# J265: METHANOGENIUM MEDIUM


## Main sol. J265

0.355	g	KCl	
4	g	MgCl2 x 6 H2O	
3.45	g	MgSO4 x 7 H2O	
0.25	g	NH4Cl	
0.14	g	CaCl2 x 2 H2O	
0.14	g	KH2PO4	
18	g	NaCl	
10	ml	**Wolfe's mineral solution**	
10	ml	**Trace vitamins**	
2	mg	Fe(NH4)2(SO4)2 x 6 H2O	
5	g	NaHCO3	
1	g	Sodium acetate	
2	g	Yeast extract (BD-Difco)	
2	g	Trypticase peptone (BD-BBL)	
1	mg	Resazurin	
980	ml	Distilled water	

1. Mix components thoroughly, except NaHCO3, and adjust pH to 6.5. Bring to a boil for 5-10 sec, then, cool down under a stream of H2-CO2 (80:20, v/v) and add NaHCO3 to the medium. Dispense the medium into suitable culture vessels (e.g., 5 ml medium in Hungate tubes) under the same gas mixture and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight. Aseptically and anaerobically add per liter the following solutions (autoclaved and stored under a N2 atmosphere): 

10	ml	L-Cysteine HCl x H2O (5%)	
10	ml	Na2S x 9 H2O (5%)	

2. Pressurize the inoculated bottles to 200 kPa H2:CO2 (80:20, v/v).


## Wolfe's mineral solution

1000	ml	**Trace minerals**	
0.02	g	NiCl2 x 6 H2O	
1	mg	Na2SeO3	
0.01	g	Na2WO4 x 2 H2O	


## 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.