Live-cell imaging of swarming bacteria in a fluidic biofilm formed on a soft agar gel substrate

Abstract

Flow visualization techniques, such as particle image velocimetry and particle tracking, have become very useful tools for analyzing the collective dynamics of swarming bacteria surrounded by a thin fluidic biofilm on a substrate. In this paper, we focus on long-term live-cell imaging of Bacillus subtilis cells grown on soft agar gel substrates of different agar concentrations to determine the effect of substrate stiffness on their swarming motility inside the fluidic biofilm. The number of viable cells and velocities of swarming bacteria in colonies were measured by phase contrast imaging and multiple particle tracking techniques. We found that bacteria in the fluidic biofilm have the ability to switch between the growth and swarming phases periodically. Agar concentration between 0.5 and 2.5 % did not affect the velocities of bacteria in the swarming phase significantly but 0.5 % agar shortened the switching period compared with agar concentrations above 1 %. Furthermore, vortical flow field generated by swarming bacteria in the fluidic biofilm was visualized in detail for the first time by a correlation-based velocimetry technique with submicron fluorescent beads. The ultimate aim of this research is to uncover a novel physical factor that regulates bacterial growth and swarming dynamics on soft gel substrates.