# J232: METHANOCALDOCOCCUS JANNASCHII MEDIUM


## Main sol. J232

0.14	g	K2HPO4	
0.14	g	CaCl2 x 2 H2O	
0.25	g	NH4Cl	
3.4	g	MgSO4 x 7 H2O	
2.7	g	MgCl2 x 6 H2O	
0.33	g	KCl	
0.75	mg	NiCl2 x 6 H2O	
0.5	mg	Na2SeO3 x 5 H2O	
30	g	NaCl	
0.01	g	Fe(NH4)2(SO4)2 x 6 H2O	
10	ml	**Trace minerals**	
10	ml	**Trace vitamins**	
1	g	NaHCO3	
0.5	g	L-Cysteine HCl x H2O	
0.5	g	Na2S x 9 H2O	
1	mg	Resazurin	
980	ml	Distilled water	

1. Mix ingredients, except NaHCO3, cysteine x HCl and Na2S x 9H2O, and adjust pH to 6.0. Bring to a boil for 5-10 sec, then, cool down under a stream of H2-CO2 (80:20) and add NaHCO3 to the medium. Dispense the medium into suitable culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a stream of H2-CO2 (80:20) and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight. Separately autoclave cysteine x HCl and Na2S x 9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the cysteine x HCl and Na2S x 9H2O solutions. Pressurize the inoculated bottles to 200 kPa H2-CO2 (80:20).


## Trace minerals

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x n H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.1	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.01	g	AlK(SO4)2	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

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


## 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