# J1315: METHANOGEN MS-I MEDIUM


## Main sol. J1315

0.45	g	NaCl	
0.2	g	NaHCO3	
0.09	g	MgSO4 x 7 H2O	
0.225	g	KH2PO4	
0.225	g	K2HPO4	
0.225	g	(NH4)2SO4	
0.06	g	CaCl2 x 2 H2O	
2	mg	(NH4)2Ni(SO4)2 x 6 H2O	
2	mg	FeSO4 x 7 H2O	
0.1	ml	**Wolfe's mineral elixir**	
1	mg	Resazurin	
1000	ml	Distilled water	

1. Mix components thoroughly, except NaHCO3, and adjust pH to 7.0. Bring to a boil for a seconds, cool down under a stream of N2-CO2 (80:20, v/v) and add NaHCO3 to the medium. Dispense the medium into culture vessels (e.g., 20 ml in 120 ml serum bottles) under a H2-CO2 (80:20, v/v) gas mixture, seal with butyl rubber stoppers and autoclave. Aseptically and anaerobically add per liter the following solutions (autoclaved or filter-sterilized, and stored under N2): 

10	ml	**Trace vitamins** (*)	
10	ml	Sodium acetate (1.0 M)	
10	ml	Na2S x 9 H2O (5%)	

2. Pressurize inoculated bottles to 100 KPa H2-CO2 (80:20, v/v).


## Wolfe's mineral elixir

30	g	MgSO4 x 7 H2O	
5	g	MnSO4 x n H2O	
10	g	NaCl	
1	g	FeSO4 x 7 H2O	
1.8	g	CoCl2 x 6 H2O	
1	g	CaCl2 x 2 H2O	
1.8	g	ZnSO4 x 7 H2O	
0.1	g	CuSO4 x 5 H2O	
0.18	g	AlK(SO4)2 x 12 H2O	
0.1	g	H3BO3	
0.1	g	Na2MoO4 x 2 H2O	
2.8	g	(NH4)2Ni(SO4)2 x 6 H2O	
0.1	g	Na2WO4 x 2 H2O	
0.1	g	Na2SeO4	
1000	ml	Distilled water	

1. First adjust pH to 1.0 with diluted H2SO4, then dissolve the salts.


## 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