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.