# J1176: METHANOSARCINA JL01 MEDIUM


## Main sol. J1176

0.15	g	KH2PO4	
0.29	g	K2HPO4	
1	g	NH4Cl	
0.2	g	MgCl2 x 2 H2O	
0.1	g	CaCl2 x 2 H2O	
0.9	g	NaCl	
1	ml	**FeCl2 solution**	
1	ml	**Trace element solution**	
2	g	Yeast extract (Oxoid)	
0.5	mg	Resazurin	
2	g	NaHCO3	
1000	ml	Distilled water	

1. Mix components except NaHCO3 and bring to a boil for a few seconds. After cooling down under a stream of N2-CO2 (80:20, v/v), add NaHCO3 to the medium. Dispense the medium into culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under the same gas mixture, seal with butyl rubber stoppers and autoclave. Allow to stand the medium until re-dissolved. Then, add per liter the following solutions (autoclaved or *filter-sterilized): 

10	ml	**Trace vitamins** (*)	
10	ml	Methanol (v/v, 50%)	
10	ml	L-Cysteine HCl x H2O (5%)	
10	ml	Na2S x 9 H2O (5%)	

2. Adjust pH of the medium to 6.8-7.0, if necessary.


## 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