Main sol. 734a

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