Ridascreen verotoxin elisa kit

3 ml LB was inoculated directly from glycerol stocks and grown overnight at 37 °C. 6 ml LB was inoculated 1/100 from overnight cultures and grown to an OD_600nm = 0.6–0.8 (t = 0). Cultures were split 1:1 and phage lysis was induced in one half by the addition of Mitomycin C (MMC, 2 μg/ml). Subsequent growth/lysis in induced and un-induced cultures was monitored spectrophotometrically at OD_600nm. For Stx toxin ELISA assays cultures were grown as above and growth/lysis allowed to proceed for 24 h. After 24 h, 1 ml culture was taken and live cells and cell debris removed by centrifugation (13,000 r.p.m, room temperature (RT)). Stx toxin containing supernatants were further sterilized by syringe filtering (0.22 μm; Milipore). The level of Stx toxin in each sample was assayed using the RIDASCREEN Verotoxin ELISA kit (R-Biopharm) according to manufacturer guidelines.

In total, 3 ml LB was inoculated directly from glycerol stocks and grown overnight at 37 °C. 6 ml LB was inoculated 1/100 from overnight cultures and grown to an OD_{600 nm} = 0.6–0.8. Mitomycin C (2 µg ml⁻¹) was added and lysis allowed to proceed for 24 h. After 24 h, 1 ml culture was taken and live cells and cell debris removed by centrifugation (13000 r.p.m.). Stx toxin containing supernatants were further sterilized by syringe filtering (0.22 µm; Milipore). The level of Stx toxin in each sample was assayed using the RIDASCREEN Verotoxin ELISA kit (R-Biopharm) according to manufacturer guidelines. Differences Stx2 production was assessed by ordinary one-way ANOVA with multiple comparisons where each strain was compared with Z1723.