# 901a: THERMODESULFOBIUM ACIDIPHILUM MEDIUM


## Main sol. 901a

0.33	g	NH4Cl	
0.33	g	KCl	
0.33	g	KH2PO4	
0.33	g	MgCl2 x 6 H2O	
0.33	g	CaCl2 x 2 H2O	
2.8	g	Na2SO4	
1	ml	**Trace element solution SL-10**	
0.5	ml	Sodium resazurin (0.1% w/v)	
3	g	Yeast extract	
1	ml	**Wolin's vitamin solution (10x)**	
15	ml	**Neutralized sulfide solution 3% (w/v)**	
1000	ml	Distilled water	

1. Dissolve ingredients except yeast extract, vitamins and sulfide.  Adjust pH to 4.5 with H2SO4 and sparge medium with 100% CO2 gas for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add yeast extract, vitamins (sterilized by filtration), and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. The sulfide stock solution (3% w/v) should be neutralized before use (see medium 28). The pH of the complete medium should be at 4.5.


## 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.


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water	


## Neutralized sulfide solution 3% (w/v)

3	g	Na2S x 9 H2O	
100	ml	Distilled water	

1. The sulfide solution is prepared in a 250 ml screw-capped bottle with a butyl rubber septum and a magnetic stirrer. The solution is bubbled with nitrogen gas, closed and autoclaved for 15 min. at 121°C. After cooling to room temperature the pH is adjusted to about 7.0 by adding of sterile 2 M H2SO4 drop-wise with a syringe without opening the bottle.