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Fully standardized Methyl Green-Pyronin methods are presented. Pure Pyronin Y and purified Methyl Green or Ethyl Green are used either simultaneously in one dye bath or are used as a sequence of Pyronin Y and Ethyl or Methyl Green. Both methods, as shown by enzymatic pretreatment, give a reliable and reproducible staining in DNA with Ethyl or Methyl Green and of RNA with Pyronin Y on Carnoy fixed material. On formaldehyde fixed material it was found advantageous to use the sequential method as chromatin was hereby stained green instead of blue as seen with the simultaneous method.

New commercial samples of Methyl Green (Gurr Certistain), Pyronine G (Gurr Certistain) and Pyronin Y (Polysciences) have been investigated using spectrophotometry, thin layer chromatography and nuclear magnetic resonance, in addition to standardized simultaneous and sequential staining methods using purified Ethyl Green and pure Pyronin Y as reference dyes. The Methyl Green was found to be Ethyl Green contaminated with Crystal Violet. It did not have any advantages compared with Ethyl Green supplied by American dye companies. The Pyronine G sample was Pyronin Y with a high dye content that gave good staining results when used with purified Ethyl Green. Pyronin Y from Polysciences was found to be essentially pure Pyronin Y.

Commercial methyl green dyes were converted to tetrafluoroborate by means of NaBF4-solution, the compounds thus obtained were analytically pure. It was shown to be possible to distinguish between a "methyl" and an "ethyl" compound by means of NMR spectroscopy. The dyes are stable in buffered aqueous solution, and in crystalline form. A spectrophotometric assay is proposed.

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