An evaluation of five techniques used to detect deoxyribonuclease production by bacteria

Abstract

Five techniques for the detection of deoxyribonuclease (DNase) activity compared. The best results were obtained by growing organisms on DNase test agar containing 0.005% toluidine blue O(TBO). Flooding DNase plates with acidified mercuric chloride or a 0.05% TBO solution yielded approximately equivalent results. Both methods were significantly inferior to the use of DNase test agar containign 0.005% TBO, but did detet all DNase positive organisms. DNase agar containing methyl green was inadequate for detecting weak DNase producers due to lack of contrast between zones of weak reaction and the pale green background provided by this medium. The standard DNase test, flooding of DNase test agar with 1.0 N HCl to precipitate the DNA, was the least sensitive of the five methods and failed to detect DNase production by two DNase positive organisms. The use of TBO, incorporated in DNase test agar or flooded over the surface of DNase test agar, provided a sharp contrast between the pink zones of DNase activity and a royal blue background. This color contrast provided a clearer delineation of the DNase activity than was exhibited by any of the other three techniques. The color contrast was sharpest and most well delineated when TBO was incorporated into the test agar. Calculation of TBO concentrations based on actual dye content rather than weight of the dye preparation purchased is essential for reliable results.