No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 459 



rapidly increases in amount, spreading from the region of the 

 nucleus (which is central) to the cell periphery. In the largest 

 ovarial ova the cytoplasm is densely filled with larger and 

 smaller yolk globules ; the larger ones appear homogeneous 

 when stained with eosin (Fig. 269), but the Ehrlich-Biondi stain 

 shows them to be composite masses of small globules. 



The nucleolus rapidly increases in size, at a somewhat greater 

 proportionate rate than the nucleus itself. It is now large 

 enough for its structure to be clearly made out : it consists of 

 a homogeneous ground substance, which seems to stain more 

 deeply with eosin as it grows larger ; a limiting membrane is 

 clearly demonstrable in the largest nucleoli (Figs. 271-277, 

 279-281) after staining by the Ehrlich-Biondi method or 

 after fixation with Flemming's fluid, though it does not differ 

 chemically or in structure from the ground substance and is 

 only a thin layer of the latter in which vacuoles never occur. 

 At the close of the metaphasis of the mitosis small vacuoles 

 make their first appearance in the ground substance of the 

 nucleolus (Figs. 263 and 270). There are only a few of them at 

 the start, but their number rapidly increases as the nucleolus 

 grows larger, until there are large numbers of them in its center 

 (Figs. 268 and 269). They are always more numerous at the 

 center than at the periphery of the nucleolus, and usually first 

 appear at the former point. On preparations stained with 

 eosin the small vacuoles appear either as clear spaces or as 

 black granules, according to the focusing of the microscope ; 

 after the use of the Ehrlich-Biondi stain they become a light 

 grayish color (note the contrast, — that in the eggs of Doto and 

 Montagua the nucleoli appear as black granules only after 

 treatment with the latter stain) ; after fixation in the fluid of 

 Flemming the substance of these vacuoles is of a lighter color 

 than the ground substance. This vacuolar substance is homo- 

 geneous, and is probably of a thin, fluid nature. With the 

 growth of the nucleolus the number of the vacuoles becomes 

 very great, though their size does not seem to increase. In the 

 nucleoli of the largest germinal vesicles examined the vacuoles 

 no longer retain their original spherical form, but become mutu- 

 ally confluent to some degree, not in such a manner as to pro- 



