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Bacterial cell surface carries an electrical charge due to the myriad functional groups present, as well as assortment of ions and molecules nonspecifically adsorbed to the cell surface. Thus, solution in contact with the bacterial cell surface play a critical role in influencing the overall surface charge characteristics through conferring nonspecifically adsorbed ions and molecules. Various wash buffers are commonly used in removing nonspecifically adsorbed ions and molecules for revealing the real surface charge of the bacterium. Using electrophoretic mobility measurement of zeta potential, this study attempted to understand the surface charge characteristics of Bacillus subtilis NRS-762 (ATCC 8473) with the help of three wash buffers: deionized (DI) water, 0.1M sodium nitrate, and 9 g/L sodium chloride. Experiment results revealed that B. subtilis NRS-762 was negatively charged over the entire pH range from 1.5 to 12. Specifically, with deionized water as wash buffer, the point-of-zero-charge (pHzpc) was at pH 1.5, which indicated that large amount of negatively charged functional groups were present on the cell surface. Comparison between the zeta potential-pH profiles of B. subtilis NRS-762 cultivated at 30 oC and 37 oC revealed that the profile for growth at 37 oC was more negatively charged over the entire pH range compared to that for growth at 30 oC. This highlighted that physiological adaptation might had occurred on the cell surface for coping with growth at a higher temperature. Zeta potential-pH profiles obtained revealed that DI water could not remove significant quantities of the nonspecifically adsorbed ions and molecules. On the other hand, the zeta potential-pH profiles of cells washed with 0.1M sodium nitrate and 9 g/L sodium chloride overlapped each other substantially, and were more negatively charged over the pH range from 2 to 11, compared to that of cells washed with DI water. This revealed substantial removal of nonspecifically adsorbed ions and molecules with the use of 0.1M sodium nitrate (0.1M ionic strength) and 9 g/L sodium chloride (0.15M ionic strength), which helped reveal the actual surface charge of B. subtilis NRS-762 cells. Collectively, actual surface charge of B. subtilis NRS-762 was masked by nonspecifically adsorbed ions and molecules, which could be removed by 0.1M sodium nitrate and 9 g/L sodium chloride wash buffer. Thus, in the case of B. subtilis NRS-762, 0.1M ionic strength wash buffer was the threshold at which there was complete removal of nonspecifically adsorbed ions and molecules from the cell surface.