# 317: METHANOSARCINA THERMOPHILA MEDIUM


## Main sol. 317

1	g	NH4Cl	
0.4	g	K2HPO4	
0.2	g	MgCl2 x 6 H2O	
50	ml	**Minerals solution**	
10	ml	**Modified Wolin's mineral solution**	
1	g	Yeast extract	
0.5	ml	Sodium resazurin (0.1% w/v)	
1	g	Na2CO3	
5	ml	Methanol	
0.5	g	L-Cysteine HCl x H2O	
0.25	g	Na2S x 9 H2O	
950	ml	Distilled water	

1. Dissolve ingredients except carbonate, methanol, cysteine and sulfide, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vilas and autoclave. Add methanol, 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 6.8 - 7.0, if necessary.


## Minerals solution

6	g	KH2PO4	
6	g	(NH4)2SO4	
12	g	NaCl	
2.6	g	MgSO4 x 7 H2O	
0.16	g	CaCl2 x 2 H2O	
1000	ml	Distilled water	


## 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.