Romanowsky stain

Romanowsky staining was a prototypical staining technique that was the forerunner of several distinct but similar methods, including Giemsa, Jenner, Wright, and Leishman stains, which are used to differentiate cells in pathologic specimens.

Ehrlich had used mixtures of acidic and basic dyes for this purpose in 1879: in 1891 Romanowsky and Malakowsky independently developed a technique using a mixture of Eosin Y and oxidated Methylene Blue that was also useful for this purpose. Because the aqueous dye solutions were unstable, methanol was introduced as a solvent, and Leishman (in 1901) and Wright (in 1902) advocated use of methanol as a fixative prior to staining. Giemsa in 1902 improved this technique by standardizing the dye solutions and adding glycerol to increase solubility and stability.

The oxidation of Methylene Blue in aqueous solution using heat and alkali produces a mixture of Azure A, Azure B, Methylene Violet and Methylene Blue. Eosin Y is then added to produce a "neutral" dye. The precipitate is then dissolved in a mixture of methanol and glycerol to form a stock solution: this is diluted with water or an aqueous buffer to form a working solution that is used in the preparation of pathology specimens.