# J945: MB MEDIUM FOR METHANOBACTERIUM


## Main sol. J945

1. Mix components thoroughly, and autoclave under a N2-CO2 (80:20, v/v) gas atmosphere. After cooling, aseptically and anaerobically add the following solutions (filter-sterilized): 

0.15	g	KH2PO4	
0.3	g	K2HPO4	
0.6	g	NaCl	
0.3	g	(NH4)2SO4	
0.12	g	MgSO4 x 7 H2O	
0.08	g	CaCl2 x 2 H2O	
10	ml	**Trace minerals solution**	
1	mg	Resazurin	
932	ml	Distilled water	
48	ml	NaHCO3 (8%)	
10	ml	**Trace vitamins**	

2. Distribute the medium into culture vessels (leave >80% gas phase by volume) under a H2-CO2 (80:20, v/v) gas mixture, seal with butyl rubber stoppers. Finally, add per liter the following solutions from anaerobic stocks (autoclaved and stored under a N2 atmosphere): 

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

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


## Trace minerals solution

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x n H2O	
0.1	g	FeSO4 x 7 H2O	
0.1	g	CoSO4 x 7 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
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. Then proceed to add minerals. Adjust final pH to 7.0.


## 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