Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Summary

Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses.

Abstract

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time.

This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process.

The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers' effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.

Introduction

Bacterial swarming motility refers to the collective migration of bacterial cells across the surface of a substance. In addition to semisolid agar plates specially prepared in the laboratory¹, this phenotype is also observed on some soft substrates such as animal tissues², hydrated surfaces³, and plant roots⁴. While a semisolid surface is considered one of the fundamental conditions for bacterial swarming, some species also require an energy-rich medium to support their swarming motility⁵. Flagella rotation powers both swimming and swarming motility-....

Protocol

1. Preparation of gradient swarm plates

1. Preparation of swarm medium
   NOTE: See the discussion section for a brief comparison of different medium viscosities; 0.7% (w/v) agar concentration of swarm medium was used in this protocol.
   1. Prepare Lysogeny broth (LB) powder with agar in two conical flasks; each flask contains 2 g of tryptone, 2 g of sodium chloride, 1 g of yeast extract, and 1.4 g of agar. Add double-distilled water (ddH₂O) and stir the suspension using a magnetic stir bar. Adjust the final volume to 200 mL by adding additional ddH₂O.
   2. Autoclave the solution at 121 °C for 20 mi....

Results

The workflow consisting of the preparation of gradient swarm plates, inoculation, and incubation is shown in Figure 1B. To generate gradient swam plates, the bottom-layer medium is poured into propped-up dishes at ~4.3° from the horizontal plane (Supplemental Figure S3), followed by pouring the upper-layer medium after the bottom layer is completely solidified. The composition of the double-layer medium is shown in Table 1. Then, bacterial culture cultured o.......

Discussion

Investigation of bacterial swarming motility on semisolid gradient plates can be a challenging task^18,19,20, as it involves multiple factors such as substrate viscosity, humidity, and medium components. Among these factors, agar concentration plays a decisive role in determining microbial reversion to either swimming or swarming motility. As the agar concentration increases from 0.3% (w/v) to 1% (w/v), bacterial swimming motilit.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	Sigma-Aldrich	V900500	500 g
Ampicillin	Solarbio	A8180	25 g, ≥ 85% (GC)
Centrifuge tube	Corning	430790	15 mL
Cryogenic vial	Corning	430488	2 mL
Dimethyl sulfoxide (DMSO)	Aladdin	D103272	AR, > 99% (GC)
L(+)-Arabinose	Aladdin	A106195	98% (GC), 500 g
Petri dishes	Bkman	B-SLPYM90-15	Plastic Petri dishes,circular,90 mm x 15 mm
Resveratrol	Aladdin	R107315	99% (GC), 25 g
Sodium chloride	Macklin	S805275	AR, 99.5% (GC), 500 g
Square Petri dishes	Bkman	B-SLPYM130F	Plastic Petri dishes, square, 13 mm x 13 mm
Tryptone	Thermo Scientific Oxoid	LP0042	500 g
Yeast extract	Thermo Scientific Oxoid	LP0021	500 g
Equipments
Biochemical incubator	Blue pard	LRH-70
Tanon 5200multi imaging system	Tanon	5200CE
Thermostatic water bath	Jinghong	DK-S28