| Mineral base 1* | 700 | ml |
| Mineral base 2** | 300 | ml |
| MgSO4·7H2O | 0.25 | g |
| NH4Cl | 0.2 | g |
| Bacto Yeast Extract (Difco) | 0.02 | g |
| Coenzyme M | 0.14 | g |
| Vitamin solution*** | 10 | ml |
| Trace element solution**** | 10 | ml |
| Methanol | 3 | ml |
| Na2S·9H2O | 0.25 | g |
|
| | Mix mineral base 1 with mineral base 2 in 7:3 propotion in tightly closed vessels under a N2 atmosphere. Separately autoclave concentrated MgSO4·7H2O, NH4Cl, Bacto Yeast Extract, coenzyme M and Na2S·9H2O under a N2 atmosphere in tightly closed vessels. Add the concentrated solutions, and filter-sterile vitamin solution, trace element solution and methanol. After re-exchanging with N2, add 1 drop of filter-sterile 10% dithionaite solution in 1 M NaHCO3 by syringe. Final pH of the complete medium should be 10. |
| | | | | | |
| | *Mineral base 1 |
| | | Na2CO3 | 95 | g |
| | | NaHCO3 | 15 | g |
| | | NaCl | 16 | g |
| | | K2HPO4 | 1 | g |
| | | Distilled water | 1 | L |
|
| | Mix ingredients and autoclave under a N2 atmosphere. Because some precipitate appears on the bottom for 1 day after autoclaving, use only supernatant for making the medium. Final pH should be 10. |
| | | | | | |
| | **Mineral base 2 |
| | | Na2CO3 | 15 | g |
| | | NaHCO3 | 20 | g |
| | | NaCl | 3 | g |
| | | K2HPO4 | 1 | g |
| | | Distilled water | 1 | L |
|
| | Mix ingredients, adjust pH to 9.5 with HCl and autoclave under a N2 atmosphere. Final pH should be 9.5. |
| | | | | | |
| | ***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 |
| | | | | | |
| | ****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. |