322 .
METHANOSPHAERA MEDIUM
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Equipment needed:
Autoclave
Centrifuge
Filter
Gassing station
Hungate tubes
Final pH:
6.7 - 6.9
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Clarified rumen fluid | 100.00 | ml | ||
|
|
Trypticase peptone
(BD BBL) |
2.00 | g | ||
|
|
Yeast extract | 2.00 | g | ||
|
|
Na-acetate | 0.50 | g | ||
|
|
Na-formate | 0.50 | g | ||
|
|
Modified Wolin's mineral solution | 10.00 | ml | ||
|
|
Na2SeO4
(0.1% w/v) |
1.90 | ml | ||
|
|
NiCl2 x 6 H2O
(0.1% w/v) |
0.70 | ml | ||
|
|
FeSO4 x 7 H2O solution (0.1% w/v) | 3.00 | ml | ||
|
|
K2HPO4 | 0.60 | g | ||
|
|
KH2PO4 | 2.80 | g | ||
|
|
(NH4)2SO4 | 0.30 | g | ||
|
|
NH4Cl | 1.00 | g | ||
|
|
NaCl | 0.60 | g | ||
|
|
MgSO4 x 7 H2O | 0.15 | g | ||
|
|
CaCl2 x 2 H2O | 0.08 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
NaHCO3 | 4.00 | g | ||
|
|
Methanol
(50% v/v) |
10.00 | ml | ||
|
|
Wolin's vitamin solution (10x) | 2.00 | ml | ||
|
|
DL-Dithiothreitol | 0.50 | g | ||
|
|
Distilled water | 900.00 | ml | ||
| 1 Dissolve ingredients except bicarbonate, methanol, and dithiothreitol (DTT), then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate, adjust pH of medium to 6.8 - 7.0, then dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add methanol (50% v/v solution), vitamins, and DTT from sterile anoxic stock solutions prepared under 100% N2 gas. Vitamins and DTT should be sterilized by filtration. Prior to use check pH of complete medium and adjust to 6.7 - 6.9, if necessary. | |||||
| 2 After inoculation add sterile 80% H2 and 20% CO2 gas mixture to 1 bar overpressure. | |||||
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. |
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. | |||||
Wolin's vitamin solution (10x) #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 20 | mg |
|
|
Folic acid | 20 | mg |
|
|
Pyridoxine hydrochloride | 100 | mg |
|
|
Thiamine HCl | 50 | mg |
|
|
Riboflavin | 50 | mg |
|
|
Nicotinic acid | 50 | mg |
|
|
Calcium D-(+)-pantothenate | 50 | mg |
|
|
Vitamin B12 | 1 | mg |
|
|
p-Aminobenzoic acid | 50 | mg |
|
|
(DL)-alpha-Lipoic acid | 50 | mg |
|
|
Distilled water | 1000 | ml |
FeSO4 x 7 H2O solution (0.1% w/v) #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
FeSO4 x 7 H2O | 1 | g | ||
|
|
H2SO4
(0.1 N) |
1000 | ml | ||
| 1 The ferrous sulfate solution is not stable and should be freshly prepared. | |||||