KP and AS wrote and revised the manuscript. KP performed the experiments and the analysis. This work was supported by the National Institutes of Health (NIH) Career Transition Award (K22AI112816) to AS.

All experimental procedures were carried out at the University of California, Irvine.
1. Buffers and Solutions
<ol>
	<li>Prepare Lysogeny Broth (LB) in a 1 L glass bottle by adding 25 g of LB-Miller mix in 1 L of double-distilled water (ddH2O). For Petri dishes, add an additional 20 g of agar. Autoclave to sterilize. Pour 25 mL of molten agar medium into 10 cm-diameter Petri dishes and allow to solidify at room temperature. Store liquid media at room temperature and agar plates at 4 &#176;C.</li>
	<li>Prepare Glucose Yeast Peptone (GYP) plates in a 1 L glass bottle by mixing 1 g of D-glucose, 2 g of peptone, 0.25 g of yeast extract, 4.2 g of KH2PO4, 5.1 g of Na2HPO47H2O, and 25 g of agar in 1 L of ddH2O. Autoclave to sterilize. Pour 25 mL of molten agar into 10 cm-diameter Petri dishes and allow to solidify at room temperature. Store at 4 &#176;C.</li>
	<li>Prepare Peptone S (PS) medium in a 1 L glass bottle by mixing 10 g of peptone, 7 g of yeast extract, 15 g of D-glucose, 0.12 g of Na2HPO47H2O, 1.4 g of KH2PO4, 40 &#181;g of vitamin B12, and 80 &#181;g of Folic acid in 800 mL of ddH2O. Calibrate an electronic pH meter using pH 4 and pH 7 standards if the pH meter has not been used within a day.
	<ol>
		<li>Measure the pH of the PS medium using the pH meter. Add either 5 M KOH or 5 M H3PO4 to adjust the pH to 6.5. Adjust the final volume to 1 L using ddH2O. Filter sterilize using 0.22 &#181;m vacuum filter and store at 4 &#176;C. 
		NOTE: Proceed cautiously with the pH adjustments by wearing protective equipment including gloves, protective clothing, eye protection, and face protection.</li>
	</ol>
	</li>
	<li>Prepare 10x Development Buffer Prime (DB&#39;) buffer in a 1 L glass bottle by adding 3.4 g of KH2PO4, 13.4 g of Na2HPO4 7H2O, and ddH2O to a total volume of 800 mL. Calibrate an electronic pH meter using pH 4 and pH 7 standards if the pH meter has not been used within a day.
	<ol>
		<li>Measure the pH of the buffer using the electronic pH meter. Adjust the pH to 6.5 by gently adding either 5 M KOH or 5 M H3PO4 as needed. Adjust the final volume to 1 L using ddH2O and sterilize by autoclaving. Store at room temperature. 
		NOTE: Proceed cautiously with the pH adjustments by wearing protective equipment including gloves, protective clothing, eye protection, and face protection.</li>
	</ol>
	</li>
	<li>Make 1x Development buffer (DB) by mixing 100 mL of 10x DB&#39; buffer with 1 mL of sterilized 2 M MgCl2, and 1 mL of sterilized 1 M CaCl2. Adjust the final volume to 1 L in a 1 L glass bottle using an autoclaved sterilized ddH2O. Store at room temperature.</li>
	<li>Prepare PS:DB medium in a 1 L glass bottle by mixing 100 mL of sterilized PS medium, 100 mL of sterilized 10x DB&#39; buffer, and sterilized ddH2O to a total volume of 1 L. Supplement with 1 mL of sterilized 2 M MgCl2 and 1 mL of sterilized 1 M CaCl2. Store at 4 &#176;C.</li>
	<li>Prepare the calcein-AM stock solution by adding 50 &#181;L of DMSO to 50 &#181;g of calcein-AM in the plastic container supplied by the manufacturer. Store at -20 &#176;C.</li>
</ol>
2. Growth and Maintenance of Amoebae
<ol>
	<li>Grow E. coli strain B/r17 as a food source for amoebae during the initial growth period. Streak E. coli B/r from a frozen stock stored at -80 &#176;C onto an LB agar plate and incubate at 37 &#176;C overnight to obtain single colonies.</li>
	<li>Inoculate a single colony of E. coli B/r into 2 mL of LB medium and incubate in a roller drum or a shaker at 37 &#176;C overnight.</li>
	<li>Bring the overnight E. coli culture to the room temperature. Using a wooden stick, inoculate amoebae from a frozen stock kept at -80 &#176;C into 750 &#181;L of the overnight culture. 
	NOTE: Use D. discoideum strain AX3, which is capable of axenic growth18. This step does not require axenic growth, but subsequent steps will have his requirement.</li>
	<li>Immediately add 700 &#181;L of the amoebae-E. coli mixture onto a Glucose Yeast Peptone (GYP) plate. Spread the culture evenly on the surface of the plate by tilting it back-and-forth and side-to-side as needed. Avoid the use of a spreader.</li>
	<li>Place the GYP plate with agar facing up in a box that maintains high humidity. Use two disposable plastic containers (183 cm x 183 cm x 91 cm) stacked on the top of each other. Fill the bottom container with approximately 25 mL of water and place the top container with several 0.5 cm-diameter holes drilled through the flooring of the container to allow moisture to move from the water reservoir to the top container.</li>
	<li>Incubate the GYP plate and box at 22 &#176;C for 4 - 5&#160;days until amoeba spores are observed growing near the surface of the plate (Figure 2). Use a refrigerated incubator to incubate plates rather than incubation at room temperature. 
	&#8203;NOTE: After spores have grown, they remain viable on plates for several weeks when stored at 4 &#176;C. Store the plates with agar side facing up. After 3 - 4 days of incubation, zones of clearing within the bacterial lawn can be observed, which indicate the onset of amoeba sporulation.
	<ol>
		<li>Observe small amoebae spores above the plate surface after 4 - 5 days of incubation (Figure 2). 
		NOTE: Do not grow the amoebae for longer than a week, as the&#160;quality of the spores decreases beyond this time period.</li>
	</ol>
	</li>
	<li>Collect amoebae spores to prepare for the axenic growth of the amoebae by sweeping a sterile 1 mL pipette tip parallel to the surface of the plate. Collect spores from half of a 10 cm Petri dish. 
	NOTE: Avoid touching the tip to the surface of the agar plate as this motion will collect large numbers of E. coli.</li>
	<li>Resuspend the amoebae on the tip of the pipette by immersing the tip into 1 mL of PS medium and gently pipetting up and down.</li>
	<li>Transfer the inoculated mixture into a 250-mL flask containing 25 mL of the PS medium supplemented with the antibiotic-antimycotic solution at 0.25x concentration. 
	NOTE: Store the antibiotic-antimycotic solution as 250 &#181;L aliquots at -20 &#176;C. Avoid repeated cycles of thawing and freezing of this solution as this may decrease its efficacy.</li>
	<li>Grow amoebae axenically at 22 &#176;C in a rotating shaker at 100 rpm. For the virulence assay in step 5, grow cells to an optical density measured at 600 nm (OD600) of 0.2 to 0.5. 
	NOTE: This step requires 3 - 4 days of growth from the time of inoculation (step 2.7). If cells have grown to a higher density, dilute the culture back in PS medium to an OD600 of 0.05 or lower and grow back to an OD600 of 0.2 to 0.5.</li>
</ol>
3. The Growth of P. aeruginosa
<ol>
	<li>Pick a single colony of P. aeruginosa from an LB plate, inoculate it into a 2 mL PS:DB culture, and grow overnight at 37 &#176;C to saturation. 
	NOTE: Do not add the antibiotic-antimycotic solution to the PS:DB media.</li>
	<li>Dilute the overnight culture 1:100 or 1:1,000 into a 6 cm-diameter Petri dish using PS:DB. If the virulence of planktonic cells is solely being assayed, grow the cultures using conventional culture tubes instead. Shake the culture on a benchtop rotator set at 100 rpm at 37 &#176;C.</li>
	<li>Harvest cultures at specific time points to ensure reproducibility. At 30 min to 1 h prior to assessing virulence, prepare an agar pad as described in section 4. 
	NOTE: Virulence in P. aeruginosa is induced by approximately 8 h of growth on surfaces.</li>
	<li>To isolate the surface-attached cells, remove the liquid medium from the Petri dish, wash immediately with 1 mL of DB buffer to remove planktonic cells, and proceed immediately to step 5.1. 
	CAUTION: Do not wash longer than 30 s as this will perturb and detach surface-attached cells and may affect the virulence measurement.</li>
	<li>To isolate planktonic cells, transfer 10 &#181;L of the culture from the Petri dish to a clean Petri dish and proceed immediately to step 5.1.</li>
</ol>
4. Microscopy - Preparation of the Agar Pad
<ol>
	<li>Prepare agar pads about 30 min to 1 h before amoebae are ready to be mixed with P. aeruginosa.</li>
	<li>Microwave 50 mL of 1% (w/v) agar in DB buffer in a 250 mL flask. Mix every 20 s until clumps of agar disappear.</li>
	<li>Cool the molten agar by placing it in preheated 55 &#176;C water bath for 15 min.</li>
	<li>Add 15 &#181;L 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.</li>
	<li>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.</li>
	<li>Keep the gel hydrated in a humid box until it is ready for use.</li>
</ol>
5. Microscopy - Preparation of the Bacteria-amoeba Sample for Imaging
<ol>
	<li>To assay the virulence of surface-attached bacterial cells, add 10 &#181;L of amoeba cells from step 2.10 to surface-attached bacteria from step 3.4. 
	NOTE: This step describes the mixing of amoebae with bacteria.</li>
	<li>To assay the virulence of planktonic cells, mix 10 &#181;L of amoeba cells from step 2.10 with 10 &#181;L of planktonic bacteria from step 3.5. 
	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.</li>
	<li>Immediately place the 1.5 cm x 1.5 cm calcein-AM agar pad from step 4.5 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, immobilized and that both cell types are confined to the same plane.</li>
	<li>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.</li>
	<li>Cover with the Petri dish with a lid and incubate the immobilized amoeba-bacteria mixture at room temperature for an additional 40 min. Proceed to step 6.1. 
	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.</li>
</ol>
6. Microscopy - Image Acquisition
<ol>
	<li>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&#160;(&#8805;0.3 numerical aperture) objective to image the amoebae. 
	NOTE: The calcein-AM in the agar will cause dying amoebae to fluorescence in the green fluorescence channel. Healthy amoebae are otherwise not fluorescent.</li>
	<li>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.</li>
	<li>Acquire images for at least 100 amoeba cells in order to obtain good statistics for calculating the host killing index.</li>
</ol>
7. Image Analysis and Host Killing Index
<ol>
	<li>Perform image analysis using ImageJ.</li>
	<li>Open the phase contrast image file in ImageJ by clicking on File | Open and selecting the image. Adjust the threshold by clicking on Image | Analysis | Threshold. Adjust the lower and upper threshold to select only the intensity peak (Figure 3A).</li>
	<li>Convert the image to binary by clicking Process | Binary | Make Binary. Fill holes by selecting Process | Binary | Fill Holes. 
	NOTE: Using the image in Figure 1 as the source, steps 7.1 - 7.2 will produce an image in which the amoebae and surface-attached bacteria appear as dark regions (Figure 3B).</li>
	<li>Ensure that the Area and Mean gray value boxes are checked in Analyze | Set Measurements. Measure the approximate size of the amoebae by selecting the oval tool on the main toolbar. Click Analyze | Measure and note the area of representative amoebae.</li>
	<li>Create masks for the amoebae by clicking Analyze | Analyze Particles. In the size box, enter the area that will include the amoebae but exclude the bacteria. Select Masks in the dropdown menu bar for the Show option. Ensure that the Add to Manager box is checked.</li>
	<li>Click OK and an image showing only the amoebae and an ROI Manager dialog will appear (Figure 3C).</li>
	<li>Open the fluorescence image using File | Open and selecting the appropriate file. Select all the regions in the ROI Manager by holding down the Shift Key and Clicking on each region in the list (Figure 3D).</li>
	<li>Click the Measure button in the ROI Manager. This will open a Results window displaying the mean intensity value of each amoeba.</li>
	<li>Copy the fluorescence values for each amoeba into a spreadsheet program and calculate the host killing index by taking the average of all fluorescence values.</li>
</ol>

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The authors have nothing to disclose.

This protocol describes a rapid and quantitative method to assay virulence in P. aeruginosa. This protocol may be tested with other bacteria. However, it is important to keep in mind that the growth medium should be compatible with the amoeba growth conditions. In particular, we have optimized the protocol using PS:DB as the bacterial growth medium. If other media are used, it may be necessary to perform a growth media-only control in which there are no bacterial cells present to verify that the medium is compat...

Bacterial infection is one of the leading causes of mortality in human and animals1,2. The ability to measure virulence of bacteria in cultures or biofilms is important in healthcare and research settings. Here, we describe a versatile, rapid, and relatively simple method to quantify bacterial virulence. The eukaryotic organism Dictyostelium discoideum (amoeba) is used as the model host organism. D. discoideum has been used as a host to identify virulence factors in Pseudomonas aeruginosa (P. aeruginosa)3,4,5 and other bacteria6,7,8 and is susceptible to largely the same virulence factors that kill mammalian cells including type III secretion9,10. Previous virulence assays using D. discoideum have involved prolonged exposure of bacteria with D. discoideum cells over the course of hours3,4,5. The protocol, here, presents a rapid method of determining virulence using this amoeba. This protocol (Figure 1) describes how to: (1) grow the amoebae axenically (in the absence of bacteria), (2) grow bacteria for the assay, (3) prepare bacteria and host cells for microscopy, (4) perform epifluorescence microscopy, and (5) analyze amoeba fluorescence.
Amoebae are initially streaked out from frozen stocks and grown on a lawn of Escherichia coli (E. coli), where the amoebae produce spores. These spores are picked and inoculated into an enriched medium for axenic growth. The amoebae are maintained through axenic growth in nutrient-rich conditions until they are ready to be mixed with bacteria for the assessment of bacterial virulence. The survival or the death of the amoebae is quantified by measuring the fluorescence of calcein-acetoxymethyl (calcein-AM), which is cleaved by intracellular esterases and, thereby, activated for fluorescence11,12. Live amoebae exhibit little or no fluorescence whereas stressed and dying cells fluoresce intensely. This result is due to a little or no incorporation of calcein-AM into healthy amoebae and incorporation and cleavage of the substrate in stressed amoebae13. This behavior is notably distinct from calcein-AM fluorescence in mammalian cells11,14,15,16.
Bacteria that will be assessed for virulence are grown separately. Here, we describe how to measure the virulence of the opportunistic pathogen P. aeruginosa and detail how to quantify the virulence of planktonic (swimming) and surface-attached sub-populations. This protocol may be adapted to test the virulence of other bacteria. In the Representative Results section, we show that virulence is activated in surface-attached cells and is low in planktonic cells, which was reported previously13. Virulence-activated surface-attached P. aeruginosa kills amoebae while non-virulent planktonic cells are consumed by the amoebae. If the virulence of planktonic bacteria is solely being assayed, bacteria can be cultured in ordinary culture tubes rather than using Petri dishes as described in the protocol.
The growth of amoebae and P. aeruginosa cultures must be coordinated such that P. aeruginosa cultures reach the intended growth phase while the amoebae are growing at steady state in nutrient-rich conditions. This condition typically requires amoebae cultures to be diluted at least 1 day prior to when they are mixed with bacteria. Amoebae and bacteria are immobilized using agar pads, are co-incubated for 1 h, and imaged using a low resolution (10X, numerical aperture 0.3) objective, green fluorescence protein (GFP) filters, and an imaging camera. Analysis can be performed using freely-available ImageJ software or customized image analysis software. Our analysis was performed using our own software written using a scientific analysis package13. The software should create a mask using the phase contrast image and extract fluorescence values from the masked areas in the fluorescence image. Fluorescence values are averaged over at least 100 cells, resulting in a numerical host killing index.

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			<td height="21" style="height:21px;width:220px;">Reagents</td>
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			<td height="21" style="height:21px;width:220px;">Bacto agar, dehydrated</td>
			<td style="width:108px;">BD Difco</td>
			<td style="width:91px;">214010</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Antibiotic-Antimycotic (100X)</td>
			<td style="width:108px;">Life Technologies</td>
			<td style="width:91px;">15240062</td>
			<td style="width:297px;">Aliquot &#60; 1 mL and store at -20 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Calcein-acetoxymethyl ester (calcein-AM)</td>
			<td style="width:108px;">Life Technologies</td>
			<td style="width:91px;">C34852</td>
			<td style="width:297px;">Calcein Acetoxymethyl (AM)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Calcium chloride, anhydrous</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">C1016</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">D-Glucose</td>
			<td style="width:108px;">Fisher Chemical</td>
			<td style="width:91px;">D16500</td>
			<td style="width:297px;">Dextrose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Dimethyl sulfoxide</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">D5879</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Folic acid</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">F8758</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">LB-Miller</td>
			<td style="width:108px;">BD Difco</td>
			<td style="width:91px;">244620</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Magnesium chloride</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">M8266</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Yeast extract</td>
			<td style="width:108px;">Oxoid</td>
			<td style="width:91px;">LP0021</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Special peptone</td>
			<td style="width:108px;">Oxoid</td>
			<td style="width:91px;">LP0072</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Potassium hyroxide</td>
			<td style="width:108px;">Fisher Chemical</td>
			<td style="width:91px;">P250</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Potassium phosphate monobasic&#160;</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">P0662</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Sodium phosphate dibasic heptahydrate</td>
			<td style="width:108px;">Fisher Chemical</td>
			<td style="width:91px;">S373</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Vitamin B12</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">V2876</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Strains</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:220px;">Dictyostelium discoideum</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">Strain AX318</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:220px;">Escherichia coli</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">Strain B/r17</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:220px;">Pseudomonas aeruginosa</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">PA14</td>
			<td style="width:297px;">PA14 strain19</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Pseudomonas aeruginosa &#916;lasR</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">AFS20.1</td>
			<td style="width:297px;">PA14-derived strain20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Supplies</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">0.22 &#181;m filter&#160;</td>
			<td style="width:108px;">Millipore</td>
			<td style="width:91px;">SCGPT01RE</td>
			<td style="width:297px;">For filter sterilization</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:220px;">Conical tube, 15 mL</td>
			<td style="width:108px;">Corning</td>
			<td style="width:91px;">352097</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Glass storage bottles</td>
			<td style="width:108px;">Pyrex</td>
			<td style="width:91px;">13951L</td>
			<td style="width:297px;">250 mL, 500 mL, 1000 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Petri dish, 6 cm diameter</td>
			<td style="width:108px;">Corning</td>
			<td style="width:91px;">351007</td>
			<td style="width:297px;">60 x 15 mm polystyrene plates</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:220px;">Petri dish, 10 cm diameter</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">FB0875712</td>
			<td style="width:297px;">100 x 15 mm polystyrene plates</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:220px;">Plastic containers with lid</td>
			<td style="width:108px;">Ziploc</td>
			<td style="width:91px;">2.57E+09</td>
			<td style="width:297px;">Square 3-cup containers</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:220px;">Glass plates</td>
			<td style="width:108px;">Bio-Rad</td>
			<td style="width:91px;">1653308</td>
			<td style="width:297px;">For preparing agar pads. Other glass plates may be used with similar dimensions.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:220px;">Wooden sticks</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">23-400-102&#160;</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Equipment</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Eclipse Ti-E microscope&#160;</td>
			<td style="width:108px;">Nikon</td>
			<td style="width:91px;">MEA53100</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">10X Plan Fluor Ph1 objective&#160; 0.3 NA</td>
			<td style="width:108px;">Nikon</td>
			<td style="width:91px;">MRH20101</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Fluorescence excitation source</td>
			<td style="width:108px;">Lumencor</td>
			<td style="width:91px;">Sola light engine</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:220px;">Fluorescence filter set</td>
			<td style="width:108px;">Semrock</td>
			<td style="width:91px;">LED-DA/FI/TX-3X3M-A-000&#160;</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Orca Flash 4.0 V2 Camera</td>
			<td style="width:108px;">Hamamatsu</td>
			<td style="width:91px;">77054098</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">ImageJ</td>
			<td style="width:108px;">NIH</td>
			<td style="width:91px;">v. 1.49</td>
			<td style="width:297px;">Software for image analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">MATLAB</td>
			<td style="width:108px;">Mathworks</td>
			<td style="width:91px;">R2013</td>
			<td style="width:297px;">Software for image analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Orbital shaker incubator</td>
			<td style="width:108px;">VWR</td>
			<td style="width:91px;">89032-092</td>
			<td style="width:297px;">For growth of bacteria at 37 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Platform shaker</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">13-687-700</td>
			<td style="width:297px;">For growth of amoebae at 22 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Spectrophotometer</td>
			<td style="width:108px;">Biochrom</td>
			<td style="width:91px;">Ultrospec 10</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Undercounter refrigerated incubator</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">97990E</td>
			<td style="width:297px;">For growth of amoebae at 22 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Isotemp waterbath&#160;</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">15-462-21Q</td>
			<td style="width:297px;">For cooling media to 55 &#176;C</td>
		</tr>
	</tbody>
</table>

a rapid image-based bacterial virulence assay using amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

We grew wild-type P. aeruginosa strain PA1419 or a &#916;lasR strain20 in the same PA14 background in 6 cm-diameter Petri dishes and assayed the virulence of planktonic and surface-attached cells. Cultures were inoculated from single-colonies into PS:DB cultures, grown overnight in culture tubes in a roller drum at 37 &#176;C to saturation, diluted 1:100 into PS:DB, grown for 8 h in 6 cm-diameter Petri dishes shaking at 100...

Watch this Scientific Journal Video about A Rapid Image-based Bacterial Virulence Assay Using Amoeba at JoVE.com

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

a-rapid-image-based-bacterial-virulence-assay-using-amoeba

Research

JoVE Journal

Bioengineering

8.1K Views.  University of California. Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

Video: A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis of the intrinsic photothermal response of hemoglobin molecules, the sensor enables high-sensitivity, chemical-free measurement of [Hb]. [Hb] detection capability with a limit of 0.12 g/dl over the range of 0.35 - 17.9 g/dl has been demonstrated previously. The method can be readily implemented using inexpensive consumer electronic devices such as a laser pointer and a webcam. The use of a micro-capillary tube as a blood container also enables the hemoglobin assay with a nanoliter-scale blood volume and a low operating cost. Here, detailed instructions for the PT-AS optical setup and signal processing procedures are presented. Experimental protocols and representative results for blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl) are also provided, and the measurements are compared with those from a hematology analyzer. Its simplicity in implementation and operation should enable its wide adoption in clinical laboratories and resource-limited settings.

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...