314 METHODS OF IDENTIFICATION AND STUDY 



bate 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 C. to destroy all vegetative forms. 



3 . Employing that percentage solution of the disinfec- 

 tant determined in the previous experiment, and com- 

 plete the investigations as detailed therein, steps 7 to 

 14, increasing the interval between planting the sub- 

 cultivations 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 disin- 

 fectant 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 

 gerrnicidal 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 hun- 

 dredth 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 neu- 

 tralise 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 through- 

 out 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. 



