Preparation, imaging, and quantification of bacterial surface motility assays.
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.
Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame; Eck Institute for Global Health, University of Notre Dame.
Nydia
N
Morales-Soto
Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame.
Morgen E
ME
Anyan
Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame.
Anne E
AE
Mattingly
Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame.
Chinedu S
CS
Madukoma
Department of Applied and Computational Mathematics and Statistics, University of Notre Dame.
Cameron W
CW
Harvey
Department of Applied and Computational Mathematics and Statistics, University of Notre Dame.
Mark
M
Alber
INRS-Institut Armand-Frappier.
Eric
E
Déziel
Department of Biology, Indiana University.
Daniel B
DB
Kearns
Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame; Eck Institute for Global Health, University of Notre Dame; Department of Biological Sciences, University of Notre Dame; joshua.shrout@nd.edu.
Joshua D
JD
Shrout
D000362:Agar; D001412:Bacillus subtilis / Q000737:chemistry / Q000502:physiology; D018407:Bacterial Physiological Phenomena; D001431:Bacteriological Techniques / Q000379:methods*; D003470:Culture Media; D008163:Luminescent Measurements / Q000379:methods; D016941:Myxococcus xanthus / Q000737:chemistry / Q000502:physiology; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000502:physiology; D015203:Reproducibility of Results; D013550:Swimming; D059008:Time-Lapse Imaging / Q000379:methods*
D003470:Culture Media; D000362:Agar