# 732: DEHALOBACTER RESTRICTUS MEDIUM (TCE)


## Main sol. 732

870	ml	**Solution A**	
100	ml	**Solution B**	
10	ml	**Solution C**	
1	ml	**Solution D**	
2	ml	**Solution E**	
10	ml	**Solution F**	
15	ml	**Solution G**	

1. Sparge solution A with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic serum vials (e.g., 9 ml medium in 50 ml bottle). Pressurize closed bottles with 80% H2 and 20% CO2 gas mixture to 0.5 bar overpressure and autoclave. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solutions C, D and F are autoclave under 100% N2 gas atmosphere.  Solution E is prepared under 100% N2 gas atmosphere and sterilized by filtration. Prepare solution G by filling 13.5 ml hexadecane into a 50 ml serum bottle, then sparge with 100% N2 gas to make it anoxic and autoclave. Add 1.50 ml anoxic autoclaved tetrachloroethene to the sterile anoxic hexadecane solution by syringe. To complete the medium add appropriate amounts of solutions B to F to the sterile solution A in the sequence as indicated. The pH of the medium before inoculation should be at 7.2.
2. Add solution G only after inoculation of the medium!


## Solution A

0.65	g	K2HPO4	
0.17	g	NaH2PO4 x H2O	
0.1	g	Peptone (BD Bacto)	
0.46	g	Na-acetate	
0.5	ml	Sodium resazurin (0.1% w/v)	
870	ml	Distilled water	


## Solution B

0.27	g	(NH4)2CO3	
3.73	g	NaHCO3	
100	ml	Distilled water	


## Solution C

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


## Solution D

1	ml	**Trace element solution**	


## Solution E

1	ml	**Wolin's vitamin solution (10x)**	
1	ml	**Seven vitamins solution**	


## Solution F

0.3	g	Na2S x 9 H2O	
10	ml	Distilled water	


## Solution G

13.5	ml	Hexadecane	
1.5	ml	Tetrachloroethene	


## Trace element solution

0.5	g	Na2-EDTA	
2	g	FeCl2 x 4 H2O	
70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
10	mg	AlCl3	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve EDTA in distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add remaining compounds.


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water	


## Seven vitamins solution

100	mg	Vitamin B12	
80	mg	p-Aminobenzoic acid	
20	mg	D-(+)-biotin	
200	mg	Nicotinic acid	
100	mg	Calcium pantothenate	
300	mg	Pyridoxine hydrochloride	
200	mg	Thiamine-HCl x 2 H2O	
1000	ml	Distilled water