1743 .
GHIORSEA MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Serum bottle
Final pH:
6.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 27.50 | g | ||
|
|
MgCl2 x 6 H2O | 5.38 | g | ||
|
|
MgSO4 x 7 H2O | 6.78 | g | ||
|
|
KCl | 0.72 | g | ||
|
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NH4Cl | 0.50 | g | ||
|
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CaCl2 x 2 H2O | 1.40 | g | ||
|
|
KH2PO4 | 0.05 | g | ||
|
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Trace element solution | 10.00 | ml | ||
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NaHCO3 | 1.70 | g | ||
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Wolin's vitamin solution (10x) | 1.00 | ml | ||
|
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Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients (except vitamins), then sparge medium with 80% N2 and 20% CO2 gas mixture to make it anoxic and until a pH of 6.5 is reached. Dispense medium under same gas atmosphere into anoxic serum vials to 20% of their volume and autoclave. After autoclaving add vitamins from a sterile stock solution prepared under 100% N2 gas atmosphere and sterilized by filtration. Prior to inoculation add 5% (v/v) sterile H2 and 5% (v/v) sterile air to the headspace by using syringes. Check pH of the complete medium and adjust pH to 6.5, if necessary. Incubate without shaking. | |||||
| 2 Note: After growth has started cell density of the culture can be increased by replenishing H2 and air every two days. | |||||
Trace element solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Na2-EDTA x 2 H2O | 0.50 | g | ||
|
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CoCl2 x 6 H2O | 0.15 | g | ||
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MnCl2 x 4 H2O | 0.10 | g | ||
|
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FeSO4 x 7 H2O | 0.10 | g | ||
|
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ZnCl2 | 0.10 | g | ||
|
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AlCl3 x 6 H2O | 40.00 | mg | ||
|
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Na2WO4 x 2 H2O | 40.00 | mg | ||
|
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Na2SeO3 x 5 H2O | 30.00 | mg | ||
|
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NiCl2 x 6 H2O | 20.00 | mg | ||
|
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CuCl2 x 2 H2O | 20.00 | mg | ||
|
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H3BO3 | 10.00 | mg | ||
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Na2MoO4 x 2 H2O | 10.00 | mg | ||
|
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Distilled water | 1000.00 | ml | ||
| 1 First dissolve EDTA in distilled water and adjust pH to 7 using 2 N NaOH, then dissolve remaining compounds. | |||||
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 |
|
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Vitamin B12 | 1 | mg |
|
|
p-Aminobenzoic acid | 50 | mg |
|
|
(DL)-alpha-Lipoic acid | 50 | mg |
|
|
Distilled water | 1000 | ml |