FIXING, IMBEDDING, AND STAINING 51 



1. A 50 per cent solution of alcohol containing a trace of nigrosin and acetic 

 acid. 



2. Mayer's alcoholic paracarmine. 



3. 5 per cent glacial acetic acid in 50 per cent alcohol, to which is added 

 sufficient nigrosin to make it bluish black in the bottle. 



4. 50 per cent alcohol strongly acidulated with acetic acid. 



The sections on the slide are mordanted for a few minutes in 

 I. They are then somewhat understated in solution 2, the 

 superfluous stain washed off in 50 per cent alcohol, and the slide 

 placed in 3. In this last it remains until examination shows it to 

 be slightly overstained. It is then washed and decolorized to the 

 desired degree in the 50 per cent alcohol strongly acidulated with 

 acetic acid (4). 



Mayer's alcoholic paracarmine, used in this connection, is made 

 by using carminic acid I gram, chloride of ammonium 0.3 grams, 

 chloride of calcium 4 grams, and 100 cc. of 70 per cent alcohol. 

 Dissolve the carminic acid by heat if desired for immediate use, 

 then allow it to settle, and filter. 



Flcmming triple stain. When material has been fixed in chromic 

 acid solutions, particularly in the Flemming chrom-osmo-acetic, then 

 the Flemming triple stain is one of the two most valuable with the 

 fungi, as with nearly all other plant tissues similarly fixed. This 

 stain requires safranin, gentian violet, and orange. The usual 

 method is to stain on the slide for several hours to a day in a 

 strong alcoholic solution of safranin, rinse in 95 per cent alcohol 

 until very little color remains, stain for a few minutes to several 

 hours in gentian violet, wash for a very short time in water, and 

 plunge into a strong solution of orange G for a few seconds. The 

 slide is then treated with absolute alcohol in order to wash out the 

 surplus gentian. Differentiation is effected by treatment with clove 

 oil or with clove oil first, and finally with xylol for fixing, before 

 being mounted in damar balsam. Almost any safranin stain may 

 be used in this combination, but it will often be found that the 

 safranin may be entirely omitted with advantage. By this means 

 the process is also greatly shortened. Perhaps the best gentian 

 violet which may be used in this process is that of Ehrlich, con- 

 sisting of : 



Gentian violet . . i part A nil in oil . . . . 3 parts 

 Alcohol .... 15 parts \Vatc-r 80 parts 



