Two DNase detection techniques in which the metachromatic dye toluidine blue O (TBO) is used have been improved, and a potential source of difficulty for personnel attempting to use TBO-related methods has been identified. Reducing the concentration of TBO in the Streitfeld plate-flooding method from 0.1 to 0.05% resulted in easier control of staining intensity, less masking of DNase-positive reactions due to overstaining, sharper delineation of zones of DNase activity, and more sensitive detection of weak DNase reactions. Incorporation of 0.005% TBO in DNase agar, rather than the recommended 0.01%, allowed growth and expression of DNase activity by gram-positive as well as gram-negative bacteria. The reduced dye content in the agar also enhanced expression of DNase activity by some organisms and provided sharper delineation of DNase-positive reactions. Because optimum expression of DNase activity depends upon exact TBO concentrations in both the flooding and agar incorporation techniques, strict attention must be paid to the dye content of commercially available TBO dye powders. TBO concentrations must reflect actual dye content; therefore, calculations must include a conversion factor that accounts for the true dye content of the commercial preparation. The conversion factor that we developed is determined by dividing 100 by the percentage of dye in the commercial powder. The grams of commercial dye powder required per 100 ml of dye mixture is calculated by multiplying the percentage of dye required in the dye mixture by the conversion factor.