Swarming Behavior of Pseudomonas aeruginosa Under Antibiotic Stress

Samenvatting

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-down overnight at 37 °C. Store LB-agar plate at 4 °C for up to 1 week.
4. Pick a single colony from the Petri dish with a sterile loop or wooden stick, inoculate it into 2 mL LB medium, and incubate the culture to saturation overnight (16–18 h) at 37 °C in a roller drum set at 100 rpm.
5. Pipet 5 µL of overnight culture from step 1.4 using a P20 pipet and spot at the center of the swarming agar plate by approaching the pipet tip at an angle (10–45°) 2.5 cm above the spotting area, pipetting down to the first stop, and touching the agar with only the liquid drop (Figure 1B).
   1. Avoid touching the agar with the pipette tip, as this can damage the agar. Use a template to position the spot consistently across different swarming agar plates.
6. For antibiotic treatments, mix 30 µL overnight culture P. aeruginosa from step 1.4 with 6 µL of 3 mg/mL gentamycin, 10 µL of 100 mg/mL kanamycin, or 7.5 µL of 100 mg/mL fosfomycin. Proceed immediately to the next step.
   1. Pipet 6 µL of antibiotic-treated P. aeruginosa using a P20 pipette and spot at 6 equidistant satellite positions on a 2.8 cm radius concentric circle about the center of the dish by approaching the pipet tip at an angle (10 to 45°) 2.5 cm above the spotting area, pipetting down to the first stop, and touching the agar with only the liquid drop (Figure 1D).
   2. Avoid touching the agar with the pipette tip, as this can damage it. Use a plating template for consistency.
7. Replace the clear Petri dish lids with black lids (Figure 2A).
8. Place the swarming agar plates on a scanner in an incubator set at 37 °C with a 10 L water bath to maintain 75% humidity (Figure 1E, Figure 2B).CAUTION: Do not disturb spotted cells on the swarming agar plates. Keep plates facing up at all times.

Resultaten

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

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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