220 E. E. JUST 
B. Observations on the sectioned egg 
Observations of the phenomena of fertilization in the living 
ege were supplemented with a study of sectioned material. 
Technique. Eggs were fixed in Meves’ fluid for thirty minutes, 
one hour, or twelve hours. Aceto-osmic-bichromate mixtures 
(Mathews,! 99; Bensley); Bouin’s fluid, modified by the addition 
of an equal volume of water; and Gilson’s mercuric-nitric mix- 
ture were likewise used. Although very destructive to the yolk 
and oil, the modified Bouin proved helpful in the study of cer- 
tain details in connection with sperm penetration. 
The difficulties of fixation, which are great in this egg, as in 
Nereis, may in large measure be overcome by the subsequent 
treatment. The following methods were used after fixation 
with Meves: 
(a) Clearing with double distilled anilin oil from 80 per cent 
alcohol. 
(b) Clearing in cedar oil from 95 per cent alcohol. 
(ec) Clearing in cedar oil from 95 per cent alcohol after treat- 
ment with glycerine (eggs put in 70 per cent alcohol plus an equal 
amount of glycerine). 
(d) Clearing in xylol from 95 per cent alcohol or from absolute 
alcohol. 
In all cases xylol was used before imbedding in paraffin or 
in paraffin with some admixture of Johnston’s rubber-asphalt 
mass. It was found that avoidance of absolute alcohol left the 
eggs less brittle and therefore less refractory in cutting. By 
far the most natural contours of both the Platynereis and the 
Nereis eggs are preserved through the use of aniline oil after 80 
per cent—a clearing agent that I have used successfully for 
several years. Staining was with iron hematoxylin alone. 
Sections were cut four micra thick. 
Spermatozoa, after fixation, were studied for the most part 
unstained after the methods of Koltzoff, de Meyer, etc. The 
iodine mixture recommended by Mayer for Volvox proved in- 
1From the legend of Mathews’ figures it appears that he used aceto-osmic- 
bichromate mixtures. 
