# 1001: BASAL MEDIUM


## Main sol. 1001

2.5	g	NaHCO3	
0.25	g	NH4Cl	
0.6	g	NaH2PO4 x H2O	
0.1	g	KCl	
10	ml	**Vitamin mixture**	
10	ml	**Mineral mixture**	
980	ml	Distilled water	

1. This medium should not be exposed to direct sunlight! Add all basal medium ingredients. Bring the final volume of the medium to 1.0 liter. Dispense to appropriate culture containers. Bubble the medium with 80:20 N2:CO2 (final pH sould be 6.8 to 7.0) - approximately 10 ml of media (anaerobic culture tube) should be gassed for 5 min in the aqueous phase (bubbled) and the headspace gassed for one minute prior to sealing the container. Sterilize per usual procedure. Add electron donor (Acetate-final conc. of 10 mM-recipe below) and electron acceptor (Fe(III)NTA-final conc. of 10 mM-recipe below) from sterile, anaerobic stock solutions using a sterile syringe and needle flushed with anaerobic gas. Store out of direct light.


## 1.0 M Acetate Stock Solution

13.6	g	Sodium acetate	
100	ml	Distilled water	

1. Add 13.6 g sodium acetate to ca. 80 ml distilled water. Bring to final volume of 100 ml. Bubble with anaerobic gas (nitrogen) for 45 min. Seal and sterilize.


## 500 mM Fe(III)NTA Stock solution

8.2	g	NaHCO3	
12.8	g	Nitrilotriacetic acid	
13.5	g	FeCl3 x 6 H2O	

1. Add 8.2 g NaHCO3 to ca. 70 ml distilled water. Add 12.8 g of Na3Nitrilotriacetic acid (NTA) to ca. 70 ml distilled water. Add 13.5 g FeCl3 x 6 H2O to ca 70 ml distilled water. Adjust pH to 6.5 using 10N NaOH. Bring solution to final volume of 100 ml. Ingredients will go into solution after stirring for ca. 15 min. Bubble with anaerobic gas (N2) for 45 min and then filter sterilize (0.2 µm filter) into a sterile, anaerobic, serum bottle.


## Vitamin mixture

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Riboflavin	
5	mg	Thiamine	
5	mg	Nicotinic acid	
5	mg	Pantothenic acid	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	Thioctic acid	
1000	ml	Distilled water	


## Mineral mixture

1.5	g	NTA	
3	g	MgSO4	
0.5	g	MnSO4 x H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	CoCl2 x 6 H2O	
0.13	g	ZnCl2	
0.01	g	CuSO4 x 5 H2O	
0.01	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.025	g	Na2MoO4	
0.024	g	NiCl2 x 6 H2O	
0.025	g	Na2WO4 x 2 H2O	
1000	ml	Distilled water