Uncontrolled aggregation of bacterial cells is a significant disadvantage of electrophoretic separations. Various aspects of the electrophoretic behavior of different strains of Gram-positive Bacillus cereus, Bacillus subtilis, Sarcina lutea, Staphylococcus aureus(1), and Micrococcus luteus bacteria and Gram-negative Escherichia coli bacteria were investigated in this study. Our findings indicate that bacteria can be rapidly analyzed by CZE with surface charge modification by calcium ions (Ca2+). Bound Ca2+ ions increase zeta potential to more than 2.0 mV and significantly reduce repulsive forces. Under the above conditions, bacterial cells create compact aggregates, and fewer high-intensity signals are observed in electropherograms. The above can be attributed to the bridging effect of Ca2+ between bacterial cells. CE was performed to analyze bacterial aggregates in an isotachophoretic mode. A single peak was observed in the electropherogram.