# J1221: MODIFIED NITRATE  MINERAL SALTS MEDIUM-2


## Main sol. J1221

1	g	MgSO4 x 7 H2O	
0.2	g	CaCl2 x 2 H2O	
0.25	g	KNO3	
1	ml	**FeCl2 solution**	
1	ml	**Trace element solution**	
2	ml	HEPES (pH 7.0, 1 M)	
4	mg	Ammonium ferric citrate	
1000	ml	Distilled water	

1. Mix components thoroughly and autoclave. After cooling, add the following solutions (autoclaved) to the medium: 

2	ml	**Phosphate buffer solution**	
10	ml	**Vitamin solution**	

2. Methane: Aseptically distribute the medium into sterile serum bottles (leave &; 80% gas phase by volume) and seal with rubber stoppers. Replace the gas phase with an air-methane gas mixture (75:25, v/v). Cultivate with shaking.
3. Adjust pH to 6.8, if necessary. Cultivate with methane or methanol as a substrate as described below:
4. Methanol: Aseptically distribute the medium into culture vessels (e.g., 20 ml in 60 ml serum bottles), supplement with 4.0 ml per liter filter-sterilized methanol and seal with rubber stoppers.


## Phosphate buffer solution

1.4	g	KH2PO4	
3.6	g	Na2HPO4 x 2 H2O	
100	ml	Distilled water	


## Vitamin solution

1000	ml	**Trace vitamins**	
20	mg	Vitamin B12	
5	mg	L-Ascorbic acid	
5	mg	Nicotinamide	


## 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