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DSMZ

DSMZ 201 . DESULFONEMA LIMICOLA MEDIUM

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Equipment needed: Autoclave Filter Gassing station Hungate tubes
Final gas composition: 80 % N2 20 % CO2 read more
Final pH: 7.6
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ml
Compound Amount Unit
Solution A 930 ml
Solution B 1 ml
Solution C 30 ml
Solution D 10 ml
Solution E 1 ml
Solution F 1 ml
Solution G 20 ml
Solution H 10 ml
1 Solution A is sparged with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to reach a pH of around 6, then distributed under same gas atmosphere into anoxic Hungate-type tubes and autoclaved.
2 Solutions B, D, E, G, and H are autoclaved separately under 100% N2 gas. Solution C is autoclaved under 80% N2 and 20% CO2 gas atmosphere. Solution F is prepared under 100% N2 gas and sterilized by filtration. 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 the complete medium to 7.6, if necessary.
3 Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution freshly prepared under N2 and filter-sterilized) just before inoculation may stimulate growth at the beginning. For transfers use 5 - 10% inoculum.
4 Cultures of Desulfonema spp. show usually improved growth in media containing an artificial light sediment of aluminum phosphate. Alternatively, a viscous medium prepared with 1 - 2 g washed agar per liter can be used (Widdel et al., Arch. Microbiol. 134:286-294 (1983)): Agar (Difco) is washed three times for one hour with distilled water at room temperature. Finally, the agar is suspended (c. 20.0 g/l) in distilled water and autoclaved. 50 ml of the hot agar is added to the hot sterile medium part A (when preparing part A calculated for 1 liter, subtract the volume of the agar solution). After cooling to room temperature complete the medium by adding part B to I. For mass cultures the aluminum phosphate as artificial sediment can be used.

Solution A #

930
Compound Amount Unit
Na2SO4 3.00 g
NaCl 15.00 g
MgCl2 x 6 H2O 2.20 g
CaCl2 x 2 H2O 0.15 g
KCl 0.50 g
KH2PO4 0.20 g
NH4Cl 0.30 g
Sodium resazurin
(0.1% w/v) 		0.50 ml
Distilled water 930.00 ml

Solution B #

1
Compound Amount Unit
Trace element solution SL-10 1 ml

Solution C #

30
Compound Amount Unit
Na2CO3 1.5 g
Distilled water 30.0 ml

Solution D #

10
Compound Amount Unit
Na-acetate x 3 H2O 2.5 g
Distilled water 10.0 ml

Solution E #

1
Compound Amount Unit
Disodium succinate 0.1 g
Distilled water 1.0 ml

Solution F #

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

Solution G #

20
Compound Amount Unit
Clarified rumen fluid , clarified 20 ml

Solution H #

10
Compound Amount Unit
Na2S x 9 H2O 0.4 g
Distilled water 10.0 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.

Clarified rumen fluid #

1000
1 Rumen fluid from cow or sheep (obtained from fistulated animals or abattoir refuse) is filtered through muslin, autoclaved at 121°C for 15 min and then centrifuged at 27,000 g for 20 min. The supernatant is made anoxic by sparging with 100% N2 gas for 15 min, dispensed under same gas atmosphere into anoxic serum vials to 30% of volume and then stored frozen at -20°C.

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