Reduced Methylene Blue as a Stain for Crustacean Nerves

Effective in situ staining of crustacean nerves was achieved with leuco methylene blue reduced with either ascorbic acid or sodium hydrosulfite (Na₂S₂O₄). A stock solution of methylene blue, 0.4% (ca. 0.001 M), and the reductants, ascorbic acid or sodium hydrosulfite (0.01 M), were prepared in van Harreveld's crayfish physiological solution. Methylene blue stock solution was mixed with either of the reductants in the approximate ratio of 1:10, v/v, and titrated to the end point. Ascorbic acid reduction is light catalyzed and requires intense illumination during titration. The cleared or leucomethylene blue stock solution is suitable for immediate use as a working nerve stain. With either reductant, the working solution oxidizes on standing in air, but can be titrated repeatedly without loss of staining properties. Dissected nerve trunks or tissue were immersed in the working stain for 20 min at room temperature and the staining process observed until suitable contrast developed. Excess dye was decanted and the tissues flooded with crayfish physiological solution. Contrast could sometimes be enhanced by flooding the stained area with 1% hydrogen peroxide in van Harreveld's solution. When permanent mounts were prepared, tissues were dehydrated with tertiary butyl alcohol in preference to ethyl alcohol series. For anatomical and neurophysiological studies of nerve distribution in crustaceans, the alternative use of either ascorbic acid or sodium hydrosulfite, as reductants for methylene blue, was preferable to the more complicated rongalit-technique and characterization of neural elements was fully as satisfactory.