1596 .
METHANOCALCULUS ALKALIPHILUS MEDIUM
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* Medium for Methanolobus sp. DSM 27087
Supplement the medium with 15 ml/l methanol solution (15% v/v). Omit Na-acetate and Na-formate.
Growth conditions: 7-14 days
anaerobic
30 °C
Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final gas composition:
100 % N2
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Final pH:
9.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 3.00 | g | ||
|
|
K2HPO4 | 1.00 | g | ||
|
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NaHCO3 | 20.00 | g | ||
|
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Na2CO3 | 15.00 | g | ||
|
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NH4Cl | 0.20 | g | ||
|
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MgSO4 x 7 H2O | 0.50 | g | ||
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Trace elements solution (Pfennig & Lippert,1966) | 1.00 | ml | ||
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Selenite-tungstate solution | 1.00 | ml | ||
| Na-formate* | 3.40 | g | |||
| Na-acetate* | 0.16 | g | |||
|
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Yeast extract
(OXOID) |
0.02 | g | ||
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Wolin's vitamin solution (10x) | 1.00 | ml | ||
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Na2S x 9 H2O | 0.25 | g | ||
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Methanol
(15% v/v) |
15.00 | ml | ||
|
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Distilled water | 1000.00 | ml | ||
| 1 Dissolve sodium chloride and hydrogenphosphate, then sparge solution with 100% N2 gas for at least 30 - 45 min to make it anoxic. Add and dissolve hydrogencarbonate and carbonate while gassing the head space only, then dispense under 100% N2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving add ammonium chloride, magnesium sulfate, trace elements, formate, acetate, yeast extract, vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Vitamins should be sterilized by filtration. The pH of the complete medium should be 9.5. | |||||
| 2 Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) may stimulate growth at the beginning. For transfers use 5 - 10% inoculum. | |||||
Trace elements solution (Pfennig & Lippert,1966) #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
EDTA | 5.00 | g | ||
|
|
FeSO4 x 7 H2O | 2.20 | g | ||
|
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ZnSO4 x 7 H2O | 0.10 | g | ||
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MnCl2 x 4 H2O | 0.03 | g | ||
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H3BO3 | 0.03 | g | ||
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CoCl2 x 6 H2O | 0.20 | g | ||
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CuCl2 x 2 H2O | 0.03 | g | ||
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NiCl2 x 6 H2O | 0.03 | g | ||
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Na2MoO4 x 2 H2O | 0.03 | g | ||
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Distilled water | 1000.00 | ml | ||
| 1 pH 3.0-4.0 | |||||
Selenite-tungstate solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
NaOH | 0.5 | g |
|
|
Na2SeO3 x 5 H2O | 3.0 | mg |
|
|
Na2WO4 x 2 H2O | 4.0 | mg |
|
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Distilled water | 1000.0 | ml |
Wolin's vitamin solution (10x) #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 20 | mg |
|
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Folic acid | 20 | mg |
|
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Pyridoxine hydrochloride | 100 | mg |
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Thiamine HCl | 50 | mg |
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Riboflavin | 50 | mg |
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Nicotinic acid | 50 | mg |
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Calcium D-(+)-pantothenate | 50 | mg |
|
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Vitamin B12 | 1 | mg |
|
|
p-Aminobenzoic acid | 50 | mg |
|
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(DL)-alpha-Lipoic acid | 50 | mg |
|
|
Distilled water | 1000 | ml |