The bacteria in Asiatic cholera

THE LIBRARY

OF

THE UNIVERSITY
OF CALIFORNIA

PRESENTED BY

PROF. CHARLES A. KOFOID AND
MRS. PRUDENCE W. KOFOID

THE BACTERIA

IN

ASIATIC CHOLERA

THE BACTERIA

IN

ASIATIC CHOLERA

BY

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

LECTURER ON GENERAL ANATOMY AND PHYSIOLOGY IN THE MEDICAL SCHOOL OF

ST. P.ARTHOLOMEW'S HOSPITAL j PROFESSOR OF BACTERIOLOGY AT THE

COLLEGE OF STATE MEDICINE, LONDON

Honfcon
MACMILLAN AND CO.

AND NEW YORK
1889

The Right oj Translation and Reproduction is Reserved

>

RICHARD CLAY AND SONS, LIMITED
LONDON AND BUNGAY.

n

TO

GEORGE BUCHANAN, ESQ., M.D., F.R.S.,

MEDICAL OFFICER OF THE LOCAL GOVERNMENT BOARD,
THIS VOLUME IS

BY

THE AUTHOR.

M374360

PREFACE.

THE present volume is a reprint of a series of articles
published in the Practitioner (October 1886, to May 1887).
Since that time a considerable number of contributions have
been made to the knowledge of the comma-bacilli of Koch.
These have been added to the present volume ; but besides
these no noteworthy addition has been made to our know-
ledge of the bacteria in Asiatic cholera. This need not at
all surprise us, considering that with few exceptions most
Continental pathologists consider the comma-bacilli of Koch
as being the cause of cholera/ that is to say as being the
real cholera microbes. They consider the chapter of
the etiology of Cholera Asiatica as closed, and there is
therefore no need to look for any other and new cholera
microbe. I have ventured to differ from this opinion
when writing the above articles in the Practitioner, and after
all the observations published by various pathoiogists since
then, I still differ from the proposition that Koch's comma-
bacilli have been satisfactorily proved to be the cause of
cholera.

viii PREFACE.

As then, so also now I hold that the comma-bacilli of
Koch do not fulfil the conditions which the cholera microbe
ought to fulfil. As then, so also now I agree with the pre-
vailing opinion that the comma-bacilli of Koch are an
important diagnostic guide.

I have been credited by various writers with the contrary
statement; I have in the articles, as they first appeared,
tried to correct such statements, though without effect, and
I must therefore again state, that I fully agree with Koch
and others as to the constant presence of the comma-bacilli
in the cholera intestine and cholera discharges during the
early stages, but that here our agreement ends. That the
comma-bacilli of Koch are not accepted as the proved
cause of cholera, in this I am not alone, as, for instance,
Professor Baumgarten seems to think ; von Pettenkofer,
acknowledged to be the greatest living authority on the
etiology of cholera, holds this view, viz. that the comma-
bacilli are not the proved cause of cholera; Dr. D. D.
Cunningham of Calcutta, who during many years had
exceptional opportunities of studying this disease, is of
the same opinion. The English Cholera Commission
(Professor Roy, Dr. Sherrington and Dr. Brown); Dr.
Shakespeare of Philadelphia, and Dr. v. Emmerich of
Munich, are the most noteworthy observers who have arrived
at the same conclusion after special study of cholera.

E. KLEIN.

CONTENTS.

PAGE

INTRODUCTION . i

CHAPTER I.

THE HISTORY OF THE COMMA-BACILLUS 5

CHAPTER II.

THE DISTRIBUTION OF COMMA-BACILLI 22

CHAPTER III.

MORPHOLOGY OF THE CHOLERAIC COMMA-BACILLI 40

CHAPTER IV.

CHARACTERS OF THE COMMA-BACILLI IN ARTIFICIAL

CULTIVATIONS . 6 1

x CONTENTS.

CHAPTER V.

PAGE

VARIOUS SPECIES OF COMMA-BACILLI 82

CHAPTER VI.

DIAGNOSTIC VALUE OF CHOLERAIC COMMA-BACILLI 112

CHAPTER VII.

EXPERIMENTAL PRODUCTION OF CHOLERA IK)

CHAPTER VIII.

THE INFECTIVENESS OF CHOLERA 14!

CHAPTER IX.

OTHER BACTERIA IN CHOLERA l66

LIST OF ILLUSTRATIONS.

FIG. PAGE

1. FROM A PREPARATION OF FRESH MUCUS-FLAKES FROM

A CHOLERAIC EVACUATION 24

2. PREPARATION OF MUCUS-FLAKES FROM THE ILEUM OF

AN ACUTE CASE OF CHOLERA 25

3. PREPARATION OF MUCUS-FLAKES FROM THE ILEUM OF AN

ACUTE CASE OF CHOLERA 26

4. FROM A PREPARATION OF FRESH MUCUS-FLAKES FROM

THE LOWER PART OF THE ILEUM OF A TYPICAL
RAPIDLY FATAL CASE OF CHOLERA 27

5. FROM A SECTION THROUGH THE ILEUM OF AN ACUTE

CASE OF CHOLERA 31

6. FROM A PREPARATION OF MUCUS-FLAKES FROM THE

LOWER ILEUM, WHICH HAD BEEN ALLOWED TO
UNDERGO PUTREFACTION FOR THREE DAYS 42

7. PREPARATION OF CHOLERAIC COMMA-BACILLI STAINED

WITH GENTIAN-VIOLET, AND AFTERWARDS WELL
WASHED 45

xii LIST OF ILLUSTRATIONS.

FIG. PAGE

8. ARTIFICIAL CULTIVATION OF CHOLERAIC COMMA-BACILLI

IN ALKALINE PEPTONE GELATINE 47

9. ARTIFICIAL CULTIVATION OF THE SAME COMMA-BACILLI
AS IN PRECEDING FIGURE IN ALKALINE PEPTONE
BROTH GELATINE 48

10. ARTIFICIAL CULTIVATION OF THE SAME COMMA-BACILLI

IN ALKALINE BEEF BROTH 49

11. FROM A CULTIVATION OF CHOLERAIC COMMA-BACILLI

IN LIQUEFIED GELATINE, AFTER SEVERAL WEEKS . 51

12. PREPARATION OF A PURE CULTIVATION OF CHOLERAIC

COMMA-BACILLI IN AGAR-AGAR MEAT-EXTRACT PEP-
TONE, SEVERAL MONTHS OLD 53

13. PREPARATION OF A CULTIVATION OF CHOLERAIC COMMA-

BACILLI IN EGG-ALBUMEN AND AGAR-AGAR, TEN
DAYS OLD 54

14. FROM AN ARTIFICIAL CULTIVATION OF CHOLERAIC

Mucus FLAKES ON DAMP LINEN 55

15. PREPARATION OF A CULTIVATION OF CHOLERAIC COMMA-

BACILLI ON DAMP LINEN AFTER THIRTY-SIX HOURS 56

16. F"ROM A RECENT ARTIFICIAL CULTIVATION OF CHOLERAIC

COMMA-BACILLI IN ALKALINE AGAR-AGAR JELLY. . 57

17. FROM AN ARTIFICIAL CULTIVATION OF CHOLERAIC

COMMA-BACILLI ON NEUTRAL AGAR-AGAR JELLY AT
ORDINARY TEMPERATURE, AFTER A FEW WEEKS . . 58

LIST OF ILLUSTRATIONS. xiii

FIG. PAGE

18. FROM A SIMILAR PREPARATION 58

19. P'ROM A SIMILAR PREPARATION 58

20. PLATE-CULTIVATION IN GELATINE FORTY -EIGHT HOURS

OLD, SHOWING YOUNG COLONIES OF CHOLERAIC
COMMA-BACILLI 62

21. PLATE CULTIVATION WITH COLONIES OF CHOLERAIC

COMMA-BACILLI SEVENTY-TWO HOURS OLD 66

22. FROM A CULTIVATION OF CHOLERAIC COMMA-BACILLI IN

GELATINE IN A GLASS DISH FOUR DAYS AFTER
INOCULATION IN SPOTS 67

23. PLATE CULTIVATION OF CHOLERAIC COMMA-BACILLI IN

GELATINE AFTER FOUR DAYS AT i9°C 68

24. A COLONY OF CHOLERAIC COMMA-BACILLI IN GELATINE,

SEVENTY-TWO HOURS OLD 71

25. CULTIVATIONS OF CHOLERAIC COMMA-BACILLI .... 74—77

26. STABCULTURES OF CHOLERAIC COMMA-BACILLI IN NUTRI-

TIVE GELATINE AFTER ONE, THREE, FOUR, FIVE,
SEVEN, AND TEN DAYS RESPECTIVELY 79

27. COVER-GLASS SPECIMEN OF TINKLER'S COMMA-BACILLI

FROM A GELATINE CULTURE 83

28. GELATINE PLATE CULTIVATION OF FINKLER'S COMMA-

BACILLI AFTER INCUBATION FOR FORTY-EIGHT
HOURS AT 20°C 86

xiv LIST OF ILLUSTRATIONS.

FIG. PAGE

29. CULTIVATION OF FINKLER'S COMMA-BACILLI IN NUTRI-
TIVE GELATINE (10 PER CENT.) AFTER FOUR DAYS'
INCUBATION 88

30. COVER-GLASS SPECIMEN OF MUCUS-FLAKES FROM A

MONKEY SUFFERING FROM DIARRHCEA ...... 91

31. COVER-GLASS SPECIMEN OF CONTENTS OF CAECUM FROM

A NORMAL GUINEA-PIG 93

32. COVER-GLASS SPECIMEN FROM A CULTIVATION IN 10 PER

CENT. NUTRITIVE GELATINE OF THE NON-LIQUEFYING
VARIETY OF COMMA- BACILLI FROM A CASE OF NOMA
IN A CHILD 97

33. PLATE-CULTIVATION OF THE SAME NON-LIQUEFYING

COMMA-BACILLI OF NOMA AS IN FIG. 32 99

34, 35. SAME NON-LIQUEFYING COMMA-BACILLI GROWING IN
10 PER CENT. NUTRITIVE GELATINE ; SEVERAL
WEEKS OLD 102

36. CULTIVATION IN 10 PER CENT. NUTRITIVE GELATINE OF

MlCROCOCCUS ISOLATED FROM THE BLOOD OF THE
FINGER OF A PERSON AFFECTED WITH SCARLATINA . 106

37. CULTIVATION IN GELATINE (10 PER CENT.) OF SAME

MlCROCOCCUS AFTER THREE WEEKS, SHOWING A
LARGE FUNNEL-SHAPED OPENING ON SURFACE WITH
AN OCCLUDING AlR-BUBBLE . IO6

LIST OF ILLUSTRATIONS. xv

FIG. I'AGE

38. SPECIMEN OF MUCUS-FLAKES FROM A MONKEY 164

39. FROM A PREPARATION OF FRESH MUCUS-FLAKES FROM

THE ILEUM OF A TYPICAL RAPIDLY FATAL CASE OF
CHOLERA 168

40. FROM A PREPARATION OF FRESH MUCUS-FLAKES FROM

THE ILEUM OF ANOTHER TYPICAL RAPIDLY FATAL
CASE OF CHOLERA 169

THE

BACTERIA IN ASIATIC CHOLERA

INTRODUCTION.

IN the following pages I propose to give an account of
the present state of our knowledge of the etiology of Asiatic
cholera, gained chiefly in the time that has elapsed since the
first communications of Koch on this disease. I do not
mean to imply that the observations made by Koch, and
others since, are to be regarded as the only valuable
addition to our knowledge of this very dire plague ; for I
am quite aware that all that is of importance in our
knowledge of the mode of its spreading and propagation, of
the various conditions of soil, air, temperature, water, which
affect it — all in fact that has helped us to combat the malady,
was gained many years before bacteria and disease-germs
had emerged from the region of mystery, long before the
recognition of and experimentation with disease-germs had
become a branch of exact science. I have only to refer to
the works of v. Pettenkofer, of Bryden, Bellew, Cunningham,
Budd, Parkes, Snow, and many other experienced observers
in India and Europe, to remind the reader, that all that has

B

2 THE BACTERIA IN ASIATIC CHOLERA. [INT.

proved of value from an epidemiological and general sanitary
point of view has been gained irrespective of any knowledge
of bacteria. The laws governing the spread of cholera are
and have been well understood by sanitarians ; the fact that
cholera, like other infectious diseases, is a communicable
disorder spreading from a focus of infection has been long
known ; and the measures required (and now almost
everywhere admitted as necessary) in order to check its
spread have been carried out for many years, and without
any exact knowledge of diseased-germs. True it was always
felt, and a reference to the theoretical parts of the papers by
v. Pettenkofer, Bryden, Budd, Parkes, and others fully bears
this out, that the knowledge thus gained was of a purely
empirical kind, no direct or exact experiment being possible
to demonstrate the truth or fallacy of the various measures
recommended and employed ; and this again was entirely
due to the fact that the nature and character of the infective
essence of the disease, or the contagium (used here in its
wider sense), was unknown. While on the one hand there
can be no doubt that the discovery of the cholera-germ
(which I may at once say has not yet been made) could
not in any way alter the nature and application of the
general laws of sanitary science, as specially applied to the
group of infectious diseases dependent on filth — and accord-
ing to the general consensus of experienced observers,
Asiatic cholera belongs to this group — there can be, on the
other hand, no doubt that the identification of a cholera-
germ, the knowledge, which from such a discovery would
inevitably follow, of its nature and mode of spread, of its
mode of alteration by temperature, soil, and season, would
unquestionably lead to a more specific application of means
to ends than has hitherto been the case. Besides, by an
exact knowledge of the cholera-germ, we should be enabled

INT.] THE BACTERIA IN ASIATIC CHOLERA. 3

accurately to determine the mode of invasion of the human
body by the cholera-virus, the distribution of this latter in
the body, the changes it undergoes, the manner in which it
leaves the body, and many other very important questions,
which would at once emerge from the region of debatable
points wherein they at present are ; in other words this
knowledge would give us a thorough insight into the whole
etiology and pathology of the disease, which at present we
do not possess. One important series of facts would by the
discovery of the cholera-germ at once become plain, viz. the
mode of its entrance into the human body and the mode of
its exit. At present opinion is divided on both these most
essential questions. How does the cholera-virus enter the
human body ? Does it enter by the alimentary canal only,
as is maintained by many authorities, or does it enter also
by the respiratory organs ? How does it leave the infected
body ? Is it present in the vomit and discharges from the
bowels, as is maintained by most observers ; or is it present
in these not as an actual but as a potential virus, as is
maintained by other equally great authorities ? It is obvious
that, according to either one of these theories, the mode of
our action in combating the spread of the disease ought to
become exact and specific.

Now, it is maintained by Koch, and many others who
confirm or accept Koch's statements, that in cholera asiatica
the intestines (chiefly the ileum of the small intestine) of a
person affected with the disease is the seat of the rapid
growth and multiplication of a definite bacterium (comma-
bacillus), which is not present in the blood or any other
part of the body, which by its multiplication in the intestine
produces a special chemical poison ; this poison is absorbed
into the system and sets up the whole chain of disturbances
of the nervous, vascular, and respiratory organs character-

B 2

4 THE BACTERIA IN ASIATIC CHOLERA. [INT.

ising cholera ; and that therefore the bowel discharges
containing those specific bacteria are par excellence the
vehicle of the cholera-germ. It is obvious, that if this comma-
bacillus of Koch is in reality the cholera-germ, is really the
cholera-bacillus — as truly as the anthrax-bacillus present in
the blood of the whole body of an animal affected with or
dead from anthrax, and the tubercle-bacillus of Koch
present in the tuberculous deposits of an animal or human
being affected with tuberculosis, are the germs of these
diseases — then the whole series of measures required for
checking the spread of cholera would be as simple as they
would be efficient. For if it be true that this comma-
bacillus, which as we shall see is present only in the
contents of, and of course in the discharges from, the
bowels of a person affected with cholera, is really and truly
the cholera-germ, then the destruction of all the bowel
discharges of a cholera-patient, the prevention of bowel
discharges gaining access by food, drink, or otherwise to the
alimentary canal of others, would be no doubt an effectual
and almost the exclusive mode of checking the spread of the
disease. Other questions : such as the relation of this comma-
bacillus to various conditions of temperature, soil, season,
&c., although important from a scientific point of view,
would be insignificant compared with this cardinal and
fundamental fact. Has it, then, been proved that the
comma-bacillus of Koch is the real cholera-germ ? This is
the question to answer which the following chapters are
devoted.

CHAPTER I.

THE HISTORY OF THE COMMA-BACILLUS.

A GOOD many statements of microscopists are on record
concerning the occurrence of various forms of bacteria in
the dejecta of cholera-patients,1 but since these statements
referred to gross morphological characters only they were
not considered of great value. To say that there occurred in
the dejecta of cholera-patients micrococci, bacilli, and
vibrios, is not one whit more than to say that in human faecal
matter occur these same forms of bacteria. Mr. Fowke 2
claims for Brittan and Swain to have shown in 1849 the
occurrence in the choleraic dejecta of the comma-bacilli in
the shape of peculiar circular and semicircular corpuscles,
which were declared by them not only to be peculiar, but
also to have a causal relation to cholera morbus. Looking
at the drawings and descriptions reproduced by Mr. Fowke,
it does not impress me that these corpuscles are identical,
as they are claimed to be, with Koch's comma-bacilli, but I
am rather inclined to think that what is there depicted and
described are altered and decolourised blood-discs. There

* Hassall, Bristowe, Klob, Lewis and Cunningham, and others.
2 British Medical Journal , March 21, 1885.

6 THE BACTERIA IN ASIATIC CHOLERA. [CH.

can be, however, no question about this, that if these
corpuscles are really the same as Koch's comma-bacilli,
their discoverers did not establish, and by reason of the
then crude state of bacteriological research could not
have established, that they represented a definite species.

The first account of the presence in the cholera-intestine
and cholera-dejecta of a definite species of bacteria character-
istic of cholera was given by Koch. We give his own
words.1

" When we examined the intestine and its contents under
the microscope, it was seen that, in some cases, especially in
those in which the Peyer's glands were red at the edge, an
invasion of bacteria corresponding to this redness had taken
place. The bacteria had partly forced their way into the
utricular glands, partly pushed themselves between the
epithelium and the basement-membrane, thereby lifting the
epithelium as it were. In other parts it was seen that they
had forced their way deeper into the tissue. Then cases
were found in which, behind these bacteria, which had a
special appearance with regard to size and shape, so that one
could distinguish them from other bacteria and devote
special attention to them, various other bacteria forced their
way into the utricular glands and the surrounding tissue, e.g.,
large thick bacilli and very thin bacilli. Thereby conditions
were produced similar to those in necrotic diphtheritic
changes of the mucous membrane of the intestine and in
typhoid ulcers, where afterwards other nonpathogenic
bacteria force their way into the tissue rendered necrotic by
pathogenic bacteria. We were, therefore, from the very be-
ginning, obliged to look upon these first-mentioned bacteria

1 Conferenz zur Erorterung der Cholerafrage^ Berliner klin. Woch .31,
1884. Translated in the British Med. Journal, August 30 and
September 6, 1884.

I.] THE HISTORY OF THE COMMA-BACILLUS. 7

as not altogether unimportant for the cholera-process, whilst
everything else gave the impression that it was something
secondary ; for the bacteria first described always advanced
beyond the others, they forced their way farther in, and gave
one the impression that they had smoothed the way for the
other bacilli.

" With regard to the contents of the intestine, at first no
clear idea could be formed, as the only cases which came
before us for examination were not suitable ; in these, also,
the contents of the intestine were already putrid and bloody.
There were an enormous quantity of various bacteria in
these contents, so that there was no possibility of attending
to the real cholera-bacilli. Not till I had dissected a couple
of acute and uncomplicated cases, in which no haemorrhage
had as yet set in, and in which the contents of the intestines
had not yet turned to putrid decomposition, did I recognise
that the purer and fresher the cases the more did a special
kind of bacteria prevail in the contents of the intestines also,
and it was soon clear that these were the same bacteria
which I had seen in the mucous membrane. This discovery
naturally turned my attention more and more to this kind
of bacteria. I investigated them in all kinds of ways in order
to establish their special peculiarities ; and am able to give
the following information regarding them.

" These bacteria, which I have called comma-bacilli on
account of their peculiar shape, are smaller than the tubercle-
bacilli. One scarcely forms a correct idea of the thickness,
length, and breadth of bacteria by giving their dimensions in
numbers ; I therefore prefer to compare the dimensions of
bacteria with other objects, so that one can immediately form
a tolerably good idea. As the tubercle-bacilli are known to
everybody, I will compare the cholera-bacteria with them.
The cholera-bacilli are about half, or at most two-thirds, as

8 THE BACTERIA IN ASIATIC CHOLERA. [CH.

long as tubercle-bacilli, but much more bulky, thicker, and
slightly curved. This curve is generally not more marked
than that of a comma ; but sometimes it is larger, becoming
semicircular. In other cases it is seen that the curve is
doubled, that one comma is attached to another, but in an
opposite direction, so that it forms the shape of S- I think
that, in both cases, two individual ones after being divided
have remained stuck together, and accordingly give the
appearance of a more marked curve. But in the artificial
cultivations, besides these, another very remarkable form of
development of the comma-bacillus is to be found, which is
very characteristic of it.

" The comma-bacilli frequently grow in threads of longer
or shorter length. But they do not then form straight threads,
like other bacilli, for instance, anthrax-bacilli, or, as it ap-
pears in the microscopic picture, simple wavy threads, but
very long slender spirals, which, as far as their length and
the rest of their appearance are concerned, bear the closest
resemblance to the spirochaetae of relapsing fever. I could
not distinguish one from the other if I had them side by side.
Owing to this peculiar form of development, I am also
inclined to the view that the comma-bacillus is not a genuine
bacillus, but that it is, properly speaking, a transition-form
between bacilli and spirilla. Perhaps, indeed, we have here
to deal with a genuine spirillum, of which we have a fragment
before us. It is seen also in other spirilla — for instance, in
spirillum undula — that very short specimens do not form
the complete thread of a screw, but only consist of a short
little staff, which is more or less curved.

" The comma-bacilli can be cultivated in meat-broth.
They grow in this liquid extremely quickly, and in great
numbers ; and this property of theirs can be utilised for
studying their other qualities, by examining with a strong

I.] THE HISTORY OF THE COMMA-BACILLUS. 9

magnifying power a small drop of meat-broth cultivation on
a cover-glass. It is seen then that the comma-bacilli move
in a very lively manner. When they are collected together
at the edge of the drop, and are moving about amongst one
another, they look like a swarm of dancing midges, and
those long spiral threads appear also moving in an animated
manner, so that the whole affords a strange and extremely
characteristic picture.

" But the comma-bacilli also grow in other liquids, and
especially, in great abundance and speedily, in milk. They
do not make milk curdle, and do not precipitate the casein,
which many other bacteria, which can also be raised in milk,
do. Hence the milk looks quite unchanged ; but if you take
a small drop from the surface, and examine it under the
microscope, it teems with comma-bacilli. They also grow in
the serum of blood, in which they also very quickly develop
and multiply in great numbers. A very good soil for the
reproduction of comma-bacilli is also nutritive gelatine.
This gelatine also serves for facilitating and securing the
discovery of comma-bacilli ; for the colonies of comma-
bacilli assume in the gelatine a most characteristic and
definite form, which, so far as I can discern, and as far as
my experience reaches, no other kind of bacteria assumes in
like manner.1

" The colony looks, when it is very young, like a very
pale and tiny little drop, which is, however, not quite cir-
cular, the shape generally assumed by these bacteria-colonies
in gelatine ; but it has a more or less irregularly bordered,
hollowed out, in parts also rough or jagged, shape. It also
has, at a very early stage, rather a granulated appearance,
and is not of such regular character as in other colonies of
bacteria.

1 This is not strictly correct, as will be shown later.

io THE BACTERIA IN ASIATIC CHOLERA. [CH.

"When the colony becomes somewhat larger, this granu-
lation becomes more and more evident ; at last it looks like
a little heap of strongly refracting granules. I might best
compare the appearance of such a colony to the appearance
of a little heap of pieces of glass. As they grow, the
gelatine liquefies in the immediate neighbourhood of the
bacteria-colony, and this latter sinks down at the same time
deeper into the mass of gelatine. A funnel-shaped cavity is
thus formed in the gelatine, in the midst of, which the colony
is seen as a little whitish point. This appearance is also
quite peculiar ; it is seen, at least in this manner, in very few
other kinds of bacteria, and, as far as I know, never so
marked as with the comma-bacilli. The sinking of the
colonies can be best observed when carrying out an artificial
cultivation. A suitable colony is selected on the gelatine-
plate under a microscope with a glass of slight magnifying
power; it is touched with a platinum-wire, previously heated;
the bacilli -are transferred by the wire into a test-tube with
gelatine, and this is closed with sterilised wadding. A
cultivation of this kind then grows in the same manner as
the colony on the gelatine-plate. I am in possession of a
numerous collection of artificial cultivations of bacteria
made in this manner ; but I have never seen in their case
such changes as the comma-bacilli cause after being trans-
ferred into the gelatine. Here, also, as soon as the cultiva-
tion begins to develop you see a little funnel, which marks
the point where the inoculation took place. By degrees, the
gelatine liquefies in the neighbourhood of this point of
inoculation ; then the little colony is plainly seen, extending
itself more and more ; but a deep spot, sunken in, always
remains, which looks, in the partially liquefied gelatine, as if
an air-bubble were hovering over the colony of bacilli. It
almost gives one the impression that the bacillary growth not

I.] THE HISTORY OF THE COMMA-BACILLUS, u

only causes a liquefaction of the gelatine, but also a speedy
evaporation of the liquid formed. We already know a
number of other kinds of bacteria which, in quite the same
manner, gradually liquefy the gelatine in test-tubes, starting
from the point of inoculation. But in these cases there is
never such a cavity, nor this bubble-like hollow space.1 I
must further mention that the liquefaction of the gelatine,
starting from a single isolated colony (the best way of
observing it is in a layer of gelatine, which is spread out on
the glass plate), never spreads very wide. The diameter of
the liquefied district of a colony may be estimated at one
millimetre.2 Other kinds of bacteria can liquefy the gelatine
to a much greater extent, so that a colony attains a size of
one centimetre in diameter, and more. In the cultivations
of comma-bacilli made in test-tubes, the liquefaction of the
gelatine extends by degrees and very slowly, starting from
the point of inoculation ; and continues in such a manner
that, after about a week, the whole contents of the tube
have become liquid. Unimportant as all these qualities
seem in themselves, special weight is to be laid on them,
because they serve to distinguish comma-bacilli from other
kinds of bacteria.

" Comma-bacilli can also be cultivated on Ceylon moss
(Agar-agar), to which meat-broth and peptone are added.
This agar-agar jelly is not liquefied by the comma-bacilli.
They can also be raised on boiled potatoes — a fact which
is very important for certain questions. They grow on
potatoes exactly like the bacilli of glanders. The cultiva-
tions of comma-bacilli, when grown on potatoes, look like
those of glanders-bacilli, but are not coloured so intensely
brown, rather a light greyish-brown.

1 As will be shown later, this is not correct.

2 This also is not correct.

12 THE BACTERIA IN ASIATIC CHOLERA. [CH.

"Comma-bacilli flourish best at temperatures between
30° and 40° Cent. (86° to 104° Fahr.), but they are not very
susceptible to lower temperatures. Experiments have been
made on this point, which show that they can grow very
well at 17° Cent., though more slowly. Below 17° Cent.
the growth is very slight, and seems to cease below 16°.
In this point the comma-bacilli remarkably resemble
anthrax-bacilli which also have this minimum temperature
as the limit for their growth-power. Once I made an
experiment to test the influence of lower temperatures
on comma-bacilli, and to see if, at a very low temperature,
they are not only hindered in their development but also
if they cannot possibly be killed. For this purpose
an artificial cultivation was exposed for an hour to a
temperature of 10° Cent, below zero; during this time it
was completely frozen. When part of it was put into
the gelatine, there was not the least difference visible in
the development or growth, so that they bear frost very
well. It is not the same with the withdrawal of air and
oxygen. They immediately cease to grow when deprived
of air, and accordingly belong, if the division into aerobic
and anaerobic bacteria be held as good, to the aerobic class.
Any one can convince himself of this very simply, by laying
a piece of talc or mica over the glass plate, when the
portion of the artifical cultivation has been placed on it in
liquid gelatine, and when the gelatine is beginning to stiffen ;
the talc or mica must be as thin as possible, and must cover
at least one-third of the gelatine surface in the middle. The
piece of mica, owing to its elasticity, adheres completely
to the surface of the gelatine, and thus cuts off the air from
the portion covered. Then, as soon as the development
of the colonies follows, it is seen that the development only
takes place where the gelatine is not covered, and only a

1.] THE HISTORY OF THE COMMA-BACILLUS. 13

trifle, about two millimetres, under the mica plate, up to
which point the air has been able to force its way. But
under the mica-plate itself nothing grows. Extremely small
colonies, invisible to the naked eye, do, it is true, appear,
which probably owe their origin to the oxygen existing in
the gelatine, but they do not increase in size afterwards.
An experiment was made in another mariner. Little glasses
containing nutritive gelatine, which had been inoculated
with comma-bacilli, were placed under an air-pump, and
others prepared in the same manner were kept outside the
air-pump. It was then seen that those under the air-pump
did not grow, but only those outside it. But when those
that had been under the air-pump were again placed in the
air, they began to grow. Hence they had not died ; they
only wanted the necessary oxygen to be able to grow.
The same occurs when the cultivations are brought into
an atmosphere of carbonic acid. Whilst the cultivations
that have been kept for comparison outside the carbonic
acid atmosphere grow in the usual manner, those that are
in a stream of carbonic acid remain undeveloped. But
in this case, also, they do not die ; for, after having been
for some time in the carbonic acid, they begin to grow
immediately after they have come out of it.

" On the whole, comma-bacilli, as I have repeatedly
observed, grow extremely rapidly. Their vegetation very
speedily reaches a maximum, at which it only remains
stationary for a short time, then diminishes again very
speedily. The comma-bacilli, when wasting away, lose
their shape ; they appear at one time shrivelled, and at
another time swollen, and in this state they are not at all,
or only slightly, susceptible to colour. The peculiar con-
ditions of vegetation of comma-bacilli can be best observed
by bringing substances which are rich in comma-bacilli, but

14 THE BACTERIA IN ASIATIC CHOLERA. [CH.

also contain other bacteria, e.g., the contents of a cholera-
intestine or cholera-dejecta, in contact with moist earth, or
by spreading them out on linen, and keeping them in a
damp condition. Comma-bacilli then increase visibly in
a very short time, and in an extraordinary manner in twenty-
four hours. Other bacteria that exist with them are at first
stifled by the comma-bacilli, a natural pure culture is
formed, and, on examining with the microscope the mass
that is taken from the surface of the damp earth or linen,
preparations can be obtained which show almost exclusively
comma-bacilli.

"But this luxuriant growth of comma-bacilli does not
last long. After two or three days they begin to die off.
and other bacteria then increase. The conditions become
the same as in the intestine itself. There also a rapid
multiplication takes place ; but when the real vegetation-
period, which only lasts for a short time, is over, and
especially when exudations of blood into the intestine take
place, the comma-bacilli disappear, and the other bacteria,
especially putrefaction-bacteria, commence to develop in
their place. I am, therefore, almost inclined to believe
that, if the comma-bacilli were brought at first into a
putrefying liquid which contained a great deal of the pro-
ducts of vital changes of other bacteria, and especially of
putrefaction-bacteria, they would not come to development,
but would soon die off. But so far sufficient experiments
have not been made on this point ; it is only a supposition
which I make, supported by my experiences of other bacteria-
cultivations. This point is important, because it is not a
matter of indifference whether the comma-bacilli, if they
come into a sink or sewer, find a good or a very bad soil
for reproduction. In the first case, they would multiply,
and would have to be destroyed by methods of disinfection ;

I.] THE HISTORY OF THE COMMA-BACILLUS. 15

but in the latter case they would die off, and there would be
no necessity for disinfecting. I am inclined to hold the
latter view, as borne out by all the experience I have so
far had.

" In these cultivation-experiments it was further seen that
the nutritive substances — at least, the gelatine and meat-
broth — must not be acid. As soon as the gelatine shows
only a trace of acid reaction, the growth of the comma-
bacilli is very stunted. If the reaction be in a marked
degree acid, the development of the bacilli completely
ceases. It is at the same time noteworthy that it is not
all acids that seem to be unfavourable to the comma-
bacillus ; for the surface of a boiled potato, where it is
cut, is known to have an acid reaction, in consequence, if,
I am not mistaken, of its containing malic acid. Never-
theless, comma-bacilli grow very luxuriantly on potatoes.
Hence, one cannot say, straight off, that all acids hinder
the growth ; but, in any case, there are a number of acids
which have this effect. In meat-broth it is probably lactic
acid, or an acid phosphate.

" In these experiments on the influence of substances in
arresting the development of comma-bacilli, the striking fact
was evident that comma-bacilli die off extremely easily when
dried. These experiments were made by letting a very
small drop of a substance containing bacilli dry on a cover-
glass, and a large supply of these cover-glasses was immedi-
ately prepared for a series of experiments. A drop of the
liquid which was to be examined was then placed upon
such a cover-glass, and left for development in the hollow
glass slide. Having proceeded in this manner, in no single
preparation did anything grow that had received meat-broth
as nutritive fluid, nor in a striking manner in the test-
preparations either. At first I did not know what caused

16 THE BACTERIA IN ASIATIC CHOLERA. [CH.

the absence of growth, and thought that the broth must be
the cause of it, for I have never met with anything like this
before in the case of other bacteria. For instance, anthrax-
bacilli can be kept in store for a long time dry on cover-
glasses ; they retain vitality from half a week to nearly a
whole week in this manner. As, however, the meat- broth
on examination proved to be unexceptionable, we had to
examine whether the comma-bacilli had not probably died
off owing to being dried upon the cover-glass. In order to
obtain certainty on this point, the following experiment was
made. A number of cover-glasses were provided with a
small drop of substance containing bacilli. The drop dried
up in a few minutes. One cover-glass was now charged
with a drop of meat-broth after an interval of a quarter of
an hour, another after an interval of half an hour, another
after an interval of an hour, and so on. Then it was seen (and
I made several series of experiments) that the comma-bacilli
did come to development on the dried glass plates that had
lain a quarter, a half, and a whole hour, but after two hours
they sometimes died off; after three hours, I could not keep
the bacilli alive in these experiments. Only when compact
masses of bacilli-cultivations — for instance, when the pulpy
substance of a cultivation made on potatoes was dried — did
the bacilli retain vitality for a longer time ; clearly because
in this case complete desiccation followed much later. But
even under these conditions I have never succeeded in
preserving the bacilli alive in a dried state longer than
twenty-four hours.

" This result was so far important, that by its means it
could easily be tested whether the bacteria have a resting
state. We know that other pathogenic bacteria — for
example, anthrax-bacteria, which form spores — can be
preserved for years in a dry state on a cover-glass without

I.] THE HISTORY OF THE COMMA-BACILLUS. 17

their dying. We know also of other infectious substances
with whose nature we are not yet accurately acquainted, for
example, the infectious matters of small-pox and of vaccine,
which can be kept in a dried state for several years, still
retaining their power of infection. If now the comma-
bacilli, which, as such, are very speedily killed by drying,
pass into a resting condition under some circumstances,
that would very soon be shown during the process of
drying.

" This is always one of the most important questions in
the etiology of an infectious disease, and especially so of
cholera. The investigation of this point has therefore been
made in the most careful manner possible, and by every
possible method, and I hardly think that anything more can
be done on this point. Above all, cholera-dejecta and the
contents of the intestines of cholera-corpses were left in a
damp condition on linen, in order that the comma-bacilli
might develop under the most favourable circumstances.
After certain intervals of time, pieces of the linen were
dried — for example, after twenty-four hours, after a few
days, after several weeks — to see if during this period any
condition of permanence had been established. For
infection through cholera-linen affords the only undisputed
example of the presence of an effectual infectious substance
which adheres to a special object. If there were a permanent
or resting state to be found anywhere, it must have been
found on cholera-linen.

"But in none of these cases was a permanent state
discovered. When the dried objects were examined, it was
seen that the comma-bacilli had died off. Then, further,
the dejecta were placed in earth, being either mixed with
earth or spread on the surface, which was either kept dry
or moist ; they were mixed with marsh- water ; and were

c

i8 THE BACTERIA IN ASIATIC CHOLERA. [CH.

also left to decay without anything being added to them.
In gelatine-cultivations, the comma-bacilli have been
cultivated up to six weeks, also in serum of blood, in milk,
and on potatoes, on which anthrax-bacilli are known to form
spores extremely rapidly and in great abundance. But we have
never obtained a permanent state of the comma-bacilli. As
we know that the majority of bacilli have a permanent or
resting state, this result appears very striking. But I will
remind you here of what I mentioned before, that we have
most probably to deal with a micro-organism which is not a
genuine bacillus at all, but is more allied to the group of
screw-shaped bacteria, or spirilla; but we do not know of
any permanent state of spirilla as yet. Spirilla are bacteria
which depend for their existence exclusively on liquids, and
do not, like anthrax-bacilli, vegetate under certain conditions
in which they have for once to endure a dry state. It
therefore seems to me, as far at least as my experience goes,
that there is no prospect of rinding a permanent state of
comma-bacilli.

" In accordance with the cholera-material that I have so
far examined, I think I can now assert that comma-bacilli
are never found absent in cases of cholera ; they are something
that is specific to cholera.

"As a test, a considerable number of other corpses,
dejecta from patients and persons in good health, and other
substances containing bacteria, were examined to see if
these bacilli, which were never missing in cases of cholera,
might, perhaps, occur elsewhere also. This is a point of
the greatest importance in judging of the causal connection
between comma-bacilli and cholera.

" Amongst these objects for investigation was the corpse of
a man who had had cholera six weeks before, and had
afterwards died of anaemia. There was no farther trace of

I.] THE HISTORY OF THE COMMA-BACILLUS. 19

comma-bacilli to be found in his intestines. The dejecta of a
man who had had an attack of cholera for eight days previously
were also examined ; his stools were already beginning to be
consistent ; in this case also comma-bacilli were absent.

"I have also thoroughly examined more than thirty
corpses, in order to convince myself more and more that
these bacilli are really only found in cases of cholera.
Corpses of those who had died of affections of the
intestines, e.g., of dysentery or of those catarrhs of the
intestine frequently mortal in the tropics, were chiefly
selected for this purpose ; also cases with ulceration in the
intestine, a case of enteric fever, and several cases of bilious
typhoid.

" In the last-named disease, the modifications in the
intestines are at first sight very similar to those which take
place in severe cases of cholera, in which haemorrhage of the
intestine takes place. The small intestine is in its lower
section infiltrated by haemorrhage ; but, strange to say, this
change in bilious typhoid affects mostly the Peyer's patches,
whilst in cholera these are very little changed.

" In all these cases, where we had to deal chiefly with
diseases of the intestine, no trace of comma-bacilli was to
be found. Experience teaches that such affections of the
intestine make people especially liable to cholera. So one
might have pre-supposed that comma-bacilli, if they were
to be found anywhere else, must be found in these cases.
Besides these, dejecta of a large number of dysenteric
patients were examined without the comma-bacilli ever
being met with. I continued these investigations afterwards
in Berlin, together with Dr. Stahl, my untiring fellow-
labourer, a man who promised much for the investigation of
bacteria, had not an early death unhappily put an end to
his work. We examined a considerable number of various

c 2

20 THE BACTERIA IN ASIATIC CHOLERA. [CH.

dejecta, especially of children's diarrhoea, as well as that of
grown-up persons ; saliva also, and the mucus that adheres
to the teeth and tongue, which abounds in bacteria, for the
purpose of finding comma-bacilli, but always without success.
Various animals were also examined with this view.
Because a complication of symptoms very similar to those
of cholera can be obtained by arsenical poisoning, animals
were poisoned with arsenic, and afterwards examined. A
great number of bacteria were found in the intestines, but no
comma-bacilli. Nor were they found in the sewage from
the drains of the town of Calcutta, in the extremely polluted
water of the River Hooghly, in a number of tanks which lie
in the villages and between the huts of the natives and
contain very dirty water. Everywhere, where I was able to
come across a liquid containing bacteria, I examined it in
search of comma-bacilli, but never found them in it. Only
once did I come across a kind of bacterium which, at first
sight, bore a strong resemblance to comma-bacilli, and that
was in the water which, at high-tide, floods the margin of the
salt-water lake that lies to the east of Calcutta ; but, on a
closer inspection, they appeared larger and thicker than
comma-bacilli, and their cultivations did not liquefy
gelatine.

" Besides these observations, I have had a considerable
experience in bacteria, but I cannot remember ever having
seen bacteria resembling the comma-bacilli. I have spoken
to several people who have made a great number of
cultivations of bacteria, and have also had experience, but all
have told me that they have not as yet seen such bacteria.
I therefore think I may say positively that the comma-bacilli
are constant concomitants of the cholera-process , and that they
are never found elsewhere"

These statements are very definite and precise, and the

i.] THE HISTORY OF THE COMMA- BACILLUS. 21

description give by Koch of the distribution, morphological
characters, and cultivation of the comma-bacilli are very
detailed and clear, and it cannot be said that any other
observer has been able to add anything of importance since
Koch's publications. The statements by Von Ermengem,
Babes, Watson Cheyne, and others on the morphological
and culture characters of the comma-bacilli, are therefore to
be regarded as repetitions of those first made by Koch.
Nothing new is brought forward by these observers. I
think I may therefore be excused from referring to the
statements of these authors so far as they treat of this part
of the subject.

CHAPTER II.

THE DISTRIBUTION OF COMMA-BACILLI.

IN the preceding account by Koch we see, then, that (a)
the comma-bacilli occur in the intestinal dejecta during the
acute stage of the disease : (b) the comma-bacilli are present
in the mucus-flakes and in the fluid of the contents of the
small intestine, chiefly and most numerously in the lower
portion of the ileum ; towards the upper part of the ileum
their number decreases, and in the jejunum they become
very scarce — hence the vomit is as a rule free from them,
and when they are present it is no doubt owing to
regurgitation : (c) the comma-bacilli are present in the tissue
of the mucous membrane of the lower ileum, in the epi-
thelium of the surface, in the lymphatic tissue of the
mucosa, within the cavity of the crypts of Lieberkiihn as
well as between the epithelium lining these crypts and their
limiting membrana propria, but especially in the lymph-
follicles of the Peyer's patches ; these according to Koch
are in pure acute typical cases visible as swollen hypersemic
structures, the blood-vessels of the marginal portion being
distended and filled with blood, and hence strongly marked ;
in these blood-vessels Koch states that he found them in
great abundance : (d) the more acute and typical a case of

CH.IL] DISTRIBUTION OF COMMA-BACILLI. 23

cholera, the more numerous are the comma-bacilli found in
the lower ileum, so much so that in the very acute cases,
marked by the whole chain of symptoms characteristic of a
typical case of cholera, the lower ileum contains the comma-
bacilli "almost in pure cultivation": (e) no comma-bacilli
occur in the blood of the general circulation, in the mesenteric
glands, or any other organ.

The observations which I have made with regard to the
general distribution of the comma-bacilli enable me to say
this, that while agreeing with Koch in some, I differ from
him in other very essential, points. We shall take the above
statements seriatim.

(a) There can be no question about this important fact,
that in every case of acute cholera during the first days, i.e.
while the patient suffers from severe purging, the intestinal
discharges contain the comma-bacilli ; but there does not
exist, according to my experience, extending over a consider-
able number of cases, any definite relation between the
number of comma-bacilli present in the stools and the
severity of the disease. I have examined a good many stools
of patients during the first day or first two days of illness, all
the symptoms characteristic of typical cholera being present
— severe vomiting and purging of watery fluid containing
mucus-flakes, great fall of temperature, voice and face that
of cholera, suppression of urine, respiration very irregular
and oppressed — and yet the most careful examination of
fresh preparations and of preparations stained in the usual
manner revealed a few comma-bacilli only. In one instance
only have I come across a stool containing very numerous
bacilli. This stool was almost clear watery fluid in which
were suspended minute greyish flakes ; under the microscope
a great many comma-bacilli were found, and but few other
bacteria, the small mucus-flakes being almost like a pure

24 THE BACTERIA IN ASIATIC CHOLERA. [CH.

cultivation of the comma-bacilli. I must however state here,
that stools more or less fluid and of a faecal character (i.e. not
simply watery fluid containing mucus-flakes in suspension,
but with finely distributed particles of faecal matter) never
contained comma-bacilli in conspicuously large numbers.
They were present amongst crowds of other bacteria, either
as isolated, slightly curved commas, as semicircular or
circular corpuscles, some conspicuous by their small size, as
§-shaped or dumb-bell-shaped particles, and as shorter or
longer spirals. Only a few groups of them were present.

FIG. i. — FROM A PREPARATION OF FRESH MUCUS-FLAKES FKOM A CHOLERAIC
EVACUATION.

Showing large numbers of comma-bacilli and a good many minute straight bacilli.
Amongst the comma-bacilli there are a few small semicircular ones. Magnifying
power about 700.

(b] A much better insight into the distribution of the
comma-bacilli is obtained by examining the intestinal con-
tents taken directly from the small intestine, which of course
can only be done on making the post-mortem examination.
In some acute typical cases on opening the abdominal cavity
the small intestine appears much congested and distended,
and in its interior is present a grumous fluid not large in
quantity and of a brownish colour, containing amongst
particles of faecal matter mucus-flakes and small clots of
blood. In these cases the mucosa is streaked and dotted
with blood. In other cases of rapid death the whole of the
small intestine, including the upper part of the jejunum and

li.] DISTRIBUTION OF COMMA-BACILLI. 25

duodenum, is of a rosy tint ; here and there the colour is in
patches and more pronounced, the intestine is distended,
and on opening it a large amount of watery fluid escapes in
which are suspended very numerous flakes of various sizes.
The internal or mucous surface is pale, and the epithelial
layer is in many places loose or separating in larger or
smaller flakes. Placing a piece of the intestine under water
this loosened or detached condition of the epithelium
becomes very conspicuous. In the lower ileum rarely and

FIG. 2.— PREPARATION OF MUCUS-FLAKES FROM THE ILEUM OF AN ACUTE
CASE OF CHOLERA.

1. Masses of single comma-bacilli.

2. Circular forms.

3. Semicircular forms.
Magnifying power about 1,400.

then only in a few places are present small particles of faecal
matter adhering to the mucous membrane, but as a rule only
watery fluid and mucus-flakes are present. Such cases
correspond to Koch's " pure " cases ; the symptoms during
life are always vehement and well pronounced, and death
ensues generally during the first twenty-four hours. There
is much purging with rice-water stools. In such cases,
according to Koch, the lower ileum is almost a pure cultiva-
tion of comma-bacilli. But I cannot confirm this statement.

26 THE BACTERIA IN ASIATIC CHOLERA. [CH.

I have seen several such pure cases of acute cholera in
which the mucus-flakes suspended in the fluid of the cavity
of the small intestine, and the epithelial flakes loosened but
still adhering to the mucous membrane, were very carefully
examined in stained specimens, but there was rarely such a
condition as an almost pure cultivation of comma-bacilli.
There were present in the fluid and in the flakes crowds of
bacteria ; in some instances and in some flakes the comma-
bacilli were extraordinarily numerous, and almost in a state
of purity, in others they were scarce — and in fact there were

FIG. 3.— PREPARATION OF MUCUS-FLAKES FROM THE ILEUM OF AN ACUTE
CASE OF CHOLERA.

1. Bacterium termo, probably v. Emmerich's bacillus.

2. Comma-bacilli.

3. Minute straight bacilli.

Magnifying power about 1,400.

some cases where there was difficulty in finding them.
What always appeared to me a curious point and difficult to
understand was this : in several post-mortem examinations
of pure acute cases the anatomical characters of the whole
of the small intestine, jejunum and ileum, and its contents
were very much the same, yet on microscopic examination
of the fluid and the mucus-flakes it was generally found
that the number of bacteria present in the jejunum were
very small, and gradually increased towards the lower ileum;
and this held good not only for the comma-bacilli but also

n] DISTRIBUTION OF COMMA-BACILLI. 27

for the other bacteria present. In addition to this a com-
parison of the different cases which came under my observa-
tion showed this significant fact, that in many instances in
which the post-mortem examination was delayed, the number

FIG. 4. — FROM A PREPARATION OF FRESH MUCUS-FLAKES FROM THE LOWER
PART OF THE ILEUM OF A TYPICAL RAPIDLY FATAL CASE OF CHOLERA.
(Duration of illness nine hours and a half, post-mortem examination after
one hour.)

The forms here delineated are met with in the same mucus-flakes.

a. Masses of minute comma-bacilli.

b. Masses of typical choleraic comma-bacilli.

c. Minute circular and semicircular comma-bacilli.

d. Large thick comma-bacilli.

e. Masses of the minute straight bacilli.
/. Micrococcus and thick straight bacilli.

Magnifying power about 700.

of comma-bacilli and other bacteria was likely to be greater
than when the examination was made immediately or almost
immediately after death.

28 THE BACTERIA IN ASIATIC CHOLERA. [CH.

In the following (copied from the Report on Cholera by
the English Cholera Commission : An Enquiry into the
Etiology of Asiatic Cholera] is given a tabular statement of
the occurrence of bacteria in the mucus-flakes taken from
the lower part of the ileum of typical rapidly fatal cases, the
ileum being slightly reddened and filled with clear fluid in
which were numerous typical flakes. The numbers attached
to the cases indicate the number in the total series of cholera
cases examined in Bombay and Calcutta.

1. Case 2. — Death after 40 hours. Post-mortem made
after four hours. Comma-bacilli abundant, small and large
straight bacilli.

2. Case ii. — Death after 18 hours. P.m. after half an
hour. Comma-bacilli tolerably numerous ; they vary in
length, and particularly in thickness. Large straight bacilli
exceedingly numerous ; minute straight bacilli.

3. Case 14. — Death after 12 hours. P.m. after half an
hour. Comma-bacilli very scarce. Few other bacteria.

4. Case 1 6. — Death after 18 hours. P.m. after three-
quarters of an hour. Very few comma-bacilli. Exceedingly
numerous small straight bacilli, singly and in clumps.
Other kinds of bacteria.

5. Case 23. — Death after 20 hours. P.m. after one and a
half hours. Various species of bacteria ; micrococcus, bac-
terium termo. Very few comma-bacilli ; they are distinctly
thinner than those of other cases. Minute straight bacilli
in clumps.

6. Case 32. — Death after 27 hours. P.m. after two hours.
All kinds of straight bacilli in great numbers. The small
straight bacilli numerous. Comma-bacilli tolerably numerous ;
they are of different lengths and thicknesses.

7. Case 35. — Death after 13 hours. P.m. after a quarter
of an hour. Comma-bacilli tolerably numerous ; large

II.] DISTRIBUTION OF COMMA-BACILLI. 29

straight bacilli tolerably numerous. The small straight
bacilli exceedingly numerous.

8. Case 48. — Death after 14 hours. P.m. after half an
hour ; great abundance of comma-bacilli, and also numerous
minute straight bacilli.

9. Case 51. — Death after 9^ hours. P.m. after one hour;
various kinds of bacilli. The minute straight bacilli in
extraordinary numbers. Comma-bacilli of three different
kinds distinguished by their various thicknesses, some
exceedingly minute, others five and six times as big, and a
third variety corresponding in length and thickness to the
typical comma-bacilli of other cases. The first variety in
very large numbers, forming continuous masses. Numerous
small semicircular commas, corresponding in size to the
small variety of the above commas.

All these organisms were numerous in the free flakes, as
well as in those still on the mucous membrane.

Other cases, which were typical and rapidly fatal, but in
which the ileum did not contain the clear watery fluid with
mucus- flakes, are not included here.

Drs. Weisser and Frank ascribe to me in the Archiv. f.
Hygiene iii. 1. p. 380 the assertion: "That in very rapid
cholera cases the comma-bacilli are missed." That this is
not my assertion I have stated above ; where these gentlemen
got hold of it I cannot say, unless, like Koch, they got their
information about my statements at second hand. Examin-
ing the tables published by Weisser and Frank (I.e. pp. 382-
389) — in which they give an account of the examination of
cover-glass specimens sent them by Dr. Dissent of Calcutta,
and made of the contents of the intestine in numerous cases
dead of typical cholera — it will be seen that out of thirty-
one cases dead within twenty-four hours (seven to twenty-
four hours), in fourteen the comma-bacilli were scarce

30 THE BACTERIA IN ASIATIC CHOLERA. [CH.

( wenige or sparliche ), in one none could be found; then
out of twenty-four cases dead between twenty-four and forty-
eight hours there was one without the comma-bacilli, one
was questionable, and in nine cases the comma-bacilli were
scarce ( sparlich or wreinzelt ). These facts then of Drs.
Weisser and Frank do not seem to agree with the con-
clusions they draw (I.e. p. 390), but they singularly harmonise
with my own statements, viz., that there does not exist any
definite and uniform relations between the severity and
rapidity of the disease and the number of comma-bacilli
present in the intestine, as was maintained by Koch.

(c] The statement of Koch that the comma-bacilli in the
acute stages are present in the tissue of the mucous
membrane of the ileum requires the most serious considera-
tion. If it were true that in the acute stages of the disease,
the comma-bacilli are constantly present in the tissue of the
mucous membrane in the definite manner described and
figured by Koch, i.e. in the epithelium and superficial mucosa,
around the Lieberkiihn's follicles, and in the peripheral
zones of the lymph-follicles of the Peyer's glands, then a
very important point in the chain of evidence would thereby
be established. One of the most essential and generally
acknowledged requirements in proving the connection
between a definite species of bacterium and the causation
of an infectious disease is the constant presence of this
particular species in the diseased tissues. Although complete
proof is not thereby given, yet it must be obvious that the
constant presence in large numbers of a definite species in
the diseased tissues cannot be of an indifferent nature. In
all those cases of infectious disease in which a definite
species of bacterium has been unequivocally proved to be
the cause of the disease, this constant presence of that
definite species of bacterium has been established.

II.]

DISTRIBUTION OF COMMA-BACILLI.

Take anthrax and relapsing fever, glanders and tuber-
culosis, erysipelas and leprosy. In the first two the blood
and spleen are the seat of the bacilli anthracis or of the
spirilla Obermeieri respectively, in the second two the mor-
bid deposits contain the bacilli of glanders or bacilli tuber-
culosis respectively, in erysipelas the lymphatics at the mar-

FIG. 5. — FROM A SECTION THROUGH THE ILEUM OF AN ACUTE CASE OF CHOLERA.

In this case no comma-bacilli were present anywhere in the tissue of the
mucous membrane.

1. Cavity of a crypt of Lieberkiihn, lined with columnar epithelium.

2. Nuclei of the membrana propria.

3. Space between the detached epithelium and the membrana propria ;

in it numerous straight bacilli.

Magnifying power about 1,400.

gin of the inflamed skin contain the specific micrococci,
and in leprosy the cells and tissue of the leprous nodules
and the lymph-spaces around them contain the bacilli leprce.
And the same holds good for other infectious diseases,
vaccinia, variola, swine-fever, swine-erysipelas, septicaemia,
pyaemic abscesses, &c. £c. If then in cholera the tissue of

32 THE BACTERIA IN ASIATIC CHOLERA. [CH.

the intestine, say the ileum, constained constantly in the acute
stages of the disease numbers of comma-bacilli, I should con-
sider this as of fundamental importance, and I should go so
far as to say that one of the most important links of the
chain of proof that they are the cause of the disease had
been established. I have therefore paid particular attention
to this point, and from a very careful examination of an
enormous number of preparations made of the ileum and
other parts of the intestine of acute and typical cases of
cholera, I am able most positively to assert that nothing of
the sort occurs1. Fresh sections and sections of the tissues
hardened in alcohol or Miiller's fluid, stained with the
different aniline dyes by the usual methods employed for the
demonstration of bacteria in tissues, were prepared and
examined ; the cases were typical and atypical ; some were
cases that died before the first day was over, all the symp-
toms during life were very characteristic, on post-mortem

1 Koch, in his second paper, "Further Researches on Cholera,"
(Zweite Conferenz zur Erorterung der Choltrajrage, Berlin, May, 1885)
says of myself: — " . . . Even before he went to India his judgment of
my statements was formed. He attempted, at that time, to show that
I had contradicted myself ; that I had, in Egypt, compared the bacteria
found in the wall of the small intestine with the bacilli of glanders,
but that the latter were not curved, but straight bacilli ; then all at
once, in India, the straight bacilli had become curved ones." What
may have prompted Koch to write this I am unable to say, but this
much I can positively say, that at no time or place, neither before I
went out to India, nor in India, nor since my return, have I said or
written anything of the sort. Koch has evidently been misinformed,
and after this I am under the impression that Koch has entered on
a criticism of my work without having read what I said. I am sorry to
think that he ascribes to me anything so absurd ; for it would no doubt
be absurd on my part to try to make out that the glanders-bacilli were
not curved after having myself figured them as curved (see my Micro-
organisms and Disease, Fig. 62, third edition, 1886) ; and it would be
equally absurd and incorrect on my part to say that Koch had stated
while in Egypt that the cholera-bacilli were straight, but that all at
once, in India, they became curved. I am sure such criticisms
would not have been applied by Koch to myself if he had read what
I did say.

ii.] DISTRIBUTION OF COMMA-BACILLI. 33

examination the condition of the intestine was such as would
merit Koch's term of pure acute cholera ; the post-mortem was
made as soon as possible after death, in some instances as
early as a quarter of an hour, in others one and a half to
two hours, but I did not find anything that showed the
presence of comma-bacilli in the intestinal mucous mem-
brane, not even in the superficial epithelium where this had
kept its position. Such appearances as are described and
figured by Koch,1 or anything approaching them, were not
met with in one single instance. But there were cases under
observation in which the tissue did contain a few comma-
bacilli besides other bacteria, and I will describe these here
more in detail.

In two cases only were there present in sections through
the Peyer's glands near the ileo-caecal valve comma-bacilli
in some places around Lieberkiihn's crypts, and also scat-
tered here and there amongst the superficial parts of the
lymph-follicles. But besides the comma-bacilli, and in greater
numbers, were straight bacilli, which with the comma-bacilli
could be traced from the broken surface into the depth of
the mucosa. As one of these is a good example of comma-
bacilli being found in the mucosa, but accompanied by a
larger number of straight bacilli, we will give the history of
this case. The patient, set. thirty, was attacked with vomit-
ing and purging at 4.30 p.m. on the 6th October, he was
admitted into the J. J. Hospital, Bombay, at 7.30 p.m. on
7th October. When admitted he was deeply collapsed,
pulse imperceptible, features sunken, extremities cold, no
urine. He died at 6 a.m. on 8th October. Post-mortem at
8.30 a.m. The patient was evidently moribund from 7.30
p.m. of 7th October till 6 a.m. of 8th October, i.e. for
1 Loc. cit. p. 6, fig. i.

D

34 THE BACTERIA IN ASIATIC CHOLERA. [CH.

nearly twelve hours; in addition to this the post-mortem
was made two hours and a half after death ; the tempera-
ture of the air was above 75° F. No wonder that under all
these circumstances the tissue of the bowels should have
become invaded by micro-organisms. In another case of
acute typical cholera, where the post-mortem had been
made fourteen minutes after death, but where the patient
had been moribund from 9 a.m. till 3 p.m., sections through
the hardened Fever's glands andmucosaoftheileum showed
the epithelium of the surface as well as that lining the Lieber-
kiihn's follicles bodily loosened and raised from the mucosa,
but fixed in position during hardening. While there was
total absence of comma-bacilli here or anywhere else in the
mucous membrane and lymph-follicles, there were neverthe-
less in some places on the surface minute groups of putre-
factive bacillus subtilis, and from here they could be traced into
the spaces resulting from the detachment of the epithelium
of the Lieberkiihn's follicles from the membrana propria.
And even capillary blood-vessels of the lymph-follicles near
the denuded surface were found crowded with putrefactive
bacilli and micrococci. In a third typical case (death after
ten hours, post-mortem after half an hour), there were pre-
sent numbers of straight putrefactive bacilli in the tissue of
the villi and around the bottom of the Lieberkiihn's follicles,
but only here and there could a comma-bacillus be found
close to the epithelium of the surface.

From this then we conclude that comma-bacilli as well
as other bacteria can find entrance into the tissue of the
intestine, but that this in a measure depends on the state
of disorganisation of the intestine, and the time that elapses
between the stage of " agony " and actual death. That the
comma-bacilli take the lead in penetrating the tissue, both

II.] DISTRIBUTION OF COMMA-BACILLI. 35

as regards depth and number, as is maintained by Koch in
regard to the acute stage of cholera, is not borne out even
in those cases that are particularly favourable to the immi-
gration of bacteria from the surface into the depth of the
tissue.

To say then as Babes does,1 that in the material (intes-
tine) from a case of cholera, which had been preserved in
alcohol for some years, he found comma-bacilli in the tissue,
means nothing whatever, since it is not stated how long after
death the intestine had been left before being removed to
the hardening fluid. The same negative value is to be
attached to the statement of Mr. Watson Cheyne, who says
that after repeated examinations he found the comma-bacilli
in the tissue, though he at first missed them. As has been
mentioned just now, I have myself examined such prepara-
tions, but there was always clear evidence that the tissue was
invaded also and more numerously by other bacteria, or that
the tissue in which the comma-bacilli were present was near
the surface, and in a state of necrosis or profound alteration.
Judging from the numerous examinations of sections of
cholera-intestine that have been made by various observers
during the last few years, there has not been one confirming
their usefulness for the purpose of diagnosis. Klebs, Van
Ermengem, Von Emmerich, Buchner and others, have all
questioned the statement of Koch.

A point of importance in interpreting the occurrence of
bacteria in the diseased intestine is, that it is necessary to
bear in mind that bacteria can penetrate during life into the
tissue of even a perfectly healthy intestine. Bizzozero was
the first to show 2 that in the tissue of the lymph-follicles
constituting the Fever's glands in the rabbit there occur,

1 Virchmtfs Archiv, 1881.

2 Centralblatt f. d. med. Wiss. 1885.

D 2

56 THE BACTERIA IN ASIATIC CHOLERA. [CH.

even during life, and in the perfectly normal state, bacteria
which can be proved to have penetrated there from the free
surface. These bacteria are present between the young
cells or enclosed in large lymph-cells, these playing prob-
ably the part of scavenger-cells (or phagocytes of Metchni-
koff), inasmuch as they swallow and destroy the bacteria
that had penetrated into the lymphatic tissue. I have re-
peated these experiments of Bizzozero, and I can fully con-
firm his observations. The very first rabbit examined with
this object yielded positive results. A perfectly normal and
healthy rabbit is killed by decapitation, the abdomen
opened, with clean and sterile forceps the serous coat and
outer muscular coat are gradually stripped off the so-called
vermiform process, which is in reality the beginning of the
caecum ; this as is well known is really one continuous mass
of lymph-follicles. Having exposed the deepest part of the
mucous membrane containing the lymph-follicles, with the
sterilised blade of a scalpel a scraping is taken of the deep
part of the lymphatic tissue, and with this scraping cover-
glass specimens are made, dried, and stained in gentian-
violet. On microscopic examination small bacilli are found
scattered and isolated between the nuclei of the lymph-cells,
and here and there a huge lymph-cell — five to eight times
the size of an ordinary lymph-cell — is met with, the proto-
plasm of which is crowded with the same small bacilli.

From this it follows then, that while in the perfectly
normal state bacteria can make their way from the free
surface, or internal cavity abounding with bacteria, into the
tissue of the mucous membrane, their penetration will be no
doubt considerably facilitated if the wall of the intestine is
in a state of disease and disorganisation ; for, as is well
known, living and healthy tissues do not favour, but on the
contrary are inimical to, the existence of septic bacteria.

ii.] DISTRIBUTION OF COMMA-BACILLI. 37

An important line of research is hereby opened up, namely
to enquire to what extent in the human subject do the
bacteria normally present in the cavity of the intestine
penetrate into the wall of the intestine ? It must be clear
from the above that statements as to the occurrence of one
or other species of bacteria in the tissue of a diseased
intestine, e.g. in typhoid fever, cannot claim that significance
which has hitherto been attributed to them. In typhoid
fever the disorganisation of the intestinal wall is very
profound, and lasts days and weeks ; there is no reason why
bacteria, particularly motile bacilli, such as the so-called
typhoid bacilli are, should not penetrate deep into the
intestinal wall and thence into the mesenteric glands,
particularly into the foci of inflammation and necrosis
always present in these glands in typhoid fever, and even
further into the necrotic foci of the spleen. And the same
applies to the choleraic intestine ; in some cases, particularly
those remaining for some hours in articulo merits, or kept
for some time after death before examination is made, the
motile comma-bacilli and other motile bacilli of the internal
cavity can penetrate into the tissue of the intestine, par-
ticularly as the mucous membrane is in a profound state of
disorganisation, and, as has been noticed and described by
various observers, also into more distant localities, e.g. the
liver and gali-bladder.

(d) As I have mentioned under (b), I cannot confirm the
statement of Koch — that the purer and the more typical and
acute a case of cholera, the more does the lower part of the
ileum contain an almost pure cultivation of the comma-
bacilli. Although I have found in some typical acute cases
that the mucus-flakes of the contents of the lower ileum
contained comma-bacilli in large numbers and continuous
masses, I have seen others where these comma-bacilli were

33 THE BACTERIA IN ASIATIC CHOLERA. [CH.

few and far between ; and besides whenever the comma-
bacilli were very abundant various other forms of bacteria
were also present in great numbers. I refer to the list of
cases given on a former page (p. 28).

(e) I can fully confirm Koch's statement that no comma-
bacilli or other bacteria occur in the blood and in the viscera.
The blood was examined fresh from living patients in various
stages of the disease, in some the disease had not lasted
more than six hours, in others sixteen hours, in others twenty-
four hours and more. But there were no bacteria of any
known forms present in the blood.

Sections of the kidney, liver, spleen, mesenleric glands,
made fresh and after hardening, and stained with the usual
dyes, were made in large numbers, but no organisms of any
known characters were met with. In the gastric discharges
(vomit) of cholera patients the comma-bacilli are rarely
present, as Koch has already shown ; I have myself examined
six cases, but have not found them more than once, and then
only in very small numbers indeed.

As a result of this part of the subject we find then (i)
that comma-bacilli are constantly present in the intestinal
contents in acute cases of cholera;1 (2) their number
cannot be said to have any definite relation to the acuteness
and severity of the illness, since in some typical and acute

1 Koch charges me in his paper read at the Second Conference on
Cholera, held in Berlin on May 4, 1885, with having had to admit that
true cholera-bacilli occur in all cases of cholera. From this it might be
inferred, as is also definitely stated by Drs. Weisser and Frank (see
above), that I at first did not admit such a fact, but finally bad to admit
it. This is another instance of the manner in which Koch criticises
those who differ from him, for I am not aware of having ever said that
the choleraic comma-bacilli do not occur in all cases of cholera ; there
was therefore no occasion for me to alter my statement.

IL] DISTRIBUTION OF COMMA-BACILLI. 39

and pure cases they were present only in small numbers
together with multitudes of other bacteria ; (3) there are
no comma-bacilli or other bacteria present as a rule in the
tissue of the intestine of acute typical cases of cholera,
although in some cases they may penetrate from the cavity
into the wall of the intestine.

CHAPTER III.

MORPHOLOGY OF THE CHOLERAIC COMMA-BACILLI.

A. — The Comma-bacilli of Cholera-stools and of the Intestinal
Contents.

As has been described by Koch, the single comma-bacillus
is a minute rod more or less curved, being a portion of a
small or large circle. Owing to this shape Koch named it
the comma-bacillus, a name which I think unfortunate and
inappropriate. As Koch has shown, and as we shall see
more fully below, there can be no doubt about the single
comma-bacillus being in its full development an element of
a vibrio or spirillum, and for this reason it is not appropriate
to speak of the species as Bacillus ; and for the same reason
the name cholera-bacillus is not more acceptable ; it implies,
besides, that this form of bacterium is peculiar to cholera ;
now although, as will be shown below, this peculiar shape of
the organism was unquestionably in the mind of Koch (see
loc. cit. p. 25) when he described his observations in Egypt,
India, and France, it is now known that these so-shaped
organisms, i.e. comma-bacilli, occur under several conditions
other than cholera.

The comma-bacillus, then, occurs in the intestinal dis-
charges and in the contents of the lower ileum, chiefly as

CH. in.] CHOLERAIC COMMA-BACILLI. 41

single and S-snaPed roc^s> tne single rods varying in length
between 0*6 and 1*2 /x : their thickness is about o-2 p. The
greatest differences exist as regards the amount of curvature.
While some, particularly the short examples, show only a
slight curve, noticeable more at the ends, there are close by
in the same particle of the mucus-flake numbers of others
which are unmistakably curved. Then one always finds in
the same flake, particularly in cases in which the comma-
bacilli are abundant, numbers of comma-bacilli in clumps or
in streaks, where the majority of the elements show only
just an indication of curvature, while others show a distinct
curve, some being curved as much as one-half to two-thirds
of a circle. Several such instances were examined, in which,
in the mucus-flakes, large numbers of semicircular and still
more curved elements were present. As regards the mucus-
flakes, this can be stated with certainty — that the more rapidly
the multiplication of the comma-bacilli proceeds, that is to
say, the more they occur in groups, patches, or streaks, the
less pronounced is the curvature in the single elements.
And the same may be said as to the length of the single
elements : the more rapid their multiplication in the flakes,
the shorter are the majority of the elements.

Great differences occur also in the thickness of the
comma-bacilli in the intestinal contents. While the typical
comma-bacilli are about the thickness of 0*2 ju,, there are
always present some that are twice and thrice as thick, and
there can be no question about this fact, that in many
specimens taken from the intestinal contents of typical
acute cases, there occur comma-bacilli differing from one
another in length and thickness within such limits that one
might well doubt their belonging to the same species. I
refer in this respect to the case 51 described on a former page
and to Fig. 4. Here the individuals constituting the groups

42 THE BACTERIA IN ASIATIC CHOLERA. [CH.

and masses of short, thin, very slightly-curved comma-bacilli
represented at a are conspicuously different from the typical
well-curved ones represented at b and c.

Very interesting forms of the comma-bacilli are those in
which the curvature amounts to half or two-thirds of a circle,
or almost a whole circle. These forms are scarce in some
typical stools and mucus-flakes, in others they are tolerably

FIG. 6. — FROM A PREPARATION OF MUCUS-FLAKES FROM THE LOWER ILEUM,

WHICH HAD BEEN ALLOWED TO UNDERGO PUTREFACTION FOR THREE DAYS.

Magnifying power 700.

abundant. I have specimens of the stools of a patient ill
with cholera a few hours only, in which the circular and
semicircular forms were the only conspicuous forms ; they
were of two different sizes, some about half the size of others.
Then I have specimens of the mucus flakes from the ileum
of cases that died within the first day, in which these forms
are very scarce, while in one case dead of typical acute
cholera in 9-^- hours the number of small circular and semi-

in.] CHOLERAIC COMMA-BACILLI. 43

circular forms is very conspicuous. Here also some were
larger than others.

I have for a long time searched for an explanation of these
forms, and I think I have found it. One cause of their
abundant appearance seems to be this : the comma-bacilli,
by transverse division, and by remaining joined end to end,
occasionally form marked spirals, in which the elements are
so much curved that the spiral possesses very close turns ;
when such a spiral is broken up into its constituent elements,
we obtain semicircular and almost circular commas. That
this is so I have ascertained in several instances, by com-
paring fresh specimens with dried and stained ones. While
in the perfectly fresh specimens in these cases a good many
spirals with closely-twisted turns were to be made out, after
drying a thin film on the cover-glass, staining and mounting
it, spirals were absent, being evidently broken up into the
numerous semicircular forms then present. Another mode
of the formation of these will be given below. Why there
should be numerous close spirals in some cases and not in
others I cannot say ; but it seems to me that the solidity and
resistance of the medium in which they grow has something
to do with it, i.e. where the material in which they grow is
and remains solid and owing to the resistance offered to .the
dividing comma-bacilli, these are limited in the area of their
expansion ; hence those which after continued division re-
main joined endways, become pushed more closely together,
become more curved, and form spirals of closer turns.
This, I am induced to think, is in some cases a true explana-
tion ; for not only do these forms abound in the depth of
solid nutritive material, such as Agar-agar mixture, which
remains solid, or in albumen-mixtures, but also in the solid
particles taken from the ileum of typical cholera cases. I do
not mean to say that other conditions do not determine the

44 THE BACTERIA IN ASIATIC CHOLERA. [CH.

presence of these forms, for I shall show below that the age
of a culture has also something to do with it.

All comma-bacilli when examined fresh show a rapid
rotatory movement ; this is obviously the more pronounced
the more curved they are ; it is very characteristic in the
" dumb-bells," in which the two elements are so arranged
that their curvature is directed in opposite ways, and in the
chains of close or open spirals. The dumb-bells just
mentioned are the result of the successive division of a
comma-bacillus ; they are generally S-snaPed and are very
characteristic and generally present along with the single
commas. But some dumb-bells also occur in which the two
elements are curved in the same direction, being more of the
shape of -^'~\ the figure of a flying bird. In the fresh stools
and fresh mucus-flakes chains longer than dumb-bells are
not frequent, although, as mentioned above, they do occur
isolated, and then chiefly in fresh specimens ; in dried and
stained specimens they are absent, evidently owing to the
facility with which they are broken up into single elements
and g-shaped forms during the act of preparation. Of these
chains and spirals more will be said below.

The single elements are rounded at their ends ; in some
cases, however, a slight thinning at the ends can be made
out with very high powers, such as a -f^ oil-immersion.
In dried and stained specimens the comma-bacilli of the
stools and intestinal contents appear uniformly stained, but
on careful washing after staining, it can be shown that they
consist of a delicate sheath with protoplasmic contents. One
peculiarity the comma-bacilli possess is that the stain is easily
taken out of them by alcohol, more easily than is the case
with many other bacteria. On spreading out on a cover-
glass a thin film of the mucus- flakes of the ileum, or of a rice-
water stool, drying well over the open flame, then staining it

in.] CHOLERAIC COMMA-BACILLI. 45

for five to ten minutes in a 2 per cent, watery solution of
Spiller's purple, or Weigert's gentian-violet anilin-oil, then
washing it in water, then just rinsing it once only with spirit,
then with water, drying and then mounting in Canada bal-
sam, many comma-bacilli of the typical lengths will be met
with which show this distinction between a faintly-tinted
sheath and the protoplasmic contents in the shape of two
stained particles, one at each end, with a faintly stained
interval, the lumps being rod-shaped and slightly curved ; the
curved state can be only made out with -^ oil-immersion lens.
I conclude from this that a single typical comma-bacillus is

<-»

.«*.

j

FIG. 7.— PREPARATION OF CHOLERAIC COMMA-BACILLI STAINED WITH GENTIAN-
VIOLET, AND AFTERWARDS WELL WASHED.

The differentiation between the sheath and protoplasm generally collected at the end
of each comma-bacillus is well seen. Magnifying power about 1,400.

composed of two slightly rod-shaped elements held together
in a common sheath. But there are numerous short comma-
bacilli, which contain only a single rod-shaped protoplasmic
element, situated at one end or occasionally also in the middle.
The longer the comma-bacillus the longer the protoplasmic
element. This enables us to say then, that the element is a
protoplasmic granule, more or less rod-shaped, and according
to its state of growth or elongation the comma-bacillus is
longer or shorter ; and further that when this element has by
transverse division given origin to two protoplasmic elements,

46 THE BACTERIA IN ASIATIC CHOLERA. [CH.

we have a comma-bacillus consisting of a common sheath,
and in it, at each end, a protoplasmic particle : finally, when
this sheath becomes divided transversely, the single comma-
bacillus has divided into two comma-bacilli. The shortest
comma-bacilli such as are present in all stools and mucus-
flakes, and which particularly abound where rapid multiplica-
tion occurs, are' composed of a sheath and a single proto-
plasmic element ; the longer and typical examples contain
two longer protoplasmic elements. The same structure can
by careful staining and washing be ascertained also in the
comma-bacilli of artificial cultures, not only of the choleraic
ones, but also of other species of comma-bacilli, as will be
mentioned below.

B. — Comma-bacilli in Artificial Cultivations.

The finest and most typical forms of comma-bacilli are
obtained by placing, after the manner of Koch, a few mucus-
flakes of the contents of the ileum of a fresh case of cholera
on linen, and keeping this in a glass dish and under a bell-
glass, on the inside of which a piece of moist blotting-paper
has been fixed ; in other words, in a moist chamber, at a
temperature of about 2o°-25°C. After twenty-four to
thirty-six hours the comma-bacilli have enormously increased ;
numerous S"snaPe(i and spirillar forms are met with. They
differ in no respect from those described in the fresh
intestinal contents. From an acute typical case of cholera,
dead within twelve hours, in the ileum of which there were
present large numbers of large and small mucus-flakes
suspended in a little watery fluid, masses of mucus-flakes
were taken and placed in a clean glass dish and covered up
with a glass plate, and left standing for three days.
Examination showed large numbers of various species of

in.] CHOLERAIC COMMA-BACILLI. 47

bacteria, and also crowds of comma-bacilli. Comparing
the comma-bacilli of such preparations with those made of
the same flakes while fresh, a marked difference was noticed :
in the former the comma-bacilli were obviously three or four
times longer and thicker than in the latter, and besides
there were present many long chains of comma-bacilli, wavy
but not spiral. On growing the comma-bacilli in 10 per
cent, gelatine and beef-broth, in plate-cultivations, after
three or four days the comma-bacilli are very finely curved
and all of about the same length, either single or

7rJ( ^

FIG. 8. — ARTIFICIAL CULTIVATION OF CHOLERAIC COMMA-BACILLI IN ALKALINE
PEPTONE GELATINE.

Magnifying power 700.

in fact, the uniformly finest and most typical forms I have
ever obtained were seen in plate-cultivations in (10 per
cent.) gelatine and beef-broth. In 10 per cent, gelatine,
beef-extract and peptone (i per cent.), or gelatine (10 per
cent), beef-broth and peptone (i per cent), the comma-
bacilli are also fine, but not so uniformly curved and typical
as in the former medium. In alkaline or neutral beef broth
during the first two or three days of growth at 35°-37°C.,
large numbers of very short comma-bacilli are met with
amongst longer ones and g-shaped ones ; but the curvature is
well-pronounced only in few, most of them are only slightly

48 THE BACTERIA IN ASIATIC CHOLERA. [CH.

curved. By this time there are also present a few long
wavy and spiral forms ; these latter increase in numbers as
growth proceeds ; the longest spirals, some extending over
one-fourth or one-third of the field of the microscope
(Zeiss's eye-piece 3, obj. D), I have found in alkaline
broth-cultures that had been growing at 35°C. for a week.
Some of them are spirally twisted, others more wavy, in
some one portion is a spiral, while another portion is wavy,
or apparently almost straight. In some well-washed stained
specimens the individual commas constituting the wavy or
spiral form can be recognised, in others there seems no

FIG. 9. — ARTIFICIAL CULTIVATION OF THE SAME COMMA-BACILLI AS IN-
PRECEDING FIGURE IN ALKALINE PEPTONE BROTH GELATINE.

The comma-bacilli are not so large and not so well curved.
Magnifying power 700.

differentiation to be made out. During the drying and
staining of the specimens many long spirals break up into
short spirals. In cultivations carried on in broth (alkaline)
at 35°-37°C. a pellicle makes its appearance already after
36-48 hours, this thickens and becomes more connected as
growth proceeds ; this pellicle is made up almost entirely of
longer or shorter spirals.

In Agar-agar mixture, alkaline and neutral (Agar-agar i per
cent., peptone i per cent., both dissolved in beef-broth), the
comma-bacilli are not so well curved and uniform in size as
in gelatine mixture. On growing the comma-bacilli in Agar-

in.] CHOLERAIC COMMA-BACILLI. 49

agar mixture for from several days to several weeks at
35°-37°C., a good many spiral forms are met with; some
have very close turns, others are more wavy, some contain
as many as ten to twenty and more turns, others one or two ;
a good many S-shaped ones are also present.

In an alkaline mixture of egg-albumen and Agar-agar
the comma-bacilli assume a very striking morphological
character ; they are all of a uniform appearance as regards
length and thickness, but there is hardly any curvature to be
noticed ; that they are not quite straight is seen when they

\ '.'

rt J

; J

FIG. io.— ARTIFICIAL CULTIVATION OF THE SAME COMMA-BACILLI IN ALKALINE
BEEF BROTH.

Magnifying power 700.

are in groups : looked at singly or situated at intervals many
look only slightly or not at all curved, and it would not
be easy to identify them as comma-bacilli. They are dis-
tinctly pointed at the ends, and longer than the normal
ones. A preparation from a gelatine-culture compared with
one from this medium shows apparently two totally different
organisms; no one would recognise them as the same
organism (see Fig. 13). Yet there can be no question
about it, since on changing the medium by transferring
them from nutritive gelatine into egg-albumen and Agar-agar,

£

50 THE BACTERIA IN ASIATIC CHOLERA. [CH.

or vice versa, they change their morphological characters
accordingly. I have examined numerous specimens from
such a culture-tube of egg-albumen and Agar-agar, after
several days' to several weeks' growth at 35°-37°C., and
although there was copious growth present I did not see any
spiral forms. The reason is obvious, namely, that since the
individual comma-bacilli are almost straight, or at all events
only very slightly bent, their chains are more like leptothrix :
a spiral can of course only be produced by a chain of
well-curved organisms ; and just as a leptothrix is made up
of a chain of straight bacilli, so a spiral is one of curved
organisms. This holds good also for the S~snaPed forms
of the comma-bacilli ; in the egg-albumen and Agar-agar
mixture there are numerous dumb-bells, but they bear not
much resemblance to the typical S~snaPed forms of the
comma-bacilli, since the elements in the former are more
or less straight.

There can, then, be no doubt about the fact that in the
different artificial media the comma-bacilli show distinctions
as regards size and curvature. The most striking difference
is that shown by comma-bacilli cultivated in an alkaline
mixture of egg-albumen and Agar-agar.

Of very great importance, both from a morphological and
from a physiological point of view, in the life-history of all
bacterial species, is the question of spore-formation.
Hitherto in those species only which are known as bacilli
has real spore-formation been observed. In some species
of bacillus the single rod or part of a chain or leptothrix-
thread is capable of producing at the expense of its
protoplasm bright glistening spores, spherical, or more
generally oval in shape ; and these spores when fully
developed leave the sheath of the bacillus and represent the
real seed, for they are capable of retaining vitality for an

in.] CHOLERAIC COMMA-BACILLI. 51

indefinite time, and when planted in suitable soil germinate
again into rods, and these by elongation and transverse divi-
sion give origin to two new bacilli, each of which continues
to multiply by division. The formation of such spores,
possible only under certain favourable conditions, such as
free access of oxygen, suitable temperature, and moisture,
constitutes the final step in the life-cycle of a bacillus, as it
does in that of the higher fungi and higher plants.

Where owing to the nature of the bacilli or to unfavour-
able conditions (such as the absence of free oxygen in the

S

FIG. n. — FROM A CUITIVATION OF CHOLERAIC COMMA-BACILLI IN LIQUEFIED
GELATINE, AFTER SEVERAL WEEKS.

case of Bacillus subtilis and anihrads] spore-formation
does not set in, the bacilli, having multiplied as long as
the nutriment lasts, undergo finally a retrograde change,
consisting in the breaking-up and disintegration and death
of their protoplasm. This is probably due to the poisonous
nature of chemical substances produced by the bacilli
themselves. Such a culture ultimately becomes devoid of
living protoplasm, and is incapable of starting new growths.
These conditions are well-known and have been studied

E 2

52 THE BACTERIA IN ASIATIC CHOLERA. [CH.

and ascertained by many competent observers, amongst
whom particularly Cohn and Koch may be mentioned.

If we then find, as is the case with the various species of
micrococci and Bacterium termo, that a culture of an
organism, kept under the most favourable conditions for the
formation of spores, loses after some time the power of
starting a new crop in a suitable medium, we are justified in
saying that in such a culture no living or life-giving particle
is present, no spore has been formed. Looked at from
this point of view, I am in agreement with Koch, who from
experimental observations (described on a former page)
denies the formation of spores in the comma-bacilli and
spirilla. I have had during the last four years a large
number of culture-tubes of comma-bacilli in gelatine and
Agar-agar mixture, which after several months proved barren
of all life, although they were once good and active cultures.
I have had cultures made in Bombay during September
1884, which for some months contained a copious crop
of living choleraic comma-bacilli. After my return to Eng-
land in January 1885, they were tested and were found
capable of starting active and good fresh cultures of comma-
bacilli. And so they were found till May 1885, i.e. after
nine months. But after this time all life in them became
extinct. Subsequent experiments carried on on a large
scale have confirmed this ; many tubes tested in this respect
were, before the end of twelve months or earlier, proved
barren of all life. As regards gelatine-tubes, such a
condition, i.e. death of the growth, sets in in many instances
after four to five months, in others after six to eight months.
(Compare also E. Weibel's observations of other vibrios.)

Now from a morphological point of view it can be easily
ascertained that in almost all culture-tubes, say in gelatine
tubes after two to three weeks, in Agar-agar tubes kept at

in.] CHOLERAIC COMMA-BACILLI. 53

35°-37° C. earlier, many comma-bacilli and spirilla are met
with in which the protoplasm has become granular, and they
can be traced into forms in which the granules have become
free, leaving the pale sheath behind. A careful examination
leaves no doubt that these granules which in old cultures
are met with in masses, are the debris of dead organisms.
Hueppe describes the presence in the single comma-bacilli,

FIG. 12. — PREPARATION OF A PURE CULTIVATION OF CHOLERAIC COMMA-BACILLI
IN AGAR-AGAR MEAT -EXTRACT PPETONE, SEVERAL MONTHS OLD.

1. Semicircular forms.

2. Circular forms.

3. Spirilla.
Magnifying power about 1,400.

and in the spiral forms, and in a free state, of granules which
he maintains to be spores (Arthrospores), having, he states,
observed them in a few instances to germinate again into
comma-bacilli. Bearing in mind that, as we have already
shown (p. 45), the comma-bacilli in all forms show at the
outset a differentiation between protoplasmic contents and

54 THE BACTERIA IN ASIATIC CHOLERA. [CH.

sheath, and that the size, or rather length, of these proto-
plasmic elements depends on the phase of growth, part of
the above assertion of Hueppe — that those elementary
masses of protoplasm are capable of elongating into well-
formed comma-bacilli—is perfectly intelligible, without any
need for ascribing to them the character of spores. The
above observation of the final death of the cultures seems
to me to prove that they are not spores. The morphological
test alone is therefore unsatisfactory ; that is to say, the
presence of elementary or young masses of protoplasm in
the single comma-bacilli, or in those forming a spirillum or
in a free state, does not seem to me conclusive proof that

FIG. 13.— PREPARATION OF A CULTIVATION OF CHOLERAIC COMMA-BACILLI in

EGG-ALBUMEN AND AGAR-AGAR, TEN DAYS OLD.

The comma-bacilli show only a slight curvature, many of them are almost straight ;
all of them are d.slinctly pointed at the ends. Magnifying power about 1,400.

they are spores in the same sense as are the well-known and
well-characterised spores of, say, Bacillus subtilis or Bacillus
anthracis, since we recognised that condition as both intim-
ately bound up with the normal structure of a comma-
bacillus and as the result of disintegration.

With regard to the alleged spore-formation in the cholera-
bacilli, Koch says (loc. cit.) : — " As the question of the
presence of a resting-form of cholera-bacilli is down on our
programme, I will say a few words with regard to it. On
account of the importance of this question, I have done my
utmost to find something which could be looked on as a

in.] CHOLERAIC COMMA-BACILLI. 55

resting-stage of the cholera-bacilli, analogous to the spore-
formation of other bacilli, but I have, just as in my former
investigations on this point, obtained only negative results.
All the statements as yet made by other observers on the
resting-forms and spore-formation depend evidently on
errors." (Compare also Koch's experiments mentioned on
pp. 1 6 and 17.)

The complex process of spore-formation of the choleraic
spirilla described by Ferran, namely by the formation of a
peronospora, has not been confirmed ; and it is difficult to
come to any other conclusion as regards them than that
Ferran's cultivations were impure, and that accompanying

FIG. 14.— FROM AN ARTIFICIAL CULTIVATION OF CHOLERAIC MUCUS-FLAKES ON

DAMP LINEN.
Comma-b&cilli of the ordinary type. Amongst them are some much thicker containing

a vacuole.
Magnifying power about 700.

the comma-bacilli there were probably present other forms
as an accidental contamination, or that he mistook degener-
ative forms for indications of spore-formation.1

A curious degenerative change, besides the granular dis-
integration already mentioned, is observed in some Agar-
agar tubes, consisting in the swelling up of one end of the
comma-bacilli, whereby the ordinary comma-bacilli become
changed into pear-shaped or club-shaped, straight or more
or less curved and twisted, corpuscles. But these ultimately

1 Carillon, Ceci and others describe spherical enlargements and
vacuolation as spore- formation in the comma-bacilli.

56 THE BACTERIA IN ASIATIC CHOLERA. [CH.

undergo granular disintegration, so that their pale sheaths
alone are left.

An interesting modification is noticeable in the 'comma-
bacilli in the stools and mucus-flakes taken from the ileum
and grown on linen, by placing mucus-flakes on a piece of
linen and keeping this in a capsule under a bell-glass, inside
of which is a piece of wet blotting-paper, at a temperature
of 2o°-25° C. On examining microscopic preparations after
twenty-four, thirty-six, or forty-eight hours, the comma-bacilli
are found to have increased very greatly, and amongst the

FIG. 15.— PREPARATION OF A CULTIVATION OP CHOLERAIC COMMA-BACILLI
ON DAMP LINEN AFTER THIRTY-SIX HOURS.

1. Typical comma-bacilli.

2. Vacuolated comma-bacilli.

3. Circular and oval forms produced during the process of vacuolation.

Magnifying power about 1,400.

typical forms — single commas, Shaped and spiral forms —
numerous examples are found which are conspicuously
thicker, and include smaller or larger vacuoles, singly in the
centre or in a row of two or more. According to the size
of this vacuole the comma-bacilli are no longer convex
concave, but plane-convex or bi-convex. If the vacuole is
large they appear more or less circular. There can be no
question about this change, since almost in every field we
find every intermediate form between a typical comma-
bacillus of the normal thickness and a circular or oval form.

in.] CHOLERAIC COMMA-BACILLI. 57

In a stool from an acute case of cholera I have seen these
forms in enormous numbers ; in some fields of the micro-
scope the transitional forms between complete circles and
typical commas being very numerous. In neutral Agar-agar
mixture kept at the temperature of the room (between 18°
and 20° C.) after three, four, and more weeks, I have
seen this change occurring in many of the commas ; in
alkaline Agar-agar mixture I have also noticed it, but more
rarely. The best specimens I possess were made from
cultures in neutral Agar-agar about four to six weeks old.
Preparations made of such cultures show in some fields of
the microscope the typical comma-bacilli only in a few

W

FIG. 16. — FROM A RECENT ARTIFICIAL CULTIVATION OF CHOLERAIC
COMMA-BACILLI IN ALKALINE AGAR-AGAR JELLY.

Magnifying power about 700.

examples, most of them showing in some stage or other the
above vacuolation and transformation into circular or oval
organisms. Some of these, although already completely
rounded, nevertheless do not yet show in their interior a
complete cavity, but two or three small cavities separated by
remnants of protoplasm.

As the comma-bacilli from which these rounded or oval
forms take their origin are of different lengths, so do also
the sizes of these rounded or oval forms differ, the small
commas giving origin to small, the large commas to large,
rounded or oval forms. The question is, whether these

58 THE BACTERIA IN ASIATIC CHOLERA. [CH.

rounded forms are discoid or spheroidal. I am inclined to
think that some of them, at any rate, are discoid, because
in the fresh state, and suspended in fluid, I have noticed
under the microscope that when they float and roll they

O

FIG. 17.— FROM AN ARTIFICIAL CULTIVATION OF CHOLERAIC COMMA-BACILLI ON

NEUTRAL AGAR-AGAR JELLY AT ORDINARY TEMPERATURE, AFTER A FEW WEEKS.

Some of the comma-bacilli have become converted into circles, which by division

give origin to two semicircular comma-bacilli.

Magnifying power about 700.

present alternately a broad and narrow surface ; but there is
evidence that some are distinctly spheroidal, since when
rolling they always show a circular outline. The next and
important question is : What is the meaning of this change ?

*>*** ° * * f

FIG. 18.— FROM A SIMILAR FIG. 19.— FROM A SIMILAR

PREPARATION. PREPARATION.

Koch, in a somewhat impatient way, says that these forms
described by me (which, as will be shown presently, are the
first stages of a longitudinal division) are a supposed dis-
covery " resting on an erroneous interpretation of the in-
volutional (i.e. degenerative) forms of the cholera-bacilli."

in.] CHOLERAIC COMMA-BACILLI. 59

I do not think Koch has taken the trouble to examine more
carefully what I did say, for I distinctly stated that some of
these forms when examined fresh show as active a movement
as the comma-bacilli themselves. Surely this proves that
they are living. And by careful examination it can also be
shown that in many of these forms a single or double
thinning leading to deficiency of the wall of the round
organism is present, by which the organism divides into
two more or less semicircular comma-bacilli.

In mucus-flakes and fresh stools from some acute cases
such forms — namely completely circular organisms, and
circular organisms with one or two gaps in the wall at
opposite poles — are very numerous, and from the above
there can be little doubt that these owe their origin to the
same process of vacuolation and subsequent division. I
have got specimens of linen-cultures, of fresh stools, and of
Agar-agar cultures where the whole chain of changes is so
marked and so well illustrated that on a careful examination
one cannot help arriving at the conclusion just stated.
Some of these circular or oval forms show even three gaps
or fractures, so that one circle gives origin to three comma-
bacilli. And, finally, there are some in which the whole
protoplasm thins, and ultimately breaks away and disappears,
except a short rod or granule at one spot of the circum-
ference, while at the same time a discoloured or circular pale
sheath remains. I have before me specimens made from
fresh and active linen-cultures, where almost all and every
comma-bacillus is thus changed ; to speak of such specimens
as indicating degenerative changes seems to me unwarranted.
These forms are well shown in Fig. 15.

On the whole, then, I think I am justified in maintaining
that these circular and oval forms represent the initial stages
of a mode of division differing from the ordinary mode of

60 THE BACTERIA IN ASIATIC CHOLERA. [CH. in.

transverse division. This ordinary or typical mode, as
observed in various bacilli, consists in the elongation of a
single bacillus, and the subsequent transverse fission of this
into two new rod-shaped elements. That this is also the
ordinary mode of propagation of the comma-bacilli is proved
(a) by the different lengths in which comma-bacilli occur,
(b} by the S-shaped forms, (c) by the chains, wavy and more
or less spiral, representing in reality a series of comma-
bacilli placed end to end. But in our instances we have to
deal with a mode of propagation essentially different from
the above; for here a single comma-bacillus does not
elongate, but by vacuolation becomes thicker, and changes
finally into a more or less rounded or oval form, giving then
origin to two new comma-bacilli. This mode may be
described as the atypical mode of division, or division after
vacuolation.

CHAPTER IV.

CHARACTERS OF THE COMMA- BACILLI IN ARTIFICIAL
CULTIVATIONS.

THE choleraic comma-bacilli possess in artificial media
certain well-established characters, by which they can be
readily recognised. Koch has clearly pointed out this fact,
and has minutely described the appearances. An idea seems
to have got abroad that while in India I denied this simple
truth. I said then that the choleraic comma-bacilli do not
differ in this matter of artificial cultivation from other septic
bacteria ; and I still say so now, after admitting and confirm-
ing the correctness of Koch's observations as to the behaviour
of the choleraic comma-bacilli in nutritive gelatine. No one
who has carefully followed recent research, and has suffi-
cient practical experience in the artificial cultivation of
bacteria, can for a moment doubt that the different bacteria,
be they septic or pathogenic, have morphological and cultural
characters of their own, which in some instances are more,
in others less, pronounced ; but a good many species are
known in which these differences in mode of growth are
sufficiently striking to be of diagnostic value even to the
unaided eye. Take, for instance, micrococci derived from
the air. There are a good many species of micrococci

62 THE BACTERIA IN ASIATIC CHOLERA. [CH.

present in the air of ordinary city laboratories, which can be
made visible by simply exposing a number of sterilised glass
dishes, in which previously a thin layer of nutritive gelatine
or Agar-agar mixture liquefied by warmth has been poured
out and allowed to set over a layer of cold water. Each glass
dish is kept covered up with another glass dish, and the whole
kept in a moist chamber, or in other words on a glass plate
covered up by a bell-glass to which a piece of wet blot-
ting-paper has been fixed. This for all practical purposes
represents a good plate-cultivation after Koch's method.
On exposing for from several seconds to several minutes the
solidified layer of the nutritive medium, by lifting off the
upper covering glass dish, then closing it again and placing
the bell-glass in position and keeping the whole at a tempera-
ture of i9°-22c C. in the case of gelatine, or 35°-36° C. in the

FIG. 20. — PLATE-CULTIVATION iv GELATINE 48 HOURS OLD, SHOWING YOUNG
COLONIES OF CHOLERAIC COMMA-BACILLI.

case of Agar-agar mixture, for two, three, or more days,
various spots or colonies will show themselves on the surface
of the plate-cultivation, some small, others large, some round,
others irregular, some spherical, others disc-shaped, some
whitish or greyish, others yellow, orange, or of other colours,
some not liquefying the gelatine, others liquefying it ; of the

iv.] ARTIFICIAL COMMA-BACILLI. 63

latter some liquefy it with a smooth circular outline, others
with an irregular serrated line ; in some the liquefied gelatine
is clear throughout the circumference and extent of the colony
except for a central opaque speck ; in others the liquefied
area is uniformly turbid, and so on. By careful examination
and re-inoculation of nutritive gelatine or Agar-agar on
plates and in tubes the different species characterised by
the different appearances just named can be isolated and
studied.

The same can be observed in the case of bacilli and
bacteria, i.e. a different mode of growth as regards rapidity
of increase or size of the colonies after a certain time, and as
regards colour, aspect, outline, and microscopic characters
of the organism constituting the colonies. To say, therefore,
that such or such an organism in plate-cultivation and in
tubes presents such and such peculiar characters, means
nothing more than that such and such an organism is of a
definite species, and, as we have said, the greater majority
of the bacterial species are possessed of such special charac-
ters. I never said that the choleraic comma-bacilli cannot in
cultivation be distinguished from other bacteria. If any one
thinks I did, I can only answer that he has misunderstood
my meaning, and at the same time has failed to apprehend
the simple fact demonstrated by Koch himself, that almost
all the different species of bacteria show under cultivation
different cultural characters, by which they are more or less
easily distinguishable one from another. I say the possession
of cultural characters is not peculiar to comma-bacilli. This
is something quite different from saying that the cultural
characters of comma-bacilli are the same as those of septic
bacteria.

What are, then, the characters shown by the choleraic
comma-bacilli in artificial cultivations ?

64 THE BACTERIA IN ASIATIC CHOLERA. [CH.

On a former page it has been mentioned that very success-
ful cultivations of comma-bacilli can be made by placing
mucus-flakes of the rice-water stools or of the cholera in-
testine on linen, and keeping this damp in a covered glass
dish at 35°-37° C. After twenty-four hours crowds of comma-
bacilli are available, from which plate-cultivations can be then
easily made.

But this is only the case if the mucus-flakes initially
contain the comma-bacilli in considerable numbers. Another
method is this : with a particle of a mucus-flake or with a
minute droplet of the rice-water stool, a test-tube containing
sterile salt solution or sterile broth is inoculated, well shaken,
and from this a tube containing sterile solid nutrient gelatine
is inoculated, liquefied in warm water, and from it one or
more plate cultivations are established ; or if the comma-
bacilli are tolerably numerous in the stools a tube containing
sterile broth is inoculated with a trace of those materials and
then incubated at 35°-37° C. Owing to the rapid power of
growth of the comma-bacilli, after twenty-four hours the
fluid contains them very abundantly, and now a tube con-
taining sterile salt solution or broth is inoculated, well shaken,
and from it nutrient gelatine is inoculated with a trace of a
droplet (with the end of a platinum wire or capillary pipette) ;
this is liquefied and used for plate-cultivations. As will be
stated further below, when other bacteria are numerous but
the comma-bacilli scarce in the original materials (stool or
mucus-flakes) a number of plate-cultivations (after the usual
method of dilution) will have to be made in order to obtain
a few colonies of the comma-bacilli.

The appearances presented by these comma-bacilli when
grown in gelatine plates, in gelatine tubes, in Agar-agar
mixture, and on potato, in broth, milk, &c., are sufficiently
characteristic for all practical purposes of diagnosis.

iv.] ARTIFICIAL COMMA-BACILLI. 65

(a) In broth. — Faintly alkaline beef-broth, or broth to which
i per cent, of peptone is added and rendered faintly alkaline,
or meat-extract peptone (i large tin of Brand's meat-extract,
10 grms. of peptone in 1000 ccm. of distilled water made
faintly alkaline) are very good fluid media, and in them the
comma-bacilli multiply with great rapidity. A flask or test-
tube of these materials inoculated with the comma-bacilli
and kept in the incubator at 35°-37° C. is already slightly
turbid in twenty-four hours, every drop contains multitudes
of single commas and S-snaPed forms. After three to four
days the turbidity has greatly increased, and a slight powdery
precipitate is noticed. On the surface of the broth a thin
loose pellicle is noticed already after 2-3 days, this becomes
more complete as growth proceeds ; on shaking the tube the
pellicle easily breaks up into flakes or thin scales, many of
them remaining on the surface. The fluid remains thin, but
has distinctly changed its reaction, having become acid ; there
is no offensive smell, but rather a slight aromatic flavour:
The fluid cannot yet be considered exhausted, since even
after a week the turbidity remains unaltered, while the
precipitate increases. After about a fortnight the fluid
begins to clear, as it were in layers, beginning at the sur-
face and gradually extending to the depth. The precipitate
meanwhile increases, and the fluid becomes after the lapse
of weeks almost clear (in the upper part) to the unaided eye,
but on examining a droplet under the microscope moving
comma-bacilli, singly, but chiefly S~snaPe(i anc^ spiral
forms, can be easily detected. The precipitate, from its
first appearance, is made up of living comma-bacilli and of
the granular debris of dead ones ; in these the outline and
sheath can in many instances still be recognised, either with
or without granules ; besides these there are smaller and
larger clumps and masses of granular debris.

F

66 THE BACTERIA IN ASIATIC CHOLERA. [CH.

The comma-bacilli grow equally well in neutral brcth. I
have seen them grow fairly well even in faintly but distinctly
acid broth, but the amount of turbidity and of precipitate
was far less than in alkaline and neutral broth.

(b) In milk the comma-bacilli grow well, but not so
luxuriantly as in broth ; the appearance of the milk (as has

^fll

•

FIG. 21. — PLATE CULTIVATION WITH COLONIES OF CHOLERAIC COMMA-BACH. LI,
SEVHNTY-TWO HOURS OLD. MAGNIFIED FIFTY TIMES. FROM A PHOTOGRAPH
AFTER PLAGGE.

been pointed out by Koch) remains practically unaltered,
and the casein is not precipitated. Mr. Warrington (Journal
of the Chemical Society, 1888) has noticed curdling of milk to
take place by the comma-bacilli when growing at 3o°C.

(c) In Agar-agar mixture (Brand's meat-extract i tin, dis-
solved in ico ccm. of distilled water, to which is added 10

iv.] ARTIFICIAL COMMA-BACILLI. 67

grms. of Grubler's peptone, made faintly alkaline, boiled and
filtered ; add to this a filtered boiled solution of 10 grms. of
Agar-agar in 900 ccm. of water). Faintly alkaline beef
broth or beef infusion, to which are added i per cent. Agar-
agnr and i per cent, peptone, is also very good.

The comma-bacilli, sown at a point of the surface, grow
at 35-37° C. in the course of a few days into a translucent
film, with rounded or slightly indented outline ; this film
gradually enlarges in extent and becomes more or less
terraced, i.e. arranged in superimposed layers differing in
extent ; radiating lines probably due to shrinking of the
Agar-agar mixture are now to be noticed ; the central portion

FIG. 22. — FROM A CULTIVATION OF CHOLERAIC COMMA-BACILLI IN GELATINE IN
A GLASS DISH FOUR DAYS AFTER INOCULATION IN SPOTS. NATURAL SIZE.

is thickest and therefore more opaque than the periphery ;
the whole growth after a few weeks to a few months shows
in transmitted light a yellowish-brown tint with denser
brownish spots, in reflected light it looks whitish grey.
In streak cultivation a film starts from the line of inoculation
which rapidly (in two to three days) spreads out in breadth.

Sown into the depth of the medium, into a channel made
with the pointed end of a capillary glass tube or the
platinum wire, and kept at a temperature of35-37°C., the
growth is always noticed after two or three days as a whitish
line ; this gradually increases in thickness, and the growth
seen under a lens appears more or less granular.

(d) In vegetable albumen and Agar-agar mixture (Grubler's

F 2

68

THE BACTERIA IN ASIATIC CHOLERA. [CH.

vegetable albumen, such as the white substance of Brazil nut,
extracted with dilute alkali, filtered, precipitated, and then
dissolved in dilute alkali ; to the solution is added Agar-
agar in the proportion of i p. c.) forms a translucent solid
nutritive medium. The comma-bacilli grow in this medium
fairly well, but not so well as in Agar-agar and meat-extract
peptone. They form a thin transparent greyish film on the

FIG. 2§.— PLATE-CULTIVATION OF CHOLERAIC COMMA-BACILLI IN GELATINE

AFTER FOUR DAYS AT 19° C. NATURAL SIZE.

Many of the colonies have become confluent.

surface with an irregular outline ; when sown into the depth
they form a greyish line indicating the channel of inocula-
tion. But in neither case does the growth even after several
weeks reach considerable dimensions, and in this respect the
medium is very inferior to the Agar-agar and meat-broth
peptone.

(e)Egg-albumen and Agar-agar is much preferable and is

iv.] ARTIFICIAL COMMA-BACILLI. 69

nearly as good as Agar-agar meat-broth peptone. The
white of an egg, about 25 to 30 ccm., is dissolved in 220 ccm.
of distilled water to which 30 ccm. of liquor potassae is pre-
viously added. Boil till all is quite dissolved ; and after this
add 4 grms. of acid phosphate of potassium, whereby the
alkalinity is reduced but not quite neutralised ; then add i
p. c. of Agar-agar ; dissolve by boiling, filter, and decant into
test-tubes, which for sterilising are treated in the usual
manner. This egg albumen and Agar-agar mixture is of
good solid consistency even at 50° C., is beautifully trans-
parent, and is a very good solid nutritive medium for the
comma-bacilli, besides being very much cheaper (one egg
being greatly cheaper and easier to obtain than a pint of beef
broth or a tin of Brand's meat-extract) than the Agar-agar
meat-extract peptone. In this medium the comma-bacilli
grow in the same matmer as in the Agar-agar meat-extract
peptone mixture.

(/) On linen. — As Koch has pointed out, if the mucus-
flakes from the ileum of an acute case are placed on linen,
kept damp in a closed glass chamber at any temperature
between 20° and 36° C., a very excellent cultivation of
comma-bacilli is obtained. On examining these mucus-flakes
after twenty-four to thirty-six hours or a little later, crowds of
comma-bacilli are seen. In a fresh preparation many of
these are wavy or spiral chains or g-shaped forms, but on
making a permanent specimen by drying and staining most
of them are found broken up into single comma-bacilli.
T. R. Lewis has pointed out this difference (Appendix to
Report on Asiatic Cholera). After forty-eight hours, owing
to overgrowth of other bacteria present, the comma-bacilli
cannot be easily obtained pure ; but if originally the mucus-
flakes contain few other bacteria besides comma-bacilli, a
linen cultivation such as that described is a ready and

70 THE BACTERIA IN ASIATIC CHOLERA. [CH.

excellent method of obtaining the comma-bacilli in almost
a pure cultivation, or at any rate sufficiently pure to prepare
from it successfully pure cultivations either in plates or in
test-tubes. But if at the outset other bacteria are present in
great numbers, one's success in obtaining anything like a
satisfactory cultivation of comma-bacilli in test-tubes is
doubtful. (See above.)

fe) ?n gelatine the comma-bacilli show the best-marked
characters. Proceed after Koch's method by inoculating a
test-tube containing nutritive gelatine (beef-broth or beef-
infusion, gelatine 10 p. c., peptone i p. c., common salt i p.
c.,) with the platinum wire or the pointed end of a capillary
glass pipette charged with a tiny particle of a mucus-flake
from the cholera-stool or from the contents of the ileum of
an acute case of cholera (or — what for the purpose of class-
demonstration is much easier — with a trace of a culture con-
taining comma-bacilli pure or impure) ; then liquefy the
gelatine and shake it gently but sufficiently so as to distribute
well the introduced germs, and pour it out on a sterilised
glass or plate, or better still, into a flat-bottomed sterilised
glass dish sufficiently large to allow the gelatine to spread out
into a thin layer ; cover this immediately with a glass plate
or glass dish, and let the gelatine rapidly set over ice or
cold water, or by placing the glass dish on stone or metal
in a cool place ; place the dish in a moist chamber under a
bell-glass as mentioned in a former page, and keep it at a
temperature between i8°and 22° C. Within this range of
temperature the comma-bacilli develop sufficiently well,
while the gelatine remains solid.

After two, three, or four days, according to the tempera-

(If the number of comma-bacilli and other bacteria should be
great, it is best first to distribute a particle of the mucus-flake in
sterile salt solution or broth, and from this then to inoculate gelatine for
making plate-cultivations, as described on a former page.)

iv.] ARTIFICIAL COMMA-BACILLI. 71

ture, the first indications of the colonies of the comma-bacilli
make themselves visible as small round depressions some-
what greyish in colour. In another day each colony is a
circular or oval, translucent or pearly-looking speck from the
size of a millet seed to that of a small pea ; the smallest when
looked at obliquely still show the central pit, surrounded by a

FIG. 24.— A COLONY OF CHOLERAIC COMMA-BACILLI IN GELATINE, PEVENTY-TWO
HOURS OLD. MAGNIFIED 170 TIMES. FROM A PHOTOGRAPH AFTER KOCH.

greyish areola of liquefied gelatine, the larger ones showing no
central pit but a white granule in the centre, while the areola
of liquefied gelatine is greyish and translucent. Under a
magnifying glass this greyish liquefying colony appears more
or less uniformly and finely granular. In another day, while
the colony enlarges in diameter — considerably larger than is
stated by Koch, its outline is still smooth, circular or oval,

72 THE BACTERIA IN ASIATIC CHOLERA. [CH.

and just at the margin marked by a whitish line that looks,
on magnification, granular. The central opaque spot en-
larges, and is granular, and gradually shades off, as it were,
into the clear areola. In some plate-cultivations the outline
of such colonies is not smooth, but more or less serrated.
(Mr. Watson Cheyne incorrectly describes the irregular out-
line of the colonies as of constant character.) It is not
correct to say that the colonies have always a serrated
or irregular outline, nor that they have always a smooth out-
line. Nor is it correct to say that the colonies at their first
appearance have already a central white spot — a precipitate
of comma-bacilli in the centre — for I have seen in the same
plates colonies which were of the size of a pea, but did not
show the central spot, while others that were smaller
possessed it. If the number of colonies that make their
appearance in a given plate-cultivation is large, the contigu-
ous colonies soon become confluent, and then we obtain an
appearance something like that in Fig. 23, when the outlines
of the original circular or oval colonies with their central
spot and clear areola are still easily distinguishable. If the
colonies were originally of irregular outline, by their con-
fluence a correspondingly altered appearance is produced.
But the general character of the colonies is their central
depression and granulation and their peripheral more or less
translucent areola of liquefied gelatine. When the growth
has proceeded far enough and the original number of colo-
nies is sufficiently great, the whole plate cultivation will be
found liquefied in four or five days. There is in such
liquefied plates a sediment of greyish powder, and here and
there an attempt at something like a loose filmy pellicle ;
this latter does not, however, extend over the whole surface
of the plate, but is only present here and there in the form
of greyish scales. On examining under the microscope a

iv.] ARTIFICIAL COMMA-BACILLI. 73

droplet from any part of an early or advanced colony, there
are always seen multitudes of actively moving comma-bacilli
single or g-shaped and in spirals. A trace taken from the
central granular spot or precipitate shows multitudes of §-
shaped, wavy, or spiral forms more or less intimately matted
together in larger or smaller masses. The spirals on drying
and staining easily break up into single commas and S"
shaped forms.

Owing to the comparative slowness with which the
colonies of comma-bacilli in gelatine plate-cultivations make
their appearance, it is clear that if other more rapidly
growing bacteria, micrococci, and bacilli, which by their
growth are capable of liquefying the gelatine, have been
introduced in large numbers into the cultivation, the colonies
of the comma-bacilli will be difficult to demonstrate. This
is especially the case when one wishes to demonstrate by
gelatine plate-cultivation the presence of comma-bacilli in a
choleraic stool or in the contents of the ileum of an early
cholera case in which the comma-bacilli are originally only
sparingly present and are accompanied by multitudes of
other bacteria ; and we have already mentioned that this is
not at all a rare thing, but on the contrary is more common
than the reverse. In such cases the demonstration of the
colonies of comma-bacilli in gelatine plates is not very easy
of achievement. It is therefore necessary in such cases to
dilute considerably with sterilised neutral fluid — salt-solution
or broth — the particle of matter taken from the stool,
and from this dilution to inoculate with a droplet the
gelatine tube. It is obvious that owing to the relatively
small number of comma-bacilli originally present it is
necessary for success to start at the same time a series
of plate-cultivations. I have seen cases of cholera where
out of a dozen of plate-cultivations made by this method,

74 THE BACTERIA IN ASIATIC CHOLERA. [CH.

only one showed evidence of a colony of choleraic comma-
bacilli. The demonstration by plate-cultivation therefore of
the presence of choleraic comma-bacilli in a given sample of
a cholera-stool, or of the contents of the ileum, is in many
cases not such a simple matter as is represented by Koch

(A) Cultivation, in solid gelatine peptone broth, of straight mobile bacilli from the
fluid of the mouth after four days' growth, showing a funnel-shaped depression,
the lower part of the funnel filled With liquefied gelatine containing the growth
of the bacilli. Semi-profile view.
B) Same tube viewed in profile.

(C and D) Cultivations in alkaline gelatine of choleraic comma-bacilli after five
days' growth. In both tubes the inoculation had been made within a few
seconds from the same stock. The surface shows the well-known depression;
the channel in which the inoculation was made contains the growth of the
comma-bacilli, the gelatine is here liquefied. At the bjttom of the channel is a
whitish precipitate of masses of comma-bacilli.

and others, for its success depends in a great measure on
the relative number of comma-bacilli originally present.
In some cases of undoubted cholera the result of such an
examination is negative, while in others it is achieved only
by making numerous plate-cultivations at the same time. It
is however true that in some cases, namely where the

iv.] ARTIFICIAL COMMA-BACILLI. 75

comma-bacilli are originally present in very large numbers
in the mucus- flakes of the rice-water stools, a couple of
plate-cultivations reveal the presence of numerous colonies
of the comma-bacilli.

In nutritive gelatine contained in tubes the comma-bacilli
grow in a typical manner. On inoculating by first dipping

F G H

FIG. 25 continued.

(E) Cultivation of comma- bacilli from the fluid of the mouth (healthy).
F) Cultivation of choleraic comma-bacilli. In both E and F the medium is the
same (alkaline gelatine peptone broth), and the inoculations were made within
a few minutes.

(G) Cultivation of mouth comma-bacilli in gelatine peptone broth.
(H) Cultivation of choleraic comma-bacilli in alkaline gelatine peptone broth; the
inoculation was made on the surface.

the platinum wire or the pointed end of a capillary glass
pipette into a pure culture of comma-bacilli, and then
pushing it into the (solid) nutritive gelatine, and exposing
the tubes to a temperature of about 20° C, pure cultivations
of the comma-bacilli are obtained in a few days. After the
lapse of a couple of days the channel of inoculation be-
comes marked as a greyish line, this as growth proceeds

76 THE BACTERIA IN ASIATIC CHOLERA. [CH.

broadens, particularly near the surface ; the surface itself
then shows a drawing inwards or depression of the gelatine,
and as development proceeds this pit is converted into a

L M

FIG. 25 continued.

(I) Cultivation of choleraic comma-bacilli in alkaline gelatine peptone broth.
(K L and M) Cultivation of choleraic comma-bacilli in alkaline gelatine peptone.
In K and L an air-bubble occludes the upper end of the channel of inoculation.

funnel the mouth of which is occupied by an air-bubble.
The rest of the channel of inoculation is a greyish canal
filled with translucent liquefied gelatine, and at the bottom
of the canal is a whitish precipitate. During the subsequent

IV.]

ARTIFICIAL COMMA-BACILLI.

77

days the canal of liquefied gelatine broadens, the precipitate
increases, and at the end of ten to fourteen days the

• FIG. 25 continued.

(N O and P) Cultivation of choleraic comma-bacill in alkaline Agar-agar peptone
and meat-extract.

growth has the form of a wide cone or a cylinder filled wit
liquefied gelatine ; not only is there prseent a voluminous

78 THE BACTERIA IN ASIATIC CHOLERA. [CH. IV.]

whitish precipitate at the deep end, but also at the sides of
the liquefied gelatine there are present small granules easily
recognised under a lens, while the bulk of the liquefied
gelatine is translucent and almost clear. On the surface the
funnel-shaped depression occluded by an air-bubble is still
noticeable, but gradually diminishes and disappears as the
growth extends more and more laterally. The growth at
the end of three to five weeks has almost entirely liquefied
the gelatine through the whole depth of the original channel
of inoculation, and now the liquefaction proceeds deeper
and gradually invades the deepest parts of the gelatine, the
lower boundary of liquefaction being always marked by a
voluminous whitish precipitate. While the bulk of the
liquefied portion is tolerably clear there is present in most
tubes on the surface a kind of loose whitish film ; indeed I
have seen very few tubes in which the surface remained free
from it. Under the microscope the film is composed of
granular debris, and moving spirilla more or less matted
together. The above-mentioned funnel-shaped depression
of the gelatine, and the occlusion of it by an air-bubble
during the first week or two, are however not invariably
present ; they are not present if the nutritive gelatine is
weaker than TO per cent, gelatine; in 2 to 7 per cent,
gelatine the funnel-shaped depression and the occluding air-
bubble is wanting, the liquefaction proceeding rapidly, and
the surface end is from the outset marked by a drop of
liquefied gelatine. The progress of the growth is consider-
ably greater in such gelatine than in 10 per cent, gelatine.
On inoculating from the same culture of the comma-bacilli
two sets of tubes, one containing 5 per cent, the other 10
per cent, nutritive gelatine, and keeping them then under
precisely the same conditions at 20° C., a marked difference
will be found on inspecting the tubes after three to five days.

£§

£ a

II

8o THE BACTERIA IN ASIATIC CHOLERA. [CH.

Those containing 10 per cent, gelatine show the characters
above described, the channel cf inoculation is a thin greyish
line of liquefied gelatine, funnel-shaped at the top, and with
an occluding air-bubble, and at the bottom a small amount
of precipitate; the other containing 5 per cent, nutritive
gelatine, contains the growth in the shape of a broad cone
of liquefied gelatine, broadest at the top, but with no
funnel-shaped depression. The air-bubble is present in
10 per cent, nutritive gelatine, if during inoculation the
comma-bacilli are well pushed down into the channel, as is
sometimes the case when the inoculation is performed with
a trace only, and by the platinum wire. With such a method
of inoculation, most of the comma-bacilli are deposited at
the bottom, few or none remain in the superficial parts,
hence when active multiplication sets in, most of the growth
takes place in the depth and away from the surface. By
using a capillary glass pipette containing the comma-bacilli
for inoculation, it can easily be arranged that the channel of
inoculation receives comma-bacilli in its whole length. This
can be achieved by holding the bulb of the pipette with the
fingers, and thereby warming it, before withdrawing the pipette
from the gelatine; not only is hereby a deposit of comma-
bacilli ensured in the superficial layer also, but as a rule more
comma-bacilli are deposited here than in the depth, because
owing to the shape of the capillary pipette the channel of
inoculation is at the outset broader on the surface than in
the depth. When all these conditions are successfully
fulfilled, it will be found that only a slight funnel-shaped
depression will be noticeable after a few days, and con-
sequently no air-bubble ever marks the mouth of the
channel. The air-jbubfele, then, occluding the mouth of the
funnel cannot be ^regarded as something quite typical and
characteristic o? a gelatine tube-cultivation of choleraic

iv.] ARTIFICIAL COMMA-BACILLI. Si

comma-bacilli, as many observers state, but is dependent on
the strength of the gelatine, and particularly on the method
of inoculation; no doubt in 10 per cent, nutritive gelatine,
and under the same conditions of inoculation, e.g. by using
a trace for inoculation and by the platinum wire, a con-
spicuous funnel-shaped depression of the surface and
occlusion of it by an air-bubble will be noticed in most
instances, but this is absent if the gelatine is weaker than
10 per cent., and if the inoculation is carried out in a
different manner. Furthermore, I have seen very conspicu-
ous funnel-shaped depressions and occluding air-bubbles in
10 percent, nutritive gelatine tubes containing other than
choleraic comma-bacilli ; certain micrococci and several
species of bacilli that possess the power of liquefying
gelatine, when inoculated by means of a platinum wire, have
in many instances shown the same funnel-shaped depression
and the occluding air-bubble.

(//) On potato. — On boiled potato kept under a moist bell-
glass the choleraic comma-bacilli grow readily when kept at
temperatures varying between 32° and 37° C. From the
place of inoculation the growth extends in the shape of a thin
light-brown translucent film, in which, as time goes on, say
after one to two weeks, thicker brownish spots and patches,
due to local increase of the growth, appear. At tempera-
tures varying between 18° and 22° C., even after several
days, only a trace of growth can be made out with a
lens.

CHAPTER V.

VARIOUS SPECIES OF COMMA-BACILLI.

WHEN, in 1883, Koch first announced from Egypt that in
all cases of Asiatic cholera examined he had discovered in
the dejecta and intestinal contents a species of bacilli which,
"owing to their peculiar form, were called comma-bacilli,"
he was, according to his own showing,1 not yet acquainted
with their peculiarities in gelatine cultures ; and after
he had concluded his observations in Egypt, India, and
France, in 1884, he stated2 that in a large number of cases
of intestinal disease the contents of the intestine had been
examined, but " never was there found any trace of comma-
bacilli." The intestinal discharges of dysentery and of in-
fantile diarrhoea, the saliva of the mouth of various animals
and the intestinal contents from various animals poisoned
by arsenic, were searched, but no comma-bacilli were ever
found. " Wherever I could get hold," he says,3 " of a fluid
containing bacteria, I examined it for comma-bacilli ; but
never have I found them. Only once I found in water from
a salt-water lake in Calcutta a species of bacteria which at

1 Conferenz zur Erorterung d. Cholera/rage: Berlin, July 26, 1884,
p. 22.

* Loc. cit. p. 24. 3 Loc. cit. p. 25

CH. v.] VARIOUS SPECIES OF COMMA-BACILLI. 83

first sight presented a certain resemblance to the cholera-
bacilli, but on more careful examination they were some-
what thicker and did not liquefy nutritive gelatine." From
this it is quite evident that, with the exception of this last
instance, Koch had failed to find comma-bacilli anywhere
except in cases of cholera. If Koch had known at that
time that comma-bacilli occur in the saliva of the mouth
and in various intestinal discharges, he would have no doubt
added these instances to the one of the salt-water lake in
Calcutta; but from his giving this as the only exception,
there can be no question that he had failed to meet with

FIG. 27.— COVER-GLASS SPECIMEN OF FINKLER'S COMMA-BACILLI FROM A

GELATINE CULTURE.
Magnifying power about 600.

comma-bacilli in any of the substances that were said to have
been examined. He therefore felt justified, he thought,1 in
pronouncing that the comma-bacilli are constantly present
in cholera Asiatica, but do not occur anywhere else. As is
now well known, comma-bacilli are not so rare as Koch
thought, but on the contrary are of rather common occur-
rence.

If in Egypt, while as yet unacquainted with the peculiar
and distinguishing characters of the comma-bacilli in cultures
in nutritive gelatine, and while, therefore, relying solely on

1 Loc. cit. p. 25.

G 2

84 THE BACTERIA IN ASIATIC CHOLERA. [CH.

morphological appearances, Koch had found, as he might
have done by more careful examination, that in the fluid of
the mouth of man, in the intestinal contents of various in-
testinal disorders in man and animals, and in the normal con-
tents of the intestines of some animals, comma-bacilli iden-
tical in morphological respects with the comma-bacilli in
Asiatic cholera were constantly present, I am inclined to
think that he would have adhered to his opinion formed be-
fore he went out to Egypt, viz., that the distribution of the
comma-bacilli in the intestine in cases of cholera (sent him to
Berlin from India some time previously), proves them to be
septic organisms.

It is, I think, necessary to go back to this history of the
discovery 'of the choleraic comma-bacilli, in order to show
that the importance ascribed to them by Koch in relation to
cholera may have had a good deal to do with his inability to
find comma-bacilli anywhere else except in cholera cases.
Koch says that since comma-bacilli occur only in cholera,
and since he has failed to find them in the normal intestine,
he concludes that the organisms and the disease stand in a
direct relation. Now from his having failed to find comma-
bacilli in a variety of localities where we now know that they
constantly occur, his positive statement that they do not
occur in the normal intestine loses a good deal of its value ;
for, as will be pointed out below, even if they did occur in
very small numbers amongst the myriads and myriads of
bacteria normally inhabiting the alimentary canal, it would
be well-nigh impossible to demonstrate them ; it would at
any rate require long and exhaustive examination by many
workers to establish their presence or absence on a reliable
basis. If any one who has failed to find comma-bacilli in a
variety of localities in which he has searched for them, but in
which they have been shown by others to exist, tells us that

v.j VARIOUS SPECIES OF COMMA-BACILLI. 85

in the normal human intestine no comma-bacilli occur, I
think we shall be justified in showing some hesitation before
accepting his dictum. What we do know of the characters,
morphological and cultural, of the innumerable species of
bacteria occurring in the different parts of the normal human
intestine, is utterly insignificant as compared with what we
do not know of them, and a great deal of work will have to be
done still before any one is justified in saying that such and
such a form present in a certain disorder of the intestine is
not present in the normal state. Of course, if there are other
evidences, such as the direct test of physiological action on
the animal body, a conclusion can be drawn with certainty,
but in the absence of such a test — and as I shall show this
test is not forthcoming as regards the cholera-bacilli and
cholera — it is quite unjustifiable for any one to pronounce so
definitely as Koch did. Supposing that a particular patho-
logical state of the ileum, particularly the mucus-flakes
therein, were to favour the multiplication of the comma-bacilli
already present in very small numbers in the normal state,
then we should not be surprised to find that in choleracomma-
bacilli are abnormally numerous (see v. Emmerich's statements
in the Archiv f. Hygiene, Band III.).

Mr. Watson Cheyne, in the discussion on Asiatic cholera
at the Medico-Chirurgical Society (March 1885), was
thought by some present to have made a great point when
he asked : How is it that if you want to demonstrate Koch's
cholera-bacilli you have only to examine the intestinal con-
tents of cholera cases, be they in India, in Egypt, France, or
anywhere else, while you cannot find them in other cases of
disease ? No doubt, cholera being a disease peculiar to the
human species only, and assuming that the cholera-intestine
favours the multiplication of the comma-bacilli, it would
follow that they are easily demonstrable in such cases ; but

86 THE BACTERIA IN ASIATIC CHOLERA. [CH.

that does not necessarily imply that they were not already
present in very small numbers before the disease set in, and
if so, of course their being found in India, Egypt, or France
would make no difference to the conclusion so long as we
have to deal with the human species.

I presume very few will dissent from the proposition that
in many a putrid fluid crowded with all kinds of bacteria,
it would be well-nigh impossible to discover Saccharomyces,
although we know Torula is one of the most common organ-
isms contaminating the air, and every fluid exposed to the
air would receive and contain a good many examples of it.

FIG. 28. — GELATINE PLATE-CULTIVATION OF FINKLER'S COMMA-BACILLI
AFTER INCUBATION FOR FORTY-EIGHT HOURS AT 20° C.

But owing to the putrid fluid being an unfavourable soil, and
particularly owing to the luxuriant growth in it of saprophytic
bacteria, the comparatively few Torulce. at the outset present
will not multiply. But transfer some of that fluid into a new
medium containing besides traces of proteid material a good
deal of sugar, and after a few days you will have no difficulty
in showing the existence in this new medium of Saccha romyces.
The conclusion to be drawn from this is obvious. Neither
Koch, nor anybody else, has sufficiently and systematically
examined the normal human intestine, and, as I have shown

v.] VARIOUS SPECIES OF COMMA-BACILLI. 87

before, there is very little known about the nature and
characters of the many species of bacteria present in the
normal intestine : therefore the off-hand statement made by
Koch and his adherents, viz. that comma-bacilli identical
with those found in Asiatic cholera do not exist in the nor-
mal intestine is, to say the least, premature and not justified
by their own limited observations.

In this connection, and as a confirmation of what has just
now been said, I will quote Dr. Koch's own words, uttered
in the discussion that followed1 the reading of his paper.
He says : lt The question as to whether there exists any other
disease, or any other condition in the human subject, where-
in this same (comma) bacillus occurs, cannot be at present
solved ; it will take years for its solution, and it will be
necessary from time to time to examine in this direction any
new disease that occurs. A strictly scientific decision (as to
whether these same comma-bacilli belong exclusively to
Asiatic cholera) is therefore at present impossible." In June
1884, a preparation of Koch's from the mucus-flakes of the
ileum of a case of Asiatic cholera was shown in London to
me and a number of others interested, by a gentleman who
was indirectly associated with Koch in Egypt ; in this speci-
men the comma-bacilli were easily recognised. I mentioned
on that occasion that I possessed specimens of the intestinal
contents from an epidemic of bad diarrhoea that had occurred
in Cornwall in the autumn of 1883, in which the same forms
of comma-bacilli occur ; to this the gentleman answered with
a smile and a shake of the head, so convinced was he from
the teaching of Koch that comma-bacilli are present in
Asiatic cholera exclusively.

I. In 1884 Finkler and Prior demonstrated and described
1 Loc. cit. July 29, 1884, p. 55.

88

THE BACTERIA IN ASIATIC CHOLERA. [CH.

before the meeting of Naturalists and Physicians at Magde-
burg, specimens of the intestinal dejecta of cholera nostras in
which comma-bacilli occurred abundantly. They had cul-
tivated them, and had thus obtained them in large numbers.
True, the dejecta from which the comma-bacilli were ob-
tained had been kept for some days, and the methods of cul-
tivation employed by Finkler and Prior were not free from

FIG. 29. — CULTIVATION OF TINKLER'S COMMA-BACILLI IN NUTRITIVE GELATINE
(lO PER CENT.) AFTER FOUR DAYS* INCUBATION.

objection, and as a matter of fact their cultivations were at
that time not pure cultivations of comma-bacilli ; still, what-
ever might be urged against their description of the morpho-
logical changes they ascribed to those comma-bacilli, the fact
remained and could not be criticised away, that veritable
comma-bacilli possessing each and all of the morphological
characters of the choleraic comma-bacilli — single commas

v.] VARIOUS SPECIES OF COMMA-BACILLI. 89

more or less curved, and S'sriaPed and spiral forms — had
been found elsewhere than in Asiatic cholera. Doubtless
this was not at all a welcome discovery to Koch and his
adherents, who had so frankly stated that notwithstanding
their careful and exhaustive examination, they had " never
seen any bacteria resembling comma-bacilli." The only
thing to be done was to show that the comma-bacilli of
Finkler and Prior were a different species from those occur-
ring in Asiatic cholera.

Finkler and Prior then have proved the existence of
comma-bacilli in a disease other than Asiatic cholera.

I have been fortunate enough to receive from them a
mounted slide and a gelatine tube of the comma-bacilli, and
there can be no question that while these comma-bacilli
are in general respects similar to the choleraic comma-
bacilli, they nevertheless present certain well-marked dif-
ferences. The characters shown by these comma-bacilli are :
(a) They occur as single commas, some more, others less
curved, as S'snaPed forms, and as wavy or more distinctly
spiral forms ; (b) they are possessed of motility, exactly like
the choleraic comma-bacilli ; (c) they show in well-stained
and well-washed specimens the same distinction between
sheath and protoplasmic contents, generally accumulated
at the ends, as the choleraic comma-bacilli ; (d) they are
distinctly thicker and longer than the choleraic comma-
bacilli ; although this may not be striking when the com-
parison is made under a low power (say 300-400), it is
conspicuous when a specimen of choleraic comma-bacilli
made from a gelatine culture is compared under a high
power with one of Finkler and Prior's comma-bacilli grown
in a like medium ; (e) cultivated on Agar-agar mixture, in
broth, or vegetable albumen and Agar-agar, and on egg-
albumen and Agar-agar, the characters of the growth are

90 THE BACTERIA IN ASIATIC CHOLERA. [CH.

very much the same as those of the choleraic comma-bacilli
— they grow rapidly and well at temperatures of from 30° —
37° C. ; (/) on boiled potato Finkler's comma-bacilli grow
well even at temperatures of i8°-22° C., and from the
spot of inoculation a smeary greyish-brown film soon ex-
pands, and gradually thickens ; in a few days at 20° C, the
growth is copious ; in this respect then a marked difference
exists between the two comma-bacilli ; (g) in nutritive
gelatine in plate-cultivation the character and aspect of the
colonies of Finkler's comma-bacilli is very similar to those
of the choleraic comma bacilli, except that the colonies
make their appearance much sooner, and having appeared,
grow much more rapidly than those of the choleraic com ma-
bacilli; (h) they liquefy the gelatine, but the liquefied
gelatine both in plate-cultivation and in tubes is less clear
than is the case with the choleraic comma-bacilli ; on com-
paring a gelatine tube of Finkler's comma-bacillus with one
of the choleraic comma-bacilli, after a week or more the
liquefied gelatine in the latter is more clear than in the
former, although in both it is not quite clear ; gelatine tubes
in which Finkler's comma-bacillus has been growing, and
in which the gelatine with the exception of the deepest parts
has become completely liquefied, show the same whitish
or greyish-brown precipitate as similarly advanced culture-
tubes of choleraic comma-bacilli, the liquefied gelatine being
only slightly opaque and in the upper layers almost trans-
lucent, while in similarly advanced culture-tubes of choleraic
bacilli the liquefied gelatine is a little less opaque, and in
many tubes there is present on the surface a granular
whitish filmy pellicle (p. 78) ; (z) the best test of distinction
is no doubt that pointed out by Koch, namely, the mode
of growth and the rapidity with which the two kinds of
comma-bacilli grow in nutritive gelatine of 10 per cent.

v.] VARIOUS SPECIES OF COMMA-BACILLI. 91

strength. The appearance of a series of gelatine-tubes
inoculated by means of the platinum wire or capillary
glass pipette with Finkler's comma-bacillus, and of another
series of similar tubes inoculated in the same manner with
choleraic comma-bacilli and kept at 20° C. from three to
four days, leaves no doubt that they contain two different
species. While the cultures of the choleraic comma-bacilli
are only in their early stage, showing the channel of inocu-
lation as a thin greyish line of liquefied gelatine with a trace
of whitish precipitate at the bottom, and a distinct funnel-

c

« '/ .,
!, *

'ff :

Fib. 30. — COVER-GLASS SPECIMEN OF MVCUS-FLAKES FROM A MONKEY SUFFERING

FROM DlARRH<EA.

i. Straight bacilli containing bright oval spores.
Magnifying power about 600.

shaped depression of the surface with an occluding air-
bubble, those of Finkler's comma-bacillus show a broad
conical growth ; the liquefied gelatine occupying almost
one-third of the breadth of the tube, and being uniformly
turbid. In the earlier stages, say after thirty to sixty hours,
the surface shows the funnel-shaped depression as well as
the air-bubble, but after three days as a general rule the
liquefaction has so far progressed that of the funnel-shaped
depression and occluding air-bubble little is left.

A curious fact which I have repeatedly observed is

92 THE BACTERIA IN ASIATIC CHOLERA. [CH.

this : if, instead of 10 per cent, gelatine, nutritive gelatine
of the strength of 3 to 6 per cent, is substituted, the
mode and rapidity of growth of the two kinds of comma-
bacilli are indistinguishable, and it seems to me probable
that Finkler and Prior's statement that the two kinds of
comma-bacilli do not differ in their mode and rapidity of
growth in nutritive gelatine must be due to their having used
gelatine of less strength than 10 per cent. I am quite sure
that Koch is right in saying that as a general rule 10 per cent,
nutritive gelatine shows the well-marked differences men-
tioned above. But I do not think Koch is right when he
says that while the growth in tubes of choleraic comma-bacilli
possesses an aromatic smell, those of Finkler's have a putrid
smell ; for there is little difference noticeable in this respect
between the two, except that in the latter there is certainly
not that distinct aromatic smell as in the former.

The question whether Finkler's comma-bacilli stand in
any definite relation to cholera nostras, as is maintained by
Finkler and Prior,1 has been definitely set at rest by the fol-
lowing considerations : — (i) The observations made by Koch
and Frank have failed to demonstrate the presence of these
comma-bacilli in typical cases of cholera nostras, therefore
their number cannot at all events be remarkably great in
this malady; (2) Dr. Miller of Berlin has proved by cul-
tivation that comma-bacilli apparently identical with those
of Finkler and Prior occur in the mouth in connection with
caries of the teeth;2 (3) Kuisl succeeded in isolating by
cultivation from normal human faecal matter comma-bacilli
which morphologically and in culture are identical with
Finkler and Prior's comma-bacilli.8

1 Ergiinztmgshefte zum Centralblatte f. alJgem. Gesundheitspflcge, vol.
i. parts 5 and 6.

- These bacilli of Miller are however not considered quite identical
with Finkler's. 3 Aerzll. Intdligenzblatt, 36 and 37, 1885.

V.] VARIOUS SPECIES OF COMMA-BACILLI. 93

II. The late Dr. T. R. Lewis was the first to point out1
the occurrence, in the fluid of the mouth, of comma-bacilli
morphologically identical with those of Asiatic cholera. They
are of the same size, of the same kind of curvature, single
or S-snaped, and they move like the choleraic comma-
bacilli. This simple fact, which is easily verified, had

@ f CS

v* ' *

FIG. 31.— COVER-GLASS SPECIMEN OF CONTENTS OF C^CUM FROM A NORMAL
GUINEA-PIG.

1. Small comma-bacilli.

2. Spirilla of same.

3. Larger comma-bacilli.

4. Large coiled organisms.
Magnifying power about 700.

entirely escaped Koch. He said 2 that he had examined
the saliva of the mouth and had not seen anything like
comma-bacilli. It seems, therefore, to say the least, very
remarkable that some of Koch's adherents should have
made so light of Lewis's discovery ; they appeared to take
1 Lancet, September 20, 1884. 2 Loc. cit. p. 25.

94 THE BACTERIA IN ASIATIC CHOLERA. [CH.

this occurrence of comma-bacil.U as a matter of course, which
"every one knew before." It must be plain to every im-
partial reader that not even Koch knew this until Lewis
pointed it out.

The comma-bacilli of Lewis are not the same as those
afterwards described and isolated by Miller, since the latter
have been isolated and grown in 10 per cent, alkaline gela-
tine, and behave similar to those of Finkler and Prior, while
those of Lewis do not behave in this way. Neither Lewis
nor Koch, nor any of the many workers in Koch's labora-
tory who have tried to grow them in alkaline 10 per cent,
gelatine, have succeeded. It occurred to me that inasmuch
as the fluid of the mouth often has a neutral or even faintly
acid reaction, it might be possible to grow these comma-
bacilli in neutral or faintly acid nutritive gelatine. I examined
the fluid of my own mouth, and as had been already shown
by Lewis, I found that comma-bacilli vary greatly in numbers
at different times : sometimes 1 could find in every specimen
(made by drying on a cover-glass a thin film, and staining
it afterwards in gentian-violet or Spiller's purple) several
examples of comma-bacilli ; at other times only in one or
the other could a few be found, while at other times
again I found numbers of them in groups and as isolated
examples. On such occasions I made a number of
plate-cultivations in 5 per cent, neutral nutritive gelatine,
and in one instance after several fruitless attempts I did
get a colony of liquefying comma-bacilli, which in their
manner of growth in the plate-cultivation seemed indis-
tinguishable from the choleraic comma-bacilli; from such
a colony 10 per cent, alkaline nutritive gelatine in test-tubes
was then inoculated, and the growths produced therein were
not distinguishable from those of choleraic comma-bacilli.
I have afterwards, on many occasions, repeated the original

v.] VARIOUS SPECIES OF COMMA-BACILLI. 95

experiment, and have had, after a great many failures,
one other successful colony produced in plate-cultivation
in neutral nutritive gelatine from which a series of tubes of 10
per cent, alkaline nutritive gelatine were started. These I
have kept growing for many generations, and their behaviour
in Agar-agar mixture, in broth, in gelatine, and in potato was
carefully noted and compared with cultures of choleraic
comma-bacilli ; all I can say is that they appear to me
identical. The only difference that I can find is that the
comma-bacilli from the colony in the plate-cultivation
appeared slightly larger, that is to say, thicker than the
choleraic comma-bacilli, but this difference was not so
striking in the cultivations in 10 per cent, alkaline gelatine.
I maintain then that in the normal fluid of the mouth
there occur at least two kinds of comma-bacilli, one very
similar to the choleraic comma-bacillus, the other (isolated
by Miller) similar to Finkler's.

III. Deneke described comma-bacilli which he found in
stale cheese, and which he afterwards isolated and cultivated.
Morphologically, and also in gelatine cultures, they appear
almost identical with those of Asiatic cholera, so much so
that he failed to observe any describable difference ; in
fact, he states that so far as he was able to observe, the only
distinction seems to be their different action when injected
into guinea-pigs. He afterwards, however, modified this
view, inasmuch as Fliigge showed that there are slight
differences from the choleraic comma-bacilli in their mode
of growth in nutritive gelatine, in which respect they stand
about midway between the choleraic and Finkler's comma-
bacilli. At any rate this much seems certain, that the
differences existing between them and Koch's comma-bacilli
are not very great, not so great, in fact, as those between

96 THE BACTERIA IN ASIATIC CHOLERA. [CH.

Finkler's and Koch's comma-bacilli. On potato they do not
grow at all. I have myself had an opportunity, thanks to
Dr. Crookshank, of examining and cultivating these cheese-
comma-bacilli or cheese-spirilla, and am able to say that in
gelatine cultures they are difficult to distinguish from the
choleraic comma-bacilli.

IV. As has been already stated, Koch in his first pamphlet
embodying the results of his investigations on cholera in
Egypt, India, and France, showed that he was then unaware of
the existence of comma-bacilli other than those in Asiatic
cholera, with the exception of one instance — that of a salt-
water lake in Calcutta, in which he found comma-bacilli
that looked like choleraic comma-bacilli, but did not behave
like them in. cultivation — he had never seen any bacteria
that looked like the comma-bacilli. This being the case,
our finding comma-bacilli in the intestinal contents of cases
of diarrhoea, dysentery, and phthisis seemed not without
interest. Hence the strong adverse criticism expressed by
Mr. Watson Cheyne during the discussion on cholera at the
Medico-Chirurgical Society in 1885, at my having mentioned
such a fact without saying how these comma-bacilli behave
under cultivation in gelatine, was not quite justified. The
statement made by me in the preliminary Report of the
English Cholera Commission, that comma-bacilli do occur
-in intestinal diseases other than Asiatic cholera, was in
relation to Koch's statement that in no other disease of the
alimentary canal, nor in any other circumstance — except the
above-quoted salt-water lake in Calcutta — had he ever seen
bacteria that looked like the comma-bacilli. The comma-
bacilli which we saw in the intestinal contents of cases of
diarrhoea, dysentery, and phthisis, looked thicker and longer
than those in cholera, and their mode of growth in gelatine

v.] VARIOUS SPECIES OF COMMA-BACILLI. 97

was not then ascertained. But I have since my return to
England ascertained that comma-bacilli obtained from a case
of severe diarrhoea in an adult do grow in 10 per cent,
gelatine ; in weaker gelatine (5 per cent.) they grow well, but
their mode of growth is no doubt altogether different from
that of the choleraic comma-bacilli ; although they liquefy
the gelatine, they do so very rapidly, and the liquefied
gelatine is turbid, thick, and smells offensively. A very
striking case of the occurrence of crowds of comma-bacilii
in the intestine in diarrhoea I have met with in a monkey.

FIG. 32. — COVER-GLASS SPECIMEN FROM A CULTIVATION IN 10 PER CENT.
NUTRITIVE GELATINE OF THE NON-LIQUEFYING VARIETY OF COMMA-BACILLI
FROM A CASE OF NOMA IN A CHILD.

1. Single comma-bacilli.

2. Spiral forms.

3. Wavy forms.
Magnifying power about 700.

These animals in the summer months are very often affected
with diarrhoea. We have had during several years at the
Brown Institution several cases of severe diarrhoea in
monkeys ; some died, others recovered under treatment.
One animal was killed thirty hours after diarrhoea set in.
The stools were liquid, yellow, and contained granules of
faecal matter; in the large intestine was a quantity of the
same yellow thin fluid, in it were suspended numerous
mucus-flakes. I may state that this animal had been pur-
chased in good health about a fortnight before, and had been

98 THE BACTERIA IN ASIATIC CHOLERA. [CH.

kept, like the other monkeys, in a separate stall, uniformly
warm and well-ventilated. The food was copious and of the
ordinary kind — potatoes, rice, and milk.

Preparations made of the mucus-flakes of the caecum
revealed, besides straight thick spore-bearing bacilli, slightly
pointed at the ends, large numbers of motile comma-bacilli.
In Fig. 30 I have given an accurate representation of a
number of these comma-bacilli present in the same place in
the mucus-flake. The identity in morphological appearances
and in size with the choleraic comma-bacilli is very striking
indeed ; there are the same single commas, and g-shaped,
circular, and semi-circular forms. I possess a good many
preparations (stained and mounted) of the mucus-flakes of
the ileum of cases of acute typical cholera in which the num-
ber of comma-bacilli is not by any means so great as in
the case of this monkey. I have made a number of plate-
cultivations from the contents of the caecum of the monkey,
but owing to the hot weather then prevailing (June) the
above-mentioned straight bacilli had in the course of twenty-
four hours so increased in numbers and so pervaded the
gelatine that the whole became liquefied and crowded with
them. Owing to this the cultivations became altogether
useless. In another monkey that died with diarrhoea, the
contents of the csecurn were also crowded with comma-
bacilli, many of these S-snaped and spiral ; but these were
conspicuously thicker than those in the former case.

Of other cases of comma-bacilli in the contents of the
caecum in monkeys I shall have something to say later on.

V. In his second paper1 Koch says he has repeated,
with positive results, the experiments made first by Nicati and
Rietsch on guinea-pigs (namely, injection of cultures of
1 Deutsche med. Woch. 45, 1884.

v.] VARIOUS SPECIES OF COMMA-BACILLI. 99

choleraic comma-bacilli direct into the duodenum) ; and in
his third paper l he gives the results of a series of successful
experiments on guinea-pigs. In both instances mention is
made of the multiplication of the injected choleraic comma-
bacilli in the intestines of the guinea-pigs experimented upon,
but I find no evidence that he had first sought for the
presence of comma-bacilli in the contents of the intestines
of normal guinea-pigs.

FIG. 33. — PLATE-CULTIVATION OF THE SAME NON-LIQUEFYING COMMA-BACILLI

OF NOMA AS IN FlG. 32.

The plate-cultivation is several weeks old. The drawing represents the colonies as
seen under a lens. The colonies marked by uniform shaping are situated in
the depth.

Van Ermengem 2 givesa figure (PI. XI. Fig. i) of comma-
bacilli in the intestinal contents of a guinea-pig that died
after intraduodenal inoculation, and he describes them
(p. 372) as " grandes virguhs fortement incurvees et d1 aspect
assez anormal" He tells us3 that he found comma-bacilli
in the large intestine of guinea-pigs, but they are much

1 Conferenz zur Eroi'teriin^ der Cholera/rage : Berlin, May
- Recherckes sur le microbe du Cholera Asiatique.
3 Loc. cit. p. 87.

H 2

TOO THE BACTERIA IN ASIATIC CHOLERA. [CH.

larger than Koch's and their cultures in gelatine plates are
altogether different. While I have no doubt that his grandes
virgules fortement incurvees correspond not to Koch's comma-
bacilli, but to certain forms normally present, I have grave
doubts about his colonies of these normal comma-bacilli
from the guinea-pig. I have tried over and over again to
isolate these by plate-cultivation, but have never succeeded
in growing them.

A somewhat similar statement is made by Mr. Watson
Cheyne on p. 13 of his pamphlet (a reprint of a series of
articles that had appeared in the British Medical Journal
of April 25, May 2, May 16, and May 23, 1885) : "One of
the most peculiar forms (of comma-bacilli) which I have
seen was found in the contents of the large intestine of
guinea-pigs, which died after injection of cholera-bacilli.
I tested the fluid by cultivation at the time very carefully,
and found that it contained almost a pure cultivation of
cholera-bacilli ; there was certainly not more than one
other kind of bacilli for every hundred cholera-bacilli. The
appearance of this material, on microscopical examination,
after staining, is shown in the accompanying figure (Fig. 5).
Large, fat, coiled, almost worm-like organisms will be seen,
which, as I know by cultivation, are cholera-bacilli, but
which could not be recognised by the microscope alone."
Now it is a fact, easily verified, that what Mr. Watson
Cheyne here describes and figures, are forms present in the
contents of the large intestine of every normal guinea-pig,
as I pointed out in a note in the British Medical Journal
(May 9, 1885).

Those organisms figured and described by Mr. Watson
Cheyne, which he thought he identified by cultivation as
cholera- bacilli, can be easily demonstrated by spreading on
a cover-glass a thin film of the contents of the caecum of any

v.] VARIOUS SPECIES OF COMMA-BACILLI. 101

normal guinea-pig, previously diluted slightly with salt-
solution, and then drying and staining in the usual manner.
Crowds of these large, fat, coiled organisms seen by Mr.
Watson Cheyne are met with, together with other smaller
ones that look very much like choleraic comma-bacilli. It
is, however, only fair to state that Mr. Watson Cheyne, be-
coming no doubt aware of his error, has afterwards inserted
on p. 28 the following corrective statement: — "I think it
most probable that these bodies [shown in his Fig. 5] are
the cholera-bacilli which were found on cultivation to be
present in enormous numbers, because there were no other
markedly curved organisms present, and because they
seemed to show all gradations between small slightly-curved
rods, and the large, coiled bodies shown in the drawing."
Now, this is exactly what is the case in the material taken
from the csecum of every normal guinea-pig that I have
examined ; small comma-bacilli, single and S~snaPed and
spiral, of the size and appearance of the choleraic comma-
bacilli, and the large, fat, coiled organisms mentioned above,
and all intermediate gradations.

In Fig. 31, I have given an accurate representation of
the appearances in a specimen, prepared, stained, and
mounted, of the contents of the caecum of a normal guinea-
pig, and it is evident from this that Mr. Watson Cheyne's
former and later descriptions are equally applicable to this
preparation. The above-mentioned large, fat, curved, and
coiled organisms seem to me more like flagellate infusoria
than comma-bacilli.

Messrs. Cornil & Babes have recently l given a drawing

and description of the comma-bacilli present normally in the

intestine of guinea-pigs. Their drawing does not exactly

represent the appearances, and they do not state, as they

1 Microbes, Second Edition.

102 THE BACTERIA IN ASIATIC CHOLERA. [CH.

ought to have done, that they are not the discoverers of
these comma-bacilli in the intestine of normal guinea-pigs.

I have repeatedly tried to grow these normal comma-
bacilli in 10 per cent, alkaline nutritive gelatine in
plate-cultivations, but have not succeeded : there was not

FIG. 34 and 35.— SAME NON-LIQUEFYING COMMA-BACILLI GROWING IN ic
PER CENT. NUTRITIVE GELATINE; SEVERAL WEEKS OLD.

FIG. 34. — On the surface.

FIG. 35. — After inoculation by stabbing.

even an attempt at growth, only straight bacilli and
micrococci were thus obtained from the intestinal contents.
Of comma-bacilli or spirilla forms, shown in Fig. 31, there
was never any trace. Mr. Watson Cheyne, in his

v.] VARIOUS SPECIES OF COMMA-BACILLI. 103

plate-cultivations made from the guinea-pig that had been
infected per duodenum with choleraic comma-bacilli, obtained
numerous colonies of the true choleraic comma-bacilli ; this
is of course proof that the choleraic comma-bacilli injected
were still present and had multiplied in the intestinal
contents.

Nicati and van Ermengem have also found comma-bacilli
in the intestinal contents of the pig, rabbit, horse, and other
animals, but they are said to differ from Koch's comma-bacilli.

VI. Comma-bacilli, in morphological respects indentical
with the choleraic comma-bacilli, have been found by my
colleague, Mr. Alfred Lingard, in a case of noma in a
child. A noma, including the whole thickness of the
mucous membrane of the lip of the mouth, was excised by
the house-surgeon at University College Hospital for Mr.
Lingard. On removal, cultivations were made from the
depth of the tissue by means of the platinum-wire, and
from the fresh tissues sections were cut in the ordinary way,
stained and mounted.

In the sections there were found at the point of
demarcation between the healthy (or rather inflamed) and
the necrotic tissue, but situated in the former, numbers of
comma-bacilli pervading the tissue in clumps and streaks.
The cultivations in Agar-agar proved to be growths of the
same comma-bacilli, but in an impure state. I then, by
Koch's method of gelatine plate-cultivation, isolated the
comma-bacilli and found that they belonged to two
well-defined species, one smaller and one slightly larger
variety. The smaller variety behaved in plate-cultivations,
and in the cultivations in gelatine tubes established from
these, very much like the choleraic comma-bacilli ; the
slowness of the colonies' growth, the manner in which they

104 THE BACTERIA IN ASIATIC CHOLERA. [CH.

liquefied the gelatine, the aspect and nature of the colonies,
the manner in which they grew in gelatine tubes, and the
microscopic appearances of fresh specimens and of dried
and stained specimens, were similar. The other and
slightly larger variety behaved altogether in a different way.
This did not liquefy the gelatine, it formed in plate-
cultivations single greyish rounded specks (see Fig. 33)
which only slowly enlarged, and even after a week or ten
days were not larger than a few millimetres in diameter,
their outline being irregular, their colour greyish, and their
aspect under a lens more or less granular. In gelatine
tubes (10 per cent, alkaline gelatine) inoculated by the
platinum-wire in the depth — stab-culture, there appeared
after several (three or four) days the first traces of the
growth in the shape of a greyish streak, slowly thickening
and broadening, and assuming a more or less granular
aspect; on growing it on the surface of 10 per cent, alkaline
nutritive gelatine in streak culture, a greyish film appears,
gradually expanding in breadth, but even after several weeks
it is only a few millimetres in breadth ; in the streak of
inoculation the growth is thickest, and assumes after two or
three weeks a slight reddish-brown tint, while the rest is
greyish and thin. After some months the film is about half
an inch broad, thin and grey in the peripheral part, thicker
and of a reddish-brown tint in the middle. The outline
of the film is smooth in some places, slightly crenate in
others. At no time, not even after some months, is there a
trace of liquefaction of the gelatine. These comma-bacilli
grow well on alkaline Agar-agar mixture, also, but not so
copiously, on vegetable albumen and Agar-agar mixture, or
egg-albumen and Agar-agar mixture, forming on all these
media a greyish film, which as time goes on assumes
in its thickest parts a brownish tint. In microscopic

v.] VARIOUS SPECIES OF COMMA-BACILLI. 105

specimens made from gelatine tubes several weeks old
there are found single commas and S-snaPed forms, and
wavy and spiral organisms. The latter are very interesting,
inasmuch as many are made up of several very closely-
twisted turns in the middle, while the ends are only slightly
wavy. Examples of these forms are shown in Fig. 32.

VII. Dr. Emil Weibel has during the last two years
described a number of different species of comma-bacilli
(Centralblatt fur Barter iologie und Parasitenkunde, II. Bd.,
No. 16., IV. Bd., Nos. 8, 9, 10).

Von Emmerich (Archivfilr Hygiene, Bd. Ill, p. 3 5 8) had
already drawn attention to the prevalent occurrence of
comma-bacilli or vibriones in various substances rich in mucus
(the mucous faeces of helix) and he thought that probably
owing to the presence of the mucus-flakes in cholera- A siatica
the comma-bacilli multiply so readily in this disease.

Weibel has followed this up and has isolated and
cultivated comma-bacilli from the nasal mucus. He
describes and figures this vibrio and found that it does
not liquefy the gelatine. Similarly he describes and figures
two species of comma-bacilli, which he isolated and cul-
tivated in pure cultures made of putrid hay infusion, vibrio
saprophyles a and /3 ; neither of them liquefy the gelatine.

Another species which did not liquefy gelatine was
isolated by Weibel from the mucus of the tongue ; further
he isolated a vibrio saprophyles y from mud of drains, and
finally he isolated three species whose growths are
conspicuous by their yellow colour: vibrio aureus, vibrio
flavus, and vibrio flavescens.

Gamalei'a (Annales de V Institut Pasteur, 1888, No. 9,
p. 482) states that an acute fatal disease affecting fowls in
Odessa during the summer months, similar to, but not

io5 THE BACTERIA IN ASIATIC CHOLERA. [CH.

identical with Fowl-cholera, is caused by a species of
comma-bacilli, which he calls " Vibrio Metschnikovi." This
vibrio is said to be indistinguishable in morphological and

FIG. 36. — CULTIVATION IN TEN PER
CENT. NUTRITIVE GELATINE OF A
MlCROCOCCUS ISOLATED FROM THE
BLOOD OF THE FlNGER OF A PER-
SON AFFECTED WITH SCARLATINA.

The cultivation is one week old, and
very much resembles a cultivation of
choleraic bacilli.

FIG. 37. — CULTIVATION IN GELATINE

(lO PER CENT.) OF SAME MlCRO-
COCCUS AFTER THREE WEEKS,
SHOWING A LARGE FUNNEL-SHAPED
OPENING ON SURFACE WITH AN
OCCLUDING AIR-BUBBLE.

The main part of the growth is liquefied
gelatine with numerous granules at the
side and within it ; the depth is not
liquefied.

cultural characters from Koch's comma-bacilli of human
Asiatic cholera.

We see then that the number of the different known
species of comma-bacilli is already considerable and is
constantly increasing.

v.] VARIOUS SPECIES OF COMMA-BACILLI. 107

As has been stated in the foregoing pages, the comma-
bacilli found by Koch in Asiatic cholera have certain
definite characters in culture media (particularly in nutritive
gelatine) by which they can be easily distinguished from
other species, although not from all. Certain other
characters at first attributed to them are not exclusive.

In his first publications Koch gave us to understand that
a notable character of the choleraic comma-bacilli is that
they require for good growth an alkaline medium. Now this
of course cannot and does not mean that they alone require
for good growth an alkaline medium, or that they do not
grow well in any but an alkaline medium. In the first place,
all the different species of comma-bacilli described in pre-
vious pages, and other species of bacteria also (various
micrococci and bacilli), grow very luxuriantly in an alkaline
medium, some much more luxuriantly in alkaline than in
neutral or faintly acid media. In the second place the
comma-bacilli of Asiatic cholera live and grow well also in
neutral and even faintly acid media. Thus the rice-water
stools and the intestinal contents have been in several in-
stances tested as to their reaction, and have been found of
neutral, or when kept for several hours, even of faintly acid
reaction, and nevertheless there were present in them
numerous comma-bacilli in active motion and multiplying
rapidly. I have repeatedly grown the choleraic comma-
bacilli in neutral nutritive gelatine, in neutral Agar-agar
mixture, and in neutral broth, and have thus obtained good
and active cultures. Koch has shown that notwithstanding
that the reaction of potato is acid, the comma-bacilli of
cholera grow well on it.1 Culture media (gelatine broth,
broth peptone), though at starting faintly alkaline, become,
when choleraic comma-bacilli grow in them, faintly but
1 Loc. cit. pp. 1 8, 19, and 20.

io8 THE BACTERIA IN ASIATIC CHOLERA. [CH.

distinctly acid, even after a few days only, and long before
the climax of growth is reached. I have in one instance
convinced myself that they are capable of growing (though
of course not copiously, any more than in the case of many
other bacteria), even when the nutritive medium (broth) was
at the outset just faintly acid.

Another assertion made by Koch was that the comma-
bacilli are killed by acid, provided this be of the requisite
strength. While there can be no question after the detailed
experiments of Mr. Watson Cheyne that hydrochloric acid
of the strength of 0-2 per cent, kills without fail the comma-
bacilli, if allowed to act on them for a few minutes, I do not
hesitate to say that with the exception of the spores of
bacilli all other bacteria — micrococci, bacteria, sporeless
bacilli, and vibriones and spirilla — are alike killed under
similar circumstances by acid: of such strength.1 And just
as a more dilute acid does not affect in a few minutes' action
the vitality of many other bacteria, so also the comma-bacilli
remain unaltered by it. I have mixed choleraic comma-
bacilli from a pure culture with a mixture of one part of
commercial hydrochloric acid in 1,000 parts of water for
from ten to thirty minutes, and on inoculating broth with
the medicated comma-bacilli I have obtained normal
growths of them. Other media known to have a noxious
effect on bacteria in general, have the same effect on
the choleraic comma-bacilli. The experiments of Koch
and also of Mr. Watson Cheyne ought, however, to be
accepted with a certain restriction after Dr. Macfadyan's
experiments carried out on the living animal.2 Dr.
Macfadyan has shown that if a dog be kept without food

1 Compare also a paper by Mr. Laws in the Report of the Medical
Officer of the Local Government Board, 1884.
* Journal of Anatomy and Physiology, vol. 21, 1887.

v.] VARIOUS SPECIES OF COMMA-BACILLI. 109

for twenty-four hours, and then water containing the
choleraic comma-bacilli be introduced into the stomach, the
comma-bacilli can be recovered by cultivation from the
contents of the jejunum and ileum four hours after, proving
that the comma-bacilli have passed the stomach in a living
state. If, however, they are given to the animal in milk
under the same condition, they cannot be recovered by
cultivation from the small intestine.

Koch in his first memoir has given us the results of
numerous systematic experiments made with regard to the
influence on the growth of the comma-bacilli of the absence
and presence of oxygen, with regard to the influence of
sufficient or deficient nutriment, and especially with certain
substances, such as alcohol, iodine, carbolic acid, cupric
sulphate, quinine, corrosive sublimate, &c. Van Ermengem
in his book states that he has repeated these experiments.

Hueppe has grown the choleraic comma-bacilli within the
hen's egg and has found that they, as well as the comma-bacilli
of Finkler, as also those of Deneke, are capable of growing
luxuriantly under these conditions, as also when growing
after Reichert's method, i.e. exclusion of all free oxygen, thus
showing that the comma-bacilli can grow well anaerobically.
One striking result of Hueppe's experiments was the
demonstration that all these three species of comma-bacilli
form sulphurated hydrogen when grown in egg.

Another remarkable fact to be again referred to further
below was the production in egg by the three kinds of
comma-bacilli (Koch, Finkler, Deneke,) of a highly toxic
chemical virus, far more distinct and striking than what is
taking place in gelatine cultures or broth cultures (see below.)

I have myself made some experiments on the choleraic
comma-bacilli and other bacteria with phenyl-propionic and
phenyl-acetic acid, with perchloride of mercury and with

I io THE BACTERIA IN ASIATIC CHOLERA. [CH.

iodate of calcium. A noteworthy result of these experi-
ments (published in the Report of the Medical Officer of the
Local Government Board for 1885) was this — that while the
killing and restraining power of corrosive sublimate on the
choleraic comma-bacilli, which by the way is greatly inferior1
to what was found by Koch, is very much the same as on
Finkler's comma-bacilli, and some notoriously saprophytic
bacteria, it is not so great on them as on some notoriously
pathogenic bacteria (exclusive of spore-bearing forms).

Strong solutions of iodate of calcium, while possessed of
antiseptic action on micrococci, as well as on pathogenic
sporeless bacilli, have no effect on the choleraic comma-
bacilli ; when kept mixed with the solution the comma-bacilli
retain their motility and their power of multiplying unimpaired.
Sea-water has likewise no disinfecting action on them.

With regard to the aspect and character of the colonies in
gelatine plate cultivations, although they are well-marked in
the case of choleraic comma-bacilli (see p. 71), they are not
exclusively confined to them ; I have seen colonies of
micrococci (obtained accidentally from necrotic tissue, and
from urine) which very much resembled them in general
aspect, and in the mode of liquefaction of the gelatine, but
they grew more rapidly.

Finally as to the character of the growth in gelatine tubes,
particularly the funnel-shaped depression of the surface and
the occluding air-bubble, I have observed these in the case
of some other bacteria, as has been already stated. But the
general appearances as growth proceeded became somewhat
different from those presented by the choleraic comma-bacilli.

Dr. Odo Bujwid pointed out 2 that the cultures of choleraic

1 Koch gives the restraining power of perchloride of mercury on
choleraic comma-bacilli as measured by I in 100,000 of water ; I find
that not even I in 30,000 has such a power.

2 Zeitschrift fur Hygiene, Bd. II. I, p. 52.

v.] VARIOUS SPECIES OF COMMA-BACILLI. in

comma-bacilli in broth at 37° C. show already after twelve
hours' incubation, on the addition of the amount of 5 to 10
per cent, of common hydrochloric acid (also nitric or sulphuric
acid) a characteristic pink colouration which rapidly increases
in intensity during the first half hour ; it lasts for a few
days, and exposed to light changes into a brownish tint.

Dr. E. K. Dunham has shown 1 that for this reaction the
presence of peptone is essential, and that the pink colour
becomes more pronounced and more rapidly evident if
instead of HC1, concentrated sulphuric acid be employed.
A previous addition of a drop of nitric acid enhances the
reaction, which under this condition can be obtained also
with Finkler's arid Deneke's comma-bacilli. Salkowski had
previously shown that this reaction is due to the formation
of indol and a trace of nitrite.

Zeitschr. fur Hygiene, II. 2, p. 337.

CHAPTER VI.

DIAGNOSTIC VALUE OF CHOLERAIC COMMA-BACILLI.

So far we have seen that various species of comma-bacilli
are known, and that of these the choleraic comma-bacilli
possess certain definite characters in cultivations on nutritive
gelatine which are not possessed either by those of Finkler
and Prior, Miller, Kuisl, Deneke, Weibel and others, by one
form of those observed in noma, or by those which I
observed in the diarrhoea of man, and in the contents of the
caecum of the guinea-pig. Those which I observed in the
contents of the caecum of the monkey suffering from diarrhoea
have not yet been cultivated. But those which occur in the
fluid of the mouth, and which are probably those observed
by T. R. Lewis, I think I have in two instances out of many
succeeded in cultivating, and they appear to me to be in
their manner of growth in nutritive gelatine strikingly similar
to the choleraic comma-bacilli. Lastly, one of the two species
observed in noma was found to grow in gelatine in the same
manner as the choleraic comma-bacilli. Now it has been
often stated, and is by many held, that two kinds of organisms,
morphologically alike, and growing in a like manner in the
various artificial media commonly in use, must of necessity
be one and the same species. There would be no more

CH.VL] VALUE OF CHOLERAIC COMMA-BACILLI. 113

justification for a general statement of this kind than if one
were to lay it down that two species notoriously different in
chemical and physiological respects must always show
different characters in the artificial cultivations commonly
employed; for the contrary has been proved as regards
several species. It has for instance been shown that the
capsulated oval coccus first discovered by Friedlander in
connection with croupous pneumonia is not the only species
that in morphological respects and in artificial cultures
possesses the characters he described. Of other bacteria,
e.g. streptococcus (some of those studied by Rosenbach, by
Uffendi and others), the same holds good.

Comparing for instance the behaviour of some of the non-
pathogenic micrococci studied by these last two observers in
artificial cultures with that of some of the pathogenic micro-
cocci (e.g. of erysipelas and of vaccinia) the similarity is very
striking; nevertheless the physiological action is totally
different. The same is to be said of the bacillus of typhoid
fever, and of some of the species of the proteus of Hauser. If
two kinds of bacteria differ in mode of growth, cateris paribus,
in the same culture media, I think it will be admitted that
they are probably different species. While then it will be ad-
mitted that Koch's comma-bacilli must be of a different species
from those of Finkler and Prior, of Miller, and of the species
described above as found in noma, it does not follow that
they are identical with those cultivated by Deneke or by
myself from the mouth, from a case of noma, and (as I shall
hereafter mention) from a monkey that had been subjected
to certain definite experiments. And it seems to me that
Koch and his adherents go too far in trying to make
out first, that the comma-bacilli belong exclusively to
Asiatic cholera, and then, after this had been disproved,
that they differ from all other comma-bacilli in mode of

i

H4 THE BACTERIA IN ASIATIC CHOLERA. [CH.

growth in gelatine, and must therefore be different from
all others.

I maintain that notwithstanding the fact that the choleraic
comma-bacilli in their mode of growth are not quite so
unique as is maintained of them, even when compared with
the few species that have already become known — to say
nothing about the many species of comma-bacilli that may
be discovered if observations are continued and extended —
it does not follow that they do not stand in a causal relation
to Asiatic cholera. If by clear and definite experiments,
imitating as much as possible the methods of infection obtain-
ing in nature. Asiatic cholera can be induced artificially by
a pure cultivation of the choleraic comma-bacilli, then it
must be considered as proved beyond doubt that they are a
vera causa, or, in other words, the contagium of cholera.

In a subsequent chapter I shall describe and review all the
experiments that have been made in this direction ; for the
present I shall content myself with the brief statement that
such proof is not forthcoming, and that what has been given
as proof is highly unsatisfactory.

No true cholera infection, as is understood in pathology
and as is proved with some other bacteria, has as yet been
produced ; all that has been hitherto shown is that the comma-
bacilli, like some other notoriously saprophytic bacteria, are
capable of producing chemical susbtances acting toxically, or
in some animals are capable of producing septicaemia.

One thing, however, may be said with certainty, namely,
that as far as our limited knowledge at present goes, in no
intestinal disorder in man have comma-bacilli behaving in
artificial cultures like those of Asiatic cholera been yet found
in the intestinal evacuations. This of course does not mean
that no intestinal disorder exists in which the same comma*-
bacilli are not present to a similar extent as in Asiatic cholera,
for, as has been pointed out on a former page, our experience

vi.] VALUE OF CHOLERAIC COMMA-BACILLI. 115

hitherto, and the observations at present available, are ex-
tremely limited ; but there can, I think, be no doubt, and in
this I fully concur with Koch, that in Asiatic cholera comma-
bacilli can be with comparative facility detected by the
microscope and by cultivation. Hence I agree to the
proposition that if in any case of diarrhoea the choleraic
comma-bacilli can be shown both by the microscope and by
culture-experiments to exist, then the suspicion that it may
be a case of Asiatic cholera is quite justified. And it must
be clear from this that the discovery by Koch of the choleraic
comma-bacilli is, on practical diagnostic grounds, of the
utmost importance. For if it should be found that in a
locality which is in communication by sea or land with an
infected country one or more cases of suspicious diarrhoea
had occurred, the demonstration by culture-experiments of
the presence in the intestinal discharges of the choleraic
comma-bacilli would fully justify us in regarding such cases
with grave suspicion, as being probably, though not neces-
sarily, choleraic. At all events sanitary officers, for the sake
of the public weal, would be justified in treating these cases
as cases of cholera, and in taking measures of isolation and
disinfection.

But there is another side not to be lost sight of — it is this.
We have seen that not in all cases of marked Asiatic cholera
do the comma-bacilli occur in large numbers, since we have
mentioned cases, acute and in all respects typical, where the
rice-water stools and intestinal contents harbour comparatively
few comma-bacilli, scattered amongst crowds of other bacteria.
Now in such cases the demonstration by the gelatine-culture
test does not invariably yield positive results. The reasons
are obvious. For demonstrating by the culture-test the
existence of Koch's comma-bacilli there exists at present no
better method than that employed by Koch, namely, the
gelatine plate-cultivation. Given a mixture of various species

j 2

ii6 THE BACTERIA IN ASIATIC CHOLERA. [CH.

of bacteria, and amongst these some species of bacilli and
micrococci characterized by rapid growth, and by the capability
of rapidly liquefying the gelatine, even if in such a mixture
a small number of choleraic comma-bacilli be present, there
will be great difficulty in rearing them as colonies in a
plate-cultivation. To establish a successful plate-cultivation
the number of bacteria introduced into one plate must of
course be limited, and the chances are small indeed that
more than one or two comma-bacilli are present in a trace
of a droplet of a mixture that teems with different species of
other bacteria. In such a plate-cultivation, owing to the very
slow growth of the choleraic comma-bacilli as compared with
many other putrefactive bacteria, and owing to the fact that
some of these latter rapidly permeate and liquefy the gelatine,
the appearance of the characteristic colonies of the choleraic
comma-bacilli will be interfered with. A case like this, then —
and it is by no means rare — does not offer very great prospect
of success even at the hands of experts in the method, and
the microscopic demonstration of comma-bacilli alone can
of course be of no diagnostic value, as every one with Koch
will readily admit. I have made a series of observations
which prove to me that although choleraic comma-bacilli are
present in a bacterial mixture, their demonstration by the
gelatine culture-test is extremely difficult, or well-nigh im-
possible. From normal human faecal matter a mixture was
made in 200 to 300 ccm. of distilled water, the mixture
being of thickish semi-fluid character. This mixture in every
trace of a droplet teemed with bacteria ; to it were added ten
drops of a pure culture in nutritive gelatine of choleraic
comma-bacilli. A series of twelve plate-cultivations was
then established after the usual methods of dilution, but in
none could any colonies of choleraic comma-bacilli be
detected. After a few days in all the plates the gelatine
was liquefied and was seething with bacilli and micrococci.

vi.] VALUE OF CHOLERAIC COMMA-BACILLI. 117

In another series, in which to 300 ccm. of fecal mixture one
cubic centimeter of the same choleraic culture was added,
out of twelve gelatine plates only in one was there a colony
of the comma-bacilli detected. In a third series to 300 ccm.
of faecal mixture 2 ccm. of the pure culture of choleraic
comma-bacilli were added; of a series of twelve gelatine
plates, two plates showed colonies of choleraic comma-bacilli;
one had one colony, one had three out of about thirty
colonies of other bacteria. It is evident from this that
although crowds of undoubted choleraic comma-bacilli may
be present, they are not easy of demonstration if they are
present only in comparatively small numbers amongst a
great majority of other quickly-growing bacteria.1 And that
this actually obtains in a certain percentage of cases of true
Asiatic cholera no one who has had large experience can
doubt. I will readily admit that plate-cultivation in a number
of cases, particularly those with typical rice-water stools and
mucus-flakes, yields positive results if the mucus-flakes are
usad ; but it is well known to all who have had to deal with
cholera epidemics that such cases are not by any means
common in the early stages of an epidemic, and it is precisely
at those stages, when no other symptom besides diarrhoea is
present to guide us, and when the bowel discharges are
simply fluid faecal matter, that the detection of the choleraic
comma-bacilli would be of the greatest importance. Later
on, when the cases have become more numerous, and the
symptoms more pronounced and typical of cholera, the

1 Kitisato shows (Zeitschrift f. Hygiene, v. 3, p. 487) that the comma-
bacilli when kept in a mixture of faecal matter undergo death : the
more rapid, the greater the comparative amount of faecal matter and the
more delayed the examination. This inimical influence of faecal matter
on the comma-bacilli does not affect our argument, since in our cases
the examination was proceeded with immediately after the mixture was
made. But it helps to explain the disappearance of the comma-bacilli
from the stools of cholera patients in the later stages of the disease,
pointed out by Koch (loc. cit.) and easily confirmed.

iiS THE BACTERIA IN ASIATIC CHOLERA. [CH. vi.

diagnostic value by means of the gelatine culture-test is of
necessity less important.

The same applies also to cultures made on linen. Koch
is easily confirmed when he says that the examination of the
mucus-flakes of the bowel discharges, with which the linen of
a cholera patient becomes soiled, very often yields a good
result, for this represents in some instances what is practically
an artificial cultivation. If such mucus-flakes are taken off
the linen and kept damp for twenty-four hours they will be
found crowded with the comma-bacilli, and their true
character can then easily be determined by plate-cultivation.
But this result is not always achieved, for if originally but
few comma-bacilli and many other bacteria are present, the
latter will, particularly in hot weather, have so enormously
increased that of the original comma-bacilli very little or
nothing is left.

Bujwid's and Dunham's colour test, described on page 1 1 1,
is also to be mentioned in connection with the diagnosis
and detection of the comma-bacilli.

Bujwid1 shows that the reaction is the more pronounced
the purer the culture in choleraic comma-bacilli is ; pure
cultivations of these bacilli in broth containing pure peptone
show after twenty- four hours' growth at 37° C. intensive purple
reaction with crude HC1.

Gaffky2 gives an account of an isolated outbreak of a
suspicious choleraic disease amongst some of the inhabitants
in Gonsenheim and Fimhen (near Mayence) during September
and October 1886 ; Dr. A, Pfeiffer had ascertained by micros-
copic examination and by the culture test the presence, in the
stools and in the contents of the intestine of some cases dead
of the disease, of comma-bacilli, having -all the characters
of the choleraic comma-bacilli. Gaffky confirmed this.

1 Centralblatt f. Bacteriologie, IV. p. 494.

2 Asbdtur ans d. A'ais. Gesund/mtsamte, II. Bad. I, 2, Heft. p. 39.

CHAPTER VII.

EXPERIMENTAL PRODUCTION OF CHOLERA.

KOCH in his first pamphlet (p. 27) told us that he has
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 Burden
Sanderson, were repeated by Koch over and over again on
fifty white mice fed with fresh 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 experiments with the cultivations of comma-bacilli ;
these were given as food in all stages of development.
When experiments were made by feeding animals with large
quantities of comma-bacilli, on killing them and examining
the contents 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 reached

the intestinal canal The comma-bacilli had been

destroyed in the stomachs of these animals. . . . The

120 THE BACTERIA IN ASIATIC CHOLERA. [CH.

experiment was therefore modified by introducing the
substances direct into the intestines of the animals. The
belly was opened, and the liquid was injected immediately
into the small intestine with a Pravaz's 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 that purgatives were previously
administered to the animals in order to put the intestine into
a state of irritation, and then the infecting substance wasgiven,
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 animals in large quan-
tities ; and it is not the same as in other experiments
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
infected with cholera, I made inquiries everywhere in India
as to whether similar diseases had ever been remarked
amongst animals. In Bengal I was assured such a pheno-
menon had never occurred. This province is extremely
thickly populated, and there are many kinds of animals there
which live together with human beings. One would suppose,
then, that in this country, where cholera exists in all parts
continually, animals must often receive into their digestive

vii.] PRODUCTION OF CHOLERA. 121

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 experi-
ments, 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."

I have quoted thus at length from Koch, to show how care-
fully he had investigated from all points of view the question
of the communicability of cholera to animals, and how
precise and definite is his conclusion arrived at in this matter.
This is the more important since his advocates and imitators
leave this entirely out of sight. After Koch had thus
declared himself (at the Cholera Conference in Berlin,
July 1884), von Pettenkofer — acknowledged to be a high
authority on cholera — refused on epidemiological grounds,
to be described below, to accept Koch's comma-bacillus as
the active cause of cholera ; in fact he was so convinced of
the contrary that he offered with other medical men, under
any conditions to be determined in committee, to swallow
any amount of pure cultivation of Koch's comma-bacilli.
Fortunately, about that time Nicati and Rietsch had published
certain experiments by which they thought they had suc-
ceeded in artificially producing cholera in dogs and guinea-
pigs. These gentlemen, struck by the fact that in cholera
cases with rice-water dejecta the intestinal contents are free
from bile, thought the exclusion of bile from the intestine
probably formed a conditio sine qua non for the success of
the experiment. Any one who has had any experience of a
choleraepidemic must be struck with the extravagance of such
an idea. Is it not known that many and many a light case of
cholera occurs, in which the intestinal contents during the
onset of the disease is still mingled with bile, and that as

122 THE BACTERIA IN ASIATIC CHOLERA. [CH.

the symptoms increase in vehemence so the bile-secretion,
like the other secretions — urine, saliva, gastric and pancreatic
juice — ceases ? One could understand the reasonableness of
such an idea, if the premonitory symptoms of cholera were
characterised by suppression of bile-secretion, but this is not
the case ; most of the secretions of the liver, kidney, saliva,
stomach, and pancreas, cease after the disease has well set
in. One might just as reasonably suppose that ligaturing
the ureter would be a conditio sine qua. non for the success of
the experiment of artificially producing cholera, on occount
of the suppression of secretion of urine in cholera. As a
matter of fact Nicati and Rietsch, in their experiments on
guinea-pigs, Koch in his experiments on dogs and guinea-
pigs, van Ermengem in his experiments on guinea-pigs, did
not employ this mode of experimentation.

Well, then, Nicati and Rietsch opened the abdomen of
dogs, ligatured the bile-duct, and injected then into the
duodenum cultivations of the choleraic comma-bacilla. The
animals died ; their intestine contained fluid with flakes of
detached epithelium ; and the comma-bacilli were found to
have increased enormously in numbers. This was put down
as cholera, due to the increase and action of the comma-
bacilli. Was anything more extraordinary ever heard of in
experimental pathology? The bile duct is ligatured, the
peritoneal cavity and intestine are exposed, inflammation of
the bowels ssts in, with fluid evacuations and detached
epithelium, and this is put down as cholera, and as due to
the comma-bacilli introduced. I venture to say that these
symptoms can be as readily reproduced without the comma-
bacilli. The comma-bacilli are found to have increased in
numbers, but surely this should be the natural and inevitable
result if they are introduced into an intestine in which disease
is set up : such an intestine is their natural breeding-

VIL] PRODUCTION OF CHOLERA. 123

ground ; from a diseased intestine they had been originally
derived.

Koch repeated (Deutsch. med. Woch. 45, 1884) these
experiments of Nicati and Rietsch on dogs, without pre-
viously ligaturing the bile-buct. The fluid, a fraction of a
drop of a cultivation of comma-bacilli greatly diluted, was
injected into the duodenum, of course after opening the
abdominal cavity. " With few exceptions the animals so
experimented upon died after one and a half to three days.
The mucous membrane of the small intestine was reddened,
its contents watery, colourless, or slightly reddish, and at
the same time flakey. In the intestinal contents the comma-
bacilli were present in pure cultivation and in enormous
numbers. We have then here the same appearances as in
the cholera intestine in acute cases." To these experiments
I have to apply exactly the same criticism as I applied above
to those of Nicati and Rietsch. There is absolutely no
guarantee that the peritoneum and bowels of an animal
under such an experiment, leaving out the comma-bacilli,
would not become inflamed, and in this state the comma-
bacilli would readily and copiously multiply. From some
experiments made later by Koch, and hereafter to be
described, this assumption will appear very feasible.

I recommend to the notice of van Ermengem, and particu-
larly to Mr. Watson Cheyne and Dr. Workman, the following
statements on those points made by Koch himself, after a large
number of experiments. These gentlemen thought it quite
unwarranted and thoughtless on my part to accuse the
operation as having anything to do with the result of the
experiments. Here is the passage of Koch's that I refer to,
as given in his address to the Second Conference on Cholera
held in Berlin, May 1885 : l

1 Brit. McL Journ. Jan. 9, 1886, p. 62.

124 THE BACTERIA IN ASIATIC CHOLERA. [CH.

" In these experiments it struck me that, the better the
operation was performed, and the less extensive the manipu-
lations, the less chance was there of the animals dying of
cholera;" and further "in this set of experiments also (with-
out ligature of the bile-duct) the results are so much the less
positive the less disturbance and the less the intestine is
squeezed or torn in searching for and pulling forward the
duodenum. Hence the experiment succeeds only excep-
tionally, when one limits oneself to opening the abdominal
cavity only to a small extent, and making the injection into
the coil of intestine first exposed, instead of into the deep-
lying duodenum. Of six guinea-pigs which were operated
on in this way, only one died of cholera, the rest remained
alive. The same experiment was then performed on three
rabbits, without any of them dying or even becoming ill."

Well, by these experiments Koch thought himself quite
justified in refusing von Pettenkofer's challenge; he said,
" Under these circumstances it is more advisable to decline
the offer of those persons who recently proposed to swallow
pure cultivations of comma-bacilli, and for the present to
continue the experiments on guinea-pigs and other animals."

The most exact and carefully-conducted experiments have
been made by van Ermengem, and they contrast, as to care
and precision and number, as well as in the character of the
experiments, with those published by Babes, Doyen,
Watson Cheyne, and others. I shall therefore for the
present refer only to the experiments of van Ermengem.
The most noteworthy result made known by him is this,
that guinea-pigs inoculated per duodenum with relatively
large doses of choleraic comma-bacilli cultivated in serum
(one gramme to half a gramme) died in the course of between
two and eighteen hours. The symptoms presented by these
animals were those of acute chemical poisoning, for what

vii.] PRODUCTION OF CHOLERA. 125

van Ermengem describes on p. 77 of his paper is quite
compatible with this. If proof were needed it is furnished
by the fact that some of the animals succumbed in a few
hours ; this could hardly be ascribed to anything else except
chemical poisoning, as contrasted with a real infection with
an incubation-period such as is known in the case of other
infectious diseases. There are now known a good many
cases of acute poisoning in the human subject (sausage
poisoning, mackerel poisoning, poisoning by over-ripe fruit,
tinned salmon, tinned sheep's tongue, &c.) investigated on
various occasions within recent years, in which the symptoms
of acute gastro-enteric disturbance set in after a few hours.
Other symptoms, as the stadium algidum, cramps, pain
in abdomen, fall of temperature, disturbed respiration,
were all present in these cases. Brieger in his pamphlet
on the Ptomaines (Berlin, 1885) has produced these symp-
toms in animals by the various alkaloids, analysed and
isolated by him from various food-stuffs, that had undergone
putrefaction. The guinea-pigs thus experimented upon by
van Ermengem showed on post-mortem examination of their
intestines amongst numerous septic bacilli and micrococci a
few of the choleraic comma-bacilli. Two observations made
by van Ermengen (I.e. pp. 78, 80) deserve special notice, (i.)
On keeping the intestinal contents of such a guinea-pig for
twenty-four hours in a moist chamber, it became crowded
with the choleraic comma-bacilli. (2.) One of several animals
which had received fractions of a drop of a serum-culture into
the duodenum remained well. This animal, nine days after
the inoculation, voided in its stools still living choleraic
comma-bacilli; from this it follows that the comma-
bacilli, although they must have lived and multiplied in
this animal's intestine for nine days, were unable to produce
any disease.

126 THE BACTERIA IN ASIATIC CHOLERA. [CH.

Another series of experiments, not less important, were
made by van Ermengem as follows : by a Chamberland filter
or by heating, he eliminated from his cultures (chiefly in serum)
the comma-bacilli themselves, and then injected into the
duodenum the remaining fluid only ; he used this in various
quantities, but in most instances produced results identical with
the above, i.e. death of the animals with the symptoms just
described, which he regarded as indicating cholera. But there
was this striking difference, that the symptoms and death were
retarded in direct proportion to the quantity injected. These
observations are in harmony with those of Nicati, Klebs,
and other?, who have found that in artificial cultivations
of the choleraic comma-bacilli there is present a chemical
poison, which in guinea-pigs produces acute poisoning
similar to ptomaine-poisoning. But whether from this it can
be concluded that when these comma-bacilli are introduced
into the intestine, they, by their multiplication necessarily
produce therein the same chemical poison, is open to
question ; moreover, from van Ermengem's observations, I
am inclined to conclude the contrary, namely, that this
chemical poison is not necessarily produced by them, and that
the comma-bacilli can live and • thrive in the intestine
without producing any such chemical poisoning.

Another important point is this : the production by the
comma-bacilli of a chemical poison setting up, when injected
into a dog or guinea-pig, symptoms identical with ptomaine
poisoning, is by no means the exclusive property of the chole-
raic comma bacilli, since various other septic bacteria have
the same power,1 and this has been proved experimentally by
Berdez.2 This gentleman found in artificial cultivations in
broth of Finkler's comma-bacillus, and of thejequirityj9^///2/.y
subtilis, the same chemical poison. As regards Finkler's

1 See Brieger, loc. cit. 2 Brit. Med. Journ. Nov. 7, 1858.

vii.] PRODUCTION OF CHOLERA. 127

comma-bacillus I shall return later on to experiments made
by Finkler.

Hueppe has by recent experiments shown that the hen's
egg is the best and readiest means to obtain this toxic sub-
stance, by cultivating within it the choleraic comma-bacilli :
already after twenty-four hours a considerable quantity of this
substance (cadaverin) becomes available. In this respect
there is a decided difference between broth and gelatine
cultures and those carried on in egg. Further : not only the
choleraic comma-bacilli produce in this condition the toxic
substance but also Finkler's and Deneke's comma-bacilli
when cultivated in this same medium ; and that there exists
between these different species a difference only in the
quantity of the poison produced.

We see, then, that there can be no question about the
presence in certain artificial cultures of the choleraic comma-
bacilli (particularly in serum-cultures four days old and in
egg-cultures) of a chemical ferment capable of producing
acute poisoning in animals, but the symptoms thus produced
are comparable with ptomaine-poisoning ; and further about
the fact that the production of such a ferment does not
appertain exclusively to cultures of choleraic comma-bacilli,
but also to other saprophytic organisms.

That this ferment is not present in the mucus-flakes of
the cholera intestine, although swarming with the comma-
bacilli, that it is not present in sufficient quantities in broth
and gelatine cultures or in Agar-agar cultures, I have con-
vinced myself by a large number of experiments. Mucus-
flakes from a fresh cholera intestine swarming with the
comma-bacilli were injected in considerable quantities (one
half to one whole Pravaz syringe) into the small intestine of
dogs, monkeys, cats, and rabbits, into the jugular vein, and
into the peritoneal cavity, but without any result of poisoning.

128 THE BACTERIA IN ASIATIC CHOLERA. [CH.

Numbers of experiments were made at the Brown Institution
by Mr. Dowdeswell and myself, by injecting into the
duodenum of dogs and guinea-pigs considerable quantities
of cultures, recent and old, of the choleraic comma-bacilli
in nutritive gelatine and in Agar-agar, but with no result.

I have also, after the manner of Ferran,1 injected sub-
cutaneously into guinea-pigs large quantities (3-5 ccm.) of
cultures of the comma-bacilli in nutritive gelatine and in
broth. Two guinea-pigs received each subcutaneously 4ccm.
of a culture of Koch's comma-bacillus in beef-broth kept at
37° C. for three days. The fluid was crowded with the
comma-bacilli. No result whatever followed.

Two guinea-pigs were similarly inoculated each with 4 ccm.
of nutritive gelatine liquefied by, and crowded with the
choleraic comma-bacilli ; two other guinea-pigs were similarly
inoculated, each with 4 ccm. of nutritive gelatine liquefied
by Finkler's comma-bacillus. All four animals were dead
in twenty-four hours. The symptoms during life were those
of ptomaine-poisoning. Post-mortem examination showed
that the whole of the subcutaneous tissue of the chest and
abdomen was dark red and cedematous, the viscera much
congested, the spleen small. The heart's blood contained
in the first two animals Koch's comma-bacilli, in the two
second animals Finkler's comma-bacilli, as was proved by
cultivations. Cultivations made of the intestinal contents —
fluid grumous mucus — yielded no comma-bacilli. Experi-
ments made with small quantities J — i ccm. of the above
gelatine-cultures, produced no result. Thus it is seen that
gelatine-cultures contain the same chemical ferment, but
only in relatively small quantity, since huge doses of such
cultures are required to produce the effect, small doses being
without effect ; and it is further seen that this ferment is not
1 Comptes Rendus, 1885.

VIL] PRODUCTION OF CHOLERA. 129

exclusively present in the cultures of the choleraic comma-
bacilli.

Dr. D. D. Cunningham has published (in "Scientific
Memoirs by Medical Officers of the Army of India," Part II.
1886, pp. i to 14,) a very interesting series of observations
made by injecting subcutaneously in the thigh into guinea-
pigs cultivations of comma-bacilli directly derived from a
case of cholera. Some of the animals thus experimented
upon died in two to three days, and showed symptoms of
effusion spreading from the seat of inoculation over the
lower half of the abdomen of the same side ; peritonitis was
present, and a sticky secretion was found on the serous covering
of the intestine ; comma-bacilli were obtained by cultivation
from the subcutaneous effusion, from the peritoneal exuda-
tion, from the intestinal contents, and from the cardiac blood. v
These results are then comparable to septicaemic infection,
such as was the case in Koch's experiments on mice, in
Ferran's and my own experiments on guinea-pigs.

I have found, what has been also noticed by others, that in
a large number of animals broth and gelatine cultures of
advanced pedigree when injected in large quantities produce
no result, and that therein they contrast markedly with
cultures of recent pedigree. I have received directly from
Dr. Cunningham of Calcutta fresh cultures of the choleraic
comma-bacilli and have been able to compare them in this
respect with subcultures that have been kept going for two
years and more.

Another important fact capable of throwing some light on
this question is this. The cases of what is called ptomaine-
poisoning may be grouped into two distinct classes : the
one comprises cases in which an alkaloid, the product of
putrefaction and putrefactive organisms, is introduced into
the system and produces acute poisoning with gastro-enteritic

130 THE BACTERIA IN ASIATIC CHOLERA. [CH.

symptoms (vomiting, purging, inflammation of the intestine,
its cavity filled with mucus, cramps, fall of temperature,
obstructed breathing, &c.). In these cases the introduction
of the chemical poison by the stomach seems ineffective,
subcutaneous and intravascular introduction, and (what
comes to the same) injection into the small intestine, seems
a conditio sine qua non. If this be the case, the gastric
juice would have a destructive effect on the chemical
poison. In another class of acute chemical poisonings,
also set down as ptomaine-poisoning, and in which the
symptoms are very much the same, the introduction of
the poison into the stomach is perfectly effective. Such
are the cases of poisoning by sausages, meat, fish, jelly,
pie, &c. Hardly a year passes in which numerous cases of
this kind of poisoning do not occur.1 While then in the
first class of cases we have to deal with ptomaines, such as
have been investigated by Selmi and others, and particularly
by Brieger, being the products of putrefaction, we have in
the second class to deal with special fermentative processes,
the products of which must be of a different nature from
the first, since they are unaffected by the gastric juice.

I have had to do with the investigations of such an out-
break of veal-pie poisoning observed by Dr. Thursfield, of
Shrewsbury, and I have shown that there was present a
species of Bacterium termo, which is incapable of life and
multiplication in the normal animal body, but when cultivated
at the ordinary temperature (18-20° C.) in nutritive gelatine
or in broth, rapidly multiplies and produces a chemical fer-

1 In both instances the rapidity with which the symptoms set in clearly
points to an unorganized or chemical poison, in the general acceptation
of the term, as distinct from an organized poison requiring incubation,
such as we have to deal with in infectious diseases, where certain bacteria
are introduced into the system and by their multiplication and life-action
give rise to the symptoms.

VIL] PRODUCTION OF CHOLERA. 131

ment, which, for want of a better term, I called paraptomaine ;
this ferment, when introduced into the stomach of mice, pro-
duced acute gastro-enteritis, such as the veal-pie did in the
human beings who partook of it.

Considered in this light there seems a striking analogy
to exist between the chemical poison produced in certain
artificial cultures of the choleraic comma-bacilli and the
ptomaines produced in putrefactive processes such as have
been investigated by Brieger. In both the chemical fer-
ment does not pass unscathed through the gastric juice.
Series of experiments have been made by a number of
workers by introducing into the stomach cholera stools or the
contents of cholera intestine and artificial cultures, but
without any result. Numbers of people continually partake
of substances (meat, game, &c.) that probably contain,
judging from the number of putrefactive organisms present
in them, considerable quantities of ptomaines, yet no dis-
turbance occurs, while in other cases (mackerel, sausage, &c.)
serious mischief is produced ; these latter cases cannot be
simply due to ptomaines produced by putrefaction in the
ordinary acceptation of the term. But let the ptomaines
obtained by putrefactive processes be introduced subcu-
taneously, by the vascular system or otherwise, and inde-
pendently of the stomach — and such experiments have been
repeatedly made by a large number of workers (see Dr.
Brunton's recent work on the Disorders of Digestion) — and
the result is acute poisoning. And such is evidently also
the nature of the chemical poison present in certain cultiva-
tions of the choleraic comma-bacilli. That only certain
cultivations of the comma-bacilli contain this poison in a
concentrated form, while others contain little or none, is, as
shown in the experiments by van Ermengem, Hueppe, and
others above mentioned, quite in harmony with Brieger's

K 2

132 THE BACTERIA IN ASIATIC CHOLERA. [CH,

observations, who could obtain some of his ptomaines only
from certain substances, not from others.

Koch having very likely felt that experiments such as
those which he and van Ermengem made after the method
of Nicati and Rietsch were not free from objection, inasmuch
as they involved severe surgical operations (see p. 124), and
inasmuch as they, unlike all other experiments employed in
bacteriological research, did not imitate the methods of
infection as they occur under natural conditions, devised a
method of experiment which, though far removed from the
first, was not quite free of the second criticism. 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 neutralizing or
making alkaline the contents of the stomach, and then
introducing per os the comma-bacilli. He therefore kept
guinea-pigs for twenty-four hours without food, and injected
then into their stomach per os 5 ccm. of a five per cent,
watery solution of carbonate of sodium. 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) after-
wards he introduced by catheter 10 ccm. of a cultivation of
the comma-bacilli in meat-infusion.

The result is noteworthy. Seven guinea-pigs thus experi-
mented upon remained perfectly well ; " they were killed after
twenty hours, and the contents of their stomach, intestine,
and caecum, were examined by gelatine plate-cultivations. In
six of the seven animals, the cholera-bacteria could be de-
monstrated in the small intestine. The experiment had thus
in so far succeeded, that the cholera-bacilli had passed unin-

vii.] PRODUCTION OF CHOLERA. 133

jured 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 re-
mained quite healthy. Finally four guinea-pigs were similarly
experimented upon (5 ccm. of solution of sodium carbon-
ate, then 10 ccm. 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 ex-
tremities came on, the respiration was weak and slow, the
head and extremities were cold, and the animal died in this
condition. On post-mortem examination the small intestine
was markedly reddened and full of a flakey, watery, colour-
less fluid. The stomach and caecum contained a large
quantity of fluid. "The examination with the microscope
and with gelatine-plates 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 tem-
porary relaxation with arrest of peristaltic movement ; and
thus had rendered it possible for the comma-bacilli to remain
longer and gain a footing in the intestine." This conclusion
appeared to Koch justifiable, inasmuch as by direct experi-
ment he thought he had proved that the contents of the

134 THE BACTERIA IN ASIATIC CHOLERA. [CH.

stomach pass too rapidly through the small intestine, and
since the comma-bacilli could only unfold their poisonous
action, i.e. could produce the chemical poison, if they had
time to remain there and to multiply. Consequently 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. To this
method of reasoning I must take exception. Koch shows
by direct experiment that even twenty hours after injection
the comma-bacilli can be recovered from the small intestine
in a living state. Now the most important character of all
pathogenic bacteria is this, that when introduced into the par-
ticular tissues suitable for their propagation they set up their
pathogenic power. How is it then, one might reasonably
ask, that the comma-bacilli, if even only for a few hours in
the small intestine, do not invade in swarms the epithelium
and superficial layers of the mucous membrane ? Koch does
not, and of course cannot, deny that all absorption of the chyle
must take place in the small intestine, and since the comma,
bacilli are much smaller than the large chyle globules, and
are possessed of spontaneous mobility, it follows of necessity
that the comma-bacilli can and must readily pass into the
epithelium and the superficial layers of the mucous mem-
brane ; and since the epithelium and the superficial mucous
membrane, according to Koch's own statement and belief, are
the suitable nidus for the multiplication and action of the
comma-bacilli, all conditions would therefore here exist
which are required for their settling down and acting. Add to
this that 10 ccm. of a broth culture of comma-bacilli containing
millions and millions of comma-bacilli, are subject to absorp-
tion by the small intestine for twenty hours (see the above-
mentioned observations of Koch), and that such vast crowds
of comma-bacilli in a few hours kept at the body-temperature

vii.] PRODUCTION OF CHOLERA. 135

ought to yield a most formidable host of descendants,
and grave doubts must arise as to the tenability of Koch's
explanation.

But to continue. 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 peri-
toneal cavity after the introduction of the sodium carbonate
and the cultivation of the comma-bacilli : this answered well
for achieving positive results. Immediately after the adminis-
tration of the 10 ccm. of the culture of the comma-bacilli, i
ccm. of German tincture of opium for every 200 grms. of the
animal's body-weight were injected into the peritoneal cavity ;
the animal became narcotized 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 : (i) According to Koch's own
showing it cannot be the narcosis which is essential, even
allowing for the present that relaxation of the intestine may
have been produced by the intraperitoneal injection of
opium-tincture, since alcohol alone was injected 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 narcosis of the animal be
produced by opium without furthering in the least the process
of the experiment? This has been tried over and over
again ; watery extract of opium is injected into the perito-
neal cavity, and narcosis lasting for one hour is produced,
but the animals remain well ; tincture of opium is sub-
cutaneously 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

136 THE BACTERIA IN ASIATIC CHOLERA. [CH.

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 were beyond question or doubt the choleraic-
comma-bacilli. (3) It is not proved that injection of
tincture of opium into the peritoneal cavity produces
relaxation of the intestine and arrest of the peristaltic
movement ; there is no proof given for this by Koch
as regards the guinea-pig ; on the contrary, there are
experiments on record made on the dog, when the re-
sult of such injection was quickening of the peristaltic
movement.

From all these considerations it appears to me unwarranted
to conclude as Koch does 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 movement 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 the mucous membrane but eventu-
ally 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

vii.] PRODUCTION OF CHOLERA. 137

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 small intestine are quite innocuous, but they are
rendered capable of great multiplication if the intestine is
previously, from some cause or another, diseased. The
chemical products of such multiplication act as poisons
analogous to the ptomaines obtained from other 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 ob-
tained 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 J published a large series of experiments, in which,
with his comma-bacilli and after the method of experimenta-
tion employed by Koch, he produced results identical with
those gained by Koch with the choleraic comma-bacillus.
There can be no doubt, from what has been shown above,
that Finkler's comma-bacillus has nothing to do with cholera
nostras, nor 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 experi-
ments, to have been proved to have a causal relation to
chohra Asiatica, any more than has Finkler's comma-bacillus,
or any of the other species of septic bacteria that are
capable of producing chemical poisons analogous to pto-
mai'nes. All that can be said is — provided that conditions
1 Erganzungsheft z. Centralb. /. allg. Gesundh. i. 5 and 6.

138 THE BACTERIA IN ASIATIC CHOLERA. [CH.

are established by which the choleraic comma-bacilli are
enabled to grow and multiply in the intestinal canal, these
chemical poisons may be produced. A very instructive and
parallel case is found in the so-called typhoid-bacillus. As
is now generally held, the experiments published by Fraenkel
and Simmonds, in which they maintained to have produced
typhoid fever and death in rabbits after injection of large
quantities of cultivations of the typhoid bacilli do not prove
any real infective action of the typhoid bacillus for the
rabbit ; it has been conclusively proved that this result is
entirely due to certain chemical substances generated by the
typhoid bacillus in the cultivations (Sirotinin, Beumer and
Peiper). This pathological condition can be produced
entirely apart from the bacilli by chemical substances
(Typhotoxin) produced by them in cultivations, and as is the
case in other similar toxic substances the severity of the
abnormal state depends on the quantity injected. Moreover
it has been shown by Beumer and Peiper that by injection
of a small quantity of the chemical substance a refractory
state against an otherwise fatal dose of the same substance
can be produced. (Beumer and Peiper, Zdtschr. f. Hygiene,
ii. I, p. no.)

Of course it cannot be expected that all septic bacteria
will behave, both as regards power of multiplication and
particularly as regards chemical products, in the same
manner as those mentioned above, for it is well known that
some do not do so. Hence experiments with these latter do
not yield any result, and cannot, therefore, have any value
for testing or controlling purposes. In these respects Koch's
comma-bacilli do not attain to the dignity of, or at any rate
do not surpass certain notorious saprophytic bacteria, which,
occurring in normal putrid substances or the human body,
are capable when inoculated in small doses into rodents

vii.] PRODUCTION OF CHOLERA. 139

of producing a true septicaemia infection. Thus certain
species of bacterium or micrococcus occurring in the
fluid of the human mouth were found by Pasteur and
Sternberg to act virulently when subcutaneously injected
into rabbits, other examples being the Bacterium termo
found by Brieger in normal faecal matter, the bacillus
isolated by Bienstock from normal human faecal matter,
and the bacillus occurring in the faeces of milk-fed infants.
These, as it were normal saprophytic organisms, are capable
on inoculation in very minute doses of producing a true infec-
tious disease, a sort of septicaemia, in various rodents, some
in rabbits, others in mice, and others again in guinea-
pigs. Gamaleia asserts * that using cultures of the choleraic
comma-bacilli after their passage through the guinea-pig,
(see above) and infecting pigeons with them in successive
series the blood of these becomes gradually the proper
breeding ground of the bacilli, and that fatal infection in
the pigeons can then be produced by the injection into
pigeons of a small dose of such blood. If such be the case
this would only prove that a septicaamic virus has hereby
been reared. Gamale'ia also states that, as in the case of the
experiments of Beumer and Peiper on the typhoid bacillus, by
the chemical products of the comma-bacilii immunity can be
produced in pigeons against the virulent cultures. Lowenthal2
found that by carrying on subcultures for some time, the
comma-bacilli lose their virulence on mice,butcanbe acquired
again by cultivation in a special medium. Mice infected
first with weak cultures are found for a time refractory
against virulent culture. I have injected into the pectoral
muscle of pigeons several cubic centimetres of a recent
broth culture of choleraic comma-bacilli ; after 24 hours

1 Semaine Medicate, No. 34.

2 Ibid., No. 35.

140 THE BACTERIA IN ASIATIC CHOLERA. [CH. vn.

the muscle and the blood of the heart were used for
establishing a very large number of plate cultivations and
also for inoculating with considerable quantities broth in
tubes, but in no single instance could any growth whatever
be produced, nor did the microscopic examination show any
organisms. The comma-bacilli had therefore been killed
already in 24 hours in the tissues of the pigeon. The animal
showed of course no disturbance of any kind.

From these considerations it follows then : (i) the presence
of the choleraic comma-bacilli occurs in dead tissues only, as
we have shown above, namely the fluid and mucus-flakes of the
diseased and disorganized intestine in cholera, (2) the pro-
duction by the comma-bacilli as by other notorious saprophy-
tic bacteria (Finkler's and Deneke's vibrios) of sulphuretted
hydrogen, (see Hueppe's experiments of cultivation of the
comma-bacilli in egg) ; (3) the production by the comma-
bacilli of Indol in broth that contains peptone (Salkowski,
Bujwid) ; (4) the absence of comma-bacilli in the living
tissue of the intestine or other organs, (5) the septicsemic
character of the disease produced by the comma-bacilli in
certain rodents,1 and the toxic character produced by the
chemical products of certain cultures alike of choleraic
comma-bacilli and other comma-bacilli notoriously saprophy tic.
From these facts the conclusion seems to me to be justified
that the comma-bacilli in these respects do not differ from
saprophytic bacteria, and their relation to the causation of
Asiatic cholera becomes therefore very doubtful.

1 For a criticism of the recently published researches of Messrs.
Macleod and Milles, see the concluding chapter of this work.

CHAPTER VIII.

THE 1NFECTIVENESS OF CHOLERA.

IN the foregoing chapters we have pointed out that under
certain conditions large numbers of comma-bacilli may pass
in the living condition through the stomach into the intestine,
without producing serious results in the latter organ unless
it be previously diseased. Millions may pass thus through
the healthy small intestine, as is shown by the numerous
experiments of Koch above quoted, without producing any
result whatever. And this fact, I think, disposes of the
idea that they can be the cause of Asiatic cholera in the
human subject. Can any one doubt, who has reflected on
the actual conditions obtaining in an epidemic of cholera,
that under natural conditions of infection this cannot be so ?
Is it not one of the most terrible facts known and constantly
observed in cholera epidemics, that in a locality where a
cholera epidemic has broken out, young and old, healthy
and unhealthy, alike are liable to infection ? Is it possible,
is it in the least justifiable, to assume that in all these
persons the state of the stomach at the time of infection was
such that its contents were not of the acidity sufficient to
kill the few comma-bacilli — for in natural infection it can
only be a question of very minute particles of contagium —

142 THE BACTERIA IN ASIATIC CHOLERA. [CH.

that had got access to it ; or stranger still, that in ail these
persons the intestine is in a state of disease, thus favouring
the settling and multiplication of the comma-bacilli ? I think
all who have witnessed an epidemic of cholera will agree
that such an assumption is out of the question. If such were
the conditions under which infection takes place, I am sure
cholera would be an extremely rare disease. For even
assuming that the comma-bacilli had got entrance into the
stomach, say before breakfast, when the stomach is sup-
posed to be perfectly empty, and its reaction supposed to
be neutral or alkaline, they would pass unscathed and in a
living state into the small intestine ; but in order to multiply
in appreciable numbers the intestine itself would have to be
in a state of disease, otherwise they could not multiply in it.
Such conditions would unquestionably reduce cholera cases
to an insignificant number. The fact, repeatedly observed
during the epidemics of 1884-1886 in Spain and Italy and
France, that in a population of 10,000 or 12,000 inhabitants,
in the course of two to three weeks 1,200 to 1,500 people
were struck down with cholera, disposes of the above
assumption. It is true, and it has been observed over and
over again, that when cholera appears in a locality it attacks
at first and with obvious predilection those persons whose
digestive organs, or whose general health for the matter of
that, are weakened or in a state of disease ; but this is
known to be the case in other infectious maladies also, the
unhealthy being as a rule more susceptible to infection than
the strong. But when an epidemic has well set in, no such
exemption of the strong and vigorous is noticeable ; pro-
vided the active contagium is present and has in this state
access to their system, it does not matter what their general
condition or that of their digestive organs is, they are struck
down by the plague. And in this respect there is no line

VIIL] THE INFECTIVENESS OF CHOLERA. 143

of distinction to be drawn between cholera and other in-
fectious diseases. Other infectious diseases, such as typhus,
typhoid fever and relapsing fever, also attack with predilec-
tion the poor, ill-nourished, and weak ; other infections
spread by filth and uncleanliness of person, of air and water,
but just as in these, so also in cholera, a healthy state of
the intestine does not ensure immunity against infection.

It will probably be argued that once the comma-bacilli
have settled in the small intestine and multiplied, the fact
will not be disputed that they can produce a chemical poison
which by absorption produces the symptoms of cholera, and
that this at least can be inferred from the experiments on
animals ; and it will be further argued that this is exactly
what Koch maintains. In other words, owing to the
absence of comma-bacilli from the blood and the tissues,
and owing to their presence in the alimentary canal only, it
must be assumed that they produce there a chemical fer-
ment which being absorbed produces cholera, and such an
inference is supported by the positive experiments on
animals. While admitting that the comma-bacilli, like
some other saprophytic bacteria, are under certain
favourable conditions of growth capable of producing a
chemical ferment analogous to ptomaines, I do not admit
that this is applicable to cholera. Certain important con-
siderations previously mentioned, for example, that the
comma-bacilli can only multiply in an intestine previously
diseased, offer an unsurmountable primary difficulty to this
assumption ; moreover, there is the fact that the comma-
bacilli pass only with great difficulty unscathed through the
normal stomach, or through a stomach in which the contents
are always acid or perhaps of more than normal acidity as
in many cases of dyspepsia ; whence it would follow that
the number of persons subject to cholera ought to be quite

144 THE BACTERIA IN ASIATIC CHOLERA. [CH.

insignificant. But there are not less important difficulties of
another kind to be settled. Supposing the comma-bacilli
in the intestine of a cholera patient produce this chemical
poison, say some kind of ptomaine, does that mean that
before the disease has set in the comma-bacilli have been
already so numerous in the intestine as to produce this
poison in sufficient quantity to set up the disease ? There
exists no proof that because in a person already ill with
cholera the stools and the contents of the ileum contain
numerous comma-bacilli, they were present there in large
numbers before the disease set in, and there is no proof
that when in a well-pronounced acute case the comma-bacilli
are very numerous, their chemical products are being
absorbed and thereby produce the symptoms. This would
be tantamount to placing the cart before the horse. In
some, not by any means in all, cases of acute typical cholera
that are brought into the post-mortem room, the mucus-
flakes of the lower ileum contain large numbers of the
comma-bacilli. Does any one mean to say that this state
of things existed before the disease set in, or that such an
intestine filled with watery fluid is capable of absorbing
anything from its cavity ? And how about those cases of
acute and typical cholera that are brought into the post-
mortem room, in which nothing of the sort obtains, i.e.
where the intestine, amongst crowds of other bacteria,
contains only a few comma-bacilli ? If it were true, as was
first maintained by Koch, that the invasion of the super-
ficial strata of the mucous-membrane of the ileum and its
Peyer's glands is the beginning of the disease, and that here
the poison is elaborated by the rapidly-multiplying organisms
and absorbed, this aspect of the argument would unquestion-
ably lend considerable support to Koch's view, but this I
have clearly shown is not the actual case ; an invasion of

viii.] THE INFECTIVENESS OF CHOLERA. 145

the superficial layers of the mucous membrane of the ileum
and of the Peyer's glands cannot be demonstrated in a
number of typical acute cases. All that can be said,
therefore, is this — in some cases the comma-bacilli are
found numerously present in the mucus-flakes of the
ileum (i.e. in dead tissue), in other equally acute and typical
cases they are there present only sparingly amongst crowds
of other bacteria ; further, when they are present in large num-
bers in the rice-water discharges, i.e. when the chemical poison
is being elaborated in large quantities, the disease has already
well set in, and in this state the intestine is pouring
out quantities of fluid and therefore cannot be said to be
capable of absorption. From these considerations it seems
to me to follow that the presence of numerous comma-bacilli
in the contents of the cholera-intestine is a result of the
peculiar pathological state of the intestine, just as was the
case in the above-mentioned successful experiments on
guinea pigs. Supposing, as has been suggested on a former
page, that the comma-bacilli are already present in the
normal human intestine, but being subject to unfavourable
conditions remain very limited in numbers ; it may be that
in cholera the conditions having become favourable we
should find them greatly increased in numbers! True, no one
has as yet shown that in the normal human intestine comma-
bacilli identical with Koch's comma-bacilli do exist, but
then few observers have as yet systematically examined the
different species of bacteria that do exist in the normal
intestine. Koch and others say that they have by plate-
cultivations carefully examined the normal contents of the
human intestine and have never found them, but it
must be remembered that they failed to find any comma-
bacilli, and yet we now know that Finkler's comma-bacilli do
occur normally. I do not mean hereby to imply that these

L

146 THE BACTERIA IN ASIATIC CHOLERA. [CH.

observers have not carefully examined the contents of the
normal intestine by gelatine plate-cultivations ; I only wish to
point out that a negative result obtained in one set of experi-
ments by one operator does not preclude a positive result being
obtained in another set by another operator. Again I have
shown on a former page that even when choleraic comma-
bacilli are added in comparatively small quantity to a large
quantity of bacterial mixture, it is extremely difficult, nay,
in many experiments impossible, to recover them by plate-
cultivation. I say, therefore, that the presence of a few
comma-bacilli even in the normal intestine is not impossible,
nor even improbable ; and that a good many further obser-
vations are required to settle this point. I am quite aware
that many are content with the facts already at hand ; many
say — " Here we have a disease in which it is admitted
that a peculiar species of bacteria does occur, that this
species has not been demonstrated as yet to exist
in either normal or morbid states of the intestine ;
further, that experiments performed on animals prove that
these comma-bacilli introduced into the small intestine can
under certain conditions multiply there ; death with lesions
similar to those of cholera ensuing ; this is quite sufficient
to show that these comma-bacilli are connected with the
causation of the disease Asiatic cholera." I fully admit
that these facts cover a good deal of ground and that these
arguments are without question of considerable strength.
But I do not think we ought to be satisfied with them. I
cannot overlook the fact that, notwithstanding the constant
presence of the comma-bacilli in the cholera discharges and
cholera-intestine, they are only present in dead tissues (fluid
and mucus in the cavity of the intestine) ; I cannot overlook
that Asiatic cholera would in this respect make one great
exception to all other infectious diseases in which at present

viii.] THE INFECTIVENESS OF CHOLERA. 147

a causal relation to definite bacteria has been fully estab-
lished ; I cannot overlook the fact that our knowledge of
the different species existing in the normal intestine and in
the cholera-intestine is far too incomplete to warrant our
assuming anything of so definite a nature ; I cannot overlook
the extremely great difficulties in harmonizing the conditions
under which the positive experiments on animals have been
carried out with the actual conditions of infection obtaining
in nature ; I cannot overlook the great discord that exists
between what is known of the comma-bacilli as regards their
behaviour in gastric juice, and their extremely limited
capability of multiplying in the normal intestine on the one
hand, and the fearful susceptibility to cholera infection of
healthy and vigorous persons in cholera epidemics on the
other.

But these are not the only difficulties. In all bacterio-
logical inquiries referring to infectious diseases the results
of such inquiries, if they are to be accepted as well-
established, must be in harmony with the well-founded
facts discovered by epidemiology ; the bacteriological portion
of the inquiry is no doubt a very important one, but unless
it well harmonizes with well-established other facts not
appertaining to bacteriology, it cannot claim our full
confidence. Now, I maintain with von Pettenkofer that
some important parts of our knowledge concerning comma-
bacilli do not harmonize with well-established epidemio-
logical facts. Some of these have been already discussed,
others will be discussed now. Some of the facts as to the
spread of cholera difficult of explanation are those pointed
out by von Pettenkofer in reference to the dependence of
epidemics on locality and season. Certain localities appear
to enjoy a special immunity against the spread of cholera.
Versailles and Lyons, Birmingham and other towns, are

L 2

148 THE BACTERIA IN ASIATIC CHOLERA. [CH.

notorious examples. Persons have carried infection into
these localities from surrounding parts, were taken ill with
cholera in Versailles or Lyons, and although many such
cases were carried thither yet cholera gained no footing in
either town. During the last epidemic in the south of
France in 1884-1885 numbers of persons coming from
Marseilles and Toulon must have carried infection into
Lyons, yet this city remained free of an epidemic. The
position of Lyons cannot account for this, for it is not, like
Rome or Madrid, situated on high ground, where one
might suppose the cholera contagium would not easily
lodge, but would be gradually carried downward into a lower
situation by the natural drainage from a high level into low
ground. Lyons, on the contrary, is, as regards its moisture
and its situation in the Rhone valley, as badly off as any
notorious cholera locality ; its cleanliness, its water-supply,
its crowded poorer quarters are not a bit better than those
of other big cities not enjoying such immunity. This is
only one example. In India there are many such localities
known ; Dr. Cuningham, the late Sanitary Commissioner
with the Government of India, in his most instructive
book On Cholera (Calcutta, 1885), has mentioned several
of them.

Von Pettenkofer has minutely dealt with these facts in
his various well-known pamphlets and writings on the
dependence of cholera on locality, and it is not necessary
to enter further into this question. Now this immunity of
a given locality, for instance Lyons, seems to me
irreconcilable with the facts known about comma-bacilli.
A few cases of cholera imported into this locality would
yield innumerable masses of comma-bacilli ; the nature and
position of such localities is, as compared with other
cholera-localities, very favourable for the spread of cholera ;

viii.] THE INFECTIVENESS OF CHOLERA. 149

the habits of the poor, the water-supply, the abundance of
filth, all combine to make them a good breeding-ground ;
the comma-bacilli are known to multiply with enormous
rapidity, they have been proved capable of growing and
multiplying in almost everything that contains animal
and vegetable matter, and yet no cholera epidemic
seems to result. The effective method adopted in India
of moving the troops out into camp and away from a
locality in which cholera has broken out proves the same
fact. Often soldiers carry infection into such a camp, are
there taken ill with cholera, yet with such exceptions
no other cases occur. Millions and millions of comma-
bacilli are present in camp, still they do not produce
infection.

The same holds good with regard to season. A few
cases of cholera occur in Calcutta all the year round ; there
is hardly a month in the year in which isolated cases do not
occur. Yet anything like an epidemic is unknown between
June and December. The number of cases begin to rise in
December, about Christmas time, steadily go on increasing
till March and April, then decrease again. The comma-
bacilli are available all the year round ; the habits of the
natives as regards the use of the water from the tanks for all
and every purpose remain the same all the year round. If
a case of cholera occurs, say in October, in one of the huts
or bustees surrounding a tank, the dejecta invariably find
entrance into the tank, for this is the natural sewer of the
huts ; along the shore of these tanks there is any amount of
decaying animal and vegetable matter, and there exists here
therefore a good and sufficient nutritive medium for the
comma-bacilli. In all tanks the natives of the bustees can
be seen at all times and seasons performing their external
and internal ablutions, washing their linen and their cooking

ISO THE BACTERIA IN ASIATIC CHOLERA. [CH.

utensils, and in many cases using the water even for drinking.
This latter is not the rule, since many fetch their drinking-
water from the hydrants, of which there are many in all
parts of the city. But there can be no doubt, as inquiry
proves, that the water of the tanks is used also for drinking
purposes. And yet isolated cases may occur in one of
these bustees during parts of the year without being followed
by other cases. Calcutta is not an isolated instance, the
same holds good of almost every city in Bengal ; in Bombay
and Benares, when I happened to be there in September
and October 1884, I had ample opportunity of studying
these facts, and they have been mentioned in the Report of
the English Cholera Commission, published by the India
Office (pp. 28, 29).

There is not a locality in India, in which, owing to the
just-mentioned habits of the natives, cholera, once imported,
might not be expected to develop into an epidemic; yet
this is often not the case. Cases of cholera have been
imported by pilgrims and others coming from an infected
locality, and while at a certain time of the year, say from
March till June, it led to an epidemic, during other times of
the year it did no such thing. The same dependence on
seasons as regards Europe is well known, and has been very
fully discussed and demonstrated by von Pettenkofer.

The notorious dependence of the spread of cholera on
season is, I think, irreconcilable with the facts that are
known concerning the comma-bacilli. The comma-bacilli
grow and multiply well at all temperatures between 16° and
40° C. I have had good cultures growing at 16° C., and
therefore the months of August, September, October, and
November in India would be extremely favourable. In the
south of Europe March and October, or even February and
November, would be quite favourable, yet these are, as a

vin] THE INFECTIVENESS OF CHOLERA. 151

rule, the very months when epidemics of cholera are rare ;
when they do occur, they occur as a rule between the end
of April and October. The epidemic in Egypt in 1883, the
very epidemic that preceded those of Toulon and Marseilles
in 1884, approached its end by the end of October.

If the comma-bacilli were possessed of the power of
forming spores, and if only in this state they were capable of
producing infection, one could understand that this forma-
tion of spores, as is the case with some other bacilli, might
be dependent on certain definite conditions, amongst which
might be a certain locality and a certain season. But such
is not the case ; Koch is very definite about it, and others
who have devoted special attention to this point are equally
definite ; I have in a previous chapter discussed this point
in detail, and have explained certain appearances which
Hueppe thought sufficient for assuming the existence of
spore-formation in comma-bacilli. When spore-formation
does occur there is no difficulty in demonstrating it ;
but in the case of the comma-bacilli such a phase is not
demonstrated.

Again, if the comma-bacilli were dependent on some
special nutriment obtainable only during certain parts of the
year and not in others, or if the habits of the people differed
as regards cleanliness, water-supply, &c. in certain parts of
the year, a difference in the spread of the disease might be
then accounted for : but the comma-bacilli live and thrive
wherever and whenever there is nitrogenous material — in
fact, they are in this respect conspicuous by their small
selective power, they grow in all localities, in all climates,
and in all seasons.

Then there is the question of the infective power of the
cholera-dejecta. If, as many believe, the fresh cholera-
dejecta were possessed of infective power, then it would

152 THE BACTERIA IN ASIATIC CHOLERA. [CH.

be quite impossible to understand how it happens that the
attendants, nurses, and physicians of cholera-patients, those
that handle the cholera-dejecta, and the friends and relatives
living in the same room with the sick, remain so often
exempt. As von Pettenkofer has pointed out, the fact that
when in any locality cholera has assumed the epidemic
character and the attendants do become liable to cholera
does not prove that they contract it from the cholera-patient ;
for another explanation, namely, that the cholera-virus has
by that time become universally distributed in the locality,
is a good explanation. If the fresh cholera-dejecta
contained the materies morbi, or if, for instance, the
comma-bacilli were the cholera-microbes, then one case of
cholera should be sufficient to infect all those coming near
it. The experiments of Koch prove that the comma-bacilli
become inert and dead by perfect drying, e.g. when dried in
a thin layer on a coverglass or on silk threads ; but the
particles of cholera-dejecta thrown on the floor, on the
bed-clothes, &c., do not become so dry that the comma-
bacilli are dead. Indeed this is far from being the case.
Every one who is working in a laboratory knows that
accidental contamination with micrococci floating about in
the air is a constant source of annoyance ; expose a layer
of sterile nutritive gelatine in a glass dish for a few minutes
to free air-contamination, particularly during the summer
months, then cover it up ; or mix the gelatine first with a
little dust taken up from any part of the floor of the room
and make with it a plate cultivation and you will find in a
few days colonies of microbes— bacilli, and particularly
micrococci. Yet micrococci are killed by perfect drying.
This proves that those contaminating micrococcus germs in
the air and dust were not dry. And the same holds good
of the comma-bacilli contained in the particles of the

viii.] THE INFECTIVENESS OF CHOLERA. 153

cholera-dejecta, for herein the comma-bacilli do not become
perfectly dry, and therefore are not dead. Convalescent
and weakly persons in hospitals, side by side with cholera-
patients voiding innumerable masses of comma-bacilli on to
the bed-clothes and the floor, would have very little chance
of escaping infection. Yet this immunity is observed over
and over again. In the Medical College Hospital at
Calcutta I have noticed this, that cholera patients were
placed in the general ward side by side with other patients :
the native non-cholera patients, like other natives, eat their
meals with their fingers, using no spoons or forks, yet I
have not heard of any of these convalescents, or the nurses,
or anybody else among the attendants, having become
infected with cholera. The same thing has been ex-
perienced in London and other places during various
epidemics. When in India in any city or village a case of
cholera occurs, except in the cholera-season, the disease
does not spread, yet amongst the natives the constant and
close attendance of the relatives on the sick is notorious ;
no special precautions against contamination with cholera-
dejecta are taken, yet no infection occurs. These are
conditions which obtain everywhere, and which have been
pointed out and demonstrated over and over again in India
during non-cholera seasons. If the fresh cholera-dejecta
or if the comma-bacilli were the infective agents, such things
could not be. Again, as has been already pointed out, the
water of the tanks becomes constantly contaminated with
cholera-dejecta and therefore also with abundance of the
comma-bacilli, and although the water of these tanks is
universally used by the people living around them, yet in
non-cholera seasons no spread of cholera occurs. I will
here give two such instances that came under my own
observation in Calcutta in 1884.

154 THE BACTERIA IN ASIATIC CHOLERA. [CH.

It will be remembered that Koch, while in Calcutta,
reported to his Government, the substance of which appeared
in the Englishman of Calcutta, on the i8th February 1884,
that cholera having broken out in one of the bustees
surrounding a tank in a suburb of Calcutta, he visited
this bustee and found numerous comma-bacilli in its
tank. On a second visit, a week later, the epidemic being
on the decline, he found much fewer comma-bacilli in the
water, and this seemed to him and the Englishman to
furnish a positive and remarkable proof that these comma-
bacilli stood in an intimate relation to the cause of the
cholera outbreak. It is known to all who have been in
India, and it has been mentioned on a former page, that
the natives use the water of every tank, ditch, and pool,
however dirty it may appear to a European, for all kinds of
purposes, — bathing, washing of mouth, washing of domestic
utensils, washing of clothes and linen, not even drinking
excepted. This particular tank visited by Koch, is, like
most other tanks, surrounded by huts, and is used as a sort
of common reservoir into which the evacuations of man and
beast, and every kind of domestic filth, find access. That
the water of such a tank, around which cholera cases occur,
and into which the evacuations of cholera patients find
access, and in which the clothes soiled with cholera-dejecta
are washed, should contain the same comma-bacilli that
are present in the choleraic evacuations is what one
would naturally expect, and likewise that the number of
these comma-bacilli should be fewer the fewer the cholera
cases, i.e. the smaller the number of comma-bacilli thrown
into the water. But to conclude, as Koch does, that because
there are comma-bacilli in the water cholera cases occur
amongst the people using the water, and as soon as the
number of the comma-bacilli decreases in the water the

viii.] THE INFECTIVENESS OF CHOLERA. 155

number of cholera cases becomes less,1 is manifestly illogical.
That Koch should have used an argument of this nature to
build up his theory is only intelligible if we remember how
little convinced some of the medical public appeared to be
of Koch's theory and that it required, as it were, a much
stronger argument to support it. This discovery of the
comma-bacillus in the water of that tank was considered
such an argument, as is clear from the manner in which at
the time the daily and some of the medical papers wrote
about it.

That the cholera virus, whatever this is, can find entrance
into a person by being conveyed there by water is in perfect
harmony with the known facts, and that pure drinking water not
contaminated with any extraneous material is of the greatest
importance finds many a good illustration in the Reports of
the Privy Council Office, in the Broad Street Pump cases in
London, in Dr. Macnamara's work on Cholera, and in the
various Indian Sanitary Reports.

Another curious illustration how even a very experienced
observer like Koch sometimes becomes unable correctly to
interpret plain facts, is furnished in the same reports sent to
the German Government. Koch stated that at Fort William,
in Calcutta, cholera abated as soon as a good water-supply to
the fort was introduced, and takes this of course as proof
that, previous to the introduction of the good water-supply,
many cholera cases were due to contaminated water. Now,
had he taken the precaution as he might easily have done,

1 This last statement of Koch's requires a certain amount of correc-
tion. The tank of \\hich Koch speaks was visited by him on the I3th
February, and again on the 2Oth February. During the week the
comma-bacilli had greatly diminished, but in the records of the police
office I find that the epidemic in the bustees surrounding this tank broke
out on the 2ist of January, and lasted till the 27th of April. It lasted,
therefore, fully two months more after this conspicuous diminution of
the comma-bacilli.

156 THE BACTERIA IN ASIATIC CHOLERA. [CH.

of looking at the records, he would have found that such a
conclusion was not in harmony with the actual facts, for he
would have found by studying the records, that cholera cases
diminished in a very marked degree some years before the
introduction of the better water-supply, and that this
diminution, but no greater one, was kept up afterwards.

The Indian Medical Gazette of November 1884 repub-
lished, on page 332, the official statistics as to the course of
cholera in Fort William from 1856 to 1876. In 1863 there
occurred a sudden decrease of cholera, and this decrease
was kept up till 1876. But the new and pure municipal
water-supply was not introduced in 1862 or 1863, but in
1872, i.e. nine years later than the conspicuous decrease
of cholera happened.

I had the opportunity in connection with Dr. D. D.
Cunningham to make an examination of the water of some
of the tanks in Calcutta, with reference to this very question
of the comma-bacilli. The same tank that plays such a
conspicuous part in Koch's report above mentioned was
visited by us on the 26th November. It is situated in
Sahil Bagan, a suburb of Calcutta, and it is surrounded by
native huts, in which altogether about 200 families are living.
There had occurred one case of cholera in one of these
huts about the first week of the month of November. The
water of this tank was very dirty, particularly all along the
shore, and the people around the tank, as is customary,
made use of the water for all and every kind of domestic
and other purposes, including drinking.

A sample of this water was taken from near the shore
where it appeared particularly impure, about twenty yards
from the house in which the cholera case had occurred,
and the microscopic examination revealed living comma-
bacilli which, as was proved by cultivation, were identical in

VIIL] THE INFECTIVENESS OF CHOLERA. 157

every respect with those found in choleraic dejecta. Not-
withstanding their presence in this water, and notwithstand-
ing the extensive use the 200 families were constantly
making of it, there has been no outbreak of cholera.
Now we have in this instance an experiment performed by
nature on a scale large enough to serve as an absolute and
exact one. This water had been unquestionably and
notoriously contaminated with choleraic evacuations, and
therefore also with the comma -bacilli, and was used exten-
sively by many human beings for several weeks ; if we say
with Koch that the comma-bacilli were the cause and essence
of cholera, how is it that not one person amongst so many,
up to the middle of December and afterwards, contracted
the disease ? Clearly because the water did not contain the
active cholera virus, and because this latter cannot be
identical with the comma-bacilli.

It might be said, and Koch has said so, as a matter of
course, in criticising my observations, that perhaps the
comma-bacilli present by the end of November were not the
same as the cholera-bacilli; but it must be remembered
that a case of undoubted cholera having here occurred,
owing to the conditions obtaining and owing to the habits of
the people, large quantities of comma-bacilli must of
necessity have been thrown and carried into this tank ; along
the shore the water contained abundance of decaying
animal and vegetable nitrogenous material to form a very
good and suitable nourishing medium for the bacilli, and
they must have had ample opportunity to multiply, and
consequently there must have been large numbers of them
present, sufficient for hundreds of human beings : neverthe-
less no case of cholera occurred.

An equally striking illustration of the innocuousness of
the comma-bacilli is furnished by a tank situated near

158 THE BACTERIA IN ASIATIC CHOLERA. [CH.

Teleepara Lane, in Calcutta. Between the i4th and i6th of
November 1885 there occurred nine cases of cholera in three
houses of Teleepara Lane, Nos. 3, 4, and 34. No. 34 had
three cases, No. 3 had three, and No. 4 had three cases.
The people of No. 34 are rich Hindoos, and those of No. 3
and No. 4 are also well-to-do. Two of these three houses
have their own hydrant, and from it they have a good supply
of very clear water, such as is supplied to all good houses in
the town. There is no condition common to all three houses,
except that just in front of each of them there appears to be
a communication with the street sewer. A narrow passage
leads from Teleepara Lane to a bustee surrounding a large
tank. As is usual the people (low caste) living in this bustee
make extensive use of the water of this tank, but the people
of those three houses, being well-to-do and having their own
drinking water, never went near this tank. In one of the
huts of this bustee lives a milkman, who supplied amongst
others, house No. 34 of Teleepara Lane, but not No. 3 or
No. 4. The water of the tank, as usual, is very dirty, espe-
cially near the shore, and a sample of it examined under
the microscope revealed the comma-bacilli. These proved
on cultivation to be identical with the choleraic comma-
bacilli. How they got there I am unable to say, but it is
highly probable that linen from the house No. 34 was
washed in the tank ; and on inquiry it was ascertained that
this was actually the case with the linen of all the houses in
the neighbourhood. Amongst the people of this bustee
there had not occurred a single, case of cholera during the
whole year.

It is quite clear from all this that the statement of Koch
and his adherents as to the importance of the comma-bacilli
in the water in producing cholera is not borne out by these
observations.

VIIL] THE INFECTIVENESS OF CHOLERA. 159

It must not however be supposed that I mean to ques-
tion the statements that cholera dejecta have produced in-
fection, or that water contaminated with cholera dejecta has
produced cholera. Such cases of infection are well estab-
lished. Dr. Snow has minutely described one such epidemic,
—the noted Broad Street Pump epidemic, and this is only
one among many noticed in former and recent epidemics in
Europe. As soon as a certain impure water-supply was
stopped cholera cases ceased ; to such a water-supply — a
river or a well — cholera dejecta had probably had access.
This question of the importance of drinking-water as a
vehicle of contagion may I think be considered settled. But
what is not at all settled is the question whether cholera
dejecta when fresh have any power to produce infection, or
whether some stage or change has to be passed through by
them in order to become infective. At any rate sufficient
evidence has been brought forward to show that fresh cholera
dejecta have not produced cholera even when mixed with
water used for various domestic purposes including drinking.
And I presume it is this consideration which led Lieber-
meister in his recent article on cholera l to say that the
choleraic comma-bacilli must possess the power of forming
spores, since only thus can the theory be brought into har-
mony with the obvious fact that fresh cholera dejecta have
often proved harmless. But to this the answer is obvious,
viz., that since the comma-bacilli do not possess, as has
been shown on former pages, this power of forming spores,
and since they have in many cases of direct and natural ex-
periment on a large scale failed to produce infection, it
follows that they cannot be the true cholera germ. A
similar criticism is applicable to all that is said by Koch
and the contagionists with regard to the infective power
1 Speciette Pathologic et Therapie, i. p. 83.

160 THE BACTERIA IN ASIATIC CHOLERA. [CH.

of linen soiled with cholera dejecta. There can be no
manner of doubt that cholera infection has been started
from linen soiled with cholera dejecta. These instances are
notorious and numerous, and are known from former and
recent cholera epidemics; they do not require any special
discussion. Now Koch says, and others repeat it with
greater or less emphasis, that it is easy to show the existence
of the comma-bacilli in the mucus-flakes of the dejecta soil-
ing the linen and clothes of a cholera patient even after days
and weeks, provided the linen be kept in a more or less
damp state, so as not to dry up and kill the comma-bacilli,
and the comma-bacilli are the only bacteria that can be at all
considered as playing any part in giving infective power to
such linen. While fully agreeing with the first part I totally
dissent from the second. It is in harmony with the known
observations that the comma-bacilli remain in a living state
and therefore are capable of multiplication in such linen, but
it is absolutely incorrect to say that they are the only bacteria
present.

As I have pointed out in former pages, it is extremely rare
to find the mucus-flakes of a cholera stool free of bacteria
other than the comma-bacilli. As a matter of fact 1 have
not failed to find in them certain small straight bacilli cap-
able of forming spores ; von Emmerich and Buchner always
found the bacterium which von Emmerich first pointed out
and isolated by cultivation. Whether, as is very probable,
such linen harbours still other bacteria no one knows.
Koch has not thought it necessary to inquire into this, nor
have others, who merely repeat what Koch says. It so
happens that the comma-bacilli are very conspicuous by their
shape — a fact which first attracted Koch's attention to
them1 — and by their mode of cultivation, as he afterwards
1 Loc. cit. p. 6.

VIIL] THE INFECTIVENESS OF CHOLERA. 161

ascertained ; but surely this does not make them necessarily
the more important. Kern described l a bacillus under the
name of Dispora caucasica, which is very peculiar, and which
he found in the Caucasus ; it is used, as he first thought, as
a ferment to produce from cows' milk a peculiar drink, called
kephir or hippo. This bacillus is quite peculiar and dis-
tinguished from all other bacilli ; it is constantly present in
such fermented milk, and is constantly present in the
material taken from fermented milk and used by the natives
to infect fresh milk. Yet, Kern himself afterwards showed
that it is not the Dispora caucasica at all which is the fer-
ment, but quite a commonplace Saccharomyces, which is also
always present, and which he did not at first consider to be
the ferment, owing to its commonplace characters. In-
stances of the simultaneous presence of two or more series
of organisms in the same materials or tissues not necessarily
connected with the cause of the disease are far more
numerous than where the _ disease microbe is the solitary
inhabitant.

Koch has stated in his last paper 2 that amongst the
hundred and odd medical men attending the special course
to study the choleraic comma-bacilli, one gentleman became
affected with ' cholerine ' ; he voided watery stools, and in
them the choleraic comma-bacilli were recovered by gelatine
plate-cultivation. This Koch considers as proof that the
comma-bacilli by careless handling had found access to the
intestine of this gentleman, and there multiplied and produced
the 'cholerine'. Von Pettenkofer, who was present at this
conference, declined to accept this explanation, but per-
sisted in saying that this ' cholerine ' might have been

1 Biologisches Centralblatt, ii.

2 Zweiie Conferenz zur Erorterung der Cholera/rage, Berlin, May
1885.

M

162 THE BACTERIA IN ASIATIC CHOLERA. [CH.

produced in some other manner, and that the presence of
the comma-bacilli in the stools was a result and not a cause
of the state of the alimentary canal. After reading the
details of the case, I am quite of the opinion of von
Pettenkofer. The gentleman in question had been five
days in Berlin when he was attacked by slight digestive
disturbance, accompanied with diarrhoea. Surely it is not a
very remarkable occurrence, that a gentleman coming from
some healthy country place to a big city like Berlin should,
under altogether new conditions of water, diet, and mode of
life, be attacked by digestive trouble and diarrhoea. To put
that at once down as due to his having swallowed a few
comma-bacilli is going a little too far. A person thus altered
may, apart from other considerations, accidentally have
swallowed in Koch's laboratory the choleraic comma-bacilli,
which, arriving in a diseased intestine, found the necessary
conditions of multiplying : but the disease itself probably was
antecedent. Amongst the hundreds, nay, thousands of
persons who, in the various laboratories in Europe and else-
where, ever since the first epidemic outbreak of cholera in
France in 1884, have handled the choleraic comma-bacilli
in large quantities for the sake of study and experiment, has
there been any other case ? No ; and the above instance,
quoted by Koch, is therefore single and isolated; the expla-
nation of ' cholerine ' in this gentleman as given by Koch
remains therefore unsupported ; and it would be going far
beyond the necessities of the case if we were to accept the
belief of Koch as to the method of infection in this case.
That a pathological state of the intestine has a good deal to
do with the multiplication of comma-bacilli I have proved by
direct experiment. In a monkey, which had received the
previous day a dose of castor-oil, and had diarrhoea therefrom^
the abdomen was opened under the spray, a loop of the

viii.] THE INFECTIVENESS OF CHOLERA. 163

lower ileum, just above the ileocaecal valve, and about 4 — 6
inches long, was ligatured above and below, care being taken
not to include in the ligatures the large vessels. With a
Pravaz syringe a droplet of mucus was withdrawn from the
interior of the loop, and on examination no comma-bacilli
could with certainty be discovered. With another syringe
about 2 ccm. of a saturated solution of magnesium sulphate
was rhen injected, the loop replaced, and the wound stitched
up and dressed antiseptically, the whole operation being
done under the spray. Immediately afterwards the animal
received subcutaneously one gramme of chloral hydrate
dissolved in one to two ccm. of distilled water. This whole
experiment was done after the method first employed by
Moreau, and repeated by the Committee of the British Medi-
cal Association on Cholera.1 Our animal was killed after
48 hours ; on post-mortem examination the ligatured loop was
found much injected, its cavity filled with and distended by
mucus, containing streaks of blood and numerous flakes.
On microscopic examination these flakes contained, besides
amorphous mucus and detached epithelial cells, longer or
shorter straight thickish bacilli, single or in dumb-bells ;
these were more or less pointed at the extremities, and many
of them included an oval bright spore. There were present
numerous comma-bacilli, some single, others in dumb-bells,
either S~shaped or with the curve in the same direction, i.e.
like the outline of a bird on the wing ; many were spirals of
three or four turns. In some places these comma-bacilli
were so numerous and crowded together that the material
looked almost like a pure cultivation of them (see Fig. 38).
On microscopic comparison it was found that they were of
the same character as the choleraic comma- bacilli, except

1 See the reprint of the Report of the Committee in the Practitioner,
1884.

M 2

1 64 THE BACTERIA IN ASIATIC CHOLERA. [CH.

perhaps that they looked a trifle smaller than those in the
choleraic mucus-flakes. Cultivations were made with them
in six gelatine plates, and in one of these after three days
there were no doubt a few colonies which corresponded with
those of the choleraic comma-bacilli ; this was proved to be
the case after two more days. Cultivations in gelatine tubes
and 'Agar-agar tubes yielded growths indistinguishable from
the cholera comma-bacilli. In the other plate-cultivations
the gelatine was found on the second day liquefied and
crowded with the above-mentioned straight bacilli.

TN

- /S

'•>/>>•

FIG. 38. — SPECIMEN OF MUCUS-FLAKES FROM A MONKEY.

1. Spiral forms of comma-bacilli.

2. Couples of comma-bacilli.

Magnifying-power 600.

In another monkey, in which the above experiment had
been repeated, the animal died during the second day; the
loop was found much distended, and filled with watery fluid,
bright red, and containing mucus-flakes. On microscopic
examination a few comma-bacilli of the same appearance
as those in the first animal could be discovered amongst
crowds of straight bacilli. Plate-cultivations did not succeed,
the straight bacilli multiplied too rapidly, and in the course

viii.] THE INFECTIVENESS OF CHOLERA. 165

of twenty-four to forty-eight hours the gelatine became quite
liquefied. Four other monkeys experimented on in the
same manner yielded no results ; neither microscopically nor
by cultivation could comma-bacilli be detected. I conclude
from the above successful experiment that owing to the
pathological process set up in the intestine, the comma-
bacilli, present already before the operation, but in far too few
examples to be recognised in the microscopic specimens, had
so rapidly multiplied that their demonstration was then
comparatively easy.1

1 Messrs. Macleod and Milles, in their paper on Asiatic Cholera,
say on p. 168, in reference to the above positive experiment in the
monkey : " Klein's experiment itself, if it proves anything, proves that
he was dealing with an example of spontaneous generation ! Farther,
it is not quite clear whether Klein claims that he was dealing with
Koch's organism, or only with one identical as to characters given."

From this it is quite clear to my mind that these observers missed
altogether the drift of my argument, or did not read the above concluding
passage.

Other criticisms made by these gentlemen will be dealt with at the
conclusion of the next chapter.

CHAPTER IX.

OTHER BACTERIA IN CHOLERA.

KOCH does not describe other bacteria, for he does not
think them of any importance ; the only ones which he
considers important are the comma-bacilli, and on these he
first fixed his attention on account of their shape and because
in acute pure cases they were in the majority.

In the small intestine, and particularly about the ileocsecal
valve, one finds in acute cases of cholera, dissected imme-
diately or very soon after death, freely-floating glassy-looking
clumps of mucus, which slightly differ from the ordinary
epithelial flakes detached from the surface of the mucus
membrane or floating in the clear fluid. They resemble
clumps more than flakes, and are more transparent ; when
examined under the microscope they prove to consist chiefly
of mucous or lymph corpuscles, and of a few epithelial cells
embedded in a hyaline mucous matter. But the same
lymph-corpuscles may occur also, only not so numerously^
in the ordinary flakes. These lymph-corpuscles are always
numerously present in those peculiar clumps, provided the
examination is made very soon after death. After an hour
and a half or two hours one misses them, since they easily
become macerated and disintegrated in the intestinal fluid.

CH. ix.] OTHER BACTERIA IN CHOLERA. 167

They can be found also amongst the flakes of the rice-water
stools, provided these are quite fresh, but then they are ob-
tained only in a fragmentary state. But the sooner the
post-mortem examination is made the more numerously they
are found in those glassy clumps. Lewis and Cunningham
in their reports on cholera have noticed them, and they
correctly state that in order to see them the material must
be fresh, i.e. examined very soon after death. One misses
any mention of them in Koch's paper, whether it be that his
attention was chiefly or wholly directed to the comma-bacilli,
or, what seems more probable, his dissections were not made
sufficiently soon after death. That this is the more likely ex-
planation appears from the fact that when stained with ani-
line dyes many of these corpuscles contain some interesting
things, as will appear presently ; and had those corpuscles
been present in Koch's specimens he could not have failed
to notice their contents. Examining these mucous corpuscles
in preparations dried (after the Weigert-Koch method in thin
layers) and stained with gentian-violet, or Spiller's purple, or
methyl-blue, they present themselves as spherical, oval, or
irregular corpuscles of about the diameter of ordinary white
blood corpuscles, or larger, if swollen up. Each contains two
or three deeply-tinted oval, spherical, or angular nuclei.
Their protoplasm is more or less hyaline, and they vary in
size, inasmuch as many of them show signs of being swollen
up or are even in the act of disintegration, as is indicated by
their faint or broken outline respectively. The best preserved
spherical corpuscles are completely filled with very minute
straight bacilli. Those that are slightly swollen show the
bacilli more isolated, but still in many places in groups, and
in those that are much swollen up and at the point of disin-
tegration the bacilli are seen very loosely and irregularly
scattered through the protoplasm or on the point of leaving

i68 THE BACTERIA IN ASIATIC CHOLERA. . [CH.

the corpuscle altogether. The accompanying figures (39 and
40) illustrate all these points. In the surrounding fluid one
always neets with the same minute bacilli scattered about.
The appearances presented by these mucous copuscles filled
with the bacilli and by those that have swollen up and in
which the bacilli are loosely scattered, are extremely striking,
since the bacilli are stained deeply, whereas the cell-sub-
stance appears homogeneous. These lymph-corpuscles are
always to be met with in the glassy clumps and under the
conditions mentioned above ; but not in all instances does

FIG. 39. — FROM A PREPARATION OF FRESH MUCUS-FLAKES FROM THE ILEUM OF
A TYPICAL RAPIDLY FATAL CASE OF CHOLERA.

(a) An epithelial cell.

(b) Lymph corpuscles filled with the minute straight bacilli.

(c) A. mass of small bacilli and a few commas.

(d) Comma-bacilli.

Magnifying power about 700.

one find that they contain the same abundance of the small
bacilli, for in some cases these latter were missed in most of
the well-preserved corpuscles, and found only in those that
had slightly swollen up or were on the point of disintegra-
tion. But in all instances the same small bacilli are found
scattered amongst the detached epithelial and lymph cells.
There has not been a single case examined in which they
were not found in the mucus-flakes ; in cases in which the

IX.]

OTHER BACTERIA IN CHOLERA.

169

comma-bacilli were very scarce, the small bacilli were not
scarcer. In most cases they were met in larger or smaller
groups and as isolated examples.

[As one amongst several cases interesting as regards the
occurrence both of comma-bacilli and the small straight
bacilli is the following : — E., aged 25, had been purging
and vomiting since twelve o'clock in the night of i5th
November ; was admitted into the Medical College Hospital,

FIG. 40. — FROM A PREPARATION OF FRESH MUCUS-FLAKES FROM THE ILEUM OF
ANOTHER TYPICAL RAPIDLY FATAL CASE OF CHOLERA.
Lymph corpuscles containing the minute straight bacilli.
Magnifying-power about 700.

Calcutta, on the i6th November, at 10 a.m., with symptoms
typical of the acute stage of cholera. Died at 1.45 p.m., i.e. a
little over twenty-five hours after the first attack. Post-mortem
examination at 2.20 p m. Ileum contains clear watery fluid,
with glassy mucus-flakes, and numerous epithelial flakes.
In the mucous membrane a few minute hsemorrhagic spots,
not bigger than the point of a pin ; Payer's glands not visible.

i/o THE BACTERIA IN ASIATIC CHOLERA. [CH.

In the mucus-flakes were large numbers of lymph-corpuscles,
some perfect and small, others swollen up ; many of them
contain the small straight bacilli in great numbers ; besides
these there were numerous coherent masses entirely com-
posed of the small bacilli, but comma-bacilli were also
everywhere to be found, though the small bacilli were in
the majority. Cultivations made on linen from these
mucus-flakes yielded after twenty-four hours large crops
both of comma-bacilli and of the small straight bacilli.]

These bacilli are of extremely small size, about half to
two-thirds the thickness of the typical comma-bacilli, and
about one-third their length. They are straight and appear
pointed at each end ; generally they are single, but occasion-
ally they form a chain of two elements. In the well-preserved
mucus corpuscles they lie closely packed together, appar-
ently all single ; in the large swollen corpuscles there are
some in couples ; and amongst those occurring free around
and between the lymph-corpuscles and epithelial cells there
are a good many in couples and in small groups. It is not at
all a rare occurrence to meet with mucus-flakes from rice-
water stools in which the corpuscles were found almost com-
pletely disintegrated ; there were nevertheless found many
groups of the small bacilli, from six to twenty and more in
each group.

Two questions present themselves in connection with these
lymph-corpuscles; (i) where do they come from? and (2)
where do they get the bacilli from ? There can be no diffi-
culty in answering the first. It is well known that in all
those places where the highly-vascular lymphatic tissue
reaches the free epithelium of a mucous membrane, e.g. the
tonsils of the palate and pharynx, the lymph-follicles of the
pyloric end of the stomach and the duodenal part of the in-
testine, the solitary andagminated lymph-follicles of the ileum,

ix.] OTHER BACTERIA IN CHOLERIA. 171

and those of the Peyer's glands of the lower part of the ileum
and ileocaecal valve, lymph-corpuscles pass (migrate) easily
through the surface epithelium, and are discharged on to the
free surface. This is the case in the normal condition to a
certain extent, and to a greater extent in the pathological
state. The mucous corpuscles found in the fluid of the mouth
are those that have passed out from the superficial lymphatic
tissue of the tonsils. In the Peyer's glands of the ileum
one constantly meets these same corpuscles on their way
through the epithelium of the surface.

The second question is more difficult to answer. From
the fact that the bacilli are found inside and outside the
mucous corpuscles, it might be said that the mucous
corpuscles being endowed with amoeboid movement while,
and immediately after, passing out of the mucosa, are
probably capable of swallowing the bacilli just as lymph-
corpuscles are capable of swallowing other granular matter ;
but against this might be urged, that the mucous corpuscles,
having passed out of the mucosa, probably do not long
retain their amoeboid power; proof being afforded by the
rapidity with which they swell up and become disintegrated
in the watery contents of the intestine. The fact that the
better the corpuscles are preserved the more numerous the
bacilli, might be an argument either way ; and, besides,
several cases have been examined with this view, and only in
one were the bacilli found plentiful within the well-preserved
corpuscles : they were absent, or almost absent, in the well-
preserved corpuscles of other cases, but were present in
small numbers in those that had already swollen up or com-
menced to disintegrate. There is one other point which
must be mentioned in connection with this, — it is the fact
that, although these bacilli are not endowed with locomotion,
it is not impossible that they settle on these corpuscles,

i?2 THE BACTERIA IN ASIATIC CHOLERA. [CH.

and penetrate by active growth into them, finding in their
protoplasm a good soil.

A very careful examination of fine microscopic sections of
different parts of the intestine, well-preserved and well-stained
in the different aniline dyes, was made in order to trace, if
possible, these small bacilli, isolated or enclosed in cells, from
the lymphatic tissues of the mucous membrane outwards,
but all in vain. No trace of them could be found in the
lymph-corpuscles or any other part of the mucous membrane
either in the stomach, intestine, mesenteric glands, blood, or
any other tissue.

On the whole, then, although these bacilli looked very
promising at first as regards their connection with the dis-
ease, they had nevertheless to be abandoned, and had to be
regarded like the comma-bacilli, as something extraneous,
present only in tissues practically dead in the cavity of the
alimentary canal. But if any one wishes to urge that these
small bacilli are probably connected with the disease, there
would exist for such a view at least as much, if not more,
justification than for Koch's comma-bacillus, since these
small bacilli are found in some elements derived from the
tissue of the intestine (the comma-bacilli are not), and are
always present in the mucus-flakes and in the intestinal
contents, at any rate in acute cases, and if post-mortem
examination be made soon enough, as often and as
numerously as the comma-bacilli. In the watery vomit,
when copious, of acute cases, these small bacilli are generally
present, chiefly as isolated individuals or in small groups.

And in the same way, one might further urge that they are
quite capable of forming some kind of chemical ferment,
which, when absorbed, produces the disease. All this could
be said, with the same justification, of these small bacilli as
Koch has said it of the comma-bacilli, and such a theory

ix.] OTHER BACTERIA IN CHOLERA. 173

would rest on a basis not a bit weaker than the one on which
Koch's theory of the comma-bacillus rests.

These small bacilli have been cultivated in the same way
as the comma-bacilli, on linen kept moist by filter paper
under a bell-glass, on mixtures of Agar-agar, meat extract
and peptone, alkaline and neutral, and their characters have
thus been studied. They grow well at ordinary temperature
(75° to 82° F.), so that after twenty-four to forty-eight hours
considerable masses become available ; of course they grow
much more rapidly at higher temperatures (90° to 102° F.),
and they grow like the comma-bacilli and other bacilli
much better and more copiously in alkaline than in neutral
media.

The appearances presented after inoculation with them of
Agar-agar material in test-tubes are very much like that
presented by the comma-bacilli : from the point of inocula-
tion the growth spreads in the form of a flattened or filmy
rounded whitish mass, its outlines uneven or knobby. Pre-
parations made of culture on linen and on Agar-agar mixture
(solid), show the bacilli singly or very often in chains of
two or dumb-bells ; the single bacilli are of the same small
size as those mentioned above, but many of them grow to
somewhat greater length in the cultivation than in the fresh
material. After twenty-four to forty-eight hours' growth (at
90° — 102° F.) some of them begin to show the formation of
spores in the shape of a bright glistening spherical granule,
the substance of the bacillus gradually becoming pale, not
staining, and ultimately altogether fading away, so that only
the spore is left. After several days' growth many of the
bacilli, which have not formed spores, become pale, stain
very faintly, and gradually fade altogether away. This
change, indicating the degeneration and death of the bacilli,
differs in no way from what was observed of the comma-

74 THE BACTERIA IN ASIATIC CHOLERA. [CH.

bacilli, and described on a former page. Growing in
nutrient gelatine they do not liquefy the material, and the
channel of inoculation after several days' growth is occupied
by streaks of granules and droplets of a whitish appearance.

Experiments were made with cultures of these small bacilli on
monkeys, rabbits, dogs, and cats, by feeding, by intravascular
and subcutaneous injection, and by introducing them directly
into the small intestine. But no result was produced and
no result was to be expected, since the experiments with the
mucus-flakes taken directly from the ileum of acute cholera
cases mentioned on a former page proved without result.

Von Emmerich stated that in cases of cholera examined
by him in the epidemic in Naples in 1884, he found a ba-
cillus which is constantly present in the dejecta, in the tissue
of the intestine, liver, spleen, lymphatic glands, and blood.
This bacillus was found to be virulent when inoculated from
cultivations into guinea-pigs, producing death in a day or
two with choleraic symptoms. In his later researches carried
on with Buchner in Palermo in 1885. he corrected some
of his original statements in so far that the presence of this
"cholera-bacillus" in the blood and tissues was not confirmed.
But its constant presence in the stools and intestinal contents
of acute cholera cases and in the mucus of the bronchial
tubes, as also its virulently poisonous action on guinea-pigs,
was maintained by these observers. I first thought that von
Emmerich's bacillus was the same as the minute straight
bacillus described by me, but from further more detailed
description and from information given me by my friend Dr.
Shakespeare of Philadelphia, who had seen and possessed
specimens of von Emmerich's bacillus, it is clear that this
latter is much larger and thicker than the minute straight
bacillus mentioned by me. At the same time von Emmer-
ich's bacillus appears more like a species of Bacterium termo^

IX.] OTHER BACTERIA IN CHOLERA. 17

and as such is also regarded by Koch and others. Although
I do not of course doubt that this bacterium does occur
in the contents of the cholera intestine and the choleraic de-
jecta, and although there can be no doubt that it possesses
those pathogenic characters on guinea-pigs which are stated
by von Emmerich and Buchner, yet I cannot for one
moment accept it as proved that it has a causal relation to
cholera Asiatica. Koch and Brieger maintain that the same
bacterium occurs in the normal intestinal contents, and the
latter observer and Weisser1 have proved that a bacterium
identical with von Emmerich's in morphological and cultural
characters occurs in normal human faeces and other localities,
and is possessed of the identical pathogenic properties on
guinea-pigs, these animals after inoculation dying from a
form of septicaemia. All the difficulties that the comma-
bacillus of Koch offers in trying to explain the known facts
of cholera are likewise attached to this bacterium of von
Emmerich's, and I quite agree with those who say that of
the two Koch's comma-bacillus has undoubtedly a stronger
claim to be considered as the cholera microbe than von
Emmerich's, Of course if it had been confirmed that von
Emmerich's bacterium is present in the blood and tissues of
acute cholera cases there would have been strong fir zmafaae
evidence for its being causally connected with cholera, but
this presence in the blood and tissues not having been
proved on further examination its claim to be considered as
the cholera microbe rests on a very slender basis.

A commission consisting of Professor C. Roy, Dr. Graham Brown,
and Dr. Sherrington of Cambridge, was sent out to Spain in 1885, to
decide between the contradictory statements as to the facts concerning
the comma-bacilli of Koch. These gentlemen have come to the con-

1 Zeitschr. f. Hygiene, i. 2.

176 THE BACTERIA IN ASIATIC CHOLERA. [CF.

elusion that the comma-bacilli are not constant in cholera. They in
their Report (printed in the Proceedings of the Royal Society, No. 247,
p. 173) state that they are unable to accept the comma-bacillus of Koch
as causally connected with cholera Asiatica. They look upon the
comma-bacilli as probably connected with the premonitory diarrhoea ;
but these gentlemen furnish no proof for this assumption. Messrs. Roy,
Brown, and Sherrington describe and figure in sections through the
mucous membrane of the cholera intestine preserved for some months
hyphse or mycelial threads which they were told by Mr. Gardiner were
the hyphse of Chitridiacea, and they are not disinclined to look upon
these as causally connected with cholera Asiatica. I have good reasons
for saying (see Nat^^re for December 23, 1886, and the British Med.
Journal vt December 25, 1886) that what these gentlemen figured and
described (in Proceedings of the Royal Society, No. 247) are the hyphoe
of common mould which must have grown into the tissue during the
process of preserving the material.

It is fair to state that Mr. Gardiner has subsequently (Native, January
20, 1887) altered his view, inasmuch as he considered the organism
shown to him in Professor Roy's specimens, i.e. moniliform threads with
terminal nodular swellings, to resemble an involution form of a bacterium.
Still later (Nature, February 3, 1887) he implied that to harmonise
what he saw in Professor Roy's specimen with what has been figured by
Roy, Brown, and Sherrington in their Report (Proc. Roy. Society, 247,
p. 173), i.e. distinctly branched mycelial threads, both might belong to
a form similar to Cladothrix dichotoma. I have not the least doubt
from actual observation that the branched mycelial threads figured in
the Report of Messrs. Roy, Brown, and Sherrington are threads of
common mould.

Such appearances cannot be found in sections through the cholera
intestine preserved under the necessary precautions in alcohol, as for
instance if small bits of the intestine taken out soon after death are
placed at once in a large quantity of strong alcohol. As a matter of
fact Messrs. Roy, Sherrington, and Brown missed these forms in cover-
glass specimens made of the contents of the cholera intestine. Messrs.
Sherrington and Rouse, who studied cholera in Italy in 1886, failed to
find any of these hyphae in the cholera intestine. Dr. Shakespeare of
Philadelphia studied in 1886 cholera in Spain and India, and in his
Report to his Government, arrived at the conclusion that while Koch's
comma-bacilli are always present in the early stages, and therefore are
of great diagnostic value, their causal relation to cholera has not been
satisfactorily established.

ix.] OTHER BACTERIA IN CHOLERA. 177

In a paper, " Abstract of the Results of an Inquiry into
the Causation of Asiatic Cholera" (reprinted from the Pro-
ceedings of the Royal Society of Edinburgh, 'and published in
the Reports from the Laboratory of the Royal College of
Physicians, Edinburgh, vol. i. p. 161), Messrs. Neil Macleod
and Walter J. Milles state that they have repeated Koch's
experiments, and have arrived at the same results. In
criticizing my statements they make certain strictures on
me which are quite unwarranted.

On p. 173 they say: "Klein's experiments, in which he
gave the opium in other ways than by the peritoneal cavity,
and then injected the cholera-bacillus with negative results,
are inconclusive, as he never made a control experiment
with his material to see whether he was able to produce
Koch's results under Koch's conditions." This is in so far
an unwarranted statement, as I have made control experi-
ments under Koch's conditions with the results described
by Koch. After the eighty-five successful experiments
recorded by Koch, I do not see the necessity for me or
any one else to emphasize the correctness of Koch's
observations. What I wished to point out was, that in
order to achieve those positive results, viz. multiplication of
the comma-bacilli in the intestine and consequent death of
the animals, it is necessary to induce a diseased state of the
intestine, and this is done by injecting the tincture of opium
into the peritoneal cavity. For I showed that if the narcosis
be produced otherwise — e.g. by subcutaneous injection of
opium tincture or watery opium extract, or by intra-
peritoneal injection of watery extract of opium — no result
follows the introduction of cultures of comma-bacilli into
the intestine after soda injection. So that, provided the
intestine be not injured, which assuredly it is by the
injection of tincture of opium or alcohol into the peritoneal

N

i;8 THE BACTERIA IN ASIATIC CHOLERA. [CH.

cavity, the comma-bacilli are unable to multiply in the
intestine, and therefore are unable to produce a fatal
result.

But while I have made the necessary control experiments
which Messrs. Macleod and Milles charge me with having
omitted, I do not find anywhere in Messrs. Macleod and
Milles's paper .a reference to control experiments of their
own. Amongst the numerous experiments made by these
gentlemen, some partly in repetition of Koch's, partly of Van
Ermengem's experiments, I do not find any experiments to
show that my contention is wrong. Surely if any one
maintains, as they do on p. 177, 3, that "the means used
to introduce the comma-bacillus into, and those used to
lessen the peristalsis of the small intestine of the guinea-pig,
cannot be regarded as causing appearances like those ot
Asiatic cholera, or as causing the death of the animal, "-
we should require proof by control experiment, i.e. we
should require proof that by inducing narcosis otherwise
than by intraperitoneal injection of opium tincture we
nevertheless obtain the same positive . results. But such
control experiments do not seem to have been made by
them.

Nor do I find anywhere in Messrs. Macleod and Milles's
paper a reference to the important experiments made on
a large scale by Finkler with Finkler's comma-bacillus, and
described in a former chapter (p. 137). As has been stated,
Finkler experimenting on guinea-pigs with cultures of his
comma-bacillus after Koch's method produced results
identical with those produced by Koch's comma-bacillus.
I think this proves conclusively that the action of the
choleraic comma-bacillus thus experimented with in the
guinea-pig cannot possibly be said to be identical with
cholera-asiatica ; for if so, then both Finkler's and Koch's

ix.J OTHER BACTERIA IN CHOLERA 179

comma-bacillus would have to be regarded as the cause of
cholera, which would be an absurdity.

In criticizing an argument of mine as to the exemption
from cholera of attendants and those who constantly are
brought in contact with cholera dejecta, Messrs. Macleod
and Milles compare on p. 177 the mode of spread of the
cholera contagium with that of syphilis ; this, I think, is
scarcely necessary for me to seriously consider.

RTCHARD CLAY AND SONS, LIMITED, LONDON AND BUNGAY.

RETURN TO:

CIRCULATION DEPARTMENT
198 Main Stacks

LOAN PERIOD 1
Home Use

2

3

4

5

6

ALL BOOKS MAY BE RECALLED AFTER 7 DAYS.

Renewals and Recharges may be made 4 days prior to the due date.
Books may be renewed by calling 642-3405.

DUE AS STAMPED BELOW.

FORM NO. DD6
50M 4-04

UNIVERSITY OF CALIFORNIA, BERKELEY
Berkeley, California 94720-6000