1210b .
PYRODICTIUM DELANEYI MEDIUM
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Equipment needed:
Autoclave
Centrifuge
Filter
Gassing station
Hungate tubes
Final pH:
5.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 18.00 | g | ||
|
|
MgCl2 x 6 H2O | 4.00 | g | ||
|
|
KCl | 0.33 | g | ||
|
|
CaCl2 x 2 H2O | 0.33 | g | ||
|
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(NH4)2SO4 | 0.50 | g | ||
|
|
Trace element solution SL-10 | 1.00 | ml | ||
|
|
Selenite-tungstate solution | 1.00 | ml | ||
|
|
Sodium resazurin
(0.1 % w/v) |
0.50 | ml | ||
|
|
Na2CO3 | 1.00 | g | ||
|
|
KH2PO4 | 0.33 | g | ||
|
|
FeCl2 x 4 H2O | 0.52 | g | ||
|
|
Yeast extract | 0.20 | g | ||
|
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Wolin's vitamin solution (10x) | 1.00 | ml | ||
|
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L-Cysteine HCl x H2O | 0.10 | g | ||
|
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Amorphous Fe(OH)3 | 100.00 | ml | ||
|
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Distilled water | 900.00 | ml | ||
| 1 Dissolve ingredients except carbonate, hydrogenphosphate, ferrous chloride, yeast extract, vitamins, cysteine and ferric iron hydroxide sludge. Suspend pellet of ferric iron hydroxide in medium and sparge with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Thereafter, dispense suspension under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave for 30 min. Add hydrogenphosphate, ferrous chloride (dissolved in 0.1 N HCl), yeast extract, cysteine and vitamins 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 mixture. Vitamins and ferrous chloride should be sterilized by filtration. The pH of the complete medium should be at 5.5. | |||||
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 | ||
|
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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 | ||
|
|
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. | |||||
Selenite-tungstate solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
NaOH | 0.5 | g |
|
|
Na2SeO3 x 5 H2O | 3.0 | mg |
|
|
Na2WO4 x 2 H2O | 4.0 | mg |
|
|
Distilled water | 1000.0 | ml |
Amorphous Fe(OH)3 #
400
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
FeCl3 x 6 H2O
(60.00 g/l) |
320 | ml | ||
|
|
NaOH
, adjust if required
(10% w/v) |
80 | ml | ||
| 1 Slowly titrate 320 ml of a FeCl3 x 6 H2O stock solution (60.00 g/l) with 10% (w/v) NaOH to pH 8.0-8.5 under agitation (use magnetic stirrer). Total amount of added NaOH approx. 80 - 100 ml. The precipitated Fe(OH)3 should be stored at room temperature overnight with surface covered with water. Thereafter, centrifuge at 2000 rpm for 5 min and discard the supernatant. Wash several times with distilled water. Resuspend the pellet in medium as described above. For storage autoclave under 100% N2 atmosphere. | |||||
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 |