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Patterns of hemolysis produced by staphylococci isolated from milk were investigated on media prepared with bovine, sheep, horse, and rabbit erythrocytes under a variety of cultural circumstances. Hemolytic patterns were sharper and easier to interpret on erythrocyte agar plates when incubated at 37 degrees C in an atmosphere containing 30% CO(2.) Alpha-hemolysin production was greatly enhanced in this environment and was not detected at times when cultures were grown in air only. This was also true of deltahemolysin but to a lesser extent. Very satisfactory identification of alpha, beta- and delta-hemolysins was obtained by streaking BEA plates with the unknown strains perpendicularly to a strain producing beta-hemolysin and incubating in 30% CO(2.) This procedure avoided use of erythrocytes of different species of origin and use of staphylococcal alpha-antitoxin. Prior action of the beta-hemolysin on bovine cells was found to completely inhibit hemolysis from alpha-hemolysin but the reverse was not true. A strain inhibiting beta-hemolysis in air failed to exhibit its antihemolytic properties for 24 hours when incubated in 30% CO(2.) Patterns of hemolysis within areas subject to multiple hemolysins were found to reflect the nature and relative strength of contributing hemolysins or antihemolysins and could be modified by the time sequence of exposure to certain hemolysins or antihemolysins.

Observations were made on the value of certain selective media for isolation and identification of staphylococci from milk obtained from individual quarters and farm bulk tanks Aerobic fermentation of mannitol salt was unreliable for identification of coagulase positive strains. Coagulase positive strains tended to clump when grown in tryptose serum broth. Tellurite glycine agar plates were not entirely reliable for the selection of coagulase positive strains. Fibrinogen-tellurite-glycine plates were very useful for the isolation of staphylococci from milk in the presence of other microorganisms and for the simultaneous identification of coagulase positive strains. Plate coagulase tests utilizing fibrinogen tellurite glycine agar corresponded almost perfectly with conventional tube tests. Fibrinolytic strains were not uncommon and were found also among beta hemolysin producing coagulase positive strains.