231 .
METHANOBACTERIUM THERMOAUTOTROPHICUM MEDIUM
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Equipment needed:
Anaerobic workbench
Autoclave
Final pH:
7.2
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
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KH2PO4 | 0.30 | g | ||
|
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(NH4)2SO4 | 1.50 | g | ||
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NaCl | 0.60 | g | ||
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MgSO4 x 7 H2O | 0.12 | g | ||
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CaCl2 x 2 H2O | 0.08 | g | ||
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FeSO4 x 7 H2O | 4.00 | mg | ||
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K2HPO4 | 0.15 | g | ||
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Na2CO3 | 4.00 | g | ||
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Trace vitamins | 10.00 | ml | ||
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Trace element solution | 10.00 | ml | ||
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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 | 980.00 | ml | ||
| 1 Mix ingredients, except cysteine x HCl and Na2S x 9H2O, and adjust pH to 7.2. Bring to a boil for 5-10 sec, then, cool down under a stream of H2-CO2 (80:20). Dispense the medium into suitable culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a stream of H2-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. Prior to inoculation, aseptically and anaerobically add the cysteine x HCl and Na2S x 9H2O solutions. Pressurize the inoculated bottles to 100 kPa H2-CO2 (80:20). | |||||
Trace element solution #
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
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Na2-EDTA | 0.640 | g | ||
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MgSO4 x 7 H2O | 6.200 | g | ||
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MnSO4 x n H2O | 0.550 | g | ||
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NaCl | 1.000 | g | ||
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FeSO4 x 7 H2O | 0.100 | g | ||
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CoCl2 x 6 H2O | 0.170 | g | ||
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CaCl2 x 2 H2O | 0.130 | g | ||
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ZnSO4 x 7 H2O | 0.180 | g | ||
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CuSO4 x 5 H2O | 0.050 | g | ||
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AlK(SO4)2 x 12 H2O | 0.018 | g | ||
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H3BO3 | 0.010 | g | ||
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Na2MoO4 x 2 H2O | 0.011 | g | ||
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NiCl2 x 6 H2O | 0.025 | g | ||
| 1 Dissolve EDTA x 2Na first in 500 ml of distilled water. Then add the remaining salts and adjust volume to 1000 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 |