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Medium No.	994
Medium	Methanosaeta concilii medium
Composition
Sodium acetate 4.1 g   (NH4)2SO4 0.45 g   K2HPO4 2.19 g   KH2PO4 1.5 g   NaCl 0.5 g   MgSO4·7H2O 0.12 g   CaCl2·2H2O 0.06 g   Resazurin 1 mg   Trace element solution* 10 ml   Vitamin solution** 10 ml   Rumen fluid*** 10 ml   FeSO4·7H2O**** 0.01 g   Na2CO3 3 g   Cysteine-HCl 0.25 g   Na2S·9H2O 0.25 g   Distilled water 1 L     Mix ingredients except for vitamin solution, rumen fluid, FeSO4, Na2CO3, cysteine-HCl and Na2S·9H2O, and autoclave under N2/CO2 (80/20) condition. A white precipitate that appears in the medium after autoclaving should dissolve again in about 2 days. Separately autoclave cysteine-HCl and Na2S·9H2O as a 5% solution under a N2 atmosphere. Vitamin and Na2CO3 (as a 10% solution) are sterilized by filtration. Aseptically and anaerobically add vitamin, rumen fluid, FeSO4, Na2CO3, cysteine-HCl and Na2S·9H2O. Adjust the pH of the medium to 7.3-7.5. Use 20% culture as an inoculum.                   *Trace element solution       Nitrilotriacetic acid (NTA) 4.1 g       FeCl3·6H2O 0.36 g       MnCl2·4H2O 0.09 g       CoCl2·6H2O 0.15 g       CaCl2·2H2O 0.018 g       ZnCl2 0.09 g       H3BO3 0.017 g       Na2MoO4·2H2O 0.009 g       NiCl2·6H2O 0.018 g       Na2SeO3 1.6 mg       Na2WO4 1.6 mg       Distilled water 1 L     First dissolve nitrilotriacetic acid and adjust pH to 6.5 with NaOH, and then add minerals. Final pH 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                   ***Rumen fluid:     Remove solid particles from a fresh rumen fluid by filtration and centrifugation, and then autoclave under a N2 atmosphere. Stock at 4°C.                   ****FeSO4·7H2O:     FeSO4·7H2O is dissolved in 0.1 N H2SO4 as a 0.2% solution and then autoclaved under a N2 atmosphere.
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