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were found to have undergone considerable growth, the fluid being milky in appearance. The typhoid
tube remained clear. A platinum loop was dipped into each tube and cultures made on nutrient
gelatine; the two B. coli tubes yielded very numerous colonies, the typhoid tube yielded only one
colony after a long interval.
From the three sewage tubes, after the lapse of two days, three fresh sewage tubes were
inoculated and incubated at 37° C. The typhoid tube was sterile; the B. coli tubes grew well,
and from them third and subsequently fourth generations in sterile sewage were carried on. The
fourth generation, three weeks after the commencement of the experiment, yielded very numerous
colonies of B. coli communis when tested by inoculation on gelatine. It was also ascertained that
after growing for four days in sewage, the second generation of B. coli communis still retained its
powers of coagulating milk, of forming gas bubbles on gelatine, and producing indol in broth.
It thus appears that the harmless colon bacillus is able to grow and multiply abundantly in
sewage sterilised by heat, when incubated at 37° C, and that it may be so cultivated for several
generations ; the typhoid bacillus, on the contrary, is unable to grow, and quickly perishes under these
conditions. This constitutes a new and important distinction between these allied organisms.
It remained to test more accurately the duration of life of the typhoid bacillus in sterilised
sewage, kept at 37° C.
Experiment III.—A tube of sewage sterilised by heat was inoculated with three drops of a
broth culture of the typhoid bacillus (four days old). The cultivation was tested at intervals by
removing a platinum loopful and spreading it over the surface of a gelatine tube. A sub-culture thus
made immediately after its inoculation with the typhoid bacillus yielded more than 100 typhoid
colonies. After the lapse of 21 f hours a sub-culture yielded 15 colonies. After 50 hours only one
colony was obtained, and after 73f hours none. The typhoid bacillus thus appears to die speedily
when cultivated in sewage sterilised by heat and incubated at 37° C. death occurring within a few days.
A temperature of 37°0. is not, however, a normal condition in sewage, and we next attempted
to determine the duration of vitality of the typhoid bacillus in sewage kept at the ordinary temperature
of the air—20° C. The two following experiments show that at this temperature life is maintained for
a longer period, though probably not beyond a fortnight.
Experiment IV.—A tube of sewage sterilised by heat was inoculated with the typhoid bacillus
as in the preceding experiment, and incubated at 20° C. It was tested in the same way by sub-cultures
on gelatine at suitable intervals, a control sub-culture being made at the outset, immediately after
inoculation. After 24 hours a slight increase in the number of colonies was noticed, as if slight
multiplication had occurred. The diminution in the number of colonies was thenceforward steady,
till at the end of ten days a large capillary pipetteful of the sewage yielded only 30 or 40 colonies, and
in 13 days none at all were found.
Experiment V.—A tube of sewage sterilised by heat was inoculated with one drop of a fresh
broth culture of B. typhosus, and incubated at 20° C. A small quantity was removed on a platinum
loop and transferred to gelatine immediately after inoculation to determine the number of colonies
originally present, and the process was repeated at stated intervals. The results were as follows:—
Immediately after inoculation 225 colonies arose
Alter 24 hours 250 „ „
„ 68 „ 140 „
5 days 48
7 ,, 13 „ ,,
13 „ 0 „
It is to be observed that the removal of so small a quantity of fluid as can be conveyed on a
platinum loop is no fair test of the total absence of living bacilli from a culture, and it is quite possible
that the removal of larger quantities might have shown living bacilli to be present after 13 days.
Similar, or approximately similar, amounts would nevertheless be removed by a platinum loop on each
occasion, so that the experiment clearly shows that although an attempt at growth may occur during
the first 24 hours, the typhoid bacillus gradually but surely dies out in sewage, and is quite incapable
of any active growth therein.
Sewage, therefore, even in the absence of the normal micro-organisms which it contains is
clearly an unfavourable medium for the growth of the typhoid germ, whereas the colon bacillus can
grow and multiply freely in it. It might be anticipated that in competition with other organisms able
to grow well in sewage, the typhoid bacillus would die out even more speedily. The problem is a
complex one, and in order to gain some idea of the influence which v arious non-pathogenic bacteria
normally present in sewage would exert upon the life and growth of the typhoid bacillus when growing
side by side with it, we selected certain of the organisms which we had frequently found during the
progress of our observations, and experimentally tested their effect on the duration of vitality of the
typhoid bacillus. In order to simplify the problem we confined our attention to those organisms
which are incapable of growing at a temperature of 37° C., by which means it became easy to separate
out the typhoid bacilli from any other organism or organisms to which it had been added, and to
determine the number still alive after any given interval. When the agar-agar plate cultivations
were incubated at 37° C., only the typhoid bacilli could grow. The organisms we selected were all
capable of growing freely in sterile sewage at the ordinary temperature of the air, and were as
follows—Bacillus fluorescens liquefaciens, bacillus albus putidus, and two of the species which we have
described above as bacillus fluorescens stercoralis and bacillus cloaca; fluorescens. In the first two
experiments the sewage was sterilised by boiling.
Experiment VI.—The typhoid bacillus grown in sterile sewage with bacillus fluorescens stercoralis
and B. albus putidus respectively.
To two tubes each containing 10 c.c. of sterilised sewage, three drops of a fresh typhoid broth
culture were added. To one of these tubes three drops of a fresh culture of B. fluorescens stercoralis, and