243 .
METHANOTHRIX 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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KH2PO4 | 0.30 | g | ||
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NaCl | 0.60 | g | ||
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MgCl2 x 6 H2O | 0.10 | g | ||
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CaCl2 x 2 H2O | 0.08 | g | ||
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NH4Cl | 1.00 | g | ||
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Trace element solution | 10.00 | ml | ||
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Trace vitamins | 10.00 | ml | ||
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Sodium acetate | 6.80 | g | ||
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KHCO3 | 4.00 | g | ||
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L-Cysteine HCl x H2O | 0.50 | g | ||
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Na2S x 9 H2O | 0.50 | g | ||
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Resazurin | 1.00 | mg | ||
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Distilled water | 1000.00 | ml | ||
| 1 Mix ingredients, except Trace vitamins, KHCO3, L-cysteine x HCl x H2O and Na2S x 9H2O, and adjust pH to 7.0. Bring to a boil for 5-10 sec, then, cool down under a stream of N2-CO2 (80:20) and add KHCO3. Dispense the medium into suitable culture vessels (e.g., 50 ml of the medium in 120 ml serum bottles) under a stream of N2-CO2 (80:20) and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight. Separately autoclave cysteine x HCl and Na2S x 9H2O as 5% solutions, respectively, under a N2 atmosphere. Filter-sterilize Trace vitamins under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add Trace vitamins, L-cysteine x HCl x H2O and Na2S x 9H2O solutions. Pressurize the inoculated bottles to 100 kPa N2-CO2 (80:20). | |||||
Trace element solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
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Nitrilotriacetic acid | 12.800 | g | ||
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FeCl3 x 6 H2O | 1.350 | g | ||
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MnCl2 x 4 H2O | 0.100 | g | ||
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CoCl2 x 6 H2O | 0.024 | g | ||
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CaCl2 x 2 H2O | 0.100 | g | ||
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ZnCl2 | 0.100 | g | ||
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CuCl2 x 2 H2O | 0.025 | g | ||
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H3BO3 | 0.010 | g | ||
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NaCl | 1.000 | g | ||
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NiCl2 x 6 H2O | 0.120 | g | ||
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Na2SeO3 x 5 H2O | 0.026 | g | ||
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Distilled water | 1000.000 | ml | ||
| 1 Dissolve nitrilotriacetic acid first in 200 ml of distilled water, and adjust pH to 6.5 with KOH. Then add the remaining salts and adjust volume to 1.0 L. Readjust pH to 7.0 with KOH. | |||||
| 2 Comment: For strain JCM 10134, incubate with a 10-20% inoculum. | |||||
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 |