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Zeta potential, defined as the electric charge at the shear plane, is widely used as a proxy parameter 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 (by charge screening) ions and charged molecules that bind nonspecifically to the cell surface. Using Escherichia coli DH5α grown in LB Lennox (with 2 g/L glucose), experiment data showed that 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 LB Lennox 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. Zeta potential-pH profiles for E. coli grown in a semi-defined medium (with a high capacity phosphate buffer system), on the other hand, 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 deviation 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 the importance of wash buffer ionic strength in affecting removal efficiency of non-specifically absorbed ions on bacterial cell surface, where a threshold exists (0.15M) for charge screening to be effective. At the upper bound, 0.6M ionic strength might remove cations intrinsic to the cell envelope, leading to possible cell surface damage and erroneous measurements.
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Supplementary information of zeta potential of bacterial cells
Enlarged figures of the ones presented in the preprint.