Zeta potential, defined as the electric charge at the shear plane, is widely used as a proxy for bacterial cell surface charge. Nonspecific adsorption of ions or polyelectrolytes onto the cell surface, however, alters the value and polarity of the measured zeta potential, leading to erroneous results. Multiple wash and centrifugation steps are commonly used in preparing cells for zeta potential analysis, where various wash buffers (such as 9 g/L NaCl, 0.001M KCl, and 0.1M NaNO3) are routinely used for removing (via charge screening) ions and charged molecules that bind nonspecifically to the cell surface. Preliminary data revealed that, for Escherichia coli DH5α grown in LB Lennox (with 2 g/L glucose, LBG hereafter), the zeta potential-pH profile was not significantly different over the pH range from 2 to 12 for deionized water, 9 g/L NaCl, and phosphate buffer saline (PBS) wash buffers. As LBG is a low salt medium without a phosphate buffer, it was likely that the extent of nonspecific adsorption of ions on the cell surface was not severe, and the different wash buffers would, correspondingly, not exert much effect on measured zeta potential. For E. coli grown in a semi-defined medium (with a high capacity phosphate buffer system), the zeta potential-pH profile was significantly different over the pH range from 1 to 12 for deionized water, 9 g/L NaCl, 0.1M NaNO3, 0.1M sodium acetate, and 0.1M sodium citrate wash buffers with the extent of difference positively correlated with wash buffer’s ionic strength. Furthermore, the point of zero charge (pHzpc) for E. coli grown in the semi-defined medium varies between 1.5 and 3, in an ionic strength-dependent manner, for the various wash buffers tested. Collectively, this preliminary study highlights that wash buffer ionic strength plays important roles in affecting removal efficiency of nonspecifically absorbed ions on bacterial surfaces, where a threshold exists (0.15M) for charge screening to be effective, while 0.6M ionic strength might result in intrinsic cations removal, leading to possible cell surface damage and erroneous measurements.