# 1042: VULCANIBACILLUS MEDIUM


## Main sol. 1042

30	g	Sea Salt (SIGMA, must not show signs of caking)	
1	g	NaNO3	
2	ml	**Wolfe's mineral elixir**	
0.5	ml	VOSO4 x 5 H2O (0.01% w/v)	
0.5	ml	Sodium resazurin (0.1% w/v)	
0.1	g	FeSO4 x 7 H2O	
1	g	NaHCO3	
0.5	g	Yeast extract	
1	g	Na-pyruvate	
2	ml	**Wolin's vitamin solution (10x)**	
1000	ml	Distilled water	

1. Dissolve ingredients (except ferrous sulfate, bicarbonate, yeast extract, pyruvate and vitamins), adjust pH to 7.0 and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense the medium under same gas atmosphere into Hungate-type tubes or serum vials and autoclave. Add ferrous sulfate, bicarbonate, yeast extract, pyruvate and vitamins from sterile anoxic stock solutions prepared under 100% N2 gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Prepare the stock solution of ferrous sulfate by dissolving 1% (w/v) FeSO4 x 7 H2O in 0.1 N H2SO4. The stock solutions of pyruvate and vitamins should be sterilized by filtration. Adjust pH of the complete medium to 6.8-7.0.
2. Note: It may be necessary to add 10-20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under 100% N2 and filter-sterilized), if the medium is not completely reduced after inoculation.


## 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