CONTROL OF CULTURES 463 



tory. Perhaps its chief drawbacks are that it is Hmited to negatively geo- 

 tropic organisms and to those types which are relatively powerful 

 swimmers, and that the flasks must be specially built, as well as the car- 

 riage, and that the whole is rather expensive. However, in those labo- 

 ratories in which an extensive program of investigation requiring sterile 

 Protozoa is being carried on, the purchase or the building of this appara- 

 tus will be found advantageous. 



D. Bactericidal agents. — Many methods have been tried in which 

 agents were employed to kill bacteria without killing the Protozoa under 

 investigation. Inhibition of bacterial overgrowth has been reported on 

 numerous occasions by the use of various chemicals (Zumstein, 1900; 

 Kofoid and Johnstone, 1929; and others) but sterility has rarely been 

 obtained. Cleveland (1928) states that he used numerous chemicals in 

 the hope that some would prove less injurious to Tritrichomonas than to 

 the associated bacteria. His results were entirely negative, and he states 

 that this type of investigation "appears to be almost a hopeless under- 

 taking" (p. 256). 



We do have, however, a few reports which indicate the possibility of 

 obtaining sterile Protozoa after treatments with various chemicals which 

 are more toxic to the bacteria than to the Protozoa. In all cases the bac- 

 tericidal agents were used on protozoan cysts, not on the trophic forms. 

 Frosch (1897) was the first to report the sterilization of cysts by chemi- 

 cal means. By the immersion of old cysts of Amoeba nitrophila in satu- 

 rated sodium carbonate for a period of three days, Frosch claims to have 

 killed all the associated non-spore-forming bacteria and to have recov- 

 ered some of the amoebae. This method was repeated by Walker (1908) 

 and the results confirmed. Oehler (1924) examined the possibility of 

 treating ciliate and amoebae cysts with a variety of disinfectants, acids, 

 alkalies, and salts but these results were far from encouraging. Severt- 

 zoff (1924) investigated the action of toluene, chlorine, and calcium 

 sulphide on the cysts of "soil amoebae" and found that the cysts were 

 able to withstand the deleterious effects of these chemicals better than 

 the associated bacteria (non-spore-forming types). He claims to have ef- 

 fected complete sterility by the use of calcium sulphide, but was unable 

 to establish pure cultures from the resulting cysts. Glaser and Coria 

 (1930) were unable to obtain sterile Euglena proxima by their washing 

 methods, but succeeded by treating "round or encysted stages" (p. 803) 



