# J251: METHANOTHERMUS FERVIDUS MEDIUM


## Main sol. J251

37.5	ml	**Mineral solution 1**	
37.5	ml	**Mineral solution 2**	
3	mg	(NH4)2Ni(SO4)2 x 6 H2O	
2	mg	FeSO4 x 7 H2O	
2	g	Yeast extract (BD-Difco)	
2	g	Trypticase peptone (BD-BBL)	
10	ml	**Trace vitamins**	
10	ml	**Trace minerals**	
3.4	g	Na2SO4	
2	g	NaHCO3	
1	mg	Resazurin	
0.5	g	Na2S x 9 H2O	
0.5	g	L-Cysteine HCl x H2O	
920	ml	Distilled water	

1. Mix ingredients, except NaHCO3, L-cysteine x HCl x H2O and Na2S x 9H2O, and bring to a boil for 5-10 sec. Cool down under a stream of H2-CO2 (80:20), and add NaHCO3 to the medium. Dispense the medium into culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a stream of H2-CO2 (80:20) gas mixture and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight and adjust pH to 6.5, if necessary. Separately autoclave L-cysteine x HCl and Na2S x 9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the L-cysteine x HCl and Na2S x 9H2O solutions. Pressurize the inoculated bottles to 200 kPa H2-CO2 (80:20).


## 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	


## Trace vitamins

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine HCl	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	Calcium pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	Lipoic acid	
1000	ml	Distilled water	


## Trace minerals

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x n H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.1	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.01	g	AlK(SO4)2	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH solution. Then proceed to add minerals. Adjust final pH to 7.0.