DEVELOPMENT OF THE MIRACIDIUM OF PARAGONIMUS. 181 



the same length of time after leaving the cold chest as was required for 

 its original control at room temperature. 



It would appear, therefore, that while temperatures above 15° C. 

 are required for the development of Paragonivius ova, temperatures as 

 low as 10° do not destroy or, apparently, even impair their vitality. 



Ova from the fresh sputum, when frozen solid and immediately thawed, 

 apparently developed as well as did their controls. Likewise, ova frozen 

 solid for five or six minutes seemed uninjured, and, although at times 

 the proportion of undeveloped ova seemed somewhat larger than in the 

 unfrozen controls, the difference certainly was very slight and the greater 

 number of the eggs developed apparently as promptly and as normalty 

 as those in the controls. Ova frozen longer than five to six minutes, 

 however, began to show injury, in that development was apt to be delayed 

 and an unmistakably higher proportion failed to develop. Nevertheless, 

 we have had cultures which were frozen for ten minutes develop ap- 

 parently as well as the control ones, though, perhaps a few days later; 

 other cultures, frozen approximately the same length of time, have shown 

 a high percentage of degenerate ova. Ova frozen fifteen, twenty, and 

 twenty-five minutes showed with fair consistency an increasing proportion 

 of ova which failed to develop, and those frozen thirty minutes revealed 

 only a few motile miracidia and these required about forty days for 

 development. Xo ova after being frozen solid for over one-half hour 

 gave any signs of development. 



The results obtained from freezing the developed miracidia were very 

 similar. The actively motile organisms in their shells could be frozen 

 solid for five minutes, with impunity, resuming their activity when 

 thawed. If frozen for ten minutes, however, only about one-half remained 

 motile, and if for fifteen to twenty minutes, perhaps one-third, or less. 

 Motile miracidia frozen twenty-fi\'e minutes or longer have consistently 

 failed to retain their motility when thawed, and have invariably died and 

 degenerated. 



If the freezing be repeated after once thawing, the effect is more 

 pronounced. A culture of motile miracidia, frozen, immediately thawed, 

 refrozen for five minutes and again thawed, showed but a few motile 

 organisms remaining, and these appeared to have died by the next day, 

 when no motile miracidia could be found. 



DEVEr;OPMENT UNDER VARIOUS CONDITIONS OF LIGHT. 



Ova exposed to direct sunlight rapidly degenerated, and ova from 

 which liglit was absolutely excluded developed practically step by step 

 with the controls which were exposed to the refiected light of the labora- 

 tory room. 



Referring to the observation of Looss (1890), tliat the miracidia of the 

 conical amphistome {Amyliistoma cervi) of cattle and slieep escape from 



