# J1135: METHANOBACTERIUM MEDIUM FOR STRAIN CAN


## Main sol. J1135

0.5	g	NaCl	
0.5	g	NH4Cl	
0.1	g	K2HPO4	
0.1	g	KH2PO4	
0.1	g	CaCl2 x 2 H2O	
0.1	g	MgSO4 x 7 H2O	
1	ml	**FeCl2 solution**	
1	ml	**Trace element solution**	
2	g	Sodium formate	
1	g	Sodium acetate	
2	mg	FeSO4 x 7 H2O	
0.1	g	L-Cysteine HCl x H2O	
0.5	mg	Resazurin	
1000	ml	Distilled water	

1. Mix components thoroughly, bring to a boil for several seconds, and cool down under a stream of N2-CO2 (80:20, v/v). Distribute the medium into culture vessels (e.g., 5 ml in the Hungate tube) under the same gas mixture, seal with butyl rubber stoppers and autoclave. Add per liter the following sterile solutions from anaerobic stocks (autoclaved or filter-sterilized): 

10	ml	Tris base (30%)	
20	ml	Na2CO3 (8%)	
10	ml	**Trace vitamins**	

2. Prior to inoculation, replace the gas phase with a gas mixture of H2-CO2 (80:20, v/v), and add per liter the following solution (autoclaved and stored under a N2 atmosphere): 

8	ml	Na2S x 9 H2O (5%)	

3. Pressurize the inoculated culture vessels to 200 kPa H2-CO2 (80:20, v/v).


## FeCl2 solution

10	ml	HCl (7.7 M, 25%)	
1.5	g	FeCl2 x 4 H2O	
990	ml	Distilled water	


## Trace element solution

70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 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