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Main sol. 1273

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Internal Solution-ID: S2543

Equipment needed: Autoclave Hungate tubes Filter Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
KH2PO4 0.14 g 0.138 1.018
NH4Cl 0.54 g 0.534 9.986
MgCl2 x 6 H2O 0.20 g 0.198 0.973
CaCl2 x 2 H2O 0.15 g 0.148 1.009
Yeast extract
(OXOID) 		0.20 g 0.198 -
Na-acetate 0.80 g 0.791 9.646
Modified Wolin's mineral solution 10.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 4.9e-4 0.002
Na2CO3 1.00 g 0.989 9.332
Wolin's vitamin solution (10x) 1.00 ml - -
L-Cysteine HCl x H2O 0.50 g 0.495 2.816
Na2S x 9 H2O 0.50 g 0.495 2.059
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except carbonate, vitamins, cysteine and sulfide) and sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. The vitamin solution should be sterilized by filtration. Adjust pH of complete medium to 6.8, if necessary.
2 After inoculation pressurize vials to 1 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture.
1273 METHANOSPIRILLUM LACUNAE MEDIUM