# J911: METHANOCELLA CONRADII MEDIUM


## Main sol. J911

0.5	g	KCl	
0.4	g	MgCl2 x 6 H2O	
0.2	g	KH2PO4	
0.1	g	NH4Cl	
0.1	g	CaCl2 x 2 H2O	
0.082	g	Sodium acetate x 3 H2O	
0.3	g	L-Cysteine HCl x H2O	
0.2	g	Yeast extract	
1	ml	**FeCl2 solution**	
1	ml	**Trace element solution**	
1	ml	**Tungstate solution**	
0.5	mg	Resazurin	
1000	ml	Distilled water	

1. Mix components thoroughly, bring to a boil and cool down under a stream of N2-CO2 (4:1, v/v) gas mixture. Distribute the medium into culture vessels (e.g., 5 ml in the Hungate tubes) under the same gas mixture, seal with butyl rubber stoppers and autoclave. Aseptically and anaerobically, add per liter the following solutions (filter-sterilized): 

30	ml	NaHCO3 (8%)	
1	ml	**Vitamin solution**	
1	ml	**Thiamine solution**	
1	ml	**Vitamin B12 solution 0.05 g/L**	

2. Prior to inoculation, replace the gas phase with a H2-CO2 (4:1, v/v) gas mixture and add per liter the following solution from an anaerobic stock (autoclaved and stored under a N2 gas atmosphere): 

5	ml	Na2S x 9 H2O (5%)	

3. Pressurize the inoculate culture vessels to 150 kPa H2-CO2 (4:1, v/v).


## Tungstate solution

0.4	g	NaOH	
7	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. Comment: For strain JCM 17849, inoculate with a 10% inoculum. When the cellular density increases, re-pressure the culture vessels to 150 kPa with the same gas mixture.


## FeCl2 solution

10	ml	HCl (7.7 M, 25%)	
1.5	g	FeCl2 x 4 H2O	
990	ml	Distilled water	


## Trace element solution

70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	


## Vitamin solution

4	mg	p-Aminobenzoic acid	
1	mg	Biotin	
10	mg	Nicotinic acid	
5	mg	DL-Calcium pantothenate	
10	mg	Pyridoxine hydrochloride	
100	ml	Sodium phosphate buffer (10 mM, pH 7.1)	


## Thiamine solution

10	mg	Thiamine HCl	
100	ml	Sodium phosphate buffer (25 mM, pH 3.4)	


## Vitamin B12 solution 0.05 g/L

5	mg	Vitamin B12	
100	ml	Distilled water