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remain in the 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 kills off the 
organisms present. Consequently, 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 Io c.c. of sterile water (tube 1) 
and well stirred round ; with the same loop a loopful of this 
dilution is carried into another 10 c.c. of sterile water (tube 2), 
and so on to a third (tube 3). A loopful from tubes 2 and 3 
is 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 Appendix 
C. 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 organisms, 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 put the plates into the incubator upside down for 
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) 1s placed in the centre of these plates 
and wiped over the entire surface. The plates are incubated 
at a temperature of 37°C 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 
