Ehrlich had used mixtures of acidic and basic dyes for this purpose in 1879: in 1891 Romanowksy[?] 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.
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.
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