| | NaCl | 12 | g |
| | MgSO4·7H2O | 0.5 | g |
| | PIPES | 3.4 | g |
| | KCl | 2 | g |
| | NH4Cl | 0.1 | g |
| | CaCl2·2H2O | 0.025 | g |
| | KH2PO4 | 0.02 | g |
| | Trace minerals* | 10 | ml |
| | Vitamin solution** | 10 | ml |
| | Bacto Yeast Extract (Difco) | 0.5 | g |
| | Bacto Peptone (Difco) | 1 | g |
| | Resazurin | 1 | mg |
| | Na2S·9H2O*** | 0.5 | g |
| | Distilled water | 1 | L |
|
| | | Mix ingredients except vitamin solution and Na2S·9H2O, adjust pH to 7.0 with NaOH and autoclaved under a N2 atmosphere. Separately autoclave and neutralize Na2S·9H2O as 5% solution under a N2 atmosphere. Anaerobically add filter-sterile vitamin and Na2S·9H2O solutions. |
| | | | | | | |
| | | *Trace minerals |
| | | | 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 |
| | | | H3BO3 | 0.01 | g |
| | | | Na2MoO4·2H2O | 0.01 | g |
| | | | NiCl2·6H2O | 0.025 | g |
| | | | Na2SeO3·5H2O | 0.8 | mg |
| | | | Na2WO4 | 0.8 | mg |
| | | | KAl(SO4)2·12H2O | 4 | 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). |
| | | | | | | |
| | | **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 |
| | | | | | | |
| | | ***The sulfide is prepared in a vessel sealed with a butyl rubber stopper. Autoclave under a N2 atmosphere. After cooling, the pH is adjusted to about 7.3 by adding of sterile 4-0.2 N H2SO4 drop-wise with a syringe without opening the bottle. Appearance of yellow color indicate the pH is about 8. The vessel should be shaken continuously to avoid precipitation of elemental sulfur. The final solution should be transparent yellow. |