"test" organisms upon nutrient
agar in a culture bottle, and incubate under optimum conditions, for
three days, for the formation of their spores.

2. Transfer the emulsion to a sterile test-tube and heat in the
differential steriliser for ten minutes at 80 deg. C. to destroy all
vegetative forms.

3. Employing that percentage solution of the disinfectant determined in
the previous experiment, and complete the investigations as detailed
therein, steps 7 to 14, increasing the interval between planting the
subcultivations to two, three, or five hours if considered advisable.

NOTE.--Where it is necessary to leave the organisms in
contact with a strong solution of the disinfectant for
lengthy periods, some means must be adopted to remove every
trace of the disinfectant from the bacteria before
transferring them to fresh culture media; otherwise,
although not actually killed, the presence of the
disinfectant may prevent their development, and so give rise
to an erroneous conclusion. Consequently it is essential in
all germicidal experiments to determine first of all the
inhibition coefficient of the germicide employed. Under the
circumstances referred to above it is usually sufficient to
prepare the subcultures in such a volume of fluid nutrient
medium as would suffice to reduce the concentration of the
germicide to about one hundredth of the inhibition
percentage, assuming that the entire bulk of inoculum was
made up of that strength of germicide employed in the test.
In some cases it is a simple matter to neutralise the
germicide and render it inert by washing the organisms in
some non-germicidal solution (such for example as ammonium
sulphide when using mercurial salts as the germicide). When,
however, it is desired to remove the last traces of
germicide proceed as follows:

1. Transfer the suspension of bacteria to sterile
centrifugal tubes; add the required amount of disinfectant,
and allow it to remain in contact with the bacteria for the
necessary period.

2. Centrifugalise thoroughly, pipette off the supernatant
fluid; fill the tube with sterile water and distribute the
deposit evenly throughout the fluid.

3. Centrifugalise again, pipette off the supernatant fluid;
fill the tube with sterile water; distribute the deposit
evenly throughout the flui