# 1028: THERMINCOLA MEDIUM (CO)


## Main sol. 1028

1	g	NH4Cl	
0.33	g	MgCl2 x 6 H2O	
0.1	g	CaCl2 x 2 H2O	
0.33	g	KCl	
0.5	g	KH2PO4	
1	ml	**Wolfe's mineral elixir**	
0.2	g	Na-acetate	
0.5	ml	Sodium resazurin (0.1% w/v)	
0.5	g	NaHCO3	
0.5	g	Na2CO3	
1	g	Na2S x 9 H2O	
0.2	g	Yeast extract	
2	ml	**Wolin's vitamin solution (10x)**	
1000	ml	Distilled water	

1. Dissolve ingredients except carbonates, sulfide, yeast extract and vitamins, then sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Add carbonates and sulfide to the medium, dispense under 100% carbon monoxide gas atmosphere into anoxic Hungate-type tubes or serum vials (e.g., 10 ml medium in 50 ml bottles) and autoclave. Prior to inoculation add yeast extract and vitamins (sterilized by filtration) from sterile anoxic stock solutions prepared under 100% N2 gas. If the medium is not completely reduced after autoclaving (remains pink) add 0.30 g/l sodium sulfide from a sterile anoxic stock solution prepared under 100% N2 gas atmosphere. Adjust pH of complete medium to 8.0 with a sterile anoxic solution of 1 N HCl.


## Wolfe's mineral elixir

30	g	MgSO4 x 7 H2O	
5	g	MnSO4 x H2O	
10	g	NaCl	
1	g	FeSO4 x 7 H2O	
1.8	g	CoCl2 x 6 H2O	
1	g	CaCl2 x 2 H2O	
1.8	g	ZnSO4 x 7 H2O	
0.1	g	CuSO4 x 5 H2O	
0.18	g	AlK(SO4)2 x 12 H2O	
0.1	g	H3BO3	
0.1	g	Na2MoO4 x 2 H2O	
2.8	g	(NH4)2Ni(SO4)2 x 6 H2O	
0.1	g	Na2WO4 x 2 H2O	
0.1	g	Na2SeO4	
1000	ml	Distilled water	

1. First adjust pH to 1.0 with diluted H2SO4, then add and dissolve the salts.


## 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