613 .
THERMOTOGA TF(C) MEDIUM
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* Medium for Thermotoga neapolitana DSM 4359
Replace glucose with 0.50 g/l soluble starch added to the autoclaved medium from a sterile anoxic stock solution prepared under 100% N2 gas.
Growth conditions: anaerobic
85 °C
Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final gas composition:
100 % N2
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Final pH:
6.5 - 6.8
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NH4Cl | 0.50 | g | ||
|
|
K2HPO4 | 1.60 | g | ||
|
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Na2HPO4 x H2O | 1.00 | g | ||
|
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NaCl | 7.00 | g | ||
|
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MgCl2 x 6 H2O | 1.40 | g | ||
|
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MgSO4 x 7 H2O | 1.96 | g | ||
|
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Modified Wolin's mineral solution | 10.00 | ml | ||
|
|
Yeast extract
(OXOID) |
2.00 | g | ||
|
|
Trypticase peptone
(BD BBL) |
2.00 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
| D-Glucose* | 3.00 | g | |||
|
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CaCl2 x 2 H2O | 0.06 | g | ||
|
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Wolin's vitamin solution (10x) | 1.00 | ml | ||
|
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L-Cysteine HCl x H2O | 0.30 | g | ||
|
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Na2S x 9 H2O | 0.30 | g | ||
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Starch | 0.50 | g | ||
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Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients (except glucose, calcium chloride, vitamins and reducing agents), adjust pH to 6.5 - 6.8 and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add glucose, calcium chloride, vitamins (sterilized by filtration), sulfide and cysteine from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. | |||||
Modified Wolin's mineral solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid | 1.50 | g | ||
|
|
MgSO4 x 7 H2O | 3.00 | g | ||
|
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MnSO4 x H2O | 0.50 | g | ||
|
|
NaCl | 1.00 | g | ||
|
|
FeSO4 x 7 H2O | 0.10 | g | ||
|
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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 | ||
|
|
H3BO3 | 0.01 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.01 | g | ||
|
|
NiCl2 x 6 H2O | 0.03 | g | ||
|
|
Na2SeO3 x 5 H2O | 0.30 | mg | ||
|
|
Na2WO4 x 2 H2O | 0.40 | mg | ||
|
|
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. | |||||
Wolin's vitamin solution (10x) #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 20 | mg |
|
|
Folic acid | 20 | mg |
|
|
Pyridoxine hydrochloride | 100 | mg |
|
|
Thiamine HCl | 50 | mg |
|
|
Riboflavin | 50 | mg |
|
|
Nicotinic acid | 50 | mg |
|
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Calcium D-(+)-pantothenate | 50 | mg |
|
|
Vitamin B12 | 1 | mg |
|
|
p-Aminobenzoic acid | 50 | mg |
|
|
(DL)-alpha-Lipoic acid | 50 | mg |
|
|
Distilled water | 1000 | ml |