976 .
THERMOFLEXUS HUNGENHOLTZII MEDIUM
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Equipment needed:
Anaerobic workbench
Autoclave
Filter
Final gas composition:
1 % O2
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Final pH:
6.0
Select a strain (optional):
ml
| 1 Mix components thoroughly and adjust pH to 6.75 with NaOH. Distribute the medium into culture vessels (e.g., 5.0 ml in 25 ml serum bottles/Balch tubes) under a stream of N2, seal with butyl rubber stoppers and autoclave. After cooling, aseptically add per liter the following solutions (filter-sterilized and stored anaerobically): | |||||
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
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NaCl | 3.000 | g | ||
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KCl | 0.150 | g | ||
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Na2SO4 | 0.300 | g | ||
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MgSO4 x 7 H2O | 0.123 | g | ||
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CaCl2 x 2 H2O | 0.015 | g | ||
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NH4Cl | 0.107 | g | ||
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NaH2PO4 | 1.380 | g | ||
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Mineral solution | 5.000 | ml | ||
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Distilled water | 1000.000 | ml | ||
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Trace vitamins | 10.000 | ml | ||
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Peptone
(w/v, 10%) |
10.000 | ml | ||
| 2 Before inoculation, add filter-sterilized O2 gas or air, at a final concentration of 1% O2 in the gas phase. | |||||
Mineral solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
EDTA | 3.7 | g | ||
|
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FeSO4 x 7 H2O | 1.1 | g | ||
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MnCl2 x 4 H2O | 98.4 | mg | ||
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ZnSO4 x 7 H2O | 28.8 | mg | ||
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CoCl2 x 6 H2O | 23.8 | mg | ||
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CuCl2 x 2 H2O | 17.2 | mg | ||
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Na2MoO4 x 2 H2O | 24.2 | mg | ||
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H3BO3 | 31.0 | mg | ||
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Distilled water | 1000.0 | ml | ||
| 1 Adjust pH to 6.0 with KOH. | |||||
| 2 Comment: Consistent growth in serum tubes requires pretreatment of butyl rubber stoppers by boiling in 1% sodium sulfide solution (1 hr) followed by boiling in distilled H2O (1 hr, twice). | |||||
Trace vitamins #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 2.0 | mg |
|
|
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 |