distinguish in a given specimen,
stained after the ordinary methods, the spores which are not
stained from vacuoles, and in these cases other methods of
staining must be resorted to.

In order, then, to decide whether or not spores are pre-
sent in a bacillary species, the morphological investigation,
fresh aspect, special methods of staining, drying, and heating
have to be resorted to.

The spore formation is associated with supply of oxygen
in all bacteria that generally live well under access of air ;
in some species this is more pronounced than in others, for
in some species — e.g. bacillus anthracis, and bacillus fila-
mentosus, spores are only formed —cceteris paribus — if oxygen
has free access, and no spores are formed if there is no free
supply of oxygen — e.g., deep in the fluid ; while in other
species, though spore formation is greatly enhanced by the
free access of oxygen, it nevertheless takes place to a certain
limited extent deep in the fluids. Thus, in the case of
bacillus subtilis and various other bacilli a pellicle soon
makes its appearance on the surface of the fluid (broth, &c.).

1 his pellicle is made up of filaments and bacilli matted
together, and in them copious spore formation is going on,
but also in the depth there arc a few spore containing bacilli

10S MICRO-ORGANISMS AND DISEASE [chap.

to be noticed. When such a pellicle is broken up by
shaking, it in most instances falls to the bottom of the fluid,
and then after another day’s growth a new pellicle appears,
and in this also copious spore formation is noticed ; and
this can be repeated for several days till the nutriment is
exhausted. The same can be seen in hay infusion, in the
case of bacillus subtilis. In neutral or faintly alkaline hay
infusion kept at 370 C. spores of bacillus subtilis are present
in the pellicle as early as the second day, and continue to
be formed till the end of eight to ten days. The view has
been expressed by some observers, amongst them Buchner,
that the spore formation in bacilli occurs on exhaustion of
the nutritive material, but it seems that the facts just men-
tioned as to the continuous and successive pellicle and spore
formation occurring in broth are incompatible with that
assertion ; and, besides, the formation of spores in other
bacilli can be shown to take place long before any exhaustion
of the nutritive matter is noticeable — e.g., in anthrax bacilli,
in the tetanus bacillus, and others. In the bacillus filamen-
tosus, growing on Agar or on potato, the spore formation is
apparent even before the first day is over and long before
the active growth and multiplication of bacilli all round
is finished. In some species, however, that do not thrive
under free access of air (e.g., oedema bacillus, tetanus ba-
cillus) spore formation does not take place if free oxygen is
present. A temperature of at least 16° C. is required for the
formation of spores, though spore formation occurs in all
temperatures between that and 450; at least spore formation
has been seen to occur in bacillus anthracis even at 450 C.
The mode of spore formation hitherto described is called
that of endo-spores, and it ought to be here stated that many
species of bacilli exist in which no spore formation can be
demonstrated — in this statement we rely on the morpho-

Vi] GENERAL CHARACTERS OF BACTERIA lod,

logical as well as the experimental test — e.g., typhoid fever
bacillus, bacillus of glanders, of diphtheria, of fowl cholera,
of fowl enteritis, and many others. So far as actual demon-
stration is concerned no other mode of spore formation can
be accepted at present. A mode of formation of spores is
described by Hueppe to occur in certain spirilla, according
to whom the comma-shaped elements and the spirilla form
special aggregations of protoplasm in the shape of terminal
granules, to which the value of spores is ascribed, and which
are called arthro-spores. But the evidence and proof for
this is quite unsatisfactory, and, judging these appearances
in the light of the character of well-ascertained spores of
other bacilli, they are contrary to the assumption of spores.
These arthro-spores of Hueppe do not look like spores, do
not behave in staining like spores, and do not behave in
drying and heating experiments like spores. In the first
place they do not differ in aspect from ordinary protoplas-
mic granules observable in some of these bacilli under all
conditions ; they stain in the ordinary dyes and after the
ordinary methods like the ordinary protoplasmic contents of
bacteria ; and they are killed by drying and exposure to 6o°
C. for five minutes.

It can be easily shown that artificial cultures of these
comma bacilli growing under conditions very favourable
for the formation of real spores in other bacilli — e.g., a good
supply of oxygen, temperature, soil, and moisture — contain
after some weeks and months those granules or supposed
arthrospores in enormous numbers ; in fact, there is almost
nothing else left, and yet no subcultures can be established
from such a culture, it being barren of all life. Structures have
been described also in the typhoid bacilli as occurring in
potato cultures, which can be, however, shown by special
modes of staining to be different from real spores, and the

no MICRO-ORGANISMS AND DISEASE [chap.

experimental test of drying and heating conclusively proves
that they are not comparable to spores.

On the other hand the tubercle bacilli have been shown
experimentally (by Koch and others) to possess spores,
although it seems difficult to identify them under the micro-
scope. True, there are present in microscopic specimens made
of fresh material — e.g., tubercular sputum — bright granules
within many of the bacilli which might be taken for spores,
and in specimens stained after the customary method of
staining for tubercle bacilli numerous stained granules.occur
in the bacilli— the bacilli appearing beaded — but, as has
been stated above, most of them are merely elementary
masses of protoplasm segregated in the bacilli. They occur
in some tubercle bacilli more numerously than in others —
e.g., in the tubercle bacilli of the human subject they are
common in the tubercular material of the fowl and in
artificial cultures they are sometimes seen with great regu-
larity, but there is no means available of identifying these
granules with spores. But by thorough drying of tubercular
material it can be shown that the tubercle microbes remain
uninjured, and that heating them up to ioo° C. for a minute
leaves the tubercle bacilli unharmed.

Spores have been described also of some micrococci, but
here again certain differentiation of structure cannot betaken
as proving the existence of spores. The writer has exa-
mined very numerous preparations of the most varied cul-
tures of different species of micrococci, and the test of
drying and heating to 70° C. proves them barren of any-
thing comparable to the well-known spores present in some
species of bacilli. He also examined experimentally various
species of spirilla and agrees with Koch that no spore for-
mation can be demonstrated in them. We arrive then at
the conclusion that real spore formation, or the formation

VI] GENERAL CHARACTERS OF BACTERIA in

of permanent seeds capable of retaining life under very
adverse conditions, and under favourable conditions capable
of germinating and of giving origin to a new brood of the
same species, can be shown to exist only in certain limited
species of bacilli, but not in micrococci or spirilla. These
real spores are endo-spores, they are formed within the
protoplasm of the bacillary elements under favourable con-
ditions, and they have certain definite morphological and
experimental characters of their own, at the same time re-
presenting as it were the last phase in the life-history of these
bacilli. Bacilli or bacteria not capable of this power of
producing spores, though they go on multiplying as long as
the conditions of nutriment, temperature, chemical by-
products, competition, &c., permit of it, ultimately degen-
erate, some sooner, some later. To propagate their species
they must as living bacilli find access to new soil before the
stage of degeneration is reached, whereas in the spore-bear-
ing bacteria their spores can remain dormant, but possessing
potential life for indefinite periods.

The statement has been occasionally made that spores
are capable of dividing, and thus giving origin to two new
spores. The writer has not been able to detect anything of
this sort ; he has never seen any appearances that would
indicate such a division. True, in bacillus anthracis, in
bacillus filamentosus, and in some species of “ potato
bacillus,” spore formation may be going on so copiously that
at some places every element constituting the threads con-
tains a spore, some of the spores closely adjoining one
another, sometimes so closely that it looked as if they were
the two elements of a dumb-bell, and here a division of one
spore into two could be thought of ; but in these places the
elements of the threads were extremely short and the spore
occupied the main part of each element. It is also a fact

i 12 MICRO-ORGANISMS AND DISEASE [chap.

that spherical globules occurring in some bacilli and being
in aspect and staining power comparable to young phases of
spores, are occasionally met with as dumb-bells within the
same element, but as regards the oval, bright, unmistakable
spores so prominent in the bacilli and their threads (bacillus
anthracis, bacillus subtilis, bacillus filamentosus, bacillus
mesentericus, &c.) it is very doubtful whether they are
capable of dividing or of undergoing any other change than
that of germination into bacilli when they are transferred to
new soil.

Spores when placed under suitable conditions germinate
again into bacilli. This is easily observed if, for instance,
of any culture-material containing spores a trace is placed
on a cover-glass, then covered with a tiny droplet of
gelatine which is made rapidly to set, or in a droplet of
broth (“ suspended drop ”) and is then observed under the
microscope, particularly in the latter medium, which can be
kept on the warm stage heated to 370 C.

Spores while fresh have a conspicuously sharp and dark
outline, their general aspect is glistening, and it is supposed
by Cohn that they are possessed of a double envelope, an
inner one of a fatty and an outer one of a gelatinous
nature : it is particularly the former which provides the
spores with their great resistance to drying and to heat.
The first indication that the spores are going to germinate
is shown by their outline becoming less sharp at one point.
This is generally at one of the poles, as in the case of
the spores of bacillus anthracis, bacillus filamentosus, and
bacillus subtilis ; or it is at one point of the long side e.g.,
in bacillus amylobacter, and also in the spores of some of
the species collectively spoken of as “potato bacillus” ; the
investment seems to become thinner at that point and a
slight pale knob appears there ; this knob gradually elongates

vi] GENERAL CHARACTERS OF BACTERIA 113

in the form of a pale rod thinner than the spore itself ; as it
elongates it protrudes more and more from the rest of the
spore, its free end being rounded, while at the same time
the rest of the spore outline becomes thinner and less dark ;
ultimately the whole spore has been consumed as it were in
the formation of the rod, which now looks like a cylindrical
bacillus of the same character and aspect as the bacilli from
which originally the spore had been derived. The bacillus
once formed divides and then continues to grow and
multiply. The time required for the production of a
bacillus from a spore varies with the different species.
Koch observed the germination of the spore into a bacillus
anthracis to be completed in about an hour ; the writer has
observed the time required for the complete formation of a
bacillus from a spore of the bacillus filamentosus in broth,
in the “ suspended drop,” at 37° C. to be certainly less than
one hour ; that of bacillus anthracis between one hour and
a half and two hours ; that of the bacillus subtilis of hay
infusion to be more than one half but less than one hour.
Occasionally one meets in these observations with motile
bacilli to which a spore which has not yet commenced to
germinate is attached and is dragged about by the former :
this evidently indicates that of two spores originally joined
by interstitial material {see spores in threads) only one has
already changed into a motile bacillus — the other has not
yet so changed.

Motility d — One of the most interesting phenomena shown
by bacteria is the power of active locomotion possessed by
some species. When examined under the microscope in a fluid
medium all bacteria show the kind of oscillation known as
Brownian molecular movement; but in some species there is

1 Copied from Klein’s article in Stevenson and Murphy, vol. ii.,
p. 13 and passim.

1 14 MICRO-ORGANISMS AND DISEASE [chap.

an active locomotion, by which the individual bacteria are
enabled to move actively and to change their place; this move-
ment shows itself either by the bacteria darting with great
rapidity across the field of the microscope in one or another
direction, or spinning round with greater or lesser velocity,
or briskly moving like a screw in one direction and then
back again. Observing a single straight bacillus in its move-
ment, either a darting or spinning movement in one direc-
tion is noticed ; when two such bacilli are connected end-
wise, but bent one to another under an angle, then often,
with a forward or backward movement of the one, a spinning
movement of the other is noticed, the former not really
actively moving but being simply propelled by the spinning
movement of the latter bent under an angle. When comma-
bacilli or spirilla move, the motion is always more or less
spiral.

When longer chains or leptothrix of bacilli move, the
movement is always more or less serpentine. The loco-
motion of bacilli is either rapid or slow ; the latter may be a
character of the species, that is to say, the individuals as a
rule show only a relatively slow movement — e.g., typhoid
bacilli generally move comparatively slowly, and the longer
bacilli move in a serpentine manner. The mobile indivi-
duals do not continue to move indefinitely, since often an
individual which has been spinning round or darting about
gradually comes to rest and remains so for some time ;
besides this, all motile bacilli during the phase of division
are at rest, and when they form groups — i.e., when they are
in an active state of division — they do not move. But of
such groups here and there an individual may be seen to
separate itself from the margin and to move briskly away ;
on breaking up a group, crowds of motile bacilli sally forth.
The writer has watched single bacilli of the human Middles-

Vi] GENERAL CHARACTERS OF BACTERIA 115

brough pneumonia spinning round with great velocity with-
out much changing their place. One and the same bacillus
was noticed to spin round for five minutes without any
diminution in its velocity ; then this gradually lessened, and
ultimately, after further five minutes, the organism came to
rest. When a drop of broth was added the spinning round
commenced again with great vigour. Some mobile bacilli
show motility under a certain condition and not under
others; others again show it under all conditions. Thus,
for instance, many individuals of the bacillus of the Middles-
brough pneumonia show active locomotion in specimens
made of gelatine and Agar cultures ; made of broth cultures
the motility of many individuals is observable only while
the broth cultures are of recent date — 24-48 hours old ;
later on only very few motile individuals are met with.
The loss of motility may be and sometimes is due to
chemical by-products in the cultivation (see bacillus of
grouse disease and of pneumonia). Some species of motile
bacilli when growing on a solid medium are capable by
their locomotion of distributing themselves from a given
point rapidly over and through the medium — e.g., certain
species of bacilli known as proteus of Hauser, certain
species of the potato bacilli, &c. ; this phenomenon is
spoken of as “swarming,” thus, when a colony of such
bacilli appears on gelatine, Agar mixture, or potato,
irregular streaks and lines and patches of the growth are
soon seen extending in different directions, this being due
to the swarming of the bacilli from the first colony and by
the establishment of new colonies by the former. There
exist great differences in this respect between different
species of motile bacteria, for while some species do not
swarm at all and their colonies on solid media remain
localised and more or less well defined, though they

MICRO-ORGANISMS AND DISEASE [chap.

i 16

Fig 18. — Typhoid Bacilli, showing Flagella.

X 1000.

Fig. iq.— Bacillus Coli, showing Flagella-
X joqo.

Vi] GENERAL CHARACTERS OF BACTERIA 117

gradually enlarge — e.g, bacillus subtilis, bacillus fluorescens,
some species of proteus, and many spirilla ; other species
possess this swarming propensity and therefore the first
colonies do not remain well defined, but gradually extend
in lines and irregular streaks in different directions. But it
is not correct to conclude that a bacillus is motile if its
colonies do not remain defined, that is, if they extend
in the shape of threads or irregular streaks on or through
the medium, for there exist several well studied species
which do this (e.g., bacillus anthracis, bacillus filamentosus),
though their bacilli are not motile, as will be more
minutely described when speaking of the cultural char-
acters of bacilli.

When certain bacilli show only slight motility it may be
extremely difficult to distinguish this from Brownian mole-
cular movement, but no locomotion can be ascribed to
bacilli unless one or the other individual can be distinctly
seen to show a darting or spinning movement. As men-
tioned above, the easiest and best way to see locomotion is
to examine the fresh bacilli in a fluid, as sterile broth or
sterile salt solution in the “ suspended drop.”

The motility of bacilli and spirilla is due to their pos-
sessing at one, and occasionally at both ends, or also over the
general surface, fine flagella or cilia, the movement of which
causes the motility of the microbe. Where two or more
microbes are connected into a chain or thread, only the
terminals have the flagella. Although the flagellum has not
been stained and photographed hitherto in all bacilli and
spirilla, there can be no doubt that all motile organisms do
possess the flagellum, for without it motility would not be
possible. Micrococci are not possessed of motility, but
recently Ali-Cohen has isolated from drinking-water a
species of micrococcus (Micrococcus agi/is) which forms an

ii8

MICRO-ORGANISMS AND DISEASE

[chap.

Fig. 20. — Choi.era Vibrio of Cultivation, showing Flagella, x iooo.

Figs. 20. 21, 22, 23 are from specimens prepared by Dr. Kanthack.

vi] GENERAL CHARACTERS OF BACTERIA 1 19

exception, since this species is motile {Centralbl. filr Bad.
VI. 2).

In aerobic bacilli and spirilla which are possessed of
motility this is intimately connected with a supply of
oxygen. Though some species seem to obtain this readily
even when in deep fluids (e.g. bacillus of hay, certain species
of proteus), many others cease to move when the supply of
oxygen becomes insufficient. Engelmann has made some
very interesting experiments with certain motile bacilli,
showing the direct influence of oxygen on their motility.
When motile bacilli, owing to insufficient oxygen or after
the consumption of the oxygen previously present, come to
rest, by adding to them new oxygen in a drop of fresh fluid
containing air, the motility is resumed. On removing the
oxygen and adding carbon dioxide or hydrogen gas, am-
monia, chloroform, or ether, the movement ceases, but on
removing these gases and replacing them again by oxygen
(or air) the movement is again resumed.

Motile bacilli and spirilla when growing in a fluid medium
have a great tendency to seek the surface of the fluid — i.e.
move towards the part where they can obtain oxygen, and
here form more or less coherent pellicles, in which they are
in a resting state, and in which a rapid multiplication goes
on ; but it is quite incorrect to assume that an organism
which in a fluid medium forms a pellicle is a motile
organism, since some species which form a pellicle are not
motile, and some species of motile organisms do not form a
pellicle.

On making a comparative study of the presence of
flagella best by v. Ermengem’s method, two things will be
found of interest : (1) that there are flagella present even
in bacilli which in the fresh state show no locomotion
or only a very feeble one ; and (2) that the length and

120

MICRO-ORGANISMS AND DISEASE [chap.

Fig. 23. — Bacillus Tetani, showing Flagella, x 1000.

vi] GENERAL CHARACTERS OF BACTERIA 12 1

number of flagella stand in no definite relation to the
intensity of the movement Tetanus bacilli of a culture
examined in the hanging drop show at best only sluggish
motility, and yet on staining for flagella the astounding fact
(see Kanthack’s specimens) will appear that most of the
bacilli possess at one or both ends, and on the sides, long
flagella, these sometimes in bundles. I have isolated a
spore-forming virulent anaerobic bacillus (bacillus enteritidis
sporogenes) from the fluid evacuations of cases of epidemic
diarrhoea, which is closely related to the bacillus butyricus
of Botkin ; it shows only feeble motility ; in fact, in an
ordinary fresh preparation made from a sugar gelatine
culture amongst the many rod-shaped or cylindrical bacilli
there is rarely one met with that shows motility. And yet
when staining for flagella numerous bacilli possess flagella ;
one, two, three, or more, at one or both ends, some
short individuals possess a bunch of flagella of extreme
length (many times longer than the bacillus itself) at one
end, and a few long cilia at the other. In fact, no greater
misproportion between feeble motility of only a few bacilli
and the frequency and number of flagella can be imagined.
As to (2), from the intensity of the motility of the fresh
microbes no conclusion can be drawn as to the number and
length of the flagella. To mention a few examples : the
cholera vibrio of a culture, though motile in a most extra-
ordinary manner, possess only one short spiral flagellum ;
the very motile bacilli of proteus vulgaris possesses only one
flagellum at one end ; some varieties of bacillus coli
extremely motile possess only two flagella, while other
varieties less motile possess two, three, up to ten flagella ;
the tetanus bacillus and the bacillus enteritidis sporogenes
are good cases in point.

CHAPTER VII

CHEMISTRY OF BACTERIA

Some of the most interesting and important manifesta-
tions of bacterial life are the chemical changes which are
brought about by bacteria. They are so manifold, many of
them of such a complicated character and so little under-
stood, that it is at present impossible to arrange them in a
system, or to classify them in any comprehensive scheme.
All that is at present possible is to give an outline of the more
obvious chemical manifestations observable during the
growth of certain species or of groups of them.

i. One chemical change frequently exhibited is the power
of bacteria to peptonise nutritive gelatine ; this exhibits itself
as more or less rapid liquefaction of the nutritive gelatine
in which growth is taking place, and as the growth proceeds
liquefaction of the whole nutritive medium is effected.
Many bacteria have this power : those occurring in water, in
the air, in the soil : bacillus fluorescens liquescens, bacillus
subtilis, bacillus mesentericus, micrococcus liquescens albus
and aureus, several species of sarcina, bacillus prodigiosus,

bacillus pyocyaneus, proteus vulgaris ; then many disease

germs : bacillus anthracis, the (anaerobic) bacillus of symp-
tomatic charbon, of Koch’s malignant oedema (anaerobic), of

chap, vii] CHEMISTRY OF BACTERIA

123

tetanus (anaerobic), bacillus enteritidis sporogenes (anaero-
bic), bacillus butyricus (anaerobic), Koch’s cholera vibrio,
vibrio of Finkler ; actinomyces, aspergillus and penicillium,
&c. Some liquefy the gelatine extremely slowly, the liquefied
gelatine being more of the consistency of thick syrup, e.g.
bacillus of swine-erysipelas and of Koch’s mouse-septi-
caemia.

In the case of all aerobic microbes, which have the power to
liquefy (peptonise) nutritive gelatine, this power is intimately
bound up with a free supply of oxygen (air) ; it proceeds from,
and is conspicuous on the surface, it is greatly retarded when
air is excluded, and in some cases is only noticed where the
growth occurs on the surface in contact with air.

But there are a good many microbes which do not
peptonise, do not liquefy the gelatine : all the species forming
the group of bacilli causing haemorrhagic septicaemia in
the rodents : bacillus of fowl cholera, of swine fever, of fowl
enteritis, all varieties of bacillus coli, bacillus of typhoid
fever, bacillus of “ Wildseuche,” & c. — all or nearly all (a few
species excepted) species of streptococci, a number of chro-
inogenic cocci, &c.

2. Another widespread manifestation is that of producing
acid or alkali ; when growing in a neutral medium, as in
Petruschki’s neutral whey, of turning this acid or alkaline, as
the case may be, the latter being more often met with than
the former (Petruschki, Centralbl. f. Bakt. a?id Parasit.
1889 and 1890.)

Buchner has first suggested a method which is very easy
of employment, and which demonstrates conspicuously
whether a microbe during its growth produces acid or alkali
or is neutral — viz., by mixing with the nutritive medium,
before steaming, a small amount of litmus tincture,
sufficient to stain it bluish. The nutritive gelatine, slightly

124

MICRO-ORGANISMS AND DISEASE [CHAP.

alkaline (see a former chapter), is then inoculated with the
microbe and incubated. During the growth, on inspection
the gelatine next to the growth will be found to have become
violet and then red if the microbe produces acid, and the
more rapidly and conspicuously so, the more rapidly and
more acid it produces. If the gelatine remains bluish, then
no acid has been produced. In this case a neutral nutritive
gelatine is prepared and mixed with neutral litmus and then
inoculated with the microbe. On incubation, as the growth
appears, if the violet colour of the gelatine has turned blue
next to the growth then the microbe is an alkali-producer, if
the gelatine remains neutral then the microbe does not
produce either acid or alkali. As mentioned above, it is
common to find that the microbe produces acid, some rapidly
and distinctly ( e.g . bacillus coli and typhoid), others only
slowly and in small amount (e.g. some varieties of the
vibrio of cholera). An interesting phenomenon is that many
microbes — even highly specialised microbes like the glanders
bacillus — grow well on potato (steamed), although the reac-
tion of this is acid (mallicacid) — in some potatoes very pro-
nounced, in others only very slight. Now the curious thing
about it is that some of the bacteria that show rapid and good
growth on potato show only very feeble or no growth if
planted on an acid medium, e.g. acid broth or acid
gelatine.

3. Some microbes have the power to liquefy and pep-
tonise such resisting substances like solid agar and solid
blood-serum, though this power is possessed only by few
species. Most of the species that are capable of liquefying
and peptonising gelatine leave the agar and blood-serum
unaltered. The bacillus of Koch’s malignant oedema, the
vibrio of Finkler, the vibrio of cholera (Koch), rapidly liquefy
blood-serum, but do not alter solid agar.

CHEMISTRY OF BACTERIA

125

VII]

4. A further not uncommon phenomenon is the formation
of gas (methan gas or marsh gas). This is best shown by
making the inoculation into deep gelatine or by inoculating
the gelatine, then melting it, shaking it, and letting it again
solidify — “ shake culture.” On incubation every colony that
appears in the depth of the gelatine is associated with
a gas bubble. A shake culture of ordinary nutrient gelatine
after inoculation with bacillus coli gives a very character-
istic appearance, being in its deeper layers crowded with
small gas bubbles. After some days they become fewer,
most of them escaping to the surface. In the cultures in
deep sugar-gelatine of bacillus of Koch’s malignant
oedema, of the bacillus of symptomatic anthrax, of tetanus,
the formation of gas is a conspicuous feature. Some species
of bacillus coli form copiously gas bubbles in deep
nutrient agar cultures and even in broth cultures as the
growth becomes conspicuous, eg. after twenty-four to thirty-
six hours at 370 C. ; on watching the culture numerous small
gas bubbles are seen to ascend to the surface.

5. A number of microbes have the power to produce in
special materials specific chemical changes representing
specific fermentations. The alcoholic fermentation of sugar
by yeast is the best-known and longest-established instance ;
the acid fermentation (oxidation of alcohol) by bacterium
aceti and mycoderma aceti, the change of lactic sugar into
lactic acid by various species of bacterium lactis and other
bacilli, is a widespread one ; so also is the formation of butyric
acid by bacillus butyricus (van Tighem). The hydration of
urea and conversion into ammonium carbonate by micro-
coccus ureae, the dextrose fermentation, the mannit fer-
mentation, are further instances. In this category must be
included the conversion of albumen into peptone, previously
described. In all these instances a particular substance,

126

MICRO-ORGANISMS AND DISEASE [chap.

glycose or grape-sugar, alcohol, lactic sugar, urea or gum, &c.,
as the case may be, are by the growth of particular microbes
changed in the manner of fermentation into other sub-
stances.

6 Many bacteria have the power to produce pigments :
these appear either on all media on which their growth
occurs, or only on particular media. In the first case the

Fig 24.— Surface (Streak) Culture on Gelatine of the Common Bacterium

LACTIS.

pigment formation is real, in the second only apparently
so. Thus a variety of bacilli, e.g. bacillus subtilis, bacillus
mesentericus, bacillus coli, bacillus of glanders, when grow-
ing on potato, form a brownish or yellowish-brown smeary
layer, but do not produce any pigment on other media ; the
bacillus anthracis turns agar brownish after the growth has
reached a certain long duration, &c.

VI ij

CHEMISTRY OF BACTERIA

12 7

Fig. 25. A Stained Film Specimen of Bacterium Lactis.
x 1 ocx).

Fig. ^.-Micrococcus Urea;, from a Gelatine Culturc.
X 1000.

128

MICRO ORGANISMS AND DISEASE [chap.

True pigment bacteria form pigment on all media; this
pigment is either diffuse or is limited to the bacterial
bodies themselves ; thus, bacillus fluorescens liquescens,
bacillus fluorescens putidus, bacillus pyocyaneus, form a
diffuse bluish-green pigment, while bacillus prodigiosus,
staphylococcus aurantiacus, spirillum rubrum, &c., &c., form
pigment limited to the bacterial bodies themselves. The

Fig. 27. — Bacillus Phosphorescens, Film Specimen from a Culture on Gela-
tine Broth and Asparagine.

X 1000.

meaning of the pigmentation is not understood, though a
large variety of pigmented species are known and com-
prise almost every tint : red, pink, orange, ochre, yellow,
lemon-yellow, green, greenish-blue, blue, violet, purple.
Some of them liquefy gelatine, e.g. bacillus prodigi-
osus, staphylococcus aureus, bacillus fluorescens liquescens
and pyocyaneus ; others are non-liquefying, as micrococcus
aurantiacus, spirillum rubrum, bacillus fluorescens putidus.

VI.]

CHEMISTRY OF BACTERIA

129

7. Winogradski and Warrington have shown that by
nitrification ammonium salts in the soil are converted
into nitrites by one set of short bacilli, and these nitrites
into nitrates by another set of bacilli ; the two species differ
from one another in their motility and general morphology.
The nitrates thus produced are the forms of nitrogen which
serve as nitrogenous food for plants. This proposition as
to the necessity of intervention of special bacteria to nitrify
ammonium salts was first enunciated and experimentally
established by Schlosing and Muntz, and they were more
accurately investigated by Winogradski, Warrington, and
Percy Frankland.

8. The power of certain bacteria to become phosphorescent
and to give the medium in which they grow the character of
phosphorescence has been first noticed by Pfluger (phos-
phorescence of putrid fish, menthol wood). Katz, Fischer,
and Beyrinck have described various species of phosphor-
escent bacteria ; particularly the latter has studied them in
pure culture (broth, salt, asparagin) and has described
various species. Elwers and Dunbar have described vibrios
that have the power of phosphorescence.

9. The series of changes produced by some species of
bacteria, called putrefaction of albuminous substances, con-
sist chiefly in the decomposition of albumin into lower
nitrogenous principles associated with the evolution of
sulphuretted hydrogen and ammonia, and the formation of
alkaloidal bodies known as ptomaines of Selmi. Brieger,
who has first isolated a number of alkaloids (cholin, neurin,
cadaverin), has shown that, while some have poisonous
action on the animal system, others have not. The fact
that injection — either directly into a vein or indirectly into
the subcutaneous tissues of animals — of putrid fluids in
sufficient doses causes acute poisoning : rise of temperature

130 MICRO-ORGANISMS AND DISEASE [CHAP.

at first, vomiting, purging, spasms, great fall of temperature,
collapse, and death, has been known since Panum, Schmidt,
Billroth, and others ; this constitutes what is now known as
saprsemia, or septic or putrid intoxication caused by the
ptomaines of Selmi and Brieger. And further, research has
shown that all the pathogenic bacteria, that is those which
when introduced into a suitable body multiply therein,
produce infection and cause a series of symptoms charac-
terising the particular infectious disease, do so by virtue
of their producing specific chemical poisons, toxins, within
the body. Not only in the animal body, but also in
artificial cultures, do these specific bacteria elaborate these
toxins, which, if injected into an animal, set up the same
symptoms of disease as if produced by the multiplication
of the microbes within the animal. These toxins have
been investigated for a series of specific microbes : septi-
caemia (Roux and Chamberland), typhoid fever (Brieger),
diphtheria (Roux and Yersin, Sidney Martin), tetanus
(Behring and Kitasato), anthrax (Hankin, Sidney Martin),
and others. These toxins are considered by Fraenkel and
Brieger to be of the nature of proteids and are called
tox-albumins, while Roux has given good evidence that
some (particularly the diphtheria toxin and the tetanus
toxin) are more of the nature of ferments. Hankin has
shown that in anthrax a poisonous albumose is formed,
while Sidney Martin has obtained, besides poisonous
albumoses, certain alkaloidal bodies having poisonous ac-
tion. In diphtheria Sidney Martin obtains alike from diph-
theria cultures and the diphtheritic membrane and spleen
in human diphtheria, besides a poisonous ferment (the
toxin), also albumoses, alkaloidal and acid bodies acting
poisonously. The fact is then established that the specific
or pathogenic bacteria produce in artificial nutritive media,

VII] CHEMISTRY OF BACTERIA 131

as also in the body affected with the disease, specific
toxins.

10. Many species of bacteria include in their proto-
plasmic bodies substances which when injected in sufficient
doses into the subcutaneous tissue — or, better still, into the
peritoneal cavity — of rodents, produce symptoms of disease
and death. Bacteria of various kinds, and not having any
connection with infectious disease— in fact, harmless and
non-pathogenic — can, when injected in sufficient doses into
the peritoneal cavity of guinea-pigs, set up acute intensive
peritonitis and death in 16 to 20 hours. If, for instance,
{ to J- of an Agar surface culture (6 cm. by 2 cm.) 1 of
bacillus prodigiosus, bacillus subtilis, bacillus coli, bacillus
proteus vulgaris, vibrio of Finkler — all microbes which have
no connection with any infectious disease of man or
animals — be injected into the peritoneal cavity of a healthy
guinea-pig, the animal shows decided illness already after a
few hours : first rise, then decided fall, of temperature ;
it is quiet, refuses food ; later on, its movements become
impaired, and it may be found dead in 18 to 24 hours.
The rapidity with which death takes place depends on the
size of the animal and on the quantity injected. After death
extensive and intensive peritonitis is found : solid lymph on
the peritoneum, pseudo-membranes on the liver, spleen, and
omentum ; the intestine is as a rule greatly congested, and
there is more or less copious peritoneal exudation, either
turbid or sanguineous. If the culture has been injected as
living culture, the peritoneal exudation is crowded with

1 The culture is made by rubbing over the whole slanting surface of
the agar a platinum loop dipped previously into the active culture,
then incubating at 37° C. for forty-eight hours. A definite quantity
of broth (sterile) is then added, and the growth rubbed down with the
platinum loop ; the turbid emulsion is poured off and used for injection.

K 2

132 MICRO-ORGANISMS AND DISEASE [chap.

the microbes injected ; occasionally also the blood yields,
in culture, colonies of the microbes, but far less numerously
than the peritoneal exudation ; if the animal survives
36 to 48 hours it as a rule recovers. The same fatal acute
peritonitis is produced by the bouillon mixture previously
sterilised at 70° C. for 5 to 10 minutes, only in this case a
larger dose is required than of the living mixture in order
to produce a fatal result.

The same disease and the same fatal result are produced
by other bacteria, as the vibrio of cholera, bacillus of typhoid
fever, staphylococcus aureus, and bacillus pyocyaneus.
Bacillus coli and bacillus prodigiosus act in this respect
more virulently than the others, so that a smaller dose of the
former is required to produce the fatal peritonitis than of the
latter.1

Since all these microbes act in the same way and produce
the same disease and post-mortem appearances, whether used
as living culture or as sterile culture, and since in these experi-
ments only the bacilli are used (the growth is scraped from the
surface of solid Agar), it follows that the microbes above
mentioned contain in their bodies similar or the same
poisonous substances — intracellular poisons. The curious
thing is that some noted pathogenic bacteria do not contain
these intracellular poisons, e.g., sporeless anthrax bacilli,
bacillus of fowl cholera, and bacillus diphtheria; can be
introduced as sterile bacilli in large quantities — far larger
than in the case of the above microbes — without producing
poisonous effects. Moreover the living bacillus diphtherias
from gelatine culture can be introduced in large quantities

1 Subcutaneous injection of large doses produces a local swelling and
cedema, which may lead to suppuration and necrosis ; in the case of
protcus vulgaris and bacillus coli it may lead to acute general infection
and death.

CHEMISTRY OF BACTERIA

133

vn]

(£ to \ of a culture) into the peritoneal cavity of guinea-
pigs — highly susceptible to this microbe when subcutaneously
injected — without producing disease or death.

Injecting, then, the bacilli of a particular species, dead or
living, in fair quantities into the peritoneal cavity, and pro-
ducing thereby disease and death, does not prove in the least
that this species is, strictly speaking, pathogenic, since some
notoriously non-pathogenic bacteria (bacillus prodigiosus,
vibrio of Finkler, bacillus subtilis) do the same, while some
notoriously specific bacteria (bacillus of fowl cholera — sterile ;
bacillus diptherise — living or dead ; and bacillus anthracis —
dead) do not produce such a result. All that can be said
in such cases is that the bacillary bodies do or do not
contain the intracellular poison that causes fatal peritonitis,
or contain it in small amount, or contain it very abundantly.
Whether a given species is or is not pathogenic — can or cannot
produce in the natural or artificial culture media specific
toxins — is a question totally separate from the above. Voges
separated by watery extract from growths of bacillus pro-
digiosus a substance which causes on injection a temporary
rise of temperature in guinea-pigs ; this is evidently a sub-
stance distinct from the intracellular poisons that cause the
above-mentioned fall of temperature and fatal peritonitis.

The intracellular poisons present in many, absent in
some, species of bacteria are thus of a distinctly different
order from the specific toxins elaborated by pathogenic
bacteria : the former are present in the bacillary bodies as
such, no matter whether dead or living ; the latter are the
products of metabolism, i.e. results of chemical changes
induced in the culture media by the growth and multiplica-
tion of the specific bacteria. Any specific change that the
living body undergoes, any specific reaction that it is
capable of acquiring after the growth in it of the living

134 MICRO-ORGANISMS AND DISEASE [ch. vn

bacilli, is, partly at least, a result of the specific toxins
created by the bacteria in it ; while the change that is
produced in the peritoneal cavity, into which the intra-
cellular poisons of dead bacilli had been previously intro-
duced in less than fatal dose, may be, and as a matter of
fact is, a local one and different from that produced by
the previous growth of the living bacilli and elaboration of
their specific toxins in the peritoneal cavity (see a later
chapter).

CHAPTER VIII

MICROCOCCI

By the specific term micrococcus is understood a minute
spherical or slightly oval organism (spherobacterium, Cohn)
that, like other bacteria, divides by fission (schizomycetes).
and that as a rule does not possess any special organ, cilium
or flagellum, by which it would be capable of moving freely
about. Excepted herefrom is the micrococcus agilis dis-
covered by Ali-Cohen and mentioned in a previous chapter.
Micrococci, like other granules when suspended in a fluid
medium, show (Brownian) molecular movement. Micro-
cocci propagate always by division ; any other mode, e.g.
gemmation and spores, is unknown. All assertions to the
contrary must as far as present knowledge goes be considered
as unproven. All micrococci, like other bacteria, possess a
delicate membrane of cellulose, and, owing to this, resist the
action of alkalies and acids. The contents are homogeneous
and highly refractive while active, pale when inactive.
They consist, like those of other bacteria, of mycoprotein
(Nencki). The size of micrococci varies within considerable
limits, say o-5 to 2 //., or even a little more. Micrococci
vary greatly as regards both size and mode of growth.
All multiply by slightly elongating and then dividing by
a transverse constriction into two : a dumb-bell ; each of

136 MICRO-ORGANISMS AND DISEASE [chap.

these again divides into two, either transversely or in the
same direction as before. The new elements of succes-
sive divisions may remain connected linearly, forming a
chain ; or they separate into single organisms or dumb-bells
or form smaller or larger connected masses. In some species
there is a pre-eminent tendency to form chiefly dumb-bells
or diplococcus of Billroth, in others to form shorter or
longer chains generally more or less curved, streptococcus
(Billroth), and in still others to form connected masses,
staphylococcus (Ogston).

Such exquisite chains one meets with sometimes in serum
of blood exposed to the air for some days, and in pleural
and peritoneal exudations of animals dead for a few days.
I have seen in an artificial culture made by my friend Mr.
A. Lingard from a blister in a rabbit’s ear the most ex-
quisite convolutions of threads of micrococci. Similarly
the streptococcus pyogenes and that of erysipelas form in
fluid media long, twisted, and convoluted chains.

In the dividing cocci the single cells are generally more or
less crescentic ; this is particularly noticed in staphylococcus
aureus and albus and in gonococcus ; it is not marked in
others, as in diplococcus pneumoniae and in the streptococci.

Some species are specially characterised by this that, having
divided into a dumb-bell, each of the elements divides again
transversely into a dumb-bell, thus forming a group of four
(tetrade or sarcinaform). Some species are occasionally
met with, particularly in products of air-contamination, in
which the four individuals are closely pressed against one
another, and then each assumes more or less the shape of a
cube, a true sarcina. But each of these cubes divides into
four small micrococci arranged as a small sarcina, so that a
sarcina-within-sarcina form results (sarcina lutea, sarcina
ventriculi).

l^i iS-

MICROCOCCI

137

VIIl]

As has been pointed out in Chapter VI. under Growth and
Division, in some species the cocci when growing on solid
media enlarge many times the size of the typical unit before
division commences, others only enlarge slightly and then
at once divide.

In many instances the individual members resulting from
division remain closely adherent without any definite arrange-

Fig. 28.— Micrococcus from a Gelatine Culture, showing various Phases of

Growth.
x 1000.

ment, and thus form smaller or larger clusters (staphylo-
coccus), a kind of zooglaa or colonies, in which the indi-
viduals appear embedded in a hyaline gelatinous matrix ; the
amount of this varies in the different species ; in some there
is little of the matrix actually visible, the micrococci being
in close juxtaposition, in others it is easily recognised, the
interstices between the individuals being measurable.

In some of the pigmented species (see below) the interstitial

-33

MICRO-ORGANISMS AND DISEASE [chap.

matrix contains the pigment. Zooglcea masses always present
themselves as uniformly granular, the granules or micrococci
being either of the same size or differing considerably.

True micrococci never elongate to form rods, although in
certain rod-like bacteria the individual elements owing to
rapid division have the shape of spherical elements (see
below).

Some species of micrococci form after some days a pellicle
on the surface of fluid nourishing material, although there is
also an abundance of these micrococci in the depth of the
nourishing material. This pellicle is composed of zoogloea,
and after some time bits of it, or the whole, sink to the
bottom of the fluid medium. Micrococci that thus form
pellicles are pre-eminently aerobic (Pasteur), i.e. require
a great deal of free oxygen, which they receive from the air
to which they are exposed on the surface of the nourishing
material. Other species do not require free oxygen
(anaerobic, Pasteur), and therefore grow well in the depth
and do not form a superficial pellicle. There is a marked
distinction in this respect between different species. The
micrococci occurring in connection with disease are
facultative anaerobic.

When cultivated in suitable fluids they produce after a day
or two general turbidity ; growing in solid nutritive gelatine
some produce liquefaction of the gelatine, others do not,
and it is with micrococci as with other bacteria that identi-
fication of different species is possible by their mode of
growth in and on solid media and in fluids, in plate cultiva-
tions, in their power of liquefying gelatine, and in their
behaviour in the animal body.

Besides those mentioned in connection with certain
special fermentative changes (micrococcus urea;), and others
to be mentioned in connection with disease, various species

VIll]

MICROCOCCI

139

of micrococci occur in air, in water, in dust, in soil, and in
all organic materials in which decomposition occurs, differing
from one another in size and in their cultural characters.
To the same class belong many of the micrococci found in
the normal fluid of the oral cavity and on the surface of the
tongue and mucous membrane of the tonsils and pharynx —
these are probably derived from the outer air ; similarly in
the bronchial and nasal secretions in catarrhal inflamma-
tion, on ulcerated surfaces, in the epidermis of the normal
skin, in the contents of the large intestine in health and
disease.

Fig. 29. — From the Base of an Ulcer of the Mucous Membrane of the
Larynx in a Child that Died of Acute Scarlatina.

1. Nuclei and fibres of the tissue.

2. Zoogloca of micrococci.

In all cases of diarrhoea the secretions of the bowels swarm
with micrococci. In typhoid fever clumps of micrococci
may be found very extensively on the ulcerations of the
bowels and in the mucous membrane surrounding the
ulcerations, and may be even traced into the mesenteric
glands and the spleen.1

In dead tissues within the living body, such as occur after
embolism, and in the case of various infectious maladies,
micrococci may be found in colonies, i.e. as zooglcea.

1 Klein, Reports of the Medical Officer, 1876. Letzei ich, Sokoloff,

Fischel, &c.

- A.

140

MICRO-ORGANISMS AND DISEASE [chap.

Ascococcns. — Billroth first described certain peculiar sphe-
rical, oval, or knobbed masses of minute micrococci, which
he found in putrid meat infusion. Each of the masses is
enveloped in a resistant, firm, hyaline capsule of about o-oio
to o-oi5 mm. thickness. The masses are of various sizes,
from o'02 to C07 mm. in diameter, and are composed of
small spherical micrococci. Cohn found them also in his
(Cohn’s) nourishing fluid (see Chapter II.), where they
produce the peculiar smell of cheese. They are capable

Fig. 30.— Ascococcus Billrothi (after Cohn).

of changing acid nourishing material into alkaline. Cohn
ca|led the organism ascococcus Billrothi.

Sarcina Ventriculi — Goodsir was the first to describe
in the vomit of some patients packets of four cubical
cells, with rounded edges, and closely placed against one
another. These sarcincc ventriculi are of a greenish or
reddish colour. The diameter of the individual cells
is about 4 ix. They are found in the contents of the
stomach of man and brutes in health and disease, where the

viu]

MICROCOCCI

Fig. 31.— Film Specimen op Pus from Acute Abscess : amongst Masses of
Nuclei, numerous Cocci, single, Diplococci, and Groups.

X 1000.

As stated just now, it is widely distributed in air, occasionally
is also found in open waters.

Micrococci connected with disease, or capable of pro-
ducing disease in man and animals : —

1. Staphylococcus pyogenes (Ogston). — In most purulent
acute inflammations there pccur numerous cocci which

groups of four cells form smaller and larger aggregations.
Occasionally small sarcinee occur on boiled potatoes, egg
albumen, and gelatine that have been exposed to the air.
The cocci of these sarcinae are smaller than those of the
sarcina ventriculi ; on cultivation the growth is of a yellow
colour and represents the species known as sarcina lutea.

142

MICRO-ORGANISMS AND DISEASE [chap.

when cultivated prove to belong to two well-defined species :
staphylococcus pyogenes aureus and albus.

Staphylococcus pyogenes aureus. — This organism is com-
mon in acute suppurations and ulcerations, alike those
in the skin or mucous membranes, serous membranes, or
parenchymatous organs ; it is met with abundantly also in
acute external imflammations, ulcerations [e.g. after vaccinia

Fig. 32. — Film Specimen of Peritoneal Exudation of a Gltnea-pig, dead
from Acute Peritonitis after Intraperitoneal Injection of Culture
of Staphylococcus Aureus.

Four lymph-cells filled with the cocci, x 1000.

and variola, in diphtheritic inflammation of the fauces,
in some cases of ulcerative endocarditis). In purulent in-
flammations (abscesses acute and chronic) this organism is
present in large numbers in the pus as single cocci, as
dumb-bells, and as large and small connected clusters.
Many of the dumb-bells and connected masses show the in-
dividuals as crescents — that is as divided. A film of pus dried
on a cover-glass, heated and stained in methyl-blue or gentian

VIIl]

MICROCOCCI

i43

violet shows the cocci as above, between, and also on the
surface and in the interior of the pus cells. In catarrhal in-
flammation of the fauces they occur in numbers adhering
to the surface of the detached scaly epithelial cells.

In gelatine plate cultivation kept at 20° C. the colonies
are minute whitish dots, visible already after 24 hours ; after
36 to 48 hours each dot is already of a yellowish tint, sunk in,
as it were, into a pit of clear liquefied gelatine. The liquefac-
tion now proceeds rapidly, each liquefied area containing a
central yellowish granular mass which is made up of clusters
of cocci. In gelatine stab cultures the line of inoculation
is soon (after 24 hours) marked as a connected lineal mass
of growth ; liquefaction commences generally at the top and
rapidly proceeds into the depth, the liquefied gelatine being
fairly clear or very slightly turbid ; at the bottom of the
liquefied channel or funnel the main part of the growth is
accumulated in the form of a yellowish powdery precipitate.

On agar it forms a characteristic yellow, pale orange
yellow, or golden-yellow moist growth— hence its name.
On subculture from generation to generation it will be found
that the colour becomes paler than is the case at starting ;
the condensation water is uniformly turbid with granules
and flocculi.

Although not invariably local suppuration and multiplica-
tion of the cocci are produced in rodents by injecting sub-
cutaneously some of the growth, it nevertheless sometimes
succeeds; it succeeds easier by injecting at the same time a
10 per cent, sugar solution.

The subcutaneous injection of a culture (broth culture)
of staphylococcus aureus in large doses is occasionally fol-
lowed by acute and general infection and death ; the blood
contains then a crop of the cocci ; the serous membranes
are inflamed, and their exudation is full of the cocci ;

144 MICRO-ORGANISMS AND DISEASE [CHAP.

occasionally, if the disease lasts a few days, disseminated
purulent abscesses are found in some of the viscera.

2. Staphylococcus pyogenes albus is also often present in
purulent matter, particularly of acute abscess, either alone
or associated with aureus. The liquescens albus differs from
the aureus morphologically and culturally only in this that
its growth on Agar possesses no colour, but forms a whitish
mass. It liquefies rapidly gelatine, and the liquefied gela-
tine is fairly clear or slightly turbid, and at the bottom is a
whitish, powdery, granular precipitate consisting of continuous
masses of cocci, which in morphological respects cannot be
distinguished from aureus. Its pathogenic action on sub-
cutaneous injection into animals is the same as in the case
of aureus ; also occasionally a general acute infection with
lethal end is producible in rodents.

Both aureus and albus grow rapidly in beef-broth, making
it strongly and uniformly turbid with a powdery and
flocculent granular precipitate.

The enormous rapidity with which staphylococcus aureus
is able to grow at 370 C. has been detailed in a former
chapter.

3. Occasionally in purulent and acute inflammatory foci is
found a coccus which forms a distinctly white growth on
Agar and on gelatine, and does not liquefy gelatine ; this
is the staphylococcus albus non-liquescens. A variety of this
forms flat, white, rapidly spreading dry colonies and growth,
and represents staphylococcus cereus albus. I have met
with both these varieties in purulent matter of the sores
after vaccination, also from variola in the suppurative stage.

4. Streptococcus pyogenes albus. — This is the microbe of
acute phlegmon ; it is also present in chronic abscess,
in acute serous effusions. The principal morphological
characters of this as also of other species of streptococci

MICROCOCCI

vm]

are that the cocci by repeated division form linear series,
thus producing shorter or longer chains, the latter more or
less twisted and wavy; when growing in fluid media at
370 C. — broth, condensation fluid of solidified Agar, or
blood-serum — the chains are rapidly formed and attain great
length. On solid media — gelatine, Agar, blood-serum— the

chains are not so long, occasionally only composed of six or
eight cocci. Examining the long chains of fluid media, one
always notices an inequality in the size of the cocci, some-
times one or the other coccus — in the middle, or oftener at
the end of the chain — being twice and thrice as big as the
average coccus ; in some chains, wholly or in part, the cocci

146 MICRO-ORGANISMS AND DISEASE [chap.

are distinctly arranged as a series of dumb-bells, in others
there is no such distinct arrangement.

Streptococcus pyogenes forms in nutrient gelatine at 20° C.
already after twenty-four to thirty-six hours minute, dot-like,
grey, translucent, round colonies, which after two to three
days’ growth are large enough to show under a magnifying
glass a darker, thicker centre and a thin, rounded, translucent
periphery ; after about a week or two the outline becomes
irregular, to one side more than to the other, thus forming
a more or less fan- or fern-shaped patch. It does not liquefy
the gelatine. In streak culture on solid gelatine, blood-serum,
or Agar, the line of inoculation becomes marked as a
line of separate, rounded, translucent, or more or less whitish-
grey colonies, which as a rule, unless very thickly sown, do
not coalesce. In fluids — broth, condensation fluid of Agar or
of serum — the growth causes slight turbidity of the fluid and
is more in the form of stringy, flaky masses, these being
composed of continuous long chains much interwoven. On
potato the growth is not visible. Streptococcus pyogenes
as obtained from acute phlegmon, from chronic purulent
matter, from purulent and serous exudations of the viscera
and cavities, does not constitute a single variety, but
belongs to varieties differing from one another slightly
in the size of the cocci, in the rapidity of the growth
on gelatine, and in the length of the chains. Similarly
varieties of streptococci are known to occur in the
various normal secretions — fauces, bronchi, intestinal con-
tents, soil, &c. — which in some or all the above respects more
or less resemble the streptococcus pyogenes. The strepto-
coccus pyogenes cultivated from pus shows on inoculation
of a rabbit or mouse in many instances a tendency to form
inflammation and abscess ; in some instances, particularly
on injecting large doses, general acute septicemic infection

MICROCOCCI

v 1 1 1 ]

and death, with plugging of capillaries in the parenchyma-
tous viscera with masses of streptococci, are observed ; the
blood yields on culture numerous colonies of streptococci.

Streptococci resembling in morphological and cultural
respects the streptococcus pyogenes are found in connected
masses in the ulcerated tissue and on the villous out-
growths of the cardiac valves in some forms of ulcerative
endocarditis. In other cases of ulcerative endocarditis
masses of staphylococcus aureus only occur. Also in puer-
peral septicaemia a streptococcus is cultivable from the blood
and spleen which in cultural respects resembles the strepto-
coccus pyogenes except that it is more virulent, producing on
injection into the subcutaneous tissue of the rabbit’s ear an
extensive blush and occasionally acute septicaemic infection.
It is difficult to say whether this streptococcus is a virulent
variety of streptococcus pyogenes or a less virulent variety
of the streptococcus erysipelatos.

It is an easily ascertained fact that the streptococcus pyo-
genes cultivated from phlegmon and various purulent exuda-
tions when tested on the animal (notably the rabbit’s ear)
does not behave in a uniform manner, inasmuch as in some
instances it acts virulently, causing distinct and spreading
blush and purulent exudation and even general infection,
whereas in others it has no appreciable pathogenic action
under the same conditions ; and it is likewise a fact that a
streptococcus, which is pathogenic at first, by repeated sub-
culture loses this action.

5. Streptococcus erysipelatos. — Fehleisen first isolated this
microbe from the progressing margin of erysipelas ; it is
a microbe which, as sections through the erysipelatous
skin show, is abundantly present in the distended lymph-
spaces and lymph-vessels of the marginal part. The mor-
phological and cultural characters coincide with that of the

l 2

1 48

MICRO-ORGANISMS AND DISEASE [chap.

Karlinski gives as the result of a large number of observations on
purulent matter of man the following list ( Cenlralbl f. Bad. und
Parasit., VII., No. 4, p. 115) : —

Disease

Staphylococcus
pyogenes aureus

- n

§>
T- V

C C

'0 v
« £

Staphylococcus
pyogenes albus

Streptococcus

pyogenes

Micrococcus

tetragonus

’u

2 1

<r.

Mastitis, 36 cases
Subcutaneous abscess, 30

cases ....

—

Phlegmon, 24 cases

—

Furuncle, 20 cases

—

Bubo, 17 cases .
Subperiosteal abscess, 1 6

—

cases ....
Panaritium cutaneum, 16

cases ....

—

Abscess of gums, 10 cases .

—

Hordeolum, 10 cases .

—

— :

Otitis media, 4 cases .

—

Carbuncle, 4 cases

—

Osteomyelitis, 3 cases .

—

Summary .

streptococcus pyogenes and other streptococci ; 1 this great
similarity in morphological and cultural characters of most
species of streptococci is no justification for assuming that
the two are the same, and that they are mutually inter-
changeable. The streptococcus erysipelatos taken direct
from the erysipelatous skin of a man or rabbit (serum
squeezed out of the progressive margin), or from cultures
on serum, or Agar, broth or gelatine, particularly the first,
when inoculated into the skin of the root of the rabbit’s ear,
produces typical progressive erysipelas : after twenty-four
hours there is distinct blush and swelling, starting from about

1 The streptococcus erysipelatos forms more pronounced chains, even
on solid media, than does the streptococcus pyogenes.

MICROCOCCI

149

viii]

the seat of inoculation, and gradually extending towards the
tip of the ear ; in three to four days the whole, ear is red,
swollen, hot, and pendulous j later on, when the process retro-
grades, the epidermis is raised in blisters and peels just as
in erysipelas of man. The process is sometimes so seveie tha-.
the ear sloughs, or general septicsemic infection occurs :
occasionally not only the ear but also the skin of the neck

Fig. 34 —Film Specimen of Streptococcus Scarlatina from a Fluid
Culture. X 1000.

becomes involved. Sometimes on subcutaneous injection
of the culture at the root of the ear an acute septicaemic
infection is at once produced, the animal dying in twenty-
four to thirty-six hours, and the blood containing the strepto-
cocci in large numbers. Cultivations with a droplet of the
serum from the erysipelatous ear always yield numerous
colonies of the streptococcus.

After repeated subcultures on gelatine or Agar the virulence

150 MICRO-ORGANISMS AND DISEASE [chap.

— that is, the power to produce typical erysipelas in the rabbit’s
ear — becomes less and less and ultimately is lost. But
by starting a fresh culture on solidified blood-serum and
using then a somewhat large dose erysipelas in the ear can
be again produced, the lymph of this ear and its cultures
again being capable of producing typical erysipelas. Until
streptococcus pyogenes obtained from abscess or common
phlegmon can be shown to produce in the rabbit’s ear the
same typical progressive erysipelas as can the lymph of
erysipelas and the culture therefrom it must be held that the
two are distinct species. The facts that streptococcus pyo-
genes in its virulent varieties can produce a phlegmon in the
rabbit’s ear, and that streptococcus erysipelatos by subcultures
loses so much of its virulence as to produce not erysipelas
but only phlegmon, do not justify considering the two
as interchangeable ; as far as I am aware, streptococcus
pyogenes has not been so changed as to be capable of pro-
ducing erysipelas in the rabbit’s ear, whereas the attenuated
form of streptococcus erysipelatos can be readily brought back
to its former virulence, i.e. the power to produce typical
erysipelas. Streptococcus erysipelatos occurs as a complica-
tion in typhoid fever in perforation ; then the peritoneal
fluid and the blood contain numerous streptococci the culture
of which produces in rabbits typical erysipelas. The name
“ streptococcus erysipelatos ” must therefore be reserved for
that species of streptococcus which is found in genuine human
erysipelas, and which can set up in the rabbit’s ear typical
spreading erysipelas, and must not be mixed up with strepto-
coccus pyogenes, however much morphologically and cultur-
ally the two approach one another.

6. The same may be said of the streptococcus, which I
described, of the contents of the vesicles and of the ulcers
in foot-and-mouth disease of sheep. In culture it resembles

MICROCOCCI

vi 1 1]

151

streptococci in general, inclusive of the streptococcus pyo-
genes, although on gelatine its colonies are markedly trans-
parent, and it grows much slower than those of streptococcus
pyogenes. Cultures injected into the skin of sheep produced
a vesicle, and from it the same streptococcus was cultivated.
Schottelius described a chain-coccus in foot-and-mouth
disease which seems to me indistinguishable from the one
which I described.

7. The streptococcus which I cultivated in a certain per-
centage of cases of scarlatina from the blood of patients
during the acute febrile stage belongs to this group ; when
injected into rodents it produces in a large percentage acute

Fic. 35. — Colonies of Streptococcus of Foot-and-Mouth Disease as seen
on the Surface of Gelatine under a Magnifying Glass.

septicemic infection. That this streptococcus is of a
secondary character and capable of producing the purulent
and other additional phlegmonous changes indicating
secondary infections in scarlatina, as is maintained by
several observers, remains to be shown. As far as my obser-
vations go, I found the streptococcus in the blood of patients
in the early febrile stages of pure scarlet fever in which of
secondary infection nothing could be seen.

The same streptococcus was found in connection with an
eruptive (ulcerative) disease on the teats and udder of milch
cows at Hendon in 1886, to the consumption of whose
milk an extensive outbreak of scarlet fever in the north of
London was definitely traced (see Mr. Power’s report for

152

MICRO-ORGANISMS AND DISEASE [chap.

1886 to the Local Government Board). This intimate
relation between an eruptive (ulcerative) disease of the teats
and udder of milch cows to the cause of human scarlet
fever was subsequently to 1886 demonstrated in several
other localities (Glasgow, New Cross). In the Hendon
cows, above referred to, there was in addition disease of
the lungs and kidneys, from which the streptococcus was
obtained by culture. Cultivations of the streptococcus
from the blood of human scarlet fever or from the eruption
on the teats of cows produced in mice and calves a
definite general infection ; in healthy milch cows the injec-
tion of the streptococcus produced the eruption with sub-
sequent ulceration on the teats and udder, as also the
visceral disease observed in the Hendon cows. (. Reports
of the Medical Officer of the Local Government Board for
1886, 1887, 1S88.)

S. Loffler 1 showed that in faucial diphtheria, and asso-
ciated with the diphtheria bacilli, occur streptococci, some
of which, at any rate, play an important part in the secon-
dary infections — swollen and suppurative glands — as also in
septictemic infection. These streptococci when injected into
animals cause occasionally disseminated inflammatory foci,
principally in the joints, and general septicemic infection.

9. Membranous exudations in, and inflammation of, the
fauces occur which are not accompanied by diphtheria
bacilli, and which therefore are not true diphtheria ; they do
not lead to post-diphtheritic paralysis and terminate in
recovery ; they resemble mild cases of diphtheria. Such
cases represent the cases of pseudo- or cocco-diphtheria. The
exudation is found to be crowded with cocci, often in
larger or smaller masses, numerous leucocytes being also
present. When cultivated one obtains colonies of staphylo-
1 Mittheilungcn aus d. k. Gesundheilsamte , I I.

VII i]

MICROCOCCI

i53

coccus aureus and albus and two species of strepto-
cocci— one in which the chains are made of cocci of the
size of those of streptococcus pyogenes, and another of
much smaller cocci and forming shorter chains.

10. Schiitz 1 discovered that acute pharyngeal abscess in
the horse (“ Druse ”) is caused by a streptococcus which
culturally differs from the streptococcus pyogenes principally

Fig. 36.— Film Specimen of Capsulated Diplococcus Pneumonias in Rusty
Sputum of Acute Croupous Pneumonia.

X 1000. (A. Pringle.)

in this that the former does not grow below 22° C. ; it acts
virulently on rodents, and its culture produced in horses the
typical pharyngeal abscess.

11. Cases of acute pneumonia occur which are associated
with the copious presence of streptococci in the blood-vessels
as also in the air-cells ; they are considered by Finkler ( Die
Lungenentziindungen , &c.) to have caused the pneumonia.

1 Archiv f. wiss. und prakl. Thierheilk. vol. 14, No. 3.

'54

MICRO-ORGANISMS AND DISEASE [chap.

12. The pneumococcus or diplococcus pneumonia of
Fraenkel and Weichselbaum. The principal morphological
character of this microbe is that it occurs chiefly as dumb-
bells or short chains of dumb-bells of cocci ; the dumb-bells
are invested in a gelatinous capsule easily stained when
obtained directly from animal tissues. It occurs occa-

Fig. 37. — Film Specimen of Bronchial Sputum from a case of Acute
Influenza, showing Capsulated Diplococcus Pneumonite.

X 1000.

sionallv, but sparingly, also in normal bronchial expecto-
ration ; in the fluid of the mouth and nose (rarely) , in
the rusty sputum and the fluid of the lung in the acute stage
(red hepatisation) of croupous pneumonia (large percentage
of cases) ; in the peritoneal exudation in some cases of peri-
tonitis ; in the pericardial and pleural effusions in acute
pericarditis and pleurisy; in the effusion in cerebro-spinal
meningitis; in the purulent matter in inflammation ot the

MICROCOCCI

155

vm]

middle ear ; in some cases of ulcerative endocarditis in which
thevalvescontain massesof this diplococcus ; in the bronchial
sputum in influenza, and in catarrhal bronchitis. This diplo-
coccus does not grow below 220 C. (i.e. not on ordinary
nutrient gelatine solidified) ; it grows well above 28° C-, best
at 35° to 38° C. On Agar or on blood-serum it forms at
370 C, already after twenty-four hours, minute, translucent,
round colonies, which after two to three days appear raised,
moist-looking, whitish-grey, and round. In culture the
capsule around the diplococci is absent altogether or only
slightly indicated.

On account of its presence in large numbers— sometimes
in pure culture — in the rusty sputum and in the blood-juice
of the lung in the stage of red hepatisation in the great
majority of cases of croupous pneumonia, prior to the height
of the disease, it must be assumed that it has an intimate
relation to the cause of this disease ; that it is not the only
cause of croupous pneumonia is shown by the fact that in
some cases only streptococci are present. In some epidemics
(Middlesbrough) a motile bacillus was found in pure
culture in the lung-juice in the red hepatised lung. But,
assuming with most pathologists that in the majority of
cases of genuine acute croupous pneumonia it is intimately
related to the causa vera, it is not easily seen why the same
microbe (the same in respect of morphological, cultural, and
physiological characters) should in one instance cause
croupous pneumonia, in another ulcerative endocarditis, in a
third peritonitis, and in a fourth suppuration of the middle
ear ; or why it should be found fairly abundantly in some
cases in the bronchial sputum (bronchitis, influenza) with-
out producing pneumonia. All this is obscure and un-
intelligible if the diplococcus pneumoniae be considered as
the essential and sole cause of croupous pneumonia.

156

MICRO-ORGANISMS AND DISEASE [chap.

Recent cultures of the diplococcus made from pneumonic
sputum or other exudations (mentioned above) inoculated into
mice or rabbits produce as a rule fatal septicaemic infection ;
the viscera are greatly congested, and the blood and viscera
contain abundantly the microbe. The same result is pro-
duced in the rabbit by injecting it with the rusty sputum of
croupous pneumonia prior to the fifth or sixth day. In the
blood and tissues of a mouse or rabbit that succumbed to

Fig. 38.— Lung Juice of Guinea-pig dead after Infection with Micrococcus

Tf.tragenus.

X 1000. (A. Pringle.)

infection, the diplococci are capsulated, and the capsules
can be as easily stained as those in the sputum or bronchial
exudation with eosin after the cocci themselves had been
stained with methyl-blue. Staining with gentian-violet in
alcoholic solution, and then carefully washing in water, shows
the cocci stained deep purple, the capsules light violet.

Cultures that have been carried on for some generations
gradually lose the power to produce infection in the rodents,

MICROCOCCI

157

vi n]

but on growing them again on serum or in broth to which
a piece of boiled white of egg has been added the cultures
regain virulence.

The capsulated, oval, rod-shaped, or cylindrical microbe de-
scribed first by Friedlander as being the cause of croupous
pneumonia occurs in the sputum only in a small percentage
of cases, certainly not more than five per cent. ; it occurs
also occasionally in the bronchial secretions not connected
with croupous pneumonia, and even in the fluid of the
mouth in health. This bacillus of Friedliinder is most prob-
ably identical with the capsulated microbe of the fluid of
the mouth described by Sternberg. Inoculated in largish
quantities into the rabbit, it causes acute septiccemic infection
and death ; in the blood and various viscera the microbe is
then abundantly present.

13. Micrococcus tetragenus. — This microbe, related to
sarcina-like cocci, was found by Gaffky in pulmonary
tubercular expectoration and in the tissue of the tubercular
lung. It occurs in groups of four cocci surrounded
by a capsule. Cultivated in gelatine plates, it forms
already after twenty-four hours minute white dots which
during further incubation enlarge into prominent white moist
discs. In streak cultures it forms a narrow, white, sticky
growth along the line of inoculation. White mice are very
susceptible to infection by subcutaneous injection of small
quantities of culture. The animals begin to show illness
after two days and generally die after three to six days. The
blood and the spleen contain the microbe in large quantities.
Also guinea-pigs are susceptible, but less so, since as a rule a
local abscess is formed only, and occasionally a general
fatal infection.

14. Micrococcus of acute infectious osteomyelitis. — Dr. Becker
has made, in the laboratory of the Berlin Imperial Sanitary

<58

MICRO-ORGANISMS AND DISEASE [chap.

Office, a series of important experiments on the micro-
cocci discovered by Schuller and Rosenbach. He collected
pus from five cases of acute osteomyelitis in which the
abscesses had not been opened, and cultivated the micro-
cocci on sterilised potatoes, coagulated serum, and gelatine-
peptone. After 3-5 days the punctures made by the
needles assumed the appearance of white streaks, around
which the gelatine gradually liquefied and took an orange
colour. The culture injected into the jugular vein
caused acute septicaemia and death ; but nothing abnormal
was found in the bones in either case. A small quantity
was then injected into the jugular veins of fifteen rabbits,
after having, some days before, fractured or bruised the
bone of one of the hind legs. At the end of the first week
a swelling was formed at the seat of the bruise or fracture ;
the animals lost flesh and died after a few days. On
dissection, large abscesses were found around and in the
bones, and in several cases metastatic abscesses had formed
in the lungs and kidneys. Numerous colonies of micrococci
were discovered in the blood, which are identical with the
staphylococcus pyogenes aureus.

15. Koch1 described various kinds of micrococci inti-
mately connected with certain pysemic processes in mice and
rabbits, (a) Micrococcus of progressive necrosis in mice.
Injecting into the ear of mice putrid fluids, he observed a
necrosis of the tissues of the ear (skin, cartilage) starting
from the point of inoculation and gradually spreading on to
the surrounding parts and killing the animal in about three
days. As far as the necrosis reaches, the tissue is crowded
with micrococci, chiefly in the form of chains and zooglcea.
The individual cells are spherical, of about 5 //. in diameter.

1 Untersuchungen iiber die Aeliologie d. IVundinfections- k'rankheiten,
Leipzig, 1 8 78.

MICROCOCCI

159

viii]

I have inoculated a number of white mice subcutaneously
in the tail with a small micrococcus, due to accidental
contamination. These micrococci, having been cultivated
through several generations, were used in small doses for
the inoculation of the mice. In two instances the
inoculation was followed after two or three days by puru-
lent inflammation at the seat of inoculation, but apparently
not spreading beyond it. But, as time went on, inflam-
mation and abscess in the lungs set in and the animals

Fig. 39. — From a Section through the Tail of a Moure inoculated into
the Subcutaneous Tissue of the Tail with artificially cultivated
Micrococcus.

The part here illustrated is a good distance from the ulceration,
i A capillary blood-vessel filled with blood-corpuscles.

2. Fat cells.

3. Groups of micrococci filling the lymph-spaces of the connective tissue.

died after about a week. On making longitudinal sections
through the tail, it was found that in most of the lymph-
spaces and lymph-vessels of all parts of the cutis and
subcutaneous tissue, far away from the seat of inflammation,
there were densely crowded masses of the same minute
micrococci as were used for inoculation. And these crowds
of micrococci could be traced to the seat of inflammation,
where they extended amongst the inflammatory products in
great masses. The abscesses in the lungs were filled with

i6o

MICRO-ORGANISMS AND DISEASE [chap.

the same micrococci. Inoculated into the skin of fresh
mice, it again produced death by pyaemia. This micro-
coccus may therefore be called the micrococcus pyaemia, of
mice. ( b ) Micrococcus causing abscesses in rabbits. Putrid
blood injected into the subcutaneous tissue of the rabbit
often produces suppurative abscess which, spreading, kills
the animal in about twelve days. In the wall of the abscess
are found continuous masses of zooglcea of micrococci.
The pus is infectious. The micrococci are spherical, and of
a very minute size, measuring only about o-oooi5 mm. in
diameter. ( c ) Micrococcus causing pyamia in rabbits.

Skin of a mouse was macerated in distilled water for two
days, and of this fluid a hypodermic syringeful was injected
under the skin of the back of a rabbit. After two days
the animal began to lose flesh and died after 105 hours.
Purulent infiltration spread from the seat of inoculation into
the subcutaneous tissue ; peritonitis ; spleen much enlarged ;
slight pneumonia. A hypodermic syringeful of the blood
of this animal was injected under the skin cf a second
rabbit, and this died after forty hours. Post-mortem exa-
mination showed the same lesions as in the first case. In
the blood-vessels of the affected parts were present micro-
cocci, single, as dumb-bells, and in zoogloea ; they were
spherical, about o-ooo25 mm. in diameter. ( d ) Micrococcus
causing septicamia in rabbits. An infusion of meat was
prepared ; this was left to putrefy, and of this fluid a quantity
was injected under the skin of the back in two cases. Ex-
tensive gangrene with much cedematous exudation followed,
and death ensued in two days and a half. The blood, the
capillaries of the kidney, and the enlarged spleen contained
numerous oval micrococci. Two drops of the cedematous
exudation-fluid were injected under the skin of the back 01
another rabbit. Death followed in twenty-two hours. There

VIII] MICROCOCCI 161

was no gangrene here ; but cedema was present, spreading
from the seat of the inoculation. Sub-serous haemorrhages
appeared in the intestines ; and minute haemorrhages were
also present in the cedematous tissue and in the muscles of
the thigh and abdomen. The oedematous fluid, the cuta-
neous veins, the capillaries in the kidney, especially those
of the glomeruli, in the lung, and in the spleen, contained
numerous oval micrococci, singly, in dumb-bells, and in
zooglcea. The micrococci measured about o-8 to i /z
in their long diameter. These micrococci (taken with
the blood) produced in another rabbit and in a mouse the
same fatal disease.

1 6. Micrococcus bombycis (Microzyma bombycis, Bechamp).
—Oval micrococci, of about i '5 /z in length, present
in large numbers, singly, and as dumb-bells and chains
(straight or curved), in the contents of the alimentary canal
and in the gastric fluid of silkworms dead of the “ maladie
de mortsblancs, flacker ie.”— Micrococcus ovatus, Nosema
bombycis. Present in large numbers in the blood and
organs, ova included, of silkworms affected with the disease
called “maladie des corpuscules,” “ pebrine,” or Cornalia’s
disease. Cornalia first saw them, afterwards Lebert and
Nageli. Pasteur proved definitely that ingestion as well as
inoculation of the silkworms with the micrococci produces
the disease. The micrococci are comparatively large, 0-003
to 0-004 mm. long, 0 002 mm. broad ; they are very bright
and occur singly, or in dumb bells, or in small groups.

17. Micrococcus of gonorrhoea { gonococcus). Neisser was
the first who pointed out the constant presence, in the
exudation in gonorrhoea, of peculiar micrococci, which
occur as dumb-bells and as masses of dumb-bells, either

1 62 MICRO-ORGANISMS AND DISEASE [chap.

free in the serum, or frequently within the protoplasm of
the pus cells, or adhering in smaller or larger numbers to
the epithelial cells : these cocci he called gonococci.
They are i-25 /x in length as diplococci, o'6-o-8 /x in trans-
verse diameter, and they occur, as just stated, in the form
of diplococci and as groups of four ; the cocci are cres-

Fig. 40 — Film Specimen of Gonorrhoeal Pus. In the Centre two Pus-
Cells CONTAINING IN THEIR INTERIOR NUMEROUS GONOCOCCI.

X 1000. fE. C. Bousfield.)

centic and in this respect do not differ from many other
species of cocci. Besides these diplococci, cocci often occur
in the pus of gonorrhoea which are spherical and probably
belong to the staphylococcus species (liquescens albus and
liquescens aureus).

The gonococcus does not grow on nutrient gelatine, on

MICROCOCCI

1 63

vi 1 1]

Agar mixture, or potato, and herein differs materially from
the ordinary cocci occurring in pus. Bumm has proved
that the gonococcus grows only on blood-serum, and Loffler
and Krause have also succeeded in growing it on serum.
In streak cultures on moderately solid blood-serum kept at
320 C., well moistened, the gonococcus, according to Bumm,
grows in the form of a thin, narrow, greyish-yellow film
1-2 mm. in breadth, with smooth and moist-looking surface.
The growth does not proceed for more than a few days and
then dies. Animals are refractory against the gonococcus
or the gonorrhoeal secretion ; dogs, rabbits, monkeys, horses,
show no reaction, neither on the conjunctiva nor on the
urethra. Bumm has, however, succeeded in producing in
the human subject real gonorrhoea by inoculating, from a
culture of the gonococcus, the urethral mucous membrane.

There can be no doubt about the fact that the gono-
coccus, which, as mentioned above, grows well on serum, is
peculiar to gonorrhoea and cannot, therefore, be confounded
with other pus micrococci. Probably Neisser’s gonococcus
was only a pus coccus, since it grew also on other media.

CHAPTER IX

bacillus ( Desmobacterium , Cohn)

GeJieral Characters. — Bacilli are cylindrical or rod-shaped
bacteria, which are rounded or square-cut at their extremi-
ties ; they are longer in proportion to their thickness, and
divide by fission, forming straight, curved, or zigzag chains
of two, four, six, or more elements. Many species of bacilli
in suitable nourishing material grow by repeated divi-
sion into longer or shorter chains of bacillus — filaments or
leplothrix — while other species have little or no tendency to
form filaments. These appear straight or wavy and twisted,
isolated or in bundles ; and, although in the fresh condition
they appear of a homogeneous aspect, when suitably pre-
pared, as by drying and staining with aniline dyes, they show
themselves composed of shorter or longer cubical, cylindrical,
or rod- shaped protoplasmic elements, contained in linear
series within a general hyaline sheath ; between many of the
elements is a fine transverse septum. The isolated bacilli
are likewise composed of a membrane and protoplasmic
contents. These latter appear homogeneous or finely
granular and, when stained with aniline dyes, absorb the
dye very easily and retain it better and longer than the
sheath.

CHAP. IX]

BACILLUS

165

The protoplasm is either uniformly stained, or, as is not
uncommon, shows at the ends of each rod much deeper
staining than in the middle — that is to say, there is denser
protoplasm at the ends of the rods than in the middle. In
the short individuals this often gives a very characteristic
appearance, inasmuch as each rod appears made up of three
parts of equal size : two terminal stained granules and a
middle clear unstained part. As just stated, this is not

Fig. 41. — Bacillus Subtilis grown in Pork Broth.

At 1, ihe elements are thickened. The preparation had been dried and stained with

aniline purple.

peculiar to any one species, but can be noticed in all species ;
it is particularly conspicuous in those in which the young
elements are short, e.g. fowl cholera, fowl enteritis, septi-
caemia of rabbit, swine fever, &c., &c. But also amongst
the longer, i.e. cylindrical, elements the middle part of the
rod appears very often unstained and clear, while the proto-
plasm at the end is denser and stained ; the middle clear
part is at the same time more or less well marked off with

1 66 MICRO-ORGANISMS AND DISEASE [chai>.

rounded outline, spherical or oval in shape, and represents a
vacuole ; occasionally the stained protoplasm is central,
while the unstained parts, the vacuoles, are terminal. Such
vacuoles are very common in all species of bacilli ; they
(vacuoles) are, however, more frequently met with under
conditions which imply want of sufficient nutritive material,
as, for instance, when bacilli grow on solid media (gelatine,
Agar mixture, potato) and when, owing to the continued
growth into the depth of the medium, the first-formed or
superficial layer becomes gradually removed from the nutri-
tive material ; in this superficial layer the vacuoles in the

c='

**-•*»,

5*=s,

Fig. 42.— The same Bacillus as in preceding figure.

At 1, spores have made their appearance.

rods are very conspicuous ; in preparations made of thread-
forming bacilli under the above conditions of growth these
appearances, i.e. of the presence of vacuoles regularl)
disposed in the individual rods, are very striking.

But, as stated before, the presence of vacuoles in the rods
is -also found under other than the above conditions, in
some species more numerously than in others, and more
often where rapid growth takes place than where this is not
the case. This vacuolation is not indicative of any degenera-
tive change, any more than it is in the mycelial threads of
fungi where it is well known and typical, but seems, in
some cases at any rate, to be due to the medium in which

m A, . -..4. . i

BACILLUS

.X]

167

the bacilli grow containing comparatively less nutritive
material : not only in bacilli, but also in the individuals
composing a spirillum, are these vacuoles to be observed.
In cylindrical bacilli these vacuoles may be, and sometimes
have been, mistaken for spores.

The ends of bacilli are generally rounded, occasionally
straight, and less frequently more or less pointed or conical
at one or both ends. In bacillus anthracis the ends are
generally more or less straight ; in the bacillus of diphtheria
grown on gelatine many bacilli show one end pointed, the
other rounded or straight and thick.

According to the stage and the rapidity of their growth,
the bacilli vary much in length ; this is the case not only
with the single bacilli and short chains, but also in an
eminent degree with the elements of a bacillus filament or
leptothrix. In each case, indeed, it is possible to ascertain
that all lengths occur, from the cubical or spherical element
to the cylinder or rod. The former elongate into the latter
and then divide. According to whether the division occurs
in a short or long element, the daughter elements are
cubical or spherical in the former, cylindrical or rod-shaped
in the latter case. This applies to single bacilli, to short
chains, and to the leptothrix forms.

There are a great many species of bacilli, differing
morphologically from one another in the shape of the
elements, in motility, in the power of forming filaments or
leptothrix, and particularly in the thickness and length of
the elements.

There are some species of bacilli — e.g. hay-bacillus,
anthrax-bacillus, bacillus mesentericus, proteus vulgaris,
bacillus of malignant oedema (Koch), & c. — in which in
the single bacilli and in the chains and filaments the size
of the elements varies from that of a cubical or spherical

1 68

MICRO-ORGANISMS AND DISEASE [ch. lx

mass of protoplasm not more than o-5 or o-8 /a in diameter to
that of a cylinder or rod several times as long as it is thick.
In some species (e.g. stained tubercle-bacilli) the elements
of a chain arc almost spherical. There are, on the other
hand, other species (e.g. bacillus typhosus) where the ele-
ments are always rods or cylinders. In these cases of short
bacilli it sometimes becomes difficult to say whether an

Fig. 43.— Chains of Bacilli (Bacillus Filamentosus) in a Stained Film

Specimen.

individual is or is not a bacillus, but the growth of the
bacilli into cylinders and leptothrix, and particularly their
power of forming spores, is decisive, although neither of
these events may happen, owing to peculiar conditions.

Flagella and motility of bacilli have been treated in a
former chapter, and we need therefore not specially further
concern ourselves about them.

Not all bacilli are capable of forming leptothrix-filaments.

Fig. 44. — A Colony of Filamentous Bacilli (Bacillus Anthracis) as seen
under Magnifying Glass.

Fig. 45.— Same seen under a low Magnifying Power.

170 MICRO-ORGANISMS AND DISEASE [chap.

This power is possessed in an eminent degree by certain
species, such as the hay-bacillus, the anthrax-bacillus, the
bacillus of malignant oedema, the bacillus found on the
surface of the mucous membrane lining the cavity of the
mouth and tongue (leptothrix buccalis). Other bacilli ( e.g .
bacillus coli, leprosy-bacillus, tubercle-bacillus, &c.) generally
do not, though exceptionally they do, form leptothrix.

Different species show great differences in the thickness
of the bacilli, some being very fine, e.g. bacillus of mouse-
septicaemia, bacillus of influenza; others thick and plump —
bacillus amylobacter, bacillus megaterium ; but it is also
noticed that the bacilli of the same species growing in
different culture media show in some cases considerable
differences in this respect, in one medium forming thin
bacilli, whereas in another medium the bacilli may be twice
and thrice the thickness. The same may even occur in the
same medium (see Fig. 41).

Many bacilli and bacillus-filaments (e.g. hay-bacillus, an-
thrax-bacillus) degenerate on growing old, the protoplasmic
elements becoming granular and breaking down altogether
into debris. This may occur to single elements within a
chain or leptothrix ; and then the corresponding part of the
sheath of the chain, owing to the subsequent disappearance
of the debris, becomes empty and devoid of protoplasm.
Longer or shorter portions of a chain or leptothrix may thus
degenerate and become deprived of protoplasm, the sheath
only persisting. These portions become at the same time
thicker, the sheath having swollen up.

Another mode of degeneration consists in the elements
and sheath curling up, swelling up, and ultimately breaking
down into debris. According to Cohn,1 bacilli do not form
zoogloea in the same way as micrococcus and bacterium do.

1 Beitr. z. Biologie d. PJlanzen, vol. ii.

IX]

BACILLUS

171

With all due deference to the authority of Cohn, I must
hold that some bacilli possessed of motility are capable of
forming a true zoogloea. When one inoculates a fluid
nourishing medium (e.g. broth) with hay-bacillus or other
motile bacillus (e.g. bacillus mesentericus), after keeping it
for twenty-four hours in the incubator one notices that the
surface of the fluid is covered with a whitish film ; this, as
incubation goes on, thickens into a thick, resistant, not very
friable pellicle. By shaking the fluid the pellicle becomes
detached from the glass wall and sinks to the bottom of the
fluid ; after another day or two a new pellicle is formed, and
so on until the material is exhausted.

Any part of this pellicle examined under the microscope
shows itself to be a zoogloea in the true sense of the word,
vast numbers of shorter or longer bacilli crossing and inter-
lacing and lying embedded in a gelatinous hyaline matrix.
As with proteus vulgaris, one occasionally notices at the
margin of the mass one or other bacillus wriggling itself free
and darting away. And in the case of non-motile bacilli,
putrefactive and others, I have also seen distinct formations
of zoogloea, having the shape of spherical or oval lumps of
various sizes composed of a hyaline jelly-like matrix, in which
are embedded the bacilli in active multiplication.

In those species in which the bacilli are capable of forming
leptothrix (leptothrix buccalis, hay-bacillus, anthrax-bacillus)
the filaments may form dense convolutions. When in these
convoluted filaments spores are formed, and the sheaths of
the filaments swell up and become agglutinated into a
hyaline jelly-like substance, the spores appear to form a sort
of zoogloea.

Bacilli are killed by drying, but it is necessary to bear in
mind that they must be exposed to the drying process in
thin layers (Koch). At the temperature of boiling water

l7- MICRO-ORGANISMS AND DISEASE [ch. ix

they are invariably killed, but not their spores. Even heat-
ing them from half an hour to several hours at a temperature
above 550 or 6o° C. kills them. Freezing also kills them,
but not their spores. Carbolic acid, corrosive sublimate,
thymol, &c., kill them.

1 he formation of spores and the germination of these

Fig. 46— Threads of Bacii.li (B. Anthracis) showing in Parts, or as a

WHOLE, THE EMPTY SHEATH WITHOUT ANY STAINED BACILLARY PROTO-
PLASM.

X 600.

have been already described in a former chapter, and it now
remains to describe the methods of staining them. When
spores, either free or in bacilli, are stained in the usual way
in film specimens, the spores do not take the stain, but
remain conspicuous as clear oval bodies ; in order to make
them take the dye it is necessary, after drying in the usual

Fig. 47 — Spore-bearing Bacilli stained in the ordinary manner
(Bacillus of Symptomatic Charbon,), the Spores being unstained.

x IOOO.

Fig. 48. — Spore-bearing Filaments (Bacillus Anthracis) ; the Spores,
stained after boiling in Carbol Fuchsin, are deeply stained, the
rest of the Filaments only faintly so.

X 600.

174 MICRO-ORGANISMS AND DISEASE [ch. ix

way, to boil the film cover-glass specimen in the dye (methyl- jj
blue, gentian violet, or carbol fuchsin) ; hereby the spores
become deeply stained and on subsequent good washing
retain the dye with great persistence. By careful washing
the point may be so hit off that the spores appear deeply
stained, whereas the bacillary substance is only faintly so.

The finest specimens are obtained by boiling the dried film
specimen in carbol fuchsin ; then wash well in water ; then
place the specimen in methyl-blue anilin water for half a
minute to one minute ; wash again well ; dry and mount in
xylol balsam : the bacillary substance appears blue, the spores
bright red.

It has been shown by Engelmann that the presence and
renewal of oxygen as well as a certain concentration of the
nutritive material are essential for the motility of those bacteria
that are possessed of cilia, i.e. that are possessed of locomo-
tion and which normally grow aerobically. This, of course,
does not apply to the motile anaerobic bacilli, e.g. bacillus
of malignant oedema, tetanus, or butyricus.

As long as the bacteria are living, their protoplasm does
not combine (stain) with nitrate of silver solution, only after
death does this become possible. Hereby an index is
furnished for ascertaining whether, and which, bacteria in a
given sample are living, and which are dead. There is no
difference in this respect, i.e. in respect of the different
reaction of nitrate of silver on living and dead protoplasm,
between the protoplasm of bacteria and that of other vege-
table or animal tissues.

All aerobic bacteria, pathogenic and n on-pathogenic,
requiring for their growth and multiplication oxygen, obtain
this from the medium in which they grow, and which oxygen
is dissolved in those media, or after this is consumed or
absent it is obtained by the bacteria in the process of the
chemical decomposition of the carbohydrates and proteids

Fic. 49 —Impression Specimen from a recent Gelatine-plate Culture
of Bacillus Anthracis. X 1000.

Fic. 50 —Impression Specimen from a rfcfnt Gelatine-plate Culture
of Bacillus Diphtherias, x 1000.

176

MICRO-ORGANISMS AND DISEASE [chap.

present. Dr. Duprd 1 has shown that the presence or
disappearance of oxygen (air) dissolved in water is a precise
gauge, in the first case of the absence, in the second of the
growth, of microphytes.

In many species one or both ends of the rods, or the free
end of the rods forming the terminals in a chain, are swollen
and thick, spherical, pear-shaped, or club-shaped (Fig. 50) :

Fig. si. — Film Specimen of Tubercle-Bacilli from a Glycerine-Agar
Culture some weeks old; showing Branched Mycelial-like Fila-
ments with Club-shaped Sproutings. .

x 1000.

occasionally there are some elements, in the middle of a
shorter or longer chain, swollen, spherical, or oval. Such
forms are considered as involution forms, but I have good
grounds for doubting this, and the reasons will be stated
later on in connection with the evolution of bacteria. When
in a chain of rods, i.e. in a thread, the individual rods
1 Report of the Medical Officer of the Local Government Board, 1884.

IX]

BACILLUS

177

become so changed, an organism results which is totally
unlike the typical thin smooth thread, but appears more like
a varicose thread in which the individuals are torula-like,
spherical, or oval cells, connected one with another by thin
bridges, the cells being three or more times as thick as the
typical rods (Fig. 49). In connection with this and the
former appearance, another appearance deserves notice, viz.
the segregation of the protoplasm in a chain or in individual
rods as separate spherical or oval granules, whereby the rods
and chains become transformed into varicose rods or fibres ;
in these the granules take and retain the dye easily, whereas
the bridges between them are less stained ; e.g., in tubercle
bacilli, leprosy bacilli, diphtheria bacilli, and others this
appearance is sometimes very regular and characteristic.

Besides the above torula-like chains of the bacilli with or
without terminal club-shaped or pear-shaped enlargements,
another curious appearance deserves notice, that is the
branching that is observed in threads of tubercle bacilli
when grown for some time on glycerine Agar ; we find
here, besides torula-like threads, with club-shaped terminals,
others which show distinct sprouting and gemmation of
lateral cells,1 these latter elongating into threads themselves
with club-shaped terminal enlargement. This suggests that
the tubercle bacilli are probably originally evolved from a
mycelial fungus and under certain conditions have a ten-
dency to revert to this state (Fig. 5r).

1 Report of the Medical Officer of the Local Government Board, 1890-91.

CHAPTER X

BACrLU : SPECIAL

Before describing the various species of bacilli which in
man or animals, or both, are associated with infectious
disease we will describe the most common non-specific
bacilli, as, owing to their wide distribution, they not un-
commonly are found associated with the former.

These are the most widely distributed species of bacilli : —

(i) Bacillus subtilis, or hay-bacillus ; (2) bacillus

mesentericus vulgatus ; (3) proteus vulgaris ; (4) proteus
Zenkeri ; (5) bacillus fluorescens liquescens; and (6) bacillus
coli.

1. Bacillus subtilis (hay-bacillus). — The elementary rods
are of various lengths from o-oo2 to o'oo6 mm., and are
about o'oo2 mm. in thickness. According to Cohn,
at a temperature of 210 C. division into two requires
about one hour and a quarter, at 35° C. only about twenty
minutes.

The bacilli are capable of forming leptothrix filaments.
The bacilli when single are possessed of one flagellum, or
sometimes of two, one at each end. After division the
individual bacilli remain connected, each possessing a
flagellum at the free end. Each ot them divides again
into four, so that a chain of four is formed. But they may

CHAP. X]

BACILLI : SPECIAL

1 79

separate again or may go on dividing, remaining united,
and thus forming a longer or shorter filament. Not all
bacilli possess motility, many of them being for a time in a
resting state.

The bacilli form a dense resistant pellicle on the surface
of the nourishing medium, and in this copious spore-
formation takes place. If shaken when growing in a fluid,

Fig. 52. — From a Culture of Bacillus subtilis (Hay- Bacillus).
Various forms between single bacilli and leptothrix.

Magnifying power about 700.

the pellicle falls to the bottom, and soon a new pellicle is
formed.

Spore-formation is independent of any deficiency of
nourishing material. The spores are oval, bright, of about
0 001 to o'oo2 mm. in length, and about o-ooo6 to o'ooi
mm. in thickness. They do not stain in ordinary dyes,
and hence form a great contrast to the bacilli.

This bacillus is very common and widely distributed ; it

N 2

i8o

MICRO-ORGANISMS AND DISEASE [chap.

occurs in organic substances left exposed to the dust of air.
The best material is hay-infusion. An infusion, cold or
hot, of hay is made in a beaker or flask ; the fluid is
neutralised, then filtered, covered with a glass plate, and
left to stand in a warm place. After a day or two it
swarms with bacillus subtilis, which is also called hay-
bacillus, since ordinary hay contains multitudes of its
spores. For this reason even boiling of the fresh infusion
for a few minutes does not sterilise it.

The bacillus grows well in every fluid that contains the
necessary salts and nitrogenous compounds ; thus all kinds

Fig. 53. — From a Culture of Bacillus Subtilis (Hay-Bacillus), with
Copious Formation of Spores.

1. Mass of spores embedded in hyaline matrix.

2. Bacilli.

3. Single bacilli containing each a spore : the sheath of the bacilli is well seen.

Magnifying power about 700.

of broth, all kinds of animal- fluids (hydrocele, blood-
serum, &c.), gelatine, peptone solution, &c., are suitable
nourishing media.

The spores of the hay-bacillus are widely distributed in
the air, and contaminations by dust are due to its spores.

Hay-bacillus is an aerobic microbe:

In gelatine plates it forms liquefying colonies showing
characteristic threads radiating from the centre. In stab
and streak gelatine cultures it grows rapidly and liquefies
the gelatine ; solidified blood-serum is liquefied by the

BACILLI : SPECIAL

1 8 1

growth. On potato it forms a thick, whitish, creamy growth
rapidly covering the inoculated surface ; litre, as also on
fluid media, it forms copious spores in a resistant, corrugated
surface-pellicle.

In hay-infusion (neutralised) that had been kept in the
incubator at 370 C. the spores which appear are not all
belonging to the bacillus subtilis ; those in the surface-
pellicle are spores of this bacillus, but in the depth of the
fluid spores occur which resemble the above in aspect,
shape, and size, but which belong to the bacillus amylo-
bacter or bacillus butyricus of Prazmovski, a strictly

4 8

1 1 if

a? &

0 $

C=»

Fig. 54. — Germination of Spores into Bacilli.
a Spores of a small kind.
b. Spores of a larger kind of bacillus subtilis.
Magnifying power about 700.

anaerobic motile bacillus liquefying grape-sugar gelatine.
Aerobic gelatine plates made of such an infusion, or of the
surface pellicle, after heating to 8o°C. from five to ten minutes,
bring forth the colonies of the hay-bacillus only. Anaerobic
cultures in grape-sugar gelatine made of the fluid taken
from the bottom yield growth of the bacillus amylobacter ;
the chief morphological character distinguishing it from the
hay-bacillus and from other anaerobic bacilli (e.g. bacillus
butyricus of Hueppe and of Botkin) is its change in shape
during sporing ; the cylindrical bacilli, as spores develop and
grow in them, change into spindle- or tadpole-shaped
forms three and more times thicker around the spore —
Clostridium.

1 8 2

MICRO-ORGANISMS AND DISEASE [chap.

2. JSacillus mesentericus vulgatus , potato bacillus (Loffler).
This bacillus is spore forming, aerobic, very motile, and is
thicker than the former (bacillus subtilis) ; it occurs singly or
in chains of two or more rods ; it and its spores have a
wide distribution ; it is common in dust of air, and in many
putrid organic substances (potato, milk) ; in milk and other
organic fluids that have been exposed to air contamination
it is often present.

It differs from bacillus subtilis, first, by the greater thick-
ness of the bacilli, and, secondly, in the aspect of its colonies
in gelatine plates : these being round, liquefying, and con-
taining in the centre a membrane-like accumulation, but no
radiation of fibres. Sown in broth and incubated at 370 C.,
it forms already in twenty-four hours a conspicuous, coherent,
wrinkled pellicle, the broth remaining limpid. The pellicle
is a network of filaments in which oval glistening spores
soon make their appearance ; the spores are of the size of
those of hay-bacillus, but slightly thicker. On potato it
forms rapidly a sticky, greyish-yellow mass, on nutrient Agar a
wrinkled membranous growth ; growing on the surface of
gelatine, it liquefies this rapidly, forming, however, a coherent,
wrinkled, membranous mass.

3. Proteus vulgaris (Hauser). — This is an aerobic motile
non-sporing bacillus which, as Hauser has shown, is the
microbe of putrefaction. It is found in all putrid organic sub-
stances ; it is the principal microbe which is found in the putrid
bodies of dead animals and man. It is present normally in
the large intestine and from here after death soon extends
(grows) through the walls of the intestine into the abdominal
cavity, into the abdominal organs, then into the thoracic
viscera, and through the blood-vessels into all other parts.
It rapidly liquefies gelatine, and peptonises and destroys
animal matter. In gelatine plates (at 20° C.) its colonies

BACILLI : SPECIAL

Fig. 56 — Impression Specimen of “Swarmeks” of a Young Colony of
Proteus Vulgaris. X icoo.

Fig. 35.— Young Colony in Gelatine Plate of Proteus Vulgaris.
As seen under a Magnifying Glass.

1 84

MICRO-ORGANISMS AND DISEASE [chap.

appear after sixteen to eighteen hours as small greyish dots ;
when looked at under glass they are irregular in outline,
possessing longer or shorter angular filamentous projections.
These are composed of motile bacilli and are the forerunners
— swarmers — for further outgrowths, so that after twenty-four
hours or later many neighbouring colonies are connected by
these filaments and coalesce, the older colonies showing rapid

lique

hour

stab,

whic

occu

gelatine being fairly translucent, at the bottom of the liquefied
mass is seen a floccular, granular, white precipitate. On the

BACILLI: SPECIAL

185

surface of Agar (streak) at 370 C. the growth is moist, sticky,
and grey. Broth is made uniformly turbid in twenty-four
hours; later on, an imperfect sort of pellicle is noticed. Film
specimens (impression) made of young colonies on gelatine,
before liquefaction has set in, show beautiful filaments of
bacilli, some of considerable length and unsegmented, others
made up of short rods ; the filaments are straight or twisted
and at their ends show rapid division into cylindrical
bacilli. Fig. 56 shows such an impression film of the
swarmers of a young colony (sixteen hours old) ; Fig. 55
an impression of a colony twenty-four hours old, the centre
already liquefied. When the liquefaction has well pro-
gressed (say after two to three days) and a drop is examined
fresh under the microscope most of the bacilli are actively
motile, either short ovals— single or in dumb-bells — or cylin-
drical and even filamentous. There are also individuals so
short that they cannot be distinguished from cocci — single
cocci and diplococci ; and, further, some of the cylindrical
bacilli are more or less curved like vibrios, while some of
the filaments are wavy and even spiral-like. It is because
the microbe appears in such older cultures under all known
shapes (i.e. protean) that Hauser gave it the name of
“ proteus.” Proteus vulgaris is not, however, a single species.

The liquefied gelatine and the broth cultures possess dis-
tinctly a putrid smell. The bacilli possess a single short
spiral flagellum, and it is astonishing how briskly they move
in the fresh state and therein stand in striking contrast with
some other bacilli, e.g. bacillus coli, which, though some
individuals are provided with several flagella, show only
very feeble movement in the fresh state. In some varieties
the bacilli possess quite a number of flagella.

4. Proteus Zenkeri. — This is an aerobic, non-sporing,
motile bacillus of about o-4 //. thickness and 1 to 1*5/* length ;

186 MICRO-ORGANISMS AND DISEASE [chap.

it occurs frequently in putrid organic matter; in meat that
has been exposed to air and is undergoing putrefaction it is
often associated with proteus vulgaris. Its colonies in
nutrient gelatine are very characteristic (Fig. 58) : already
after twenty-four, better after forty-eight, hours’ incubation,
whitish dots are seen which are made up of numerous

Fig. 58. — Impression of a Colony in Gelatine Plate of Proteus Zenkeri.

Magnified with a glass.

bundles of more or less beaded filaments radiating from a
shorter or longer line situated in the depth ; in addition to
this, irregular grey grdups of plate-like masses seem to pass
out and to spread from the central mass on the surface. The
mass of threads resemble a mycelium of fungus ; the presence
of the grey plate-like masses makes it at once distinct. Under

BACILLI : SPECIAL

187

the microscope, in stained specimens the growth is made up of
threads which consist of rows — generally more than one — of
short bacilli ; in many places the bacilli form clusters in the
threads (Fig. 59). It does not liquefy gelatine. On the
surface of gelatine it forms a filamentous expansion, the
filaments growing from the central streak of inoculation like
the filaments in the fan of a feather ; the same kind of

Fig. 59.— Impression Specimen of the Filaments of Proteus Zenkeri.

X 300.

growth, only not so distinct, is formed by the microbe on
Agar ; it grows better at 20° than at 37° C.

5. Bacillus fluorescens liquescens. — This is a typical water
bacillus; it occurs in most waters — river, lake, pond, well — -
and in all or most organic substances to which such water had
been added. It is a motile, aerobic, non-spore-forming
bacillus, liquefying gelatine rapidly and producing a fluor-
escent greenish or diffuse greenish-blue colouration. Its

1 88

MICRO-ORGANISMS AND DISEASE [chap.

character in gelatine plates is sufficient to identify it : after
twenty-four hours at 20° C. it first forms grey, circular colonies,
already depressed and liquefying; after forty-eight hours the
colonieshave much increased and are now liquefied, depressed,
turbid, circular patches with a distinct greenish tinge of
colour. When the colonies are numerous and closely placed
the plate may by this time be altogether liquefied, the fluid
gelatine turbid and of greenish, fluorescent tint. In gelatine
stab culture the liquefaction proceeds from the upper part
of the stab, the lower part being made up of a row of
greyish-white dots. The appearance of a plate culture and
of a stab culture after twenty-four to thirty-six or forty-eight
hours’ incubation, as also of the individual bacilli seen under
the microscope, looks exactly like those figures of the Bacillus
radicicola mentioned in a former chapter (Chapter VI), the
liquefied gelatine being fluorescent, greenish. Soon the
liquefaction extends throughout the whole culture. On
Agar also the greenish, fluorescent colouration is pronounced,
the surface growth itself being brownish, translucent. Under
the microscope the bacilli are thin and cylindrical, motile,
singly or in dumb-bells, or in filaments ; they do not form
spores. They grow best at lower temperatures up to 22° C.,
but grow also at 370 C., only not so well in comparison.

6. Bacillus coli communis (Escherich). — The typical
bacillus of faecal matter, of the intestinal contents of man
and animals ; and occurs also in all solids and fluids to
which intestinal discharges have had access. It is sometimes
present in nasal, oral, and pectoral discharges. It occurs
(due to secondary invasion from the intestine) in abdominal
inflammatory processes : abscess of the liver, spleen,

peritoneum ; in pulmonary and bronchial suppurations ; in
ulceration and abscesses of the skin and mucous membranes
open to contamination with filth. Its primary home ap-

BACILLI : SPECIAL

189

pears to be the normal large intestine ; in acute and chronic
diseases of the small intestine it may be very copiously
present in the ileum.

Bacillus coli is a motile, aerobic (facultative anaerobic),
non-sporing, non-liquefying rod ; it is killed by thorough
drying and by a temperature of 66° C. in five minutes.

Fig. 60. — Surface Growth on Gelatine of Bacillus Coli, showing
Isolated, Confluent Colonies.

The length of the individuals varies between o-8 /x and
1 '5 fj.- 3 fi, though in later stages in culture longer or shorter
filaments are met with ; its thickness is about o'4-o'5 /x.
When examined fresh from the intestinal contents in health
and disease only a minority are as a rule found to be pos-
sessed of motility, though in some cases (English cholera)

190 MICRO-ORGANISMS AND DISEASE [chap.

motility may be observed on many individuals. The same
holds good for artificial cultures — plates, surface gelatine
and suiface Agar, broth and milk cultures for here also in
young cultures, as a rule, only a minority show motility, in
old cultures the motile individuals are rare.

Bacillus coli forms typical colonies on the surface of gela-
tine at 20 C.; after twenty-four hours they are recognisable

Fig. 6i.— A Stab Culture and a Shake Culture in Gelatine of Bacillus
Coli, with Gas Bubbles.

as flat, translucent, greyish, roundish, but angular patches,
slightly thickened in the middle part or near one margin ;
after forty-eight hours the patches are considerably enlarged,
angular, thin and filmy, and translucent in the marginal,
thick and less translucent in the middle part. The whole
patch is dry, whitish in reflected light, and under a magni-
fying glass appears fairly homogeneous, though after several

BACILLI : SPECIAL

>9*

days it commences to show some kind of concentric differen-
tiation.

The colonies in the depth of the gelatine appear as
spherical small dots, white in reflected, brownish in trans-
mitted light. Fig. 72 is a good illustration of a gelatine
| plate culture of bacillus coli ; compare also Fig. 60.

Fig. 62. — Film Specimen of a Variety of Bacillus Coli, the individual
Bacilli chiefly Oval Rods, some few Cylindrical.

X 1000.

Equally characteristic is the streak culture on the slanting
surface of gelatine ; after twenty-four hours a greyish band,
thicker in the line of inoculation — grey, filmy, knobbed, or
crenated in the marginal part — after forty-eight hours it has
spread considerably in breadth, but has retained the above
aspect, except that the middle part is more thickened, and

192 MICRO-ORGANISMS AND DISEASE [chap.

the whole growth appears more white in reflected light.
After 3-4 days the band has spread over the greater part of
the surface of the gelatine, but is still dry, filmy, crenate,
and irregular in the marginal part ; the whole band examined
under a glass appears more or less homogeneous.

When ordinary nutrient gelatine is inoculated from a
culture, then melted and shaken and allowed to set again,

Fig. 63. — Impression Specimen of the Marginal Part of a Colony of
Bacillus Coli ; most of the Bacilli are Cylindrical.
x 1000.

and incubated, it will be found after 24-36 hours that this
shake culture is permeated by minute spherical colonies in all
its depth, and in connection with each colony is a spherical or
lenticular gas bubble (methan gas) ; this gives to the culture a
very characteristic aspect ; the same is observed if, instead of
ordinary nutrient gelatine, grape-sugar gelatine is used for
the shake culture. In gelatine stab culture the stab becomes
on incubation marked as a row or rows of minute dots,

BACILLI : SPECIAL

193

white in reflected, brownish in transmitted light ; on the top
of the stab is a translucent, plate-like expansion of the
growth. After two, three, or four days’ incubation this ex-
pansion covers the whole upper surface of the gelatine,
while in connection with the stab there are a few large flat
gas bubbles hanging on, as it were, to the growth in the stab.

The gas bubbles in the upper layers of the shake culture
gradually break through and escape on to the free surface,
so that after a time only the deeper layers still contain gas
bubbles. Neutral litmus-whey is turned red by the growth
of bacillus coli (Petruschki).

s On the surface of Agar the growth is a grey, dry film, not
possessing any special character.

On potato it forms a light yellowish-brown expansion.
Alkaline broth becomes strongly and uniformly turbid at
3 70 C. already after twenty-four hours ; later, while the
turbidity increases, a whitish, floccular, granular precipitate
appears in the depth, and on the surface an attempt at the
formation of a white, ‘imperfect pellicle.

If after 3 — 5 days’ incubation a few drops of potassium
nitrite solution, and then a small quantity of nitric acid, are
added to the broth culture, a characteristic pink colouration
appears, due to nitroso-indol, the typical bacillus being a
strong decomposer of albumen, forming thereby indol. In
milk incubated at 370 C. the typical bacillus coli grows
copiously, and clots and solidifies the milk already in
30 — 48 hours, or latest three days ; after clotting a separation
of the clot from the whey takes place. These are the
principal morphological and cultural characters of the
typical bacillus coli, and it remains to be added that,
stained for flagella after van Ermengem’s method, the
bacilli contain at one or both ends several flagella — two,
three, up to eight altogether; the flagella are wavy, whip-

i94

MICRO-ORGANISMS AND DISEASE [chap.

like, or even spiral, but not very long. Bacillus coli grows
well in gelatine and broth to which phenol has been added
to the amount of 0-05 per cent. While these are in
general the characters of the typical bacillus, such as can
be isolated from stools normal and pathological, there occur
in the intestinal contents and discharges, as also in various
other substances — pathological secretions, dust, water,
sewage, &c. — bacilli which, examined as regards all the
above points, coincide in some, but differ in others.
Owing to their general morphological similarity — rods of
the shape and size of bacillus coli, and flagella, two to eight —
and owing to the non-liquefaction of gelatine and the power
to grow well in phenolated gelatine and broth, and the
identical appearances and rapidity of growth in gelatine
plates and in gelatine streak and stab, on Agar, potato, and
broth cultures, they must for the present be considered as
bacillus coli, but on account of their differing from the
typical bacilli in respect of gas-production in gelatine shake
culture, clotting of milk, and indol-reaction, they must be
considered as varieties of bacillus coli. (1) As to size, the
figures given above are open to considerable alterations,
since there are varieties of bacillus coli of which the
elementary rods as taken from a young colony on gelatine
or Agar appear distinctly and uniformly cylindrical, whereas
in some other varieties the great majority are under the
same conditions very short ovals. (2) As to motility, there
exist also great differences. While in some, e.g. the typical
bacillus coli of the intestine taken from a young colony, only
here and there a bacillus shows motility — darting to and
fro, and spinning round — there are varieties of which almost
all the bacilli, at any rate the majority, show active
motility. And similarly as to the number of flagella : for,
while in some two or three flagella at one or both ends are

BACILLI : SPECIAL

'95

discoverable, in others their number mounts up to eight
or even in single cases to ten flagella. The production of
gas bubbles in shake cultures notifies great differences.
While the typical bacillus coli forms gas bubbles copiously and
rapidly in 24 — 48 hours, there are varieties which produce
gas bubbles under these conditions later, or very late —
8 — 10 days or not at all. The same holds good as to
milk curdling : varieties exist which either curdle milk at
370 C. after several days, or after many days — as late as
20 — 25 days. And, finally, the indol reaction of broth
cultures is in some varieties to be obtained after many
v days’ growth, and in others, otherwise behaving like typical
bacillus coli, is not at all obtainable.

Mr. Mervyn Gordon, who has devoted in my laboratory
special attention to these varieties, has isolated from the in-
testinal contents in health and disease, from waters, and from
sewage, a number of varieties which in respect of length,
motility, and number of flagella, of the power of gas-
formation, of the power of curdling milk, and of the power
of indol-formation in broth cultures, furnish quite a
respectable number. Thus he found varieties which in all
respects compare with the typical bacillus coli except that
it has eight flagella, or that it is pronounced cylindrical,
or that it does not form gas, or that it does not curdle
milk till very late, or that it does not form indol in broth
culture; then he found varieties which, except in two
of these characters combined, have all other characters ;
and so on to a variety which by the mode of growth in
plate and streak and on potato, and by the flagella, is bacillus
coli, but has no other character of bacillus coli, in that it does
not form gas, does not curdle milk, and does not form indol.

The typical bacillus coli is a strong producer of acid.
This can be shown very strikingly by using for culture

o 2

196 MICRO-ORGANISMS AND DISEASE [chap.

medium ascitic fluid made strongly alkaline ; then glycerine
is added, and the whole sterilised before inoculation with
the microbe. Incubating the cultivation at 370 C., it will
be found to have become completely solidified in forty-eight
hours, this solidification being due to neutralisation and
further coagulation of the alkali albumen. The same
phenomenon is observed with the typhoid bacillus sown in
the alkaline ascitic fluid and glycerine. So that both these
microbes are strong producers of acid ; and yet there exists
this striking difference between the typical bacillus coli and
the typical bacillus of typhoid with which the above rapid
coagulation of alkali albumen is produced that the former
curdles milk in 36 — 48 hours while the latter does nothing
of the kind ; no coagulation of milk can be produced with
this particular typhoid bacillus that was used for the above
experiment. The conclusion which I think can be drawn
from these facts is that the curdling of the milk so con-
spicuous in the case of bacillus coli cannot be due solely
to the acid formed, but must be due to ferment action, and
further that those varieties of bacillus coli which have the
power of curdling milk in an imperfect degree (very late
curdling), or not at all, owe this deficiency to a want, not of
acid-production, but of ferment-production. A species of
non-sporing, non-liquefying aerobic bacillus occurs in a
small percentage of intestinal discharges and in a somewhat
larger percentage (30 per cent.) of sewage, which in so far
is of interest and importance as its distribution seems to be
limited to these two materials ; at any rate I have not met
with them otherwise, and I have met with them in water
which had received in a conspicuous degree sewage, and for
this reason I am inclined to think that, if this species be found
in water, such water has most probably been polluted with
sewage ; and, further, I think the presence of this species in

BACILLI : SPECIAL

197

water is of even greater importance than that of the bacillus
coli. For it must be obvious that, since bacillus coli is
often present in many materials besides sewage, its presence
alone in water, particularly in limited numbers, does not
justify the conclusion that such water had been directly
polluted with sewage. If, however, bacillus coli and proteus
vulgaris should be present in considerable numbers, such
a conclusion as to probable sewage pollution would be most
probably a correct one. The bacillus which I am about to
describe being of rarer distribution outside sewage, and
being present in sewage, it is clear that for diagnostic
\ purposes it is of importance. Now, this bacillus has certain
characters in cultivation in common with bacillus coli, and
from the aspect of its colonies in gelatine and in streak
cultures on gelatine might be mistaken for it : it grows as
rapidly as, if not more so than, bacillus coli, and forms the
same kind of flat, dry, translucent, angular, patch-like colonies ;
in gelatine streak it forms the same kind of translucent band
with filmy, irregular, or crenate and knobbed margin. Like
bacillus coli, it grows well in phcnolated gelatine and in
plwiolated broth , it differs, however, from bacillus coli in the
following respects : —

It grows quicker in plates and in streak culture in
gelatine; its colonies are flatter and show, when examined
with a magnifying glass, already after twenty-four hours,
better after forty-eight hours, in reflected light very charac-
teristic white granules scattered through the middle part of
the patch ; the same white granules are noticed along the
middle of the band in streak culture ; later, say after three
days, the number of the granules increase considerably and
extend from the middle to near the margin both in the
colonies of the plate as also in the band of the streak, so
that thereby the growth becomes whitish in reflected, opaque

198 MICRO-ORGANISMS AND DISEASE [CHAP.

and brownish in transmitted light. This bacillus is non-
motile ; it is markedly cylindrical, forming short and long
chains and filaments, the above white granules in the young
colonies being due to collections of such chains and fila-
ments ; it does not curdle milk, does not form gas-bubbles
in gelatine shake cultures, and does not form indol in broth :

Fig. 63a. — Film Specimen of Bacillus Pyocyaneus.
X 1000.

it is, therefore, easily distinguishable from bacillus coli. It
approaches the proteus Zenkeri inasmuch as in streak culture
in gelatine after some days it forms threads and filaments
radiating from the centre of the streak which recall the
growth of proteus Zenkeri ; we therefore call it (in the
laboratory) the sewage variety of proteus Zenkeri, though,
as mentioned above, it resembles more the bacillus coli than

BACILLI : SPECIAL

199

the proteus Zenkeri ; from this latter it differs in almost all
other respects, our bacillus being more cylindrical, not
motile, and its colonies on gelatine being filmy, translucent,
granular patches.

Another bacillus which I found occasionally, but rarely, in
sewage, and which I have not found elsewhere, is a bacillus
which on account of its eminent tendency to form long "

Fig 64. — Strei'tothrix Foersteri Fig. 65. — Cladothrix Dichotoma

(after Cohn). (after Cohn).

threads I have called bacillus filamentosus ; this is a
strongly aerobic, non-motile microbe, consisting of cylindrical
bacilli with square ends like bacillus anthracis ; in stab and
streak it forms a marked feathery, filamentous growth ; it
liquefies gelatine very rapidly, the liquefied gelatine being
quite limpid, and it rapidly forms bright, glistening, oval
spores in size, shape, and position the same as bacillus
anthracis.

200

MICRO-ORGANISMS AND DISEASE [chai>.

7. Of less common occurrence is the Bacillus prodigiosus,
forming a characteristic bright-red or bright-pink growth.
This microbe occurs occasionally in air, in water, and in
soil. On Agar plates and Agar surface it forms round
colonies which have a bright pink colour ; on gelatine the
colonies appear round, at first faintly red and rapidly
liquefying, making the liquefied gelatine turbid and of a
pale-red tint. On potato it grows rapidly, forming a bright
pink expansion. The microbe grows best at 20° C. ; it does
not grow at 370 C. The growth is composed of non-motile,
oval, or even spherical or cylindrical rods, singly or in
dumb-bells, or in short chains. The pink colour is noticed
only in aggregations of the microbe. I have seen a whole-
sale infection of food-stuffs (beef, mutton, fish) occurring in
a City establishment next to which an old churchyard had
been disturbed, owing to old graves having been dug up
previously ; the larder in which the infection occurred was
overlooking the said churchyard. By means of alcohol or
chloroform the pigment can be easily extracted.

8. Bacillus pyocyaneus is the microbe found in blue-green
pus — in fact, it is the organism which produces the blue-
green colour. Gessard and Charrin (Gessard : These de
Paris, 1882. Charrin : Communication a la Societe Ana-
tomique, December 1884) first described the microbe.
Gessard particularly isolated the blue pigment produced by
it, pyocyanin. When isolated by gelatine plates the microbe
grows as translucent colonies irregular in outline and show-
ing a fine radial striation, the gelatine gradually assuming a
greenish colour. The gelatine is liquefied and of a uni-
formly greenish colour; on Agar it forms a white film, while
the Agar becomes tinted greenish ; on potato it forms a
brownish film, while the substance of the potato underneath
assumes a greenish colour. It has pathogenic action on

m ■»>» -

BACILLI : SPECIAL

201

guinea-pigs.1 Under the microscope it is an extremely
minute and thin cylindrical rod (see Fig. 63^7).

This microbe has a much wider distribution than green
pus, for I have isolated it several times from the contents of
the intestine both in acute diarrhoea and in cholera. Dr. F.
W. Andrewes has made some interesting experiments with
the blue pigment, showing that a solution of it turns bright
red on the addition of acid, and it assumes again the deep
blue colour on adding sufficient alkali.

I append here, as morphologically interesting forms, three
micro-organisms of which the position amongst bacteria is
v not definitely determined yet.

(a) Streptothrix. — Cohn 2 found in a concretion of the
human lacrymal canals long, pale, smooth, apparently
branched threads, either straight or twisted ; they were
finer than the threads of leptothrix buccalis ; he called them
Streptothrix Foersteri.

(b) Cladothrix dichotoma (Zopf). — This occurs in pond-
water containing decomposing organic matter. It consists
of long whitish threads fixed on chlorophyll-containing algte.
The threads when fresh appear smooth, pale, occasionally
granular, and on staining they are seen to be composed of
shorter or longer bacilli, just like the leptothrix form of
bacillus subtilis ; but they are thicker than the bacillus
subtilis. Occasionally the ends of the threads are seen, not
as linear series of bacillar rods, but, like bacillus anthracis,
as chains of torula-like spherical elements. From the threads
single motile bacilli are seen to come off. The threads are

1 When a few divisions up to half a Pravaz syringe of the broth
culture is injected subcutaneously, the animals become ill and die in
from two to four days, showing peritonitis, pericarditis, and pleuritis,
with copious membranous and purulent exudation, which contains
abundantly the bacilli.

2 Beitr. z. Biol. d. PJlanzen , vol. i. p. 186.

202

MICRO-ORGANISMS AND DISEASE [ciiap.

only apparently branched, since the branches are threads
merely stuck on to other threads sideways at an acute angle.
A bacillus may be seen to stick to a thread and then to

Fig 66. — Threads of Cladothrix Dichotoma highly magnified and
STAINED WITH SpiLLER’s PURPLE.

1. Threads of bacilli.

2. Torula-forms.

The sheath is everywhere well seen.

grow out by continuous divisions into a long chain of
bacilli, thus forming, as it were, a side-branch. Some of
the threads are wavy and curved \ most of them are, how
ever, straight. Zopf1 states to have observed that the
i Zur Morphologic dcr Spaltpflaiizcn, Leipzig, 1SS2 ; see also
Cienkowski.

BACILLI : SPECIAL

203

threads of the cladothrix gave rise to micrococcus, bacte-
rium, bacillus, and spirillum ; and further that each of
these is again capable of growing into the threads of the
cladothrix.

(c) Beggiatoa. — In stagnant water, particularly in sulphur-
containing water, peculiar oscillating colourless threads are
' met with of the thickness of o'oooi to o-oi6 mm.; they
contain highly refractive granules, which Cohn ( Beitrdge zur
Biol. d. Pfl. i. 3) has shown to be composed of sulphur.
After dissolving these granules it is seen that each thread is
septate, being composed of a sheath and transverse septa at
I regular intervals, by which the threads appear made up of a
series of short cylindrical elements. There are a number
of species varying from one another in the thickness of the
threads.

CHAPTER XI.

BACILLI SPECIFICALLY PATHOGENIC TO MAN OR ANIMALS.

Group A. — Amongst these a group of bacilli is first
to be considered which comprises several species, all of
which have certain characters in common, (i) All of them
arc short oval rods, some more cylindrical than others, :
occurring singly, in dumb bells, or even in short chains.

(2) They do not liquefy gelatine and do not form spores.

(3) They produce uniform turbidity of broth already after 24-
36 hours at 37° C., although the amount of turbidity varies
in the different species, and also as regards presence or
absence of a pellicle. (4) They are killed by a temperature of
6o° C. in five minutes. (5) They all produce in one or the
other rodent, on subcutaneous injection of small quantities of
culture — recent broth culture best — or of blood and tissues
containing the microbe, acute septiaemic infection, the
blood of the general circulation of the infected rodent con-
taining more or less copiously the injected microbe; the
viscera are hypersemic, the spleen, the liver, the lungs, the
kidneys, and particularly the peritoneum, containing small
haemorrhages with peritoneal, pericardial, and pleural exuda-
tion. They differ from one another (1) in the species of
animals in which originally they are associated with acute

CH. xi] BACILLI : SPECIFICALLY PATHOGENIC 205

specific disease ; (2) in the rodent in which they produce the
acute haemorrhagic septicaemia ; (3) in the rapidity of growth,
aspect, and size of their colonies on gelatine in the plate and
streak culture ; and (4) in the presence or absence of motility.

To this group belong (1) Bacillus of Davaine Septi-
caemia. (2) Bacillus of Fowl Cholera. (3) Bacillus of
Frettchenseuche. (4) Bacillus of Duck Cholera. (5)
Bacillus of Fowl Enteritis. (6) Bacillus of Grouse Dis-
ease. (7) Bacillus of Swine Fever, or Hog Cholera (and
Swine Plague). (8) Bacillus of Wildseuche. And (9)
Bacillus of Oriental Plague of Man.

The following short account is copied from Klein’s article,
Infectious Diseases, in Stevenson and Murphy, II., pp. 97,
98, 103, 104, 105, 106, 107, and 108 : —

1. Bacillus of Davahic septicamia. — This is a septicaemia
which Davaine first produced by injecting into rabbits
putrid ox’s blood. It is known now that a small motile
bacillus is the microbe, which by its great multiplication
and universal distribution in the circulating blood causes
the disease and death. The microbe is present in the
blood in great numbers, nearly as great as that of the blood'
corpuscles ; in stained specimens the rods, which are short
and oval, show a stained granule at each end with a clear
space in the middle; the length of the rods is about
in thickness about half. The rods are motile, and from the
heart’s blood and all other tissues pure cultures can easily be
made. In plate cultures after about two days minute white
dots are visible ; under a glass they appear as flat circular
discs, white in reflected, yellow brown in transmitted light.
After several days the colonies are larger, and appear thicker
and broader in the centre than in the periphery, which
itself appears more or less concentric owing to regular
differences in thickness. At maximum growth the

206

MICRO-ORGANISMS AND DISEASE [chap.

colony does not exceed one to two millimetres. In stab
culture the stab is occupied by a whitish line ; under a
glass this is seen to be made up of minute droplets and dots,
whitish in reflected, yellow-brown in transmitted light. In
streak cultures the streak is represented by a narrow whitish
band of irregular outline and thicker in the middle than at
the margin. Gelatine is not liquefied by the growth.

Rabbits, mice, fowls, pigeons, and sparrows are very
susceptible (Koch) to the inoculation of very minute doses
of culture or of blood of an animal previously dead of the
disease ; guinea-pigs and rats are unsusceptible (Koch).
When inoculated with a trace of the blood of a rabbit dead
of the disease, or with a trace of culture, rabbits show already
after ten to twenty-one hours a distinct rise of temperature ;
in severe cases the animals show spasms, rapid fall of tempera-
ture, already before the end of the first sixteen hours, and are
dead before the day is over ; but in some cases, particularly
after inoculation with minute traces of culture, death does
not take place before thirty-six to forty-eight hours. The
bacilli are found very numerously in the blood-vessels of all
organs. Spleen and liver, lymph glands and lungs, are
highly congested, so also the intestines ; extravasations are
only rarely found, and then only in the omentum and lungs ;
peritonitis is only noticed in a small percentage of cases,
and then only when the omentum shows the extravasations ;
the serous coverings of the intestines are greatly injected.
As a rule, these symptoms are greatly more pronounced if
death does not occur before the second day.

A bacillus closely related to this is the one which causes
acute septicaemia in guinea-pigs and mice, and which I
obtained from the pleural exudation of mice and guinea-pigs
that had died spontaneously from septicaemia— that is to say,
in which no primary cause could be assigned, and in which

xij BACILLI : SPECIFICALLY PATHOGENIC 207

the post-mortem appearances showed the symptoms of
septicaemia : viz., great congestion of the lungs, liver, and
kidney, inflamed peritoneum, pleural and pericardial exuda-
tion, the spleen dark and slightly enlarged in the mice, the
intestines relaxed, congested in the mucous and serous coats,
the cavity of the small intestine filled with sanguineous
I mucus. Inoculation of guinea-pigs or mice with the gela-
tine cultures proved fatal in the mice within one, two, or
three days ; in the guinea-pigs larger doses had to be used to
produce death in a day or two. When small doses are used
there is noticed already in twenty-four hours, about the seat
of inoculation, a firm thickening which gradually extends
! into wider areas ; and death ensues after several days to a
week. In all cases the bacilli can be easily demonstrated in
the heart’s blood and in the congested organs by cover-glass
specimens and by culture. In sections through the liver and
kidney the bacilli are found in masses occluding like emboli
the capillary blood-vessels ; in the liver the central vein of a
lobule and numerous capillaries leading into it are found
filled with and distended by continuous masses of the
bacilli, the surrounding liver tissue being in a necrotic
state ; in the kidney numerous capillaries between the con-
voluted tubes of the cortex and in the glomeruli are found
occluded by the bacilli. The bacilli taken from the blood
are rounded at their ends, and motile ; in cultures, notably
in broth or other fluids, some of the bacilli are short like
cocci, others are oval, others again cylindrical ; there are
also numerous longer and shorter chains, which show active
motility ; in these chains the joints or elements are of all
shapes cylindrical, oval, or coccus-like. That all these
forms belong to the same species can be easily proved by
plate cultivation ; for in these all colonies are of exactly the
same kind.

MICRO-ORGANISMS AND DISEASE [chap.

Fig. 67 — Film Specimen of
Cholera. Numerous Oval
Red Blood Discs.

Ieart's Blood of Fowl mad op Fo'vl
3acilli with Polar Staining amongst the

p Pelatine Pl*te Cultivation of Bacillus of Fowl Cholera

Fig. 68,-GelatineFi ^Hreb Days’ Incubation at 20 C.

Natural size.

xi] BACILLI : SPECIFICALLY PATHOGENIC 209

2. Fowl cholera. — This disease causes great devastation
amongst poultry. The malady, well known by the researches
of Perroncito, Toussaint, Pasteur, Kitt, and others, affects
fowls, pigeons, and rabbits. In the fowl, after an incubative
period varying between sixteen or eighteen hours to twenty-
four hours, the disease declares itself by diarrhoea of fluid,
greenish evacuations, great drowsiness, and sleepiness of the
animal. In about twenty to forty-eight hours the animals
are found dead ; the blood in the heart and general circula-
tion, and in the vessels of all organs, the intestinal contents,
and evacuations teem with short, oval, non-motile bacilli
measuring i-i'2 yu, in length; in stained preparations they
show at each end a stained granule, while the middle part
is clear and unstained. On post-mortem examination the
viscera are found greatly congested and containing hoemor
rhages ; the mucous membrane of the upper part of the
intestine is found congested ; often small haemorrhages occur
in its mucous membrane ; the contents are fluid faeces, the
spleen is enlarged. Fowls, rabbits, and pigeons inoculated
with a droplet of the blood of a fowl dead of the disease, or
inoculated with the artificial culture of the bacilli, die of the
disease in between thirty-six to forty-eight hours, the blood
teeming with the bacilli. Feeding with the intestinal con-
tents of fowls, the disease is reproduced in them. From
this the conclusion is justified that also under natural con-
dition infection is carried out by the healthy fowls picking
up the contagium with the food from soil tainted with the
evacuations of diseased animals.

Cultures of the bacilli show the following characters : In
plate cultivations the colonies appear before forty-eight
hours as minute yellowish-white dots, irregularly outlined
or round ; seen under a glass they are discs faintly granular ;
the centre is yellow and transparent, then follows a brown

210 MICRO-ORGANISMS AND DISEASE [chap.

zone, and then a transparent marginal part. In stab culture
the line of inoculation becomes marked as a white line
made up of more or less confluent yellowish-white droplets; ,
on the surface of the stab is a thin, irregularly outlined
plate ; in streak cultures the growth appears after two or
three days as a yellowish-white band with irregular or
knobbed outlines, thin in the centre and margin, thicker
and brownish in the intermediate parts; on potato the \
microbe grows only at higher temperatures, 28°-38° C.

It grows slowly and forms a waxy grey-white film.

By inoculation of minute quantities, a drop of culture
into the subcutaneous tissue, or by feeding of fowls, rabbits,
mice, or pigeons with culture, the disease is easily repro- \
duced. In guinea-pigs and sheep it produces a local abscess
at the seat of inoculation.

By keeping broth cultures for some months Pasteur has ;
succeeded in producing by inoculation of fowls a local
oedematous inflammation ; the animals became only slightly
affected, but recovered and showed themselves refractory
against a second inoculation. Pasteur thought that the
influence of the oxygen of the air produced the attenuation ;
it is now proved, however, that this is not so (Kitt), but that
Pasteur had impurities (accidental microbes) in his broth
cultures, which at first attenuated the bacilli of fowl cholera
and, as time went on, altogether suppressed these ; hence the
broth cultures of Pasteur after the lapse of some months
proved barren of all pathogenic action.

Pasteur has shown that by injection of large quantities of
broth cultures from which the bacilli of fowl cholera have
been previously removed by filtration a transitory illness can
be produced, and that such animals show themselves after-
wards refractory against inoculation with virulent material.
Marchiafava and Celli showed that the microbe passes from

XI] BACILLI : SPECIFICALLY PATHOGENIC 211

the mother to the foetus, probably owing to ruptures (haemor-
rhages) in the vessels of the maternal placenta.

3. Eberth and Schimmelbusch ( Fortschritte d. Median,
Bd. VI., No. 8, p. 295) described an acute infectious disease
in mustela furo — Frettchenseuche — chiefly showing itself as
pneumonia with enlarged spleen ; in the heart’s blood, in
the inflamed lung, the liver, and enlarged spleen there are
present numerous motile bacilli, similar in many repects to
the bacillus of swine fever, fowl cholera, and Wildseuche.
The cultures act very virulently on sparrows, less virulently
on pigeons; fowls are refractory; in rabbits the inoculation
produces only a local inflammation of a temporary char-
acter, and the same results are obtained, only milder, in
guinea-pigs.

4. Duck cholera. — As such, Cornil describes a fatal
infectious disease affecting ducks, and in its symptoms and
causation similar to fowl cholera ; but there is this difference
between them, that the disease of the duck is not trans-
missible to the fowl. The bacilli are, however, similar in
many respects to those of fowl cholera.

5. Foud enteritis. — This is an acute fatal infectious
disease affecting fowls, but not pigeons and rabbits, and by
this alone its differentiation from fowl cholera is established ;
besides the microbe and its distribution, the course and
symptoms of the disease are quite distinct from fowl cholera.
I have met with the fowl enteritis on a poultry farm in
England, where it caused great mortality. The disease has
been prevalent also in Ireland during the last few years.
The fowls when affected show diarrhoea of fluid greenish
evacuations, are quiet, but never show sleepiness or drowsi-
ness. In a day or two after the diarrhoea has set in they
are found dead. The mucous membrane of the intestine is
found congested, but without haemorrhage ; the internal

p 2

212

MICRO-ORGANISMS AND DISEASE [chap.

surface of the mucous membrane is coated with grey or
yellowish mucus, which under the microscope contains
numerous leucocytes and detached epithelial cells ; the
liver is congested and brittle, the spleen much enlarged, the
lungs are normal. In the heart’s blood are present relatively
few bacilli, which are a little longer and thicker than in fowl
cholera; the spleen contains the bacilli numerously, and

Fin. 6q. — Film Specimen of Intestinal Mucus of a Fowl dead of Fowl
Enteritis. Pure Culture of Bacillus of Fowl Enteritis.

X icoo.

also the vessels of the liver; the mucus of the intestine
contains the bacilli in almost pure culture. In cultural
respects the microbe resembles the bacillus of fowl cholera,
except that its colonies are disc-like when growing on the
surface ; further, that the microbe of fowl enteritis is more
cylindrical, and that in gelatine streak culture it grows much
faster and forms a broader, less translucent band than that

XI] BACILLI : SPECIFICALLY PATHOGENIC 213

of fowl cholera. Pigeons are unsusceptible, rabbits only
very slightly susceptible. By feeding of fowls with the con-
tents of the intestine the disease can be reproduced ; by
subcutaneous inoculation the disease can be produced, both
with the blood or spleen tissue of a fowl dead of the disease
as also by artificial cultures of the microbe. In all cases

X 1000.

ithe animals do not show any illness till the third or fourth
day (this is also an important distinction from fowl cholera),
or more generally till the fifth day : they suffer then from
diarrhoea and are quiet ; on the sixth or seventh day most
of them are found dead, rarely do they survive till the eighth

2 14 MICRO-ORGANISMS AND DISEASE [chap.

day, nor do they die before the fifth day. The course of the
disease, the symptoms and the appearances after death, the
morphology and cultural characters of the microbe, dis-
tinguish this disease from fowl cholera.

Fig 71. — Plate Cultivation of the Bacillus of Fowl Enteritis, show-
ing NUMEROUS DOT-LIKE COLONIES IN THE DEPTH OF THE GELATINE, AND
disc-shaped Colonies on the Surface, thus showing a striking Con-
trast to Fig. 68 of a Plate Culture of Bacillus of Fowl Cholera.

Natural size.

6. Grouse disease. — The fatal disease which affects red
grouse, and known as the grouse disease, is an acute infec-
tious disease, of which the chief, and we may say the essen-
tia], pathological character is that of pneumonia, the lungs
being greatly congested, and sometimes one or the other
portion almost in a state of red hepatisation with engorge-
ment of the blood-vessels and extravasation of blood into
the air-spaces ; the serosa and mucosa of the intestine show
patchy redness ; the liver is greatly congested and dark ; the

xij BACILLI : SPECIFICALLY PATHOGENIC 215

spleen is not enlarged. In the diseased lung and in the
liver there occur in the vessels and in the extravasated
blood numerous bacilli singly, or more commonly in larger
or smaller groups, sometimes forming emboli in the capil-

Fig. 72. — Plate Cultivation of Bacillus of Grouse Disease, showing
dot-like Colonies in the Depth, patch-like Colonies on the
Surface.

This illustration may serve also to show the character of a gelatine-plate culture
of the typical Bacillus coli.

Natural size.

lary blood-vessels. These bacilli belong to one and the
same species ; they are motile, either oval or even coccus-like ;
some few are rod-shaped. By cultivation on gelatine they
can be easily obtained in numerous colonies from the san-
guineous juice of the lung and liver ; only in few cases are
they to be seen in the heart's blood, both in cover-glass

2 16 MICRO-ORGANISMS AND DISEASE [ciiap.

specimens and in culture. The morphological and cultural
characters of the microbe are shown in Figs. 72 and 73.

The microbe when examined from a cultivation is often
rod-shaped — more often than in the tissue of the grouse.
The motile forms are common in recent cultivations ; in
cultivations some days old most of the microbes are non-

F IG. — Film Specimen of Blood of Grouse in Grouse Disease, showing
'i he Nuclei of Red Elood Discs and a Number of the Bacilli.

X 1000.

motile. Cultures inoculated into mice and guinea-pigs
produce general infection, and rapidly death, mice being
more susceptible than guinea-pigs : in both animals the
disease produced is a double-sided pneumonia. Sparrows
are also susceptible, but less so than the common bunting
and yellow-ammer, which animals are highly susceptible;
also in these the disease produced is a double-sided pneu-
monia. The microbe is present in numbers in the heart's
blood, but particularly in the diseased lung.

Xi] BACILLI : SPECIFICALLY PATHOGENIC 217

Fowls, pigeons, and rabbits are unsusceptible to the
disease.

As far as the appearances in gelatine plate and gelatine
streak go, there is a considerable similarity between the
microbe of grouse disease and bacillus coli ; this is also
strengthened by the fact that the former, like the latter,
forms gas bubbles in shake culture and curdles milk, the
difference being chiefly this — that the microbe of grouse
disease on subcutaneous injection is highly virulent to mice,
and particularly to the yellow-ammer ; less so to guinea-pigs.

Fic. 74. — From a Section through the enlarged Inguinal Lymph-Gland
of a Pig dead of Swine Fever.

1. A capillary blood-vessel filled with bacilli.

2. Reticulum of adenoid tissue.

3. A lymph-cell.

Magnifying power 700.

7. Bacillus of swine fever. — This disease prevails largely
in this country ; in America it is known as hog cholera, on
the continent of Europe as swine plague. It is a highly
infectious disease, spreading from animal to animal by air,
food, water, the lungs and bronchi and the intestines being
the chief places of disease, and containing the virus. The
infection is, under natural conditions, attributable to the
virus being derived from and spread by the expectoration of
the lungs and evacuations of the bowels. Alike by feeding,

2 l8

MICRO-ORGANISMS AND DISEASE [chap.

respiration, and by inoculation with the diseased particles
of lung and intestine the disease is easily reproduced in
healthy swine. After an incubation period varying from
between two days and six to seven days the animals are
quiet and refuse food, the body temperature shows slight
rise, red patches of transitory nature are noticed on the
belly and thighs ; cough and occasionally diarrhoea of fluid
evacuations declare themselves soon ; the inguinal lymph-
glands appear enlarged. In severe cases the diarrhoea

Kig. 75. — From a Section through the Kidney of Rabbit dead of Swine
Fever, showing a Malpighian Corpuscle, the Capillaries of the
Glomerulus being transformed into hyaline impermeable Cylinders.

1. Bacilli.

Magnifying power 500.

increases, the fever continues, the cough becomes more
pronounced ; this state lasts for a few days, seldom more
than a week, and under general prostration the animal suc-
cumbs. In a large percentage (50) of cases the animals
recover. In mild cases, representing a considerable per-
centage, the disease is diagnosed only with difficulty ; the
rise of temperature is only slight and transitory, lasting only
a day or two ; the animals feed fairly well ; show only, very
occasionally at long intervals, a slight cough ; the inguinal
glands are slightly enlarged. These symptoms are so slight

„

xi] BACILLI : SPECIFICALLY PATHOGENIC 219

and so little marked that it requires careful examination to
diagnose the disease ; nevertheless, on auscultation of the
chest distinct lung disease may be recognised. On post-
mortem examination of such slight cases the symptoms of
the disease of the lung are easily confirmed.

In the well-pronounced cases dying naturally the post-
mortem examination shows the following. The lungs of both
sides show severe, extensive, lobar pneumonia, involving
sometimes the greater part of the lung ; the lobules show

Fig. 76.— Blood of fkesh Spleen of a Mouse that died of Swine Fever.

1 Blood discs.

2 A large nucleus.

3. Groups of minute bacilli.

4. Long bacilli.

5. Dumb-bells of bacilli.

Magnifying power 700. (Stained with gentian violet.)

in recent cases all stages between congestion (punctiform
haemorrhages) and hepatisation ; the lobes that are longer
affected show more consolidation, and, the older this is, the
niore grey, and solid, and necrotic, dry and friable, is this
part of the lung ; the septa between the lobes are cedcma-
tous and well-marked ; the bronchi and trachea contain
grey and sanguineous muco-purulent matter ; the endocard
of the left and occasionally the right ventricle, near the

220 MICRO-ORGANISMS AND DISEASE [chap.

atrio-ventricular valves, and also these latter, show patchy
and punctiform haemorrhages ; the liver is congested and
occasionally shows dark-red patches due to haemorrhage ;
the spleen is enlarged and dark ; the colon and caecum,
particularly the former, contain punctiform haemorrhages ;
in most cases they contain prominent round or oval,
isolated, and in severe cases more or less confluent, ulcers
(necrosis), showing an infiltrated base, and are stained
greenish-black by altered bile-pigment ; between a few small
round ulcers near the ileo caecal valve to very numerous
extensive long ulcerations, comprising occasionally exten-
sive areas of the mucous membrane of the caecum and colon,
all intermediate stages can be noticed. ( See Klein, in the
Report of the Med. Off. of the Loc. Gov. Board for 1878.)

In the stomach occasionally haemorrhagic patches can be
seen. The lymph glands along the bronchi, the mesenteric
and pelvic glands, are swollen, juicy, dark red, in part or
wholly, and contain haemorrhage. The peritoneum is in-
flamed, and on its surface are clumps of solid lymph com-
posed of leucocytes. Owing to the lungs and intestines
being found constantly affected, the disease has been desig-
nated by me pneumo-enteritis ; but in Germany (Sehiitz)
and in America (Salmon) it is asserted that the above dis-
ease is really two : one a disease of the lung, the other of
the intestine ; but from experiments made on a large scale
with diseased lung and with diseased intestine, and from
the post-mortem appearances in well-defined localised out-
breaks that I have made, I am of opinion that this division
cannot be maintained, but that the swine fever in this
country is one single disease, viz. pneumo-enteritis. Micro-
scopic examination of the lung and intestine shows that the
disease really commences with congestion, stasis, and haemor-
rhage, leading to infiltration and necrosis of the affected parts.

XI] BACILLI : SPECIFICALLY PATHOGENIC 221

The cause of the disease is a bacillus, which in the affected
tissues of the pig appears, as a rule, as a short rod, often
constricted in the middle ; in fluid cultivations (broth) and
in animals (rabbits, mice) as a cylindrical rod, singly or in
dumb-bells, occasionally growing to considerable length, and
forming longer or shorter chains ; but there can be always
found short forms almost like oval cocci, rods, and cylin-
drical bacilli. Cover-glass specimens and cultures of the
lung, spleen, lymph glands, and the sub-mucous tissue of the
affected intestine demonstrate the presence of the bacilli.
These bacilli are motile, though the motility is observed in
a minority ; in cultivations of broth, gelatine and Agar Agar
many of the bacilli are motile during the first few days, but
lose their motility later.

In plate cultivations the colonies are first noticed as
greyish dots just visible to the eye already after twenty-four
hours ; in two or three days they are already conspicuous as
whitish, round, angular specks of about the size of a large pin’s
head ; in transmitted light they appear brownish, granular.
In stab culture the stab of inoculation becomes marked as
a white line made up (when seen under a glass) of minute
globules closely placed side by side ; on the surface of the
stab is a small, irregularly outlined, whitish plate. In streak
culture the line of inoculation is occupied in a few days by
a grey band, knobbed or crenated in its outline. On Agar
the growth (at 370 C.) is a greyish-brown smeary film,
rapidly spreading over the surface of the Agar. In alkaline
broth at 370 C. uniform turbidity is produced ; after a few
days a voluminous greyish-white precipitate is noticed at
the bottom of the tube. No distinct pellicle is formed on
the surface.

Inoculation of swine with cultures produces the disease,
but this does not lead to death, and such animals after re-

222 MICRO-ORGANISMS ANI) DISEASE [chap.

covery show themselves refractory against inoculation with
material of the diseased lung or intestine.

Inoculations into guinea-pigs with material from the
diseased swine produce at the seat of inoculation haemor-
rhagic infiltration and thickening, sometimes leading to death
in two or three days ; often, however, the thickening passes
off in a week or so ; cultures injected subcutaneously in
guinea-pigs produce thickening at the seat of inoculation,
but rarely death.

Inoculation into mice of minute particles of material of
the diseased lung, or intestine, or of gelatine, or broth cul-
ture of the bacillus of swine fever causes disease and death
in four to eight days ; the spleen is found enlarged and
dark ; the liver is mottled with grey dots, streaks, and patches
of necrotic tissue ; the peritoneum is inflamed, and so are
the kidneys and both lungs. Cover-glass specimens and
cultures from the heart's blood and liver, kidney, and parti-
cularly the spleen, demonstrate the presence of large num-
bers of the bacilli (see Fig. 76). Among the bacilli in the
spleen numerous long cylindrical rods can be seen. In the
kidneys many of the capillaries of the glomeruli are plugged
by the bacilli, so also in the liver.

In the rabbit inoculation produces disease and death in a
few days : the spleen is slightly enlarged, the lungs are in-
flamed, the kidney is much congested in the cortex. Here
also the bacilli can be easily demonstrated in the heart’s
blood, the liver, and the kidney ; in this latter many Mal-
pighian corpuscles show the capillaries of the glomeruli
plugged with masses of the bacilli.

8. Bacillus of Wildseuche. — A disease amongst cattle
(Rinderseuche) and horses, and amongst deer (Wildseuche),
manifesting itself in diffuse pneumonia and haemorrhagic
enteritis, but without necrotic change (consolidation and

XI] BACILLI : SPECIFICALLY PATHOGENIC 223

dryness) of the lung, and without ulceration of the intestine,
has been first recognised by Bollinger. Kitt has shown that
this affection is caused by a bacillus in many respects (mor-
phological and cultural) similar to that of fowl cholera,
rabbit septicaemia, and swine fever ; and Kitt and Hueppe
maintain, indeed, the identity of all these microbes ; but the
evidence to prove this is not sufficiently satisfactory. True,
rabbits inoculated with the microbes obtained from Davaine’s
septicaemia, fowl cholera, swine fever, or Wildseuche suc-
cumb under the symptoms of Davaine septicaemia ; it is like-
wise true that pigeons inoculated with cultures derived from
either of these diseases succumb to fowl cholera ; still a
great deal remains yet to prove the identity as regards the
action on swine of the bacteria of rabbit septicaemia, fowl
cholera, and Wildseuche. To mention only one series of
difficulties. Fowls, as mentioned above, are highly suscep-
tible to the microbe of fowl cholera, but they are unsuscep-
tible to the microbes of swine fever or Wildseuche. Billings
(Texas fever, Lincoln, Nebraska, 1888) describes a species
of small motile bacilli closely related to the bacilli of swine
fever, alike as to morphology, cultural, and pathogenic
characters, as the cause of the cattle plague in Texa's and
southern countries of the States.

Loftier (Cefitralbiatt f. Bald, und Par as it., vol. xi., p. 134)
described a fatal epidemic amongst mice kept in the labora-
tory. From the enlarged spleen of the dead mice a motile
short bacillus was isolated, which evidently belongs to this
group of swine fever— Wildseuche bacilli. Its culture
proved very virulent on tame as well as wild mice, producing
on subcutaneous inoculation, as also on ingestion, acute
fatal septiaemia, the blood and the enlarged spleen par-
ticularly teeming with the microbe. On account of the
microbe bearing a certain cultural resemblance to the

224

MICRO-ORGANISMS AND DISEASE [chap.

bacillus of human typhoid on gelatine, Agar, in milk and
potato, Loffler called it bacillus typhi murium. Successful
experiments with cultures were made in Thessaly to produce
wholesale infection and destruction of field mice then
infesting the agricultural districts of that country.

Of the same nature appears to be the bacillus isolated by
H. Laser ( Centralblatt f. Bakt. und Parasit., vol. xi., p. 184),
and which he found in a fatal epidemic amongst field mice
kept in the laboratory. The morphological and cultural
characters of the microbe, its virulence on mice, and the
post-mortem appearances in these animals coincide with
Loffler’s bacillus typhi murium.

9. Bacillus of Oriental or bubonic plague. — This is at
present the only known species of this group which affects
the human subject.' As shown by Kitasato and Yersin, the
bacillus of the inflamed lymph-glands (bubo) and also of the
blood, but principally the former, contain in pure culture an
abundance of short rod-like bacilli, which in shape and size, in
cultural characters, in plate and in streak on gelatine, and in
their effect on rodents belong clearly to the above group of
non-sporing, non-liquefying bacilli. The bacillus is non-
motile, and its effect on the rodent (guinea-pig) is to produce
acute hcemorrhagic, septicaemic infection and death.

Group B. — A second group comprises species which in
many points resemble the bacillus coli,1 but differ from it in
this particular that they are capable of producing acute
infection and death of the animal body. Like bacillus coli,
the microbes of this group grow rapidly in gelatine plate
and gelatine streak and stab, and the appearances herein

1 It must be distinctly understood that I do not and cannot say
whether the various species I am about to describe are or are not
varieties of bacillus coli, for the characters by which _ this last is
identified are, after all, only comparatively few, besides being artificial;
it is more for convenience that we speak of “ varieties ” of b. coli.

XI] BACILLI : SPECIFICALLY PATHOGENIC 225

produced are not essentially different from those of bacillus
coli ; they also form gas-bubbles in gelatine shake culture,
curdle milk, and produce indol in broth. The appearances of
the growth on Agar and on potato are the same as those of the
bacillus coli. As to flagella, they possess two or three flagella,
and taken from recent culture many individuals show active

Fig. 77.— Film Specimen of the Juice of a Bubo in Oriental Plague;

BESIDES A FEW NUCLEI THE Fll.M CONTAINS THE BACILLUS OF PLAGUE IN

Pure Culture.

x IOOO.

locomotion. Morphologically they occur as short ovals,
singly and in dumb-bells, or as cylindrical individuals with
tendency to form chains. As stated just now, the chief
difference lies in the fact that, whereas bacillus coli injected
subcutaneously into guinea-pigs and mice in small doses
causes transitory local swelling only, and in large doses

226 MICRO-ORGANISMS AND DISEASE [chap.

general infection, the microbes in question cause on subcu-
taneous injection, already in small doses (a few drops of a
recent broth culture), acute septicemic infection ; the
blood contains copiously the microbe ; the lungs and liver,
and particularly the spleen, is hypersemic and enlarged,
and full of the microbe.

X 1000.

To this group belong:- — i. The bacillus that I found in
pure culture very copiously in the juice of the congested lungs
in an epidetnic of fatal pneumonia that occurred in Middles-
brough (Dr. Ballard’s Report to the M.O. of the Local
Government Board, 1S89).

xi] BACILLI : SPECIFICALLY PATHOGENIC 227

The general morphological and cultural characters are
those of bacillus coli. As stated just now, from the lung
juice pure cultures were obtained, the organism being
present in the lungs in great abundance (see Fig. 78). The
bacilli are o^-o -4/x thick, o'S-i'6/x long.

The cultures as also the lung juice act virulently on mice
and guinea-pigs, on the former more than on the latter.
Subcutaneous inoculation produces disease and death in the
course of thirty to one hundred hours. On post-mortem
examination both lungs are found intensely inflamed,
some portions in a state of red hepatisation ; generally there
are present pleurisy and pericarditis and peritonitis, with more
or less sanguineous exudation. The spleen is enlarged in
mice, but not in guinea-pigs. The bacilli can be easily
demonstrated in very large numbers both by cover-glass
specimens and cultures in the heart’s blood, the lung juice,
and the spleen of mice, and in the lung juice of guinea-
pigs.

The lung juice, or cultures derived from the tissues of
the infected mice or guinea-pigs, inoculated into further mice
or guinea-pigs, produce the same disease and death with the
symptoms just described.

While working with cultures of these bacilli on mice
and guinea-pigs there occurred amongst normal mice and
guinea-pigs kept in the same stalls as the experimental
animals an epidemic of pneumonia, leading to the death
of a great many of them j on post-mortem examination all
showed exactly the same appearances as those experimental
mice and guinea-pigs, and the juice of the inflamed lungs
contained the bacilli in crowds.

Three monkeys, kept on the same premises, and which
most probably became accidentally infected by food, died of
pneumonia. In the inflamed lungs the bacilli could be

Q 2

228 MICRO-ORGANISMS AND DISEASE [chap.

easily demonstrated by cover-glass specimens and by
culture.

2. Guinea-pigs injected intraperitoneally with large doses
of various species of non-pathogenic bacteria taken from
the surface of Agar cultures, e.g. bac. prodigiosus, bacillus
coli, vibrio of Finkler, &c., &c., succumb, as has been pointed
out in a former chapter, to acute fatal peritonitis. The more
or less copious, more or less sanguineous peritoneal fluid .
is crowded with the microbe injected, but in one or the
other such case, although rarely, contains in addition a
number of bacilli which in morphological and cultural .
respects coincide with the bacillus coli. Gartner has met
with this bacillus in the peritoneal exudation after intraperi-
toneal injection with pus coccus \ I have met with it after
prodigiosus injection. Cultures of this peritoneal bacillus •'
prove it to be bacillus coli, and I explained its presence •
in the peritoneal cavity by the nearness of the intestine —
its original habitat — to the inflamed peritoneum. This
peritoneal bacillus differs, however, from the intestinal
bacillus coli in its high virulence since small doses of
the former injected subcutaneously into guinea-pigs produce
acute general septicgemic infection.

3. In a good many cases of fatal English cholera I have
found the mucus flakes and the fluid of the small intestine
containing a bacillus copiously and almost in pure culture,
which in its general morphological and cultural characters
(gas- formation, curdling of milk, and indol production)
coincides with the bacillus coli ; it is, however, more motile
and more cylindrical than the typical bacillus coli. In the
mucus flakes it is in some cases present in continuous
streaks and masses, and not seldom arranged linearly in the
manner characteristic of the cholera vibrio in the mucus
flakes of the rice-water contents of the ileum in Asiatic

XI] BACILLI : SPECIFICALLY PATHOGENIC 229

cholera (see chapter on Cholera). The colon variety ob-
tained from these cases of English cholera possesses, how-
ever, considerable virulence or. the guinea-pig, producing in
this animal after subcutaneous injection of small or moderate
doses acute septiciemic infection and death. Of this
character appears to be the Bacillus neapolitanus isolated
by Emmerich from the intestinal fluid in cases of Asiatic
cholera.

4. The aerobic bacillus of malignant oedema , which I
obtained from recently manured garden earth, produces
on subcutaneous inoculation into guinea-pigs and mice
extensive gangrene of the skin and muscle, with san-
guineous, malodorous exudation, and death in twenty-four
to thirty-six hours — a condition similar to that produced by
the anaerobic malignant oedema bacillus of Koch (also
obtainable from manured garden earth) ; in cultural respects,
in its motility and flagella, it is not distinguishable from
bacillus coli ; the chief difference from the latter consists in
the great virulence of the former. The subcutaneous
exudation as also the skin itself is crowded with the bacilli.

5. A bacillus which in morphological and cultural
respects is closely related to the bacillus coli was found in
abundance in beef pie (. Portsmouth ), which had caused in
those who partook of it severe gastro enteritic symptoms.
By feeding mice with the pie or with the broth cultures
of the bacillus acute gastro-enteritis was produced (Report
of the M.O. of the Loc. Gov. Board, 1890-91), and
thereby its difference from the bacillus coli was established,
for such a result is not to be obtained with the cultures of
the bacillus coli derived from the intestinal contents.

6. A bacillus of which the cultural characters have not
been ascertained (it occurred at a time before solid culture
media were used), and of which therefore I am unable to

230

MICRO-ORGANISMS AND DISEASE [chap.

say to what group of bacilli it does belong, though from its
size it could not belong to the group of bacillus coli, is the

Fig 79. — From a Section through the Kidney of a Case that died
after Meat-Poisoning at .Welbeck.

The figure represents part of a glomerulus of a Malpighian corpuscle, in which
some of the capillary blood-vessels are filled with the bacilli. Magnifying
power 700.

1. Capsule of Malpighian corpuscle.

2. Capillaries filled with bacilli.

3. Capillaries empty.

4. Bacilli contained between capillaries.

microbe found in connection with outbreaks of choleraic
diarrhoea in Welbeck and in Nottingham.

Bacillus of choleraic diarrhoea from meat-poisoning. — In

xi] BACILLI : SPECIFICALLY PATHOGENIC 231

July, 1880, there occurred in Welbeck, Notts, an extensive
outbreak of diarrhoea among over seventy-two persons who
had partaken of beef and ham sandwiches sold at Welbeck
on the occasion of a sale of timber and machinery on the
estate of the Duke of Portland. The infection showed
itself after an incubation-period varying from twelve hours
or less to forty-eight hours or more. .The first symptoms
were a sudden feeling of languor, nausea, griping in the
abdomen, in some cases giddiness and fainting, and pain in
the trunk. Then followed pain in the abdomen, diarrhoea,
and vomiting, the diarrhoea being most constant. Four
cases ended fatally. On post-mortem examination enteritis
and pneumonia were most prominent. Part of the kidney

<*a> •

***> *

Fig. 80.— Isolated Bacilli in a small Artery of the same Kidney as in

PRECEDING FIGURE.

Some of the bacilli contain spores.

was examined in microscopic sections, and it was found
that many of the tubuli uriniferi contained hyaline casts ;
that the capillaries of the glomeruli of the Malpighian
corpuscles, and the afferent arterioles, contained numbers
of bacilli, some of the capillaries being distended by and
plugged with masses of bacilli densely aggregated. In
February, 1881, a similar but less extensive outbreak
occurred at Nottingham, among fifteen persons that had
partaken of certain baked pork. The symptoms were
similar to those in the Welbeck outbreak. One case
ended fatally. Post-mortem : bloody exudation in peri-
cardium, intense pneumonia, mesenteric glands enlarged,
enteritis, Peyer’s glands enlarged. Bacilli similar to those

232

MICRO-ORGANISMS AND DISEASE [chap.

of the above case were found in the blood, in the peri-
cardial exudation, in the juice and in the bloody fluid filling
the alveolar cavities of the inflamed lung, in the vessels of
the kidney, in the submucosa of the inflamed Peyer’s
glands of the small intestine, and in the blood-vessels of
the spleen and around them.

The bacilli vary in -length between 0-003 and 0-009 mm. ;
their thickness is about 0-0013 nun. They are rounded at
their extremities, single or in chains of two, and some con-
tain a bright oval spore, situated in the centre or at one end,
and about o-ooi mm. thick. This was the case with the
bacilli in the glomeruli of the kidney of the Welbeck case.
The bacilli containing spores were thicker than those without
them.

Experiments by feeding and inoculation made on dogs and
cats, rabbits, guinea-pigs, and mice with the ham that had
done the mischief in the Welbeck case produced positive
results. In all cases we found pneumonia and haemorrhage
in the liver, peritonitis in some, spleen enlarged in most.
The bacilli found in this ham were cultivated in the incubator
in white of egg, and after two days’ cultivation four white
rats and several guinea-pigs and white mice were inocu-
lated, and they became ill after twenty-four hours; they
were quiet, did not feed well, and were more or less
soporous. When killed the spleen was found enlarged,
and in the lungs were found haemorrhage and hyperEemia,
and in some cases extensive pneumonia.

Blood, pericardial exudation, and lung juice from the
fatal Nottingham case inoculated into ten animals (guinea-
pigs and white mice) produced fatal results in six, the other
four were killed : but in all there was severe pneumonia, in
eight out of the ten there was peritonitis, in four also
pleuritis, and in two in addition enlargement of the liver

XI] BACILLI : SPECIFICALLY PATHOGENIC 233

and spleen. Bacilli were found in the blood and exuda-
tions of these animals. On cultivating blood and lung
juice from the above case a crop of bacilli was produced
which on inoculation proved very poisonous in the same
way as in the previous cases.

7. Bacillus enteritidis of Gartner} — This microbe was
found in the flesh of a cow that had been killed after ailing
with diarrhoea ; and the bacillus was also found in the
spleen of a man who died twelve hours after eating of the
above beef. The morphological and cultural characters —
as far as investigated — coincide with those of bacillus coli ;
on rodents the cultures proved virulent on mice after
feeding, and on rabbits and guinea-pigs after subcuta-
neous injection. It seems to me quite probable that this is
the same microbe that I mentioned sub (5) as the beef-pie
(Portsmouth) bacillus.

8. Of the same nature, i.c. allied morphologically and
culturally to a variety of bacillus coli, is the bacillus de-
scribed by H. Laser ( Centralblatt f Bacteriologie imd
Parasit., xiii. Band, No. 7, p. 217) as a “gas-forming aerobic
bacillus,” and which was met with in and cultivated from
the lung and liver of a young calf that had died, with several
others, from some unknown malady. The characters of the
microbe in microscopic specimens and in culture on the
various media show that it belongs to the colon group.
The cultures possess on subcutaneous injection into rodents
a moderate degree of virulence and produce in a small
percentage of them septicaemic infection.

9. A bacillus possessed of the power, to a considerable
degree, of forming gas has been obtained from the dead body,
and described by Welch and Nutall under the name of

1 Correspondembldtler d. allgem. Aerztl. Vertins von T/iuringen ,

1888, No. 9.

Fig. 8i.— Film Specimen of Spleen in Typhoid Fever; Nuclei and Cells
of Spleen Pulp, two Typhoid Bacilli.

X IOOO.

Fig. 82.- From a Section through the Spleen in Typhoid Fever, showing
a Collection in the Pulp of Typhoid Bacilli.

X 1000.

CH. XI] BACILLI : SPECIFICALLY PATHOGENIC 235

bacillus cerogenes capsulatus, and by A. Fraenkel under
the name of bacillus gasoformans. This bacillus is virulent
to rodents (rabbits), producing acute septicemic infection,
and death in twenty-four hours, the blood of the general
circulation containing copiously the microbe ; so strong is the
gas-forming power of this microbe that the viscera of the dead
(experimental) animal are found permeated by gas bubbles.
My colleague, Dr. Kanthack. has also obtained this bacillus
from the human dead body ; it completely coincided with
that described by Welch and Nutall and by Fraenkel, and
after carefully investigating its morphological and cultural
characters Dr. Kanthack came to the conclusion that this
bacillus is a virulent variety of bacillus coli. In shape, size,
in its flagella; in plate, streak, and stab culture in gelatine ;
in its forming copiously gas bubbles in gelatine shake
culture ; in its power of curdling milk and of forming indol
in broth — it completely coincides with the bacillus coli, the
difference being, as stated above, that the bacillus gasoformans
is at first very virulent and forms copiously gas, but in con-
tinued subcultures assumes the character, both as to virulence
and formation of gas bubbles, of the typical bacillus coli.

Bacillus of typhoid fever in man (Eberth-Gaffky).- — In all
cases of typhoid fever, if the spleen or the mesenteric
glands are examined by film specimens or by culture,
bacilli will be found in numbers which in morphological
and cultural respects belong to the group of colon-like
species that we have been describing hitherto, viz. they are
cylindrical motile bacilli which do not liquefy gelatine,
which do not form spores, and which in gelatine plates, in
gelatine streak and gelatine stab, on Agar and broth, show
similar characters as those of the above bacilli, but, as
we shall presently show, possess, on careful analysis,

236 MICRO-ORGANISMS AND DISEASE [chap.

sufficiently well-marked differential characters, enabling us
to say that the typhoid bacillus is a well-defined species,
and to identify and distinguish it from bacillus coli and its
nearest allied varieties.

The true typhoid bacillus is constantly present in the

Fig. 83. — A Culture on the Surface of Nutrient Gelatine, obtained by
rubbing a Particle of Spleen Pulp (Typhoid Fever) over the Sur-
face of the Gelatine; showing a Confluent Mass of Colonies of
the Typhoid Bacillus.

Natural size.

tissue of the spleen and the mesenteric glands in this
disease, and in this disease only ; in the spleen it occurs
as a rule in larger or smaller groups, though it is also found
here and there in small numbers and isolated ; the same
is the case with the mesenteric glands. In the wall of the
ileum, in and around the swollen or ulcerated Peyer’s glands

Xi] BACILLI : SPECIFICALLY PATHOGENIC 237

— whenever such an examination is made by microscopic
or cultural specimens — this bacillus occurs in numbers. In
the intestinal contents and in the typhoid stools the true
typhoid bacillus can be also identified, although this
examination is only in comparatively few cases successful,
on account of the great number of bacillus coli present, and
it is for the same reason that if the typhoid bacillus is so

Fig. 84. Impression Specimen of a very young Colony of the Typhoid
Bacillus on Gelatine.
x 1000.

demonstrable it is in cases in which the Peyer’s glands
have already begun to ulcerate, i.e. in the second and third
week of the disease, that is when their number passed from the
tissue of the mucous membrane is sufficiently great. In the
blood of the general circulation in typhoid fever the bacillus
is not demonstrable, except in very rare instances, and
then only after the second week. From this the conclusion

238 MICRO-ORGANISMS AND DISEASE [chap.

is drawn that typhoid fever is not a blood disease ; that is to
say, the blood is not the proper soil for the growth and
multiplication of the microbe, but the wall of the ileum,
the spleen, and mesenteric glands (and possibly other lymph
glands) represent the localities wherein the bacillus grows and

Fig. 85. — A typical. Gelatine Plate Cultivation of the Typhoid Bacillus;

THE SMALL DOTS ARE DEEP COLONIES, THE PATCHES ARE COLONIES OK

the Surface ; the Culture is about 6-7 days old and shows the
CONCENTRIC MARKINGS OF SUPERFICIAL COLONIES.

Natural size.

multiplies and produces the toxin (typhotoxin) which causes
the symptoms of the disease. Thus typhoid fever would in
reality be the result of intoxication in its chief clinical
symptoms. Owing to the fact that the demonstration of
the typhoid bacillus in the typhoid stools, because of our

xi] BACILLI : SPECIFICALLY PATHOGENIC 239

at present imperfect methods, is in many cases negative,
Sanarelli has come to the conclusion that the pathological
changes of the intestine are as much a result of the toxin
action of the bacillus distributed in the blood and viscera as

Fig. 86. — Three Tube-Plate Cultivations of the Typhoid Bacillus:
Colonies on the Surface of Gelatine. ]n the left Tube the Colonies

ARE VERY NUMEROUS, SMALL, AND IN THE LOWER PART CONFLUENT
IN THE MIDDLE TUBE THE COLONIES ARE FEWER AND LARGER; AND IN
THE RIGHT TUBE ONLY ONE COLONY OF GREAT SlZE, AND SHOWING THE

concentric Aspect.

Compare this figure with Fig. 60 of similar cultures of bacillus coli.

Natural size. - •

are the other clinical symptoms. Wright and Semple 1 have
attempted to give support to this theory of typhoid fever
being really a blood disease by stating that in all, or almost
all, cases which they have examined — some of them early

1 The Lancet for July 27, 1895.

240

MICRO-ORGANISMS AND DISEASE [CHAP.

cases— the urine excreted by the patient contained the
typhoid bacillus in considerable numbers, and they con-
clude that contrasted with the intestinal discharges the
urine is more constantly and more highly charged with the
contagium and deserves, therefore, more attention for pur-
poses of disinfection than it has hitherto received. While

Fig. 87 —Streak Sub-culture on Gelatine of the Bacillus of Typhoid.

Natural size.

this conclusion in a general way and to a certain extent
harmonises with previous results, viz. as to the occasional
occurrence of the typhoid bacillus in the kidney and in the
urine, it differs in this essential respect that Wright and
Semple maintain the almost constant occurrence of the
typhoid bacillus in the urine, even in early cases, and for
this reason they favour Sanarelli, inasmuch as they conclude

xij BACILLI : SPECIFICALLY PATHOGENIC 241

that the normal habitat of the typhoid bacillus is the
circulating blood, hence its passage into the urine already
in the early phases of the disease. Dr. Horton Smith,
working in my laboratory, has paid special attention to this
question, and his conclusions do not confirm those arrived
at by Wright and Semple. The identification of the typhoid
bacillus in the stools, in the urine, in the spleen, in the
blood, or in the glands, & c , if it is to be considered free
from criticism, must not content itself merely with the
demonstration of a general similarity as to size, shape, and
motility, or as to the general aspect of the plate cultiva-
tion, streak and stab cultures on Agar and in gelatine— it
is precisely on account of such general observations that
many of the statements made in previous years as to the
occurrence of the typhoid bacilli in one or the other tissue,
in one or the other locality, cannot be accepted as proven.
The identification of a bacillus as typhoid bacillus must be
such as to show that as regards every one and all of the
following characters there is complete harmony between it
and the bacillus obtainable in pure culture from the typical
spleen of a typical case of typhoid fever. The characters
are these : —

1. The typhoid microbe taken from the spleen of a
typhoid case is a cylindrical bacillus measuring on the
average 2-4 fj. in length ; in gelatine or Agar cultures already
after twenty-four hours there are present longer forms, some
filamentous ; the great majority of the bacilli from a recent
culture are distinctly longer and more cylindrical than those
of a similar culture of the typical bacillus coli.

2. Examined in the hanging drop in sterile broth the
typhoid bacillus of a recent gelatine or Agar culture (16-24
hours old) is extremely motile, contrasting markedly with
a similar culture of the typical bacillus coli.

243 MICRO-ORGANISMS AND DISEASE [chap.

3. On staining flagella the typhoid bacillus of a recent
Agar culture is seen to be possessed of a large number of
long wavy or spiral flagella extending in a radial fashion on
—or rather coming off vertically from — the whole length of
the bacillus. From a considerable experience I am pre-
pared to attribute to this particular distribution, and to the

Fig. 88.— Stab Subculture of the Typhoid Bacillus.
Magnified twice.

abundance of long flagella, a very important differential
value. (See illustrations of flagella in Chapter VI.)

4. In gelatine plates the typhoid bacillus grows markedly
slower than the typical bacillus coli ; the colonies of the
former, more translucent, homogeneous, show after some days
an indication of concentric layers ; but their outlines are as

XI] BACILLI : SPECIFICALLY PATHOGENIC 243

angular and filmy as those of bacillus coli. Compare Fig. 7 2,
which, although of the grouse bacillus, is a good repre-
sentation of a bacillus coli plate, with Fig. 85, of a plate of
the typhoid bacillus. In gelatine streak and Agar streak
the growth is also markedly slower and more translucent
in the first few days than that of the bacillus coli, but its

Fiu. 89.— Streak Cultures on Nutrient Gelatine after 48 hours: on
THE LEFT, OF BACILLUS CoLI | ON THE RIGHT, OF TYPHOID BACILLUS.

Natural size.

irregular filmy margin in the gelatine streak culture is the
same as in bacillus coli.

5. In gelatine stab the line of inoculation is marked like
that of bacillus coli, as a row or rows of droplets white in
reflected, brownish in transmitted light, but on the surface
of the stab in typhoid the plate-like filmy expansion is
small, and is not well marked in the first few days, whereas

R 2

244 MICRO-ORGANISMS AND DISEASE [chap.

in those of bacillus coli this plate-like expansion is well
developed.

6. Shake cultures in ordinary and in sugar gelatine do
not develop gas bubbles, though the gelatine is in all layers
pervaded by colonies.

7. Milk is not curdled by the typhoid bacillus.

8. Broth is made rapidly turbid, and after some days an
imperfect pellicle may make its appearance, but at no time
does such broth give the nitroso-indol reaction. The typhoid
bacillus grown in milk, broth, or litmus-whey produces less
acid than the typical bacillus coli (Petruschki).

9. On steamed potato, kept after inoculation at 370 C.,
the growth is a colourless transparent film.

10. In nutrient gelatine, containing gelatine to the amount
of 25 per cent., the difference between bacillus coli and
bacillus of typhoid on incubation at 370 C. is very striking;
the (fluid) gelatine remains limpid, and its surface is covered
with a thick pellicle during the first forty-eight hours in the
case of the typical bacillus coli, but is strongly and uniformly
turbid in the case of the typhoid bacillus.

A bacillus which does not conform to all the above
points is in my laboratory rejected as typhoid. Whether or
not a bacillus which in one or the other of the above
points deviates and approaches the bacillus coli is or is not
the typhoid bacillus, has or has not originally been derived
from the typhoid bacillus, I feel neither inclined to deny nor
to affirm — in the present state of our knowledge it would not
be justifiable to do so ; but what I maintain is that since the
true typhoid bacillus taken from the spleen of a typical
typhoid case possesses all and every one of the above charac-
ters it is more justifiable to reject as typhoid those which
either in the number and character of the flagella, or in the
manner of growth in gelatine shake culture, in milk, in broth,

xi] BACILLI : SPECIFICALLY PATHOGENIC 245

on potato, and in 25 per cent, gelatine (at 37° C.) do not
correspond to the above tests. In respect of the rapidity of
growth of the colonies in gelatine plates and in gelatine
streak a latitude may be excusable, since in these respects
continued subcultures of the typhoid bacillus on artificial
media show that in respect of rate of growth it does some-
what alter with age, but not in respect of greater translucency ;
nor have I seen any alteration, after two or three years of
continued subcultivation, in the matter of flagella, of gelatine
shake culture, of milk-, broth-, potato-, or 25 per cent, gela-
tine cultures. It may be added that bacillus coli has longer
vitality, both in water and in sewage, than bacillus of
typhoid, and also that, while bacillus coli requires 65° C. for
five minutes to become killed, the typhoid bacillus is killed
already at 62° C. in five minutes.

The identification by Dr. Horton Smith of the typhoid
bacillus from the urine of cases of typhoid fever was based on
the above characters, and his results are very instructive : —

(a) In two cases of undoubted typhoid fever — mild
cases — the examination of the urine — always considerable
quantities being examined by Parietti’s method— com-
mencing from the first week of illness and carried on till
the temperature again became normal, revealed no typhoid
bacillus.

(/>) One case, dead from typhoid fever during the third
week ; the urine taken in the post-mortem room yielded
numbers of colonies of the typhoid bacillus.

(c) One case, first examined on the twelfth day of illness,
did not yield the typhoid bacillus, but, beginning with the
fourteenth day, till the twenty-second day, — the day of fatal
issue — yielded typhoid colonies.

(d) One case, examined first on the tenth day of illness
and continued through the whole of the first attack and

246 MICRO-ORGANISMS AND DISEASE [CHAP.

right through a short relapse, failed to yield typhoid
bacillus in the urine. By the thirty-ninth day, when the
temperature had become almost normal again, the urine
yielded abundance of typhoid bacilli, in fact the urine
was quite turbid, being a pure culture of the typhoid
bacillus, and, strange to say, this condition, viz. abundance
of typhoid bacilli in the urine, continued until twenty-two
days after the temperature had again been normal.

There is then in these observations no confirmation to
be found of Wright and Semple’s contention as to the
early excretion of the typhoid bacillus, on the contrary they
show that, as had been hitherto accepted, the general dis-
charge of the typhoid bacillus from the system by the
kidney is an occurrence belonging to the later stages and
cannot therefore be taken as indicating that the typhoid
bacillus is circulating in the blood in the early stages, or
that therefore typhoid fever is a blood disease, a true
infection like anthrax or septicaemia.

With regard to the effect of subcutaneous or intraperi-
toneal injection of large doses of living or sterilised culture
no differentiation can be made between the typhoid
bacillus and the bacillus coli, they both— in common with
other species, e.g. bacillus prodigiosus— act in the same
manner ; recent gelatine cultures of either act more viru-
lently on the mouse and guinea-pig injected subcutaneously
than broth or Agarculture, producing in sufficiently large
doses acute septicaemic infection. Smaller doses produce
only a transitory swelling, which, however, may lead to
local sloughing and necrosis of the skin.

The immunisation by injection of living culture of the
typhoid bacillus and the specific action of blood-serum
of immunised animals we shall have an opportunity to
discuss in a later chapter.

xi] BACILLI : SPECIFICALLY PATHOGENIC 247

Petruschki described (Centra/Matt f. Bakt. und Parasit.
No. 6/7, 1896) a bacillus, occurring occasionally in the
typhoid stools and also other putrid materials, which coin-
cides in most points with the typhoid bacillus. This is the
bacillus fcecalis alkaligeues. It differs from the typhoid
bacillus in forming alkali in litmus-whey, the latter being an
acid former.

CHAPTER XII

PATHOGENIC BACILLI : GROUP C

As belonging to this group we consider pathogenic bacilli
which in morphological and cultural respects differ from
those hitherto considered. They are fine cylindrical rods
of about o-8 to i fx long, o-i to o' 2 /x thick, forming charac-
teristic translucent, filamentous, gelatinous growths, slowly ,
liquefying gelatine and not forming spores.

1. Bacillus of mouse septicaemia of Koch. — By inoculation
of filthy water into mice Koch produced a fatal and acute
septicaemia, which owing to the peculiar microbe has great
interest. At the seat of the inoculated animals there is
found slight haemorrhage, the internal viscera are greatly
congested, the spleen is not much enlarged ; the animals
die during the second day. In the blood of all parts are
found in very large numbers exceedingly minute bacilli,
some longer than others, but all very fine ; many of the
white blood-corpuscles are quite filled with them, being at
the same time swollen up. In the lungs there is slight
haemorrhage into the alveolar tissue : everywhere one sees
the swollen leucocytes completely filled with the minute
bacilli, some of these also free owing to the disintegration
of the leucocytes. Sections stained carefully in fuchsin and

CH. XI i] PATHOGENIC BACILLI : GROUP C

249

then in methyl blue show the nuclei of the tissue and of the
leucocytes blue, the bacilli bright red.

Fig. 90. — From a Section through the Lung of a Mouse dead of Koch’s

Septicaemia.

1. Small vessel filled with blood ; the white blood-corpuscles are filled with very

minute bacilli.

2. Interalveolar tissue : in it a white corpuscle filled with the bacilli. Magnifying

power 700.

3 A white blood-corpuscle more highly magnified, 1000.

(Stained with magenta.)

Cultivations of the heart’s blood or of the juice of the
viscera yield numerous colonies ; pure cultivation in gela-
tine in test tubes can be made without difficulty directly

Fig. 91. — From a Section through the Small Intestine of a Mouse dead

of Septicemia.

The figure represents a section through a small vein in the submucous tissue, filled
with blood. At 1, there is a homogeneous substance and in it numerous bacilli,
but these bacilli are much larger than the bacilli of Koch's septicaemia in the
mouse.

Magnifying power about 700 (Stained with methylene blue and vesuvin.)

from the heart’s blood. The colonies in plate cultivations
appear after two or three days as highly translucent, gela-

250

MICRO-ORGANISMS AND DISEASE [chap.

tinous, grey, irregularly outlined, angular, minute patches ;
in the stab culture in gelatine after two or three days a very
characteristic growth is noticed : the stab is a translucent
grey line from which branch out horizontally vast numbers
of fine, closely placed, gelatinous, translucent grey threads ;
in the streak culture the streak becomes visible after two to

Fig. 92. — Film Specimen of Blood of Mouse dead of Koch’s Mouse

Septicaemia.

X 1 coo.

three days as a gelatinous, grey, translucent, thin band from
which pass out vertically numerous grey fine lines. The
growth liquefies the gelatine very slowly ; it takes generally
some days before liquefaction commences, and it proceeds
very slowly ; the gelatine is thick like syrup, is fairly limpid,
but contains greyish, translucent flakes. In Agar mixture

XII]

PATHOGENIC BACILLI : GROUP C

251

the growth is slow and very transparent. No spore form-
ation has been observed.

Specimens made of the cultures show under the micro-
scope, besides short bacilli, also a great many which are long
threads more or less curved. Inoculation produces in mice
the septic?emia with certainty.

Fig. 93. — Film Specimen of Blood of Pigeon dead after Infection with

Swine Erysipelas.
x 1000.

Loffler describes a spontaneous fatal epidemic amongst
white mice which occurred in his laboratory, and which
was caused by this same bacillus ( Ccntralbl. f. Bakt. und
Parasit., vol. xi. p. 134).

2. Bacillus of swine erysipelas (mal rouge, rouget, red
soldier). — An acute infectious disease, to which swine are

252 MICRO-ORGANISMS AND DISEASE [chap.

very susceptible, and of which about 60 per cent, succumb.
When affected the animals are quiet, the voice is hoarse,
and the temperature is much raised ; on the skin of the
neck, chest, abdomen, and thighs extensive red patches of
swollen cedematous skin are noticed ; under convulsions—

Fig. 94 — Stab Culture in Gelatine of the Bacillus of Swine Erysipelas.

Natural size.

occasionally paralysis of the hind extremities — the animals
die from between twelve hours and three or four days since
the first symptom. On post-mortem examination is found
haemorrhage in the affected patches of the skin, the lymph
glands are swollen and much congested, the peritoneum is
inflamed ; the mucous membrane of the intestine is much

XU]

PATHOGENIC BACILLI : GROUP C

253

injected and cedematous ; the Peyer’s glands are swollen, the
spleen and liver are much congested and slightly enlarged.

The blood of the heart, and particularly the juice of the
lymph glands and the spleen, contain fine bacilli very similar
to those of the mouse septicaemia (Schiitz) (o*6~r8 //. long).
On microscopic sections through the liver, spleen, kidney,
and lymph glands the bacilli are easily demonstrated in the
capillary blood-vessels, either isolated between the blood
corpuscles or enclosed in the swollen leucocytes. As re-
gards cultural characters they completely resemble those of
the mouse septicaemia (Koch).

e Swine fed or inoculated with the blood or tissues of a
pig dead of the disease become also affected. Mice 1
and pigeons are very susceptible to the disease ; guinea-
pigs and fowls are refractory ; rabbits show generally only a
local effect ; mice die in two or three days, pigeons in three
to four days ; in both the blood of the general inoculation
and of the organs contains abundantly the bacilli. In the
pigeon numerous white blood-corpuscles in the vessels of
the viscera are filled with the bacilli.

Pasteur has shown that the virus in its passage through a
series of pigeons increases in virulence, both as regards the
pigeon as well as the pig ; on its passage through a series of
rabbits it increases in virulence as regards the rabbit, but
decreases in virulence as regards the pig. Pigs inoculated
with blood of the last rabbit of the series become ill, but
recover, and are then found refractory to inoculation with
the virulent disease.

3. 1'he bacillus of Egyptian ophthalmia : catarrhal con-
junctivitis (Koch). — Koch ( Cholerabericht , 1883) has shown

1 Mice die with congested and enlarged spleen, greatly congested
lung- ; the intestines are relaxed and filled with sanguineous mucus ;
the kidney and liver are enlarged and congested.

254

MICRO-ORGANISMS AND DISEASE [chap.

that what is spoken of as “ Egyptian ophthalmia ” is really
several kinds of infectious ophthalmise : one is an acute
blennorrhoea or purulent ophthalmia, and does not differ
from that known to occur in consequence of infection with
gonorrhoeal exudation. A second, the true Egyptian oph-
thalmia, is however of an altogether different etiological

Fig. ys.— Film Specimen of Egyptian Ophthalmia, Catarrhal Conjunc-
tivitis (Koch), showing many Pus-cells containing the specific
Bacilli in their Protoplasm.

x 1000.

character, though in its symptoms and pathology it is
similar to, but not identical with, the blennorrhoea. This
second one, the “ catarrhal conjunctivitis ,” is associated,
not with the gonococcus, but with a minute fine bacillus,
very similar in morphological respects to the bacillus of
Koch’s mouse septicaemia. In this ophthalmia the bacillus

XII] PATHOGENIC BACILLI: GROUP C 255

is present in the purulent exudation of the conjunctiva as
isolated examples, and more commonly enclosed within the
pus cells, whose protoplasm is sometimes found crowded
with them (see Fig. 95), in the same way as we saw the

Fig 96.— From a Section through a Lymphatic Gland of Man dead of

Septicaemia.

1 A blood-vessel which at one place is distended by and filled with minute bacilli.

2. Lymph corpuscles.

3. Degenerated lymph-corpuscles.

Magnifying power 700. (Stained with gentian violet.)

leucocytes in the mouse septicaemia crowded with the small
bacilli. The cultivation of these bacilli carried out by
Kartulis shows that there exists a great difference between
them and the mouse septicaemia bacillus.

Kartulis, besides describing their symptoms and course

256 MICRO-ORGANISMS AND DISEASE [chap.

( Centralb /. f. Bakt. und Parasit., Band I., No. 10, pp.
289-293), and the differential characters existing between
blennorrhoea of the conjunctiva and catarrhal ophthalmia,
succeeded in cultivating the bacilli of the catarrhal or true
Egyptian conjunctivitis. He showed that they do not grow
on peptone or gelatine; on blood-serum or on Agar they
grow well between 28-36° C., forming in thirty to forty
hours small white punctiform colonies, prominent over the
surface of the medium ; when closely sown (e.g., in streak
culture) they soon coalesce into a whitish-grey band of a
fatty, glistening appearance ; the margin of the band is wavy
or crenated. Animals inoculated on the conjunctiva with
the conjunctival secretion or with the culture prove refrac-
tory ; but Kartulis succeeded in producing with the culture
the typical catarrhal conjunctivitis in one out of six cases.
The pus corpuscles resulting in this case were crowded with
the characteristic bacilli. This one case was that of an in-
dividual twenty-five years old.

I append here the illustration of a bacillus of septiccemia oj
man. In several cases of human septicaemia I have found
in the blood-vessels of the swollen lymphatic glands large
numbers of minute bacilli, slightly thicker than those just
mentioned. They form continuous masses, both in the
capillaries and in the minute veins, amounting in some
cases to veritable emboli. They occur isolated or in short
chains, their length about 1 /x to 2'5 fx, their thickness about
0-3 ix to 0-5 fx ; no cultivations having been made, the
characters of these bacilli could not be ascertained.

The bacillus of influenza} — R. Pfeiffer (. Deutsche Med.
Wochenschnft, No. 2, 1892) was the first who made the

1 The following account is taken from my Report to the Medical
Officer of the Local Government Board : Further Report on Influenza,
1S89-92.

Xll]

PATHOGENIC BACILLI : GROUP C

257

announcement that in all cases of influenza there are
present in the characteristic grey purulent bronchial secretion
enormous numbers of minute non-motile bacilli. He de-
scribes these as occurring only during the acute stages and
gradually diminishing in numbers as the disease abates.
The bacilli, he tells us, are very minute, about the thickness
of the well known bacilli of Koch’s mouse septicaemia, but
only half their length ; they stain with some difficulty in
anilin dyes, requiring a somewhat prolonged application of
the dye. In stained specimens these bacilli have a charac-
teristic appearance, inasmuch as their protoplasm is segre-
gated into a stained granule at each end while the middle
portion remains unstained and show's only the outline of the
sheath. Thus the bacillus looks like a diplococcus, and
where two such bacilli are placed end to end they look like
a chain (streptococcus) of four spherical cocci. In the
sputum these bacilli occur in smaller and larger masses,
occasionally almost as a pure culture. In severe cases they
form continuous masses in the peribronchial tissue and also
in the subpleural lymphatics, and they are also met with
inside the leucocytes of the sputuni. As the disease passes
off, so the bacilli disappear from the sputa. These bacilli
are constantly present in influenza, but do not occur in the
bronchial secretion of other bronchial or pulmonary affec-
tions.

Kitasato, in the same paper, gives his observations on the
cultivation of these bacilli of Pfeiffer, and records that they
have cultural characters by which they can be readily dis-
tinguished from other bacilli : that they are, in fact, a
definite species not occurring in any disease except in
influenza. They do not thrive at temperatures below 28° C.,
that is to say at temperatures at which nutrient gelatine
still keeps its solid condition. They grow well in broth and

258

MICRO-ORGANISMS AND DISEASE [chap.

on glycerine Agar at 370 C. or thereabouts. The broth
does not become turbid, but remains limpid. The growth
in broth appears as whitish small granules and flocculi ; on
glycerine Agar the bacilli form minute translucent colonies
like droplets, having no tendency to coalesce as growth
proceeds. The cultures are also characterised by this fact
that they soon die, and therefore sub-cultures cannot easily
be carried on through many generations. In stained
specimens grown in cultures the bacilli retain the same
characters observed in the bacilli of sputum, viz., they show
the characteristic bipolar staining.

These statements and observations of Pfeiffer and
Kitasato are very definite, and if confirmed would afford
strong reason for believing that in these bacilli we had
found the special microbe of influenza. The life-history of
this microbe would conform with what we believe to be the
facts about the contagium of influenza, its being spread and
received by the organs of respiration, and the reception of
the infection by the same channel ; the presence in most
cases of influenza of some kind of bronchial disturbance
more or less pronounced, showing itself at the outset of the
disease or a few days later, and increasing after the febrile
stage of the complaint had been passed.

From our own observations of a large number of cases,
we find ourselves in a position to confirm the statements of
Pfeiffer and Kitasato in all essential points ; and accordingly
we have arrived at the conclusion that the particular bacilli
as described by them ought to be regarded as the specific
microbe of influenza.

The bronchial expectoration was examined in twenty
cases from the living patient ; of these, five were cases of
genuine influenza-pneumonia, that is of pneumonia setting
in very soon, a few days, after the attack of influenza

Xi i] PATHOGENIC BACILLI : GROUP C 259

commenced, and where the history showed that the pneu-
monia was to be regarded as a part of the disease and not
as a secondary complication.

The result then of these examinations confirms fully the
assertions of Pfeiffer, viz., that the characteristic influenza
bacilli are constantly present in the bronchial sputum of

Fic. 97. — Film Specimen of Pulmonary Expectoration of an Acute Case
of Influenza Pneumonia; Nuclei of Leucocytes and the Influenza
Bacilli in Pure Condition.

x 1000.

influenza cases ; that in well-marked cases they occur in
. great abundance, singly, in small groups, and in larger
: masses, and in some portions of the sputum almost as a
I pure culture. The results also go to confirm Pfeiffer’s
I statement that as the disease abates, as the patients get
I better and as the sputum becomes scantier, the number of
| the bacilli also rapidly diminishes ; this was the case in the

s 2

26o

MICRO-ORGANISMS AND DISEASE [chap.

sputum from patients having pneumonia of influenza :
before the height of the disease is passed the number
of the characteristic bacilli is very great, after the height of
the disease it diminishes. Also in the cases of bronchitis
the number of the characteristic bacilli is found at first
to be considerable, but when the disease abates and the
patient improves their number becomes greatly diminished.

Fig. 98.— Film Specimen of Blood of a Case of Influenza, showing Blood
Discs and Minute Bacilli.

x IOOO.

It deserves notice, as a matter of no small practical
importance, that in cases of acute influenza with bronchial
expectoration the fluids of the mouth contained abundance
of influenza bacilli. Thus cover-glass specimens of such
bronchial expectoration that had not been washed at all (or
at best not well washed) showed scaly epithelial cells,

xn] PATHOGENIC BACILLI: GROUP C 261

derived from the oral cavity or fauces, literally crowded
with masses of what, when duly stained, looked exactly
like the typical influenza bacilli.

1. Culture in broth. — Broth-tubes containing a pure
culture of the influenza bacillus remain quite limpid ; at
the bottom of the fluid there are noticed already after
twenty-four hours, but better after forty-eight hours, a few
whitish-grey, irregular granules or flocculi, which during the
next two or three days increase in size and number and
form at the bottom of the tube greyish-white nebulous fluffy
masses ; when shaken they break up into whitish-grey
granules and flocculi, but soon again settle at the bottom
of the fluid, leaving the rest of the broth perfectly limpid.
In four or five days (at 370 C.) the growth has reached its
maximum. Sub-cultures show the same characters, but we
generally noticed that as the number of removes increases
the broth has a tendency to show slight turbidity after one,
two, or three days’ incubation, minute granules sticking to
the wall of the tube and showing themselves also in various
layers of the fluid.

Furthermore, in successive sub cultures it is noticed that
the amount of growth (floccular masses) at the bottom of
the fluid is not invariably the same, being decidedly less in
the later than in the earlier sub-cultures.

A point of great interest is the comparatively rapid death
of the bacillar elements in the broth cultures. Unless the
transmission is carried on within two, three, or four, up to
seven days, it will be found that the sub-cultures are sterile;
broth cultures from eight to ten days old are very uncertain,
broth cultures a fortnight old yield no living organisms to
subsequent sub-cultures. But if the sub-cultures are set up
every two or three days we did not find a limit to the
number of generations to which some of our cultures could

262 MICRO-ORGANISMS AND DISEASE [chap.

be carried on ; although in other cases after about a dozen
generations in broth no living sub-cultures could be made
in broth.

2. Culture on Agar. — The cultivations and sub-cultiva-
tions were (a) on beef broth (not beef infusion), Agar (i p.c.),
peptone (r p.c.), and salt (i p.c.) ; and (&) on glycerine
Agar, that is the ordinary Agar plus glycerine (6 p.c.).
The growth on our ordinary Agar is rather more easily
observed than on glycerine Agar, being a little more
copious (the colonies being somewhat larger) and a little
less translucent, and therefore more readily noticeable.

The colonies on the surface of both these media can be
discerned under a glass after twenty-four hours’ incubation
at 370 C. They then have the appearance of extremely
minute translucent flat droplets, and these during the next
day or two increase somewhat in size, but even at their
largest are but small — not exceeding three millimetres in
breadth, and only just visible to the eye as translucent
circular flat droplets — -on further incubation becoming
flatter (Fig. 99). Under a lens their margin is seen to
be slightly crenated and their centre darker than the rest.
The crenated margins show no tendency to coalesce, even
when the colonies are thickly planted.

In Agar stab-culture the stab is indicated after two or
more days as ti grey line, this being made up of granules
densely and closely placed : viewed under a glass, minute
club-shaped and pear-shaped projections are seen to
extend from the dark line of inoculation. In stab-
cultures, as in surface growths, the several colonies are a
little more copious and less translucent when our ordinary
Agar is used for the cultivations than when glycerine has
been added.

The condensation water in the Agar tubes (of ordinary

xiij PATHOGENIC BACILLI : GROUP C 263

as well as of glycerine Agar tubes set with slanting surface)
show, in the course of one or two days, a copious floccular
or granular whitish precipitate, the condensation water
itself remaining limpid. The amount of this precipitate
increases till about the fifth or seventh day, when it has
reached its maximum.

Agar tubes inoculated with the influenza bacillus support
life in the organism longer than broth tubes, particularly if
the Agar tubes be inoculated by stab-culture. We have
successfully carried on sub-cultures from Agar cultures
through many generations, in fact we have some cases
that have reached already the twentieth generation, and
we see no reason why there should be any limit
placed at all, provided each successive sub-culture be
established within a week — after that time the result
becomes uncertain.1 But if the culture tube after five or
six days’ incubation at 370 C. be then kept at the ordinary
temperature (capped and protected from drying) the life of
the culture can be preserved for a much longer time ; we
have as a matter of fact found it living after two weeks ;
this would certainly not have been the case if any culture
of the series had been kept at 37° C. for a fortnight.

3. Culture 071 potato. — No visible growth is to be obtained.

The vitality of the cultures is considerably prolonged if
nutrient gelatine after inoculation is incubated at 370 C. ;
herein good growth occurs, and the growth remains alive for
at least three to four weeks.

1 We add here that under the above conditions we have carried on
the sub-cultures on Agar from the sputum through more than thirty
generations.

264

MICRO-ORGANISMS AND DISEASE [chai\

Microscopic Examination of the Cultures.

With the cultures above described cover-glass specimens
may be made in the usual way, i.e. a thin film of the fluffy
or floccular precipitate from the broth cultures, or of the
precipitate from the Agar condensation fluid, is prepared byi
drying and staining ; and this is found to exhibit the bacilli
in long twisted chains and threads, aggregated so as to
form dense networks and convolutions or frequently forming
bundles (Fig. 100). Many of the threads are found to
measure several millimetres in length, while some are
broken up into shorter bits. The threads are formed by
the individual bacilli placed end to end, the sheaths of the
bacilli forming a continuous sheath for the thread ; in the
stained specimens each element is marked either as a
minute rod or more commonly as a dumb-bell of granules,
this appearance being due to the polar granules of the
individual bacilli being very strongly marked : or, by stain-
ing this dried film in carbolmethyl-blue for about half to one
hour, and then washing in water, drying, and mounting in
balsam, the character of the bacilli in the threads may be
very well seen.

In recent cultures the threads are either wholly or
partially made up of bacilli which stain at the two
poles ; such elements as do not show this character ap-
pearing as uniform rods about 0-4 /x in thickness, o'S to
1 -2 /x in length. Cultures several days old show many of
the threads already degenerating ; that is to say, shorter or
longer portions being empty of protoplasm showing only the
faintly stained sheath with here and there indistinct granules
in it. But in all specimens made of however recent a
culture there are threads, in which here and there a bacillus

xi l] PATHOGENIC BACILLI : GROUP C 265

is swollen up into a spherical or oval ball, many times
thicker than the typical element ; the number of these
enlarged elements is greater in later than in recent cultures,
and the largest of them often show a vacuole in their centre
or at one side. From these facts it is probable that these
enlarged elements are involution forms.

Fig. 99 — Translucent Colonies of the Influenza Bacilli on the
Surface of Agar.

Magnified twice.

Preparations made of the colonies grown on the surface
of the Agar or glycerine Agar show the bacilli exactly of the
same aspect and character as those grown in fluid media,
namely as threads or else as large clumps ; in these the bi-
polarly-stained bacilli are very typical, and such clumps

266 MICRO-ORGANISMS AND DISEASE [chap.

resemble in every respect the clumps seen in the bronchial
sputum.

Ry staining a cover-glass film of the young colonies first
with rubin and afterwards with methyl-blue the sheath of
the threads is well differentiated as of pink colour from the
polar granules, or the rod-shaped protoplasm in the sheath.

Fig. ioo. — Film Specimen from a Broth Culture of Influenza Bacilli.

X IODO.

The same result is obtained from the growth in broth, but
the most satisfactory specimens for microscopic observation
were obtained from Agar cultures. We possess specimens
from Agar cultures stained double as mentioned above, and
the rod-shaped character of the elements of considerable
portions of a thread is very strikingly marked by being
stained blue in the pink general sheath.

On looking at the threads or clumps of any growth with

Xii] PATHOGENIC BACILLI: GROUP C 267

a moderately high power they are seen to resemble strepto-
cocci, but with an oil-immersion lens there is no difficulty
in recognising the elements constituting the threads or
clumps as really being bacilli, the protoplasm being either
rod-shaped and stained uniformly, or else being segregated
as a granule at each end and then receiving the stain at the
two poles.

The description which we have here given of the character
of the growth in the different media and of their microscopic
aspect coincides in every essential with that given by Pfeiffer
and Kitasato in their paper already quoted, except that in
that paper sufficient prominence is not given to the thread-
like nature of the growth ; this, however, may be entirely
owing to their communication having the character of a
preliminary short account of their results.

The result of the examination of the blood of influenza
cases was this : —

Of forty-three cases of blood examination, no bacterial
forms could be discovered in thirty-seven. In the other
six cases cover-glass specimens revealed the presence of
one and the same kind of minute bacillus; in one the
bacilli were numerous, in two they were fairly numerous,
and in the other three they were very sparse.

In the thirty-seven cases the temperature in the majority
was higher than normal, in a minority it was normal or sub-
normal. In some cases blood was taken at two different
periods : during and after the fever ; or both times during
the period of raised temperature : or when the tempera-
ture had again fallen. But in these respects no definite
relation as to the presence or absence of the bacilli could
be made out.

It is then clear from these observations that neither
during the febrile stage nor after the temperature has again

268 MICRO-ORGANISMS AND DISEASE [chap.

fallen do the bacilli occur in the blood with anything like
constancy, considering that in thirty-seven out of forty-three
cases no bacilli could be found, in each case at least four
cover-glass specimens, in some six and even eight, being
made, stained by the appropriate methods, that is to say b\
methods by which they are readily shown in the positive
cases. But also the extremely varying number in which
the bacilli occurred in the six positive cases indicates that
their presence in the blood cannot be of the same essential
value for the disease as is the case in the typical acute
infectious diseases — “ blood-diseases,’’ of which the various
known septicaemias, anthrax, and fowl cholera are types.
This view is strongly borne out by the consideration that in
all six cases in which the bacilli were found in the blood in
the cover-glass specimens they could not be demonstrated
in culture, the media used for these cultures being (as
will presently be shown) perfectly suitable for the living
bacilli of the bronchial sputum. This would appear to
indicate that the bacilli found in the six affirmative cases
were not living, and that any bacilli of influenza that may
gain access to the circulation lose here their vitality and
are present in the blood only as dead bacilli. Canon
( Deutsche Med. IVociunschr ., No. 2, 1S92) states that

he found in all cover glass films of blood of influenza a
particular kind of bacillus present in numbers varying from
five to twenty, the bacilli and the nuclei of the white cells
being stained blue, the blood discs and the body 01 the
leucocytes pink. Now, our observations do not bear out
this statement of Canon, since by the same methods as ne
used we stained the specimens for even a longer time
than he did we failed to find bacilli in thirty-seven out
of forty three cases, and we therefore, in opposition to
him, do not consider the presence of these bacilli as 01

ui] PATHOGENIC BACILLI : GROUP C 269

■>athognomonic value, or their absence as of diagnostic
mportance.

The same conclusion is arrived at by Pfuhl ( Centralbl.
f Bakt. und Parasit. xi, No. 13) and by Pfeiffer and Beck
'( Deutsche Med. Woch., May 26th, 1892).

A large number of experiments were made on rabbits
and monkeys by using either bronchial sputum of influenza
cases containing an abundance of the Pfeiffer influenza bacilli
— the majority of the experiments — or of cultures of these
bacilli, ancjjby introducing such materials under the skin or
into the trachea, or by direct injection into the vein
(rabbits), but it has not been practicable to arrive at any
definite production of influenza disease in monkeys or in
rabbits. Only in one monkey out of eighteen was a
definite disease of the lungs produced by such injection,
and there (but in company with other bacilli) clumps of
influenza bacilli were found ; while among thirty rabbits
injected with like materials there was no single instance of
a disease recognisable as influenza in nature having resulted
from the experiment.

Now the question has repeatedly been raised, and indeed
has been repeatedly answered in the affirmative, viz., whether
the disease of influenza, such as prevailed in this country,

: on the Continent of Europe, and in most other parts of the
world in 1889-1890 and in 1891-1892, is a disease to which
also the domestic and other animals are subject. It has
been particularly asserted that in this country influenza
was common amongst horses antecedently to and during
the prevalence of influenza in man.

Though we have not made intentional experiments upon
horses or other animals beyond those mentioned in these
pages, we have not the less been on the watch during the
time that we carried on our inquiry (February to April, 1892)

2/0

MICRO-ORGANISMS AND DISEASE [cii. xn

for indications of any influenza-like disease affecting the
lower animals. We could not get evidence of horses being
affected with any complaint identical with influenza in
man, nor, as regards other animals which live amongst
human habitations, are we aware of any evidence proving
that amongst them influenza or any similar disease was rife
during the periods of the influenza epidemic. Under these
circumstances we have made inquiries at the Zoological
Gardens in London, and Mr. Beddard has kindly given us
the facts as to the condition of illness and deaths amongst
the mammals kept there. From his record we learn that
the incidence of disease and death at the Zoological
Gardens was not unusually heavy during the years of the
influenza epidemic in the metropolis. As regards the
monkeys in particular, kept at the Zoological Gardens, we
also understand from Mr. Beddard that no increased sick-
ness was observed amongst them during these periods.
The fact conforms with the results from our experimental
observations on monkeys above recorded. It can hardly
be supposed that if monkeys were, as a class, susceptible
to the infection of human influenza the creatures living
in the monkey-house in Regent’s Park, frequented by
many thousands of people a month while influenza was
abundant in the London population, would have kept free
from the complaint. And from the general experience of
the Gardens of the Zoological Society it would appear
that few mammalia share with the human subject a
susceptibility to epidemic influenza. At all events, few of
them are liable to receive the infection by the method
which habitually obtains in man, through the respiratory
passages.

CHAPTER XIII

THE MICROBES OF MALIGNANT ANTHRAX, OF DIPHTHERIA,
AND OF GLANDERS

Bacillus anthracis. — Pollender,1 Brauell,2 Davaine,3 and
then Bollinger 4 recognised in the blood of animals dead of
malignant anthrax the presence of stiff short and long rods,
which Davaine called bacteridie du charbon. They were
identified by Cohn 5 as bacilli in morphological respects
similar to bacillus subtilis, except that the bacilli anthracis
are non-motile.

Koch6 showed the ubiquitous distribution of these bacilli
in the blood of the organs, and especially of the spleen. He
succeeded in cultivating the bacilli artificially, by placing a
bit of such a spleen in a drop of aqueous humour, and
watching the growth of the bacilli under the microscope.
In this manner he ascertained that the rods multiply by
division, and that they grow into long, homogeneous-looking,
straight or twisted filaments in which after some time, and

1 Viertelj. f. Gericht. Med., 1855.

2 Virchow's Archiv , vol. xiv. 1858.

3 Comples Rendus , lvii. 1863.

4 Med. Centralblatt, June, 1872.

5 Beitr. 2. Biol. d. PJlamen, vol. ii,

« Ibid., vol. ii.

272 MICRO-ORGANISMS AND DISEASE [chap.

with free access of air, bright oval spores make their appear-
ance, while the filaments become homogeneous and swollen.
These spores become free, and when artificially cultivated or
injected into a rodent animal germinate into the character-
istic bacilli ; these elongate and divide, and in artificial

Fig. ioi.— Film Specimen of Blood of Guinea-pig dead of Malignant
Anthrax, showing Blood Discs and Bacillus Anthracis in Chains.

X about 700.

cultures again grow into the long leptothrix filaments, which
again form spores. Koch 1 saw in preparations of aqueous
humour kept at 35° C. in the incubator the spores germin-
ating after three to four hours. The single bacilli as they
present themselves in the blood measure between o-oo5 and

1 Beilr z. Biol. d. Pflanzen , vol. ii. part ii. p. 2S8.

273

xi 1 1] MICROBES OF MALIGNANT ANTHRAX

0 02 mm. in length, and o'ooi to o-ooi2 in thickness ; they
are truncated.1 The spores produced by growing the bacilli
with free access of air are about o*ooi mm. thick, and about
o‘oo2 to 0*003 nim. long. They are not stained by the
ordinary dyes and differ herein from the bacilli.

Fig. io2. — From a Section through the Spleen of a Guinea-pig dead of
Malignant Anthrax, showing numerous Bacilli Anthracis in the
Spleen Pulp.

X about 700.

In the human subject malignant anthrax occurs as “ woolsorter’s
disease ” ; for the aetiology and pathology of this malady see Spears
(A 'sports of the Medical Officer of the Local Government Board , 1881 and
1882) and Greenfield (ibid. 1881). It occurs also in sorters of hides
and rags.

All rodents and herbivorous animals are susceptible to anthrax ;

1 It is generally assumed that the bacilli are the same in all animals
affected with splenic fever, but this is most undoubtedly not the case,
as has been already pointed out by Huber (Deutsche Med. Woch. 1881) ;
the bacilli of the guinea-pig are thicker than those of the mouse
or sheep, and these again are thicker than those of the rabbit.

274 MICRO-ORGANISMS AND DISEASE [chap.

adult rats are, however, infected with difficulty, pigs are not very
susceptible, and dogs and cats are very insusceptible. Infection of
animals can be produced by inoculation into the skin and subcutaneous
tissue, intraperitoneal or intravascular injections, and by inhalation and
ingestion of spores. In woolsorter’s disease the usual mode of infection
is by inhalation of spores adhering to the wool of the fleeces of animals
(sheep, goats) dead of anthrax. As in rodents infected with anthrax,

Fig. 103. — From a Section through the Liver of a Guinea-pig dead of
Malignant Anthrax. The Capillarv Blood-vessels contain Chains
of Bacilli Anthracis.

X about 700.

so also in man, the blood-vessels of all organs contain the bacilli, and
extravasations of the infected blood are frequent in many parts of the
body. The presence of bacilli in the extravasations into the mucous
membrane of the trachea and bronchi does not necessarily mean that
these parts represent the points of entrance of the bacilli into the
system, as Greenfield seems to regard as self-evident ( Reports of 1 he
Medical Officer of the Local Government Board, 1SS1). As a matter of
fact I find in every lung of mouse, rabbit, and guinea-pig, dead after

x 1 1 1] MICROBES OF MALIGNANT ANTHRAX 275

subcutaneous inoculation with anthrax, bacilli anthracis in the alveolar
cavities and in the smaller and larger bronchi. Ingestion of bacillar
material is sometimes followed by anthrax, but in these cases abrasions
in the mucous membrane of the mouth, pharynx, or gut may have
been the real place of entrance. Mice fed with fresh anthrax material
do not become infected (Klein, ibid. 1881). But the reported cases
of intestinal mycosis (see, for the literature of this subject, Koch,

!• ig. 104. Section through the Pustule of Malignant Carbuncle in
Man. The Blood-vessels of the Skin are filled with Bacilli
Anthracis.

Low magnification.

“Auiologie d. Milzbrandes,” Mittheil. a. d. k. Geswidheilsamte,
1881) indicate that infection with spores by the alimentary canal is
not excluded. Compare also Falk, Virchow's Archiv, vol. xciii.
From the observations by Koch and Gaffky it has become clear that
infection of sheep by the alimentary canal can be produced with spores.

Normal frogs are insusceptible to anthrax.

Frogs and adult rats are however susceptible if they are
subjected to chloroform narcosis and the injection is made

T 2

276

MICRO-ORGANISMS AND DISEASE [CHAP.

during or shortly before or after narcosis (Klein and
Coxwell). Petruschki has shown that by keeping frogs at the
temperature of the warm-blooded animal it becomes suscepti-
ble to anthrax. Normal fowls are insusceptible, but Pasteur
showed that by lowering their temperature they become
susceptible.

Fig. 105.— Impression Specimen op the Edge of a young Colony op
Bacillus Anthracis on Gelatine. Threads of Bacilli made up of
Cylindrical Bacilli.

Magnified about 700.

Besides general infection of human beings by spores of
anthrax (wcolsorters, hidesorters, and ragsorters) they are
able to contract severe local carbuncle by inoculation
(through a cutaneous abrasion or wound) with anthrax
blood of an animal (sheep, cattle, or horses).

Rodents inoculated with the bacillus of the blood or

XIII] MICROBES OF MALIGNANT ANTHRAX 277

spleen of an animal dead of anthrax, or with the bacillus or
spores of an artificial culture, die generally within forty eight
hours ; in some instances in twenty-four to thirty hours,
in other instances after forty-eight to sixty hours. The
blood in all instances contains the bacilli, the spleen is
large and full of bacilli, and so are the blood-vessels of most
other organs, the exudations, and the urine. In the placenta
of a pregnant guinea-pig dead in consequence of inoculated
anthrax, I have seen that the bacilli kept strictly as a rule

Fig. io5. — Fro.i a Preparation- of Heart’s Blood of a Guinea-pig dead

of Anthrax.

1. Red blood discs.

2. White corpuscle.

3. Bacilli anthracis, showing well their sheath.

Magnifying power 700. (Stained with Spiller's purplo.)

within the maternal blood-vessels, and are wholly absent in
the blood of the vessels of the foetus. Subcutaneous in-
oculation or injection into the cutis of minute quantities
of bacillus containing material (blood or virulent culture)
invariably produces death. Subcutaneous injection of
bacillus-containing material in the guinea-pig almost always
produces a characteristic oedema, spreading sometimes over
a large area. The oedematous fluid is clear and contains
only a few bacilli.

Any neutral or faintly alkaline material containing pro-

278 MICRO-ORGANISMS AND DISEASE [chap.

teids is a suitable nutrient medium for the bacilli ; they
grow abundantly at all temperatures between 150 and 43°C,
best between 250 and 40° C. They elongate and divide
rapidly, and the bacilli grow out into long curved and
peculiarly twisted filaments which often form bundles, the

Fig. 107.— Impression Specimens of young Colonies of Bacillus Anthracis

on Gelatine.

Low magnification.

individual filaments being twisted round one another like
the strands of a cable.

The bacillus anthracis offers some very characteristic
features in cultivations. In gelatine plate cultivations
made of the blood (previously well diluted with neutral
salt solution or broth, on account of the large number of
bacilli present in the blood) already after twenty-four to

XIII] MICROBES OF MALIGNANT ANTHRAX 279

thirty-six hours the first signs of colonies can be made out
in the form of translucent, grey, angular, dots ; after forty-
eight hours to three days they are conspicuous by their size,
and by their margin being distinctly made up, to the naked
eye, of filaments, either straight or bending like loops.
Under the microscope the filamentous nature of the

Fig. 108 — Stab Culture in Gelatine of Bacillus Anthracis. Lique-
faction on the Surface has already commenced.

colonies is distinctly seen ; the filaments looked at
under a magnifying glass are more or less in bundles
twisted like cables, and extending sometimes like radii
from a centre ; at the margin this is particularly con-
spicuous. At the same time the colony is seen to be sunk
in the middle, being situated in a slight depression of the

200

MICRO-ORGANISMS AND DISEASE [chap.

gelatine due to commencing liquefaction. Looked at
obliquely, the gelatine looks pitted by the colonies. As
growth proceeds the colony enlarges ; the marginal loops
and bundles of twisted filaments project more or less
irregularly ; some project for longer, others for shorter dis-
tances, sometimes not much beyond the margin of the
colony, and the gelatine surrounding the colony becomes
more and more liquefied, but remains clear in the liquefied
part. In stab cultures made from a culture or from the
blood the stab is noticeable after a day or two as a whitish
line made up of closely placed dots ; in another day or two,
from each dot a lot of fine whitish filaments are seen ex-
tending, often like rays from a centre. When the dots are
closely placed in linear series the white filaments projecting
mostly in horizontal direction from them give to the stab
a characteristic appearance, like the vane of a grey feather,
the stab being the middle rib ; liquefaction has by this time
set in on the surface, i.e. on the upper end of the stab, and
there is here a more compact plate-like mass of filaments ;
the liquefaction gradually proceeds into the depth while the
surface patch of the growth increases in bulk ; the liquefied
gelatine is clear, and the original surface growth occupies
always the deepest part of the liquefied gelatine. When
the surface patch while spreading remains adhering to the
glass wall of the test-tube, spore formation is observed in
the threads of the bacilli, but when the growth is in the
depth of the liquefied gelatine no spore formation ever
takes place. After ten to fourteen days at 19-20° C. the
upper half of the gelatine in the tube is quite liquefied, the
liquefied gelatine is clear, and the whole growth is at the
bottom of the liquefied part in the form of whitish-grey
fluffy masses.; when shaken, the mass breaks up into whitish
nebulous flocculi.

xiii] MICROBES OF MALIGNANT ANTHRAX 281

In streak culture on gelatine the streak of inoculation is
marked after twenty-four to forty-eight hours as a whitish-
grey line ; then a number of whitish fine threads shoot out
horizontally from this line, liquefaction at the same time
commencing and proceeding slowly and gradually; the
line thickens and broadens, and after a week is made up of
masses of threads twisted and convoluted, and forming a
thick, white, filmy patch, which as liquefaction proceeds
sinks to the bottom of the liquefied gelatine, forming here
a whitish grey fluffy mass.

In neutral or faintly alkaline broth kept at 36-38° C.
there is,*if the broth be thin, uniform slight turbidity after
thirty-six to forty-eight hours : flakes small and large then
appear at the bottom of the fluid, while this latter remains
fairly clear. As growth proceeds, about the end of the
week, there are contained at the bottom of the fluid
characteristic greyish, fluffy, loose, nebulous masses, which
are masses of anthrax threads matted together ; these
masses increase in bulk and extend as it were from the
bottom of the fluid towards the upper parts. If during
the first few days some of the flakes remain adhering to the
glass at the surface of the fluid, these flakes enlarge and
form on the glass, on a level with the surface of the fluid,
a sort of whitish ring, somewhat like a pellicle ; in this
copious spore formation takes place ; but in the tubes, in
which all the growth is limited to the deeper parts of the
fluid, no spore formation occurs at any time, since for the
formation of spores a free and copious supply of oxygen is
required.

On Agar mixture at 36-38° C. a greyish, thick film is
noticed after two days along and beyond the line of in-
oculation. This rapidly increases in breadth till the whole
surface of the Agar is covered with a sticky, pasty, greyish

282

MICRO-ORGANISMS AND DISEASE [chap.

layei > this after some days shows some patches thicker
than others, is light brown, and in some patches even dark
brown.

On potato at 35-37° C. a thick cohesive layer like paste
is formed ; this is of a brownish colour ; the growth is ex-

•:c

ft \

•

b /

f 'v

® •»

•of

*•

if A

nt i jh }\

/ If ,#/ / (
* // /? •' / 1

#7 ^ ■ ''

- $ / t /

Fig. 109.— From an Artificial Culture of Bacillus Anthracis, carried
on at Ordinary Temperature and on Solid (Gelatine) Material.
Torula Form.

Magnifying power 450. (Stained with Spiller’s purple.)

tensive after a few days. Both on nutrient Agar and on
potato the film is a mass of threads matted together, and
after two to three days copious spore formation is noticed
in many threads ; at the end of ten days to a fortnight the
whole of the film is a mass of spores ; little of the original
bacilli is recognisable

XIII] MICROBES OF MALIGNANT ANTHRAX 283

Bacilli anthracis when growing at ordinary temperatures
on a solid medium (e.g. a mixture of gelatine and broth, or
Agar-Agar and peptone) show a very peculiar modification,
inasmuch as some of the elements assume a spherical, oval,
or spindle shape, a torula-form, and as such they multiply
by division and form clusters or arrange themselves in
chains. By-and-bye each of these spherical elements

Fig. 110.— Spores forming in Threads of Anthrax.
x 700.

elongates into a rod, and when all elements have under-
gone this change we have the typical smooth filament of
the leptothrix form. Some of the elements in such a
filament remain for a long time of a spherical shape, and
are much larger, looking like the sporangium of a nostoc-
alga. The most interesting forms are those where an
ordinary smooth filament of anthrax-bacillus at its growing
ends shows itself to be composed of a chain of torula

284

MICRO-ORGANISMS AND DISEASE [chap.

elements. Such torula forms occur also in ordinary
cultivations in fluid media at temperatures of 20° to 30° C.,
but not by any means so often as at ordinary tempera-
tures and in a solid medium. Compare also the chapter
on General Characters of Bacilli.

After a few days’ incubation, no matter what the tempera-
tuie is, many of the bacilli and their leptothrix-filaments
show signs of degeneration, consisting in the granular disin-
tegration and absorption of the protoplasmic contents of the
bacilli and their filaments, at first only here and there, but

Fig hi.— From an Artificial Culture of Bacillus Anthracis in Broth
AFTER MANY Days' INCUBATION.

The threads are swollen and curled up, and in many places the protoplasm has
disappeared, leaving the sheath and septa distinct.

Magnifying power 700. (Stained with Spider’s purple.)

by-and-bye over longer pieces. Such bacilli and leptothrix-
filaments appear in such places as if empty. This is also
noticed in the bacilli of the blood and spleen of an animal
inoculated with anthrax, even at the point of death or soon
after death, if the number of bacilli is great.

Another form of degeneration consists in the filaments of
bacilli becoming much curled and swollen, and finally
disintegrated into an amorphous debris.

As long as the bacilli grow in the depth of a fluid they
never form spores, but when grown on the surface with free

XIII] MICROBES OF MALIGNANT ANTHRAX 285

access of air, or on solid media ( e.g . serum gelatine, gelatine
broth, Agar-Agar, potato, &c.), the bacilli, having developed
into filaments, proceed to form spores. But they may form
spores even in fluid media if by some accident, either by
sticking to the glass vessel containing the fluid or by means
of a cotton-wool fibre, some of the bacilli remain on the
surface of the fluid. This formation of spores is not due to
exhaustion of the nourishing medium, as has been already
discussed on a former page, but represents the last stage in
the life-history of the bacilli, provided they have an ample
supply of oxygen. If this latter condition is not fulfilled,
as wheirHhey are grown at the bottom of a fluid, the bacilli
gradually degenerate as mentioned above.

Spore-formation occurs, cccteris paribus , at all tempera-
tures between 180 and 450 C. Koch found 15° C. the
lower limit. Under the most favourable conditions, each
cubical or rod-shaped mass of protoplasm includes one
spore, in which case the bacillar filament contains an almost
unbroken row of spores ; but in other cases only an elemen-
tary mass here and there contains a spore, the rest breaking
down and becoming absorbed. In the first case, also, the
protoplasm of the elements almost entirely disappears, the
sheath swelling up and becoming hyaline, and only the
bright spores remaining. Their linear arrangement, however,
still indicates that they were formerly contained in one
filament.

If bacilli grow in the depth of a fluid medium, they do
not form spores, as has been stated above ; and, as we have
also seen, as new bacilli appear, or the old filaments increase
in length, degeneration sets in. This degeneration gradually
affects greater and greater numbers, and when the fluid is
exhausted for the formation of new bacilli it necessarily
follows that the whole growth gradually becomes involved in

286

MICRO-ORGANISMS AND DISEASE [chap.

the process of degeneration, the whole mass becoming
smaller, and finally only debris is left. Such cultures,
namely those in which the degeneration involves the whole
mass of the bacilli, are quite innocuous when inoculated into
animals or into fresh nourishing media. But as long as

Fig. ii2.— From an Artificial Culture in Neutral Pork-Broth of
Bacillus Anthracis, with copious Formation of Spores.

Magnifying power 700. (Stained with Spiller s purple )

there are any good protoplasmic elements of the bacilli left
the culture is virulent to rodents, with the exception of mice,
as will be referred to presently; and it is capable, when
transferred to new suitable nourishing media, of starting new
cultures that prove virulent to all rodents and sheep.

XIII] MICROBES OF MALIGNANT ANTHRAX 287

The same holds good of the bacilli in the blood and organs
of an animal dead of anthrax, provided the animal be not
opened, and its organs, exudations, or urine be not exposed
to the free air ; for the bacilli not exposed to the air gradually
degenerate, and the blood and organs of such an animal,
although at first deadly poison to other susceptible animals,
become at length quite innocuous. Systematic observation
has shown me that small animals, such as mice and guinea-
pigs, when kept unopened or buried in earth, become quite
innocuous after five to eight days, the anthrax-bacilli having
by this time, by degeneration, altogether disappeared from the
blood, spleen, and other organs. Pasteur’s statement that
in animals dead of anthrax and buried the bacilli form
spores, and that these spores are taken up by earthworms and
carried to the surface of the soil, where they are deposited
with their castings and thus are capable of infecting animals
grazing or sojourning on this soil, is not borne out by the
above observations. And, further,, Koch has proved 1 by
direct experiment that spores of anthrax-bacilli when mixed
with earth in which worms are present are not taken up by
these creatures.

Drying bacilli of the blood or of a culture in a thin layer
invariably kills them, but the spores remain unaffected.

The bacilli of the blood of a rodent dead of anthrax are
always thinner than the bacilli cultivated in a neutral fluid
medium.

Cultivation of the blood-bacilli at temperatures varying
between 20° and 40° C. in any suitable nourishing material,
solid or fluid, however many transferences (new cultivations
or so called new generations) be made, always yields a crop
of virulent bacilli. It is quite incorrect to say, as Buchner2

] MiUkeil. a. d. k. Gesundkeitsamte, 1881.

Ucber d. Erzeug. des Milzbrandes, Munich, 1S80.

288 MICRO-ORGANISMS AND DISEASE [chap.

and Greenfield 1 maintain, that continued transference
weakens and ultimately destroys the action of the bacilli ;
as long as the cultures remain pure, not contaminated and
finally suppressed by accidental innocuous bacilli, the
anthrax-bacilli retain their virulence.

Cultures of the blood-bacilli at 20° to 38° C. in neutral
broth, during the first or second week, are virulent to mice,
guinea-pigs, and rabbits ; but after that they lose their
power on mice, provided the growth takes place only in the
depth and no spores are formed ; but they retain it as
regards guinea-pigs and rabbits, as long as they contain good
bacilli at all.'2 But fresh cultures made of such bacilli
invariably produce a growth which is fatal to all rodents
during the first or second week.

The first observations that bacillus anthracis can become
attenuated in its action without losing its morphological and
biological characters were recorded by Toussaint, who
found that heating anthrax blood up to 550 C. for a few
minutes incapacitates such blood from producing anthrax on
inoculation. Chauveau then found that the same attenua- i
tion and destruction of virulence occur when the virulent
bacillus anthracis, e.g. the blood, is subjected to the action of
5 per cent, carbolic acid for a few minutes. Pasteur was
the first who showed that when bacillus anthracis is culti-
vated in broth at high temperature (42‘5° C.) it gradually
loses its full virulence, and when such cultures are inoculated
into sheep and cattle a mild and transitory form of anthrax
is produced ; animals so treated withstand successfully the
further inoculation of virulent materials, and are therefore
protected by the inoculation with the attenuated cultures.

1 Proceedings of the Royal Society, June 17, 1SS0.

2 Klein, Reports of the Medical Officer of the Local Government Board, ,
1881.

XIII] MICROBES OF MALIGNANT ANTHRAX 289

Pasteur has shown by a large number of experiments
carried out in France and elsewhere that, by inoculation of
such attenuated cultures, protective inoculation can be
effected on sheep and cattle. He used two kinds of culture,

Fig. 113. — Network of Capillaries filled with Bacillus Anthracis ; from
the Omentum of a Rabbit dead of Anthrax.

1. Extravasation of the bacilli.

2. Capillaries filled with the bacilli.

Magnifying power 350.

for protective inoculation : (a) premibre vaccine : this is a
culture of anthrax bacillus in chicken broth kept at 42-5° C.
for fourteen days ; when inoculated into sheep or cattle it
produces only a slight local tumour ; after about twelve days

290 MICRO-ORGANISMS AND DISEASE [chap.

the animals are inoculated with (h) deuxieme vaccine : this
is chicken broth culture kept at 42-3° C. for a week only. This
culture produces also a local effect with slight constitutional
disturbance, more pronounced than after the inoculation of
the premiere vaccine ; but the disturbance is only.transitory
and the animals recover. Up to nine months such animals
are refractory against inoculation with virulent anthrax
blood.

If the deuxieme vaccine is used for the first inoculation,
the effect is more severe and may lead to fatal general
anthrax ; this deuxieme vaccine having been grown for one
week only at 42 '5° C. is therefore stronger, and is of a higher
degree of virulence than the premiere vaccine, which had
been grown at the high temperature for a fortnight.

In all experiments with the anthrax bacilli it is necessary
to bear in mind that by passing the bacilli through different
species of animals they become endowed with different
qualities, and that bacilli which are fatal to some are not
fatal to all animals. While, for instance, the blood-bacillus
of sheep or cattle dead of anthrax invariably produces death
when inoculated into sheep or cattle, after passing through
white mice1 it loses this virulence for sheep and cattle.
The blood of white mice dead of anthrax does not kill
sheep ; it produces only a transitory illness, and the animals
are, for a time at least, protected against virulent anthrax.
The blood of guinea-pigs dead of anthrax produces illness,
sometimes death, in cattle, but as a rule does not kill
(Sanderson and Duguid), and the blood of the biscachia of
South America does not kill cattle, while it gives them a
transitory illness, and after this immunity for a time.2 Again

1 Klein, Reports of the Medical Officer of the Local Government Board,
1882.

2 Roy, Nature , December, 1883.

XIII] MICROBES OF MALIGNANT ANTHRAX 291

Pasteur’s “ vaccine,” which does not kill sheep or cattle,
is fatal to rodents.1 From all this it follows that as regards

Fig. 114. From a Section through the Kidney of a Rabbit dead of

Anthrax.

The capillaries of the cortex are naturally injected with the Bacillus anthracis.

1. A glomerulus.

2. Capillaries surrounding the convoluted uriniferous tubules not shown here.

Magnifying power 450. (Spider’s purple.)

virulence the bacilli anthracis differ in the different species
of animals, and in them acquire different qualities.

00 ^eln’ Reports of the Medical Officer of the Local Government Board,
1SS2. Similar results have been obtained by Gaffky [Mitt hell. a. d k
Gesundheitsamte, 1882).

U 2

292 MICRO-ORGANISMS AND DISEASE (chap.

Bacillus anthracis is capable, as we have seen, of growing
well outside the body, and, when well supplied with oxygen
from the air, of forming spores which represent the per-
manent seeds. Thus if animals, such as sheep and cattle,
die of anthrax in a field the effusions of such animals
(e.g. urine, blood, sanguineous effluvia from the mouth and
nostrils) always contain numbers of the bacilli, and these will
be able to grow indefinitely on the surface of the soil, there
being always present a large amount of suitable nourishing
material, as vegetable and animal decaying matter, and since
free access of air is always ensured they will eventually form
spores. Such soils, owing to the presence of these spores,
will remain a permanent source of infection to sheep and
cattle sojourning on them (Koch).

Acute infection of rag-sorters with anthrax has been ob-
served several times (Paltauf, Wiener Klin. Wochens., 1888,
Nos. 18-26), but not all acute infectious diseases contracted
by the sorters of old rags are anthrax, as has been shown by
Bordoni Uffreduzzi ( Zeitschr . f. Hygiene , ///, 2, p. 333).
From a fatal case, in which the post-moj-tem examination
showed enlarged spleen, congestion, and haemorrhage of the
lung, lymph glands, and serous membranes, this observer
isolated a non-sporing motile bacillus which in many points
resembles the proteus of Hauser. Bordoni Uffreduzzi calls it
p7-oteus liominis capsulatus ; it does not liquefy gelatine and
acts virulently on dogs and mice, rabbits and guinea-pigs
being less susceptible.

Bacillus of ulcerative stomatitis in the calf. — In the Lancet
of May, 1883, A. Lingard and E. Batt described pecu-
liar bacilli in ulcerations occurring on the tongue and
buccal mucous membrane of the calf. “The typical ulcer
in advanced cases consists of a sore with free overhanging
edges. On section through the sore the tongue is found

Kill] MICROBES OF MALIGNANT ANTHRAX 293

necrosed to a considerable depth.’ “ Whenever the sore
touches any other part of the mouth or cheek, the disease is
communicated and rapidly spreads. In some cases similar
necrotic changes had taken place in the lung. The line of

Fig. 115.— From a Section through necrosed and adjoining inflamed Parts
of the Ear of a Rabbit, inoculated with Matter taken from
Ulcerative Stomatitis of the Calf.

1. Necrosed part.

2. Inflamed tissue.

3. Bundles of bacilli.

Magnifying power 700. (Stained with magenta.)

junction of the necrotic with the healthy tissues was found
to be occupied by a dense mass of bacilli having the appear-
ance of a dense phalanx advancing upon the healthy tissues.
The disease has been proved capable of transmission (to the
rabbit and mouse) by injection of the bacilli in question,

294 MICRO-ORGANISMS AND DISEASE [char

which are equally numerous and virulent after passing
through several generations by inoculation.”

The disease often ends fatally in calves.

The best method of staining the bacilli was found to be
this ; The sections, both those prepared from the ulcerations

Fig. ii6.— From a Section through Tongue of Calf, Ulcerative

Stomatitis.

x. Muscular fibres.

2. Inflamed tissue.

3. Bundles of the bacilli.

Magnifying power 700. (Stained with magenta.)

of the calf’s tongue and from the inoculated tissues of the
rabbit, are immersed in a mixture of magenta and methyl-
blue, then washed in spirit, and after clarifying in clove-oil
are mounted in Canada-balsam solution. The bacilli are
stained deep pink, the inflamed tissue blue. The bacilli

XIII] MICROBES OF MALIGNANT ANTHRAX 295

appear as thin rods in rows, thus forming a leptothrix-like
growth. In some of the long filaments the individual bacilli
are not well shown. The filaments are either straight or
more or less curved. The length of the single bacilli varies
from 4 fx or less to 8 /x or more ; the thickness is about 1 //..

Fig. 117. — From a Section through the Cartilage of Rabbit’s Ear in which
Ulceration had been produced by Inoculation with necrosed Matter
of Calf’s Tongue.

1. Cartilage capsules.

2. Bundles of good bacilli.

3. Bundles of degenerating bacilli.
Magnifying power 700. (Stained with magenta.)

Many of them contain spores. In the ear of the rabbit
they invade the connective tissue as well as the cartilage
over the whole extent of the ulceration and its neighbour-
hood. Lingard found the same bacilli, having the same
arrangement, in a case of noma in the human subject.

296

MICRO-ORGANISMS AND DISEASE [chai*.

Bacillus diphtheria.1 — This acute infectious disease, to
which childien and young individuals are particularly
prone, show's itself in most instances as a severe inflam-
mation and fibrinous infiltration of the mucous membrane
of the fauces and pharynx, or also the larynx and trachea,
leading to, and early in the disease consisting in, a necrosis

Fig. 118. — Film Specimen of the deeper Layer of the Diphtheritic
Membrane, showing numerous Leucocytes and the Diphtheria Bacilli.

X 1000.

of the superficial part of the mucous membrane, and thereby
changing this into a tenacious, whitish pseudo-membrane,
the “ diphtheritic membrane.” In most cases only the
mucosa of the fauces (tonsils, palatine arches, velum palati
and uvula, upper part of pharynx) shows this change, i.e.
into whitish-grey “ diphtheritic membranes ” ; in other

1 Pnrt of the following account is copied from Klein’s Etiology and
Pathology of Infectious Diseases in Stevenson and Murphy’s Treatise
on Hygiene, vol. ii.

XIII] MICROBES OF MALIGNANT ANTHRAX 297

cases this necrotic change extends over the whole of the
pharynx into the larynx, and even the trachea ; in still other
cases it starts in the larynx and invades this and the trachea
— croup. In some cases a similar inflammation and the
formation of diphtheritic membranes are observed in the
stomach, in the intestines, in the urinary organs, and inde-
pendently and primarily on wounds. In addition is to

Fig. 119.— Film Specimen from the Superficial Lavers of the Diphtheritic
Membrane, showing the Diphtheria Bacilli in pure Culture.

X 1000.

be mentioned myocarditis diphtheritica. The microscopic
character of typical or membranous diphtheria is generally
this, that the mucous membrane is the scat of a severe in-
flammation and necrosis : engorgement of, and extravasation
from, the superficial capillaries and veins, with stasis of
blood in them, and swelling due to infiltration of the
mucosa with fibrine and round cells ; the epithelium as

298 MICRO-ORGANISMS AND DISEASE [chap.

a whole is lost ; the affected mucosa itself becomes necrosed
and changed into a whitish-grey coagulated mass, in which
fibrin, a close network of threads and septa, and in the
superficial parts lymph cells, may be recognised, this ne-
crosed or coagulated portion forming the diphtheritic mem-
brane ; close to that part which comprises the necrosed
mucosa the outlines of blood-vessels filled with stagnated
and coagulated blood, and extravasated blood, as also
dense infiltration with lymph cells, may be recognised.
When the process continues into the depth and breadth,
this inflamed portion also becomes necrosed, and a part of
the diphtheritic membrane. After the process passes the
acme, the inflamed tissue, not necrosed by the exudation,
gradually detaches the diphtheritic membrane above it, and
an ulcer is left behind, which, like other healing ulcers,
gradually contracts and becomes covered with healthy
membrane and epithelium.

A section through a diphtheritic membrane shows a few
nuclei in a dense, more or less fibrinous, reticulated or
hyaline matrix, more or less ill-preserved ; some of these
take the staining, i.e. are not dead ; in others already dead
the outlines can be barely recognised. In the superficial
parts of the diphtheritic membranes a number of larger or
smaller loculi are always seen, which are filled with clumps
of bacteria (see illustration). These clumps of bacteria are
of various kinds : generally staphylococci and at least two
kinds of streptococci, thick and long septic bacilli, and
groups of minute bacilli which we will call the diphtheria
bacilli. These latter are found in larger and smaller masses
on the surface, forming sometimes a continuous layer ; in
some cases sections show that in the middle, and occasion-
ally, but rarely, even in the deep parts, they are the only
bacteria present ; here they are in small clusters, or they

XIII] MICROBES OF MALIGNANT ANTHRAX 299

form large masses (see Fig. 121). In the mucous mem-
brane next to, but not part of, the diphtheritic membrane
the writer has found them occasionally in small numbers ;
in the inflamed mucous membrane of the depth these diph-
theria bacilli are, as a rule, rarely to be found. In the
blood and in the viscera the bacilli are generally absent ;
n<5r are other micro-organisms to be found as constant
inhabitants. In cases of diphtheria ending fatally, even
if the disease only lasted a few days, the lungs are the
seat of severe bronchial catarrh, lobular or broncho-
pneumonia, with numerous diphtheria bacilli ; the kidney
is congested and shows distinct parenchymatous nephritis :
the epithelium of many convoluted tubes of the cortex is
granular, disintegrating, and fatty ; in the liver fatty
degeneration of the liver cells is generally present. The
one species of bacteria that is constant and can be easily
isolated in many cases in almost pure cultivation from the
superficial and even middle layers of the fresh diphtheritic
membrane consists of non-motile minute bacilli : some are
curved, most are straight, some slightly swollen at each end or
knob-shaped at one end, many of them pointed at one end ;
in fact, this latter may be regarded as the typical bacillus.
These bacilli occur either singly or in dumb-bells, or aggre-
gated in continuous masses ; many show a segregation of their
protoplasm into granules or rods of unequal size ; amongst
these “ granular ” forms one or both terminal granules are
occasionally club-shaped. Some of the single bacilli in well-
stained specimens show' a deeply stained granule at each end.
I he bacilli of Agar cultures show' the same appearances as
those in the diphtheritic membrane ; in gelatine culture the
bacilli are shorter, thicker, and many are conical (see Fig. 1 23).
These bacilli were first seen by Klebs, and by Loffler were
regarded, owing to their constancy, as pathognomonic and

300 MICRO-ORGANISMS AND DISEASE [chap.

pathogenic for diphtheria ; Loffler had first isolated them by
culture on blood-serum, but he, and then Hoffmann, found
a morphologically similar bacillus in the normal discharges
of the fauces. Now Loffler has shown that, while the
former or the “ diphtheria bacillus ” is pathogenic for ani-
mals, the latter or pseudo-diphtheria bacillus is not so ; but
this, although not accepted by all, nevertheless corresponds
to the facts.

Roux and Yersin ( Annales de l' Institut Pasteur, iv., p. 409)
state that from simple sore throat, as also from normal throat,
the pseudo-diphtheria bacillus was isolated by them, which
in morphological and cultural respects is identical with the
true diphtheria bacillus, but which is not pathogenic to
guinea-pigs. They further conclude that this pseudo-diph-
theria bacillus is really the diphtheria bacillus after it has
lost its virulence.

As to the virulence of the diphtheria cultures directly
derived from the human diphtheritic secretion or membrane
and tested on the guinea-pig (see below), this does not stand
in any definite relation to the severity of the human case,
for extremely virulent (for the guinea-pig) bacilli may be
obtained from mild cases, while from severe or fatal cases
bacilli are cultivated which are less virulent for the guinea-
pig, inasmuch as of the former the subcutaneous injection
of less culture material will produce a fatal result in the
guinea-pig than of the latter. Similarly the length of the
diphtheria bacilli in the membrane and in the cultures ob-
tained from this is no index of their virulence ; as a rule
when the membrane contains the diphtheria bacilli in al-
most pure culture the great majority are relatively short rods.

Besides, in true diphtheria of the fauces the diphtheria
bacilli can be demonstrated in many cases of fibrinous rhinitis,
fibrinous croup, and in diphtheria following scarlatina, but

XU i] MICROBES OF MALIGNANT ANTHRAX 301

not in so-called scarlatinal diphtheria, that is in necrotic
change in the fauces occurring simultaneously with scar-
latina. (Loftier, Kolisko and Paltauf, Tangl, Klein.)

As a result of recent investigations the opinion is well

tic. 120. — Cultivations of the Bacillus Diphtheria on the slanting
Surface of Nutrient Gelatine: on the left, Streak Culture; in

THE MIDDLE, A TUBE-PLATE CULTURE WITH NUMEROUS MINUTE COLONIES;
ON THE RIGHT, A XUBE-PLATE CULTURE WITH A LIMITED NUMBER OF

Diphtheria Colonies ; in all, the Centre thicker, less transparent,
THE PERIPHERAL Part .MORE FILMY.

Natural size.

founded that also in cases of “simple sore throat,” if the
presence of the true diphtheria bacilli can be demonstrated
in the secretion, those cases are diphtheria; and conversely, if
in any case of sore throat, no matter whether it is or is not
associated with membranous exudation, the true diphtheria

302 MICRO-ORGANISMS AND DISEASE [chap.

bacillus cannot be demonstrated, such case cannot be con-
sidered as diphtheria. As a matter of fact it has now been
amply shown that the after-events prove the correctness of
these statements, for it has been shown that the former
cases, apart from their being the centre of an outbreak
of cases of true membranous diphtheria, develop occasion-

F ig. i2i. — Section through a diphtheritic Membrane showing connected
Masses of the Diphtheria Bacilli extending from the Surface of the
Membrane into its deeper Layers. The Tissue of the Membrane is
not shown ; as Cultures proved, all the Masses (black) are Masses
of pure Diphtheria Bacilli.

ally post-diphtheritic paralysis, while the latter (non-diph-
theritic) cases do not lead to post-diphtheritic sequelae
These cases of faucial inflammation not associated with the
true diphtheria bacilli, and therefore not true diphtheria, are
associated with, and probably caused by, either staphylococci
(staph, aureus) or streptococci, and are therefore regarded as

Low magnification.

XIII] MICROBES OF MALIGNANT ANTHRAX 303

“ cocco diphtheria.” The streptococci are at least of two
kinds : the same as are occasionally also found as compli-
cating severe cases of true faucial diphtheria. Non-
diphtheritic membranous exudations of the fauces are
brittle and composed of leucocytes, whereas the diphtheritic
membrane is tough, coherent and poor in leucocytes, con-
taining principally the above-mentioned reticulated mass.

In some epidemic sore throats thrush fungus or saccharo-
myces is present in large numbers.

As a further result of recent investigations it is admitted
that the true diphtheria bacilli occur in the fauces of per-
sons who, themselves free of diphtheria, have however been
in contact with diphtheria cases, and further that, even weeks
after in a diphtheria case recovery had taken place, the
mucous membrane of the fauces may still harbour true diph-
theria bacilli. In the majority of cases of faucial diphtheria,
however, the bacilli disappear two or three weeks, or even
earlier, after the mucous membrane had assumed its normal
condition.

The bacillus of diphtheria isolated by Loffler forms
colonies of definite characters on serum and Agar plates
kept at 35~37°C. : round white colonies, thickest in the
middle and gradually assuming here a yellowish-brown tint.
According to Ldffler it does not grow on gelatine, but the
writer has shown that abundance of growth takes place on
gelatine at 2o-2i°C. ; on potato it shows no visible growth.
Loffler found this particular bacillus in a large percentage,
but not in all, of the diphtheritic membranes ; Kolisko and
Paltauf, Roux and Yersin, Zarniko and Escherich, found
this microbe in all cases of diphtheria, and owing to its
peculiar pathogenic action (see later) they definitely re-
garded it as the microbe of diphtheria. The writer has
shown that there occur occasionally in diphtheritic mem-

304 MICRO-ORGANISMS AND DISEASE [chap.

branes two species of bacilli, similar in morphological
respects and in the mode of growth on and in Agar plates,
on serum, and on potato ; but one species is not constant,
and is probably tbe pseudo-diphtheria bacillus of Hoffmann
and Loffler, while the other is present in all cases, and in
some almost in pure culture ; it is pathogenic on guinea-
pigs. It grows abundantly on broth at 370 C., making this
uniformly turbid already in 24 hours ; this increases during
the next day, while a whitish, powdery precipitate appears,
and on the surface a filmy membranous-like pellicle.

On gelatine the colonies are at first rounded, white,
prominent dots, which enlarging in breadth thicken in the
middle and become here slightly yellowish, dark brown i*n
transmitted light, the peripheral part being thin, plate-
like, and angular (Fig. 120). In the streak cultivation on
gelatine the streak becomes marked as a white band, at first
made up of droplets, but soon becoming confluent into a
uniform band ; at the margin the droplets and knob-like
expansions can still be recognised ; the middle is thick and
prominent ; in stab culture in gelatine the stab becomes
indicated by a line of droplets, white in reflected, brownish
in transmitted light ; the upper point of the stab is occupied
by a crenate, convex, white plate. Of course on gelatine, at
19-210 C., the growth is much slower than on Agar-Agar at
3 5-3 70 C. In milk kept at 20° C. our bacillus grows
luxuriantly and produces already after three days, or even
less, slight curdling of the milk, minute flakes of coagulated
casein; at 37°C. the growth is curiously less abundant in
the same space of time, and the curdling far less. The
diphtheria bacilli are killed by heating to 6o° C. for five
minutes ; they do not form spores. The diphtheria bacilli
when transmitted through several subcultures acquire the
power to grow more and more rapidly on gelatine at 20-2 1 °C.,

XIII] MICROBES OF MALIGNANT ANTHRAX 305

as also they appear to form longer rods and chains than at
first. The club-shaped forms and the chains of granules
and rods with spindle-shaped and clubbed ends also appear
sooner in the cultures : these forms have nothing to do with
involution forms, as they can be demonstrated already in the
active and early phases of the development of the colonies.

Fig. 122,-Film Specimen of an Agar Culture of Bacillus Diphtheria-

AFTER A FEW DAYS GROWTH ; CHAINS AND CLUBS ARE WELL SHOWN.

For the isolation of the diphtheria bacillus, serum (pure
blood-serum or, better, Lofflcr’s serum) or nutrient Agar is used,
for then the colonies if present can be recognised already after
twenty-four hours. As stated above, in some cases of mem-
branous diphtheria numerous colonies, occasionally in pure
culture, of the diphtheria bacillican be easily obtained eitherby
rubbing a particle of the membrane over the slanting surface

3°6 MICRO-ORGANISMS AND DISEASE [CHAP.

of the solid medium or by first shaking up a particle of the
membrane in sterile salt solution and rubbing a droplet of this
over the culture surface. But, unfortunately, in a large per-
centage of doubtful cases the diphtheria bacilli are mixed
up in the exudation with numerous cocci : in such cases it
is necessary to use serum cultures. On this medium the
diphtheria bacillus grows better than the cocci, and therefore

Fig. 123. — Film Specimen of a Gelatine Culture after several Days

Growth.

X 1000.

after 24-36 hours its colonies can be recognised. Another
plan which I found useful is to melt over the flame sterile
nutrient Agar or Glycerine Agar and to pour it out into
sterile plate dishes ; after it has set herein a particle of the
suspected secretion or membrane is rubbed over the whole
surface of the solid Agar, and the plate is incubated at 370 C.
After twenty-four hours, by means of a magnifying glass or
simple microscope, the colonies are carefully examined, and

XIII] MICROBES OF MALIGNANT ANTHRAX 307

those which resemble diphtheria colonies are subjected to
microscopic examination in stained film specimens and to
subcultures. I have thus succeeded in finding a few diph-
theria colonies amongst crowds of colonies of cocci, whereas
serum tubes have failed to give a positive result. But by far
the best method is the Ascites Agar fluid set with slanting
surface, which was mentioned in a former chapter as Kan-
thack’s serum Agar; for by means of this medium the diph-
theria colonies can be demonstrated far more certainly than
with any other medium. I have seen this in cases in which
the diphtheria bacilli were scanty and much mixed up with
cocci, and yet a particle of the secretion on the membrane
rubbed over the slanting surface of Kanthack’s serum
Agar, and incubated at 37° C., produced in twenty-four
hours a pure crop of diphtheria colonies.

Recent cultures of the diphtheria bacillus on Agar, on
gelatine, on serum, and in broth prove virulent on guinea-
pigs, but this virulence decreases with the age of the culture.
A broth culture of which after forty-eight hours’ incubation
at 370 C. 0-25 to 0-3 cc.is capable of killing in thirty to forty
hours one kilogramme body-weight of guinea-pigs is con-
sidered of normal virulence (Behring). Of gelatine subculture
made from a normal broth culture (three streaks on a slanting
surface six centimetres by two centimetres) incubated at
20-21° C. for seven to ten days, the growth being then
scraped down and suspended in sterile broth, one-sixth of
the total growth is sufficient to kill one kilo, guinea-pig in
thirty to forty hours. In an Agar culture made in the same
way, incubated at 37° C\, the same amount of virulence is
found during the first three or four days ; later the virulence
decreases, as does also often that of a broth culture after the
first six or seven days.

Loffler has shown that with cultures of the diphtheria

x 2

308 MICRO-ORGANISMS AND DISEASE [chap.

bacillus definite pathological results — inflammation, with
something like diphtheritic necrotic membrane — can be
obtained by rubbing them into an abraded surface of the
mucous membrane (mouth, trachea) of rabbits, fowls, or
pigeons, and Roux and Yersin found the same; but such
results are not easily and constantly obtainable either with
human diphtheritic membranes or with the cultures of the
diphtheria bacillus. By subcutaneous inoculation of
guinea-pigs with diphtheritic membrane, and particularly
with cultures of the bacillus diphtherise, definite results are
obtained. After subcutaneous inoculation with cultures a
few days old the result is very rapid and more striking than
with diphtheritic membrane ; for obtaining very acute
results only a small particle, not more than what can be
removed from a colony with the end of a platinum loop,
often suffices. In the severe cases produced by injecting
several minims of a recent broth culture (forty-eight hours
old) the animals are very quiet already after twelve or
sixteen hours ; a soft, painful swelling is found at the seat
of inoculation. During the second day the hair is erect, the
eyes are small, the temperature is raised ; the animals are
tremulous and refuse food ; the condition grows rapidly
worse, movement ceases, the body temperature rapidly falls,
and they are found dead before thirty to forty hours are
over. In other cases the illness lasts two to three days; in
still others as long as five days, or even more. The
younger the culture the more active it is, and the more
bacilli are injected the shorter the illness. On post-mortem
examination we find haemorrhage and oedema in and about
the place of inoculation, in the subcutaneous and muscular
tissue, extending sometimes over considerable areas ; when
inoculation is made into the groin the changes (haemorrhage
and oedema) extend over the thigh, abdomen, and even

XII l] MICROBES OF MALIGNANT ANTHRAX 309

chest of the inoculated side; the inguinal glands of the
inoculated side are deeply congested. The lungs are
congested, sometimes more, sometimes less ; sometimes the
greater part of one lobe or another is deep purple ; pleuritis
and pericarditis are often found; the liver is slightly or not
at all congested, is even pale ; the spleen is not enlarged ;
the serous covering of the stomach and intestines is con-
gested ; the suprarenals are deep red ; the kidney is con-
gested in the medullary part. Neither from the heart’s
blood nor from the lung, liver, spleen, or kidney can as a
rule any organisms be cultivated, but occasionally the lungs
and the omentum yield positive results ; from the sub-
cutaneous tissue of the inoculated part, particularly from
the congested inguinal glands, the bacilli can be obtained
in pure cultivations, some tubes showing a limited number
of colonies, others showing them abundantly ; but not in all
animals is the culture test successful, though in most it is so.

While guinea-pigs are very susceptible to subcutaneous
inoculation, they show considerable resistance to in-
traperitoneal injection. It has been mentioned in a
former chapter (Chapter vii.) that, while a number of
species of bacteria possess in their protoplasm substances
which act poisonously on the animal body (protein poisons,
intracellular poisons) when introduced in sufficient doses as
bacterial bodies, living or sterilised, into the peritoneal
cavity — e.g., vibrio of Finkler and cholera, bacillus pro-
digiosus, bacillus coli and typhosus, proteus vulgaris,
&c. — causing acute fatal peritonitis, and while further
some notoriously pathogenic bacilli — e.g., anthrax, fowl
cholera, and diphtheria — do not contain these intracellular
poisons, at any rate large doses of the bacterial bodies
(sterilised) can be injected intraperitoneally without pro-
ducing the acute fatal peritonitis. Now it is a strange fact

3io

MICRO-ORGANISMS AND DISEASE [CHAP.

that the diphtheria bacillus does not cause this peritonitis
even if injected into the peritoneal cavity in a living state. I f
from an active gelatine culture (slanting surface) the growth
is scraped off and distributed in sterile bouillon, and of this
suspension one-sixth is injected subcutaneously into a
guinea-pig of 500-700 grammes weight, the typical tumour
is produced, and death occurs in thirty or thirty-six
hours with certainty, but the same dose of the same
culture injected into the peritoneal cavity of a guinea-pig
half that weight does not cause fatal illness. If from the
peritoneal fluid a little is withdrawn two, three, four, and
six hours after the intraperitoneal injection of the large dosj
of living diphtheria bacilli, and examined, it will be found
that most of the bacilli are dead already after two hours, and
that no living bacilli (no successful subculture) can be estab-
lished after four to six hours. Such guinea-pigs as had been
once intraperitoneally injected with more than about a
double, otherwise fatal, dose of living gelatine culture can
repeatedly at intervals be injected with increasing amounts
— at the fifth injection as much as one-third of a living
gelatine culture can be introduced intraperitoneally — that
is to say, an otherwise fourfold fatal dose — without producing
any illness. Moreover, such guinea-pigs appear also immu-
nised against an otherwise fatal dose of living diphtheria
bacilli subcutaneously injected, no tumour and no disease is
hereby produced. By these and other similar experiments to
be mentioned in the chapter on Immunity, I have been able
to show that the specific immunising or germicidal power
against a bacterial species which the blood-serum of repeatedly
intraperitoneally injected (immunised) guinea-pigs acquires
(R. Pfeiffer) is related to substances derived from the bacilli
themselves that had been introduced into the peritoneum,
and had been used for the immunisation.

X 1 1 1] MICROBES OF MALIGNANT ANTHRAX 31 1

Roux and Yersin 1 have separated certain chemical
products (toxins) from broth cultures, and shown that
those products themselves act poisonously in the proportion
in which they are injected. Roux and Yersin have also
observed in experimental animals, after inoculation with
small doses of broth culture or of the diphtheria toxin
separated by filtration from broth cultures, the same kind
of paralysis as occurs also in human diphtheria in the later
stages, that is after the acute symptoms have passed away.
Sidney Martin has published an account of the chemical
nature of the poisons occurring in the human diphtheritic
membrane ; these same poisonous principles (ferment,
organic acid, albumoses) were also obtained from albumen
cultures of the diphtheria bacilli. Dr. Martin shows that
with the chemical products the same diphtheritic paralysis
can be produced, and he further shows that this paralysis is
due to degeneration of the peripheral nerves. (Reports of
the Medical Officer of the Local Government Board, 1891-
1892.) According to Roux and Yersin 2 the toxin of broth
cultures is a ferment and when injected into guinea-pigs pro-
duces the same cedematous haemorrhagic tumour and death
as the living culture. Roux and Yersin3 have further shown
that by growing the diphtheria bacilli in broth under constant
supply with fresh oxygen a toxin can be obtained of high
degree of virulence, 0-2 gramme being capable of producing
a tumour and fatal result in forty-eight hours in one kilo, of
guinea-pig. Loffler, Roux and Yersin, and others have
therefore justly concluded that in diphtheria we have to
deal with a chemical poisoning, the chemical poison being
produced by the living bacilli in the diphtheritic membrane

1 Aimales de P Institut Pasteur, December, 1888,

- Ibid., June, 1889.

3 Ibid., vol. iv., p. 421.

312 MICRO-ORGANISMS AND DISEASE [chap.

of the human mucous membrane, and in the case of the ex-
perimental guinea-pigs, at the seat of inoculation, and absorbed
by the system, produces the whole set of general disease
symptoms in the lung, liver, kidney, and nervous system,
associated with and characterising diphtheria ; the absence
generally of the bacilli from the circulation and all affected
organs, and their localised presence in the diphtheritic mem-
brane, suggests this already. From this it follows that if
the growth and multiplication of the bacilli in the diph-
theritic membrane could sufficiently early be prevented or
checked — by cautery or otherwise — the amount of the
poison would be small, and the disease would cease. Diph-
theria is then not a real infection but more of the nature of
intoxication. ^

It has been asserted by various authors that a necrotic,
chronic, infective process observed in the mucous mem-
brane of the mouth and pharynx in fowls, calves, and pigeons
is intimately connected with human diphtheria ; but Loffler 1
has shown this is not the case, since these necrotic pro-
cesses are both as to the pathology and the microbe
altogether different diseases.

Cats, however, have unquestionably been observed 2 to
suffer in connection with human diphtheria; in houses where
human diphtheria obtained, cats have been known either
antecedently, or coincidently, or subsequently to become ill ;
they appear to have some kind of throat illness and cannot
swallow ; as a rule, bronchial mischief is already noticed
early, and if the disease is protracted through several weeks,
as it generally is, they become much emaciated and die.
On post-mortem examination the lung is found to be full of

1 Mittheil. aus d. k. Gesundh., vol. ii., p. 482.

2 Dr. George Turner, Dr. Bruce Low, Dr. C. T. Renshaw, Dr. A.
Downes, Dr. Thursfield ; see the writer’s Report in the Volume of the
Medical Officer of the Local Government Board, 1889, p. 162.

XIII] MICROBES OF MALIGNANT ANTHRAX 313

grey, consolidated, lobular patches, and the kidneys are
always enlarged and white ; on a section the whole cortex is
found to be fatty degenerated, while the medulla shows con-
gestion. Further, I have ascertained that an infectious
disease with the same symptoms and leading to the same
result exists naturally amongst cats ; the animals have severe
lung trouble, emaciate, and die with the same pathological
-appearances, notably on the part of the kidney. In one case
I have seen such a cat after several weeks’ illness showing
paresis of the hind extremities.

When cats are inoculated subcutaneously in the groin
with a particle of human diphtheritic membrane they
become very ill, show already after twenty-four hours a
painful swelling in the groin, have high temperature, and
refuse food. In the severe cases these symptoms increase
in intensity during the next days, and the animals die before
the end of the week. On post-mortem examination the
subcutaneous and muscular tissue at and near the seat of
inoculation are found to contain haemorrhage and oedema,
and the tissue is separated into layers, which are more or less
necrotic. The viscera show much congestion, particularly
the lungs, also the serous covering of the stomach and
intestine as well as the peritoneum ; the kidney is large and
7o/iite, the medulla congested, while the cortex is more or
less uniformly fatty. This condition is more marked the
longer the illness ; when the animals die in three to four
weeks, or later, the condition of the kidney is very striking,
and then also the lungs show lobular patches of grey
consolidation. Still more striking is the result when a small
quantity, 1 cc., of a virulent culture of our bacillus diph-
theria is subcutaneously inoculated. If a fresh culture —
one twenty-four to forty-eight hours old — is used, the
animals are very ill already after twenty-four hours : they

314 MICRO-ORGANISMS AND DISEASE [chap.

are quiet, refuse food, the temperature is raised, and at the
seat of inoculation is a painful swelling ; some animals die
after two, three, or four days, others live to the end of the
week. On post-mortem examination the same appearances
of the viscera, notably of the lungs and kidney, are found ;
and here also the fatty white kidney and the pneumonia are
the more marked the longer the duration of the disease ; in
animals that die forty-eight to seventy-two hours after inocu-
lation with culture the subcutaneous and muscular tissues
about the seat of inoculation show much haemorrhage, in
many parts the tissues are almost gangrenous. On the
death of the animal, the bacillus diphtheriae can be re-
covered by cultivation in numerous colonies, but no bacilli
can be demonstrated in the lungs, liver, or kidney.

The dog is similarly affected by subcutaneous injection of
virulent diphtheria culture.

Different animals offer, however, different degrees of
resistance to infection with living culture or with toxin
produced by Roux and Yersin’s method in broth culture and
separated by filtration with a Chamberland filter. Thus
the sheep and goat, the ass and the horse, offer different
degrees of susceptibility ; the sheep and goat react well
(Behring), the ass better, and the horse as a rule least
(Roux) ; in the latter animal the relative dose of living
culture or toxin can be taken greater than in the ass in
order to produce a positive result, but also amongst horses
the resistance varies in different animals. On subcutaneous
injection of a non-fatal dose a tumour is formed at the
seat of inoculation, the body temperature is raised next day
(by o'5— 2° C. according to the dose and virulence ot the
material), the animals are quiet and do not feed quite in the
normal manner. But they soon again recover their normal
temperature, feed again well, the local tumour becomes

XIII] MICROBES OF MALIGNANT ANTHRAX 315

smaller and in a few days has almost entirely disappeared.
By reinjection after the lapse of a week to a fortnight the
dose of culture or toxin can be made a little larger or the
virus a little more potent without again producing more
than the former transitory result. In this way Behring1
was the first to show that the resistance of the animal can
be gradually more and more increased, inasmuch as after
fepeated injections it is capable of resisting (except for the
transitory tumour and rise of temperature) larger and larger
and more potent doses of the virus, doses which at a former
stage would have at once produced fatal results. Behring
has thus succeeded in “ immunising” sheep and goats to a
very high degree, that is to say that after many injections
with increasing amounts and potency the animals are
capable of resisting a dose of virus many times the former
fatal dose. Roux 2 uses for this purpose the horse, and he
succeeds after many injections (over thirty, extending over
nearly three months) in enabling this animal to at last
resist the intravenous injection of the prodigious amount
of 250 cc. of the most potent toxin.

As is well established, diphtheria is a highly contagious
disease, transmissible from person to person, its contagium
belonging to the group called fixed contagia. But it is
likewise well established that milk infected from a human
source has, in several epidemics, been the means of pro-
ducing diphtheria in the consumers (Ballard). It is further
known that a room in which a diphtheria case has once
existed may for years harbour the contagium of diphtheria,
so that any new-comer or inhabitant may contract the
disease ; moreover, it is known that in a locality in which
diphtheria has once been rife the disease may at any time
reappear, and in these instances the transmission of the

1 Behring, Deutsche Med. Wocheitschrift , 1890, No. 50.

* Roux, Annales de P Institut Pasteur, September, 1894.

3>6

MICRO-ORGANISMS AND DISEASE [chap.

contagium from sewers is maintained by some sanitarians.
Lastly, it has been shown by Mr. Power, Dr. Mason, and
Dr. Philpott that in certain epidemics of diphtheria (York-
town and Camberley, Barking, Croydon), while the milk
was the vehicle of infection, the milk did not receive its
infective power from a human source.

Several epidemics of milk diphtheria, in which fouling of
the milk with human diphtheritic material could not be
demonstrated, but, on the other hand, could be excluded,
have of late years become known, and in these cases the
suspicion attached itself to the cows, for it could be shown
that there existed on the farms concerned no other condition
which in any way could account for the infectivity of Jhe
milk ; besides, this infectivity was inherent to the milk over
a certain period. In the case of the Yorktown and Cam-
berley epidemic (see Mr. Power’s Report in the volume of
the Medical Officer of the Local Government Board for
1 886) the cows were certified by a veterinary surgeon to
have been in good health, though even several days after the
human diphtheria cases had ceased to occur two of the
cows showed some slight signs of “ chaps ” on their teats.
Mr. Power saw at the farm one cow which had suffered from
chapped teats in October, 1886 (the month in which the
epidemic occurred), and which still had at the beginning of
November a scab or crust at the site of a “chap.” At
Barking the cows whose milk produced the diphtheria (in
1888) suffered from a distinctly contagious eruptive disease
on the teats and udder, showing itself in sores covered with
brown black crusts. The same was noticed in connection
with an outbreak of diphtheria (through milk) at Croydon,
November, 1S90. The question which was therefore con-
sidered important to decide was this : Can cows be infected
with the bacillus diphtherias ? During the years 1S89, 1890,

xml MICROBES OF MALIGNANT ANTHRAX 317

and 1S91 I made experiments on eight milch cows (which
had calved some weeks previously), which strikingly showed
that this is really the case. The results of some of these
experiments are so definite and so important in connection
with milk derived from such cows being charged with the
diphtheria contagium that we may be excused for giving two
of these experiments somewhat in detail.1
' A broth culture was made of the bacillus diphtherias
derived from a human diphtheritic membrane, but passed
through several gelatine subcultures ; the broth culture had
been growing for two to three days at 370 C., and was very
virulent on the guinea-pigs.

One cubic centimetre of the culture was injected under
the skin into the subcutaneous tissue of the left shoulder in
each of two cows. These animals were, at the time of the
experiment, in very fine condition (teats and udder quite
clean, copious milk secretion), and had been so during eight
to ten days, during which they had been under observation.
During the second and third days after inoculation the
body temperature showed a slight rise (to 4o-6°), and they
did not feed well on those two days ; but afterwards the
temperature went down to the normal state, and the
animals became all right again otherwise. But at the seat
of the inoculation there was a painful large soft tumour to
be felt and seen. On the fifth to the sixth day, for the first
time, there was noticed on the udder and on one teat in one
cow an eruption of about half a dozen firm papules : red
and injected, projecting above the surface of the skin, the
subcutaneous tissue indurated with a nodule. In addition
to the papules about half a dozen vesicles and two round
patches covered with brown crusts could be seen on the
udder.

1 Report of the Medical Officer of the Local Government Board for 1 889,
p. 168.

3<B

MICRO-ORGANISMS AND DISEASE [chap.

Some of the vesicles contained clear lymph, others were
pustular, i.e. purulent.

On the seventh day new papules and vesicles were
found ; those of the previous day had already become
changed into dark brown crusts. On the eighth day a new
crop of vesicles could be noticed on this cow’s udder, and
on that day for the first time about half a dozen were also
seen on the udder of the second cow. Some were vesicular,
others pustular, and still others covered with brown-black
crusts ; the vesicles and pustules were round and prominent,
with a narrow margin of injected skin, the crusted places
irregular. The whole thickness of the skin and subcuta-
neous tissue felt hard, nodular. For two or three days (ninth
to twelfth day) did this go on in the first cow ; that is, Aew
vesicles appeared : those that were vesicles with clear lymph
one day were pustular the next, and crusted the following
day. The crusts did not remain long ; after two or three
days they became loose, and left a dry healing sore behind,
but when recent, on removal, showed a bleeding sore of the
corium underneath.

We have, then, here a new eruptive disease on the teats
and udder of the cow : a disease marked by papule, vesicle,
pustule, sore and crust, but of a very rapid progress, since
the crusts fell off and the sore healed in less than seven to
nine days since its first appearance, the skin being at the
same time much indurated. This eruptive disease on the
udder, be it well observed, was produced by inoculating
the animals subcutaneously in the region of the left shoulder
with a culture of the bacillus diphtherias.

As stated above, in both animals on the second and third
days there was a painful soft tumour to be felt at the seat
of inoculation. From day to day the tumour became
larger ; about the end of the week it was as large as a man’s
fist ; after this time it gradually became firm : but about the

XIII] MICROBES OF MALIGNANT ANTHRAX 310

fifth and sixth days it was still soft, felt like oedema, and on
pressure a quantity of clear serum could be squeezed out
from it. After the death of the animals (one died after a
fortnight, the other was killed on the twenty-fifth day) the
tumour was examined, and it was found to be located in
the subcutaneous tissue, but was firmly connected both to
the skin above and the muscular tissue below, and was
-surrounded by oedematous tissue. It was streaked white,
was firm, but on section clear serum could be pressed out
from it. Under the microscope the tissue of the tumour
was found to be of the same nature as diphtheritic material :
a general matrix of reticulated necrotic tissue in which rem-
nants of nuclei, outlines of blood-vessels, and remnants of
extravasated blood could be recognised ; this tissue shaded
gradually both into the cutis and into the surrounding muscles.

Both animals showed normal temperature to the end, but
they both coughed and gradually fell off from feeding and
did not take any water. One of them by the end of the fort-
night suddenly became worse : it took no food or water, its
milk failed, its evacuations became scanty and dry, its breath-
ing became very rapid, and after a sudden collapse it died.
The other animal after twenty-four days (since inoculation)
grew much worse, and was therefore killed.

In both animals the lymph glands nearest the left shoulder,
i.e. close to the tumour, were much enlarged, very oedema-
tous, and contained haemorrhage ; no change in the organs
of the throat ; both lungs showed extensive congestion, in
fact almost amounting to red hepatisation of the upper
lobes and the upper portion of the middle lobe, petechiae,
and haemorrhagic patches under the pleura ; the pleural
lymphatics were everywhere in the congested portions con-
spicuous and distended, either with clear lymph, or, as was
the case in the second cow, tinged with blood. Cutting into

320

MICRO-ORGANISMS AND DISEASE [chap.

the congested portions, the lung was seen to be highly
cedematous, a large quantity of blood-tinged serum flowing
from and accumulating at the cut end; the lobules were
well mapped out, and there was also sharp demarcation by
cedematous connective tissue between the normal lung
tissue and the deeply congested lobules, as also between
groups of lobules and individual lobules in the congested
areas ; haemorrhage appeared as spots and patches on the
parietal and visceral pericardium. The liver showed yellow-
grey necrotic patches, the spleen showed grey, necrotic
streaks in the capsule; both kidneys showed congestion of
the medulla, and fatty patches in the cortex. We have,
then, in both these animals a striking result, completely
coinciding with the disease in the cat. ^

The next important point ascertained in these cows had
reference to the distribution of the diphtheria bacilli inocu-
lated. In the tissue of the tumour in both animals after
death, i.e. after fourteen and twenty-four days respectively,
the diphtheria bacilli could be demonstrated without any
difficulty under the microscope in the sections and by
culture. On sections the necrotic tissue of the tumour
contained great numbers of the bacilli in clumps ; culture
experiments on gelatine and on Agar with a particle of the
tissue of the firm tumour produced innumerable colonies of
the diphtheria bacillus ; when examined under the microscope
they resembled the human diphtheria bacillus in all respects.
They were also tested on guinea-pigs and found to act
extremely virulently, causing death of the animals under
the typical appearances in thirty to fifty hours. But neither
in the heart’s blood nor in the lung or liver of these cows
could any microbes be demonstrated in microscopic speci-
mens or by culture. So far, then, there is complete analogy
between the cows, guinea-pigs, and cats, that is to say the

XIII] MICROBES OF MALIGNANT ANTHRAX 321

diphtheria bacilli introduced in the subcutaneous tissue pro-
duce here by growth and multiplication the chemical poison,
setting up the general disease in the viscera. The presence
of the diphtheria bacillus in the eruption of the cow could be
demonstrated both microscopically and by culture during
the vesicular and pustular stages ; in the latter also numerous
pus cocci.

„ That in the cow the diphtheria bacillus as such passed into
the system of the animal and appeared, though not in the
viscera, but on the udder, was demonstrated conclusively
by the fact that before the end of five days after inoculation,
in the milk of the cow collected under all precautions, the
presence of the diphtheria bacillus could be demonstrated
with certainty by microscopic and culture observation ;
the number of bacilli present on that day in the milk
amounted to thirty-two per cubic centimetre. It need
hardly be added that these results throw a great deal of
light in understanding certain epidemics of milk diphtherias,
such as at Camberley and Yorktown, Enfield, Barking, and
Croydon.

This positive result of udder eruption was also obtained
on two of further six experimental milch cows, and in one
of two cases the bacillus was demonstrated in the milk
about the end of the first week after inoculation. In all
cases, however, the culture used was very virulent broth
culture.

With cultures not of the virulent character — e.st. Agar
cultures or broth cultures of some standing, inoculation
produces a transitory tumour and smaller in extent without
the visceral disease, and the animals soon recover. Such
was the case in some of my own milch cows and in those
experimented upon by Abbott ( Journal of Pathology and
Bacteriology , vol. ii., 1893, p. 35).

322 MICRO-ORGANISMS AND DISEASE [chap.

Von Emmerich isolated short thick rods from diphtheritic membranes,
with which he produced a fatal disease in pigeons, rabbits, and mice.
He found that, inoculated into the mucous membrane of the trachea of
rabbits, the microbe produces death in sixty hours, with grey fibrinous
membranes on the mucous membrane ; the bacilli are present in the
mucous membrane, blood, and viscera.

LbiTler 1 showed that the so-called diphtheritic deposits in the mucous
membrane of the fauces, larynx, and conjunctiva of fowls and pigeons is
not the same as human diphtheria ; in the pigeon it is different from
that of fowls, while in the former it is caused by minute bacilli, thinner
and a little longer than those of rabbit’s septicaemia (Davaine, Koch) ;
he also showed that the so-called diphtheria of calves is not the same as
human diphtheria, since it is caused by long bacillary threads. Lingard
and Batt have found previously the same bacilli in the necrotic masses
in the mouth in calves ; they have described the disease as a chronic
ulcerative necrotic stomatitis. Dr. Lingard has shown that it is trans-
missible to the rabbit’s ear, w'herein the characteristic bacilli produce
the same chronic necrotic ulcerative process.

As to the necrotic deposits in the fauces and mouth of fowls, not at
all rare amongst poultry, and regarded by some as identical with human
diphtheria, Loffler has already pointed out that it is different from the
similar disease in the pigeon ; it certainly is not due to the same bacteria
as those shown by Loffler to be the cause of the pigeon’s disease. The
writer has cultivated from the caseous yellow-white deposits in the
pharynx and mouth of such a fowl an organism w'hich wras present in
almost pure culture. The yellow-white deposits are dry and brittle,
and are made up of epithelial cells and debris. There are present various
species of microbes in the superficial layers ; but in the deeper parts was
present predominantly one species of minute more or less constricted
rods, of the same size as those of fowl cholera, but differing from these
latter by the fact that on potato they form rapidly a characteristic deep
yellow growth ; on gelatine they form already after twenty-four to forty-
eight hours white, round, prominent dots, which become more yellowish
and project over the surface like little buttons, and are easily lifted oft
bodily ; they are very tenacious, and do not break up when shaken in
fluid.

Bacilli resembling the diphtheria bacilli in some of the morphological
characters have been obtained from various materials. Besides the
non-pathogenic-pseudo-diphtheria bacillus of Hoffmann, Loftier, Klein,

1 Miltheil ans. clem k. Gesundh., vol. ii.

xm] MICROBES OF MALIGNANT ANTHRAX 323

l Roux and Yersin, isolated from the fauces of the healthy as also of in-
• flamed throat, there occur bacilli which form very pronounced clubs
and threads with segregated protoplasm with terminal knobs, but which
: in cultural respects differ from the pseudo-diphtheria and the true
> diphtheria bacilli.

Thus, from milk taken directly from the teats of the cow I have

Fig. 124.— Film Specimen of a Colony on Agar of a Bacillus obtained
from Milk of a Cow ; the Colony was distinctly yellow. Markedly

CLUB-SHAPED BACILLI.

X IOOO.

t ...

isolated a bacillus forming exquisite clubs (see Fig. 124), but which in
cultural respects markedly differs from the diphtheria bacillus ; it forms
on Agar yellow round colonies and grows much faster than the diph-
theria bacillus, besides being non-pathogenic. Similarly from putrid
beef I have isolated bacilli which in morphological respects bear a great
resemblance (see Fig. 125) to the diphtheria bacillus but do not grow
at 37* C.

Y 2

324

MICRO-ORGANISMS AND DISEASE [chap.

The Bacillus of Glanders. — In 1882 Schiitz and Loftier1
demonstrated the constant occurrence of definite bacilli in
the characteristic deposits and nodules of the nasal mucous
membrane and internal organs, such as the lung, spleen,
and liver of horses dead or dying from glanders. This
bacillus is called bacillus mallei or glanders bacillus. The

Fig. 125. — Film Specimen from a Colony on Gelatine of a Bacillus

OBTAINED FROM I’UTRID BEEF. Many BACILLI SHOW SEGREGATED PROTO-
PLASM and Clubs similar to the Diphtheria Bacillus.

x 1000.

bacilli occur generally isolated, and in small groups between,
and also enclosed in, the cells of the nodules : they are
more numerous in the nodules which have not become
purulent ; after the nodules have become purulent the
number of the bacilli in them diminishes. The bacilli are non-
motile rods, of about 1-5 to 3-5 n in length, that is the

1 Deutsche vied. Wochcnschnft, 52, 1S82.

XIII] MICROBES OF MALIGNANT ANTHRAX 325

same size as tubercle bacilli, but a little thicker, rounded
at their ends, straight or sometimes more or less curved ;
this latter is especially noticed when they lie in groups ;
' their substance is either homogeneous or, like that of the
tubercle bacilli, shows segregation of the protoplasm into
granules within the sheath. The bacilli stain best in
alkaline methylene-blue and then washed in acidulated water
(acetic acid 1 per cent.) ; also in alkaline fuchsin of Ehrlich,
or in gentian violet aniline water. The bacilli are easily
cultivated at 35-38° C. on blood serum, Agar mixture, and

Fig. 126.— Pus of a Pulmonary Abscess in a Horse dead of Glanders.

1. The nuclei of pus cells.

2. The glanders-bacilli.

Magnifying power 700. (The preparation has been stained with methylene-blue.)

potato. On boiled potato at 35° C., they form a charac-
teristic yellow-brownish amber-coloured sticky film. On
solid blood-serum at 37° C., after three days, one notices
small translucent droplets slightly projecting over the
general surface. These are the youngest colonies. On
Agar culture the colonies are also translucent greyish
I droplets, gradually flattening and becoming dark in the
centre.

According to Raskina the glanders bacilli grow also at
18-20" C., on gelatine, milk, serum, and white of egg.

1 Kranzfeldt grew them also on glycerine Agar mixture.

326 MICRO-ORGANISMS AND DISEASE [chap.

There is no difficulty in obtaining good cultures in the
ordinary beef broth peptone gelatine kept at 20-21° C\,
as also on potato at this temperature. They form on
ordinary gelatine whitish-grey, flat, round, disc-shaped
colonies. The gelatine is only very slowly liquefied.

Loffler and Schiitz proved that the artificial cultivations
inoculated into horses and asses produced typical glanders.
On most white mice the bacilli do not act, nor does fresh
glanders material directly taken from the horse 1 ; wild mice
(field mice), however, are very susceptible to inoculation
with the cultures ; they die within eight days, and their
spleen and liver are riddled with yellowish-grey minute
nodules containing numerously the glanders bacilli. In the
rabbit subcutaneous inoculation produces generally a positive
result ; in most cases, however, only a local abscess is
formed which leads to a sore rapidly healing. In guinea-
pigs both the fresh glanders material, as also the culture,
produce a characteristic disease : on the third or fourth
day a sore is found at the seat of inoculation, which soon
involves the nearest lymphatics, these being found swollen
and congested ; further the testis or ovary become much
swollen, congested, and the seat of minute glanders nodules,
so does the skin and the nasal mucous membrane, leading
to purulent infiltration and, after the discharge of the pus,
to ulceration. The spleen contains white nodules. The
glanders bacilli are present everywhere in the deposits.

Glanders bacilli of cultures are killed by prolonged drying
(in about fourteen days) ; the glanders material directly
from the horse becomes innocuous after a few days’ drying,
which facts seem to indicate that the bacilli do not form

1 H. Leo ( Zeilschrift f Hygiene, VII. 3) succeeded in giving glanders
to white mice after feeding them for days with phloridzine, whereby
their tissues contained much sugar.

xm] MICROBES OF MALIGNANT ANTHRAX 32 7

spores. With this agrees also the observation of Loffler,
that the cultures of the bacilli die after a few months, and
Carde'al and Malet found that putrefaction destroys the
bacilli, though only in many days. Loffler studied also the
resistance of the bacilli to heat, and he found that, for
instance, ten minutes’ heating to 55° C. completely killed

Fig. 127. — Film Specimen of a Pulmonary Nodule of a Horse dead of
Glanders ; numerous Glanders Bacilli are shown.

X 1000.

the bacilli of the cultures ; in this respect the glanders
bacilli are even less resistant than many other non-spore-
bearing bacilli. Further Loffler found that perchloride of
mercury 1 : 5,000 kills the bacilli in two minutes, carbolic
acid (3 to 5 per cent.) in five minutes. All these facts
strongly point that no spore formation took place.

Under natural conditions the general mode of infection

328 MICRO-ORGANISMS AND DISEASE [chap.

seems to be that of inoculation. It appears to be doubtful
whether the direct transmission of the glanders material on
to the intact nasal mucous membrane can produce infection,
since such a mode yields experimentally no result; but
cutaneous and subcutaneous inoculation in horses and asses
is always followed by the characteristic disease of the nasal
mucous membrane.

Fig. 128.— Film Specimen of the Glanders Bacilli from a Potato Culture.

x 1000.

Horses and asses are very susceptible ; of carnivorous
animals glanders has been observed in feline animals (lions
and tigers fed on flesh of glandered horses) ; cats, dogs, and
sheep are only very slightly susceptible, but in goats
glanders has been observed ; in cattle glanders is unknown.
Rodents are easily infected by inoculation (see the experi-
ments of Loffler and Schiitz).

In man glanders occurs after infection from the horse,

XIII] MICROBES OF MALIGNANT ANTHRAX

329

generally through a cutaneous wound ; it generally runs
an acute course, characterised by the appearance of purulent
infiltration about the seat of infection of the skin, par-
ticularly the muscular tissue, further the lung and respiratory
mucous membrane ; metastatic purulent infiltration occurs
also in the joints, the liver, spleen, kidneys, and testis.

Within recent times it has been shown by a series of
observations, carried out by a number of workers,1 that the
chemical products in the artificial cultures of the glanders
bacilli (Mallein) injected into horses produces a definite
reaction— viz., a decided rise of temperature, if the animals
are affected with glanders ; but no reaction follows in
healthy horses. So that in doubtful cases the injection of
the Mallein determines the diagnosis. The Mallein is
prepared in the same way as Koch’s tuberculin (see below),
and is a further instance of the vast importance of the study
of the chemical products of pathogenic bacteria.

Mallein at present used is either an extract of old potato
cultures of bacillus malli with dilute glycerine, filtered and
sterilised by steam (potato culture extract, Preusse) ; or
Roux establishes cultures of virulent glanders bacilli in broth,
incubated at 370 C. for four weeks; by heating to no0 they
are sterilised, then inspissated at low temperatures to one-
tenth bulk, filtered, and finally before use diluted with ten
times its bulk of o-5 per cent, carbolic (Bouillon mallein).

Foth 2 obtains Mallein in the form of a powder (dry
Mallein) : virulent bacilli of glanders are grown in broth to
which 4 ’5 per cent, glycerine is added and incubated for

1 Ilelman, Veterinary Society, St. Petersburg, April, 1890 ; Kalning,
Archiv f. Veteriniirwiss I. 1891, St. Petersburg; Preusse, Berliner
Thierdrztl. Wochenschr., No. 29, 1891 ; Heyne, Berl. Thierdrztl.
Wochenschr., 1891, Nos. 33 and 39 ; Pearson, Zeitschr. f Veteriniirk.,
No. 5, 1891 ; Sohne, Sticks. Vet. Jahresbericht, 1891, p. 56.

2 Fortschritte der Mcdizin, No. 16, 1895, p. 639.

330

MICRO-ORGANISMS AND DISEASE [chap.

twenty days at 37'5° C. The culture is then inspissated at
8o° C, to one-tenth its volume and filtered. From this fil-
trate by addition of a thirty-fold volume of 99 per cent,
alcohol a white voluminous precipitate is produced, which
dried in vacuo over calcium chloride yields a white powder,
easily soluble in water. o'oq to 0^05 grain of the powder is
a dose for a horse ; of the fluid preparation above men-
tioned (Preusse’s potato culture extract) and (Roux’s broth
culture Mallein) 1 cc.

If horses are injected subcutaneously with the Mallein
those affected with glanders react with great swelling
and rise of temperature from i°-2-5° C. or more; those
without glanders do not react as a rule, but 1° C. rise of
temperature may occur also in normal horses. The enor- /
mous number of observations on the diagnostic value of
Mallein in all countries leave no doubt that although not
infallible in all cases, it has, nevertheless, in an overwhelm-
ing number of trials proved of the greatest value.

Bacillus of Syphilis. — Lustgarten described {Med. Jalirb.
dcr lz. k. Gesellsch. d. Acrste , Vienna, 1885) peculiar bacilli
as occurring in syphilitic products. They resemble in size
and aspect very much the tubercle-bacilli ; their ends are
slightly thickened, and they often show nodosities ; these
bacilli are never found free between the tissue elements, but
always inclosed in cells, generally singly or in couples, or
rarely in groups, but their total number in a given section
is always small. The peculiarities they show in their mode
of staining have been mentioned in a former chapter.

Doutrelepont and Schiitz {Deutsche Med. TVoch. 1885,
No. 19) have also demonstrated the occurrence of these
same bacilli by simply staining sections made of syphilitic
tissues in a watery 1 per cent, solution of gentian-violet with
subsequent contrast staining by safranin.

Xlli] MICROBES OF MALIGNANT ANTHRAX 331

On the other hand Cornil, and particularly MM. Alvarez
and Tavel, state that a bacillus identical in mode of stain-
ing, size, and aspect with the one described by Lustgarten
as the specific syphilis-bacillus, has been found by them in
some normal secretions { Brit . Med. Jour n., Oct. 17, 1885).
Klemperer, Zeissl, Baumgarten, and others have failed to find
Lustgarten’s bacilli in syphilis materials.

Bacillus of Foulbrood. — Messrs. F. Cheshire and Watson
Cheyne described (Microsc. Journ., August, 1885) a peculiar
bacillus, bacillus alvei , which occurs in the tissues and juices
of bees, and especially their larvae, which sometimes in bee-
hives become affected with, and die of, the disease known
as “ foulbrood.” This bacillus shows certain peculiarities
in its mode of growth in nutritive gelatine and Agar-Agar,
and is capable of forming spores. With such cultivations
the disease was reproduced in healthy bees.

Bacillus of Rliinoscleroma. — A. von Frisch 1 was the first
to show that in the tissue of rliinoscleroma, particularly in
the large hyaline cells, known as “ Mikulicz cells,” there
occur small oval bacilli, either singly or as dumb-bells. He
cultivated them and used them for inoculations on animals,
but without result. Cornil and Alvarez 2 then showed that
the rhinosclerom bacilli possess a gelatinous capsule, and
therefore resemble the pneumonia bacilli of Friedliinder (see
a former chapter). Dittrich has then made extended
experiments and observations on these rhinosclerom bacilli,
and showed that morphologically and culturally they are
distinguishable, but only with difficulty, from Friedlander’s
bacilli ; though he maintains that in some minute details
as to staining and as to appearance in gelatine cultures the
two can be distinguished from each other. This is, how-

1 Wiener Med. Wochenschrift, No. 32, 1SS2.

2 Archives de Physiologic not male et path., vi., 1885.

332

MICRO-ORGANISMS AND DISEASE [ch. Xin.

ever, not admitted by many observers. Alvarez, Paltauf
and Von Eiselsberg, Wolkowitsch and Dittrich found these
bacilli also in the lymphatics of the surrounding tissue.
Paltauf and Von Eiselsberg, then Dittrich, Babes, and others,
produced in guinea-pigs, mice, and rabbits a septicaemic
infection similar to that producible by Friedlander’s bacilli,
but no chronic nodular disease.

The constant presence, then, of the capsulated rhino-
sclerom bacilli in the scleromatous tissue, particularly the
Mikulicz cells, is a fact of which there can be no doubt, but
it is equally a fact that they are identical with the bacilli of
Friedlander ; their causative relation to the rhinoscleroma-
tous process is, therefore, more than doubtful, or at any
rate not sufficiently supported.

CHAPTER XIV

BACILLUS TUBERCULOSIS AND BACILLUS LEPRA£ 1

Bacillus Tuberculosis. — The first decisive experimental
proof that tuberculosis is a communicable disease has been
given by Klencke and Villemin, the latter showing that by
inoculation of tubercular matter, such as sputum derived
from a tuberculous patient, into guinea pigs a chronic
disease is produced, which had the distinct characters of
disseminated tuberculosis in the lymph glands, the lungs,
the serous membranes, the liver, and spleen. The deposits
are at first minute and gray, not larger than a pin’s head ;
they gradually enlarge and caseate in the centre, which
caseation spreads over the whole tubercle. Chauvau,
Wilson Fox, Burdon-Sanderson, Klebs, Cohnheim, and
many others have repeated and confirmed these experi-
ments. Inoculations with bovine tubercular matter were
also made on guinea-pigs and rabbits, and true dis-
seminated tuberculosis was produced. Feeding of calves,
pigs, guinea-pigs, and rabbits with tubercular matter, both

1 The greater part of the following account is taken from Klein’s
article in Stevenson and Murphy’s Treatise on Hygiene, vol. ii. , p. 210
el ttassim.

334

MICRO-ORGANISMS AND DISEASE [chap.

human and bovine, produced disseminated tuberculosis.
The tubercular deposits of all such experimental animals
transferred to normal animals again produced the same
tuberculosis.

When inoculation into the subcutaneous tissue of the
groin of guinea-pigs is carried out with a minute particle of
human tubercular material, after a lapse of about twelve
days, more or less, the lymph gland nearest the seat of
inoculation can be easily felt, being a firm swollen nodule
of the size of a pea ; after a lapse of a further ten or twelve
days the first gland is much enlarged (size of a bean or
filbert) and may have become already changed into an
abscess firmly fixed to the skin, but one or the other lymph
gland near it can now be felt as a firm swollen nodule. The
abscess soon opens and discharges thick creamy pus, a sore
is established which persists, and though it may from time
to time become covered with scab or crust, the accumula-
tion of thick pus underneath soon causes again its being
opened. The other enlarged lymph glands about the
seat of inoculation also become converted into abscesses.
When killing the animal after about four to six weeks, we find
at the seat of inoculation an open sore discharging thick pus,
and the subcutaneous connective tissue around and for some
distance is hypersemic and oedematous. In connection with
the sore we find a chain or a packet of swollen firm lymph
glands (from the size of a split pea to that of a bean) con-
taining cheesy, yellow deposits. When cutting into such a
gland we find it very juicy, and containing larger or smaller
yellowish masses ; in the largest gland some of these masses
are already changed into thick creamy pus. At or about
this stage, i.e. fouWto six weeks, in most instances either no
tubercles visible to the unaided eye are yet found in the
lungs, or only very few minute punctiform nodules; in the

xiv]

BACILLUS TUBERCULOSIS

335

spleen, which is enlarged, we find already numbers of
minute granules projecting above the surface of the capsule,
thus making the surface uneven and rough. In the liver
there are numerous minute, gray, punctiform nodules, which
in some places have a tendency to confluence : on section
grayish streaks are recognised under a glass between the
normal red liver tissue ; the whole organ is slightly en-
larged. The omentum shows also numerous minute opaque
patches, which are only more numerous and larger than
those normally found. The lymph glands in the porta
hepatis are large and firm, so also those in the hilum of the
spleen ; the mesenteric glands are large and firm. In the
marrow of long bones gray and even caseous tubercles
can be distinguished. If the animal is allowed to live, it
will be found gradually getting thinner towards the third or
fourth month ; it dies generally not before the end of the
third or later than the end of the fifth month, the average
duration being ioo to 120 days after inoculation.

Rabbits inoculated subcutaneously in the inguinal region
with human tubercular sputum show very much less pro-
nounced disseminated tuberculosis than guinea-pigs ; after
many weeks — twelve to sixteen or more weeks — the animal
is found much emaciated ; the lymph glands of the inguinal
region enlarged, caseous ; in the lungs few or no tubercles,
in the liver a few tubercles, some gray, others yellow ; the
spleen is enlarged and contains many tubercular deposits ;
the mesenteric and other abdominal lymph glands swollen,
firm, caseous ; the process, on the whole, is very distinctly
less intensive and extensive than in the guinea-pigs. I
have seen numerous cases in which, after twelve to sixteen
weeks, the only organ containing numerous tubercles was
the spleen, the liver contained only few, the lungs none.

Feeding guinea-pigs and rabbits on human tubercular

336

MICRO-ORGANISMS AND DISEASE [chap.

matter produces tuberculosis, but with this difference, that
while in the guinea-pig it leads to general disseminated
tubercular deposits, it is far less so in the rabbit. In the
guinea-pig, if the animal be killed after six to eight weeks,
we find distinct tubercular deposits in the wall of the small
intestine, the tubercles are situated in the Peyer’s glands of
the ileum and ileo-csecal valve, are of various sizes, and

Fig. 129. — From a Preparation of Human Tuberculous Sputum, stained
AFTER THE EhRLICH-WeIGERT METHOD.

Nuclei and the tubercle-bacilli. Magnifying power 700.

more or less caseous in the centre ; the mesenteric glands
are always enlarged and contain firm caseous deposits. The
liver also shows already grey tubercular nodules and streaks,
the spleen is slightly enlarged and granular. The whole
process, judging from the amount and progress of the
changes, started in the lymphatic follicles of the ileum and
spread from here into the mesenteric lymph glands, liver,
and spleen. The lungs show by this time no tubercles yet.

xiv]

BACILLUS TUBERCULOSIS

337

If the disease is allowed to run its course, the animal be-
comes greatly emaciated and dies in about four to five
months or later, and then we find tubercles in all lymph
glands, in the viscera, and in the marrow of bone and
serous membranes, but the changes in the abdominal viscera
are the most extensive, those of the thorax considerably
less.

Fig. 130.— Film Specimen of Human Pulmonary Tubercle-Sputum. Numerous
long Tubercle Bacilli with Segregated Protoplasm. (A. Pringle.)

x 1000.

In the rabbit, on the other hand, feeding with human
tubercular matter produces considerably less result ; in a
large percentage of cases, even after many weeks, caseous
tubercles are found only in the lower ileum and mesenteric
glands, the spleen, liver, and lungs appear free, only in a
few cases are also these organs involved but to a small
degree, viz., containing only few tubercles.

In the fowl, both by subcutaneous inoculation and by

.33^

MICRO-ORGANISMS AND DISEASE [chap.

feeding with human sputum, tuberculosis can be produced,
although not all animals are equally susceptible. In most
cases tubercles of the spleen, in others of the spleen and
liver are the result ; the intensity of the process in both
these organs is striking only in a very few successful cases ;
in these cases we find both those organs enlarged and con-
taining numerous spherical, firm, white nodules, from the
size of a millet seed to that of a pea. They project oyer
the capsule when superficial. In many other cases tubercles
are found only in the spleen. The remarkable fact is that
in most instances, notwithstanding the tuberculosis going on
in their spleen, the animals are very fat ; when, however,
the liver becomes involved to a large extent, the animal is
found emaciated.

In the fowl occurs natural tuberculosis, but as Koch
has shown ( Iniernat . Med. Congress , Berlin, 1890) this
disease in the morphology and cultural characters of the
tubercle bacilli is not identical with human or bovine
tubercle. Mafucci ( Archiv f Hygiene und Inf. vol. xi.)
has more in detail described this natural tuberculosis in the
fowl.

Tuberculosis can be produced in animals (guinea-pigs,
rabbits) by inhalation. By a spray producer tubercular matter
finely divided can be distributed in the air in which guinea-
pigs sojourn ; the majority of these will become affected with
general tuberculosis in the usual lapse of time, the lungs
being here most advanced in the tubercular process. I
have had guinea-pigs kept in their cages in the ventilating
shaft at Brompton Hospital, and have thereby produced
general tuberculosis in the great majority of these : caseous
tubercles in the lungs, in the lymph glands, spleen, liver,
pelvic glands, were the result ; thus proving that the air of
any place where tuberculous persons sojourn contains the

xiv]

BACILLUS TUBERCULOSIS

339

tubercle virus, and must therefore be considered as not free
from danger.

In guinea-pigs, rabbits, and fowls, in all tubercular de-
posits giant cells are numerously met with.

Tuberculosis is a common disease of the bovine species :
the number of tubercular animals is astonishingly great. In

Fig. 131. — From a Preparation of Caseous Matter from Pulmonary
Deposits in Bovine Tuberculosis, stained as in preceding figure.

Magnifying power 700.

many instances, on slaughtering them, only the lungs
are found diseased, presenting a peculiar and characteristic
appearance, viz., the surface of the lung, of the pleura and
diaphragm presenting numerous oval, spherical or irregular
shaped nodules, some with short broad basis, others with
long thin basis or stalk fixed on to the organs, sometimes
clusters of them projecting from the general surface ; these
appearances have caused the disease to be called “the
grapes,” in German “ Perlsucht.” Not only the surface of
the lung, but also the interior contains numerous such
nodular deposits. They differ considerably in size, some

Bovine Tuberculosis

l. ,

z 3

340

MICRO-ORGANISMS AND DISEASE [chap.

not larger than a split pea, others as big as a filbert or
walnut, or larger. Some of these nodules are filled with
thick, creamy pus, others are yellow and caseous but firm,
still others contain calcareous matter. Under the micro-
scope the nodules contain in the periphery round cells in a
fibrous matrix, amongst them very numerous giant cells of
all different sizes, from one only twice or thrice the size of
an ordinary leucocyte to that of a real giant, with twenty to

Fig. 132. — From a Section through Tuherculous Deposits in the Lung of

a Cow.

Two giant-cells and two small cells containing tubercle-bacilli.

Magnifying power 700.

thirty and more nuclei all regularly disposed near a peri-
pheral zone of the cell. Near the caseous portion these
huge giant cells are very conspicuous ; the caseous part may
still show the outline of the giant cells, but their nuclei do
not take the stain, and the whole tissue of the caseous por-
tion is a granular debris. In pronounced and advanced
cases, freely projecting nodules, as also nodules within the
substance, having a great tendency to suppurate, are met
with in the lymphatic glands, in the spleen, liver, and even

xiv]

BACILLUS TUBERCULOSIS

341

in the milk gland. In this latter, the condition assumes an
important practical aspect, since the udder of a cow may
contain tubercular nodules without these being easily
diagnosed, and which may give to the milk infective pro-
perties. Although in light cases the milk gland is found
free of tubercles, yet in many advanced cases purulent
tubercular deposits have been demonstrated in the udder.

In the lymph glands, spleen, and liver, the character of
the nodules is the same as in the lung, and giant cells form
a very conspicuous feature.

Infection with general tuberculosis of guinea-pigs and
rabbits by bovine tubercular matter, both by feeding and sub-
cutaneous inoculation, is easily achieved ; the result is more
intense and much more rapid than by infection with human
tubercular matter. Guinea-pigs subcutaneously inoculated
develop disseminated tuberculosis of the lymph glands,
lungs, liver, spleen, serous membranes, and marrow of bone
in less than half the time ; in some cases the animals die in
about five to six weeks with remarkably widespread and
advanced tubercular deposits. Also as regards rabbits, the
process is much more rapid and more intensive ; for while
these animals, as mentioned above, after inoculation with
human tubercular matter, develop, as a rule, only a more or
less mild form of tuberculosis, limited chiefly to some lymph
glands, spleen, and perhaps the liver, after inoculation with
bovine tubercular matter they show very numerous tubercular
deposits in the lungs, liver, and spleen, all lymph glands,
and even the kidneys. The same results are obtained
by feeding rabbits and guinea-pigs with bovine tubercular
matter. Here also the process starts with tubercles of the
ileum, then spreads to the mesenteric glands, the pelvic
glands, the omentum, spleen, and liver, and finally the
lungs and sternal and bronchial lymph glands. The differ-

342

MICRO-ORGANISMS AND DISEASE [chap.

once in the intensity and duration of the process is decidedly
more pronounced with bovine than human tubercular matter,
and also in the rabbit the difference between feeding with
bovine and with human tubercular matter is striking; so
that there can be no question that bovine tubercular
matter acts in a conspicuous degree more virulently than
human tubercular matter, both in guinea-pigs and rabbits.

Fig. 133.— A single Giant-cell in Bovine Pulmonary Tuhf.rcle containing
numerous Tubercle Bacilli ; around them the characteristic Ring of
the Nuclei. (A. Pringle.)

X 1000.

Feeding calves with milk derived from an udder con-
taining tubercular deposits produced tuberculosis in these
calves, but milk coming from a healthy udder (though the
cow had tubercles in the lung) failed to produce tubercle.

Hirschberger ( Experim . Be it rage zur Inf der Milch tuber-
culoser Thieve, Miinchen, 1889) finds that at least five per

xiv]

BACILLUS TUBERCULOSIS

343

cent, of milch cows are tubercular, and though in many
cases their milk is not different from the milk of normal
cows, and no tubercle bacilli can be detected, the same
milk injected into the peritoneal cavity of guinea-pigs never-
theless produced miliary tuberculosis in the peritoneum,
spleen, and liver. Out of twenty series of experiments only
once could the tubercle bacilli be demonstrated in the milk,

Fig. 134. — A single Giant-cell, from a similar Specimen as in preceding

figure.

The ring-like arrangement of the tubercle bacilli and the nuclei around them are well

shown. (A. Pringle.)

X 1000.

and yet in ten such experiments in which the milk did not
show tubercle bacilli, it nevertheless produced tuberculosis
on intra-peritoneal injection. Hirschberger explains these
results by assuming that though tubercle bacilli were not
present in the milk as bacilli, their spores must have been
present.

344

MICRO-ORGANISMS AND DISEASE [chap.

The comparatively numerous cases of miliary tuberculosis
in children suggest the probability that they are due to the
consumption of cows’ milk containing the tubercular virus
derived from a tubercular udder. Dr. Sims Wood head’s
explanation that the numerous cases of tabes mesenterica
(tuberculosis of the intestines and mesenteric glands) of

Fig. 135. — From a Section through a Tubercle of the Lung from a Case
of Acute Miliary Tuberculosis in a Child.

Several alveoli are seen filled with debris ; in the centre of this are numerous nuclei,
and amongst them the tubercle-bacilli. Magnifying power about 350.

children are attributable to the consumption by these chil-
dren of milk derived from the tubercular udder of the cow
seems a feasible one.

Cohnheim and Salamonson were the first to show that in
all tubercular material there is present a specific virus. By
injecting a small particle of such matter into the anterior
chamber of the eye, they noticed that after the first result due
to the injury has passed off, the introduced particle gradually

XI v]

BACILLUS TUBERCULOSIS

345

undergoes diminution to almost complete disappearance,
but in about a fortnight or three weeks there occur in the
iris a crop of minute gray nodules which in reality are typical
young tubercles ; these gradually enlarge, and like all ordi-
nary tubercles undergo caseation ; while the tubercular

Fig. 136. — From a Section through a Tubercle in the Liver of a Rabbit

INFECTED INTRAVENOUSLY WITH CULTURE OF TUBERCLE BACILLI.

Two giant-cells full of tubercle bacilli are seen in the tissue of the tubercle.

x 1000.

process is at first localised to the iris, it gradually spreads to
the cervical lymph glands, and ultimately leads to general
tuberculosis of the other lymph glands and viscera exactly
as after subcutaneous inoculation. This typical production
of a crop of gray tubercles on the iris by tubercular matter
enabled Cohnheim and Salomonson to differentiate tuber-

346

MICRO-ORGANISMS AND DISEASE [chap.

cular from non-tubercular matter, and they have formulated
this fact by saying that only matter that is derived from tu-
bercle is capable of producing tubercle, and that whenever
any substance is found capable of producing this iris tuber-
culosis, it is derived from tubercle. By this clear proof for
the first time a means was offered to make a definite differ-
ential diagnosis between tubercular and non-tubercular
matter, a diagnosis which those who, at former times, were
engaged in work on tubercle, found extremely difficult. As
is well known from clinical observation, the diagnosis of
tuberculosis of the lungs is sometimes associated with diffi-
culties : the physical examination and symptomatology do
not always insure a correct diagnosis. It is true that Ville-
min had proved by experiment that tuberculosis is inocu-
lable, and Wilson Fox had insisted on the specificity of
tuberculosis by numerous experiments which he himself had
carried out, yet there were authorities who did not draw this
sharp distinction, but were rather inclined to the view that
artificial tuberculosis is due to infective matter derived from
a variety of sources not necessarily always tubercular. For
that period, therefore, the exact proof given by Cohnheim and
Salomonson marked a very important step, though the exact
nature of this specific tubercular virus remained undeter-
mined. The next discovery was that of Koch, who showed
what this nature is ; he demonstrated a particular species of
bacilli, now familiar to all pathologists as the tubei'de-bacillus,
which he found only in tubercle and in no other disease, a
bacillus so peculiar and so constant that its important dia-
gnostic value was at once recognised. No matter whether
it is a nodule in any tissue or organ that does or does not
present the typical pathological (gross and minute) charac-
ters of the classical tubercle, no matter whether in man or
the bovine species, in the sheep, in the monkey, dog, cat,

XI v]

BACILLUS TUBERCULOSIS

347

rat, mouse, rabbit, or guinea-pig, fowl or ostrich, if in such
a nodule the bacilli characteristic of tubercle can be
demonstrated, that nodule is tubercle , and the disease tuber-
culosis. The discovery by Koch of this fundamental fact
marks one of the most brilliant and most practical dis-
coveries of modern medical science ; the diagnosis of tubercle,

Fig. 137.— Three Test-tube Cultivations of Tubercle Bacilli on Glycerin
Agar after several Weeks.

Natural size

once so difficult to make with certainty, is now, by
means of the demonstration of the presence of the tubercle
bacilli, one of the easiest and at the same time one of the
most important helps in the formation of a correct diagnosis
in many otherwise doubtful cases. Koch further proved
that not only are these particular bacilli present in all and
every tubercle of man and brutes, but he also showed that

348

MICRO-ORGANISMS AND DISEASE [chap.

these bacilli can be artificially cultivated outside the animal
body, and with such cultures by inoculation typical and
general tuberculosis can be produced, the tubercles thus pro-
duced again containing the same tubercle bacilli ; in short
he conclusively established that these bacilli are the vera causa
of the disease tuberculosis. The whole problem concerning
one of the most widespread, fatal, and little understood
diseases of man and animals was by these researches at
once cleared up, and considering the difficulties in the solu-
tion of the problem and the necessity of having had to
invent special methods by which this research was carried
to a successful issue, I have no hesitation in saying that
this discovery of Koch marks one of the most important, if
not the most important, landmark in pathology. Many are ^
the workers who since Koch have contributed towards fur-
thering details as to this question, but without any intention
of minimising the importance of any and every contribution
of facts towards a clear understanding of disease and its pre-
vention, I am, I think, within the limits of absolute correct-
ness in saying that Koch’s publications on tuberculosis
( Deutsche vied. Woch., 1881 ; and Mitth. aus d. K. Gcsund-
heitsamte, ii.) contain almost the complete solution of the
problem. The best method, and always used with success,
is the staining of film-specimens or of sections with Ziehl’s
carbol fuchsin from twenty to thirty minutes at 35°to 40° C.,
then wash in water for a second or two, then for a few
seconds (five to ten) in 30-33 per cent, nitric acid, wash
again in water and place in methyl-blue aniline oil for five
to ten minutes, wash and treat in the usual way, according to
whether cover-glass specimen or section. I have never seen
failure with this method : the tubercle bacilli are always
brought out with striking clearness.

A great many modifications for staining the tubercle

xiv]

BACILLUS TUBERCULOSIS

349

bacilli have been published, which are all good to a lesser
or greater degree, but the one just mentioned is as good
and in many instances has proved simpler and better. The
tubercle bacilli always occur in tubercular nodules, more
numerously where caseation has already set in than in the
earlier stages. They occur isolated or in groups between
the cells constituting the tubercle, or they are found singly
or in small groups within the larger cells ; when present in
giant cells they are found in large numbers forming a sort
of zonular ring around the central portion {see Figs. 133
and 134). In some giant cells their number is sometimes
very limited, and Koch has concluded from this fact that
the tubercle bacilli suffer death in the giant cells and hence
disappear from them. In human tubercle the tubercle
bacilli are, as a rule, between the elements constituting the
tubercle, but as just mentioned they also occur within the
cells uni- and multi-nucleated. In bovine tubercle, however,
the rule is that they are mostly present in the uni- and multi-
nuclear cells, and only when these degenerate and break
up do they become free ; in the caseous matter they are
present in groups in the granular debris. In tubercle of
rabbit (lung and liver) produced by inoculation with bovine
tubercular matter, or with artificial culture derived from
bovine tubercle, the presence of tubercle bacilli within the
cells — small, large, and giant cells — is very conspicuous,
and yields very remarkable specimens (see Fig. 136).

The tubercle bacilli in human tubercle are delicate
cylindrical rods measuring i‘5-4 /a; many are straight, with
rounded ends, but others are slightly curved ; in pre-
parations (sputum, purulent matter or sections) stained in
the above manner the bacilli always appear composed of
granules, that is to say, within a faintly stained sheath the
protoplasm is segregated into deeply stained, cubical, spheri-

350

MICRO-ORGANISMS AND DISEASE [chap.

cal, or rodshaped granules; between the granules the
sheath is empty, but these empty places are not to be
taken for bright spores, as is done by some observers, nor is
it proved that the above granules are spores. That the
tubercle bacilli contain spores is proved by numerous
experiments of drying and heating, to be detailed below,
but what the character of these spores is, and how they
appear in the bacilli, has not been satisfactorily shown. In
bovine tubercular matter prepared in the same manner 'the
tubercle bacilli are distinctly shorter and thinner, and though
I do not for a moment question the fact that some tubercle
bacilli of human tubercle are as short and thin as those of
bovine tubercle, I am confident from numerous observations
that the majority of the human tubercle bacilli of sputum
are longer and thicker than those of bovine tubercle
besides, in preparations stained in the above manner alike,
the segregation of the protoplasm within the sheath, though
also present in many tubercle bacilli of bovine tubercle, is
not so general and uniform as in those of human tubercle.
But these minute differences need mean nothing more than
differences due to the different soil on which the bacilli
were reared. Such morphological differences in size and
aspect in one and the same species of microbes are well
known to occur in other instances if the microbe be
cultivated in different soils. When the tubercle bacilli from
whatever source (bovine, human, or from articially infected
animals) are passed through the rabbit or the guinea-pig, in
these animals the new crop of bacilli all appear to be
morphologically the same.

xiv]

BACILLUS TUBERCULOSIS

35'

Scrofula and Lupus

Koch and many other observers have shown that, both in
scrofula and lupus, tubercle bacilli occur, and that with both
these materials general tuberculosis can be induced in
guinea-pigs. But since these two diseases are in the human
subject well-marked disorders, distinct from pulmonary
tuberculosis, it is necessary to assume that the tubercle
bacilli in the three diseases possess some functional differ-
ences. To say that lupus is a form of tuberculosis of the
skin does not cover the facts, since real tuberculosis of the
skin does occur, and is totally different from lupus ; so also
scrofula is not merely tuberculosis localised in the cervical
lymph glands, since, in many instances, it does not lead to
pulmonary and general tuberculosis, whereas the true tuber-
culosis of lymph glands does do so. It is quite feasible to
assume that both lupus and scrofula are tuberculosis, but
that in origin and virulence their tubercle bacilli are different
from the bacilli, causing true tuberculosis. That the virulence
of the virus of lupus and scrofula cannot be the same as
that of the material of human and bovine pulmonary
tubercle is proved by experiments of Dr. A. Lingard,1 who
showed that the duration and extent of the disease induced
by inoculation of lupus or scrofula into guinea-pigs are quite
different from that induced by pulmonary tubercular matter,
and, further, that if a guinea-pig is made tubercular with
scrofulous matter, and the tubercle of such an animal is
again transmitted by inoculation through several generations
of fresh guinea-pigs, the disease thus produced gains
gradually in shortness of duration and intensity, until after

1 Reports of the Medical Officer of the Local Government Board ,
1888-89, P- 462.

352

MICRO-ORGANISMS AND DISEASE [char

several generations the same effect of general tuberculosis is
produced as that directly by matter of pulmonary human
tuberculosis.

The tubercle bacilli show definite characters in cultivation.
Koch succeeded in cultivating them on solid blood-serum.
Inoculating the slanting surface of the solid serum with
tubercular matter, and provided no other bacteria are
introduced, Koch noticed the first signs of growth in ten to
fourteen days. Koch used for inoculations of the serum
tubes the tubercular deposits of a swollen lymphatic gland
of a guinea-pig, three to four weeks previously inoculated
with tubercular matter, clean sterile instruments being used.
After ten to fourteen days the first signs of the growth of
the tubercle bacilli show themselves in the form of whitish
points and patches, resembling dry scales. On further /
growth they enlarge, and where close together they coalesce
into dry whitish scaly masses with irregular outline. From
such primary cultures subcultures on serum were then
carried out. But under a magnifying glass, or better under
the microscope, the growth and multiplication of the
tubercle bacilli can be seen already before the end of the first
week. Peculiar curved, or convoluted, or S-shaped whitish
lines, which prove to be strands of tubercle bacilli, are noticed
even at this early stage. On Agar broth the growth is very
limited, so also in broth. But Roux and Nocard showed that
by adding six per cent, glycerine to Agar meat infusion,
or to meat broth, the tubercle bacilli can be brought to rapid
and extensive multiplication. On glycerine-Agar-beef broth
the tubercle bacilli grow very rapidly, the growth being
already visible after six to eight days, and after several
weeks covers the whole surface as a whitish, peculiarly
wrinkled, dry film (Fig. 137) extending as a pellicle over the
condensation water at the bottom of the tube. In order to

XIV]

BACILLUS TUBERCULOSIS

353

obtain good and copious growth it is necessary to keep the
tubes capped from the outset, and in this way I have
obtained very copious growths in alkaline broth, to which a
piece of boiled white of egg is added. In such tubes the
broth kept at 370 C. remains clear for four to five days, the
minute flocculi and granules appear at the bottom and along

Fig. 138. — From a- Section through the Kidney of a Rabbit dead of
Artificial Tuberculosis.

a. Blood-vessel filled with caseous matter, and in it numerous tubercle-bacilli.

b. Nuclei of cells of the tuberculous new growth.

c. Capillary vessel in cross section.

Magnifying power 700.

the wall of the tube where it is in contact with the broth ;
after a fortnight the growth is abundant, and on shaking the
broth is made turbid by the numerous flocculi. On potato
moistened with broth it is likewise possible to get growth.
Temperatures between 36° to 38° C. are most favourable
for the growth ; below 30° or above 420 C. no growth can
be noticed.

A A

354

MICRO-ORGANISMS AND DISEASE [chap.

Koch has shown that by subcutaneous inoculation, by
inhalation, inoculation into the peritoneum, the anterior
chamber of the eye, &c., of artificial subcultures far
removed (by many generations) from the original source,
typical tuberculosis is produced in all animals susceptible to
tubercle (guinea-pigs, rabbits, dogs, rats, mice), and that of
course the tubercular deposits in these experimental animals
again contain abundantly the tubercle bacilli ; thus the final
and exact proof that the tubercle bacilli are the vera causa
of the tubercular process was definitely established. The
intravascular and intra-peritoneal injection produced the
most striking and rapid results.

Although the growth on glycerine Agar mixture is copious,
it yet has this drawback, that by continued subculture the
virulence of the bacilli is worn off. The first subcultures 4
act virulently, inasmuch as they produce on inoculation into
guinea-pigs general tuberculosis ; thus even with a fourth
and fifth subculture I have succeeded in producing the
same results as by directly using sputum or bovine tubercle,
but after the eighth or tenth generation I have not succeeded
in producing general tuberculosis and death of the guinea-
pigs by inoculation. I have found that if from an Agar-
glycerine culture, which, owing to age or subcultures, has
lost completely its virulence, new cultures are established in
alkaline beef broth, to which a piece of boiled white of egg
is added, these acquire rapidly again a somewhat virulent
character.

Also on Agar ascites fluid with glycerine ( see a former
chapter) the original virulence can be re-established. It
ought to be also stated that by continued subculture in
glycerine broth or in glycerine Agar the growth becomes
more abundant and makes its appearance in much shorter
time,

XIV]

BACILLUS TUBERCULOSIS

355

The subcultures on glycerine Agar show after several
months besides the typical forms of cylindrical and granular
tubercle bacilli also some filaments made up of rods and
granules. Some of these filaments are remarkable by their
being undoubtedly branched like the mycelium of a
hyphomycetes, and, further, that some are club-shaped at
the end or beaded in their course ; these club-shaped and
branched filaments (see Fig. 138) are the more numerous
the older the culture. Although the club-shaped and
beaded condition might correspond to involution of the
threads, the branched condition cannot, and therefore the
club-shaped forms may well represent the growing ends
of the threads of a mycelium ; the two together, i.e., club-
shaped and branched threads, would, therefore, indicate
that the typical tubercle bacillus is a phase only in the
development of an organism, which under certain conditions
(glycerine Agar) declares its true nature and origin, being,
namely, comparable to a fungus having a mycelial stage
(see Klein in the Reports of the Medical Officer of the
Local Government Board, 1889-90, Plate XXVII., Figs.
61, 62, 63).

Later, Mafucci (Archiv f Hygiene und Infect., xi. p. 445)
described the same forms in the culture of the tubercle
bacilli of the fowl, and Fischel (Fortschr. d. Med., Bnd. x.
No. 22, p. 908) also of the human tubercle cultures; and
this latter observer arrived at the same conclusion as
myself — viz. that we are dealing with forms which are
comparable to a mycelial fungus.

That the tubercle bacilli in one phase or another do
contain spores has been shown by Koch, who found that
tubercular sputum when thoroughly dried maintains its
virulent character. This has been confirmed by other
observers.

A A 2

356

MICRO-ORGANISMS AND DISEASE [chap.

Now, if the tubercle bacilli had no spores, they would
not in all cases survive thorough drying ; no sporeless
bacillus is known that can survive thorough drying ;
whereas all bacilli in the stage of spore-bearing survive this

Fig. 139. — From the same Kidney as in preceding figure.

a. Large artery filled with caseous matter, and in it numerous tubercle-bacilli.

b. Coat of artery.

c. Nuclei of the tuberculous new growth. d. A Malpighian corpuscle.

Magnifying power about 500.

process. Further, tubercular matter and cultures of tubercle
bacilli survive temperatures up to ioo° C. Non-spore-
bearing bacilli and micrococci are killed by being exposed
for five minutes to a temperature of 60-70 C., whereas

xiv]

BACILLUS TUBERCULOSIS

357

spores of other bacilli withstand much higher temperatures.
Tubercular sputum distributed in salt solution does not
lose in the least its virulence by being kept at ioo° C.
from one to two minutes. Nor does a solution of per-
chloride of mercury kill the tubercle bacilli in the way it
does sporeless bacilli. Dr. Lingard found (Report of
Medical Officer of Local Government Board for 1885-86,
p. 183) that solution of perchloride of mercury, one grain
of mercuric bichloride to 960 grains of water, that is to say,
about one in i,ooo, although it kills the bacilli in human

Fig. 140. — From the Juice of Lupus-Tissue prepared after the Koch-
Weigert Method of drying a thin Layer on a Cover-glass.

Magnifying power about 700.

tubercular matter when acting on it for four hours, does not
do so in the case of bovine tubercular matter, since not
even eight hours’ exposure to the solution is sufficient to
neutralise the infective power of that material. This also
shows what has been mentioned already on a former page,
viz., that bovine tubercular matter is of a higher degree
of virulence than human tubercular matter.

The Royal Commission on Tuberculosis issued in 1895
their Report (Part I.) and from the evidence given before
them and the extensive researches made for them by

358

MICRO-ORGANISMS AND DISEASE [chap.

Dr. Sidney Martin and Dr. Sims Woodhead they arrived
unanimously at the following conclusions (p. 20) :

“ We have obtained ample evidence that food derived
from tuberculous animals can produce tuberculosis in
healthy animals. The proportion of animals contracting
tuberculosis after experimental use of such food, is different
in one and another class of animals ; both carnivora and
herbivora are susceptible, and the proportion is high in
pigs. In the absence of direct experiments on human
subjects, we infer that man also can acquire tuberculosis
by feeding upon materials derived from tuberculous food-
animals.

“ The actual amount of tuberculous disease among
certain classes of food-animals is so large as to afford to
man frequent occasions for contracting tuberculous disease A
through his food. As to the proportion of tuberculosis
acquired by man through his food or through other means
we can form no definite opinion, but we think it probable
that an appreciable part of the tuberculosis that affects
man is obtained through his food.

“ The circumstances and conditions with regard to the
tuberculosis in the food-animal which lead to the production
of tuberculosis in man are, ultimately, the presence of
active tuberculous matter in the food taken from the
animal and consumed by the man in a raw or insufficiently
cooked state.

“Tuberculous disease is observed most frequently in
cattle and in swine. It is found far more frequently in
cattle (full grown) than in calves, and with much greater
frequency in cows kept in town cow-houses than in cattle
bred for the express purpose of slaughter. Tuberculous
matter is but seldom found in the meat substance of the
carcase, it is principally found in the organs, membranes,

Xivj BACILLUS TUBERCULOSIS 359

and glands. There is reason to believe that tuberculous
matter, when present in meat sold to the public, is more
commonly due to contamination of the surface of the meat
with material derived from other diseased parts, than to
disease of the meat itself. The same matter is found in
the milk of cows when the udder has become invaded by
tuberculous disease, and seldom or never when the udder
is not diseased. Tuberculous matter in milk is exception-
ally active in its operation upon animals fed either with the
milk or with dairy produce derived from it. No doubt the
largest part of the tuberculosis which man obtains through
his food is by means of milk containing tuberculous matter.

“ The recognition of tuberculous disease during the life
of an animal is not wholly unattended with difficulty.
Happily, however, it can, in most cases, be detected with
certainty in the udders of milch cows.

“ Provided every part that is the seat of tuberculous
matter be avoided and destroyed, and provided care be
taken to save from contamination by such matter the actual
meat substance of a tuberculous animal, a great deal of
meat from animals affected by tuberculosis may be eaten
without risk to the consumer.

“ Ordinary processes of cooking applied to meat which
has got contaminated on its surface are probably sufficient
to destroy the harmful quality. They would not avail to
render wholesome any piece of meat that contained tuber-
culous matter in its deeper parts. In regard to milk we
are aware of the preference by English people for drinking
cows’ milk raw, a practice attended by danger, on account
of possible contamination by pathogenic organisms. The
boiling of milk, even for a moment, would probably be
sufficient to remove the very dangerous quality of tuber-
culous milk.”

360

MICRO-ORGANISMS AND DISEASE [chap.

In August 1890, on the occasion of the International
Medical Congress held in Berlin, and in subsequent publi-
cations, Koch announced that by experiment on guinea-
pigs he had ascertained that when guinea-pigs, previously
made tubercular by subcutaneous inoculation, be inoculated
again with extracts (glycerine extract) of sterilised tubercle
cultures, the growth itself from the surface of serum or
glycerine Agar being rubbed down and extracted with
dilute glycerine, or with the filtrate of glycerine broth cul-
tures (previously sterilised), the tubercular glands undergo a
rapid necrosis and elimination, brought about by an acute
reactive inflammation setting in in the tissues around the
tubercle, but the tubercle bacilli themselves are not affected
by it. He then applied this method of injecting glycerine
extract of tubercle cultures — tuberculin — in very small doses, ^
o-ooi-o'oi gramme, on the human subject, lupus, bone
tuberculosis, early pulmonary tuberculosis. The result was
remarkable, since most patients affected with one or another
form of tuberculosis reacted very conspicuously to such
injection, high temperature, great local congestion and
inflammation in lupus and bone tubercle ; persons not
affected with tubercle, as a rule, not showing any reaction
to such small doses. In lupus, bone and joint tubercle,
the tubercular tissue becomes necrotic, is either sponta-
neously eliminated, as in lupus, by the reactive inflammation
of the surrounding tissue, or can be removed by surgical
aid, as in tuberculosis of bone. Tuberculin is, then, a
distinct means of diagnosing tubercle, otherwise not easily
diagnosed.

Weyl has analysed the tuberculin, and found that the
essential portion of it is a substance related to mucin, not
to albumin.

Good therapeutic results have been obtained with the

Xiv] BACILLUS TUBERCULOSIS 361

tuberculin in lupus, bone tuberculosis, and in early pul
monary tuberculosis; in advanced pulmonary tuberculosis
the injection of tuberculin has in some cases produced a
dissemination of the tubercle bacilli and acute miliary
tuberculosis in the lung and other viscera (Virchow). The
same or similar results had been obtained in tuberculised
guinea pigs by Baumgarten after injection of tuberculin.

Fig. 141. — From a Section through Leprous Skin, showing numerous
Leprosy Bacilli in Cells and between them.
x 500.

With Koch's tuberculinum a large number of observations
have been made in all countries as to its diagnostic value in
bovine tuberculosis, and the result is overwhelmingly in
favour of it, since by the positive reaction (raised tempera-
ture and constitutional disturbance) produced in a given
animal after subcutaneous injection of tuberculinum, it is
possible to diagnose tuberculosis even when other (physical

362

MICRO-ORGANISMS AND DISEASE [chap.

and clinical) signs are wanting. And although the results
of the use of tuberculinum for diagnostic purposes are not
absolutely uniform, they are nevertheless sufficiently striking
to consider such use as of paramount importance.

Bacillus Leprcc. — Armauer Hansen 1 first ascertained the
existence of large numbers of minute bacilli in the peculiar

Fig. 142. — From a Section through Leprous Shin, showing the Leprosy
Bacilli. (E. C. Bousfield.)

X 1000.

large leprosy-cells of Virchow, which constitute the nodules
of leprous patients. Neisser2 confirmed this, and con-
siderably extended our knowledge of the bacilli, showing
that they can be readily stained with fuchsin or with
Ehrlich’s acid solution of eosin-hsematoxylin. The bacilli

1 Virchow’ s Archiv , vol. Ixxix. ; and Quart. Journ. of Micro. Sci.
1S80.

2 Breslauer drztl. Zeitschr xx. and xxi., 1877, and Virchow’ s Archiv,
84.

xiv]

BACILLUS TUBERCULOSIS

363

are stiff rods about 4 to 8/j. long and less than 1 fj. thick.
They are pointed at their ends, and always occur in masses
within the large leprosy-cells of the leprous tubercles of the
skin and internal organs. But they are also present in the
interstitial tissue of the nervous branches in the anaesthetic
variety of the disease.1 Some bacilli are motile, others

Fig. 143. — From a Section through the Larynx of a Patient dead of

Leprosy.

Huge cells in fibrous connective tissue ; the cells are filled with the leprosy bacilli.
Magnifying power 600. (Stained with magenta and vesuvin.)

not ; some are more or less granular and beaded, owing
to local collections of the protoplasm within their sheath.

Cover-glass specimens made from a scraping of a leprous
nodule or the discharge of a leprous ulcer by spreading out

1 Compare also Comil, Union Medicale, 1881, Nos. 178, 179, and
Babes, Archives de Physiologic , July, 1883.

3<M

MICRO-ORGANISMS AND DISEASE [chap.

a thin film on a cover-glass, drying and heating then stain-
ing after Ehrlich’s method of staining for tubercle bacilli
(in carbol fuchsin for twenty to thirty minutes at 350 C.,
then washed in water, then for a few seconds in 33 per
cent, nitric acid, washed again in water, dried, and mounted)
show the leprosy cells, some small, some very big, all
crowded with the stiff, thin, and relatively long bacilli
leprae. Many cells are in a state of disintegration, or
broken down into granular de'bris and in accordance with
this numerous bacilli are found free, isolated, or in groups.
The large and middle-sized cells are particularly interesting,
since their substance is almost entirely occupied with the
bacilli arranged in bundles, which bundles often lie towards

Fig. 144. — From an artificial Culture of Bacillus of Leprosy.

(After Neisser.)

one another under sharp angles, and hereby produce a
very striking effect. Sections through a leprous tubercle
stained in the above manner (in carbol fuchsin passed
through 33 per cent, nitric acid, washed in water, then
counter-stained in methyl blue anilin water for fifteen to
thirty minutes) show the nuclei of the tissue blue, the cells
forming the leprous nodule red, owing to the fact that their
substance is crowded with the (red) leprosy bacilli ; in such
sections nothing can be seen of the nuclei or substance
of the leprosy cells, the cells being marked merely as
groups of densely aggregated leprosy bacilli (fig. 143).
While, then, the lepra bacilli have characters in staining by
which they resemble the tubercle bacilli, they differ accord-

XIV]

BACILLUS TUBERCULOSIS

365

ing to Baumgarten and others in this, that they stain in
alkaline methylene blue with conspicuously greater difficulty
than the tubercle bacilli.

Neisser has shown that the characteristic leprosy cells are
only wandering cells or leucocytes modified by the growth
and multiplication in them of the bacilli. In the blood the
bacilli do not occur, and they spread probably only by way
of the lymphatics.

Fig 145.— From a Section through a Nodule of the Liver of Rhea.
j. Cells of various sizes filled with minute bacilli ; owing to the smallness of the
bacilli and to their being crowded in the cells and owing to the comparatively
low magnifying power (300) the bacilli appear like dots.

(Stained with fuchsin and methyl-blue.)

Inoculation experiments on domestic animals and monkeys
have hitherto failed.1 Damsch 2 maintains, however, that
he was able, by inoculation with leprous tissue into the
< peritoneal cavity and into the skin, to produce in cats a
distinct increase and sprouting of the bacilli.

1 Kobner, Virchow's Arckiv, vol. lxxxviii. ; Hansen, ibidem, vol. xc.

2 Virchow's Archiv, vol. xcii.

366

MICRO-ORGANISMS AND DISEASE [chap.

Neisser maintained that he had succeeded in cultivating
the lepra bacilli, but the evidence he adduced is not deemed
sufficient. Bordoni-Uffreduzzi ( Zeitschrift f. Hygiene, Hi. ,
p. 178) maintains, however, positively that he has pro-
duced artificial cultures from the leprosy nodules of bone
marrow, on glycerine serum to which peptone and salt had
been added, kept at 35-3 70 C. The line of inoculation
became marked as a yellowish irregularly outlined band ;
the serum was not liquefied. On glycerine Agar, inoculated
with considerable quantity of leprous material, the same
kind of growth took place. In glycerine Agar plates the
colonies that grew on the surface and in the depth, seen

Fig. 146. — Two Cells of the Leprosy (?) Noddles in the Liver of a

Bird (Rhea).

The cell-substance is crowded with minute bacilli, similar to leprosy-bacilli.

Magnifying power 700. (Stained with magenta.)

under a magnifying power of 100-200, were rounded reti-
culated patches, with dark thick centre.

In a section through the liver of a bird {Rhea) that
died in the Zoological Gardens in London, prepared by
Dr. Gibbes after his method of staining for tubercle-bacilli,
there were seen innumerable aggregations of larger and
smaller pink masses (visible to the unaided eye as dots of
the size of a pin’s point to that of a pin’s head or millet-
seed, and larger). Under the microscope these pink masses .
were seen to be composed of cells of various sizes, each
filled with an enormous number of what appeared under a
high power very short bacilli, much shorter than tubercle-

XIV] BACILLUS TUBERCULOSIS 367

bacilli. But they gave the same reaction as tubercle-bacilli.
Here and there isolated cells of various sizes could be seen
filled with the bacilli. In the large cells the cell-outline
was becoming indistinct, and in some the cell-substance was
seen to break down, whereby the bacilli became free. In
these respects, in the size, distribution, and character of the
bacilli, there exists a remarkable similarity between the
nodules in leprosy and the nodules just mentioned.

CHAPTER XV

ANAEROBIC BACILLI

The group of microbes which we now proceed to describe
comprises several species of specific pathogenic bacilli which
have the following characters in common: — (fyThey are
obligatory anaerobic, growing therefore best, in fact growing
only, when not in contact with air (oxygen) ; (2) they are
strong gas-formers — methan or marsh gas ; (3) they are
cylindrical bacilli, more or less capable of forming chains
and filaments ; (4) they are motile, and possessed of several
and sometimes numerous flagella ; (5) they form bright oval
spores (endospores) thicker than the bacilli, which spores
just like those mentioned of other bacilli (bac. subtilis, bac.
mesentericus, bac. anthracis) have a great resisting power to
heat, so that while the sporeless bacilli are killed by heat of
65-70° C. in ten or five minutes respectively, the spores do
not lose their germinating power by being heated up to
8o°-85° for ten or fifteen minutes ; (6) they grow well in
the depth of grape-sugar gelatine and liquefy this.

They differ among themselves in (a) the nature of the
disease they cause in the animal body, (b) their distribution
in the body of the infected animal, (c) the nature and
rapidity of their growth in artificial media, (d) the rapidity

CHAP. XV]

ANAEROBIC BACILLI

3 r>9

of the liquefaction of grape-sugar gelatine, ( e ) the size of the
bacilli and spores and the position of the latter in the former,
and (/) the distribution and number of flagella and the
greater or lesser tendency of the bacilli to form filaments.

To this group belong : the bacillus of malignant oedema
of Koch, the bacillus of tetanus, the bacillus of symptomatic
charbon, quarter evil, or Rauschbrand, and the bacillus
enteritidis sporogenes.

Material, containing the spores of either of the above
microbes, is transferred to sterile grape-sugar gelatine con-
tained in a test-tube plugged with sterile cotton-wool — the
gelatine to the height of about four inches — the test-tube is
then placed in water of which the temperature is main-
tained at 78-80° C. for from ten to fifteen minutes, then in
cold water so as to allow the gelatine to set, and after
sealing the top with gutta-percha paper is finally incubated
at 20-21° C. After twenty-four hours or latest after thirty-six
or forty-eight hours a number of spherical colonies are
noticed in the deeper parts of the sugar gelatine, the
rapidity with which these grow, their general aspect, and the
rapidity and nature of the liquefaction differ for the dif-
ferent species. As the colonies increase in size they become
gradually confluent, and the liquefaction of the gelatine
extends till the whole is liquefied, at the same time more or
less copious gas evolution takes place, the gas bubbles being
on the top of the liquefied gelatine, and when the liquefac-
tion has extended to the top the gas bubbles escape into the
space between the gelatine and cotton-wool plug.

In order to obtain uniform and characteristic growths in
sugar gelatine tubes from an already established pure
gelatine culture, the method of inoculation described in a
former chapter as the capillary pipette method is by far the
most reliable and best : by means of a freshly drawn-out

B B

370 MICRO-ORGANISMS AND DISEASE [chap.

capillary glass pipette a droplet of the liquefied culture
material is drawn up and allowed to ascend into the end of
the capillary glass pipette that had been pushed down into
the liquefied culture, or if it does not ascend is drawn up by
gentle aspiration at the other end of the capillary pipette ;
this so charged capillary tube is then withdrawn and pushed

Fig. 147. — Stab Culture of Bacillus of Malignant CEdema in the Depth of
Sugar Gelatine incubated for three Days at 20° C.

The growth is indicated by a cylinder of liquefied, slightly turbid gelatine ; on the top
of the growth a gas bubble.

Natural size.

down into the lowest part of the fresh sugar gelatine tube,
and a trace of the material is by blowing forced out, the
capillary tube is withdrawn, the plug replaced, and the top
part of the new gelatine liquefied by holding this part of the
culture tube over the flame till the gelatine at this point
bubbles ; the tube is then placed in an upright position in

XV]

ANAEROBIC BACILLI

3/1

cold water in order to set the top layers of the gelatine
quickly; after this the tube is sealed with gutta-percha
paper and placed in the incubator. The result will be
apparent in twenty-four to forty-eight hours by the appearance
of a linear growth in the deep parts, which, as time proceeds,
enlarges and shows all the differential characters of aspect,
progress, and liquefaction.

Fig. 148. — Stab Culture of Eacili.us Enteritidis Sporogenes in deep Sugar
Gelatine, incubated for forty-eight Hours at 20° C.

Liquefaction has proceeded very rapidly ; the liquefied gelatine is fairly translucent ;
at the bottom is a fluffy floccular mass, on the top is a gas bubble.

Natural size.

In Figures 147, 148, 149, and 150 four such stab cultures
in the depth of grape sugar gelatine are shown in which the
inoculation had been carried out by the capillary glass
pipette method, and from these will be seen the uniformity
of this method and the striking differences noticeable
between the four species here dealt with. In all four tubes

b b 2

372 MICRO-ORGANISMS AND DISEASE [chap.

liquefaction is proceeding along the line of growth, but at
greatly different rates.

From a liquefied sugar gelatine culture a subculture is
easily made by rubbing a liberal amount of the material
over the slanting surface of solidified grape sugar Agar and
o'5 per cent, formate of soda (Kitasato and Weyl) ; this is

Fig. 149.— Stab Culture of Bacillus Tetani in the Depth of Sugar
Gelatine, incubated for three Days at 20° C.

The growth is indicated by a spindle-shaped mass of threads extending laterally ; the
gelatine is liquefied to the extent of the growth.

Natural size.

then placed after Buchner’s method in a glass tube contain-
ing for each gramme of pyrogallic acid one cc. of liquor
potassse closed by a well-fitting indiarubber plug (see a
former chapter) and incubated at 37° C. As soon as
colonies make their appearance on the Agar surface, a little
of it can be withdrawn by the platinum needle or loop, and

xv]

ANAEROBIC BACILLI

373

used for flagella staining ( see a former chapter), or the
culture can be left to go on for some time till spores have
made their appearance ; in some of the above microbes
spore-formation does not occur in the grape sugar gelatine
(malignant oedema, symptomatic charbon) and for this reason
cultures on solid media (stab culture in grape sugar Agar, or,

Fig. 150. — Stab Culture of Bacillus of Symptomatic Charbon in Sugar
Gelatine, incubated for three Days at 20° C.

The growth is a cylinder of turbid liquefied gelatine with lateral outgrowths.

Natural size.

better, the just mentioned culture on the slanting surface of
sugar Agar) must be resorted to.

Besides the above media, glycerine broth peptone and
broth peptone (ordinary nutrient broth) are useful for
obtaining copious growths. These are employed when it is
required to obtain the toxins produced by the microbe
during its growth. Thus in the case of bacillus of tetanus

374

MICRO-ORGANISMS AND DISEASE [chap.

important experiments have been made by Behring and
Kitasato, Kitasato, Roux and Vaillard as to the nature and
action of the tetanus-toxin (see Immunisation and Antitoxin),
obtained in the filtrate of broth cultures.

Different kinds of flasks and test tubes have been
designed which permit replacing the air above the broth

Fig. 151.— Film Specimen of Subcutaneous (Edema Fluid of a Guinea-pig
dead of Malignant (Edema ; Filamentous Forms of the Bacillus of
Malignant (Edema.

X 1000.

(after inoculation) by hydrogen gas — generally the flask or
test tube possesses a lateral tube so as to allow of this replace-
ment being easily effected. I find, however, that the
anaerobic microbes grow7 well in ordinary flasks and test
tubes, provided there is a large amount of the broth (three-
quarters or more of the volume) and the flask or test tubes

XV]

ANAEROBIC BACILLI

375

are sealed with gutta-percha paper immediately after
inoculation.

i. Bacillus ad em at is maligni (Koch). — This disease has
been produced by Koch in guinea-pigs by the subcutaneous
injection of recently manured garden earth. An extensive
oedema occurs at and about the seat of inoculation • the
oedema is accompanied by haemorrhage into the sub-

Fig. 152.— Bacilli of Malignant CEdema containing Spores, chiefly

SITUATED IN THE MIDDLE PART OF THE BACILLI.

X IOOO.

cutaneous tissue, and is of an offensive odour ; it spreads
during the second day, leads to gangrene of the sub-
cutaneous and muscular tissues with the formation of gas
bubbles, and the animals die in from twenty-four to forty-
eight hours : the spleen is found congested, so also are the
liver, kidney, lungs, and intestines. In the oedematous
exudation and in the spleen long mobile bacilli are present,
either singly or in filaments and long chains ; their number

376

MICRO-ORGANISMS AND DISEASE [chap.

in the blood is comparatively small immediately after death,
but they soon multiply therein and if the examination of the
blood (heart’s blood) is delayed, the bacilli may be found
present in considerable numbers. The bacilli do not stain
after Gram. The size of the short bacilli is 2-3 ’5 //. in length,
and 1 //. in thickness ; their ends are more or less rounded.
If the flagella are stained, it is seen that the bacilli possess
several (4-5) flagella attached laterally near the ends of the
bacillus. Many bacilli are in the form of chains and filaments.
The cedematous fluid, and the blood, inoculated into fnjsh
guinea-pigs produce the fatal disease.

Rabbits are also very susceptible to the disease ; and at
the seat of inoculation oedema is produced. Mice are very
susceptible, and die before the end of the first day, but no
oedema is present at the seat of inoculation. All the viscera
are congested, and the spleen is enlarged ; the blood of the
spleen, the exudation of the peritoneum, and the pleura,
contain the bacilli. A sure diagnosis, and differentiation
from anthrax bacilli, to which the oedema bacilli bear a
certain likeness, can with certainty be made by cultures.

The cultural characters of this bacillus show that it is
altogether different from that of bacillus anthracis ; although
in size and general aspect in the fresh state, and in stained
cover-glass specimens, it is not unlike, in its action on
animals, in the condition of the spleen of the inoculated
animals, and in its small numbers in the blood of these
it is quite different from bacillus anthracis. When culti-
vated it shows the following characters : The oedema
bacillus is anaerobic, since it does not show growth on
the surface of nutritive media ; it grows only when planted
in the depth ; in grape sugar gelatine (in the depth) it
forms characteristic globular colonies of different sizes,
opaque and liquefied, their margin more opaque than the
centre and finely striated. The growth and liquefaction

xv] ANAEROBIC BACILLI 377

proceed gradually and slowly, till all the gelatine is lique-
fied; at the bottom of this is a voluminous greyish-white
filamentous mass. It grows best in gelatine to which 1-2
per cent, of grape sugar has been added. In solidified
sugar Agar it grows well, producing uniform turbidity all
through the medium, with floccular condensations and

Fic. 153. — Stab Culture of Sugar Gelatine with the Bacillus of Malignant
CEdema ; numerous Gas Bubbles are shown above and in the Liquefied
Growth.

Natural size.

numerous gas bubbles. Solidified blood-serum is liquefied
by the bacillus. The cultures act virulently on animals,
provided comparatively large quantities are injected.

Oval bright spores are formed in the short bacilli, either
in the middle or at one end ; the spores are thicker than
the bacilli themselves ; and some of the bacilli in the

378

MICRO-ORGANISMS AND DISEASE [chap.

cedematous fluid contain spores, particularly if the examina-
tion be delayed after death. The oedema bacillus is of great
importance, since by the observations and experiments of
Chauveau and Cornevin, Brieger, and others, it has been
shown that surgical gangrene (progressive gangrenous em-
physema) in the human subject is caused by the same bacillus.
It seems that many a soil containing putrid animal substances,
such as hay dust, rag dust, offensively smelling filth of dustbins,
offensively smelling exudations, gangrenous discharges, See.,
contains the oedema bacillus or its spores. Horses, pigs,
and sheep are susceptible to this malignant oedema, pro-
vided large doses are inoculated ; cattle are not susceptible.
As mentioned above, guinea-pigs are the best experimental
animals, since inoculation produces a typical, emphysemat-
ous, spreading oedema, with fatal result.

Pasteur has studied this septicsemia on guinea-pigs, and
it is also called Pasteur’s septicsemia, and the bacillus is
called by him vibrio septique. Roux and Chamberland
have demonstrated in the broth cultures of this microbe
chemical substances which separated by filtration from the
bacilli and injected into animals cause a transitory illness
proportionate to the amount injected, and hereby confer
immunity against the injection of the virulent bacilli them-
selves. But this immunity does not last long, and is not
produced if too small quantities are used.

Fliigge has isolated from recently manured garden earth
a pseudo-malignant oedema bacillus which resembles Koch’s
malignant oedema bacillus, but is non-pathogenic.

2. Bacillus tetani. — Carle and Rattone ( Giorn . dell. r.
Accad. d. Med. Toj'ind, 1884) were the first to show that
tetanus is a communicable disease. They succeeded in
producing typical tetanus, terminating fatally, by inoculating
into rabbits pus taken from the ulceration of a human

xv]

ANAEROBIC BACILLI

379

being in whom tetanus had set in. Purulent exudation
was taken in these rabbits from the place of inoculation and
transferred to fresh rabbits, and here typical tetanus was
again produced. In human tetanus the place of infection
(in the skin of the hand, foot, or other part, by a tainted
splinter, earth, or other material) becomes marked as a
purulent inflammation leading to ulceration ; the tissue
surrounding the ulceration is much infiltrated, and there is
always hcemorrhage in it. After death the membranes of
the brain and cord are found much injected, and so also the
grey matter of the medulla and cord ; occasionally there is
a slight accumulation of red and white blood-corpuscles
around the vessels.

Nicolaier ( Tuattgural Diss., Gottingen, 1885) made the
important discovery that earth taken from superficial layers
of the soil is often capable of producing, when inoculated
into the subcutaneous tissue of the mouse, rabbit, or guinea-
pig, a local suppuration and haemorrhagic effusion about the
seat of inoculation, rapidly followed by typical tetanus and
death. In that earth and in the pus and exudation of the
seat of inoculation he demonstrated the constant presence
of fine, straight “ drumstick ” bacilli, which he considered
as the teta?ius bacilli. The purulent matter containing these
bacilli, inoculated into fresh mice, rabbits, or guinea-pigs
again produces tetanus. Rosenbach ( Archiv f. klin.
Chirurgie , Band xxxiv., 1886) showed that the same bacilli
exist in the exudation at the place of infection in human
tetanus. Hochsinger, Beumer and Peiper, Bonone, Shake-
speare, Raun, and many others have confirmed the existence
of these bacilli in tetanus, but no one of these succeeded in
cultivating them in pure cultivations. Though numerous
cultivations have been established, and tetanus been pro-
duced in animals with them by the aid of foreign bodies —

380 MICRO-ORGANISMS AND DISEASE [chap.

cotton-wool, splinters soaked with the cultures — yet
these cultivations were always in an impure state, until
Kitasato ( Zeitsclirift f. Hygiene , Band vii., p. 225) has
succeeded in cultivating the tetanus bacillus of Nicolaier in
pure cultivations and in producing tetanus with such pure
cultures. Minimal doses inoculated into mice produced
tetanus in twenty-four hours, death in two to three days.

Fig. 154. — Film Specimen of Bacillus Tetani from a Culture in Sugar
Gelatine; some of the Bacilli show a terminal Spore, “Drumsticks.”

X 1000.

In the case of rats, rabbits and guinea-pigs the dose had to be
somewhat larger, 0-3-0 ‘5 cc. of broth culture. Rats and
guinea-pigs are ill with tetanus already after twenty-four to
thirty hours, rabbits not before two to three days. On
post-mortem examination of such animals there is no sup-
puration at the seat of the inoculation, but only hypersemia ;
hence the suppuration observed in other cases is not an
essential feature, and in former experiments and in the case

xv] ANAEROBIC BACILLI 381

of human beings is probably only due to the presence of the
foreign bodies themselves (earth, splinters, &c.) which were
the vehicles of the tetanus bacilli; in the internal organs
there is no definite change. In the organs there are no
bacilli present, nor was it possible to produce tetanus in
other animals by inoculating them with the cord, nerves,
blood or spleen of the animals dead of tetanus. In rabbits

Fig. 155.— Similar Specimen as in preceding figure.
x 1000.

Kilasato produced typical tetanus by injection of o-5 cubic
centimetre of broth culture of the tetanus bacillus into
the vein of the ear. Also by injection of the culture after
trephining into the dura mater Kitasato produced typical
tetanus ; but neither in the brain nor in the cord, nor in the
blood or other viscera of these animals, could the tetanus
bacilli be found.

382

MICRO-ORGANISMS AND DISEASE [chap.

The tetanus bacilli are straight cylindrical bacilli, in
culture varying between ip. and 4/;., occasionally forming
longer chains and filaments ; in the fresh state they show slug-
gish motility, but in suitably stained specimens show numerous
flagella, often arranged in bundles (see Figs. 21, 22, 23).
They form rapidly (already in 30 hours at 370 C.) a terminal
spore which gives them the shape of a “ drumstick ” ; the
spores are at first spherical, later oblong, bright and glisten-
ing. Pus containing the tetanus virus preserves its virulence
in the dried state for many months, owing to the presence, of
the spores (Kitt).

The bacilli stain well after Gram.

The success of obtaining pure cultures of the tetanus
bacilli was achieved by Kitasato by cultivating tetanus pus
anaerobically ; preliminarily to this he watched Ifer the time
when in ordinary cultures of the tetanus pus on serum or
Agar amongst the different species of bacteria present he
found such which by their peculiar shape and containing a
terminal thick spore — “ drumstick forms ” — he recognised as
the tetanus bacilli. By exposing such impure cultures three-
quarters to an hour in water at 8o° C. all bacteria were killed
except those spores. With material thus treated he made
gelatine plate cultures, and gelatine tube cultures, but in such
a way that the air was excluded by filling them with hydrogen
gas or by planting the bacilli into the depth of the gelatine.
Under these anaerobic conditions he obtained pure cultures.

It appears, then, from these exact researches that the
introduction of the tetanus bacilli under the skin is followed
by the production by them of a chemical virus, which, as it
is being produced at the seat of inoculation,' is absorbed
into the system and sets up the disease ; but the bacilli
themselves appear to remain limited to the seat of inocula-
tion, and do not live in the blood or any other tissue, and

xv]

ANAEROBIC BACILLI

383

therefore only the seat of the inoculation contains the
infective principle, i.e., the bacilli ; for this reason brain,
cord, nerves, blood and viscera have no power to produce
infection.

The disease tetanus is then, like that of diphtheria, not a
true infection but intoxication.

Brieger has, as a matter of fact, isolated from the exuda-
tion at the seat of infection in human tetanus a toxic
principle, tetanin , the injection of which produces tetanus
symptoms in animals ; and Kitasato showed this to hold
good also for the tetanin obtained from the cultures of the
tetanus bacilli.

In his “Experimental Researches on the Poison of Teta-
nus ” ( Zeitschr . f Hygiene , x. 2) Kitasato gives a full account
of the influence of light, heat, drying and of various chemical
substances on the tetanus poison.

Behring and Kitasato 1 have shown that by repeated in-
jection of non-fatal doses — using at first small doses of
active culture or tetanus toxin, or by using attenuated virus
(by the addition of trichloride of iodine, carbolic acid) — it
is possible gradually to increase the amount of virulence of
the dose without causing a fatal issue in the experimental
animals (rabbits).

Hereby the animals were rendered insusceptible to fatal
doses of tetanus bacilli or tetanus toxin, and further it was
shown that the blood-serum of such (artificially) highly
immunised animals when injected into an otherwise sus-
ceptible animal (mouse) possesses the remarkable power of
neutralising the effect of a fatal dose of tetanus bacilli or
tetanus toxin injected before or after into that animal
(mouse). It is from these researches that the scientific

1 Deutsche Med. IVock., 1890, No. 49, and Behring, Zeitschrift f.
Hygiene und Infekt. xii.

384

MICRO-ORGANISMS AND DISEASE [chap.

basis for the use of blood-serum of animals, artificially im-
munised against tetanus, for the cure of human tetanus
— the antitoxic power of that blood-serum — is derived ;
researches carried on by Behring, by Roux and Vaillard
[A f males de V Institut Pasteur, 1893), and others have led
to the obtaining of tetanus antitoxin blood-serum both for
protective and curative purposes ( see Chapter on Im-
munity).

Tizzoni and Cattani, in a series of memoirs, had already
demonstrated the means by which animals possessed natu-
rally of slight or great susceptibility respectively can be
made altogether insusceptible to tetanus. Further, the
blood-serum of animals, made previously insusceptible,
injected into animals possesses a decided antitoxic action.
They have isolated from such blood-serum this substance —
the tetanus antitoxin — by precipitating with alcohol, drying
in vacuo, and dissolving in water. In four cases of human
tetanus, by the injection of the antitoxin of Tizzoni the
disease was arrested and the patients recovered [Centra lb l .
f. Bact. und Parasit., Band x., No. 24, p. 785.)

3. Bacillus of symptomatic charbon [quarter evil, Rausch-
brand ). — This disease affecting young cattle and sheep
occasionally produces great mortality amongst them, par-
ticularly amongst the former. Owing to its involving
chiefly one of the hind extremities in the form of a large
subcutaneous tumour, in which, on incision, a quantity of
sanguineous, discoloured, almost black fluid is shown, the
disease is called quarter evil or black leg. Owing to its
slight resemblance to anthrax (large tumour containing
serous sanguineous fluid) it is called in France charbon
symptomatique ; in Germany it is called Rauschbrand on
account of the emphysematous nature of the tumour, and
on account of the gangrenous nature of the infiltrated

xv]

ANAEROBIC BACILLI

385

tissues. The disease, when it appears, rapidly spreads
amongst young cattle and sheep ; rare amongst horses, it
is unknown amongst swine or poultry. The animals
affected are quiet, do not feed, and show high temperature ;
on one or the other quarters — generally one of the hind —
there appears a large diffuse swelling, on account of which

Fic. 156. — Film Specimen of Blood of a Guinea-pig dead after subcutaneous
Inoculation of the Bacillus of Symptomatic Charbon.

Blood discs and long chains of bacilli.

X 1000.

the animal is lame and cannot move that extremity. In
the course of thirty-six to forty-eight hours death takes
place. On post-mortem examination the tumour is seen to
be located subcutaneously ; here the connective and
muscular tissues are dark, almost black, gangrenous, and
contain a large quantity of sanguineous serum and a large
quantity of gas bubbles (said to be C02 and methane).

c c

386

MICRO-ORGANISMS AND DISEASE [CHAP.

The infiltration with sanguineous serous fluid extends for
some distance into the adjacent parts in the muscular tissue ;
in the viscera : congestion of the liver, spleen, kidney, and
particularly the subcutaneous lymph glands ; these, begin-
ning from near the tumour, are found swollen, dark, and on
incision a sanguineous fluid oozes out from them. The
spleen is only very slightly enlarged. Cover-glass specimens
of the subcutaneous and muscular infiltration at or near the
tumour, particularly of the subcutaneous lymph glands,
show in considerable numbers small motile bacilli 3-5 / x
long, and about 0-5 fx thick : they are rounded at their ends,
and some contain terminally a bright oval spore ; others
possess a terminal enlargement without spore (Bollinger,
Arloing, Cornevin, and Thomas1).

Immediately after death the bacilli are not easily demon-
strable in the heart’s blood because present only in small
numbers, though they can be shown to be present in the
liver, spleen, and kidney, but always more numerously if
some hours are allowed to elapse after death.

The exudation of the tumour or the surrounding muscular
tissue injected subcutaneously into guinea-pigs in compara-
tively large quantities (|— 1 Pravaz syringe) produces the same
kind of emphysematous gangrenous change with sanguineo-
serous exudation at or near the place of inoculation ; the
animals die between twenty-four to sixty hours, the internal
viscera show great congestion ; in the subcutaneous tumour,
in the blood of the heart, and in the juice of the viscera the
bacilli can be easily demonstrated ; in the blood and viscera
they are fairly numerous if some hours have elapsed after
death. Rabbits are far less susceptible than guinea-pigs.

If only a drop or two are injected the guinea-pigs, though
they become affected with the local disease, do not succumb,

1 Bull, de r Acad. Franfaise, 18S1.

xv]

ANAEROBIC BACILLI

387

but show themselves refractory against infection with large
quantities, such as in control animals would invariably
produce death.

Arloing, Cornevin, and Thomas have brought to light
various important facts connected with the action of the
bacilli. These authors cultivated the bacilli in broth, but
they found that the bacilli grow best in chicken broth,

Fig. 157. — Film Specimen of Spleen Juice of a Sheep dead of Symptomatic
Charbon, showing a few Nuclei and the Specific Bacilli.

X 1000.

glycerine and sulphate of iron, provided oxygen (air) is
excluded ; they are, therefore, true or obligatory anaerobic
bacteria. They grow well in grape sugar gelatine, but
must be inoculated into the depth of it. The character of
the growth in a stab culture in sugar gelatine has been
described already, and is shown in Fig. 150. Though
similar to that of the anaerobic bacillus of malignant oedema

c c 2

388

MICRO-ORGANISMS AND DISEASE [chap.

it differs from it in growing slower than this latter, the lique-
faction proceeds slower, and there are not present the
voluminous fluffy masses at the bottom of the liquefied
gelatine. The spores in the bacillus of symptomatic
charbon are generally situated terminally in the bacilli.

Arloing, Cornevin, and Thomas have shown that if small
quantities of the fluid of the natural tumour (muscle fluid)

Fig. 158. — Film Specimen of a Culture of Bacillus of Symptomatic Charbon

SHOWING THE OVAL Sl’ORES, ONE IN EACH BACILLUS SITUATED TERMINALLY.

X 700.

be injected subcutaneously into cattle only a local though
typical tumour is the result ; the animals recover, and then
are possessed of immunity against further inoculation with
otherwise fatal doses.

Further they found that three to five drops of the tumour
fluid injected into the vein of cattle — but without inocula-
ting the subcutaneous tissue around the vein — produce only
a transitory febrile disturbance ; the animals quickly

xv]

ANAEROBIC BACILLI

389

recover, and show themselves refractory against subcu-
taneous fatal doses. A safe mode of protective inoculation
used by these observers successfully on a large scale is this :
The tumour fluid (the juice of the gangrenous muscular
tissue) is rapidly dried at 32-350 C., then it is rubbed up
with water and heated to ioo° C. Another lot is treated in
the same way, but heated only to 85° C. ; the first lot
represents a first vaccine ( premier vaccin ), the second lot a
second vaccine ( deuxibne vaccin ) ; both can be dried and
sent to distances ; when required for use the dried matter is
rubbed up in 100 parts of water, and of this 1 cc. per animal
is subcutaneously injected. The premier (weaker) vaccine
must be used first ; after the lapse of about ten or twelve
days the deuxibne (stronger) vaccine is injected. Animals
thus twice vaccinated proved themselves completely pro-
tected against a fatal and virulent dose taken from the
natural tumour.

Though there exists, both as regards the pathology and
the microbes, a certain resemblance between the malignant
oedema and the charbon symptom at ique, this resemblance
is only superficial, and there can be little doubt that the
two diseases in their pathology, in their microbes, and their
transmissibility or non-transmissibility to certain animals
are totally different diseases. The differences between the
non-motile aerobic bacillus anthracis and the motile anaerobic
bacillus of symptomatic charbon morphologically, culturally,
and in their effect on the guinea-pig are very conspicuous.

4. Bacillus Enteritidis sporogenes.1 — During the night of
2 7th-28th October, 1895, there occurred suddenly an
epidemic of severe diarrhoea among the patients in the
wards of St. Bartholomew’s Hospital ; the number of cases

1 Illustrations of the morphology of this bacillus could not be got ready
in time for this edition.

39o

MICRO-ORGANISMS AND DISEASE [char

amounted to fifty-nine, of the twenty-eight wards of the
hospital fifteen were attacked.

The first cases occurred about midnight, the majority
about 2 a.m., and a few between 5 and 6 a.m. of October
28th. By noon of the 28th, the epidemic was practically
over, no further cases occurring. Dr. Andrewes, the sani-
tary officer of the hospital, has investigated the clinical and
etiological facts of this epidemic, and from his notes I
gather that the cases were not of the choleraic type, vomit-
ing and cramps being conspicuously absent. In all cases
the onset was sudden and consisted in abdominal pains
followed by copious watery evacuations with numerous
mucus flakes ; in the severe cases, the discharges were con-
siderable in amount and frequency and contained much
blood ; in such cases there was also prostration and even
collapse, all cases recovered.

Examining specimens of the evacuations under the micro-
scope, they were found to contain numerous red and white
blood corpuscles and crowds of bacteria. Amongst these a
very large number of oval glistening spores attracted atten-
tion ; these were either free, isolated and in continuous
masses, or they were contained within cylindrical bacilli,
each of these bacilli containing one spore nearer to one end.
These spores and spore-bearing bacilli were found abund-
antly in every one of the evacuations that had been
examined. As the occurrence of such an abundance of
spores and spore-bearing bacilli in the human intestine is an
unusual feature, special attention was directed to them and
cultivations were made to isolate them. The only two
known species of spores and spore-forming bacilli in the
intestine that had to be here considered were (1) the aerobic
Bacillus mesentericus and (2) the anaerobic Bacillus amylo-
bacter. The last-named could be at once excluded, from

xv]

ANAEROBIC BACILLI

39'

the fact that in the above microscopic specimens no Clos-
tridia could be discovered, Bacillus amylobacter being
noted for the clostridia forms of its sporing bacilli. The
first species, viz., the Bacillus mesentericus, could with pro-
bability be excluded from the microscopic examination of
fresh specimens alone, since its bacilli show conspicuous
motility, whereas in our cases the motility of the bacilli was
extremely feeble and could be recognised only on very few
examples. But the culture test soon proved that our spores
were not those of Bacillus mesentericus. Aerobic gelatine
and Agar plates, surface cultures on gelatine and Agar,
brought forth the colonies of Bacillus coli only ; the Bacillus
mesentericus being aerobic, if it had been present in the
evacuations, would undoubtedly have made its appearance
in these cultures. More direct proof, however, was ob-
tained by placing a mucus flake of the evacuation in gelatine
or Agar, heating these to 78-80° C. for ten to fifteen minutes,
then preparing ordinary aerobic plates and incubating them
at 20° and 37° C. respectively : no colonies of any descrip-
tion made their appearance. The spores of Bacillus mesen-
tericus, like other well-known spores (of Bacillus subtilis,
of Bacillus anthracis, of tetanus, of quarter evil, of malignant
oedema, &c.) when heated to 80° C. for ten or fifteen
minutes do not hereby lose their power of subsequent ger-
mination, although all non-sporing bacilli — e.g. , Bacillus
coli — are thereby killed. Since then in the aerobic plates of
the heated gelatine and Agar no growth took place, there
could not have been any Bacillus mesentericus present.
Besides the above aerobic, also anaerobic cultures were
made of the evacuations ; a flake was placed into grape-
sugar gelatine and grape-sugar Agar, heated to 78-80° C.
for ten to fifteen minutes, then allowed to set and incubate.
In both the sugar gelatine and sugar Agar cultures already

392

MICRO-ORGANISMS AND DISEASE [chap.

after twenty-four hours numerous colonies were noticeable
in the depth, those in the sugar gelatine were spherical
translucent masses of liquefied gelatine, those in the sugar
Agar whitish small dots not liquefying the Agar; at the
same time gas bubbles were present in connection with the
colonies, particularly in the sugar Agar. After forty-eight
hours the growth had so advanced, and the liquefaction of
the gelatine had become so extensive, that the lower half in
the test tube was completely liquefied, very slightly turbid
by the growth ; on the surface of the liquefied growth gas
bubbles may be present or they may be altogether absent,
in the depth whitish cloudy flakes (see Fig. 148).

Examining under the microscope such a liquefied culture
after two or three days’ growth it was found to be made up
of rod-shaped or cylindrical bacilli, generally singly or in
chains of two, three or more rods ; they were mostly appa-
rently stationary, but here and there feeble locomotion could
be noticed, consisting in a wobbling or rolling slightly progres-
sive movement ; but only few such motile bacilli could
be seen. In some of the bacilli there was present a bright
oval spore, occasionally in the middle, but oftener near one
end. In the floccular masses at the bottom of the culture
tube spore-bearing bacilli were numerous, and even occa-
sionally a free spore. After three or latest after four days
the whole of the sugar gelatine in the tube had become
liquefied by the growth, on the top there were gas bubbles ;
in the depth a white powdery precipitate which on micro-
scopic examination shows numerous free spores. When
such a culture is opened it has a distinct smell of butyric
acid ; when the liquefied gelatine is disturbed by moving in
it a platinum or glass rod, numerous gas bubbles rise up ;
the liquid when sucked up in a capillary pipette emits a con-
siderable amount of gas bubbles. When the culture tubes

xv]

ANAEROBIC BACILLI

393

are exposed to the light, numerous gas bubbles rise up and
collect on the surface. A large number of subcultures in
various media were made from the primary anaerobic sugar
gelatine cultures and of the results the following deserve
special mention.

(a) The rapidity of liquefaction of the sugar gelatine by
the growth stands in an inverse ratio to the amount of gas
bubbles escaping through the gelatine as the growth pro-
ceeds. If, after inoculation of the sugar gelatine by stab,
there are found, after one or two days’ incubation, nume-
rous gas bubbles distributed in the upper part of the
gelatine and escaping to the free surface, it may be pre-
dicted with certainty that the growth in, and the liquefaction
of, the gelatine in such a culture will proceed very slowly ;
and conversely, if after one or two days’ incubation the
progress of liquefaction in the depth (after inoculation of
the depth) is conspicuous, there is very little or nothing to
be seen of gas bubbles on the surface.

(£) The formation of spores stands in direct relation to
the rapidity of liquefaction ; in tubes in which the growth
and the liquefaction proceed very slowly, there are at no
time spores formed in the bacilli ; in old cultures of this
kind the bacilli are found as longer or shorter threads, some
undergoing involution and death by granular disintegration.
Whereas, in tubes in which liquefaction proceeds rapidly —
the whole of the gelatine liquefied in two to three days —
there is always copious spore formation.

(c) Milk inoculated with the bacillus and incubated at
370 C. shows, as a rule, already after twenty-four hours,
sometimes a little later, distinct changes consisting in the
separation of flocculi of coagulated casein from the slightly
turbid whey, numerous gas bubbles being present in the
creamy layer on the surface ; after forty-eight hours the

394

MICRO-ORGANISMS AND DISEASE [chap.

separation is complete, most of the casein fiocculi are on
the surface mixed with numerous gas bubbles, the cream
being so altered that only a thin layer of fluid yellow oil is
present on the surface of the culture. Examined under the
microscope, the clear whey is full of short cylindrical bacilli.
Spore formation in milk cultures is observed only when the
culture is made strictly anaerobically and there is no marked
spontaneous evolution of gas bubbles ; under these condi-
tions, whitish cloudy fiocculi are found in the whey, which
are full of spores.

(, d ) The spores do not lose their power to germinate -if
exposed to 8o° C. for fifteen minutes ; they are, however,
killed if immersed in boiling water for two minutes.

( e ) The cultures in gelatine, as also in milk, have a distinct
smell of butyric acid, this is more pronounced the older the
culture. ^

Cultures in sugar gelatine, as also cultures in milk, while
young, not more than a week old, when injected into the
subcutaneous tissue of guinea-pigs or mice prove virulent.
Half to three-quarters of a cubic centimetre of the liquefied
gelatine culture, or of the whey of a milk-culture, per 200
grammes body-weight of guinea-pig, injected under the skin
of the groin, causes distinct illness already in six to eight
hours : the animals are quiet, do not feed, they have
cedematous swelling about the seat of injection, and the
body temperature is lower than normal ; their muscular
movements become gradually greatly impaired, and they are
found dead between twenty and twenty-four hours. Smaller
quantities produce the fatal result in two, or even three
days, and very small quantities cause only temporary illness
and transitory local swelling.

On post-mortem examination, the subcutaneous and
muscular tissue of the groin, of the whole of the abdomen

XV]

ANAEROBIC BACILLI

395

and chest, and even of the neck, are found deeply con-
gested, separated from the skin by accumulations of gas,
and the tissues infiltrated with copious sanguineous mal-
odorous exudation. This, under the microscope, is densely
filled with rod-shaped or cylindrical bacilli, few of these
motile, most of them without motility. While the local
appearances produced in the animal by our cultures bear a
considerable general resemblance to those produced by
injection of Koch’s bacillus of malignant oedema, which, as
is well known, is also an anaerobic microbe, there exist
marked differences between the two ; in malignant oedema
the sanguineous exudation contains, besides cylindrical
bacilli, numerous characteristic, thread-like bacilli, in our
cases these threads are quite absent, besides, the bacilli of
malignant oedema are generally longer than those in our
case ; in malignant oedema most of the bacilli are actively
motile, in our cases very few are motile, and these only
feebly so. A further difference is brought out by the
examination of microscopic specimens, both of the cultures
and of the subcutaneous exudation, in which the bacilli
have been submitted to the process of staining after Gram.
While the bacillus of malignant oedema after staining with
the dye is decolourised by Gram, our bacillus retains the dye
well. The bacillus of malignant oedema does not cause the
rapid curdling of milk, as our bacillus does.

Another noteworthy difference between the two microbes
is the rapidity of liquefaction of sugar gelatine in anaerobic
cultures : although the colonies in sugar gelatine look alike
for both these microbes, our bacillus liquefies the gelatine
conspicuously faster than the bacillus of malignant oedema^
and the gelatine liquefied by the former is less turbid than
that by the latter. Also, in respect of flagella, a marked
difference is noticed between the two microbes. The

39^

MICRO-ORGANISMS AND DISEASE [chap.

bacillus of malignant oedema possesses numerous flagella
fastened along its cylindrical body, our bacillus possesses
flagella only near the rounded ends ; the short rods possess,
as a rule, flagella at both ends, one, two, or three at one, a
bundle of three to eight at the other end, and the flagella
are always attached at one point laterally to the rounded
end ; the cylindrical bacilli have one to three flagella at one
end. Some of the flagella are very long — six to ten times
the length of the bacillus — and spiral, others are shorter and
wavy. In a preparation in which the flagella are success-
fully stained (by Van Ermengem’s modification of Lofflej’s
method), besides those that are still attached to the bacillus
there are numerous flagella— single or in bundles, wavy or
spiral — which are free, that is, had become detached during
the process of preparation. It is certainly very surprising to
find in such specimens what a large number of1 bacilli do
possess numbers of long spiral flagella, and to compare with
this the extremely feeble motility shown by the few in the
fresh state ; from a flagella-stained specimen one would
conclude that the majority of the bacilli are possessed of
brisk motility, such a conclusion is, however, very con-
spicuously contradicted by actual observation.

A further difference between our bacillus and that of
malignant oedema is the distribution and morphology of the
bacilli in the infected animal : while in animals that suc-
cumb after infection with the malignant oedema bacillus
numerous bacilli are present in the spleen, many of them
as the characteristic threads, in our case the spleen contains
the bacilli very sparingly, and then only as short rods, and
considerable masses of spleen tissue have to be used for ob-
taining successful cultures ; the same applies to the blood of
the circulation. But in the size and the position of the
spores in the bacilli our bacillus closely resembles the

xv]

ANAEROBIC BACILLI

397

bacillus of malignant oedema. As in the case of the bacillus
of malignant cedema so also with our bacillus, larger doses
of culture are required for infection of guinea-pigs than of
the subcutaneous exudation, this latter on subcutaneous in-
jection proving more virulent than the artificial culture. In
our case, subcutaneous injection of five minims of the sub-
cutaneous exudation suffices to produce fatal infection within
twenty to twenty-four hours in a guinea-pig of 200 grammes
weight.

Spores alone, or cultures five to seven days old in which
spore-formation is nearly completed, do not act as virulently
as young cultures when injected subcutaneously into the
guinea-pig, larger doses of the former being required to pro-
duce the same result as smaller doses of the latter. Doses,
which taken from recen: cultures produce fatal results in
twenty to twenty-four hours, when taken from old cultures full
of spores produce only a transitory local swelling and transi-
tory constitutional disturbance. Neither mice nor guinea-pigs
are susceptible to infection by feeding with spores.

Injected into the peritoneal cavity of the guinea-pig the
bacilli of young cultures produce fatal results in six to eight
hours ; the peritoneal cavity containing after death copious

sanguineous exudation full of the bacilli.

M-

The size of the bacilli is, length 1 '6 to 4 8

,, ,, thickness o-8

,, free spores is, length i-6

,, thickness o-8 to 1

In size, shape, feeble motility, in the rapid liquefaction of
sugar gelatine, in the characteristic changes produced in
milk, our bacillus resembles the anaerobic Bacillus butyricus
described by Botkin,1 but Botkin’s bacillus differs from our

1 Botkin, “ Ueber einen Bacillus butyricus,” Zeitschrift f. Hygiene u.
Infeclionskrank., bd. xi. p. 421.

393

MICRO-ORGANISMS AND DISEASE [chap.

microbe by the character and aspect of its young colonies in
gelatine1 and Agar, and by not being pathogenic. Our
organism is as strongly pathogenic as that of malignant
oedema, from which, however, as pointed out above, it
differs, both morphologically and culturally, in several
important points.

Bacillus variola — vaccinia. — In the Report of the Medical
Officer of the Local Government Board for 1892-1893 I
described a peculiar extremely minute bacillus as occurring
in the calf-lymph and in human variola lymph during
the early phases; in the calf-lymph 72 to 96 hours after
vaccination, in the human variola during the third or fourth
day ; in both instances the lymph was collected aseptically
and only clear lymph and as much as possible without
any epidermal adnexa was used for film specimens ; after
heating and treatment with 30 p.c. acetic acid, for some
minutes, were subjected to prolonged staining in alcoholic
gentian violet. Some of the films of calf-lymph (collected
after removal of the epidermis as a whole) showed an
abundance of these minute bacilli, generally in small and
large masses ; some of the specimens look like film speci-
mens of an artificial culture (Figs. 159 and 160). Lymph
of early human variola vesicles showed the same bacilli, but
not so abundantly. Calf-lymph of later stages (five or six
days old) showed no bacilli or only here and there a trace.
In the bacilli, when abundant, forms may be recognised in
which some globules of the nature of spores were present,
in Fig. 16 1 this is shown in the bacilli magnified 2000. The
presence of these spore-like bodies and the absence of the
bacilli in the lymph of later stages led me to the conclusion
that we have here to deal with a spore-forming bacillus, and

1 The colonies of Botkin’s Bacillus butyricus grow slower, and are
in their early phases more opaque and distinctly filamentous.

XV]

ANAEROBIC BACILLI

399

that after the multiplication of the bacilli in the early phases
has reached its climax spores begin to be formed, and it is
these which prevail in the lymph of the later phases. This
would well accord with the known facts concerning the
preservation of the active principles, for it is established
that the active principle of vaccine is preserved in glycerine,
although as is also known pure glycerine acting for long
times is a germicide for cocci and sporeless bacilli ; likewise
lymph dried in thin layers on points preserves its efficacy
for long periods, although such prolonged drying would kill
all but spores.

Large numbers of species of microbes, have been described
as occurring in vaccine lymph : cocci, bacilli, torula, and
there is no difficulty in demonstrating by film specimens and
particularly by culture their occurrence in lymph collected
in the usual fashion — i.e. without special precautions in
avoiding surface or epidemic admixtures. My experience,
extending over a very considerable number of experiments,
is this, that from carefully and properly collected vaccine
lymph (humanised) such as is sent out by the Vaccine
Department of the Local Government Board, and such as
it is possible to collect aseptically from a calf-vesicle (after
scraping off the crust) from a percentage of tubes containing
proved active vaccinia, no cultures are obtainable in the
ordinary media (nutrient gelatine, nutrient Agar, sugar
gelatine, sugar Agar, solidified serum) although the cocci and
bacilli which are present and have been described in many
samples of vaccinia are easily cultivable in these media :
staphylococcus albus and cereus, staphylococcus aureus,
bacillus mesentericus, torula, &c. From my own observa-
tions, which are in complete accord with those of Dr. Cope-
man, I maintain that none of those ordinarily cultivable
microbes are an essential inhabitant in vaccinia, and can have

400

MICRO-ORGANISMS AND DISEASE [chap.

anything to do with its active principle. Now, the above
minute bacilli which I have described above as occurring
abundantly in early phases in calf-lymph — in some instances
so abundant that the lymph looks like a culture of them —
are not cultivable in the ordinary culture media. The very
lymph from which the specimens of Figs. 159 and 160 were
derived was tested by culture and by transference to the calf,

Fig. 159. — Film Specimen of Calf-Lymph 72 hours, Clumps of Minute

Bacilli.

How minute these bacilli are, is shown by the fact that the photogram is taken at a

magnification of 1000.

and while in this latter it produced typical vaccinia it failed to
produce any growth whatever in the culture media (solidified
blood-serum, glycerine Agar, ordinary Agar, sugar gelatine,
and ordinary gelatine). From these observations I concluded
that the above minute bacilli are most probably the microbes
of vaccinia. Dr. Copeman, who has worked at the same
subject, has completely confirmed the presence of these

xv]

ANAEROBIC BACILLI

40 1

bacilli in active lymph and their inability to grow in the
ordinary culture media.

L. Pfeiffer (Die Protozoen als Krankheitserreger, Jena,
1S90) describes the presence of coccidia in the epithelium
in variola, vaccinia, varicella, herpes zoster, and other
vesicular eruptions. From his description and the illus-
trations given by him (Figs. 28-34, pp. 88-99) he has no
doubt that they occur in the substance of the epithelial

Fig. 160. — From a similar Specimen as the preceding figure.

x 1000.

cells ; that here they (the coccidia) multiply by division,
and form in their interior the spores. It can be easily shown
that certain peculiar bodies do occur in the epithelial cells
in these affections, which bodies are not the typical ordinary
nuclei, and which can be brought out by various dyes, and
thereby can be differentiated both from the cell-protoplasm
and from the ordinary cell nucleus. In sections through
the vesicles of sheep-pox, as also of human small-pox, stained

D D

402

MICRO-ORGANISMS AND DISEASE [chap.

first with rubin and then with methyl blue, many of the
epithelial ceils in the region of the vesicle contain each an
oval or spherical homogeneous body, which by its pink colour
is well marked off both from the cell protoplasm and the
swollen and hydropic cell nucleus, both these being stained
blue ; but it is extremely difficult — and it seems premature
and improbable— to identify them as of the nature of
extraneous parasites, viz., coccidia : on the contrary, these
bodies look extremely like derivatives of the cell nucleus.

Fig. 161. — From a similar Specimen.
x 2000.

In 1892 Guarneri describes after inoculation of the cornea
of rabbits with variola and vaccinia in the epithelial cells of
the cornea a peculiar parasite, called by him Citofyctes. L.
Pfeiffer, J. Clarke, and Sicherer confirmed their occurrence
in the corneal epithelium under the same conditions.

Quite recently Ernst Pfeiffer ( Ccntralblatt f. Bald. u?id
Parasitenk. xviii., No. 25) describes the same bodies :

XV]

ANAEROBIC BACILLI

403

spherical, oval, crescentic, granular, spindle-shaped or
threadlike bodies of about the size of red blood discs, or
larger and smaller fractions of them, after a few hours to a
few days after insertion of the lymph, in the tissue of
the corneal substance or in the corneal epithelium. These
bodies stain in dyes somewhat like red blood discs or
haemoglobin masses, and to my mind, after reading the
description and illustrations given by E. Pfeiffer, the proba-
bilities are very great that these bodies are in reality blood
discs or part of. such, as well as nuclei of leucocytes, that
had been introduced into the cornea with the vaccine lymph.
What must appear extremely curious is — (1) the indefinite
shape and size of these bodies, and (2) the fact that accord-
ing to these observers they should be capable of growth and
multiplication in the rabbit’s cornea, which animal is well
known to be insusceptible to vaccinia.

It cannot be said from the facts adduced that we have
really to do with a living parasite ; it seems tc me for
the above reasons more probable that these bodies are not
parasites at all.

D D 2

CHAPTER XVI

VIBRIO AND SPIRILLUM

Vibriones are called those bacteria which have the shape
of a more or less curved cylindrical rod-comma bacilli,
and when after division the two new individuals remain
joined end to end they form a characteristic S-shaped mi-
crobe. Vibriones elongate and by repeated divisions and
the new elements remaining joined end to end produce
wavy, spiral, or corkscrewlike filaments or spirilla. Spirilla
may be uniform without being composed of jointed commas
or they may be composed of separate vibrios. Some
species of vibrios form uniform unsegmented spirilla, others
may have less tendency to do so or may produce short seg-
mented spiral chains. When growing in fluid some species
form readily long spirilla apparently showing no segmenta-
tion. There exist considerable differences both with regard
to the length of the spirals and the amount of curvature,
for in some media or in some species the comma bacilli or
vibrios form readily well twisted spirals, while in another
medium or of another species the spirilla are short, or if
long are only slightly wavy. Many of the species of vibrios
and spirilla are distinctly motile, and where flagella staining
had been applied have been seen to be possessed of one or

CH. XVl]

VIBRIO AND SPIRILLUM

405

two fine spiral or wavy flagella. Owing to the curved shape
their movement is always characteristically corkscrewlike,
and therefore already by observing their movement in the
fresh specimen (hanging drop) they can be recognised as
comma bacilli or vibrios. This is particularly striking in
the S-shaped forms.

The individual comma bacilli in stained and well-washed
specimens show the same distinction into sheath and proto-
plasm as was mentioned of the bacilli, and also the presence
of a vacuole in the middle of the individual comma bacilli
and the terminal easily stained collections of protoplasm.
Though in some species of bacilli, e.g., bacillus of glanders,
bacillus of diphtheria, there exist rods which are more or
less curved, they do not form spirals, and their curved
character is not permanent ; but in the true vibrios and
spirilla, however slight the curvature of some elements —
and in some species and under some media the curvature
of some of the elements is very slight indeed — they never-
theless are capable of forming spirals. Above all only
vibrios and spirilla form S-shaped forms, and the presence
of these is as typical a character as the formation of spirals
themselves. Anthrax bacilli growing on alkaline gelatine
assume occasionally a curved shape, while Finkler’s spirilla,
or those found in noma and in cholera Asiatica, appear in
some media only to show the very slightest curve ; but from
subcultures of the above anthrax bacilli in broth or gelatine
the typical straight anthrax bacilli result, while of the above
spirilla subcultures made in broth, in gelatine, &c., the
typical spirilla will be the result. This shows that the first,
though they may occasionally become curved rods, are not
spirilla but bacilli, and the latter, though the individuals
may occasionally appear almost straight, are not bacilli but
spirilla.

406

MICRO-ORGANISMS AND DISEASE [chap.

Similarly, some of the bacilli of proteus vulgaris, of
diphtheria, and of glanders are of a curved shape, but they
do not form S-shaped forms or spirilla. Cohn 1 has de-
scribed a number of vibrios and spirilla occurring in various
decomposing fluids.

(a) Vibrio rugula consists of rods of about 8 to 16 // in
length, and curved either like a C or like an S. They are
single, or form chains of two. Their protoplasm is always
slightly granular. They are found in putrefying organic

Fig. 162. — Vibrio Rugula
(after Cohn).

Fig. 163. — Vibrio Serpens,

ISOLATED (AFTER Cohn).

substances, and often form continuous masses, the indi-
viduals interlacing in all directions.

(b) Vibrio serpens. — This is also a septic organism, much
thinner and longer than the previous one, more wavy, as a
rule, curved into a single or double wave. The length
varies between 11 and 25 fj.. It is motile; and also
forms continuous masses, the individuals interlacing in all
directions.

1 Beitrdge s. Biol. d. PJlanzen, vol. ii.

XVI]

VIBRIO AND SPIRILLUM

407

spiral. Its length varies between 2 and 5 n ; it often forms
continuous felted masses ; it is motile.

Occasionally the spirilla grow to a great length — two,
three, and more of them forming a chain ; the individual

Fig. 164. — Vibrio Serpens in Swarms (after Cohn).

spirilla are not arranged in a linear series, but folded into a
zigzag. This form, which in reality is not a special kind of
spirillum, is called by Cohn 1 spirochceta plicatilis. The

Fig. 165. — Spirillum Tenue, (i) singly and (2) in Swarms (after Cohn).

spirillum found in the tartar of the teeth is of this form,
spirochceta dentico/a. But there exist all intermediate forms
between a single spirillum tenue and a spirochaeta. In
stained specimens the construction of the spirochaeta from

Beit rage zur Biologie d. PJlanzen , vol. ii

4o8 MICRO-ORGANISMS AND DISEASE [chap.

several spirilla tenua is very distinct in some, though not in
others.

(d) Spirillum undula is much thicker and shorter than
the former ; there are all forms between such as are only
half a turn to such as are of a whole turn of a spiral. It is
motile and forms chains of two or more elements, occurring

Fig. 167. — Spirillum Volutans
(after Cohn).

also in continuous masses, occasionally held together by a
hyaline interstitial substance.

(e) Spirillum volutans. — These organisms are giant
spirilla; long and thick, with granular protoplasm; 25 to
30 [x long ; motile, and with a flagellum at each end.

(f) Spirillum rosaceum. — I have seen on paste a spirillum,
morphologically identical with spirillum undula; it is of a

XV i]

VIBRIO AND SPIRILLUM

409

pale pink or rosy colour.1 It is motile, and forms a kind
of zooglcea, the individuals being closely placed and there-
fore producing a rosy colour of a more decided tint. Where
they form continuous masses, the naked eye can detect the
rosy tint.

(g) Spirillum sanguineum ( Ophidotnonas sanguined, Ehren-
berg). — This was observed by Cohn and Warming 2 in
pond-water. Morphologically it is identical with spirillum
volutans. It is motile, with a flagellum either at one or
both ends. Warming occasionally saw two and three
flagella at one end. It is about 3 yu. thick ; all forms occur
between such as have half and such as have two and a half
turns of a spiral. Lankester also saw the same kind of
organism among his peach-coloured bacteria.3

Ui) Spirillum rubrum (von Esmarch 4) forms long, very
motile spirilla, possessed of numerous flagella attached to
the sides of the spirilla ; it does not liquefy gelatine. Its
colonies are of a deep red colour.

(i) A variety of species of vibrios have been described
by Weibel 5 as occurring in sewage and on cultivation formed
coloured growths : vibrio aureus, flavescens and flavus ; none
of them liquefy the gelatine and are apparently not possessed
of motility.

(/') Elwers and also Dunbar have isolated a vibrio or
spirillum phosphorescens which in cultivation has the
power to form phosphorescence ; it liquefies gelatine, and
is motile.

(k) Dr. Lingard has found in, and I have isolated from,

1 “On a Rose-coloured Spirillum,” Quar. Journ. of Micr. Sci.,
vol. xv. New Series.

* Beitr. z. Biol. d. BJlanzen, vol. i.

3 Quarterly Jour, of Micr. Science, vol. xiii. New Series.

4 Centralbl. f. Bakt. und Parasil. , vol. i. , p. 225.

6 /bid. , vol. iv. , p. 258.

4io

MICRO-ORGANISMS AND DISEASE [chap.

the necrotic tissue of the tumour in noma of a child a motile
vibrio, which does not liquefy gelatine ; it forms on it in
streak a moist brownish growth ; in film specimens the
vibrios are found as commas, as S-shaped forms, and as
wavy or corkscrewlike longer or shorter spirilla. It grows
well at 37c C. on Agar and forms also here in streak a
brownish moist filmy growth. In gelatine plate it forms

Fig. 168. — Film Specimen of a Flake of a Rice-water Stool, showing the
Vibrios in Linear Rows ; one shows a Flagellum.

X 1000.

greyish round colonies which slowly enlarge, and after a
week or ten days are not more than a few millimetres in
diameter. {Bacteria in Asiatic Cholera. Macmillan, 1889,
p. 103.)

(/) Vibrio or spirillum cholera Asiatics (Koch), comma
bacillus of Koch. — Examining microscopically the intestinal
discharges of acute cases of cholera one notices, besides

XV l]

VIBRIO AND SPIRILLUM

4i 1

detached epithelial cells and lymph-corpuscles, numerous
bacteria belonging to different species of micrococci and
bacilli. Some there are amongst them which are comma-
shaped, t'.e., curved, cylindrical rods, single or double, or
S-shaped ; they are motile, spinning round or moving in a
spiral ; they are of different lengths and of different amount of
curvature, but, as cultivation experiments show, belong all to

X jooo.

the same species : namely, the comma bacilli, or vibrios or
spirilla of Koch, discovered by him as constantly present in
the acute stages of cholera Asiatica, and as showing definite
cultural characters.1 There exist, however, considerable
differences with regard to the number of these comma bacilli

1 Conferenz zur Erorterung tier Cholerafrage, Berliner kl. JVoch.
31, 1884.

412

MICRO-ORGANISMS AND DISEASE [chap.

present. In some acute cases the mucus flakes of the typical
rice-water stools or of the intestinal fluid contain these comma
bacilli in enormous numbers, almost to the exclusion of
other bacteria ; such is the case in some typical cases in
the mucus flakes taken directly from the watery contents
of the ileum, though the mucus flakes taken in the same

Fig. 170. — Film Specimen of a Flake of the Rice-water Fluid of a

further Case

N umerous flagella are seen.

X 1000.

body from the jejunum, in all other respects identical,
contain but few of these comma bacilli. In other equally
typical acute cases they are mixed up with other bacteria.

There is no definite relation between the number of
Koch’s vibrios in the intestinal fluid and the severity,
acuteness or purity of the case. Some cases there no
doubt are in which the mucus flakes of the rice-water

XVl]

VIBRIO AND SPIRILLUM

4'3

stools or of the contents of the lower ileum are crowded
with the comma bacilli, but in a considerable percentage
of typical cases this condition does not obtain ; there are
comma bacilli present, but they are mixed up sometimes to
a considerable amount with other bacteria. The epithelial
flakes detached and suspended in the contents of the
ileum, as well as the epithelial flakes loosened but not
quite detached from the mucous membrane, both of the
villi as also of the mouth of the Lieberkiihn’s follicles,
contain comma bacilli as well as other bacteria. In sections
through the hardened mucous membrane of the ileum one
can find sometimes comma bacilli as well as other bacteria
within the tissue of the superficial mucosa denuded of
epithelium, in the cavity of the Lieberkiihn’s follicles and
in spaces artificially produced by the loosening and detach-
ment of the epithelium of the Lieberkiihn’s follicles, but
their presence in these localities is due to immigration from
the free surface into a disorganised mucous membrane, and
neither bears any relation to the onset nor to the severity of
the illness. Where the comma bacilli are scarce in the
intestinal contents, they, or other bacteria, are altogether
missed from the mucosa, where they are abundant in the
contents and on the surface they may penetrate from the
surface into the mucous membrane.

In Figs. 168 and 169 film specimens of mucus flakes of
typical acute cases of Asiatic cholera are represented, in
which the cholera vibrios are present in fairly pure state,
and it will be noticed that as Koch has pointed out they are
arranged more or less in linear rows, “ fish-in-stream arrange-
ment” ; this condition and distribution of comma bacilli in
mucus flakes of watery stools is so characteristic of cholera
asiatica that it alone is sufficient to make a correct diagnosis,
although as a matter of routine further experiments of culti-

4M Tabular Statement of Bacterioscopic Examination of

No.

Derivation of Material.

Microscopical
Characters of Stool
or of Intestinal
Contents.

General Characters
of Cultures.

Cholera

Red

Reaction.

Hull, No. i -

Typical -

Positive -

Distinct -

ir.

Grimsby, No. i

iii.

Grimsby, No. 2

9 9

IV.

Hull, No. 2 -

Rotherham -

VI.

Westminster -

IX.

Boston -

Not altogether
typical

Morton (Gainsboro’

R.)

99 99 99

99 ~ j

XII.

Leicester

Typical -

„ - ■*-

XIII.

Handsworth -

Doubtful -

XIV.

Retford

Typical -

9 9

XV.

Fulham

Not typical

XVII.

Kennington (Lam-
beth)

9 9

XVIII.

Ashbourne -

XXII.

Croydon Borough -

9 9

XXIV.

Derby -

Typical -

9 9

XXVII.

Accrington -

9 9

XXX.

Ilkeston

Not typical

9 9

XXXIII.

Appleton-le-Street,
No. 1

Fairly typical -

XXXVI.

Great Yarmouth,
No. 1

Typical -

9 9

XXXVIII.

Tividale (Rowley
Regis)

Fairly typical -

XXXIX.

Southwark (St.

George the Martyr)

Not typical

Not liquefying
gelatine in stab,
slowly liquefy-
ing in plate cul-
ture

XL.

Great Yarmouth,
No. 2

Typical -

Positive -

XLI.

Liverpool

Not typical

XLIII.

Coton Hill (Staf-
ford R.)

Fairly typical -

XLV(a).

North Bierley,No.2

Typical -

9 9

9 9 j

LI.

Balby(DoncasterR. )

9 9

LII.

Rawmarsh

LIV.

Bingley (Township)

Not typical

9 9

LV.

Keighley

Doubtful -

”

Intestinal Materials of Cases of Cholera in England, 1893.

4i5

Growth in Gelatine Stab
Culture.

Growth on Potato
Culture at 37° C.

Milk Culture at 37” C.

Amount of
Agar Culture
required
for production,
by Intra-
peritoneal
Injection, of
Fatal
Result in
Guinea-pigs.

Liquefied fairly quick ; good

No growth after 10-14

Coagulated after 1 1

^ of a tube.

pellicle

days.

99 99 99

Light yellow after 14
days.

Fluid after 14 days

i 99

Liquefies quickly; no pellicle

Nogrowth after 14 days

Coagulated after 6 ,,

ii i >>

99 99 99

99 99

,, 11 ,,

H > 9

,, ,, slight pellicle

99 14 99

Fluid after 14 ,,

Not tested.

99 9 9 99

9 9 *4 >>

99 14 99

1 of a tube.

Fairly quick ; good pellicle -

99 14 99

99 J4 99

Not tested.

,, no pellicle

Light yellow after 14
days.

Coagulated after 5 ,,

Moderate ; good pellicle

Light brown after 5
days.

’ 99 5 99

g of a tube.

Fairly quick ; slight pellicle

No growth after iqdays

Fluid after 14 ,,

Not tested.

Moderate ; slight pellicle

99 14 9 9

Coagulated after 5 , ,

9 9

Fairly quick ; good pellicle -

99 14 99

Fluid after 14 ,,

i, J of a tube.

Quick ; slight pellicle -

99 14 99

Coagulated after 6 ,,

1 1
~i 99

Slow ; no pellicle

>> 14 >>

99 IO ,,

1 1

H> T >>

Quick ; good pellicle -

!! J4 >>

99 6 ,,

Not tested.

,, no pellicle

9 9 ^4 9 9

Fluid after 14 ,,

4 of a tube.

Slow ; good pellicle -

Light yellow after 3 ,,

99 14 99

9 9

99 99

9 9 5 9 9

99 X4 99

Not tested.

Fairly quick ; good pellicle

Nogrowth after 14 ,,

Coagulated after 10 ,,

9 9

99 99 99

!) H !>

,, 11 ,,

^ of a tube.

Slow ; no pellicle

99 14 99

99 5 99

Not tested.

Not liquefying -

99 *4 99

Fluid after 14 ,,

y of a tube.

Quick ; slight pellicle

99 m 14 99

Coagulated after 11 „

sj i 99

Fairly quick ; good pellicle

99 T4 99

99 5 99

Not tested.

Moderate ; good pellicle

Light yellow after 5 ,,

99 99

£ of a tube.

Quick ; no pellicle

N 0 growth after 1 4 , ,

,, 14

Not tested.

,, slight pellicle -

>) 14 >>

99 5 99

,, good pellicle -

99 14 99

99 5 99

Very quick ; no pellicle

99 T4 99

,» 6 .»

9 9

99 99

Light yellow after 14

99 699

5, \ of a tube.

days.

416 MICRO-ORGANISMS AND DISEASE [chap.

vation are resorted to for confirmation. Unfortunately such
a condition is present only in a percentage of cases ;
amongst the fifty odd cases of Asiatic cholera occurring in
England in September and October of 1893 ( see Tabular
Statement) such a condition was found in fifteen cases, that
is to say, when from the number and distribution of the
vibrios in the flakes of the intestinal contents alone the
diagnosis could be made.

Koch’s cholera vibrios or Koch’s comma bacilli can -be
demonstrated in almost all cases of cholera Asiatica, begin-
ning with those that show as yet only diarrhoea, more or
less profuse, up to those that have shown all the typical
characters, with vomiting and purging of copious rice-water
evacuations. After the acute stage has passed, and the
typhoid stage has set in, the comma bacilli become less
numerous, and gradually disappear, so that when after three,
four, or five days the evacuations assume again the character
of faeces the comma bacilli are either only found with diffi-
culty or are altogether missed ; in fact, in cases in which
they are scarce at the earlier stage they are not to be seen
later than the third day.

If cholera stools, particularly rice-water stools, are kept for
a day or so, one meets with comma bacilli which have
formed spirilla ; some wavy threads, others distinctly cork-
screw-shaped, some short, others long ; in dried and stained
preparations many of these spirilla are seen to be chains of
comma bacilli ; spirilla are found occasionally already in
the fresh stools or fresh mucus flakes, but as a rule the
comma bacilli are present as single vibrios or as dumb-bell
vibrios, i.e., S-shaped forms. As regards the amount of
curvature and length of the individuals there exist varia-
tions. Moreover as cultures prove and as has been already
mentioned (see also Tabular Statement of Cholera Cases in

XVI]

VIBRIO AND SPIRILLUM

417

England in 1S93) the commas derived from different
undoubted cases of cholera represent different varieties, that
is to say they are in their general characters and reactions
cholera vibrios, but in the details of the appearances of
their growth in the different media they differ in a definite
manner, which are not merely of an accidental or transitory
character but are differences maintained by them in sub-
culture through a number of successive transferences.
These facts fully confirm the statements first made
by D. D. Cunningham (Scientific Memoirs) derived from
observation of cholera in Calcutta, and although at first
doubted (as for instance by Hueppe and Gruber at the
International Congress of Hygiene held in London in 1891)
they are now admitted, by no one more so than by Hueppe
and Gruber. In this I am not referring to changes which
are well known to occur in individual varieties in course of
many transferences, e.g. the gradual decrease or increase in
rapidity with which the gelatine is liquefied, or the differences
that can be observed in subcultures through many trans-
ferences as regards the more or less distinct alteration in the
formation of a pellicle on gelatine or on broth, &c., but I
am referring to pronounced differences present from the
outset on the different commas of different stock, and per-
sisting for many generations unaltered.

For the object of demonstrating in a rapid manner the
presence of the cholera vibrios in the evacuations, even when
present in very small numbers, the method of Dunham is
the best : a flake or a loopful of the dejecta or contents of
the ileum is placed in a watery solution of pure peptone
1 per cent., common salt o‘5 per cent. After incubation at
37° C. already after 10-12 hours, better after 16-24 hours,
greater or lesser turbidity (according to the number of
comma bacilli present in the original intestinal material) is

418

MICRO-ORGANISMS AND DISEASE [chap.

noticed in the culture-tube ; with a platinum loop a droplet
is taken from the superficial layers of the culture fluid and
examined in the living state (hanging drop) or in stained
film specimens. In the former the individual commas and
the characteristic S-shaped forms can be easily recognised
under the microscope both by their shape and by the
peculiar corkscrewlike movement ; in the stained film speci-
men the presence of commas and particularly of S-shaped
forms is of importance.

From these peptone cultures subcultures in Agar plates
(at 370 C.) or in nutrient gelatine plates are then made for
further isolation, and if the peptone culture on micro-
scopic examination (stained film specimen) be found fairly
pure the addition of a few drops of pure sulphlfric acid to
the peptone culture produces the nitroso-indol reaction of
Bujwid 1 and Dunham 2 3, i.e. a pink colouration of the cul-
ture— cholera-red reaction. If the cholera vibrios are,
however, mixed with other bacteria (bac. coli or proteus)
then they must be first purified by plate cultures, and from
the colonies of cholera vibrios of these plates pure peptone
cultures can be made for the Bujwid-Dunham test.

Loftier, as has already been stated in a former chapter, was
the first to stain the flagella of the cholera vibrios, and he
found that each comma bacillus possesses one spiral flagellum
at one end ; but it can be shown by van Ermengem’s modifica-
tion that, though this is the rule, occasionally more than one
such flagellum is present. I have shown 8 that by staining
the flakes of a typical rice-water (cholera) stool with gentian
violet the flagella of the cholera vibrios can be demonstrated
as stained wavy or spiral appendages, and in some cases I

1 Zeitschrift f. Hygiene , vol. ii. i, p. 52.

2 Ibid, , vol. ii. 2, p. 337.

3 Centralbl. f, Bakteriol. und Parasit ., vol. xiv. No. 19.

xvi]

VIBRIO AND SPIRILLUM

4*9

have seen these flagella attached more than as a single
flagellum for each vibrio, sometimes they were present as
bundles (Fig. 170), still attached or free (detached in the
course of preparation). Abel, Aufrecht, and others have
described “ fine faintly stained spirilla ” in addition to the
typical vibrio in cholera stools, and Abel thinks that what I
considered to be detached free flagella were really only these

Fig. 171. — Film Specimen op a recent Agar Culture of Cholera Vibrios

X IOOO.

“fine spirilla.'’ Such “ fine faintly stained spirilla” can be
seen in every fiagella-stained film specimen of bacillus coli,
particularly of the typhoid bacillus taken from a pure Agar
culture of these microbes, and I have seen free flagella and
flagella attached to bacillus coli from flakes in the watery
evacuations of severe acute diarrhoea, they resembled the
above “fine faintly stained spirilla.” Neither Abel nor
anybody else has succeeded in cultivating the above “ fine

E E 2

420

MICRO ORGANISMS AND DISEASE [CHAP.

faintly stained spirilla,” and until this is done I maintain that
they are detached flagella (probably of bacillus coli) which
have become stained, and that there exists something in the
watery stools which acted like a mordant and which makes
the flagella susceptible of becoming stained.

The comma bacilli occur in cholera as a rule only in the
cavity of the small and large intestines, chiefly the lower
part of the ileum and large intestine ; no bacteria occur in
the blood or other tissues. Comma bacilli and also other
bacteria may and sometimes do immigrate into the tissue of
the wall of the ileum, and in a few cases have been traced
even as far as the liver and gall-bladder ; but in the large
majority of cases the comma bacilli are limited to the con-
tents of the ileum and large intestine and the^superficial
parts of the internal surface of the mucous membrane of the
ileum. For this reason Koch maintained that the disease
is an intoxication, that is, it is caused by a chemical poison
which, being elaborated by the comma bacilli within the
intestine, is absorbed into the blood, and hereby sets up the
disease cholera.

The comma bacilli of Asiatic cholera show on cultivation
in nutrient gelatine well-defined appearances, which enable
us to recognise them, so much so that in suspicious cases
of cholera their demonstration by cultivation in the evacua-
tions is of diagnostic value. But in connection with this it
must be borne in mind that in some cases or in non-typical
cases their demonstration by the gelatine culture test, owing
to the vast predominance of other bacteria, is a matter of
some difficulty. Where they are present in large numbers
their demonstration by the gelatine culture test is a matter
of comparative ease. All that is necessary is to place a
small flake of the evacuation into a few (8-10) cubic centi-
metres of sterile (well-boiled) salt solution, shake it well up,

xvi]

VIBRIO AND SPIRILLUM

421

and then with a droplet of this inoculate nutrient gelatine,
contained in a test tube, liquefy this in warm water, shake
up and then pour it into sterile glass dishes for the object
of plate cultivation. A particle of a mucus flake of a rice-
water stool rich in the comma bacilli first diluted in several
cubic centimetres of sterile salt solution and a trace of this
mixture being used for plate cultivation yields large numbers
of colonies of the comma bacilli. These show themselves (at
200 C.) already after thirty to forty hours as greyish-white
minute specks just visible to the unaided eye; after two to

Fig. 172. — Plate Cultivations in Nutritive Gelatine, after three Days
Growth at 2o”C., seen with the unaided Eye.

Colonies of cholera comma-bacilli.

The clear part is due to liquefaction of the gelatine.

three days they are distinctly visible as clear, circular de-
pressions, due to liquefaction of the gelatine within this
depression. In the centre of the depression is a round,
greyish mass surrounded by clear, liquefied gelatine ; looked
at under a magnifying glass this mass appears like a mass of
minute glass splinters, with a more or less uneven margin ;
in the centre of the mass is a more opaque larger granule.
Each of the colonies gradually enlarges ; the zone of clear,
liquefied gelatine becomes broader, and the whitish central
granular patch enlarges ; where the colonies lie closely to-
gether at the outset, the progressing liquefaction produces

422 MICRO-ORGANISMS AND DISEASE [chap.

soon a coalescence of the adjoining colonies, and then we
get a number of circular zones of clear, liquefied gelatine,
each with a central gray granular mass, the zones being
fused at the points of contact. When during the further
growth the gelatine becomes liquefied over extensive areas,

Part of a test-tube containing gelatine-peptone ; in it pure cultivation of choleraic
comma-bacilli. The funnel-shaped opening of the channel in which I he growth
of the comma-bacilli is going on contains a long air bubble.

the outlines of the original colonies are lost, and on the
surface of the clear, liquefied gelatine are thin, filmy flakes,
and at the bottom are minute whitish granules. In all stages
before and after the liquefaction of the gelatine has become
well pronounced, there are found under the microscope

Fig. 173.

xvi]

VIBRIO AND SPIRILLUM

423

rapidly motile vibrios, single commas, S-shaped dumb-bells
and numerous longer or shorter spirilla, some wavy chains cf
commas, others uniformly spiral; the above-named “gran-
ules and “flakes” are masses of commas and spirals inti-
mately matted together, and when examined in the hanging
drop look like so many clumps rapidly revolving.

In stab culture in gelatine the characters of these comma
bacilli are also well marked ; they are accurately repre-
sented in Fig. 176, and need not further be described,
except that there are considerable differences as regards
the rapidity with which the growth causes liquefaction in
the gelatine ; in all cases however it starts from the surface.

After several days to a fortnight there is noticed a dis-
tinct pellicle on the surface of the liquefied gelatine in
some cases, in others such a pellicle is absent : the gelatine
is clear, but contains a few whitish granules marking the
outline of the funnel-shaped channel of liquefied gelatine.

Alkaline broth (at 36-38° C.) is slightly turbid already
after twenty-four hours’ growth : this increases during the
succeeding days. After a week or so the superficial layers
become gradually clearer, and this extends gradually and
insensibly towards the deeper layers ; hand in hand with
this goes the deposit of a grayish-white powdery precipi-
tate ; a more or less distinct pellicle is noticed already after
a few days, and this gradually increases in thickness. In
some cases the pellicle is distinct and complete, in others
it is absent. Under the microscope the comma bacilli in
the fluid and in the pellicle are seen to be connected into
beautiful spirilla, some of these measuring great lengths,
some as many as twenty to thirty turns, the long spirilla
more or less plicated and bent.

The growth on Agar mixture is not characteristic, being
in the form of thin, translucent patches and films with

424

MICRO-ORGANISMS AND DISEASE [chap.

rounded or knobbed outline, assuming as growth goes on,
i.e., after some days, a slight brownish tint.

On boiled potato the comma bacilli grow only at tem-
peratures above 25° C. ; at 36° they form after a few days a
thick, smeary, brown film. In some cases the growth is
a transparent film, in others no growth takes place on
potato. Comma bacilli grow well and rapidly, if mucus
flakes of a cholera intestine containing numerous comma
bacilli are placed on linen kept damp. After tweniy-
four hours the comma bacilli have increased to an enormous
extent, almost to the exclusion of other bacteria originally
present, provided these were at the outset less numerous
than the comma bacilli.

Cholera vibrios show rapid growth at 370 C. Oh solidified
blood-serum, which becomes liquefied by the growth.

In cultivations of the comma bacilli one meets with
forms which in so far differ from the typical curved,
cylindrical vibrios, as they are much thicker, plano-convex,
or bi-convex, or even approaching the spherical shape with
a clear vacuole in the middle. In well-stained and well-
washed specimens also the most typical comma bacilli show
within a sheath the protoplasm collected at the ends — as a
granule at each end — whereas the middle part remains
clear. The above atypical forms are merely a further develop-
ment of their normal constitution, being derived from
them by enlargement of the central clear space or vacuole.
Such atypical forms are to be met with in all cultures ;
they are as actively motile as the typical commas; their
number, however, varies greatly with the character of the
culture. If comma bacilli, originally derived from the
cholera intestine, are carried through many successive sub-
cultures in gelatine, say one or two dozen, the number of
such atypical bi-convex or spherical forms is found larger.

XVI] VIBRIO AND SPIRILLUM 425

Comma bacilli when in culture rapidly undergo degenera-
tion into granular debris ; in fact, a good deal of the white
deposit in gelatine and broth cultures is due to debris of
comma bacilli. Degeneration goes on comparatively more
rapidly in Agar culture than in gelatine cultures. It is a
notorious fact that on the surface of Agar cultures the
whole of the growth is found dead after from a few to several
months, so that no new culture can be started from such an
old culture. This degeneration and death occur sooner or
later in all cultures after the lapse of some time ; this alone
proves sufficiently that the comma bacilli do not form per-
manent seeds or spores. Koch has proved by many
experiments of drying that the comma bacilli are invariably
killed by drying, unlike spore bearing bacilli, and at no time
do the comma bacilli form spores. Heating cultures (old
or recent) of comma bacilli to 6o° to 65" C. for five minutes
invariably kills the cultures — proof that no spores are formed.
The assertion of Hueppe that the terminal granules ob-
served in comma bacilli are spores,/ viz., arthrospores, is
definitely negatived by the above direct experiments.

Comma bacilli of cholera mucus flakes or of cultures,
recent or old, are killed by acids, e.g., a fluid containing
o*2 per cent, hydrochloric acid,1 so that the normal acid
fluid of the stomach kills the comma bacilli ; also this is
opposed to there being present spores in the comma bacilli.

Comma bacilli grow well and luxuriantly between 170
and 40° C., on almost anything— paste, boiled egg, turnip,
cucumber, cabbage, bread, meat, various fruits, &c. They
grow best at 35— 3 70 C., if the medium is faintly alkaline,
they nevertheless grow also on neutral medium, and even
on some media like potato and fruit, which are slightly
acid. I have seen comma bacilli which, having started on
1 Koch, l.c. ; Watson Cheyne, Brit. Med. Journal, 1885.

426

MICRO-ORGANISMS AND DISEASE [chap.

nutrient gelatine kept for a few days at 20° C, continued to
grow slowly but steadily after the gelatine was then kept at
1 5-1 6° C. Comma bacilli gradually die off if nutri-
ment is insufficient, eg., in water ; they are gradually killed
in faecal matter (Kitasato) ; and they do not grow well when
oxygen is absent from the culture (Koch).

Comma bacilli obtained from typical cases of Asiatic
cholera grow well in milk at 3 70 C., they herein rapidly
multiply and in some cases cause no visible change, while
in others they cause coagulation of the milk ; but also in
regard to this latter phenomenon there exist considerable
differences, for while some varieties cause coagulation after
five or six days others take several weeks. Most varieties
of cholera vibrios (derived from cases of AsiafFb cholera)
produce alkali in culture media {eg., in Petruschki’s
neutral whey), but some varieties undoubtedly produce
slight acid. Another difference noticed between the
vibrios derived from different cases of Asiatic cholera refers
to their action when injected subcutaneously into guinea-
pigs. Koch 1 had already succeeded in producing acute
septicsemic infection of mice by intraperitoneal injection of
large doses {see later), with rapid multiplication of the
vibrios in the blood ; Ferran and D. D. Cunningham 2
have succeeded in producing septicsemic infection by sub-
cutaneous injection into guinea pigs ; after death the blood,
the smeary exudation on the serous covering of the intestine
and the intestinal contents containing an abundance of
the cholera vibrios. I have produced this effect both with
gelatine cultures of cholera vibrios as also of Finkler’s
vibrios, using 0-5-2 cc. of the liquefied culture per guinea-

1 Conferenz zur Erorterung d. Cholerafrage, Berl. klin. Woch. 31,
1884.

2 Scientific Memoirs, Calcutta, 1S91.

xvi] VIBRIO AND SPIRILLUM 427

pig ; the animals died in thirty to forty hours, the blood
and the intestine, liver, and spleen containing numerous
vibrios. Now, when testing the cultures of cholera vibrios
derived from different cases of undoubted cholera asiatica
and grown on the slanting surface of solidified nutrient
Agar for a day or two it will be found that they possess
different degrees of virulence. Of some varieties J- or 1 of a
culture produces distinct tumour at the seat of inoculation and

55 M

Fig. 174.

From an Artificial Cultivation of chole-
raic Comma-bacilli in Gelatine
Peptone. Magnifying power 700.
Most of these are single curved
bacteria, a few are joined end to
end in twos, thus forming S-shaped
organisms ; and a few are in chains
of several placed end to end.

Fig. 175.

From an Artificial Cultivation of chole-
raic Comma-bacilli in Agar-Agar
Peptone at the ordinary tempera-
ture of the room after several
weeks. The Comma-bacilli change
by vacuolation into plano-convex,
then biconvex organisms. Magni-
fying power about 700.

death in thirty to forty-eight hours with all the appearances
of general septioemic infection, while with other varieties
double and treble this dose produces only a transitory
tumour with transitory constitutional disturbance ; after
several days the animals completely recover, or at most
ulceration of the skin about the seat of the tumour and
ultimate recovery takes place. R. Pfeiffer and Metschnikoff
have had cultures of cholera vibrios which in small doses
produced general septicsemic infection of the guinea-pig
after subcutaneous injection. The greater or lesser virulence
of the cholera vibrios (tested by subcutaneous injection of

CH. XVl]

VIBRIO AND SPIRILLUM

429

the guinea-pig) stands in no definite relation to the severity
of the cholera case from which they are derived.

Haffkine has on the other hand shown that by successive
transference from guinea-pig to guinea-pig of the peritoneal
exudation produced in the first of the series by intraperi-
toneal injection of a fatal dose of cholera (Agar) culture,
after as many as twenty and more transferences the cultures
of cholera vibrios obtained from the peritoneal fluid of the last
guinea-pig reach a high degree of virulence, so much so
that minute quantities of such a culture injected intraperi-
toneally are capable of causing fatal general septiccemic
infection of the guinea-pig.

It has been shown by Sabolotny {Central, f Bakt. 11.
Paras, vol. xv. p. 1 50) that the marmot is particularly sus-
ceptible to subcutaneous injection with the vibrio, acute
septicaemic infection and death being the result.

Similarly also for the guinea-pig the virulence of a given
stock of vibrios can be materially increased by adding to
the culture medium potassium nitrate, or even a larger pro-
portion of sodium chloride.

The same holds good for the degrees of virulence shown
by the cholera (Agar) cultures when injected intraperi-
toneally into guinea-pigs. Of some varieties TU of an
Agar culture is sufficient to produce a fatal result in a
guinea-pig of 300 grammes weight in twenty to twenty-four
hours, while of others as much as J or even \ of a culture
tube is required. The slanting surface of nutrient Agar is
inoculated over its whole extent, then incubated at 370 C.
for forty-eight hours. By this time the whole surface (six
centimetres by two) is covered with a translucent gray film
of growth ; to the culture tube are then added four, five,
or six cc. of sterile bouillon, and by means of a sterile plati-
num loop the growth is rubbed completely down into the

430 MICRO-ORGANISMS AND DISEASE [chap.

bouillon ; this distribution is then poured into a sterile watch-
glass or capsule, and J, £, TV, or TV or less of the culture, as
the case requires, is drawn up into a hypodermic sterile syringe
and injected intraperitoneally into a guinea-pig of known
weight. The result is always that according to the virulence
and the relative proportion of the dose and body-weight ’the

Fig. 177.— Film Specimen of the Peritoneal Fluid of a Guinea-pig dead
from Acute Peritonitis after intraperitoneal Injection of Cholera
Vibrios.

X 1000.

guinea-pig is distinctly ill after from a few to several hours,
the animal is quiet, does not feed, its coat becomes rough,
the temperature gradually falls, movement becomes more and
more impaired, and the animal is found dead after sixteen,
eighteen, twenty, twenty-four hours, or as late as thirty-six
hours. If it does not die after thirty-six hours it as a rule
again recovers,

XVl]

VIBRIO AND SPIRILLUM

431

The fatal dose — producing death in or within twenty-four
hours — differs according to the initial virulence and the size
of the animal. The fatal dose of living vibrios from an
Agar culture is always a little smaller than if the dose to be
injected is first sterilised, either by boiling or, as I generally
do, by heating it to 70° C. for five or ten minutes, or, as was
done by R. Pfeiffer, by killing the vibrios by chloroform.

Fig. 178.— Film Specimen of an Agar Culture of Cholera Vibrios, a few
Weeks old, showing numerous long Spirilla.

About 400.

On post-mortem examination the peritoneum is found
intensely inflamed : hypcrtemia of the serous covering and
of the wall of the intestine, sanguineous copious fluid or
slightly viscid peritoneal exudation, turbid by being densely
crowded with the living motile vibrios (if the culture injected
was not previously sterilised), flocculi of lymph on the
omentum, on the intestine, and particularly the surfaces of

432 MICRO-ORGANISMS AND DISEASE [chap.

the liver. The intestine is relaxed, and filled occasionally
but not always with sanguineous mucus. The same symp-
toms and the same post-mortem appearances are observed
if the culture injected was first sterilised, only, as stated
above, the dose has to be a little larger to produce fatal
issue. Examining by cultivation, the peritoneal fluid (after
living culture had been injected) is found crowded with

X IOOO

Fig. 179 — Film Specimen of the same Culture as in previous figure,
SHOWING A LONG SPIRILLUM.

living cholera vibrios ; from the heart’s blood as a rule
colonies of cholera vibrios can be recovered by culture, in
some cases fairly abundant, in others relatively sparingly.
In some cases the intestinal cavity contains fluid mucus
filled with the cholera vibrios. All these results, as I have
shown, are obtained, of the same kind and the same degree,

VIBRIO AND SPIRILLUM

433

Xvi]

by vibrio of Finkler, by proteus vulgaris, by bacillus pro-
digiosus, by bacillus coli, and bacillus of typhoid, and I
found that bacillus prodigiosus, coli, and typhoid are in this
respect more virulent than vibrio of cholera or of Finkler.

The result of the intraperitoneal injection of living or
dead vibrios taken from Agar cultures does not therefore in
any way throw any light on the specific action of the cholera
vibrio, it being an action due to the presence of poisonous
substances, intracellular poisons or protem poisons , within
the bodies of the vibrios or in those of many other microbes
mentioned. Under all these injections of the bodies of the
most varied microbes the same disease and the same patho-
logical changes are produced.

That there are different degrees of virulence amongst
different cultures of the same species, amongst the different
varieties of a species and amongst the different species
themselves, has been already mentioned.

A dose of living microbes need be smaller than of dead
microbes of the same culture in order to produce a fatal
result. This is easily explained by remembering that in the
case of dead microbes no further addition is made after
introduction into the peritoneal cavity, whereas if the
microbes are injected in a living state their multiplication
within the peritoneal cavity — as mentioned above, the
peritoneal exudation is found crowded with them — adds
considerably to the original intracellular poisons as also the
metabolic products, specific toxins, produced in conse-
quence of this multiplication act towards bringing about
a fatal result. That the cholera vibrios create poisonous
metabolic products, toxins, in a culture is well established ;
in some fluids not much of it — e.g. in broth or in ordinary
gelatine — but in aqueous humour or in serum the cholera
vibrio produces this toxin rapidly and in considerable quan-

F F

434 MICRO-ORGANISMS AND DISEASE [chai\

lity, as has been first shown by Van Ermengem ; 1 and
McLeod and Mills 2 found this toxin in a very effective and
concentrated form produced in the intestinal fluid of the
guinea-pig infected per os after Koch’s method ( see below).

The effect of non-fatal doses of cholera vibrios (dead or
living) or of other microbes injected intraperitoneally into
the guinea-pig will be considered later on in connection
with artificial immunisation, at present suffice it to say
that the fatal effect of cholera vibrios injected intraperi-
toneally into the guinea-pig proves nothing whatever as
to any specific action any more than is the case with
bacillus prodigiosus, and that the greater or lesser virulence
of one microbe as compared with another (as judged by
the relative amount injected intraperitoneally) proves
nothing whatever for or against intrinsic specific action.
I have shown 3 that by repeated intraperitoneal injection of
sterilised vibrios (of cholera, of Finkler) or of sterilised
bacilli (coli, prodigiosus, typhoid, proteus) taken from the
surface of recent Agar cultures and used in non-fatal doses,
the guinea-pigs become furnished with a high degree of re-
sistance against a subsequent intraperitoneal injection of
fatal doses of living vibrios or bacilli respectively. In the
case of the cholera vibrios, starting for the first injection
with a sterilised culture, and increasing the dose up to
i, then £, -|, and finally L sterilised culture, allowing
eight to ten days to intervene between each two injections,
it will ultimately be found that such a prepared animal does
not react any further to the intraperitoneal injection of a
double or even treble otherwise fatal dose of living Agar

1 Rccherches stir le Microbe dti ChoUra asiatique, Bruxelles, 1S85.

2 Reports from the Laboratory of the Royal College of Physicians,
Edinburgh , vol. i.

3 Centralbl. f Baht, tind Parasit., 1893, and Report of the Medical
Officer of the Local Government Board for 1893.

VIBRIO AND SPIRILLUM

435

xv i]

culture. It follows from this that in this animal from the
purely intercellular substances— only dead bacillary bodies
having been used— substances have been produced which
can immunise— i.e., can act germicidally against the intra-
peritoneal growth and multiplication of the cholera vibrio.
Testing the blood-serum, “cholera serum,” of such an
animal immunised by dead bacillary bodies only as to its
immunising or germicidal action against living cholera
vibrios after the method of Pfeiffer — i.e. mixing a definite
amount of “ cholera serum ” with an otherwise fatal dose
of living cholera vibrios, and injecting the mixture into the
peritoneal cavity of a fresh guinea-pig, at the same time
injecting into a control guinea-pig of the same weight the
same dose of living vibrios without the cholera serum—
it will be found that that serum exhibits in the peritoneal
cavity marked and definite germicidal power. R. Pfeiffer1
has described numerous experiments, by which it was
clearly established that by repeated intraperitoneal in-
jections of doses of living cholera vibrios, starting with
non-fatal doses and gradually increasing the dose till no
reaction follows any longer, and the animal after the last
injection again gains in body-weight, the blood-serum of
such an artificially or “actively” immunised guinea-pig
has potential, powerful, germicidal power, inasmuch as
injected into the peritoneal cavity of a fresh guinea-pig,
together with an otherwise fatal dose of living cholera
vibrios, it produces a rapid alteration and crumbling away
of the vibrios,2 no multiplication of them and no disease
follows — that is to say, the addition of the serum of an

1 Zeilschr. f. Hygiene u. Infekt. vol. xvi.

2 The peculiar alteration produced in a suspension of cholera vibrios
by the addition of such “ cholera serum ” (Bordet and Durham) will be
described and discussed later on.

F F 2

436

MICRO-ORGANISMS AND DISEASE [chap.

“ actively immunised ” animal is capable of giving im-
munity (“passive immunity”) to another guinea-pig
against the cholera vibrio injected intraperitoneally. There
is no difficulty in confirming this discovery of Pfeiffer as
to the presence of potential germicidal substances in the
blood-serum of an actively immunised guinea-pig. What
I have, however, to add, is that according to the above
experiments of immunising against living vibrios by means
of the intracellular substances only, I conclude that for the
production of germicidal serum it is not necessary that
there should be produced in the animal body toxins — by
the multiplication of the living vibrios injected — for the
immunising substances in the above experiments could
have been derived solely from the dead bodies of the
vibrios used for immunisation. I may state here also that
the same evidence I have obtained in showing that
germicidal serum of typhoid immunised guinea-pigs against
living typhoid bacilli, as also of germicidal serum of
diphtheria immunised guinea-pigs against living diphtheria
bacilli, is obtainable by using for immunisation the bacillary
bodies only, without allowing these microbes to undergo
multiplication and production of toxins within the peri-
toneal cavity. We shall return to this subject more in
detail when treating of immunity.

Guinea-pigs that by repeated intraperitoneal injections
of dead cultures of cholera vibrios have acquired re-
sistance by which they can withstand an otherwise fatal
dose of living cholera vibrios in their peritoneum, the
vibrios not being now able to live and multiply in such a
peritoneal cavity, are, however, not proof against cholera
toxin. I have made experiments 1 to show that guinea-pigs

1 Reports of the Medical Officer of the Local Government Board for
1S94.

XV i]

VIBRIO AND SPIRILLUM

437

well immunised against living vibrios by previous repeated
injection of dead vibrios succumb to a dose of toxin pro-
duced by cholera vibrios in serum cultures. (The same
also holds good for vibrio Finlder and the toxin produced
in serum cultures of this vibrio.) So that the distinction
on which I have always insisted, 1 between the action of
intracellular poisons of a microbe and that of the toxins
produced by the microbe as a result of its metabolism is
well founded.

Koch 2 in his first pamphlet on cholera told us 3 that he
had made every imaginable effort to produce cholera in
animals experimentally. The experiments of feeding white
mice with cholera dejecta, first made by Tiersch and then
by Burdon Sanderson, were repeated by Koch over and
over again on fifty white mice fed with this material (dejecta
of cholera patients, and the contents of the intestine of
cholera corpses) and with choleraic material after it had
begun to decompose, but no result whatever followed ; the
mice remained healthy. “ We then made experiments on
monkeys, cats, poultry, dogs and various other animals
that we were able to get hold of, but we were never able
to arrive at anything in animals similar to the cholera
process. In precisely the same manner we made experi-
ments with the cultivations of comma bacilli ; these were
given as food in all stages of development. When experi-
ments were made by feeding animals with large quantities
of comma bacilli, on killing them and examining the con-
tents of their stomachs and intestines with a view to find
comma bacilli it was seen that the comma bacilli had
already perished in the stomach, and had usually not

1 Ibidem, 1892, 1893, and 1894.

2 The following is copied from my Bacteria in Asiatic Cholera.

3 Conference zur Erorlcrung der Cholerafrage , Berlin, 1884, p. 27.

438 MICRO-ORGANISMS AND DISEASE [chap.

reached the intestinal canal. . . . The comma bacilli had
been destroyed in the stomachs of these animals. . . . The
experiment was therefore modified by introducing the sub-
stances direct into the intestines of the animals. The belly
was opened, and the liquid was injected immediately into
the small intestine with a Pravaz syringe. The animals
bore this very well, but it did not make them ill.
We also tried to bring the cholera dejecta as high as
possible into the intestines of monkeys by means of a
long catheter. This succeeded very well, but the animals
did not suffer from it.” “ I must also mention,” says
Koch, “ that purgatives were previously administered to
the animals in order to put the intestine into a state of
irritation, and then the infecting substance was given,
without producing any different result. The only experi-
ment in which the comma bacilli exhibited a pathogenic
effect, which therefore gave me hope at first that we should
arrive at some result, was that in which pure cultivations
were injected directly into the blood-vessels of rabbits or
into the abdominal cavity of mice. Rabbits seemed very
ill after the injection, but recovered after a few days. Mice,
on the contrary, died from twenty-four to forty-eight hours
after the injection, and comma bacilli were found in their
blood. Of course they must be administered to the animals
in large quantities ; and it is not the same as in other ex-
periments connected with infection, where the smallest
quantities of infectious matter are used, and yet an effect
is produced. In order to arrive at certainty as to whether
animals can be affected with cholera, I made inquiries every-
where in India as to whether similar diseases had ever been
remarked amongst animals. In Bengal I was assured such
a phenomenon had never occurred. This province is ex-
tremely thickly populated, and there are many kinds of

VIBRIO AND SPIRILLUM

439

xv i]

animals there which live together with human beings.
One would suppose, then, that in that country, where
cholera exists in all parts continually, animals must often
receive into their digestive canal the infectious matter of
cholera, and in just as effective a form as human beings,
but no case of an animal having an attack of cholera has
ever been observed there. Hence I think that all the
animals on which we can make experiments, and all those,
too, which come into contact with human beings, are not
liable to cholera, and that a real cholera process cannot be
artificially produced in them.”

Koch,1 starting from the idea that the comma bacilli are
killed by the gastric juice, and that in order to develop their
pathogenic powers they have to get unscathed and living
into the small intestine — their natural breeding-ground — it
occurred to him that this difficulty might be obviated by
first neutralising or making alkaline the contents of the
stomach, and introducing per os the comma bacilli. He
therefore kept guinea-pigs for twenty-four hours without
food, and then injected into their stomach per os 5 cubic
centimetres of a 5 per cent, watery solution of carbonate of
soda. This does not noticeably injure the stomach, and,
as direct observation proved, kept the contents of the
stomach in an alkaline condition for three hours. Some
minutes (twenty) afterwards he introduced by catheter 10
cubic centimetres of a cultivation of the comma bacilli in
meat infusion.

The result is noteworthy. Seven guinea-pigs thus ex-
perimented upon remained perfectly well: “They were
killed after twenty hours,” says Koch, “ and the contents of
their stomach, intestine, and caecum were examined by
gelatine plate cultivations. In six of the seven animals the
1 Second Conference on Cholera , Berlin, May, 1885.

440 MICRO-ORGANISMS AND DISEASE [chap.

cholera bacteria could be demonstrated in the small in-
testine. The experiment had thus in so far succeeded that
the cholera bacilli had passed uninjured through the
stomach, but they had not set up any disease in the
animals.” Similar experiments were then made on eight
other guinea-pigs. These animals also remained quite
healthy. Finally four guinea-pigs were similarly experi-
mented upon (5 cc. of solution of sodium carbonate, the
10 cc. of cultivation of the comma bacilli in meat infusion) ;
three remained well, the fourth appeared ill next day, looked
shaggy and did not eat ; on the following day it was very-
ill ; paralytic weakness of the posterior extremities came
on, the respiration was weak and slow, the head and ex-
tremities were cold, and the animal died in this condition.
O xv post-mortem examination the small intestine was markedly
reddened and full of a flaky, watery, colourless fluid. The
stomach and caecum contained a large quantity of fluid.
“ The examination with the microscope and with gelatine
plates,” says Koch, “showed that the contents of the small
intestine contained a pure cultivation of the choleraic
comma bacilli.” “That this one animal only should have
died, out of a series of nineteen, uniformly experimented
upon, suggested some peculiar condition that had obtained
in this one animal, and as a matter of fact on examination
it was ascertained that this animal had aborted immediately
before the injection, and on post-mortem examination it was
found that the abdominal walls were very flaccid and the
uterus still greatly enlarged. This led me to the idea that
either the abortion per se, or perhaps its unknown cause,
had acted on the other abdominal organs, more especially
on the small intestine, in such a way as to produce a
temporary relaxation with arrest of peristaltic movement ;
and thus had rendered it possible for the comma bacilli to

XVl]

VIBRIO AND SPIRILLUM

441

remain longer and gain a footing in the intestine.” This
conclusion appeared quite justifiable, inasmuch as by direct
experiment it had been proved that the contents of the
stomach pass too rapidly through the small intestine, and
since the comma bacilli could only unfold their poisonous
action, i.e ., could multiply and produce the chemical poison,
if they had time to remain there and to multiply. Conse-
quently if they were not delayed on their passage through
the small intestine they would not multiply there, and once
in the caecum, where the reaction is acid, they would
become harmless.

In order to produce a condition similar to the one in the
above single successful experiment on the guinea-pig, Koch
injected tincture of opium into the peritoneal cavity after
the introduction of the sodium carbonate and the cultiva-
tion of the comma bacilli : this answered well for achieving
positive results. Immediately after the administration of
the 10 cc. of the culture of the comma bacilli, 1 cc. of
German tincture of opium for every 200 grms. of the
animal’s body-weight were injected into the peritoneal cavity :
the animal became thereby narcotised for half an hour, and
died after one and a half to three days, with the same
symptoms as the above guinea-pig. “Eighty-five guinea-
pigs have been infected in this way with cholera.”

Now the following criticisms can, I think, be justly
applied to these experiments: (1) According to Koch’s
own showing, it cannot be the narcosis which is essential,
even allowing for the present that relaxation of the in-
testine may have been produced by the intraperitoneal
injection of opium tincture, since alcohol alone was in-
jected by Koch into the peritoneal cavity, and he says that
thereby “ we were most successful in making the animals
susceptible to the cholera infection. (2) Can naicosis

442 MICRO-ORGANISMS AND DISEASE [chap.

of the animal be produced by opium without furthering
in the least the process of the experiment ? 1 This has
been tried over and over again ; watery extract of opium is
injected into the peritoneal cavity, and narcosis lasting for
one hour is produced, but the animals remain well ;
tincture of opium is subcutaneously injected, the animals
fall into narcosis, lasting for from forty to eighty minutes,
but no result is obtained from the previous introduction of
the comma bacilli ; in fact, the experiment as designed by
Koch was repeated by me on a large number of guinea-
pigs, thirty in all, but instead of producing narcosis by
injection of tincture of opium into the peritoneum I
produced it by intraperitoneal injection of watery extract
of opium, or subcutaneous injection of tincture of opium
and watery extract of opium, but all in vain. The comma
bacilli used .were of recent broth culture, or of gelatine
culture, and 1 were beyond question or doubt the choleraic
comma bacilli.

From all these considerations it appears to me un-
warranted to conclude that the multiplication of the
comma bacilli in the small intestine, and their fatal action
by the chemical products they elaborate, takes place on
account of a relaxation and arrest of the peristaltic move-
ment by the opium. Another explanation appears to me
much more probably correct. It is this — provided the
intestine is first made diseased, either in consequence of
slight peritonitis, as was probably the case in the guinea-pig
that had aborted, or in the experiments when tincture
of opium is injected into the peritoneal cavity, or from
other reasons, the comma bacilli that are present in the
intestinal cavity undergo rapid multiplication, and by
their chemical products not only increase the disorder of
1 The Practitioner, 18S6 and 1SS7.

VIBRIO AND SPIRILLUM

443

xv i]

the mucous membrane, but eventually poison the animal.
And from this I conclude, further, that a multiplication
of the comma bacilli can and does take place only when
the intestine is previously brought into a diseased state.
Under this view all Koch’s and Van Ermengem’s results
become at once intelligible.

I maintain, then, that the living choleraic comma bacilli
per se, however large their number, when introduced into
the normal small intestine of the guinea-pig are quite
innocuous, but they are rendered capable of great multi-
plication if the intestine is previously, from some cause or
another, diseased. The chemical products, the toxins, of
such multiplication act as poisons analogous to the
ptomaines obtained from putrefactive bacteria.

That this is the true explanation I find proof in some of
Koch’s experiments with other bacteria, notably with
Finkler’s and Deneke’s comma bacilli. With both these
organisms on experimenting in the above manner he
obtained positive results ; not so constantly, it is true, but
still he did obtain positive results, not identical, but
similar. Of course it is not to be expected that, seeing
these are three different species, they would act in the same
manner. Finkler published a large series of experiments,
in which, with his comma bacilli, and after the method of
experimentation employed by Koch, he produced results
identical with those gained by Koch with the choleraic
comma bacillus. There can be no doubt, as will be
mentioned later, that Finkler’s comma bacillus has nothing
to do with cholera nostras, or with any other infectious
disease, but that it is simply a putrefactive organism. And
on the same grounds Koch’s comma bacillus cannot be
said by these experiments on the guinea-pig to have been
proved to have a causal relation to cholera asiatica or that

444 MICRO-ORGANISMS AND DISEASE [chap.

the disease so produced in the guinea pig is cholera, any
more than has Finkler’s comma bacillus, or any of the
other species of bacteria that are capable of producing
chemical poisons analogous to ptomaines. All that can be
said is, provided that conditions are established by which
the choleraic comma bacilli are enabled to grow .and
multiply in the intestinal canal, these chemical poisons are
produced.

This method of experimentation introduced by Koch
cannot therefore be held to prove a specific action of the
cholera vibrio on the guinea-pig, since after this^method the
same result is produced with other bacteria, in no way con-
nected with cholera asiatica. Metchnikoff (Annales de
.l’lnstitut Pasteur, 1895) has shown that by choosing very
young rabbits, almost immediately after birth, it is possible
in a large percentage to produce by ingestion of culture of
the cholera vibrio rapid multiplication of the vibrios within
the alimentary canal and death of the animal in 24—48
hours ; I have repeated these experiments and can con-
firm them, but I have to add that the same result is ob-
tained with the vibrio of Finkler.

While then the position of affairs, viz., whether the
vibrio of cholera (Koch) is or is not the real causa causans
of Asiatic cholera, is not altered by all these experiments on
the guinea-pig (subcutaneous, intraperitoneal, and intro-
intestinal injection), there have been made numerous
observations within the last three or four years (since the
Hamburg epidemic in 1892) which materially alter the
circumstances from what they were previously. Since 1886,
and up to that date the fundamental fact discovered by Koch
that the particular vibrio found by him in Asiatic cholera is
peculiar to cases of Asiatic cholera and to no other disease of
the intestine, its demonstration being therefore of the greatest

xvi]

VIBRIO AND SPIRILLUM

445

importance for diagnostic purposes, had been conceded and
confirmed on almost all sides (see this 3rd edition and my
Bacteria in Asiatic Cholera, 1886 and 1887), the proof, how-
ever, as to the experimental production of cholera in the
guinea-pig, as we have shown above, was far from a satis-
factory kind.

Experiments by ingestion of cultures of cholera vibrios in
the human subject have been made in Munich (Pettenkofer
and von Emmerich), in Vienna (Strieker), and in Paris
(Metchnikoff), and the results of these, though not un-
equivocal, were sufficiently instructive to strengthen the
position of Koch’s view as to the causal relation of the
cholera vibrio to Asiatic cholera.

In a considerable percentage of these experiments it was
shown that the ingestion of cultivation of cholera vibrio,
that had been kept up through many subcultures in the
laboratory, produced more or less severe diarrhoea with the
presence of the cholera vibrios in the evacuations as shown
by the culture test. In a few the effect was tolerably severe
(Pettenkofer and Emmerich), and in one case (a boy)
observed by Metchnikoff it was a very good imitation of
genuine Asiatic cholera, including the rice-water stools with
crowds of the cholera vibrios. It is well established by the
older researches of v. Pettenkofer and fully confirmed by
the observations made in reference to cholera in India and
in Europe down to the most recent times, viz., that in the
production of cholera the predisposition of the individual,
season, and locality are important factors besides the real
causa causans or the cholera microbe — the x, y, and z
of sanitarians. If then in the above precise and de-
liberate experiments with pure cultures of Koch’s cholera
vibrio by ingestion fair results — even few in number—
at a time and locality when and where no cholera exists, are

446

MICRO-ORGANISMS AND DISEASE [chap.

brought about, the inference, it must be admitted, that the
cholera vibrio is the microbe of cholera is extremely near.
It cannot be expected that in a number of healthy persons
the ingestion of laboratory cultures, which, as experiments
on the guinea-pig show, are liable to become less and less
virulent, should be productive of severe and typical attacks
of cholera ; even at the beginning and towards the end of
an epidemic of cholera we see occasionally a considerable per-
centage of mild attacks — practically only more or less severe
diarrhoea ; therefore, that in the above experiments there
should have been a percentage of positive results — cases with
fairly severe diarrhoea — and in the one case of IvFetchnikoffs
series a severe result should have been actually brought about,
and that in all these positive cases the comma bacilli introduced
should have multiplied in the intestine and their presence
been demonstrated by microscopic and culture test, is
in itself a very strong link in the evidence as to the causative
relation between the vibrio and the disease cholera.

Another important link of evidence was brought forward
by showing that the blood-serum of a person who had
recovered from an attack of Asiatic cholera possesses the
power to confer passive immunity to guinea-pigs against the
action of the cholera vibrio (Klemperer, Botkin, Wasser-
mann), that is to say that serum possesses immunising action
against the cholera vibrio. If the cholera vibrio is really
the cause of cholera, one can understand that, just as in
other communicable diseases a first attack alters or adds
something to the blood, so as to furnish this with immunising
power, a like effect should be produced by the cholera
vibrio after it has been growing and multiplying within the
affected individual, that is to say that the blood-serum of
such an individual should possess a specific immunising
action against the cholera vibrio. And this is actually the

xvi]

VIBRIO AND SPIRILLUM

447

case from the observations recorded. This harmonises well
with R. Pfeiffer’s discovery of the germicidal action of the
serum of actively immunised guinea-pigs against fatal doses
of the cholera vibrio. By itself this link of the evidence is
not very strong, but it proves this that the blood-serum of
an individual after an attack of cholera possesses a specific
immunising action against the cholera vibrio, and it is per-
missible to conclude that: this power of the serum was
brought about by the action of the cholera vibrio just as is
the case in Pfeiffer’s active immunisation of guinea-pigs. Not
that it proves that the disease produced in the guinea-pig
by intraperitoneal injection is a process comparable to cholera
in man, but it shows that within the blood of the living body
the cholera vibrio is capable of creating specific immunising
substances, and taken together with the analogous observa-
tions with the bacillus of diphtheria, the bacillus of tetanus,
the pneumococcus, the bacillus of septicaemia, and other
specific microbes it becomes extremely probable that also
in cholera of man the production of immunising serum, is
due to a like cause, i.e. to the cholera vibrio.

Haffkine in a long continued series of observations has
established that by transmission of the peritoneal exudation
of a guinea-pig, dead after intraperitoneal injection of living
cholera culture, through a large number of successive guinea-
pigs ultimately the vibrios present in such exudation (see a
former page) assume increasingly greater virulence, so much
so that cultures made from the peritoneal exudation of the
last animal of the series (twenty to thirty transmissions)
yield extremely virulent vibrios, a tenth or a twentieth or
less of the dose of that with which the series was started,
being now sufficient to produce fatal results in sixteen to
twenty hours when injected intraperitoneally. Such cul-
tures of “exalted virulence” injected subcutaneously into

448

MICRO-ORGANISMS AND DISEASE [chap.

guinea-pigs cause even as small doses intensive effects :
tumour and, haemorrhage, constitutional illness, and, if the
dose is not too small, death ; if the dose is small enough
the effect passes off, the tumour leads in most instances to
sloughing, but ultimately the skin heals. A second injection
has less effect, and a third still less. After a time, i.e. when
the animal has again quite recovered, it is found to be immu-
nised against the intraperitoneal injection of multiple fatal
doses of even the powerful virus.

In order to mitigate the effect of the first injection
Haffkine attenuates the virulent living vibrios by the
addition of phenol — first vaccine — and only orf second or
even third injection uses the full virulent living vibrio —
second vaccine. Having preliminarily tested the effects by
subcutaneous injection of these vaccines into (willing)
human subjects (himself, Hankin, and others at the Pasteur
Institute), and producing well marked tumour and con-
stitutional symptoms more or less rapidly passing off, he
proceeded to India to test these “ vaccines ” in reference
to protective subcutaneous inoculations of the human sub-
ject— two, and in some instances three, separate injections
being made — against cholera.

Now, it ought to be here distinctly understood that
before Haffkine started on this work in India he was
convinced that guinea-pigs successfully protected, “actively
immunised,” by subcutaneous injection with his “ vaccines ”
against a subsequent intraperitoneal injection of fatal doses
of virulent vibrios, were also protected against ingestion of
the vibrios administered after Koch’s method described on
a former page, and therefore concluded that a similar effect
might be produced also in human beings by previous sub-
cutaneous injection of his vaccines — that is to say, such
persons might be protected, actively immunised, against

XVI]

VIBRIO AND SPIRILLUM

449

natural infection with cholera per os. It ought to be
further stated, however, that according to the observations
of Wassermann and Pfeiffer (ZeitscAr. f Hygiene u?id Infect.
vol. XIV., and according to my own observations (Reports
of the Medical Officer of the Local Government Board
for 1893 and 1894), such intestinal protection of guinea-
pigs is not by any means uniformly observed even after
intraperitoneal active immunisation. R. Pfeiffer, as a result
of his recent experiments, finds that guinea-pigs passively
immunised by the intraperitoneal injection of “cholera
serum” are still susceptible to intestinal infection after
Koch’s method.

But, be this as it may as regards the guinea-pig,
Haffkine has in India, during 1894 and 1895, made a large
number of double and treble vaccinations, and has collected
a large body of statistics as to cholera-vaccinated persons
that have been exposed to cholera in India living in the
same locality and conditions side by side with non-vaccinated
persons. The latest statistics published in India, and in the
British Medical Journal , during the last months of 1895 by
medical men who had assisted in these vaccinations and
had observed the results are of a most encouraging nature ;
when seeing it stated that in a large body of un vaccinated
persons of a given locality (tea plantations) the incidence of
attacks is enormous, and in an equally large body of vac-
cinated persons living side by side and under the same
conditions with the former the incidence of attacks is
incomparably smaller, in fact in some of the latest statistics
is very small indeed as compared with that in unvaccinated
persons— that is to say, while of unvaccinated persons the
disease kills off many, of the vaccinated persons only few —
seeing all these statements one cannot help arriving at the
conclusion that the protective inoculations practised by

G G

450

MICRO-ORGANISMS AND DISEASE [chap.

Haffkine in India with cultures of cholera vibrio have had
positive results, and further that these observations form a
strong link in the chain of evidence that the cholera vibrio
is the cause of cholera. The evidence that Koch’s vibrio is
the microbe of Asiatic cholera, and as such forms an
essential — though not the only — factor in the production

Fig. 180. — Plate Cultivation in Gelatine of Vibrio Finkler — Prior,

INCUBATED AT 20°C. FORTY-EIGHT HOURS ; THE COLONIES ARE ROUND, LIQUE-
FIED, TURBID, SOME ISOLATED, OTHERS CONFLUENT.

Natural size.

of cholera asiatica— disposition, locality, season, being other
factors — is then a chain in which the individual links taken
separately are open to criticism, but when all are taken to-
gether— notably : the diagnostic value of the cholera vibrio
for cases of Asiatic cholera, the capability of the vibrio to pro-
duce powerful toxin, the result of experiments on ingestion of
cultures of cholera vibrio on human beings, the immunising

xvi]

VIBRIO AND SPIRILLUM

45i

action of cholera serum of human beings against the cholera
vibrio, taken together with Pfeiffer’s germicidal action of
cholera serum of immunised guinea-pigs, and the results
of Haffkine’s protective inoculations on human beings — form
as strong a body of evidence as can be expected — seeing
that animals are not subject to cholera — to confirm Koch’s
original view as correct, viz., that the vibrio is the microbe

Fig. 181. — Film Specimen of peritoneal Exudation of a Guinea-pig dead

AFTER 1NTRAPERITONEAL INJECTION OF CULTURE OF VlBRIO FlNKLER-

Prior.

X IOOO.

of cholera, that the growth and multiplication of this within
the cavity of the intestines produces toxins which absorbed
into the system cause the disease cholera. Consequently
consumption of articles of food, water and solids, con-
taminated with cholera vibrios, derived directly or indirectly
from the discharges of a cholera case, as also direct con-
tamination and introduction of cholera vibrios into the
alimentary canal, are capable of causing cholera.

G G 2

452 MICRO-ORGANISMS AND DISEASE [chap.

A vibrio was isolated by Finkler and Prior 1 from decom-
posing stools of a case of sporadic cholera, cholera nostras
(English cholera) ; this was done at a time when Koch’s
discovery of the comma bacillus in Asiatic cholera was but
recent, and when for the first time a vibrto was isolated from
the human intestinal discharges. The vibrio of Finkler-
Prior (or Finkler comma bacillus, or Finkler vibrio, or Finkler
vibrio proteus) is a comma bacillus which, in many points
resembling the vibrio of Koch, was thought by its discoverers
to be causally related to cholera nostras, but this view has
not been supported by subsequent investigation. Frank
and Kartulis have missed them in all cases of sporadic
cholera which they investigated, and I have myself not yet
come across the vibrio of Finkler in the intestine of a
considerable number of fatal cases of sporadic or English
cholera which I have had the opportunity of examining
during 1894 and 1895.

The points in which the Finkler-Prior vibrio resembles the
cholera vibrio are : (1) it liquefies gelatine, (2) and it is a
motile vibrio, also S-shaped forms and spirilla, but there
never was any difficulty in distinguishing Koch’s cholera
vibrio from the vibrio of Finkler-Prior by the following
characters : the vibrio of Finkler-Prior is distinctly larger —
longer and thicker— than the cholera vibrio, and it grows
incomparably faster at 20° C. in gelatine, and liquefies this
incomparably quicker than the cholera vibrio. Besides, the
colonies in the gelatine plate are always round and the
liquefied gelatine is uniformly turbid ; in these respects the
Finkler vibrio compares well with the proteus vulgaris ; in
the stab gelatine the vibrio of Finkler forms already after
forty-eight hours considerable growth and liquefaction, and
the liquefied gelatine is uniformly turbid ; also herein it
1 Centralblatt fur allg. Gesiindheitspjlcge, vol. i., Nos. 5 and 6.

XVI] VIBRIO AND SPIRILLUM 453

resembles the proteus vulgaris. Gelatine liquefied by the
cholera vibrio has no smell, whereas in the case of vibrio
Finkler it has a more or less putrid smell, just like that of a
growth of proteus vulgaris.

The cultures of vibrio Finkler act on the guinea-pig in
the same way as the cholera vibrio, subcutaneously, intra-
peritoneally, and by ingestion ; there is no difference gene-
rally in this respect between the two vibrios. There are,
as stated on a former page, particularly virulent cultures of
the cholera vibrio which in intensity of action on the guinea-
pig surpass the vibrio of Finkler, but they surpass also other
less virulent cultures of undoubted cholera vibrios.

Vibrio Finkler grows best at 20-2 1° C. ; it does not at all
grow well at 37° C., that is at a temperature when the
cholera vibrio grows best. If the peptone salt solution is
inoculated in one set of tubes with the cholera vibrio, in a
second set with the vibrio of Finkler, and of each set one
tube is kept in the incubator at 20° C., and likewise of each
set one tube is kept at 370 C., a very marked difference will
be observed between the two species of vibrios after twenty-
four hours, viz. the peptone culture of cholera vibrio incu-
bated at 20° C. is only very slightly turbid, there is just an
indication of growth having taken place, while the peptone
culture of vibrio Finkler shows marked turbidity, good
growth having taken place ; whereas the peptone culture of
cholera vibrio incubated at 370 C. shows uniform good
turbidity, the peptone culture of Finkler vibrio shows no
turbidity. The same holds good for cultures in broth pep-
tone. By incubating the peptone cultures at 370 C. for even
from twelve to eighteen hours the difference between the
two species is marked. Also on growing on the slanting sur-
face of Agar at 370 C. vibrio Finkler shows faint growth after
twenty-four or even after forty-eight hours, while the vibrio of

454

MICRO-ORGANISMS AND DISEASE [chap.

cholera has produced already in twenty-four hours a con-
spicuous film.

If to a peptone salt culture of pure cholera vibrio, as
soon as it shows turbidity (no matter whether incubated at
20° C. or at 370), a few drops of pure sulphuric acid are
added, as was mentioned on a former page, a distinct
rose-red tint, cholera red, is produced ; a peptone salt culture
of vibrio Finkler which in order to produce turbidity had
been incubated at 20° C. — it does not become turbid at
3 7C C.— treated with a few drops of pure sulphuric acid gives
no cholera red reaction. The assertions to the contrary are
based on the sulphuric acid used not being pure but con-
taining nitrites ; with such impure sulphuric acid also in a
peptone culture of proteus vulgaris a red reaction is
obtainable.

Another point in this connection worth mentioning is
that for the demonstration of the pure cholera red reaction
the peptone used for peptone salt culture ought to be pure
and free from nitrites. Pestana of Lisbon, who isolated
from the intestinal discharges of cases of cholerine that
occurred in epidemic form in Lisbon in 1894 a vibrio ( see
below), has shown that a culture of it in peptone salt, when
the peptone used was free from nitrites, gives no cholera red
reaction, but a culture of it in peptone salt made with
nitrite-containing peptone gives a faint but distinct cholera
red reaction.

Soon after Koch’s discovery Deneke1 isolated from stale
cheese a spirillum — spirillum tyrogenum , which in morpho-
logical and cultural respects bore a very great resemblance
to Koch’s cholera vibrio, in fact, looked at in the light of
the present knowledge of different varieties of cholera vibrio,
cannot be distinguished from this latter. In size, shape,
1 Deutsche Mcdicin. Woehenschrift , 1885, No. 3.

xvi]

VIBRIO AND SPIRILLUM

455

motility, growth in peptone salt, and cholera red reaction,
in gelatine, on Agar, on blood-serum, in its action on the
guinea-pig (administered per os after Koch), it is difficult
to distinguish it from the cholera vibrio ; perhaps it grows a
little faster on gelatine in the plate and in the stab, but, as
has been stated on a former page, such differences are
also observed between the individual varieties of the cholera
vibrios.

The same has to be said of a number of vibrios and
spirilla that have been isolated in the course of the last
three or four years by various observers in different waters :
vibrio berilonensis, vibrio danubicus, vibrio of Warsaw,
vibrio Nordhafen, vibrios of the Elbe, various species of
vibrios isolated from water (Seine and other rivers near and
around Paris) by Sanarelli ( Annales de I’Institut Pasteur ,
November 1893). With the exception of the vibrio phos-
phorescens of Elwers and Dunbar, most of the others differ
from the typical vibrio of Koch so little and in so few
details — in fact, less so than do the individual varieties of
vibrios isolated from noted cases of cholera — that from their
morphological and cultural characters, including the cholera
red reaction which they all show to a greater or lesser
degree, and from their intraperitoneal pathogenic action on
the guinea-pig, they cannot be distinguished from the
different varieties of the true cholera vibrios. And for this
reason I think Sanarelli’s contention that, inasmuch as the
water vibrios which he found in the Seine and other rivers
in France, that had been subject to notorious pollution with
the dejecta of cholera cases which had occurred in Paris, its
suburbs, and elsewhere in France, during the preceding years,
resemble in many respects the cholera vibrio, those water
vibrios are genetically related to the cholera vibrio, this con-
tention, I say, does not deserve to be set aside in the off-

456

MICRO-ORGANISMS AND DISEASE [chap.

hand manner that R. Pfeiffer does when criticising Sanarelli’s
results. Nor do I think that the discoverers of the various
water vibrios (berilonensis, danubicus, Dunbar’s vibrio found
in the Elbe in 1894, and other similar finds) are justified
by the small differences observable between these vibrios
and the typical cholera vibrio in denying a genetic relation.
I do not for a moment intend to imply that any or all were
so related, but because the waters, in which these vibrios
were found, did not produce cholera in the consumers, is
not sufficient arugment, as for the production of cholera it
would require a virulent cholera vibrio and various other
factors (mentioned on a former page), and alt* these may
have been absent in these cases.

I have isolated a vibrio from drain water (Hull, Sutton
drain) which was described on page 193 in the Cholera
Report of the Medical Officer of the Local Government
Board, 1894; the cultural characters of this vibrio were in
some respects distinctly different from the typical cholera
vibrio, in others they were identical, but in etiological
respects there was strong evidence that the water of that
Sutton drain had an important relation to causing cholera
asiatica (see Dr. Theodore Thomson’s report, ibidem, pp.
101, 102).

On the other hand, in certain filth-polluted well-water, to
the consumption of which an epidemic of Asiatic cholera at
Ashbourne in September, 1893, had been clearly traced (see
Dr. Bruce Low’s report, ibidem , p. 127), I have found in
the floccular suspended matter crowds of comma bacilli
(Fig. 182) which in morphological and cultural characters com-
pletely resembled the typical cholera vibrio (ibidem, p. 194).

The vibrio isolated by Pestana (Centralbl. f. Bald, und
Parasitenkwide, 1894) from the flakes of the dejecta of
cases of epidemic cholerine in Lisbon grows much slower

xvi]

VIBRIO AND SPIRILLUM

457

in gelatine than the typical cholera vibrio ; on Agar the
growth is also slower and much more transparent, it does
not give the cholera red reaction with pure sulphuric acid
when grown in pure peptone salt, and when injected into the
peritoneal cavity of the guinea-pig is far less virulent than
the typical cholera vibrio. The epidemic of cholerine in
Lisbon had a very low mortality — few cases of death out

Fig. 182. — Film Specimen of a Flocculus from the Water of the polluted
Well of Ashbourne that had caused an Epidemic of Asiatic Cholera.

x 1000.

of over 140 attacks, the normal mortality in an epidemic of
cholera being 50 per cent, and sometimes more — all these
facts justified Pestana in declaring that the vibrio is not the
cholera vibrio and the disease is not Asiatic cholera.
Recent observations, which I have carried out for the
Medical Department of the Local Government Board, and
which will be published in their Reports in 1896, seem to show

453 MICRO-ORGANISMS AND DISEASE [chap.

however, that Pestana’s and similar conclusions, notably
those arrived at with regard to the non-choleraic origin of the
water vibrios previously mentioned, are not to be accepted
unconditionally. The observations to which I refer were made
on oysters kept in sea-water tanks to which previously culture
of the typical cholera vibrio had been added. These vibrios
had been originally derived from the flakes of the rice-water
contents of a typical fatal case of cholera that had occurred
on board of a steamer arriving in the Thames in August
1894, from a cholera-infected port; these vibrios had in cul-
tivation all the characters of the typical Koch’s vibrio, and
tested on the guinea-pig’s peritoneum showed a^onsiderably
high degree of virulence — and, be it also noted, having been
carried on in subcultures through many generations showed
both the Pfeiffer’s test in corpore as also the Bordet-Durham
test in vitro with “ cholera serum.”

From the slanting surface of an Agar culture — incubated
for two days at 370 C. — the growth was scraped off and dis-
tributed in sterile salt solution, and then added to the sea
water in a tank in which oysters fresh from the oyster-beds
had been previously deposited. Several of these oysters as
also the sea water had for control been previously carefully
examined by the culture test for the presence of vibrios. In
the case of the oysters, after well brushing off under a stream
of water from the tap the exterior of the shell, the latter was
dried with a clean towel, opened with a sterile knife, and of
the liquor and the mashed-up substance of the oyster a
number of peptone salt cultures were made. Of the sea water
90 cc. were placed in a sterile flask, to it were added 10 cc. of a
10 per cent, peptone, 5 per cent, salt solution, and the whole
was incubated at 37° C. In neither case, the interior of several
oysters as also the sea water, were any vibrios discovered.

After the addition of cholera culture to the sea water 111

xvi]

VIBRIO AND SPIRILLUM

459

the tank, as previously stated, oysters kept therein for four
days and for nine days respectively — the tank being daily well
irrigated with fresh filtered sea water — yielded in peptone
salt cultures from their interior liquor and body substance
positive results,1 that is to say, yielded cultures of vibrios, but
though in many respects they resembled the cholera vibrios
added to the tank water, yet in some important points they
differed markedly from them as also from one another, and
retained these differences constant through subcultures.

In another series in which oysters were kept for four
days in cholera-infected sea water the peptone culture
yielded vibrios which possessed distinct differences, retaining
them in subcultures, not only from the original vibrios
employed for the experiment, but also from those
obtained from the previous two sets of oysters. The

conclusion which these observation-s justify seems to be
that in the bodies of oysters vibrios, which had an un-
doubted cholera origin, become markedly altered and be
come possessed of certain apparently permanent characters
not possessed by the vibrios previously. I cannot here
enter into the details of these observations, as these will
be published in the Reports of the Medical Officer of the
Local Government Board, and must content myself with
the statement that I think a permanent alteration of the
characters of the cholera vibrio had been established.
If this be so, then the differences noted in many of the
vibrios discovered in various waters (Spree, Danube, Elbe,
Seine, &c.) after the visitation by cholera of the respective
countries, as also those discovered by Pestana in cholerine,
need not indicate that these vibrios were not originally cholera

1 The water of the tank was examined in the above-named manner
and was found to yield positive peptone cultures after four and after ten
days respectively.

460

MICRO-ORGANISMS AND DISEASE [chap.

vibrios ; their cultural and other differences may, just as
was the case with the above oyster-vibrios, have been
acquired and established through the environment, through
their sojourn for some time under abnormal conditions.

R. Pfeiffer, in a series of well-known papers published in
the Zeitschrift f. Hygiene und Infekt. during 1894 and 1895,
has established the important fact that the blood-serum of
guinea-pigs highly immunised by repeated intraperitoneal
injection of living cholera vibrios ( see a former page)
possesses potentially specific immunising or ^germicidal
action in corpore, that is to say, when in a certain pro-
portion mixed with an otherwise fatal dose of cholera
vibrios and injected into the peritoneal cavity of a guinea-
pig, it kills the vibrios, and no disease follows, the animal re-
mains alive and well and is “ passively immunised.” This was
then extended by Pfeiffer also for the obtaining of “ cholera
serum,” i.e. of immunising serum, from the highly immunised
goat, and was shown to hold good also for “ typhoid serum,”
i.e. for a potential specific germicidal action of blood-serum
of animals highly immunised by intraperitoneal injection of
cultures of the typhoid bacillus against an otherwise fatal
dose of the typhoid bacillus. Several observers, I myself,
have been able to confirm — as indeed is easily done —
Pfeiffer’s fundamental discovery.

Bordet ( Anna/es de F Institut Pasteur , June, 1895) and
recently Durham (. Proceedings of the Royal Society, January
23, 1896) show that also in vit?'o “cholera serum” shows a
definite separating action, inasmuch as when added in
definite proportion (sometimes alone, sometimes with
normal serum — Bordet, alone — Durham) to a suspension
of living cholera vibrios contained in a. test-tube it makes
the vibrios become matted together in clumps, settling
at the bottom of the test-tube while the suspending

xv i] VIBRIO AND SPIRILLUM 461

fluid becomes clear, and that after some time the
motility of the vibrios becomes impaired and ceases, al-
though living colonies can still be cultivated from them.
Durham shows this action to take place also when “ typhoid
serum ” is added to a suspension of typhoid bacilli.

We shall speak of Preiffer’s germicidal action of the
cholera serum in corpore as of Pfeiffer’s test, of the Bordet-
Durham separation test in vitro as of the Bordet-Durham test.

As stated just previously, the fundamental fact discovered
by Pfeiffer as to the pronounced germicidal action of
“ cholera serum ” or “ typhoid serum ” on cholera vibrios
and typhoid bacilli, respectively, is well established. Now,
Pfeiffer shows by numerous experiments that the “cholera
serum,” that is, the blood-serum of animals highly im-
munised by living vibrios of an undoubted cholera origin,
possesses this pronounced germicidal action in corp07-e on
all samples of vibrios — several hundred — which he and
others got hold of from undoubted cases of Asiatic cholera,
but that it fails to exhibit this action on vibrios of doubtful
derivation, like the various water vibrios, the vibrio Nord-
hafen, &c. — that is to say, on vibrios which are not directly
and notoriously derived from undoubted cases of Asiatic
cholera — and he therefore feels justified in concluding that
any species of vibrio which submitted to Pfeiffer’s test
succumbs is a cholera vibrio, any species which does not
succumb to Pfeffer’s test is not a cholera vibrio. The same
is applied by Pfeiffer to the typhoid test mutatis mutandis.
Bordet and Durham imply through their test in vitro a
somewhat similar conclusion ; but though their test was of
positive differential value in the case of the cholera serum
and cholera vibrio it was not so unequivocal, according to
Durham, in the case of colon serum and colon bacillus.
Koch has shown ( Zeitsclir . /. Hygiene , vol. xii.) that if in

462

MICRO-ORGANISMS AND DISEASE [chap.

any case of suspected cholera the flakes of the intestinal
fluid or evacuation contain the vibrios in the typical distribu-
tion and in almost a pure condition such a case can
without further hesitation be declared as cholera asiatica;
those who have had sufficient experience of microscopic and
cultural experiments of numerous cases of cholera can but
confirm Koch’s statement. Subsequent cultivations con-
firm the primary diagnosis. It must be obvious that if
there be sporadic, not typical cases, from which by the
culture test vibrios are isolated which in many respects
resemble, in others slightly deviate from, the typical Koch’s
vibrio, an unfailing test by which these vibrios could be
shown to be or not to be the true cholera vibrios would be
invaluable, and in much higher degree would such a test be
invaluable in the case of vibrios which like the above-quoted
water vibrios cannot be shown to have been directly derived
from cholera cases, and which owing to slight cultural
differences are declared not to be cholera vibrios. Pfeiffer
maintains that his test does furnish this important and unfail-
ing evidence : the vibrios, no matter what their slight cultural
differences be, that are derived from true cholera cases, give
his test, therefore are true cholera vibrios ; the vibrios, how-
ever, no matter how similar they be in morphological and
cultural respects to the Koch’s vibrio, that are not derived
from cholera cases, do not give his test, are therefore not
cholera vibrios. Without wishing in the least to deny
Pfeiffer’s justification in formulating so definitely his con-
clusions, nor wishing to accept unconditionally and in full
— for reasons presently to be stated — Pfeiffer’s statement, it
is at the outset only fair to draw attention to the following
hitherto unexplained facts of the Massowah vibrio.

Pasquale had a few years ago sent to Pfeiffer and to
Metchnikoff cultures obtained from case% assumed to be

XVI] VIBRIO AND SPIRILLUM 463

cholera that had occurred in Massowah. Pfeiffer accepted
this Massowah vibrio as the cholera vibrio — notwithstanding
its slight deviations in cultural respects from the typical
Koch’s vibrio, no doubt influenced by the knowledge gained
that the vibrios derived from undoubted cases of cholera do
not all coincide in all cultural characters — and his earlier
experiments and statements on cholera were admittedly
made with, and refer to this Massowah vibrio. Metchnikoff
also accepted, after study, the Massowah vibrio as the true
cholera vibrio ; many of his experiments and observations
on animals and human beings were made with this vibrio.
Now, unfortunately this Massowah vibrio does not give
Pfeiffer’s test, and therefore is declared by Pfeiffer not to be
cholera vibrio at all. This is a difficulty, though like all
such difficulties it need not deter us from altering an initial
wrong conclusion. But there are other and greater diffi-
culties. Pfeiffer cannot deny the possibility that vibrios
originally derived from true cholera, but living afterwards
under abnormal conditions of temperature, soil and others, for
considerable periods, could so alter as to change some of their
original cultural characters as also their physiological reactions.

This, for instance, seems to me to have been the case
with Sanarelli’s water vibrios, with Pestana’s vibrio, and I
have already given direct evidence of such being the case
with my oyster vibrios. There is nothing extraordinary or
new in such an assumption ; it is borne out by laboratory
observations on a number of microbes, altering their charac-
ters permanently, cultural and chemical, by the influence of
medium, temperature, the animal body, &c. One could
therefore well assume or at any rate admit the possibility —
it would be no exaggeration even to say the probability
that cholera vibrios living in water might or would so alter
that the nature of their behaviour under Pfeiffer’s test might

464

MICRO-ORGANISMS AND DISEASE [chap.

or would be altered. As a matter of fact, I have found that
of guinea-pigs immunised by repeated intraperitoneal injec-
tion with one variety of living cholera vibrios— -derived from
an undoubted typical fatal case of Asiatic cholera in one
locality in England in 1893 — a certain percentage did not
prove themselves resistant against a subsequent intraperi-
toneal injection with a fatal dose of living cholera vibrios
derived from an undoubted and typical fatal case of Asiatic
cholera that occurred in another locality in England in 1893.
The animal that so died had acute peritonitis and only few
vibrios in the peritoneal exudation, but the intestine was full
of grumous fluid that contained the cholera vibrios in almost
pure culture (Reports of the Medical Officer of the Local
Government Board for 1894).

All these results seem to me to show that the apodictic
announcement that such and such a vibrio is not a cholera
vibrio because it does not succumb to the “ cholera serum
obtained by immunisation with a particular cholera vibrio
is not sufficiently established, although it may be conceded
that a vibrio which does answer in positive fashion to
Pfeiffer’s test is a cholera vibrio. For this last reason Pfeiffer’s
test is undoubtedly of exceedingly great value both with
reference to cholera and typhoid, but it should not extend
its differential value to the negative cases.

(m). Vibrio Metchnikovi. — Gamale'ia 1 describes an acute
fatal disease — gastro-enteritis cholerica — affecting fowls in
Odessa during the summer months ; the disease in its
symptoms and its fatality is very similar to fowl cholera,
but it differs in this essential respect that it is not caused by
the bacillus of fowl cholera ; it is caused by a vibrio present
in large numbers in the blood. In its morphology, motility,
size, and shape, and formation of S-shaped and spiral forms,

1 Annales de V In stihit Pasteur , No. 9, 18SS.

VIBRIO AND SPIRILLUM

465

xvi]

as well as in its cultural character it resembles, but is not
quite identical with, Koch’s cholera vibrio. Inoculated sub-
cutaneously into pigeons or guinea-pigs it proves very virulent,
producing acute disease and death ; fowls can be infected by
ingestion. On post-mortem examination of the infected animals
the intestines are found greatly congested and contain in their
cavity grumouss anguineous fluid. The spleen is not enlarged.

Fig. 183.— Film Specimen of a Culture of Vibrio Metchnikovi.

X 1000.

The vibrio is copiously present in the intestinal fluid and in
the blood.

Gamale'ia made the statement that the vibrio Metchnikovi
and the cholera vibrio are mutually protective for the pigeon ;
that is to say, that a pigeon that has survived disease caused
by the injection of anon-fatal dose of one vibrio is protected
against a subsequent injection of a fatal dose of the other.

H H

466

MICRO-ORGANISMS AND DISEASE [chap.

But Pfeiffer1 has shown that while the vibrio Metchnikovi
is virulent for the pigeon the vibrio of cholera is not, and
further that a pigeon that had received first a large dose of
cholera vibrios succumbs to a further injection of the vibrio
Metchnikovi just like any normal pigeon. That pigeons are
insusceptible to subcutaneous and intermuscular injection
can be easily shown ; I have injected into the pectoral
muscle as much as 2-3 cc. of recent active broth culture, and
on searching by the culture test and film specimens twenty-
four hours afterwards for comma bacilli no trace of them
could be discovered. The pigeons were and remained
perfectly normal.

Vibrio Metchnikovi differs then from the vibrio of cholera
as regards the guinea-pig, the former being very virulent
injected subcutaneously. Metchnikoff has shown that
guinea-pigs can be immunised by successive inoculations
of non-fatal doses of culture, and that the blood-serum of
such animals also possesses germicidal action and can confer
passive immunity to guinea-pigs against an otherwise fatal
dose of the vibrio ; and further that the germicidal action of
the serum of an immunised animal exhibits this germicidal
action against the vibrio Metchnikovi already in vitro.

(n.) Spirillum Obermeyeri of relapsing fever. — Obermeyer
{Centralbl. f. d. vied. Wiss ., 1873, No. 10) discovered in the
circulating blood of patients affected with this fever, during
the febrile stage, innumerable spirilla actively motile : they
disappear from the blood immediately preceding the end of
the febrile stage.

The spirilla are very thin and about 20-30-40 \x. long ;
their movement is that of rapidly' progressing spirals. Koch
has demonstrated by photography of dried and stained speci-
mens the presence of the flagella in the spirilla. Weigert

1 Zeitschrift f. Hygiene, vii. 3.

xvi] VIBRIO AND SPIRILLUM 467

has shown that, unlike other bacteria, they are barren of a
cellulose sheath, since dilute liquor potassae dissolves the
whole substance of the spirilla. By drying and staining
cover-glass specimens it has been shown that the spirilla are
uniform spirals, and do not show anything that might be
interpreted as being made up of shorter elements, comma
bacilli or vibrios. The spirals when long are often plicated,
but their turns are always close, and more or less in the
manner of a corkscrew. Immediately preceding the febrile
stage they appear in the blood, grow more and more numer-
ous during the fever, and disappear again completely from
the circulating blood before the fever quite ceases. During
the non-febrile stage they most probably are present in the
spleen and marrow of bone — Birch-Hirschfeld found many
of them in the necrotic foci of the spleen — where perhaps
they undergo germination and reproduction. It is feasible
to assume that when during the febrile stage they reach the
acme of their development they gradually break down,
leaving spores in the shape of granules behind : these are
carried into the spleen and bone marrow where they accu-
mulate. During the non-febrile stage these spores germinate
here again and gradually grow into the spirilla, which when
ripe and motile gradually find their way again into the blood.
Such a view would well harmonise with the facts of the case
and also with what has been shown of the plasmodium
malaria:.

As a matter of fact the spirilla in the blood often show
bright granules in their interior, which might well be spores.

Koch has shown that in artificial culture the spirilla are
capable of growing out into long spiral filaments matted
together, but no real artificial cultures have been as yet pro-
duced. That the spirilla are the real microbes of relapsing
fever is proved by the experiments of Vandyke Carter

H H 2

468

MICRO-ORGANISMS AND DISEASE [chap.

( British Medical Journal , October, 1881), who was the first
to produce typical relapsing fever in the ape after injection
of blood of a patient taken during the febrile stage and con-
taining the spirilla. The disease produced in the ape was

Fig. 184. — Blood of Relapsing Fever (Human).
Blood-corpuscles and spirilla Obermeyeri.
Magnifying power 700. (After Koch).

true relapsing fever, and the animal’s blood contained
during the febrile stage the identical spirilla in large
numbers. Koch, Heydenreich, and Metchnikoff have con-
firmed this. Motschutkowsky (Deutsches Archiv f. klin. Med.,

XVI] VIBRIO AND SPIRILLUM 469

Band xxiv.) has produced relapsing fever in the human
subject by inoculation of blood containing the spirilla.

Metchnikoff ( Virchow's Arc/iiv, Band cix., 1887) main-
tains that the disappearance of the spirilla from the system,
i.e. recovery, is due to phagocytes, that is to say, that the

Fig. 185. — Blood of Ape inoculated with Blood shown in preceding.

Figure.

Blood-corpuscles and spirilla.

Magnifying power 700. (After Kocli).

white cells of the spleen swallow and destroy the spirilla and
thus purge the system of them. It is a fact that the spirilla
are found enclosed within the white cells of the spleen, but
it does not follow that Metchnikoffs view is correct, for

470

MICRO-ORGANISMS AND DISEASE [ch.xvi

Baumgarten has justly pointed out that as in other diseases
so also in relapsing fever the enclosure of the spirilla by
leucocytes may be only a result of the microbes having pre-
viously been killed by other agencies, and that after this
they have been taken up by the white cells just like other
dead formed matter.

CHAPTER XVII

YEAST FUNGI : TORULACEvE, SACCHAROMYCES

Yeast, torn la (Pasteur), or saccharomyces, is not a bac-
terium, but belongs to an altogether different order of fungi
— the Blastomycetes. It consists of spherical or oval cells,
very much larger than the largest micrococci, and as in the
case of these each cell consists of a membrane and contents.
The contents are either homogeneous or finely granular proto-
plasm ; in the latter case there are generally present one,
two, or more small vacuoles.

There are a great many species of Torula , varying from
one another morphologically chiefly in their size, and
physiologically by their action on various fluids {see below).

The cells multiply in suitable media by gemmation, a
minute knob-like projection appearing at one side of the cell,
and enlarging till it reaches nearly the size of the original or
mother-cell. It finally becomes altogether constricted off
from this latter, or having reached its full size remains
fixed to the mother-cell, and each cell again producing
by gemmation a new cell. In this way aggregations
of four, six, eight, or more cells are formed, which may
be arranged either as a chain when the production proceeds

472

MICRO-ORGANISMS AND DISEASE [CHAP.

in a linear manner, or as a group if the gemmation takes
place laterally.

Under varying conditions of growth, e.g. on transplanting
ordinary yeast growing in sugar-containing fluids on to potato,
but sometimes also in the same nutritive fluid, it is observed
that some of the yeast cells enlarge twice, thrice, and more
times ; they then form in their interior two, three, or more
small cells by endogenous formation ; these new cells are

Fig. 186.— Torula, or Saccharomyces.

In the lower part of the figure an ascospore and four isolated spores (after Rees)

are shown.

Magnifying power about 700.

regarded as spores 1 — the mother-cell being an ascospore
— and become free by finally bursting the membrane
of the mother-cell. On sowing these new cells into
sugar-containing fluids they multiply by the process of
gemmation.

Classifying them according to physiological function there
are various species of torula or saccharomyces. They all

1 T. de Seynes, Comptes Rendus , 1S66 ; Rees, Bot. Zeilschr. 1S69 ;
Hansen, Carlsberg Laborat . 1883.

XVI l]

YEAST FUNGI

473

have the power to split up sugar into alcohol and carbonic
acid, but this power is not possessed by all to the same
degree.

(a) Saccharotnvces cerevisicc (torn la cerevisice). — This is the
ordinary yeast used in the production of beer. The in-
dividual full-grown cells vary in diameter from o’ooS to
o'oi mm. ; they form beautiful long chains. They produce
ascospores.

(h) Saccharomyces vini is very common in the air, and
produces alcoholic fermentation of grape-juice ; it is
therefore the proper yeast of wine-production. Its cells
are elliptical, slightly smaller than the former ; it forms
ascospores.

(c) Saccharomyces pastorianus is of various kinds (Hansen) :
in some the cells are about o-oo2 to 0 005 mm. in diameter,
in others larger. Some form ascospores, others do not.
Most of them can be found in wine-fermentation and in
cider-fermentation, but only after the first alcoholic fermen-
tation is completed. They are very common in the air. I
have sown a saccharomyces, which was contained in ordinary
water, on solid nourishing media (gelatine and gelatine
and broth). It grew up copiously and formed groups of a
distinct pink colour. When growing in the depth of the
nourishing medium it grew as a colourless torula, no
ascospores were formed, multiplication taking place by
gemmation only.1

(d) Saccharomyces mycoderma ( mycoderma vini). — This
yeast is found forming the scum or pellicle on the surface of
wine, beer, and fermented cabbage ( Sauerkraut ) ; its cells
are oval, about o'oo6 mm. long and 0-002 broad. It forms
chains ; the ascospores are two or three times larger. It has
nothing to do with the alcoholic fermentation, and is not to

1 Quart. Journ. of Micr. Science, 1883.

474

MICRO-ORGANISMS AND DISEASE [chap.

be confounded with mycoderma aceti} which is a bacterium
and the efficient cause of acid fermentation in wine and
beer.

The saccharomyces mycoderma does not grow well in the
depth of liquids, but when sown into a liquid of acid re-
action and containing but little sugar Cienkowsky saw the

Fig. 187. — Saccharomyces mycoderma, or Oidium albicans.
(After Grawitz.)

From an artificial cultivation in dilute nourishing material.

d. Branched mycelium.
fa. Torula stage.
ffi. Mycelial stage.

cells elongating into cylindrical elements ; each of which by
gemmation produced a new cell which also elongated, and
so on till a linear series of cylindrical cells was formed,
separated from one another only by a thin septum ; a mass
of filaments very much resembling a mycelium was thus

1 Nageli, see chapter viii. 2.

XVII]

YEAST FUNGI

475

formed. The cylindrical cells give origin by gemmation to
spherical and elliptical torula-cells.

Such a growth, in which the torula-cells are capable of
forming a sort of mycelium, was formerly called o'idium , and
as o'idium albicans is recognised as the cause of “ thrush ” :
the well-known white patches which occur on the mucous

Fig. 188.— Film Specimen of Thrush Fungus.
(Bousfield.)

X 700.

membrane of the mouth and pharynx in suckling infants
and debilitated patients.

Grawitz 1 has proved by observations on artificial cultures
that this fungus is identical with the o'idium variety of Sac-
charomyces mycoderma ; the cells are spherical or cylindrical,
the former about o'oo3 to 0-005 rnni. in diameter, the latter
1 Virchow's Archiv, vol. Ixx.

476

MICRO-ORGANISMS AND DISEASE [ch.xvii

up to o-o3 or o-o5 mm. long. As above described it forms
mycelium-like filaments from which, by lateral and terminal
gemmation, spring spherical or oval torula-cells. It also
forms ascospores containing four to eight spores.

It grows well on gelatine at 20° C. and on Agar at 370 C.,
forming white round colonies from which extend radially
fine threads ; it does not liquefy the gelatine.

CHAPTER XVIII

MOULD-FUNGI: HYPHOMYCETES OR MYCELIAL FUNGI

Of this class of fungi only those are of special interest to
-the pathologist which in some way or other are connected
with disease. The fungi consist of branched and septate
threads or hyphse ; each filament or hypha is composed of a
row of cylindrical cells, consisting of a membrane and clear
protoplasm, the individual cells being separated from one
another by a thin transverse septum ; they increase in
number by fission, and in this way the filaments increase in
length. The growing ends of the hyphte are filled, not with
transparent, but with highly-refractive protoplasm. Some
cells, by budding out laterally, produce cylindrical threads,
which subdivide into a series of cylindrical cells, these by
division and lengthening forming a new branch-hypha. The
filaments form by their branches an interlacing feltwork,
called thallus or mycelium. The mycelial fungi which in-
terest us belong to the order known to botanists as the
Ascomycetes. They are characterised by the fact that one or
other branch of the mycelial hyphae produces at its end a
series of spherical or oval cells — the conidia spores or con-
idia. In addition to this some of the hyphae form peculiar
large mother-cells, or sporangia , in the interior of which

478 MICRO-ORGANISMS AND DISEASE [chap.

spores are formed by endogenous formation. When these
sporangia are cylindrical or club-shaped, they include eight
spores, and are called asd ; the spores being ascospores. All
conidia or spores by germination grow into the mycelial
threads which become septate or subdivided into a row of
cylindrical cells ; these by division cause the lengthening of
the mycelial threads.

(a) O'idium ladis. — Here the mycelium is composed of
septate branched filaments of various thicknesses. Some
branches of the mycelium at their ends or laterally at a sep-
tum produce by division a series of spherical or oval conidia-
spores, about 0*007 to 0 01 mm. long. These ultimately
become isolated, and then germinate into a short cylindrical

XVI 1 1]

MOULD-FUNGI

479

filament, which subdivides by transverse septa into a series
of cylindrical cells ; these by continued growth and division
give origin to the ordinary septate branch-hyphse. The
formation of conidia proceeds at the ends of these in the
same manner as before. The o'idium lactis forms a whitish
mould on milk, bread, paste, potato, &c.

Fig. 190 — Fungi from a Favus-Patch (Neumann).

Favus, Herpes tonsurans, and Pityriasis versicolor of man
and animals, are, according to the researches of Grawitz,1 due
to a fungus in morphological respects identical with o'idium
lactis. In favus it is known as Achonon Schoenleim, in
Herpes tonsurans as Trichophyton tonsurans , in Pityriasis
versicolor as Microsporon furfur. Grawitz has shown by
artificial cultures on gelatine that the spherical or oval

1 Virchow's Archiv, vol. Ixx. p. 560.

480

MICRO-ORGANISMS AND DISEASE [chap.

conidia germinate into shorter or longer cylindrical filaments,
which by subdivision form septate mycelial hyphae. These
and their branches give origin in their turn to spherical or
oval spores or conidia. They, as well as the hyphae, differ
in size in the various species.

Malcolm Morris and G. C. Henderson,1 on the other hand,
maintain that by artificial cultivation of the spores of
Trichophyton in the substance of gelatine-peptone, at tem-
peratures varying from 150 to 250 C, these grow into
branched septate mycelial filaments, which by their mode of
fructification are seen to be identical with the mycelium of
Penicillium. Compare also with Babes.2

( b ) Aspergillus. — Some of the branches of the mycelium
of this fungus assume an upright position, are thicker and
not at all, or only slightly, septate, and at their end form
flask-shaped enlargements, from which grow out radially
short cylindrical cells — basidia ; and these again at their
distal or free ends produce chains of spherical spores or
conidia. This is a very common mould, and according to
differences in the colouration of the mycelium and spores is
subdivided into different species : A. glaucus, candidus,
flavescetis , fumigatus , 6-v.

Besides this mode of spore-formation (asexual), there is
another (sexual), which according to De Bary consists in
this : some terminal branch of the mycelium becomes
twisted like a spiral, this is considered the female organ of
fructification or carpogonium ; from the same thread branches
grow towards the carpogonium ; one of these branches
becomes fused with the terminal portion of the carpogonium
called the ascogonium, while the others — the pollinodia —
branch and surround the carpogonium like a capsule : the

1 Journal of the Royal Microscopical Society, April II, 1SS3.

2 Archives de Physiologic, 8, 1883, p. 466.

XVIII]

MOULD-FUNGI

481

A. Hypha, the end of which, c, bears
st. The basidia.
as Ascogonium.

Fig. 192.— E. Perithecium, highly magnified.
as. Ascogonium.
w. Cells of the pollinodia.

I I

482

MICRO-ORGANISMS AND DISEASE [chap.

whole organ is now called a perithecium. Finally the asco-
gonium by rapid division gives origin to a number of oval
septate tubes, inside of which by endogenous formation
numerous spores make their appearance.

Grohe 1 was the first to show that the introduction of the
spores of some species of aspgrgillus into the vascular
system of rabbits sometimes produces death, with symptoms
of metastasis into the various organs due to localised foci,
where these spores grow into mycelial filaments. Lichtheim 2
showed that such mycoses in rabbits cannot be produced by
the spores of Aspergillus glaucus , but easily by those of
Aspergillus flavescens and fumigatus , less in degree by
Aspergillus niger. Grawitz 3 studied this process more
minutely, and found that, no matter whether the spores
• are injected into the vascular system or into the peritoneal
cavity, there are established in the kidneys, liver, intestines,
lungs, muscles, and occasionally in the spleen, marrowbones,
lymphatic glands, nervous system, and skin, minute meta-
static foci, due to the growth of the spores into mycelial
filaments with imperfect organs of fructification, but no
spore-formation. Grawitz thought that the spores of ordi-
nary moulds (penicillium and aspergillus) are capable of
assuming these pathogenic properties if cultivated at higher
temperatures (39° to 40° C.), and in alkaline media. These
fungi, as is well known, grow well at ordinary temperatures
and in acid media, and are then innocuous when introduced
into the animal body ; but by gradual acclimatisation they
can also be made to grow at higher temperatures and in
alkaline media, when they assume pathogenic properties,
becoming capable of resisting the action of living tissues
and of growing in them. This view has been proved to be

1 Berl. Jdin. IVoch. 1871. 2 Ibid. 9 and 10, 1S82.

3 Virchow's Archiv, vol. lxxxi. p. 355.

XVIIl]

MOULD-FUNGI

4§3

Fic. 193. — From a Section through the Kidney of a Rabbit dead thirty-

six HOURS AFTER THE INJECTION OF SPORES INTO THE JUGULAR VEIN.

Fat droplets. T. Tyrosin crystals.

In the upper part of the figure is a metastatic focus composed of Aspergillus spores
and mycelium. In the lower half of the figure the urinary tubules and two
Malpighian corpuscles are seen. (After Grawitz.)

incorrect by Gaffky,1 Koch,2 and Leber.3 Those spores
that do exert such pathogenic properties are not at all

1 Miltheil. a. d. kais. Gesundheitsamte, 1880.

2 Berl. /din. Woch. 1881. 3 Ibid. 1882.

I I 2

484 MICRO-ORGANISMS AND DISEASE [chap.

dependent on such acclimatisation, and are not ordinary
moulds, but a distinct species of aspergillus (Lichtheim),
which grows well at higher temperatures (38° to 48° C.),
and the spores of which under all conditions of growth are
capable of producing in rabbits the mycosis in question.

Several cases of aspergillus infection in man (Pneumono-
mycosis) have been recorded, see R. Boyce, Journal of
Pathol, and Bacteriol. , No. 2, 1892.

(c) Penicillium. — In this fungus hyphae, which are not sep-
tate, grow out from the mycelium ; from the end of each of
these arise like the fingers of the hand a number of short
branched cylindrical cells, which give origin to chains of
spherical spores.

The following two fungi belong to the order of fungi
called Phy corny cetes.

(d) Mucor is characterised by this, that from the mycelium
hyphse grow out which are not septate, and at the end of
these a large spherical cell originates, sporangium , in which
by endogenous formation a large number of spherical spores
are developed ; the wall of the sporangium giving way, the
spores become free.

An important case of general '■'■mycosis mucorina ” in
man, ending in death, has been recently described by Dr.
Paltauf (Virchow’s Archiv , vol. 102, 3, p. 543). From the
alimentary canal of the patient an invasion of the internal
organs by the mycelium and spores of a kind of mucor
occurred, leading to the formation of metastatic inflam-
matory foci in the Peyer’s glands, lungs, pharynx, larynx,
cerebrum, and cerebellum. In these organs were found foci
of inflammation caused by mycelial threads and sporangia,
belonging to the group of mucor. Mucor rhizopodiformis
and corymbifer were shown by Lichtheim to be pathogenic
when injected into the vessels of the rabbit.

XVIIl]

MOULU-FUNGI

Fig. 194.— Saprolegnia of Salmon Disease.

A sporangium filled with zoospores ; in connection with them several young
mycelial threads.

(?) Saprolegnia ; colourless tubular threads, forming gela-
tinous masses on living and dead animal and vegetable
matter in fresh water. The cylindrical or flask shaped ends

of the threads— zoosporangia — form in their interior numbers
of spherical or oval spores — zoospores , possessed of locomo-

486 MICRO-ORGANISMS AND DISEASE [chap.

tion (one flagellum at each pole) and which finally escape
from the threads. These zoospores after some time become
resting, surround themselves with a membrane, and finally
germinate into a cylindrical mass which becomes transformed
into the mycelium. Besides this asexual there is, however,
a second or sexual mode of fructification, consisting in -this :
At the end of a mycelial thread a cell grows up into a
spherical large ball, the oogonium. From the same thread,
thin threads — antheridia — grow towards the oogonium, with
the protoplasm of which they merge. This latter then
differentiates into a number of spherical masses, the oospores ,
which become invested with a membrane. These become
free and then germinate and grow into a mycelium.
Saprolegnia grows on the skin of living fish, and causes
here severe illness often terminating in death. Thus the
salmon disease, as Professor Huxley has shown,1 is caused
by this parasite. The zoospores of this salmon saprolegnia
are, however, as Huxley has shown, as a rule non-motile.
The hyphae of the fungus traverse the epidermis in the
diseased patches of the salmon, and they bore through the
superficial layer of the derma, a stem-part being situated in
the epidermis, and a root-part in the derma ; each of these
elongates and branches out. “ The free ends of the stem-
hyphae rise above the surface of the epidermis and become
converted into zoosporangia, more or fewer of the spores of
which attach themselves to the surrounding epidermis and
repeat the process of penetration.” In saprolegnia associated
with the salmon-disease Professor Huxley observed only the
asexual mode of fructification.

(/) Actinomyces, or ray fungus. — Bollinger 2 first showed
that various tumours in cattle leading to chronic suppuration,

1 Proceedings of the Royal Society. No. 219, 1882.

2 Centralbl. f. d. vied. JViss., 1877, No. 27.

XV 111]

MOULD-FUNGI

487

e.g., in the jaw, the tongue, pharynx and skin, are one and
the same disease, due to a parasite which he constantly found

A nodule is shown composed of round cells ; in the centre is the clump of aclinomyces
surrounded by large transparent cells. Magnifying power 350.

Fig. 195. — From a Section through the Tongue of a Cow dead of
Actinomycosis.

Fig. 196. — a Clump of Actinomyces more highly magnified, 700.

in the tumours in the shape of yellow granules, and which,
when examined under the microscope, consisted of radially

488

MICRO-ORGANISMS AND DISEASE [char

arranged fibres and clubs which Professor Harz designated
actinomyces , or ray fungus ; the disease it causes is therefore
called actinomycosis. Israel 1 next observed a disease in the
human lung, in which he found a mycelial fungus; this was
afterwards identified by Pomfick as the same ray fungus
seen by Bollinger in the tumours of cattle. Pomfick himself
published 2 several cases of actinomycosis in man. Since
these observations a large number of cases in cattle, pigs,
and in man have been published, in which tumours, abscesses,
and suppurations, &c., were found in one or the other of
the following organs : in the jaw, skin, tongue, pharynx,
larynx, lung, intestine, liver, brain, and which proved to be
due to the same parasite — actinomyces.

In cattle the disease manifests itself by firm tumours in
the jaw, in the alveoli of the teeth, and particularly by a
great enlargement and induration of the tongue — “ woodeti
tongue On making sections through this latter organ there
are found present in all parts microscopic tumours of small-
cell growth. In the centre of each tumour is a clump of
actinomyces. This clump is surrounded by a zone of largish
cells, with one to four nuclei. The periphery of the tumour
is made up of a fibrous capsule, with spindle-shaped cells.
Occasionally the tumours are to be seen also in the skin and
in the lung ; in the latter organ they appear as whitish
nodules, easily mistaken for tubercles.

As the central fungus by active growth enlarges, so the
tumour enlarges by new infiltration with round cells spread-
ing into the surrounding tissues. In a later stage the central
portion softens and becomes purulent : an abscess is thus
formed which, Opening on to the surface, or into the nearest
cavity, soon discharges copious pus; when the abscess opens

3 Virchow's Archiv, Band lxxiv.

2 Die Aciinomykose des Menschen, Berlin, 1SS2.

XVIIl]

MOULD-FUNGI

489

on to the free surface, e.g., jaw, skin, pharynx, larynx, lung,
intestine, an ulcer is established, which, as the infiltration in
the periphery proceeds, enlarges. In the discharge of the
abscess a number of yellowish minute granules can be found ;
these granules looked at under the microscope are a mass of
the ray fungus. It has now been established that carious teeth
may represent the point of entrance for the fungus ; in these
cases the alveolar process of the jaw becomes the place for
the growth of the fungus, leading to the formation of a hard
tumour, gradually becoming converted into an abscess and
ulcer. The infection, i.e. invasion by the fungus, then spreads
to the lymph glands and skin nearest to the affected jaw, and
here produces tumour, then suppuration and ulcer ; or it
invades the tonsils and the pharynx, either primarily or after
it has once taken root in the jaw, tongue, or cheek. Or it
appears primarily in the larynx, trachea, and lung ; in these
cases the fungus has evidently been introduced by the air
during inspiration. In the case of the lung extensive inter-
stitial inflammation is set up, leading to abscesses perforating
into a bronchus. Or it invades primarily the alimentary
canal and leads here to abscess and copious suppuration,
and even to perforation of the part ; in the case of the
alimentary canal the fungus may have entered with the food.
From the alimentary canal the disease spreads to the mesen-
teric glands and the liver ; in this latter organ it produces
abscess, which may open through the peritoneum into the
peritoneal cavity, or, if previously an adhesion with the
abdominal wall had been established, may perforate out-
wards. In all these instances the discharged pus contains
the yellow granules, i.e. groups of the ray fungus.

In the case of the skin the fate of the tumours is suppura-
tion and formation of abscess, and this opening on the
surface leads to the formation of a sore. The primary

490

MICRO-ORGANISMS AND DISEASE [chap.

infection of the skin by actinomyces has been proved (E.
Muller, Mi/th. aus d. chirurg. Klinik , Tubingen, Band iii.,
3) in a case in which a wood splinter in the skin had
evidently been the means of providing an entrance for the
fungus. Both in man and cattle these various ways of
infection with actinomyces have been observed in many
cases.

The various ways above mentioned in which the fungus
invades the organism at once suggest that it has its usual
habitat in the outside world, i.e. that it is an organism which
is introduced into the animal or human body from the out-
side, and is not directly derived from an infected animal or
man. It is a prevalent opinion that the natural habitat of
the ray fungus is on cerealia, that it lives on these parasiti-
cally, and through and from these enters the animal body
through wounds, abrasions, &c. Johne ( Centralbl f. d.
med. Wiss. 1881, No. 15) has shown that actinomyces
occurs normally in the pits and the loculi in the tonsils of
the pig ; in these instances there are always present bits of
ears of barley covered with what appeared to be ray fungus.
Jensen (. Deutsche Zeitschrift f Thiermed.) observed an epi-
demic of actinomycosis in cattle fed on barley ; and Piana
described actinomyces nodules in the tongue of cattle, where
in the midst of some of the nodules there were present
portions of vascular fibre tissue of corn surrounded by ray
fungus. Finally, Soltmann {Breslauer drztl. Zeitschrift , 1885,
No. 3) made the remarkable observation of an actinomyces
abscess in man in the region of the dorsal vertebral column,
which was caused by the penetration (during swallowing) of
an ear of barley ; the abscess opened and the ear was
discharged. Fischer ( Centralbl . fiir Chirurgie , No. 22,
1890) describes a similar case: a labourer on chewing barley
pierced his tongue with a portion of the awn. Eight days

XVI 1 1]

MOULD-FUNGI

491

later a swelling appeared on the punctured spot, and after a
fortnight a tumour of the size of a filbert could be distinctly
felt. After eighteen days an incision was made into the
tumour, and the examination of the scanty pus and the
tissue of the tumour revealed the presence of numerous
yellow granules — actinomyces. Also the fragment of the
awn was removed from the interior of the tumour, and
on examining it under the microscope was found covered
with clumps of actinomyces. So that from all this the con-
clusion appears justified that actinomyces is a fungus having
its habitat on cerealia, and with and by them is introduced
into cattle and man.

As mentioned above, the tumours and abscesses occurring
in one or the other organ contain peculiar minute granules
and clumps, visible already to the unaided eye, generally of
a yellowish, occasionally of a yellow-greenish tint. Under
the microscope they appear made up of a central mass of
fine granules, or of a distinct trellis-work of fine branched
threads : next is a zone of coarser granules, which granules
do not look unlike cocci ; but when this or the central zone
is teased out it can be shown that the granules are not really
granules, but in reality are densely aggregated and twisted
branched fine fibres, the “ granules ” being only due to
optical sections of the fibres ; at the periphery of the mass
are glistening densely and radially aggregated flask-shaped
or club shaped bodies called the “clubs” (Fig. 198). The
central mass is occasionally found in a state of calcification :
this is not seldom the case in cattle.

That these clubs are an important and characteristic
feature in the morphology of the fungus is shown by the
name of ray fungus and by the fact that, what is commonly
observed— at any rate in cattle it is common — all the actino-
myces nodules and abscesses contain one or more central

492

MICRO-ORGANISMS AND DISEASE [chap.

mass or masses of these radiating aggregations of clubs.
But also in the human disease the clubs are with few
exceptions present, though there are cases described in
which the fungus was said to have been represented only by
a dense felt-work of branched fine threads.

Examining sections of hardened actinomyces nodules,
after suitable staining (rubin, 2 per cent, watery solution,

for several hours, then washed in water and stained in
methyl-blue anilin water for fifteen to thirty minutes) the
ray fungus appears in the middle of the nodule as an
irregular, spherical, or, more commonly, particularly when
large, a lobed mass, composed of a central, faintly stained,
homogeneous, or faintly granular mass ; around this is a
zone deeply stained in blue, and owing to its being com-
posed of densely aggregated and twisted branched threads

XVIII]

MOULD-FUNGI

493

looking not unlike cocci. The peripheral part is made up
of conical or cylindrical or club-shaped corpuscles of
different length and thickness, deeply stained pink, closely
placed side by side, and all radiating by longer or shorter
thin, pink, filamentous stalks from the next, the blue or
‘•granular-’ zone; each of the “clubs'’ possesses a faintly
stained homogeneous sheath. In human actinomycosis,

Fig. 198. — From a similar preparation, more highly magnified.

and also in actinomycosis of cattle, the central mass is not
seldom recognisable as a dense felt-work of fine branched
threads ; from the periphery of the mass longer threads
project, each or only some of which possess a terminal en-
largement ; in other cases the terminal club-shaped enlarge-
ment is the only distinct portion.

Now, some observers consider the clubs as indicating an
involution or a degeneration phase of the threads, and,

494

MICRO-ORGANISMS AND DISEASE [chap.

further, the above granules of the second zone as indicating
a coccus phase of the threads, and for this reason consider
the ray fungus as belonging to the species of cladothrix, a
polymorphous fungus, in which the threads may break up
into or develop from cocci and shorter or longer rods or
bacilli. Now, I quite agree with Crookshank in not accept-
ing this view, for I find constantly in actinomycosis of cattle
some of the smaller, i.e. younger, tumours contain fine clubs
in isolated examples or in small groups, without any fila-
mentous or granular centre ; in the preparations stained
successfully as above I find appearances which place me in
full agreement with Crookshank (Trans actions of the Med.
Chir. Soc ., 1889): single clubs very conspicuous by their
deep red staining attached to a short single or branched
stalk free or enclosed within a nucleated cell. Further,
there is, free or enclosed within a larger mass of protoplasm,
a small homogeneous mass from which are budding out
two, three, or four clubs of different lengths and with very
short stalks, these structures being stained bright pink stand
out very conspicuously from the blue ground. Further, I
find spherical or oval globules recognisable by their deep
pink staining becoming constricted off from the free end of
the clubs. Putting these features together there can be no
difficulty in recognising a striking likeness between the ray
fungus and a mycelial fungus : the fine branched threads
being the mycelium, the clubs being the growing ends of the
hyphae, such as are common to most hyphomycetes ; these
clubs, with their power of sprouting and giving off conidia
(the above spherical or oval globules), would render this
view easily intelligible. Further, the central part is the
only part which in any way can be said to represent the
part which is actually degenerating, since it often contains
lime deposits. This view of considering the clubs as the

XVIII]

MOULD-FUNGI

495

sprouting parts and conidia-bearing ends, the threads as
analogous to the mycelium of a mould-like fungus (Bollinger,
Israel, and others) is the view which stands better in
harmony, I think, with the actual facts than the view that
the ray fungus belongs to a species of cladothrix (Bostrom,
Paltauf, Afanassiew). Israel has shown that the ray fungus
can be artificially cultivated, but Bostrom was the first to
have succeeded in artificially producing good cultures of
this fungus. On blood-serum, on Agar at 33~37°C., the
fungus forms whitish granules, which rapidly enlarge ; they
show a yellow or reddish, round, knobbed centre, from
which start fluffy nebulous branched masses. After five to
six days the growth has reached its height. The presence
under the microscope of the mycelial branched threads and
of the clubs was established in these cultures. Paltauf and
Afanassiew have confirmed these observations.

O. Bujwid has cultivated the actinomyces fungus
anaerobically ( Centralbl. f. Bakt. u. Parasit., vol. vi. p. 630)
and succeeded in obtaining an actively growing mycelium
and clubs.

Actinomyces “ granules ” planted in the depth of glycerin-
bouillon at 37° C. grow well, the yellowish granules in-
crease in size and number and from their margin a fine
mycelium is seen to project. On the slanting surface of
grape-sugar gelatine actinomyces (taken from a previous
culture) grows well at 20° C, the tube having been sealed
up after inoculation ; it forms at first minute whitish-yellow
dots which gradually — in the course of a few weeks — enlarge
to yellow or yellow pink, dry, firm, tough patches and warts
which by enlarging coalesce so as to form a coherent, un-
even, knobby membranous expansion, the gelatine gradually
liquefies and the growth sinks in, and after liquefaction has
extended to the deep layers the growth falls to the bottom of

496

MICRO-ORGANISMS AND DISEASE [chap.

the culture tube ; the liquefied gelatine is limpid, thick like
syrup and of a brownish colour. When a part of the
growth is examined under the microscope, having been
previously stained, then well separated and teased out with
needles, it is seen to be composed of a delicate mycelium

Fig. 199. — From a teased-out specimen of the actinomyces growth on sugar

gelatine.

X 1000.

of fine threads, some uniform, others containing within the
sheath granules, rods, and cylinders ( see Fig. 199).

An important fact established by Israel, Bostrom, Rotter,
and others is this, that the ray fungus of man can by in-
oculation produce typical actinomycosis in cattle, and there
is therefore the greatest probability that the inverse also
holds good.

xv m]

MOULD-FUNGI

497

The chronic necrotic disease occurring in India, and
known as the Madura disease (Mycetoma), or the fungus
disease, has been investigated by Kanthack and found to be
caused by a fungus resembling in many respects actinomyces.
This was found to be the case in the yellow or pale variety,
as also in the black or melanoid kind (Pathological Society
of London, January 19, 1892).

Boyce and Surveyor, however, find different parasites in
the two forms of the disease (i.e. in the white and in the
black variety). After carefully describing the naked-eye
and low-power appearances of the “ roe-like particles ” in the
white and black variety, they find on microscopic examina-
tion that the fungus neither in the white nor black variety
is comparable to actinomyces, but to a mycelial hypho-
mycetes which is different in the two varieties of mycetoma.
(Phil. Transactions , 1895, B, part i. p. 1 and passim.)

K K

CHAPTER XIX

PROTOZOA CAUSING DISEASE

i. Plasmodium Malaria. — Laveran {Comp/es Rendus, 1 882.
No. 17) and Richard ( ibidem , No. 8) were the first who dis-
covered in the blood of malaria cases in the febrile stages
peculiar bodies, spherical or crescentic, consisting of a pale,
homogeneous substance and enclosing clumps of pigment
granules ; these bodies are possessed of cilia by which they
are enabled to perform rapid movement. Laveran con-
sidered these bodies as the true cause of malaria and
identified them as protozoa. This discovery was a few
years later (1885) confirmed and considerably amplified by
Marchiafava and Celli. The credit of the important dis-
covery of the malaria parasite belongs therefore unquestion-
ably to Laveran, and the observations of Marchiafava and
Celli 1 have amplified by a good deal our knowledge of them,
they called the parasite Ilamoplasmodium Malaria or
Plasmodium Malaria.

Marchiafava and Celli showed that during the beginning
of the febrile stage the parasite invades the red blood-
corpuscles as small, globular, pale, homogeneous corpuscles

1 Untersuchungen iiber die Malaria-Infection, Fortschritte d. Med.,
1SS5, p. 339 and 787.

CH. xix] PROTOZOA CAUSING DISEASE

499

9 9 9

8 0 10

Reduced from Golgi’s Plate III. in the Fortschrittc der Med., vol. iv.

i — 16 show the plasmodium malaria: within the red blood discs, gradually enlarging
at the expense of the substance of the blood disc ; pigment granules in 13 — 16
derived from hmmatin.

19 — 32 show the successive changes of the plasmodium towards final segmentation in
sporules.

35 — 41. — Laveran’s corpuscles from atypical cases of malaria.

K K 2

500

MICRO-ORGANISMS AND DISEASE [chap.

measuring not more than a fifth to a seventh of the diameter
of a red blood-corpuscle ; in this host the parasite performs
active amoeboid movement, hereby changing continually its
shape ; but it gradually increases in size and consumes the
substance of the red blood-corpuscle, leaving black pigment
granules— iron-free melanin — in the disc. These pigment
granules, as the parasite grows to the size of the original'red
blood disc, are now contained within the body of the
parasite, in which they appear uniformly distributed. When
the disc of the red blood-corpuscle is entirely consumed by
the growth of the parasite this latter appears free in the
blood plasma, its substance filled with the melanin granules ;
some of these free parasites have cilia by which they move
actively — these are the corpuscles seen by Laveran. Next,
the pigment granules aggregate in the central part of the
parasite and the peripheral, pale, homogeneous portion
gradually undergoes a more or less regular mode of seg-
mentation, in the course of which small globular particles
or sporules become constricted off from the main body :
when this segmentation has been completed the young
gemmae or sporules all disappear from the blood, so also the
pigmented central parts, and are stored up in the spleen,
liver, and bone marrow ; this terminates one febrile attack.
The next febrile attack is caused by the sporules again in-
vading the blood-corpuscles of the general circulation, and
herein undergoing the same series of changes as just
described. So that each febrile stage comprises the in-
vasion of the blood-corpuscles by the sporules, the germina-
tion, amoeboid movement and growth of these latter within
and at the expense of the former, then the gemmation and
segmentation into a new crop of sporules, and finally the dis-
appearance of these from the general circulation. Golgi 1
1 Fortscliritte d. Medizin, 1889, No. 3.

Xix] PROTOZOA CAUSING DISEASE 501

by his numerous researches was able to show that the
various forms of malarial fever are due to various species of
the parasite, at any rate that in the different forms of inter-
mittent fever the time in which the parasite passes through
all the above-mentioned phases of its development is
different, and stands in a definite relation to the form of the
fever. Thus Golgi found that in the febris quartana the
parasite from its first appearance in the red blood-corpuscle,
that is, from the onset of a febrile attack, through the com-
plete segmentation of the full-grown parasite into the
sporules, and to the disappearance of these from the
general circulation, /.<?., till the end of the febrile stage, re-
quires three days, whereas in the febris tertiana it requires
only two days. Besides, there are certain slight morpho-
logical differences between the parasite in the febris quartana
and in that of the tertiana, as also differences in the mode of
segmentation (r^Figs. 200 and 201). As to the parasite in
the fever of irregular type, Golgi shows that also in this the
time occupied for passing through its phases is irregular,
either too rapid or too slow. The crescentic form of the
parasites mentioned by Laveran and Marchiafava and Cclli
are present only in fever of irregular type, and are really an
atypical form in the development of the parasite. So also
the flagellate forms seen by Laveran are atypical forms.

Whether in these different forms we have really to deal with
different species of the same group of parasites, as Golgi inclines
to think, or rather with differences in the life-history of the
same species caused by unknown conditions, e.g., individual
person, different tissue, season, locality, &c., is not decided.

Canalis (Studi della Infezione malarian, Torino, 1889)
studied the atypical forms of malarial fever, characterised by
longer or shorter febrile intervals. He found in these cases
an endoglobular form of the plasmodium malaria;, which has

502

MICRO-ORGANISMS AND DISEASE [cHAF.

been signalised already by Golgi, viz., a crescentic form ; but
also here the commencement of the attack is characterised by
the amoeboid endoglobular forms, and the life cycle of the
parasite becomes completed by the division of it into sporules.

Danilewsky, Grassi and Feletti, Kruse, Pfeiffer, Celli and
Sanfelice, and others describe the occurrence of similar
parasites in the red blood-corpuscles of a number- of
different animals, frogs and birds (see Fortschritte d. Med.,
Band ix., Nos. 12 and 13, 1891).

2. A mceba Coli of dysentery. — Losch ( Virchow's Archiv
f pathol. Anatomic , 1875, Band Ixv., p. 196) was the first
who discovered the amoeba coli in great numbers in a case
of ulcerated large intestine in the human subject. This
case, in all its clinical and pathological symptoms, resembled
true dysentery.

Kartulis ( Centralbl . fur Fact, und Farasit., vii., 2) has
shown that in the cases of tropical dysentery which he
examined there were present in the characteristic sanguineous
stools numerous amaebcz (amoeba coli of Losch) showing
active amoeboid movement, and he gives good reason for
considering these the cause of the dysentery, though others
who had met with similar amoebse in intestinal diseases in
Russia (Massiatin, Centralbl. fiir Fact. u?id Farasit., vi.,
Nos. 16 and 17) did not think so.

Further, Kartulis has shown that in twenty cases of
abscess of the liver complicating dysentery he found in
every one of them the same dysentery amobae ; they could
be seen in sections through the wall of the abscess, but in
the pus of the abscess cavity he did not find them.

A considerable amount of literature exists at present on
the occurrence of amoebse in certain forms of dysentery,
chiefly those that run a chronic course, and on their absence
and the presence of various species of bacilli in other

XIX]

PROTOZOA CAUSING DISEASE

503

forms of acute dysenteric inflammation of the large intestine.
While some have confirmed Kartulis (Osier, Councilman,
Maggiora, and others) others have missed the amoeba, but
describe various species of bacteria as connected with the
disease; from the careful bibliography collected by Maggiora
(1 Centralblatt f Bad. und Pa rasitenku fide, xi., Nos. 6 and 7)
there can be little doubt that what is clinically spoken of as
dysentery is not one single disease in etiological respects,
since some dysenteric affections are, others are not, caused
by the amoeba coli.

3. Flagellate protozoa. — Many species of flagellate infusoria
are known to inhabit the body of invertebrate and verte-
brate animals ; of these the group known as Monadinse are
in so far of interest as some of them have been found in
vertebrates in connection with disease. The genus Tricho-
monas'1 has been found by L. Pfeiffer in the oral and
pharyngeal mucus of pigeons affected with the chronic
necrotic thickening of the mucous membrane, called also
“ diphtheria,” 2 and which this observer considers to be
connected with the cause of the disease. But Lbffler (see
the chapter on Diphtheria) has shown that the disease in the
pigeon is due to a specific bacillus, and in this he is fully
confirmed by Babes.3 Pfeiffer in the monograph just quoted
still maintains his original assertion, that the disease is due
to trichomonas invading and ultimately destroying the
epithelial cells. Pfeiffer, however, differs, as regards the life-
history of this protozoon, from all other observers and writers
on protozoa (Leuckart, Butschli, Dallinger and Drysdale),
inasmuch as he describes the formation of spores within the
substance of the trichomonas.4 It ought also to be mentioned

1 Leuckart, Die Parasitcn des Menschen, 2te Auflage, p. 31 1.

2 L. Pfeiffer, Die Protozocn a/s Krankheilserreger , 1890 (Jena),
p. 85.

* Zeitschr. f. Hygiene, Band x. 4 Lor. cit. p. 85, fig. 26.

504

MICRO-ORGANISMS AND DISEASE [chap.

that the seemingly identical trichomonas is frequently found
in the pharyngeal mucus of perfectly normal pigeons. I
have had the opportunity of examining a case of this so-
called diphtheria in the pigeon, and found the presence of
the parasite in the pharyngeal mucus, but on comparing with
it a perfectly healthy pigeon the same trichomonas was found
abundantly also here in the pharyngeal mucus. Davajne 1
mentions the genus Circomonas, much smaller than Tricho-
monas, being minute club-shaped, ciliated protozoa, possessed
of no envelope, having a pointed prolongation at one end
and a long, fine flagellum at the other ( Circomonas ititesti-
nnlis hominis), as occurring in the stools of cases of acute
Asiatic cholera, and once he also found them in the stools of
a patient in typhoid fever. Lambl already in 1859 described
them as occurring in the stools of children in diarrhoea, and
Losch found them also in the stools in cases of dysentery.
The writer has had the opportunity of finding in a mouse,
spontaneously dead, the peritoneal cavity and almost the
whole of the intestine distended by, and filled with, a
grumous milky fluid, in which, besides leucocytes and
micrococci, there were present trichomonas and innumerable
circomonas ; in fact, the main part of the corpuscular
elements was made up of circomonas, many of them very
rapidly moving

A certain species of flagellate monadinse was first described
by T. Lewis in 1877 as occurring in the blood of normal
horses, dogs, and rats ; by Evans in 1880 as occurring in
the blood of horses in Madrid ; by Wittich and R. Koch in
1881 as occurring in the blood of normal badgers. These
protozoa are known as the Herpetomonas Lewisii : the body
is cylindrical, often spiral ; the flagellum extends as a delicate
membrane all along the body of the creature ; anteriorly the
1 Traite des Eiitozoaires, p. xxiii.

xix] PROTOZOA CAUSING DISEASE 505

body terminates as a pointed rigid process. A haematozoon
which, according to Lewis and Crookshank,1 is identical with
that occurring in the healthy rat has been discovered by
Evans, but first assumed to be a spirillum and considered by
this observer as the cause of the surra disease, a deadly
malady affecting in India, in an epidemic form, horses,
mules, and camels. Crookshank 2 gave good photographs of
them ; he believes them to belong to the genus Trichomonas.
Lingard in his exhaustive reports to the Government of India
on surra disease had besides making a thorough investigation
into the clinical and pathological aspects of the disease
described the nature of the contagion, the life-history of the
parasite in its relation to the various phases of the disease.

4. Psorospennia or Coccidia. — (a) Coccidium oviforme.
The class of protozoa known as sporozoa — unicellular
parasites of fixed form of body, surrounded by a capsule,
forming within their body a number of spores, each sur-
rounded by a cuticle, the spores becoming free after the
bursting of the capsule, and giving rise to a new parasite — -
comprise a group which is important to the pathologist, oval
psorospermia or coccidia. These are capsulated, uni-
nuclear, oval, protoplasmic corpuscles, in the interior of
which out of the protoplasm a number of spores are
developed ; many of these coccidia are endo-epithelial
parasites, and as such are the causes of a chronic
hypertrophy of the epithelium. The coccidium best studied
is the Coccidium oviforme, causing in the liver of the
rabbit a chronic disease of the epithelium of the bile ducts,
by which the bile ducts become greatly distended, their
epithelium much hypertrophied and their coats thickened ;
in consequence of this whitish-grey nodules appear in the

1 Journal of the Roy. Micr. Society, No'’. 10, 1886.

2 Ibidem, loc. cit.

506 MICRO-ORGANISMS AND DISEASE [chap.

liver composed entirely of the hypertrophied folded and
fringed wall of the bile ducts.

Coccidium oviforme occurs also in the epithelial cells
lining the mucous membrane of the intestine in the rabbit.
The coccidia are oval corpuscles about 33-37 / >. in length,
15-20 /x broad ; each possesses a distinct capsule or cuticle,
which at one (thinner) pole contains a minute opening or
micropyle. The body of the parasite is a granular proto-
plasm, in its fully formed state completely filling the space
within the capsule, and containing an oval, clear nucleus.
In this condition they are numerously found amongst the
epithelium, and also free in the cavity of the intestine and
the hypertrophied bile ducts respectively ; but the majority
of forms seen in the epithelium are almost spherical, less
oval than the above, and possess a thinner capsule ; in some
the capsule is hardly recognisable, they contain a more
coarsely granular protoplasm, and within it a clear,
spherical nucleus. These are found in great numbers in
the epithelium, replacing at points almost completely the
epithelial cells, some being distinctly situated within the
body of the epithelial cells. The same is the case in the
epithelium of the enlarged bile ducts in the liver nodules.

It is not at all easy to decide what is the exact relation-
ship between these smaller, granular, indistinctly capsulated,
spherical bodies and the large, granular, oval, distinctly
capsulated coccidia. According to Leuckart, the former
would represent young coccidia just germinated from the
spores ; but this can hardly be correct, considering that in
all nodules, particularly the large ones, the small spherical,
indistinctly capsulated coccidia abound, and, further, con-
sidering that spores are not formed in the coccidia within
the animal body. It is therefore more probable that the
small spherical coccidia are derived by division from the

xix]

PROTOZOA CAUSING DISEASE

507

large oval forms, and in their turn, on their ripening, grow
into these latter.

It can be shown that the coccidia first appear in the
epithelium of the intestine, and from here they find their
way into the epithelium of the hepatic duct and gradually
into the bile ducts within the liver. Here their multiplica-
tion produces saccular, tubular, and cystic enlargements of
the interlobular bile ducts, the wall of which becomes
thickened by connective tissue, and folded in many ways.
In this manner numerous whitish irregularly shaped firm
nodules and cysts are formed in the liver, which, when cut
into, show a cavity with the thick white wall folded
inwards.

The columnar epithelial cells lining the hypertrophied
bile ducts, which harbour the coccidia, are, in fact, the soil
at the expense of which the coccidia grow and ripen ; these
latter in their turn, and for their own purpose, cause a
continuous multiplication of the epithelial cells.

Leuckart in his work, Die Parasiten des Menschen, 2te
Aufl. i., gives an exhaustive account of the life-history of
the coccidium oviforme, the mode of passing into new
animals, and the changes and distribution of it. In the
human subject nodules of the liver have been observed
which were caused by coccidia, probably coccidium ovi-
forme. Gubler, Leuckart, Dressier, and Peris ( see Leuckart,
loc. cit. p. 281) have observed such cases. Besides the
rabbit coccidium oviforme has been found in the intestines
of the dog, cat, sheep, guinea-pig, and pheasant.1

1 Miescher’s coccidia lubes occur in the muscles of the mouse occasion-
ally ; they are noticed as fine white lines which under the microscope are
tapering cylindrical granular masses, the latter in reality densely packed
crescentic or kidney-shaped pale corpuscles, about O'OI mm. long and
considered to be spores. (Leuckart, loc. cit.)

508

MICRO-ORGANISMS AND DISEASE [chap.

{b) Another disease in which the presence of psoro-
sperms,1 or at any rate of parasites morphologically related
to them, causes a chronic thickening and hypertrophy
of epithelial structures has been described by L. Pfeiffer
( Die Protozoen ah Krankheitserreger, Jena, 1890) as occur-
ring in the fowl. This animal is occasionally found to show
on its skin in various parts of the body large and, small
prominent nodules which consist of greatly hypertrophied
epidermis (stratum Malpighii) with corresponding infiltration
of leucocytes and distension of the blood-vessels of the
subjacent corium. The disease in question — epithelioma
cotitagiosum of the fowl — is not at all of rare occurrence,
and when it occurs on a farm it generally spreads to other
fowls. I have myself met with several such instances.
Sections made through the nodules show an enormous local
hypertrophy of the stratum Malpighii of the epidermis ;
and amongst the epithelial cells, in many places within these
cells, are found oval bodies, mostly capsulated, which are
distinctly of the nature of psorosperms. On staining the
sections in fuchsin, and then washing in alcohol or dilute
nitric acid, the epithelium is decolourised and leaves the
psorosperms stained pink ; in this manner the psorosperms,
with their pink protoplasm, their capsule and clear nucleus,
can be easily recognised. It is not a question of finding
such a psorospermic corpuscle here and there ; the epi-
thelium is pervaded by them in almost continuous masses.
They are found isolated within the epithelial cells, destroying
the substance of these latter ; or, when the epithelial cells
are almost destroyed over extensive areas, the psorosperms
are found to have entirely replaced them. There is not the

1 The following account is an abstract of my Report on “ Psorosperms
in their Relation to the Etiology of Cancer” in the Reports of the Medical
Officer of the Local Government Board for 1893-1S94, pp. 479j &c.

xix]

PROTOZOA CAUSING DISEASE

5°9

slightest difficulty in recognising these psorosperms and in
differentiating them from the epithelial cells. This disease
in the fowl is, like the psorospermosis of the liver in the
rabbit, essentially a chronic hypertrophy of the epithelium
caused by the process of growth and multiplication of the
psorosperm parasite.

(c.) Cancer parasites. — There are recognised in the human
subject a number of chronic diseases which consist essen-
tially in a chronic hypertrophy, with ultimate destruction,
of the epithelium of the skin, and of various mucous
membranes. The principal diseases known as such are :
Darrier’s disease, molluscum contagiosum, Paget’s disease
of the nipple of the breast, and last, but not least, various
forms of epithelioma and cancer of the skin, mucous
membranes, & c. The number of observers who have
searched for and found in these various chronic epithelial
disorders parasite-like bodies comparable to psorosperms is
legion ; but it is equalled, if not surpassed, by the number
of other observers, who, though they have searched for these
alleged psorosperms in these diseases with equal care and
perseverance, have utterly failed to identify them.1 While,
however, it is one of the simplest and easiest things to
demonstrate, by a microscopic examination of the above-
mentioned nodular disease of the rabbit, not only the
existence of the psorosperms but their relation also to the
hypertrophy of the epithelium, it is quite another affair to
obtain anything like clear evidence of similar conditions in
the above-named human diseases. In the first place,
amongst the number of observers who affirm that they have
discovered psorosperms in epithelioma of the human subject
there are scarcely two who describe the same* parasite ;

1 An excellent summary of all these researches is given by Strcebe in
the Centralblatt Jiir Allg. Pathol ., &c., 1894, Nos. 1, 2, and 3.

5io

MICRO-ORGANISMS AND DISEASE [chap.

while not one amongst them is able to do more than draw
attention to certain morphological appearances in the
epithelium which are put forward as denoting the presence
of something extraneous to the typical epithelial cells. No
one has in regard of cancer ever succeeded in isolating the
parasite, as is easily enough done in the psorospermosis of the
rabbit ; and, notwithstanding all assertions to the contrary, the
evidence amounts only, as I have said, to the description of
certain bodies which, after certain methods of staining, can
be demonstrated within or between the epithelial cells in the
above human affections. Amongst these bodies, however,
none are typical psorosperms. Under these circumstances
the critics of those who affirm a parasitic cause of cancer
have an easy task ; they can with perfect justification
demand, where— if such-and-such indefinite and mysterious
bodies do occur in cancerous epithelium — is the proof that
they are of a parasitic nature. To this the others can only
reply that they are most probably parasites, because they
are not typical epithelial cells. But how do you know that
they are not part of the cell or nucleus ? again ask their
critics. Because they stain differently from epithelial cell
substances or epithelial nucleus, answer the upholders of
parasitic cause. But surely, retort their critics, you have no
right to call those bodies psorosperms merely because you
are unable to think of them as derivatives of the substance
of the epithelial cells or their nuclei. And so on, and so on.

I have I think thus given sufficient account of the general
nature of a great part of the controversy ; at least it is not
necessary for the present to go further into details, as will
duly appear later on.

Before coming to the discussion of the alleged parasites
of cancer and of other chronic epithelial diseases in man
that I have referred to, it is necessary to have a clear view

XIX] PROTOZOA CAUSING DISEASE 51 1

of the elements of the problem that has to be dealt with ;
indeed, to every one who has devoted any considerable
amount of attention to pathological histology in general, and
to the changes of the epithelium in health and disease in
particular, it will be clear that insistence on this necessity
is by no means uncalled for. By going carefully over the
descriptions and illustrations of the “cancer parasites” put
forward by different observers, it is quite obvious that a
considerable number of them have thought it sufficient,
having made a few sections of cancer material, and having
stained and mounted them, to at once declare without
hesitation — and regardless of the histology, normal and
pathological, of epithelium — that such-and-such a particle
or corpuscle present in the substance of the epithelial cell
or in its nucleus is a parasite. As to this, I illustrate my
meaning as follows

Podwyssozki and Sawtschenko published in the
Centralblatt f. Bakt. und Parasit. vol. xi., Nos. 16, 17, and
18, a paper on cancer psorosperms, and they add two
coloured plates (Plates VII. and VIII.) illustrating the
presence of the alleged sporozoa within and between the
epithelial cells. Now, any one who, after staining them, has
examined sections of well-preserved epithelial structures,
growing and proliferating under normal and under patho-
logical conditions, will recognise in the figures given by
these authors appearances very commonly met with : viz.,
bodies, variously shaped and variously sized, contained
within the cells ; bodies, indeed, which take the stain
differently from the typical nucleus of the epithelial cell.
These bodies are commonly and justly considered to be
derivatives of the nuclear substance, particularly of that
portion commonly called chromatin. Appearances such as
are shown by the authors in their Figures 1-7, 11-15, 16-20,

512 MICRO-ORGANISMS AND DISEASE [chap.

&c., arc to be met with within the epithelial cells in sections
through normal glands, as also in sections of the skin and
of oral mucous membrane, in normal and (better still) in
pathological states. The epithelium of cancer is not
required for demonstration of these bodies, though in
cancer — owing no doubt to extensive multiplication of the
epithelial cells — they are met with sometimes, but by no
means always, as copiously as these authors would imply.
Why, therefore, they should consider these bodies to be
coccidia, is not easy to understand.

Another and perhaps more striking illustration of the
same tendency is afforded by Soudakewitsch in Centralblatt f.
Bakt. und Parasit. vol. xiii. page 415, Plate 1. He
describes there, as parasites, intracellular nucleus-like
bodies, which most histologists, with experience of normal
and pathological epithelium, would have no difficulty in
identifying as chromatic and other derivatives of epithelial
nuclei.

To quote one more instance: In vol. i., p. 198, of the
Journal of Pathology and Bacteriology , Drs. Ruffer and
Walker describe and figure epithelial cells, in which the
main part of the cell substance is occupied by a vacuole,
while within this vacuole lie three round clear cells, each
with several nuclei. Most histologists recognise these at
once as vacuolated epithelial cells containing common
leucocytes, such indeed as are commonly found in epithelium
under pathological conditions, and normally in certain
localities, e.g., fauces, tonsils, and tongue. Such vacuolated
epithelial cells containing leucocytes have been familiar to
histologists for many years ; they were the very cells about
which, seventeen or eighteen years ago, a considerable
amount of discussion arose. The question then under
debate was : whether they are endogenously developed

XIX]

PROTOZOA CAUSING DISEASE

5*3

within the inflamed epithelium (Strieker, Rindfleisch, and
others); or whether, as Cohnheim and others maintained,
and as is now universally believed, they are immigrants into
the epithelium and into the epithelial cells themselves. As
a matter of fact, they are found easily in the epithelium in
gonorrhcea and in the conjunctiva in catarrhal (blennorrhceal)
inflammation.

Messrs. Ruffer and Walker meeting, in cancer, with
vacuolated epithelial cells enclosing several leucocytes, seem
to assume that there has originally been a parasite within
each such epithelial cell, which has been eaten up by the
leucocytes. And in this way it could be shown that, in a
considerable number of instances, belief has arisen in the
existence of coccidia, psorosperms, and other parasitic forms
in cancer epithelial cells. Those enunciating such belief
would seem to be wanting in a clear understanding of the
elements of the problem with which they are concerned ;
for in dealing with purely morphological questions, such as
the presence or absence of certain bodies in the hyper-
trophied epithelium constituting cancer and similar chronic
epithelial diseases, it is obvious that a critical apprehension
of what appertains to cancerous epithelial new growths atid
to 7io other epithelium in health or disease is necessary as an
elementary condition for proper study of the subject. And
let me here at once say, in making this statement, that I am
in no way disinclined to regard molluscum contagiosum,
Paget’s disease, and cancer as belonging to the group of in-
fectious diseases that are of parasitic origin ; all that I wish
to insist on is that many, if not most, of the assertions as
to coccidia, psorosperms, and similar parasites in cancer
and in allied diseases are not founded on admissible
evidence. See Mr. D’Arcy Power’s demonstration 1 of some
1 Journal of Pathology and Bacl. viii. No. I, p. 124..

I L

5*4

MICRO-ORGANISMS AND DISEASE [chap.

of the so-called “ cancer parasites ” in epithelium experi-
mentally inflamed, but not of the nature of cancer.

From the various considerations I have adduced, it must
be evident that a number of assertions as to the parasitic
nature, in cancer, of various bodies in the substance of the
epithelial cells, of nuclear-like bodies between the epithelial
cells, of encysted nucleated bodies, of hyaline nucleated
cells, of multi-nucleated cells within epithelial cells, and of
stained particles within the epithelial nuclei, take no ac-
count of the presence of similar bodies and of similar change
in epithelium that is not cancerous ; and until such com-
parative study of the epithelium in health and in disease
has been undertaken statements to the above effect cannot
claim that value which their authors attribute to them.

But I have not completely exhausted the list of difficulties
which beset a theory of parasitic cause for cancer. Not
least among those that remain is the circumstance that few
observers describe the same kind of parasite. One might
almost say that there seem to be as many kinds of cancer
parasites as there are writers thereon ; and, further, that the
morphological characters of most of these alleged parasites
do not conform with the characters of any of the known
psorosperm species.

An exception is however to be made in the case of
L. Pfeiffer. Plis numerous researches 1 testify not only to
his thorough and extensive knowledge of the subject of
undoubted parasitism of sporidia in the animal kingdom, but
also in a very high degree to his systematic investigations of
the characters and occurrence of these and similar bodies.
Whatever, therefore, he has to say on this subject should

1 Die Protozoen als Krankheitserreger , Jena, 1890; Untersuchinigen
iiber den Krebs , Jena, 1893; and “ Der Parasitismus des Epithelcar-
cinoins,” Centralblatt f. Bab/, nnd Parasilcnk. , vol. xiv., page 11S.

XIX]

PROTOZOA CAUSING DISEASE

5i5

command universal and respectful attention. We may differ
from him, and may hold that his generalisations, notably
with reference to the same kind of parasitism in variola,
vaccinia, varicella, herpes, variola ovina, and in other
vesicular diseases, are by no means satisfactorily established
by the evidence he adduces ; nevertheless he makes a
genuine scientific and systematised attempt at throwing light
on what is shrouded in darkness, and what is considerably
complicated by the many less extensive investigations and
somewhat hasty assertions of a great number of young
pathologists.

Omitting Pfeiffer’s studies of the nature, character, and
distribution of coccidia, klossia, and eimeria, and of the
sporidia (myxo-, sarco-, and micro-sporidia), let us turn to his
observations on amoebo-sporidia in cancer in the human
subject. According to him the parasite of cancer is found
either within the epithelial cells in the form of an intra-
cellular encysted spore, or around the epithelial cells as a
free and growing cell-like germ which, when enlarging,
resembles a nucleated protoplasmic cell ; such cells as are
always found numerously infiltrating the connective tissue
around the true cancer epithelial masses. That is to say
Pfeiffer considers the various intracellular bodies as spores,
and the leucocytes infiltrating the connective tissue as the
growing cancer parasites. Views of this kind, which, on the
one hand, cannot be accepted as based on anything like
evidence, but rather as a sort of ipse dixit , cannot, on the
other hand, be directly disposed of. In just the same way
it is open to any one to affirm that the white blood cells
found in inflammation, &c., are amoebo-sporidia, though
other persons may prefer to call them inflammatory cells,
exudation cells, or leucocytes. They all are, whatever we
call them, living independent organisms which grow and

L l 2

5 1 6

MICRO-ORGANISMS AND DISEASE [CHAP.

multiply. If it be contended that they, being normal con-
stituents of the healthy body, cannot be the parasites of
cancer, it might be answered that the amoebo-sporidia of
Pfeiffer are not identical with them ; that the normal white
blood cells are one species, and that the amcebo-sporidia are
a different pathogenic species. I am merely showing the
kind of argument that Pfeiffer could bring forward if he
chose to go beyond his ipse dixit. And nothing is gained
by studying Pfeiffer’s photograms (1. c., Plate i, Figures
i — 4), representing sections through epithelial cancer of the
pectoral muscle and the lip of man, which are submitted by
him as illustrating amoebo-sporidia. What is represented by
him under a low magnifying power ( x 60) may be any-
thing ; whereas the appearances shown (Figure 4) under a
magnifying power of 600 are nothing else than a cluster of
nuclei, which may be those of epithelial cells or of leuco-
cytes.

Pfeiffer’s researches, indeed, though systematic and ex-
haustive so far as they refer to the nature and distribution
of coccidia and allied psorosperms, and to the various
sporidia in the animal kingdom, are extremely fragmentary
with reference to cancer in man ; there is practically no
satisfactory evidence of the presence of what Pfeiffer calls
spore forms in the cancer epithelial cells, or of the presence
of amoeba forms around them.

One of the most striking facts in the large mass of the
literature on the subject of coccidia and psorosperms in
cancer is the absence of any well-authenticated sickle-like
bodies ; that is to say, of those characteristic bodies which,
according to the unanimous testimony of all those who have
investigated the life-history of the various parasitic and non-
parasitic coccidia and psorosperms (Leukart, Biitschli,
Eimer, L. Pfeiffer, and others), constitute one of the most

xix]

PROTOZOA CAUSING DISEASE

5*7

typical phases in the life cycle of a coccidium — the phase,
namely, of commencing germination of the spores. True,
there have been a few observers (Soudakewitsch, for in-
stance), who assert the occurrence in cancer of sickle-like
bodies, but most other observers deny their relation to
cancer, and regard them as altered nuclei. There is no
difficulty, whatever, in finding in cancer, as also in other
chronically and acutely changed epithelium, nuclei of the
epithelial cells which resemble, or rather possess the shape
of, crescentic or sickle-like bodies ; nuclei, that is, which
are so changed that they appear swollen or hydropic, with
their chromatin collected at one side in the form of a cres-
centic body. So that the only typical phase, /.<?., sickle-like
germs, of a coccidium or psorosperm, the constancy of
which would represent a certain morphological evidence for
the presence of coccidia or of psorosperms in cancer, is
absent. As a consequence, therefore, the acceptance of
other phases of the alleged coccidia in cancer constantly
met with in the writings of parasitologists is beset with very
grave difficulties.

Briefly reviewed the following are the conditions which
have been described as indicating psorosperms or zoospores
in cancer : —

i. There is, in the first place, the occurrence of encysted
nucleated protoplasmic bodies among the epithelial cells of
cancer, which resemble, to a limited extent, similar bodies
in, for instance, the coccidia in the rabbit's liver. With
reference to these encysted cells it has to be said that such
forms do undoubtedly occur in cancer. I have examined
a considerable number of sections through cancer — of the
lip and tongue, of the penis, of the liver, of the omentum,
of the breast, of the bladder, and of the oesophagus — and
have met with such encysted cells. But I have met the

5>8

MICRO-ORGANISMS AND DISEASE [chap.

same bodies in stratified epithelium of a variety of tissues in
normal, and particularly in pathological, conditions which
have nothing to do with cancer. The same is shown by
D’Arcy Power. Hence I am justified in denying their
parasitic nature, not only because they occur in normal
stratified epithelium, but also, and chiefly, from the fact that
in every respect they resemble epithelial cells wherein the
main body of the protoplasm has shrunk around the nucleus,
leaving a peripheral portion surrounding it like a capsule.
Whether this apparent encysting of nucleated epithelial cells
is connected with and dependent on the structural differ-
ences of the marginal and central portions of the cell proto-
plasm, as is suggested by Heidenhain’s researches, I am
unable to say ; but as evidence at least tending in this
direction, I may state that, however partial or extensive
coagulation-necrosis occurs in stratified epithelium, such
apparent encysting of the main coagulated mass of the
epithelial substance does occur. To this class of appear-
ances belong epithelial cells in which the nucleus is shifted
to one side and compressed, owing to the presence within
the central part of the cell of an almost homogeneous
spherical body ; and in this remnants of granular matter
may be often recognised. These clear intracellular globules
are represented by some observers as the cancer parasites ;
but, apart from their remarkable dissimilarity to anything
resembling a sporozoon, the possibility of their being indica-
tive of and due to a hydropic or colloid change of the cell
protoplasm must not be lost sight of. Moreover, the pre-
sence of fluid colloid fatty matter or other material within
the cell protoplasm is not at all of rare occurrence in
epithelial and other cells in various pathological states.

2. The presence of well-outlined, more or less clear,
intracellular bodies showing a more or less distinct peri-

xix] PROTOZOA CAUSING DISEASE 519

pheral radial striation with a central deeply stained granule
or granules (Soudakewitsch, Ruffer). These strikingly re-
semble altered nuclei of the epithelial cells : that is, in a
hydropic condition with accumulation in the centre of the
main part of the chromatin, remnants of the mitoma still
attaching the chromatin to the nuclear membrane. I do
not miss these forms in cancer, in fact they are by many
considered to be the most characteristic forms. But in
some cancers that I have examined they are not numerous ;
and I do not see that the evidence as to their parasitic
nature is at all satisfactory, the less since bodies closely
resembling such forms are to be met with in epithelial
structures under other conditions, as already stated. It is
quite possible that in cancer the changes of the nuclei in
epithelium are of a chemical nature different from those
obtaining in other conditions, and that hence they are more
easily met with in cancer than in other diseases ; but this is
no reason why such changes should be considered as
indicating the presence of parasites. In passing, it may be
mentioned that these bodies do not occur in the coccidia,
say, of the rabbit’s liver.

3. The presence of “ spores ” and “ spore-like ” bodies in
the cell substance and in the nuclei of the epithelial cells in
cancer, either isolated or in groups. To these, Sjobring,
Soudakewitsch, Ruffer, and Walker have devoted particular
attention ; and they undoubtedly represent good types of
the so-called cancer parasites which can be easily studied.
But, as I have stated, such “ spores ” cannot be distinguished
from masses of cell protoplasm or of nuclear substance
respectively, separated from the main cell substance, and
owing to chemical change taking dyes often differently.
Besides such spore-like bodies are met with in epithelium
under conditions (sheep-pox, foot-and-mouth disease, chronic

520

MICRO-ORGANISMS AND DISEASE [chap.

inflammation of skin, &c.) that have nothing whatever to do
with cancer. It must, however, he left open whether this
interpretation is or is not a correct one ; though amongst
the figures given by the different observers as showing
intracellular spores (Sjobring, Ruffer, and Walker) there are
some that cannot be distinguished from vacuoles in the
protoplasms of epithelial cells.

4. As to the presence of rounded transparent cells with
one, two, or more nuclei amongst, or even within, the
epithelial cells, there is nothing to distinguish them from
ordinary leucocytes. They are met with in cancer, and
they are met with in the normal epithelium of the palate,
tonsil, and back of the tongue. To assume with L. Pfeiffer
that these are amoebo-sporidia seems quite gratuitous. The
same applies to the occurrence of kerato-hyaline cells which
are said to be a phase of the cancer coccidium.

5. Perhaps the most important bodies that have been
adduced as cancer parasites are those described by Korotneff
in Centralblatt f. Bakt. and Parasitenk ., vol. xiii., p. 373.
Under the name of rophalocephalus carcinomcitosus, Korotneff
describes a pedunculated and band-like protoplasmic mass,
consisting of a spheroidal or pear shaped nucleated “ head,”
and, directly continued from it, a band-like longer or shorter
protoplasmic mass. The whole is without a sheath and is
situated partly within and partly without the epithelial cells
of a cancer ; and the band-like stalk is several times the
diameter of the individual epithelial cells. In this form the
“parasite” is considered as an adult one, whereas smaller
uni-, duo-, or pluri-nucleated masses without stalks are con-
sidered young or growing forms — gregarina forms. This
parasite was found not only in carcinoma labii but also in
carcinoma mammae, maxillae, &c. Kurloff in Centralblatt f
Bakt. und Parasit ., vol. xv., p. 341, adduces confirmatory

xix]

PROTOZOA CAUSING DISEASE

521

evidence as to the existence of these forms : namely, adult
band-like masses with a nucleated head, and also smaller
spherical, oval, or pear-shaped masses (i.e., growing forms)
in a case of carcinoma of the skin of the hand.

These band-like masses with nucleated pear-shaped head
are from their shape and their size so unlike anything else
amongst the epithelium that it is quite out of the question
to refer them to anything oelonging to the epithelial or to
other known cells, and the question therefore arises as to
their nature and as to their relation to carcinoma. Both
Korotneff and Kurloff show that by special staining they
can be easily demonstrated ; and in this I agree, so far as
their presence in some carcinomata is concerned. I have
seen identical bodies in sections of a carcinoma of the
oesophagus, which were stained first in fuchsin or rubin and
then in methyl-blue. They were conspicuous not only by
their shape but also by their deep pink stain (fuchsin or
rubin), which they take up and retain with great persistence.
They appeared as partly intracellular, more generally inter-
cellular, filamentous, or band-like deeply stained masses
with a spheroidal or pear-shaped nucleated enlargement ;
and in this form they are easily recognised. Besides such
forms there occur similar deep-pink spheroidal or oval
nucleated intracellular masses ; and it is difficult to decide
whether all these or only some of them, notably the larger
ones, are merely truncated enlargements of the former.
The smaller of such forms are, most probably, young grow-
ing forms, as is maintained by Korotneff and Kurloff; but
it must be obvious that the “ rophalocephalus ” may appear
in the specimen in longitudinal, in oblique, or in transverse
section, and as a matter of fact gradations between the long
band -like adult (so called) forms and the spherical or oval
nucleated bodies can be easily observed. I myself cannot

522

MICRO-ORGANISMS AND DISEASE [chap.

say whether the smaller uni-, bi-, or multi-nucleated intra-
cellular corpuscles referred to by these authors are in reality
what they appear to be, namely, young and growing forms,
or whether they are merely the “ heads ” of the band-like
forms seen in optical or in real transverse section. If the
former view, viz., that of Korotneff and Kurloff, be the right
one, some of these young amoeba-like nucleated bodies
(gregariniform) have been described by other observers,
notably by Sawtschenko and Ruffer.

It may not be amiss to mention here that it seems to me
that some, at any rate, of the “ fuchsin bodies ” first described
by Russell, British Medical Journal, 1891, belong to this
category ; and it may also be mentioned that other of
Russell’s fuchsin bodies are red blood-corpuscles, for in
some carcinomata red blood-corpuscles deeply stained with
fuchsin can be found between the deep epithelial cells.
That they are red blood-corpuscles can be recognised by
their size and shape, and by the fact that the capillaries of
the papillae contain them. But some of the larger “ fuchsin
bodies ” of Russell seem to me to be undoubtedly the
above parasite-like bodies.

There can be then no doubt that there occur in cancer
certain bodies which can be distinguished as separate and
different from epithelial cells, from nuclei, or from leucocytes ;
and the question is, Of what nature are they? The two
Russian authors consider some of them as rophalocephalus
carcinomatosus in its adult stage, while the smaller nucleated
bodies they consider as young growing gregarinous forms.
It seems to me, however, that it is not necessary to accept
this view of a new species, one which by the way does not
coincide in its characters with any known gregarinse, It
appears more probable that the band-like pedunculated
knobbed nucleated mass of protoplasm represents simply a

xix]

PROTOZOA CAUSING DISEASE

523

large amceba that has thrown out a long stalk, and that
when the amceba, after the nature of amoebae, divides it
gives origin to smaller nucleated protoplasmic masses. If
only the latter were present in the sections, it would, owing
to their special staining and their intracellular position, not
be difficult to mistake them for epithelial cells or leucocytes ;
but the presence of the large pedunculated knobbed masses
is of the utmost importance as proving that we are dealing
with something quite different from either epithelial cells or
leucocytes.

I have searched for these pedunculated amoebae in a
large number of carcinomata ; but, excepting a single case
of carcinoma of the oesophagus, I have not come across
them. As already stated, however, Korotneff has in this
respect been more fortunate.

To sum up, then, we have to exclude from the evidence
adduced by the various authors, as indicating cancer
parasites, the following bodies : —

(a) Nucleated epithelial cells which have undergone a
kerato-hyaline change. These are observable as spheroidal
or oval corpuscles, generally situated away from the deepest
epithelial cells : that is, from the cells immediately in con-
tact with the connective tissue matrix. They are of about
the size of ordinary epithelial cells, stain like keratin of the
superficial cells, and possess a relatively small deeply stained
shrunken nucleus, such as is found in many other examples
of chemically or acutely inflamed epithelium.

(/>) Spherical transparent cells with one, two, or more
nuclei, which in aspect, size, nuclei, and in their mode of
staining, cannot be distinguished from ordinary leucocytes.
They are found between the epithelial cells, or have im-
migrated into the substance of the latter — as in the case
of vacuolated epithelial cells enclosing leucocytes. This

524 MICRO-ORGANISMS AND DISEASE [chap.

appearance is found, not only in the epithelium of cancer
but in many other normal or pathological conditions of
epithelium. There is nothing to distinguish these small
bodies from leucocytes.

(c) Small and large particles, vacuoles singly and in
clusters, situated in a more or less distinct cavity in the
cell substance. They occur singly or several together in
an epithelial cell ; but similar cell enclosures occur in
many other epithelial structures besides those of cancer,
and the fact that they occasionally stain differently from the
main cell substance may merely indicate a chemical change
which this part of the cell substance has undergone : it is
not a proof of their being spores of parasites.

(d) Encysted nucleated epithelial cells. These are not
uncommon in cancer epithelium, but they also occur in
other epithelial structures. The envelope is not a real
capsule, but owes its origin to a separation and shrinkage
of the main part of the protoplasm around the nucleus,
whereby a peripheral part remains detached and resembles
a capsule. Further, in epithelial cells in which the central
part has undergone a hyaline change (hydrops, colloid), the
nucleus of the epithelial cells is pressed to the side.

(e) Nuclear well-defined bodies, containing one or even
several small granules, with, in the periphery next to the
surrounding membrane, a more or less distinct radial
striation. These bodies are nuclei of epithelial cells, the
epithelial cell substance having become destroyed and the
nucleus become swollen and hydropic. The granules and
striae are remnants of chromatin. Such bodies occur not
only amongst the epithelial cells of cancer, but also in
other rapidly growing epithelium.

(/) Nuclear bodies situated within the epithelial cell
next to the normal nucleus ; the latter slightly swollen and

xix]

PROTOZOA CAUSING DISEASE

525

staining differently from the former. Such occur in many
epithelial and other cells — as paranuclei — both in
normal and in pathological conditions. These secondary
“ nuclei ” are probably derivatives of the chemically changed
chromatin substance of the original nucleus.

All that, therefore, remains and cannot be placed to the
account of either epithelial cells or their nuclei, or of
leucocytes, are the large pedunculated protoplasmic bodies
with a nucleated knobbed enlargement, contained within
epithelial cells, that were first seen and described by
Korotneff, as rhophalocephalus carcinomatosus. These
seem to me to be large amoebae-like bodies, which, by
reproduction, bring forth small nucleated protoplasmic
amoebae, generally also contained within epithelial cells.
These small amcebic offsprings, just like the parent amoeba,
are conspicuous by their staining, and by their apparent
direct connection with the pedunculated large amoebae.
Whether many of the nucleated cells enclosed within the
epithelial cells of cancer, seen and described by other
observers (Soudakewitsch, Ruffer, and others) as con-
spicuous by their staining, are or are not the young amoebae
in question, cannot be easily determined.

Lastly, it has to be mentioned that the above pedun-
culated amoebae have been found by myself in one case
only, that of cancer of the oesophagus ; I could not find
them in many other cancers. Korotneff, however, asserts
that he has found them in a variety of cancers. It is
quite possible that this condition, viz., that of the pedun-
culated form, may be more difficult to meet with, or may
be more rare ; the form of smaller, rapidly dividing amoebae
being more frequent. But at all events even these latter
forms are in many cancerous epithelial growths only
sparingly to be met with ; in some I have missed them

526

MICRO-ORGANISMS AND DISEASE [ch xix

altogether, while in others several sections had to be
examined in order to find one or the other nucleated
bodies resembling them. From this it would appear
hazardous to assign to them a definite causative relation
to the rapid growth and multiplication of the epithelial
cells constituting carcinoma.

CHAPTER XX

ANTAGONISM AMONGST BACTERIA

That the chemical products of some species of microbes,
while acting inimically on the further multiplication of this
species, are not inimical to that of another species has been
proved by various observations, but it has also been proved
that an inimical action is undoubtedly exerted by the growth
of particular species on that of others. It is well known
that a number of species of bacteria can exist and thrive
under conditions under which other bacteria cannot so
exist ; take, for instance, the water bacteria, i.e., the bacteria
inhabiting common drinking water ; these are capable of
living and of multiplying on the very small amount of
nutritive material present in ordinary drinking water, nay,
micrococcus aquatilis and bacillus erythrosporus (Fltigge)
and others, as mentioned above, multiply even in distilled
water (Meade Bolton, Niessen, Percy Frankland); whereas
numerous species of bacteria non-habitually in water cannot
do so under the same conditions ; therefore the water
bacteria will persist and even multiply, whereas others added
to the water, or accidentally finding entrance into the water,
will perish, some sooner, some later. Numerous observa-
tions have been put on record by Meade Bolton, Wolffhiigel
and Riedel, and others to show in what way and to what

528

MICRO-ORGANISMS AND DISEASE [chap.

extent various bacteria — the bacillus anthracis, cholera
spirilla, the typhoid bacillus, micrococcus tetragenus, and
staphylococcus aureus gradually die off when kept in
ordinary drinking water, i.e., water very poor in nutritive
materials. (The results of Meade Bolton are published
in the Zeitschrift fit? ■ Hygiene , i. 1, p. 76 ; those of Wolff-
hiigel and Riedel in the Mittheil. aus dem k. Gesundheitscunte,
Berlin, i. p. 455. See also G. and P. Frankland’s Handbook
on Water Examination .)

It need hardly be said that if even small amounts of
nutritive material be added to water these bacteria will
have a better chance of survival and of multiplication, and
this chance will be proportionate to the amount of nutritive
material added. Similarly De Giaxa ( Zeitschrift f Hygiene,
vi. 2, p. 162) made observations with reference to the con-
ditions of existence of various bacteria in sea water, and his
results are parallel to those made on ordinary drinking
water. It need not be specially insisted on that neither
ordinary nor sea water in themselves have any killing power
on bacteria, but that where such an inhibitory power is
observed it is due to the want of sufficient nutritive material,
and that the greater the dependence of bacteria on organic
material, and the poorer the water in such material, the
more unfavourable is such water for the existence and
multiplication of those bacterial species.

Next we have to consider the relations between two or
more species simultaneously present in the same medium
with sufficient nutritive material. Here more rapid multi-
plication will naturally depend, cceteris paribus, on the
greater assimilative power ; the greater this is, the more
predominating will the species become. Thus, for instance,
if in any organic material, say dead animal tissues, saprophytic
bacteria are present together with bacillus anthracis, this

xx] ANTAGONISM AMONGST BACTERIA

529

latter has not much chance of growing and multiplying ;
and hence in any part of an animal dead of anthrax, at
first full of the bacillus anthracis, as soon as putrefaction
has actively set in, the anthrax bacilli will be gradually
killed off by the saprophytes, so that such material becomes
deprived of producing anthrax infection. The same obtains
with other highly specialised bacteria, e.g, the streptococci,
the typhoid fever bacillus, and others. While this process
of killing off of the more specialised and less assimilative
bacteria by the more rapidly growing and more assimilative
bacteria is essentially a survival of the fittest in the struggle
for existence, there is another factor to be considered that
not immaterially helps to bring about that result : it is the
inimical influence the chemical products of the saprophytic
bacteria have on the more sensitive and more highly speci-
alised pathogenic bacteria. If, for instance, a filtered solution
is made of a putrid albuminous substance, the putrefactive
bacteria being all removed, and of this solution a consider-
able amount is added to an otherwise favourable nutritive
material, e.g., alkaline broth, it will be found that this
mixture is unfavourable for the growth of some species, in
some cases more than in others. To the same class of
inimical influences belongs the influence of fecal matter on
various species of bacteria, e.g., anthrax bacilli, cholera
spirilla, typhoid fever bacillus investigated by Kitasato.
His results on the death of cholera spirilla in fecal matter
are instructive. They are published in the Zeitschrift f.
Hygiene, v. p. 487.

Of a similar character are the observations recorded by
Garre ( Correspond . f. schweizer. Aerzte, xvii. 1887), who
showed that nutritive gelatine which has served already for
the growth of bacillus fluorescens putidus — a common sapro-
phyte in water and putrid fluids — is no more capable of

M M

530

MICRO-ORGANISMS AND DISEASE [chap.

serving the growth of some bacteria : bacillus of Friedlander,
typhoid bacillus, pink torula, staphylococcus pyogenes
aureus ; while others are capable of growing in such gelatine,
though slightly retarded : cholera spirillum ; and still others
grew normally : Finkler’s spirilla, bacillus anthracis.' To-
wards the former, therefore, the bacillus putidus has a
decided antagonistic action, while with the latter it is sym-
biotic. But this antagonism, when existing, is not neces-
sarily mutual, for while the typhoid bacillus renders the
gelatine also unfit for the growth of the bacillus fluorescens
putidus — these two species being mutually antagonistic — it
is not so with the bacillus of Friedlander or the staphylo-
coccus aureus. Diphtheria bacilli grow well in broth pre-
viously exhausted by proteus vulgaris.

To the same category belong the observations of Soyka
and Bandler, who studied the manner in which certain bac-
teria are capable of growing in media previously exhausted
by other bacteria ( Fortschritte der Med. 1S88, p. 76).

Cash has made similar observations with bacillus anthracis
and certain micrococci when growing simultaneously. He
found that the growth of the bacillus anthracis does go on
to a certain extent, but that the virulence of it is impaired
by the growth of the micrococci. This subject deserves a
more exhaustive study than it has hitherto received ; it is
mainly of importance to ascertain whether and to what
•extent an inimical influence is exerted by one species on the
other capable of growing simultaneously in the same me-
dium. There is good reason for supposing that hereby, in
some cases at any rate, one species is capable of attenuating
the virulence of another. Thus in the cultures which Pasteur
used as attenuated cultures for producing protective inocu-
lation in fowl cholera it was not, as Pasteur believed, the
prolonged exposure to air that produced the attenuation of

xx] ANTAGONISM AMONGST BACTERIA

53r

his cultures, but the impurity of his cultures (Kitt), and
likewise the attenuated condition of the culture fluids that
Pasteur used for protective inoculations against swine ery-
sipelas was probably caused by the impurity of the culture
fluid (Schiitz), there being present in Pasteur’s fluid, be-
sides the true bacillus of swine erysipelas, a contaminating
micrococcus.

Watson Cheyne, von Emmerich ( Archiv . f. Hygiene , vi.
1SS7), and others showed that the streptococcus erysipelatos
possesses such an attenuating influence on the bacillus an-
thracis, for by inoculating simultaneously pure cultures of
the two microbes into rabbits they were able to show that
the bacillus anthracis was unable to produce fatal anthrax,
though when separately inoculated they exerted their full
virulence. It depends, however, to a considerable degree
how much of the one and how much of the other microbe
is injected in order to produce this inhibitory effect, for if too
little of the streptococcus be injected the bacillus anthracis
will exert its full virulence, or vice versa. This whole subject
is obviously a very important one from a practical point of
view — from the point of view of finding antidotes against the
action of pathogenic bacteria— and it deserves greater atten-
tion than it has hitherto received.

The writer has himself made some experiments with regard
to injecting simultaneously two species. In one series the
bacillus of fowl enteritis was grown in broth with the swine
fever bacillus ; in the other, the bacillus of swine erysipelas
with that of swine fever, but neither in the amount of multi-
plication nor in the virulence of the swine fever bacillus
could any change be noticed. He has, however, succeeded
in neutralising the fatal effect on mice of the grouse bacillus,
if at the same time the aerobic malignant oedema bacillus
{sec later) be injected.

M M 2

532

MICRO-ORGANISMS AND DISEASE [chap.

Bouchard, Charrin, Woodhead and C. Wood have shown
that there exists a strong antagonism between the bacillus
pyocyaneus or its products and the bacillus anthracis ; so
much so that upon the injection of the former, simultaneously
with or immediately after the latter, into an animal susceptible
to anthrax, this latter disease does not take place at all,
whereas a control animal not treated with the pyocyaneus
succumbs to anthrax.

It is well known that certain infectious diseases, of which
infection occurred simultaneously in the same body, do not
take place simultaneously, but that the one probably has to
wait, as it were, till the other has gone through its course.
In other cases one disease has clearly an inimical influence
on another. Take, for instance, the observations repeatedly
made by surgeons that erysipelas has a curative influence on
certain tumours ; Fehleisen had by direct experiment with
pure cultures of streptococcus erysipelatos proved that certain
sarcomata can be made to disappear and a cure effected by
producing erysipelas in the skin of the part.

But there is a converse side to this, namely the question
whether, and if so, to what extent, one condition, one species of
bacteria or its products, enhances the power of multiplication
and the action of another. Monti (Ac. d. Line., October 6,
1889) pointed out that the culture of the diplococcus pneu-
moniae— which, as is well known, gradually (by age and by
continued subcultures) loses its virulent action on animals —
regains the virulence if injected simultaneously with broth
culture of the common saprophyte proteus vulgaris, from
which the bacilli themselves are previously removed or killed
by heat. This increased virulence of the pneumococcus may
be achieved either by injecting this and the proteus culture
at the same place, or at distant places simultaneously or soon
after one another. Similarly I found that cultures of strepto-

XX] ANTAGONISM AMONGST BACTERIA

533

coccus of erysipelas, which had lost their action on rabbits,
regained virulence if injected mixed, i.e, simultaneously,
with broth culture (four days old) of the proteus vulgaris ;
and it made no difference whether the latter culture was or
was not previously sterilised. The virulence on the guinea-
pig of the bacillus diphtherias is, as I have shown, greatly
enhanced by a simultaneous inoculation of the bacillus
pyocyaneus.

CHAPTER XXL

THE RELATION OF SAPROPHYTIC TO PATHOGENIC ORGANISMS.1

There is hardly any question which to the pathologist and
sanitary officer can be of greater importance than the relation
of saprophytic to pathogenic or parasitic organisms. To the
pathologist the life-history of a micro-organism, outside and
within the animal body, must ever remain an important field
of inquiry ; to the sanitary officer all conditions affecting the
life and death of those organisms which produce, or at least
are intimately bound up with, infectious diseases, such as the
distribution and growth of these micro-organisms outside the
animal body, the agencies which affect it in a favourable and
unfavourable sense, are the points which he has particularly
to consider in dealing with the spread and prevention of
infectious maladies. Now, it is known of many micro-
organisms, both those that are associated with putrefactive
processes as well as those that are bound up with infectious
disease, that temperature, the character of the medium in
which they grow, presence and absence of certain chemical
compounds, &c., are capable of materially affecting them. I
need not for this purpose enumerate all that is known already

1 Part of this chapter is copied from an interim report by myself to
the Medical Officer of the Local Government Board, 18S4.

CH. XXl]

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535

by direct experiment, but will only limit myself to reference to
the researches of Schroter, Cohn, and Wernich on that group
of micro-organisms known as pigment bacteria, i.e. bacteria
which only under certain conditions, notably temperature and
soil, produce definite pigments (Cohn’s Beitriige zu?- Biologie
d. Pflanzen ) ; to those of Hansen (Carlsberg Laboratory) on
yeast; to those of Neelsen on the bacilli producing the blue
colour of milk, the bacillus syncyanus ( Beitr . zur. Biol. d.
Pflanzen , iii. 2, p. 187) ; to the works of Toussaint, Pasteur,
Chauveau, Koch, and others on the bacillus anthracis ;
Arloing, Thomas, and Cornevin on the bacillus of symptomatic
charbon ; of Koch on the bacillus of tuberculosis ; of Israel
on actinomyces, and many others ; and particularly would I
refer to the many valuable suggestions and considerations
expressed by v. Nageli in these respects in his book, Die
niederen Pilze, Miinchen, 1877 and 1882.

While from these observations it would appear that both
saprophytic and parasitic micro-organisms are capable of
suffering modifications in their morphological and physio-
logical behaviour, sometimes small, sometimes great and
pronounced, it is nevertheless still an open question whether
an organism which under ordinary conditions is only
associated with septic changes in dead organic material,
and which cannot under these ordinary conditions grow and
multiply within the living body, can, under certain extra-
ordinary circumstances, acquire the nature of a parasite,
become endowed with the power of growing and multiplying
within the body of a living animal, creating there a patho-
logical condition, inducing there an infectious disease.

It is a common laboratory experience that many specific
microbes, owing to medium, temperature, &c., or to successive
subcultures, while retaining their general morphological
characters nevertheless gradually change their physiological
action, becoming more and more attenuated, and ultimately

536 MICRO-ORGANISMS AND DISEASE [chap.

lose their specific action altogether. Examples illustrating
this have been mentioned on various previous occasions
and they are familiar to every bacteriologist : the attenuated
anthrax vaccines obtained by Pasteur by growing bacillus
anthracis in chicken broth at 42' 50 C., and successfully used
for protective inoculation, the attenuation of the bacillus of
fowl cholera by Pasteur, of the bacillus tuberculosis grown
on Glycerin Agar, of the pneumonococcus, of the bacillus
of malignant oedema, of streptococcus of erysipelas, and
many other microbes. Similarly it is a common experience
that a specific microbe which possesses a low virulence, or
which has altered or lost its specific pathogenic action, can
by altering the soil (artificial medium or animal body)
become more virulent or recover its former virulence respec-
tively. All these phenomena are constantly met with in all
bacteriological work, and very few microbes are exempt from
such changes.

Now, the questions that to the sanitarian are of great
importance are these : (1) can a parasitic microbe which
although at first derived from a virulent animal source, but
existing under abnormal conditions inside or outside the
animal body, alter its physiological nature so as to cease to
be any longer capable of being a pathogenic or parasitic
microbe? (2) can such a degraded microbe, i.e. once patho-
genic but now living as a saprophyte again, under altered con-
ditions resume its virulence ? and (3) can a true saprophyte,
that is a microbe not at any time connected with patho-
genicity, owing to certain peculiar conditions under which it
has been living when introduced into the animal assume the
nature of a parasite?

From what has been stated previously, laboratory experi-
ence justifies us in answering questions (1) and (2) in the
affirmative, but it is more difficult to give a decided
answer to question (3), for it is quite possible to imagine, it

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has indeed been shown by experimental investigation, that
there are a good many microbes, not derived from, or
associated with, any infectious disease of man or animals,
but generally carrying on a saprophytic existence, which
under certain conditions are capable of producing decided
pathogenic action in the animal body. This question has
to be considered under two aspects : (a) is there any
evidence to show that a true saprophyte can, owing to
alteration in the conditions of its growth in outside nature,
acquire pathogenic action ? and (fi) can a true saprophyte,
previously non pathogenic, become pathogenic in the animal
body owing to conditions within the animal body ? It
must be clear that for the sanitarian the first aspect is of the
first importance, for if a true saprophyte could so alter in
outside nature as to be capable of eventually starting an
epidemic of infectious disease his views of the specific
nature of infectious diseases will have to undergo a com-
plete alteration. We will illustrate this by the following
instances : G. Roux and Bordet have asserted that the
bacillus coli, which as we have shown is a common sapro-
phyte in the human and animal intestine, when sojourning
in sewage — into which it naturally and commonly finds its
way — is capable of becoming changed into the typhoid
bacillus. This assertion was made on quite insufficient
bacteriological evidence ; moreover it was made at a time
when the distinction between bacillus coli and bacillus of
typhoid fever was not as easy or as well established as it
now is. We can now dismiss this statement, viz., the
conversion into and interchange of bacillus coli and bacillus
of typhoid, as contrary to bacteriological experience.

Another instance of this kind is that adduced by Buchner
on the experimental conversion of bacillus subtilis or hay
bacillus into the bacillus anthracis (“ Ucber die experim.

538

MICRO-ORGANISMS AND DISEASE [chap.

Erzeugung des Milzbrandcontagiums,” Sitzungsb. d. inath.-
phys. Classe d. K. Bair. Akad. d. Wiss. 1880, iii. p. 369).
We have in a former chapter described in detail the morpho-
logical and cultural characters of these two microbes and
have shown them to be sufficiently striking to- be readily
distinguishable one from the other.

Buchner states that the bacillus anthracis when passed
through a large number of successive cultures at a tempera-
ture of 350 to 370 C. gradually loses its pathogenic properties.
In a Report to the Medical Officer of the Local Govern-
ment Board for 1881-1882 I have shown that, even
assuming that Buchner has had in all his cultures the true
bacillus anthracis, but for which there is no definite proof,
as Koch has so ably pointed out in his critical review of
Buchner’s work ( Mittheilungen aus dem k. Gesundheitsamte,
Berlin, 1881, Bnd. I.), Buchner, having tested his cultures
on white mice only, has fallen into a serious error, for, as I
have shown (Reports for 1881-1882), a culture of bacillus
anthracis may have become quite harmless to white mice,
but be still virulent to other animals. In fact, therefore,
Buchner’s result does not require for its achievement more
than one culture, provided this has been kept for several
days or weeks without spore-formation, as was the case in
Buchner’s experiments.

As regards Buchner’s statement that by successive cul-
tivation of bacillus anthracis at 35° to 37° C. this assumes
the morphological and physiological characters of hay
bacillus, I agree with Koch in regarding this as a complete
error. If the cultures are kept safe from contamination,
nothing of the sort ever happens. It is of course clear that
if by any accidental contamination, say at the time of inocu-
lating a fresh tube, a motile septic non-pathogenic bacillus,
with which, or with the spores of which, the air sometimes

XXI]

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539

abounds, is introduced, every new culture established from
this one will abound in this bacillus, and as it grows quicker
and more easily than.the bacillus anthracis, the next cultiva-
tions become barren of all the bacilli anthracis and only
the non-pathogenic motile bacillus will be found present.
This criticism has been applied by Koch to Buchner’s
experiments, and I must fully endorse it.

But there is a much more serious statement of Buchner’s
— serious, because if true in nature it is dreadful to contem-
plate to what amount of anthrax man and brute may become
subject — viz., he maintains to have succeeded in trans-
forming the hay bacillus into bacillus anthracis, by carrying
the former through many generations under ever varying
change of soil. It is needless to detail here all these experi-
ments of Buchner, since I do not attach any great value to
them, and I should not have troubled myself much about
them, were it not that one meets in mycological literature,
particularly on the part of botanists, an acceptance of
Buchner’s statement that hay bacillus can change into the
pathogenic bacillus anthracis (see Zopf, Die Spaltpi/ze,
Breslau, 1883).

I have repeated Buchner's experiments on rabbits, guinea-
pigs, and white mice. I have grown the hay bacillus in
various kinds of broth, in gelatine -broth mixtures, in hydro-
cele fluid, in peptone fluid, in Agar-Agar and peptone, at
temperatures varying between 30° and 38° C., and I have,
to put it shortly, never seen that it shows the least tendency
to change its general morphological characters, or that it
ever assumes the morphological or physiological characters
of the bacillus anthracis. I consider this a perfectly hope-
less task, and I feel sure any one might as soon attempt to
transform the bulb of the common onion into the bulb of
the poisonous colchicum.

540 MICRO-ORGANISMS AND DISEASE [chap.

A further instance in which the transformation of a com-
mon saprophyte into a specific or pathogenic organism has
been experimentally achieved, or I should rather say has
been stated to have been achieved, is the jequirity bacillus.
In 1882 the well-known ophthalmologist M. L. de Wecker
in Paris drew attention to the therapeutic value of the seeds
or beans of Abrus precatorius , a leguminosa common in
India and South America. The people of Brazil use it
under the name jequirity as a means to cure trachoma, or
granular lids. De Wecker after many experiments found
that a few drops of an infusion made of these seeds causes
severe conjunctivitis, in the course of which, no doubt,
trachoma is brought to disappearance and cure, and it is
accordingly on the Continent and in this country now used
for this therapeutic object. [I was informed by the late
Dr. T. Lewis, formerly of India, then pathologist at the
Netley Army Medical School, that the people in some parts
of India know the poisonous properties of these seeds,
and use them for inoculating cattle subcutaneously ; in
consequence a severe inflammation is set up, and the
animals die of some sort of septicaemia. This is done for
the sake of simply obtaining the hides of the beasts.]

Sattler, in a very important and extensive research ( Wiener
medic. Wochenschrift , N. 17-21, 1883, and Klin. Monatsbl. f.
Augenheilk. June 1883), ascertained that when an infusion of
the jequirity seeds is made of the strength of about half per
cent, this infusion after some hours to a few days contains
numerous bacilli, motile, capable of forming spores, and in
most respects identical with the bacillus mesentericus. The
bacilli are about 0-00058 mm. thick, and from c-oo2 to
0-0045 nim. long. They form a pellicle on the surface of
the infusion, and in the bacilli of this pellicle active spore
formation is going on. The bacilli grow and multiply well

XXI] PATHOGENIC ORGANISMS 541

at a temperature of about 35° C., but also, only slower,
at ordinary temperature. Sattler cultivated artificially the
bacilli on blood-serum gelatine and meat extract peptone
gelatine, both solid media, and continued their growth
through several successive cultivations. Both the infusions
of the jequirity and the bacilli taken from these artificial
cultures inoculated into the conjunctiva of healthy rabbits
produce severe ophthalmia, leading to the production of
great oedematous swelling of the conjunctiva and eyelids,
and temporary closure of the latter, and to the secretion of
purulent exudation. Both the exudation and the swollen
lids are said to contain infective bacilli and their spores.
Sattler ascertained by many experiments that none of the
bacilli and the spores distributed in the atmosphere had
those specific properties, viz., to excite ophthalmia, as long
as they grow in other than jequirity fluid, but having had
access, i.e. having entered the jequirity infusion, assume
here this specific power. There is no doubt that Sattler
worked the whole problem with great care, worked out all
points connected with it in great detail, and for this reason
his work was considered to have for the first time un-
mistakably established that a harmless saprophyte, to wit the
bacillus mesentericus, owing to the particular soil in which
it grew, assumes definite specific or pathogenic properties.
To me this jequirity bacillus had a great interest, since I was
particularly anxious to get hold of such an organism, in
order to see whether and how far it can again be made
harmless. For if ever there was a good case, a case in
which a previously harmless saprophyte had by some
peculiar conditions become specific, this was a case ; and
therefore it must be here possible by altering its conditions
of life again to transform it into a harmless being. The
theoretical and practical importance of such a case must be

542 MICRO-ORGANISMS AND DISEASE [CHAP.

evident to every one who has at all devoted any thought to
the relation of micro-organisms to disease. The whole
doctrine of the specificity of infectious diseases, I might
almost say, is involved in such a case, for if in one case it
can be unmistakably proved that a harmless bacterium can
be transformed into a pathogenic organism, i.e. that an
infectious malady can originate de novo, then we should at
once be relieved of searching for the initial cause in the
outbreak of an epidemic. But in that case we should be
forced to contemplate, as contained in the air, in the water,
in the soil, everywhere, numbers of bacteria which, owing to
some peculiar unknown condition, are capable at once to
start any kind of infectious disorder, say anthrax (Buchner),
infectious ophthalmia (Sattler), and probably a host of
other infectious diseases, and thus to form the starting-
point of epidemics. And the only redeeming feature,
if redeeming it can be called, in this calamity would be
the thought that the particular bacterium would by-and-
bye, owing to some accidental new conditions, again become
harmless.

These were the reasons, and good reasons I think they
were, which prompted me to inquire into the jequirity
bacillus and jequirity ophthalmia, and after a very careful
and extensive series of experiments, to be described presently,
I have proved beyond any doubt that the jequirity bacillus,
per se, has no more power to create an infectious ophthalmia
than Buchner’s hay bacillus had of creating anthrax.

The following experiments prove this conclusively : —

The seeds of jequirity ( Abrus precatorius) are crushed and
powdered, the perisperm is removed, and of the rest an
infusion is made of about the strength of half per cent,
with distilled -water, previously boiled and contained in a
flask previously sterilised (by heat) and plugged with sterile

XXI]

PATHOGENIC ORGANISMS

543

cotton-wool. The infusion is made while the water is still
tepid. After half an hour the infusion is filtered into a
fresh sterile flask, plugged with sterile cotton-wool, the access
of air being limited as much as possible. This is effected
by keeping the cotton-wool in the mouth of the flask around
the end of the glass filter. The filtered fluid is of a slightly
yellowish-green colour, and is almost neutral and limpid.
A small quantity is withdrawn with a capillary glass pipette
freshly drawn out, and from this several test-tubes containing
sterile nourishing material (peptone solution, broth, Agar-
Agar and peptone) are inoculated ; and from the same
pipette, and at the same time, several eyeballs of healthy
rabbits are inoculated, by placing a drop or two of the
infusion under the conjunctiva bulbi. The test-tubes are
placed in the incubator and kept there at 350 C. After
twenty-four hours all eyeballs are intensely inflamed, the
eyelids closed and swollen, and a large amount of purulent
secretion is present in the conjunctival sac, but all the test-
tubes remain perfectly limpid ; no growth has made its
appearance, and they remain so.

In a second series the infusion prepared in the above
manner is used fifteen minutes after it is made and used as
above, for inoculation of test-tubes and eyeballs. The fluid
in the test-tubes after incubation remains limpid, the eyeballs
all become inflamed. In both series the amount of fluid
inoculated into the test-tubes is more than twice as great
as that injected into the eyeballs. From this it is quite
clear that the fluid used for inoculation of the test-tubes was
barren of any micro-organisms, and nevertheless it possessed
a powerful poisonous principle. I do not mean to say that
the infusion as a whole contained in the flask contains no
organisms, but that the small quantity of the fresh infusion
that was used for the inoculation of the test-tubes and eyeballs

544

MICRO-ORGANISMS AND DISEASE [chap.

contained none is absolutely certain. When such a flask
is placed in the incubator, after twenty-four to forty-eight
hours or later there are found in it large quantities of bacilli,
the spores of which must have entered from the air during
the process of preparing the infusion. The bacilli are such ]
as described by Sattler ; they soon form spores in the usual
way. Such an infusion is very poisonous, just like the fresh
one. Sattler has shown, and this is easily confirmed, that
the spores of these bacilli stand boiling for a few minutes
without losing their power to germinate. Consequently, if
such a poisonous infusion full of bacilli and spores be boiled
for half a minute the spores are not killed ; proof for this :
that if with a minute dose of this spore containing boiled
infusion any suitable sterile nourishing material in test-tubes
be inoculated, and then these test-tubes be placed in the
incubator at 350 C., after twenty-four to forty-eight hours
the nourishing fluids are found teeming with the jequirity
bacilli ; but no amount of this material produces the least
symptom of ophthalmia. Every infusion of jequirity loses its
poisonous activity by boiling it a short time , -l- to 1 minute,
and hence the above result.

In this respect the poisonous principle of jequirity infusion
comports itself similarly to the pepsin ferment, which, as is
well known, is destroyed by short boiling.

If an infusion is made as above, and after fifteen minutes
it is filtered and then subjected to boiling for ^ to 1 minute,
it will be found to have become absolutely non-poisonous,
but not sterile : placing it in the incubator after twenty-four to
forty-eight hours, vast numbers of the jequirity bacillus are
found in it. But no amount of this fluid is capable of
producing the slightest symptom of ophthalmia.

A large percentage of the rabbits, whose conjunctiva has
been inoculated with the fresh unboiled poisonous infusion,

XXI]

PATHOGENIC ORGANISMS

545

die after several, three to eight, days. The eyeballs and
eyelids are intensely inflamed, as stated above, the skin and
subcutaneous tissue of - the face, neck, chest, and even
abdomen, are found enormously cedematous, the pericar-
dium, pleura, lungs, and peritoneum very much inflamed,
their cavities filled with a large quantity of exudation.
The exudations of the conjunctiva, pericardium, peri-
toneum, the cedematous skin and subcutaneous tissues contain
no infective property, and as a rule no bacilli or spores of
any kind, if examined in the living animal or immediately
after death.

There is one point which requires careful consideration ;
it is this : Sattler states that he has cultivated the bacillus,
taken from a poisonous jequirity infusion, through several
successive generations on solid material, and with the new
cultures he was able to produce the jequirity ophthalmia.
I have no doubt whatever that this is really the case, but it
bears an interpretation different from the one Sattler gave
it. Sattler, and many others, would, of course, say this : if
any micro-organism taken from a soil that possesses infective
properties be carried through many successive artificial
cultivations, all accidentally adhering matter would hereby
become so diluted that it may be considered as practically
lost ; that is to say, the organisms of the further generations
have become altogether free of that matter. If the
organisms of these further generations still possess the same
poisonous property as the original material, then we must
conclude that this poisonous principle is identical with the
micro-organism. I do not agree with this whole chain of
propositions although I agree with some parts. If a micro-
organism be carried through several successive cultivations,
always using for inoculation of a new culture an infinitesimal
dose, then, no doubt, carrying on the cultivations through

N N

546

MICRO-ORGANISMS AND DISEASE [chap.

four, five, or six successive cultures, any accidentally adhering
original matter becomes practically lost, and if then the
organism still possesses the same specific action as the original
material, then no doubt the conclusion that organism and
poison are in this case identical becomes inevitable. But
this is not the case with the jequirity bacillus. Taking from
a poisonous jequirity infusion full of the bacilli one to two
drops, and inoculating with it a test-tube containing about
four to five cc. of nourishing fluid, and using this at once
without previous iticubation, we find that even a few drops of
this so diluted fluid still possess poisonous action. Precisely
the same result is obtained when taking from a perfectly fresh
jequirity infusion, i.e. before any organisms have made their
appearance, one to two drops, and diluting them with four
to five cc. of distilled water, and using of this diluted fluid
one to two drops for inoculating the conjunctiva of healthy
rabbits : severe ophthalmia will be the result. Carrying
on the cultivation of these bacilli started from a poisonous
infusion, for a second generation in fluid medium, no trace
of any poisonous action can be now detected, any quantity
of such a cultivation is incapable of producing ophthalmia.
Sattler used in his cultivations solid nutritive gelatine on the
surface of which he deposited his drop of poisonous jequirity
infusion containing the bacilli ; after some days’ incubation,
the bacilli having become greatly multiplied and having
liquefied the top layer of the gelatine, he took out from this
second culture a drop, and transferred it to a new culture-tube
of solid material, and so he went on : every one of these
cultures possessed poisonous action. Clearly it would, since
he always used part of the original fluid deposited on the
surface of the solid nourishing material. Part of this (being
gelatine) became by the growth liquefied, but considering that
Sattler started with infusions of considerable concentration

XXI]

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547

— he left the seeds for many hours and days in the infusion
— it is not to be wondered at that this would bear a con-
siderable amount of dilution, and still retain its poisonous
properties. From all this we see, then, that the jequirity
bacillus/e’rjtf has nothing to do with the poisonous principle
of the jequirity seeds, but that this principle is a chemical
ferment in some respects (in its inability to withstand boiling)
similar to the pepsin ferment.

Messrs. Warden and Waddell published in Calcutta
during 1886 a most valuable memoir, detailing a large
number of observations on the jequirity poison, which are
in complete harmony with my own observations. They
have definitely proved that the active principle is a proteid
— abrhi — closely allied to native albumen ; that its action is
similar to that of a soluble ferment, that it can be isolated,
and that it is contained not only in the seeds but also in
the root and stem of Abrus precatorius.

Sidney Martin has also published important facts con-
cerning the chemical nature of abrin, according to him it is
allied to an albumose. Ehrlich considers it as a tox-
albumin and he has shown the remarkable fact that by small
and repeated doses of abrin an animal (rabbit) can be
immunised against a fatal dose ( see later).

The second question which we put, viz. : Can a true
saprophyte become pathogenic in the animal body owing to
conditions within the animal ? is more difficult to answer,
and is intimately bound up with the further question, viz. :
What is and what is not a specific or pathogenic microbe ?

Specific or Pathogenic Microbes. — If under a specific microbe
is understood a microbe that is connected with and is the
causa causans of a definite infectious disease belonging to
the group of communicable diseases occurring in nature and
affecting man or animal or both, then the number of

N N 2

548

MICRO-ORGANISMS AND DISEASE [CHAP.

pathogenic microbes is a limited one, and the number of
such microbes known is less extensive than the number
of known infectious diseases, since of some of them the
specific microbe has not been discovered yet, e.g., hydro-
phobia, syphilis, measles, whooping cough, &c., &c._; if, I
however, under a pathogenic microbe is understood one
that is capable of living, under one condition or another,
parasitic in the animal body and causing therein disease or
a diseased condition, then their number is practically un-
limited and no line of demarcation can be drawn between
specific or parasitic and non-specific or saprophytic microbes.

It is well established that microbes like bacillus prodigiosus,
bacillus subtilis, proteus vulgaris, bacillus coli, and others,
i.e. microbes living generally as saprophytes, when injected
subcutaneously into the guinea-pig in small quantities, such
as in the case of specific microbes capable of producing
a specific disease, cause no disturbance, because they are
not capable of living and multiplying in the subcutaneous
tissue. I have shown that if these same saprophytes be
injected in considerable quantities into the peritoneal cavity
of a guinea-pig ( see a former chapter) they are capable of
living herein, of multiplying and causing acute peritonitis and
death ; after death the peritoneal exudation is found teeming
with the living microbe, and they can be demonstrated in a
living state in the blood and in the cavity of the inflamed
intestine. Under this aspect of pathogenicity many a
species occurring in nature ordinarily as saprophytes and
connected with no infectious disease does cause acute fatal
peritonitis and is capable of multiplying within the peritoneal
fluid, whereas I have likewise shown that a notoriously
specific microbe, like the diphtheria bacillus (from a gelatine
culture), while virulent in the subcutaneous tissue of the
guinea-pig, when injected in large doses into the peritoneum

XXI I

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549

foils to cause disease, it becoming soon killed herein. If then
we are to judge of the nature of a microbe by its behaviour
in the peritoneal cavity of the guinea-gig, i.e. whether patho-
genic or non-pathogenic, we should have to include among
the pathogenic class a large number of microbes which are
generally pure saprophytes, while we should have to exclude
from that class microbes which, as a matter of fact, are
notoriously specific. If, on the other hand, we distinguish
the microbes by the presence or absence of poisonous
substances in their bodies (intracellular poisons or proteins)
or elaborated (or secreted) by them while growing and multi-
plying—toxins — we do not get much further either ; because,
as I have shown and as has been mentioned in a former
chapter, some notoriously specific microbes have not these
intracellular poisons (anthrax, fowl cholera, diphtheria),
while other notoriously saprophytic bacteria possess them
(vibrio of Finkler, bacillus prodigiosus, bacillus coli, &c.).
Again, if we judge by this whether a microbe does or does
not produce in the course of its growth and multiplication
toxins, i.e. poisonous metabolic substances, we would not
get at a true definition either, because some microbes con-
nected with putrefactive changes (proteus vulgaris) produce
well-specialised toxic principles, while other microbes,
connected with infectious diseases, do not, as for as one can
judge from experiments, produce any specialised toxin, e.g.
the whole group of microbes which cause in the rodent
haemorrhagic septicaemia ; then there are other microbes, true
specific or pathogenic, which although producing highly
specialised toxin, i.e. toxin which injected into the animal
causes the same disease as when we inject the living
microbe (tetanus, diphtheria), do not as a rule live in the
blood or in the tissues : the bacillus diphtherias, the bacillus
tetani, lives chiefly at the seat of inoculation, where it produces

55o MICRO-ORGANISMS AND DISEASE [chap.

its toxin ; in the blood or tissues it as a rule does not seem
capable of existing. Further : there are microbes which are
capable of producing specialised toxins when growing in one
kind of medium but not in another; bacillus anthracis is a
good case in point : it would be extremely difficult — in fact
it has not succeeded hitherto — to obtain from a broth
peptone culture of bacillus anthracis, however luxuriant, any-
thing like the specific toxin that was obtained by Wooldridge
in cultivations in fluid alkali albumen, by Hankin in fibrin,
by Sidney Martin in albuminous fluid. Or take the cholera
vibrio. This microbe does not produce toxic substances to
any appreciable degree in broth culture, but in albuminous
fluids (van Ermengem) it produces it in a concentrated form.
I have found the same to be the case with the vibrio of
Finkler, bacillus coli, and others. From all this it follows
that the presence or absence in the microbic bodies of
principles poisonous to the animal body or of poisonous
principles produced in culture is no guide in distinguishing
a pathogenic from non-pathogenic microbes. Equally un-
satisfactory is the distinction into microbes which can and
such as cannot grow and thrive in the tissues of an animal,
the former being generally considered pathogenic, the latter
non-pathogenic. We have already mentioned the fact that
some notorious saprophytes not connected with any specific
disease — e.g. bacillus prodigiosus, bacillus coli, proteus
vulgaris, vibrio Finkler-Prior, bacillus subtilis, and others —
can live and multiply in the peritoneal cavity of a guinea-
pig, provided they are injected therein in comparatively
large quantities, whereas a notorious specific or virulent
microbe, e.g. the bacillus diphtherias taken from a gelatine
culture, cannot live in the peritoneal cavity even if intro-
duced in large quantities ; it rapidly in the course of a few
hours degenerates and dies, whereas of the same culture a

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much smaller dose injected subcutaneously causes tumour
and fatal issue in thirty to thirty-six hours. But also in the
subcutaneous tissue of the guinea-pig the above saprophytes
can live and multiply, provided they are injected in large
doses ; a swelling appears, which is as a rule only of a
temporary nature, lasting for from a few days to a week,
according to the dose, and sometimes leading to abscess or
purulent infiltration and necrosis. While the tumour lasts
the microbe injected can be demonstrated in a living state
by the culture test. In very large doses they may even
produce rapidly general infection and death. On the other
hand we see that even in the case of well-recognised specific
microbes the subcutaneous injection may produce no
appreciable result or only a slight and transitory tumour
and recovery.

We have mentioned in several instances such results
having been obtained by using attenuated cultures ; it is
this result which represents the essence of protective inocu-
lations. A temporary tumour can be therefore produced
by a specific microbe of attenuated virulence or by an
otherwise virulent microbe if it be injected in too small a
dose, or into an animal which is not very susceptible ; ac-
cording to any or all of these factors the effect of inoculation
may be nil, or very slight and rapidly passing, or it may be
moderate and slowly passing off, or it may be conspicuous and
leading to death. So that the microbe introduced, although
specific and pathogenic, may degenerate and be killed
rapidly in the tissue, or it may multiply slightly, or it may
multiply rapidly and easily and cause general infection.

From these considerations it follows then both in the
case of true saprophytic as of true pathogenic microbes that
all gradations of their capability to live and thrive in the
animal tissues may be shown to be demonstrable, these

MICRO-ORGANISMS AND DISEASE [chap.

552

gradations in both classes of microbes depending (a) on the
initial character (or virulence), some species acting in smaller
doses than others, {/>) on the quantity injected, ( c ) on the
greater or lesser reactivity, smaller or greater resistance of the
tissue in particular or the animal in general.

This brings us now to the consideration of the subject
of what constitutes this resistance or immunity, what is the
cause of this natural resistance or spontaneous insuscepti-
bility, observed at the outset.

It must be clear from what has been just stated that the
greater or lesser immunity or resistance of the tissue or of
the animal are relative quantities. Under spontaneous or
natural resistance is meant the natural capability of a tissue
to withstand the growth and multiplication of a microbe and
to destroy or kill the latter. And this spontaneous immunity
must be distinguished from acquired or secondary immunity.
We have shown that while one tissue is capable of destroying
the microbes brought in contact with it, another tissue of the
same animal does not achieve this result, or while an animal
in one condition is found resistant it is found susceptible
under another condition, or again while an animal is sus-
ceptible towards a microbe in an unaltered virulent condition
it is possessed of resistance against an altered or attenuated con-
dition of the same microbe, and lastly, while a particular tissue
or the animal is insusceptible and found resistant against
a small dose it is found susceptible against a large dose. We
will mention here a few examples illustrating these points: —

(a) The cholera vibrio, the vibrio of Finkler, the bacillus
prodigiosus, the bacillus coli, and many other microbes when
injected into the peritoneal cavity of a normal guinea-pig
in sufficient doses live and grow well and produce acute peri-
tonitis and death, while when introduced in the same amount
into the subcutaneous tissue or into the blood they soon

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degenerate and die off ; the diphtheria bacilli of a gelatine
culture injected into the subcutaneous tissue of a guinea-pig
— one-fifth of a culture per one kilo animal weight — produce
typical tumour and death in thirty to thirty-six hours ; when
from the same stock one-third or one-fourth of the culture
per 300 — 400 grms. guinea-pig is injected into the peritoneal
cavity, nothing happens, the bacilli very soon degenerate
and are killed.

When hay bacillus, staphylococcus taken from septic
fluids, bacillus mesentericus, bacillus coli, or other sapro*
phytes are injected into the vascular system of a healthy
animal, no disease follows, the microbes soon disappear ; but
if in such an animal a focus of inflammation, necrosis, or
ulceration has been previously established this focus becomes
easily the soil for the growth and multiplication of those
saprophytes (Wyssokovitch, Zeitschr.f. Hygiene , i.). Of the
same nature are the observations constantly to be made
of micrococci of various kinds, pneumococcus, and of
bacillus coli or proteus vulgaris being found present, as a
secondary invasion, in inflamed portions of various organs —
liver, lung, -spleen, kidney — in which the inflammation had
been caused by some other antecedent disease.

Even in the case of an animal highly susceptible to a
particular specific microbe it will be noticed that not all
tissues of such animal are favourable for the life and growth
of the microbe. While in the so-called blood diseases :
septicaemias of different kinds, anthrax, fowl cholera, &c., the
microbe lives and thrives well in the blood and blood-vessels
of all tissues, this is not the case in many other instances, e.g.
diphtheria, tetanus, cholera, tubercle, typhoid fever, and others.

(, b ) A normal frog is insusceptible to anthrax infection,
but if it be kept heated to the temperature of a warm-
blooded animal it is susceptible (Petruschki). A normal

554 MICRO-ORGANISMS AND DISEASE [chap.

frog or a normal adult white rat is insusceptible to anthrax
infection, but if it be subjected to narcosis with ether-
chloroform it becomes susceptible (Klein and Coxwell).
Fowls are insusceptible to anthrax, but if cooled they
become susceptible (Pasteur).

Charrin and Roger (La Semaine Med., 1890, No. - 4)
show that while normal rats are, as is known, very little
susceptible to anthrax, they become highly susceptible if by
working at a treadmill they are made fatigued, and H. Leo
( Zeitschrift f. Hygiene , vii. 3) finds that by the presence of
much sugar in the blood and tissues the susceptibility to
anthrax and tubercle is not increased, while for glanders it
becomes greatly enhanced. Phloridzin is administered in
small doses with the food, sugar thereby becoming present
in the tissues. Rats thus prepared resist anthrax as much
as unprepared rats, guinea-pigs first prepared with phloridzin
and then inoculated with tubercle do not show more
intensive or more rapid tuberculosis. While normal white
mice are almost insusceptible to glanders, they become
highly susceptible to such infection if prepared with
phloridzin. Maya and Sanarelli give an account (Fortschr.
d. Med., ix. No. 22) of a large number of experiments, in
which by introducing acetylphenylhydrazin into an animal
insusceptible to a particular disease this animal becomes
thereby susceptible. This substance is known to produce
destruction of the red blood-corpuscles (Gottstein) and
luemoglobinsemia ; pigeons and rats thus prepared proved
susceptible to anthrax.

(c) A normal guinea-pig when injected subcutaneously
with a moderate dose of cholera vibrio not of high virulence
from the outset, or that had owing to subculture for many
generations lost its virulence, fails to show any result, but
when the dose is transmitted through the peritoneal cavity of

PATHOGENIC ORGANISMS

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XX l]

the guinea-pig for some successive transmissions it becomes
virulent even for subcutaneous injection ; the same applies
to bacillus coli, bacillus of typhoid, vibrio of Finkler, and
others. Particularly bacillus coli can in this way so much
increase in virulence that small doses injected subcutaneously
cause acute septictemic infection.

Some very striking phenomena are shown in these respects
by the bacillus coli. The typical bacillus coli cultivated
from the intestinal contents of a guinea-pig does not possess
towards the guinea-pig greater pathogenic power (as shown
by subcutaneous or intraperitoneal injection) than the typical
bacillus cultivated from the human intestine.

Now, occasionally on intraperitoneal injection of a fatal
dose of one or the other microbe (bacillus prodigiosus,
staphylococcus aureus, vibrio Finkler, or vibrio cholerae)
after death of the animal (in sixteen to twenty-four hours
according to the dose and virulence) the peritoneal fluid
contains besides the microbe injected also an abundance of
a rapidly motile cylindrical bacillus which in culture proves
to be the typical bacillus coli (Klein, Gartner). That this
could have been derived from the interior of the intestine
only seems clear, but whether it got through the diseased
but uninjured wall of the intestine (there has been established
severe peritonitis by the microbe injected) or whether during
the intraperitoneal injection the intestine has been injured
by the cannula of the syringe it is difficult to say, at any
rate there is no visible puncture of the intestine to be found.
But it must be obvious that if on injection into the peritoneal
cavity of say a pure culture of bacillus prodigiosus there
should after the death of the animal be found in the peri-
toneal exudation, besides the bacillus prodigiosus, a bacillus
which possesses all cultural characters of the bacillus coli
the conclusion that this latter has got into the peritoneal

556

MICRO-ORGANISMS AND DISEASE [chap.

cavity from the interior of the intestine is the only one that
can be admitted. Now this peritoneal accidental bacillus
coli after cultivation proves highly virulent for the guinea-pig,
a small dose injected subcutaneously produces acute
haemorrhagic septicaemia easily transmissible from guinea-
pig to guinea-pig.

Bacillus anthracis of one source or another possesses
different degrees of virulence (as has been mentioned in the
chapter on Anthrax), thus if a comparatively large dose of
anthrax bacilli in the blood of a mouse be injected into a sheep
perhaps only transitory illness will be the result, the sheep
possessing a certain amount of resistance against the mouse-
bacilli, but if a few drops of blood of sheep dead of anthrax
be used for subcutaneous injection of a normal sheep fatal
anthrax will be the result.

(d) That the amount, i.e. the number, of the microbes
introduced plays an important part has been mentioned on
various previous occasions ; here are a few more examples :
A guinea-pig is susceptible to virulent anthrax if only a
few bacilli are injected subcutaneously (Watson Cheyne,
Lubarsch), while for the rabbit to achieve this result a con-
siderably greater number is required, and in the case of a
dog not even large doses suffice to produce infection.

The bacillus of fowl cholera taken from a drop of the
blood of a fowl dead of the disease, injected subcutaneously
into a rabbit or a pigeon, produces acute fatal infection, in
the guinea-pig such a dose produces no result. A small
particle of a glanders nodule of the horse injected subcu-
taneously into a guinea-pig or a field mouse produces fatal
infection, in a rabbit it produces local abscess, and in a
normal white mouse produces no result or only a slight
transitory tumour.

These, as stated above, are only a few examples amongst

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the large mass of observations that have been made on the
relation of the degrees of natural immunity of different
tissues and different animals in their normal and abnormal
states against saprophytic and specific microbes in different
conditions (of virulence) and introduced in different
amounts.

Now, what is the cause of this spontaneous immunity? At
the outset it is necessary to keep this question separate from
that of acquired immunity, that is immunity which is pro-
duced by one or more previous mild or transitory attacks ; it
has been known as long as infectious diseases have been
recognised as such that in some at any rate one attack, mild
or severe, protects against a second severe attack, and the
inoculations against small-pox (brought from the East),
Jenner’s vaccination against small-pox, “ vaccination ” against
anthrax, fowl cholera, swine erysipelas (Pasteur), are based
on that experience. The important fundamental observa-
tions that have been made in this field, subsequently to
Pasteur’s work, by Salmon, Roux and Yersin, Klemperer,
Behring and Kitasato, Behring, R. Pfeiffer, and many
others show that a specific immunity or resistance of
different degrees can be produced against a specific microbe
or its toxin according to the strength (virulence) and amount
of the living microbe or its toxin previously introduced.
Moreover this same principle of “ active immunisation ” by
previous toxin injections holds good also for other than
microbic toxins : Ehrlich produced this active immunity
against an otherwise fatal dose of Ricin and Abrin respec-
tively by previous repeated administration of subfatal and
gradually increasing doses of these toxins, Calmette and
Fraser the same against snake venom. The point that at
present concerns us is the meaning and cause of spon-
taneous or natural resistance or immunity of one or another
tissue or the animal body against one or another kind of

558 MICRO-ORGANISMS AND DISEASE [chap.

microbe, be this cither a saprophyte or a pathogenic
microbe.

Spontaneous or natural immunity. — The first who at-
tempted an explanation based on experiment was Metchni-
koff, who ascribed to tire leucocytes, lymph- or white
blood-corpuscles the power of taking up, destroying, and
neutralising the microbe introduced into the tissue and
thereby protecting the tissue and the body from infection,
inasmuch as the microbes thus destroyed cease to exist,
to multiply, and to produce their toxic effects. This view
was based on the fundamental observation of Metchnikoff
that when in a normal frog anthrax bacilli are introduced
into the dorsal lymph sac, leucocytes soon rush, as it were,
and are attracted to the place, eat up the bacilli, and thus
protect the animal against infection, preventing the bacilli
from living, growing, multiplying, and causing disease.
This process of “phagocytosis,” as it was called, is therefore
an essential feature in natural immunity ; it is in the first
instance a purely mechanical process, effected by the amoeboid
movements and capability of the leucocytes to embody and
swallow up and digest and destroy the invading enemy.
In a large number of instances of known immunity — of
greater or lesser degree — Metchnikoff and his pupils have
sought and found this process of mechanical phagocytosis,
and have explained to their own satisfaction every case
of immunity of one or another animal or its tissues
against one or another kind of microbes, be they true
saprophytes or true parasites.

This theory relies on the following facts : (i) leucocytes
are well known to be capable in the course of their amoeboid
movements of embodying and swallowing particulate matter,
(2) leucocytes generally accumulate at, i.e. are attracted to, a
locality into which foreign particles en masse are introduced
or injected, and (3) it is notorious that in cases of immunity

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of tissues or the animal the leucocytes present do contain
in their interior the bacteria, some more, some less degener-
ated. The first and third points may be taken to represent
phagocytosis in a mechanical sense, the second point may be
considered as leucocytosis owing to positive chemiotaxis.

Now, while this represents the positive side of immunity,
the reverse, viz., the greater or lesser inability of the leuco-
cytes to rush to the bacteria, the greater or lesser inability
to take them up, and the greater or lesser inability to destroy
them, represent the negative side of complete or imperfect
immunity; that is to say: if the leucocytes are of this nature,
no immunity is present, the introduced microbes are not
interfered with, they live, thrive, and multiply and cause
infection and the disease. This is in essence the sum total
of the views and observations that Metchnikoff and his
school have put forward as sufficient to explain immunity
complete and incomplete. For a fairly complete literature
and history of this view see Lubarsch, Centralbl. f Bakt.
und Parasit., vol. vi., No. 20.

First as to the phenomenon of leucocytosis : it is notorious
that in many instances when microbes are injected into the
subcutaneous tissue of an insusceptible animal, or are intro-
duced in an attenuated form or in too small a number to
maintain themselves in the struggle for existence against the
living tissue, such leucocytosis does take place ; this is the
case when, for instance, a dose of anthrax bacilli is injected
subcutaneously into a normal adult rat, or a dog, or into
the lymph sac of a normal frog, or if a small dose of bacillus
of symptomatic charbon is injected into the subcutaneous
tissue of the little susceptible rabbit, or if a fair dose of
moderately virulent bacillus typhosus or of vibrio cholerae is
injected subcutaneously into the guinea-pig. But this is by
no means universally the case. Take, for instance, the case

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MICRO-ORGANISMS AND DISEASE [chap.

of bacillus prodigiosus, or bacillus coli, bacillus typhosus, or
cholera vibrio, in its relation to the subcutaneous tissue of
the guinea-pig. If a small dose of either of these living
microbes (taken from the slanting surface of an Agar cul-
ture), say one-fifteenth or one-twentieth of a culture of an
ordinary not exceptionally virulent stock distributed in
sterile salt solution or sterile bouillon, be injected subcu-
taneously into a normal guinea-pig of about 300 grammes,
the result is nil , no tumour is noticed, no leucocytosis ; if
the dose be larger, say one-tenth to one-eighth of a culture,
there is noticed next day a more or less distinct swelling
and leucocytosis, with general constitutional disturbance ;
the swelling increases for a day or two, then diminishes and
becomes firmer, and may ultimately lead to suppuration
and ulceration of the skin. While the tumour grows and
increases, and even when it has begun to decrease and to
become firmer, the microbes injected can be recovered by
culture in a living state. If the dose be still more increased,
say a quarter to a third of a culture, the result is more
pronounced, the tumour and leucocytosis are greater and in
some cases in two or three days may be followed by general
infection and death.

So that the capability or incapability of a microbe to per-
sist in a tissue, and to maintain its life and multiply therein,
stands in no necessary relation to the existence or non-
existence of a leucocytosis.

Moreover, even in the case of a particular microbe, eg.
bacillus anthracis, its introduction into the subcutaneous
tissue of an insusceptible animal, say an adult rat or dog,
is by no means necessarily followed by leucocytosis, and
yet no infection ensues ; this is noticed in the case when
a small dose is injected. A further important fact to be
mentioned in this connection of leucocytosis preceding

XX i] PATHOGENIC ORGANISMS 561

phagocytosis is this : after the introduction of a particular
microbe, say bacillus anthracis, into the tissue, say the
dorsal lymph-sac of a normal frog, the ensuing leucocytosis
develops comparatively slowly and late ; if, for instance, a
dose of bacillus anthracis or its spores, or of bacillus prodi-
giosus, be injected into the dorsal lymph-sac, numbers of
these bacilli or the spores are rapidly absorbed into the
blood of the general circulation, and can be there demon-
strated by culture already ten minutes after the injection
(Klein). If the animal be killed ten, thirty minutes, two
or six hours after injection, the heart opened and a drop of
blood rubbed over the slanting surface of gelatin or Agar
respectively, and incubated, twenty-four to forty-eight hours
later a large number of typical colonies of bacillus anthracis
or prodigiosus respectively will be found on the gelatine or
Agar respectively.

The “fight” ensuing between the bacilli and their spores
introduced into the dorsal lymph-sac and the leucocytes,
which are supposed to rush to the seat of the battle, i.e. the
dorsal lymph-sac, must be considered a very hollow affair,
if before the defending army can reach the seat of war a
host of the invaders have already escaped all over the
country as it were. Besides, before the defenders can
amass their legions at the seat of battle, many hours must
elapse, and it has been shown that many of the invaders
are already dead in the lymph-sac before there is any sign
of attack by the defenders, any sign of phagocytosis (Fischel,
Fortschr. d. Med. ix. 2), and that the lymph of the lymph-sac
free of leucocytes destroys the bacilli (Sanarelli, Centr. f.
Bakt. und Parasit. ix. 14). More than that, Kanthack and
Hardy show conclusively that prior to any phagocytosis, i.e.
prior to the accumulation of leucocytes which are able to take
up the microbes, the cells which are present or which aggre-

o o

562

MICRO-ORGANISMS AND DISEASE [chap.

gate in the first place are certain cells which do not take up
the microbes, which do not act as phagocytes, the latter
coming only late into the field. Those non-phagocytic
first-comers are the eosinophyle granular cells, which have
the power to destroy the anthrax bacilli with which they
come in contact, probably by secreting some matterobnoxious
to the bacilli, and that only after this work of injuring
the bacilli had been accomplished, the later comers,
i.e. the ordinary pale leucocytes, commence to take the
bacilli up, to act as phagocytes. These observations of
Kanthack and Hardy are very clear and easily verified,
and appear to me of the utmost importance, inasmuch as
they prove a first process of a change of the bacilli, followed
by a second process of scavenging by leucocytes.

One of the weak points in Metchnikoff’s theory of
phagocytosis being the primary cause of spontaneous im-
munity is the notorious fact that while in some cases of
immunity such mechanical phagocytosis — i.e., swallowing of
the microbes by leucocytes — cannot be demonstrated, there
are other cases not connected with immunity at all, in fact,
just the reverse, in which a mechanical phagocytosis is a
conspicuous phenomenon : we have in former chapters
repeatedly mentioned that if a fairly large dose of active
and otherwise virulent diphtheria bacilli (taken from the
slanting surface of gelatine), such as would more than
suffice to produce tumour and death if injected into the
subcutaneous tissue, be injected into the peritoneal cavity
of a normal guinea-pig, as a rule no disease or no death
follows, the diphtheria bacilli soon disappear from the
peritoneal cavity, in fact their degeneration and breaking up
can be demonstrated already a few hours after injection.
But occasionally in a percentage if the dose be too large, or
if instead of gelatine Agar culture is used, disease and death

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do follow. This is particularly the case if recent Agar or
serum cultures be used instead of gelatine culture, or if in
addition to the gelatine cultures diphtheria toxin — from an
active broth culture — be injected. In such a case the animal,
presumably on account of the toxin present (evidently not
present in the gelatine growth), soon sickens, and is found
dead in thirty-six to forty-eight hours or later. On examining
the peritoneal fluid in such a case it will be seen that very
few diphtheria bacilli are demonstrable, either in cover-glass
specimens or by culture ; in the latter case a drop of the
fluid yields only a few colonies. But if we look to the
omentum next to the large curvature of the stomach we
find masses of lymph, which examined under the micro-
scope show aggregations of leucocytes all filled with diph-
theria bacilli, some well preserved, others in fragments ;
between the leucocytes are also large numbers of free
bacilli. In these cases then, when the resistance of the
peritoneum — so perfect against the bacilli of gelatine cul-
ture— has broken down and been overcome, presumably by
the additional introduction of toxin, we find numbers of
phagocytes, whereas in the other case when the resistance
of the peritoneum has successfully been maintained — e.g., in
the case of using bacilli of gelatine culture — there is no sign
of phagocytes.

To the same group of phenomena belongs the occurrence
of numerous phagocytes, i.e., leucocytes filled with the
microbe, in cases when the injection has produced fatal
infection and where there is just the reverse of immunity
either of a particular tissue or of the animal as, for instance,
in fatal mouse septicxmia, in fatal swine erysipelas, when the
presence of leucocytes filled with living bacilli is a con-
spicuous feature. Add to this the same condition in the
leucocytes of the purulent secretion in Koch’s Egyptian

002

564 MICRO-ORGANISMS AND DISEASE [chap.

ophthalmia, in gonorrhoea, in tubercle, and particularly in
the leprosy cells of the leprous tubercles. The presence of
the microbes in the interior of cells in these cases means
just the reverse of a destruction of the microbes by the cells,
it means a destruction of the cells by the microbes, the
latter multiplying in the former and thereby producing their
(the cells’) ultimate destruction.

The occasional local leucocytosis observed in connection
with immunity, i.e. occurring at the seat of introduction of
bacteria, is explained by a remarkable attraction which the
introduced microbes seem to exert on the leucocytes.

Pfeffer made the first observations as to the remarkable
power possessed by different chemical substances towards
bacteria and other micro-organisms, substances which either
attract or repel bacteria, these phenomena being spoken of
as chemiotaxis, the former as positive, the latter as negative
chemiotaxis. Pfeffer ( Uuters. a. d. dot. Inst. Tubingen ,
1887, p. 582) found that motile organisms (bacteria, flagel-
lata, and volvocinea) are stimulated by many organic and
inorganic substances in solution — positive chemiotaxis. To
mention only a few of the substances, the salts of potas-
sium have a great “stimulating” power, likewise peptone,
glycerine, morphine. Alcohol, free acids, and free alkalies
have a negative chemiotactic action, i.e., repel the microbes.
Ali Cohen ( Centr. f. Bakt. und Parasit., viii. 6) made
systematic observations on this same subject with various
kinds of bacteria.

Gabritschevsky, Massart and Bordet (A finales de V I nstitut
Pasteur , 1891, iv. 6), and others tested then the action of
bacteria on leucocytes, introducing chemical substances in
capillary glass tubes into the living body of animals, and
then examining these capillary tubes and seeing whether
they attracted leucocytes or not ; in this way they found

XXI] PATHOGENIC ORGANISMS 565

that chemical substances either attract or do not attract
leucocytes. Thus, for instance, Massart and Bordet found
the lactic acid acting powerfully — negative chemiotaxis ;
Buchner found collagen, alkali albumen, gluten casein
acting powerfully — positive chemiotaxis. Now, Buchner
argues, and I think with justice ( Centralb 1. f Pakt. und
Para sit., x. 22 and 23), that when in an insusceptible
animal leucocytosis does occur at the seat of inoculation
this leucocytosis is not an expression of the commencing
battle between the microbes and the leucocytes, as is main-
tained by Metchnikoff and his followers, but is due to a
positive chemiotactic action on the part of the bacteria
(dead or alive), by which the leucocytes are attracted.
Extensive leucocytosis (suppuration) has been shown by
Koch to occur after injection of tuberculin containing the
products of the tubercle bacilli previously killed ; suppura-
tion (miliary abscesses) has been produced by Prudden and
Hodenpel in the rabbit after injection into the vascular
system of the substance of the tubercle bacilli, previously
sterilised ; also inserting sterilised tubercle culture by means
of capillary glass tubes into the subcutaneous tissue of the
rabbit proves positive chemiotactic attractions of the dead
bacilli towards leucocytes. This chemiotaxis is brought
about by substances — protein — derived from the bacteria
themselves, and is dependent on the previous inimical action
on the bacteria by the tissue per se. Where the tissue per se
possesses this action the bacteria are either only weakened or
destroyed, and only under this condition does their sub-
stance— protein — become available to attract the leucocytes ;
in such cases the weakened and also the killed bacteria are
easily taken up by the leucocytes, and these then help to
remove them. Under this theory the phagocytosis observed
at the seat of the inoculation is therefore dependent on the

566

MICRO-ORGANISMS AND DISEASE [chap,

preceding alteration of the bacteria. But in the case of a
susceptible animal, that is, when the introduction of the
bacteria produces general infection and no local leuco-
cytosis at the seat of inoculation, the bacteria, because they
remain vigorous and because they withstand the action of
the tissue, do not yield the chemiotactic substance —
protein — and therefore no leucocytes are attracted to the
seat of the inoculation.

In connection with the phenomena of chemiotaxis it
ought to be borne in mind that just as certain bacteria
exert an attraction to the leucocytes, so also is it imaginable
that the cells and tissues exert chemical attraction on cer-
tain bacteria, just as in the case of Pfeffer’s experiments.
This at any rate offers a ready explanation of the conspi-
cuous attraction that one or the other tissue seems to exert
towards certain specific microbes. It is well known that in
the acute exanthemata the skin is the tissue which pre-
eminently exerts such a positive chemiotaxis on the specific
microbes. In anthrax, in typhoid fever, in malaria, in
relapsing fever, the spleen has a conspicuous attractiveness
for the microbes ; in tuberculosis it is the lymphatic tissues
and the spleen. In this disease the lymph-cells seem to be
the particular nidus for the growth and multiplication of the
bacilli. It is quite possible that the presence of saprophytes
in the lymph-cells of the superficial parts of the tonsil,
pharynx, and Peyer’s glands (Bizzozero, Ribbert, Ruffer) is
to be explained in this way, viz., that these cells possess a
chemiotactic action, being a more favourable nidus for the
growth of the bacteria.

The conclusion which we think justified in making is that
the phenomenon of mechanical phagocytosis in Metchni-
koff’s original sense is in some cases unquestionably a sign
of weakening and destruction of the microbes, but it cannot

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567

xxi]

be the primary and essential part to which the resistance and
immunity of the tissue or the animalis arc due. This does not
deny the possibility that leucocytes do and can take up
microbes still in a living and even active state, but, what
seems from all that has been said highly improbable, that such
phagocytosis is the first phenomenon in the destruction and
neutralisation of the microbes introduced into a tissue, or
that it is sufficiently extensi ve or sufficiently early to account
for the rapid and complete destruction of the microbes intro-
duced that in some cases is noticeable. This forces us to
assume that spontaneous resistance or immunity is primarily
and essentially due to an inimical action of the blood and
tissue or tissues per se on the microbe, a view which as we
shall see harmonises well not only with the facts concerning
natural immunity, but in a still more marked manner with
acquired, active, or artificially produced immunity.

The first definite proof as to the germicidal power of
blood was given by Fodor ( Deutsche vied. Wochevschrift ,
1887, No. 34), then Nutall (Zeitschrift f Hygiene , t 888, iv.
p. 353), Niessen ( Zeitschr . f. Hygiene , 1889, vi. p. 487),
Behring ( Centralbl. f Klin. Med. 1888, No. 38), and par-
ticularly Buchner ( Centralbl . f. Bakt. u. Parasit., 1889,
vol. v. p. 25, vol. vi. pp. 14, 21 ; Archiv f. Hygiene , 1890,
p. 85), and others have shown that the plasmatic fluids of
the body — lymph and blood — have in their fresh and living
state the power to destroy and kill bacteria brought into
contact with them. The experiments of Buchner, Nutall,
and Niessen have shown that the fresh blood plasma used
in the test-tube has a remarkable power of doing this,
although this power differs considerably as regards different
species. Thus, micrococcus aquatilis, cholera spirillum,
anthrax bacillus, typhoid bacillus, and the bacillus of P'ried-
lander are easily killed after a few minutes (five to twenty

568

MICRO-ORGANISMS AND DISEASE [chap.

minutes), while others, e.g., staphylococcus pyogenes aureus
and albus, streptococcus erysipelatos, bacillus of fowl cholera
and swine fever, and proteus hominis, are only very slightly
affected by it ; on proteus vulgaris, bacillus fluorescens
liquescens, bacillus aquatilis, and bacillus prodigiosus it has
no appreciable effect. But also in the cases where the fresh
blood exerts its inimical action this only takes place if 'the
relative number of bacteria added is limited, for the killing
power of a given quantity of fresh blood is limited, so that
if the number of bacteria introduced be too large the killing
power of the blood does not extend to all bacteria; and
having been consumed and exhausted in killing a certain
number of them, others escape, and these, then, are capable
of rapidly multiplying, as in any other medium. The power
of the blood to kill certain bacteria rests with the plasma,
and it is the same power that also kills the leucocytes.
There is a remarkable parallelism between blood plasma
and leucocytes on the one hand and blood plasma and
bacteria on the other, for when the blood plasma kills the
leucocytes it also kills bacteria, e.g., fresh blood and blood
plasma ; but fresh peptonised blood and peptonised plasma,
which have not this power on the former, have it not on the
latter. When blood is heated to 520 or 58° C. for twenty
to thirty minutes (Nutall) it loses the power of killing
bacteria, which it otherwise killed ; blood mixed with mag-
nesium sulphate loses the killing power ; when blood is
kept for several hours it also loses this power. Blood to
which bacteria had been added and thereby killed coagulates
quicker (Grohmann), just as blood which kills the leucocytes
coagulates quicker.

Buchner ( Centralbl '. f. Bakt. mid Paras//., vi.) has made
very extensive observations on the germicidal power of
blood plasma and blood serum ; he points out an important

xxi]

PATHOGENIC ORGANISMS

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antagonistic action vested in these fluids, on the one hand,
as to their power of being nutritive , and, on the other, as to
being germicidal', the first depends on materials no longer
living, e.g., dissolved or broken-down blood-corpuscles, the
latter on the “ living ” or “ active ” condition of albumen.
Buchner shows that the circulating blood possesses the
germicidal property in a higher degree than blood after
removal from the body : evidently the former contains in a
much smaller degree the particular nutritive elements than
the latter, which of course contains the products of the dead
or broken-down blood-corpuscles. Buchner further shows
that the germicidal power of the blood is not directly
dependent on the leucocytes, and further that it depends on
the albumen present in the plasma or serum, as long as this is
in combination with salt, or, as he terms it, is in an “active ”
state. Plasma or serum free of cells acts germicidally ; if
from it, by dialysis, the salt is removed, it loses its germi-
cidal power ; the salts of the plasma or serum themselves
possess, however, no germicidal power. Lubarsch ( Fort -
schritte d. Medizin , Bd. viii., No. 17) thinks it probable
that the germicidal action and inhibitive power of the living
tissues may in a large measure depend on the chemical activity
of the tissue cells, that is, on chemical substances excreted
or produced by the cells ; hence the battle against bacteria
is essentially of a bio-chemical nature, as has been ably
demonstrated by Petruschki in a series of papers.

The substance or substances to which the plasma, serum,
or tissue iuices owe their germicidal power are called by
Buchner Alexines (d\e£eir, to protect). It must however be
clear that, whatever the exact nature of these alexines, they
cannot be the same, either in all animals or for the different
pathogenic bacteria. The alexines against anthrax in an in-
susceptible animal, e.g., rat, frog, cannot be the same as the

57°

MICRO-ORGANISMS AND DISEASE [chap.

alexines in glanders in an almost insusceptible animal, as
the tame mouse. Nor can the alexines which are present
in the tissues, and which act germicidally on saprophytic
bacteria, be the same as the alexines protective in insuscep-
tible animals against specific bacteria. Again, the alexines
protecting a naturally insusceptible animal against a specific
microbe cannot be the same as the substances protecting
against a second infection a susceptible animal which has
passed through one mild attack ; that is to say, the natural
immunity of an individual cannot be due to the same kind
of protective substance as the acquired immunity.

Moreover, it has been shown that the inhibitory power
possessed by the blood (serum), though it can be greatly
increased and rendered specific against a particular species
of microbes by previous injections of a particular animal
with this microbe (artificial immunisation), may be and
sometimes is already naturally present in the normal
animal : Roux (Annates de P Inst. Pasteur , September, 1894),
for instance, finds the blood-serum of a normal horse pos-
sessed of a certain high degree of resisting or inhibitory
power against diphtheria, Loffler ( Centralbl. f. Bakteriologie ,
February, 1896) finds the blood of a normal dog possessed
of inhibitory power against the typhoid bacillus, Cobbett
( Journal of Pathology and Bacteriology, January, 1896) finds
the blood-serum of a normal horse possessed of a certain
amount of inhibitory power against diphtheria toxin as also
against the living diphtheria bacilli.

The essential and primary element in the resistance or
immunity of a tissue or of an animal against the growth and
multiplication of a microbe is the power of the tissue juices
(plasma, serum, or lymph) to injure or destroy the microbe
by virtue of its alexines, that then the so altered microbes
may be easily taken up by leucocytes (attracted there) and

XXl]

PATHOGENIC ORGANISMS

57i

further broken up and removed — phagocytosis. It is clear

that a compromise between the two views : (a) Mctchnikoffs
of phagocytosis and (/>) Buchner’s of alexines, is easily possible ;
and, Kanthack’s and Hardy’s researches having indicated
such a compromise, it is satisfactory to find that Metchnikoff
himself has already placed himself more in harmony with the
ascertained fact of acquired immunity by suggesting that
the inimical or inhibitory power of the blood (plasma, serum,
and lymph) in acquired immunity is due to the presence in
the blood of substances secreted or elaborated by the tissue
cells. This is in so far a welcome admission as we can
easily extend it to natural immunity by saying that in
insusceptible tissues or an insusceptible animal the alexines,
like other substances, are secretions, or products, or what-
ever we like to call them, of the living tissue cells, and this
would also well harmonise with Kanthack and Hardy’s de-
monstration of a direct process of destruction of anthrax
bacilli by the secretions or the products of living cells
(eosinophile cells). Phagocytes, i.e., cells which actually
are capable of embodying bacteria living, injured, or dead
in the process of the destruction and removal of the
microbes from the insusceptible tissue or insusceptible
body, are in no way opposed to the theory of alexines, since
the alexines themselves are substances produced by, and
freed from, the living cell protoplasm, and it would make
little difference whether the inhibitory or germicidal action
by alexines takes place within the cell protoplasm of some
cells, or by the alexines originally produced by the cells
but now free in the tissue juices.

Acquired or artificial immunity. — The observation that in
some infectious diseases one attack protects against a second
underlies, as stated on a previous page, the whole theory
and practice of protective inoculations, but only within

572 MICRO-ORGANISMS AND DISEASE [chap.

recent years has it been possible to demonstrate experi-
mentally the intimate nature and causes of the immunity
and resistance acquired by a first attack. Salmon and
Theobald Smith, Beumer and Peiper, Ehrlich and Fraenkel,
Roux and Chamberland, Roux and Yersin, showed that an
animal acquires immunity against a particular infection not
only by Pasteur’s method, i.e. by a first infection with miti-
gated or attenuated microbes — by a mild attack — but that
such immunity can be acquired also by previous injection
or injections of the ready-made specific toxins. Behring,1
Behring and Kitasato,2 Roux,3 and others followed this up
by showing by more exact methods (Diphtheria and Tetanus)
that a definite relation exists between the degree of resistance
acquired and the amount and virulence of the infecting
material (both microbes and specific toxin) used for the
antecedent injection or injections ; further that it is possible
to raise this resistance up to more or less complete immunity
by intermittent, repeated, and gradually increasing doses
used in these antecedent injections, allowing the animal time
to recover completely before the next injection.

Starting with a small dose or a mitigated virus —microbe
or toxin — the mitigation being achieved by heat, chemical
reagent, or method of cultivation — the first injections produce
slight reaction, if the animal is possessed at the outset of a
certain spontaneous resistance ; the reaction is greater, large
tumour in subcutaneous injections, constitutional disturbance
in most cases, if the animal is at the outset more susceptible.
The less the initial resistance of the animal or the greater or
more virulent the first dose, i.e. the. greater the reaction on
the part of the animal, the greater as a rule is the resistance

1 Deutsche vied. Woch. No. 49, 1S90.

'l Ibid., No. 50, 1890.

51 Annates de /’ Institut Pasteur, September, 1894.

xxi]

PATHOGENIC ORGANISMS

573

acquired, the sooner the point of acquired immunity is
reached, or in other words the greater or more virulent the
subsequent dose that the animal can bear. By repeated
injections of gradually increasing doses a high degree of
resistance is ultimately reached. While by the method of
conferring this acquired or active immunity against a specific
disease, used by Behring, Behring and Kitasato, Klemperer,
Roux, R. Pfeiffer, and others, the animal is allowed to recover
from the previous injection before a further injection of the
increased dose is administered, Loffier shows ( Centralblatt
f Bakt. und Parasit., February, 1896) that as regards the
typhoid bacillus, by injections of small doses of virulent
bacilli administered in very short intervals of a few hours,
immunity can be acquired already in a few days.

The fact that a specific immunity can be thus acquired
by injections of specific toxin, that is to say, by the repeated
injections of the pure toxin elaborated by a particular
microbe, and separated from the latter by filtration, e.g.
diphtheria toxin, tetanus toxin, typhoid toxin, &c., proves
conclusively that the condition of this immunity cannot
be due to a phagocytic action of the leucocytes ; no
microbes being used for the injections, there are no
microbes to be swallowed up and destroyed.

Now, the most striking fact that was first demonstrated
by Behring and his co-workers is this : the blood or blood-
serum of an animal actively immunised, or, generally speak-
ing, of the animal body which has acquired immunity in one
way or another against a particular infectious disease, pos-
sesses a definite and measurable power to confer immunity,
passive immunity , against that particular disease if injected
into a normal animal ; more than that : it is capable of
modifying or even completely neutralising — curing — the

574

MICRO-ORGANISMS AND DISEASE [chap.

effect of an already-established infection in a normal (unpre-
pared) animal.

This immunising and curative power of the blood-serum
of the actively immunised animal body is commensurate with
the degree of immunisation, so that the blood-serum of an
animal in which the active immunisation has been carried
to a high degree has itself measurably greater immuni-
sing and curative power than the blood-serum of an
animal not immunised to the same degree (Diphtheria,
Tetanus).

Behring uses as the standard for measuring (in diphtheria)
this potency of the blood-serum by taking as unit the
amount of serum required to completely neutralise a dose of
pure toxin that would produce death in ten guinea-pigs
each of about 300 grams weight in thirty to thirty-six hours.
As was mentioned in the chapter on Diphtheria, Roux and
Yersin, who first separated the diphtheria toxin elaborated
by the diphtheria bacilli in broth cultures, showed that the
injection into the subcutaneous tissue of the guinea-pig of
a fatal dose of this pure toxin produces the same tumour at
the seat of injection and the same subsequent symptoms
and death of the animal as does the injection of the active
and living diphtheria bacilli. Behring^s unit of potency of
diphtheria serum is the amount of serum required to inject —
antecedently or simultaneously, or shortly after — in order to
neutralise, i.e. prevent from producing tumour, disease, and
death, a tenfold fatal dose of toxin in a guinea-pig of 300
grams weight. This measure of the serum is then its
antitoxic potency. Behring has carried the active immuni-
sation against diphtheria of animals : sheep, goat, to
such a high degree that the (diphtheria) antitoxic
potency of the serum of these animals reaches the high

PATHOGENIC ORGANISMS

575

XX l]

figure of about 90, 130, and even 200 units, or, more
accurately stated, 7 5 cc. of serum possess 600, 1,000, or
1,500 antitoxic units respectively. Roux submits horses —
as a rule not possessed of great susceptibility for diphtheria at
the outset — to repeated injections with pure and powerful
diphtheria toxin, starting by injecting subcutaneously small
doses of attenuated toxin, then gradually increasing the
dose of the pure toxin, till after many injections the intra-
vascular injection of enormous doses — 250 cc. — of the
most powerful toxin do not produce more than a transitory
result. After three months’ immunisation he obtains an
antitoxic serum which is possessed of great potency : one
cubic centimetre being capable of neutralising a fatal dose
of pure toxin for 10,000, 20,000, 50,000, 100,000 and even
200,000 grams guinea-pig, or, put differently, T^,

30^, and even of a cubic centimetre of the antitoxic
serum can completely neutralise the effect of a fatal dose of
toxin injected into a guinea-pig of 200 grams weight. But
while this serum possesses the high antitoxic power, i.e. the
neutralising power of toxin, both when injected into the
unprepared animal shortly before or simultaneously with,
or some hours — six or even twelve hours — after the toxin,
its germicidal potency, i.e. its action against the living
microbes (Diphtheria bacilli), is considerably less, though
it is for a time at least considerable. Thus, for instance, of
Behring’s diphtheria antitoxic serum, marked 600 units, ^
of a cubic centimetre is required to completely neutralise
for a guinea-pig of 500 grams weight a fatal dose of living
culture of the diphtheria bacilli, Tj of a cc. does not pre-
vent the formation of a tumour, although the animal does
not die, but recovers after some days, of a cc. neither
prevents the formation of a tumour nor the fatal issue.

I have succeeded in obtaining serum of considerable anti-

576

MICRO-ORGANISMS AND DISEASE [chap.

toxic and immunising power by subjecting the horse to
repeated injections with large doses of living diphtheria
bacilli; in one horse already after three weeks, in another
after four weeks, the serum had the same immunising power
as in horses done after Roux’s method with pure toxin for a
considerably longer period.

The immunising power of antitoxic diphtheria serum,
i.e. the power of the serum when injected into a normal
guinea-pig to protect the animal against subsequent in-
fection with the diphtheria bacilli, is only of a temporary
character, being of short duration, generally from a few
days to a few weeks, and depends on the amount of serum
injected. The immunising action of the injection into a
guinea-pig of a subfatal dose of living culture of bacillus
diphtherias is of considerably longer duration, but it must
be added that in that of the bacillus of diphtheria, un-
like with some other microbes — anthrax, cholera, typhoid,
colon, &c. — the resistance of the guinea-pig against new
and further infection is comparatively limited.

It is clear from the facts above recorded that during the
process of “ active immunisation ,” as first practised by
Behring and Kitasato, Behring, Roux, and others, the blood
(and blood-serum) of the immunised animal contains sub-
stances, antitoxins or antibodies , as a result of the antecedent
injections of toxin. Of what nature are these bodies ? Are
they a result of the activity of the cells and tissues, a re-
active secretion of new substances (ferment) by the cells
in consequence of successful and effective stimulation by
the toxin (Roux), or are they the original toxin modified
and chemically altered by the tissue cells (Buchner) ? Are
they of the nature of albumins like the toxalbumins, or are
they bodies more resembling ferments ?

As regards the diphtheria antitoxins Aronson’s mode of

XX.]

PATHOGENIC ORGANISMS

577

separating them and obtaining them in a concentrated form
from the serum of immunised animals suggests that they are
of the nature of ferments like the diphtheria toxin itself of
Roux and Yersin, the diphtheria antitoxin also in other
ways comporting itself like a ferment, e.g. the diminution
and final destruction of its potency by heat of 65 to 70° C.

One of the most remarkable results of immunisation
against particular toxins was achieved by Ehrlich1 with
Ricin and Abrin. By feeding animals with one or the
other of these poisons he was able to gradually im-
munise them, and just as in Behring’s experiments was
able to achieve a high degree of immunity ; moreover the
blood of these animals possessed antitoxic (antiricin or
antiabrin respectively) potency (immunising and curative)
commensurate with and proportionate to the amounts of
the antecedent toxins used for the immunisation.

And last but not least Sc-wall,2 having shown that
immunity against rattlesnake poison can be conferred on an
animal by antecedent subfatal doses of this poison,
Calmette3 was able to produce antitoxic serum (in the
rabbit) by immunising with repeated injections of at first
small subfatal and gradually increasing doses of snake venom
so much so that the serum of highly immunised animals is
capable of conferring protection or passive immunity and
even exert curative action against snake venom in normal
unprepared animals. Fraser4 has confirmed these obser-
vations.

We may take it then as a general law that an animal can

1 Deutsche med. Woch., 1891, Nos. 32 and 44.

2 Journal of Physiology, 1887, p. 203.

3 Annates de I’lnstitul Pasteur , May 1894, April 1895.

4 Royal Society of Edinburgh, June 3 and July 15, 1895.

P P

578

MICRO-ORGANISMS AND DISEASE [chap.

be immunised against a particular toxin, and that its blood
and blood-serum thereby acquire a proportionate specific
antitoxic potency.

Antitoxic diphtheritic blood-serum does not act anti-
microbically or germicidally in vitro , for, as Wright has
shown, antitoxic blood-serum of a diphtheria-immunised
horse forms a good artificial medium for the growth of
the diphtheria bacilli.

This production of acquired or active immunity by
toxin, is apparently not the same as is created in the animal
body under natural conditions, that is when the animal body
acquires immunity against a particular infectious disease by
a previous attack of the disease. In the natural condition
when the animal body is subject to an attack the specific
microbe, having found entrance, lives and multiplies within
the infected body and causes the particular disease, and
after the body recovers and the microbes again disappear it
is found some time afterwards that it has acquired the
power to resist a new infection with the microbe, or if this
be injected in an otherwise sufficient dose the animal and
its tissues resist it, the microbes cannot now live in such a
tissue or such an animal body, they degenerate and die
and produce no disease. Evidently during the first attack
something was formed in the animal which after the disease
has passed off is present in the blood and tissues and which
acts inimically, germicidally against the same microbe. This
germicidal substance does not appear immediately on re-
covery (in Fraenkel’s experiments on diphtheria in the
guinea-pig it requires two to three weeks for its appearance) ;
the same holds good for pneumonia, for cholera, typhoid,
and others ; further this germicidal or immunising action of
the blood and tissues does not in all cases last for an

XXI]

PATHOGENIC ORGANISMS

579

indefinite time, in some, e.g. variola, scarlet fever, and the
acute exanthemata in general, it seems as a rule to persist
for lifetime, in others, e.g., erysipelas, diphtheria, it is of
a more limited duration. We said above that this naturally
acquired immunity is apparently not of the same character as
that producible by repeated toxin injections, but in reality it
may be the same, since also in the naturally acquired
immunity against a particular infectious disease by a
previous attack this attack is caused by the toxin elabor-
ated by the microbe in the infected body, so that after all
the difference in the two methods is merely this, that in the
one, the Behring’s method, the toxins are prepared outside
the animal body in artificial cultures, while in the other, i.e.
the immunity acquired under natural conditions or by
Pasteur’s method of protective inoculations, the toxins are
elaborated by the microbe within the infected animal
body.

But is there really no difference between the immunity
acquired in the two methods ? We have already indicated
that the antitoxic power of diphtheria serum prepared after
Behring or Roux by toxin injections is incomparably greater
than the immunising, or germicidal, or antimicrobic power,
and it can be further shown that while an animal can by re-
peated injections of dead bacterial bodies be well immunised
and protected against an otherwise fatal dose of the same
bacterial bodies in a living state it is not protected against
an otherwise fatal dose of the specific toxin. A guinea-pig
is repeatedly intraperitoneally or subcutaneously injected
with dead cholera vibrios or dead vibrios of Finkler, bacillus
coli or bacillus of typhoid, bacillus prodigiosus or proteus
(scraped from the slanting surface of an active Agar culture,
then distributed in sterile bouillon and finally thoroughly

p p 2

580 MICRO-ORGANISMS AND DISEASE [chap.

sterilised by being heated to 60-70° C. for ten or twelve
minutes). The dose for subcutaneous must be larger than
for intraperitoneal injection ; the dose is at first subfatal, but
sufficient to produce distinct illness, then after a week or
ten days a second injection is made with a larger dose, then
a third, a fourth, a fifth, and a sixth injection, till no reaction
at all follows, If ultimately, a fortnight after the last injection,
such a prepared animal be tested with a dose of living
microbes more than sufficient to kill an unprepared control
guinea-pig, it will be found that the prepared animal shows
no reaction whatever, and that the living microbe very soon
after its injection degenerates and disappears.

From these and similar experiments as also from experi-
ments such as immunisation of guinea-pigs by intraperitoneal
repeated injections with diphtheria bacilli, it seems feasible
to assume that the immunising or germicidal or antimicrobic
potency of the blood-serum in naturally acquired immunity
as also in immunity produced by injection of living or dead
microbes owes its origin principally or in part to substances
derived from the bacterial bodies.

Now, in the case of the vibrio choleive or of vibrio Finkler,
by cultivating them in solidified blood-serum, which is
liquefied by the growth, it will be found that after some
weeks’ growth at 37° C. a powerful toxin is produced in
these cultures, which when used free of the living bacilli (or
after sterilisation by heat) affects and kills guinea-pigs pre-
viously immunised by dead vibrios against living cultures in
the same way and to the same degree as unprepared
animals.

Acquired or artificial immunity against a specific toxin or
a specific microbe may be limited to a single tissue or it
may involve the whole body, thus Cobbett and Melsome

PATHOGENIC ORGANISM S

58.

XXlJ

show ( Journal of Path, and Bad., November, 1894) that
only a local immunity against the streptococcus of erysipelas
or its toxin is produced in the rabbit in one ear previously
the seat of erysipelas, and further that also in the process of
immunisation of horses against diphtheria the region of a
former inoculation acquires resistance against the new dose,
whereas a new region of the skin is more suitable for the
purpose (Cobbett, Journal of Path, and Bait., January,
1896).

Again, the peritoneum of a guinea-pig may be immunised
against the living or dead cholera vibrio by repeated pre-
vious intraperitoneal injections of cholera vibrios, without
its alimentary canal being immunised against the growth and
multiplication of the cholera vibrio (R. Pfeiffer and VVasser-
mann, Klein). Koch and Gaffky showed that sheep
successfully vaccinated after Pasteur’s method of subcuta-
neous protective inoculation are still subject to anthrax by
ingestion of spores.

R. Pfeiffer in a series of publications (already referred to
in the chapter on Cholera) has demonstrated that by re-
peated intraperitoneal injections of guinea-pigs with living
cholera vibrios, at first in small non-fatal, then gradually
rising doses, the blood and blood-serum of the animal, as the
immunity becomes greater and greater, possesses higher and
higher germicidal or immunising potency against cholera
vibrio : the higher the degree of immunisation the greater
the germicidal power of the blood-serum. When a definite
quantity of this “ cholera serum ” is mixed with a definite
otherwise fatal quantity of living cholera vibrios and injected
into an unprepared animal no result follows, the animal
survives and remains well ; already after a short time, in
twenty minutes or so, after injection the vibrios degenerate

582

MICRO-ORGANISMS AND DISEASE [chap.

and break up into globules and granules. This germicidal
action of the cholera serum in corporc has been already
spoken of as Pfeiffer's test , it does not take place in vitro.
The same law holds good more or less for other microbes :
erysipelas, typhoid, Finkler, colon, prodigiosus, so that it
seems to possess general application, but it must be added
that it is by no means so absolute as is represented by
Pfeiffer.

Bordet and Durham {loc. cit ) show that a “ potent serum ”
acts specifically ( specialised , Durham) on its particular
microbe or races of microbes also in vitro , inasmuch as in
definite quantity and definite time a potent serum causes a
more or less perfect separation, aggregation, and precipita-
tion and loss of motility of the microbe contained in a
suspension, without however destroying the microbe, for
even long after the microbes have separated active cultures
can still be produced with them.

The germicidal action of the serum as shown by Pfeiffer’s
test is on the whole but not without exception specific,
that is to say it is only exerted against the microbe with
which the animal had been actively immunised. As
is now well known an animal, say a guinea-pig, can be
protected intraperitoneally against a fatal dose of the living
microbe, e.g. vibrio of cholera, by previous repeated intra-
peritoneal injections of the living or of the dead microbes
(Klein), but this immunisation is of a. comparatively tem-
porary nature, and does not yield specific germicidal serum
unless often repeated and with considerable doses.

A certain resistance, non-specific in nature, of the tissues
against microbic action has been produced in various ways :
thus Wooldridge showed that the injection of thymus extract

xxi]

PATHOGENIC ORGANISMS

533

may protect rabbits against anthrax ; Kossel, Vaughan,
McClintock, produced a refractory condition to microbe
infection by the administration of nuclein and nucleinic
acid.

A very transitory local immunity of the peritoneum of the
guinea-pig has been produced by Pfeiffer and Issaeff ( Archiv
f. Hygiene, vol. xvi. part 2) by intraperitoneal injection of
normal serum, saline solution, nucleo-albumin and other
substances.

INDEX

Abscess, chronic, the microbe of, 144
Aceti, mycoderma, 474
Achorion Schoenleini , 479
Acid fermenation by bacterium aceti and
mycoderma aceti, 125
Actinomycosis : or ray fungus, 486
disease of, 488
granules of 491, 495
nodules of, 492.
clubs. 493

rErobic bacteria, 89
rErogenes capsulatus, 235
Agar-Agar :

nutrient nature of, 35
grape sugar, 36
glycerine, 37
slanting tubes, 44

degrees of virulence of cholera cultures
of, 429

Air :

contamination, how to avoid, 63
examination of, 83
Albumose, how formed, 130
Alcoholic fermentations, 125, 473
Alkali albumen, coagulation of, 196
Alkaloids :

cholin, 129
neurin, 129

cadaveriii-formation of, 129
Ammonium carbonate, formation of, 125
Amoeba coli, 502
sporidia, 515

Amylobacter, bacillus of, 391
Anaerobic :

cultivation, methods of, 86
bacteria, 89
bacilli, 369 — 403
Aniline dyes:

importance of, 9

list of the most useful in the examina-
tion of animal tissues, 1 3
oil for preparing dyes, 13
watery solution of. 12
animalculi found in London waters, 81

Animals, food :

tuberculous disease among, 358
effects of food derived from, 358
in cattle and swine, 358
Antagonism amongst bacteria, 527
Antheridia (fungi), 486
Anthracis, bacillus of, 271
Anthrax :

microbes of malignant, 27 1 et seq.
spores of. 283
rag-sorters’, 292
Arthro spores in bacteria, 109
Ascococcus microbes, 140
Ascogonium, nature of, 480
Ascomycetes, an order of fungi, 477
Ascospore, the mother-cell, 472
Aspergillus, fungi, 480 :
glaucus, 482
flavescens, 482
fumigatus, 481
niger, 482

Asexual and sexual spore- formation, 480

Aurens, vibrio, 409

Autoclave, description and use of, 4 1

Baciu.us :

typhoid fever, 23, 235
vibrio cholera: Asiatics, 23
in water, 75
coli, 188, 224

-coli in London waters, 80
radicicola, 91
termophilus, 95
tetanus, 121 et seq., 181,
enteritidis sporangenes, 121 ct seq.
butyricus, 121 et seq.
of Friedlander, 157
leptothrix of, 164
subtilis of, 165, 178
vacuoles of, 166
leptothrix filaments, 168
buccalis, 170
megaterium, 170

586

INDEX

Bacillus :

segregation of the protoplasm of, 177
torula-like chains of, 177
club-shaped terminals of, 177
germinationof spores of in hay infusion,
188

mesentericus, 182
proteus vulgaris, 75, 182
fiuorescens liquescens, 187
colonies of typical, 190
clots and solidifies milk, 193
filamentosus, 199
prodigiosus, 200
pyocyanetts, 200
steptothrix, 201
Foersteri. 201
cladothrix dichotoma, 201
beggiatoa, 203
pathogenic, 204, 535— 583
Davaine septicaemia, 205
fowl cholera, 208
enteritis, 214
grouse disease, 215
swine fever, 217
wildseuche, 222

oriental, or bubonic, plague, 224
Texas fever, 223
typbi murium, 224
aerobic of malignant oedema, 229
beef-pie (Portsmouth), 229
choleraic diarrhoea, 230
gas-forming atrobic, 233
aerogenes capsulatus, 235
gasoformans, 235
faecalis alkaligenes, 247
erysipelas, swine. 251
Egyptian ophthalmia, 253
septicaemia in man, 256
in mouse, 248
Pasteur’s, 378
influenza, 256, 259
anthracis, 271

Buchner's experiments with, 538,
539

ulcerative stomatitis (calf ), 292, 294
diphtheria, 296
pseudo, 300
cultures of, 307

glanders, called mallei, 324, 329

syphilis, 330

foulbrood, 331

rhinosclerma, 331

tuberculosis, 333

tubercle, 346

lepra:, 362

tetani, 372, 378, 379. 380
charbon, symptomatic, 373, 383, 384
cedematis maligni (Koch’s), 375 — 378
“ drumstick,” 379

enteriiidis sporogenes, 371, 389, 397
amylobacter, 391,
variolte-vaccinse, 398
calf lymph 398 — 400

Bacillus :

jequirity, Saltier’s researches regard-
r, ing, 540, 547
Bacteria :

staining and treating, methods of, 7 et
scq.

ingredients adapted for, 10—15
Eherlich’s method for tubercle and
leprosy, 17
Gram’s, 16
Koch’s, 17
Lustgarten’s, 17
examination of air for, 83
of ice for, 85
of milk for, 85
of soil for, 85

methods of studying in the living state,
66

of anaerobic cultivation, 86
pyrogallic acid, use in, 87
characters, general of, 88 — 121
composition of, 88
two kinds — aerobic ; anaerobic, 89
phosphorescent, 93
spores, vitality of, 95
growth and division of, 97
multiplication, rapidity of, 97
division, mode of, 101
spores of, formation of, 103, 105, 106, 107
endo-spores in, 108
arthro-spores in, 109
tubercle-spores in, no
germination spores in, 112
motility of, 113

Brownian, molecular movement in,ii3
“swarming” of, 115
flagella, uses of to, 117
chemistry of, 122, 123
chemical changes wrought by, 122
nutritive gelatine, power of to pepto-
nise, 122

whey, neutral, they produce acid or
alkali in, 123

litmus tincture in staining, 123
gas. formation of, by, 125
fermentations, various specific, pro-
duced by, 125

pigments, power to produce, 126 — 128
researches on, 535

phosphorescent, power to become, 129
putrefaction, power to produce, 129
ptomaines, power to form, 129
evolution of, 176
vibriones, 404
antagonism amongst, 527
water, 527

micrococcus aquatilis, 527
cry throsporus, 527

faical matter, influence on various
species of, 529
action on, of leucocytes, 564
Bacteridic du charbon , 271
Bacterioscopic examination of water, 67

INDEX

587

Bacterium :

photometricum, a, 89
desmo, of Cohn, 164
Balsam, Canada, solution, 10
Basidia (fungi) nature of, 480
Beef-pie, bacillus of (Portsmouth), 229
Bcggiatoa, 203

Berkefeld pressure-filter, use of, 77
Bismarck-brown, 13

watery solution of, 14
Blastomycetes, an order of fungi, 471
Blcnorrhcea, acute, 254
Blood serum :

Koch’s, 30

anti-toxic power of, 384
germicidal power of, 568 — 571
Blood, typhoid bacillus in, 241
Blue-methyl, 13
Bordet -Durham test, 458
Bouillon Malleln, 329
Bovine tuberculosis, 339
giant cell in, 342

diagnostic value of Koch’s tubercu-
linum in, 361
Broth :

meat, 27
nutrient, 29
glycerine, 30
phenolated, 78, 197
Brown, Bismarck, 13

Brownian molecular movement of bacteria,
112

Buchners fluid, composition of, 30

his experiments with bacillus anthracis,

„ .538.539

Butyric acid, formation of, 125
Butyricus bacillus, 121, 181, 397

Cadaverin, 129
Calf, the:

ulcerative stomatitis in, 292
lymph, 398, 399
bacilli of, 400

Cambridge rocker for cutting ribbon-sec-
tions from paraffin-embedded ma-
terial, 18, 19

Canada-balsain, solution, 10

Cancer parasites, 509

Capillary glass pipette, how used, 42

Carbol-fusin, prepared after Ziehl, 14

Carpogoniuin in fungi, 480

Caseous tubercle, in the guinea-pig, 336

Catarrhal conjunctivitis, 253

Cathcart's microtome, 18

Cats, throat illness of, 312

Cellulose, nature of, 88

Cerebro-spinal meningitis, the microbe of,

Charbon, Symptomatic, bacillus of, 373,
384. 385

Cbeuiiotaxis, phenomena of, 564 ct seq.

Cholera :

vibrios in water, how to detect, 82
in fowl, 208, 209

experiments by ingestion of, 443

in duck, 21 1

English, 228

Asiatic, 410

Koch’s vibrios in, 416

stools, 416

red reaction, 418

comma bacilli of, 420

power of serum of, 435

toxin, 436

microbe of, 446

protective inoculations of vaccines
against, 448

vaccinated persons, statistics of, 449
sporadic, 452
nostras, 452
serum of, 460

Choleraic diarrhoea, bacillus of, 230
Cholerine, epidemic at Lisbon, 457
Cholin, 129

Circomonas intestinalis hominis, 504

Citoryctes, 402

Cornalia’s disease, 161

Corymbifer (fungi), 484

Cows, eruptive disease of milch, 151, 317

Cocco-diphtheria, 303

Croup, fibrinous, 300

Croupous pneumonia, microbe of, 154

Cladothrix dichotoma, 201

Club-shaped terminals, 177

Coccidia :

in the epithelium, 401
Miescher’s, 507
Coccidium oviforme, 505
Cohn’s fluid, 31
Coli, amoeba, 502

bacillus in London waters, 80
communis :
bacillus, 188

forms typical colonies, 190, 224
Comma bacillus :
or vibriones, 404
Koch’s. 410
varieties of, 417
Asiatic cholera, 420, 421
colonies of cholera, 421
stab-culture, 423

experiments with the cultivations 0^437
Commas, different varieties of, 417
Condensor, substage, use of a, 7
Conidia spores (fungi), 477
Contagia, fixed, group of, 315
Con tag in m vibum , doctrine of, 4
Contrast dyes, 15
Copper ovens, their uses, 44
Cultivations :

of tubercle bacilli, 347
staining of, 348
definite characters of in, 352
spores in, 355

588

INDEX

Culture media :

for inoculation, 45 — 52
test tubes most suitable for, 45
india-rubber caps and gutta-percha
paper caps, use of in, 52
MATERIAL :

preparation of, 24 et scq.
fluids : 27

nourishing material, 27
broth, 27

flasks for Containing media, peptone
and salt solution, 29
nutrient broth, 29
glycerine broth, 30
blood serum, 30
Buchner’s, 30
hydrocele, 30
ascites, 30
milk whey, 31
Pasteur’s, 31
Cohn's, 31
solids :

boiled potato, 32
white of egg, 32
paste, 32

blocks of potato, 32
gelatine, 32
nutrient gelatine, 33
solidified blood serum, or hydrocele
fluid, 34

solidified ascites, 34
fluid and Agar-Agar, 34
Lofller’s serum with condensation
water, 34

Kanthack's serum, 35
nutrient, Agar-Agar, 35
grape-sugar gelatine and grape-
sugar Agar, 36
glycerine Agar, 37

VESSELS AND INSTRUMENTS USED
IN, 38—44

Fletcher’s burner, plugged with
sterile cotton-wool, 38, 40
cotton wool, uses of, 40
stab of comma bacilli, 423
degrees of virulence of the cholera,

„ , 429

Cultures :

fixing of, 64
hanging drop, 65
of bacillus :
diphtheria, 307 ,
leprosy, 364
oedema, malignant, 376
enteritidis sporogenes, 371
tetani, 372
comma, 437

cholera vibrios, experiments by in-
gestion, 445

“ exalted ’’ virulence. 447
Pasteur’s attenuated, inutility of,
33°. 53i

attenuated, results of, 551

Barrier's disease, 509
Davaine septicaemia bacillus, 205
Decolourising re-agents, uses of, 14
Decomposition, proteid, effects of, 2
De Giacomi methods of dealing with
syphilis material, 18
Desmobacterium of Cohn, 164
Deuxieme vaccine, 290
Dextrose fermentation, 125
Diarrhoea :

bacillus of, 121

epidemic of, at St Bartholomew’s
Hospital, 380
choleraic, bacillus of, 230
Diphlococcus, pneumoniae, 154
Diphtheria :
faucial, 162
pseudo, 152
cocco, 152, 303
bacillus of, 296
membraneous, 296, 297
cultures of bacillus of. 307
toxin, 311

serum, Vehring's experiments with, 574
Diphtheritic and necrotic deposits in fowls,
3.23

Disseminated, tuberculosis, how produced,
333

Double-staining, best methods of, 15
“ Drumstick ’’ bacilli, 379
Duck cholera, 21 1
Dumb-bells, or diphlococcus, 136
Dyes, aniline :

importance of, 9

list of most useful in the examination
of animal tissues, 13
Dysentery :

tropical, 502
amccba;, 502

Ehrlich’s method fordemonstratingtuber-
cle-bacilli and leprosy-bacilli, 17
Egyptian ophthalmia, bacillus of, 253, 254
Emphysema, progressive gangrenous, 37S
Encysted nucleated epithelial cells, 524
Endocarditis, ulcerative, the microbe of,i47
Endo spores in bacteria, 108
Endoglobular form ofplasmodium malaria;

English cholera, 228, 432
Enteritis :
fowl, 211
microbe of, 212
bacillus of, 214

Enteritidis sporogenes, culture of bacillus
of, 371, 389

Eosin, alcoholic solution of, 14
Epidemic :

diarrhoea bacilli, 121
Middlesbrough, bacillus of, 156, 226
Lisbon, vibrio in cholerine. 456

INDEX

589

Epithelioma, 509
Epithelium, coccidia in the, 401
Eruption of papules and vesicles in a
cow, 317, 321

“ Exalted virulence,” cultures of, 447
F

Facultative anxrobic and icrobic bac-
teria, 90

Fxcal matter, influence of on various
species of bacteria, 529
Favus herpes tonsurans, 479
Fermentations :

power of bacteria to form various
specific, 123
poisonous, 130

Fever, relapsing, spirillum Obermeyeri of,
466

Fibrinous croup, 300
rhinitis, 300
Film specimens, 11
Filter, hot water, a, 42
Fixed contagia, group of, 315
Finkler- Prior, vibrio of, 452
Flagella :

demonstration of with aid of tannin
and ferro sulphate solution, 21
spirilla-like, 21

staining of, with osmic acid, acetate
of soda and potassium bichromate,
22

uses of to bacteria, 117
possessed by typhoid bacillus, 242
of cholera vibrios, 4x8
Flagellate motiadime, 504
Flasks for fluid media, 28
F'lavus, vibrio, 409
Flavescens, vibrio. 409
Fluorescens liquescens, bacillus, 187
Food animals:

tuherculous disease among, 358
effect- of food derived from, 358
in cattle and swine, 358
Foot and mouth disease, microbe of, 150
Formalin, use of the fumes of, 65
Foulbrood, bacillus of, 331
Fouls :

dipthcr.tic and necrotic deposits in,
322

natural tuberculosis in, 338
cholera, 209
enteritis, 21 1

epithelioma contagiosum of, 508
Fresh specimens, importance of, 8
Frettchenseuche, disease of, 21 1
Friedlander's bacillus, 157
Fuchsin. 73
bodies, 522
Fungi :

veast, 471 — 476
torula, 471
blastomycetes, 471

gemination of 472
ascospores of, 472
torula cerevisiaj, 473
saccharomyces, 471
cerevisim, 473
vini, 473
pastorianus, 473
mycoderma, 473
ntycoderma vini, 473
aceti, 474

Sidiuin albicans, 474
thrush, 475
mould, 477—497

hyphomycetes, or mycelial, 477

hyphae, 477

thallus, 477

mycelium, 477

ascomycetes, 477

conidia, 477

sporangia, 477

oidium lactis, 478

favus, 479

herpes tonsurans, 479
pityriasis versicolor, 479
Achorion schoenleini. 479
Trichophyton tonsurans, 479
nticrosporon furfur, 479
Aspergillus, 480

glaucus, 480, 482
flavescens, 480, 482
niger, 482
basidia, 480
candidus, 480
fumigatus, 480, 482
spore formation — asexual, sexual,
480

carpogonium, 480
pollinodia, 480
ascogonium, 480
perithecium, 482
pneu mono-mycosis, 484
pencillium, 484
phycomycetes, 484
mucor, 484
corymbifer, 484
rhizopodiformis, 484
“ mycosis mucorina,” 484
saprolegnia, 485
zoosporangia, 485, 486
oogonium, 486
antheridia, 486
oospores, 486
salmon disease, 4S6
actinomyces, or ray fungus, 48S
actinomycosis. 488
wooden tongue, 488
granules, actinomyces, 492, 49;
nodules, ,, 492

clubs, „ 493

mycelial branched threads, 495
mycetoma- madura disease, 497
varieties of, 497

590

INDEX

Gangrene, surgical, 378
emphysematous, 386
Gas-forming rerobic, bacillus of, 233
Gasoformans, 235
Gelatine phenolated :
uses of, 78

power of bacteria to neutralise nutri-
tive, 122

batillus coli grows well in, 197
Gemmation, nature of, 472
Gentian-violet, 13

aniline water of, 14
Giant cells :

in tubercular deposits, 339
in bovine pulmonary tubercle, 342
Guinea-pig :

power of blood-serum in an actively
immunised, 435
passive immunity, 436
Glanders, bacillus of, 324, 328, 329
Glands, typhoid bacillus in, 241
Glass cell, use of, 66
Gonococcus, 161
Gonorrhoea, micrococcus of, 161
Gottstein’s method of dealing with
• syphilis material with the aid of
. liquor ferri, 18

Gram’s method for staining bacteria 16
“ Grapes, the,” the disease called, 339
Grawitz, researches of, 479 and «.
Gregarina forms, 520
Grouse disease, 214
bacillus of, 215
Gum mucilage, use- of, 19

Hay infusion, germination of spores in,i8i
Hatmoplasmodium, malaria, 498
Hmmatozoon, 505
Hearson's incubator, 26
Hepitisation, red, of the lung, microbe of,
155 • „

Herpes, favus, tonsurans, due to a fungus,
479

Herpetomonas Lewtsit, 504
Hide sorters’ disease, 276
Hog cholera, 217

Horse, pharyngeal abscess in the, strepto-
coccus of, 153
Humboldt’s red dye, 13
Hydrocele fluid, Koch's, 30
Hyphae, or threads of fungi, 477
Hyphomycetes, or mycelial fungi, 477

Ice, examination of, 85
Immersion, use of oil, 7
Incubators, for preparation of culture-
material, 24 — 27

Indol, how formed, 193
reaction, J94

Infection caused by toxins, 130
I n halation, tuberculosis produced in animals
]>y> 338

Influenza, bacillus of, 256, 259
its culture in broth, 261
Injection, intraperitoneal of vibrios, 433
Inoculations :

methods of, 53 — 87
plate cultivation for isolation in, 57
JPetri’s dishes, use of in, 57 «.
moist chamber, use of in, 58
stab culture, 54
streak culture, 54

fractional cultivation and dilution,
methods of in, 55
pure sub-cultures, how to start, «p
test-tube plate cultivation, use of in, 61
with blood juices and tissues, 61
air-contamination to be avoided in, 63
fixing of cultures, 64
formalin, use of in, 65
hanging drop cultures, 66
glass cell, use of in, 66
studying bacteria in the living state,
importance of in, 66
bacterioscopic examination of water
in, 67

glass pipettes use of in, 69 n.
number of microbes in water in, 68
character of the microbes in, 74
bacillus coli and proteus vulgaris in
water in, 75

sewage pollution of water in, 76
Berkefeld or Pasteur pressure-filter,
use of in, 77

Parietti’s method in phenolated gela-
tine or broth, use of in, 78
protective, 389

of vaccines against cholera, 448
of rabbits by vaccinia, 402
parasite produced by, 402
Instruments and vessels used for cultiva-
tions, 38 — 44
Iris, tubercles in the, 345

Jequirity bacillus, Sattler’s researches
regarding the, 540—547

Kanthack’s serum, composition of, 35
K la t sc lip rcrla ra tc of the Germans, 12
Koch:

his method of staining bacilli with the
aid of carbonate of potash, 17
his hydrocele fluid and blood-serum, 30
his gelatine, 32
his malignant oedema, 96

INDEX

59i

Koch :

his tuberculinum, 361

his cedematis inaligni bacillus, 375

comma bacillus of, 410 it seq.

Lactic acid, formed by bacterium lactis,
. ,2S .

Lactis, oTdium, 478
I.epr:e, bacillus of, 362
Leprosy :

Virchow’s cells, 362
nodule 363
bacilli, 364
Leptothrix :

filaments of bacillus, 165, 168
buccal is, 170
sheath of, 170

Lcucocytosis, phenomenon of, 559 et sea.
Lewisii herpetomonas, 504
Liquor potassx, use of, 13
Litmus tincture for staining bacteria, 123
Ld filer :

his methyl-blue, 14

his serum for cultivation of diphtheria
bacillus, 34
London waters :

bacillus coli found in, 80
animalculi found in, 81
Lustgarten's method of demonstrating the
syphilis-bacilli with the aid of per-
manganate of potash, 1 7
Lymph :

calf and vaccine, 398, 399
microbes in, 399

Magenta, 13
Malaria, plasmodium, 498
Mallein, bouillon and dry, 329
Mallic acid, how formed, 124
Mannit fermentation, 125
Massowah. vibrio, the, 462
Meat poisoning, choleraic diarrhoea from,
230

Megaterium, bacillus, 170
Membrane :
pseudo, 296
diphtheritic, 296, 300
Membraneous diphtheria, 297
Meningitis, cerebro spinal, microbe, of, 154
Mesenterica tabes in children, 344
MetchnikotTs theory of phagocytosis, 562
Metchnikovi vibrio, 464
Methan gas, formation of, 123
Methods of inoculation, 53 — 87
Methyl :
blue, 13
Loftier s, 14
violet, 13

M ice :

micrococcus of progressive necrosis
and pyaemite in, 158, 160
septicxmia in, 248
Microbes :

anthrax, malignant, 271, et seq.
cholera, 446

fermentations of various and specific,
125

lymph vaccine, 399
nitrifying, 90

poisons in, intracellular, 132
typhoid, 241

specific or pathogenic, 547 et seq.
spontaneous or natural immunity from
. 557.. 558

Micrococci :

ascococcus, 140
nature of, 135

various forms assumed by, 136
pyogenes albus, 144
aurens, 142

pyogenes stephlococcus, 141
sarcina ventriculi, 140
albus non liquescens, 144
streptococcus pyogenes albens, 144
bombycis, 161
ovatus, 161
Micrococcus :
agilis, 1 17

necrosis in mice, 158
osteomyelitis, acute infections of, 157
pytemia: in mice and rabbits, 160
septicaemia and abscesses in rabbits,
160

tetragenus, 157 «

gonorrhoea, 16 1
aquatilis, 527

Microtomes in common use, 18
Microsporon furfur , 479
Micrezyma bombycis, 161
Mikulicz cells, 331
Milk:

hoiv sterilised, 31
examination of, 85
how clotted and solidified, 193
tuberculous matter in, 359
Miliary tuberculosis in children, 344
Millei, or glanders bacillus, 324, 328, 329
Minot's microtome, 18
Moist chamber, use of, 58
Molluscum Contagiosum, 509'

Mortsblanc ftachcrie, disease of, in silk-
worms, 161

Motility of bacteria, 1x3
Mould-fungi, nature and history of, 476 —
497.

Mucor rhizopodiformis (fungi), 484
Mucor (fungi), 484

Mucus flakes in typical rice-water stools,
412

Muller’s fluid for hardened material, 19
Mycelial fungi, 477

INDEX

592

Mycelial fungi :

branched threads, 495
mycosis mucorina, 484
Mycetoma, or madura disease, 497
varieties of, 497
Mycoderma aceti, 474
saccharomyces, 474
Mycoprotein, nature of, 88

Necrotic and diphtheritic deposits in
fowls, 322
Neurin, 129
Nitrifying microbes, 90
Nitroso-indol, 193
reaction of, 418

Noma tumour of a child, vibrio in, 410
Nosema bombycis, 161
Nutrient Agar-Agar, nature of, 35
Nutrient-gelatine, 33
meat infusion, 34

CEdema :

serobic of malignant bacillus of, 229
culture of, 370, 378
Koch’s maligni bacillus of, 375
O'idium :

albicans the cause of thrush, 475
lactis, 478

Oil:

immersion, use of, 7
aniline, 13

Oogonium, (fungi), 486
Oospores (in fungi), 486
Ophthalmia :

Egyptian, 253, 254

jequirity, experiments regarding, 542
ct scq.

purulent, 254

Osteomyelitis, acute infectious, microbe of,
157

Oysters, vibrios in, 458, 459

Paget’s disease, 109
Papules, eruption of, in a cow, 317
Paraffin, embedding in with aid of paraffin
block and rocking microtome, 20
Paralysis, post-diphtheritic, 302
Parietti’s method of cultivation, 78
Pasteur’s fluid, 31

his pressure filter, 77
his vaccine, 291
his yeast torula, 471
his attenuated cultures, inutility of,
53°. 53:

Pathogenic, bacilli, 204

organisms, relations of saprophytic to,
. 535 583

Pdbrine, or Cornalta's disease, 161
Pencillium, 484
Peptone arid salt solution, 29
Pericarditis, microbe of, 154
Peritonitis, acute, 131
Perithecium, nature of, 482
“ Perlsucht," the disease called, 339
Petri's dishes, use of in inoculation, 57K.
Pfeiffer’s test, 458

Phagocytosis MetchnikofFs theory’ of, 562
Phenolated :

gelatine and broth, 78
bacillus coli grows well in, 197
Phenomena :

leucocytosis, 559 ct scq.
chemiotaxis, 564 et scq.

Phlegmon, acute, the microbe of. 144
Phosphorescens, spirillum, 409
Phosphorescent bacteria, 129
Phycomycetes, 484
Pigeons, diphtheritic deposits in, 322
Pigments :

formation of, 126
bacteria, 128
researches on, 535

Pipette, capillary glass, how used, 42
Pityriasis versicolor, due to fungus, 479
Pleurisy, microbe of 154
Plague :

swine, 217

oriental, or bubonic, 224
Plasmodium malaria, 498, 501
Plate cultivation in inoculation, 57,
Platinum needles, loops and lancets, uses
. of, 44

Poisons, intracellular, 132
Pollinodia, nature of, 480
Potato bacillus, 182
Pravaz sy'ringe, the, 41
Pneumonia :

acute, streptococci of, 153
croupous, 154

fatal epidemic of (Middlesbrough),
226

Pneumo-coccus, the 154

enteritis, disease of, 220
mycosis, 484
Premiere vaccine, 289
Prodigiosus bacillus, 200
Proteid decomposition, effects of, 2
Proteus :

vulgaris, 75, 182
Zenkeri, 185,
sewage, variety of, 198
hominis capsulatus, 292
Protoplasm, segregation of the, 177
Protozoa :

Plasmodium malaria, 498

true course of malaria, 408
haunoplasmodium malaria:, 498

INDEX

593

Protozoa :

malarial fever, 501
endoglobular form of the plasmo-
dium malaria:, 501
Amoeba coli, 502

tropical dysentery, 502
dysentery amoeba, 502
Flagellate protozoa, 503
trichomonas, 503

circomonas intestinalis hominis,
5°4

flagellate monadinae, 504
herpetomonas Lewisii, 504
haematozoon, 505
surra disease, 505
Psorospermia, or coccidia, 505
sporozoa, 505
coccidium oviforme, 505
Miescher's coccidia, 307
epithelioma contagiosum of the
fowl, 508

psorospermosis, 509
Cancer parasites, 509

Darrier's disease, 509
molluscum contagiosum, 509
Paget’s disease, 509
epithelioma, 509
cancer of the skin, 509
coccidia, 515
cimeria, 515
amoebo-sporidia, 515
klossia, 515

rophalocephaius carcinomatosus,
5=° .

gregarina forms, 520
fuchsin bodies, 521
encysted epithelial cells, 524
Pseudo membrane, 296
Psorospermia, or coccidia, 505
Ptomaines :

nature and action of, 2
formation of by bacteria, 129
Puerperal septicarmia, microbe of, 147
Purple dye, Spiller's, 13
Pyocyaneus, 200

Pyrogallic acid for culture tubes, 87

Quarter-fvil, disease of, Rauschbrand's,

384

Rabbits :

micrococci of abscesses, pyaemia: and
septica:mia in, 160

effects on of feeding with tubercular
matter, 937

Rag-sorters' disease, 276, 292
Rauschbrand's quarter-evil, disease of,

384

Ray fungus, actinomyces, or, 486
Red dye, Humboldt’s, 13
Relapsing fever, spirillum Obermeyeri of,
466

Rhinitis, fibrinous, joo
Rhinoscleroma, bacillus of, 331
Rice-water stools :
typical, 412
mucus flakes in, 416
Rinderseuche, disease of, 222
Rophalocephaius carcinomatosus, 52c
Rosaceum, spirillum, 408
Rosaniline, 13

Rubin, watery solution of, 14
Rubrum, spirillum, 409
Rugula, vibrio, 406

Saccharomyces :

alcoholic fermentation, 473
cerevisia: (torula cere visa:), 473
mycoderma (mycoderma vini), 473
pastorianus, 473
oldium albicans, 474
Saline solution, advantage of, 8
Salmon disease, fungi of, 486
Sanarelli’s water vibrios, 463
Sanguineum, spirillum, 409
Saprmmia, or putrid intoxication, cause of,
130

Saprolegnia (fungi), 483
Saprophytes, power of over the anthrax
bacilli, 529 et seq.

Sarcina lutea and ventriculi microbes, 136,
140

tattler’s jequirity bacillus, researches re-
garding, 54°— 547
Scarlatina, streptococcus of. 151
Schizomycetes, nature of, 88
Schoenleini , achorion , 479
Sea-water, vibrios in, 458
Seeds of jequirity, experiments with, 542 —

„ . 547 . .

Septic intoxication, 130
Septicaimia :

puerperal, microbe of, 147
in man, 256
Pasteur’s, 378
bacillus of, 378
infection, 147
acute microbe of, 226
Davaine bacillus, 205
Serpens, vibrio, 406
Serum :

blood, 30
solidified, 34
inspissator, the, 44
anti-toxic power of, 384
cholera, power of from an “actively'
immunised guinea-pig, 435
cholera, 460

Q Q

594

INDEX

Sexual and asexual spore formation, 480
Shake culture, 125

Sheath of the chain, or leptothrix, 170
Skin, cancer of, 509
Soil examination of, 85
Solution, saline, advantage of, 8
Sore throat, simple, 301
Specimens :

fresh, importance of, 8
permanent, how made, 10
film, 11

cover-glass, how treated, n
impressions Klatsch-prmparate of the
Germans, how to make, 12
Spider’s purple dye, 13
Spirilla produced by vibriones, 404 ; fine,
419

Spirillum :
tenue, 406
undula, 408
volutans, 408
rosaceum, 408

sanguineum (Ophidomonas sanguinea,
Ehrenberg), 409
eubrum (von Esmarch’s), 409
phosphorescens, 409
tyrogenum, 454

Obermeyeri of relapsing fever, 466
vibrios, spirilla and, found in different
waters, 455

Spirochceta plicatilis, Cohn’s, 407
denticola, 407

Spleen, typhoid bacillus in, 241
Sporadic cholera, 452
Sporangia (fungi), 477, 484
Spores :

bacilli, vitality of, 95
resistance of to heat, 96
power of bacteria to form, 103 — 112
germination of, in hay infusion, 181
of malignant anthrax, 283
in tubercle, 355
conidia, 477, et seq.
formation, asexual and sexual, 480
Sporozoa, 505
Staining :

cover-glass specimens and sections, list
of most useful dyes for, 13
of tubercle-bacilli, 348
Staphlococcus : 136
pyogenes, 141
pyogenes aureus, 142
Steamer for sterilizing, 43
Stomatitis, ulcerative in the calf, 292, 294
Stools, typhoid bacillus in, 241
Streptococcus :

streptococcus, 136
various kinds of, 144, et seq.
pyogenes albens, 144
erysipelatos, 147, 531
foot and mouth disease, 150
scarlatina, 15 1

eruptive disease in milch cows, 151

Streptococcus :

diphtheria, faucial, 132
pharyngeal abscess in the horse, 153
pneumonia, acute, 153, 144
croupous, 154
pericarditis, 154
pleurisy, 154

meningitis, cerebro-spinal, 154
endocarditis, ulcerative. 155
lung, red hepitisation of. 155
in some epidemics (Middlesbrough),
1 55

influence of on bacillus anthracis, 531
Streptothrix bacillus, 201
Foersteri, 201

Sub-cultures, pure, how to start, 59
Substage-condensor, use of a, 7
Subtilis, the bacillus, 165, 178
Surra disease, 505
“Swarming” of bacteria, 115
Swine fever :

bacillus of, 217
erysipelas in, 231

Syphilis-bacilli, various methods of dealing
with, 17, 18, 330

Syringes used for inoculations, 41
T

Tabes, mesenterica, in children, 344
Tests :

Pfeiffer’s, 458
Bordet-Durham’s, 458
Test-tubes :

for culture media, 45
for plate cultivation, 61
Tetani :

culture of bacillus of, 372
bacillus of, 378 — 380
Tetanin, a toxic principle, 383
Tetanus :

bacilli, 121
toxin, use of, 383

artificially immunised against, 384
anti-toxin, 384
Tetrade microbes, 136
Tetragenus micrococcus, 157
Texas fever, bacilli of, 223
'Phallus (fungi), 477
Throat :

simple sore, 301
illness of cats, 312
Thrush fungus, 475

Tongue, wooden- a disease in cattle, 488
Torula-like chains, 94, 177
yeast, 471

Tox-albumins or ferments, 130
Toxins :

caused by specific bacteria, 130, 133
cholera, 436

Trichomonas, the genus, 503
Trichophyton tonsurans, 479
Tubes, test, for culture media, 45

INDEX

595

Tubercle :

spores in bacilli, 1 10
caseous, in the guinea-pig, 336
in the iris, 345
Tubercle-bacillus :

cultivation of, 346, 347
staining of, 348
in cultivation, 352
spores in, 355
Tubercular matter :

feeding rabbits with, 337
giant cells in, 339
Tuberculin :

nature of, 360
in lupus, 361

Tuberculinum, Koch’s 361
Tuberculosis :

bacillus of, 333

disseminated, how produced, 333
natural, in fowl, 338
can be produced in animals by
inhalation, 338
bovine. 339

miliary, in children. 344
disease among food-animals, 358
Tuberculous disease :

among food-animals, 358

effects of food derived from, 358
in cattle and swine, 358
matter in milk, 359
Typhi murium, bacillus of, 224
Typhoid-fever, bacillus of, 235, 241

. U

■ Udder eruption in milch cows, 321
Ulcerative stomatitis in the calf, 292, 294
Unduta, spirillum, 408
Urea, hydration of, 125
Urine, typhoid bacillus in, 241

Vaccines :

premiere. 289
deuxieme, 290
Pasteur's, 291
bacillus of, 398
variola, 398

protective inoculations of, against
cholera, 448, 449
Vacuoles of bacilli, 166
Varied®, vaccine, 398

Vehring’s experiments with diphtheria
serum, 574

Vesicles, eruption of, in a cow, 317
Vessels and instruments used for cultiva-
tions, 38—44
Vesuvin, 13 .

Violet, methyl, 13
gentian, 13
Vibrio :

septique, 378
rugula, 406

Vi brio :

serpens, 406
aurens, 409
tlavescens, 409
flavus, 409

Asiatic® choleras, 410, 426
in noma tumour of a child, 410
Koch's cholera, 416
flagella of, 418
of Finkler-Prior, 452
of epidemic cholerine in Lisbon, 456
Massowah, the, 462
Sanarelli's water, 463
Metchnikovo, 464
Vibriones ;

bacteria, 404

called “comma bacilli’’ from their
shape, and produce spirilla, 404
Vibrios :

Asiatic cholera, effect of when injected
subcutaneously, 426
intraperitoneal injection of, 433
experiments by ingestion of cultures
of, 445

found in different waters, 455
in oysters, 458
in sea water, 459

Water :

bacterioscopic examination of, 67
number of microbes in, 73
characters of the microbes in, 74
bacillus coli in, 75, 196
proteus vulgaris in, 75
sewage pollution of, 76
vibrios found in different, 455
bacteria, 527

Welbeck, choleraic diarrhoea at, 230
Whey as an admixture, 31
Wildseuche, bacillus of, 222
Williams's :

microtome, 18

mucilage must be used with, 19
Wooden-tongue, a disease in cattle, 488
Wool-sorters’ disease, 273

Xylol, or clove-oil, 10
Y

Yeast- fungi. 472—476
Z

Zenkeri, proteus, 185
sewage variety, 198
Zoogloca microbes 173
Zoosporangia (fungi), 485, 486

Richard Clay and Sons, Limited,

LONDON AND BUNGAY.

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