468 CONTROL OF CULTURES 



plates to keep the plates for at least ten days and it is safer to keep them 

 two weeks. The plating method is usually a better criterion of conditions 

 within a culture than the turbidity test, for another reason. When dealing 

 with ciliates, normally bacteria-feeders, it is often the case that the bac- 

 teria are eaten out of the media almost as fast as they multiply. This is 

 more likely to happen when the ciliate is a voracious feeder and multi- 

 plies rapidly and when the contaminant is one of the slow-growing va- 

 riety. It should not be supposed, however, that all of the bacteria will 

 be eaten, although two such cases are on record (Elliott, 1933; Johnson, 

 1935). Some of the bacteria will almost invariably escape and be carried 

 along from transplant to transplant. On the solid media, however, the 

 ciliates do not move about, and colonies of bacteria develop unhampered. 

 Although they are not prevalent in wild infusions, tests should always 

 be conducted for anaerobic bacteria. The simplest test and one which 

 will usually determine their presence or absence is the following: Tubes 

 containing not over 3 ml. of nutrient broth plus a two to two-and-a-half- 

 inch layer of paraffin oil, are plugged with cotton and autoclaved for 

 twenty minutes at fifteen pounds' pressure. Rubber stoppers are sterilized 

 at the same time. Immediately after sterilization the rubber stoppers are 

 fitted into the tubes, which are then allowed to cool. When the broth is 

 cool, inoculations are accomplished by injecting the material to be tested 

 through the paraffin oil into the broth, the rubber stopper being im- 

 mediately replaced. This type of culture will allow even obligatory an- 

 aerobes to multiply, although not necessarily to the height of their 

 capacity. Enough growth is obtained, however, to determine the pres- 

 ence of anaerobic contaminants. The obligatory anaerobic bacteria, it 

 must be admitted, do not form an important group for our consideration, 

 as they occur so infrequently. The facultative anaerobes may be detected 

 by more common procedures. 



Establishment of Sterilized Protozoa in Culture 



The sterilization of Protozoa is, after all, only a means to an end. It 

 is of very little value to the investigator if, after going to the trouble to 

 rid a species of Protozoa of their associated bacteria, the Protozoa fail to 

 live. For the most perfect control of a protozoan culture for experimental 

 work, pure cultures are necessary. This means that the protozoan under 

 investigation must be established in a medium containing no other living 



