52 .
CLOSTRIDIUM KLUYVERI MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
6.8 - 7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
K-acetate | 10.00 | g | ||
|
|
K2HPO4 | 0.31 | g | ||
|
|
KH2PO4 | 0.23 | g | ||
|
|
NH4Cl | 0.25 | g | ||
|
|
MgSO4 x 7 H2O | 0.20 | g | ||
|
|
Trace element solution SL-10 | 1.00 | ml | ||
|
|
Selenite-tungstate solution | 1.00 | ml | ||
|
|
Yeast extract | 1.00 | g | ||
|
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Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
Ethanol absolute | 20.00 | ml | ||
|
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Na2CO3 | 1.00 | g | ||
|
|
Seven vitamins solution | 1.00 | ml | ||
|
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L-Cysteine HCl x H2O | 0.25 | g | ||
|
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Na2S x 9 H2O | 0.25 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve all ingredients (except ethanol, carbonate, vitamins, cysteine and sulfide) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add ethanol, then dispense medium under same gas atmosphere in Hungate-type tubes or serum vials and autoclave. After autoclaving, add carbonate from a sterile stock solution prepared under 80% N2 and 20% CO2 gas atmosphere and vitamins from an anoxic and filter-sterilized stock solution prepared under 100% N2 gas. Before inoculation, add cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Adjust pH of medium to 6.8 - 7.0, if necessary. | |||||
| 2 Note: If reduction of medium is not complete after inoculation, add 10 - 20 mg/l sodium dithionite from a 5% w/v solution freshly prepared under 100% N2 gas and filter sterilized. | |||||
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 | ||
|
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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 | ||
|
|
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. | |||||
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 |
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 |