# 342: METHANOBACTERIUM ALCALIPHILUM MEDIUM


## Main sol. 342

792	ml	**Solution A**	
210	ml	**Solution B**	
10	ml	**Solution C**	

1. Dissolve ingredients of solution A (except cysteine), bring to the boil, then cool to room temperature under 100% H2 gas. Add cysteine and adjust pH to 7.0, if necessary. Dispense medium under 100% H2 gas atmosphere into anoxic Hungate-type tubes or serum vials vials to 30% of their volume and then autoclave. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solution C is prepared under 100% N2 gas atmosphere. Complete the medium by adding appropriate amounts of solutions B and C to the sterile solution A. Adjust pH of complete medium to 8.3 - 8.4, if necessary.
2. After inoculation add sterile H2 gas to 1 - 2 bar overpressure.


## Solution A

1	g	NH4Cl	
0.1	g	MgCl2 x 6 H2O	
0.1	g	CaCl2 x 2 H2O	
0.4	g	K2HPO4	
2	ml	Na2SeO3 x 5 H2O (0.01% w/v)	
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)	
0.5	g	L-Cysteine HCl x H2O	
780	ml	Distilled water	


## Solution B

10	g	NaHCO3	
0.5	g	Na2CO3	
210	ml	Distilled water	


## Solution C

10	ml	Tris-HCl buffer (pH 8.4, 2 M)	


## 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.