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Two stains long used in exfoliative cytology, the hematoxylin-eosin Y and Papanicolaou stains, have not been standardized even today. Some dozens of hematoxylin and eosin and Papanicolaou staining recipes have been recommended in the literature. Consequently, the staining pattern of hematoxylin and eosin, and Papanicolaou stained cytological material varies from laboratory to laboratory. To a certain degree this is due to batch-to-batch variations of commercial samples of the natural dye hematoxylin (C.I. 75290). The present paper describes a simple, standardized and reproducible procedure using thionin bromide to replace hematoxylin in the hematoxylin and eosin stain.

A method is described for the purification of the dye azure B in quantities sufficient for biological staining experiments on a larger scale. The method is based on the use of column chromatography. Two columns are employed. In column A with silica gel as adsorbent the azure B fraction is isolated from a suitable substrate ('technical' azure B gained by a modification of Bernthsen's synthesis of methylene blue, or polychrome methylene blue) using an acetate-formate mixture as eluent. In column B, on an Amberlite polymeric adsorbent (XAD-2) the acetate-formate anions are exchanged in chloride. Regeneration of both columns is possible: KMnO4, Na2S2O4 and water are run through column A; 5% NaOH, methanol and water through column B. Purification of azure B on economic terms is thus attained. The opinion is expressed that this method is also applicable to the purification of other cationic dyes.

The Giemsa stain is one of the most widely used staining techniques in cytology, especially in hematology. A standardized Romanowsky-Giemsa staining procedure using pure cationic azure B (C.I. 52010) and anionic eosin (C.I. 45380) has been described by Wittekind et al (1982). A revised standard Giemsa staining procedure was recently published (Wittekind and Kretschmer 1987). Usually the Romanowsky-Giemsa stain is applied to air dried and methanol fixed cytological material, e.g. blood smears and bone marrow films (ICSH 1984).