A Two-stage Method for Vital Staining with Rongalit Methylene Blue

A new method is described for vital staining of nervous tissue with methylene blue; it involves impregnation of whole embryos or tissue-fragments with the rongalitreduced dye under anaerobic conditions at pH 5, followed by development of the stain in an oxygenated neutral saline solution. Determinations of the solubility of leucomethylene blue and experiments on vital staining with it and with the oxidized dye at different pH values are consistent with the hypothesis that the dye penetrates cell membranes and tissues mainly as the leucobase, but may pass along nerve (and muscle) fibres in the oxidized form. Penetration of the leucomethylene blue below pH 5 is hindered by its ionization; above pH 5 the penetration falls off because of reduced permeability of the cell membranes.