Motility Assay for Comparing Vibrio cholerae Strains Using Triphenyltetrazolium Chloride

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 a soft agar assay using triphenyltetrazolium chloride to assess and compare the motility of Vibrio cholerae strains by visualizing metabolic activity through red zone formation around actively migrating cells.

Protocol

1. Motility Assay
   1. Prepare overnight culture(s) of Vibrio cholerae strains in liquid LB (Luria-Bertani) broth.
   2. Pellet 1.8 mL of the overnight culture for 2 min by centrifugation (≥8,600 x g) in a sterile 2 mL microcentrifuge tube and remove the supernatant using a pipette. Resuspend the cell pellet in 1.8 mL 1x PBS (phosphate-buffered saline) by pipetting.
   3. Repeat washing procedure three times, followed by a final resuspension in 1.8 mL 1x PBS.
   4. Inoculate motility agar plates by inserting an inoculating stab into the washed liquid culture and stabbing vertically into the media. In between each inoculation, sterilize the wire stab using a Bunsen burner, and when stabbing agar, ensure the inoculating stab does not bend, as this can alter results.
   5. Incubate plates lid-side up for 14 to 24 h at 37 °C. After 14 h, monitor motility plates closely to prevent overgrowth of cultures.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	Becto, Dickinson and Co.	214030	http://catalog.bd.com/nexus-ecat/getProductDetail?productId=214030&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3D214030%26typeOfSearch%3DproductSearch
Benchtop centrifuge	Eppendorf	5424	Any centrifuge for 1.5ml eppendorf tubes
Le Stab	Decon Labs Inc.	MP186-5	http://deconlabs.com/products/lestab/
LB broth (Miller)	Thermo/Gibco	10855021	For static bacterial culture and OD measurement
Petri Dishes (150 mm x 15 mm)	Fisher Scientific	FB0875714	https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875714?searchHijack=true&searchTerm=FB0875714&searchType=RAPID
PBS 1x			For static bacterial culture and OD measurement
Triphenyltetrazolium chloride	Alfa Aesar	A10870	https://www.alfa.com/en/catalog/A10870/