A Rapid Image-Based Assay for Screening Surface-Induced Bacterial Virulence

Abstract

Source: Perinbam, K., and Siryaporn, A. A Rapid Image-based Bacterial Virulence Assay Using Amoeba. J. Vis. Exp. (2018)

This video demonstrates a rapid, image-based method to assess bacterial virulence by incubating amoebae with surface-attached or planktonic bacteria, applying a calcein-acetoxymethyl ester (calcein-AM) agarose pad, and imaging fluorescence to detect host cell damage.

Protocol

1. Microscopy - Preparation of the agar pad

1. Prepare agar pads about 30 min to 1 h before the amoebae are ready to be mixed with Pseudomonas aeruginosa.
2. Microwave 50 mL of 1% (w/v) agar in development buffer (DB) in a 250 mL flask. Mix every 20 s until clumps of agar disappear.
3. Cool the molten agar by placing it in a preheated 55 °C water bath for 15 min.
4. Add 15 µL of the calcein-AM stock solution to 15 mL of the molten agar in a conical tube. Mix by gently inverting the tube 4 - 5 times, and proceed immediately to the next step.
   NOTE: Perform this step in a polypropylene 15 mL centrifuge tube. Do not use a thick glass container, as this will cause the agar to solidify too quickly.
5. Pour the molten agar mixture on top of a 12 cm x 10 cm glass plate and allow the agar to solidify for 10 minutes at room temperature. Cut the solidified agar pad into smaller 1.5 cm x 1.5 cm sections by placing the glass plate over a printed grid and cutting along the grid using a metal ruler.
6. Keep the gel hydrated in a humid box until it is ready for use.

2. Microscopy - Preparation of the bacteria-amoeba sample for imaging

1. To assay the virulence of surface-attached bacterial cells, add 10 µL of amoeba cells to surface-attached bacteria.
   NOTE: This step describes the mixing of amoebae with bacteria.
2. To assay the virulence of planktonic cells, mix 10 µL of amoeba cells with 10 µL of planktonic bacteria.
   NOTE: Do not wash the axenically-grown amoeba cells (at an OD₆₀₀ of 0.2 to 0.5) before mixing with the bacteria. No E. coli growth will be observed in the amoeba culture due to the use of the antibiotic-antimycotic solution.
3. Immediately place the 1.5 cm x 1.5 cm calcein-AM agar pad from step 1.5 on top of the bacteria-amoeba mixture on the Petri dish surface.
   NOTE: Place the agar pad on top of the mixture using a quick, smooth motion. Do not lower the pad slowly onto the mixture, as this motion tends to push cells towards the periphery and away from the underside of the pad. This step will ensure that bacteria and amoebae are evenly mixed, immobilize,d and that both cell types are confined to the same plane.
4. Remove excess liquid by gently pipetting out any remaining liquid surrounding the agar pad. Allow the Petri dish to dry for 20 min at room temperature.
5. Cover the Petri dish with a lid and incubate the immobilized amoeba-bacteria mixture at room temperature for an additional 40 min. Proceed to step 3.1.
   NOTE: It is important to incubate all samples for the same amount of time, as the background fluorescence increases over time. An incubation time of 1 h is recommended. Incubations for longer than 1 h are less reproducible as amoebae cells may begin to burst.

3. Microscopy - Image Acquisition

1. Image amoebae using a fluorescence microscope capable of acquiring brightfield and green fluorescence images. For green fluorescence, use green fluorescence protein (GFP) filters with 474/27 nm and 525/45 nm for excitation and emission, respectively. Use a 10X (≥0.3 numerical aperture) objective to image the amoebae.
   NOTE: The calcein-AM in the agar will cause dying amoebae to fluoresce in the green fluorescence channel. Healthy amoebae are otherwise not fluorescent.
2. Adjust acquisition times for the phase contrast, GFP channels to limit phototoxicity and optimize the dynamic range of the image intensities. Substitute the differential interference contrast (DIC) phase contrast if necessary.
   NOTE: Use 100 - 200 ms exposures for phase contrast and GFP fluorescence if possible. Keep the exposure and instrument settings unchanged throughout the entire experiment. This is critical for computing the host killing index.
3. Acquire images for at least 100 amoeba cells in order to obtain good statistics for calculating the host killing index.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Bacto agar, dehydrated	BD Difco	214010
Antibiotic-Antimycotic (100X)	Life Technologies	15240062	Aliquot < 1 mL and store at -20 °C
Calcein-acetoxymethyl ester (calcein-AM)	Life Technologies	C34852	Calcein Acetoxymethyl (AM)
Calcium chloride, anhydrous	Sigma-Aldrich	C1016
D-Glucose	Fisher Chemical	D16500	Dextrose
Dimethyl sulfoxide	Sigma-Aldrich	D5879
Folic acid	Sigma-Aldrich	F8758
LB-Miller	BD Difco	244620
Magnesium chloride	Sigma-Aldrich	M8266
Yeast extract	Oxoid	LP0021
Special peptone	Oxoid	LP0072
Potassium hyroxide	Fisher Chemical	P250
Potassium phosphate monobasic	Sigma-Aldrich	P0662
Sodium phosphate dibasic heptahydrate	Fisher Chemical	S373
Vitamin B12	Sigma-Aldrich	V2876
Strains
Dictyostelium discoideum	Siryaporn lab	Strain AX318
Escherichia coli	Siryaporn lab	Strain B/r17
Pseudomonas aeruginosa	Siryaporn lab	PA14	PA14 strain19
Pseudomonas aeruginosa ΔlasR	Siryaporn lab	AFS20.1	PA14-derived strain20
Supplies
0.22 µm filter	Millipore	SCGPT01RE	For filter sterilization
Conical tube, 15 mL	Corning	352097
Glass storage bottles	Pyrex	13951L	250 mL, 500 mL, 1000 mL
Petri dish, 6 cm diameter	Corning	351007	60 x 15 mm polystyrene plates
Petri dish, 10 cm diameter	Fisher	FB0875712	100 x 15 mm polystyrene plates
Plastic containers with lid	Ziploc	2.57E+09	Square 3-cup containers
Glass plates	Bio-Rad	1653308	For preparing agar pads. Other glass plates may be used with similar dimensions.
Wooden sticks	Fisher	23-400-102
Equipment
Eclipse Ti-E microscope	Nikon	MEA53100	Microscope setup
10X Plan Fluor Ph1 objective 0.3 NA	Nikon	MRH20101	Microscope setup
Fluorescence excitation source	Lumencor	Sola light engine	Microscope setup
Fluorescence filter set	Semrock	LED-DA/FI/TX-3X3M-A-000	Microscope setup
Orca Flash 4.0 V2 Camera	Hamamatsu	77054098	Microscope setup
ImageJ	NIH	v. 1.49	Software for image analysis
MATLAB	Mathworks	R2013	Software for image analysis
Orbital shaker incubator	VWR	89032-092	For growth of bacteria at 37 °C
Platform shaker	Fisher	13-687-700	For growth of amoebae at 22 °C
Spectrophotometer	Biochrom	Ultrospec 10
Undercounter refrigerated incubator	Fisher	97990E	For growth of amoebae at 22 °C
Isotemp waterbath	Fisher	15-462-21Q	For cooling media to 55 °C