Biofilm analysis by confocal laser scanning microscopy

Sample preparation was performed as described previously⁸⁶ with some modifications. Inoculum was prepared as described above for biofilm assay. Cells were diluted to OD₆₀₀ = 0.2 and 1 ml of the cells was added to each well of a 12‐well microtiter plate (Greiner Bio‐one) containing a circular glass coverslip. Plates were incubated for 24 h in the anaerobic chamber. Then glass coverslips were gently washed with distilled water to remove free cells and stained with the Invitrogen Live/Dead BacLight Bacterial Viability Kit (Thermo Fisher Scientific). For staining each coverslip, 1 µl of SYTO 9 and 1 µl of propidium iodide (PI) were mixed and added to 1 ml of distilled water. The coverslips were treated with the staining solution for 15 min, followed by washing with 1 ml of distilled water. The coverslips were then removed, placed face down on a glass microscope slide, and biofilms were visualized by a laser scanning confocal microscopy on a Leica SP8 confocal microscope (Leica Microsystems Inc.), using 507/545 nm (SYTO 9) and 614/745 nm (PI) lasers, and ×20 objective. Images were analyzed using IMARIS image analysis software (Bitplane AG).