Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response

Summary

We detail a simple method to produce high-resolution time-lapse movies of Pseudomonas aeruginosa swarms that respond to bacteriophage (phage) and antibiotic stress using a flatbed document scanner. This procedure is a fast and simple method for monitoring swarming dynamics and may be adapted to study the motility and growth of other bacterial species.

Abstract

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations of bacteria move over large distances in characteristic tendril-shaped communities over the course of hours. Swarming is sensitive to several factors including medium moisture, humidity, and nutrient content. In addition, the collective stress response, which is observed in P. aeruginosa that are stressed by antibiotics or bacteriophage (phage), repels swarms from approaching the area containing the stress. The methods described here address how to control the critical factors that affect swarming. We introduce a simple method to monitor swarming dynamics and the collective stress response with high temporal resolution using a flatbed document scanner, and describe how to compile and perform a quantitative analysis of swarms. This simple and cost-effective method provides precise and well-controlled quantification of swarming and may be extended to other types of plate-based growth assays and bacterial species.

Introduction

Swarming is a collective form of coordinated bacterial motility that increases antibiotic resistance and production of virulence factors in the host^1,2,3. This multicellular behavior occurs on semi-solid surfaces that resemble those of mucous layers covering epithelial membranes in the lungs^4,5. Biosurfactants are commonly produced by swarming populations to overcome the surface tension on surfaces and the production of these is regulated by complex cell-cell signaling systems, also known as quorum sensing

Protocol

1. Preparing Swarming Agar Plates for P. aeruginosa Swarming Time-lapse Imaging

1. Prepare 1 L of 5x M8 minimum media in a glass bottle by adding 64 g of Na₂HPO₄•7H₂O, 15 g of KH₂PO₄, and 2.5 g of NaCl in 500 mL double-distilled water (ddH₂O). Adjust the final volume to 1 L with additional ddH₂O. Autoclave to sterilize and store liquid media at room temperature.
2. Prepare 100 mL of 1 M MgSO₄ (magnesium sulfate) in a glass bottle by adding 24.6 g of MgSO₄•7H₂O in 50 mL ddH₂O. Adjust the final volume to 100 mL wit....

Results

The steps to grow P. aeruginosa, stress the cells, and image the swarming agar plates are represented in Figure 1. We inoculated a single colony of wild-type P. aeruginosa UCBPP-PA14 strain from an LB-agar plate in 2 mL of LB broth overnight at 37 °C and spotted 5 µL in the center of the swarming agar plate. Time-lapse imaging of this plate reveals initial growth in the form of a colony at the center and then spreading of tendrils radially from the colony (Video 1). For.......

Discussion

This protocol focuses on minimizing the variability in swarming agar plates and providing a simple and low-cost method to acquire time-lapse images of P. aeruginosa swarming and responding to stress. This procedure can be extended to image other bacterial systems by adapting the media composition and growth conditions. For P. aeruginosa, although M9 or FAB minimal medium can be used to induce swarming^16,21, the protocol presented here uses M8 me.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Bacto agar, dehydrated	BD Difco	214010	For LB-agar plate and swarming agar plate
Casamino acids	BD Difco	223050	For swarming media
D-Glucose	Fisher Chemical	D16500	Dextrose. For swarming media
Fosfomycin disodium salt	Tokyo Chemical Industry	F0889	Stock concentration: 200 mg/mL. Dissolved in ddH2O
Gentamycin sulfate	Sigma-Aldrich	G1914	Stock concentration: 3 mg/mL. Dissolved in ddH2O
Kanamycin sulfate	Sigma-Aldrich	60615	Stock concentration: 100 mg/mL. Dissolved in ddH2O
LB-Miller	BD Difco	244620	For LB broth and LB-agar plates
Magnesium sulfate heptahydrate	Sigma-Aldrich	230391	For swarming media
Potassium phosphate monobasic	Sigma-Aldrich	P0662	For 5x M8 media
Sodium chloride	Sigma-Aldrich	S9888	For 5x M8 media
Sodium phosphate dibasic heptahydrate	Fisher Chemical	S373	For 5x M8 media
Strains
Pseudomonas aeruginosa	Siryaporn lab	AFS27E.118	PA14 strain
DMS3vir	O'Toole lab	DMS3vir20	Bacteriophage
Supplies
Aluminium oxide sandpaper	3M	150 Fine	For black lids
Black fabric	Joann	PRD7089	Black fabric
Black spray paint	Krylon	5592 Matte Black	For black lids
Erlenmeyer flask	Kimax	26500	250 mL
Glass storage bottles	Pyrex	13951L	250 mL, 500 mL, 1,000 mL
8 inches zip ties	Gardner Bender	E173770	For attaching black matte fabric
Petri dishes (100 mm x 15 mm)	Fisher	FB0875712	100 mm x 15 mm polystyrene plates
Wooden sticks	Fisher	23-400-102	For streaking and inoculating bacteria
Equipment
Autoclave	Market Forge Industries	STM-E	For sterilizing reagents
25 mL pipette	USA Scientific, Inc.	1072-5410	To pipet 20 mL for swarming agar plates
Dehumidifier	Frigidaire	FAD704DWD 70-pint	For maintaing room relative humidity at about 45%
ImageJ	NIH	v1.52a	Software for image analysis
Incubator	VWR	89032-092	For growth of bacteria at 37 °C
Isotemp waterbath	Fisher	15-462-21Q	For cooling media to 55 °C
Laminar flow hood	The Baker Company	SG603A	For drying plates
P-20 pipet	Gilson	F123601	Spotting on swarming agar plates
Pipette Controller	BrandTech	accu-jet	To pipet 20 mL for swarming agar plates
Roller Drum	New Brunswick	TC-7	For growth of bacteria at 100 rpm
Scanner	Epson	Epson Perfection V370 Photo	Scanner for imaging plates
Scanner automation software	RoboTask Lite	v7.0.1.932	For 30 min internals imaging
Scanner image acquisition software	Epson	v9.9.2.5US	Software for imaging plates