Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

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.

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

“…The culturing conditions described here apply for B. subtilis and the exact parameters for expansion on agar media might require optimization for other species or strains 20 . Placing the samples in an incubation chamber while imaging permits the experimenter to follow the population dynamics in time, although attention should be given to the humidity level within the chamber during the incubation.…”

“…Therefore, the described analysis of the images determines the spatial distribution, but not the abundance of the strains within a certain location. Previous protocols described the sample preparation for swarming 20 or fluorescence imaging of population dynamics in bacterial colonies 38 , but our protocol combines these techniques. Other microscopy techniques that permit the observation of three dimensional resolution of the population structure (e.g.…”

“…Strategies of surface colonization by bacteria can be easily manipulated under laboratory conditions by modifying the agar concentration in the growth media. At low agar levels (0.2-0.3%), bacteria harboring active flagella are able to swim, while semi-solid agar (0.7-1% agar) facilitates flagellum driven community spreading, called swarming [19][20][21] . In the absence of flagellum, certain bacterial strains are able to move over semi-solid medium via sliding, i.e.…”

Multimodal chemical imaging of molecular messengers in emerging Pseudomonas aeruginosa bacterial communities

“…Sterile medium (7.5 mL) was gently pipetted into 60 mm polystyrene Petri dishes and allowed to cure for 30 min. After drying, the plates were stab-inoculated with an overnight FAB-glucose broth culture (OD = 1 at 600 nm) and incubated at 30 °C for 48 h. 20 …”

Metal-assisted polyatomic SIMS and laser desorption/ionization for enhanced small molecule imaging of bacterial biofilms

“…45 Aliquots (200 ll each) of the resulting culture (optical density of 1 at 600 nm h) were transferred to 2 cm  2 cm sterilized silicon shards in a Petri dish. Following 10-15 min of incubation, 18 m of fresh culture medium with 30 mM glucose was gently added, and the bacteria were allowed to grow at 37 C for 24 h. The liquid medium was carefully removed from the dish, and the resulting colonies were allowed to dry for 1 h in a sterile hood.…”