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DSMZ

DSMZ 861 . DESULFOFRIGUS MEDIUM

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Equipment needed: Autoclave Filter Gassing station Serum bottle
Final gas composition: 80 % N2 20 % CO2 read more
Final pH: 7.0 - 7.2
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ml
Compound Amount Unit
Solution A 952 ml
Solution B 30 ml
Solution C 10 ml
Solution D 1 ml
Solution E 10 ml
Solution F 1 ml
1 Solution A is sparged with 80% N2 and 20% CO2 gas mixture to reach a pH below 6 (at least 30 - 45 min), then dispensed under the same gas atmosphere into serum vials (use at least bottles of 50 ml volume filled with 30 ml medium) and autoclaved. Solutions B and F are prepared under 80% N2 and 20% CO2 gas atmosphere and sterilized. Sterile solutions C, D and E are prepared under 100% N2 gas. Solutions D and F are sterilized by filtration. To complete the medium appropriate amounts of solutions B to F are added to the sterile solution A in the sequence as indicated. Final pH of the medium should be 7.0 - 7.2.
2 Note: For transfer of cultures use at least 10% (v/v) inoculum.

Solution A #

952
Compound Amount Unit
NaCl 20.00 g
Na2SO4 4.00 g
KH2PO4 0.20 g
NH4Cl 0.25 g
MgCl2 x 6 H2O 3.00 g
CaCl2 x 2 H2O 0.15 g
KBr 0.09 g
KCl 0.50 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 950.00 ml

Solution B #

30
Compound Amount Unit
Na2CO3 1.5 g
Distilled water 30.0 ml

Solution C #

10
Compound Amount Unit
Na-DL-lactate 2.5 g
Distilled water 10.0 ml

Solution D #

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

Solution E #

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

Solution F #

1
Compound Amount Unit
Na-dithionite solution (5% w/v) 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

Na-dithionite solution (5% w/v) #

10
Compound Amount Unit
NaOH
(1 N) 		1.0 ml
Na2S2O4 0.5 g
Distilled water 9.0 ml
1 Add 1 N NaOH to distilled water and make the solution anoxic by sparging with 100% N2 gas. Then dissolve the Na-dithionite and filter sterilize the solution into anoxic Hungate tubes. Store the prepared solution in a dark and cool place. Prepare only small amounts of stock solution, as Na-dithionite decomposes rapidly.