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Medium No.	912
Medium	Methanogenium Medium
Composition
KCl 0.355 g   MgCl2·6H2O 4 g   MgSO4·7H2O 3.45 g   NH4Cl 0.25 g   CaCl2·2H2O 0.14 g   KH2PO4 0.14 g   NaCl 18 g   Trace element solution* 10 ml   Vitamin solution** 10 ml   Fe(NH4)2(SO4)2·6H2O 2 mg   NaHCO3 5 g   Sodium acetate 1 g   Bacto Yeast Extract (Difco) 2 g   Trypticase Peptone (BBL) 2 g   Na2S·9H2O 0.5 g   Cysteine-HCl 0.5 g   Distilled water 1 L     Mix ingredients except NaHCO3, Na2S·9H2O and cysteine-HCl, and adjust pH to 6.5. Bring to a boil for 5-10 seconds, then cool under a stream of H2/CO2 (80/20) and add NaHCO3 to the medium. After autoclaving under a H2/CO2 atmosphere, stand the medium overnight. Separately autoclave cysteine-HCl and Na2S·9H2O as 5% solutions under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the cysteine-HCl and Na2S·9H2O solutions. Pressurize the inoculated bottles to 200 kPa with H2/CO2.                   *Trace element solution       Nitrilotriacetic acid 1.5 g       MgSO4·7H2O 3 g       MnSO4·xH2O 0.5 g       NaCl 1 g       FeSO4·7H2O 0.1 g       CoSO4·7H2O 0.1 g       CaCl2·2H2O 0.1 g       ZnSO4·7H2O 0.1 g       CuSO4·5H2O 0.01 g       AlK(SO4)2 0.01 g       H3BO3 0.01 g       Na2MoO4·2H2O 0.01 g       NiCl2·6H2O 0.025 g       Na2SeO3·5H2O 0.3 g       (NH4)2Ni(SO4)2·6H2O 2 g       Na2WO4·2H2O 0.01 g       Distilled water 1 L     Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH. Then add minerals. Adjust final pH to 7.0.                   **Vitamin solution       Biotin 2 mg       Folic acid 2 mg       Pyridoxine-HCl 10 mg       Thiamine-HCl 5 mg       Riboflavin 5 mg       Nicotinic acid 5 mg       Ca-pantothenate 5 mg       p-Aminobenzoic acid 1 mg       Vitamin B12 0.01 mg       Distilled water 1 L
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