# 203: METHANOTHERMUS MEDIUM


## Main sol. 203

37.5	ml	**Mineral solution 1**	
37.5	ml	**Mineral solution 2**	
1	ml	NiCl2 x 6 H2O (0.1% w/v)	
2	ml	FeSO4 x 7 H2O (0.1% w/v in 0.1 N H2SO4)	
10	ml	**Modified Wolin's mineral solution**	
3.4	g	Na2SO4	
0.5	ml	Sodium resazurin (0.1% w/v)	
1	g	Na2CO3	
2	g	Yeast extract (OXOID)	
2	g	Trypticase peptone (BD BBL)	
1	ml	**Wolin's vitamin solution (10x)**	
0.5	g	Na2S x 9 H2O	
0.5	g	L-Cysteine HCl x H2O	
920	ml	Distilled water	

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.


## Mineral solution 1

6	g	K2HPO4	
1000	ml	Distilled water	


## Mineral solution 2

6	g	KH2PO4	
6	g	(NH4)2SO4	
12	g	NaCl	
2.4	g	MgSO4 x 7 H2O	
1.6	g	CaCl2 x 2 H2O	
1000	ml	Distilled water	


## Modified Wolin's mineral solution

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.18	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.02	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 H2O	
0.4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.


## 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