320 .
CLOSTRIDIUM CELLULOVORANS MEDIUM
What is this page?
Equipment needed:
Autoclave
Centrifuge
Filter
Gassing station
Hungate tubes
Final pH:
7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
K2HPO4 x 3 H2O | 1.00 | g | ||
|
|
NH4Cl | 1.00 | g | ||
|
|
KCl | 0.50 | g | ||
|
|
MgSO4 x 7 H2O | 0.50 | g | ||
|
|
Trypticase peptone
(BD BBL) |
0.50 | g | ||
|
|
Yeast extract | 0.50 | g | ||
|
|
Clarified rumen fluid | 20.00 | ml | ||
|
|
Sludge fluid
, alternative
optional |
20.00 | ml | ||
|
|
Trace element solution SL-10 | 1.00 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
L-Cysteine HCl x H2O | 0.15 | g | ||
|
|
Na2CO3 | 1.00 | g | ||
|
|
Cellobiose | 5.00 | g | ||
|
|
Cellulose
, MN 301
optional |
10.00 | g | ||
|
|
Na2S x 9 H2O | 0.15 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients (except cysteine, carbonate, cellobiose and sulfide), bring medium to the boil, then cool to room temperature under 80% N2 and 20% CO2 gas mixture and add cysteine. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add sulfide from a sterile anoxic stock solution prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Sterilize cellobiose separately by filtration under 100% N2 gas. Adjust pH of the complete medium to 7.0, if necessary. | |||||
| 2 Note: Some strains can be adapted to cellulose as substrate using 10.0 g/l cellulose powder MN 301 (MACHEREY-NAGEL). | |||||
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. | |||||
Sludge fluid #
1000
| 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. | |||||
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. |