# J230: METHANOSARCINA MEDIUM


## Main sol. J230

0.227	g	KH2PO4	
0.348	g	K2HPO4	
0.5	g	NH4Cl	
0.5	g	MgSO4 x 7 H2O	
0.25	g	CaCl2 x 2 H2O	
2.25	g	NaCl	
2	mg	FeSO4 x 7 H2O	
2	g	Yeast extract (BD-Difco)	
2	g	Casitone (BD-Difco)	
10	ml	**Trace vitamins**	
1	ml	**FeCl2 solution**	
1	ml	**Trace element solution**	
0.85	g	NaHCO3	
10	ml	Methanol	
0.3	g	L-Cysteine HCl x H2O	
0.3	g	Na2S x 9 H2O	
1	mg	Resazurin	
980	ml	Distilled water	

1. Mix ingredients, except NaHCO3, methanol, cysteine x HCl and Na2S x 9H2O, and bring to a boil for 5-10 sec. Cool down under a stream of N2-CO2 (80:20), and add NaHCO3 and methanol to the medium. Dispense the medium into suitable culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a stream of N2-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.


## 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	


## 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