| HEPES | 23.8 | g | | NaCl | 19 | g | | MgCl2·6H2O | 2.6 | g | | CaCl2·2H2O | 0.15 | g | | Na2SO4 | 4 | g | | NH4Cl | 0.25 | g | | KH2PO4 | 0.2 | g | | KCl | 0.5 | g | | Trace elements solution* | 1 | ml | | Vitamin solution** | 1 | ml | | NaHCO3 | 2.52 | g | | Iron granule (1-2 mm)*** | 150 | g | | Cysteine-HCl | 1 | g | | Distilled water | 1 | L | | | | Mix ingredients, except vitamin solution, NaHCO3, iron granule and cysteine-HCl. Dispense the medium into suitable culture vessels under a stream of N2/CO2 (80/20), seal with butyl rubber stoppers, and autoclave for 20 min at 121°C. Sterilize vitamin and 8.4% NaHCO3 solutions by filtration. 5% Cysteine-HCl solution and iron granule are separately sterilized by autoclaving for 20 min at 121°C under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add vitamin, NaHCO3, iron granule and cysteine-HCl. | | | | | | | | | | *Trace elements solution | | | | Nitrilotriacetic acid (NTA) | 12.8 | g | | | | FeCl3·6H2O | 1.35 | g | | | | MnCl2·4H2O | 0.1 | g | | | | CoCl2·6H2O | 0.024 | g | | | | CaCl2·2H2O | 0.1 | g | | | | ZnCl2 | 0.1 | g | | | | CuCl2·2H2O | 0.025 | g | | | | H3BO3 | 0.01 | g | | | | Na2MoO4·2H2O | 0.024 | g | | | | NaCl | 1 | g | | | | NiCl2·6H2O | 0.12 | g | | | | Na2SeO4 | 0.004 | g | | | | Na2WO4 | 0.004 | g | | | | KAl(SO4)2·12H2O | 0.02 | g | | | | Distilled water | 1 | L | | | | First dissolve nitrilotriacetic acid and adjust pH to 6.5 with NaOH, and then add other 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 | | | | | | | | | | ***Alfa Aesar A Johnson Matthey Company, Massachusetts, USA. |
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