HEMATOXYLIN-STAINED, 48-HOUR CHICK 93 



The result is that the outer surface of the embryo becomes adequately stained 

 while the inner structures do not, but this defect is difficult to detect until the 

 embryo is finally cleared for mounting. When the embryos are removed from 

 the stain, at which time they should appear a deep purple, they should be 

 transferred to a large finger bowl of distilled water, where they are rocked 

 gently backward and forward until most of the stain has been removed from 

 the paper to which they are attached. Each embryo should then be taken 

 separately and placed in 0.1 per cent hydrochloric acid in 70 per cent alcohol. 

 The color will immediately start to change from a deep purple to a pale 

 bluish pink, and the embryos should remain in this solution until, on exami- 

 nation under a low power of the microscope, all the required internal structures 

 appear clearly diffisrentiated. Most people differentiate too little, forgetting that 

 the pale pink of the embryo will be changed back to a deep blue by sub- 

 sequent treatment and that the apparent color will also increase in density 

 when the embryo is cleared. No specific directions for the extent of the differ- 

 entiation can be given beyond the general advice to differentiate far more than 

 you would anticipate to be necessary. After the embryos have been sufficiently 

 differentiated, each one should be placed in alkaline tap water, either as it 

 occurs in nature or rendered alkaline with the addition of sodium bicarbonate. 

 Here it should remain until all the acid has been neutralized and the embryo 

 itself has been changed from a pink back to a deep blue coloration. It may 

 then be dehydrated in the ordinary manner through successive alcohols. It is 

 the author's custom to remove it from its paper only when it is in the last 

 alcohol and before placing it in the clearing agent. Some persons place it in 

 the clearing agent attached to its paper and remove it only before mounting. 

 Any clearing reagent may be tried at the choice of the operator; the author's 

 preference for chick embryos is terpineol which has the advantage of not 

 rendering these delicate structures as brittle as do many other reagents. The 

 cleared embryo is mounted in balsam, preferably with the coverslip supported 

 in the manner described on page 41. 



Summary 



1. Incubate fertile eggs at 103° F. for 48 hours. 



2. Remove shell under surface of warm 0.9 per cent sodium chloride. 



3. Detach embryo from surface of yolk with bold cuts and wash in clean 

 saline solution. 



4. Strand embryo in watch glass. 



5. Cut hole slightly larger than embryo in filter-paper rectangle, soak paper 

 and drop it over embryo. 



6. Fix in Gilson's fluid 1 to 6 days. 



