This study investigates the Voges-Proskauer (VP) reaction in different biotypes of Vibrio cholerae, focusing on the wild-type El Tor's ability to produce acetoin. The experiment highlights the biochemical differences among classical, wild-type El Tor, and El Tor variant biotypes.
Take separate cultures of Vibrio cholerae, a pathogenic bacterium, containing three different biotypes: classical, wild-type El Tor, and El Tor variant, each representing genetically distinct subgroups.
Inoculate each culture into tubes filled with a specialized medium to perform the Voges-Proskauer (VP) assay.
Incubate with shaking to facilitate bacterial proliferation.
The wild-type El Tor ferments glucose in the medium and produces acetoin, whereas classical and variant biotypes do not.
Next, add α-naphthol and potassium hydroxide to each tube, mix thoroughly, and incubate.
In the presence of potassium hydroxide, atmospheric oxygen catalyzes the oxidation of acetoin to diacetyl.
Diacetyl reacts with compounds containing guanidine groups in the medium to form a pinkish-red colored product, whose intensity is enhanced by α-naphthol.
Color development indicates a positive VP reaction, characteristic of the wild-type El Tor biotype, while the classical and variant biotypes show no color formation.
Inoculate 4 milliliters of Methyl Red Voges-Proskauer or MR-VP broth by pipetting 10 microliters of previously prepared overnight culture into 4 milliliters of MR-VP broth in a sterile culture tube and incubate with aeration in a shaker incubator at 225 rpm for 12-16 hours at 37 degrees Celsius. Next, add 150 microliters of 5% weight per volume of Alpha-naphthol and 50 microliters of 40% weight per volume potassium hydroxide to 1 milliliter aliquots of the MR-VP overnight culture in sterile culture tubes, respectively.
Then, briefly vortex the tubes. Allow the tubes to stand at room temperature for up to four hours until color change develops.
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