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Main sol. 1257

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Internal Solution-ID: S2490

Equipment needed: Hungate tubes Filter Autoclave Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
NaCl 1.0 g 0.998 17.077
MgCl2 x 6 H2O 0.4 g 0.399 1.964
CaCl2 x 2 H2O 0.1 g 0.1 0.679
NH4Cl 0.1 g 0.1 1.866
KH2PO4 0.2 g 0.2 1.467
KCl 0.5 g 0.499 6.694
Trace element solution 1.0 ml - -
Yeast extract
(OXOID) 		0.1 g 0.1 -
Na-acetate 0.1 g 0.1 1.217
Sodium resazurin
(0.1% w/v) 		0.5 ml 5.0e-4 0.002
Na2CO3 1.5 g 1.497 14.124
Wolin's vitamin solution (10x) 1.0 ml - -
L-Cysteine HCl x H2O 0.3 g 0.299 1.705
Na2S x 9 H2O 0.3 g 0.299 1.247
Distilled water 1000.0 ml - -
1 Dissolve ingredients (except carbonate, vitamins and reducing agents), then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic and adjust pH to 6.0. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Prior to inoculation add vitamins, cysteine and sulfide from sterile anoxic stock solution prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Vitamins are sterilized by filtration. If necessary, adjust the pH of the complete medium to 7.0.
2 After inoculation pressurize vials to 1 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture.
1257 METHANOCELLA MEDIUM