# J231: METHANOBACTERIUM THERMOAUTOTROPHICUM MEDIUM


## Main sol. J231

0.3	g	KH2PO4	
1.5	g	(NH4)2SO4	
0.6	g	NaCl	
0.12	g	MgSO4 x 7 H2O	
0.08	g	CaCl2 x 2 H2O	
4	mg	FeSO4 x 7 H2O	
0.15	g	K2HPO4	
4	g	Na2CO3	
10	ml	**Trace vitamins**	
10	ml	**Trace element solution**	
0.5	g	L-Cysteine HCl x H2O	
0.5	g	Na2S x 9 H2O	
1	mg	Resazurin	
980	ml	Distilled water	

1. Mix ingredients, except cysteine x HCl and Na2S x 9H2O, and adjust pH to 7.2. Bring to a boil for 5-10 sec, then, cool down under a stream of H2-CO2 (80:20). Dispense the medium into suitable culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a stream of H2-CO2 (80:20) and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight. Separately autoclave cysteine x HCl and Na2S x 9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the cysteine x HCl and Na2S x 9H2O solutions. Pressurize the inoculated bottles to 100 kPa H2-CO2 (80:20).


## Trace element solution

0.64	g	Na2-EDTA	
6.2	g	MgSO4 x 7 H2O	
0.55	g	MnSO4 x n H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.17	g	CoCl2 x 6 H2O	
0.13	g	CaCl2 x 2 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.05	g	CuSO4 x 5 H2O	
0.018	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.011	g	Na2MoO4 x 2 H2O	
0.025	g	NiCl2 x 6 H2O	

1. Dissolve EDTA x 2Na first in 500 ml of distilled water. Then add the remaining salts and adjust volume to 1000 ml.


## 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