At the end of the experiment, the medium was discarded, and non-adherent bacteria were removed by three washes with sterile PBS. Quantification of bacterial adhesiveness and biofilm formation on polystyrene was assessed by a spectrophotometric method, as previously described by Christensen et al. [43], with minor modifications. Briefly, after washing, attached bacteria were fixed for 1 hour at 60°C and then stained with Hucker crystal violet solution for 5 minutes. After washing with water to remove the excess of stain, the plates were dried for 30 minutes at 37°C. The color produced by attached bacteria (indirect index of adhesiveness

or biofilm formation) was measured spectrophotometrically at OD492. A low cut-off corresponding to 3 standard deviations (SDs) above the mean of control wells not seeded with bacteria was chosen [43]. Co-infection assays Co-infection assays were performed using S. maltophilia strain selleck compound OBGTC9 and P. aeruginosa strain PAO1. Briefly, confluent IB3-1 cell monolayers were first infected for 2 hours

at 37°C with P. aeruginosa PAO1 (MOI 1000). At that time, non-adherent bacteria were removed by three washes with PBS, and monolayers were then infected with S. maltophilia strain OBGTC9 (MOI 1000) and incubated for further 2 hours. At the end of the experiment infected IB3-1 cells were removed by a treatment with 0.25% Ferrostatin-1 mw trypsin/EDTA, vortexed, serially diluted and plated on MH agar to determine the number (cfu chamber-1) of the two bacteria bound to IB3-1 cells. P. aeruginosa PAO1 and S. maltophilia OBGTC10 colonies were easily differentiated on the basis of their colonial morphology. As controls we used IB3-1 cell monolayers infected separately with each of the two bacterial strains. Motility tests Swimming

motility assays were performed with single well-isolated colonies grown overnight on MH agar plates, according to a modification of the technique described by Rashid et al. [44]. Briefly, tryptone swim plates (1% tryptone, 0.5% NaCl, 0.3% agar; Oxoid) were click here inoculated with bacteria at the surface by using a sterile needle. Plates were incubated for 24 hours at 37°C. Motility was assessed by calculating the diameter (mm) of the circular turbid zone formed by bacterial cells migrating away from the point of inoculation at the agar surface. Scanning electron MK-1775 clinical trial microscopy Biofilm formation was assessed by scanning electron microscopy (SEM). Samples were air-dried, and fixed with a solution of 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 90 minutes. After washing with buffer, samples were post-fixed in osmium tetroxide and then dehydrated in a series of aqueous ethanol solutions (30 to 70%). Specimens were mounted on aluminum stubs with conductive carbon cement, allowed to dry for 3 hours, and coated with 15-nm Au film with an agar automatic sputter coater.