450 CONTROL OF CULTURES 



can be determined only by experimentation), then cultures may be estab- 

 lished by suspending yeast cells in spring water, distilled water, or bal- 

 anced salt solutions, and introducing the desired ciliate. Often abundant 

 growth will result and after a few subcultures have been made, the ratio 

 of bacteria to Protozoa will be reduced. When serious attempts at sterili- 

 zation are then carried out, the yeast cells will be found relatively easy 

 to eliminate. It should not be supposed that it will be possible to elimi- 

 nate the contaminating bacteria in this way, as they will be multiplying 

 slowly all the while. In fact, long-continued cultures of this type are apt 

 to show a decided increase in non-nutritive bacteria over those which 

 were present at the start (see Kidder and Stuart, 1939). Therefore it 

 is advisable to make from one to three subcultures only, and then to start 

 the sterilization procedure. 



In general it can be said that the larger the protozoan the more diffi- 

 cult it will be to sterilize. This fact becomes apparent from an examina- 

 tion of the literature, and was noted by Hetherington (1934). Physical 

 properties likewise play a role in ease of sterilization. Holotrichous and 

 heterotrichous ciliates, possessing large numbers of closely set cilia, are 

 apt to retain a few of their associated bacteria even after repeated wash- 

 ing, while hypotrichous ciliates may be washed free of bacteria more 

 readily. Flagellates, being for the most part smooth in surface, are rela- 

 tively easy to sterilize. Activity is also important, both as to movement 

 and metabolism. Highly motile forms may usually be freed of bacteria 

 more readily than sluggish types. Those possessing a high rate of me- 

 tabolism tend to utilize or defecate the contents of their food vacuoles 

 more rapidly than the slow-growing types, and do not tend to carry over 

 viable spores to contaminate later cultures. Many other characters which 

 will influence the facility with which sterilization may be accomplished 

 might be mentioned, but these will become apparent when we examine 

 some of the procedures. 



2. GENERAL METHODS OF STERILIZATION 



In order to rid the Protozoa of their associated bacteria, workers have 

 made use of three principles. The first and most generally useful is simple 

 washing in sterile fluid. This is the dilution method whereby the bacteria 

 are diluted out of the solution. The Protozoa must be retained of course, 

 and a number of different manipulations have been devised to insure this. 



