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. |
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 |
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. |
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. |
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 |