# 508: CARBOXYDOTHERMUS MEDIUM (HETEROTROPH)


## Main sol. 508

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	
10	ml	**Trace element solution SL-4**	
0.5	ml	Sodium resazurin (0.1% w/v)	
1	g	Na2CO3	
2.5	g	Na-pyruvate	
0.05	g	Yeast extract	
1	ml	**Seven vitamins solution**	
1	ml	**Wolin's vitamin solution (10x)**	
0.3	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except carbonate, pyruvate, yeast extract, 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. Add pyruvate, yeast extract, 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. Stock solutions of pyruvate and vitamins are sterilized by filtration. After completing the medium adjust pH to 6.8-7.0 with a sterile anoxic stock solution of Na2CO3 (5% w/v), if necessary.


## Trace element solution SL-4

0.5	g	Na2-EDTA	
0.2	g	FeSO4 x 7 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.03	g	MnCl2 x 4 H2O	
0.3	g	H3BO3	
0.2	g	CoCl2 x 6 H2O	
0.01	g	CuCl2 x 2 H2O	
0.02	g	NiCl2 x 6 H2O	
0.03	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve EDTA in distilled water and adjust pH to 7.0 using 2 N NaOH; then add other compounds.


## Seven vitamins solution

100	mg	Vitamin B12	
80	mg	p-Aminobenzoic acid	
20	mg	D-(+)-biotin	
200	mg	Nicotinic acid	
100	mg	Calcium pantothenate	
300	mg	Pyridoxine hydrochloride	
200	mg	Thiamine-HCl 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