# J1248: MINERAL SALTS MEDIUM WITH TCE


## Main sol. J1248

1	g	NaCl	
0.5	g	MgCl2 x 6 H2O	
0.2	g	KH2PO4	
0.3	g	NH4Cl	
0.3	g	KCl	
0.015	g	CaCl2 x 2 H2O	
1	ml	**FeCl2 solution**	
1	ml	**Trace element solution**	
1	ml	**Selenite-tungstate solution**	
0.34	g	Sodium formate	
0.41	g	Sodium acetate	
0.25	mg	Resazurin	
1000	ml	Distilled water	

1. Mix components thoroughly, bring to a boil and cool down under a N2-CO2 (4:1, v/v) gas mixture. Add the following solutions: 

30	ml	NaHCO3 (8%)	
6	ml	L-Cysteine HCl x H2O (5%)	
10	ml	Na2S x 9 H2O (5%)	

2. Adjust pH of the medium to 7.0-7.2, if necessary. Dispense the medium into culture vessels (e.g., 100 ml in 120 ml-serum bottles) under the same gas mixture and seal with butyl rubber stoppers or Teflon-lined stoppers. Then, anaerobically add per liter the following solution: 

60	μl	Tetrachloroethene	

3. Finally, add per liter the following solution from an anaerobic stock (filter-sterilized): 

10	ml	**Trace vitamins**	

4. Dissolve tetrachloroethene with ultrasound. Autoclave in a closed basket. Do not remove the culture vessels from autoclave before temperature of the media has reached below 70C.


## FeCl2 solution

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


## Selenite-tungstate solution

0.4	g	NaOH	
6	mg	Na2SeO3 x 5 H2O	
8	mg	Na2WO4 x 2 H2O	
1000	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