Study of 3D bacterial motion and biofilm formation by digital holographic microscopy*

Digital Holographic Microscopy (DHM) was applied to study the 3D bacterial motion and biofilm formation.The bacterial sample was placed in a closed chamber with a thickness of 180 µm and the bacterial motion recorded at 50 frames per second continuously for 40 s at every 1 hour. The experiment began at a low bacterial concentration of 500 cells/mm³ and ran for a duration of 4 hours by which the concentration increased to 6000 cells/mm³. The holograms were post-processed to generate 3D trajectories of bacteria. Two strains of Shewanella sp. were studied, one showing preferential growth near the surface (i.e., creating biofilm), while the other exhibiting similar growth rates throughout the chamber (i.e., no detectable biofilm formation). We analyzed and compared the velocity and mean-square-displacement (MSD) of the two strains. We found that in the near-wall region, both bacterial strains have small velocity and small MSD. While in the bulk region, the strain creating biofilm has a much large velocity (50 µm/s) and a large MSD compared to the one showing no preference growth. The large velocity may indicate that the bacteria are actively searching for solid surface. Our result suggests that biofilm formation is related to the bacterial swimming behavior in the bulk region.