792 .
PYROLOBUS FUMARII MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Serum bottle
Final pH:
5.8
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Solution A | 989 | ml | ||
|
|
Solution B | 10 | ml | ||
|
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Solution C | 1 | ml | ||
|
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Solution D | 10 | ml | ||
| 1 Sparge solution A with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic serum vials (e.g., 20 ml medium in 100 ml serum bottles) and autoclave. Solutions B and D are autoclaved separately under 100% N2 gas atmosphere and solution C is prepared under 100% N2 gas and sterilized by filtration. To complete the medium appropriate amounts of solutions B, C and D are added to solution A. Adjust pH of the complete medium to 5.8 using a sterile anoxic stock solution of 5% (w/v) Na2CO3, if necessary. | |||||
| 2 After inoculation pressurize vials to 1 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture. | |||||
| 3 Note: Solution A has to be prepared freshly. It cannot be stored for a long period of time. | |||||
Solution A #
989
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
NaCl | 13.85 | g |
|
|
MgSO4 x 7 H2O | 3.50 | g |
|
|
MgCl2 x 6 H2O | 2.75 | g |
|
|
CaCl2 x 2 H2O | 0.38 | g |
|
|
KCl | 0.33 | g |
|
|
NaBr | 0.05 | g |
|
|
H3BO3 | 15.00 | mg |
|
|
SrCl2 x 6 H2O
(0.1% w/v) |
7.00 | ml |
|
|
KI
(0.01% w/v) |
0.50 | ml |
|
|
Wolfe's mineral elixir | 1.00 | ml |
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml |
|
|
Double distilled water | 980.00 | ml |
Wolfe's mineral elixir #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
MgSO4 x 7 H2O | 30.00 | g | ||
|
|
MnSO4 x H2O | 5.00 | g | ||
|
|
NaCl | 10.00 | g | ||
|
|
FeSO4 x 7 H2O | 1.00 | g | ||
|
|
CoCl2 x 6 H2O | 1.80 | g | ||
|
|
CaCl2 x 2 H2O | 1.00 | g | ||
|
|
ZnSO4 x 7 H2O | 1.80 | g | ||
|
|
CuSO4 x 5 H2O | 0.10 | g | ||
|
|
AlK(SO4)2 x 12 H2O | 0.18 | g | ||
|
|
H3BO3 | 0.10 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.10 | g | ||
|
|
(NH4)2Ni(SO4)2 x 6 H2O | 2.80 | g | ||
|
|
Na2WO4 x 2 H2O | 0.10 | g | ||
|
|
Na2SeO4 | 0.10 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 First adjust pH to 1.0 with diluted H2SO4, then add and dissolve the salts. | |||||
Seven vitamins solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Vitamin B12 | 100 | mg |
|
|
p-Aminobenzoic acid | 80 | mg |
|
|
D-(+)-biotin | 20 | mg |
|
|
Nicotinic acid | 200 | mg |
|
|
Calcium pantothenate | 100 | mg |
|
|
Pyridoxine hydrochloride | 300 | mg |
|
|
Thiamine-HCl x 2 H2O | 200 | mg |
|
|
Distilled water | 1000 | ml |