Chemical Basis for the Histological Use of Safranin O in the Study of Articular Cartilage

Interpretation of the histological behavior of metachromatic dyes requires information regarding their absorption spectra in solution and in histological sections. This information, previously unavailable for the interaction of safranin O with chondroitin sulphate or keratan sulphate in solution is provided by the work reported. The orthochromatic and metachromatic absorption spectra of safranin O in solution have been compared with the spectra of safranin O in histological sections of cartilage mounted under water or permanently mounted after dehydration through alcohol and xylene. Under water, safranin O is bound to cartilage mucopolysaccharide in the metachromatic form; in permanently mounted sections, safranin O is transformed to the orthochromatic form. In permanently mounted sections, safranin O seems to bind only to tissue polyanions and not to collagen. Chemical studies of the interaction of safranin O with chondroitin 6-sulphate and keratan sulphate in solution indicate that essentially one molecule of the dye is bound to each negatively charged group of both mucopolysaccharides. Of two cationic dyes studied, safranin O most nearly begins to meet three criteria proposed as a basis for a simple quantitative histochemical method for following changes in the concentration of mucopolysaccharides in cartilage ground substance.