# 1030: SMITHELLA MEDIUM


## Main sol. 1030

1	g	NH4Cl	
1	g	MgCl2 x 6 H2O	
0.4	g	CaCl2 x 2 H2O	
0.4	g	KH2PO4	
1	ml	**Wolfe's mineral elixir**	
2	g	Yeast extract	
2	g	Trypticase peptone (BD BBL)	
0.5	ml	Sodium resazurin (0.1% w/v)	
1	g	Na2CO3	
20	ml	**Na-crotonate solution (1 M)**	
0.5	g	2-Mercaptoethanesulfonic acid (coenzyme M)	
1	ml	**Wolin's vitamin solution (10x)**	
0.3	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except carbonate, crotonate, coenzyme M, 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 Hungate-type tubes or serum vials (e.g., 5 ml medium in Balch-type tubes) and autoclave. Add crotonate, coenzyme M, 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 crotonate, coenzyme M and vitamins should be sterilized by filtration. Adjust pH of the complete medium to 7.0 - 7.2.
2. Note: Use 10% (v/v) as inoculum.


## 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.


## Na-crotonate solution (1 M)

86	g	Crotonic acid (ALDRICH 113018)	
100	ml	NaOH (10 N)	
900	ml	Distilled water	

1. Dissolve crotonic acid in 800 ml distilled water, add around 100 ml of 10 N NaOH and adjust pH to around 7. Then add water to reach a volume of 1000 ml. Sterilize by filtration under 100% N2 gas atmosphere.


## 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