Effects of Temperature, Poststaining Rinses and Ethanol-Butanol Dehydrating Mixtures on Methyl Green-Pyronin Staining

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Abstract
Methyl green GA (Chroma) and pyronin GS (Chroma) were used. Procedure recommended: Stain for 1 hr at 37 C in a purified 0.5% aqueous methyl green, buffered to pH 4.1 with Walpoles acetate buffer, and containing 0.2% pyronin; rinse for 1-2 sec in ice-cold distilled water; blot sections evenly, and rinse with vigorous agitation in t-butanol; dehydrate in 2 changes of t-butanol for 5 min each; clear in xylene and mount. This technique results in a consistent staining pattern for qualitative nucleic acid differentiation, whereas older methods have been only partly satisfactory. Rinsing in ice-cold water is a critical step; t-butanol was superior to n-butanol and to ethanol-butanol mixtures for dehydration. Staining at 25-27 C is feasible hut less effective.