# J576: DESULFATIBACILLUS OLEFINIVORANS MEDIUM


## Main sol. J576


## Solution A

3	g	Na2SO4	
0.2	g	KH2PO4	
0.3	g	NH4Cl	
7	g	NaCl	
0.5	g	KCl	
0.15	g	CaCl2 x 2 H2O	
0.1	g	Yeast extract (BD-Difco)	
1	ml	Trace element solution SL12 (see below)	
1	ml	**Selenite-tungstate solution**	
2.38	g	HEPES	
1	mg	Resazurin	
960	ml	Distilled water	

1. Mix ingredients, adjust pH to 7.5 and autoclave under a N2 atmosphere. After cooling, add the following stock solutions (filter-sterilized or *autoclaved) to the solution: 

10	ml	MgCl2 x 6 H2O (*, 15%)	
30	ml	NaHCO3 (8%)	
1	ml	**Vitamin solution**	
1	ml	**Thiamine solution**	
1	ml	**Vitamin B12 solution 0.05 g/L**	


## Substrate

2.8	g	Sodium palmitate	

1. Autoclave in culture vessels.
2. To complete the medium, combine Solution A and substrate in the culture vessels under a N2-CO2 (4:1, v/v) gas mixture and seal with butyl rubber stoppers. Finally, reduce the medium with 0.01 volume of 5% Na2S x 9H2O solution (autoclaved and stored under a N2 atmosphere).


## Trace metal solution SL12

3	g	Na2-EDTA	
1.1	g	FeSO4 x 7 H2O	
190	mg	CoCl2 x 6 H2O	
50	mg	MnCl2 x 4 H2O	
42	mg	ZnCl2	
24	mg	NiCl2 x 6 H2O	
18	mg	Na2MoO4 x 2 H2O	
300	mg	H3BO3	
2	mg	CuCl2 x 2 H2O	
1000	ml	Distilled water	


## Selenite-tungstate solution

0.4	g	NaOH	
6	mg	Na2SeO3 x 5 H2O	
8	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## Vitamin solution

4	mg	p-Aminobenzoic acid	
1	mg	Biotin	
10	mg	Nicotinic acid	
5	mg	DL-Calcium pantothenate	
10	mg	Pyridoxine hydrochloride	
100	ml	Sodium phosphate buffer (10 mM, pH 7.1)	


## Thiamine solution

10	mg	Thiamine HCl	
100	ml	Sodium phosphate buffer (25 mM, pH 3.4)	


## Vitamin B12 solution 0.05 g/L

5	mg	Vitamin B12	
100	ml	Distilled water