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 odcyte 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 microtuberculatus. 
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- 
