188 Specific Examples of Slide Making 



ternal structures appear clearly differentiated. Most people differentiate 

 too little, forgetting that the pale pink of the embryo will be changed back 

 to a deep blue by subsequent treatment and that the apparent color will 

 also increase in density when the embiyo is cleared. No specific direc- 

 tions for the extent of the differentiation can be given beyond the general 

 advice to differentiate far more than you would anticipate to be necessary 

 (Fig. 132 is a good guide). After the embryos have been sufficiently 

 differentiated, each one should be placed in tap water, 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 cus- 

 tom to remove it from its paper only when it is in the last alcohol and 

 before placing it in the clearing agent. Some people 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. 



SUMMARY 



1. Incubate fertile eggs at 103° F for 33 hr. 



2. Remove shell under surface of warm 0.9% 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. 



7. Wash in running water overnight. 



8. Stain overnight in Delafield's hematoxylin. 



9. Differentiate in acid 70% alcohol until internal anatomy is shown 

 clearly. 



10. Treat with alkaline tap water until blue. 



11. Dehydrate. 



12. Detach embryo from paper and clear in terpineol. 



13. Mount in balsam. 



