# 479: METHANOHALOPHILUS MEDIUM


## Main sol. 479

87	g	NaCl	
1.5	g	KCl	
6	g	MgCl2 x 6 H2O	
0.4	g	CaCl2 x 2 H2O	
1	g	NH4Cl	
0.4	g	K2HPO4 x 3 H2O	
10	ml	**Modified Wolin's mineral solution**	
2	g	Yeast extract (OXOID)	
2	g	Trypticase peptone (BD BBL)	
0.5	ml	Sodium resazurin (0.1% w/v)	
1.5	g	Na2CO3	
2	g	Trimethylamine-HCl	
0.2	g	2-Mercaptoethanesulfonate (coenzyme M)	
0.25	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except carbonate, trimethylamine, coenzyme M and sulfide. Sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add trimethylamine, coenzyme M 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. Adjust pH of complete medium to 7.0 - 7.2, if necessary.


## 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.