172 BULLETIN OF THE 



A very large per cent of eggs kept in this way remain in good condi- 

 tion until hatching, which, in a moderately warm room, occurs between 

 the twenty-second and twenty-seventh day. 



The best reagents for killing embryos were found to be either chromic 

 acid, 0.33%, or Perenyi's fluid. The chromic material when well stained 

 with alcoholic borax-carmine shows the differentiation of nerve cells and 

 nuclei excellently, but it is more difficult to stain sufficiently chromic 

 material than such as has been preserved in Perenyi's fluid. The latter 

 may be stained with alcoholic borax-carmine or picrocarminate of lithium. 

 Good results for the study of cell division have also been obtained by 

 staining with Czoker's cochineal. The picrocarminate of lithium is par- 

 ticularly valuable in the older stages, because it brings out the nerve 

 fibres, the latter being stained yellow, while the ganglionic cells are 

 colored red. 



To obtain the embryos in an uninjured condition, it is advisable, in 

 using the chromic-acid method, to remove only the outer envelope 

 before killing. The egg may be held between the thumb and forefinger 

 of the left hand, while with a finely pointed stick, somewhat like a 

 wooden toothpick, the outer membrane is gently punctured ; the probe 

 should be run under the membrane a little way, to make a larger open- 

 ing, and the egg carefully pressed with the thumb and forefinger, 

 whereupon the albumen, containing the embryo and surrounded by the 

 inner membrane, will come out in a perfect condition. This may be 

 dropped at once into water, if several are to be treated together, for it 

 is more convenient to put them all into the chromic acid at the same 

 time. When all have been shelled, they should be put into 0.33% 

 chromic acid for two or three minutes only, simply to kill the embryo 

 without hardening the albumen. Then they should be transferred to a 

 watch-glass of water, to which a few drops of the acid have been added. 

 While in this fluid, the inner membrane may be removed with needles. 

 To accomplish this, it is advisable, in the very young stages, to make as 

 large an opening in the membrane as possible, and then with a needle 

 gently to press the embryo out, even if the albumen adheres to it, for 

 the albumen becomes slightly coagulated in the weak acid, and then 

 can easily be washed off". In the stages from the tenth to the sixteenth 

 day, the large size of the pulsating sacs of both head and foot regions 

 makes it extremely difficult to extract the embryos uninjured; great 

 care must therefore be taken, and no pressure used. While employing 

 one of the needles to hold the membrane, the other should be forced 

 through the membrane, which may then be ruptured and turned back 



