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: (i) 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 



