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Main sol. 119b

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Internal Solution-ID: S211

Equipment needed: Autoclave Hungate tubes Gassing station Centrifuge
Compound Amount Unit Conc. [g/L] Conc. [mM]
KH2PO4 0.50 g 0.498 3.66
MgSO4 x 7 H2O 0.40 g 0.398 1.616
NaCl 0.40 g 0.398 6.817
NH4Cl 0.40 g 0.398 7.448
CaCl2 x 2 H2O 0.05 g 0.05 0.339
Trace element solution SL-10 1.00 ml - -
Selenite-tungstate solution 1.00 ml - -
FeSO4 x 7 H2O solution (0.1% w/v) 2.00 ml - -
Yeast extract
(OXOID) 		1.00 g 0.996 -
Na-acetate 1.00 g 0.996 12.142
Na-formate 2.00 g 1.992 29.292
Sludge fluid 50.00 ml - -
Fatty acid mixture 20.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.0e-4 0.002
Methanol
(15% v/v) 		10.00 ml 7.888 246.191
NaHCO3 1.00 g 0.996 11.856
L-Cysteine HCl x H2O 0.50 g 0.498 2.835
Na2S x 9 H2O 0.50 g 0.498 2.073
Distilled water 940.00 ml - -
1 Dissolve ingredients except methanol, bicarbonate, cysteine and sulfide. Adjust pH of medium to 7.2 and sparge with 100% N2 gas for 30 - 45 min to make it anoxic. Then dispense medium under same gas atmosphere into anoxic Hungate-type tubes to 30% v/v of their volume and autoclave. Add methanol (from 15% v/v stock solution), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Prior to use check pH of complete medium and adjust to 7.6, if necessary.
2 After inoculation, add sterile 80% H2 and 20% CO2 gas mixture to 0.5 bar overpressure. After growth becomes visible overpressure of 80% H2 and 20% CO2 gas mixture can be increased to 1 bar.
3 Note: Use 10% (v/v) as inoculum.
119b METHANOMASSILIICOCCUS MEDIUM