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Main sol. 769

Internal Solution-ID: S1567

Equipment needed: Autoclave Hungate tubes Filter Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
Sea Salt
(SIGMA)
12.0 g 11.988 -
NH4NO3 0.1 g 0.1 -
Yeast extract
(BD Bacto)
1.0 g 0.999 -
Trypticase peptone
(BD BBL)
0.4 g 0.4 -
Sodium resazurin
(0.1% w/v)
0.5 ml 5.0e-4 0.002
NaHCO3 1.0 g 0.999 11.892
Na-phosphate buffer
(0.2 M, pH 6.5)
20.0 ml - 4
Lactose 2.0 g 1.998 5.837
Wolin's vitamin solution (10x) 1.0 ml - -
L-Cysteine HCl x H2O 0.3 g 0.3 1.706
Na2S x 9 H2O 0.3 g 0.3 1.248
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.
769 CLOSTRIDIUM VINCENTII MEDIUM