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The various theories of the nature of the act of staining of animal cells, tissues and products, which have been put forward by different writers fall into three groups: (i) that the process of staining is a chemical reaction between the dye on the one hand, and the cell or tissue elements on the other; (2) that in staining the dye is adsorbed by the tissue elements stained; and (3) that the dye is present in the form of a solid solution in the stained material. The problem remains, however, still unsettled, further data respecting individual stains being much to be desired.' For studying the nature of an individual staining act methylenblue-eosin was selected as being a dye which is peculiarly suitable for the purpose. This substance is a salt which is formed by the union of the basic radical of methylenblue and the acid radical of eosin. When it is allowed to act upon cells or tissues some of the elements stain blue, others red. For example, with an alcoholic or aqueous solution of methylenblue-eosin a polynuclear leucocyte exhibits blue staining of the chromoplasm of the nucleus, while at the same time the oxyphile granules of the cytoplasm stain deep red, precisely similar blue staining being obtained with methylenblue and the red staining with eosin. In the case of methylenblue-eosin, provided that hydrolysis of the dye can be excluded, the act of staining can only be explained as due to a chemical reaction between dye and cell elements. If hydrolysis is not avoided then both adsorption and solid solution of the free acid and the free base may possibly occur and produce the staining effect.

The Staining Act: An Investigation into the Nature of Methylenblue-Eosin Staining