507 .
CARBOXYDOTHERMUS MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
6.8 - 7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
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KCl | 0.33 | g | ||
|
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MgCl2 x 6 H2O | 0.52 | g | ||
|
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CaCl2 x 2 H2O | 0.29 | g | ||
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NH4Cl | 0.33 | g | ||
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KH2PO4 | 0.33 | g | ||
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Trace element solution SL-11 | 1.00 | ml | ||
|
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Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
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NaHCO3 | 1.00 | g | ||
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Yeast extract | 0.05 | g | ||
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Wolin's vitamin solution (10x) | 1.00 | ml | ||
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Neutralized sulfide solution 3% (w/v) | 20.00 | ml | ||
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Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients except bicarbonate, yeast extract, vitamins and sulfide, then sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add yeast extract, vitamins (sterilized by filtration) and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Neutralize the sodium sulfide with sterile H2SO4 (see medium 29). After completing the medium adjust pH to 6.8-7.0. | |||||
| 2 Inoculated vessels are pressurized with sterile carbon monoxide gas to 2 bar overpressure. | |||||
Trace element solution SL-11 #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Na2-EDTA x 2 H2O | 5.2 | g | ||
|
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FeCl2 x 4 H2O | 1.5 | g | ||
|
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ZnCl2 | 70.0 | mg | ||
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MnCl2 x 4 H2O | 100.0 | mg | ||
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H3BO3 | 6.0 | mg | ||
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CoCl2 x 6 H2O | 190.0 | mg | ||
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CuCl2 x 2 H2O | 2.0 | mg | ||
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NiCl2 x 6 H2O | 24.0 | mg | ||
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Na2MoO4 x 2 H2O | 36.0 | mg | ||
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Distilled water | 1000.0 | ml | ||
| 1 Dissolve EDTA in 800 ml distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds. Finally adjust pH to 6.0 and bring volume to 1000 ml. | |||||
Wolin's vitamin solution (10x) #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 20 | mg |
|
|
Folic acid | 20 | mg |
|
|
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 |
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p-Aminobenzoic acid | 50 | mg |
|
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(DL)-alpha-Lipoic acid | 50 | mg |
|
|
Distilled water | 1000 | ml |
Neutralized sulfide solution 3% (w/v) #
100
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Na2S x 9 H2O | 3 | g | ||
|
|
Distilled water | 100 | ml | ||
| 1 The sulfide solution is prepared in a 250 ml screw-capped bottle with a butyl rubber septum and a magnetic stirrer. The solution is bubbled with nitrogen gas, closed and autoclaved for 15 min. at 121°C. After cooling to room temperature the pH is adjusted to about 7.0 by adding of sterile 2 M H2SO4 drop-wise with a syringe without opening the bottle. | |||||