Biofilms are bacterial aggregates, encased in extracellular matrix and attached to biological or abiological surfaces. They represent an important bacterial lifestyle and protect encased bacterial cells. Biofilms represent a survival strategy that helps bacteria survive unfavourable conditions and/or colonize different ecological niches. In B. subtilis early stage of biofilm formation is associated with transformation of planktonic cells, which lose mobility and align into chains. Transformation stages into sesile mode of growth and especially formation of floating biofilms (pellicles) that form at liquid-air interface are poorly understood at the microscopic level. Here we developed a method to temporally monitor cellular distribution during pellicle formation at different depths of microscopic specimen using the model bacterium B. subtilis PS-216. In addition to the wild type strain we also evaluate its mutant Δeps, lacking matrix polysaccharide Eps. Results show that B. subtilis pellicle formation is a very dynamic process and that biofilm form either on the surface of the growth medium or at the bottom of the petri dish. Propidium iodide staining has further shown that cells at the bottom have impaired membranes and lysed relatively soon after the onset of incubation. A very different scenario was observed for the Δeps mutant that transiently formed but did not retain the pellicle at the surface of the growth medium.