Inclusion of this indicator made it easier to

see the small recombinant colonies. Plates were seeded with 5 μl H. pylori liquid culture (forming a circle with 3 mm diameter) standardised to an OD600 nm of 1.0 and were incubated at 37°C for up to 7 days under the conditions described above. The motility halos were recorded using a digital camera and the area of each halo was measured using a GS-800 Calibrated Densitometer (Biorad). Motility analysis was also carried out by direct observation under phase-contrast microscopy using a Nikon Eclipse E600 after cells were grown in co-culture conditions as used by Wand et al. [24]. Briefly, co-cultures of H. pylori-human gastric adenocarcinoma (AGS) cells were prepared selleck compound (described below). After 24 h, 10 μl culture was placed onto a microscope slide and covered with a coverslip and freely-motile H. pylori cells were analysed under the microscope. Plate motility bioassay using chemically defined media (CDM) The liquid chemically defined media were prepared as previously described [15, 28]. 60 ml of sterile chemically defined media were added to 40 ml of molten 1% Oxoid No. 1 agar base to make 0.4% semi-solid chemically defined agar. Cysteine supplemented

plates (CSP) were made by adding cysteine to the selleck kinase inhibitor molten agar, shortly before it set. The final concentration of cysteine was 1.0 mM, which was non-limiting for H. pylori growth. The centre of each plate was seeded with one-day incubated H. pylori cells and was incubated for 5 PAK5 days under the conditions described above. The motility halos were recorded using a digital camera and the area of each halo was measured using a GS-800 Calibrated Densitometer (Biorad). Motility assay with AI-2 complementation AI-2 was synthesised enzymatically as described previously using purified proteins LuxS

E. coli and Pfs E. coli [8]. For complementation of the ΔluxS Hp motility phenotype, soft motility agar plates (0.4% w/v) were made as previously described. Bioluminescence activity of the AI-2 product was quantified using the V. harveyi bioassay and compared to CFS from H. pylori wild-type broth culture standardised to an OD600 nm of 1.0 at the time point in the growth curve that maximal AI-2 activity was measured. 1/400 diluted in vitro synthesised AI-2 product shows the same level of bioluminescence as seen in the H. pylori wild-type CFS in the V. harveyi bioassay. Therefore, in the complementation experiment AI-2 was added to motility plates to a final concentration of 0.25% (v/v). 24 h H. pylori cultures were seeded individually onto the centre of each motility plate and incubated for 5 days. The area of outward migration was recorded with a digital camera and measured using a GS-800 Calibrated Densitometer (Biorad). Tissue culture and bacterial co-culture All chemicals were obtained from Gibco, UK.