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DSMZ

DSMZ 833 . DEHALOSPIRILLUM MEDIUM

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Equipment needed: Autoclave Filter Gassing station Hungate tubes
Final gas composition: 100 % N2 read more
Final pH: 7.3 - 7.6
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ml
Compound Amount Unit
Solution A 892 ml
Solution B 10 ml
Solution C 2 ml
Solution D 35 ml
Solution E 20 ml
Solution F 40 ml
Solution G 3 ml
Solution H 1 ml
1 Sparge solution A with 100% N2 gas for 30 - 45 min to make it anoxic, then distribute under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solutions B and H are autoclaved separately under 100% N2 gas. Solutions C, E, F and G are prepared under 100% N2 gas and sterilized by filtration. Solution D is autoclaved under 80% N2 and 20% CO2 gas atmosphere. To complete the medium appropriate amounts of solutions B to H are added to the sterile solution A in the sequence as indicated. Adjust pH of complete medium to 7.3 - 7.6, if necessary.
2 Note: Before inoculation, the medium must be completely reduced, i.e. resazurin must be colorless. The addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) can help to reduce the medium.

Solution A #

892
Compound Amount Unit
Na2SO4 0.70 g
KH2PO4 0.20 g
NH4Cl 0.25 g
NaCl 0.25 g
MgCl2 x 6 H2O 0.40 g
KCl 0.50 g
CaCl2 x 2 H2O 0.15 g
Yeast extract 2.00 g
Trace element solution SL-10 1.00 ml
Selenite-tungstate solution 1.00 ml
Sodium resazurin
(0.1% w/v) 		0.50 ml
Distilled water 890.00 ml

Solution B #

10
Compound Amount Unit
Potassium phosphate buffer
(0.1 M, pH 7.5) 		10 ml

Solution C #

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

Solution D #

35
Compound Amount Unit
Na2CO3 1.75 g
Distilled water 35.00 ml

Solution E #

20
Compound Amount Unit
Na-pyruvate 4.5 g
Distilled water 20.0 ml

Solution F #

40
Compound Amount Unit
Na2-fumarate 6.4 g
Distilled water 40.0 ml

Solution G #

7
Compound Amount Unit
FeSO4 x 7 H2O 30 mg
H2SO4
(0.1 N) 		3 ml

Solution H #

1
Compound Amount Unit
L-Cysteine HCl x H2O 50 mg
Distilled water 1 ml

Trace element solution SL-10 #

1000
Compound Amount Unit
HCl
(25%) 		10.0 ml
FeCl2 x 4 H2O 1.5 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
NiCl2 x 6 H2O 24.0 mg
Na2MoO4 x 2 H2O 36.0 mg
Distilled water 990.0 ml
1 First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml.

Selenite-tungstate solution #

1000
Compound Amount Unit
NaOH 0.5 g
Na2SeO3 x 5 H2O 3.0 mg
Na2WO4 x 2 H2O 4.0 mg
Distilled water 1000.0 ml

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