380 .
MAGNETOSPIRILLUM MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
6.7
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
KH2PO4 | 0.68 | g | ||
|
|
NaNO3 | 0.12 | g | ||
|
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L(+)-Tartaric acid | 0.37 | g | ||
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Succinic acid | 0.37 | g | ||
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Na-acetate | 0.05 | g | ||
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Yeast extract | 0.10 | g | ||
|
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Modified Wolin's mineral solution | 5.00 | ml | ||
|
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Fe(III) quinate solution 0.01 M | 2.00 | ml | ||
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Agar
, for semi-solid medium
(BD Bacto) optional |
1.30 | g | ||
|
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Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
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Na-thioglycolate | 0.05 | g | ||
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Seven vitamins solution | 1.00 | ml | ||
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Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients (except thioglycolate and vitamins) in the order given and adjust pH to 6.75 with NaOH. | |||||
| 2 Preparation of liquid medium: Sparge medium with 100% N2 gas for 30 -45 min and dispense under the same gas atmosphere into anoxic Hungate-type tubes to 50% of their volume. Seal vials with screw caps and gas tight butyl rubber closures. Autoclave at 121°C for 15 min. Before inoculation add thioglycolate and vitamins from stock solutions prepared under 100% N2 gas and filter-sterilized. Then add sterile air (with hypodermic syringe through the rubber closure) to a concentration of ca. 1% (v/v) O2 in the vial (e.g., add 1 ml air to a Hungate-type tube of 16 ml total volume). | |||||
| 3 Preparation of semi solid medium: Supplement medium with agar, bring medium to the boil and cool under 100% N2 gas atmosphere. Dispense under same gas atmosphere aliquots of 10 ml semi-solid medium into Hungate-type tubes. Prior to inoculation add thioglycolate from a 0.5% (w/v) stock solution, freshly prepared under 100% N2 gas and filter-sterilized. Then add sterile air (with hypodermic syringe through the rubber closure) to a concentration of ca. 1% (v/v) in the vial. | |||||
| 4 Note: Prior to inoculation media should be slightly pink in color. Strongly reduced conditions will not support growth of microaerophilic Magnetospirillum species. Use as inoculum 10% (v/v). Incubate tubes with medium without agitation in an inclined position. During growth O2 will be consumed and the pH will increase. For cultivation of magnetic cells we recommend preparation of liquid medium, while semi-solid medium is more suitable for demonstration of microaerophilic band formation and storage. | |||||
Modified Wolin's mineral solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid | 1.50 | g | ||
|
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MgSO4 x 7 H2O | 3.00 | g | ||
|
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MnSO4 x H2O | 0.50 | g | ||
|
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NaCl | 1.00 | g | ||
|
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FeSO4 x 7 H2O | 0.10 | g | ||
|
|
CoSO4 x 7 H2O | 0.18 | g | ||
|
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CaCl2 x 2 H2O | 0.10 | g | ||
|
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ZnSO4 x 7 H2O | 0.18 | g | ||
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CuSO4 x 5 H2O | 0.01 | g | ||
|
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AlK(SO4)2 x 12 H2O | 0.02 | g | ||
|
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H3BO3 | 0.01 | g | ||
|
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Na2MoO4 x 2 H2O | 0.01 | g | ||
|
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NiCl2 x 6 H2O | 0.03 | g | ||
|
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Na2SeO3 x 5 H2O | 0.30 | mg | ||
|
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Na2WO4 x 2 H2O | 0.40 | mg | ||
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Distilled water | 1000.00 | ml | ||
| 1 First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH. | |||||
Seven vitamins solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Vitamin B12 | 100 | mg |
|
|
p-Aminobenzoic acid | 80 | mg |
|
|
D-(+)-biotin | 20 | mg |
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Nicotinic acid | 200 | mg |
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Calcium pantothenate | 100 | mg |
|
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Pyridoxine hydrochloride | 300 | mg |
|
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Thiamine-HCl x 2 H2O | 200 | mg |
|
|
Distilled water | 1000 | ml |
Fe(III) quinate solution 0.01 M #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
FeCl3 x 6 H2O | 4.5 | g | ||
|
|
Quinic acid | 1.9 | g | ||
|
|
Distilled water | 1000.0 | ml | ||
| 1 Sterilize by filtration under 100% N2 gas atmosphere. | |||||