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Main sol. 203

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Internal Solution-ID: S431

Equipment needed: Autoclave Filter Gassing station Serum bottle
Compound Amount Unit Conc. [g/L] Conc. [mM]
Mineral solution 1 37.5 ml - -
Mineral solution 2 37.5 ml - -
NiCl2 x 6 H2O
(0.1% w/v) 		1.0 ml 9.9e-4 0.008
FeSO4 x 7 H2O
(0.1% w/v in 0.1 N H2SO4) 		2.0 ml 0.002 0.007
Modified Wolin's mineral solution 10.0 ml - -
Na2SO4 3.4 g 3.37 23.723
Sodium resazurin
(0.1% w/v) 		0.5 ml 5.0e-4 0.002
Na2CO3 1.0 g 0.991 9.351
Yeast extract
(OXOID) 		2.0 g 1.982 -
Trypticase peptone
(BD BBL) 		2.0 g 1.982 -
Wolin's vitamin solution (10x) 1.0 ml - -
Na2S x 9 H2O 0.5 g 0.496 2.063
L-Cysteine HCl x H2O 0.5 g 0.496 2.821
Distilled water 920.0 ml - -
1 Dissolve ingredients except carbonate, yeast extract, Trypticase peptone, vitamins, sulfide and cysteine, then adjust pH to 6.0 with sulfuric acid. 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 serum bottles to 30% of their volume and autoclave. Thereafter, add by injection yeast extract, Trypticase peptone, vitamins (sterilized by filtration), sulfide and cysteine from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Adjust pH of complete medium to 6.5.
2 After inoculation pressurize bottles to 2 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture.
203 METHANOTHERMUS MEDIUM