CONTROL OF CULTURES 467 



under the conditions of wild cultures, sterile Protozoa could be obtained 

 with any regularity by this method. 



The Importance of Adequate Sterility Tests 



It is, of course, obvious that any method for ridding Protozoa' of bac- 

 teria must be carried out under the rigid rules of bacteriological tech- 

 nique. Bacteria are so varied in form and activity that special pains must 

 be taken to check the results of any method before the treated Protozoa 

 may be pronounced sterile. Microscopic examination is of little or no 

 use, at least until time has been given for any accompanying bacteria 

 to multiply. We must therefore give any possible contammant every 

 conceivable chance to multiply, thereby revealing its presence. 



Sterility tests are usually made in two ways, by inoculation into fluid 

 media and by spreading on nutrient solid media. In the fluid media 

 (broth) contamination shows itself when the broth becomes turbid. The 

 turbidity test is sufficient, when the contaminant is such a one as will 

 distribute itself through the media. In other words, turbidity denotes 

 contamination. But lack of turbidity does not always denote sterility. 

 Some bacteria grow very slowly in broth and form small clumps which 

 sink to the bottom of the test tube, leaving the broth clear. This experi- 

 ence was reported by Hetherington (1933, 1934) and by Stuart, Kidder, 

 and Griffin ( 1939) . A macroscopic examination of a tube was not enough 

 to reveal the presence of these organisms. 



Plating, either by pipetting fluid from the culture to be tested or by 

 streaking the surface of solid media (nutrient agar) with a needle dipped 

 into the culture, is usually more satisfactory than the turbidity test. The 

 plates are allowed to incubate, and the surface is examined for bacterial 

 colonies. The usual contaminants from wild infusions will appear in 

 from twenty-four to forty-eight hours. But this method has its limitations. 

 Some bacteria grow very slowly at room temperatures, but well at higher 

 temperatures. Others are the reverse. Duplicate sets of plates should 

 always be made, one set to be incubated at room temperature and the 

 other at temperatures from 30° to 37° C. The time factor should be 

 carefully considered. The slow-growing types (such as the Mycobac- 

 terhun reported by Stuart, Kidder, and Griffin, 1939) may not appear 

 until many days after the inoculation. It is necessary in all tests with agar 



