# 507: CARBOXYDOTHERMUS MEDIUM


## Main sol. 507

0.33	g	KCl	
0.52	g	MgCl2 x 6 H2O	
0.29	g	CaCl2 x 2 H2O	
0.33	g	NH4Cl	
0.33	g	KH2PO4	
1	ml	**Trace element solution SL-11**	
0.5	ml	Sodium resazurin (0.1% w/v)	
1	g	NaHCO3	
0.05	g	Yeast extract	
1	ml	**Wolin's vitamin solution (10x)**	
20	ml	**Neutralized sulfide solution 3% (w/v)**	
1000	ml	Distilled water	

1. Dissolve ingredients except bicarbonate, yeast extract, vitamins and sulfide, then sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. 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 (sterilized by filtration) and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Neutralize the sodium sulfide with sterile H2SO4 (see medium 29). After completing the medium adjust pH to 6.8-7.0.
2. Inoculated vessels are pressurized with sterile carbon monoxide gas to 2 bar overpressure.


## Trace element solution SL-11

5.2	g	Na2-EDTA x 2 H2O	
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	
1000	ml	Distilled water	

1. Dissolve EDTA in 800 ml distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds. Finally adjust pH to 6.0 and bring volume to 1000 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.