Toluidine blue binding by developing muscle tissue: assay and data on the mechanism involved.

The correlation of the basophilia of tissues with their nucleic acid content is one of the oldest and most widely applied chemical interpretations of histological staining reactions. The interaction of basic dyes with tissue constituents has been regarded for a long time, and without explicit qualification, as a salt formation. This interpretation was based essentially on the observation of stoichiometric relations between the maximum combining capacities of proteins with dye ions, as in the experiments of Schmidt (1). The limitations of such an oversimplified explanation soon became evident; a clear distinction (2) between various types of forces which participate in these reactions seems to provide a much wider possibility of experimentation and make more concrete the interpretation of results. With increasing knowledge of the reaction mechanism of dye combination, this type of reaction can be used as a quantitative assay as well as a sensitive indicator for changes in the properties of the reacting groups. A study of the interaction of dye cations and nucleoproteins was attempted by Hammarsten et al. (3). More recently, Kelley and Miller (4) and Michaelis (5) studied the metachromatic changes in the spectral properties of some basic dyes on combination with nucleic acids. Pischinger (6) and his followers have shown how biological materials depend upon the pH and the dissociation of the substrate. This development has been reviewed and subjected to detailed criticism in a noteworthy report by Levine (7). Later this use of dyes was expanded in numerous contributions by Dempsey, Wislocki, and Singer (8), and others. An interesting series of experiments on the interaction of basic dyes with nucleoproteins after treatment with various fixing agents was carried out by Kelley (9) in continuation of his studies with pure nucleic acids. Brachet (10) used the uptake of basic dye to assay ribose nucleic acid in the cytoplasm of developing cells, controlling his observations by comparing tissue slices before and after treatment with ribonuclease. He also attempted a quantitative determination of dye after extraction from the tissues and a correlation of dye uptake with analytical data for nucleic acid content