CONTROL OF CULTURES 449 



It is the purpose of this section to devote some time to methods or 

 techniques of protozoan sterihzation, in order to bring before the reader 

 some of the many problems which must be dealt with in work of this 

 kind. As may be supposed, the nature of such a discussion makes it 

 necessary to assume at least a rudimentary knowledge of bacteriological 

 technique. And above all there must be a thorough appreciation of the 

 potentialities of many different types of bacteria to resist even the most 

 careful methods of irradication, potentialities which express themselves 

 in some cases only after prolonged periods of apparent sterility. 



In addition to an outline of techniques for sterilization, the question 

 of acceptable tests for the sterility of cultures will be considered, and 

 finally some of the problems and conditions arising from the establish- 

 ment of sterile Protozoa in culture. 



The Problem of Protozoan Sterilization 



1. general material 



Protozoa from natural waters, soil, and so forth, are, and have been 

 throughout their existence, in association with bacteria. This does not 

 mean that the bacterial flora of their surroundings has remained constant 

 either as to numbers or types. The flora is probably continually changing. 

 This very change is one of the most important factors in the succession 

 of microscopic animals in ponds and streams. The variety of bacterial 

 types one would expect to encounter in any extended survey of natural 

 ponds is practically limitless. Therefore it is impossible to do more than 

 discuss the general factors to be taken into account in dealing with bac- 

 teria. 



To attain successful sterile cultures of Protozoa, it is, of course, de- 

 sirable to have rather large numbers of healthy organisms with which to 

 work. It is usually possible to isolate single organisms into fresh infusion 

 and obtain from fair to good growth. If they are bacteria-feeders (the 

 great majority of free-living ciliates are), enough food organisms are 

 brought over in the isolation to insure, at least in a high percentage of 

 cases, against starvation. As the bacteria multiply they, in turn, are uti- 

 lized by the Protozoa. 



In dealing with ciliates from the wild, a partial substitution method 

 may be attempted. For a number of species, this method facilitates later 

 sterilization. If the ciliate to be used will feed on living yeast (and this 



