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.