The life history of Diplodiscus temporatus Stafford. 597 



taken from the canal during the spring months also contained sporo- 

 cysts in which cercariae were developing. 



All of the adult worms obtained during the early part of this 

 study came from artificially infected tadpoles kept in the laboratory, 

 or from frogs which had been fed Avith the intestines from such 

 tadpoles; but in the spring many adult worms were obtained from 

 frogs taken from ponds near the canal where infected snails were 

 abundant. 



The material both for sections and whole mounts was fixed 

 with a number of reagents : Flemming's stronger and weaker solutions. 

 Hermann's fluid. Boveri's picro acetic, picro sulfuric and sublimate 

 acetic were used. Hermann's fluid and Picro acetic gave the best 

 results for material to be sectioned. For material to be used as 

 whole mounts the picric acid mixtures were the most useful. 



When intended for sectioning the worms were fixed while still 

 within the liver of the snail, as it was found that there was less 

 likelihood of distortion of the tissues when this method was followed, 

 than when they were separated from the snail liver and preserved 

 by themselves. 



The material for whole mounts was shaken free from the snail 

 tissue in a dish of water and killed in one of the picric acid mix- 

 tures. For staining this material Conklin's Picro-Haematoxj'lin gave 

 the best results. The snail livers containing the parasites were im- 

 bedded in paraffin in the usual manner and serial sections 2, 3 and 

 5 micra in thickness were cut from them. The first mentioned 

 thickness (2 jt<) was the one used for all of the material which 

 contained the earliest stages in the development of the germ cells. 



After a few unsuccessful trials of other stains, Heidenhain's 

 Iron Alum Haematoxylin, followed by a counter stain of Eosin in 

 95 7o alcohol, was used for all the sections, whatever had been the 

 fluid used for their fixation. 



For the study of the youngest germ cells smear preparations 

 made from a fragment of tissue from the broad end of the sporocyst 

 were attempted, but none of these preparations gave as satisfactory 

 results as were obtained from the sections, so this method was 

 abandoned. 



For studying the living material of all stages aqueous methylin 

 blue and neutral red were sometimes useful, although these stains 

 were of little use in diff'erentiating tissues other than the nervous 

 and o'landular structures. 



