817 .
THERMOSPHAERA MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
6.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
MgCl2 x 6 H2O | 2.2 | g | ||
|
|
NaCl | 0.9 | g | ||
|
|
KCl | 17.0 | mg | ||
|
|
NH4Cl | 12.0 | mg | ||
|
|
CaCl2 x 2 H2O
(0.1% w/v) |
7.0 | ml | ||
|
|
K2HPO4 x 3 H2O
(0.1% w/v) |
7.0 | ml | ||
|
|
FeCl3
(0.01% w/v in 0.2 N HCl) |
0.5 | ml | ||
|
|
Yeast extract
(BD Bacto) |
1.0 | g | ||
|
|
Peptone
(BD Bacto) |
1.0 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.5 | ml | ||
|
|
Na2CO3 | 0.5 | g | ||
|
|
Wolin's vitamin solution (10x) | 1.0 | ml | ||
|
|
Na2S x 9 H2O | 0.5 | g | ||
|
|
Distilled water | 1000.0 | ml | ||
| 1 Dissolve ingredients except carbonate, vitamins and sulfide. Sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Vitamins should be sterilized by filtration. The pH of the complete medium should be adjusted to 6.5. | |||||
| 2 After inoculation pressurize tubes or bottles (heavy walled) to 2 bar overpressure with sterile 80% N2 and 20% CO2 gas mixture. | |||||
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 |
|
|
Calcium D-(+)-pantothenate | 50 | mg |
|
|
Vitamin B12 | 1 | mg |
|
|
p-Aminobenzoic acid | 50 | mg |
|
|
(DL)-alpha-Lipoic acid | 50 | mg |
|
|
Distilled water | 1000 | ml |