In search of the malarial parasite: biographical sketches of the blood stain contributors.

Methylene blue was synthesized by Caro in 1876 at BASF, a chemical company. Six years later, Koch employed methylene blue when he discovered the tubercle bacillus. In 1880, Ehrlich described what he termed "neutral" dyes: mixtures of acidic and basic dyes for the differentiation of cells in peripheral blood smears. Bernthsen prepared in 1886 a relatively pure dye, obtained by decomposition of methylene blue, and called it methylene azure. In 1891, Malachowski developed a method which used mixtures of eosin and "ripened" methylene blue that not only differentiated blood cells, but also demonstrated the nuclei of malarial parasites. Romanowsky later performed the same feat with an unrepeatable method. A number of "ripening" (polychroming) techniques were investigated by different groups (Nocht 1899) but the aqueous dye solutions produced were unstable and precipitated rapidly. Subsequently, methanol was introduced as a solvent for the dye precipitate (Jenner 1899) and techniques were developed that utilized the fixative properties of the methanolic solution prior to aqueous dilution for staining (Wright 1902). Giemsa (1902) further improved these techniques by developing more precise methods of methylene blue demethylation and adding glycerol as a stabilizing agent to the methanol solvent. Today, the Malachowski-Wright-Giemsa stain continues to be regarded as the world's standard diagnostic technique for malaria.