# J944: MW MEDIUM FOR METHANOSARCINA


## Main sol. J944

1	g	NaCl	
0.5	g	KCl	
0.4	g	MgCl2 x 6 H2O	
0.25	g	NH4Cl	
0.15	g	CaCl2 x 2 H2O	
10	ml	**Trace elements solution**	
1	mg	Resazurin	
946	ml	Distilled water	

1. Mix components thoroughly, and autoclave under a N2-CO2 (80:20, v/v) gas atmosphere. After cooling, aseptically and anaerobically add the following solutions (autoclaved or *filter-sterilized): 

2	ml	KH2PO4 (10%)	
32	ml	NaHCO3 (*, 8%)	
10	ml	**Vitamin solution** (*)	

2. Aseptically distribute the medium into culture vessels under a H2-CO2 (80:20, v/v) gas mixture and seal with butyl rubber stoppers (leave >80% gas phase by volume). Finally, add per liter the following solutions from anaerobic stocks (autoclaved and stored under a N2 atmosphere): 

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

3. Pressurize the inoculated vessels to 200 kPa H2-CO2 (80:20, v/v).


## Trace elements solution

1.5	g	Nitrilotriacetic acid	
0.2	g	Fe(NH4)2(SO4)2 x 6 H2O	
0.2	g	Na2SeO3 x 5 H2O	
0.11	g	MnCl2 x 4 H2O	
0.1	g	CoCl2 x 6 H2O	
0.1	g	Na2MoO4 x 2 H2O	
0.1	g	Na2WO4 x 2 H2O	
0.25	g	NiCl2 x 6 H2O	
0.2	g	ZnSO4 x 7 H2O	
0.01	g	H3BO3	
2	mg	CuSO4 x 5 H2O	
1000	ml	Distilled water	

1. Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH. Then proceed to add minerals. Adjust final pH to 7.0.


## Vitamin solution

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