Voges-Proskauer Assay for Differentiating Biotypes of Vibrio cholerae

Abstract

Source: Brumfield, K. D., et al., Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates. J. Vis. Exp. (2017).

This video demonstrates the Voges-Proskauer (VP) test to differentiate among three biotypes of Vibrio cholerae: classical, wild-type El Tor, and El Tor variant. Each culture is inoculated into a glucose-containing medium, incubated, and treated with α-naphthol and potassium hydroxide to detect the presence of acetoin. A pinkish-red color, indicating a positive VP reaction, is observed only in the wild-type El Tor biotype, distinguishing it from the classical and variant types.

Protocol

Voges-Proskauer (VP) assay

1. Prepare overnight culture(s) in liquid LB broth.
2. Inoculate 4 mL of methyl red Voges-Proskauer, or MR-VP broth, by pipetting 10 μL of previously prepared overnight culture into 4 mL MR-VP broth in a sterile culture tube and incubate with aeration in a shaker incubator at 225 rpm for 12 to 16 h at 37 °C.
3. Add 150 μL of 5% (w/v) α-naphthol and 50 μL 40% (w/v) KOH to 1 mL aliquots of MR-VP overnight culture in sterile culture tubes, respectively.
4. Briefly vortex and let stand at room temperature for up to 4 h until color change develops.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MR-VP Broth	Difco	216300	http://catalog.bd.com/nexus-ecat/getProductDetail?productId=216300&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3Dmr-vp%2Bmedium%26typeOfSearch%3DproductSearch
KOH	Fisher Scientific	P250-500	https://www.fishersci.com/shop/products/potassium-hydroxide-pellets-certified-acs-fisher-chemical-5/p250500?searchHijack=true&searchTerm=P250500&searchType=RAPID
α-napthol	MP Biomedicals	204189	http://www.mpbio.com/product.php?pid=05204189