734a .
METHANOBREVIBACTER CUTICULARIS MEDIUM
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Equipment needed:
Autoclave
Centrifuge
Filter
Gassing station
Hungate tubes
Final pH:
7.7
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Clarified rumen fluid | 20.00 | ml | ||
|
|
NaCl | 1.00 | g | ||
|
|
KCl | 0.50 | g | ||
|
|
MgCl2 x 6 H2O | 0.40 | g | ||
|
|
CaCl2 x 2 H2O | 0.10 | g | ||
|
|
NH4Cl | 0.30 | g | ||
|
|
KH2PO4 | 0.20 | g | ||
|
|
Na2SO4 | 0.15 | g | ||
|
|
Casamino acids
(BD Bacto) |
0.50 | g | ||
|
|
Yeast extract
(OXOID) |
0.50 | g | ||
|
|
Trace element solution SL-10 | 1.00 | ml | ||
|
|
Selenite-tungstate solution | 1.00 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
Na2CO3 | 2.00 | g | ||
|
|
MOPS
(SIGMA) |
2.10 | g | ||
|
|
Seven vitamins solution | 1.00 | ml | ||
|
|
DL-Dithiothreitol
(DTT) |
0.16 | g | ||
|
|
Distilled water | 980.00 | ml | ||
| 1 Dissolve ingredients (except carbonate, MOPS buffer, vitamins and DTT), bring medium to the boil, then cool to room temperature under 80% H2 and 20% CO2 gas mixture. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add MOPS buffer adjusted to pH 7.7, vitamins and DTT from anoxic stock solutions prepared under 100% N2 gas and sterilized by filtration and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Adjust pH of complete medium to 7.7, 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. |
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. | |||||
Selenite-tungstate solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
NaOH | 0.5 | g |
|
|
Na2SeO3 x 5 H2O | 3.0 | mg |
|
|
Na2WO4 x 2 H2O | 4.0 | mg |
|
|
Distilled water | 1000.0 | ml |
Seven vitamins solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Vitamin B12 | 100 | mg |
|
|
p-Aminobenzoic acid | 80 | mg |
|
|
D-(+)-biotin | 20 | mg |
|
|
Nicotinic acid | 200 | mg |
|
|
Calcium pantothenate | 100 | mg |
|
|
Pyridoxine hydrochloride | 300 | mg |
|
|
Thiamine-HCl x 2 H2O | 200 | mg |
|
|
Distilled water | 1000 | ml |