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Main sol. 825

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Internal Solution-ID: S1673

Equipment needed: Autoclave Hungate tubes Filter Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
NH4Cl 1.0 g 0.989 18.492
K2HPO4 0.3 g 0.297 1.704
KH2PO4 0.3 g 0.297 2.181
MgCl2 x 6 H2O 0.2 g 0.198 0.973
CaCl2 x 2 H2O 0.1 g 0.099 0.673
KCl 0.1 g 0.099 1.327
NaCl 0.6 g 0.593 10.155
Modified Wolin's mineral solution 10.0 ml - -
Na-acetate 0.5 g 0.495 6.029
Yeast extract 1.0 g 0.989 -
Sodium resazurin
(0.1% w/v) 		0.5 ml 4.9e-4 0.002
NaHCO3 4.0 g 3.956 47.097
Wolin's vitamin solution (10x) 1.0 ml - -
L-Cysteine HCl x H2O 0.5 g 0.495 2.816
Na2S x 9 H2O 0.5 g 0.495 2.059
Distilled water 1000.0 ml - -
1 Dissolve ingredients except bicarbonate, vitamins, cysteine and sulfide. Sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate, adjust pH to 7.0, then dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add vitamins (sterilized by filtration), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Prior to use check pH of complete medium and adjust to 6.8 - 7.0, if necessary.
2 After inoculation pressurize cultivation vessels to 2 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture.
825 METHANOBACTERIUM II MEDIUM