294 ox SOME TREMATODR PARASITES OP AUSTRALIAN FROfJS, 



kills them quickly, but they contract a little more. In both cases, 

 subsequent investigation shows tlie tissues to be excellently fixed. 



Sections were cut in three directions; in the case of Hat worms, 

 the horizontal longitudinal sections, that is, cut parallel to the 

 dorsal or ventral surface, gave particularly pleasing pictures; for 

 these sections, the worms were fixed under coverglasses, with just 

 enough pressure to keep them fairly flat. Whei'e the amount of 

 material allowed, lai'ge numbers of each of the species examined 

 (20-30 worms) were sectioned. The sections were cut in thick- 

 nesses varying from 5 to 10 /a. 



For staining whole-mounts, hasmatoxylin gave, on the whole, 

 the best results, though safranin, borax-carmine and paracarmine 

 were very good. Safranin was specially useful for staining yolk- 

 glands in those specimens where the yolk-glands ai'e more or less 

 hidden from view by other organs, for the .safranin still stained 

 these glands brilliantly when it was almost completely washed 

 out of all the rest of the tissues. For staining the sections, 

 Ehrlich's ha^matoxylin followed by eosiu gave uniformly good 

 .stains; paracarmine was also very useful. Neither of these stains, 

 however, was any good for Flemming-fixed material, and for this 

 iron-alum hi«matoxylin was used. Very good results were 

 obtained by using, on Flemming-fixed material, a modification of 

 a process suggested by Dr. Arnold of the Institute of Tropical 

 Medicine, Livei'pool. The stains used in this method are safranin, 

 methylene blue and orange G. From 50% alcohol, the sections 

 are brought into an iodine mordant (40 c.c. abs. ale, 2 gm. iodine 

 crystals, 60 c.c. distilled water, 5 gm. potassium iodide) for 5 

 minutes, washed in tapwater for 30 seconds, and placed in 

 safranin (the aniline-water formula) for 24 hours; wash in tap- 

 water and put into methylene blue for 30-45 minutes; wash in 

 tapwater one minute, run through 30, 50, 70, 90 per cents, 

 alcohol, 30 seconds in each, absolute alcohol 2 or 3 miimtes; 

 orange G in clove oil n-10 minutes; xylol; and mount in Canada 

 balsam. 



By this process, the methylene blue stains the chromatin blue, 

 the safranin stains the egg-shells and the parts of the yolk-glands 

 that furuLsh the material for the egg-shelLs (Goldschmidt, 26) a 



