died from anthrax, either during or after treatment;
whereas the mortality previous to the introduction of
this method was 10 per cent, on the average. Of
438,000 cattle inoculations only 0.33 per cent, have
died; the previous mortality from anthrax was 5 per
cent. These figures would seem to indicate the prac-
tical value of Pasteur's method of inoculation, notwith-
standing the arguments which have been put forward in
opposition to it. It is, however, not unattended with
danger, as some of the animals succumb to the after-
effects of the attenuated culture.
Differential Diagnosis. The differential diagnosis of
the anthrax bacillus is ordinarily not difficult, as this
organism presents morphological, biological, and patho-
gen ical characteristics which distinguish it from all other
bacteria. In the later stages of the disease, however,
the bacilli may be absent or difficult to find, and culti-
vation on artificial media and experimental inoculation
in animals are not always followed by positive results.
Even in sections taken from the extirpated pustule it is
sometimes difficult to detect the bacilli. In such cases
only a probable diagnosis of anthrax can be made. It
should be remembered that the bacilli are not found in
the blood until shortly before death, and then only in
varying quantity; thus blood examinations often give
negative results, though the bacilli may be present in
large numbers in the spleen, kidneys, and other organs
of the body. The suspected material should be inocu-
lated in nutrient gelatin and agar in Petri plates and
in mice.
Among other bacteria which may possibly be mis-
BACILL US ANTHRA CIS SYMPTOMA T1CL 563
taken for anthrax bacilli are the bacillus subtilis and
the bacillus of malignant oedema. The former is dis-
tinguished by its motility, by various cultural peculi-
arities, and by being non-pathogenic. The latter differs
from the anthrax bacillus in form and motility, in being
decolorized by Gram's solution, in being a strict anaer-
obe, and in various pathogenic properties.
The diagnosis of internal anthrax in man is by no
means easy, unless the history points definitely to infec-
tion in the occupation of the individual. In cases of
doubt cultures should be made and inoculations per-
formed in animals. According to Cornil and Babes,
some of these cases may possibly be caused by organ-
isms other than the bacillus of anthrax.
BACILLUS ANTHRACIS SYMPTOMATICI.
(Bacillus of Symptomatic Anthrax.)
Like the bacilli of anthrax and of malignant oedema,
both of which it resembles in other respects also, the
bacillus of symptomatic anthrax is an inhabitant of the
soil. It is found as the chief cause of the disease in
animals principally cattle and sheep affected with
what is known as " black leg," " quarter evil," or
symptomatic anthrax (German, rauschbrand; French,
charbon symptomatique), a disease which prevails in
certain localities in summer, and is characterized by a
peculiar emphysematous swelling of the subcutaneous
tissues and muscles, especially over the quarters.
Morphology. Bacilli having rounded ends, from
0.5/j. to O.Qfj. broad and from 3// to 6/J. long; mostly
isolated, also occurring in pairs, joined end-to-end,
but never growing out into long filaments, as the an-
564 SA CTERIOL OGY.
thrax bacilli in culture and the bacilli of malignant
oedema in the bodies of animals are frequently seen to
do. Under the hanging drop the bacilli are observed
to be actively motile, and in stained preparations flagella
may be demonstrated surrounding the periphery. The
spores are elliptical in shape, usually thicker than the
bacilli, lying near the middle of the rods, but rather
toward one extremity. This gives to the bacilli con-
taining spores a somewhat spindle shape.
Stains with the ordinary aniline dyes, but not with
Gram's method, or only with difficulty and after long
treatment or intense colors.
Biological Characters. Like the bacillus of malignant
oedema this is also a strict anaerobe, and cannot be cul-
tivated in an atmosphere in which oxygen is present.
It grows best under hydrogen, and does not grow under
carbonic acid. This bacillus develops at the room-tem-
perature in the usual culture media, in the absence of
oxygen, but it grows best in those to which 1.5 to 2 per
cent, of glucose or 5 per cent, of glycerin has been added.
Growth on Agar. The colonies on agar are some-
what more compact than those of malignant oedema, but
they also send out projections very often. In agar stick
cultures, in the incubator, growth occurs after a day or
two also some distance below the surface, and is accom-
panied by the production of gas and a peculiar disagree-
able acid odor.
Pathogenesis. The bacillus of symptomatic anthrax
is pathogenic for cattle (which are immune against ma-
lignant oedema), sheep, goats, guinea-pigs, and mice;
horses, asses, and white rats when inoculated with a cul-
ture of this bacillus present only a limited reaction; and
rabbits, swine, dogs, cats, chickens, ducks, and pigeons
BACILLUS ANTHEACIS SYMPTOMATIC!. 565
are, as a rule, naturally immune to the disease. The
guinea-pig is the most susceptible of test animals.
When susceptible animals are inoculated subcutane-
ously with pure cultures of this organism, with spores
attached to a silk thread, or with bits of tissue from
the affected parts of another animal dead of the disease,
death ensues in from twenty-four to thirty-six hours.
At the autopsy a bloody serum is found in the subcuta-
neous tissues extending from the point of inoculation
over the entire surface of the abdomen, and the muscles
present a dark-red or black appearance, even more in-
tense in color than in malignant oedema, and there is a
considerable development of gas. The lymphatic glands
are markedly hypenemic.
The disease occurs chiefly in cattle, more rarely in
sheep and goats; horses are not attacked spontaneously
i. e. y by accidental infection. In man, infection has
never been produced, though ample opportunity, by
infection through wounds in slaughter-houses and by
the ingestion of infected meat, has been given. The
usual mode of natural infection by symptomatic an-
thrax is through wounds which penetrate not only the
skin but the deep intercellular tissues; some cases of
infection by ingestion have been observed. The patho-
logical findings present the conditions above described
as occurring in experimental infection.
Symptomatic anthrax, like anthrax and malignant
oedema, is a disease of the soil, but it shows a more
limited endemic distribution than the first, and is differ-
ently distributed over the earth's surface than the sec-
ond of these diseases, being confined especially to places
over which infected herds of cattle have been pastured.
It is doubtful whether the bacilli are capable of devel-
566 BACTERIOLOGY.
opment outside of the body like anthrax. In the form
of spores, however, reproduction may take place; and
by contamination with these, through deep wounds ac-
quired by animals in infected pastures, the disease is
spread. Possibly also it may originate through infec-
tion of the mouth and by feeding which would account
for the cases of symptomatic anthrax occurring in stall-
fed cattle (Hafner).
It is well known to veterinarians that recovery from
one attack of symptomatic anthrax protects an animal
against a second infection. Artificial immunity to in-
fection can also be produced in various ways : by intra-
venous inoculations; or, in guinea-pigs, by inoculations
with bouillon cultures which have been kept for a few
days and as a result have lost their original virulence,
or with cultures kept in an incubating oven at a tem-
perature of 42 to 43 C.; or by inoculations made into
the extremity of the tail; or by inoculations with filtered
cultures, or with cultures sterilized by heat. For the
production of immunity in cattle, Arloing, Cornevin,
and Thomas recommend the use of a dried powder of
the muscles of animals which have succumbed to the
disease, and which have been subjected to a suitable
temperature to ensure attenuation of the virulence of
the spores contained in it. Two vaccines are prepared,
as in anthrax a stronger vaccine by exposure of a por-
tion of the powder to a temperature of 85 to 90 C.
for six hours, and a weaker vaccine by exposure for the
same time to a temperature of 100 to 104 C. Inocu-
lations made with this attenuated virus (into the end of
the tail) the weakest first and later the stronger give
rise to a local reaction of moderate intensity, and the
animal is subsequently immune from the effects of the
BA CILL US ANTHRA CIS S YMPTOMA TICI. 567
most virulent material and from the disease. Fourteen
days are allowed to elapse between the two inoculations.
The results obtained from this method of preventive in-
oculation seem to have been very satisfactory. Accord-
ing to the statistics of Hafner, Luchanka, Hess, Strebel,
etc. (1885-93), including many thousand cattle treated,
the mortality, which among 22,300 non-inoculated cattle
was 2.20 per cent., has been reduced to 0.16 per cent,
in 14,700 animals inoculated.
To recapitulate briefly, the principal points of differ-
entiation between this bacillus and the bacillus of malig-
nant oedema, which it closely simulates, are smaller; it
does not develop into long threads in the tissues; it is
more actively motile, and forms spores more readily in
the animal body than does the bacillus of malignant
oedema. It is pathogenic for cattle, while malignant
oedema is not; and swine, dogs, rabbits, chickens, and
pigeons, which are readily infected with malignant
oedema, are not, as a rule, susceptible to symptomatic
anthrax.
CHAPTER XXXIII.
SPIRILLUM CHOLERA ASIATICS (KOCH ? S COMMA
BACILLUS OF ASIATIC CHOLERA).
IN 1883, Koch separated a characteristically curved
organism from the dejecta and intestines of cholera
patients the so-called " comma bacillus." This he
declared to be absent from the stools and intestinal con-
tents of healthy persons and of persons suffering from
other affections. The organism was said to possess cer-
tain morphological and biological features which readily
distinguished it from all previously described organisms.
It was absent from the blood and viscera, and was found
only in the intestines; and in greater number, it was
said, the more acute the attack. Koch also demonstrated
an invasion of the mucosa and its glands by the comma
bacilli. The organisms were found in the stools on
staining the mucous flakes or the fluid with methylene-
blue or fuchsin, and sometimes alone; by means of cul-
tivation on gelatin they were readily separated from the
stools. During his stay in India, in Egypt, and at Tou-
lon, Koch had examined over one hundred cases, and
other investigators confirmed his statements. Numer-
ous control observations made upon other diarrhoeic de-
jecta and upon normal stools were negative; the comma
bacillus was found in choleraic material only, or in mate-
rial contaminated by cholera. Soon other observers,
however, described comma-shaped organisms of non-
SPIRILLUM CHOLERA ASIATICS. 569
choleraic origin; Finkler and Prior, for instance, found
them in the diarrhceic stools of cholera nostras, Deneke
in cheese, Lewis and Miller in saliva. All of these
organisms, however, differed in many respects from
Koch's comma bacillus, and since then it has been
proved that none of them was affected by the specific
serum of animals immunized to cholera; and gradually
the exclusive association of Koch's vibrio with cholera
FIG. 74. FIG. 75.
Contact smear of colony of Contact spirilla preparation from plate
cholera spirilla from agar. culture of cholera. X 800 diameters.
X 700 diameters. (DUNHAM.) (DUNHAM.)
became almost generally acknowledged. It is now re-
garded by bacteriologists everywhere to be the specific
cause of Asiatic cholera.
Morphology. Curved rods with rounded ends which
do not lie in the same plane, from 0.8// to 2/* in length
and about 0. 4// in breadth. The curvature of the rods
may be very slight, like that of a comma, or distinctly
marked, particularly in fresh unstained preparations
of full-grown individuals, presenting the appearance
of a half -circle. By the junction of two vibrios
S-shaped forms are produced, and under unfavorable
570 BACTERIOLOGY.
conditions of growth they may develop into long, spiral
filaments, which may consist of numerous spiral turns
in which it is impossible to recognize any connection
with the individual elements of which they are made
up. In stained preparations the spiral character of the
long filaments is often obliterated, or nearly so. Under
favorable conditions of growth that is, when the
growth is rapid the short-curved or almost straight
forms are commonly observed (Figs. 74 and 75). In
old cultures involution forms are frequent.
Stains with the aniline colors usually employed, but
not as readily as many other bacteria; an aqueous solu-
tion of carbol-fuchsin is recommended as the most reli-
able staining agent with the application of a few minutes'
heat. It is decolorized by Gram's method. The motile
organs exhibit one or two long, fine, spiral flagellse
attached to one end of the rods.
Biological Characters. An aerobic (facultative ana-
erobic), liquefying, motile spirillum. Grows readily in
the ordinary culture media, best at 37 C., but also at
the room-temperature (22 C.); does not grow at a tem-
perature above 42 or below 8 C. Does not form
spores.
In gelatin plate cultures, at 22 C., at the end of
twenty-four hours, small, round, yellowish-white to yel-
low colonies may be seen in the depths of the gelatin,
which later grow toward the surface and cause liquefac-
tion of the medium, the colonies lying at the bottom of
the holes or pocket thus formed. The zone of lique-
faction, which increases rapidly, remains at first clear,
then becomes cloudy, mostly gray, as the result of the
growth of the colonies. In many cases after a time
concentric rings, which increase from day to day, appear
SPIRILLUM CHOLERA ASIATICS. 571
in the zone of liquefaction. (See Figs 76 and 77.)
Examined under a low-power lens, at the end of sixteen
to twenty-four hours, the colonies appear as small, light
yellow, round, coarsely granular disks, with a more or
less irregular outline. In many cases at this stage an
FIG. 76. FIG. 77.
Cholera colonies in gelatin ;
Cholera colonies in gelatin; twenty-four thirty-six to forty-eight hours'
hours' growth. (DUNHAM.) growth. X about 30 diameters.
ill-defined halo is seen to surround the granular colony,
which by transmitted light has a peculiar reddish tint.
The older the colonies become the more the granular
structure increases, until a stage is reached when the
surface looks as if it were covered with little fragments
of broken glass (Koch). Liquefaction continues around
the colonies, their structure appears fissured and coarsely
granular in texture, and occasionally a hair-like border
is formed at the periphery (Fig. 78), or a gray trans-
parent zone, until the entire colony breaks up into frag-
ments. Sometimes the colonies may be retained as com-
pact masses in the zone of liquefaction, and then they are
572 BACTERIOLOGY.
dark-yellow or brown in color, and forms occur which
are absolutely unlike the typical cholera colonies. In
gelatin stick cultures the growth is at first thread-like
and uncharacteristic. At the end of twenty-four to
thirty-six hours a small, funnel-shaped depression ap-
pears on the surface of the gelatin, which soon spreads
out in the form of an air-bubble above, while below
this is a whitish, viscid mass. Later, the funnel in-
creases in depth and diameter, and at the end of from
four to six days may reach the edge of the test-tube;
PIG. 78.
Cholera colony in gelatin. X 30 diameters. (DUNHAM.)
in from eight to fourteen days the upper two-thirds of
the gelatin is completely liquefied. (See Fig. 79 and
Fig. 31, page 230.) Freshly isolated cholera vibrios
liquefy gelatin more rapidly than old laboratory cul-
tures; a certain variation in the characteristic liquefac-
tion of the gelatin even in fresh cultures under some
circumstances should be borne in mind in making a
diagnosis. Such variations in cultural peculiarities
occur also with other bacteria, and only the sum of all
the characteristics taken together enables a positive
diagnosis to be established.
Upon the surface ofagar the comma bacillus develops
a moist, shining, grayish-yellow layer. Blood-serum
is rapidly liquefied at the temperature of the incu-
SPIRILLUM CHOLERA ASIATICS.
573
bator. In bouillon the growth is rapid and abun-
dant; in the incubator at the end of ten to sixteen hours
the liquid is diffusely clouded, and on the surface a
wrinkled membranous layer is often formed. In gen-
FIG. 79.
A characteristic series of cholera cultures in gelatin ; one, two, three, four,
and six days' growth. (DUNHAM.)
eral the spirillum grows in any liquid containing a
small quantity of organic matter and having a slightly
alkaline reaction. An acid reaction of the culture me-
dium prevents its development, as a rule; but it has the
power of gradually accommodating itself to the pres-
ence of vegetable acids, and grows upon potatoes, in
the incubator only, which have a slight acid reaction.
Abundant development occurs in bouillon which has
been diluted with eight to ten parts of water and in
574 BACTERIOLOGY.
simple peptone solution, and it has been shown by ex-
periment that it also multiplies to some extent in steril-
ized river or well-water, and preserves its vitality in
such water for several weeks or even months. Koch
found in his early investigations that rapid multiplica-
tion may occur upon the surface of moist linen, and also
demonstrated the presence of this spirillum in the foul
water of a tank in India which was used by the natives
for drinking purposes.
The comma bacillus belongs to the class of aerobic
organisms, inasmuch as it grows readily only in the pres-
ence of oxygen, and that it develops active motility
only when a certain amount of oxygen is present. It
does not grow in the total absence of oxygen, but a
small quantity of oxygen is all that is required for its
development, as in the intestines.
Temperature is also of considerable importance in the
growth of cultures. Active growth does not begin
until a temperature of 22 to 25 C. is reached, though
the optimum growth is between 30 and 40 C.
The vitality of cultures of the comma bacillus is
quickly destroyed by desiccation. If a culture be
spread on a cover-glass and exposed to the action of
the air at room-temperature the bacilli are dead at the
end of two or three hours, unless the layer of culture
is very thick, when it may take twenty-four hours or
more to kill all the bacilli. This fact indicates that
infection is not produced by means of dust or other dried
objects contaminated with cholera bacilli. The trans-
misson of these organisms through the air, therefore,
can only take place for short distances, as by a spray of
infectious liquids by mechanical means as, for instance,
SPIRILLUM CHOLERA ASIATICS. 575
the breaking of waves in a harbor, on water-wheels,
etc., or in moist wash of cholera patients.
The cholera bacillus is also injuriously affected by the
abundant growth of saprophytic bacteria. It is true
that when associated with other bacteria, if present in
large numbers, and if the conditions for their develop-
ment are particularly favorable, the cholera bacillus
may at first gain the upper hand, as in the moist linen
of cholera patients, or in soil impregnated with chol-
era dejecta; but later, after two or three days, even in
such cases, the bacilli die off and other bacteria gradu-
ally take their place. Thus Koch found that the fluid
contents of privies twenty-four hours after the introduc-
tion of comma bacilli no longer contained the living
organisms; in Berlin canal-water they were not demon-
strable for more than six to seven days, as a rule. In
the dejecta of cholera patients they were found usually
only for a few days (one to three days), though rarely
they have been observed for twenty to thirty days, and
on one occasion for one hundred and twenty days. In
unsterilized water they may also retain their vitality for
a relatively long time ; thus in stagnant well-water they
have been found for eighteen days, and in an aquarium
containing plants and fishes, the water of which was
inoculated with cholera germs, they were isolated sev-
eral months later from the mud at the bottom. In
running river-water, however, they have not been ob-
served for over six to eight days. Even in cultures the
comma bacillus is one of the shorter-lived bacteria.
They have been observed, however, in pure bouillon
cultures for three to four months, and in agar cul-
tures for six months, and occasionally in one-year-
old cultures when they were protected from desicca-
576 BACTERIOLOGY.
tion. In these they occurred only in involution
forms.
The comma bacillus is killed by exposure to moist
heat at 60 C. in ten minutes. The bacilli have been
found alive in ice kept for a few days, but ice which
has been preserved for several weeks does not contain
living bacilli.
Chemical disinfectants readily destroy the vital-
ity of cholera vibrios. For disinfection on a small
scale, as for washing the hands when contaminated with
cholera infection, a 0. 1 per cent, solution of bichloride
of mercury or a 2 to 3 per cent, solution of carbolic acid
may be used. For disinfection on a large scale, as for
the disinfection of cholera stools, strongly alkaline milk
of lime, according to PfuhFs experiments, is an excel-
lent agent. The wash of cholera patients, contaminated
furniture, floors, etc., may be disinfected by a solution
of 5 per cent, carbolic acid and soap-water. The dis-
infecting action of mineral acids, particularly of sul-
phuric acid, has been advantageously employed for the
disinfection of entire systems of water-works into which
cholera bacilli had gained access.
Pohl, Bujivid, and Dunham have shown that when
a small quantity of chemically pure sulphuric acid is
added to a twenty-four-hour bouillon culture of the
cholera bacillus containing peptone a reddish-violet
color is produced. Brieger separated the pigment
formed in this reaction the so-called cholera-red and
showed that it was indol, and that the reaction was
nothing more than the well-known indol reaction. Sal-
kowski and Petri then demonstrated that the cholera
bacilli produced in thin bouillon cultures, along with
indol, nitrites by reduction from the nitrates con-
SPIRILLUM CHOLERA ASIATICS. 577
tained in small quantities in the culture media; and
showed that it is the setting free of nitric acid, upon
the addition of sulphuric acid to the culture, which
gives with indol the red body upon which the cholera
reaction depends. For a long time it was believed that
this nitroso-indol reaction was peculiar to the cholera
bacillus, and great weight was placed on it as a diag-
nostic test. It has since been shown, however, that
there are a number of other vibrios which, under sim-
ilar conditioDS as the cholera vibrio, give the same red
reaction. The reaction is, nevertheless, a constant and
characteristic peculiarity of this spirillum, and is of
unquestionable value. It is even more valuable as a
negative than as a positive test, as the absence of the
reaction enables one to say of a suspected organism that
it is not the cholera spirillum. There are, however,
certain precautions to be observed in its use. It has
been shown that the reaction may be absent, for in-
stance, when the culture contains either too much or
too little nitrate. It is, therefore, advisable not to em-
ploy a bouillon culture the composition of which is
uncertain, but a distinctly alkaline solution of peptone,
containing 1 per cent, pure peptone and 0.5 per cent,
of pure chloride of sodium (Dunham's solution). With
such a solution constant results can be obtained.
Pathogenesis. Since none of the lower animals is nat-
urally subject to cholera, nor has ever contracted the
disease during the prevalence of an epidemic or as the
result of the iugestion of food contaminated with chol-
eraic excreta, there is no reason to expect that inocula-
tions of pure cultures of the spirillum, either subcuta-
neously or by the mouth, will give rise in animals to a
typical attack of cholera. It has been shown, more-
37
578 BACTERIOLOGY.