Abstract:Three viable counting methods, including MTT assay, ATP bioluminescence assay, and high-throughput growth curve assay, for live Vibrio parahaemolyticus were compared in a high-throughput manner on a 96-well culture plate. The standard curves and linear ranges of these three methods were determined. The results showed that the directly detected index of MTT assay is OD555 nm, which is the absorption at 555 nm of DMSO-dissolved formazan produced from MTT. The liner standard curve for viable V. parahaemolyticus between logarithmic viable counts (LgC) and the logarithmic OD555 nm (LgOD555 nm) was LgC=(1.0439±0.0200)LgOD555 nm+(8.0565±0.0125), with a correlation index R²= 0.9965, and the linear detection range is 7.8×106~2.5×108 CFU/ml. The directly detected index of ATP bioluminescence assay is the relative luminescence unit (RLU), which is produced by ATP of viable cells. The liner standard curve between LgC and LgRUL was LgC=(0.9590±0.0065)LgRLU+(0.9949±0.0366), with a correlation index R²=0.9994, and the linear detection range is 1.0×104~3.0×108 CFU/ml. The directly detected index of the high-throughput growth curve method is the spinodal time (Ts), which is the time reaching the reflection point on the growth curve. The liner standard curve between LgC and Ts was LgC=–(0.8727±0.0230)Ts+(9.0128±0.1572), with a correlation index R²=0.9924, and a linear detection range of 1.0×100~1.0×107 CFU/ml. These three methods were used to measure 10 broths of V. parahaemolyticus and comparison with the plate counting method, respectively. The results showed both the ATP bioluminescence assay and the high-throughput growth curve method had satisfying accuracy, but the MTT assay had lower accuracy. The high-throughput growth curve has the widest linear range and is the most suitable for high throughput measurement.
