# 391: METHANOCOCCUS VOLTAE MEDIUM


## Main sol. 391

500	ml	**Mineral solution 2xW**	
10	ml	**Modified Wolin's mineral solution**	
50	ml	**LIP-solution**	
1	g	Na-acetate	
0.5	ml	Sodium resazurin (0.1% w/v)	
50	ml	**TYC-solution**	
1.5	g	Na2CO3	
0.5	g	L-Cysteine HCl x H2O	
0.5	g	Na2S x 9 H2O	
400	ml	Distilled water	

1. Dissolve ingredients except TYC solution, carbonate, cystein and sulfide. Sparge medium with 80% H2 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. Before use add TYC solution, 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. Adjust pH of complete medium to 7.2, if necessary.


## Mineral solution 2xW

40	g	NaCl	
5.6	g	MgCl2 x 6 H2O	
0.7	g	MgSO4 x 7 H2O	
0.68	g	KCl	
0.5	g	NH4Cl	
0.28	g	K2HPO4	
0.28	g	CaCl2 x 2 H2O	
1000	ml	Distilled water	


## LIP-solution

5	g	L-Leucine	
10	g	L-Isoleucine	
0.1	g	Pantothenate	
1000	ml	Distilled water	

1. May be stored frozen without sterilization.


## TYC-solution

100	g	Casamino acids (BD Bacto)	
50	g	Yeast extract (OXOID)	
1	g	L-Tryptophan	
1000	ml	Distilled water	

1. Prepare and autoclave under 100% N2 gas.


## 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.