# 283: PYRODICTIUM MEDIUM


## Main sol. 283

13.85	g	NaCl	
3.5	g	MgSO4 x 7 H2O	
2.75	g	MgCl2 x 6 H2O	
0.33	g	KCl	
0.05	g	NaBr	
15	mg	H3BO3	
7	ml	SrCl2 x 6 H2O (0.1% w/v)	
10	mg	(NH4)2SO4	
5	ml	Citric acid (0.1% w/v)	
0.5	ml	KI (0.01% w/v)	
0.75	g	CaCl2 x 2 H2O	
0.5	g	KH2PO4	
2	ml	NiCl2 x 6 H2O (0.1% w/v)	
10	ml	**Modified Wolin's mineral solution**	
30	g	Sulfur (powdered)	
0.5	ml	Sodium resazurin (0.1% w/v)	
2	g	Yeast extract (OXOID)	
0.5	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Prepare the medium without sulfur, yeast extract and sodium sulfide, adjust pH to 5.0 - 5.5 with 10 N sulfuric acid, then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under 80% H2 and CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials  that contain already the appropriate amount of sulfur, only to 30% of their volume to allow for a large headspace. For sterilization of medium heat vessels for at least 1 hour to 90 - 100°C on each of 3 successive days. Do not autoclave! Complete the medium by adding yeast extract and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Adjust pH of complete medium to 5.5, if necessary.
2. After inoculation pressurize the vessels to 2 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture.


## Modified Wolin's mineral solution

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.18	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.02	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 H2O	
0.4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.