464 CONTROL OF CULTURES 



with a solution of potassium dichromate (1.25 to 2.5 percent) for from 

 fifteen to thirty minutes. Perhaps the most exhaustive study on the rela- 

 tive effects of chemicals on bacteria and protozoan cysts was carried out 

 by Luck and Sheets (1931). They investigated the lethal concentrations 

 of some eighteen substances on two-day-old cysts of Euplotes taylori and 

 their associated bacteria. They appear to have obtained sterile ciliates in 

 some cases, when silver nitrate was used in high concentrations of glu- 

 cose and sucrose. All other substances either were more toxic to the 

 protozoan cysts than to the bacteria or were uniformly lethal to both. 



This line of approach to protozoan sterility does not seem to be too 

 encouraging. As might well be supposed, the investigator is limited in 

 any case to cyst-forming types. Even these are not uniformly resistant 

 to chemical action, so that it appears that little can be learned at this 

 time from the experiences of others. It seems that the methods referred 

 to above, while they may have produced the desired results in some cases, 

 are not to be recommended for routine work. There is always the prob- 

 ability that any type of protozoan collected from the wild will have as- 

 sociated with it spore-forming bacteria, which are always highly resistant 

 to disinfectants. If a cyst-forming type of protozoan is first sterilized by 

 washing or some other method and established in culture with a single 

 known bacterium, then some of these disinfectants might be used to ad- 

 vantage later for obtaining large numbers of sterile organisms for ex- 

 perimentation. 



Of the physical bactericidal agents which have been employed, heat, 

 used in different ways but always upon cysts of various species, has been 

 most generally used. Walker (1908) reports that he was able to obtain 

 sterile "Amoeba intestinalh" by inoculating an agar plate, first with 

 concentric rings of B. coli and then, in the center, with amoebae cysts. 

 The plate was heated to 70°-75° C. for one hour, which was sufficient to 

 kill the Bacillus coli but did not kill the cysts. He then added fresh bac- 

 teria to the center of the plate and the amoebae excysted, fed, and mi- 

 grated through the rings of dead B. coli. Walker states that in their migra- 

 tion through the dead bacteria they freed themselves of all living bacteria 

 (by scraping them off.''). He was able to recover sterile amoebae at the 

 periphery of the plate. This report is surprising, in that moist heat was 

 used, and cysts of Protozoa in general, under these conditions, are usually 

 killed. 



