ease itself is evidently the same as history shows it to
have been before the time of Christ. The same is true
for tuberculosis, smallpox, hydrophobia, leprosy, etc.
Under practically unchanged conditions, therefore, as
exist in the bodies of men, bacteria which have once
become established as parasites continue so long as they
remain to retain their peculiar (specific) characteristics.
Whether new disease varieties, such as the influenza
bacillus, are coming into existence from time to time, is,
262 BACTERIOLOGY.
of course, a possibility, but not a certainty. The one
thing we can probably safely assert is that there is no
probability that any saprophytic variety now existing
can, under any possibility, develop into the now recog-
nized varieties of pathogenic bacteria. It is almost
impossible to conceive that any such variety should
start with the same characteristics and then develop
parasitic tendencies under exactly the same circum-
stances as those varieties which now produce disease.
Attenuation. It is now a well-established fact that
the great majority of parasitic bacteria can be so altered
by change of conditions, and especially by being sub-
jected to unfavorable conditions, that they, while mor-
phologically the same, lose their power of developing
in the body and of producing specific poisons. When
either or both these properties are partially destroyed
they can usually be redeveloped; but when power to
produce specific toxins is absolutely lost, it is, so far
as we now know, lost forever.
The recovery of toxin production is brought about
by developing the micro-organism for a considerable
length of time under the conditions best suited for it.
The recovery of the ability to grow in the body of any
animal species is brought about by causing the germ
to develop in a series of such animals whose resistance
has been overcome by reducing their vitality through
poisons, heat, cold, etc. Another method is to ac-
custom the micro-organism to the animal's body by
letting it remain surrounded by the animal fluids as it
rests in a pervious capsule in the peritoneal cavity.
CHAPTER XVIII.
BACILLUS OF TUBERCULOSIS (KOCH'S TUBERCLE
BACILLUS).
IT was a common belief many years ago in some
countries (kingdom of Naples, 1782) that tuberculosis
was an infectious disease; but it is only within com-
paratively recent times that the infectiousness of tuber-
culosis has become an established fact in scientific
medicine. Villemin (1868) was the first to show ex-
perimentally that tuberculosis might be induced in
healthy animals and man by inoculations of tubercu-
lous material. Others attempted to microscopically
demonstrate the origin of the disease (Ziirn, Buhl,
Klebs, Toussaint, etc.); but these investigations,
though paving the way to the discovery, which it
remained for Robert Koch to make, proved to be un-
satisfactory and incomplete. The announcement of
the discovery of the tubercle bacillus was made by
Koch, in March, 1882, at a meeting of the Physiolog-
ical Society of Berlin. At the same time satisfactory
experimental evidence was presented as to its etiolog-
ical relation to tuberculosis in man and in susceptible
animals, and its principal biological characters were
given. An innumerable number of investigators now
followed Koch into this field, but their observations
served only to confirm his original discovery.
264 BACTERIOLOGY.
The bacilli are found in the sputum of persons suf-
fering from pulmonary or laryngeal tuberculosis, either
free or in the interior of pus-cells; in miliary tubercles
and fresh caseous masses in the lungs and elsewhere;
in recent tuberculous cavities in the lungs; in tuber-
culous glands, joints, bones, mucous membranes, and
skin affections; in the lungs of cattle suffering from
pulmonary tuberculosis, and in tubercular nodules,
generally in animals which are infected naturally or
by experimental inoculations.
Morphological Characters. The tubercle bacilli are
slender, non-motile rods of about 0.2/2 in diameter by
1.5 to 4/* in length. (Plate L, Figs. 1, 2, and 3.)
Commonly they occur singly or in pairs, and are then
usually slightly curved; frequently they are observed
in smaller or larger bunches. Under exceptional con-
ditions branching forms are observed. In stained
preparations there are often seen unstained portions,
which have been improperly thought to be spores.
From two to six of these unstained spaces may some-
times be noticed in a single rod, and under moderate
magnification may give to the bacilli the appearance
of short chains of streptococci. In old cultures irregu-
lar forms may be obtained, the rods being occasionally
swollen at one end or presenting lateral projections.
The staining peculiarities of this bacillus are very
important, for by them its differentiation and recogni-
tion in microscopical preparations of sputum, etc., are
rendered possible. It does not readily take up the
ordinary aniline colors, but when once stained it is
very difficult to decolorize, even by the use of strong
acids. Koch first recognized it in a staining prepara-
tion to which an alkali had been added a solution of
PLATE I.
FIG. i.
FIG. 2.
Tubercle bacilli, in red.
Strepto-bacilli, in blue.
X i ioo diameters.
Tubercle bacilli, in red.
Tissue, in blue.
X i ioo diameters.
FIG. 3.
FIG. 4.
Very large tubercle bacilli.
Cells in specimen are in blue,
while bacilli are red.
X i ioo diameters.
Short smegma bacilli.
Bacilli in specimen are red,
rest of material in blue.
X i ioo diameters.
BACILLUS OF TUBERCULOSIS. 265
methylene-blue with caustic potash. More recently
Ehrlich devised a method of staining which proved
to be better, viz., the use of a solution of an aniline
color fuchsin or methyl-violet in a saturated aqueous
solution of aniline oil and decolorization of other bac-
teria with a solution of a mineral acid, to be followed
by a contrast stain, such as methylene-blue. (Plate
I., Figs. 1 and 2.) Various modifications of Ehrlich's
method are now commonly used. The carbol-fuchsin
solution of Ziehl is largely employed ; it has the advan-
tage of acting quickly and keeping well. The tubercle
bacilli can be demonstrated also by Gram's method of
staining, but this is not recommended for general use.
Biological Characters. The bacillus tuberculosis is a
parasitic, aerobic, non-motile bacillus, and .grows only
at a temperature of about 37 C. It has been assumed
that this bacillus is capable of forming spores. The
refractile spaces, however, are not found to possess the
regular shape and brilliancy of ordinary spores, nor have
they any greater resisting power to heat, desiccation,
etc., than the homogeneous bacilli. Exposure to 60 C.
in water destroys them in fifteen minutes. The bacilli
have, however, a somewhat greater resisting power than
most other pathogenic bacteria, since frequently the
bacilli resist desiccation at the ordinary temperatures
for months; many bacilli die, however, soon after dry-
ing. Portions of the lung from a tuberculous cow, dried
and pulverized, produced tuberculosis in guinea-pigs at
the end of 102 days (Cad&ic and Malet). They retain
their vitality for a considerable time in putrefying ma-
terial. Cold has no effect upon them. When dry the
more resistant organisms stand dry heat at 100 C. for
hours; but when moist, as in milk, they are more quickly
266 BACTERIOLOGY.
killed viz., at 55 C. in one hour, at 60 C. in fifteen
minutes, at 65 C. in fifteen minutes, at 70 C. in ten
minutes, at 80 C. in five minutes, and at 95 C. in one
minute. One reason why they appear to withstand in
milk high temperatures for a longer time than given in
the above figures is, as pointed out by Theobald Smith,
that when heated in a test-tube the cream which rises
on heating is exposed on its surface to a lower tem-
perature than the rest of the milk, and as this contains
many bacteria some of them are exposed to less heat
than those in the rest of the fluid receive.
The resisting power of this bacillus against chemi-
cal disinfectants is considerable, but not as great as it
is apt to appear, for, as in sputum, the bacillus is
usually protected by mucus or cell protoplasm from
penetration by the germicidal agent. It is not always
destroyed by the gastric juice in the stomach, as is
shown by successful infection experiments in susceptible
animals by feeding them with tubercle bacilli (Baumgar-
ten and others). They are destroyed in sputum in six
hours or less by the addition of an equal quantity of a
3 per cent, solution of carbolic acid, and in about one
hour by an equal amount of a 5 per cent, solution.
Bichloride of mercury is unsuitable for the disinfection
of sputum unless used in very strong solutions (1 : 500).
From recent experiments by Yersin upon pure cultures
of the bacillus it appears that tubercle bacilli were killed
by a 5 per cent, solution of carbolic acid in thirty sec-
onds; by 1 per cent, in one minute; absolute alcohol,
five minutes; iodoform-ether, 1 per cent., five minutes;
mercuric chloride, 1 : 1000 solution, ten minutes. Salt-
ing and smoking are said not to destroy the virulence
of tuberculous meat (Forster).
BACILLUS OF TUBERCULOSIS. 267
The tubercle bacillus when exposed to direct sunlight
is killed in from a few minutes to several hours, accord-
ing to the thickness of the layer and the season of the
year; it is also usually destroyed by diffuse daylight in
from five to seven days when placed near a window.
This fact is worthy of note, as it has an important
hygienic bearing. Thus, tuberculous sputum expector-
ated upon sidewalks, etc., being exposed to the action
of direct sunlight, will in many cases, especially in
summer, be disinfected by the time it is in a condi-
tion to be carried into the air as dust. For the same
reason, consumptive patients should occupy light, sunny
rooms and live as much as possible in the open air and
exposed to the action of direct sunlight.
The tubercle bacillus is a strict parasite that is to
say, its biological characters are such that it could
scarcely find natural conditions outside of the bodies
of living animals favorable for its multiplication. But
it has been noted that when it is cultivated for a time
in artificial media containing glycerin it may grow on
the surface of plain veal or chicken bouillon, in which
media it fails to develop when introduced directly from
a culture originating from the body of an infected ani-
mal. This would indicate the possibility of its acquir-
ing the ability to grow as a saprophyte. The experi-
ments of Nutall also show that the bacillus may multi-
ply, under favorable conditions, in tuberculous sputum
outside of the body. Notwithstanding these facts,
it is probable that the growth of tubercle bacillus out-
side of the living bodies of man and animals is so
slight as to have no practical importance in causing
infection.
On account of their slow growth and the special con-
268 BACTERIOLOGY.
ditions which they require, tubercle bacilli cannot be
grown in pure culture by the plate method on the
ordinary culture media. Koch first succeeded in culti-
vating and isolating this bacillus on coagulated blood-
serum, which he inoculated by carefully rubbing the
surface with sections of tuberculous tissue and then
leaving the culture, protected from evaporation, for
several weeks in the incubator. Roux and Nocard
afterward showed that the bacilli from man and ani-
mals occasionally grow on nutrient agar to which
glycerin has been added in the proportion of 5 per
cent.
Growth on Coagulated Blood-serum. On this medium,
which is regularly used to obtain the first culture, the
growth first becomes visible at the end of ten to four-
teen days at 37 C., and at the end of three to four
weeks a distinct and characteristic development has
occurred. Small, grayish-white points and scales first
appear on the surface of the medium. As development
progresses there is formed an irregular, membranous-
looking layer. When a tiny piece of this is removed,
placed on a cover-glass without rubbing, stained, and
then observed under the microscope the surface growth
presents a characteristic appearance, the bacilli being
arranged in parallel rows of variously curved figures.
Owing to the greater facility of preparing and steril-
izing glycerin-cigar, and the more rapid and abundant
growth of the bacilli, which have become accustomed
to growth outside the body on this medium, it is now
usually employed in preference to blood-serum for
preserving cultures. The development at the end of
fourteen to twenty-one days is more abundant than
upon blood-serum after several weeks. When numer-
BACILLUS OF TUBERCULOSIS. 269
ous bacilli have been distributed over the surface of
the culture medium, a rather uniform, thick, white
layer, which subsequently requires a slightly yellowish
tint, is developed; when the bacilli sown are few in
number, or are associated in scattered groups, separate
colonies are developed, which acquire considerable
thickness and have more or less irregular outlines.
Growth on Peptonized Veal or Beef Broth Containing
5 per cent, of Glycerin. On these media the tubercle
bacillus also grows readily if a very fresh thin film of
growth from the glycerin agar is floated on the surface.
The latter of these media is used for the development of
tuberculin. The small piece of pellicle removed from
the previous culture continues to enlarge while it floats
on the surface of the liquid, and in the course of three
to six weeks covers it wholly as a single film, which on
agitation is easily broken up and then settles on the
bottom of the flask, where it ceases to develop further.
The liquid remains clear, containing in solution the
products formed by the growth of the bacillus, and is
really a dilute crude tuberculin. A practical point of
importance, if a quick growth is desired, is to remove
for the new cultures a portion of the pellicle of a grow-
ing bouillon culture, which is very thin and actively
increasing.
The Obtaining of Cultures of the Tubercle Bacillus from
Sputa and Infected Materials for Diagnostic Purposes.
As this is a matter of great and increasing importance,
we will consider in detail the methods which have
been successfully employed. Pure cultures can be ob-
tained directly from tuberculous material ; but as
it is so difficult to get rid of the other bacteria
which are almost always present, and which grow
270 BACTERIOLOGY.
much more rapidly and take possession of the medium
before the tubercle bacillus has had time to form
visible colonies, it is best, unless human tissues can
be obtained free from other infection, first to inocu-
late a guinea-pig, both subcutaneously and intraperito-
ueally, with the * sputum, and then obtain cultures
from the animal as soon as the tubercle infection has
fully developed. From acute tuberculosis in man in
other regions than the lungs, where mixed infection
usually exists, direct cultures on blood-serum may be
made.
The animals thus inoculated usually die at the end
of three weeks to four months. It is better, however,
to kill a guinea-pig which by its enlarged glands shows
evidence of tuberculosis, and to remove, with the greatest
care as to cleanliness, one or more nodules from the
lungs, spleen, or lymphatic glands. Animals which
develop tuberculosis acutely are apt to have abundant
tubercle bacilli and give successful cultures, while the
chronic cases usually have few bacilli and give unsuc-
cessful cultures. The animals after being killed are
placed in trays, and after washing with a 5 per cent,
solution of carbolic acid, immediately autopsied. The
skin over the anterior portion of the body having been
carefully turned back, an opening is cut with a fresh
set of sterile instruments into the thoracic or abdominal
cavity; then with a sterile forceps the lymph-gland
portion of spleen or other part which it is desired to
examine is removed to a sterile covered beaker. This
tissue if suitable may be sliced in thin sections and con-
veyed directly to the surface of the solid culture medium
and gently rubbed over the surface, and then left on it,
or a part of it may first be crushed between two sterilized
BACILLUS OF TUBERCULOSIS. 271
glass slides and then transferred to the serum and
rubbed gently over its surface. Owing to the liability
of the blood -serum to become too dry for the develop-
ment of the bacillus, it is necessary to keep the cul-
ture moist by sealing the end in some way, as by
applying a rubber cap over the open end of the test-
tube, which prevents evaporation. This cap should be
sterilized in a solution of mercuric bichloride (1 : 1000)
and the end of the cotton plug burned off just before
applying it, to destroy any spores of mould fungi
present. Theobald Smith, who has had a very large
experience in growing the tubercle bacillus, gives the
following details as to his method :
"Throughout the work solidified dog's serum was
used. The dog was bJed under chloroform and the
blood drawn from a femoral artery, under aseptic
conditions, through sterile tubes directly into sterile
flasks. The serum was drawn from the clot with
sterile pipettes, and either distributed at once into
tubes or else stored with 0.25 to 0.3 per cent, chloro-
form added. The temperature required to produce a
sufficiently firm and yet not too hard and dry serum is,
for the dog, 75 to 76 C.; for horse and beef serum
it is from 4 to 5 lower. The tubes containing the
serum were set in a thermostat, into which a dish of
water was placed, to forestall any abstraction of moist-
ure from the serum. About three hours suffice for the
coagulation. This procedure dispenses with all sterili-
zation excepting that going on during the coagulation
of the serum. It prevents the gradual formation of
membranes of salts, which, remaining on the surface
during coagulation, form a film unsuited for bacteria.
Tubes of coagulated serum should be kept in a cold,
272 BACTERIOLOGY.
closed space, where the opportunities for evaporation
are slight. They should always be kept inclined.
<( The ordinary cotton-plugged test-tubes I do not
use, because of the rapid drying out permitted by
them as well as the opportunities for infection with
fungi. Instead, a tube is used which has a ground-
glass cap fitted over it. This cap contracts into a
narrow tube plugged with glass-wool; this plug is not
disturbed. The tube is cleaned, filled, and inoculated
by removing the cap. With sufficient opportunity for
the interchange of air very little evaporation takes place,
and contamination of the culture is a very rare occur-
rence. In inoculating these tubes bits of tissue which
include tuberculous foci, especially the most recent, are
torn from the organs and transferred to the serum.
Very little crushing, if any, is desirable or necessary.
I think many failures are due to the often futile
attempts to break up firm tubercles. Nor should the
bits of tissue be rubbed into the surface, as is some-
times recommended. After a stay of several weeks in
the thermostat I usually remove the tubes and stir
about the bits of tissue. This frequently is the occa-
sion for a prompt appearance of growth within a week,
as it seems to put certain still microscopical colonies in
or around the tissues into better condition for further
development. The thermostat should be fairly constant,
as urged by Koch in his classic monograph; but I look
upon moisture as of more importance. If possible a
thermostat should be used which is opened only occa-
sionally. Into this a large dish of water is placed,
which keeps the space saturated. Ventilation should
be restricted to a minimum. As a consequence, moulds
grow luxuriantly, and even the gummed labels must be
BACILLUS OF TUBERCULOSIS. 273
replaced by pieces of stiff manila paper fastened to the
tube with a rubber band. By keeping the tubes in-
clined no undue amount of condensation of water can
collect in the bottom, and the upper portion of the
serum remains moist. The only precaution to be
applied to prevent infection with moulds is to thor-
oughly flame the joint between the tube and cap, as
well as the plugged end, before opening the tube."
At the Saranac Laboratory beef blood-serum is used
in ordinary test-tubes, which are sealed by rubber caps.
The tubercles are crushed between sterile glass slides
and rubbed gently upon the serum surface. The serum
itself must not be too firm. It should just be solid
enough to stand upright. The results thus obtained
by Trudeau and Baldwin have been as good as those
reported by Smith. In our experience all methods fre-
quently fail with those unfamiliar with them, especially
when, as shown by microscopical examination, the
tubercular tissue used contains very few bacilli.
Pathogenesis. The tubercle bacillus is pathogenic not
only to man, but to a large number of animals, such as
the monkey, pig, cow, etc. Guinea-pigs are extremely
susceptible, and are much used for the detection of
tubercle bacilli in suspected material. When inoculated
with the minutest doses of the living bacilli they
usually succumb to the disease. Infection is most
rapidly produced by intraperitoneal injection. If a
large dose is given death follows in from ten to
twenty days. The omentum is found to be clumped
together in sausage-like masses and converted into hard
knots, which contain many bacilli. There is no serous
fluid in the peritoneal cavity, but generally in both
pleural sacs. The spleen is enlarged, and it, as well
18
274 BACTERIOLOGY.
as the liver and peritoneum, contains large numbers
of tubercle bacilli. If smaller doses are given the
disease is prolonged. The peritoneum and interior or-
gans spleen, liver, etc. are then filled with tubercles.
On subcutaneous injection, for instance, into the ab-
dominal wall, there is a thickening of the tissues about
the point of inoculation, which breaks down in about a
week and leaves a sluggish ulcer covered with cheesy
material. The neighboring lymph-glands are swollen,
and at the end of two or three weeks may attain the
size of hazel-nuts. Soon an irregular fever is set up,
and the animal becomes emaciated, usually dying within
four to eight weeks. If the injected material contained
only a small number of bacilli the wound at the point
of inoculation may heal up and death be postponed for
a long time. On autopsy the lymphatic glands are
found to have undergone cheesy degeneration ; the
spleen is very much enlarged, and throughout its sub-
stance, which is colored dark red, are distributed masses
of nodules. The liver is also enormously increased in
size, streaked brown and yellow, and the lungs are filled
with grayish-white tubercles; but, as a rule, the kidneys
contain no nodules. Tubercle bacilli are always found
in the affected tissues, but the more chronic the process
the fewer the bacilli that are apt to be present.
Rabbits are also quite susceptible to tuberculosis, but
considerably less so than guinea-pigs. In rabbits death
almost invariably follows inoculations of tuberculous
material into the anterior chamber of the eye. The
local effects are iris-tuberculosis and cheesy degeneration
of the pupil. The bacilli then penetrate to the neigh-
boring lymph-glands, producing softening of these, then
pulmonary tuberculosis, general tuberculosis, and finally
BACILLUS OF TUBERCULOSIS. 275
death at the end of several weeks or months. Subcuta-
neous inoculations are less effective, and in small doses
do not always kill. Intravenous and intraperitoneal
iujections usually produce general tuberculosis and
death at the end of a few weeks. The tubercles in
rabbits are smaller, as a rule, and the spleen and liver
not so much enlarged as in guinea-pigs, but the kidneys
not infrequently contain nodules of the size of a pea.
Of other susceptible animals, field-mice and cats
are readily infected by artificial inoculations of tuber-
culous material ; rats, white mice, and dogs only
when very large doses are given. All these animals
present the anatomical lesions of miliary tuberculosis.
Bollinger has produced intestinal tuberculosis in calves
by inoculating them with material taken from a tuber-
culous man. Canaries are also susceptible to inocula-
tions of the tubercle bacillus; but not sparrows. Cold-
blooded animals of various kinds, according to the
experiments of Koch, are immune, unless, as recently
demonstrated, the bacilli are first slowly accustomed
to growth at low temperatures. Fowls and pigeons