A study of the effect of aggregate size on the resuscitation of dormant M. smegmatis was conducted by constructing cell aggregates with defined sizes and shapes using dielectrophoresis and monitoring the resuscitation process under controlled laboratorial conditions in a long-term cell feeding system. Differently sized cell aggregates were created on the surface of indium tin oxide coated microelectrodes, their heights and shapes controlled by the strength of the induced electric field and the shape of the microelectrodes. Both two-dimensional (ring-patterned) and three-dimensional cell aggregates were produced. The cell aggregates were maintained under sterile conditions at 37 °C for up to 14 days by continuously flushing Sauton's medium through the chamber. Resuscitation of dormant M. smegmatis was evaluated by the production of the fluorescent dye 5-cyano-2,3-ditolyltetrazolium chloride. The results confirm that the resuscitation of dormant M. smegmatis is triggered by the accumulation of a resuscitation promoting factor inside the aggregates by diffusion limitation. © 2010 American Institute of Physics.