Visualization of Biofilms Using Confocal Laser Scanning Microscopy (CLSM)

Biofilm structure and microbial viability in biofilms were also visualized using CLSM. In brief, aBL-treated (500 J/cm²) and untreated monomicrobial and polymicrobial biofilms grown on the polycarbonate coupons of the CDC biofilm reactor were stained with SYTO 9 and propidium iodide (PI) (Invitrogen, United States). SYTO 9 is a dye that can diffuse through the membranes of microorganisms and stain DNA. On the other hand, PI can only penetrate the cells and dye DNA when the integrity of the membrane has been compromised. As a result, SYTO9 and PI work as an indicator of viability and death of microorganisms, respectively (Stiefel et al., 2015). However, PI can only be used for evaluating death of microorganisms when the membrane is damaged. In case bacteria are killed without membrane damage, death of bacteria may be understimated using this approach. After staining, the biofilms were examined with an Olympus Fluoview FV10i CLSM using the Alexa Fluor 488 nm and the 568 nm wavelength filters and the 10 × or 60 × objectives.