C difficile agar base

For strain development and in vitro analyses, C. difficile was routinely maintained on Brain heart infusion medium (Oxoid) supplemented with 5μg/ml yeast extract, 0.1% w/v L-cysteine (BHIS), C. difficile selective supplement comprising 250μg/ml D-cycloserine, and 8 μg/ml cefoxitin (Oxoid) (BHIS CC), and where necessary, an additional supplementation of 15 μg/ml thiamphenicol (BHIS CCTM). C. difficile cultures were grown at 37°C in an anaerobic workstation (Don Whitley) with an anaerobic gas mixture comprising 80% N₂, 10% H₂ and 10% CO₂.
For murine experiments, frozen stocks of wild-type and mutant strains were cultured on CDMN agar plates (C. difficile agar base (Oxoid) supplemented with 7% (v/v) defibrinated horse blood (Lampire Biological Laboratories), 32 mg/L moxalactam (Santa Cruz Biotechnology), 12 mg/L norfloxacin (Sigma-Aldrich) and 500 mg/L cysteine hydrochloride (Fischer) in an anaerobic chamber [27 (link)] at 37°C for 24 hours. Single colonies were picked and grown anaerobically for 16–18 hours at 37°C to saturation in 5 mL of pre-reduced reinforced Clostridial medium (RCM, Oxoid) for inoculation.
For hamster experiments, strains were cultured on blood agar for 5d in order to generate the spore stocks. Terminal colonization was assessed using C. difficile selective agar (Oxoid). In both instances, strains were maintained in an anaerobic workstation, as described above.

C. difficile VPI 10463 was inoculated in anaerobic chopped meat media tubes (Hardy Diagnostics) injected with vancomycin (10 μg/mL), 2-DG (1 μg/mL, 10 μg/mL or 100 μg/mL), or untreated, and incubated for 8 h at 37 °C. After incubation, samples were serially diluted and plated in C. difficile agar base (Oxoid) plates supplemented with 7% laked horse blood (Lampire Biological Laboratories) and C. difficile selective supplement (Oxoid) and incubated anaerobically. Colonies were counted 3 days after culture.