7Q EDWARD DRANE CRABB. 



sectioned tissue of virgin L. columella, L. palustns, PJiysa gyrina, 

 and P. savii were studied. Planorbis trivolvis were reared in 

 strict isolation but did not lay eggs. 



B. Methods. 



a. Fixation. The ovotestis of L. .?. appressa is easily fixed in 

 any kind of nuclear fixing solution with sufficiently good results 

 to permit the study of germ cells in a general way though further 

 study requires special technique. Eggs which have been ovi- 

 posited present difficulties that are not overcome by any ordinary 

 technique. 



In general, it was found that a modification of Flemming's 

 solution containing 2 cc. of 2 per cent, osmic acid, added at the 

 time of use to 15 cc. of a solution consisting- of 50 cc. of i per 

 cent, chromic and i cc. of glacial acetic acid gave good results on 

 both eggs and ovotestis tissue. Burkhardt's solution of potassium 

 bichromate, chromic and acetic acids (La Cellule, 1897, p. 335), 

 to each cubic centimeter of which about one half grain of cane 

 sugar or sodium chloride is added, gives fair chromosomes with 

 little cytoplasmic disturbance. This solution was used cold and at 

 30 to 85 C. When thus used, it thoroughly penetrates a whole 

 specimen of L. ,s. apprcssa in a maximum of one hour or a mass 

 of eggs in about two hours. A few minutes is sufficient for the 

 actual penetration of the eggs ; the additional time facilitates in- 

 filtration. As may be readily inferred tissue may be left in the 

 above modification of Flemming much longer than in this modi- 

 fication of Burkhardt without seriously injuring it. 



Bouin's picro-acetic and Allen's chromic acid and urea modi- 

 fication gave fair chromatin, but poor cytoplasmic fixation of 

 tissue ; however, these and other picric combinations were found 

 to be of little or no value for eggs. It was found, as had Rockling 

 ('22), on Helix, and H. Hoffman ('22) on Li max, that Zenker's 

 fluid is a dependable fixing agent for general use. Very distinct 

 mitotic figures and sperm asters were secured in ova which had 

 been recently ovulated by using this agent at 50 C. However, it 

 was useless for fixing eggs. 



b. Imbedding. In infiltrating and imbedding eggs and tissue, a 

 number of aqueous media, several celloidin methods and John- 



