# 1516: THERMOFILUM MEDIUM


## Main sol. 1516

1000	ml	**Liquid medium**	

1. Prepare the liquid medium. Boil the medium in a water bath and cool under N2 to room
temperature. Adjust to pH 5.8-6.2. Dispense the medium into Hungate tubes or serum
bottles under gassing with N2. Sterilize by autoclaving. Before inoculation add from
sterile stock solutions:

10	ml	**Filtrate of Desulfurococcus kamchatkensis (DSM18924)**	
10	ml	**Wolin's vitamin solution**	
0.3	g	Na2S x H2O	

2. Prepare the liquid medium. Boil the medium in a water bath and cool under N2 to room temperature. Adjust to pH 5.8-6.2. Dispense the medium into Hungate tubes or serum bottles under gassing with N2. Sterilize by autoclaving. Before inoculation add from sterile stock solutions: 


## Trace Element Solution

784	mg	Fe(NH4)2(SO4)2 x 6 H2O	
225	mg	NiCl2 x 2 H2O	
38	mg	CoCl2 x 6 H2O	
99	mg	MnCl2 x 4 H2O	
144	mg	ZnSO4 x 7 H2O	
95	mg	Na2SeO4	
33	mg	Na2WO4 x 2 H2O	
24	mg	Na2MoO4 x 2 H2O	
6	mg	H3BO3	
2	mg	CuCl2 x 2 H2O	
1000	ml	Distilled water	


## 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	


## Filtrate of Desulfurococcus kamchatkensis (DSM18924)

10	ml	**Liquid medium**	
2	g/l	Glucose	
0.6	g/l	Yeast extract	

1. Strain DSM 18924 was inoculated into anaerobic tube, containing 10ml of the medium
mentioned above. Glucose (2g/l) and yeast extract (0,6g/l) were added as substrate.
After 3 days of incubation culture was filtered throuth 0,22nm filter. Filtered culture
broth is used for the cultivation of the strain at a concentration of 1% as a source of
growth factor


## Liquid medium

0.33	g	KH2PO4	
0.33	g	NH4Cl	
0.33	g	MgCl2	
0.33	g	CaCl2	
0.33	g	KCl	
2	g	Glucose	
0.1	g	Yeast extract	
1	ml	**Trace Element Solution**	
1000	ml	Distilled water