# J600: G5G6 MEDIUM


## Main sol. J600


## Solution A

0.5	g	Na2HPO4 x 12 H2O	
0.65	g	KH2PO4	
1	ml	**FeCl2 solution**	
1	ml	**Trace element solution**	
0.5	mg	Resazurin	
880	ml	Distilled water	


## Solution B

0.11	g	CaCl2 x 2 H2O	
0.1	g	MgCl2 x 6 H2O	
50	ml	Distilled water	


## Solution C

0.44	g	NH4HCO3	
3.73	g	NaHCO3	
0.175	g	L-Cysteine HCl x H2O	
50	ml	Distilled water	

1. Prepare all solutions and autoclave under a N2 atmosphere. After cooling, combine the solutions and replace the gas phase with a N2-CO2 (4:1, v/v) gas mixture. Then, add the following autoclaved or *filter-sterilized solutions to the medium: 

10	ml	NaNO3 (1.0 M)	
10	ml	**Trace vitamins** (*)	
1	ml	**Vitamin B12 solution 0.05 g/L** (*)	

2. Distribute appropriate amount of the medium into culture vessels under the same gas mixture (e.g., 40.0 ml in 120 ml serum bottle) and seal with Viton rubber stoppers. Finally add per liter the following solution from an anaerobic stock (autoclaved): 

40	ml	Toluene (water-saturated, 5.0 mM)	

3. Check the final pH to be around 7.3.


## Trace element solution

1000	ml	**Trace element solution**	
10	mg	AlCl3 x 6 H2O	

1. Comment: For strain JCM 14632, incubate with a 5-10% inoculum. When toluene is depleted, re-add the toluene solution (normally every 3 to 4 days, until enough cell growth is seen).


## FeCl2 solution

10	ml	HCl (7.7 M, 25%)	
1.5	g	FeCl2 x 4 H2O	
990	ml	Distilled water	


## 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 element solution

70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
1000	ml	Distilled water