# 829b: THERMODESULFOBACTERIUM HYDROGENIPHILUM MEDIUM


## Main sol. 829b

30	g	Sea Salt (SIGMA)	
1	g	NH4Cl	
0.35	g	KH2PO4	
1.95	g	MES [2-(N-morpholino) ethane sulfonic acid]	
1	ml	**Trace element solution SL-10**	
1	ml	**Selenite-tungstate solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
0.5	g	Na2CO3	
0.5	g	Yeast extract	
1	ml	**Seven vitamins solution**	
1	ml	**Growth-stimulating factors**	
1	ml	**Na-dithionite solution (5% w/v)**	
1000	ml	Distilled water	

1. Dissolve ingredients (except carbonate, yeast extract, vitamins, growth-stimulating factors and Na-dithionite), boil medium for 1 min, then cool to room temperature under 80% H2 and 20% CO2 gas atmosphere. Adjust pH to 6.0 and dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add yeast extract, vitamins, and growth-stimulating factors from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere and carbonate and Na-dithionite  from sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Vitamins and Na-dithionite are sterilized by filtration. Adjust pH of the complete medium to 6.5, if necessary.
2. After inoculation use 2 bar overpressure of sterile 80% H2 and 20% CO2 gas mixture.


## Trace element solution SL-10

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

1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml.


## Selenite-tungstate solution

0.5	g	NaOH	
3	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## Seven vitamins solution

100	mg	Vitamin B12	
80	mg	p-Aminobenzoic acid	
20	mg	D-(+)-biotin	
200	mg	Nicotinic acid	
100	mg	Calcium pantothenate	
300	mg	Pyridoxine hydrochloride	
200	mg	Thiamine-HCl x 2 H2O	
1000	ml	Distilled water	


## Growth-stimulating factors

5	g	Isobutyric acid	
5	g	Valeric acid	
5	g	2-Methylbutyric acid	
5	g	3-Methylbutyric acid	
2	g	Caproic acid	
6	g	Succinic acid	
1000	ml	Distilled water	

1. Dissolve ingredients, adjust pH to 9.0 with NaOH, then autoclave under 100% N2 gas.


## Na-dithionite solution (5% w/v)

50	g	NaHCO3	
50	g	Na2S2O4	
1000	ml	Distilled water	

1. Dissolve NaHCO3 in water and make the solution anoxic by sparging with 80% N2 and 20% CO2 gas mixture. Then dissolve the Na-dithionite and filter sterilize the solution into anoxic Hungate tubes. Store the prepared solution in the dark and refrigerated. Prepare only small amounts of stock solution, as Na-dithionite decomposes rapidly.