378 .
ALKALIPHILUS CROTONATOXIDANS MEDIUM
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Equipment needed:
Anaerobic workbench
Autoclave
Filter
Final pH:
6.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Yeast extract
(BD-Difco) |
2.00 | g | ||
|
|
Tryptone
(BD-Difco) |
2.00 | g | ||
|
|
Crotonic acid | 0.86 | g | ||
|
|
KH2PO4 | 0.50 | g | ||
|
|
MgCl2 x 6 H2O | 0.33 | g | ||
|
|
NaCl | 0.40 | g | ||
|
|
NH4Cl | 0.40 | g | ||
|
|
CaCl2 x 2 H2O | 0.05 | g | ||
|
|
Trace metal solution | 10.00 | ml | ||
|
|
Trace vitamins | 10.00 | ml | ||
|
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NaHCO3 | 3.50 | g | ||
|
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L-Cysteine HCl x H2O | 0.50 | g | ||
|
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Na2S x 9 H2O | 0.50 | g | ||
|
|
Resazurin | 1.00 | mg | ||
| 1 Dissolve ingredients, except crotonic acid, NaHCO3, L-cysteine x HCl x H2O and Na2S x 9H2O, in 900 ml of distilled water and adjust pH to 7.0-7.5. Bring to a boil for 5-10 sec, then, cool down under a stream of N2-CO2 (80:20, v/v) and add NaHCO3 to the medium. Dispense 0.9 volume of the medium into suitable culture vessels under a stream of N2-CO2 (80:20, v/v) and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight. Add 0.1 volume of a filter-sterilized crotonic acid solution (0.86 g/100 ml, pH 7.0) to the medium. Autoclave cysteine x HCl and Na2S x 9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the solutions of crotonic acid, L-cysteine x HCl x H2O and Na2S x 9H2O to the medium. | |||||
Trace metal solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
MnSO4 x n H2O | 1.00 | g | ||
|
|
Fe(NH4)2(SO4)2 x 6 H2O | 0.80 | g | ||
|
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CoCl2 x 6 H2O | 0.20 | g | ||
|
|
ZnSO4 x 7 H2O | 0.20 | g | ||
|
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CuCl2 x 2 H2O | 0.02 | g | ||
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NiCl2 x 6 H2O | 0.02 | g | ||
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Na2MoO4 x 2 H2O | 0.02 | g | ||
|
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Na2SeO4 | 0.02 | g | ||
|
|
Na2WO4 | 0.02 | g | ||
|
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Nitrilotriacetic acid | 2.00 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Adjust pH to 6.0 with KOH. | |||||
Trace vitamins #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 2.0 | mg |
|
|
Folic acid | 2.0 | mg |
|
|
Pyridoxine hydrochloride | 10.0 | mg |
|
|
Thiamine HCl | 5.0 | mg |
|
|
Riboflavin | 5.0 | mg |
|
|
Nicotinic acid | 5.0 | mg |
|
|
Calcium pantothenate | 5.0 | mg |
|
|
Vitamin B12 | 0.1 | mg |
|
|
p-Aminobenzoic acid | 5.0 | mg |
|
|
Lipoic acid | 5.0 | mg |
|
|
Distilled water | 1000.0 | ml |