Characterizing a live shear-resistant (SR-) biofilm and its interaction with substrates of varying energy landscapes by digital holographic microscope in eChip microcosm

Recent studies reveal that biofilm can develop under severe flow shear (e.g. >10,000 s-1) and eventually becomes resistant to shear erosion. Additional anecdotal evidence suggests clear correlation between biofilm structure and its underlying substrate energy landscapes. In this study, we are to investigate systematically the effects of these two environmental factors on formation of SR-biofilm. Here, we present experimental techniques that combine a long-term ecology-on-a-chip (eChip) milli-/micro-fluidic platform to grow a live SR-biofilm and a digital holographic microscope (DHM). The newly improved eChip platform not only provides long-term well controlled environments to a live SR-biofilm but also allow DHM to track thousands of individual bacteria as they interact with the substrate. New milli-fluidics also enables the interchange of substrates (bottom wall) containing different energy landscapes (e.g. alternating hydrophilic-hydrophobic patterns). Model bacteria include E.coli.(AW405), P. aeruginosa (PAO1) and its 12 mutants. Apart from homogeneous hydrophobic and hydrophilic substates, six patterned substrates (i.e. hydrophilic micro-patches, microscale squares and stars, of 20um, 50um, 100um over hydrophobic background) are used. Interactions of bacteria with these substrates are conducted under two shear flow rates (0 &10ul/min). During each experiment, bacteria will be cultured in eChip platform and flow over the patterned substrates for observation. Thousands of individual bacteria are tracked simultaneously in 3D over 20min at 14.5 frames per seconds at 20X and subsequently 3D trajectories, from which changes of cell motility (swimming speed, reorientation motility, and their translational/angular dispersions) as well as their attachment rates, will be obtained. In this talk, we will first present the novel microfluidic approach and robust digital holography technique (recording & analysis) in measuring microbial motilities/particle mobilities, then followed by a kernel study of P. aeruginosa in quiescent fluid interacting with substrates.