| NH4Cl | 0.33 | g | | MgCl2·6H2O | 0.33 | g | | CaCl2·2H2O | 0.33 | g | | KCl | 0.33 | g | | KNO3 | 0.33 | g | | NaCl | 30 | g | | HEPES | 2.38 | g | | Yeast extract | 0.2 | g | | Tryptone | 1 | g | | Sucrose | 2 | g | | Vitamin solution* | 1 | ml | | Trace element solution** | 1 | ml | | Distilled water | 1 | L | | | | Prepare the medium anaerobically, under N2, omitting the CaCl2, MgCl2, KNO3, tryptone, yeast extract, vitamins and sucrose. The pH should be 7.0-7.5. Dispense the medium into vessels suitable for anaerobic growth (Hungate tubes or serum bottles) under an atmosphere of nitrogen and autoclave. To the sterile, cooled medium add, from sterile stock solutions the CaCl2, MgCl2, KNO3, tryptone, yeast extract, vitamins and sucrose. The CaCl2, MgCl2, KNO3, tryptone, yeast extract, and sucrose stock solutions should be autoclaved, while the vitamin solution is filter-sterilized. | | | | | | | | | | *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 | | | | Vitamin B12 | 0.1 | mg | | | | p-Aminobenzoic acid | 5 | mg | | | | Lipoic acid | 5 | mg | | | | Distilled water | 1 | L | | | | | | | | | | **Trace element solution | | | | Nitrilotriacetic acid | 1.5 | g | | | | MgSO4·7H2O | 3 | g | | | | MnSO4·2H2O | 0.5 | g | | | | NaCl | 1 | g | | | | FeSO4·7H2O | 0.1 | g | | | | CoSO4·7H2O | 0.18 | g | | | | CaCl2·2H2O | 0.1 | g | | | | ZnSO4·7H2O | 0.18 | g | | | | CuSO4·5H2O | 0.01 | g | | | | KAl(SO4)2·12H2O | 0.02 | g | | | | H3BO3 | 0.01 | g | | | | Na2MoO4·2H2O | 0.01 | g | | | | NiCl2·6H2O | 0.025 | g | | | | Na2SeO3·5H2O | 0.3 | mg | | | | Distilled water | 1 | L | | | | First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Final pH 7.0 (with KOH). |
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