MUSEUM OF COMPARATIVE ZOOLOGY. 173 



over the embryo, being drawn off like the finger of a glove. In the 

 older stages, not much care is necessary, because the embryos bear with- 

 out injury considerable handling, and there is so little albumen left that 

 their position is not readily changed while the membranes are being re- 

 moved. When freed from the membranes and as much of the albumen 

 as possible, the embryos are to be returned with a large-mouthed pipette 

 to the chromic acid (0.33%), where they may be left for an hour or 

 two ; after washing in running water for two or three hours, they may 

 be carried up to 70% alcohol by adding to the water, drop by drop, 

 35 °J alcohol ; then 50 °f alcohol, etc. This dehydration must be made 

 very carefully, to avoid shrinkage. The embryos are extremely deli- 

 cate, and must be handled with great care through every step of the 

 process. 



In using Perenyi's mixture, it is best to free the embryos while living 

 from the surrounding membranes and the albumen, removing the inner 

 membrane under clear water. When set free, they should be trans- 

 ferred at once with a pipette into a dish of Perenyi's mixture, where 

 they may remain from two to three minutes. They are then to be washed 

 thoroughly in distilled water at least five minutes, put into a 5% aq. sol. 

 of alum for thirty minutes, washed again in water, and finally car- 

 ried through the grades of alcohol as in the chromic method. It is 

 necessary to remove the embryo while living, because otherwise the 

 albumen becomes in this reagent like a jelly, and cannot be removed 

 without injury to the embryo. Material designed to be sectioned must 

 not be left in alcohol longer than a month, since the albumen in the 

 nutritive sac gradually becomes too hard to be cut, especially if pre- 

 pared in Perenyi's mixture. The stages from the tenth to the sixteenth 

 day can still be used, even if they have been thus overhardened, by re- 

 moving the nutritive sac ; but in the younger stages this is apt to 

 destroy the embryo, and in the older ones — much of the albumen hav- 

 ing been swallowed — its removal is still more certain to have the same 

 effect. Attempts subsequently to soften the albumen by prolonged 

 treatment with weak acetic acid proved to be only partially successful. 

 If the embryos are to be kept at all, they should be left unstained ; 

 but the safest way is to carry them through to embedding as soon as 

 possible. 



They can be stained whole ; but to do this successfully, they must 

 be carried gradually through successively weaker grades of alcohol until 

 a grade corresponding to the stain is reached. It is advisable to make 

 the necessary steps from the stain to the parafine as quickly as possible. 



