Swarming Behavior of Pseudomonas aeruginosa Under Antibiotic Stress

Abstract

Source: Bru, J., et al., Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response. J. Vis. Exp. (2020)

This video demonstrates the procedure for analyzing the swarming behavior of Pseudomonas aeruginosa by observing bacterial growth from a central culture toward satellite spots containing the same culture mixed with an antibiotic, using an agar plate setup and time-lapse imaging.

Protocol

1. Growth of P. aeruginosa and Plating Conditions

1. Prepare 400 mL of lysogeny broth (LB) by adding 10 g of LB-Miller powder mix to 400 mL of ddH₂O. Add an additional 8 g of agar for 2% LB-agar Petri dishes. Autoclave to sterilize.
2. Pour 20 mL of molten LB-agar medium into 10 cm-diameter Petri dishes and allow them to solidify at room temperature overnight. Store the liquid media at room temperature and the agar plates at 4 °C.
3. Streak P. aeruginosa on an LB-agar Petri dish from a frozen stock stored at -80 °C using sterile loops or wooden sticks. Incubate the Petri dish upside-dow....

Results

Figure 1: Schematic of the P. aeruginosa swarming assay and c.......

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
Strains
Pseudomonas aeruginosa	Siryaporn lab	AFS27E.118	PA14 strain
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, 1000 mL
8 inches zip ties	Gardner Bender	E173770	For attaching black matte fabric
Petri dishes (100 mm x 15 mm)	Fisher	FB0875712	100 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%
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