# 1297: NATRONIELLA SULFIDIGENA MEDIUM


## Main sol. 1297

25	g	NaHCO3	
140	g	Na2CO3	
15	g	NaCl	
1	g	K2HPO4	
0.2	g	NH4Cl	
0.2	g	MgCl2 x 6 H2O	
0.2	g	Yeast extract	
1	ml	**Trace elements solution (Pfennig & Lippert,1966)**	
1	ml	**Selenite-tungstate solution**	
2	g	Glycerol	
1	ml	**Wolin's vitamin solution (10x)**	
2	g	Sulfur (powdered)	
0.25	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve hydrogen carbonate, carbonate, sodium chloride, and hydrogen phosphate, then sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense solution under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add ammonium chloride, magnesium chloride, yeast extract, trace elements, glycerol, vitamins, and sulfide from a sterile anoxic stock solution prepared under 100% N2 gas. The vitamin solution should be sterilized by filtration. Sulfur is sterilized by steaming for 3 hours on each of 3 successive days (see medium 35) and added aseptically to the sterile medium as powder while retaining anoxic conditions. Adjust the pH of the complete medium to 9.8 - 10.0, if necessary.


## Trace elements solution (Pfennig & Lippert,1966)

5	g	EDTA	
2.2	g	FeSO4 x 7 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.03	g	MnCl2 x 4 H2O	
0.03	g	H3BO3	
0.2	g	CoCl2 x 6 H2O	
0.03	g	CuCl2 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.03	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. pH 3.0-4.0


## Selenite-tungstate solution

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


## 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