Quantitative Hemolysis Evaluation of Bacterial Strains

We determined the hemolysis types of 11 strains (10 candidate mutant strains and a wild-type control) by streaking the bacterial cells on sheep blood agar plates. We selected a strain with a positive result from the blood agar plate assay for further quantitative investigation of hemolysis. We measured the hemolysis capability of each strain using the method previously described^{49 (link),50 (link)}. Briefly, the bacterial cells were cultured overnight, then sub-cultured in 10 mL of tryptic soy broth (TSB) for 3 h at 37 °C (optical density at 600 nm (OD₆₀₀) ≈ 0.6). After centrifugation of the bacterial culture at 13,000 rpm for 1 min at 4 °C, a 100-μL aliquot of the supernatant was mixed with 900 μL of 8% sheep blood, which was subsequently washed three times in phosphate-buffered saline (PBS). The resultant mixtures were incubated at 37 °C for 3 h, then centrifuged at 1,500 g for 10 min at 4 °C. We then measured the OD₄₅₀ for each strain using a BioTek Epoch spectrometer.