st the formation of an acid
reaction and the production of gas in subcultures prepared from the
germ-germicide mixture is fairly complete evidence of the presence of
living B. coli.

The amount of medium present in each test-tube is a matter of
importance, since the medium not only provides pabulum for the test
germ, but also acts as a diluent to the germicide, to reduce its
strength below its inhibition coefficient. For routine work each
subculture tube contains 10 c.c. of medium, but it is obvious that if
germicide-x possesses an inhibition coefficient of 0.1 per cent. the
addition of 0.1 c.c. of a 10 per cent. solution to 10 c.c. of medium
would effectually prevent the subsequent growth of the test germ after a
contact period insufficient to destroy its vitality. Hence the
preliminary tests may in some instances indicate the necessity for the
presence of 12 c.c., 15 c.c. or more of the fluid medium in the culture
tubes.

_f. Incubation Temperature_, ~37 deg. C.~--

_g. Observation Period of the Subcultivations_, ~Seven Days.~--

In order to determine whether or no the test germs have been destroyed,
observations must always be continued--when growth appears to be
absent--up to the end of seven days before recording "no growth."

_h. Identification of the Organisms Developing in the Subcultivations
after Contact in the Germ + Germicide Solution._--

This is based on the naked eye characters of the growth in the bile salt
broth, supplemented where necessary by plating methods, further
subcultivations upon carbohydrate media and agglutination experiments.
The sign (+) is used to indicate that growth of the test organism
occurred in the subcultivations, and the sign (-) to indicate that the
test germs have been destroyed and no subsequent growth has taken place.

METHOD.--

_Apparatus Required_:

Sterile test-tubes (narrow, not exceeding 1.3 cm. diameter).

Test-tube rack (Fig. 219).

Sterile graduated pipettes in case, 1 c.c. (in tenths).

Sterile graduated pipettes in case, 5 c.c. (in c.c.).

Circular rubber washers, 2.5 cm. diameter with central hole,
sterilised by boiling immediately before use, then
transferred to sterilised glass double dish.

Electric signal clock or stop watch.

Sterile forceps.

Sterilised glass beads.

Shaking machine.

Grease pencil.

_Material Required_:

Percentage solutions of germicide-x (