336 .
OXOBACTER PFENNIGII MEDIUM
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Equipment needed:
Autoclave
Centrifuge
Filter
Gassing station
Hungate tubes
Final pH:
7.0 - 7.2
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Solution A | 972 | ml | ||
|
|
Solution B | 30 | ml | ||
|
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Solution C | 1 | ml | ||
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Solution D | 10 | ml | ||
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Solution E | 3 | ml | ||
| 1 Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, distribute under same gas atmosphere 8.9 ml medium per Hungate-type tube and autoclave. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solution C is prepared under 100% N2 gas atmosphere and sterilized by filtration. Autoclave solutions D and E under 100% N2 gas. To complete the medium add appropriate amounts of the solutions B to E to the sterile solution A. Adjust pH of complete medium to 7.0 - 7.2, if necessary. | |||||
Solution A #
972
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Mineral solution | 50.0 | ml | ||
|
|
Trace element solution SL-10 | 1.0 | ml | ||
|
|
Yeast extract | 2.0 | g | ||
|
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Na-vanillate | 2.0 | g | ||
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Clarified rumen fluid | 300.0 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.5 | ml | ||
|
|
Distilled water | 620.0 | ml | ||
| 1 Adjust pH to 6.9 before autoclaving. | |||||
Mineral solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
KH2PO4 | 10.0 | g |
|
|
MgCl2 x 6 H2O | 6.6 | g |
|
|
NaCl | 8.0 | g |
|
|
NH4Cl | 8.0 | g |
|
|
CaCl2 x 2 H2O | 1.0 | g |
|
|
Distilled water | 1000.0 | ml |
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 | ||
|
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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. | |||||
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. |
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 |