2.5 Analysis of hemolytic activity
The hemolytic activity in WH1 and ΔsrfA were determined with agar plates containing sheep blood cells. Briefly, 1 µL fresh broth of WH1 or ΔsrfA was used for inoculation of agar plates containing sheep blood cells, incubated at 37 oC for 48 h, then the hemolytic activity was determined to evaluate surfactin production in the strains [20].
The hemolytic activity of broth was also used to assay surfactin production in different strains [36]. The blood was collected from mice, added into 10 ml Alsever’s solution (glucose 2.05 g, sodium citrate 0.8 g, NaCl 0.42 g and citric acid 0.055 g in 100 ml water, pH 6.1), then centrifuged at 300 g for 8 min. The blood cells were collected, washed once with 10 ml Alsever’s Solution, then suspended with saline solution to 2% (v/v). The broth of different strains were centrifuged at 12,000 rpm for 5 min, then the supernatant was collected and mixed with the same volume of 2% red blood cells after serial dilution. After incubation at 37 oC for 1 h, the mixture was centrifuged at 300 g for 2 min, then the supernatant was collected for determining the OD540 value. 2% red blood cells were incubated with pure water as positive control, and incubated with the relative medium for culturing bacterial strains as negative control. The hemolytic rate was calculated to assess surfactin production in different strains as the following formula: the hemolytic rate = (OD540s-OD540n)/OD540p x 100%. OD540s: the OD540 value of sample; OD540n: the OD540 value of negative control; OD540p: the OD540value of positive control.