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Medium No.	249
Medium
Composition
Na2HPO4·12H2O 4.2 g   KH2PO4 1.5 g   Na2CO3 4 g   MgCl2·6H2O 0.4 g   NH4Cl 0.6 g   Yeast extract 1 g   Bacto Tryptone (Difco) 1 g   Na2WO4·2H2O 0.4 mg   Wolfe's modified mineral solution* 50 ml   VFA** 1 ml   Distilled water 738 ml   pH 6.5       Adjust pH to 6.5 with 10 N KOH and add the following medium.       0.1% Resazurin solution 1 ml       myo-Inositol 1 mg       Vitamin mixture*** 10 ml       Cellobiose 0.2 g       Cellulose**** 200 ml     Heat the above mixed solution at 60℃ for 5 to 10 min and then bubble with N2gas. And then add the following ingredients.       Cysteine 0.5 g       Na2S·9H2O 0.5 g       Agar (if needed) 15 g     Under anaerobic condition, the medium is autoclaved at 100℃ for 10 min.                   *Wolfe's modified mineral solution       Nitrilotriacetic acid (NTA) 1.5 g       MgSO4·7H2O 3 g       NaCl 1 g       MnSO4·H2O 0.5 g       FeSO4·7H2O 0.1 g       Co(NO3)2·6H2O 0.1 g       CaCl2 0.1 g       ZnSO4·7H2O 0.1 g       CuSO4·5H2O 0.01 g       Boric acid 0.01 g       Na2MoO4·2H2O 0.01 g       Na2SeO3 0.001 g     Nitrilotriacetic acid is suspended in 500 ml water, and dissolved by titrating with 2 N KOH until its pH stabilize at 6.5. The rest of the ingredients are added and dissolved. Finally total volume is adjusted to 1 L.                   **VFA       Acetic acid 17 ml       Propionic acid 6 ml       Butyric acid 4 ml       iso-Butyric acid 1 ml       n-Valeric acid 1 ml       iso-Valeric acid 1 ml       DL-α-Methylbutyric acid 1 ml                   ***Vitamin mixture       Biotin 0.2 mg       Ca-pantothenate 40 mg       Folic acid 0.2 mg       myo-Inositol 200 mg       Niacin 40 mg       p-Aminobenzoic acid 20 mg       Pyridoxine-HCl 40 mg       Riboflavin 40 mg       Thiamin-HCl 20 mg       Choline chloride 100 mg       Cyanocobalamin 2 μg       Distilled water 1 L                   ****3% Cellulose suspension which is ball-milled for 3 days.
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