# 727: CLOSTRIDIUM ULTUNENSE MEDIUM


## Main sol. 727

0.4	g	KH2PO4	
0.43	g	Na2HPO4	
0.3	g	NH4Cl	
0.3	g	NaCl	
0.1	g	CaCl2 x 2 H2O	
0.1	g	MgCl2 x 6 H2O	
1	ml	**Trace element solution**	
1	ml	**Selenite-tungstate solution**	
10	g	Yeast extract	
10	g	Peptone	
5	g	Lab Lemco powder (Oxoid)	
0.4	g	Starch (soluble)	
0.4	g	Na-acetate	
0.5	mg	Resazurin	
1.5	g	Na2CO3	
1	ml	**Wolin's vitamin solution (10x)**	
0.3	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except carbonate, vitamins and sulfide, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Prior to inoculation add vitamins and sulfide from sterile
anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Stock solutions of vitamins should be sterilized by filtration. Adjust pH of complete medium to 7.0, if necessay.


## Trace element solution

0.5	g	Na2-EDTA	
2	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	
10	mg	AlCl3	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve EDTA in distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add remaining compounds.


## 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