557 .
CLOSTRIDIUM SP. MEDIUM (CREATININE)
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final gas composition:
100 % N2
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Final pH:
7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
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CaSO4 x 2 H2O | 0.05 | g | ||
|
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MgSO4 x 7 H2O | 0.50 | g | ||
|
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FeSO4 x 7 H2O | 0.01 | g | ||
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MnCl2 x 4 H2O | 0.06 | g | ||
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NaCl | 1.00 | g | ||
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Trace element solution SL-11 | 1.00 | ml | ||
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Selenite-tungstate solution | 1.00 | ml | ||
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Yeast extract | 5.00 | g | ||
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Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
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KH2PO4 | 2.64 | g | ||
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K2HPO4 | 5.33 | g | ||
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NaHCO3 | 1.00 | g | ||
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Creatinine | 5.50 | g | ||
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Wolin's vitamin solution (10x) | 1.00 | ml | ||
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L-Cysteine HCl x H2O | 0.50 | g | ||
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Na2S x 9 H2O | 0.50 | g | ||
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Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients except hydrogenphosphates, bicarbonate, creatinine, vitamins, cysteine and sulfide, adjust pH to 7.0 and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Complete medium by adding hydrogenphosphates, creatinine, vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Stock solutions of creatinine (5% w/v) and vitamins should be sterilized by filtration. Adjust pH of complete medium to 7.0, if necessary. | |||||
Trace element solution SL-11 #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Na2-EDTA x 2 H2O | 5.2 | g | ||
|
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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 | 1000.0 | ml | ||
| 1 Dissolve EDTA in 800 ml distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds. Finally adjust pH to 6.0 and bring volume to 1000 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 |
|
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Distilled water | 1000.0 | ml |
Wolin's vitamin solution (10x) #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 20 | mg |
|
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Folic acid | 20 | mg |
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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 |