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332
BULLETIN No. 161
[November,
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FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY
333
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334 BULLETIN No. 161 [November,
of time that they remained alive and by the severity of the disease
produced in the guinea pigs inoculated upon the same days from
each of these samples. This difference, however, was doubtless
due mainly to the difference in drying.
3. Tubercle bacilli in the manure of a naturally infected cow
were dead within two weeks after exposure. More experimental re-
sults are necessary before trustworthy conclusions can be drawn.
4. Danger of infecting farm animals from tuberculous ma-
nure is indicated.
i
DURATION OF LIFE OF BOVINE BACILLI IN GARDEN SOIL
DIIDDOCC Some investigators state that tubercle bacilli will
KUKrUot .,,.,, , . , ,
OF TEST live in soil and in dead carcasses buried in the
soil from two to three years. If this be true, ma-
nure from tuberculous cattle when put upon cultivated fields which
are later used to pasture stock may be a source of infection for farm
animals, especially hogs. For this and other reasons it was thought
advisable to test the length of time a pure culture of bovine tubercle
bacilli will live in garden soil.
The culture of bovine tubercle bacilli was of the
CULTURE same strain as that used in the experiment forarti-
fically infecting the cow manure. About four milli-
grams of pure culture were obtained from a four-weeks' growth on
the surface of two large tubes of glycerine agar. The organisms
were carefully removed from the glycerine agar and emulsified as
described under "Cultures and Emulsions," page 315.
CA .. DI c The sample of soil with which the tubercle bacilli
oAlir*LC . . ......
OF SOIL emulsion was mixed was obtained from a garden
plot that had been in cultivation only two years.
Previous to that time, this plot of ground had been in sod for at
least fifteen years. The part from which the soil sample was ob-
tained had been well manured with horse manure the first year it
was under cultivation. The second year no manure was added but
the ground was well stirred and made into a lettuce bed. A suf-
ficient amount of this soil was obtained to fill a ^4 -inch mesh wire
basket having the dimensions of 4x5x6' inches. This required
1700 grams. This pulverized soil was placed in a large pan and
the emulsion of tubercle bacilli sprinkled over it. These were
thoroly mixed by being constantly stirred for some time. The
amount of emulsion was sufficient to make the soil quite wet and
stickv.
FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 335
PLACE OF g rams ot this infected soil were removed to
EXPOSURE be tested for tubercle bacilli as a control. The re-
maining soil was put into the wire basket and
covered with a wire gauze. It was buried in another garden that
had been under cultivation for one year and had received no ma-
nure or fertilizer of any kind. The place where it was buried had
been previously well pulverized. The basket with the infected soil
was buried six inches under the surface of the ground.
TESTS OF Samples of this infected soil were tested for tuber-
SAMPLES cle bacilli on the first day of exposure and on the
7th, 1 6th, 34th, 55th, and thereafter about once
a month for 352 days. Ten grams of soil were removed by dig-
ging down beside the mouth of the basket and with a sterile pota-
to knife making an opening to the center of the basket. After the
removal of the sample, the opening was filled by pressing. the soil
in around this opening with a potato knife. The wire gauze was
placed over the mouth of the basket and the garden earth filled in
over it to a depth of six inches. The soil sample taken to the lab-
oratory was thoroly shaken with 200 cc. of 0.8 percent salt solu-
tion in a 300 cc. flask. After standing ten minutes until the coarser
sediment had fallen to the bottom, 40 cc. were removed and placed in
two sterile centrifuge tubes and centrifuged for five minutes. The
supernatant liquid was drawn off and put into two other centrifuge
tubes and centrifuged for thirty minutes at 2000 revolutions per
minute. The supernatant liquid was drawn off and discarded. The
last 5 cc. of the liquor and sediment were thoroly mixed and in-
jected subcutaneously into guinea pigs, in graded doses, giving one
i l /2 cc., another i cc., and for the first test a third received y 2 cc.
RESULTS OF ^ e resu l ts considering the length of time that
THE TEST tubercle bacilli live in the soil are recorded in
Table 20. The guinea pigs inoculated from
samples taken on the day of exposure, and on the 7th, i6th and
34th day after exposure showed in each case, when killed and ex-
amined, severe generalized tuberculosis. Microscopical and cul-
tural tests showed the germs from the diseased tissues to be char-
acteristic of active, living tubercle bacilli. Tests after this time indi-
cated a weakening in virulence, but slight tuberculosis was produced
in the test animals from material taken on the 21 3th day of expos-
ure. Microscopic, cultural, and guinea-pig tests from the diseased tis-
sues of the original guinea pig showed the tubercle bacilli in the soil
sample to be active and virulent. Five tests were made after this
date. In every case the guinea pigs, when killed and examined,
were found to be healthy. The two testings after the last one
in which tubercle bacilli were found, viz., the ones made on the
336
BULLETIN No. 161
[November,
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Macroscopic
Tub. only in R.S. inguinal, slight
Tub. only in R.S. inguinal, slight
Generalized tuberculosis, severe
Generalized tuberculosis, severe
Generalized tuberculosis, severe
Generalized tuberculosis, severe
Generalized tuberculosis, moderate
Generalized tuberculosis, severe
Generalized tuberculosis, severe
Tub. at point of inoculation and in
R.S. inguinal
Generalized tuberculosis, not se-
vere
Generalized tuberculosis
Tuberculosis only at the point of
inoculation
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FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY
337
TABL.E 20 Continued.
+
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'The right superior inguinal enlarged in the third and fourth week and then became normal again.
+
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Acute in-
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Acute infection
Generalized tuberculosis
Slightly tuberculous
Acute infection
Acute infection
Acute infection
Acute infection
Tub. in R.S. inguinal and spleen
Healthv 1
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rO tO iO i/) O^ O\ THTH OrO VOO C>^ THTH to
THrH TH TH THTH C-1CS C^C^ C^d C1M COtO tO
338 BULLETIN No. 161 [November,
2301!], and on the 261 st days of the exposure, produced in the
third and fourth weeks an enlargement in the right superior in-
guinals of each guinea pig inoculated, which then became normal
again. Tho it cannot be surely known, it may be that the number
of live tubercle bacilli was so small as not to cause the disease, but
of sufficient number to produce an enlargement of the glands.
Three tests made later than these two showed no such enlargement
of these glands.
BOVINE TUBERCULOSIS IN A DEAD ANIMAL,
crtnorc re Besides the exposure of the pure culture of bovine
bUUKL/t \Jr * _ r
MATERIAL tubercle bacilli in garden soil, a guinea pig that
had died of bovine tuberculosis was exposed in
this same soil. This guinea pig was extensively tuberculous. It
was placed in a flower pot and covered with a screen of wire hav-
ing a mesh fine enough to exclude earthworms. The screen was
pressed down close over the guinea pig, which allowed the fine gar-
den earth to come into immediate contract with the dead body.
The abdominal and thoracic cavities had previously been opened
in making an examination of the guinea pig just before placing it
in the garden soil.
METHOD OF ^ ^ e time of the first test, made the 7ist day of
TESTING the exposure, the tissues of the guinea pig were
so decayed that the flesh and the skin were easily
torn. A part of the tuberculous lung and of the right superior in-
guinal lymphatic was removed for the test. At the next test, on
the 99th day, most of the soft tissues had been carried away by
small ants. On the i33d day only the bones, hair and some ten-
dons remained. A few pieces of bones and a bunch of hair were
obtained for this test.
The results of these tests are given in Table 21.
RESULTS Live, active tubercle bacilli were found on the
7 ist day; after this date no tuberculosis was pro-
duced in any of the guinea pigs inoculated. No doubt if the small
ants had not molested the soft tissues, tuberculosis would have
been produced in the test animals at later dates. While no final
conclusion can be drawn, it is evident that these germs live a suffici-
ent time in dead tuberculosis animals to be dangerous to stock.
FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY
339
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340 BULLETIN No. 161 [November,
DURATION OF LIFE OF TUBERCLE BACILLI IN WATER
PURPOSE AND ^ ne P ur P ose f these tests was primarily to deter-
PLAN OF TESTS mine how long bovine tubercle bacilli will live in
a watering tank such as is found on the average
stock farm in Illinois. In connection with this work it was desired
to test the length of time human tubercle bacilli will live in drink-
ing water. With this in view experiments were planned and car-
ried out with tubercle bacilli from the following sources :
1. Pure cultures, Series i.
2. Pure cultures, Series 2.
3. The diseased tissues of a tuberculous guinea pig.
4. Tuberculous sputum.
Series i. Pure Cultures
CULTURES The bovine culture was the same as that used in
the cow manure which was exposed to weather
conditions. The human culture was taken from the tuberculous
glands of the neck of a patient from the Burnham hospital and iso-
lated in this laboratory. The tissues were received May 25, 1910. A
pure culture was obtained by inoculating a guinea pig and cultur-
ing the diseased tissues of the guinea pig. This culture, which is
typical for the human type, has since been kepi upon glycerine agar.
SAMPLES OF The sam pl e f water use d to make the emulsion
WATER for exposing the bovine tubercle bacilli was ob-
tained from the large watering tank used to water
the dairy cattle of this station. This tank is 2^2x4x10 feet, and
is supplied with running water. A considerable amount of spiro-
gyra and various kinds of smaller green algae was floating in the
tank. Diatoms were also abundant. A green scum covered a
large portion of the surface of the water. The sample was ob-
tained in a 500 cc., sterile, cotton-stoppered flask. The water in
the tank was slightly agitated with a stick about as much as it
was thought the cattle Wtf'n* stir the water by drinking. The
mouth of the flask* was pi 1 iQ'ed under about six inches and then
allowed to fill. The sampl -vas at once taken to the laboratory
and the emulsion prepared aat same afternoon. The spirogyra
died soon after the emulsion was made, but the diatoms and the
smaller green algae, as well as numerous bacteria, were present at
the conclusion of the experiment December 5, 1911, 586 days after
the experiment started. The sample of water used to make the emul-
sion with the pure culture of human tubercle bacilli was taken from
the tap water of the bacteriological laboratory. There were at the
19*-']
FATE OF TUHERCLE BACILLI OUTSIDE THE ANIMAL BODY
341
time some algae and bacteria present in this water, a part of which
were still living at the last test made.
PREPARATION emulsions both of the bovine and of the
OF EMULSIONS human cultures of tubercle bacilli were prepared
in the same way. About two milligrams of a four
weeks' culture grown upon glycerine agar were emulsified in the
manner described under ''Cultures and Emulsions," page 315. The
emulsion of bovine tubercle bacilli was diluted to 350 cc. with
the tank water obtained for this purpose; 250 cc. were used to
expose in the running water, and 100 cc. to expose in standing
water. In a similar way a 250 cc. emulsion of human tubercle
bacilli was made.
EXPOSURE OF
SAMPLE
For exposing the emulsions in running water two
6-inch flower pots were obtained. The small
opening in the bottom was stopped with a cork
and this was sealed over with boiler paint. A test of the two
flower pots thus prepared showed that they held water. After
immersing them for twenty-four hours to within two inches of
the top, no water rose on the inside, tho it became very moist. The
two emulsions were now poured into these two flower pots and
FIG. 1. THE VESSELS AND THE Poor, OF RUNNING WATER IN WHICH THE
TUBERCLE BACII^I WERE EXPOSED.
The intake is at A; the outlet at B. The submerged vase C has been placed
upon the shelf with other vessels so it can be seen. The flower pot D,
which contained the tuberculous guinea pig, is placed upon a brick to
bring it into view. The two unglazed cylinders and one of the flower
pots show the effect of freezing.
342 -
BULLETIN No. 161
[November,
each flower pot placed in a gallon glass jar containing water and
immersed about three inches, so that the level of the emulsion of
tubercle bacilli on the inside was the same as the level of the water
on the outside. The water in the glass jars around the outside of
the flower pots was kept continually running. While this procedure
does not give the same condition as water running directly into the
emulsion of bacteria, it allows to some extent the circulation of the
water inside the porous flower pot with running water on the out-
side of these vessels. Another part of the emulsion of bovine tu-
bercle bacilli was kept in a cotton-stoppered glass bottle. This
bottle was partly immersed in one of the glass jars of running
water. The water could in no way circulate in this bottle, but it
was kept at approximately the same temperature as the emulsions in
the flower pots.
The samples were kept in the laboratory until April 15, 1911,
250 days after the emulsions were first made. It was then found
neither convenient nor desirable to keep them there longer. A
pool was prepared in the courtyard of the Agricultural Building,
to which they were transferred. The pool was made by sinking a
large tile three feet in diameter and filling in the bottom with
concrete. Constantly running water was maintained to a depth of
twenty inches the year round. It did not freeze any time during
the winter of 1911-12. A shelf of slate was placed four inches
under the surface of the water on which the vessels containing the
tubercle bacilli were set. .The pool was screened against flies and
other insects. (See Figs, i and 2.)
FIG. 2. THE POOL SCREENED AGAINST FLIES.
i<)i2\ FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 343
-reo-riMf TUP Nineteen tests from these samples were made for
TESTING Tnt , A .
SAMPLES the presence and for the virulence of tubercle
bacilli. These tests were made on the 9th, 23d,
and 44th days, and thereafter about once a month until the last
test, which was made December 5, 1911, 586 days from the begin-
ning. The sample of the bovine tubercle bacilli kppt in the cotton-
stoppered bottle was tested for only 202 days, since the sample was
exhausted at this time. This sample was not transferred to the
pool.
Each of the three samples was tested upon the same days and
in the same manner. The water in the two flower pots was thoroly
agitated by giving it a circular motion. With a sterile glass rod
the sides and bottom of the flower pot were scraped so as to loosen
adhering sediment that might contain tubercle bacilli. The water
in the bottle containing the bovine tubercle bacilli was thoroly
shaken before the test sample was taken. It was not convenient
nor thought necessary to rub the inside of this bottle, as was done
in the case of the flower pots. With a sterile Pasteur bulb pipette
from each of the three containers approximately 5 cc. of this in-
fected water were removed and placed in a sterile centrifuge tube
and centrifuged for thirty minutes at a high speed. Four cc. of
the supernatant liquid were removed and discarded. One or two
drops of the sediment were placed upon a glass slide and a micro-
scopic preparation made and stained for tubercle bacilli. The
remaining sediment and liquid in the centrifuge tubes, about i cc.
in quantity, was thoroly mixed and injected subcutaneously into a
guinea pig. The same precautions were taken with these tests as
in the case of the tuberculous manure to guard against mistaking
tubercles produced by dead tubercle bacilli for those produced by
living, virulent ones. Microscopic preparations and cultures were
always prepared to test the diseased tissues of the infected guinea
pig for tubercle bacilli. In a part of the cases, however, the tuber-
culosis was so extensive that it was not thought necessary to make
inoculations of diseased tissue into another guinea pig.
RESULTS OF ^ e resu ^ ts f the tests to determine the length of
THE TESTS time tubercle bacilli live in water are recorded in
Tables 22, 23 and 24. With the first four tests
of each of the three samples containing tubercle bacilli the guinea
pigs became extensively and severely tuberculous, as will be shown
by an examination of the three tables. Further tests of the dis-
eased tissue from all these guinea pigs showed the tubercle bacilli
to be living- and virulent. The fifth test, made after 126 days,
showed at least an apparent weakening of the virulence of these
germs.
344
BULLETIN No. 161
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