The dielectrophoretic behavior of active, dead, and dormant Mycobacterium smegmatis bacterial cells was studied. It was found that the 72-h-old dormant cells had a much higher effective particle conductivity (812±10 µS cm-1), almost double that of active cells (560±20 µS cm-1), while that of dead (autoclaved) M. smegmatis cells was the highest (950±15 µS cm-1) overall. It was also found that at 80 kHz, 900 µS cm-1 dead cells were attracted at the edges of interdigitated castellated electrodes by positive dielectrophoresis, but dormant cells were not. Similarly, at 120 kHz, 2 µS cm-1 active cells were attracted and dormant cells were not. Using these findings a dielectrophoresis-based microfluidic separation system was developed in which dead and active cells were collected from a given cell suspension, while dormant cells were eluted. © 2010 American Institute of Physics.