398 A. J. NICHOLSON 
witseh, Zenker, and alcoholic Bouin. Of these, Petrunkewitsch 
was by far the most useful for general purposes as its penetra- 
tion is very good, a most important consideration when dealing 
with oocytes containing a large yolk-mass, particularly when 
the egg-walls are present. lor the finer cytological detuils 
Flemming with acetic gave the best results, though Flemming 
without acetic appeared to give a more perfect fixation, but 
the latter had the disadvantage that the chromatin did not 
stain as distinctly as it did with unmodified Flemming. 
Another fixative of which I made considerable use was the 
modified Bouin described by Sheppard (27). This I used in 
conjunction with the method of staining described by the same 
author, i.e. bulk staining with carmalum and counter-staining 
with Griibler’s light green. Using this method the fixation was 
excellent, and the double staining gave very beautiful prepara- 
tions—yolk and chorion staining bright green and the proto- 
plasmic structures red. This property was very useful in 
following the branching nucleus through the yolk-mass and 
in following the production of chorion by the epithelial cells. 
The fixative had the disadvantage, however. of making the 
material brittle. 
The stain principally used was Grenacher’s haematoxylin 
counter-stained with dilute Lichtgriin picric. For the latter 
the ordinary Lichtgriin picric solution (0-2 grm. Lichtgriin 
dissolved in 100 ¢.c. of a saturated solution of picric acid in 
absolute alcohol) was diluted with about ten times the bulk 
of 90 per cent. alcohol. The counter-stainng was done under 
observation, as Lichtgriin appears to displace the haematoxylin 
and the reaction requires to be stopped when all the yolk has 
become green and the protoplasmic structures are still blue. 
Using this method the branching nucleus can easily be followed 
amongst the yolk granules. 
Heidenhain’s iron haematoxylin counter-stained with eosin 
or orange G was also extensively used and was particularly 
useful for the finer nuclear details, but as it stained the yolk- 
mass dense black it was not satisfactory for the more developed 
oocytes. 
