# 1280: METHANOREGULA (PEAT) MEDIUM


## Main sol. 1280

954	ml	**Solution A**	
10	ml	**Solution B**	
13	ml	**Solution C**	
10	ml	**Solution D**	
10	ml	**Solution E**	
10	ml	**Solution F**	

1. Dissolve ingredients of solution A, adjust pH to 5.0 with 0.1 N HCl and sparge with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense solution A under same gas atmosphere into anoxic Hungate-type tubes or serum vials (e.g., 20 ml medium in 50 ml bottles) and autoclave. Solutions B - F are prepared under 100% N2 gas atmosphere and sterilized by filtration. Prior to inoculation complete the medium by adding appropriate amounts of solutions B to F to the sterile solution A in the sequence as indicated.
2. Adjust pH of the complete medium to 5.1 with a sterile anoxic solution of 1 N HCl. After inoculation pressurize vials to 1 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture.
3. Note: Use 10-20% (v/v) as inoculum.


## Solution A

27	mg	NH4Cl	
14	mg	KH2PO4	
2	ml	KCl (0.1% w/v)	
17	mg	Na-acetate	
1	ml	**Trace elements solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
950	ml	Distilled water	


## Solution B

1.58	g	Homo-PIPES (SIGMA)	
0.18	g	NaOH (pellets)	
10	ml	Distilled water	

1. Adjust pH of buffer to 5.5.


## Solution C

7.2	ml	NaOH (0.38 M)	
4.8	ml	Nitrilotriacetate (0.5 M, neutralized with 10 N NaOH)	
0.55	ml	TiCl3 (15% w/v in HCl, RIEDEL-de HAEN)	


## Solution D

0.2	g	Yeast extract	
10	ml	Distilled water	


## Solution E

0.07	g	2-Mercaptoethanesulfonate (coenzyme M)	
10	ml	Distilled water	


## Solution F

10	ml	**Wolin's vitamin solution (10x)**	


## Trace elements solution

10	ml	HCl (25%)	
1.34	g	FeCl2 x 4 H2O	
24	mg	CoCl2 x 6 H2O	
75	mg	ZnCl2	
19	mg	H3BO3	
24	mg	NiCl2 x 6 H2O	
24	mg	Na2MoO4 x 2 H2O	
26	mg	MnSO4 x 4 H2O	
1.56	g	MgSO4 x 7 H2O	
2.34	g	CaCl2 x 2 H2O	
9	mg	CuSO4 x 5 H2O	
3.45	g	AlK(SO4)2 x 12 H2O	
1000	ml	Distilled water	

1. First dissolve ferrous chloride in the HCl, then dilute in water and dissolve the other salts. Finally make up to 1000 ml.


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water