Biofilm development of an opportunistic model bacterium analysed at high spatiotemporal resolution in the framework...

Life of bacteria is governed by the physical dimensions of life in microscales, which is dominated by fast diffusion and flow at low Reynolds numbers. Microbial biofilms are structurally and functionally heterogeneous and their development is suggested to be interactively related to their microenvironments. In this study, we were guided by the challenging requirements of precise tools and engineered procedures to achieve reproducible experiments at high spatial and temporal resolutions. Here, we developed a robust precise engineering approach allowing for the quantification of real-time, high-content imaging of biofilm behaviour under well-controlled flow conditions. Through the merging of engineering and microbial ecology, we present a rigorous methodology to quantify biofilm development at resolutions of single micrometre and single minute, using a newly developed flow cell. We designed and fabricated a high-precision flow cell to create defined and reproducible flow conditions. We applied high-content confocal laser scanning microscopy and developed image quantification using a model biofilm of a defined opportunistic strain, Pseudomonas putida OUS82. We observed complex patterns in the early events of biofilm formation, which were followed by total dispersal. These patterns were closely related to the flow conditions. These biofilm behavioural phenomena were found to be highly reproducible, despite the heterogeneous nature of biofilm.