161 .
METHANOFOLLIS MEDIUM
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Equipment needed:
Autoclave
Centrifuge
Filter
Gassing station
Hungate tubes
Final pH:
6.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Clarified rumen fluid | 300.00 | ml | ||
|
|
K2HPO4 | 0.30 | g | ||
|
|
KH2PO4 | 0.30 | g | ||
|
|
(NH4)2SO4 | 0.30 | g | ||
|
|
NaCl | 0.60 | g | ||
|
|
MgSO4 x 7 H2O | 0.13 | g | ||
|
|
CaCl2 x 2 H2O
(0.1% w/v) |
8.00 | ml | ||
|
|
Modified Wolin's mineral solution | 10.00 | ml | ||
|
|
FeSO4 x 7 H2O
(0.1% w/v in 0.1 N H2SO4) |
2.00 | ml | ||
|
|
Yeast extract
(DIFCO) |
1.00 | g | ||
|
|
Trypticase
(BBL) |
1.00 | g | ||
|
|
Fatty acid mixture | 20.00 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
NaHCO3 | 2.00 | g | ||
|
|
Wolin's vitamin solution (10x) | 1.00 | ml | ||
|
|
L-Cysteine HCl x H2O | 0.50 | g | ||
|
|
Na2S x 9 H2O | 0.50 | g | ||
|
|
Distilled water | 660.00 | ml | ||
| 1 Dissolve ingredients (except bicarbonate, vitamins, cysteine and sulfide) and sparge medium for 30 - 45 min with 80% H2 and 20% CO2 gas mixture to make it anoxic. Add and dissolve bicarbonate and adjust pH to 6.8, then distribute under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add cysteine and sulfide from sterile anoxic stock solutions autoclaved under 100% N2 gas atmosphere. Vitamins are prepared under 100% N2 gas atmosphere and sterilized by filtration. Adjust pH of complete medium to 6.5. For incubation use sterile 80% H2 and 20% CO2 gas mixture at two atmospheres of pressure. | |||||
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. | |||||
Fatty acid mixture #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Isobutyric acid | 23 | ml | ||
|
|
DL-2-Methylbutyric acid | 27 | ml | ||
|
|
Valeric acid | 27 | ml | ||
|
|
Isovaleric acid | 27 | ml | ||
|
|
Distilled water | 896 | ml | ||
| 1 Adjust pH to 7.5 with concentrated NaOH. | |||||
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 |