298b .
SYNTROPHOBOTULUS MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
7.2 - 7.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
KH2PO4 | 0.20 | g | ||
|
|
NH4Cl | 0.25 | g | ||
|
|
NaCl | 1.00 | g | ||
|
|
MgCl2 x 6 H2O | 0.40 | g | ||
|
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KCl | 0.50 | g | ||
|
|
CaCl2 x 2 H2O | 0.15 | g | ||
|
|
Trace element solution SL-10 | 1.00 | ml | ||
|
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Na-acetate | 0.20 | g | ||
|
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Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
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Na2CO3 | 1.50 | g | ||
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Na2-glyoxalate | 0.60 | g | ||
|
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Wolin's vitamin solution (10x) | 1.00 | ml | ||
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L-Cysteine HCl x H2O | 0.30 | g | ||
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Na2S x 9 H2O | 0.30 | g | ||
|
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Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients except carbonate, glyoxalate, vitamins, cysteine and sulfide, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium in anoxic Hungate-type tubes or serum vials under the same gas atmosphere and autoclave. Add glyoxalate (sterilized by filtration), vitamins (sterilized by filtration), cysteine and sulfide 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. Prior to use adjust pH of complete medium to 7.2 - 7.5. | |||||
| 2 Note: When growth has started, feed culture once or twice with the same amount of glyoxalate. | |||||
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 | ||
|
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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. | |||||
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 |