Biofilm formation in the developed media

Measurement of attached biofilm biomass by crystal violet staining

Starting from an overnight liquid culture in LB (incubated for 18 hours), bacterial cultures were diluted 1:100 in the developed media, or in LB, or M9, two widely used laboratory bacterial culture media. To minimise edge-effect, non-perimeter wells of U-bottomed polystyrene 96-well microtiter plate (Greiner U-bottomed) were inoculated with 180 μl of these dilutions and perimeter wells were used as a negative control (sterile medium). Following 3 days of growth at 37°C, the supernatant (containing non-adhered cells) was removed from each well and plates were rinsed using 200 μl sterile PBS, twice. Subsequently, 200 μl of Crystal Violet (CV) was added to non-perimeter wells and plates were incubated at room temperature for minimum 20 minutes. After 20 minutes, the excess CV was removed by washing the plates under running tap water. Finally, bound CV was released by addition of 200 μl of 95-100% ethanol (Sigma-Aldrich) to the wells with bacteria. After incubating with ethanol for 30 minutes, the absorbance was measured at 600 nm using a BMG plate reader. 95-100% ethanol was used as a blank.

Measurement of cell viability and free-floating biofilm formation by resazurin assays

Overnight cultures of PAO1 and LESB65 were diluted in LB to an OD 600 of 0.05 (±0.01). These cultures were then further diluted in the developed media (1:100) to a total volume of 10 ml in glass universal tubes. Free floating biofilm formation in respiratory tract-mimicking media was compared to that observed in LB and M9. Diluted developed media cultures were incubated under conditions appropriate for each respiratory niche, as described above. Cultures were incubated for 3 days while shaking at 75 rpm. Three glass universal tubes containing bacteria-free respiratory media, LB or M9 were used as a negative control. After the incubation, biofilms were disrupted using 250 μl of 100 mg/ml cellulase (diluted in 0.05 M citrate buffer [9.6 g/l Citrate.H ₂O (VWR)] in water and pH to 4.6 with NaOH) and further incubated under aerobic conditions at 37 °C, while shaking at 150 rpm for 1 h. After 30 minutes incubation, biofilms were further disrupted by manual pipetting to ensure complete disruption of biofilms. A portion of disrupted biofilms were then transferred to 96-well plates (Greiner U-bottomed) and to determine the metabolic activity of the bacterial cells released from the disrupted biofilms, 10 μl of 0.02 % (v/v) resazurin (Sigma-Aldrich) (diluted in distilled water) was added to each well of the 96-well plates and incubated for 1-2 hours at niche specific temperature, while shaking at 150 rpm. Following incubation with resazurin, the fluorescence of each well was measured using an excitation wavelength of 540 nm and an emission wavelength of 590 nm in a Fluostar Omega microplate reader and the MARS Data Analysis Software.