# J665: FRESHWATER IRON GEL MEDIUM


## Main sol. J665

100	mM	**Poorly crystalline iron(III) oxide**	
2.5	g	NaHCO3	
0.25	g	NH4Cl	
0.6	g	NaH2PO4 x H2O	
0.1	g	KCl	
1	ml	Na2SeO4 (1 mM)	
10	ml	**Vitamin solution**	
10	ml	**Trace mineral solution**	

1. Add components to distilled water, bring volume to 1.0 L and adjust pH to 7.0. Distribute the medium into culture vessels under a N2-CO2 (4:1, v/v) gas mixture, sparge with the same gas mixture for 10 - 15 min, seal with butyl rubber stoppers and autoclave. Before inoculation, add the following sterile solutions (autoclaved and stored under a N2 gas atmosphere) to the medium (per 10 ml of medium): 

0.1	ml	FeCl2 (130 mM)	
0.1	ml	CaCl2 x 2 H2O (0.4%)	
0.1	ml	Sodium acetate (pH 7.0, 2 M)	


## Vitamin solution

20	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine HCl	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	DL-Calcium pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	Lipoic acid	
1000	ml	Distilled water	


## Poorly crystalline iron(III) oxide

1. Dissolve 108 g FeCl3 in 1.0 L distilled water. Adjust pH to 7.0 with 10 N NaOH. Allow the suspension to sit for 30 min, then readjust pH to 7.0. Centrifuge the suspension at 5,000 x g for 20 min and pour off supernatant. Wash the precipitate with distilled water by resuspending in distilled water followed by the centrifugation. Repeat the washing at least 5 times, then, resuspend in 1.0 L distilled water. Use the Fe(III) suspension as 0.67 M solution. More precisely, check the concentration of Cl- to be less than 1 mM and determine the concentration of Fe(III) by using hydroxylamine as described by Lovley and Phillips (Appl. Environ. Microbiol. 54, 751-757, 1986).


## Trace mineral solution

1. Use Trace minerals of Medium No. 151 supplemented with 0.025 g (final) Na2MoO4 x 2H2O, 0.024 g NiCl2 x 6H2O and 0.025 g Na2WO4 x 2H2O (per liter).
2. Comment: For strain JCM 12378, inoculate with a 10% inoculum.