Compound | Amount | Unit | |||
---|---|---|---|---|---|
|
KH2PO4 | 0.50 | g | ||
|
NaCl | 1.00 | g | ||
|
(NH4)2SO4 | 0.50 | g | ||
|
MgSO4 x 7 H2O | 0.10 | g | ||
|
CaCl2 x 2 H2O | 0.10 | g | ||
|
K2HPO4 | 0.50 | g | ||
|
Clarified rumen fluid | 300.00 | ml | ||
|
Sludge fluid
, alternative
optional |
300.00 | ml | ||
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
Na2CO3 | 2.50 | g | ||
|
Cellobiose | 5.00 | g | ||
|
L-Cysteine HCl x H2O | 0.25 | g | ||
|
Na2S x 9 H2O | 0.25 | g | ||
|
Distilled water | 700.00 | ml | ||
1 Dissolve ingredients (except carbonate, cellobiose and reducing agents), bring medium to the boil, then cool to room temperature under 100% CO2 gas atmosphere. Use either clarified rumen fluid or sludge fluid as supplement. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add cellobiose, sulfide and cysteine from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Cellobiose has to be sterilized by filtration. Adjust pH of complete medium to 6.8, if necessary. |
Compound | Amount | Unit | |||
---|---|---|---|---|---|
|
Yeast extract | 4 | g | ||
|
Sludge | 1000 | ml | ||
1 Add 0.4% yeast extract to sludge from an anaerobic digester, and after gassing with nitrogen gas for a few minutes incubate it at 37°C for 24 hours. Then centrifuge the sludge at 13000 g and autoclave the resulting, clear supernatant in screw-capped vessels under nitrogen gas. The sludge fluid can be stored at 8-12°C in the dark. |
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. |