321 .
METHANOCORPUSCULUM AGGREGANS MEDIUM
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Equipment needed:
Autoclave
Centrifuge
Gassing station
Hungate tubes
Final pH:
6.8 - 7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NH4Cl | 1.0 | g | ||
|
|
K2HPO4 x 3 H2O | 0.4 | g | ||
|
|
MgCl2 x 6 H2O | 0.4 | g | ||
|
|
Minerals solution | 50.0 | ml | ||
|
|
Na-formate | 5.0 | g | ||
|
|
Na-acetate | 1.0 | g | ||
|
|
Trypticase peptone
(BD BBL) |
1.0 | g | ||
|
|
Yeast extract
(OXOID) |
1.0 | g | ||
|
|
Modified Wolin's mineral solution | 10.0 | ml | ||
|
|
Sludge fluid | 5.0 | ml | ||
|
|
Clarified rumen fluid
, alternative
optional |
5.0 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.5 | ml | ||
|
|
Na2CO3 | 1.5 | g | ||
|
|
L-Cysteine HCl x H2O | 0.2 | g | ||
|
|
Na2S x 9 H2O | 0.2 | g | ||
|
|
Distilled water | 935.0 | ml | ||
| 1 Dissolve ingredients except carbonate, cysteine and sulfide, then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add cysteine and sulfide 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. Prior to use check pH of complete medium and adjust to 6.8 - 7.0, if necessary. | |||||
| 2 After inoculation add sterile 80% H2 and 20% CO2 gas mixture to 2 bar overpressure. | |||||
Minerals solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
KH2PO4 | 6.00 | g |
|
|
(NH4)2SO4 | 6.00 | g |
|
|
NaCl | 12.00 | g |
|
|
MgSO4 x 7 H2O | 2.60 | g |
|
|
CaCl2 x 2 H2O | 0.16 | g |
|
|
Distilled water | 1000.00 | ml |
Modified Wolin's mineral solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid | 1.50 | g | ||
|
|
MgSO4 x 7 H2O | 3.00 | g | ||
|
|
MnSO4 x H2O | 0.50 | g | ||
|
|
NaCl | 1.00 | g | ||
|
|
FeSO4 x 7 H2O | 0.10 | g | ||
|
|
CoSO4 x 7 H2O | 0.18 | g | ||
|
|
CaCl2 x 2 H2O | 0.10 | g | ||
|
|
ZnSO4 x 7 H2O | 0.18 | g | ||
|
|
CuSO4 x 5 H2O | 0.01 | g | ||
|
|
AlK(SO4)2 x 12 H2O | 0.02 | g | ||
|
|
H3BO3 | 0.01 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.01 | g | ||
|
|
NiCl2 x 6 H2O | 0.03 | g | ||
|
|
Na2SeO3 x 5 H2O | 0.30 | mg | ||
|
|
Na2WO4 x 2 H2O | 0.40 | mg | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH. | |||||
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. |
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. | |||||