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Main sol. 322

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Internal Solution-ID: S598

Equipment needed: Autoclave Hungate tubes Filter Gassing station Centrifuge
Compound Amount Unit Conc. [g/L] Conc. [mM]
Clarified rumen fluid 100.00 ml - -
Trypticase peptone
(BD BBL) 		2.00 g 1.946 -
Yeast extract 2.00 g 1.946 -
Na-acetate 0.50 g 0.486 5.929
Na-formate 0.50 g 0.486 7.152
Modified Wolin's mineral solution 10.00 ml - -
Na2SeO4
(0.1% w/v) 		1.90 ml 0.002 0.01
NiCl2 x 6 H2O
(0.1% w/v) 		0.70 ml 6.8e-4 0.005
FeSO4 x 7 H2O solution (0.1% w/v) 3.00 ml - -
K2HPO4 0.60 g 0.584 3.351
KH2PO4 2.80 g 2.724 20.015
(NH4)2SO4 0.30 g 0.292 2.208
NH4Cl 1.00 g 0.973 18.186
NaCl 0.60 g 0.584 9.987
MgSO4 x 7 H2O 0.15 g 0.146 0.592
CaCl2 x 2 H2O 0.08 g 0.078 0.529
Sodium resazurin
(0.1% w/v) 		0.50 ml 4.9e-4 0.002
NaHCO3 4.00 g 3.891 46.318
Methanol
(50% v/v) 		10.00 ml 7.704 240.443
Wolin's vitamin solution (10x) 2.00 ml - -
DL-Dithiothreitol 0.50 g 0.486 3.152
Distilled water 900.00 ml - -
1 Dissolve ingredients except bicarbonate, methanol, and dithiothreitol (DTT), then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate, adjust pH of medium to 6.8 - 7.0, then dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add methanol (50% v/v solution), vitamins, and DTT from sterile anoxic stock solutions prepared under 100% N2 gas. Vitamins and DTT should be sterilized by filtration. Prior to use check pH of complete medium and adjust to 6.7 - 6.9, if necessary.
2 After inoculation add sterile 80% H2 and 20% CO2 gas mixture to 1 bar overpressure.
322 METHANOSPHAERA MEDIUM