AbstractBacterial biofilms relieve themselves from external stresses through internal rearrangement, as mathematically modeled in many studies, but never microscopically visualized for their underlying microbiological processes. The aim of this study was to visualize rearrangement processes occurring in mechanically deformed biofilms using confocal-laser-scanning-microscopy after SYTO9 (green-fluorescent) and calcofluor-white (blue-fluorescent) staining to visualize bacteria and extracellular-polymeric matrix substances, respectively. We apply 20% uniaxial deformation toPseudomonas aeruginosabiofilms and fix deformed biofilms prior to staining, after allowing different time-periods for relaxation. Two isogenicP. aeruginosastrains with different abilities to produce extracellular polymeric substances (EPS) were used. By confocal-laser-scanning-microscopy all biofilms showed intensity distributions for fluorescence from which rearrangement of EPS and bacteria in deformed biofilms were derived. For theP. aeruginosastrain producing EPS, bacteria could not find new, stable positions within 100 s after deformation, while EPS moved toward deeper layers within 20 s. Bacterial rearrangement was not seen inP. aeruginosabiofilms deficient in production of EPS. Thus, EPS is required to stimulate bacterial rearrangement in mechanically deformed biofilms within the time-scale of our experiments, and the mere presence of water is insufficient to induce bacterial movement, likely due to its looser association with the bacteria.
Visualization of extracellular polymeric substances in Aspergillus niger biofilms using lectin-conjugates and confocal laser scanning microscopy (CLSM)
