66 bulletin: museum of comparative zoology. 



gone the early cleavage while in the brood-lamelke, 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 contaiuiug 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 wlicn 

 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-lamellae 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 wasiied 

 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-lamellaj brittle, so that the eggs can easily be 

 isolated by the use of needles. In practiqe the stained pieces of egg- 

 lamellse were placed in a drop of clove oil on a glass slide. Then, using 

 a dissecting microscope, the lameUte were cut with fine needles and the 

 eggs 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. 



