# 829a: DESULFUROBACTERIUM ATLANTICUM MEDIUM


## Main sol. 829a

30	g	NaCl	
3	g	MgCl2 x 6 H2O	
0.15	g	CaCl2 x 2 H2O	
0.5	g	KCl	
0.5	g	NH4Cl	
1.95	g	MES [2-(N-morpholino) ethane sulfonic acid]	
0.2	g	KH2PO4	
0.5	ml	Sodium resazurin (0.1% w/v)	
2.5	g	Na2S2O3 x 5 H2O	
20	ml	**Neutralized sulfide solution 3% (w/v)**	
1000	ml	Distilled water	

1. Dissolve ingredients (except thiosulfate and sodium sulfide), adjust pH to 6.0, boil medium for 1 min, then cool to room temperature under 80% H2 and 20% CO2 gas atmosphere. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add sodium thiosulfate from an anoxic stock solution prepared under 100% N2 gas and sterilized by filtration. Reduce medium with a sterile, neutralized solution of sodium sulfide prepared under 100% N2 gas . Adjust pH of complete medium to 6.0, if necessary.
2. After inoculation pressurize cultivation vessels to   2 bar overpressure using sterile 80% H2 and 20% CO2 gas mixture.


## 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.