# J1258: DESULFOBACTERIUM OLEOVORANS MEDIUM


## Main sol. J1258

4	g	Na2SO4	
0.2	g	KH2PO4	
0.25	g	NH4Cl	
20	g	NaCl	
3.4	g	MgCl2 x 6 H2O	
0.25	g	CaCl2 x 2 H2O	
1	ml	**FeCl2 solution**	
1	ml	**Trace element solution**	
1	ml	**Selenite-tungstate solution**	
0.5	mg	Resazurin	
1000	ml	Distilled water	

1. Mix components thoroughly, bring to a boil and cool down under a N2-CO2 (4:1, v/v) gas mixture. Distribute the medium into culture vessels (e.g., 10 ml in Balch tubes) under the same gas mixture, seal with butyl rubber stoppers and autoclave. After cooling, aseptically and anaerobically add per liter the following solutions (autoclaved or *filter-sterilized and stored under a N2 gas atmosphere; **filter-sterilized and stored under an 80% N2-20% CO2 gas mixture): 

10	ml	**Trace vitamins** (*)	
1	ml	**Vitamin B12 solution 0.05 g/L** (*)	
10	ml	Steric solution (see below)	
20	ml	NaHCO3 (**, 8%)	
8	ml	Na2S x 9 H2O (5%)	


## Stearic solution

0.36	g	Stearic acid	
0.63	ml	NaOH (2 N)	
10	ml	Distilled water	

1. Heat the solution in a closed vial under a N2 atmosphere in a boiling water bath. Shake until stearic acid is dissolved and autoclave. Add the solution to the medium while stearic acid is dissolved. Re-melt if stearic acid is solidified.


## FeCl2 solution

10	ml	HCl (7.7 M, 25%)	
1.5	g	FeCl2 x 4 H2O	
990	ml	Distilled water	


## Trace element solution

70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 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	


## 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	


## Vitamin B12 solution 0.05 g/L

5	mg	Vitamin B12	
100	ml	Distilled water