No. 2.] COMPARATIVE CVTOLOGICAL STUDIES. 435 
what unequal in size. In this stage, accordingly, they increase 
in size (perhaps by the fusion of contiguous ones (Fig. 242), 
and decrease in number, whereas in the preceding one the 
reverse process took place. 
Fourth nucleolar stage. — Almost all the nucleoli are flattened 
against the nuclear membrane (Figs. 245 and 248), and they com¬ 
mence to show a vacuolated structure; these apparent vacuoles, 
which are unstaining globules, when stained by the Ehrlich- 
Biondi method, whereby only the ground substance of the 
nucleolus is colored, appear as refractive granules (Fig. 248). 
At the conclusion of this period the nucleoli become irregular 
in shape, granular in appearance, stain less deeply, and each 
finally breaks up into a mass of granules. In this manner they 
decrease both in number and size. 
During the third and fourth stages, while the nucleoli are 
undergoing the metamorphoses described, a small number of 
newly formed ones appear in the nucleus, which are of later form¬ 
ation than the others (Figs. 245, 247, n. 2 ). These may serve 
as nourishment for the chromatic filaments, as in Tetrastemma 
catenulatum; but in the present species I have not observed 
any distribution of them along these filaments, and further 
they are numerically scarcer than in Tetrastemma. 
No yolk is present in the cytoplasm in the first and second 
nucleolar stages. This fact is easily proved by the use of the 
Ehrlich-Biondi stain, by which the cytoplasm is stained green, 
and the yolk substance, when present, a brownish maroon color. 
Yolk first appears in the third nucleolar stage, and at the com¬ 
mencement of the following stage the whole cytoplasm is nearly 
filled with it. Further, the nucleoli stain differently from the 
yolk globules by the use of the stain mentioned. These facts 
show that the origin of the nucleolar substance is not to be found 
in the yolk substance proper, but in a cytoplasmic substance 
from which the latter may later be evolved. That the sub¬ 
stance of the nucleoli is extranuclear in origin is shown by the 
fact that the nucleoli at their first appearance lie in contact 
with the nuclear membrane (Figs. 236-238), and only later do 
they take a central position. Though I have seen no nuclei 
smaller than those figured, which could without doubt be 
