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MADE AT THE PLYMOUTH LABORATORY. 263 
many times greater than the germinal vesicle, or than all the nucleoli 
put together. There is no indication of a rapid formation of 
nucleoli, and at the end in the egg almost ripe there are still a 
number of nucleoli remaining. It is practically certain, therefore, 
that the yolk spherules are formed in situ, first on the outside of 
the egg and then progressively inwards. 
Another point in which I am unable to agree with Scharff’s 
description is his account of the division of the protoplasm of the 
egg into two layers. In this case again, in my opinion, he has been 
misled, as many others have been in microscopical researches, by 
alterations due to method of preparation and the action of reagents. 
Scharff states that the ovaries from which his sections were prepared 
were hardened with weak chromic or with picro-sulphuric acid, both 
excellent reagents which I have largely used myself. But he states 
that his preserved material was prepared not by himself, but by 
Professor McIntosh ; and it is not certain whether the ovaries were 
perfectly fresh when preserved—a very important point. My 
experience is that when preparations are made from fish obtained 
in the market which have been dead several hours, the preserving 
liquid used being Perenyi’s mixture, a division of the protoplasm 
into two zones is seen. The ovary when examined fresh appears 
perfectly unaltered, but after preparation produces a result different 
from that obtained from an ovary taken from a fish just killed. 
The outer lighter zone is frequently separated from the inner. In 
all my successful preparations from ovaries preserved immediately 
after the death of the fish there is no division of the protoplasm into 
zones in the yolkless ova, and in the older ova the only distinction 
is that between the outer layer containing yolk granules or spherules, 
and the inner layer where there is no yolk. In young yolkless ova, 
whether in sections from immature ovaries in which all the ova are in 
this condition, or in sections from ovaries in which the majority of 
ova are larger and developing yolk, the whole of the protoplasm is 
deeply stained, almost as deeply as the nucleoli, while the rest of 
the germinal vesicle is scarcely stained at all. In fact, the proto- 
plasm of the ovum from its earliest appearance is distinguished 
by its affinity for stains, which causes young ova to contrast vividly 
with the connective tissue of the sections. The staiming is less 
after the action of chromic acid, but after corrosive sublimate or 
picro-sulphuric acid it is usually intense. Yolk substance, on the 
other hand, does not stain at all, and hence in older eggs the con- 
trast between the unstained yolk layer and the inner protoplasmic 
layer is marked. In the older eggs, however, the inner unyolked 
protoplasm does not stain so intensely as the protoplasm of the 
young unyolked eggs. In some preparations the protoplasm of 
NEW SERIES.—VOL. III, NO. IV. 22 
