# 713: METHANOHALOPHILUS EUHALOBIUS MEDIUM


## Main sol. 713

0.5	g	NH4Cl	
60	g	NaCl	
2.4	g	MgCl2 x 6 H2O	
1	g	KCl	
0.4	g	KH2PO4	
0.5	g	Na2-EDTA	
2	g	Yeast extract	
10	ml	**Modified Wolin's mineral solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
1	g	Na2CO3	
10	g	Trimethylamine-HCl	
5	ml	**Wolin's vitamin solution**	
2	g	CaCl2 x 2 H2O	
0.5	g	L-Cysteine HCl x H2O	
0.5	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except carbonate, trimethylamine, vitamins, calcium chloride, cysteine and sulfide), bring medium to the boil, then cool to room temperature under 80% N2 and 20% CO2 gas mixture. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add trimethylamine, calcium chloride, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Vitamins are prepared under 100% N2 gas atmosphere and sterilized by filtration. Adjust pH of complete medium to 6.8 - 7.0, 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.


## Wolin's vitamin solution

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine HCl	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	Calcium D-(+)-pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water