SECTION SIX 



2.* Methyl green-Pyronin {Bracket) 



Methyl green, 00 chloroform washed 0-15 gm. 



Pyronin G . . . . . . . . 0-25 gm. 



Alcohol 95% 2-5 ml. 



Acetate Buffer pH 47 . . . . 97-5 ml. 



Methyl green, OO is washed repeatedly with chloroform, to extract 

 all traces of methyl violet. The washed methyl green is then dried, and 

 0-15 gm. is weighed out and dissolved in the acetate buffer and the 

 alcohol with the pyronin. 



Note: the stain is liable to deteriorate after two or three weeks. 



* See note {d) at the end of this technique. 



3. fRibonuclease o-i% in distilled water which 

 has been adjusted to pH 6-o 



t If this is not available a suitable ribonuclease extract can be prepared in the 

 laboratory as follows: 



I. Pass 0-5 to I kilo Ox pancreas through a meat mincer. 

 II. Pound it to a smooth pulp with a mortar and pestle. 

 III. Suspend the pulp in one or two volumes of N/io acetic acid for sixteen 



to twenty-four hours. 

 IV. Boil for ten minutes. 

 V. Cool; then filter. 

 VI. Adjust the pH of the filtrate to pH 6-o. 

 VII. Filter. 

 VIII. Add a crystal of thymol or camphor, as a preservative and store in the 

 refrigerator: under these conditions the solution will keep for several 

 months. 



Technique: 



Note: It is not possible to give absolute rules concerning the 

 following procedure, as the methods of fixation, the solubility 

 of the ribonucleic acid of the organ to be studied, and the activity 

 of the ribonuclease, are all possible variables. The technique 

 given below, which should be regarded as a basis for experiment, 

 may be varied to suit individual lines of research and investigation. 



I . Fresh material should be used and objects or slices of tissue, 

 which must not be more than 2-3 mm. in thickness, are fixed for 

 a maximum of one hour in one of the above fixatives. 



2a. After Zenker, wash with tap water for twenty-four hours. 



2h. After Carnoy or Serra, wash in two or three changes of 

 95% alcohol for the minimum time necessary to remove the 

 fixative. 



3. Dehydrate and embed at once, through alcohol and toluol, 

 in paraffin wax. 



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