K2HPO4 0.35g，KH2PO4 0.23g，NH4Cl 0.50g，MgSO4 x 7 H2O 0.50g，CaCl2 x 2 H2O 0.25g ，NaCl 2.25g FeSO4 x 7 H2O solution (0.1% w/v) 2.00ml Trace element solution SL-10 1.00ml Yeast extract (OXOID) 2.00g ，Casitone (BD BBL) 2.00g，Na-acetate 2.50g，Sodium resazurin (0.1% w/v) 0.50ml NaHCO3 2.00g，Methanol (50% v/v) 20.00ml， Wolin's vitamin solution (10x) 1.00ml ， L-Cysteine HCl x H2O 0.30g Na2S x 9 H2O 0.30g ，Distilled water 1000.00 ml Dissolve ingredients (except bicarbonate, vitamins, methanol, cysteine and sulfide) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Then add and dissolve bicarbonate, adjust pH to 6.5 and dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Methanol (50% v/v stock solution) and the reducing agents are each autoclaved separately under 100% N2 gas atmosphere as concentrated solutions in tightly closed tubes. Vitamins are prepared under 100% N2 gas atmosphere and sterilized by filtration. Appropriate volumes of the stock solutions are injected into the sterile medium with hypodermic syringes. Adjust pH of the complete medium to 6.8 - 7.0, if necessary. For DSM 1538, DSM 7058, DSM 9195: Replace methanol with 5.00 g/l trimethylamine-HCl. For DSM 1825, DSM 7081, DSM 11855: Supplement medium with 5% (v/v) clarified rumen fluid (see medium 1310) and adjust pH to 6.5 - 6.8. For DSM 21571: Supplement medium with 6.00 g/l NaCl. FeSO4 x 7 H2O solution (0.1% w/v) (from medium 119) FeSO4 x 7 H2O 1.00 g H2SO4 (0.1 N) 1000.00 ml The ferrous sulfate solution is not stable and should be freshly prepared. Trace element solution SL-10 (from medium 320) HCl (25%) 10.00 ml FeCl2 x 4 H2O 1.50 g ZnCl2 70.00 mg MnCl2 x 4 H2O 100.00 mg H3BO3 6.00 mg CoCl2 x 6 H2O 190.00 mg CuCl2 x 2 H2O 2.00 mg NiCl2 x 6 H2O 24.00 mg Na2MoO4 x 2 H2O 36.00 mg Distilled water 990.00 ml First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. Wolin's vitamin solution (10x) (from medium 120) Biotin 20.00 mg Folic acid 20.00 mg Pyridoxine hydrochloride 100.00 mg Thiamine HCl 50.00 mg Riboflavin 50.00 mg Nicotinic acid 50.00 mg Calcium D-(+)-pantothenate 50.00 mg Vitamin B12 1.00 mg p-Aminobenzoic acid 50.00 mg (DL)-alpha-Lipoic acid 50.00 mg Distilled water 1000.00 ml
