126 
Transactions of the Society. 
egg is surrounded by a membrane, inside of which is a semi- 
gelatinous fluid, and the whole of the eggs are contained within a 
gelatinous envelope, rendering it extremely difficult to fix such 
material. Cleavage and differentiation of the ovum takes place 
inside the gelatinous capsules, and the young mollusc does not 
launch out into the world until well advanced in development. 
Technique. 
The behaviour of the nucleolus during oogenesis and sperma- 
togenesis was studied in preparations made from gonads fixed in 
Bouin’s picro- formal-acetic, corrosive acetic or alcohol acetic. The 
stains used included eosin and haematoxylin, iron alum haematoxylin, 
Auerbach’s and Pappenheimer’s stains, Obst’s carmine and methyl 
green, and Mann’s methyl-blue eosin. The best preparations were 
obtained with corrosive acetic fixation and Mann’s methyl blue 
eosin. 
The preparation of the ova and developing embryos presented 
problems of considerable difficulty, owing to the impenetrability of 
the gelatinous membranes in which they are enclosed. 
Attempts were first made unsuccessfully to fix the egg masses 
entire in picro-formal-acetic and corrosive acetic. Then the outer 
gelatinous envelope of each egg mass was cut open, and the eggs were 
scraped out with a knife blade on to filter paper, which removed 
the adhering mucilage of the envelope. The eggs surrounded by 
the outer membrane and inner semi-gelatinous fluid were then 
transferred to various fixatives for varying periods of time. The 
fixatives tried were Bouin, corrosive acetic, Carnoy, Gilson, 
Petrunkewitsch, and a mixture of the two latter. In all cases the 
eggs failed to clear when transferred to either chloroform or xylol 
after passage through the alcohols. 
In these earlier attempts the trouble in fixation arose owing to 
the difficulty in getting the fixative to penetrate the peripheral 
layer of mucilage around each egg. Various methods were tried 
for tearing open this surrounding layer of semi-gelatinous sub- 
stance. Under a simple microscope, eggs which had been removed 
from the outer envelope of the egg cluster were stabbed with fine 
forceps and immediately dropped into the fixative. This method 
was hardly successful, as the eggs in most cases came completely 
away from their surrounding membrane, and owing to their ex- 
tremely small size it was most difficult to manipulate large 
numbers in this condition. 
It seemed best after a large number of preliminary experiments 
to prick an opening in the membrane enclosing the semi-gelatinous 
fluid around each egg — an opening sufficiently large to allow the 
fixative to penetrate freely, but not large enough to allow the egg 
to float out. At first eggs were pricked with fine steel needles to 
