After staining and washing, the CL samples were placed

onto glass slides, embedded in 10 μL Mowiol 4-88 (Polysciences Inc., Warrington, USA) and covered find more with a cover slip for observation by CLSM. Scanning electron microscopy (SEM) P. aeruginosa adhesion to CLs was also observed by SEM (DSM-940A, Zeiss, Oberkochen, Germany) at various magnifications (100×, 500×, 2000×, 5000×). All buffer solutions were passed through 0.2 μm filters to eliminate background particles. The CL samples were fixed in HEPES buffer (10 mM, pH 7.4) containing NaN3 (50 mM), 3% glutaraldehyde, and 4% paraformaldehyde for 1 h at room temperature and then overnight at 4°C. Further treatment was carried out using two different methods. They were: i. critical point drying, which consisted of 2% tannic acid for 1 h, 1% osmium tetroxide for 2 h, 1% thiocarbohydrazide for 30 min, 1% osmium tetroxide overnight, and 2% uranyl acetate for 2 h, with washing steps in between. The samples were then dehydrated by immersion in increasing concentrations of ethanol (10 – 100%) and dried in a critical point drier using amylacetate and liquid CO2; ii. sodium hydroxide drying: osmium tetroxide vapor for Saracatinib research buy 3 days; drying over sodium hydroxide disks

for 3 weeks at -20°C. All samples were mounted onto aluminum stubs and sputter-coated with gold for observation using SEM. Statistical analyses Statistical analyses were performed using analysis of variance (ANOVA) to determine the main effects of CL material and incubation time, and the interaction effect on biofilm ABT-263 mw growth in (log [CFU/cm2]). Additionally, ANOVA was performed with Tukey’s HSD post-hoc test to compare the viable bacterial cell counts in log [CFU/cm2]. Two distinct comparisons were made: i. differences between the viable cell counts at different incubation times (24, 48 and 72 h) independent of the CL materials and separately for each CL material; ii. differences between the viable cell counts on various CL materials independent of the incubation times and separately for each incubation time. P ≤ 0.05 was considered statistically significant. Results Pseudomonas GBA3 aeruginosa

biofilm growth on various contact lens materials To evaluate biofilm formation in the novel in-vitro biofilm model (Figure 1), the accumulation of viable bacterial cells over time was measured on four CLs using quantitative culturing (Figure 2). For comparison and for statistical analysis, variation between the CL materials in terms of viable cell counts in log [CFU/cm2] after 24, 48 and 72 h growth are represented separately in Figure 3. Analysis of variance showed that biofilm growth was significantly affected primarily by the incubation time, and secondarily by the CL material. The interaction effect of time and material had a comparatively minor effect (Table 3). Figure 2 Biofilm growth dynamics on contact lens materials.