# 120a: METHANOSARCINA BARKERI MEDIUM ## Main sol. 120a 0.35 g K2HPO4 0.23 g KH2PO4 0.5 g NH4Cl 0.5 g MgSO4 x 7 H2O 0.25 g CaCl2 x 2 H2O 2.25 g NaCl 2 ml **FeSO4 x 7 H2O solution (0.1% w/v)** 1 ml **Trace element solution SL-10** 2 g Yeast extract (OXOID) 2 g Casitone (BD BBL) 0.5 ml Sodium resazurin (0.1% w/v) 0.85 g NaHCO3 20 ml Methanol (50% v/v) 1 ml **Wolin's vitamin solution (10x)** 0.3 g L-Cysteine HCl x H2O 0.3 g Na2S x 9 H2O 1000 ml Distilled water 1. 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 solutions are injected into the sterile medium with hypodermic syringes. Adjust pH of the complete medium to 6.5 - 6.8, if necessary. ## FeSO4 x 7 H2O solution (0.1% w/v) 1 g FeSO4 x 7 H2O 1000 ml H2SO4 (0.1 N) 1. The ferrous sulfate solution is not stable and should be freshly prepared. ## Trace element solution SL-10 10 ml HCl (25%) 1.5 g FeCl2 x 4 H2O 70 mg ZnCl2 100 mg MnCl2 x 4 H2O 6 mg H3BO3 190 mg CoCl2 x 6 H2O 2 mg CuCl2 x 2 H2O 24 mg NiCl2 x 6 H2O 36 mg Na2MoO4 x 2 H2O 990 ml Distilled water 1. 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) 20 mg Biotin 20 mg Folic acid 100 mg Pyridoxine hydrochloride 50 mg Thiamine HCl 50 mg Riboflavin 50 mg Nicotinic acid 50 mg Calcium D-(+)-pantothenate 1 mg Vitamin B12 50 mg p-Aminobenzoic acid 50 mg (DL)-alpha-Lipoic acid 1000 ml Distilled water