55l^ CYTOCHEMICAL TESTS ON EMBRYOS 



6. Normal HCl at room temperature, rinse only. 



7. Distilled water - rinse. 



8. Sulphurous acid - 2 minutes. 



9. Leuco basic fuchsin - Ij 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 counterstaln with fast green in aque- 

 ous or alcoholic solutions. Dehydration ia accomplished either through the 

 alcohols or from water through triethyl phosphate (Nelsen, I'^k^: Stain Techn. 

 20:131) directly unto xylene. The latter is a shorter method and does not ap- 

 preciably remove the aqueous counterstaln. 



The Feulgen reaction is essentially Schiff's aldehyde teat applied to a tis- 

 sue cell. The aldehyde is the carbohydrate released from the nucleic acid com- 

 ponent of chromatin after hydrolysis with normal HCl; this carbohydrate, a 

 d-ribodesose (Levene, 1951), combines with the active principle of fuchsin- 

 sulphurous acid, an N-sulphinic acid with the formula 



H 

 .H<^ (Welland and Scheurlng, I92I) 



SO2H 



to form, a blue- red color, ofteh 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 sulphlnlc acid. If the fuchsin-sulphinic acid ia oxidized, 

 the color may be restored to act as a stain rather than as a reagent. Gardiner 

 (1955) says: "It la impossible in the Bouin material to diatinguiah the chromatin 

 from other cell constituents taking haematoxylin, but the Feulgen preparations 

 show clearly that this perinuclear substance ia not chromidlal." 



************* 



UrmA'S (1921) METHYL-GREEN FYBONINE STAIN FOB NUCLEIC ACIDS : 



Methyl green stains thymonuclelc acid green while pyronlne stains the ribo- 

 nucleic acid red. It is important that Gruebler's Pyronlne be used and this pro- 

 cedure works best on late embryonic or adult tisauea rather than the oocytes and 

 early cleavage stages. The stain is aa follows: 



Gruebler's methyl green O.I5 gm. 



Pyronlne B 0.25 gm- 



Alcohol (95^) 2 .50 cc. 



Glycerine 20.00 cc. 



Carbolic acid (0.5^ aqueous) 77-50 cc. 



Fix the tissues in 95^ alcohol, embed eind section in the usual manner, and stain 

 in the above (Unna's) stain for 20 minutes. 



The albumins and globulins are stained red by the pyronlne, while the nucleo- 

 proteins are stained blue-green with the methyl green. To separate the albumins 

 and globulins one can take advantage of the differential solubility. The albumins 

 and pseudoglobulins are soluble in water, the globulins are not soluble in water. 

 Both are soluble in salt solutions. Failure to stain with methyl green would 

 indicate the absence of nucleoproteins. 



