# J350: THERMOCOCCUS MEDIUM (pH 6.0)


## Main sol. J350

1000	ml	**Main sol. J280**	

1. After reduction of the medium, readjust pH to 6.0 with 1.0 N H2SO4.


## Main sol. J280

0.33	g	KCl	
2.8	g	MgCl2 x 6 H2O	
3.4	g	MgSO4 x 7 H2O	
0.25	g	NH4Cl	
25	g	NaCl	
0.3	g	K2HPO4	
3	g	Yeast extract (BD-Difco)	
3	g	Tryptone (BD-Difco)	
0.025	g	FeSO4 x 7 H2O	
10	mg	NaBr	
10	ml	**Trace minerals**	
10	ml	**Trace vitamins**	
10	g	Sulfur (powder)	
0.5	g	Na2S x 9 H2O	
1	mg	Resazurin	
1000	ml	Distilled water	

1. Mix ingredients except sulfur and Na2S x 9H2O, adjust pH to 7.0-7.2 and autoclave under a N2 atmosphere. Steam sulfur for 3 hr on each of 3 successive days. Neutralize Na2S x 9H2O as a 5% solution and autoclave under a N2 gas atmosphere. Distribute the medium and sulfur into culture vessels under a N2 stream, and seal with butyl rubber stoppers. Prior to inoculation, add the sterile Na2S x 9H2O solution, and readjust pH to 7.0-7.2, if necessary.
2. Comment:
3. Most of <I>Thermococcus</I> strains will grow in this medium supplemented with 5.0 g/L maltodextrin and omitted sulfur.


## 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