No. 2.] COMPARATIVE CYTOLOGICAL STUDIES . 465 
pace proportionally to that of the nucleus (Figs. 301-304). 
Then vacuoles arise in the nucleolus, these being neither very 
numerous nor very minute (Figs. 304-310, 312-316). The time 
when these vacuoles first arise is very variable, though in the 
majority of cases they do not appear before the nuclear sap 
begins to stain red. The size of the nucleolus does not 
always stand in the same proportion to that of the nucleus. 
Its ground substance is dense, stains deeply with eosin, and 
no limiting membrane is present; but by the use of the double 
stain Lyons blue and acid carmine, whereby the nucleolus 
stains blue and the chromatin red, a deep red line appears to 
surround the nucleolus: I cannot determine whether this 
line is a nucleolar membrane or a layer of chromatin, or 
whether it is merely an appearance caused by the refraction 
of the nucleolus. 
When the nucleolus first appears it is usually situated at 
that pole of the nucleus opposite the chromatin mass and is 
not in contact with the nuclear membrane (Fig. 301). In 
nuclei of intermediate size, before the nuclear sap commences 
to stain with eosin, it is most frequently in contact with the 
nuclear membrane (Figs. 302-304); but in the largest ger¬ 
minal vesicles it is never in contact with this membrane, 
though often lying excentrically in the nucleus. 
As soon as the germinal vesicle has nearly, or quite, attained 
its maximum dimensions (quite frequently, however, in those of 
still smaller size) two very noticeable changes take place in it: 
(1) the chromatin assumes a different form and stains differ¬ 
ently (these chromatin changes shall be delineated later); and 
(2) the nuclear sap, which had heretofore remained colorless or 
was merely of a light lilac shade (by the double stain haema- 
toxylin and eosin), now becomes a yellowish-red color, so that 
the nuclei in this stage may be easily distinguished from those 
of preceding ones (Figs. 304, 305, 307-310, 316). Simul¬ 
taneously two changes occur in the nucleolus: (1) it stains no 
longer a deep red with eosin, as before, but a yellowish red, 
and appears more refractive; and (2) the several vacuoles 
within it gradually fuse together and so produce a larger one, 
which has usually a central position. The fluid, structureless 
