BANCROFT: OVOGENESIS IN DISTAPLIA OCCIDENTALIS. 61 



But, as has already been said, I have not made the extended observa- 

 tions necessary to place beyond doubt the position of D. occidentalis. 



The Styela and Chelyosoma material was mostly collected by myself, 

 at various places along the California coast, during the summers of 1894 

 and 1895, and studied about a year later. Four fixing media were used, 

 — Flemming's fluid, picro-sulphuric acid, glacial acetic acid, and Pere- 

 nyi's fluid, and of these the last gave the best results. 



The investigation of the Styela and Chelyosoma material was done 

 for the most part at the University of California, and that of Distaplia 

 entirely at Harvard University during the college years of 1896-97 and 

 1897-98. 



The stains used for the last species were mostly iron haernatoxylin 

 and a combination of methyl green and acid fuchsin, adapted from 

 Auerbach's ('96, p. 414) method B a. These two methods supplement 

 each other very nicely, and render the use of others almost superfluous ; 

 the haernatoxylin demonstrating most morphological features splendidly, 

 while the chemical constitution is brought out well by the methyl 

 green and fuchsin. The iron hematoxylin was employed in the usual 

 way ; fresh solutions of the stain were found to give decidedly the best 

 results, but for some things, such as nucleoli, which take a fresh solu- 

 tion too strongly, older ones are often to be preferred. In using the 

 double stain, sections were first treated in a mixture containing two 

 parts of a 0.1% aqueous acid fuchsin solution, to which a little acetic 

 acid had been added, and three parts of a 0.1 % solution of methyl 

 green. They were left in this mixture fur about fifteen minutes, and then 

 immersed in a 0.1% solution of methyl green for a few minutes longer. 

 From the stain the sections were transferred directly to 90%, and then 

 to absolute alcohol, xylol, and balsam. No especial care is necessary in 

 order to get a satisfactory differentiation. In addition to the two stains 

 mentioned, List's ('96, pp. 480-487) potassium ferro-cyanide methods 

 were also employed, but yielded no very satisfactory results. They will 

 be discussed later. 



The sectioning of the younger ova presents no difficulties, but the 

 older ones, which, with the exception of the germinative vesicle, are pure 

 yolk, are exceedingly difficult to deal with, on account of the crumbling 

 of the yolk, and for them no satisfactory method was found. However, 

 all of the material that I had access to had been preserved in 80% or 

 90% alcohol for some time, so that I did not have an opportunity of 

 testing the effect of preservation in weaker grades of alcohol, formalin or 

 paraffine. As a rule, the whole colony was sectioned, but when dealing 



