Biofilms were stained with 1% crystal violet, washed with deionis

Biofilms were stained with 1% crystal violet, washed with deionised water and quantitated by Selleck AZD1390 adding 95% ethanol followed by measurement of the absorbance (OD 595 nm) as per Stepanovic et al. [33]. Strains with no change in O.D over the control were classified non-biofilm producers, weak- (up to a 2 fold change), moderate- (up to 4 fold change) or strong- (greater than 4 fold

change) as per Strepanovic et al. [33] All tests were carried out in triplicate and the results were averaged. P. aeruginosa strain PAO1 was included as a positive control. Biofilms in a capillary flow reactor were grown in glass capillary tubes of square cross sections under continuous flow conditions. The capillaries mTOR inhibitor had a nominal inside dimension of 900 μm and a wall thickness of 170 ± 10 μm (Friedrich & Dimmock, Millville, N.J., USA). The flow cell apparatus consisted of a vented medium feed carboy (four litre capacity), a flow break, a filtered air entry, a peristaltic pump (Watson-Marlow), the capillary and flow cell holder,

an inoculation port, and a waste carboy. The components were connected by silicone rubber tubing and were sterilised by autoclaving. A culture of gfp-P. aeruginosa was grown in LB overnight at 37°C BMN 673 purchase in a shaking incubator at 140 rpm. A 100 μl aliquot of this culture was used to inoculate 10 ml of sterile LB broth in a 250 ml conical flask to achieve good aeration and the culture was grown at 37°C with shaking at 200 rpm for 3 h. The tubing was clamped downstream of the inoculation port and the capillary flow system was inoculated with 300 μl of this fresh culture. The tubing was then clamped upstream of the glass tube and the system was allowed to stand without a flow for 19 h to allow the cells to attach to the glass capillary at 37°C. After initial attachment, the flow of medium (1/10 strength LB, to avoid blockage of the capillary due to excessive biomass production) was adjusted to

a flow rate of 20 ml h-1. Bacterial staining of mixed biofilms consisting of biofilm+ and biofilm- isolates, were stained with 300 μl of a 5 mg l-1 rhodamine B (Kodak) solution in water. The stain solution was injected into the capillary reactor through the PAK5 inoculation port and the cells allowed to stain for 5 min. Biofilms were subsequently observed by confocal scanning laser microscopy with excitation and emission wavelengths of 540 nm at 625 nm respectively for rhodamine B and 475 nm and 510 for GFP. Scanning Electron Microscopy (SEM) Prior to SEM, samples were chemically fixed as follows: A 10 μl aliquot of an overnight culture, grown in LB broth at 37°C, with shaking at 140 rpm was placed in a round glass coverslip (10 mm diameter, Chance Proper Ltd., UK) with a 10 μl of fixative (3% glutaraldehyde in 0.1% sodium cacodylate, pH 7.3). The coverslips were previously coated with polylysine (Sigma-Aldrich) to assist adherence of bacterial cells.

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