FEEDING PARAMECIUM KNOWN BACTERIA 145 



The effi.ciency of this method of washing was tested by plating 

 the contents of each watch-crystal within a washing chamber. 

 It was found that the process of plating could be simplified by 

 pouring the agar directly into the watch-crystals. In this way 

 exceedingly striking demonstrations of the effi.ciency of the 

 method were secured. It was possible to determine the exact 

 point at which the animals became free from bacteria by absence 

 of colonies on these miniature plates. Thorough testing of this 

 method showed that while colonies would not be present after 

 from three to five washings, it was not safe to rely upon this 

 number, for if the animal were plated with the washing fluid of 

 the fifth plate, an occasional colony would sometimes develop. 

 This did not occur after the sixth or seventh washing, and re- 

 peated trials convinced me that seven washings could be relied 

 upon to free Paramecium aurelia from bacteria. 



The animals were grown in pure cultures of bacteria or in 

 mixtures of such cultures in Petri-dish moist chambers containing 

 five watch-crystal cultures as just described. The safety of such 

 a method was completely demonstrated by the tests made while 

 these cultures were under observation. The method employed 

 was to take samples of the fluid from each watch-crystal of a 

 group of five, dilute these with sterile tap-water, and plate in the 

 usual way. These plates were allowed to develop at room tem- 

 perature for from three to four days according to the temperature 

 of the laboratory. In this way it was possible to know whether 

 sufficient contamination was occurring to make rewashing the 

 animals necessary. An occasional foreign colony was not con- 

 sidered evidence of serious contamination, since the great mass of 

 bacteria would still be of the original stock. 



Since tests at intervals as great as thirty days showed no 

 evidence of serious contamination, it is probable that it is not 

 necessary to wash the animals more frequently. However, in 

 actual practice it was customary to wash them as often as every 

 three or four weeks, in order to be absolutely sure that contamina- 

 tion was being kept out of the cultures. It is interesting to 

 note that a test made after actual observation of the cultures 

 had been discontinued, and the great care in handling lessened, 



