culture on glycerin-agar. These masses, floating on
the surface, give rise in from three to six weeks, accord-
ing to the rapidity with which the culture grows, to an
abundant development and to the formation of ,a toler-
ably thick and dry, white crumpled layer, which finally
covers the entire surface. At the end of four to eight
weeks development ceases, and the layer after a time
sinks to the bottom. Fully developed cultures, after
having been tested for purity by a microscopical exami-
nation, are passed into a suitable vessel and evaporated
to one-tenth of their original bulk over a water-bath
at a temperature of 70 to 80 C. The liquid is then
filtered through chemically pure sterilized filter-paper.
The crude tuberculin thus obtained contains 40 to 50 per
cent, of glycerin and keeps well, retaining its activity
indefinitely.
The method of treatment and the results obtained
from the old tuberculin have been described recently
by Koch briefly as follows : After each injection, which
should be large enough to cause a slight but not a great
19
290 BACTERIOLOGY.
rise of temperature, a noticeable improvement in the
tuberculous process results. The amount of tuberculin
injected is continually increased, so as to continue the
moderate reactions. After several months all reactions
cease, the patients having become temporarily immune
to the toxin, but not to the growth of the bacillus.
Further injections are now useless until this immunity
has passed. During the treatment the bacilli themselves
have not been directly affected, and when the treatment
is interrupted the tuberculous process is apt to progress.
Many cases, however, of pure tuberculosis become cured
or greatly benefited by several periods of treatment.
The substances produced in the body by the old tuber-
culin neutralized the tubercular toxins, according to
Koch, but were not bactericidal. After a series of ex-
periments, he considered the difficulty to be due to the
nature of the envelope of the tubercle bacillus, which
made it difficult to obtain the substance of the bacilli
in soluble form without so altering it by heat or chem-
icals that it was useless to produce immunizing sub-
stances. He conceived that immunity was not produced
in man for somewhat similar reasons possibly, the
bacilli never giving out sufficient toxin to cause cura-
tive substances to be produced. He therefore decided
to grind up the dried bacilli and soak them in water,
and thus obtain, if possible, without the addition of
heat, a soluble extract of the body-substance of the
bacilli, which he hoped would be immunizing. He
also tried to eliminate as much as possible of the toxic
products which produce fever. Biichner by a different
method, through crushing under a great pressure
tubercle bacilli mixed with sand, and thus squeezing
out their protoplasm, obtained a very similar substance.
BACILLUS OF TUBERCULOSIS. 291
The new tuberculin formed by either of these methods
is a watery extract of the soluble portions of the un-
altered tubercle bacilli. As can be readily seen, in a
preparation thus made contamination is difficult to
avoid, freedom from intact bacilli is uncertain, and the
strength of the solution prepared at different times is
variable. Twenty per cent, of glycerin is added to
preserve the tuberculin from contamination. After
three years of trial the results obtained with the new
tuberculin preparations cannot be considered to have
exerted either very different or very superior effects to
the older product.
As to the results obtained in general the reports are
as yet conflicting. Lupus seems to be decidedly bene-
fited for a time both by the old and the new tuberculin.
Relapses are, however, common. On advanced phthisis,
laryngeal tuberculosis, arid other tubercular processes
no effects have been noted, and nearly every one dis-
approves of their use in these cases as well as in those
where mixed infection is suspected ; even in cases of
beginning infection, opinions, as a whole, are not very
enthusiastic. The new tuberculin is, except when
prepared with the utmost care, .a dangerous substance,
for Trudeau, Baldwin and others found that guinea-pigs
injected with it not only did not become immunized,
but actually became infected from the living bacilli in
the fluid.
The chief use to which tuberculin has been put is as
an aid to the diagnosis of tuberculosis in cattle and
human beings, and for this purpose it has proved to be
of inestimable value. Numerous experiments made by
veterinary surgeons show that the injection of tuber-
culin in tuberculous cows in doses of 25 to 50 centi-
292 BACTERIOLOGY.
grammes produces in at least 95 per cent, a rise of tem-
perature of from 1 to 3 C. The febrile reaction occurs
in from twelve to fifteen hours after the injection. Its
intensity and duration do not depend upon the extent
of the tuberculous lesions, but is even more marked
when these are slight than in advanced cases. In non-
tuberculous animals no reaction occurs, or one much
less than in tuberculous animals, and the results ob-
tained on autopsy justify the suspicion that tubercu-
losis exists if an elevation of temperature of a degree
or more occurs from the subcutaneous injection of the
dose mentioned. For these injections the crude old
tuberculin is used, which for the convenience of admin-
istration is diluted with water. The following are the
directions for inspecting herds for tuberculosis :
(t Inspections should be carried on while the herd is
stabled. If it is necessary to stable animals under
unusual conditions or among unusual surroundings that
make them uneasy and excited the tuberculin test should
be postponed until the cattle have become accustomed
to the conditions they are subjected to, and then begin
with a careful physical examination of each animal.
This is essential, because in some severe cases of tuber-
culosis, on account of saturation with toxins, no reaction
follows the injection of tuberculin, but experience has
shown that these cases can be discovered by physical
examination. This should include a careful examina-
tion of the udder and of the superficial lymphatic glands
and auscultation of the lungs.
" Each animal should be numbered or described in
such a way that it can be recognized without difficulty.
It is well to number the stalls with chalk and transfer
these numbers to the temperature-sheet, so that the
BACILLUS OF TUBERCULOSIS. 293
temperature of each animal can be recorded in its
appropriate place without danger of confusion. The
following procedure has been used extensively and has
given excellent results :
" (a) Take the temperature of each animal to be tested
at least twice, at intervals of three hours, before tuber-
culin is injected.
" (b) Inject the tuberculin in the evening, prefer-
ably between the hours of six and nine. The injection
should be made with a carefully sterilized hypoder-
matic syringe. The most convenient point for injec-
tion is back of the left scapula. Prior to the injection
the skin should be washed carefully with a 5 per cent,
solution of carbolic acid or other antiseptic.
" (c) The temperature should be taken nine hours
after the injection, and temperature measurements
repeated at regular intervals of two or three hours
until the sixteenth hour after the injection.
" (d) When there is no elevation of temperature at
this time (sixteen hours after the injection) the exam-
ination may be discontinued; but if the temperature
shows an upward tendency, measurements must be con-
tinued until a distinct reaction is recognized or until
the temperature begins to fall.
" (e) If a reaction is detected prior to the sixteenth
hour, the measurements of temperature should be con-
tinued until the expiration of this period.
" (/) If there is an unusual change of temperature
of the stable, or a sudden change in the weather, this
fact should be recorded on the report-blank.
" (g) If a cow is in a febrile condition tuberculin
should not be used, because it would be impossible to
determine whether, if a rise of temperature occurred,
it was due to the tuberculin or to some transitory illness.
294 BACTERIOLOGY.
" (Ji) Cows should not be tested within a few days
before or after calving, for experience has shown that
the result at these times may be misleading.
" (i) The tuberculin test is not recommended for
calves under three months old.
" (j) In old, emaciated animals and in re-tests use
twice the usual dose of tuberculin, for these animals
are less sensitive.
" (k) Condemned cattle must be removed from the
herd and kept away from those that are healthy.
" (/) In making post-mortems the carcasses should
be thoroughly inspected, and all of the organs should
be examined."
Tuberculin injections are also made in man to reveal
a suspected tuberculosis. At first some believed that the
irritation aroused in the tuberculous foci by the tuber-
culin sometimes caused a dissemination of the bacilli and
an increase in the disease. When carefully used, how-
ever, in suitable cases there is probably no danger. A
drawback to its usefulness is that it does not reveal at
all the extent of the disease, nor whether the tuber-
culosis is active or dormant. It is, however, of great
value in selected cases, both surgical and medical,
where slight tuberculosis is suspected, and yet no de-
cision can be reached. I quote here Dr. Trudeau upon
the use of the test.
"In the absence of any well-defined rules founded
upon the experience of others at the time I began to
use the test, the method I adopted has been a purely
arbitrary one, and I make no claim for its being the
best or the most reliable, although, as far as my own
personal experience goes, I have as yet seen no objec-
tion to it or any reason to modify it.
BACILLUS OF TUBERCULOSIS. 295
" The range of the patient's temperature is ascer-
tained by taking it at 8 A.M., 3 P.M., and 8 P.M. for
three or four days before making the test. The first
injection should not exceed 0.5 mg., and if any fever
is habitually present should be even less, and is best
given early in the morning or late at night, as the
typical reaction usually begins, in my experience,
within six or twelve hours. Such a small dose, while
it will often be sufficient to produce the looked-for rise
of temperature, has, under my observation, never pro-
duced unpleasant or violent symptoms. An interval of
two or three days should be allowed between each of the
two or three subsequent injections it may be necessary
to give, as reaction in very rare cases may be delayed
for twenty-four or even thirty-six hours. On the third
day a second dose of 1 mg. is given, and if no effect is
produced a third, of 2 mg., three days later. In the
great majority of cases of latent tuberculosis an appre-
ciable reaction will be produced by the time a dose of
2 mg. has been reached. If no effect has been caused
by the tests applied as above I have usually gone no
further, and concluded that no tuberculous process was
present, or at least not to a degree which need be taken
into account in advising the patient or which would
warrant insisting on a radical change in his surround-
ings and mode of life. If some slight symptoms, how-
ever, have been produced by a dose of 2 mg., it may
be necessary to give a fourth injection of 3 mg. in order
to reach a positive conclusion. Nevertheless, it should
be borne in mind that in a few cases the exhibition
of even larger doses may cause reaction and indicate
the existence of some slight latent tuberculous lesion,
and the test should not, when applied within the
296 BACTERIOLOGY.
moderate doses described, be considered absolutely
infallible.
" No evidence in connection with the tuberculin test
as applied to man and animals has been forthcoming
thus far from those who have made use of it, which
would tend to sustain the general impression that this
method is necessarily dangerous and tends invariably
to aggravate the disease, and my own experience has
developed nothing which would seem to confirm this
impression. It is evident that the size of the doses
given has much to do with the limitations of this method
for usefulness and the correctness of the conclusions
reached by its application. The tuberculin used is also
a matter of some importance in determining the dosage,
as different samples vary considerably in their efficiency.
The minute amounts adopted by Grasset and Vedel
i. e., from 0.0002 to 0.0005 while they have the advan-
tage of absolute safety, may lead into error, as they
are insufficient, on the evidence of these observers them-
selves, to cause reaction in cases proven to be tuberculous
by the presence of the bacillus in the expectoration. If,
on the other hand, the test be pushed to the injection of
such large amounts as 10 mg. or more, as advocated by
Maragliano, such doses are by no means free from the
objection of occasionally causing unpleasant and some-
times dangerous symptoms; and even if the amount
given be not carried to the dose of 10 mg., which is
known to produce fever in healthy subjects, it is likely
that on account of individual susceptibility or the pres-
ence of some other morbid process in the body, reaction
will be found to occur with the larger doses when no
tuberculous process exists. The adoption of an initial
dose so small as to guard against the absolute possibility
BACILLUS OF TUBERCULOSIS. 297
of producing violent reactionary symptoms, and the
graded increase of the subsequent doses within such
quantities as are known never to produce reaction in
healthy individuals, would seem to afford the best
protection against unpleasant results and misleading
evidence. "
Antituberculous Serum. Whether serum-therapy is
destined to solve the problem of the treatment of tuber-
culosis remains for the future to decide, but up to the
present the results obtained with antituberculous serum
do not warrant our forming such an opinion. The at-
tempts to obtain from animals chiefly horses a serum
which would be protective have been carried out along
very much the same lines as Koch's experiments upon
man. The methods adopted have been as follows: Old
cultures of tubercle bacilli grown in 5 per cent, glycerin
bouillon have been filtered either with or without pre-
vious boiling, and then injected into animals, this
process being similar to Koch's with his first tuberculin.
Others have injected living virulent or non-virulent
tubercle bacilli, either alone or with their culture fluids;
others still (Biichner) have injected the bacterial proto-
plasm obtained by crushing tubercle bacilli together
with sand and squeezing them ; this, like Koch with
his new tuberculin, being an attempt to get from the
unaltered products and cell-contents of the bacilli the
formation in the body of bactericidal or immunizing
substances.
Among the many claiming good results in man or
animals thus treated may be mentioned Hericourt,
Bichet, Bernheim, Maragliano, Yiquerat, Paquin,
de Schweinitz and Dorset, McFarland, and others.
The majority claim for their serum the power to neu-
298 BACTERIOLOGY.
tralize the effect of tuberculin when injected into tuber-
culous guinea-pigs; but this test is insufficient and prob-
ably valueless, since tuberculin is not the same as the
unaltered products of the tubercle bacillus. Moreover,
it has been shown by Trudeau and Baldwin that other
substances which have no specific properties whatever
will have much the same effect as the serum under
certain conditions. Some make the further claim that
guinea-pigs injected with serum acquire an immunity
to the virulent tubercle bacilli, and that those already
infected live longer than the controls which receive no
serum; and some even claim to be able to cure animals
eighteen days after inoculation with, a culture of tu-
bercle bacilli. Very few observers, however, have suc-
ceeded in obtaining appreciable results with the serums
prepared by other experimenters. In spite of such con-
flicting testimony, it is probably safe to assert that no
serums now obtainable have any great value. Nor as
we look at the progressive nature of tuberculosis can
we see much ground to hope for the abundant de-
velopment of curative substances in the blood of ani-
mals.
Prophylaxis. Meanwhile all energies should be
directed to the prevention of tuberculosis, not only
by the enforcement of proper sanitary regulations as
regards the care of sputum, milk, meat, disinfection,
etc., but also by continued experimental work and by
the establishment of free consumptive hospitals, and
by efforts to improve the character of the food, dwell-
ings, and condition of the people in general, we should
endeavor to build up the individual resistance to the
disease. It may be years yet before the public are
sufficiently educated to co-operate with the sanitary
BACILLUS OF TUBERCULOSIS. 299
authorities in adopting the necessary hygienic measures
to stamp out tuberculosis entirely; but, judging from
the results which have already been obtained in reducing
the mortality from this dread disease, we have reason to
believe that in time it can be completely controlled.
The Tubercle Bacillus of Cattle and its Relation to
Human Tuberculosis. Among the domestic animals
tuberculosis is most common in cattle. On account
of the milk which they provide for our use, and which
is liable to contain bacilli, the relation of these to human
tuberculosis is a matter of extreme importance.
The chief seat of the lesions is apt to be the lungs
and with them the pleura; less often the abdominal
organs and the udder are affected. In pigs and horses
the abdominal organs are most often involved, then the
lungs and lymphatic glands. In sheep and goats tuber-
culosis is rare. The bovine bacillus, as the most im-
portant of the group, will be alone considered here.
The bacilli derived from cattle are on the average a
little shorter and straighter than the average human
bacillus; but there are many derived from cattle exactly
similar to those derived from man in size, shape, and
staining. In guinea-pigs, and especially in rabbits, the
bovine bacilli are more virulent than those from human
sources. Animals infected with the bacilli from cattle,
as well as those from the other domestic animals, react
to the tuberculin test. All these bacilli are, therefore,
undoubtedly from the same original stock, and at
first glance we might consider it unnecessary to prove
that those derived from cattle were capable of causing
human tuberculosis. There are facts, however, which
tend to make us doubtful of the extent to which this
infection takes place. As we investigate we find that
300 BACTERIOLOGY.
all facts tend to show that the great majority of cases,
in adults at least, come from human infection. The
cases where fairly strong proof of bovine infection has
been obtained are certainly rare.
Further, we have the undoubted fact that constant
sojourn in one species of animal tends to increase the
virulence of the germ for that animal and to lessen it
for others.
Theobald Smith has made the interesting discovery
that there is a wide difference between the culture
growth of the average bovine bacillus and the average
one from human sources, the bovine bacilli being shorter
and straighter, and growing less luxuriently than those
from man ; and, further, that the bovine bacilli are
much more virulent for rabbits. He has found these
differences persist for long periods, and believes that
the simple passage through a single person in a case of
human tnberculosis would not be sufficient to change
these characteristics. He has not yet had a chance to
examine the bacilli of any case in young children where
milk infection was strongly suspected, but in adults
not one of some half a dozen cultures showed the
bovine characteristics.
At present it seems fair to assume that bovine bacilli
are capable of infecting only those that are very sus-
ceptible, such as young children. This question is in
great need of further study, and unless proof is
brought to show that bovine bacilli never infect human
beings, no cattle which are shown to be tubercular
should be allowed to furnish milk, or at least none un-
sterilized should be used for drinking purposes. The flesh
is less harmful, as muscular tissue is seldom infected.
Bird (Avian) Tuberculosis. Tuberculosis is very com-
BACILLUS OF TUBERCULOSIS. 301
mon and infectious among fowls. The bacilli them-
selves grow more readily on artificial culture media
and produce a more even and moist growth. The
bacilli are more apt to show branching forms than the
human. In rabbits they produce very similar lesions.
They are probably from the same stock as the mamma-
lian varieties; but it is not believed that they are any,
and certainly not any great, factor in the production of
human tuberculosis.
Diagnosis. One of the most important results of the
discovery of the tubercle bacillus relates to the practical
diagnosis of tuberculosis. The staining peculiarities
of this bacillus render it possible by the bacteriological
examination of microscopical preparations to make an
almost absolutely positive diagnosis in the majority of
cases. A still more certain test in doubtful cases is
the subcutaneous or intraperitoneal injection of guinea-
pigs, which permits of the determination of the presence
of numbers of bacilli so small as to escape detection by
microscopical examination. For the animal test, how-
ever, time is required at least three weeks, and, when
the result is negative, several months before any posi-
tive conclusion can be reached, for when only a few ba-
cilli are present tuberculosis develops slowly in animals.
LABORATORY TECHNIQUE IN THE EXAMINATION
FOR TUBERCLE BACILLI AND OTHER ASSO-
CIATED BACTERIA.
I. Microscopical Examination of Sputum for the Presence
of Tubercle Bacilli.
1. Collection of Material. The sputum should be col-
lected in a clean bottle (two-ounce) with a wide mouth
and a water-tight stopper, and the bottle labelled with
302 BACTERIOLOGY.
the name of the patient or other distinguishing mark.
The expectoration discharged in the morning is to be
preferred, especially in recent cases, and the material
should be coughed up from the lungs. Care should be
taken that the contents of the stomach, nasopharyngeal
mucus, etc., are not discharged during the act of expec-
toration and collected instead of pulmonary sputum. If
the expectoration be scanty the entire amount discharged
in twenty-four hoars should be collected. In pul-
monary tuberculosis the purulent, cheesy, and muco-
purulent sputum usually contains bacilli; while pure
mucus, blood, and saliva, as a rule, do not. When
hemorrhage has occurred, if possible some purulent,
cheesy, or mucopurulent sputum should be collected
for examination. The sputum should not be kept any
longer than necessary before examination, for, though
a slight delay or even till putrefaction begins, does not
entirely vitiate the result, it is best to examine it in as
fresh a condition as possible.
2. Methods of Examination, (a) EXAMINATION FOR
TUBERCLE BACILLI. Pour the specimen into a clean,
shallow vessel having a blackened bottom a Petr
dish placed upon a sheet of dull black paper answers
the purpose and select from the sputum one of the
small, white or yellowish-white, cheesy masses or
11 balls " which it is seen to contain. From this make
a cover-glass " smear " in the usual way. Immerse
this in a solution of Ehrlich's aniline-water fuchsin (see
page 198), contained in a thin watch-glass or porcelain
dish, and steam over a small flame for two minutes.
Then remove the cover-glass from this and wash with
water. Now decolorize by immersing the stained prep-
aration in a 3 per cent, hydrochloric acid solution in
BACILLUS OF TUBERCULOSIS. 303
alcohol for from a few seconds up to one minute, re-
moving at the time when all color is just about gone
from the cover-glass smear. Wash thoroughly with
water and make a contrast stain by applying a cold
solution of Loffler's alkaline methylene-blue
Concentrated alcoholic solution of methyl blue 30 c. c.
Caustic potash (1:1 0,000 solution) . .100"
for from fifteen to thirty seconds. Wash with water;
press between folds of filter-paper; dry in the air;
mount and examine.
The tubercle bacilli are distinguished by the fact that
they retain the red color imparted to them in the
fuchsin solution, while the other bacteria present,
having been decolorized in the acid solution, take the