DEVELOPMENT OF SIMOCEPHALUS 629 
bursts, the fixation is not good, while after the blastula stage 
the reverse is the case. If the membrane bursts, it produces 
a certain amount of distortion, and with all fixatives except 
Carnoy this was so bad as to make the material useless. With 
Carnoy a variable amount of swelling was produced, but with 
the other fixatives the whole egg usually burst. If the mem- 
brane remains intact, the ege becomes very difficult to embed 
owing to the embedding material not penetrating the mem- 
brane, and so it was found extremely difficult to obtain sections 
of the segmenting egg. One method used for the earliest 
stages was to employ hot water as the fixative. The eggs 
were dissected out of the brood-pouch and flooded with 
boiling water. After thirty seconds they were transferred to 
70 per cent. alcohol. This gave fairly good results, but with 
later stages the nuclei were not well preserved and so the 
method was not of much use. Fixing in bichromate-formol 
and subsequent treatment with 5 per cent. formalin gave good 
results, but here again it was unreliable and gave results no 
better than those obtained with Carnoy’s fluid. Gilson’s 
mixture (Subl. Ac. Ale. Chloroform) gave very good fixation 
when it succeeded in fixing the embryo without producing 
excessive distortion. 
For later stages Carnoy was again used, but better results 
were obtained with hot Flemming. The embryos were placed 
in Flemming’s strong solution at 56° C. for ten minutes and 
then washed out in water. Strong picro-sulphuric gave fair 
fixation. 
The embryos were stained with alcoholic eosin before 
clearing in clove oil and embedding in clove-oil ‘ celloidine ’. 
‘This made them more conspicuous and hence easier to manipu- 
late. After hardening the celloidine they were embedded in 
paraffin at 56° C. 
Sections were cut 6» and 7 » thick and stained on the slide. 
The best stain was Ehrlich’s haematoxylin. Iron haematoxylin 
was used after Flemming fixation. Haemalum, picro-indigo- 
carmine and thionin were among other stains used which 
proved satisfactory. 
