Main sol. 825

Equipment needed: Autoclave Hungate tubes Filter Gassing station

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
NH₄Cl	1.0	g	0.989	18.492
K₂HPO₄	0.3	g	0.297	1.704
KH₂PO₄	0.3	g	0.297	2.181
MgCl₂ x 6 H₂O	0.2	g	0.198	0.973
CaCl₂ x 2 H₂O	0.1	g	0.099	0.673
KCl	0.1	g	0.099	1.327
NaCl	0.6	g	0.593	10.155
Modified Wolin's mineral solution	10.0	ml	-	-
Na-acetate	0.5	g	0.495	6.029
Yeast extract	1.0	g	0.989	-
Sodium resazurin (0.1% w/v)	0.5	ml	4.9e-4	0.002
NaHCO₃	4.0	g	3.956	47.097
Wolin's vitamin solution (10x)	1.0	ml	-	-
L-Cysteine HCl x H₂O	0.5	g	0.495	2.816
Na₂S x 9 H₂O	0.5	g	0.495	2.059
Distilled water	1000.0	ml	-	-
1 Dissolve ingredients except bicarbonate, vitamins, 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, adjust pH to 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 vitamins (sterilized by filtration), 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 pressurize cultivation vessels to 2 bar overpressure with sterile 80% H₂ and 20% CO₂ gas mixture.

825	METHANOBACTERIUM II MEDIUM