Tissue culture treated polystyrene dishes

In order to obtain sufficient material for estimating biofilm biomass (dry weight) and protein content, NTHi biofilms were formed in 20×100 mm tissue culture-treated polystyrene dishes (BD Biosciences, Bedford, MA, USA) in the presence and absence of 170 ng/mL ampicillin (3 replicas each). Antibiotics were present during biofilm formation. The culture medium was washed from the formed biofilms with PBS and the biofilm material was freeze-dried under vacuum. Dry weights were measured from batches of pooled biofilms (3 dishes each batch). Protein content was estimated using the BCA Protein Assay Kit (Thermo Scientific Pierce) on biofilm material scraped from dishes and tabulated as percent protein content per biofilm. For CFU counts, biofilms were not dried but were scraped and suspended in sBHI, serially diluted and estimates of total viable bacteria calculated after 24 hr incubation [49] (link). The results were tabulated as numbers of viable bacteria per mg dry biofilm.

For better specimen handling for processing and examination in an SEM, plastic coverslips were used as growth substrate for NTHi biofilms; hence, biofilms were prepared in 20×100 mm tissue culture-treated polystyrene dishes (BD Biosciences, Bedford, MA, USA) with Thermanox plastic coverslips (Nalgene Nunc International, Rochester, NY, USA) placed at the bottom. Then, as previously described [22] , biofilms on coverslips were fixed by plunge-freezing in liquid propane, freeze-substituted with ethanol, gradually warmed to 4°C, and critical point dried. The dried biofilms were mounted on a specimen stub, sputter coated in argon with a 18 nm-layer of platinum and examined in the XL 30 SFEG SEM operating at 5 kV in the secondary electron mode (FEI Company, Hillsboro, OR, USA). Biofilm thickness estimates were obtained from regions where the biofilm profile was visible.