# J556: METHYLOSOMA DIFFICILE MEDIUM


## Main sol. J556

0.1	g	NaCl	
0.04	g	MgCl2 x 6 H2O	
0.05	g	KCl	
0.015	g	CaCl2 x 2 H2O	
0.016	g	Na2SO4	
993	ml	Distilled water	

1. Mix components and autoclave. After cooling, add the following solutions (autoclaved or filter-sterilized) to the medium: 

5	ml	HEPES buffer (pH 7.0, 2 M)	
0.15	ml	**1 M Phosphate buffer** (pH 7.2, 1 M)	
0.544	ml	KNO3 (0.09 M)	
1	ml	**FeCl2 solution**	
1	ml	**Trace element solution**	
1	ml	**Vitamin solution** (filter-sterilized)	

2. Distribute the culture medium into culture tubes (leave >80% volume as a gas phase) and seal with butyl rubber stoppers.
3. Finally, replace the gas phase with 20% methane and 5% CO2 in air (by volume).


## 1 M Phosphate buffer

1. Mix 28 ml of 1 M KH2PO4 and 78 ml of 1 M Na2HPO4 to prepare 100 ml of 1 M phosphate buffer.


## Vitamin solution

50	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
10	mg	Biotin	
100	mg	Nicotinic acid	
25	mg	DL-Calcium pantothenate	
250	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
1000	ml	Distilled water	


## 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