Figure 3

Impact of normoxia and anoxia upon the production of anti-staphylococcal exoproducts by P. aeruginosa. (a) Protease production was determined following the addition of P. aeruginosa cell-free supernatants to wells in milk agar, prior to the diameter of the zones of clearance (mm) being measured following 24 h incubation. (b) P. aeruginosa cell-free supernatants were added to heat-killed S. aureus and staphylolytic activity was determined by measuring changes in the OD₅₉₅ after 60 min. The control consisted of heat-killed S. aureus with sterile LBN broth only. (c) Pyocyanin was extracted from P. aeruginosa cell-free supernatants using chloroform and 0.2 M hydrochloric acid. Spectrophotometric measurements were taken at OD₅₂₀ and multiplied by extinction co-efficient of pyocyanin at this wavelength (17.072) to express pyocyanin in µg/mL. (d) Pyoverdine production was quantified by measuring the relative fluorescence units (RFU) of P. aeruginosa cell-free supernatants following excitation at 460 nm and emission at 490 nm. (e) Surfactant activity was measured using the established drop collapse assay, where cell-free supernatants were serially diluted (1:1) with deionised water containing 0.005% crystal violet to aid visualisation. Surfactant scores are equal to the reciprocal of the greatest dilution at which there was surfactant activity. Data shown are the mean ± S.E.M. of three independent experiments (N = 3), each performed in triplicate. In panels A, B and D, statistical differences were determined using one-way ANOVA with Tukey’s post-hoc test *P < 0.05, **P < 0.01 and ***P < 0.001.