Relation between Nucleolus and Chromosomes. 45 



ulum scarcely visible leaves the nucleolus as an intensely black homo- 

 geneous sphere, showing no trace of the vacuoles that are at other stages 

 so conspicuous. Auerbach's stain applied at this stage also reveals a dark- 

 green homogeneous nucleolus. That vacuoles are really present is seen 

 from a study of living eggs and becomes manifest also when other stains 

 are employed. The only reasonable conclusion seems to be that the entire 

 nucleolus, vacuoles and ground-substance, are filled or impregnated with 

 chromatin all at approximately the same stage of elaboration. 



The same stains applied at earlier and later stages show vacuoles. Borax 

 carmine stains the entire oocyte red, the cytoplasm, nuclear reticulum, and 

 nucleolus showing progressively deeper shades. The nucleolus under this 

 stain always appears abundantly vacuolated. Orange G stains the nucleo- 

 lus yellow and reveals a vacuolated structure ; combined with iron hema- 

 toxylin the main portion of the nucleolus stains black, while the vacuoles 

 remain yellow. With Auerbach's stain the main body stains dark green and 

 the vacuoles red. A combination of orange G and Lyons' blue yields an in- 

 teresting result. The nucleolus stains yellow and appears vacuolated, while 

 the cytoplasm and nuclear reticulum stain blue. In these sections also the 

 nucleolus shows a very distinct dark-blue wall. This result would seem to 

 indicate that the nucleolar wall is derived from the nuclear reticulum. 



Occasionally one meets stages likes the one shown in figure 38, stained 

 with iron hematoxylin and orange G. Here one sees a dark-stained (chroma- 

 tin) mass separating from a yellow-stained (plastin) mass of similar shape 

 and size. The early maturation stages yield abundant evidence, as will 

 appear in the descriptions which follow, that the nucleolus consists of a plas- 

 tin ground-substance, throughout which, partly in the form of spherules 

 (vacuoles) and partly as a fluid imbibed by the plastin itself, the chromatin 

 is scattered in varying degrees of elaboration and condensation. Hartmann 

 (1902) likewise describes a double structure of the nucleolus in Asterias 

 glacialis, as also Guenther (1903) in Psammechinus nucrotubcrcnlatus. 

 Chubb (1906) states that in Antedon the " nucleolar material consists of two 

 substances the one acidophile and extending throughout the nucleolus, the 

 other deeply basophile and borne by the acidophile ground-substance, to 

 which its presence imparts a considerably firmer consistency." 



This gives a clue for the interpretation of the varying appearances when 

 different stains are employed the nucleoli are formed by the union of a 

 plastin ground-substance with a more or less fluid chromatin content. We 

 have thus a mixed nucleolus. There is here another fact to support the 

 view of a very intimate relation between linin (plastin) and chromatin. 

 There is evidence also to show, as I shall describe later, that chromatin is 

 capable of manufacturing its own plastin. The red vacuoles seen after 

 Auerbach's stain and the yellow vacuoles after iron hematoxylin and orange 

 G are thus the appearance of the plastin ground-substance, now visible be- 



