298 Feulgen and PAS Technics (chap. 19) 



comments: 



These two dyes distinguish between states of polymerization of 

 nucleic acids, not between the acids themselves. Highly polymerized 

 nucleic acid stains with methyl green; low polymers of both DNA 

 and RNA stain with the pyronin. The usual technics resulted in too 

 pale a methyl green, or if excess staining was tried, water rinses re- 

 moved the methyl green and acetone removed the pyronin. Dehy- 

 drating ^vith ethyl alcohol removes most of the color; isopropyl and 

 tertiary butyl are no improvement. So Kurnick developed the above 

 method, ^vhen he found that 7j-butyl alcohol differentiated the methyl 

 green, but it removed the pyronin. He, therefore, decided to leave 

 the pyronin out of the first solution (it was lost anyway) and tried 

 follo^ving the methyl green with pyronin saturated in acetone. The 

 acetone must be free of water, so always use a fresh solution. 



Later Kurnick {1953) published a method in which he did combine 

 the two stains: 



SOLUTION : 



pyronin Y, 2% (2 gm./lOO ml. water) 12.5 ml. 



methyl green, 2% (2 gm./lOO ml. water) 7.5 ml. 



distilled water 30.0 ml. 



procedure: 



1. Deparaffinize and hydrate slides to water. 



2. Stain in methyl green-pyronin: 6 miniues. 



3. Blot careftilly with filter paper. 



4. Dehydrate, r?-butyl alcohol, 2 changes: 5 minutes each. 



5. Clear in xylene: 5 minutes; transfer to cedarwood oil: 5 minutes. 



6. Mount. 



Flax and PolUster {1949) used 0.1% aqueous Azure A, and differ- 

 entiated overnight in absohite alcohol. The stain was specific for 

 nucleic acids; chromatin, nucleoli, and cytoplasmic regions high in 

 nucleoprotein concentrations, but it also stained mast cells, mucus 

 and cartilage matrix. The latter could be checked by applying ribo- 

 nuclease. 



Periodic Acid-Schiff (PAS) Reaction 



Two methods are outlined; one {A) with alcoholic solutions, the 

 other {B) with aqueous .solutions. 



