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to the other a similar amount of a fresh culture of B. albus putidus were further added. The contents of
each tube were well mixed, and they were allowed to remain at the temperature of the room screened
from the action of direct sunlight. At the outset of the experiment, and at given intervals, the number
of typhoid bacilli still living was determined, by making agar-agar plate cultivations with so much of
the sewage as was removed on the end of a platinum loop, and the plates were incubated at 37° C.
The following results were obtained-
Typhoid + B. fluorescein
stercoralis.
Typhoid + B.
albus putidus.
No. of colonies in original mixture 630 590
After 24 hours 148 570
After 76 hours 0 156
After 4 days — 141
After 5 days — 134
After 11 days — 24
Experiment VII.—This experiment was in every detail similar to the preceding, but was carried
out with B. fluorescens liquefaciens, bacillus cloacae fluoresceins and again with bacillus fluorescens
stercoralis, three drops of fresh cultures of each respectively being added to the sterile sewage at the
same time that it was inoculated with the typhoid bacillus.
The results were as follows—
Typhoid + B.
fluorescens
stercoralis.
Typhoid +
B. fluor.
liquefaciens.
Typhoid +
B. cloacae
fluorescens.
No. of colonies in original mixture 1,030 1,769 935
After 24 hours 1 949 946
After 3 days 0 1,010 235
After 4 days — 20 —
After 7 days — 0 —
After 10 days (.5 c.c.) 135 18
It will be seen from these experiments that B. fluorescens stercoralis and B. fluorescens
liquefaciens both exert a marked influence upon the duration of vitality of the typhoid bacillus in
sewage, particularly the former. In no instance is there evidence of any increase in the number of
typhoid bacilli. As no typhoid colonies arose after the third day in the case of B. fluorescens
stercoralis, and after the 7th day in the case of B. fluorescens liquefaciens, it was desirable to know
whether the typhoid bacilli had been completely destroyed or whether they had merely been so reduced
in number as to escape detection when only a minute quantity of the mixture was used for making
the plate cultivations. On making a plate culture with half a cubic centimetre of the mixture 135
colonies of the typhoid bacillus were obtained even after 10 days' exposure to the influence of B.
fluorescens liquefaciens. The above figures must, therefore, be regarded as aflording merely an indication
of the rate at which the typhoid bacilli perished, and not as fixing definitely the date of their final
destruction.
We determined to test this matter on a more extended scale, and in a more accurate manner.
Therefore in the following experiment several slight modifications and improvements were introduced.
The sewage was sterilised by filtration instead of by boiling. Larger quantities of sewage were
employed for cultivation, and much greater precision was aimed at by measuring accurately the
quantities of typhoid and other cultures used, and by employing for each agar-agar plate a carefully
measured amount of the mixtures.
Experiment VIII.—Six small wide mouthed flasks, each containing 2½ ounces of sterile sewage,
were inoculated each with 0.5 c.c. of the same broth culture of the typhoid bacillus. Four of these
flasks then received 0.2 c.c. respectively of fresh broth cultures of the following organisms:—B.
fluorescens stercoralis, B. fluorescens liquefaciens, B. albus putidus and B. cloacœ fluorescens. A fifth
flask received 0.2 c.c. of cultures of all four organisms—0.8 c.c. in all. The sixth flask, which was used
as the standard, received only the typhoid bacilli. The flasks were agitated so as intimately to mix their
contents and then kept in the dark at the temperature of the room. The number of typhoid bacilli per
cubic centimetre was determined in each flask, from time to time, by abstracting a measured quantity
of the fluid (from 1/10 c.c. to 1 c.c.) and diluting it with sterilised distilled water to a given volume, and
then making agar-agar plate cultures with 1 c.c. of the diluted material. The degree of dilution was
diminished pari passu with the decrease in the number of the typhoid bacilli present, until it was
possible to take relatively large quantities of the undiluted mixtures. The agar-agar plates were
incubated at 37° C. As all the six flasks were inoculated with an exactly similar quantity of the same
typhoid broth culture which had previously been thoroughly mixed, the number of bacilli per cubic
centimetre in the flask containing typhoid bacilli alone was regarded as representing the average for
the whole series. The results which we obtained are expressed in the following table—the number of
bacilli per c.c. being calculated from the actual numbers observed.
Time of
experiment.
Typhoid alone
No. of colonies
per c.c.
Typhoid + B.
fluorescens
stercoralis.
Typhoid + B.
fluorescens
liquefaciens.
Typhoid+B.
albus
putidus.
Typhoid + B.
cloacæ
fluorescens
Typhoid+
Mixture of all
four species.
At starting 1,048,000 — — — — —
1 day after 1,838,000 — 230,000 — — —
3 days 506,000 107,000 80,000 531,000 839,000 228,000
5 „ „ 3,000 800* 45,200 340,000 825,000 41,800
7 „ — 616* 26.400 3,000 78,000 27,800
10 „ „ 0 502* 0 0 0 0
* 0.5 c.c. of the undiluted mixture employed for each plate.