Main sol. 203

Equipment needed: Autoclave Filter Gassing station Serum bottle

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
Mineral solution 1	37.5	ml	-	-
Mineral solution 2	37.5	ml	-	-
NiCl₂ x 6 H₂O (0.1% w/v)	1.0	ml	9.9e-4	0.008
FeSO₄ x 7 H₂O (0.1% w/v in 0.1 N H₂SO₄)	2.0	ml	0.002	0.007
Modified Wolin's mineral solution	10.0	ml	-	-
Na₂SO₄	3.4	g	3.37	23.723
Sodium resazurin (0.1% w/v)	0.5	ml	5.0e-4	0.002
Na₂CO₃	1.0	g	0.991	9.351
Yeast extract (OXOID)	2.0	g	1.982	-
Trypticase peptone (BD BBL)	2.0	g	1.982	-
Wolin's vitamin solution (10x)	1.0	ml	-	-
Na₂S x 9 H₂O	0.5	g	0.496	2.063
L-Cysteine HCl x H₂O	0.5	g	0.496	2.821
Distilled water	920.0	ml	-	-
1 Dissolve ingredients except carbonate, yeast extract, Trypticase peptone, vitamins, sulfide and cysteine, then adjust pH to 6.0 with sulfuric acid. Sparge medium with 80% H₂ and 20% CO₂ gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into serum bottles to 30% of their volume and autoclave. Thereafter, add by injection yeast extract, Trypticase peptone, vitamins (sterilized by filtration), sulfide and cysteine from sterile anoxic stock solutions prepared under 100% N₂ gas atmosphere and carbonate from a sterile anoxic stock solution prepared under 80% N₂ and 20% CO₂ gas mixture. Adjust pH of complete medium to 6.5.
2 After inoculation pressurize bottles to 2 bar overpressure with sterile 80% H₂ and 20% CO₂ gas mixture.

203	METHANOTHERMUS MEDIUM