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 : (i) 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 

 75^ 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 



