Campylobacter blood free selective agar base

The isolation of Campylobacter spp. from each fecal sample was carried out with an enrichment step in tubes containing Tryptone Soy Broth (Oxoid Ltd., Basingstoke, UK) liquid medium added with 5% horse blood and incubated at 37 °C for 4 h, then at 42 °C for 24 h.
A loop of each broth culture was streaked onto dishes containing Campylobacter Blood-Free selective agar base added with CAT selective supplement (Oxoid Ltd., Basingstoke, UK). Plates were incubated at 37 °C for 48 h in microaerophilia (5% oxygen, 10% CO₂) [22 (link)].

E. coli BL21 (DE3) Star (Invitrogen) was grown at 37°C on Luria-Bertani (LB) plates or in LB broth (Biotrading) supplemented with 100 μg x ml^-1 of ampicillin. C. jejuni strain 81116 [23 (link)] and C. jejuni 81116 ΔflaAB [22 (link)] were routinely grown on agar plates with 5% saponin-lysed horse blood or in heart infusion (HI) broth (Biotrading) at 37°C or 42°C under microaerobic conditions (10% CO₂, 5% O₂, 85% N₂). The presence of Campylobacter in cloacal swabs was tested using CCDA (charcoal cefoperazone deoxycholate agar) plates containing Campylobacter blood free selective agar base (Oxoid) and CCDA selective supplement (Oxoid) according to the manufacturer’s instructions. HeLa57A cell line stably transfected with a NF-κB luciferase reporter construct [24 (link)], was generously provided by Dr. R. T. Hay (Institute of Biomolecular Sciences, University of St. Andrews, St. Andrews, Scotland, U.K.). HeLa57A cells were propagated in Dulbecco modified Eagle medium (DMEM, Invitrogen) supplemented with 5% fetal calf serum (FCS) at 37°C under 10% CO₂.

All samples were analysed within 6 h from collection. To obtain pure cultures, samples were processed as previously described by Shobo et al. [31 (link)]. Briefly, to confirm the purity of the culture, samples incubated in broth were filtered through a 0.47 µM mixed cellulose ester filter (Merck Millipore, Ireland) onto Campylobacter blood-free selective agar base (Oxoid, Hampshire, UK), supplemented with charcoal cefoperazone deoxycholate (CCD) agar-selective supplement (Oxoid, Hampshire, UK). Approximately 500 µL of the inoculum was evenly and aseptically distributed over the filter. Once the liquid had been filtered through, forceps were used to aseptically remove the filter. Plates were incubated at 37 °C in a microaerophilic atmosphere (CampyGen; Oxoid, UK) for 48 h. This was followed by sub-culturing on Tryptose Blood Agar Base (Biolab, Modderfontein, South Africa) supplemented with 5% defibrinated sheep blood and incubated at 37 °C for 42 h in a microaerophilic atmosphere. Susceptibility to nalidixic acid (30 µg Oxoid, UK) and cephalothin (30 µg Oxoid, UK) was also ascertained [33 (link)].