In this work we detail the development of insulator-based dielectrophoresis devices to be used for enrichment of cellular subpopulations with phenotypic differences in membrane composition. We present a novel insulator-based dielectrophoresis device design that incorporates coherently patterned three-dimensional channel constrictions and demonstrate continuous flow particle separation based on dielectrophoretic mobility. Experimental and numerical techniques were used to characterize the effects of channel geometry on particle motion and distilled to a set of design criteria for similar devices. We also detail further characterization of fluid motion due to electrothermal fluid body forces via numeric multiphysics simulations. These simulations model heat transport via convection through the channel and conduction through the substrate material and consider the coupling between fluid, electrical, and thermal equation systems via temperature-dependent material properties. Finally, we discuss the development of an automated electrode-based dielectrophoresis device for cell characterization and its application to Escherichia coli as well as wild-type and ethambutol-treated Mycobacterium smegmatis. We discuss these results in the context of Mycobacterial physiology and the mechanism of action of ethambutol.