# 1257: METHANOCELLA MEDIUM


## Main sol. 1257

1	g	NaCl	
0.4	g	MgCl2 x 6 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	NH4Cl	
0.2	g	KH2PO4	
0.5	g	KCl	
1	ml	**Trace element solution**	
0.1	g	Yeast extract (OXOID)	
0.1	g	Na-acetate	
0.5	ml	Sodium resazurin (0.1% w/v)	
1.5	g	Na2CO3	
1	ml	**Wolin's vitamin solution (10x)**	
0.3	g	L-Cysteine HCl x H2O	
0.3	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except carbonate, vitamins and reducing agents), then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic and adjust pH to 6.0. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Prior to inoculation add vitamins, cysteine and sulfide from sterile anoxic stock solution prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Vitamins are sterilized by filtration. If necessary, adjust the pH of the complete medium to 7.0.
2. After inoculation pressurize vials to 1 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture.


## Trace element solution

12.8	g	Nitrilotriacetic acid (NTA)	
1	g	FeCl2 x 4 H2O	
0.1	g	MnCl2 x 4 H2O	
0.03	g	CoCl2 x 6 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	ZnCl2	
0.02	g	CuCl2	
0.01	g	H3BO3	
0.03	g	Na2MoO4 x 2 H2O	
0.1	g	NiCl2 x 6 H2O	
1	g	NaCl	
0.03	g	Na2SeO3 x 5 H2O	
0.04	g	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve NTA in 200 ml of distilled water and adjust pH to 6.5 with KOH, then dissolve mineral salts. Finally adjust pH to 6.5 with KOH and make up to 1000.00 ml.


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water