Final pH: 6.7 - 6.8 Final volume: 1003 ml Mineral solution 38.00 ml K2HPO4 0.30 g Trypticase peptone (BD BBL) 2.00 g Yeast extract (OXOID) 0.50 g Volatile fatty acid mixture 3.10 ml Haemin solution (0.05% w/v) 2.00 ml Glycerol 0.50 g Sodium resazurin (0.1% w/v) 0.50 ml Na2CO3 4.00 g D-Glucose 0.50 g Maltose 0.50 g Cellobiose 0.50 g Starch, soluble 0.50 g L-Cysteine HCl x H2O 0.25 g Na2S x 9 H2O 0.25 g Distilled water 960.00 ml Dissolve ingredients (except carbonate, glucose, maltose, cellobiose, soluble starch, cysteine and sulfide), then sparge medium with 100% CO2 gas for 30 - 45 min to make it anoxic. Add the carbonate and equilibrate the medium with the CO2 gas to pH 6.8. Distribute under 100% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Thereafter, add glucose, maltose, cellobiose, soluble starch, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Adjust pH of complete medium to 6.7 - 6.8, if necessary. For DSM 17630, DSM 121616, DSM 121617, DSM 122475, DSM 122576, DSM 122577: Supplement the medium after autoclaving with sterile and anoxic rumen fluid (30 ml/l). Mineral solution (from medium 330) KH2PO4 6.00 g NaCl 12.00 g (NH4)2SO4 6.00 g CaCl2 x 2 H2O 1.60 g MgSO4 x 7 H2O 2.50 g Distilled water 1000.00 ml Volatile fatty acid mixture (from medium 330) Acetic acid 548.50 ml Propionic acid 193.50 ml Butyric acid 129.00 ml n-Valeric acid 32.25 ml iso-Butyric acid 32.25 ml DL-2-Methylbutyric acid 32.25 ml iso-Valeric acid 32.25 ml Haemin solution (from medium 78) Haemin 50.00 mg NaOH (1 N) 1.00 ml Distilled water 100.00 ml Dissolve 50 mg haemin in 1 ml 1 N NaOH; make up to 100 ml with distilled water and filter sterilize. Store refrigerated.
