Main sol. 1523

Equipment needed: Hungate tubes Filter Autoclave Gassing station

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
KH₂PO₄	0.50	g	0.499	3.667
MgSO₄ x 7 H₂O	0.40	g	0.399	1.62
NaCl	0.40	g	0.399	6.831
NH₄Cl	0.40	g	0.399	7.463
CaCl₂ x 2 H₂O	0.05	g	0.05	0.339
Trace element solution SL-10	1.00	ml	-	-
Brain heart infusion (BD Bacto)	6.00	g	5.988	-
Proteose peptone (BD Difco)	6.00	g	5.988	-
Yeast extract (OXOID)	2.00	g	1.996	-
Na-acetate	1.00	g	0.998	12.166
Na-formate	2.00	g	1.996	29.35
Sodium resazurin (0.1% w/v)	0.50	ml	5.0e-4	0.002
NaHCO₃	4.00	g	3.992	47.52
Seven vitamins solution	1.00	ml	-	-
L-Cysteine HCl x H₂O	0.50	g	0.499	2.841
Na₂S x 9 H₂O	0.50	g	0.499	2.078
Distilled water	1000.00	ml	-	-
1 Dissolve ingredients except bicarbonate, vitamins, cysteine, and sulfide, then sparge medium with 80% H₂ and 20% CO₂ gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate and adjust pH to 6.5. 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, cysteine, and sulfide from sterile anoxic stock solutions prepared under 100% N₂ gas. The vitamin solution should be sterilized by filtration. Before use, check the complete medium's pH and adjust it to 6.8 - 7.0, if necessary.
2 After growth has started and the culture is becoming turbid add sterile 80% H₂ and 20% CO₂ gas mixture to 0.5 - 1 bar overpressure.

1523	MODIFIED METHANOBACTERIUM MEDIUM