522 BACTERIOLOGY. 



proportion of 1 to 1,000,000. It is evident, therefore, that 

 in such work only one loopful should be carried over into 

 the bouillon, the volume of which should not be less than 

 10 c.c. 



7. Observation of the sub-cultures over a considerable 

 length of time. The failure of tubes to develop within 24 

 hours is not a positive indication that the organism has 

 been destroyed by the disinfectant. In the attenuated con- 

 dition the organism will grow more slowly than it would if 

 it were normal and in possession of full vitality. More- 

 over, as stated already, traces of the disinfectant which 

 are carried over in the experiment will still further tend to 

 retard the growth. For these reasons the tubes should be 

 kept under observation for at least one week before definite 

 conclusions can be drawn. 



8. Temperature at which the sub-cultures are kept. The 

 organism which has been exposed to the action of the dis- 

 infectant should be placed under conditions which are the 

 most favorable to its growth. That is to say, tlie best nu- 

 trient medium and the most suitable temperature should be 

 furnished. Transplantations made into gelatin and kept at 

 ordinary room temperature frequently fail to grow while 

 parallel bouillon and agar cultures, placed in the incubator, 

 develop. It is, therefore, desirable to make the transplan- 

 tation to the surface of inclined agar tubes or into bouillon 

 and to keep the tubes under observation at a temperature 

 of about 37 for a week or more. 



9. Negative experiments with animals inoculated with or- 

 ganisms exposed to heat, or to the action of chemicals prove 

 but little. The organism may be dead, or it may have be- 

 come attenuated and is therefore without action. In the 

 latter case it may still grow on artificial media. Thus, an- 

 thrax spores, which are exposed to the boiling temperature 



