7
put the plates into the incubator upside down for the 24 hours, so as to get the surface
of the Medium nice and dry, otherwise the colonies become confluent, and the plates are
useless. As already stated. a loopful from the diluted bile salt. broth (tubes 2 and. 3)
is placed in the centre of these plates and wiped. over the entire surface. The plates-
are incubated at a' temperature. of 42° for 24 or 48 hours. The method of dilution
given above usually produces plates containing about 10 to 20 colonies per plate from
tube 3, and something between 50 and 200 from tube 2. The object being to get
a plate which does not contain more than about 20 colonies, for if this is done
successfully, it becomes a very easy matter to take either all the colonies, or half of
them, for putting through the sugars, and by this means it obviates all selection,
so reducing the " speculative venture " as Houston rightly describes this selection,
to a minimum.* The colonies present a very great variety of appearances on this
solid medium. Some are bright red ; some have a red centre with a pale rim ; others
are white, or yellow and some have a transparent look. The red colonies usually
cause a precipitation of insoluble bile acid in the solid medium producing a haze round
the colony. The appearance of any particular colony on the medium is of little or no
use for identifying the bacillus present ; very little reliance can be placed on the
naked eye appearance. Two colonies as different as it is possible for them to be in
appearance, the one bright red with haze of precipitated bile salt round it, the other
pale yellow, will on further investigation turn out to be the same organism. The
reason for this is probably due to the fact that acid and alkali may be both produced
by colonies at different times, sometimes the one predominates, and sometimes the other.
Having obtained a good plate with, say, 10 or 12 discreet colonies on it, each
separate colony is now put through the various sugar reactions. If the colonies are
small, and are crowded together, or if, as sometimes happens, instead of getting a plate
containing 10 colonies, it contains 100, it is advisable to pick off 10 or 12 colonies, while
they are small, and inoculate agar slope tubes from each one. In doing this care
should. be taken not to select 10 colonies from all over the plate, otherwise the
tendencies to get them all of one kind is natural, and the " speculative venture " factor
comes in, but it is better to mark off a segment of the plate and take all out of the part
marked. If, on the other hand, the plates have been successful, and colonies are
separate, it is possible to inoculate the six tubes from each colony, without passing
through the stage of growing it separately on agar. If this latter method is going to
be carried out the first thing to do is to number the colonies on the back of the .plate
with a pencil, otherwise confusion will occur. Six separate tubes containing the various
materials are inoculated from each colony. The safest and quickest method of carrying
out this operation is to scoop up the whole colony with a loop, put the growth in a
small test tube (3" by ½") containing a few drops of sterile water, mix well, and place
six sterile iron wire loops in the mixture—one wire is used to inoculate each of the
six tubes containing the sugars.
The sugars that are recommended by MacConkey are saccharose, dulcit, adonit,
inulin. Two other tubes are also inoculated, the one a glucose tube for the Voges and
Proskauer reaction, and a broth tube for the indol reaction and for motility.
The four sugar media are made up with sugar free peptone, bile salt, and neutral red,
according to MacConkey's formula. The glucose tube contains neither neutral red nor
bile salt. These tubes are placed after inoculation into one rack and are incubated for
48 hours at 42°. The broth tube however is examined for motility after 18 hours'
growth of the organism. The reaction in the sugars is considered to have taken
place if gas and acid are developed. Gas is not always. very apparent. The
Voges and Proskauer reaction is completed by adding a few drops of a very concen-
trated solution of caustic potash after 48 hours' (or more) incubation. The red
colouration nearly always appears within an hour, but in some cases it is delayed.
The tubes are however kept for 24 hours after the caustic potash has been added.
The new indol reaction with benzaldehyd † is used. The method of recording the
results of these changes in the sugars will be seen from the tables given of the various
a nalyses, all tubes that give a definite reaction are marked as † those that fail — ; it is
* Houston says : "The picking out, of coli-like colonies for studying in pure culture is after all, even to the
expert, a speculative venture."
† Persulphate of potash and paramdiethyl-amido-benzaldehyd.