was rubbed up in 50 cc. of 0.8 percent salt solution, 40 cc. of the
emulsion was centrifuged and the sediment injected subcutaneously
into two guinea pigs, both of which became infected with gen-
eralized tuberculosis.
Naturally Infected Manure. The sample of naturally infected
manure was obtained from a tuberculous cow (cow No. 56 from
the dairy herd of this station) that had previously reacted to
tuberculin. The sample was taken by the method described in our
previous publication referred to above. Approximately three kilos
320 BULLETIN No. 161 [November,
of fresh manure were obtained. The manure from this cow had
been tested a number of times for tubercle bacilli by making sub-
cutaneous inoculations of I cc. of a 2 percent emulsion of the
fresh f eces. Upon four occasions such tests gave negative results ;
three other tests, made respectively August 31, 1909, July 18,
1910, and August 16, 1910, produced tuberculous guinea pigs.
The guinea pigs in none of the three tests became severely tuber-
culous ; and two of the guinea pigs, one each in the last two tests,
remained healthy. The last testing, August 16, 1910, was made
on the day of exposing this sample of manure to the weather.
Tho the infected guinea pig from this sample did not show severe
tuberculosis when killed and examined 80 days after inoculation,
a second guinea pig inoculated with the diseased tissue from this
guinea pig showed severe generalized tuberculosis when killed and
examined 38 days later.
EXPOSURE OF infected manures were taken to a secluded
SAMPLES plot of ground on the Experiment Station farm
for exposure. The manure infected with pure
culture of bovine tubercle bacilli was divided into two equal- parts.
One half was placed in a free, open place, fully exposed to the
sunshine thruout the whole day. This part, flattened out into a two-
inoh layer, was placed upon a sod with the grass cut short. A
one-inch mesh poultry netting was placed over the infected manure
in order, especially, to keep out the English sparrows, so that
they could not carry upon their feet this infected manure to the
stock upon the Experiment Station Farm.
The other part, protected from the sunshine, was placed a few
yards from the former on the north side and very near a bank of
earth six feet high. The ground upon which this manure was
exposed was first made smooth by removing the sod. The manure
was then spread in a two-inch layer just as was the part exposed in
the sunshine. To protect this sample further from the light, an em-
bankment of soil was made one foot high on three sides, and was
covered over with a bunch of weeds. Tho the soil was more
moist than in the plot where the part was exposed in the sunshine,
it was not more moist than that usually found in a shady place. The
layer of infected manure in this protected place dried on top in
a week's time to a hard crust, but trie bottom always remained
moist. The manure from the tuberculous cow was divided into
two parts; one part was placed in the sunshine, and the other in
the place protected from the sunshine, very near the two artificially
infected samples and exposed in the same way.
iyi2\ FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 321
TCCTIM^ -rue The samples were tested almost exclusively by
Tea I IINla I nt _ . . . ., J , ',
SAMPLES inoculation of guinea pigs following the method
outlined in our previous publication. Stains of
smears from the infected manure were made on a few occasions;
but since it is impossible to distinguish between dead and living
tubercle bacilli by examining stained preparations, no reliance is
placed upon this test. The largest sample possible was used so
as not to kill the guinea pigs by acute infection. The amount of
sample varied, using the centrifuge sediment from 40 cc. of a 1^2
to a 2^2 percent emulsion of dried feces in 0.8 percent salt solu-
tion. The sample from the layers of infected manures was taken
by cutting out pieces of the dried layer about one inch square in
cross section and taking all the manure in this section down to
the ground and a small layer of soil with the sample, so as to be
sure to get any tubercle bacilli that might have passed into this
top layer of soil just under the infected manure.
The testing of the sample was carried out with more than
usual precautions, keeping in mind all the time the probability of
producing tubercles in the guinea pig, with dead tubercle bacilli.
To be sure that the tubercles in the infected guinea pigs were
produced by living, virulent bacilli, the extent and rapidity of the
disease was considered, and also the diseased tissues were tested
for tubercle bacilli by staining smears, by cultures, and by inocula-
tion of another guinea pig. If the second guinea pig became
tuberculous, stains and cultures for tubercle bacilli from its dis-
eased tissues were made. If, now, all these tests were character-
istic for tubercle bacilli, it was considered reasonably certain that
the tubercle bacilli found in these diseased tissues were alive and
virulent. But when the guinea pig inoculated from the sample of
manure became in a short time severely tuberculous, it was not
thought necessary always to inoculate a second guinea pig from
the diseased tissues of the former.
WEATHER weatner conditions during the time the ma-
CONDITIONS nure infected with tubercle bacilli was exposed are
given in Table I2. 1 The weather was not marked
with any unusual occurrences. With the exception of the loss of
the sunshine records from the third to the seventh of September,
inclusive, when the electric sunshine recorder failed to work, the
records are quite complete. This table records the date on which
each test was made, the number of days of exposure to the time
1 The data for this table were furnished us from the L/aboratory of Soil
Physics of the Department of Agronomy of this station by the kindness of
Professor J. G. Hosier.
322
BULLETIN No. 161
[November,
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I<)i2\ FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 323
of the test; the number of clear days during this time and also the
number of partly and of wholly cloudy days. There is also given
the number of hours of sunshine, the amount of rainfall, the
highest maximum daily temperature and the lowest minimum daily
temperature occurring between each test; and an average daily
maximum, and an average daily minimum temperature during the
time of exposure up to the time the test was made. There is
also given the average monthly temperature occurring during the
time of exposure.
RESULTS OP THE TESTS
FROM ARTIFI- Exposed in the Sunshine. The results of testing
FECTED t ^ ie sam pl es f cow manure infected with a pure
MANURE culture of tubercle bacilli exposed in the sunshine
are given in Table 13. As previously mentioned
(page 320), the test of the sample on July 29, 1910, the day it was
first exposed, produced two severely tuberculous guinea pigs. Since
exposure seven tests have been made. On the 7th and i6th days
the four guinea pigs inoculated contracted severe generalized tuber-
culosis. Only miscroscopic and cultural tests were made from the
two guinea pigs inoculated on the /th day of exposure, since the
infection in both guinea pigs was so severe it appeared certain to
be from living tubercle bacilli. This was later shown to be true
from the culture tests. Microscopic and culture tests from the
diseased tissues of both of the tuberculous guinea pigs infected
with the sample taken on the i6th day of exposure, as well as an
inoculation test from the tissues of one of them, showed charac-
teristic tubercle bacilli. The test of the sample made 31 days since
first exposed produced in both guinea pigs inoculated only slight
tuberculosis of the right superior inguinal lymphatics. Microscopic,
cultural, and inoculation tests of the diseased tissues from these
guinea pigs showed typical tubercle bacilli. On the 49th day of
exposure (September 16, 1910) the test showed that the virulence
of the tubercle bacilli had considerably decreased, but was sufficient
to produce slight tuberculosis in one of the two guinea pigs in-
oculated. The guinea pigs Were inoculated subcutaneously with
the centrifuge sediment of 40 cc. of an emulsion made by thoroly
grinding three grams of the dried sample of infected manure in
150 cc. of 0.8 percent salt solution. The pus from the right supe-
rior inguinal of the guinea pig that became tuberculous was shown
by miscroscopic, cultural, and inoculation tests to contain charac-
teristic tubercle bacilli. The other guinea pig, when killed 53
days after inoculation, was found to be healthy. Tests made on
324
BULLETIN No. 161
[November.
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326 BULLETIN No. 161 [November,
the 8 1st, 1 23d and I7ist days of the exposure to sunshine showed
the tubercle bacilli to be dead or at least not sufficiently virulent to
produce tuberculosis when the centrifuge sediment was injected sub-
cutaneously into guinea pigs.
It thus appears from these tests that pure cultures of bovine
tubercle bacilli, when mixed with cow manure and exposed in an
open place in a pasture field, remained alive in this instance for ap-
proximately two months.
Exposed in the Shade. The part of the artificially infected
manure exposed in a place protected from sunshine was tested on
the same days and in the same manner as was the part exposed
in the sunshine. These results are given in Table 14. The parts
tested on the 7th, i6th, 3ist and 49th days were shown at each
testing to contain virulent tubercle bacilli. The infection of the
guinea pigs in the first two tests was severe. The guinea pigs in-
oculated with samples taken on the 3ist and 49th days of the
exposure were infected with tuberculosis but not so severely as
the guinea pigs inoculated on the 7th and i6th days. However,
they were much more severely infected than the guinea pigs in-
oculated with the sample taken from the part exposed in the sun-
shine and tested upon the same days. The three samples taken
on the 8ist, I23d and I7ist days were shown not to contain viru-
lent tubercle bacilli. Three of the guinea pigs inoculated with the
first two samples remained healthy until killed on the 49th and
55th days respectively after inoculation of the sample. One of
the two guinea pigs inoculated with the sample taken on the 8ist
day died five days later with an acute infection. The two guinea
pigs inoculated with the sample taken on the I7ist day died of
acute infection with no evidence of tuberculosis.
From these tests it appears that tubercle bacilli mixed with
cow manure remain virulent to guinea pigs for 49 days after ex-
posure in a place protected from the sun. All tests made later
than this date showed these organisms to have lost their virulence.
Virulence was retained longer in the shade than in the sunshine,
as shown by the production of more severe tuberculosis in the
guinea pigs inoculated from samples exposed in the shade than that
produced in the guinea pigs inoculated on the same days with sam-
ples exposed in the sunshine.
FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 327
FROM NATUR- The tests of the samples of manure from the
ALLY INFECTED tuberculous cow, both the one exposed in the
MANURE , , , . j xi i
shade and the one exposed in the sunshine, at no
time after exposure produced tuberculous guinea pigs. The re-
sults are tabulated in Tables 15 and 16. The sample exposed in
the sunshine was tested on the I3th, 34th, 63d, and io5th days
after first exposure. All the guinea pigs save one, which died of
acute infection in four days, remained healthy until killed and
examined 49 and 52 days after inoculation. The sample exposed
in the place protected from sunlight was tested on the same days
as the one exposed in sunshine, with one exception. No test was
made on the I3th day of exposure from this part of the infected
manure since there were not a sufficient number of guinea pigs
available at that time. This sample was omitted because it was
thought that the tubercle bacilli in the part protected from the
sun would be the least likely to die. It was indeed unexpected that
these bacilli would be dead in either of these two sampled at this
time. It was a hot time in August. During the exposure from
August 1 6 to 29 there was an average temperature of 72.32 F.
and a rainfall of 2.32 inches, having six clear, one wholly cloudy,
and seven partly cloudy days. A number of showers occurred,
making it an excellent time for the growth of decay organisms
found in the manure.
The killing of the tubercle bacilli in so short a time was no
doubt due partly to the antagonism of the decay organisms and
partly to the weakened virulence of these germs. Slight virulence
was shown by the producing of only localized tuberculosis in one
of the two control guinea pigs inoculated with a sample of the
fresh manure. More experimental data upon this subject is very
desirable.
328
BULLETIN No. 161
[November,
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330 BULLETIN No. 161 > [November,
Series 2
PREPARATION A second series of experiments exposing cow ma-
AND EXPOSURE nltre artificially infected with a pure culture of
OF SAMPLES , . , , t . ATA,
bovine tubercle bacilli was made, ine prepara-
tion and exposure of these samples were made in the same man-
ner as in Series I (see pages 319 and 320). The places of exposure
were within a few feet from the places where the samples were ex-
posed in the first series.
WEATHER Table 17 gives the weather conditions during the
CONDITIONS time of exposure of this series. The same plan
is followed as in Table 12 giving the weather
conditions in the first series. These conditions were just the re-
verse of those in the first series, the first series beginning in the
hot month of August, the second in the cooler and more moist
month of March. No especially unusual weather conditions oc-
curred during this period.
Manure 'Exposed in the Sunshine. The results of
RESULTS exposing this infected manure in the sunshine are
given in Table 18. The tubercle bacilli were still
alive at the third test after 45 days of exposure. Tho the tuber-
culosis produced in each of the two guinea pigs from this test was
only slight, as shown when these guinea pigs were killed and ex-
amined 53 days later, Guinea Pig 973, inoculated with the dis-
eased tissues from these two, produced severe generalized tuber-
culosis. Typical cultures were obtained from the diseased tissues
of both the original and the secondary inoculated guinea pigs.
The samples taken after this date produced no tuberculosis in the
six guinea pigs inoculated.
Manure Exposed in the Shade. The results of exposing the
sample in the shade are given in Table 19. Here we find that the
tubercle bacilli were alive for 73 days, while in the sample ex-
posed in the sun they were dead at this testing. Also the test
made on the 45th day shows more severe tuberculosis in the guinea
pigs inoculated from the sample kept in the shade than from the
sample in the sun.
i. As shown by the results in both series of ex-
CONCLUSIONS periments, a pure culture of bovine tubercle bacilli
mixed with cow manure and exposed in the sun-
shine in a pasture, remains alive and virulent for approximately
two months.
2. The virulence of the tubercle bacilli in cow manure was re-
tained in the samples protected from the sunshine longer than in
those exposed in the sun, as shown both by the increased length
1912} FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY
331
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