264 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 



method gave excellent results for all except fertilization stages. In order 

 to differentiate the sperm within the egg from the deutoplasm, the eggs 

 were stained in Delafield's haematoxylin and differentiated with a weak 

 solution of picric acid in 90% alcohol. The picric acid renders the deuto- 

 plasm reddish yellow, but leaves the sperm black. This is also a good 

 stain for the maturation stages and has been used as a control of the 

 iron-hsematoxylin method. 



During the current year a number of the preparations have been re- 

 stained with Brazilin (Hickson, : 01) , which has given results even better 

 than the iron-haematoxylin. The eggs after being sectioned and mounted 

 were mordanted in a solution of iron in 70% alcohol for periods varying 

 from thirty to sixty minutes, and then stained from thirty minutes to 

 two hours in a ^% solution of pure Brazilin in 70% alcohol. The Brazilin 

 gives a double stain: the centrosomes, chromosomes, and microsomes 

 are stained intensely black, while the cytoplasm takes a color resembling 

 that of a Bordeaux-red stain ; but the cytoplasm is not often overstained, 

 as frequently occurs with Bordeaux red. This stain has the further ad- 

 vantage over the iron-hsematoxylin that the process is shorter and the 

 tissues do not have to pass from alcohol to water. It also rarely over- 

 stains ; and even when it does the excess may be removed by simply 

 washing the preparation in 70 % alcohol, or dipping it in the mordant for 

 a few seconds. 



III. Observations. 



1. Growth op the Egg. 



The eggs of this species when full-grown lie free in the follicles of the 

 ovotestis. A follicle may contain eggs only, spermatozoa only, or both 

 eggs and spermatozoa. It is impossible to show that any part of the 

 ovotestis is exclusively concerned in the production of either of the sex- 

 ual elements. 



If an animal is examined after egg-laying, there are rarely foiind any 

 full-grown eggs, or even eggs partly filled with deutoplasm. This is in 

 striking contrast to what is found in Limax (Byrnes, '99), where rela- 

 tively few eggs mature simultaneously. 



For two reasons no attempt has been made to work out the ovogene- 

 sis or spermatogenesis : first, because the early changes take place when 

 the cells are very small ; secondly, these changes take place in the spring, 

 while the animals are living in deep water, where they would be inac- 

 cessible except by dredging, even after their habitat had been discovered. 



