193a .
PHB/PYRUVATE MEDIUM
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Selected strain: DSM 6754
This strain uses the standard medium without modifications.
Growth conditions: anaerobic
37 °C
Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
6.8 - 7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Solution A | 942 | ml | ||
|
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Solution B | 30 | ml | ||
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Solution C | 20 | ml | ||
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Solution D | 1 | ml | ||
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Solution E | 10 | ml | ||
| 1 Solution A is sparged with 80% H2 and 20% CO2 gas mixture to reach a pH below 6 (at least 30 min), then distributed under the same gas atmosphere in anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclaved. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solutions C and D are prepared under 100% N2 gas atmosphere and sterilized by filtration. Solution E is autoclaved separately under 100% N2 gas. To complete the medium appropriate amounts of solutions B to E are added to the sterile solution A in the sequence as indicated. Final pH of the medium should be 6.8 - 7.0. | |||||
| 2 After inoculation, pressurize vessels with 80% H2 and 20% CO2 gas mixture to 1 bar overpressure. | |||||
| 3 Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) may stimulate growth at the beginning. For transfers use 5 - 10% inoculum. | |||||
Solution A #
942
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Na2SO4 | 3.00 | g |
|
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KH2PO4 | 0.20 | g |
|
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NH4Cl | 0.30 | g |
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NaCl | 7.00 | g |
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MgCl2 x 6 H2O | 1.30 | g |
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KCl | 0.50 | g |
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CaCl2 x 2 H2O | 0.15 | g |
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Selenite-tungstate solution | 1.00 | ml |
|
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Trace element solution SL-10 | 1.00 | ml |
|
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Poly[(R)-3-hydroxybutyric acid]
(SIGMA) |
1.00 | g |
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml |
|
|
Distilled water | 940.00 | 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 |
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 | ||
|
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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 |
|
|
Thiamine HCl | 50 | mg |
|
|
Riboflavin | 50 | mg |
|
|
Nicotinic acid | 50 | mg |
|
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Calcium D-(+)-pantothenate | 50 | mg |
|
|
Vitamin B12 | 1 | mg |
|
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p-Aminobenzoic acid | 50 | mg |
|
|
(DL)-alpha-Lipoic acid | 50 | mg |
|
|
Distilled water | 1000 | ml |