Non-chromotropic substances such as fibrin and gelatin and most tissue and cellular structures stain orthochromatically with internal dye concentrations of such metachromatic dyes as methylene blue and toluidine blue which, if in solution, would be metachromatic. Therefore, at ordinary levels of staining these substances depress the natural tendency of these dyes to change color. However, at elevated levels of dye-binding metachromasy eventually occurs. This phenomenon is explained on the basis of the distribution of dye-binding sites. In these substrates, by contrast with chromotropic substances, many binding sites are too far removed for dye interaction, consequently the interaction frequency can become high enough to produce a color change only as saturation of the available sites is approached.

It is also shown that the destruction of color is a characteristic of metachromasy and that water molecules intercalated between approximated dye ions are responsible for the loss and change of color. A concept of metachromasy is proposed in which the interaction between water molecules and suitably approximated dye ions plays an essential role.

The experimental studies are described against a background of the history and evolution of ideas on metachromasy. The literature is reviewed and reassessed particularly from the physicochemical viewpoint.