# 716: AMMONIFEX DEGENSII MEDIUM


## Main sol. 716

0.3	g	K2HPO4 x 3 H2O	
0.22	g	KH2PO4	
0.22	g	(NH4)2SO4	
1	g	NaCl	
0.09	g	MgSO4 x 7 H2O	
0.06	g	CaCl2 x 2 H2O	
2	ml	FeSO4 x 7 H2O (0.1% w/v in 0.1 N H2SO4)	
0.2	ml	NiCl2 x 6 H2O (0.1% w/v)	
1	ml	**Modified Wolin's mineral solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
1	g	KNO3	
1.5	g	Na2CO3	
0.3	g	DL-Dithiothreitol	
1000	ml	Distilled water	

1. Dissolve ingredients (except nitrate, carbonate, and dithiothreitol), then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic serum vials (e.g., 10 ml medium in 25 ml Balch-type tubes) and autoclave. Before use, add nitrate from a sterile anoxic stock solution prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Before inoculation add dithiothreitol from an anoxic stock solution sterilized by filtration under 100% N2 gas and adjust the pH of the medium to 7.2.
2. After growth has started pressurize vials to 0.5 bar overpressure with a 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.