Revised tests and standards for Biological Stain Commission certification of alcian blue dyes

ABSTRACT

Alcian blue dyes are copper phthalocyanines with a variety of cationic side chains; they are useful for staining carbohydrate polyanions while avoiding staining of nucleic acids. The properties of the original alcian blue and of similar dyes with published chemical structures are reviewed here. Variation among samples submitted to the Biological Stain Commission (BSC) for certification has led to the recognition of two types of commercially available alcian blue at this time. The designation “alcian blue 8G or equivalent” is reserved for dyes that resemble alcian blue 8GX manufactured in the 1960s (CI 74240; ingrain blue 1). These dyes react with alkali to form an insoluble pigment that cannot be re-dissolved in acid. The name “alcian blue variant” is for similar dyes that do not form insoluble pigments; an alkali-induced precipitate, if formed, re-dissolves with acidification. For certification by the BSC, both types of alcian blue must dissolve in 3% acetic acid to make a 1% solution (pH close to 2.5), which must provide selective coloration of intestinal mucus, cartilage and mast cells, but not of nuclei. After alcian blue staining and treatment with 0.03 M Na₂CO₃ or Li₂CO₃ to convert the bound dye to a pigment, the Feulgen stain for DNA is applied. Dyes to be certified as alcian blue 8G or the equivalent must resist extraction by the 5 M HCl used in the Feulgen reaction. Dyes to be certified as alcian blue variant are not required to be convertible to acid-insoluble pigments, but they must dissolve easily in water at pH 5.7 containing 0.5 M magnesium chloride and the dye must remain in solution for at least 24 h. A critical electrolyte concentration (CEC) staining test also is described; this must be passed for certification of an alcian blue variant. Successful CEC staining is also a desirable property of alcian blue 8G or equivalent, but not essential for certification of an otherwise satisfactory batch. The spectrophotometric criteria for alcian blue dyes also are revised; a wider range of absorption maximum (605–634 nm) is allowed. The dye powders used in published staining techniques with the original alcian blue 8G were 40–60% dye, but some modern alcian blue dyes have dye content as high as 90%. The BSC’s assay for dye content is not a criterion for certification, but it should influence the amount of dye to include in a staining solution.

KEYWORDS:

• alcian blue
• Biological Stain Commission
• certified stains
• dye testing
• standardization

Acknowledgments

We thank the late Hans O. Lyon and the late Thomas Wickersham for helpful advice in the preparation of this communication. JAK thanks the Natural Sciences and Engineering Research Council of Canada and the Biological Stain Commission for grants in aid of research. We also thank Ms Saba Ali, School of Chemistry, the University of Leeds, England, for providing information concerning the alkali instability of alcian blue pyridine variant prior to formal publication, and Algy Kazlauciunas, Colour Science Analytical, University of Leeds, England, and Prof. Richard Hartley, School of Chemistry, the University of Glasgow, Scotland, for useful discussions and assistance.

Additional information

Funding

This work was supported by the Natural Sciences and Engineering Research Council of Canada [None];Biological Stain Commission [None];