666a .
CELLULOSILYTICUM MEDIUM
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Equipment needed:
Autoclave
Centrifuge
Filter
Gassing station
Hungate tubes
Final gas composition:
100 % CO2
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Final pH:
6.7 - 6.8
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ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Clarified rumen fluid | 400.00 | ml | ||
|
|
K2HPO4 | 0.23 | g | ||
|
|
KH2PO4 | 0.23 | g | ||
|
|
NaCl | 0.45 | g | ||
|
|
(NH4)2SO4 | 0.45 | g | ||
|
|
CaCl2 x 2 H2O | 0.06 | g | ||
|
|
MgSO4 x 7 H2O | 0.09 | g | ||
|
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Agar | 1.00 | g | ||
|
|
Indigocarmine | 5.00 | mg | ||
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NaHCO3 | 6.40 | g | ||
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Cellobiose | 2.50 | g | ||
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Yeast extract | 5.00 | g | ||
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L-Cysteine HCl x H2O | 0.30 | g | ||
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|
DL-Dithiothreitol
(DTT) |
0.30 | g | ||
|
|
Distilled water | 600.00 | ml | ||
| 1 Dissolve ingredients (except bicarbonate, cellobiose, yeast extract, cysteine and DTT), bring medium to the boil, then cool to room temperature under 100% CO2 gas atmosphere. Add the bicarbonate and equilibrate the medium with the CO2 gas to pH 6.8. Distribute under 100% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Thereafter, add cellobiose, yeast extract, cysteine and DTT from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Cellobiose has to be sterilzed by filtration. Adjust pH of complete medium to 6.7 - 6.8, if necessary. | |||||
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. |