Fig. 2

Hemolysis of erythrocytes through supernatants from S. aureus depends on alpha toxin and gamma toxin. a To measure percentage of hemolysis, cells were grown to stationary phase, and sterile-filtered supernatants were diluted 1:50 in 1X PBS. Absolute hemolysis was defined as the optical density of freed heme upon the addition of the natural glycoside saponin (from Quillaja saponaria) and was set at 100%. Each run of hemolysis was accompanied by parallel measurement of saponin as positive control and 1X PBS as negative control. Percentages were calculated from mean values out of three independent experiments. Groups i) to iii) are defined in the text. Recombinant wild type alpha toxin and recombinant wild type gamma toxin were diluted in 1X PBS to receive 100 ng, 10 ng, and 1 ng per well. For neutralisation, 100 ng of toxin was incubated with or without 50-fold diluted antisera (AS) in parallel for 1 h at 37 °C. b Western blot analysis of HLG1 protein levels in the supernatants of tst-positive isolates. 20 μl were taken from each supernatant (sn) and loaded undiluted. 0.1 μg/μl of recombinant wild type HLG1 was used as positive control (lane 1, grey arrow). Broad range standard (Biorad) was added to identify correct bands (lane 2). Upon blotting, membrane was cut to avoid extensive background. c Neutralisation of hemolysis of erythrocytes through antisera was assessed. To verify specificity, antiserum against alpha toxin or gamma toxin was used as control for the opposite hemolysin (100 ng per well). Antisera were applied in a final 50-fold dilution and incubated for 1 h at 37 °C. Hemolysis and percentage calculation (here for neutralisation of hemolysis) was done as described in (a)