452 GEO. T. HARGITT AND WALTER W. FRAY 



and sometimes worse than the mixed cultures. A mixture of 

 different kinds of bacteria, therefore, seems essential as a diet 

 for Paramecium grown in depression sUdes; probably the same 

 thing is true for large cultures. 



. SUGGESTIONS 



From the results obtained in this study it is seen that certain 

 precautions are demanded of those who carry on pedigree cul- 

 tures of Paramecium or other bacteria-eating Protozoa. Some 

 of these are here suggested: 



1. Whenever constancy in the matter of food is desired the 

 pipettes used must be sterile. This is not obtained by the inser- 

 tion of the pipette in bojling water for the spores of some bac- 

 teria are resistant to this temperature. The pipette should be 

 sterilized within a closed vessel placed in a hot air sterilizer, or 

 by insertion in the flame of a burner. The pipette must be 

 sterihzed each time before being used. 



2. Before starting cultures the protozoa should be sterilized. 

 Bacteriological tests show this maj^ be efficiently accomplished 

 with Paramecium by washing through five or six sterile fluids in 

 sterile depression sHdes enclosed within sterile Petri-dishes. 

 Transfer is to made each time with a sterile pipette. 



3. Where different cultures are to be critically compared all 

 the protozoa, after having- been sterihzed, should be kept in 

 identical culture fluids with identical bacteria present. So far 

 as the present data goes it suggests the inoculation of the cul- 

 ture medium from a normal hay infusion or by chance infection 

 from the air as preferable to the use of pure cultures of bacteria. 

 But the food must be identical and this can only be the case 

 when a single fluid is inoculated and some of this is added to 

 the slides in which the sterile paramecia are to grow. 



4. If the demands for uniformity of food are very strict the 

 shde cultures should be kept in a sterile moist atmosphere, pro- 

 tected from the contamination of air bacteria. This condition 

 may easily be met by placing sterile water in sterile Petri-dishes. 

 Transfers of animals or addition of fluid, and the hke, should be 



