Solution A: Brain heart infusion broth 12.50 g Trypticase (BBL 11921) 10.00 g Yeast extract 2.50 g Na-thioglycolate 0.50 g L-Cysteine x HCl 1.00 g L-Asparagine 0.25 g Glucose 2.00 g Agar 3.00 g Distilled water 1000.00 ml
Boil for 5 min, cool to 50?C while gassing with 80% N2 + 20% CO2. Distribute to anaerobic tubes under the same atmosphere and autoclave.
Solution B: Thiamine pyrophosphate 6.00 mg NaHCO3 2.00 g Distilled water 25.00 ml Volatile fatty acid solution 20.00 ml
Sterilize by filtration, add 20 ml rabbit serum, gas with 80% N2 + 20% CO2. Add 0.45 ml Sol. B to 10 ml Sol. A. Inoculate and pressurize the tubes with H2 to 0.5 bar. Volatile fatty acid solution: KOH, 0.1 N 100.00 ml Isobutyric acid 0.50 ml D,L-2-methylbutyric acid 0.50 ml Isovaleric acid 0.50 ml Valeric acid 0.50 ml For DSM 111712 omit agar from solution A.
