Equipment needed: Autoclave
Anaerobic workbench
Hungate tubes
| Compound | Amount | Unit | Conc. [g/L] | Conc. [mM] | |
|---|---|---|---|---|---|
| KCl | 0.355 | g | 0.348 | 4.669 | |
| MgCl2 x 6 H2O | 4.000 | g | 3.922 | 19.289 | |
| MgSO4 x 7 H2O | 3.450 | g | 3.382 | 13.723 | |
| NH4Cl | 0.250 | g | 0.245 | 4.582 | |
| CaCl2 x 2 H2O | 0.140 | g | 0.137 | 0.934 | |
| KH2PO4 | 0.140 | g | 0.137 | 1.009 | |
| NaCl | 18.000 | g | 17.647 | 301.969 | |
| Wolfe's mineral solution | 10.000 | ml | - | - | |
| Trace vitamins | 10.000 | ml | - | - | |
| Fe(NH4)2(SO4)2 x 6 H2O | 2.000 | mg | 0.002 | - | |
| NaHCO3 | 5.000 | g | 4.902 | 58.352 | |
| Sodium acetate | 1.000 | g | 0.98 | 11.952 | |
|
Yeast extract
(BD-Difco) |
2.000 | g | 1.961 | - | |
|
Trypticase peptone
(BD-BBL) |
2.000 | g | 1.961 | - | |
| Resazurin | 1.000 | mg | 9.8e-4 | 0.004 | |
| Distilled water | 980.000 | ml | - | - | |
| 1 Mix components thoroughly, except NaHCO3, and adjust pH to 6.5. Bring to a boil for 5-10 sec, then, cool down under a stream of H2-CO2 (80:20, v/v) and add NaHCO3 to the medium. Dispense the medium into suitable culture vessels (e.g., 5 ml medium in Hungate tubes) under the same gas mixture and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight. Aseptically and anaerobically add per liter the following solutions (autoclaved and stored under a N2 atmosphere): | |||||
|
L-Cysteine HCl x H2O
(5%) |
10.000 | ml | - | - | |
|
Na2S x 9 H2O
(5%) |
10.000 | ml | - | - | |
| 2 Pressurize the inoculated bottles to 200 kPa H2:CO2 (80:20, v/v). | |||||
Media with this solution
| J265 | METHANOGENIUM MEDIUM |