# 814: METHANOBACTERIUM SUBTERRANEUM MEDIUM


## Main sol. 814

0.4	g	NH4Cl	
0.03	g	MgCl2 x 6 H2O	
0.45	g	NaCl	
0.5	g	K2HPO4	
3	ml	FeSO4 x 7 H2O (0.1% w/v in 0.1 N H2SO4)	
10	ml	**Modified Wolin's mineral solution**	
1	g	Tryptone (BD Bacto)	
3.2	g	Na-formate	
1	g	Na-acetate	
0.5	ml	Sodium resazurin (0.1% w/v)	
1	g	Na2CO3	
1	ml	**Seven vitamins solution**	
0.25	g	L-Cysteine HCl x H2O	
0.25	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except carbonate, vitamins, cysteine and sulfide, then sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add vitamins (sterilized by filtration), cysteine 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. Prior to use adjust pH of complete medium to 8.0 - 8.5.


## 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.


## Seven vitamins solution

100	mg	Vitamin B12	
80	mg	p-Aminobenzoic acid	
20	mg	D-(+)-biotin	
200	mg	Nicotinic acid	
100	mg	Calcium pantothenate	
300	mg	Pyridoxine hydrochloride	
200	mg	Thiamine-HCl x 2 H2O	
1000	ml	Distilled water