# J1294: MMJ/KT2 MEDIUM


## Main sol. J1294

1000	ml	Sea water (modified MJ synthetic )	
1	mg	Resazurin	

1. Adjust pH to 7.0 and autoclave. After cooling, add the following solutions (filter-sterilized): 

12.5	ml	NaHCO3 (8%)	
10	ml	**Trace vitamins**	
0.2	ml	**Vitamin B12 solution 0.05 g/L**	

2. Aseptically distribute the medium into culture vessels under a stream of H2-CO2 (4:1, v/v) and seal with butyl rubber stoppers. Prior to inoculation, add per liter the following solutions (autoclaved and stored under N2): 

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

3. Pressurize the inoculated bottles to 200 kPa H2-CO2 (4:1, v/v).


## Modified MJ synthetic seawater C

25	g	NaCl	
4.2	g	MgCl2 x 6 H2O	
3.4	g	MgSO4 x 7 H2O	
0.5	g	KCl	
0.25	g	NH4Cl	
0.14	g	K2HPO4	
0.7	g	CaCl2 x 2 H2O	
0.02	g	FeSO4 x 7 H2O	
10	ml	**Trace mineral solution**	
1000	ml	Distilled water	


## Trace mineral solution

1000	ml	**Trace minerals**	
0.075	g	NiCl2 x 6 H2O	
0.05	g	Na2SeO3 x 5 H2O	
0.1	g	Na2WO4 x 2 H2O	


## 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	


## Vitamin B12 solution 0.05 g/L

5	mg	Vitamin B12	
100	ml	Distilled water	


## 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.