# J1067: FRESHWATER IRON-OXIDIZING BACTERIA MEDIUM-II


## Main sol. J1067

50	ml	**FeS solution**	
50	ml	**Modified Wolfe's mineral solution**	
1.5	g	Agar (BD-Difco),  Noble	

1. Top layer: 

1000	ml	**Modified Wolfe's mineral solution**	
10	ml	**Trace minerals**	

2. After cooling of the top layer solution, add per liter the following solutions (filter-sterilized): 

10	ml	**Trace vitamins**	
5.25	ml	NaHCO3 (8%)	

3. Bottom layer:
4. Autoclave both layer solutions. Shortly after autoclaving, pipette the bottom layer into each culture vessel (typically 5 ml in 60 ml glass vials, use pipette tips that have been cut to enlarge the opening). The bottom layer is allowed to cool for at least 30 min to ensure its solidification.
5. Add the top layer solution in 5 to 6 times the volume of the bottom layer to the culture vessels containing the bottom layer (e.g., 25 to 30 ml in 60 ml glass vials with 5 ml of the bottom layer).
6. Then, cap the culture vessels with silicone rubber stoppers, and replace the gas phase with a N2-CO2 (4:1, v/v) gas mixture.
7. Comment: The medium can be stored in culture vessels for a certain time by capping with butyl rubber stoppers, instead of silicon rubber stoppers and keeping anoxic in the vessels. Silicone rubber stopper is O2 permeable and creates a mircoaerophilic condition in the culture vessels.


## FeS solution

46.2	g	FeSO4 x 7 H2O	
39.6	g	Na2S x 9 H2O	
300	ml	Distilled water	

1. Prewarm distilled water at 50C in a beaker with a stir bar. While rapidly starring the water, add FeSO4 x 7H2O, followed immediately by Na2S x 9H2O. A thick black precipitate will form instantly. Stir continuously for 2 to 3 min. Remove the mixture to a narrow-mouthed glass bottle (500 ml) that can be tightly stopped. The bottle is filled to the top with distilled water and capped. Allow to settle for several hours, decant and replace the overlaying water. Repeat this procedure at least five times to wash the FeS precipitate. Check pH to be close to neutral. To stock the FeS solution, fill the bottle with distilled water and cap. Remove the overlaying water when prepare the bottom layer.


## Modified Wolfe's mineral solution

1	g	NH4Cl	
0.2	g	MgSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.05	g	KH2PO4	
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.


## 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