Main sol. 892

Equipment needed: Filter Autoclave Hungate tubes Gassing station

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
KH₂PO₄	0.40	g	0.396	2.907
NH₄Cl	0.90	g	0.89	16.643
MgCl₂ x 6 H₂O	0.36	g	0.356	1.751
NaCl	10.00	g	9.891	169.254
Na-acetate	0.50	g	0.495	6.029
Yeast extract (OXOID)	2.00	g	1.978	-
Trypticase peptone (BD BBL)	2.00	g	1.978	-
Trace element solution	10.00	ml	-	-
Sodium resazurin (0.1% w/v)	0.50	ml	4.9e-4	0.002
Na₂CO₃	5.00	g	4.946	46.662
Wolin's vitamin solution (10x)	1.00	ml	-	-
Na₂S x 9 H₂O	0.50	g	0.495	2.059
L-Cysteine HCl x H₂O	0.50	g	0.495	2.816
Distilled water	1000.00	ml	-	-
1 Dissolve ingredients except carbonate, 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 carbonate, adjust pH to 7.2 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 7.3 - 7.5, if necessary.
2 After inoculation add sterile 80% H₂ and 20% CO₂ gas mixture to 1.5 - 2 bar overpressure.

892	METHANOCALCULUS PUMILUS MEDIUM