484 .
MODIFIED METHANOCALDOCOCCUS MEDIUM
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Equipment needed:
Anaerobic workbench
Autoclave
Filter
Final pH:
6.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
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K2HPO4 | 0.14 | g | ||
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CaCl2 x 2 H2O | 0.14 | g | ||
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NH4Cl | 0.25 | g | ||
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MgSO4 x 7 H2O | 3.40 | g | ||
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MgCl2 x 6 H2O | 2.70 | g | ||
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KCl | 0.33 | g | ||
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NaCl | 30.00 | g | ||
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Iron stock solution | 5.00 | ml | ||
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Trace minerals solution | 10.00 | ml | ||
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Trace vitamins | 10.00 | ml | ||
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Na2S2O3 x 5 H2O | 1.00 | g | ||
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Yeast extract
(BD-Difco) |
2.00 | g | ||
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2-Mercaptoethanol | 0.40 | g | ||
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Resazurin | 1.00 | mg | ||
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Distilled water | 1000.00 | ml | ||
| 1 Mix ingredients except Na2S2O3 x 5H2O, yeast extract, Trace vitamins and 2-mercaptoethanol, and adjust pH to 6.0. Bring to a boil for 5-10 sec, then, cool down under a stream of H2-CO2 (80:20, v/v). Dispense the medium into suitable culture vessels under a stream of H2-CO2 (80:20, v/v), seal with butyl rubber stoppers and autoclave 115C for 40 min. Separately autoclave Na2S2O3 x 5H2O and 2-mercaptoethanol as 50 g/L solutions and yeast extract as 100 g/L solution under a N2 atmosphere. Filter-sterilize Trace vitamins and store under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the Na2S2O3 x 5H2O, yeast extract, Trace vitamins and 2-mercaptoethanol solutions to the medium. Pressurize the inoculated bottles to 200 kPa H2-CO2 (80:20, v/v). | |||||
Trace minerals solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
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Nitrilotriacetic acid | 1.500 | g | ||
|
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MgSO4 x 7 H2O | 3.000 | g | ||
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MnSO4 x n H2O | 0.500 | g | ||
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NaCl | 1.000 | g | ||
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FeSO4 x 7 H2O | 0.100 | g | ||
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CoSO4 x 7 H2O | 0.100 | g | ||
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CaCl2 x 2 H2O | 0.100 | g | ||
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ZnSO4 x 7 H2O | 0.100 | g | ||
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CuSO4 x 5 H2O | 0.010 | g | ||
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AlK(SO4)2 | 0.010 | g | ||
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H3BO3 | 0.010 | g | ||
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Na2MoO4 x 2 H2O | 0.010 | g | ||
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NiCl2 x 6 H2O | 0.025 | g | ||
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Na2SeO3 x 5 H2O | 0.300 | mg | ||
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Distilled water | 1000.000 | ml | ||
| 1 Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH. Then proceed to add minerals. Adjust final pH to 7.0. | |||||
Iron stock solution #
| 1 Dissolve 0.2 g of Fe(NH4)2(SO4)2 x 6H2O in 5 ml of distilled water containing 2 drops of concentrated HCl. When the Fe(NH4)2(SO4)2 x 6H2O crystal have dissolved, bring volume up to 100 ml. |
Trace vitamins #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 2.0 | mg |
|
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Folic acid | 2.0 | mg |
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Pyridoxine hydrochloride | 10.0 | mg |
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Thiamine HCl | 5.0 | mg |
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Riboflavin | 5.0 | mg |
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Nicotinic acid | 5.0 | mg |
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Calcium pantothenate | 5.0 | mg |
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Vitamin B12 | 0.1 | mg |
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p-Aminobenzoic acid | 5.0 | mg |
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Lipoic acid | 5.0 | mg |
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Distilled water | 1000.0 | ml |