Based on the metachromatic property of Toluidine Blue 0, three, convenient agardiffusion methods have been developed that enable detection of the nuclease of Staphylococcus aureus at concentrations as low as 0.005 ,ug/ml in agar and broth cultures. The interactions of agar and deoxyribonucleic acid with Toluidine Blue 0 are discussed.

Some strains of Staphylococcus epidermidis and Micrococcus sp. produce nucleases. However, thermal stability was shown to be unique to the nucleases of S. aureus. In addition, two micromethods for susceptibility testing to lysostaphin were more precise and convenient than anaerobic glucose fermentation in distinguishing between the genera Staphylococcus and Micrococcus. of S. epidermidis were recovered from milk. Strains of Micrococcus sp. were generously provided by W.

The metachromatic agar-diffusion (MAD) microslide technique was shown to detect nanogram quantities of staphylococcal thermonuclease in various foods without prior extraction, purification, or concentration.

The metachromatic agar-diffusion (MAD) microslide technique was adapted for quantitative assay for staphylococcal thermonuclease in heterogeneous systems, such as milk and broth. When an enzyme-containing solution was placed in a well cut in the agar, a bright pink halo was obtained. The diameter of the pink zone of hydrolysis was related to time and temperature of incubation and to nuclease concentration. Concentrations of nuclease as low as 0.005 ,ug/ml and as high as 2.0 ,ug/ml were conveniently determined after 3 hr at 37 C.