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Main sol. 734a

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Internal Solution-ID: S1505

Equipment needed: Filter Autoclave Hungate tubes Gassing station Centrifuge
Compound Amount Unit Conc. [g/L] Conc. [mM]
Clarified rumen fluid 20.00 ml - -
NaCl 1.00 g 1 17.112
KCl 0.50 g 0.5 6.707
MgCl2 x 6 H2O 0.40 g 0.4 1.968
CaCl2 x 2 H2O 0.10 g 0.1 0.68
NH4Cl 0.30 g 0.3 5.609
KH2PO4 0.20 g 0.2 1.47
Na2SO4 0.15 g 0.15 1.056
Casamino acids
(BD Bacto) 		0.50 g 0.5 -
Yeast extract
(OXOID) 		0.50 g 0.5 -
Trace element solution SL-10 1.00 ml - -
Selenite-tungstate solution 1.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.0e-4 -
Na2CO3 2.00 g 2 18.87
MOPS
(SIGMA) 		2.10 g 2.1 10.035
Seven vitamins solution 1.00 ml - -
DL-Dithiothreitol
(DTT) 		0.16 g 0.16 1.037
Distilled water 980.00 ml - -
1 Dissolve ingredients (except carbonate, MOPS buffer, vitamins and DTT), bring medium to the boil, then cool to room temperature under 80% H2 and 20% CO2 gas mixture. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add MOPS buffer adjusted to pH 7.7, vitamins and DTT from anoxic stock solutions prepared under 100% N2 gas and sterilized by filtration and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Adjust pH of complete medium to 7.7, if necessary.
734a METHANOBREVIBACTER CUTICULARIS MEDIUM