523 .
CLOSTRIDIUM CHARTATABIDUM 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
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Mineral solution | 38.00 | ml | ||
|
|
Clarified rumen fluid | 200.00 | ml | ||
|
|
K2HPO4 | 0.30 | g | ||
|
|
Trypticase peptone
(BD BBL) |
2.00 | g | ||
|
|
Yeast extract
(OXOID) |
0.50 | g | ||
|
|
Haemin solution
(0.05% w/v) |
2.00 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
Na2CO3 | 4.00 | g | ||
|
|
Cellobiose | 5.00 | g | ||
|
|
L-Cysteine HCl x H2O | 0.25 | g | ||
|
|
Na2S x 9 H2O | 0.25 | g | ||
|
|
Distilled water | 760.00 | ml | ||
| 1 Dissolve ingredients (except carbonate, cellobiose, cysteine and sulfide), bring medium to the boil, then cool to room temperature under 100% CO2 gas atmosphere. Add the carbonate and equilibrate the medium with the CO2 gas to pH 6.8. Distribute under CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add cellobiose, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Cellobiose has to be sterilized by filtration. Adjust pH of complete medium to 6.7 - 6.8, if necessary. | |||||
Mineral solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
KH2PO4 | 6.0 | g |
|
|
NaCl | 12.0 | g |
|
|
(NH4)2SO4 | 6.0 | g |
|
|
CaCl2 x 2 H2O | 1.6 | g |
|
|
MgSO4 x 7 H2O | 2.5 | g |
|
|
Distilled water | 1000.0 | 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. |
Haemin solution #
100
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Haemin | 50 | mg | ||
|
|
NaOH
(1 N) |
1 | ml | ||
|
|
Distilled water | 100 | ml | ||
| 1 Dissolve 50 mg haemin in 1 ml 1 N NaOH; make up to 100 ml with distilled water and filter sterilize. Store refrigerated. | |||||