THE CAUSE AND NATURE OF EN'CYSTMENT. 47 



in about a week, the controls in this case did not encyst until the 

 tenth day and then very few cysts appeared, most of the active 

 forms dying rather than encysting. Although all the cultures of 

 Z>4 (controls) were in excellent condition on the eighth day they 

 had entirely died out by the fifteenth day and few cysts were 

 present. The cultures of series D2 and D$ did not die out as 

 soon as those of ZXj. nor did they encyst as quickly. On the 

 twenty-second day of the experiment they had reached the same 

 condition in which D^ was found on the fifteenth. By the 

 thirtieth day all active forms had disappeared from the cultures of 

 Di and slight encystment had occurred. 



Not knowing what to make of this behavior, twenty-four more 

 cultures were seeded and the experiment repeated. These cul- 

 tures w r ere divided into four series Ei, 2, 3, and 4, each 

 series corresponding to the similarly numbered series of D. This 

 yielded identically the same results as D with the exception that 

 El and 2 died out without any encystment whatever. 



It is well to state here that D was seeded from a culture of Ci 

 shortly before it completely died out and E was seeded from a 

 culture of 2 after it had reached a very poor condition. No 

 cysts were transferred, only active individuals. Series C had 

 likewise been seeded from A$. In an attempt to find the 

 explanation for this loss of ability to encyst on the part of series D 

 and E, some cysts were taken from C6, placed in fresh hay infusion 

 and permitted to excyst. On the next day eighteen cultures 

 were seeded from these newly excysted ones and two days later it 

 was found that every one of the eighteen contained many cysts. 

 As a check on this, cysts were taken from Cy, washed in seventy 

 per cent, alcohol, and permitted to excyst in fresh hay infusion. 

 From these, thirty-six cultures were seeded and on the second 

 day they were found to have repeated the activity of the eighteen, 

 being largely encysted. In addition, cysts were taken from 3, 

 washed in alcohol, excysted, and twelve cultures seeded from 

 them. At the end of two days many cysts were found in all of 

 the cultures. 



By putting the last two paragraphs together, one gets a strong 

 indication that the longer Polytomella citri is removed from 

 encystment, the less is their tendency to encyst. 



