# J1068: SALTWATER IRON-OXIDIZING BACTERIA  MEDIUM-II


## Main sol. J1068

1000	ml	**Artificial saltwater**	
10	ml	**Wolfe's mineral solution**	

1. Mix components, distribute the medium into culture vessels (e.g., 10 ml in Balch-type tubes), seal with butyl rubber stoppers and autoclave. After autoclaving, aseptically add per liter the following solutions (filter-sterilized): 

10	ml	**Trace vitamins**	
12.5	ml	NaHCO3 (8%)	
10	ml	FeCl2 (freshly prepared, 0.2 M)	

2. Replace the gas phase with a N2-CO2-O2 (80:20:3, v/v/v) gas mixture and pressurize to 200 kPa with the same gas mixture.


## Artificial saltwater

27.5	g	NaCl	
5.38	g	MgCl2 x 6 H2O	
6.78	g	MgSO4 x 7 H2O	
0.72	g	KCl	
0.2	g	NaHCO3	
1.4	g	CaCl2 x 2 H2O	
1	g	NH4Cl	
0.05	g	KH2PO4	
1000	ml	Distilled water	


## Wolfe's mineral solution

1000	ml	**Trace minerals**	
0.02	g	NiCl2 x 6 H2O	
1	mg	Na2SeO3	
0.01	g	Na2WO4 x 2 H2O	


## 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	


## Trace minerals

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x n H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.1	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.01	g	AlK(SO4)2	
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 solution. Then proceed to add minerals. Adjust final pH to 7.0.