SECTION TWO 



METHYL GREEN - PYRONIN Y 

 For differentiating ribonucleic and deoxyribonucleic acids 



Solutions required: 



A. Pyronin Y . . . . . . i gm. 



Distilled water . . . . . . 50 ml. 



Extract with chloroform by shaking the above solution 

 in a separating funnel until the chloroform layer (lower 

 layer) is colourless. About 60 ml. of chloroform will be 

 required, using 20 ml. each for three extractions. 



Note: The third extraction should be almost colourless, 

 otherwise the extracting process will have to be con- 

 tinued with one or two additional 20-ml. lots of chloro- 

 form. If after that the chloroform layer is still coloured, 

 the pyronin is unsuitable for this technique. 



B. Methyl green OO . . . . 2 gm. 

 Distilled water . . . . . . 100 ml. 



Extract with chloroform by shaking the above solu- 

 tion in a separating funnel until the chloroform layer is 

 no longer coloured violet. 



Note: Use about 50 ml. of chloroform for each extrac- 

 tion. A total of about 300 ml. of chloroform will be re- 

 quired. 



C. Pyronin Y solution, chloroform 



extracted as above .. .. 12-5 ml. 



Methyl green OO solution, chlor- 

 oform extracted as above . . 7-5 ml. 



Acetate buffer pH 47 . . . . 30 ml. 



Techyiique: 



1 . Carry sections through to distilled water in the usual way. 



2. Immerse in solution C for six minutes. 



3. Blot carefully with filter paper. 



4. Immerse for five minutes in each of two changes of normal 

 butyl alcohol. 



5. Immerse in xylol for five minutes. 



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