# J1027: METHANOGENS SALINE WATER MEDIUM


## Main sol. J1027

0.54	g	NH4Cl	
0.14	g	KH2PO4	
3.5	g	MgCl2 x 6 H2O	
0.3	g	K2HPO4	
0.15	g	CaCl2 x 2 H2O	
5	g	NaCl	
2.52	g	NaHCO3	
1	ml	**Trace mineral solution**	
1	ml	**Se/W solution**	
1	ml	**Trace vitamins solution**	
1	mg	Resazurin	
1000	ml	Distilled water	

1. Mix components thoroughly and distribute the medium into culture vessels (e.g., 20 ml medium in 100 ml serum bottles or 5.0 ml medium in Hungate tubes) under a stream of H2-CO2 (80:20, v/v), seal with butyl rubber stoppers and autoclave. Allow to stand the autoclaved medium overnight. Add per liter the following solution from a sterile anaerobic stock (filter-sterilized and stored under a N2 atmosphere): 

10	ml	Trimethylamine (1 M)	

2. Prior to inoculation, add per liter the following solutions aseptically and anaerobically (autoclaved and stored under a N2 atmosphere): 

10	ml	L-Cysteine HCl x H2O (5%)	
10	ml	Na2S x 9 H2O (5%)	

3. Comments: For strain JCM 19935, replace trimethylamine with yeast extract (final 0.1 g/L) and pressurize to 100 kPa H2-CO2 (80:20, v/v). For strain JCM 19936, prepare the medium without trimethylamine. Pressurize inoculated culture vessels to 100 kPa H2-CO2 (80:20, v/v).


## Trace mineral solution

1.99	g	FeCl2 x 4 H2O	
0.198	g	MnCl2 x 4 H2O	
0.13	g	CoCl2 x 6 H2O	
0.136	g	ZnCl2	
1.7	mg	CuCl2 x 2 H2O	
6.2	mg	H3BO3	
0.024	g	Na2MoO4 x 2 H2O	
0.013	g	NiCl2 x 6 H2O	
0.013	g	AlCl3	
1000	ml	Distilled water	


## Se/W solution

1.7	mg	Na2SeO3 x 5 H2O	
3.3	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## Trace vitamins solution

4.9	mg	Biotin	
8.8	mg	Folic acid	
4.1	mg	Pyridoxine hydrochloride	
6.7	mg	Thiamine HCl	
7.5	mg	Riboflavin	
2.5	mg	Nicotinic acid	
9.5	mg	DL-Calcium pantothenate	
27.1	mg	Vitamin B12	
2.7	mg	p-Aminobenzoic acid	
4.1	mg	Lipoic acid	
1000	ml	Distilled water