APPENDIX. 635 



transverse sections are desired, and cutting should begin at the head end. The dorsal side 

 of the embryo should be turned toward the operator. The sections will then lie cranial 

 surface up, with the ventral side of the embryo away from the operator. Sections mounted 

 on the slide in the same relative position will thus be seen right side up, so to speak, under 

 the microscope. 



It is always best to mount paraffin sections, especially serial sections, on the slide without 

 delay. This is done as follows: Smear a very thin coat of egg albumen* on a slide with the 

 finger. The slide must be dean. Put several drops of distilled water on the slide, and 

 then lay the sections on the water. Warm the slide until the sections become perfectly 

 flat, but not enough to melt the paraffin. Drain off the excess of water and put the slide in 

 a dry place for several hours until all the water evaporates. The sections are then ready 

 for further treatment (see "Staining paraffin sections with hasmatoxylin and eosin'') 



VII. STAINING. 



Although it is usually advisable to stain embryological material differentially a method 

 best applied to sections it is often convenient and saves time to stain in toto. Alum-carmine 

 and borax-carmine are two of the most generally used bulk stains. 



Alum-carmine. Mix i gram best carmine with 100 c.c. of a 4 per cent, aqueous solution 

 ammonia (common) alum. Boil 15 minutes, then add enough sterile water to replace that 

 lost by evaporation. When cool, filter. 



Small embryos, after being fixed and hardened, are placed in the stain for 24 hours, then 

 washed in water, dehydrated, embedded, and cut in the usual way. 



Borax-carmine. Mix 3 grams best carmine and 2 grams borax with 50 c.c. of water. 

 Boil 20 minutes. When cool add enough water to replace that lost by evaporation. Then 

 add 50 c.c. of 70 per cent, alcohol, let stand 24 hours, and filter. 



Small embryos, after being fixed and hardened, are put in the stain for 24 hours or 

 longer; then placed in 70 per cent, alcohol, to every 100 c.c. of which 4 or 5 drops of hydro- 

 chloric acid have been added, until the specimen appears rather transparent (several hours). 

 Wash in 2 or 3 changes of 70 per cent, alcohol, dehydrate, embed and cut 



DIFFERENTIAL STAINING is most satisfactorily done with one of the haematoxylin solutions 

 followed by a plasma stain, such as eosin or acid fuchsin, or a combination of picric acid and 

 fuchsin. The following give good results: 

 Delafield's hamaloxylin. 



Haematoxylin crystals i gm. 



Alcohol 6 c.c. 



Ammonia alum, saturated aqueous solution, 100 c.c. 



Dissolve the hasmatoxylin in the alcohol, then add the alum solution. Allow the mixture 

 to stand in the light for 10 days to ripen. Filter, and add to the filtrate 25 c.c. of glycerin 

 and 25 c.c. of wood naptha. Allow to stand 3 or 4 days and filter again. It may be used 

 in full strength or diluted with water to any degree desired. For using, see "Staining with 

 hasmatoxylin and eosin." 



Weigerfs hcemaloxylin. Make up two stock solutions as follows: 

 A. i per cent, haematoxylin in 95 per cent, alcohol. 



B Hydrochloric acid (sp. gr. 1.126) 10 c.c. 



Ferric chloride, 30 per cent, solution 40 c.c. 



Distilled water 95 c - c - 



*Formula: Beat slightly the white of one fresh egg, filter (it will take 24 hours), and add an 

 equal amount of glycerin and one gram of salicylate of soda. This will keep for many months. 



