6
up to 80° for 20 minutes, and a plug of hot vaseline is run on to the surface of the milk
so as to render the conditions of growth anaerobic. The tubes are incubated at 37° for
48 hours. When sporogenes spores are present the characteristic gas formation and
clotting of the milk result.
(3) Bile salt broth.--On the recommendation of Dr. MacConkey we gave up
the glucose broth for a lactose broth, so that we are really dealing with only one group
of intestinal organisms, namely, the lactose fermenters. Varying quantities of the
water are put into these tubes. One tube receives 20 cc. of the water ; 2, 10 cc. ; 4.
5 cc. ; .5, 1 cc. ; 5, .1. cc. ; and 5, 01 cc. (for these first three tubes a concentrated
broth is used, so that the dilution, after the addition of the water, is kept approximately
equal). It will be observed that thefirst three series each make up to 20 cc., so that.,
if there is only one tube in the first seven of these which gives the reaction, it means
that faecal bacilli. are present only in 60 cc. (i.e., 3 twenties). This arrangement of
obtaining a result in 60 cc. has obvious advantages. It gives considerable information
as to the number of these organisms present i.n this quantity of water. In the latter
three groups it is of course impossible to keep the total quantity of water used the
same. Therefore, for purposes of recording results, we consider that there are fecal
bacilli. in 1 cc., if 3 out of . the 5 tubes give the reaction. Precisely the same applies
to the 1. cc. and the 01 cc. groups. This number of tubes of bile salt broth has given
very satisfactory results. It is economical on the whole, and it certainly gi.ves sufficient
detail of the quantity of fcal bacilli present in any given sample. The total number
of tube per sample used is 22, and we do not consider that it would be advisable to cut
this down to any great extent. If, however, saving of media is of great importance,
3 tubes to each of which 5 cc., 1 cc., 1 cc., and 0cc. of water be added, might be
used ; and if 2 out of 3 in a group gave the reaction, faecal bacilli are might be consi-
dered as present in that group. Only tubes that give acid and gas are counted as
giving the reaction. The bile salt broth tubes are incubated at 42° C., but 37° does
nearly as well.
The next step is the separation of the Various bacilli that have grown in the bile
salt broth, and. their identification by passing them through the sugars. The method of
doing this is as follows, and it is necessary to carry out these details with considerable
care, otherwise great variety in the results will be obtained, owing to the possibility
of causing too great acidity in the bile salt medium. Many of the bile salt
broth tubes that have had water added, will show acid and gas after about 18 or 24
hours' incubation. For sub-culturing the bacilli present a tube containing a large
quantity of the water is taken. In actual practice we usually take one tube containing
either 5 or 1.0 cc. of the water, 18 hours after it has been inoculated. If tubes which
have received smaller quantities of water are taken for this purpose, there is .a danger
that perhaps a single bacilli may have been added to the tube, and i.n that case a
pure culture of this bac fæca is obtained, this cannot represent the bacteriological flora
of the water as a whole, whereas the growth from 10 cc. is much more likely to do so.
Further, if the tube selected for sub-culture is allowed to remain in incubator more
than about 24 hours (which is always sufficiently long provided the bacilli in the
quantity taken are fairly numerous) the increasing acidity of the medium due to the
fermentation of the lactose retards the growth of certain organisms present. Conse-
quently, it is better to begin the work of sub-culturing as soon as the reaction has
declared itself, so as to keep the numerical relation of the various bacilli, originally
present in the water, as little altered as possible.
Having selected the tube for the sub-culture of the organism, one loopful of the
bile salt broth is taken out from the tube and put straight into 10 cc. of sterile water
(tube 1) and well stirred round ; with the same loop a loopful of this dilution is carried
into another 10 cc. of sterile water (tube 2), and so on to a third (tube 3). A loopful
from tubes 2 and 3 are put on the centre of a plate containing solid medium and
. spread out with a sterile glass rod.
The solid medium that is used is a bile salt, neutral red, lactose, agar. The
formula for this will be found in the appendix. This medium gives most excellent
results. The presence of the bile salt and the neutral red both tend to retard the
growth of moulds and air organism, so that it does not rapidly go bad in a warm
climate. The medium is also particularly easy to make. It is stored in the laboratory
in test tubes, melted and poured into plates 24 hours before use. It is necessary to