Name: a rapid image-based bacterial virulence assay using amoeba
Uploaded: 2018-06-27T15:00:00
Description: Watch this Scientific Journal Video about A Rapid Image-based Bacterial Virulence Assay Using Amoeba at JoVE.com

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...

<ol>
	<li>Rasko, D. A., Sperandio, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-virulence+strategies+to+combat+bacteria-mediated+disease.">Anti-virulence strategies to combat bacteria-mediated disease.</a> Nature Reviews Drug Discovery. 9 (2), 117-128 (2010).</li><li>Coburn, B., Grassl, G. A., Finlay, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella,+the+host+and+disease:+a+brief+review.">Salmonella, the host and disease: a brief review.</a> Immunology and Cell Biology. 85 (2), 112-118 (2007).</li><li>Alibaud, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+virulence+genes+identified+in+a+Dictyostelium+host+model.">Pseudomonas aeruginosa virulence genes identified in a Dictyostelium host model.</a> Cellular Microbiology. 10 (3), 729-740 (2008).</li><li>Pukatzki, S., Kessin, R. H., Mekalanos, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+pathogen+Pseudomonas+aeruginosa+utilizes+conserved+virulence+pathways+to+infect+the+social+amoeba+Dictyostelium+discoideum.">The human pathogen Pseudomonas aeruginosa utilizes conserved virulence pathways to infect the social amoeba Dictyostelium discoideum.</a> Proceedings of the National Academy of Sciences U S A. 99 (5), 3159-3164 (2002).</li><li>Cosson, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+Virulence+Analyzed+in+a+Dictyostelium+discoideum+Host+System.">Pseudomonas aeruginosa Virulence Analyzed in a Dictyostelium discoideum Host System.</a> Journal of Bacteriology. 184 (11), 3027-3033 (2002).</li><li>Pukatzki, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+conserved+bacterial+protein+secretion+system+in+Vibrio+cholerae+using+the+Dictyostelium+host+model+system.">Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system.</a> Proceedings of the National Academy of Sciences U S A. 103 (5), 1528-1533 (2006).</li><li>Steinert, M., Heuner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+as+host+model+for+pathogenesis.">Dictyostelium as host model for pathogenesis.</a> Cellular Microbiology. 7 (3), 307-314 (2005).</li><li>Hagele, S., Kohler, R., Merkert, H., Schleicher, M., Hacker, J., Steinert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+discoideum:+a+new+host+model+system+for+intracellular+pathogens+of+the+genus+Legionella.">Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella.</a> Cellular Microbiology. 2 (2), 165-171 (2000).</li><li>Matz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+uses+type+III+secretion+system+to+kill+biofilm-associated+amoebae.">Pseudomonas aeruginosa uses type III secretion system to kill biofilm-associated amoebae.</a> ISME Journal. 2 (8), 843-852 (2008).</li><li>Hauser, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+type+III+secretion+system+of+Pseudomonas+aeruginosa:+infection+by+injection.">The type III secretion system of Pseudomonas aeruginosa: infection by injection.</a> Nature Reviews Microbiology. 7 (9), 654-665 (2009).</li><li>Moore, P. L., MacCoubrey, I. C., Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+pH+insensitive,+two+color+fluorescence+viability+(cytotoxicity)+assay.">A rapid pH insensitive, two color fluorescence viability (cytotoxicity) assay.</a> Journal of Cell Biology. 111, 58 (1990).</li><li>Kaneshiro, E. S., Wyder, M. A., Wu, Y. -. P., Cushion, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+calcein+acetoxy+methyl+ester+and+ethidium+homodimer+or+propidium+iodide+for+viability+assessment+of+microbes.">Reliability of calcein acetoxy methyl ester and ethidium homodimer or propidium iodide for viability assessment of microbes.</a> Journal of Microbiological Methods. 17 (1), 1-16 (1993).</li><li>Siryaporn, A., Kuchma, S. L., O'Toole, G. A., Gitai, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+attachment+induces+Pseudomonas+aeruginosa+virulence.">Surface attachment induces Pseudomonas aeruginosa virulence.</a> Proceedings of the National Academy of Sciences U S A. 111 (47), 16860-16865 (2014).</li><li>Decherchi, P., Cochard, P., Gauthier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+staining+assessment+of+Schwann+cell+viability+within+whole+peripheral+nerves+using+calcein-AM+and+ethidium+homodimer.">Dual staining assessment of Schwann cell viability within whole peripheral nerves using calcein-AM and ethidium homodimer.</a> Journal of Neuroscience Methods. 71 (2), 205-213 (1997).</li><li>Braut-Boucher, F., Pichon, J., Rat, P., Adolphe, M., Aubery, M., Font, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-isotopic,+highly+sensitive,+fluorimetric,+cell-cell+adhesion+microplate+assay+using+calcein+AM-labeled+lymphocytes.">A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes.</a> Journal of Immunological Methods. 178 (1), 41-51 (1995).</li><li>Grieshaber, P., Lagr&#232;ze, W. A., Noack, C., Boehringer, D., Biermann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+of+fluorogold-prelabeled+retinal+ganglion+cells+with+calcein-AM:+A+new+method+for+assessing+cell+vitality.">Staining of fluorogold-prelabeled retinal ganglion cells with calcein-AM: A new method for assessing cell vitality.</a> Journal of Neuroscience Methods. 192 (2), 233-239 (2010).</li><li>Witkin, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherited+Differences+in+Sensitivity+to+Radiation+in+Escherichia+Coli.">Inherited Differences in Sensitivity to Radiation in Escherichia Coli.</a> Proceedings of the National Academy of Sciences U S A. 32 (3), 59-68 (1946).</li><li>Loomis, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+Dictyostelium+discoideum+to+nucleic+acid+analogues.">Sensitivity of Dictyostelium discoideum to nucleic acid analogues.</a> Experimental Cell Research. 64 (2), 484-486 (1971).</li><li>Rahme, L. G., Stevens, E. J., Wolfort, S. F., Shao, J., Tompkins, R. G., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+virulence+factors+for+bacterial+pathogenicity+in+plants+and+animals.">Common virulence factors for bacterial pathogenicity in plants and animals.</a> Science. 268 (5219), 1899-1902 (1995).</li><li>O'Loughlin, C. T., Miller, L. C., Siryaporn, A., Drescher, K., Semmelhack, M. F., Bassler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quorum-sensing+inhibitor+blocks+Pseudomonas+aeruginosa+virulence+and+biofilm+formation.">A quorum-sensing inhibitor blocks Pseudomonas aeruginosa virulence and biofilm formation.</a> Proceedings of the National Academy of Sciences U S A. 110 (44), 17981-17986 (2013).</li></ol>

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.

<table border="0" cellpadding="0" cellspacing="0" style="width:716px;" width="715">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Reagents</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;">Bacto agar, dehydrated</td>
			<td style="width:108px;">BD Difco</td>
			<td style="width:91px;">214010</td>
			<td style="width:297px;"></td>
		</tr>
		<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 ...

Research

JoVE Journal

Bioengineering

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

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 ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Bacterial Detection & 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 ...

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 ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

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 ...

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 ...

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 ...

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

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. ...

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 ...

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

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 ...