MICROSPECTROPHOTOMETRIC ANALYSIS OF BASIC PROTEIN RICH SITES STAINED WITH BIEBRICH SCARLET

STEVEN D. DOUGLAS ¹, SAMUEL S. SPICER ¹, and PETER H. BARTELS ¹

¹ National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Public Health Service, United States Department of Health, Education, and Welfare, Bethesda, Maryland 20014

Microspectrophotometric analyses on histologic sections stained with the acid dye Biebrich scarlet at pH 9.6 and above reveal absorption curves with maxima at 505 mµ for pink-red orthochromatic sites. Such sites of strongly basic protein include particularly spermatozoa, eosinophil and heterophil granules, Paneth cell granules, elastic fibers and seminal vesicle fluid. In contrast, absorption curves of sites with a metachromatic red-orange staining have a maximium of 485 mµ. The structures containing a strongly basic metachromatic protein invariably are the sites of a polyanion with metachromatic staining properties. Such sites include nuclei, mast cell granules, ovarian follicular fluid and various mucous acini and goblets. The absorption spectra of orthochromatic and metachromatic sites in tissue sections correspond with the spectra of Biebrich scarlet in solution alone and with polyanions, respectively. Similarly, histologic structuures containing acid mucosaccharides when stained with the basic dye safranin O at low pH displayed absorption maxima at 530 mµ pink-red orthochromatic sites and at 500 mµ for red-orange metachromatic sites.

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