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Main sol. 119a

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Internal Solution-ID: S210

Equipment needed: Autoclave Centrifuge Hungate tubes Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
KH2PO4 0.50 g 0.499 3.663
MgSO4 x 7 H2O 0.40 g 0.399 1.618
NaCl 0.40 g 0.399 6.824
NH4Cl 0.40 g 0.399 7.456
CaCl2 x 2 H2O 0.05 g 0.05 0.339
Trace element solution SL-10 1.00 ml - -
FeSO4 x 7 H2O solution (0.1% w/v) 2.00 ml - -
Yeast extract
(OXOID) 		1.00 g 0.997 -
Na-acetate 1.00 g 0.997 12.154
Na-formate 2.00 g 1.994 29.321
Clarified rumen fluid 200.00 ml - -
Fatty acid mixture 20.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.0e-4 0.002
NaHCO3 4.00 g 3.988 47.473
L-Cysteine HCl x H2O 0.50 g 0.499 2.838
Na2S x 9 H2O 0.50 g 0.499 2.076
Distilled water 780.00 ml - -
1 Dissolve ingredients except bicarbonate, cysteine and sulfide. Sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate, then dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes to 30% of their volume and autoclave. Add cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Prior to use check pH of complete medium and adjust to 6.8 - 7.0, if necessary.
2 After inoculation add sterile 80% H2 and 20% CO2 gas mixture to 1 bar overpressure.
119a METHANOBREVIBACTER MEDIUM