# J484: MODIFIED METHANOCALDOCOCCUS MEDIUM


## Main sol. J484

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	
30	g	NaCl	
5	ml	**Iron stock solution**	
10	ml	**Trace minerals solution**	
10	ml	**Trace vitamins**	
1	g	Na2S2O3 x 5 H2O	
2	g	Yeast extract (BD-Difco)	
0.4	g	2-Mercaptoethanol	
1	mg	Resazurin	
1000	ml	Distilled water	

1. Mix ingredients except Na2S2O3 x 5H2O, yeast extract, Trace vitamins and 2-mercaptoethanol, 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, v/v). Dispense the medium into suitable culture vessels under a stream of H2-CO2 (80:20, v/v), seal with butyl rubber stoppers and autoclave 115C for 40 min. Separately autoclave Na2S2O3 x 5H2O and 2-mercaptoethanol as 50 g/L solutions and yeast extract as 100 g/L solution under a N2 atmosphere. Filter-sterilize Trace vitamins and store under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the Na2S2O3 x 5H2O, yeast extract, Trace vitamins and 2-mercaptoethanol solutions to the medium. Pressurize the inoculated bottles to 200 kPa H2-CO2 (80:20, v/v).


## Trace minerals solution

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	
0.025	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 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.


## Iron stock solution

1. Dissolve 0.2 g of Fe(NH4)2(SO4)2 x 6H2O in 5 ml of distilled water containing 2 drops of concentrated HCl. When the Fe(NH4)2(SO4)2 x 6H2O crystal have dissolved, bring volume up to 100 ml.


## 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