DNase Test Agar with Toluidine Blue is differential media used for the detection of deoxyribonuclease activity to aid in the identification of bacteria isolated from clinical specimens.
SUMMARY AND EXPLANATION
The DNase test is used to detect the degradation of deoxyribonucleic acid (DNA). The test is useful for differentiating Serratia from Enterobacter, Staphylococcus aureus from coagulase-negative staphylococci, and Moraxella catarrhalis from Neisseria species.
In 1957, Jeffries et al. described a rapid agar plate method for demonstrating DNase activity of microorganisms. This procedure utilized a semi-synthetic medium with nucleic acid solution incorporated in the medium. Enzymatic activity is detected by flooding the plate with 1 N hydrochloric acid (HCl). A clear zone surrounding growth indicates a positive reaction.
DNase Test Agar is based on a medium developed by DiSalvo to adapt the rapid plate method for staphylococci. Rather than using semi-synthetic medium, DiSalvo incorporated DNA into Trypticase™ Soy Agar and subsequently reported a correlation between coagulase production and DNase activity.
DNase Test Agar with Toluidine Blue contains a metachromatic dye to eliminate the necessity of reagent addition to the agar following incubation. Toluidine blue may be toxic to some gram-positive cocci and, therefore, should be used primarily with Enterobacteriaceae.
Peptones provide amino acids and other complex nitrogenous substances to support bacterial growth. Sodium chloride maintains osmotic equilibrium. DNA is the substrate for DNase activity. DNase is an extracellular enzyme that breaks the DNA down into subunits composed of nucleotides.
Toluidine blue forms a complex with intact (polymerized) DNA. In the intact DNA complex, the toluidine blue has the normal blue color. DNase activity depolymerizes the DNA, breaking down the dye-DNA complex. In the presence of nucleotides produced from the DNase depolymerization, the dye takes on its metachromatic color, forming pink to red zones around bacterial growth. A negative test is indicated when the medium remains blue.