# 1094: METHANOSPHAERULA (PEAT) MEDIUM


## Main sol. 1094

943	ml	**Solution A**	
13	ml	**Solution B**	
20	ml	**Solution C**	
1	ml	**Solution D**	
10	ml	**Solution E**	
10	ml	**Solution F**	
10	ml	**Solution G**	

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

1.5	ml	KCl (0.1% w/v)	
13.6	ml	KH2PO4 (0.1% w/v)	
26.8	ml	NH4Cl (0.1% w/v)	
1	ml	**Trace element solution**	
900	ml	Distilled water	


## Solution B

7.2	ml	Tris-HCl buffer (1.0 M solution, pH 8.0)	
4.8	ml	Na3-NTA x H2O (0.5 M solution)	
0.55	ml	TiCl3 (15% w/v solution in HCl; Riedel-de Haen)	


## Solution C

1.95	g	2-(N-Morpholino)ethanesulfonic acid (MES)	
0.2	g	NaOH	
20	ml	Distilled water	

1. Adjust to pH 7.5 with NaOH.


## Solution D

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


## Solution E

0.08	g	2-Mercaptoethanesulfonic acid (Coenzyme M)	
10	ml	Distilled water	


## Solution F

0.41	g	Na-acetate	
10	ml	Distilled water	


## Solution G

9.6	mg	Na2S x 9 H2O	
10	ml	Distilled water	


## Trace element solution

37.23	g	Na2-EDTA	
1.34	g	FeCl2 x 4 H2O	
3.45	g	AlK(SO4)2 x 12 H2O	
2.34	g	CaCl2 x 2 H2O	
1.56	g	MgSO4 x 7 H2O	
24	mg	CoCl2 x 6 H2O	
75	mg	ZnCl2	
19	mg	H3BO3	
24	mg	NiCl2 x 6 H2O	
24	mg	Na2Mo4 x 2 H2O	
26	mg	MnSO4 x 4 H2O	
9	mg	CuSO4 x 5 H2O	
1000	ml	Distilled water	

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)

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