CONTROL OF CULTURES 453 



water was autoclaved. They claim to have effected sterihzation in a high 

 percentage of their trials, with a total time consumption per ciliate of not 

 more than five minutes. If their bacteriological tests are accepted (de- 

 velopment of bacterial colonies on agar plates, time of incubation not 

 given) then we can only conclude that they were extremely fortunate 

 in avoiding ciliates with ingested viable spores. 



A chief criticism of the work of Hargitt and Fray was given by Par- 

 part (1928). He pointed out that most of their sterility tests were con- 

 fined to the washing fluids and not to the supposedly sterilized paramecia. 

 In reinvestigating their results, he found that five washes gave sterile 

 fluid at the end, but that even after ten washes in six out of eight trials 

 the animals themselves were contaminated. He ascribed this fact to the 

 probability of the carrying over of viable spores within the vacuoles of 

 the ciliates. He suggested a simple modification of the method of Hargitt 

 and Fray, which yielded fifty sterile paramecia out of fifty trials. Instead 

 of five washes he employed ten, thereby increasing the dilution factor. 

 Time was allowed (five hours) for the ciliates to void their vacuoles 

 of possible spores in the fifth wash. 



His method is essentially as follows : A single 'Paramecium was trans- 

 ferred with a sterile pipette from a wild culture to a sterile Petri-dish- 

 enclosed depression slide containing about six drops of washing fluid. 

 After about one minute, the animal was transferred to the next similar 

 bath. At the fifth bath the Paramecium was allowed to swim about for five 

 hours and was then carried through five further washes. All of the manipu- 

 lations were carried out under a rather elaborate hood to minimize the 

 possibility of contamination from the air. 



This modification of the simple washing method has probably been 

 more generally used than any other, owing principally to its simplicity 

 of manipulation. It is admirably adapted to large ciliates which can be 

 followed with ease under the low powers of the dissecting microscope. 

 The smaller the organism the more difficult this method becomes. Of 

 course failure will most surely follow any deviation from absolutely 

 aseptic technique. 



In an attempt further to simplify the technique as outlined by Par- 

 part, especially regarding the hood under which the transfers were 

 carried on, the following procedure has yielded extremely satisfactory 

 results in our laboratory (Kidder, Lilly, and Claff, 1940). Syracuse 



