Biofilm observation by confocal laser scanning microscopy

Polystyrene coupons (1.0 × 1.0 × 0.1 cm) were first cleaned by washing with liquid neutral detergent and water, followed by rinsing with distilled water and then immersing in 70% ethyl alcohol for 1 h to remove fat. Subsequently, they were rinsed with distilled water and air-dried under UV light. Confirmation of the coupons sterility was performed by the incubation of some coupons in LB broth at 37 °C for 24 h following by turbidity verification.

For biofilm formation assays, polystyrene coupons were immersed in LB broth contained 24-well polystyrene microtiter plates and inoculated with the mixed culture of S. aureus in the presence and absence of nisin at a concentration of 2.01 mg l⁻¹, which allow the growth of all strains. Then, after 46 h at 37 °C at static condition, the coupons were rinsed twice by immersion in PBS (0.2 M; pH 7.2) and incubated in the dark for 15 min with a mixture of 20 mg l⁻¹ propidium iodide (PI) (Sigma-Aldrich, Germany) and 2 mg l⁻¹ fluorescein isothiocyanate (FITC) (Sigma-Aldrich, Germany) in PBS (0.2 M, pH 7.2) prepared immediately before use. After incubation, the coupons were washed by immersion in PBS and analyzed by a confocal laser scanning microscope model LSM 510 META (Zeiss, Germany) using argon laser with a wavelength of 458 to 514 nm. Cells with green color were considered alive and those showing yellowish or reddish color were considered dead.