| CaCl2·2H2O | 0.25 | g |
| KH2PO4 | 0.34 | g |
| NH4Cl | 0.34 | g |
| KCl | 0.34 | g |
| MgSO4 | 0.5 | g |
| NaCl | 30 | g |
| Vitamin B12 (0.002%) solution | 1 | ml |
| Trace element solution SL-12B* | 1 | ml |
| NaHCO3 | 1.5 | g |
| Na2S·9H2O | 0.4 | g |
| Resazurin | 0.5 | mg |
| Distilled water | 998 | ml |
| pH 7.3 | |
|
| | Dissolve ingredients except bicarbonate, Vitamin B12 and sulfide, dispense under a N2/CO2 atmosphere (80/20) in culture vessels, and autoclave. Add bicarbonate, vitamin and sulfide** from sterile anaerobic stock solutions prepared under N2 or N2/CO2 (bicarbonate). The bicarbonate and vitamin solutions should be sterilized by filtration. Adjust the pH of the medium with sterile HCl or Na2CO3 solutions (2 mol/L each) to pH 7.3. |
| | | | | | |
| | *Trace element solution SL-12B |
| | | Na2-EDTA | 3 | g |
| | | FeSO4·7H2O | 1.1 | g |
| | | CoCl2·6H2O | 190 | mg |
| | | MnCl2·2H2O | 50 | mg |
| | | ZnCl2 | 42 | mg |
| | | NiCl2·6H2O | 24 | mg |
| | | Na2MoO4·2H2O | 18 | mg |
| | | H3BO3 | 300 | mg |
| | | CuCl2·2H2O | 2 | mg |
| | | Distilled water | 1 | L |
| pH 6 | |
| | | | | | |
| | **Neutralized sulfide solution |
| | | Na2S·9H2O | 1.5 | g |
| | | Distilled water | 100 | ml |
|
| | The sulfide solution is prepared in a vessel sealed with a butyl rubber stopper. Autoclave at 121℃ for 15 min under a N2 atmosphere. After cooling to room temperature, the pH is adjusted to about 7.3 by adding of sterile 2 N H2SO4 drop-wise with a syringe without opening the bottle. Appearance of yellow colour indicates the pH is about 8. The vessel should be shaken continuously to avoid precipitation of elemental sulfur. The final solution should be transparent yellow. |