769 .
CLOSTRIDIUM VINCENTII MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
6.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Sea Salt
(SIGMA) |
12.0 | g | ||
|
|
NH4NO3 | 0.1 | g | ||
|
|
Yeast extract
(BD Bacto) |
1.0 | g | ||
|
|
Trypticase peptone
(BD BBL) |
0.4 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.5 | ml | ||
|
|
NaHCO3 | 1.0 | g | ||
|
|
Na-phosphate buffer
(0.2 M, pH 6.5) |
20.0 | ml | ||
|
|
Lactose | 2.0 | g | ||
|
|
Wolin's vitamin solution (10x) | 1.0 | ml | ||
|
|
L-Cysteine HCl x H2O | 0.3 | g | ||
|
|
Na2S x 9 H2O | 0.3 | g | ||
|
|
Distilled water | 980.0 | ml | ||
| 1 Dissolve ingredients (except bicarbonate, phosphate-buffer, lactose, 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 under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add phosphate-buffer, lactose, cysteine and sulfide from sterile anoxic stock solutions autoclaved under 100% N2 gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Vitamins are prepared under N2 gas atmosphere and sterilized by filtration. Adjust pH of complete medium to 6.5, if necessary. | |||||
| 2 Note: Sea salts can be replaced by 300.0 ml/l of filtered aged sea water. | |||||
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 |