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Main sol. 825a

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Internal Solution-ID: S1674

Equipment needed: Hungate tubes Filter Autoclave Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
NH4Cl 1.0 g 0.979 18.311
K2HPO4 0.3 g 0.294 1.687
KH2PO4 0.3 g 0.294 2.159
MgCl2 x 6 H2O 0.2 g 0.196 0.964
CaCl2 x 2 H2O 0.1 g 0.098 0.666
KCl 0.1 g 0.098 1.314
NaCl 0.6 g 0.588 10.056
Modified Wolin's mineral solution 10.0 ml - -
Na-acetate 0.5 g 0.49 5.97
Yeast extract 1.0 g 0.979 -
Sodium resazurin
(0.1% w/v) 		0.5 ml 4.9e-4 0.002
NaHCO3 4.0 g 3.918 46.636
Methanol
(15% v/v) 		10.0 ml 7.757 242.092
Wolin's vitamin solution (10x) 1.0 ml - -
L-Cysteine HCl x H2O 0.5 g 0.49 2.788
Na2S x 9 H2O 0.5 g 0.49 2.039
Distilled water 1000.0 ml - -
1 Dissolve ingredients except bicarbonate, methanol, vitamins, cysteine and sulfide. Sparge medium with 80% N2 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% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add methanol (15% v/v solution), 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.
825a METHANOBACTERIUM II MEDIUM (N2/CO2)