2. Transfer the emulsion to a sterile test-tube and heat in the differential steriliser for ten minutes at 80° 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 fluid, and transfer the suspension to a litre flask. 4. Make up to a litre by the addition of sterile water; filter the suspension through a sterile porcelain candle. 5. Emulsify the bacterial residue with 5 c.c. sterile bouillon. 6. Prepare the necessary subcultivations from this emulsion. PATHOGENESIS. _Living Bacteria._-- (a) Psychrophilic Bacteria: When the organism will only grow at or below 18° to 20° C., 1. Prepare cultivations in nutrient broth and incubate under optimum conditions.