Dielectrophoresis (DEP) represents a powerful approach to manipulate and study living cells. Hitherto, several approaches have used 2-D DEP chips. With the aim to increase sample volume, in this study we used a 3-D carbon-electrode DEP chip to trap and release bacterial cells. A continuous flow was used to plug an Escherichia coli cell suspension first, to retain cells by positive DEP, and thereafter to recover them by washing with peptone water washing solution. This approach allows one not only to analyze DEP behavior of living cells within the chip, but also to further recover fractions containing DEP-trapped cells. Bacterial concentration and flow rate appeared as critical parameters influencing the separation capacity of the chip. Evidence is presented demonstrating that the setup developed in this study can be used to separate different types of bacterial cells.