901 .
ACIDOLOBUS ACETICUS MEDIUM
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Equipment needed:
Filter
Gassing station
Hungate tubes
Water bath
Final gas composition:
100 % CO2
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Final pH:
3.5 - 3.8
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NH4Cl | 0.33 | g | ||
|
|
KCl | 0.33 | g | ||
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KH2PO4 | 0.33 | g | ||
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MgCl2 x 6 H2O | 0.33 | g | ||
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CaCl2 x 2 H2O | 0.33 | g | ||
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Trace element solution SL-10 | 1.00 | ml | ||
|
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Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
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Sulfur
(powdered) |
10.00 | g | ||
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Yeast extract | 3.00 | g | ||
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Wolin's vitamin solution (10x) | 1.00 | ml | ||
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Neutralized sulfide solution 3% (w/v) | 15.00 | ml | ||
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Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients except sulfur, yeast extract, vitamins and sulfide. Adjust pH to 3.5 with H2SO4 and sparge medium with 100% CO2 gas for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials containing the appropriate amount of sulfur. Heat vessels containing medium to 90°C for 1 hour on each of 3 succesive days. Add yeast extract, vitamins (sterilized by filtration) and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. The sulfide stock solution (3% w/v) should be neutralized before use (see medium 29). The pH of the complete medium should be at 3.5 - 3.8. | |||||
Trace element solution SL-10 #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
HCl
(25%) |
10.0 | ml | ||
|
|
FeCl2 x 4 H2O | 1.5 | g | ||
|
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ZnCl2 | 70.0 | mg | ||
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MnCl2 x 4 H2O | 100.0 | mg | ||
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H3BO3 | 6.0 | mg | ||
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CoCl2 x 6 H2O | 190.0 | mg | ||
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CuCl2 x 2 H2O | 2.0 | mg | ||
|
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NiCl2 x 6 H2O | 24.0 | mg | ||
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Na2MoO4 x 2 H2O | 36.0 | mg | ||
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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. | |||||
Wolin's vitamin solution (10x) #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 20 | mg |
|
|
Folic acid | 20 | mg |
|
|
Pyridoxine hydrochloride | 100 | mg |
|
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Thiamine HCl | 50 | mg |
|
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Riboflavin | 50 | mg |
|
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Nicotinic acid | 50 | mg |
|
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Calcium D-(+)-pantothenate | 50 | mg |
|
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Vitamin B12 | 1 | mg |
|
|
p-Aminobenzoic acid | 50 | mg |
|
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(DL)-alpha-Lipoic acid | 50 | mg |
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Distilled water | 1000 | ml |
Neutralized sulfide solution 3% (w/v) #
100
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Na2S x 9 H2O | 3 | g | ||
|
|
Distilled water | 100 | ml | ||
| 1 The sulfide solution is prepared in a 250 ml screw-capped bottle with a butyl rubber septum and a magnetic stirrer. The solution is bubbled with nitrogen gas, closed and autoclaved for 15 min. at 121°C. After cooling to room temperature the pH is adjusted to about 7.0 by adding of sterile 2 M H2SO4 drop-wise with a syringe without opening the bottle. | |||||