Fciihc)] Read ion 297 



\s 



For details concerning fluorescent microscope and equipment, see 

 pages 102. 



Pyronin-Methyl Green for Nucleic Acids (kurmck, 1952) 



fixation: absolute alcohol, Carnoy or cold acetone. If formalin is used, 

 it must be adjusted to pH 7 and used only briefly before the solution 

 tinns acid and produces a faint green staining of cytoplasm. Picric 

 acid depolymerizes DNA and shows no green chromatin. 



SOLUTIONS- 



Methyl green: 



methyl green, C.I. 42590 0.2 gm. 



O.IM acetate buffer (pH 4.2) or distilled water lOO.O ml. 



Before making solution, methyl gTeen should be ptirified by extrac- 

 tion with chloroform. Add approximately 10 gm. methyl green to 

 200 ml. of chloroform in 500 ml. Erlenmeyer flask and shake. Filter 

 with suction and repeat wdth smaller amoimts of chloroform tnitil 

 the solution comes off blue-green instead of lavender (usually re- 

 quires at least .S extractions). Dry and store in stoppered bottle. Puri- 

 fied dye is stable. 



Pyronin: 



pyronin }' saturated in acetone, or for lighter color in 10% acetone. 

 The British Pyronin Y for RNA (G. T. Gurr or Edward Gnrr) is 

 recommended. 



procedure: 



1. Deparaffinize and hydrate slides to water. 



2. Stain in methyl green solution: 6 minutes. 



3. Blot and immerse in n-butyl alcohol: several mintites in each of 2 

 changes. 



4. Stain in pyronin: 30-90 seconds. Shorten to a few dips if stain is 

 too dark. 



5. Clear in cedar oil, xylene, and mount. 



results: 



RNA containing cytoplasm — red 



nucleoli — red 



chromatin — brioht oreen 



erythrocytes — bro^vn . 



eosinophilic grantiles — red / 



cartilage matrix — green 



osseous matrix — pink with trace of violet 



