# 905: METHANOCALCULUS HALOTOLERANS MEDIUM


## Main sol. 905

1	g	NH4Cl	
0.3	g	K2HPO4	
0.3	g	KH2PO4	
0.17	g	KCl	
50	g	NaCl	
0.5	g	Na-acetate	
0.5	g	Yeast extract (OXOID)	
0.5	g	Trypticase (BD BBL)	
10	ml	**Modified Wolin's mineral solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
2	g	NaHCO3	
0.5	g	L-Cysteine HCl x H2O	
0.6	g	CaCl2 x 2 H2O	
3.2	g	MgCl2 x 6 H2O	
0.3	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except bicarbonate, cysteine, calcium chloride, magnesium chloride and sulfide), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve solid bicarbonate and cysteine, then adjust pH to 7.5 with 10 N KOH. Dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials (to 30% of volume) and autoclave. Add calcium chloride, magnesium chloride and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. The pH of the complete medium should be between 7.2 and 7.6.
2. After inoculation pressurize vessels with sterile 80% H2 and 20% CO2 gas mixture to 1 bar overpressure.


## 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.