| KP buffer* | 5 | ml |
| MgCl2·6H2O | 0.75 | g |
| CaCl2·2H2O | 0.15 | g |
| NH4Cl | 0.54 | g |
| NaCl | 30 | g |
| Na2SO4 | 2.4 | g |
| Na2S2O3·5H2O | 2.4 | g |
| Sodium acetate | 0.8 | g |
| Sodium pyruvate | 1.1 | g |
| Bacto Yeast Extract (Difco) | 0.5 | g |
| Fe(NH4)2(SO4)2·7H2O | 2 | mg |
| Trace elements solution** | 2 | ml |
| Vitamin solution*** | 2 | ml |
| Resazurin | 1 | mg |
| Na2CO3 | 1 | g |
| Na2S·9H2O | 0.36 | g |
| Distilled water | 1 | L |
| pH unadjusted | |
|
| | Mix ingredients except KP buffer, vitamin solution, Na2CO3 and Na2S·9H2O. Dispense the medium into suitable culture vessels under a stream of H2/CO2 (80/20) and seal with butyl rubber stoppers. Separately autoclave KP buffer and Na2S·9H2O (5% solution) under a N2 atmosphere. Sterilize vitamin and 10% Na2CO3 solutions by filtration. Prior to inoculation, aseptically and anaerobically add KP buffer, vitamin, Na2CO3 and Na2S·9H2O solutions. Pressurize the inoculated vessels to 150 kPa with H2/CO2 (80/20). |
| | | | | | |
| | *KP buffer |
| | | KH2PO4 | 119 | g |
| | | K2HPO4 | 21 | g |
| | | Distilled water | 1 | L |
| | Autoclaved under N2. |
| | | | | | |
| | **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 |
| | | 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 |