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A pour-plate method is described for the detection of coagulase production by Staphylococcus aureus. Either Brain Heart Infusion agar or yeast extract-Trypticase soy agar containing swine plasma was the best medium for the detection of coagulase by this method. The advantages of this method and its potential utilization in the clinical laboratory, in food microbiology, and in specialized studies with S. aureus are discussed.

A pour-plate method is described for the detection of coagulase production by Staphylococcus aureus . Either Brain Heart Infusion agar or yeast extract-Trypticase soy agar containing swine plasma was the best medium for the detection of coagulase by this method. The advantages of this method and its potential utilization in the clinical laboratory, in food microbiology, and in specialized studies with S. aureus are discussed.

THE PRODUCTION of coagulase is universally accepted as the most distinctive characteristic of Staphylococcus aureus. Furthermore, among the numerous extracellular proteins commonly associated with staphylococcal virulence, coagulase seems to be the most stably associated. Recently we have developed a technique whereby it is readily possible to detect one colony that produces coagulase among 30 OOO coagulase-negative colonies by means of a pour-plate technique (parisi, Baldwin and Sottile, 1973). Coagulase-producing colonies in the agar can be detected by the dense zone of precipitated fibrin around them. This technique makes feasible certain types of genetic studies on production and regulation of coagulase, as well as studies of other aspects of its possible role in staphylococcal disease. MATERIALS AND METHODSBacteriaf strains. A coagulase-negative mutant was obtained from strain 1-746 after exposure of the culture to 100 pg of N-methyl-N-nitro-N-nitrosoguanidine (Aldrich Chemical Co. Inc., Milwaukee, Wis.) per ml of 0 . 1~ phosphate buffer, pH 7.0. Transduction experiments were done with the coagulase-negative mutant of strain 1-746 and with a lysogenic variant of this mutant strain. Both parent and mutant, before lysogenisation, were susceptible to lysis by phages 80 and 81 of the International Basic Set of staphylococcal phages. Upon lysogenisation, the coagulase-negative mutant was untypable. The S. aweus strains listed in the table were used in the curing experiments. All cultures were maintained on Difco Brain Heart Infusion @HI) agar at 4°C.Lysogenisation of strain I-746. Lysogenisation of a coagulase-negative mutant of S.aureus strain 1-746 with phage 80 was carried out by the method of Blair and Carr (1961). Lysogenisation was confirmed by immunity to phage 80, a change in the phage typing pattern, and the release of a phage which lysed the propagating strain for phage 80 (PS 80) when the lysogenised culture was induced with mitomycin C (Sigma Chemical Co., St Louis, Mo.) according to the method of Yu and Baldwin (1971). Bacteriophage and transductions. Phage 80 was propagated, titrated, and maintained as described by Kasatiya and Baldwin (1967), except that 0 . 1~ phosphate buffer, pH 7.0, was used for harvesting the phage. For transduction, two 18-h BHI-agar-slant cultures of the coagulase-negative or lysogenic coagulase-negative mutant of S. aureus strain 1-746 were suspended in each of 2 ml of Trypticase Soy Broth (BBL) containing Difco Yeast Extract 0-3 % (w/v) (YETS) to which calcium chloride had been added at a concentration of 400 pg per ml. The h a l cell concentration was 1010 to 2 x 1010 c.f.u. per ml. One ml of suspension was mixed in a centrifuge tube with 1 ml of phage-80 suspension that had been exposed for 60 s. to ultraviolet (UV) irradiation from a General Electric 25-W germicidal lamp at a distance of 46 cm. This suspension which contained 2 x 109 plaque-forming units gave a multiplicity of infection of about 0.1, Control tubes contained 1 ml of bacterial suspension and 1 ml of Y...