THE CAUSE AND NATURE OF EXCYSTMKXT. 43 



Polytoniclla was associated with bacteria and yeast in all of 

 the cultures. 



EXPERIMENTAL. 



Temperature. The procedure determined upon was to elimi- 

 nate temperature first and then carry on the remainder of the 

 experiments at the optimum temperature. Accordingly, thirty- 

 six cultures were seeded and twelve of these were kept at each 

 of the following temperatures: io-ii C. (series Ai), 2i-22 C. 

 (series A2), and 22-27 C. (series ^3). Series A$ was kept in 

 the laboratory, which explains the wide variation in temperature. 

 At the end of nine days all cultures of the three series were in 

 excellent condition. Those of series Ai showed no cysts, A2 

 contained few, while some w r ere found in A$. In fourteen days 

 Ai was in good condition with no cysts; A2 was in fair condition, 

 with some cysts ; and A 3 was poor, with many cysts. In twenty- 

 one days A I was only fair and no cysts had yet appeared; A2 

 had reached the same condition that A$ was in on the fourteenth 

 day and A$ now contained no active forms and many cysts. 

 The active forms had entirely disappeared from A2by the thirty- 

 fourth day. These two series continued in the same condition 

 for the remainder of the experiment. On the thirty-fourth day 

 A i was in poor condition, without encystment. From the 

 thirty-sixth to thirty-ninth day there was no ice in the refriger- 

 ator and the temperature rose from 10 to 21 C. At the end of 

 those three days all twelve cultures were in fair condition and a 

 few cysts had appeared in five of them. On the thirty-ninth 

 day ice was again placed in the refrigerator and the experiment 

 continued for twenty-one more days. At the end of that time 

 there were practically no active forms in any of the cultures and 

 the few cysts that were in five of the cultures on the thirty-ninth 

 day were still present, but the remaining seven cultures had died 

 out without any encystment whatever. 



The first attempt showed that the cultures kept at 35 C. would 

 not develop. Consequently, to attempt to determine the effect 

 of so high a temperature, cultures were permitted to develop in 

 the laboratory for a few days and were then placed in the incu- 

 bator. It was found that at 35 thriving cultures would gradu- 

 ally die out until, at the end of two days, they would have com- 



