Toluidine Blue DNA Agar, Modified

Principle And Interpretation

Toluidine Blue DNA Agar, Modified is recommended for detection of thermostable deoxyribonuclease activity and establish speciation of S. aureus in contaminated foods in accordance with FDA BAM, 1998. Staphylococcus is a Gram-positive bacteria which includes several species that can cause a wide variety of infections in humans and other animals through infection or the production of toxins. Staphylococcal intoxication worldwide stands out as one of the main food-borne diseases, with a frequency in incidents of microbiological origin second only to salmonellosis(1). Foods that lack hygienic conditions and that are improperly stored can aid in the Staphylococcal growth and toxin production. Since the infection is caused due to toxin, Staphylococcal food poisoning is also called as 'food intoxication' (2). Toluidine Blue DNA Agar, Modified (M613F) is recommended for detection of thermostable deoxyribonuclease activity and establish speciation of S. aureus in contaminated foods in accordance with FDA BAM, 1998(3) with a slight modification in concentration of calcium chloride and toluidine blue. DNA in the medium enables the detection of DNase activity by getting depolymerized and forming a clear zone around the microbial growth. Inclusion of toluidine blue aids in detection of DNase activity by the production of a visible bright rose-pink coloured reaction due to its metachromatic properties.

Total plate count of the suspected sample is carried out using Baird Parker Agar (M043). Speciation of the organism can be confirmed using Toluidine Blue DNA Agar, Modified. Like the coagulase test, this is a highly specific test for the confirmation of the organism. The test is carried out in petriplates or microslides containing the media, prepared by spreading appropriate amount (3ml if slide) of the Toludine Blue DNA Agar, Modified medium. 10 to 12 wells of 1mm diameter are prepared in the plates/slides after solidification. Add 0.01 ml of the broth culture into these wells after heating in a boiling water bath for 15min. Incubate (in a moist chamber) for 4 hrs at 35°C. Development of bright pink halo extending at least 1 mm from periphery of well indicates a positive reaction. Tris amino methane forms the buffering system. Sodium chloride and calcium chloride provide the ions and also maintains osmotic equilibrium.