Biofilms are ubiquitous communities of bacteria that are interconnected by the extracellular matrix. The bacterium Bacillus subtilis forms floating biofilms at the water / air interphase, also called pellicles. After optimization and development, we applied the improved cultivation method to monitor the formation and dynamics of floating biofilm in situ in time under a confocal laser-scanning microscope (CLSM) in static growth conditions. We used a model strain of the wild-type bacterium B. subtilis and its mutant incapable of producing surfactin, both labeled with the constitutively expressed Yfp protein. Time-lapse CLSM imaging was perform over the entire depth of MSgg medium. By adding propidium iodide (PI), we identified dead cells and at the same time studied the effect of PI on biofilm development. The inoculum for growing biofilm was either from the cells in the exponential growth phase or from the spores. We found that regardless of the conditions tested, the population of B. subtilis, splits into two subpopulations already at the initial stages of biofilm formation. One subpopulation occupies the lower part and the other the upper part of the samples. Compared to the mutants, the distribution of the cells is shifted to the upper layers along the depth of the growth media in the wild-type strain. In addition, the dynamics of the formation and collapse of pellicle is more pronounced in the wild-type. Part of the population collapses during the formation of a floating biofilm under all tested conditions. The addition of PI to the medium has a long-term effect on the dynamic of the floating biofilm formation, and in higher concentrations leads to increased lysis and decreased final biomass. Type of the inoculum affects the formation of a floating biofilm. By spore inoculation, the time from inoculation to the formation of a mature biofilm prolongs and the rise above the initial growth medium surface and maximum thickness of the biofilm increase. In the case of the surfactin mutant, the difference is amplified, but in all cases, a mature pellicle is formed.
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