# J447: METHANOBACTERIUM ADHERENS MEDIUM


## Main sol. J447

0.15	g	CaCl2 x 2 H2O	
3	g	MgCl2 x 6 H2O	
0.25	g	NH4Cl	
0.2	g	KH2PO4	
0.5	g	KCl	
20	g	NaCl	
1	ml	**Trace element solution**	
1	ml	**Selenite-tungstate solution**	
0.082	g	Sodium acetate	
1	g	Yeast extract (BD-Difco)	
10	ml	**Trace vitamins**	
0.05	mg	Vitamin B12	
2.5	g	NaHCO3	
1	mg	Resazurin	
0.5	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Mix ingredients except NaHCO3 and Na2S x 9H2O, and bring to a boil for 5 min, then, cool down under a stream of a H2-CO2 (80:20, v/v) gas mixture. Distribute the medium into culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under the same gas mixture, seal with butyl rubber stoppers and autoclave. Filter-sterilize NaHCO3 as an 8% solution while gassing with N2-CO2 (80:20, v/v). Separately autoclave 5% Na2S x 9H2O solution under a N2 atmosphere. Aseptically and anaerobically add the NaHCO3 solution to the medium and readjust pH to 7.6 - 7.8 with a sterile 1% Na2CO3 solution (stocked anaerobically). Prior to inoculation, add the sterile Na2S x 9H2O solution. Pressurize inoculated bottles to 200 kPa H2-CO2 (80:20, v/v).


## Trace element solution

12.8	g	Nitrilotriacetic acid	
0.42	g	FeSO4 x 7 H2O	
0.01	g	H3BO3	
0.1	g	MnCl2 x 4 H2O	
0.17	g	CoCl2 x 6 H2O	
0.02	g	CuCl2 x 2 H2O	
0.21	g	ZnSO4 x 7 H2O	
0.01	g	Na2MoO4 x 2 H2O	

1. Dissolve nitrilotriacetic acid in 800 ml distilled water and adjust pH to 6.5 with NaOH. Then proceed to add the minerals and bring volume to 1.0 L.


## Selenite-tungstate solution

0.4	g	NaOH	
6	mg	Na2SeO3 x 5 H2O	
8	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## Trace vitamins

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine HCl	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	Calcium pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	Lipoic acid	
1000	ml	Distilled water