1455 .
ALTERNATIVE RUMEN BACTERIA MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final gas composition:
100 % CO2
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Final pH:
6.6 - 6.8
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
K2HPO4 | 0.23 | g | ||
|
|
KH2PO4 | 0.23 | g | ||
|
|
NaCl | 0.45 | g | ||
|
|
(NH4)2SO4 | 0.45 | g | ||
|
|
CaCl2 x 2 H2O | 0.06 | g | ||
|
|
MgSO4 x 7 H2O | 0.09 | g | ||
|
|
Brain heart infusion
(BD BBL) |
6.00 | g | ||
|
|
Yeast extract | 5.00 | g | ||
|
|
Volatile fatty acid mixture | 3.10 | ml | ||
|
|
Haemin solution
(0.05% w/v) |
2.00 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
NaHCO3 | 6.40 | g | ||
|
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Disodium succinate | 5.00 | g | ||
|
|
Wolin's vitamin solution (10x) | 1.00 | ml | ||
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L-Cysteine HCl x H2O | 0.30 | g | ||
|
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Na2S x 9 H2O | 0.30 | g | ||
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Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients except bicarbonate, succinate, vitamins, cysteine and sulfide, then sparge medium with 100% CO2 gas for 30 - 45 min to make it anoxic. Add the bicarbonate and equilibrate the medium with the CO2 gas to reach a pH between 6.5 and 7.0. Dispense medium under 100% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add succinate, vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. The vitamin solution should be sterilized by filtration. Prior to use adjust pH of complete medium to 6.6 - 6.8, if necessary. | |||||
Volatile fatty acid mixture #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Acetic acid | 548.50 | ml |
|
|
Propionic acid | 193.50 | ml |
|
|
Butyric acid | 129.00 | ml |
|
|
n-Valeric acid | 32.25 | ml |
|
|
iso-Butyric acid | 32.25 | ml |
|
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DL-2-Methylbutyric acid | 32.25 | ml |
|
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iso-Valeric acid | 32.25 | ml |
Haemin solution #
100
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Haemin | 50 | mg | ||
|
|
NaOH
(1 N) |
1 | ml | ||
|
|
Distilled water | 100 | ml | ||
| 1 Dissolve 50 mg haemin in 1 ml 1 N NaOH; make up to 100 ml with distilled water and filter sterilize. Store refrigerated. | |||||
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 |