# 975: OCEANITHERMUS PROFUNDUS MEDIUM


## Main sol. 975

0.33	g	NH4Cl	
0.33	g	MgCl2 x 6 H2O	
0.33	g	CaCl2 x 2 H2O	
0.33	g	KCl	
1	g	KNO3	
30	g	NaCl	
2.38	g	HEPES (10mM)	
0.2	g	Yeast extract	
1	g	Tryptone	
2	g	Sucrose	
1	ml	**Wolin's vitamin solution**	
1	ml	**Modified Wolin's mineral solution**	
1000	ml	Distilled water	

1. Prepare the medium anaerobically, under nitrogen, omitting the CaCl2, MgCl2, KNO3, tryptone, yeast extract, vitamins and sucrose. The pH should be 7.0-7.5. Dispense the medium into vessels suitable for anaerobic growth (Hungate tubes or serum bottles) under an atmosphere of nitrogen and autoclave. To the sterile, cooled medium add, from sterile stock solutions the CaCl2, MgCl2, KNO3, tryptone, yeast extract, vitamins and sucrose. The CaCl2, MgCl2, KNO3, tryptone, yeast extract, and sucrose stock solutions should be autoclaved, while the vitamin solution is sterile filtered. This strain grows anaerobically in the presence of nitrate, but microaerophilic growth has been reported provided the oxygen concentration does not exceed 6 % (v/v). Under anaerobic conditions a redox agent, such as sulphide is not required.


## Wolin's vitamin solution

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 D-(+)-pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water	


## Modified Wolin's mineral solution

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.18	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.02	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 H2O	
0.4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.