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Main sol. 404

Internal Solution-ID: S823

Equipment needed: Hungate tubes Filter Autoclave Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
Mineral solution 1 40.0 ml - -
Minerals solution 40.0 ml - -
Fe(NH4)2(SO4)2 x 7 H2O
(0.1% w/v)
2.0 ml 0.002 0.005
NiCl2 x 6 H2O
(0.1% w/v)
0.2 ml 2.0e-4 0.002
Modified Wolin's mineral solution 10.0 ml - -
Na-acetate x 3 H2O 3.0 g 2.973 -
Yeast extract
(OXOID)
1.0 g 0.991 -
Trypticase peptone
(BD BBL)
1.0 g 0.991 -
Sodium resazurin
(0.1% w/v)
0.5 ml 5.0e-4 0.002
NaHCO3 3.0 g 2.973 35.393
Methanol 5.0 ml 3.925 122.485
Wolin's vitamin solution (10x) 1.0 ml - -
L-Cysteine HCl x H2O 0.3 g 0.297 1.693
Na2S x 9 H2O 0.3 g 0.297 1.238
Distilled water 910.0 ml - -
1 Dissolve ingredients (except bicarbonate, methanol, vitamins, cysteine and sulfide), then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate and adjust pH to 7.0, then distribute under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. After sterilization add methanol, vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Vitamins are sterilized by filtration. Adjust pH of complete medium to 7.0 - 7.2.
2 After inoculation, pressurize culture bottles with sterile 80% H2 and 20% CO2 gas mixture to 2 bar overpressure.
404 METHANOSPHAERA CUNICULI MEDIUM