CONTROL OF CULTURES 451 



The principle of dilution takes for granted that the bacteria either are 

 suspended in the fluid or that they may be caused to become suspended. 



The second principle is one of migration. The Protozoa to be sterilized 

 are allowed or caused to swim through sterile fluid or semisolid medium 

 or over the surface of solid medium, leaving the bacteria behind. This 

 method has been used with success on a number of different types of 

 ciliates and flagellates and a few amoebae. The Protozoa may be induced 

 to migrate laterally by introducing them into one side of a flat dish of 

 sterile fluid. Or, if they happen to be negatively geotropic, they may 

 be introduced into the bottom of a vessel of sterile fluid and taken off 

 at the top. Those that are positively geotropic may be introduced at the 

 top and taken off at the bottom. Extremely active types may be able to 

 migrate through a semisolid medium and literally scrape off their adher- 

 ing bacteria. 



Combinations of the above two principles have been used with marked 

 success, and a number of ingenious pieces of apparatus have been de- 

 signed to facilitate the manipulations and to reduce the chance of ex- 

 traneous contamination. These will be described in detail later. 



The third principle that has been applied to this problem is that of 

 bactericidal agents. This method has met with questionable success and 

 then usually only in cases where resistant phases (cysts) could be ob- 

 tained. As might be expected, any agent which would kill the bacteria 

 in a culture would most surely kill trophic Protozoa. It has been shown 

 many times that the various species of Protozoa are much more suscep- 

 tible to the usual toxic agents than many of the common bacteria. (An 

 exception to the foregoing statement is indicated in the work of Brown, 

 et al., 1933, using X-rays as a sterilizing agent.) 



3. SPECIAL METHODS AND MANIPULATIONS 



This section will be devoted to a description of the procedures which 

 have been used by various investigators to rid the different types of Proto- 

 zoa of their associated bacteria. Considerable pains will be taken to de- 

 scribe the apparatus used, the manipulations performed, and the results 

 obtained. It is hoped that by so doing the reader will be able to gain 

 constructive ideas which will allow him either to utilize one of the de- 

 scribed methods or to formulate a modification which will meet his needs. 



A. Dilution. — One of the first reports of the sterilization of Protozoa 



