# 806: THERMOCOCCUS MEDIUM


## Main sol. 806

0.33	g	KCl	
2.7	g	MgCl2 x 2 H2O	
3.4	g	MgSO4 x 7 H2O	
0.25	g	NH4Cl	
0.14	g	CaCl2 x 2 H2O	
0.14	g	K2HPO4	
1	µg	Na2SeO3	
1	µg	NiCl2 x 6 H2O	
1	g	NaHCO3	
18	g	NaCl	
1	mg	Resazurin	
0.5	g	L-Cysteine HCl x H2O	
0.5	g	Na2S x 9 H2O	
1	g	Yeast extract	
1	g	Trypticase	
5	g	Sulphur	
10	ml	**Modified Wolin's mineral solution II**	
10	ml	**Wolin's vitamin solution**	
980	ml	Distilled water	

1. Prepare the medium without NaHCO3, cysteine hydrochloride, Na2S x 9 H2O, and vitamin solution. Boil the medium and cool under N2. Add the NaHCO3 to the cooled medium and adjust the pH to about pH 7.2. Sterilise the medium at 100°C for 3 hours on 3 consecutive days. Reduce the medium by adding 10% neutralised sodium sulphide and cysteine hydrochloride from sterile stock solutions. The vitamins are added from a sterile (filter sterilised) stock solution prepared under nitrogen. The final pH is 7.2.


## Modified Wolin's mineral solution II

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	
0.025	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 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.


## 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