# 332: METHANOCULLEUS BOURGENSIS MEDIUM


## Main sol. 332

1	g	NH4Cl	
0.4	g	K2HPO4 x 3 H2O	
0.1	g	MgCl2 x 6 H2O	
5	g	Na-formate	
1	g	Na-acetate	
1	g	Trypticase peptone (BD BBL)	
1	g	Yeast extract (OXOID)	
0.5	ml	Sodium resazurin (0.1% w/v)	
0.5	g	L-Cysteine HCl x H2O	
1.5	g	Na2CO3	
0.2	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except cysteine, carbonate and sulfide. Sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve cysteine, then dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture and sulfide from a sterile anoxic stock solution prepared under 100% N2 gas. Prior to use check pH of complete medium and adjust to 6.8 - 7.0, if necessary.
2. Note: After growth has started add sterile 80% H2 and 20% CO2 gas mixture to 0.5 - 1 bar overpressure.