No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 
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- 
