Characterizing Biofilm Structure and Viability

A confocal laser scanning microscope (CLSM, LSM 700, Zeiss) was used to analyze the biofilm structure and viability. Before CLSM analysis, a piece of graphite plate with biofilm (0.5 × 1 cm) was sampled from each reactor in an anaerobic glove box and was dipped in sterilized PBS to remove loosely attached planktonic cells or debris. The sample was then stained with LIVE/DEAD BacLight staining kit (Molecular Probes, Invitrogen), by which cells with damaged membrane can be stained by PI and visualized as red cells while the intact cells are stained by both PI and SYTO 9 and visualized as green cells. Randomly sampled view-fields of each biofilm were observed (n ≥ 10), pixel-based biofilm viability was analyzed and presented as described before9 (link)10 (link). A CFU count was also performed with LB agar plates. The biofilm cells were scraped into LB broth with a sterilized blade. The LB broth containing biofilm cells was then gently blended to scatter the cell clusters and observed under a microscope to avoid cell death or clustering. The blended samples were spread on the LB agar plate and the CFUs were counted after 24 and 48 hours.