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Proteins and metabolites typically adsorb to bacterial cell surface through a variety of mechanisms such as van der Waals attraction and electrostatic interactions, and forms a layer of nonspecifically adsorbed ions and molecules on the cell surface. Thus, the bacterial cell surface charge comprised the contribution from the cell wall as well as layers of nonspecifically adsorbed ions and molecules on the cell surface. This is the cell surface charge perceived by other bacterial cells in the growth medium. Given that different growth medium comprises different ensemble of proteins and metabolites that could adsorb onto the cell surface of bacteria, it is important to examine the effect of growth in different medium on the cell surface characteristics of bacterial cells using zeta potential as the proxy parameter. Defined at the shear plane, zeta potential provides a comprehensive view of the cell surface charge that include the nonspecifically adsorbed ions and molecules on the cell surface and the intrinsic electric charges in the cell wall. Using Escherichia coli DH5α (ATCC 53868) as model organism, experiments performed with deionized water as wash and resuspension buffer revealed that the zeta potential-pH profiles of cells grown in LB Lennox, and LB Lennox with 2 g/L glucose overlapped each other over the entire pH range from 2 to 9. This suggested that there was little physiological adaptation of the cell envelope of cells grown in LB Lennox supplemented with 2 g/L glucose, which indicated that the medium could be used for increasing biomass yield without affecting cell surface characteristics. Similarly, the zeta potential-pH profiles of E. coli DH5α grown in LB Lennox and LB Lennox buffered with 89 mM potassium hydrogen phosphate buffer also overlapped each other, which highlighted that the buffered medium did not elicit physiological adaptation of the cell envelope. However, supplementation of the LB Lennox (buffered) medium with 6 g/L glucose resulted in a more negatively charged zeta potential-pH profile in the pH range from 4 to 12 compared to that during growth in LB Lennox. Growth of E. coli DH5α in other media such as Tryptic Soy Broth (TSB), formulated medium, and formulated medium with 6 g/L glucose also resulted in more negatively charged zeta potential-pH profiles compared to that during growth in LB Lennox medium. However, the point-of-zero-charge (pHzpc) of cells grown in TSB and formulated medium were the same as that of cells grown in LB Lennox medium. Collectively, physiological adaptation to growth in different media as well as different ensemble of proteins and metabolites nonspecifically adsorbed to the cell surface would generally result in different zeta potential-pH profiles of bacteria cultivated in growth media of different compositions. Understanding the cell surface charge characteristics of E. coli DH5α grown in different media would thus help unveil the mysteries of cell-cell interactions in the medium.