PLATE XVIIT H E aim of this study was to detect a number of haemolytic and non-haemolytic extracellular products formed by selected strains of Staphylococcus aureus. For this purpose we used the electrophoretic localisation technique (Haque, 1967) by means of which the various components of the crude culture filtrates are first separated by electrophoresis in agar gel and then recognised by their biological activities. This technique has the advantages that interactions among the various products are minimised, and that the products can be identsed directly by their electrophoretic mobility and biological activities. We anticipated that the use of these criteria of identification would not only help to define the number and type of extracellular products but would aid us in selecting suitable cultures for future studies of the purification of these substances.
MATERIALS AND METHODSOf the 13 cultures of staphylococci used, the Wood 46-white, Wood 46-clear, Foggie, 502A, 146P, and 681C strains of S. aureus have been previously described (Haque, 1967;Murphy and Haque, 1967;Baman and Haque, 1970). S. aureus strains 233, T19, E-delta, and N-delta were supplied by Dr S. K. Maheswaran, Department of Veterinary Bacteriology Public Health, University of Minnesota, St Paul, Minnesota; strain Wood 46M was supplied by Dr A. W. Bernheimer, Department of Microbiology, New York University School of Medicine, New York, NY; and strain PG114 was supplied by Dr F. Kapral, The Ohio State University, Columbus, Ohio. The M3 strain of S. epidermidis, a glucose-fermenting, coagulase-negative staphylococcus, was isolated in our laboratory from a marmoset.All cultures were maintained on Trypticase Soy Agar (Difco) slants. Before use they were checked for haemolytic activity by streaking for isolation on rabbit, sheep, human, and horse blood agar plates.Production of cell-free filtrates. Each culture was grown on dialysis membranes placed over Brain Liver Heart Agar (Difco) plates at 37°C for 20 hours in an atmosphere containing 10-15 % C02 in air. Growth from 25 such membranes was harvested in 12.5 ml of phosphatebuffered saline (PBS), pH 7.3, centrifuged at 3000 g for 15 min. and the supernate filtered through a sterile 0.22-pm membrane-filter.Measurement of biological activities. The haemolytic activities of the filtrates were measured by the tube-titration and the substrate-agar-plate methods (Baman and Haque, 1970). Other biological activities were measured by the substrate-agar-plate method.Preparation of substrate agar. The following substrates were separately added to a sterile 1-5 % (w/v) solution of Bacto Agar (Difco) in PBS, pH 7.3, that had been cooled to