1912}
FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY
357
tuberculosis, save those that died too early with acute infection.
The two guinea pigs used in the third test, made 99 days af-
ter exposure, remained healthy. Probably only non-tuberculous
tissue was obtained for this test. Later tests taken from broken-
down tissues showed virulent tubercle bacilli. The tests were
positive up to the 32ist day; after this date the test animals
all died with acute infection, the last one, however, not too
early to have shown tubercle bacilli. On the 229th day and
later no structure of tissue could be determined except the hair
and bones, and the bones were almost as fragile as garden earth.
Sediment from the bottom of the flower pot was obtained for the
samples.
Tuberculous Sputum
To determine the length of time that tubercle
bacilli from tuberculous sputum will live in run-
ning water, a sample was obtained from an ad-
vanced case of tuberculosis. A stained smear of this sputum
showed numerous bacilli. A very small sample was inoculated into
a guinea pig the first day of exposure. This guinea pig died in
three days of acute infection.
RESULTS The results are given in Table 29. Tuberculosis
has been produced in the test animals up to the
last test, 232 days since exposure. However, only local tubercu-
losis was produced at this last test and there has not been sufficient
time to determine whether the organisms are alive or dead. The
test made just previous to the last one, 187 days after exposing
the sputum in, water, produced severe generalized tuberculosis in
Guinea Pig No. 1126 (see Fig. 4).
SOURCE OF
SAMPLE
TABLE 29. TUBERCLE BACILLI IN WATER
Kind and source of the organisms
Not killed
Killed
Human tubercle bacilli:
a. Pure culture in flower pot, Series 1
441 days
470 days
b. Pure culture in 8-inch cylinder. Series 2
c Pure culture in submerged va*e
259 days
259 days
d In tuberculous guinea pig
321 days
381 days
g JQ sputum
232 days
Bovine tubercle bacilli:
441 days
470 days
b. Pure culture in 8-inch cylinder, Series 1
c. Pure culture in cotton-stoooered bottle ..
259 days
202 davs
358
BULLETIN No. 161
[November,
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I9i-] FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 359
A summary of the results showing the length of time that
tubercle bacilli from various materials -live in water is given in
Table 29.
Conclusions
1. Tubercle bacilli live for more than a year (441 days) in
running water.
2. The length of time that human and bovine tubercle bacilli
live in water is practically the same.
3. These organisms live in sputum exposed in water for more
than 232 days.
4. They live in the tissues of a dead guinea pig exposed in
water for more than 321 days but are dead in 381 days.
5. A watering trough harboring tubercle bacilli may become a
dangerous source of infection to cattle.
6. A dead tuberculous animal in a stream on your neighbor's
farm may be a means of infecting your stock.
7. The better disposition of dead tuberculous animals is to
destroy by burning.
8. Tubercle bacilli in drinking water is one of the possible
sources of infection for man.
9. Infection is not prevented by dilution, since clumps contain-
ing a great number of these organisms may be inclosed in mucoid
material which prevents their separation and destruction.
DURATION OF LIFE OF BOVINE TUBERCLE BACILLI IN BUTTER
INTRODUCTORY Considerable attention is at present being directed
STATEMENT to the presence of tubercle bacilli in foods, more
especially milk, butter and cheese. It has been
determined by Sedgwick and Winslow and by Park that typhoid
bacilli frozen in water die very rapidly. After an hour's freezing
30 to 60 percent were destroyed, and in two weeks 99 percent were
killed. The remaining one percent lived for a number of weeks.
Tubercle bacilli in butter kept at a temperature below freezing are
not killed in this way, as determined by Mohler, Washburn and
Rogers in 1909. In order to obtain further information upon this
subject the following experiments were planned.
Butter was mixed with an emulsion of a pure culture of bovine
tubercle bacilli and placad in small vials which were stored in the
three following places:
360 BULLETIN No. 161 [November,
1. The cold storage of the Monarch Refrigerating Company,
Chicago, 111., at 10 C. below freezing.
2. The University of Illinois Dairy storeroom at 4 C. (above
freezing).
3. The basement of a dwelling in Urbana, Illinois, kept at an
average of approximately 20 C.
PREPARATION ^ pound of fresh butter was obtained from the
OF SAMPLES creamery of the University of Illinois directly
from the moulding board. It was salted as usual
for the market, one ounce of dry salt to one pound of butter.
After the butter is mixed, pressed and drained it has a salt content
of two to three percent. It was not chilled, but at once taken to
the bacterioligocal laboratory and mixed with the emulsion of
tubercle bacilli. An emulsion of 3 mg. of bovine tubercle bacilli
in 100 c.c. of 0.8 percent salt solution was made, and the butter
melted at 35 C. was thoroly shaken with this emulsion. From
about 10 to 15 cc. of this emulsion were put into small sterile glass
vials and stopped with sterile cork stoppers. Thirty such samples
were prepared, ten of which were stored in each of the three places
mentioned above.
TESTING THE ^ e sam pl es were tested when prepared and at
SAMPLES varying intervals afterward on the same day from
each of the places stored. They were brought
to the laboratory, melted at a temperature of about 38 C., and
two guinea pigs were each injected subcutaneously with i cc. of
the melted butter from each sample. The samples kept at Chicago
at 10 C. below freezing were always in excellent condition; those
kept in the basement of the dwelling became very rancid and
slightly mouldy; and those kept in the University Dairy storage
showed slight moulding in part of the bottles.
RESULTS The results of the tests are given in Tables 31, 32
33. No end point was reached. Generalized tu-
berculosis was produced in the test animals from each of the three
samples taken on the 274th day. It was noted that the tuberculo-
sis produced by the samples kept at the lower temperature was
more severe. This was probably due to the killing out of other
organisms that at higher temperatures acted antagonistically to the
tubercle bacilli.
1912}
FATE OF TUBERCLE BACILLI OUTSIDE TriE ANIMAL BODY
361
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FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY
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364 BULLETIN No. 161 [November,
GENERAL DISCUSSION
A summary of the results of the experimental work is given
in Table 34. It is seen from this table that tubercle bacilli in pure
culture, spread in thin layers on sterile glazed paper slips and ex-
posed to the direct rays of the sun, are killed in a very short time
(i to 4 minutes). In this respect tubercle bacilli may be classed
with other nonspore-bearing organisms. When exposed to desic-
cation, pure cultures of these germs in thin layers are found to be
dead in a few days. In sputum and other foul material they appear
to live longer than the other nonspore-bearers. They are known
to live long enough to be blown around so that the inhalation of
dried sputum dust causes tuberculosis in test animals. Just how
frequently people are infected by breathing dried tuberculous ma-
terial has been discussed (see page 270). That sunlight plays an
important part in the disinfection of this dried tuberculous dust
is evident. Also that our homes, factories, and places of business
should have an abundance of window space, located so as to admit
the light, is another timely lesson.
Tubercle bacilli in cow manure lived 73 days when a pure cul-
ture mixed in a sample of manure was exposed to weather condi-
tions in a pasture field in the shade, and as long as 49 days when
exposed in the sunshine. The sample from a tuberculous cow was
dead at the first test, made 13 days after exposure. We were dis-
appointed in not being able to repeat these experiments. It would
be advisable to repeat this work, especially with naturally infected
manure from several tuberculous cows that were known to be
expelling tubercle bacilli per rectum. Keep pigs from three to four
months old in a pasture with such tuberculous cows, and allow the
pigs to feed upon the cow dung. Feed other pigs by mixing with
their feed tuberculous manure which had remained in this pasture
for varying intervals of time. An examination of the internal or-
gans of these shoats would give valuable information both as to
the infectiveness of the cow manure and to the length of time that
tubercle bacilli remain alive in such manure. This would indicate,
much more completely than our experiments, the length of time
that stock should be kept from a field in which tuberculous cattle
had been pastured.
When tubercle bacilli, either in manure or in dead tuberculous
animals, become mixed in the soil, the danger may be still greater,
depending upon the opportunity for hogs to take this material
along with their food. The bacilli live longer under these condi-
tions, but the opportunity of being taken is usually less.
The danger of man becoming infected with tuberculosis from
drinking water has been discussed (see page 307). Just how likely
FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL
BODY
365
TABLE 34. SUMMARY ox RESULTS
Organisms exposed to direct sunlight
Name of organism
B. subtilis, spores
B. mesentericus vulgatus, spores.
B. subtilis, vegetative cells
B. mesenteric, vegetative cells...
B. prodigiosus
B. diphtheria
B. coli
B. ty phosus
B. violaceus . .
Not killed
180 min.
Yz min.
6 min.
2 min.
1 min.
2 min.
Killed
1 min.
% min.
after 6 min:
3 min.
2 min.
3 min.
Tubercle Bacilli Exposed to Direct Sunlight
Name of culture
Not killed
Killed
B. tuberculosis, human.
B. tuberculosis, bovine
B. tuberculosis, avian..
1 min.
1 min.
2 min.
2 min.
2 min.
4 min.
Bovine Tubercle Bacilli in Cow Manure
Kind and source of material exposed
Not killed
Killed
Bovine tubercle bacilli, pure culture:
Exposed in cow manure, in sunshine, Series 1.
Exposed in cow manure, in sunshine, Series 2.
Exposed in cow manure, in shade, Series 1 . . . .
Exposed in cow manure, in shade, Series 2
49 days
45 days
49 days
73 days
81 days
73 days
81 days
100 days
Bovine Tubercle Bacilli from a Naturally Tuberculous Cow
Tuberculous manure from this cow, in sunshine.
Tuberculous manure from this cow, in shade ....
13 days
43 days
Bovine Tubercle Bacilli in Garden Soil
Bovine tubercle bacilli, pure culture 213 days 230 days
Bovine tubercle bacilli, in tissue of a dead
guinea pig 77 days 91 days
Tubercle Bacilli in Water
Human tubercle bacilli:
Pure culture, in flower pot, Series 1 441 days 470 days
Pure culture, in 8-inch cylinder 258 days
Pure culture, in an unglazed vase, submerged. 259 days
In the tissues of a tuberculous guinea pig 321 days 381 days
In tuberculous sputum 232 days
Bovine tubercle bacilli:
Pure culture in flower pot, Series 1 441 days 470 days
Pure culture, in an 8-inch cylinder \ 259 days
Pure culture, in a cotton-stoppered bottle j 202 days
Bovine Tubercle Bacilli in Market Butter
Temperature stored Not killed Killed
10 C. below zero , . . . 274 days
4 C. above zero 274 days
20 C. above zero 274 days
366 BULLETIN No. 161 [November.
it is that cattle and other farm animals are infected with tuberculo-
sis from the presence of these germs in water is not surely known.
The common watering tank may become a source of infection. Here
the tubercle bacilli live among the algae and in the decaying organic
matter for more than a year. Calmette points out that constant
and repeated infections are the most dangerous. Cattle would thus
be subjected when a watering trough was infected.
Another source of danger to man is in the use of phosphates
made by grinding up dead tuberculous animals (which is done
rather extensively in the United States), this fertilizer often being
used in vegetable gardening. One can easily conceive how a small
piece of tuberculous tissue containing many dozens of tuberculous
germs could be made to adhere to an onion or a radish, especially
in a slightly bruised place, and be carried directly to the consumer.
That these germs would remain alive and virulent during such a
circuit there is no question.
It is seen that tubercle bacilli in butter kept at 10 C. below zero
retain their virulence longer than when kept at the higher temper-
ature. This temperature of 10 C. apparently has no injurious
effect on these germs, while the antagonism of other organisms
is largely prevented. Butter can be kept in cold storage for
months in an excellent condition, but this in no way lessens the
danger from tubercle bacilli that were originally introduced into
the butter. All such dairy products should be tested by govern-
ment officials not only for quality but also for the presence of tu-
bercle bacilli.
REFERENCES
1. Abba e Barelli, Sulla resist, del bac. tub. negli sputi sopra di-
parimenti e dentro le biancherie, Rivista d'igiene e sauita publica 12:
115, 1901. Cited by Treskinskaja.
2. Aiello und Drago, Cited by Sobernheim. G., in Kolle-Wasser-
mann Handbuch der Pathogenen Mikroorganismen 2: 28.
3. Alexander, John, Das Yerhalten des kaninchens gegeniiber den
verschiedenen Infektionswegen bei verschiedenen Typen des Tuber-
kelbacillus. Ztschr. f. Hyg. 60: 467-478. 1908.
4. Annett, H. E., Tubercular expectoration in public thoroughfare
an experimental inquiry. Thompson-Yates Lab. Reports 4: part 2,
395- 1902. Cited by Rosenau, 6th Internat. Cong, on Tuberculosis.
1908.
5. Anon, Sterilization of polluted water by ultraviolet rays at Mar-
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