66 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 
gone the early cleavage while in the brood-lamellz, was necessarily used 
for the study of later cleavages. 
Many of the fixing reagents ordinarily employed in embryological work 
have been tried, but only solutions containing picric acid have proven 
entirely satisfactory. Kleinenberg’s stronger fluid and a saturated solu- 
tion of picric acid in 35% alcohol both gave excellent fixation, but a 
saturated solution of picric acid in 5% acetic acid gave results which were 
far superior to those obtained by any other fixing solution. This fluid 
penetrated rapidly, and eggs thus prepared were very transparent when 
stained and mounted entire. This transparency was a very important 
feature in the study of all cleavage stages. The picro-acetic mixture also 
gave the best results for material which was to be sectioned. It should 
be remarked that solutions with less acetic acid lack penetrating power. 
Strong solutions of mercuric chloride in distilled water, in sea water, 
in alcohol, or combined with picric acid, gave some good results in the 
study of maturation and early cleavage stages by means of sections, but 
material thus fixed proved too opaque for preparations of entire eggs. 
Material fixed in the mercuric chloride solutions was especially valuable 
in determining the distribution of the yolk, which readily stained differ- 
entially after such fixation. In the study of all stages of development use 
was made both of sections and of entire eggs viewed as transparent ob- 
jects. The method of preparing the latter will be described first. Small 
pieces of egg-lamellz which had been fixed in the picro-acetic mixture 
were stained from one to three hours in a concentrated solution of borax- 
carmine in 35% alcohol (Grenacher’s formula). They were then washed 
in alcohol and rapidly decolorized in 70% alcohol containing 0.3% hydro- 
chloric acid. The decolorizing was watched with a compound microscope, 
and quickly checked when nuclei and cell-boundaries began to appear. 
The piece of egg-lamella was then dehydrated and, within two or three 
hours after staining, cleared. 
All the ordinary clearing oils were tried, but no other one gave results 
comparable in excellence with those obtained by the use of clove oil. 
This oil renders the egg-lamelle brittle, so that the eggs can easily be 
isolated by the use of needles. In practice the stained pieces of egg- 
lamellz were placed in a drop of clove oil on a glass slide. Then, using 
a dissecting microscope, the lamellz were cut with fine needles and the 
egos set free, but they were still surrounded by the vitelline membrane. 
All attempts at removing this membrane proved unsuccessful. After 
the greater part of the clove oil had been drained away, the eggs were 
mounted in xylol-balsam. 
