1094 . METHANOSPHAERULA (PEAT) MEDIUM

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Selected strain: Methanosphaerula palustris DSM 19958

This strain uses the standard medium without modifications.

Growth conditions: 7-14 days anaerobic 28 °C

Equipment needed: Autoclave Filter Gassing station Hungate tubes

Final gas composition: 40 % N2 40 % H2 20 % CO2 read more

Final pH: 5.7

Select a strain (optional):

	Compound	Amount	Unit
	Solution A	943	ml
	Solution B	13	ml
	Solution C	20	ml
	Solution D	1	ml
	Solution E	10	ml
	Solution F	10	ml
	Solution G	10	ml
1 Sparge solution A with 80% N₂ and 20% CO₂ gas mixture for 30 - 45 min to make anoxic. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Solutions B, C, D and G are prepared under 100% N₂ gas atmosphere and sterilized by filtration. Solutions E and F are prepared under 100% N₂ gas atmosphere and autoclaved. To complete the medium add appropriate amounts of solutions B to G to the sterile solution A in the sequence indicated and let medium equilibrate overnight. Adjust pH of complete medium to 5.7, if necessary.
2 After inoculation pressurize vials to 1 bar overpressure with sterile 80% H₂ and 20% CO₂ gas mixture.
3 Note: The complete medium is not stable and can be stored only for a short time.

Solution A #

943

Compound	Amount	Unit
KCl (0.1% w/v)	1.5	ml
KH₂PO₄ (0.1% w/v)	13.6	ml
NH₄Cl (0.1% w/v)	26.8	ml
Trace element solution	1.0	ml
Distilled water	900.0	ml

Solution B #

Compound	Amount	Unit
Tris-HCl buffer (1.0 M solution, pH 8.0)	7.20	ml
Nitrilotriacetate (0.5 M, neutralized with 10 N NaOH)	4.80	ml
TiCl₃ (15% w/v solution in HCl; Riedel-de Haen)	0.55	ml

Solution C #

	Compound	Amount	Unit
	2-(N-Morpholino)ethanesulfonic acid (MES)	1.95	g
	NaOH	0.20	g
	Distilled water	20.00	ml
1 Adjust to pH 7.5 with NaOH.

Solution D #

	Compound	Amount	Unit
	Wolin's vitamin solution (10x)	1	ml

Solution E #

	Compound	Amount	Unit
	2-Mercaptoethanesulfonate (Coenzyme M)	0.08	g
	Distilled water	10.00	ml

Solution F #

	Compound	Amount	Unit
	Na-acetate	0.41	g
	Distilled water	10.00	ml

Solution G #

	Compound	Amount	Unit
	Na₂S x 9 H₂O	9.6	mg
	Distilled water	10.0	ml

Trace element solution #

1000

	Compound	Amount	Unit
	Na₂-EDTA	37.23	g
	FeCl₂ x 4 H₂O	1.34	g
	AlK(SO₄)₂ x 12 H₂O	3.45	g
	CaCl₂ x 2 H₂O	2.34	g
	MgSO₄ x 7 H₂O	1.56	g
	CoCl₂ x 6 H₂O	24.00	mg
	ZnCl₂	75.00	mg
	H₃BO₃	19.00	mg
	NiCl₂ x 6 H₂O	24.00	mg
	Na₂Mo₄ x 2 H₂O	24.00	mg
	MnSO₄ x 4 H₂O	26.00	mg
	CuSO₄ x 5 H₂O	9.00	mg
	Distilled water	1000.00	ml
1 Dissolve EDTA in distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds.

Wolin's vitamin solution (10x) #