# 945: MARINITOGA PIEZOPHILA MEDIUM


## Main sol. 945

1	g	NH4Cl	
0.2	g	MgCl2 x 6 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	KCl	
30	g	NaCl	
0.83	g	Na-acetate	
1.95	g	MES (SIGMA)	
5	g	Yeast extract	
5	g	Trypticase peptone (BD BBL)	
0.3	g	K2HPO4	
0.3	g	KH2PO4	
0.5	ml	Sodium resazurin (0.1% w/v)	
10	g	Sulfur (powdered)	
0.3	g	L-Cysteine HCl x H2O	
0.3	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except sulfur, cysteine and sulfide. Sparge medium with 100% N2 gas for at least 30 - 45 min to make it anoxic and adjust pH to 6.0 with NaOH. Distribute the medium under 100% N2 gas atmosphere into anoxic Hungate-type tubes or serum vials which contain already the appropriate amount of sulfur. For sterilization heat the cultivation vessels containing medium plus sulfur in boiling water for 2 - 3 hours on each of 3 successive days! Prior to inoculation add cysteine and sulfide and from sterile anoxic stock solutions prepared under 100% N2 gas.
2. After inoculation pressurize tubes to 2 bar overpressure with sterile 100% N2 gas.