Spectrophotometric Characteristics and Assay of Biological Stains

Abstract

I. The chemical and spectrophotometric methods adopted in the Stain Commission laboratories for the certification of stains are described. Spectrophotometric examination is by the Beckman spectrophotometer in which the color density at the absorption maximum is determined as well as a ratio of color densities designed to detect abnormal symmetry of the absorption curve. A somewhat modified titanous chloride assay is described; eerie sulfate is used to determine the normality of the unstable titanous chloride, and the oxidant solution, which is stable, is standardized against Bureau of Standards arsenious oxide. The general approach used for re-study of the stains by these methods and the criteria necessary for adoption of the spectrophotometric method of assay are described. II. This report gives the details of the spectrophotometric examination and assay of the individual stains in the thiazin group, namely methylene blue, thionin, methylene violet, toluidine blue O, and azures A, B, and C. The spectrophotometric method of assay was adopted for the first three named; the others showed too large a variation among samples to permit adoption of this simpler type of assay. In the case of methylene violet it was necessary to do nitrogen analyses to relate purity of the dye and color density. Complete absorption curves for typical samples of each of the above stains are presented.