While live cell imaging offers advantages over traditional static imaging, this approach has been challenging for studying microbes due to the difficulty in tracking very small cells in a single focal plane during long term culture. Together with the CellASIC® ONIX2 Microfluidic System for live cell imaging, the CellASIC® Trap Plates offer unique architecture that positions nonadherent live cells in a focal plane to enable continuous imaging and simpler generational analysis of progeny over time. In addition, these plates offer special features for monitoring biofilm growth and its response to environmental perturbation. Here, we report additional capability enabled by our next generation trap plate with a proprietary design that alleviates drawbacks previously imposed by cellular overgrowth. When combined with the ONIX2 system, the new plate offers an unprecedented turnkey solution to the mechanistic investigation of the impact of environmental conditions on microbes.