No. 2.] COMPARATIVE CYTOLOGICAL 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 Tctrastefnma 

 catcmilatufn ; but in the present species I have not observed 

 any distribution of them along these filaments, and further 

 they are numerically scarcer than in Tetrastcmma. 



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 



