412 .
Fe(III)-REDUCER FRESHWATER ENRICHMENT MEDIUM
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Equipment needed:
Autoclave
Centrifuge
Final pH:
7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
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Poorly crystalline iron(III) oxide | 100.000 | mM | ||
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MgCl2 x 6 H2O | 0.330 | g | ||
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CaCl2 x 2 H2O | 0.330 | g | ||
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KCl | 0.330 | g | ||
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KH2PO4 | 0.330 | g | ||
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NH4Cl | 0.250 | g | ||
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NaHCO3 | 2.500 | g | ||
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Na2SeO4 | 18.900 | mg | ||
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Trace vitamins | 10.000 | ml | ||
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Trace mineral solution | 10.000 | ml | ||
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L-Cysteine HCl x H2O | 0.044 | g | ||
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FeCl2 x 4 H2O | 0.260 | g | ||
| 1 Add components except L-cysteine x HCl x H2O and FeCl2 x 4H2O to distilled water, bring volume to 1.0 L and adjust pH to 7.0. Distribute the medium into appropriate culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a H2-CO2 (4:1, v/v) gas mixture, sparge with the same gas mixture for 10 - 15 min, seal with butyl rubber stoppers and autoclave. Separately autoclave concentrated (100- or 200-fold) L-cysteine x HCl x H2O and FeCl2 x 4H2O solutions under a N2 gas atmosphere. Prior to inoculation, add the sterile L-cysteine x HCl x H2O and FeCl2 x 4H2O solutions. Pressurize the inoculated vessels to 100 kPa H2-CO2 (4:1, v/v) and cultivate without shaking. | |||||
| 2 Comment: For strain JCM 12379, inoculate with a 10% inoculum. | |||||
Poorly crystalline iron(III) oxide #
| 1 Dissolve 108 g FeCl3 in 1.0 L distilled water. Adjust pH to 7.0 with 10 N NaOH. Allow the suspension to sit for 30 min, then readjust pH to 7.0. Centrifuge the suspension at 5,000 x g for 20 min and pour off supernatant. Wash the precipitate with distilled water by resuspending in distilled water followed by the centrifugation. Repeat the washing at least 5 times, then, resuspend in 1.0 L distilled water. Use the Fe(III) suspension as 0.67 M solution. More precisely, check the concentration of Cl- to be less than 1 mM and determine the concentration of Fe(III) by using hydroxylamine as described by Lovley and Phillips (Appl. Environ. Microbiol. 54, 751-757, 1986). |
Trace vitamins #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
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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 |
Trace mineral solution #
| 1 Use Trace minerals of Medium No. 151 supplemented with 0.025 g (final) Na2MoO4 x 2H2O, 0.024 g NiCl2 x 6H2O and 0.025 g Na2WO4 x 2H2O (per liter). | |||||
| 2 Comment: For strain JCM 12378, inoculate with a 10% inoculum. |