Confocal laser scanning microscopy.

Biofilms formed in a 96-well plate or on sHAs by the procedure described above were stained with the LIVE/DEAD BacLight bacterial viability kit (Molecular Probes, Leiden, The Netherlands). Complementation of the biofilm morphology was observed by adding 200 μg/ml commercial inulin to TSBr medium before biofilm formation. Syto 9 and propidium iodide (PI) stain bacteria with an intact membrane (live bacteria, green fluorescence) and bacteria with damaged membranes (dead bacteria, red fluorescence), respectively. After the stained biofilm was washed with DW 2 times to remove the excess dye, it was examined by confocal laser scanning microscopy (CLSM) (LSM 700; Carl Zeiss, Jena, Germany). Biofilm images were acquired using a Plan-Apochromat 10×/0.45 M27 objective lens, and a 4,880-nm laser line and a 555-nm laser line for excitation of Syto 9 and PI, respectively. The Zen software (Zeiss) was used for biofilm analysis.