480d .
THERMOPROTEUS UZONIENSIS MEDIUM
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Selected strain: Thermoproteus uzoniensis DSM 5263
This strain uses the standard medium without modifications.
Growth conditions: 85 °C
Equipment needed:
Anaerobic workbench
Autoclave
Filter
Gassing station
Hungate tubes
Water bath
Final pH:
5.7 - 6.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Medium 480 | 1000 | ml | ||
|
|
Casitone | 3 | g/l | ||
| 1 Use medium 480, but reduce the amount of Na acetate to 1.0 g/l and add 3.0 g/l casitone. Prepare sulphide and vitamin stock solutions anaerobically. Autoclave the sulphide solution but filter sterilise the vitamins. Prepare the medium anaerobically under N2:CO2 80:20. Adjust the pH to 5.7 with HCl. Sterilise the medium by heating for 3 hours on three successive days at 90-100°C. Add the vitamins and sulphide to the sterilised medium before use. The final pH should be 5.7-6.0. Dr. Bonch-Osmoloskaya supplied the information that the strain grows better on a peptone originally available in Russia. This product is no longer available, but it appears this product gave better growth than with highly purified products from Oxoid or Difco. | |||||
Wolin's vitamin solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 2.0 | mg |
|
|
Folic acid | 2.0 | mg |
|
|
Pyridoxine hydrochloride | 10.0 | mg |
|
|
Thiamine HCl | 5.0 | mg |
|
|
Riboflavin | 5.0 | mg |
|
|
Nicotinic acid | 5.0 | mg |
|
|
Calcium D-(+)-pantothenate | 5.0 | mg |
|
|
Vitamin B12 | 0.1 | mg |
|
|
p-Aminobenzoic acid | 5.0 | mg |
|
|
(DL)-alpha-Lipoic acid | 5.0 | mg |
|
|
Distilled water | 1000.0 | ml |
Trace element solution SL-10 #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
HCl
(25%) |
10.0 | ml | ||
|
|
FeCl2 x 4 H2O | 1.5 | g | ||
|
|
ZnCl2 | 70.0 | mg | ||
|
|
MnCl2 x 4 H2O | 100.0 | mg | ||
|
|
H3BO3 | 6.0 | mg | ||
|
|
CoCl2 x 6 H2O | 190.0 | mg | ||
|
|
CuCl2 x 2 H2O | 2.0 | mg | ||
|
|
NiCl2 x 6 H2O | 24.0 | mg | ||
|
|
Na2MoO4 x 2 H2O | 36.0 | mg | ||
|
|
Distilled water | 990.0 | ml | ||
| 1 First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. | |||||
Main sol. 480 #
1011
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NH4Cl | 0.33 | g | ||
|
|
CaCl2 x 2 H2O | 0.33 | g | ||
|
|
MgCl2 x 6 H2O | 0.33 | g | ||
|
|
KCl | 0.33 | g | ||
|
|
KH2PO4 | 0.33 | g | ||
|
|
Trace element solution SL-10 | 1.00 | ml | ||
|
|
Yeast extract | 0.10 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
Sulfur
(powder) |
10.00 | g | ||
|
|
Na-acetate | 5.00 | g | ||
|
|
NaHCO3 | 2.50 | g | ||
|
|
Wolin's vitamin solution | 10.00 | ml | ||
|
|
Na2S x 9 H2O | 0.50 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients (except sulfur, acetate, bicarbonate, vitamins and sulfide) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Adjust pH to 5.9, dispense under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials containing already the appropriate amount of sulfur and sterilize by heating for 2-3 hours in a boiling water bath on each of 3 successive days. Add acetate, vitamins (sterilized by filtration)and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Prior to use adjust pH of complete medium to 6.8 - 7.0, if necessary. | |||||