# 396: METHANOSALSUM ZHILINAE MEDIUM


## Main sol. 396

2	g	Yeast extract (OXOID)	
2	g	Trypticase peptone (BD BBL)	
40	g	NaCl	
1	g	NH4Cl	
3.5	g	MgCl2 x 6 H2O	
3	g	MgSO4 x 7 H2O	
1	g	KCl	
0.4	g	K2HPO4	
5	ml	**Modified Wolin's mineral solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
0.5	g	L-Cysteine HCl x H2O	
3	g	NaHCO3	
2	g	Na2CO3	
2	g	Trimethylamine-HCl	
0.25	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except cysteine, bicarbonate, carbonate, trimethylamine and sulfide, then sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Add cysteine and dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add trimethylamine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas, whereas bicarbonate and carbonate are added from sterile anoxic stock solutions prepared under 80% N2 and 20% CO2 gas atmosphere. Adjust pH of complete medium to 9.2 - 9.4.


## 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.