over, that the comma bacillus is extremely sensitive to
the action of acids, and is quickly destroyed by the acid
secretions of the stomach of man or the lower animals
when these secretions are normally produced. Despite
the small prospects of success, however, from animal
experiments, these have been undertaken again and
again, until finally a method was found by which at
least similar processes have been produced in test ani-
mals by inoculation of pure cultures of the cholera
vibrio. Koch sought to produce infection in guinea-
pigs per vias naturales by first neutralizing the con-
tents of the stomach with a solution of carbonate of
soda 5 c.c. of a 5 per cent, solution injected into the
stomach through a pharyngeal catheter and then after
a while administered through a similar catheter 10 c.c.
of a liquid into which had been put one or two drops
of a bouillon culture of the comma bacillus. The ani-
mal then receives a dose of 1 c.c. of tincture of opium
per 200 grammes of body-weight introduced into the
abdominal cavity, for the purpose of controlling the
peristaltic movements. As a result of this treatment
the animals are completely narcotized for about half an
hour, but recover from it without showing any ill effects.
On the evening of the same or following day the ani-
mal shows an indisposition to eat and other signs of
weakness, its posterior extremities become weak and
apparently paralyzed, and, as a rule, death occurs
within forty-eight hours with the symptoms of collapse
and fall of temperature. At the autopsy the small
intestine is found to be congested and filled with a
watery fluid containing the spirillum in great numbers.
Koch experimented in this way on about one hundred
guinea-pigs. These results, however, are somewhat
SPIRILLUM CHOLERA ASIATICS. 579
weakened by the fact that experiments made with
some other bacteria viz., those isolated by Finkler and
Prior, Deneke, and Miller, and morphologically similar
to the comma bacillus of Koch occasionally pro-
duced death when introduced in the same way into
the small intestines of guinea-pigs; but while only
twelve out of fifty-one animals died when injected
with cultures of these last-mentioned bacteria, in the
cholera experiments there was 90 per cent, of deaths,
and when larger doses were administered all of the
animals died. Control experiments made with many
other bacteria gave negative results. Intraperitoneal
injections of larger quantities of pure cholera cultures
also often produce death in rabbits and mice.
There are several cases on record which furnish the
most satisfactory evidence that the cholera bacillus is
able to produce the disease in man. In 1884, a student
in Koch's laboratory in Berlin, who was taking a course
on cholera, became ill with a severe attack of cholera.
At that time there was no cholera in Germany, and the
infection could not have been produced in any other
way than through the cholera cultures which were being
used for the instruction of students. In 1892, Petten-
kofer and Emmerich experimented on themselves by
swallowing small quantities of fresh cholera cultures
obtained from Hamburg. Pettenkofer was affected
with a mild attack of cholerine or severe diarrhoea, from
which he recovered in a few days without any serious
effects; but Emmerich became very ill. On the night
following the infection he was attacked by frequent evac-
uations of the characteristic rice-water type, cramps,
tympanitis, and great prostration. His voice became
hoarse, and the secretion of urine was somewhat dimiu-
580 BACTERIOLOGY.
ished, this condition lasting for several days. In both
cases the cholera spirillum was obtained in pure cul-
ture from the dejecta. Another instance is reported
by Metschnikoff, in Paris, of a man who became
infected experimentally. In this case the algid stage
of cholera was produced, with complete suppression of
urine, cramps in the legs, contraction of the extrem-
ities, and collapse, the man's life being saved only with
difficulty. Finally, there is the case of Dr. Oergel,
of Hamburg, who accidentally, while experimenting on
a guinea-pig, had some of the infected peritoneal fluid
to squirt into his mouth. He was taken ill and died
a few days afterward of typical cholera, though at the
time of his death there was no cholera in the city.
These accidents and experiments would certainly seem
to prove conclusively the capability of pure cholera cul-
tures of producing the disease; and yet Strickler and
Hasterlick (Vienna, 1893) report negative results from
experiments on the human subject. This only goes to
show, however, that in cholera, like other infectious
diseases, there is an individual susceptibility. It is
also possible that the cultures used for experimentation
may have lost in virulence, as cholera cultures are so
liable to do when kept for any length of time.
Cholera Toxin. Koch was the first to assume, as the
result of his investigations, that the severe symptoms
of the algid stage of cholera were due to the effects of
a toxin produced by the growth of the comma bacillus
in the intestines.
In 1892, Pfeiffer published an account of his elabo-
rate researches relating to the cholera poison. He finds
that recent aerobic cultures of the cholera spirillum con-
tain a specific toxic substance which is fatal to guinea-
SPIRILLUM CHOLER^E ASIATICS. 581
pigs in extremely small doses. This substance stands in
close relation with the bacterial cells, and is perhaps an
integral part of them. The spirilla may be killed by
chloroform, thymol, or by desiccation without apparent
injury to the toxic power of this substance. It is de-
stroyed, however, by absolute alcohol, by concentrated
solutions of neutral salts, and by the boiling tempera-
ture. Secondary toxic products are formed which have
a similar physiological action, but are from ten to twenty
times less potent. Similar toxic substances were ob-
tained by Pfeiffer from cultures of Finkler and Prior's
spirillum and from the spirillum Metschnikovi.
Cholera Immunity. Koch found in his animal experi-
ments that recovery from an intraperitoneal infection
with small doses of living cholera vibrios produced a
certain immunity against larger doses, though the ani-
mals inoculated were not very much more resistant to
the cholera poison than they were originally. In 1892
Lazarus observed that the blood-serum of persons who
had recently recovered from an attack of cholera pos-
sessed the power of preventing the development in
guinea-pigs of cholera bacilli, which in these animals
are rapidly fatal when injected intraperitoneally ; while
the serum of healthy individuals had no such effect.
He attributed this to the presence in the serum of con-
valescents from cholera of antitoxic substances which
neutralized the poison produced by the cholera vibrios,
in the same manner as the antitoxins of diphtheria and
tetanus. Pfeiffer, on the other hand, maintained that
the serum contained bactericidal substances which killed
the bacilli so rapidly when injected into the animal that
they did not have time to produce their specific poison,
and that thus the death of the animal was prevented.
582 BACTERIOLOGY.
The serum is now known to be feebly antitoxic and
strongly bactericidal. This specific change in the blood
is observed to take place from eight to ten days after
the termination of an attack of cholera and reaches its
maximum during the fourth week of convalescence,
after which it declines rapidly and disappears entirely
in about two or three months. Similar antitoxic or
bactericidal substances exist also in the serum of guinea-
pigs, rabbits, and goats, when these animals are immu-
nized artificially against cholera by subcutaneous or
intraperitoneal injections of living or dead cultures.
These specific substances present in the blood of chol-
era-immune men and animals act only upon organisms
similar to those with which they were infected ; but,
as Pfeiffer showed, this specific relation, which is found
to exist between the antibacterial and protective sub-
stances produced during immunization and the bacteria
employed to immunize the animals, is not confined
alone to cholera. The discovery, moreover, of this
specific reaction of the blood-serum of immunized men
and animals when brought in contact with the spirilla,
has given us an apparently reliable means of distin-
guishing the cholera from all other vibrios, and the
disease cholera from other similar affections, both of
which have proved to be of great value, particularly in
obscure or doubtful cases, in which heretofore the only
method of differential diagnosis available viz., by
cultural tests was often unsatisfactory.
Cholera in man is an infective process of the epithe-
lium of the intestine, in which the spirilla clinging to
and between the epithelial cells produce a partial or
entire necrosis and final destruction of the epithelial
covering, which thus renders possible the absorption of
SPIRILLUM CHOLERA ASIATICS. 583
the cholera toxin formed by the growth of the spirilla.
The larger the surface of the mucous membrane infected,
the more luxuriant the development of bacilli and the
production of toxin, the more pronounced will be the
poisoning, ending fatally in a toxic paralysis of the
circulatory and thermic centres. On the other hand,
however, there may be cases where, in spite of the large
number of cholera bacilli present in the dejecta, severe
symptoms of intoxication may be absent. In such cases
the destruction of epithelium is then either not pro-
duced or so slight that the toxic substance absorbed is
not in sufficient concentration to give rise to the algid
stage of the disease, or for some reason the toxin is
not produced to any extent by the spirilla. In no
stage of the disease are living cholera spirilla found in
the organs of the body or in the secretions.
From this fact and other known properties of the
cholera bacillus, which have already been referred to,
several important deductions may be made with regard
to the mode of transmission of cholera infection. In
the first place the bacilli evidently leave the bodies of
cholera patients, chiefly in the dejections during the early
part of the disease (they have usually disappeared after
the fourth to the fourteenth day), and only these dejec-
tions, therefore, and objects contaminated by them, such
as bed and body wash, floors, vaults, soil, well-water
and river-water, etc., can be regarded as possible sources
of infection. There is a special limitation even in
these sources of infection, owing to the fact that this
spirillum is so easily destroyed by desiccation and
crowded out by saprophytic organisms. Thus, as a
rule, only fresh dejections and freshly contaminated
objects are liable to convey infection; after they have
584 BACTERIOLOGY.
become completely dry there is little danger. Farther,
we must conclude from the distribution of the cholera
bacillus in the body and from experiments upon ani-
mals that the commonest mode of infection is by way
of the mouth, and chiefly by means of water used for
drinking purposes, for the preparation of food, etc. In
recent times cholera spirilla have been found not infre-
quently in water (wells, water-mains, rivers, harbors,
and canals) which have become contaminated by the
dejections of cholera patients.
But, like other infectious diseases, not everyone who
is exposed to infection is attacked by cholera. The
bacilli have been found during cholera epidemics in the
dejections of healthy individuals without any patholog-
ical symptoms. Abel and Claussen found, for example,
in 14 out of 17 persons belonging to the families of 7
cholera patients, cholera vibrios, in some of them for a
period of fourteen days. In Hamburg there were 28
such cases of healthy choleraic individuals with abso-
lutely normal stools. It is evident, therefore, that an
individual susceptibility is requisite to produce the dis-
ease. In the normal healthy stomach the hydrochloric
acid of the gastric secretions may destroy the spirilla;
and, finally, the normal vital resistance of the tissue
cells to the action of the cholera poison may be taken
into consideration. According to the greater or less
power of this vital resistance of the body the same
infectious matter may give rise to no disturbance what-
ever, a slight diarrhoea, or it may lead to serious results.
Furthermore, it may be accepted as an established fact,
that recovery from one attack of cholera produces per-
sonal immunity to a second attack for a considerable
length of time. This does not appear to depend upon
SPIRILLUM CHOLERA ASIATICS. 585
the severity of the attack; for cases are recorded of
persons who were apparently not sick at all, and yet
in whom an acquired immunity was produced. How
long this immunity lasts is not positively known, but
probably for a month or more, so that the same person
is not likely to be taken ill again with cholera during
an epidemic.
Within the last few years Haffkine, in India, has
succeeded in producing an artificial immunity against
cholera infection by means of subcutaneous injections of
cholera cultures. In over 200,000 persons whom he
has inoculated the results obtained would undoubtedly
seem to show a distinct protective influence in the pre-
ventive inoculations. And Kolle has found that the
blood-serum of persons inoculated by Haffkine' s method
gave a similar reaction to that of persons who had re-
covered from cholera.
On the other hand, we may take it for granted that
susceptibility to cholera may be acquired or increased.
For instance, there is no doubt that gastric and intes-
tinal disorders produced by overeating, etc., may act
as contributing causes to the disease. Other predispos-
ing causes are general debility from poverty, hunger,
disease, etc.
Varieties and Variations of the Cholera Bacillus. Cun-
ningham, as a result of researches made in Calcutta
(1891), arrives at the conclusion that Koch's comma
bacillus cannot be accepted as the specific etiological
agent in this disease: First, as in many undoubted cases
of cholera he has failed to find comma bacilli; second,
because in one case he found three different species;
and, third, because in one case the indol reaction could
not be obtained. Since Cunningham's investigations
586 BACTERIOLOGY.
many other observers have reported finding varieties of
the comma bacillus. Only a few of these can be here
mentioned, of which there is any certainty that they
were derived from true cholera cases. Thus Friedreich
has accurately described and photographed a series of
forms which, however, vary but little from the original
type. But more interesting than the reports of varie-
ties are the observations of the variability of the cholera
bacillus. Claussen, in Esmarch' s institute, isolated from
fresh cholera stools vibrios which presented in plate
cultures a different appearance of the colonies, which
showed a tendency to disintegrate and having an irreg-
ular border. The nitrosoindol reaction was absent;
bouillon cultures were non-pathogenic to guinea-pigs,
and stick cultures grew slowly and uncharacteristically.
On repeated inoculation, however, a guinea-pig died
after the injection of 1 c.c. of a bouillon culture; in
the peritoneal exudate and even in the blood character-
istic bacilli were found and the cultures gave the indol
reaction. Celli and Santori, in Rome (1893), isolated
from the stools of many typical cholera cases a vibrio
which they called vibrio romanus, which was non-patho-
genic for animals, gave no indol reaction, did not coag-
ulate milk, and at 37 C. grew neither in bouillon nor
on agar. After cultivation for nine months it gave the
indol reaction and grew at 37 C., but was still almost
non-pathogenic. Bordoni-Uffreduzzi and Abb culti-
vated from a typical cholera case a short vibrio which
liquefied gelatin very rapidly and presented an abnor-
mal growth, and gave a yellow growth on potato, but
which on continued cultivation became more and more
like the cholera spirillum. Variations even greater than
occur in these varieties of cholera spirilla are met with
among diphtheria bacilli.
SPIRILLUM CHOLERJE ASIATICS. 587
Plan of Procedure for the Biological Diagnosis of the
Cholera Vibrio. A. Dejecta (fluid) or intestinal con-
tents of a cholera patient or cholera suspect.
1. Use one drop (one platinum loop) for gelatin plate-
cultures, making two dilutions. Do this in duplicate
or triplicate. Cultivate at 22 C.
2. Inoculate a couple of bouillon tubes and a couple
of Dunham's 1 per cent, peptone solution with one drop
each, and place them in the incubator (37 to 38 C.)
for six to eight hours.
3. Examine a drop of the dejecta in the hanging drop.
4. Examine a drop of the dejecta in stained cover-
glass preparation. 1
5. Make gelatin plates from one drop taken from
the surface of each of the bouillon and peptone solution
tubes and cultivate at 22 C.
6. As soon as the plates (see 1 and 5) are sufficiently
developed (thirty-six to forty-eight hours) fish the
suspected cholera colonies and use the material for the
following procedures :
7. Inoculate six or eight peptone tubes (1 per cent,
peptone, 0.5 per cent. NaCl in distilled water) and
place them at once in the incubator. Note the time.
8. Examine hanging drop for form, size, and motility
(and arrangement).
9. Make stained cover-glass preparations and exam-
ine.
1 These direct microscopical examinations of the intestinal contents are, as
a rule, very unsatisfactory, at least in those in which the symptoms are not
marked. In a few the spirals will make up from 50 to 100 per cent, of the
bacteria present. In most of the cases during the last epidemic in New York
Dunham found abundance of columnar epithelium from the intestinal mucous
membrane, numerous straight, thick bacilli, and only a few curved bacilli or
segments of spirals too few to identify. Plate cultures from these showed
from 20 to 80 per cent, of all the colonies developing to be cholera spirilla.
588 BACTERIOLOGY..
10. Then try indol reacton with the same tubes.
11. While these tubes are incubating use material
from the suspected colonies on the plates (1 and 5) for
hanging drop cultures.
12. Meanwhile make stained cover-glass prepara-
tions from other colonies of suspected cholera on the
plates (1 and 5).
13. Make gelatin tube cultures from colonies on
plates (1 and 5).
14. Make gelatin tube cultures daily for five or six
days, to study shape of growth along the line of punc-
ture to preserve the culture.
B. Suspected water.
Add to 500 c.c. or 1 litre of the water to be exam-
ined in a flask half -full enough peptone-salt solution
(20 per cent, peptone and 10 per cent. NaCl) to make
a 1 per cent, solution of peptone. Then proceed as in A.
PFEIFFER'S SERUM REACTION. All authors now
agree that the differentiation of the cholera vibrio from
other similar vibrios cannot always be made by the cul-
tural method, nor is the usual inoculation of animals
sufficient. For this purpose serum is employed either
by making intraperitoneal injections of a surely fatal
dose of the suspected spirillum along with the serum
of animals immunized to undoubted cholera cultures,
or by watching the action of the spirillum in the hang-
ing drop when added to a dilution of the above men-
tioned serum, so as to note whether immobilization
and clumping occurred.
CHAPTER XXXI Y.
SPIRILLA RESEMBLING THAT OF CHOLERA THE
SPIRILLUM OF RELAPSING FEVER.
SPIRILLUM OF FINKLER AND PRIOR.
FINKLER and Prior, in 1884, obtained from the feces
of patients with cholera nostras, after allowing the
dejecta to stand for some days, a sprillum which is
of interest mainly because it simulates the comma
bacillus of Koch, but differs from it in several cultural
peculiarities.
Morphology. More or less curved rods with an aver-
age length of 2.4/z and a breadth of 0.4 to 0.6//, some-
what longer and thicker than the spirillum of Asiatic
cholera and not so uniform in diameter, the central por-
tion being usually wider than the pointed ends; forms
sometimes S-shaped and spiral filaments, which are not
as numerous, and are usually shorter than those formed
by the cholera spirillum. Examined in the hanging
drop they are seen to be actively motile. A single
flagellum is attached to one end of the curved segments.
In unfavorable media involution forms are common.
Stains with the usual aniline colors.
Biological Characters. An aerobic and facultative
anaerobic, liquefying spirillum. Does not form spores.
Upon gelatin plates small, white, punctiforrn colonies
are developed at the end of twenty-four hours, which
590
BACTERIOLOGY.
under the microscope are seen to be finely granular and
yellowish or yellowish-brown in color; the colonies are
round with more sharply denned border, less coarsely
granular and darker in color than those of the comma
bacillus. Liquefaction of the gelatin around these col-
onies progresses rapidly, and at the end of forty-eight
hours is usually complete in plates where they are numer-
The surface colonies sink quickly into the gelatin
ous.
FIG. 80.
Spirillum of Finkler and Prior. X 1100 diameters.
and present a darker peripheral zone. The differenti-
ation between the Finkler and Prior and cholera spirilla
can readily be made in the earlier stages of their growth.
Later on, and especially when the cholera colonies are
the older, the diagnosis is not so easy. In gelatin stick
cultures liquefaction progresses much more rapidly than
in similar cultures of the cholera spirillum, and a stock-
ing-shaped pouch of liquefied gelatin is already seen
after forty-eight hours, which is filled with a cloudy
liquid. There is no bubble formation. The liquefac-
SPIRILLUM OF FINKLER AND PRIOR. 591
tion increases, and in twenty-four hours more reaches
the sides of the tube in the upper part of the medium ;
by the end of the week the gelatin is usually completely
liquefied. Upon the surface of the liquefied medium a
whitish film is seen. Upon agar there is a somewhat
more luxuriant growth than with the cholera vibrio; a
slimy, whitish-yellow layer covering the entire surface
is quickly developed. Upon potato this spirillum grows
at the room-temperature and produces a slimy, grayish-
yellow, glistening layer which soon extends over the
entire surface. The cholera spirillum does not grow at
room-temperature, and in the incubator produces a thin,
brownish layer. Cultures of the Finkler and Prior
spirillum give off a strong putrefactive odor; in media
containing sugar, according to Buchner, an acid reac-
tion is produced as a result of their growth; they do
not form indol in peptone solutions; and they have a
greater resistance to desiccation than the cholera spiril-
lum. The absence of agglutination with a dilution of
the serum of an animal immunized to the cholera
spirillum is a valuable differential sign.
Pathogenesis. When injected into the stomach of
guinea-pigs, after previous injection of a soda solution
and opium, the Finkler and Prior spirillum is some-
what pathogenic for these animals; but a smaller pro-
portion die from such injections than from those of
the fresh cultures of cholera. At the autopsy the in-
testine is pale, and its watery contents, which contain
the spirilla in great numbers, have a putrefactive odor.
This organism has been found in the dejections of
some healthy persons and of persons affected with diar-
rhoea or cholera nostras. It does not seem to have any
etiological relation, however, with this disease in man,
592 BACTERIOLOGY.
as since its disco verey, though repeatedly sought for, it
has seldom been found by subsequent investigators.
In 1884, Miller observed a curved bacillus in a hollow
tooth, which from its behavior in microscopical prepa-
rations, in cultures and animal experiments, is probably
identical with the Finkler and Prior spirillum; and
other very similar spirilla have been found by others.
DENEKE'S CHEESE SPIRILLUM.
Obtained by Deneke (1885) from old cheese, but since
then rarely met with. Morphologically and culturally
it shows greater similarity to Koch's comma bacillus
than the Finkler and Prior spirillum, but can be read-
ily differentiated from it also.
Morphology. Curved rods and long spiral filaments,
somewhat more slender than the cholera spirillum, the
turns in the spiral threads being lower and closer
together. Has a single flagelluin attached to one end.
Stains with the usual aniline colors.
Biological Characters. An aerobic and facultative