Stains and Staining 61 



Transfer from the orange G to 95 per cent alcohol, clipping the 

 slide a few times in this merely to save the absolute alcohol. Dehy- 

 drate in absolute alcohol 3 to 30 seconds. 



Clear in clove oil, as already described in the paragraph on 

 gentian-violet. Transfer to xylol and mount in balsam. 



/ 



Safranin and gentian-violet are often used without the orange. 

 In this case, transfer from the gentian-violet directly to 95 per cent 

 alcohol, and proceed as before. 



A serious objection to both these methods is that the gradual series 

 of alcohols cannot be used, because the gentian-violet washes out so 

 rapidly. While the objection may be overcome, to some extent, 

 by using the orange in 95 per cent alcohol, solutions of gentian-violet 

 in strong alcohol have not been satisfactory. We have been trying 

 a third method, with more or less success. So far, it seems better 

 than either of the two methods just described. 



C. Use the safranin solution described in B, but use the gentian- 

 violet and orange G in 1 per cent clove-oil solutions. 



For paraffin sections, transfer to safranin from 70 or 50 per cent 

 alcohol and stain as directed under B. Pass through 70, 85, 95, 

 and 100 per cent alcohol, about 5 minutes in each. Put the clove- 

 oil gentian solution on the slide with a pipette and stain 5 to 30 

 minutes. Drain off the stain (which can be used repeatedly), and 

 add the clove-oil orange solution and allow it to act 10 to 20 seconds. 

 This stains with orange and, at the same time, extracts the gentian- 

 violet. Pour off the clove-oil orange solution (which, unlike the 

 gentian solution, is not worth much for a second staining) and 

 pour on pure clove oil. Watch it until the gentian-violet is satis- 

 factory, then transfer to xvlol and mount in balsam. 



W J *J 



This method avoids the big skips of A and B, and the prepara- 

 tions seem better. 



Cyanin and Erythrosin. Make both solutions according to the 

 general formula for anilins, or make 1 per cent aqueous solutions, 

 but note what was said about cyanin in the paragraph on p. 57. 



Stain in cyanin 5 to 10 minutes or longer; rinse quickly in water 

 if using the aqueous solution, or in 35 per cent alcohol if using the 

 general formula; then stain 30 seconds to 1 minute in erythrosin. 



