No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 415 
stage. Accordingly, in this second period of the nucleolar growth 
there appears to be no increase in the amount of the true 
nucleolar substance, but merely an increase in the amount of 
the vacuolar substance. The thickened portion of the periph¬ 
eral layer of the nucleolus is at first biconvex, but as the large 
vacuole grows larger the pressure of the latter causes it to 
gradually assume a concavo-convex form (Figs. 84-86). Thus 
the shape of the large vacuole is at first concavo-convex, and 
later spherical or oval. This thickened portion of the outer 
layer of the nucleolus is usually homogeneous in structure, as 
is the remainder of the true nucleolar substance which envelops 
the vacuole ; but sometimes small vacuoles may occur within 
it also (Fig. 71). 
Two poles may be distinguished in the nucleolus at this 
second stage of its differentiation : (1) the pole at which the 
large vacuole lies ; and (2) the pole at which the thickened mass 
of the true peripheral substance is situated. From the study 
of a large number of nuclei at this period I find that in about 
75j& of them the second of these poles is directed towards the 
nuclear membrane, the first pole towards the center of the 
nucleus ; at this stage, as in the preceding, the nucleolus lies 
usually excentrically within the nucleus. 
The later differentiation of the nucleolus consists, accord¬ 
ingly, in the accumulation in it of fluid vacuoles (their substance 
identical with that of the nutritive globules of the nucleus), 
but the true nucleolar substance undergoes no change whatever, 
as far as can be determined from differential staining. There 
is no chemical union of the vacuolar with the true nucleolar 
substance, but the fluid vacuoles simply push aside this sub¬ 
stance, so that, after these numerous smaller vacuoles have 
united to form a single large vacuole, the true nucleolar sub¬ 
stance remains unchanged as a peripheral layer around this 
vacuole. The substance of the vacuoles becomes colored with 
the same stains, though always more lightly, as does the true 
nucleolar substance, so that we find in this stage a more deeply 
staining envelope of substance around a less deeply stained 
portion. This difference of staining between these two parts 
of the nucleolus is best shown by employing haematoxylin 
