Preparation, imaging, and quantification of bacterial surface motility assays

J Vis Exp. 2015 Apr 7:(98):52338. doi: 10.3791/52338.

Abstract

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more "temperate swarmers" that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. "Wettability", or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Agar
  • Bacillus subtilis / chemistry
  • Bacillus subtilis / physiology
  • Bacterial Physiological Phenomena*
  • Bacteriological Techniques / methods*
  • Culture Media
  • Luminescent Measurements / methods
  • Myxococcus xanthus / chemistry
  • Myxococcus xanthus / physiology
  • Pseudomonas aeruginosa / chemistry
  • Pseudomonas aeruginosa / physiology
  • Reproducibility of Results
  • Swimming
  • Time-Lapse Imaging / methods*

Substances

  • Culture Media
  • Agar