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CYTOCHEMICAL TESTS ON EMBRYOS 



THE PROCEDURE 



The procedure given here is based on the paper by Rafalko (1946): Hydrochloric acid 

 is necessary only to release the sulphur dioxide from the sulphites used in the for- 

 mation of the leuco-basic fuchsin and in the sulphurous acid bath following staining. 

 Both the acid and the sulphite were eliminated and direct charging of both the basic 

 fuchsin and the bath water with sulphur dioxide gas was substituted. 



Bubble sulphur dioxide gas from a small aperture in glass tubing into 100 cc. of 0. 5% 

 basic fuchsin, beneath a hood. Decolorization takes place in 1 hour and the reagent 

 is ready for use. Distilled water is similarly saturated, and may be stored in 

 tightly corked bottles for weeks. To get SOt, use simple flask-and-funnel generator 

 and sodium bisulphite and dilute sulphuric acid. 



1. Fix tissues in Zenkers (or Bouin) for 2-20 minutes. 



2. Wash not more than 20 minutes, embed and section in the usual manner. 



3. Place in distilled water - 2 minutes. 



4. Normal HCl at room temperature - 2 minutes. 



5. Normal HCl at 60°C. for 8 to 10 minutes. 



6. Normal HCl at room temperature, rinse only. 



7. Distilled water - rinse. 



8. Sulphurous acid - 2 minutes. 



9. Leuco basic fuchsin - 1-1/2 to 2 hours. 



10. Sulphurous acid bath for sufficient time to remove the free, unreacted leuco 

 basic fuchsin; three 1 minute changes should be sufficient. 



11. Tap water for 10 to 15 minutes. 



It is possible, and even advisable, to counterstain with fast green in aqueous or al- 

 coholic solutions. Dehydration is accomplished either through the alcohols or from 

 water through triethyl phosphate (Nelsen, 1945: Stain Techn. 20:131) directly unto 

 xylene. The latter is a shorter inethod and does not appreciably remove the aqueous 

 counterstain. 



The Feulgen reaction is essentially Schiff ' s aldehyde test applied to a tissue cell. 

 The aldehyde is the carbohydrate released from the nucleic acid component of chrom- 

 atin after hydrolysis with normal HCl; this carbohydrate, a d-ribodesose (Levene, 

 1931), combines with the active principle of fuchsin-sulphurous acid, an N-sulphinic 



acid with the formula 



R-H<' (Weiland and Scheuring, 1921) 



SO2H 



to form, a blue-red color, often almost purple. The validity of the test rests upon 

 the absence from the tissues of any aldehyde other than that tested, which might 

 combine with sulphinic acid. If the fuchsin-sulphinic acid is oxidized, the color niay 

 be restored to act as a stain rather than as a reagent. Gardiner (1935) says: "It is 

 impossible in the Bouin material to distinguish the chromatin from other cell con- 

 stituents taking haeniatoxylin, but the Feulgen preparations show clearly that this 

 perinuclear substance is not chromidial. " 



D. Unna's (1921) Methyl-Green Pyronine Stain for Nucleic Acids : 



Methyl green stains thymonucleic acid green while pyronine stains the ribo-nucleic 

 acid red. It is important that Gruebler's Pyronine be used and this procedure works 

 best on late embryonic or adult tissues rather than the oocytes and early cleavage 

 stages. The stain is as follows: 



Gruebler ' s methyl green 0.15 gm. 



Pyronine B 0. 25 gm. 



Alcohol 2. 50 cc. 



Glycerine 20. 00 cc. 



Carbolic acid (0. 5% aqueous) 77. 50 cc. 



