Iron Hematoxylin Chelates. I. The Weil Staining Bath

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Abstract
The procedure for the preparation of the staining solution for the Weil myelin sheath stain was systematically varied in respect to pH, concentration, time, temperature and relative proportions of the ingredients. The results were explainable on the basis of the presence of a number of iron hematoxylin chelates in the staining bath. Compounds of the form of [FenHem]m+ are nuclear stains, those of the form of [FeHemn]m± are myelin sheath stains while the precipitate is probably [FenHemy]xm±. The following procedure for the stain is recommended. Mix equal portions of a 0.25% solution of ripened hematoxylin prepared from a 10% alcoholic solution and 1% ferric ammonium sulphate and use immediately. Preferably, the solutions should be at a temperature of about 5 C and the staining done in the refrigerator, but room temperature may be used. Higher temperatures are contraindicated. Hematein should not be substituted for ripened hematoxylin; the resulting stains are too weak to be usable. The absorbance of hematein is no measure of the concentration of the component that stains myelin sheaths. Hematein apparently consists largely of a sparingly soluble highly colored inactive compound.