Figure 3.

Simulated gastro-intestinal transit of stationary phase cells. Cells were grown to stationary phase at 37°C under aerobic () or anaerobic () conditions before being subjected to ‘short’ (A, B) or ‘long’ (C, D) simulated gastric transit. For ‘short’ simulated gastric transit 4 ml of growing culture was added to 16 ml simulated gastric juice at pH 2.5 (A) and pH 1.7 (B), for final pH values of 2.5 and 3.5 respectively. Suspensions were incubated at 37°C for 60 minutes, neutralised to ~ pH 7.2 and 0.1x volumes of 3% (w/v) bile salt solution added (giving a final bile salt concentration of 0.3% w/v, as seen in vivo). Suspensions were incubated for a further 60 minutes at 37°C. For ‘long’ simulated gastric transit 4 ml of growing culture was added to 16 ml simulated gastric juice at pH 2.5 (C) and pH 1.7 (D), for final pH values of 2.5 and 3.5 respectively. Suspensions were incubated at 37°C for 120 minutes, neutralised to ~ pH 7.2 and 0.1x volumes of 3% (w/v) bile salt solution added (giving a final bile salt concentration of 0.3% w/v, as seen in vivo). Suspensions were incubated for a further 120 minutes at 37°C. Throughout testing, samples were taken at half hourly intervals, diluted in BPW and spiral plated on BHI agar. Plates were enumerated after 24–48 hours incubation at 37°C. PBS (), SGJ () and bile () controls were also included. Limit of detection = 3x10²cfu ml⁻¹. Arrows indicate pH neutralization and addition of bile salts. Data is representative of three independent experiments conducted in duplicate.