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A standardized Romanowsky stain prepared from purified dyes.

1975; BMJ; Volume: 28; Issue: 11 Linguagem: Inglês

10.1136/jcp.28.11.920

1472-4146

P. N. Marshall, Sharon A Bentley, S. M. Lewis,

Dyeing and Modifying Textile Fibers

Stains consisting of combinations of various thiazine dyes with eosin, for example, those of Giemsa, Leishman, and Wright, are used routinely to study the cellular morphology of blood and bone marrow.The major problem in the use of these Romanowsky- type stains is batch-to-batch variations in their staining properties as a result of variations in their dye composition and the presence of contaminating metal salts (Lillie, 1943 and 1944a; Lillie and Roe, 1942; Marshall et al, 1975a; Saal, 1964; Scott and French, 1924).The extent of these variations in a large number of commercially available Romanowsky-type stains was demonstrated in a previous study (Marshall et al, 1975a).In this study it was found that the simplest successful stain examined contained methylene blue, azure B, and eosin and was only mini- mally contaminated with metal salts.Accordingly, we have produced a stain from purified samples of these dyes. Materials and Methods PURIFICATION OF DYESMethylene blue and azure B were purified as des- cribed previously (Marshall and Lewis, 1975).Commercial samples of eosin (table) were freed of contaminating metal salts and volatile material and converted to the free acid by the following scheme:(a) The commercial dye was dissolved at 10 g/l in 0-1 M aqueous ammonia.Any insoluble material was removed by filtration.(b) 0-2 M aqueous hydrochloric acid was added to this solution until precipitation ceased.(c) The precipitate was removed by filtration and washed several times with dilute aqueous hydrochloric acid (ca 0'0005 M).