# 829c: DESULFUROBACTERIUM CRINIFEX MEDIUM


## Main sol. 829c

30	g	Sea Salt (SIGMA)	
1	g	NH4Cl	
0.35	g	KH2PO4	
1.95	g	MES [2-(N-morpholino) ethane sulfonic acid]	
10	ml	**Modified Wolin's mineral solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
10	g	Sulfur, powdered	
0.5	g	Na2CO3	
1	ml	**Wolin's vitamin solution (10x)**	
0.5	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except sulfur, carbonate, vitamins and sulfide), 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 under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials which contain already the appropriate amount of sulfur, only to 30% of their volume to allow for a large head space. Autoclave at a temperature of <b>110°C</b> for 20 min! Add vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Vitamins are sterilized by filtration. Adjust pH of the complete medium to 6.0 - 6.5, if necessary.
2. After inoculation use 1 bar overpressure of sterile 80% H2 and 20% CO2 gas mixture.


## Modified Wolin's mineral solution

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.18	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.02	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 H2O	
0.4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.


## 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