have been found in the tissues of the animal body
other than the point of infection, the fact remains that
in the vast majority of cases the tetanus bacillus is
localized. This toxin can be readily separated from
cultures by filtration. One-hundredth of a milligramme
of an eight-day filtered bouillon culture is sufficient, as
a rule, to kill a mouse. From this filtrate, however,
the active toxic substance has been obtained in a much
more concentrated form. The purified and dried tetanus
toxin prepared by Brieger and Cohn was surely fatal to
a 15 gramme mouse in a dose of 0.00000005 gramme.
Reckoning according to the body-weight of 75 kilo-
grammes, or 175 pounds, it would require but 0.00023
gramme, or 0.23 milligramme of this toxin, to prove
fatal to a man. By comparing this with snake-
poison, Calmette has found that dried cobra venom
requires 0.25 milligramme to kill a rabbit of 4 kilo-
grammes' weight, and according to body-weight, it
would require 4.375 milligrammes to kill a man of 70
kilogrammes. As the fatal dose of atropine for an
adult is 130 milligrammes, of strychnine from 30 to
100 milligrammes, and of anhydrous prussic acid 54
milligrammes, the appalling strength of the tetanus toxin
THE BACILLUS OF TETANUS. 393
can readily be appreciated (Lambert). What the true
composition and constitution of the tetanus poisons are
is unknown. It has been shown, however, that it pos-
sesses neither the characteristics of an alkaloid (ptomain)
nor of an albuminous body (toxalbumin); it is largely
precipitated from fluids saturated with ammonium sul-
phate.
The quantity of the toxin produced varies, even
when derived from one and the same culture, according
to the age of the culture, its composition, reaction, etc. ;
and partly it is due to the extreme sensitiveness of the
toxin, which cannot bear keeping any length of time or
exposure to light, being sensibly affected by most chem-
ical reagents and destroyed by heating to 55 to 60 C.
for any length of time. It retains its strength best in
the dry state.
Some authors (Kitasato and Sanfelice) have main-
tained that the tetanus cultures retain their viru-
lence unaltered; others, again, have observed consider-
able alteration in toxicity. Righi, for instance, has
observed that the tetanus bacillus cultivated under
aerobic conditions may entirely lose its virulence.
Certain chemical agents also produce on cultures of the
tetanus bacillus an attenuation of virulence, if only a
temporary one.
The Action of Tetanus Toxin in the Body. The parts
first to be affected with tetanus are in about one- third of
the cases in man, and usually in animals the muscles
lying in the vicinity of the inoculation for instance,
the hind foot of a mouse inoculated on that leg is first
affected, then the tail, the other foot, the back and
chest muscles on both sides, and the forelegs, until
finally there is a general tetanus of the entire body.
394 BACTERIOLOGY.
In mild cases, or when a dose too small to be fatal
has been received, the tetanic spasm may remain con-
fined to the muscles adjacent to the point of inocula-
tion or infection. According to Gumprecht, the action
of tetanus depends upon an increased reflex excita-
bility, as in strychnine-poisoning; but it is different
from strychnine in its mode of distribution, and prob-
ably takes place chiefly through the nervous system, as
in rabies. This view is supported by Brunner, Brusch-
ettini, and others. Beck has described a peculiar degen-
eration in the motor cells of the cord in animals killed
by tetanus. This degeneration does not seem to attack
the entire cells, but only a peripheral part, and seems
to be confined chiefly to the body of the cell, usually
leaving the nucleus intact. Only very late do the
nucleus and the nucleolus take part in the changes.
The changes consist in a swelling of the cell and a
homogeneous or finely granular degeneration with a
swelling, and, finally, coarse lumping together of the
chromatin This is especially evident at the tiny emi-
nence from which the axis-cylinder arises and in the
axis-cylinder itself. Beck considers this as proving
that the poison travels along the axis-cylinder, and
that, as the nucleus is the last portion affected, the
change is not a necrosis but only a modification of
cell function.
But there is also, in addition, undoubtedly a diffusion
of the poison by means of the blood and lymph. The
blood usually contains the poison, as has been proved
experimentally on animals. Neisser showed that the
blood of a tetanic patient was capable of inducing
tetanus in animals when injected subcutaneously.
Kitasato also found the serous exudates of the pleural
THE BACILLUS OF TETANUS. 395
and pericardial cavities as well as the blood of tetanic
animals would cause tetanus when transferred to other
animals. Kahlmeyer, Bruschettini, and others have
obtained similar results. The toxin has also been
demonstrated in the urine when large amounts have
been inoculated.
Courmont and Doyon believe that the so-called toxin
elaborated by the tetanus bacillus is not the true poison,
but is a ferment which forms from the poison in the
body at the expense of the organism, and is found
in the blood, sometimes in the urine, and in, especial
abundance in tetanized muscles. The action of tetanus
toxin is never suddenly produced, though when once
formed its absorption is rapid, but always requires a
certain period of incubation. These authors hold that
the substances produced by the tetanus bacillus must
undergo a chemical change in the body, because after
it is formed in the tissues it can be extracted from
them by boiling, and when injected into other animals
causes immediate tetanic symptoms without any period
of incubation. But other observers repeating these
experiments have failed to confirm Courmont and
Doyon' s results, and appear to have proved their
theory to be untenable.
Tetanus Antitoxin. Behring and Kitasato were the
first to show the possibility of immunizing animals
against tetanus infection. Here the question of immu-
nity against infection does not consist in producing an
increased power of resistance against the development
of the infecting agent, as is the case in most infectious
diseases, but similar to diphtheria, in bringing about
an immunity to the effects of the tetanus toxin. The
bacillus of tetanus, as we have seen, does not belong
396 BACTERIOLOGY.
to the septicsemic class of organisms which spreads
through the body, and by their growth and increase
produce their effects, but, on the contrary, remains
localized at the original point of infection. It pro-
duces, however, in its growth a most powerful toxin.
The treatment of tetanus is, therefore, directed against
the production of toxin and its neutralization in the
body. The methods originally proposed by Behring and
by Roux for producing a curative serum consisted
chiefly in weakening the tetanus toxin by means of
chemical, disinfectants (iodine trichloride, Gram's and
LugoPs solutions), so that when inoculated into the
test-animals they produced comparatively little reac-
tion. At the present time we inject the pure unaltered
toxin either alone in small doses or along with anti-
toxin. After the first dose of toxin the animals acquire
a certain tolerance which enables them to stand a dose
of a less attenuated toxin or of a greater amount of un-
changed toxin. Thus by gradually increasing the doses
or the strength of the toxin administered, the animals
are finally able to bear injections of large quantities of
the strongest toxin.
These immunizing experiments in tetanus have borne
practical fruit, for it was through them that the prin-
ciple of serum-therapeutics first became known the
protective and curative effects of the blood -serum of
immunized animals. It was thus shown that animals
could be protected from tetanus infection by the pre-
vious or simultaneous injection of tetanus antitoxin,
provided that such antitoxic serum was obtained from
a thoroughly immunized animal; and from this it was
assumed that the same result could be produced in natu-
ral tetanus in man; but, unfortunately, the conditions
THE BACILLUS OF TETANUS. 397
in the natural disease are very much less favorable, in-
asmuch as treatment is usually commenced not shortly
after the infection has taken place, but often only on
the appearance of tetanic symptoms, when the poison
has already diffused itself through the body.
Tetanus Antitoxin. The tetanus antitoxin is developed
in the same manner as the diphtheria antitoxin by
inoculating the tetanus toxin in increasing doses into
horses. The toxin is produced in bouillon cultures
grown anaerobically. After ten or fifteen days the
culture fluid is filtered through porcelain, and the germ-
free filtrate is used for the inoculations. The horses
receive half a c.c. as the initial dose of a toxin of which
1 c.c. kills 250,000 grammes of guinea-pig, and along
with this a sufficient amount of antitoxin to neutralize
it. In five days this dose is doubled, and then every
five to seven days larger amounts are given. The dose
is increased, as rapidly as the horse s can stand it, until
they support 700 to 800 c.c. or more at a single injec-
tion. After some months of this treatment the blood
of the horse contains the antitoxin in sufficient amount
for therapeutic use. When the animals 7 temperatures
are normal and they have recovered from the dose of
toxin last given, they are bled into sterile flasks and
the serum collected.
Technique of Testing Antitoxin Serum for Value in
Antitoxin. Tetanus antitoxin is tested exactly in the
same manner as diphtheria antitoxin, except that the
standard unit is different. The test toxin used in the
German method is one of which 1 gramme destroys
150,000,000 grammes of mouse. This is dissolved in
33 J c.c. of 10 per cent. NaCl solution. Ten times the
amount of antitoxic serum which neutralizes 1 c.c. of
398 BACTERIOLOGY.
this dilution of the test toxin contains one unit of anti-
toxin. In the French method the amount of antitoxin
which is required to protect a mouse from a dose of
toxin sufficient to kill in four days is determined, and
the strength of the antitoxin is stated by determining
the amount of serum required to protect one gramme
of animal. If 0.001 c.c. protected a 10 gramme mouse
the strength of that serum would be 1 : 10,000. Guinea-
pigs are sometimes used in place of mice. Knorr's toxin
is preserved by precipitating it with saturated ammo-
nium sulphate and drying and preserving the precipi-
tate in sealed tubes. As required, it is dissolved in 10
per cent, salt solution, as above stated. For small
testing stations the best way is to obtain some freshly
standardized antitoxin and compare serums with this.
The Persistence of Antitoxin in the Blood. Ransom
has recently shown that the tetanus antitoxin is elimi-
nated just about as rapidly from the blood of an animal
when produced by toxin injections as when injected
with antitoxin, so long as the serum was from an ani-
mal of the same race. When from a different race, it
is much more quickly eliminated. From this we see
a possible explanation of the fact that immunity in
man, due to an injection of the antitoxic serum of the
horse, is less persistent than immunity conferred by an
attack of the disease.
He found some interesting facts in testing the anti-
toxic values of the serum of an immunized mare, of its
foal, and of the milk. The foal's serum was one-third
the strength of the mare's, and one hundred and fifty
times that of the mare's milk. In two months the
mare's serum lost two-thirds in antitoxic strength, the
foal's five-sixths, and the milk one-half. Injections of
THE BACILLUS OF TETANUS. 399
toxin were then given the mare, so that it doubled its
original strength in one month. The milk increased
eightfold, but the foal's continued to lose in antitoxin,
although it was feeding on the antitoxic milk.
Eesults of the Antitoxin Treatment in Tetanus. Tetanus
is a comparatively rare disease both in man and animals,
though in some localities it is more common than in
others. In New York city there are usually fifteen
to thirty cases following every fourth of July. Most of
them are caused by infection through blank cartridge
wounds. Recovery sometimes follows from the ordi-
nary symptomatic treatment or without treatment at
all, so that the statistics of cures of the disease by the
injection of antitoxic serum must be very carefully
sifted before they can be accepted as reliable. Lambert,
however, who has recently made an exhaustive study
of tetanus, states that in a total of 114 cases of this
disease treated with antitoxin, according to published
and unpublished reports, there was a mortality of 40.35
per cent. Of these, 47 were acute cases that is, cases
with an incubation period of eight days or less and with
rapid onset, or cases with a longer period of incubation,
but intensely rapid onset of symptoms; of these the
mortality was 74.46 per cent. Of the chronic type
those with an incubation period of nine days or more,
or those with shorter incubation with slow onset there
were 61 cases, with a mortality of 1 6.39 per cent. With
a still larger number of cases the results indicate that
with tetanus antitoxin about 20 per cent, better results
are obtained than without. The new method of inject-
ing from 3-15 c.c. of antitoxic serum into the lateral
ventricles has not, in the writer's opinion, shown itself
to be superior to the intravenous or subcutaneous
400 -BA CTERIOL OGY.
methods. Some speak well of it. No bad results have
followed the injections when the serum was sterile and
the operation was performed aseptically.
The Dosage of Tetanus Antitoxin. For immunization
10 c.c. of a serum of a strength of 1:1,000,000,000
will suffice unless the danger seems great, when the
injection is repeated at the end of a week. For treat-
ment, it is well to begin with 50 c.c., and then, accord-
ing to the severity of the case, give from 20 to 50 c.c.
each day until the symptoms abate. In the gravest
cases no curative effect will be noticed from the serum.
Though these few cases are not sufficient to form a
final judgment of any treatment, Lambert concludes
that by means of the antitoxin treatment, combined
with other rational methods, the prognosis, even in
acute cases of tetanus, has been improved; but that
it still remains exceedingly grave so much so that
the preventive inoculation of serum in all cases where
dirt has been ground into serious contusions de-
serves a much more extensive consideration than has
heretofore been given it. The striking results which
have been obtained, particularly in veterinary practice,
with the prophylactic injection of tetanus antitoxin,
would seem to warrant the treating of patients with
immunizing doses of serum at least in neighborhoods
where tetanus is not uncommon when the lacerated
and dirty condition of their wounds may indicate the
possibility of a tetanus infection.
Differential Diagnosis. The differential diagnosis of
the bacillus of tetanus is, generally speaking, not diffi-
cult, inasmuch as animal inoculation affords a sure test
of the specific organism. No other micro-organism
known produces similar effects to the tetanus bacillus,
THE BACILLUS OF TETANUS. 4Q1
nor is any other neutralized by tetanus antitoxin. The
other characteristics also of this bacillus are usually dis-
tinctive, though microscopical examination alone cannot
be depended on to make a differential diagnosis. Diffi-
culty arises when other anaerobic or aerobic bacilli,
almost morphologically identical with the tetanus
bacillus, are encountered which are non-pathogenic,
such as the bacillus pseudotetanicus anaerobius, already
mentioned, and the bacillus pseudotetanicus aerobius.
It is possible, however, that both these bacilli, when
characteristic in cultures, are only varieties of the
tetanus bacillus, which, under unfavorable conditions of
growth, have lost, their virulence. These non-virulent
types do not, as a rule, have spores absolutely at their
ends, and the spores themselves are usually more ovoid
than those in the true tetanus bacilli.
26
CHAPTER XXIII.
BACILLUS TYPHOSUS (EBERTH-GAFFKY ? S BACILLUS OF
TYPHOID FEVER ; BACILLUS TYPHI ABDOMINALIS).
THIS organism was first observed by Eberth, and inde-
pendently by Koch, in 1880, in the spleen and diseased
organs of the intestine in typhoid cadavers, but was
not obtained in pure culture and its principal biologi-
cal cultures described until the researches of Gaffky,
in 1884. Its etiological relationship to typhoid fever
has been particularly difficult of demonstration, for
although pathogenic for many animals when subcuta-
neously or intravenously inoculated, it has been almost
impossible to produce infection or in any way give rise
to lesions corresponding to those occurring generally in
man. It has been recently shown, however, that
animals under certain conditions, when their power
of resistance has been reduced, as by exposure to the
influence of noxious gases, may be rendered susceptible
to infection, with the production of more or less char-
acteristic lesions. These results, together with the
specific reactions of the blood-serum of typhoid patients,
as first pointed out by Pfeiffer, Gruber, Widal, and
others, and the constant presence of the bacillus
typhosus in the intestines and in some of the organs
of the typhoid cadavers, as shown by its frequent
isolation from the spleen, blood, and excretions of the
sick during life and its absence in healthy persons,
BACILLUS TYPHOSUS.
403
unless they are convalescent from typhoid infection,
have demonstrated, on a scientific basis, that this bacil-
lus is the chief etiological factor in the production of
typhoid fever.
Morphological Characters. The typhoid bacilli are
rods of about l/i to 3/2 in length by 0.5// to Sfjt in
diameter, with rounded ends, often growing into long
threads. They are usually longer and somewhat more
slender in form than the bacilli coli communis under
similar conditions. The typhoid bacilli vary, however,
in shape when grown in different culture media. (See
Figs. 49, 50, and Fig. 6, page 39.)
FIG. 49.
FIG. 50.
Typhoid bacilli from nutrient agar.
X 1100 diameters.
Typhoid bacilli from nutrient gelatin.
X 1100 diameters.
The typhoid bacilli stain with the ordinary aniline
colors, but a little less readily than do most other bac-
teria, though there is no constant difference in staining
characteristics between these and other bacilli of this
group the colon bacilli. They are decolorized by
Gram's iodine solution. Not infrequently, particu-
larly when grown on potato, refractive granules may
404 BACTERIOLOGY.
be seen at the ends of the rods, which stain more in-
tensely, and either at the extremities or along the body
" vacuoles" are observed, which remain unstained; but
as these show even less resistant power than the homo-
geneous bacilli found in other cultures, they are cer-
tainly not spores, but probably are evidences only of
retrograde changes and effects of the drying prepara-
tory to staining.
FIG. 51.
Flagella, heavily stained, attached to bacilli.
The bacilli, when existing under favorable conditions,
are, although in various cultures to a different degree,
very actively motile, the smaller ones having often an
undulating motion, while the larger rods dart about
rapidly, with a snake-like movement. This movement
is produced by a number of delicate locomotive organs
in the form of fine, hair-like flagella, which are arranged
around the bodies of the bacilli. (Fig. 10, page 43,
and Fig. 51.) The flagella are usually from eighteen
to twenty in number, but many short rods have but a
single terminal flagellum. They are not seen in un-
stained preparations, nor are they rendered visible by
BACILLUS TYPHOSUS. 405
the ordinary methods of staining. (See Staining of
Flagella, page 205.)
Biological Characters. The typhoid bacillus is a
motile, aerobic, non-liquefying bacillus, developing
best at 37 C.; over 40 and below 30 its growth is
retarded; below 10 it ceases. It grows most abun-
dantly in the presence of oxygen, but oxygen is not
essential to its development.
Its growth on most culture media is similar to that
of the bacillus coli conimunis, but it is somewhat
slower and not quite so luxuriant.
FIG. 52.
A superficial and a deep colony of typhoid bacilli in gelatin.
X 50 diameters.
Growth on Gelatin Plates. (Fig. 52.) The colonies
growing deep down in this plate medium have nothing
in their appearance to distinguish them; they appear
as round points with a -sharp margin, of a yellowish-
406 BACTERIOLOGY.
brown color, and finely granular. The superficial
colonies, however, particularly when young, are often
quite characteristic; they are transparent, bluish-white
in color, with an irregular outline, not unlike a grape-
leaf in shape. Slightly magnified they appear homo-
geneous in structure, but marked by a delicate network
of furrows.
In stick cultures in gelatin the growth is mostly on the
surface, appearing as a thin, scalloped extension, which
gradually reaches out to the sides of the tube. In the
track of the needle there is but a limited growth, which
may be streaked, granular, or uniform in structure, and
of a yellowish-brown color. There is no liquefaction.
Growth in Bouillon. This medium is uniformly
clouded by the typhoid bacillus, but the clouding is not
so intense as by the colon bacillus. A film is frequently
formed on the surface after eighteen to twenty-four
hours' growth. A very slight amount of acid is pro-
duced.
Growth on Agar. The streak cultures on agar are
not distinctive; a transparent, grayish streak is formed.
Growth on Potato. The growth on this medium
has been held by some to be very important, but it
varies considerably. When characteristic the growth
is invisible, but luxuriant, usually covering the surface
of the medium, and when scraped with the needle offers
a certain resistance. In some cases, however, the
growth is restricted to the immediate vicinity of the
point of inoculation, not very luxuriant, and of the
same color as the potato. Again, the growth may be
quite heavy and colojred yellowish-brown, and with a
greenish halo, when it is very similar to the growth of
the colon bacillus. These differences of growth on this
BACILLUS TYPHOSUS. 407
medium appear to be chiefly due to variations in the
substance, especially in the reaction, of the potato.
Milk. The typhoid bacillus does not cause coagu-
lation when grown in sterilized milk.
Fermentation. It does not produce fermentation in
either glucose, lactose, saccharose, or glycerin bouillon,
and evolves no gas as the result of fermentation.
Lactose-litmus Agar. It grows usually as pale blue
colonies on lactose-litmus agar, but occasionally causes
slight reddening of the surrounding medium.
Indol Reaction. It does not produce indol. This
test was proposed by Kitasato for differentiating the
typhoid bacillus from other similar bacilli, such as
those of the colon group, which, as a rule, give the
indol reaction.
The reaction, being a very delicate one, requires
great care in its performance to arrive at accurate con-
clusions. (For test of indol, see page 77.) Instead of
bouillon, the simple peptone-water (which consists of
dried peptone, 1 part; sodium chloride, 0.5 part, and
distilled water, 100 parts) is to be preferred for this
purpose, because its pale color does not mask the reac-
tion*.
Pathogenic Properties. It has been extremely diffi-
cult to show experimentally that the bacillus typhosus
is specifically pathogenic for animals. A great many
experiments have been made, with the view of repro-
ducing in the tissues of lower animals the pathological
lesions of typhoid fever as seen in man, but the results
have not been completely satisfactory ; nor is this sur-
prising when one considers that this disease does not
occur naturally, so far as is known, among animals.
Sickness or fatal results without the appearance of the
408 BACTERIOLOGY.
typical pathological changes have regularly followed
animal inoculations, but in most cases they could easily
be traced to the toxaemia produced by the substances in
the bodies of the bacilli injected, not necessarily accom-
panied by the growth of the organism, rather than to
infection due to the development of the typhoid bacillus
in the tissues.
In a certain number of cases subcutaneous and intra-