Biofilm Assay

The ability of P. aeruginosa strains to attach to surfaces as an initial step of the biofilm formation was tested by using Crystal violet staining. The attached cell mass was determined by a biofilm assay published by O'Tool et al. with modifications (O'Toole, 2010). Briefly, bacterial strains were grown overnight at 37°C and were diluted in LB broth to a concentration of OD 0.8. Aliquots of 100 μl were added to wells of a 96-well plate containing a PDMS piece (3 × 15 mm) and incubated at 37°C for 3 h to allowing the bacteria to adhere to the surface. The medium and planktonic cells were removed and the PDMS slices were washed three times with 200 μl PBS. The Biofilms were fixed for 60 min at 60°C and stained using 150 μl 0.1% (v/v) crystal violet solution for 15 min. The PDMS slices were washed in distilled water, and the water was removed. The plate containing the PDMS slices was then air-dried and the dye was eluted with 110 μl 33% acetic acid. The signal was measured using a FLUOstar Omega plate reader (BMG Labtech) at a wavelength of 585 nm. Measurements were performed in three biological repetitions consisting of 4 replications each. The reduction of cell mass was assessed by comparing treated and untreated samples. As a negative control to assess the amount of background dye which is staining PDMS an additional staining was performed with wells only containing PDMS and medium without bacterial cells. This was considered the technical negative control.