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DSMZ

DSMZ 732 . DEHALOBACTER RESTRICTUS MEDIUM (TCE)

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Equipment needed: Autoclave Filter Gassing station Serum bottle
Final gas composition: 80 % H2 20 % CO2 read more
Final pH: 7.2
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ml
Compound Amount Unit
Solution A 870 ml
Solution B 100 ml
Solution C 10 ml
Solution D 1 ml
Solution E 2 ml
Solution F 10 ml
Solution G 15 ml
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 #

870
Compound Amount Unit
K2HPO4 0.65 g
NaH2PO4 x H2O 0.17 g
Peptone
(BD Bacto) 		0.10 g
Na-acetate 0.46 g
Sodium resazurin
(0.1% w/v) 		0.50 ml
Distilled water 870.00 ml

Solution B #

100
Compound Amount Unit
(NH4)2CO3 0.27 g
NaHCO3 3.73 g
Distilled water 100.00 ml

Solution C #

10
Compound Amount Unit
CaCl2 x 2 H2O 0.11 g
MgCl2 x 6 H2O 0.10 g
Distilled water 10.00 ml

Solution D #

1
Compound Amount Unit
Trace element solution 1 ml

Solution E #

2
Compound Amount Unit
Wolin's vitamin solution (10x) 1 ml
Seven vitamins solution 1 ml

Solution F #

10
Compound Amount Unit
Na2S x 9 H2O 0.3 g
Distilled water 10.0 ml

Solution G #

15
Compound Amount Unit
Hexadecane 13.5 ml
Tetrachloroethene 1.5 ml

Trace element solution #

1000
Compound Amount Unit
Na2-EDTA 0.5 g
FeCl2 x 4 H2O 2.0 g
ZnCl2 70.0 mg
MnCl2 x 4 H2O 100.0 mg
H3BO3 6.0 mg
CoCl2 x 6 H2O 190.0 mg
CuCl2 x 2 H2O 2.0 mg
AlCl3 10.0 mg
NiCl2 x 6 H2O 24.0 mg
Na2MoO4 x 2 H2O 36.0 mg
Distilled water 1000.0 ml
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) #

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

Seven vitamins solution #

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