days
Same (repeated)
120 day
150 days
Same (repeated) triturated with water,
in air
Heim"
1889
In butter
Straus and
Dubarry ' * 3
1889
In river water (Ourcq):
at 20 C
27 days
at 35 C
at 38 C
95 days
In distilled water:
at 30 C
24 days
at 35 C
25 days
at 38 C
115 days
Gartner 44
1890
7 mo
Freytag 43
1890
Tuberculous sputum in an abundance
of table salt for two weeks; then
inoculated under the skin of a
guinea pig caused generalized
tuberculosis
2 wks
Schottelius 131
1890
Buried phthisical lungs
1891
In butter
Petri 113
1891
Organs containing tubercle bacilli:
Stone 140
1891
Ransome and
Delepine 11 *
1894
Very virulent sputum exposed to very
little air in dark
L/oesner 88
1896
Tuberculous organs placed in cada-
vers of hogs and buried. ......
Schneiderlin
1897
Tubercle bacilli remained 8 years in
soil without losing their staining
property
8 vr<* l?\
Gaertner 48
Hance 5 '-
1898
1898
In manure, temperature only 40 C, . . .
Tuberculous sputum kept fluid in a
well'stoppered bottle
3^ mo.
Klein 70
1899
Buried in dead mice and guinea pigs
7 wks.
310
BULLETIN No. 161
TABLE 7. Continued
[November,
Author
Date
Conditions
Not killed
Killed
Musehold 101
1900
Tuberculous sputum mixed with river
water:
In diffuse light, room temperature.
In the dark virulence was greater. .
Sputum in canal water:
Diff use light
162 days
162 days
131 days
211 days
211 days
197 days
Kept in dark
131 days
197 days
131 days
197 days
Sputum in sewage, room temperature
Sputa in sewage:
194 days
194 days
Kept in dark .
105 days
Canal water mixed with sputa and
garden soil kept at room tempera-
ture and received mid-day sun. ...
Same as above except exposed to
frost snow and sunshine
145 days
148 days
197 days
Same as above except kept in sewage
Same as above except calcium chlo-
rid added
150 days
Peterson 111
1900
In sputum protected from the sun the
bacilli almost unchanged morpho-
\Yz mo.
Patterson 112
1903
In butter containing 4 percent salt . .
In butter containing 5 percent salt . .
In salt flesh not killed
4 wks
3% wks.
4 days
Dixon 31
Schroeder 132
1908
1908
Examined sewage from the tubercu-
lous hospital of Philadelphia.
Guinea pigs were inoculated, but
previous to the inoculation the
sewage was heated 60 to 70 C.
from 2 to IS minutes. No tubercu-
lous guinea pigs were produced. 1 .
In butter as ordinarily salted
160 days
Mohler, Wash-
burn, Doan
1909
In butter in cold storage, pure culture
or tuberculous udder
6 mo.
and Rogers"
261 days
1 Temperature sufficient to pasteurize; most likely the organisms were killed by
heating.
EXPERIMENTAL WORK
The main purpose of this work was to determine
INTRODUCTION tne length of time that bovine tubercle bacilli live
outside the animal body where they may be scat-
tered by tuberculous farm animals, especially dairy cattle. As it was
not feasible to keep tuberculous cattle at the Station, only a part of
the work originally planned with naturally infected feces was done.
In connection with this investigation some work has been done on
tubercle bacilli of the human type and on other pathogenic an4
non-pathogenic organisms for purposes of comparison,-
1912] FATE or TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 311
General Methods of Procedure
There are three points that need special attention: the sample,
the manner of exposure, and the length of time required for life
to become extinct in the culture exposed.
It is advisable that the sample of the organism
SAMPLE undergoing test be a pure culture. In some cases
where the organism is readily differentiated from
contaminating growth, this is not so essential, as, for example, when
pathogenic organisms are mixed with non-pathogenic organisms
and the latter are readily eliminated by animal inoculation, or when
a colored organism can be readily distinguished by its color char-
acteristic. Even in these cases it is usually advisable to work with
pure cultures.
EXPOSURE Much depends upon the manner of exposure; for
OF SAMPLE instance, if exposure is to light it makes a decided
difference whether the culture is exposed directly
or in a glass container, or in the presence or in the absence of
air. A complete statement as to the manner of exposure is ab-
solutely essential to a definite interpretation of the reported re-
sults.
INDEX OF most difficult point is to determine when life
DEATH becomes extinct. This is comparatively simple
with those organisms that readily grow on culture
media, when they are exposed in pure culture. Their failure to
grow gives a ready index of their death. With tubercle bacilli it
it much more difficult to tell just when these organisms are dead.
They cannot be cultivated readily from mixed growth since it is
very difficult to prevent over-growth with contaminating organ-
isms. Another difficulty is that dead tubercle bacilli produce typi-
cal tuberculosis in test animals (see page 312). It is therefore
necessary to make animal inoculations in all- cases where mixed
tuberculosis materials are used, and from the original test animal,
especially if only a local lesion is produced, a second healthy ani-
mal must be inoculated from some of the, suspected tuberculous
material. Suitable culture media should be seeded with this
material from both the original and the secondary test animal.
When all these tests prove characteristic for tubercle bacilli, it is
evident that these germs are still living and virulent; when no in-
fection, or only a local lesion, is produced in either the original
or the secondary animal, and no growth occurs on the culture
media, the tubercle bacilli are surely dead. In cases where the
tuberculosis produced in the original inoculated animal from a
small amount of material is severe and generalized, it may be con-
312 BULLETIN No. 161 [November.
sidered that these germs are living, and this should then be verified
by cultnring on media suitable for the growth of tubercle bacilli.
LESIONS PRODUCED BY DEAD TUBERCLE BACILLI
PURPOSE OF T ne purpose of these experiments was to deter-
MENTS mine the difference in effect of dead tubercle bacilli
and of living germs when injected into guinea
pigs; also to find a means of determining when lesions were pro-
duced by living and when by dead germs. In order to determine
this three experiments were made.
For the first experiment a pure culture of the
EXPERIMENT I bovine type of tubercle bacilli was used. A heavy
emulsion of fresh culture was made in sterile
distilled water. The emulsion showed a distinctly milky appear-
ance. One cubic centimeter of this fresh emulsion was injected
subcutaneously into Guinea Pig 993. The remaining portion of
this emulsion was autoclaved at fifteen pounds pressure for ten
minutes. Two guinea pigs, Nos. 994 and 995, were each injected
subcutaneously with I cc. of the autoclaved emulsion. Guinea Pig
993 was killed 49 days later and showed generalized tuberculosis.
Guinea Pigs 994 and 995, killed respectively 85 and 50 days after
inoculation, were found to be healthy. Their weights continued
to increase from the time they were inoculated until killed, and
at no time did they show any physical signs of tuberculosis.
Before the conclusion of the first experiment a
EXPERIMENT II second was started in which an eight-day culture
of the same strain of bovine tubercle bacilli was
used. One milligram was removed and rubbed up in 4 cc. of
broth. One loopful of this emulsion was diluted with 2 cc. of
broth and injected interperitoneally into Guinea Pig 1026. The
4 cc. of broth were pla,ced in streaming steam for one hour.
Guinea Pigs 1027 and 1028 were each injected interperitoneally
with 2 cc. of this steamed emulsion. Guinea Pig 1026 showed
physical signs of tuberculosis ten days after injection. No physical
signs of tuberculosis were observed in either of the two guinea
pigs (1027 and 1028) that were inoculated with the killed culture.
Thirty-five days after inoculation Guinea Pigs 1026 and 1027 were
injected with 2 cc. of the tuberculin. Guinea Pig 1026 died from
the effect of the tuberculin after eight hours; 1027 died after
eighteen hours. Autopsy of 1026 showed severe generalized tuber-
culosis ; that of 1027 showed a few lesions in the liver and a very
slight enlargement of the inguinal lymphatics. Microscopic prep-
arations stained for tubercle bacilli revealed none of these organ-
1<)I2\ FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 313
isms. Cultures on glycerine egg and the inoculation of a second
guinea pig gave negative results. Guinea Pig 1028 was killed 41
days after inoculation, and the autopsy showed a small abcess at
the point of inoculation and several lesions in the liver. All other
organs were apparently healthy. A stained preparation revealed
several tubercle bacilli a part of which showed signs of disintegra-
tion. Glycerine egg media and a guinea pig were inoculated with
pus material from the point of inoculation and a liver abcess.
Neither of these revealed living tubercle bacilli.
A third experiment was made using the same
EXPERIMENT III strain of tubercle bacilli. An emulsion was made
in sterile 0.8 percent salt solution. The emul-
sion showed a faintly milky appearance. Three tubes of glycerine
egg were seeded with this emulsion of tubercle bacilli and Guinea
Pig 1666 was injected interperitoneally with i cc. The remaining
portion of the emulsion was divided into two parts : one was
heated at 85 C. for ten minutes, the other was heated in the
autoclave at 115 C. for ten minutes. With each of these portions
three glycerine egg slants were seeded and a guinea pig was in-
oculated interperitoneally with I cc. The unheated portion gave
excellent growth and produced severe generalized tuberculosis in
Guinea Pig 1666. The part heated to 85 C. for ten minutes gave
no growth on either of the three glycerine egg tubes after six
weeks incubation at 38 C. Guinea Pig 1167, killed 67 days after
inoculation, showed local tuberculosis in the right superior inguinal
near the point of inoculation and a few small lesions in the liver.
All other organs were apparently healthy. A stained preparation
from pusl material of these lesions showed tubercle bacilli. Cul-
tures on glycerine egg and the inoculation of a second healthy
guinea pig from this material gave negative results. The part
heated to 115 C. for ten minutes neither gave growth on the
glycerine egg media nor produced any effect when I cc. was in-
jected interperitoneally into Guinea Pig 1168.
i. Dead cultures, when not killed at too high a
CONCLUSIONS temperature, produce tuberculous lesions in guinea
pigs.
2. Secondary guinea pigs inoculated from tuberculous material
from lesions produced by dead cultures always remain healthy.
3. In determining the length of time tubercle bacilli live when
exposed to various conditions outside the animal body, it is nec-
essary to inoculate a second healthy guinea pig, especially when
only local lesions are produced in the guinea pig inoculated with
the original material, in order to be sure the tubercles are not
produced by dead tubercle bacilli.
314 BULLETIN No. 161 [November,
KI'I-'KCT op LIGHT UPON SPORK- AND NoNSPOKB-BfiARii^e
ORGANISMS
A few spore-bearing and nonspore-bearing or-
CULTURE organisms were tested by exposing them to the
direct rays of the sun. The spore-bearing organ-
isms, B. subtilis and B. mesentericus vulgatus, were grown upon
peptoneless agar (made with three grams of beef extract and fif-
teen grams of agar per liter) for six days at 32 C. An examina-
tion of the cultures showed an abundance of well-developed spores.
The vegetative cells of these two organisms were obtained by re-
peated growths in beef broth. The broth cultures were kept at
37 C. and repeated every twenty-four hours for six days. A
microscopic examination at the end of this time showed no spores.
B. diphtheria cultures were obtained from twenty-four hours'
growth upon Loeffler's blood serum. All other organisms used were
from fresh broth cultures grown at the optimum temperature for
the organism tested.
MANNER OF ^^ e cu l tlires grown on the solid media were sus-
EXPOSURE pended in 0.8 percent salt solution. A concen-
tration of slightly milky appearing emulsion was
formed. Then a loop of this emulsion was spread in a very thin
smear on either a small slip of sterile glazed paper or a small sterile
glass cover. These were then exposed to the direct rays of the
sun for definite periods of time between the hours of ten in the
morning and three in the afternoon. The exposed slips, with the
exception of those containing B. diphtheria, were aseptically drop-
ped into sterile broth and incubated at a temperature of 30 C. for
one week. The exposed smears of B. diphtheria were seeded on
Loeffler's blood serum and incubated at 37 C. for one week. The
cultures were carefully examined for growth, and if growth was
evident further tests were made to see if it was the same as the
original culture or an accidental contamination.
__..._ A summary of the results is given in Table 8.
It will be noted that the nonspore-bearing organ-
isms were all killed in a few minutes. ( l /2 to 6
minutes). While the spore-bearing organisms were not killed in
the time exposed (180 minutes).
1912] FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 315
TABLE 8. ORGANISMS EXPOSED TO DIRECT SUNLIGHT
Name of organism
Not killed
Killed
B.
120 min.
B.
180 min.
B.
mesentericus vulgatus, spores 1
180 min.
B.
subtilis, vegetative cells
/^ min.
1 min.
B.
mesentericus, vegetative cells
1 niiii.
2 min.
B.
/^ min.
B.
6 min.
after 6 min.
B.
2 min.
3 min.
B.
ty phosus
1 min.
2 min.
B.
violaceus
2 min.
3 min .
'Time of exposure was not continued longer; no end point was reached.
EFFECT OF DIRECT SUNLIGHT UPON TUBERCLE BACILLI
CULTURE AND f direct sunlight was tested several
EMULSION times upon cultures from three types of tubercle
bacilli. The organisms used for exposure were
always from active cultures grown upon glycerine egg from two to
three weeks. A heavy emulsion was made by rubbing up some of
the culture on the inside of the neck of a sterile glass-stoppered
graduate flask with a sterile glass rod. From time to time a few
drops of sterile 0.8 percent salt solution were added. At first
the culture was rubbed into a fine paste with the addition of only
a few drops of the salt solution ; then about 6 cc. of 0.8 percent
salt solution were added and the contents shaken thoroly. The
emulsion then showed a decidedly milky appearance and was
filtered thru sterile glass wool to remove the larger clumps. The
examination of a stained preparation, made in a similar way as
the smears that were used for exposure to sunlight, showed the in-
dividual organisms usually well separated. There were, however,
clumps of twenty-five to thirty organisms still to be found.
PREPARATION Smears from the prepared emulsions were made
AND EXPOSURE U p O n small slips of sterile glazed paper. Pins
OF SMEARS F i , ., ,, F r ,, & ..
pushed thru the corners of these small paper slips
were then stuck into the bottom of a pasteboard box having a snug
fitting lid. The box and the slips were sterilized at 150 C. for
one hour. A small loop f til of the emulsion was smeared in a thin
layer upon each, of the sterile glazed paper slips. These were ex-
posed at once to the sun for the desired length of time. Triplicate
smeared slips for each period of exposure were seeded upon culture
media suitable for the growth of tubercle bacilli. The media iised
was 5 percent glycerine beef-juice agar or glycerine egg.
316
BULLETIN No. 161
[November,
The glycerine egg was prepared by opening, aseptically, ten
good fresh eggs and pouring the whole contents into a sterile flask.
To the well-beaten eggs there were added 200 cc. of 5 percent gly-
cerine beef broth. This emulsion was placed in sterile test tubes
by means of a sterile Pasteur's bulb pipette. It was solidified, in
a slanting position, at 73 C. for two hours. After placing the
exposed slip on the surface of the media, it was rubbed and scraped
by means of a small sterile platinum spatula so as to dislodge some
of the dried tubercle bacilli, and a small amount of the glycerine
egg or glycerine agar was smeared over this slip until it was
thoroly moistened. The cotton stoppers were flamed, dipped into
paraffine and pushed into the tubes, which were then stopped with
paraffined stoppers. The cultures were incubated at 38 C. for one
month and then carefully examined for growth.
Table 9 indicates the method of recording the re-
RESULTS suits for the separate tests. A summary of the
results of the various tests from the three types
of cultures is given in Table 10. It is seen that these germs are
killed in a very short time (i to 4 minutes). These results agree
with those of Weinzirl (see Table 2).
TABLE 9. BACILLUS TUBERCULOSIS, HUMAN TYPE EXPOSED TO SUNSHINE
(Between 11 and 12 A. M., Nov. 2, 1911)
Time of
exposure,
minutes
Conditions
Result of
growth
Remarks
%
%
Y*
i
i
2
2
2
4
4
4
6
6
6
10
10
10
C<
S
V
jntrols
II
in bri
w clc
i
<
5, not
(i
F ht> n
uds n
sxpos
< i
o cloi
ar tl
ed
+
+
+
f?
+
Excelh
it
ii
Contarr
Excelle
Modera
No gro
Con tat
:nt growth
< i
ination
nt growth
te growth
wth
ination mould
ads
?
le horizon
i
< <i
i<)i2\ FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 317
TABLE 10 SUMMARY ot? THE RESULTS, FROM EXPOSING TUBERCLE BACILLI
TO DIRECT SUNSHINE
Name of culture
Not killed,
minutes
Killed,
minutes
B.
tuberculosis, human
1
2
B.
1
2
B.
tuberculosis, avian
2
4
OF DESICCATION UPON BACTERIA
PURPOSE ^ le P ur P ses f this work was to compare theef-
OF WORK feet of desiccation upon tubercle bacilli with that
upon some nonacid-fast bacteria. Two types of
tubercle bacilli were used, human and bovine. The nonacid-fast
organisms used were two pathogenic organisms, B. typhosus and
B. diphtheria, and of the nonpathogenic germs sporebearing and
nonspore-bearing were used (see list in Table n).
CULTURES AND ^ e cu l tures were grown and the emulsions made
EMULSIONS in the same way as described in the section on the
effect of light upon bacteria. Only fresh, active
cultures were used. The tubercle bacilli were grown only upon
glycerine egg.
MANNER OF ^he manner of preparing the organisms for ex-
EXPOSURE posure was also the same as that described in the
last section, with the exception that the smears
were made only upon sterile glazed paper slips. These smeared
slips were exposed in a dark sheet-iron box that was well ventilated.
Control slips of all the organisms tested were seeded a few minutes
after the visible moisture had disappeared from the paper slips.
Thereafter duplicate slips were seeded at the end of 12 hours and
i, 2, 4, 8, 12, and 16 days. The tubercle bacilli were cultivated
upon glycerine egg at 38 C. for six weeks. In examining the cul-
tures for growth of these germs it is necessary to scrape the surface
and make stained preparations even when visible growth is not
present, for in practically every case there will be one or two cul-
tures between the evidently visible growth and the culture proven
to contain no growth by the' absence of organisms in stained pre-
parations, in which the cultures will have only a few, usually in-
visible, colonies. This condition was not noted in the cultures
from smears exposed to the sun.
The smears of B. diphtheria were cultivated upon Loeffler's
blood serum at 37 C., and those of all other organisms were placed
in broth and cultivated at the optimum temperature for each species.
318
BULLETIN No. 161
[November
RESULTS
The results are given in Table i T . These are the
average results from two testings of all organisms
used. Some other tests were made, but the re-
sults are only slightly different from the average of these two
complete series. In each of these two series the organisms were
all tested at the same time and under the same conditions, with
the exception that the cultures were grown upon different media,
as noted above. This may have made some difference in the
results. The tubercle bacilli were grown upon glycerine egg media
for approximately three weeks, while B. violaceus, for instance,
was grown in broth for 24 hours at 30^ C. The tubercle bacilli
were emulsified in 0.8 percent salt solution and transferred to the
sterile paper slips, while the B. liolaceus were transferred directly
from the broth to the slips. It may be that at least part of the
difference in the length of time required to kill these organisms
by desiccation is due to this difference of growth and age of the
cultures.
It is seen that a very little longer time is required to kill the
tubercle bacilli than other nonspore-bearing organism; and this
slight difference may readily be produced by the protection of the
glycerine clinging to these organisms from the culture media, or
more likely the protection offered by the presence of clumps of
tubercle bacilli. It is almost impossible to eliminate clumps from
an emulsion of tubercle bacilli, while other organisms repeatedly
grown in broth may be well separated.
The tubercle bacilli, therefore, cannot be put in the class of
bacteria with spores, and they are very near to nonspore-bearing
organisms, or the vegetative cells of spore-bearings organisms, as
regards the effect of desiccation. They should be classed with the
more resistant of the nonspore-bearers.
TABLE 11. EFFECT OF DESICCATION UPON BACTERIA
Organism
Not killed,
days
Killed,
days
B.
tuberculosis, human
4
8
B.
tuberculosis, bovine
8
12
B.
diphtheria
4
5
B.
typhosus
3
5
B
subtilis, spores
35
B.
subtilis, vegetative cells
1
2
B.
35
B.
vulgatus, vegetative cells
2
3
B.
coli
3
4
B.
violaceus
1
2
B.
prodi"iosus
1
9
Sar
lutea
4
5
Sar.
4
5
I9i-?] FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 319
DURATION of LIFE) OF BOVINE TUBERCLE BACILLI IN Cow
MANURE
PURPOSES OF T ne general purposes of these experiments was to
MENTS determine how long bovine tubercle bacilli will
live in cow manure when exposed to weather con-
ditions in a pasture field. Two series of tests have been made con-
cerning the resistance of these organisms when a! pure culture is
artificially mixed in cow manure exposed in the sunshine, and also
when it is exposed in a place protected from the sunshine. In a
similar way other tests have been made with naturally infected
manure from a tuberculous cow.
Series i
(Special Methods)
PREPARATION Artificially Infected Manure. The culture used
OF SAMPLES in preparing this sample of artificially infected
manure was a strain of bovine tubercle bacilli ob-
tained from Dr. Theobald Smith of the Harvard Medical School.
It was received and has since been kept upon glycerine agar. The
strain readily produces an abundant growth and is quite virulent
to test animals. *
Four milligrams of the culture from two tubes of glycerine
agar were removed and emulsified as described on page 315. The
emulsion was diluted to 200 cc. with sterile salt solution. It was
expected that the large amount of solution used would give a
more uniform mixture of the tubercle bacilli in the cow manure.
The sample of manure was obtained from two cows of the
dairy herd. It was taken by the method described in Bulletin 149
of this station; i. e., by injecting air into the rectum of the cow
until stimulated to defecate. The feces were caught in a sterile
pail and at once covered and brought to the laboratory. The 200
cc. emulsion of bovine tubercle bacilli was thoroly mixed with
1800 grams of the fresh cow manure. The infected manure was
tested for virulence by inoculation of two guinea pigs. One gram