472 CONTROL OF CULTURES 



in Syracuse watch glasses enclosed in cellophane bags, as previously 

 described. In the case of Stylonychia, it was found possible to establish 

 them on living yeast cells, suspended in distilled water, in the absence 

 of any other food material. Sterile ciliates would not live on autoclaved 

 yeast, however. Sterile ciliates would eat quantities of living T etrahymena 

 (taken from agar slants and suspended in distilled water), but would 

 not divide. Sterile ciliates, placed in suspensions of autoclaved yeast, and 

 living Tetrahyniena grew well and established flourishing cultures. Sterile 

 ciliates in dead yeast and dead Tetrahymena, failed to multiply. Additions 

 of none of the known water-soluble vitamins changed the situation. The 

 inference is, as Lilly points out (1940), that Stylonychia requires, among 

 other things, two unknown factors — one found in yeast (even after 

 autoclaving), but not present, at least in sufficient quantities, in Tetra- 

 hymena: the other what might be called a living factor, present in living 

 yeast and Tetrahymena. Both of these factors are present in certain favor- 

 able species of bacteria when the bacteria are alive, but the "living factor" 

 is destroyed with the death of the bacteria. The so-called living factor 

 is not a surprising requirement among Protozoa, as experience has shown 

 that many different types will not live without being supplied with some 

 type of living organism. The yeast factor seems to belong to the water- 

 soluble, heat-stabile group, but is not identifiable with any one of the 

 known B complex. While this factor is present in dried and pasteurized 

 yeast (Brewer's Yeast Harris), it is not present in sufficient quantities 

 in Difco dehydrated yeast extract. Concentration and partial purification 

 of the yeast factor have been carried out, but until this work is further 

 along we must content ourselves with these few facts. 



This example is one of many similar cases and serves to point out that 

 several conditions must be recognized and fulfilled, if the investigator 

 is to be successful in establishing sterile Protozoa in culture. The possi- 

 bility of supplementary factors must be considered before it can be said 

 of any type that it cannot be grown bacteria-free. Somewhat the same 

 situation was encountered by Glaser and Coria (1933) in their work on 

 Paramecium. They finally announced a complicated medium which 

 proved to be successful, and this medium contained pieces of fresh rabbit 

 kidney (possibly supplying the living factor during the early growth 

 phases of the ciliate). 



It is not the purpose of this chapter to consider in detail all of the 



