Quantitative determination of cationic dyebinding in connective tissue.

Abstract

Mucoid layer of the rooster comb and cartilage of the nasal septum of steers were used to measure the binding of cationic dyes by unfixed frozen sections. The amount of dye bound was calculated from the decrease of the optical density of the dyebath after equilibrium had been reached between the dyebath and the tissue at the saturation level. Various factors influencing this reaction, such as the tissue-dye ratio, the dyebath concentration, the thickness of the tissue sections and mechanical agitation, have been studied. In order to quantitatively determine the dyebinding, certain conditions must be fulfilled. The dyebath concentration must be high enough to be well in excess of all available binding sites of the tissue. The precipitate formed and the tissue fragments must be removed prior to optical density measurement. The electrolyte concentration of the dyebath must be kept below a certain level. The dyebath should not contain colored substances other than the free dye. Under these conditions, the amount of dye bound by azure A and other cationic dyes is constant for a given tissue, depending only on the pH of the dyebath. Electrostatic interaction between the cationic dye and the anionic sites of the tissue is primarily responsible for dyebinding. The quantitative determination of dyebinding is a measure of the free anionic sites of a tissue.