| NaCl | 20 | g |
| MgCl2·6H2O | 3 | g |
| CaCl2·2H2O | 0.15 | g |
| Na2SO4 | 4 | g |
| NH4Cl | 0.25 | g |
| KH2PO4 | 0.2 | g |
| KCl | 0.5 | g |
| Resazurin | 1 | mg |
| Fumarate solution (1.0 M)* | 10 | ml |
| Trace element mixture** | 1 | ml |
| Selenite-tungstate solution*** | 1 | ml |
| Bicarbonate solution (1.0 M)**** | 30 | ml |
| Vitamin solution***** | 1 | ml |
| Thiamine solution****** | 1 | ml |
| Vitamin B12 solution******* | 1 | ml |
| Sulfide solution (1.0 M)******** | 1.5 | ml |
| Distilled water | 953.5 | ml |
| pH 7.0-7.3 | |
| | Mix ingredients except for fumarate, bicarbonate, vitamin, thiamine, cyanocobalamin and sulfide solutions. Dispense the medium into suitable culture vessels under a stream of the N2/CO2 (80/20) mixed gas, seal with butyl rubber stoppers, and autoclave at 121°C for 15 min. Aseptically and anaerobically add other ingredients using sterile syringes. Adjust pH to 7.0-7.3 using compact pH meter. Moreover, prior to inoculation, aseptically and anaerobically add the fumarate solution (final concentration, 10 mM). |
| | | | | | |
| | *Fumarate solution (1.0 M) |
| | | Disodium fumarate | 1.6 | g |
| | | Distilled water | 10 | ml |
|
| | Sterilized by filtration and stored under N2 gas. |
| | | | | | |
| | **Trace element mixture |
| | | HCl (25%; 7.7 M) | 12.5 | ml |
| | | FeSO4·7H2O | 2100 | mg |
| | | H3BO3 | 30 | mg |
| | | MnCl2·4H2O | 100 | mg |
| | | CoCl2·6H2O | 190 | mg |
| | | NiCl2·6H2O | 24 | mg |
| | | CuCl2·2H2O | 2 | mg |
| | | ZnSO4·7H2O | 144 | mg |
| | | Na2MoO4·2H2O | 36 | mg |
| | | Distilled water | 987 | ml |
|
| | Sterilized by autoclaving. |
| | | | | | |
| | ***Selenite-tungstate solution |
| | | NaOH | 0.4 | g |
| | | Na2SeO3·5H2O | 6 | mg |
| | | Na2WO4·2H2O | 8 | mg |
| | | Distilled water | 1 | L |
|
| | Sterilized by autoclaving. |
| | | | | | |
| | ****Bicarbonate solution (1.0 M) |
| | | NaHCO3 | 8.4 | g |
| | | Distilled water | 100 | ml |
|
| | Saturated with CO2 gas. Tightly sealed in bottles filled with CO2 gas and then sterilized by autoclaving. |
| | | | | | |
| | *****Vitamine solution |
| | | p-Aminobenzoic acid | 4 | mg |
| | | Biotin | 1 | mg |
| | | Nicotinic acid | 10 | mg |
| | | Ca-pantothenate | 5 | mg |
| | | Pyridoxine-HCl | 15 | mg |
| | | Sodium phosphate buffer (10 mM; pH7.1) | 100 | ml |
|
| | Sterilized by filtration. |
| | | | | | |
| | ******Thiamine solution |
| | | Thiamine-HCl | 10 | mg |
| | | Sodium phosphate buffer (25 mM; pH 3.4) | 100 | ml |
|
| | Sterilized by filtration. |
| | | | | | |
| | *******Vitamin B12 solution |
| | | Cyanocobalamin | 5 | mg |
| | | Distilled water | 100 | ml |
|
| | Sterilized by filtration. |
| | | | | | |
| | ********Sulfide solution (1.0 M) |
| | | Na2S·9H2O | 24 | g |
| | | Distilled water | 100 | ml |
|
| | Sterilized by filtration and stored under N2 gas. |