This study was supported by the Cancer Prevention and Research Institute of Texas (CPRIT) Award RR150044, Welch Foundation Research Grant C-1930, and by the National Institutes of Health K22 AI110552 awarded to NVK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Caution: P. aeruginosa and E. faecalis are Biosafety Level 2 pathogens, and proper safety precautions must be taken to prevent accidental infection and to minimize contamination of surfaces. All media and materials that come into contact with pathogens must be sterilized and/or discarded. Further guidelines are available from the CDC publication Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th edition.
1. Preparation and Maintenance of P. aeruginosa
<ol>
	<li>Streak P. aeruginosa from a frozen stock onto an LB (Lysogeny Broth) agar plate. Incubate 16 to 24 h at 37 &#176;C 
	Note: Perform P. aeruginosa experiments inside of a BSL-2 biological safety cabinet to ensure sterility. Use appropriate safety precautions to minimize the chance of pathogenic infection or contamination.</li>
	<li>Transfer this plate to 4 &#176;C. This plate may be retained at 4 &#176;C and used to inoculate cultures for up to one week. After one week, decontaminate and discard the plate and make a new LB streak plate. 
	Note: P. aeruginosa virulence decreases after prolonged storage.</li>
	<li>Two days prior to setting up assay plates, inoculate 3 - 5 mL of sterile LB broth with a single colony of P. aeruginosa from the plate made in 1.1. Incubate at 37 &#176;C for 12 to 16 h. 
	Note: Do not incubate longer than 16 h. In rich liquid cultures, P. aeruginosa PA14 begins to lyse.</li>
	<li>Prepare sterile 10 cm plates with Slow Kill media (3 g NaCl, 3.5 g peptone, 1 mM CaCl2, 1 mM MgSO4, 25 mL of phosphate buffer (amount per liter: 132 mL of K2HPO4 (1M) and 868 mL of KH2PO4 (1M)) and 18 g agar/L). 
	Note: Prepare plates ahead of time. They can be stored for up to 3 weeks in an airtight container at 4 &#176;C.</li>
	<li>Seed each 10 cm Slow Kill plate (SK plate) with 350 &#956;L of P. aeruginosa from fresh overnight LB culture. Using a sterile bacterial spreader, spread bacteria evenly across surface of media and allow to dry in a BSL-2 biological safety cabinet.</li>
	<li>Incubate plates at 37 &#176;C for 24 h.</li>
</ol>
2. Preparation of RNAi Bacteria
<ol>
	<li>Streak out or pin-transfer desired strains of RNAi-containing bacteria onto LB agar plates containing appropriate antibiotics (carbenicillin at 100 &#181;g/mL and tetracycline at 15 &#181;g/mL for Ahringer and Vidal libraries) from a frozen stock. 
	Note: When working with nonpathogenic bacteria, use either a BSL-2 A/B biological safety cabinet or a BSL-1 laminar flow hood to ensure sterility of the cultures and to minimize the chance of cross-contamination of the library.</li>
	<li>Incubate plates for 24 h at 37 &#176;C.</li>
	<li>Store the plates at 4 &#176;C for up to two weeks.
	<ol>
		<li>After two weeks, discard the plates and replace them with freshly made plates.</li>
	</ol>
	</li>
	<li>Test multiple RNAi strains in parallel by individually inoculating a single colony from each clone into 4 mL of carbenicillin-supplemented LB in a single well of a 24-well deep-well plate or into a sterile test tube. 
	Note: Tetracycline has been reported to reduce the efficiency of RNAi. Do not use it in this media.</li>
	<li>Place 24-well deep well plates into a shaking incubator optimized for multiwell plates and incubate at 37 &#176;C for 16 h while shaking at 950 rpm. 
	Note: It is hard to obtain uniform bacterial growth in multiwell plates using a conventional shaking incubator. The shaking incubator needs to be able to shake at a minimum of 750 rpm in order for the cultures to grow properly. In the absence of a specialized shaker, it is possible to grow each culture in an individual tube. Cultures can be transferred into the 24-well plate for centrifugation afterward, if desired.</li>
	<li>Collect bacteria by centrifuging for 5 minutes at 2,000 x g.</li>
	<li>Decant supernatant by inverting the plate and shaking vigorously. Resuspend RNAi bacteria in 100 &#181;L of S Basal (5.85 g NaCl, 1 g K2HPO4, 6 g KH2PO4 dissolved into 1 L of ultrapure water and sterilized by autoclave).</li>
	<li>Pipette resuspended bacteria into an appropriate number of wells of a multiwell NGM plate (Nematode Growth Media, 3 g NaCl, 2.5 g peptone, 1 mM CaCl2, 1 mM MgSO4, 25 mL of phosphate buffer (amount per liter: 132 mL of K2HPO4 (1M) and 868 mL of KH2PO4 (1M)), and 18 g agar per liter of water) supplemented with IPTG (1 mM final concentration) and carbenicillin (100 &#956;g/mL final concentration).
	<ol>
		<li>Use 3.5 mL of NGM media per well of 6-well plate, 1 mL per well of a 24-well plate, or 150 &#181;L per well of 96-well plate.</li>
		<li>Use 100 &#956;L/well of bacteria for 6-well plate; 50 &#956;L/well for 24-well; or 20 &#956;L/well for 96-well plate. Allow to dry. 
		Note: Drying is normally performed in a sterile flow hood to promote rapid, uniform drying. Uniform drying is very important. Avoid over-drying, as cracks in the agar promote burrowing of worms. This leads to worm losses and potential clogging of liquid-handling systems.</li>
	</ol>
	</li>
	<li>Use the prepared plates immediately or store them at 4 &#176;C for up to two weeks for later use. 
	Note: To prevent plates from drying out, they can be sealed with plastic paraffin film or placed in a tightly-sealed container with damp paper towels.</li>
</ol>
3. Maintenance and Preparation of C. elegans
Note: Before initiating experiments, generate a synchronized population of gravid, adult hermaphroditic worms as follows.
<ol>
	<li>Wash gravid adults (i.e., with multiple embryos visible within the gonad) from 4 - 6 10-cm NGM plates into a 15 mL conical tube by adding 4 mL of S Basal to plate, swirling gently, and then pouring into a 15 mL conical tube.</li>
	<li>Pellet worms via centrifugation at 1,000 x g for 30 seconds.</li>
	<li>Aspirate supernatant, taking care not to disrupt the worm pellet.</li>
	<li>Add 3.5 mL of worm bleach solution (final concentration 0.5M NaOH, 1% sodium hypochlorite, in sterile water) to the worm pellet, and mix by inverting for 1 minute.</li>
	<li>Briefly vortex and centrifuge at 1,000 x g for 30 seconds.</li>
	<li>Aspirate or decant the supernatant, taking care not to disturb the worm pellet.</li>
	<li>Add 3.5 mL of fresh worm bleach solution to the worm pellet.</li>
	<li>Vigorously mix worms in bleach solution. Periodically (approximately every ten seconds) visually inspect worms under a dissecting microscope. Watch for worm cuticles to break, releasing eggs. Once most of the adult worms have been dissolved (~95%), dilute bleach to 15 mL with S Basal to stop digestion. 
	Note: Egg shells are more resistant to bleach than adult tissues, but they can be dissolved during prolonged exposure. To avoid egg dissolution, do not expose eggs to worm bleach solution for longer than 7 minutes. If egg shells are excessively damaged, the embryos will die.</li>
	<li>Pellet embryos at 1,000 x g for 30 seconds and aspirate or decant the supernatant without removing the pellet of embryos.</li>
	<li>Resuspend worms in S basal to a final volume of 15 mL to dilute remaining bleach solution.</li>
	<li>Repeat the washing steps (3.9&#160;- 3.10) three more times for a total of 4 consecutive washes.</li>
	<li>After the fourth wash, aspirate supernatant and add up to 5 or 6 mL of sterile S Basal into the 15 mL conical tube. The goal is to resuspend embryos to a concentration of ~50 worms per &#181;L. 
	Note: The amount of S Basal depends upon the size of the egg pellet; the larger the pellet, the more S Basal is necessary.</li>
	<li>Incubate the conical tube overnight on a table-top rotator at room temperature or 48 h at 15 &#176;C. Larvae will hatch, but larval development will arrest at the L1 stage (L1 diapause) due to nutrient deprivation.</li>
	<li>Examine the embryos under a dissecting microscope to verify that most of them have hatched and arrested at the L1 stage of development.</li>
	<li>Determine the worm count by taking 20 &#181;L of worm prep and diluting it with 80 &#181;L of S Basal. Pipette 10 &#181;L of diluted worm solution directly onto a blank NGM plate. Repeat 2 more times.
	<ol>
		<li>Count the larvae in each spot and determine the average number. Divide this average by 2 to obtain an approximate count of worms per &#181;L in the worm prep. Worm preps can then be used for propagation plates or for experiments. 
		Note: After 4 - 5 days, starved larvae will begin to die; discard the prep at this point.</li>
	</ol>
	</li>
	<li>For propagating the strain, pipette an appropriate number of L1 worms (5,000 - 6,000) onto 3 - 4 10-cm NGM plates spotted with concentrated OP50 E. coli &#8211; &#8220;superfood&#8221; (E. coli OP50 spotted at 25X concentration (i.e., 1 L of overnight OP50 culture grown in LB yields 40 mL of 25X OP50 superfood); 2 mL of this concentrated bacterial suspension are spotted onto each 10 cm NGM plate).</li>
	<li>To prepare plates for experiments, do one of the following (for &#8220;Basic Protocol&#8221; use 3.17.1 setup, for the &#8220;RNAi screen set up&#8221; use steps 3.17.2 - 3.17.4 as appropriate):
	<ol>
		<li>Pipette ~ 5,000 worms/10 cm plate seeded with RNAi or OP50 superfood.</li>
		<li>Pipette ~ 500 worms/well in a 6-well plate seeded with RNAi.</li>
		<li>Pipette ~300 worms/well in a 24-well plate seeded with RNAi.</li>
		<li>Pipette ~100 worms/well in a 96-well plate seeded with RNAi. 
		Note: For instructions on how RNAi was seeded please refer to steps 2.7 - 2.9 in Preparation of RNAi Bacteria.</li>
	</ol>
	</li>
	<li>If a fertile strain of worms is being used, incubate worms at 25 &#176;C for ~44 - 48 h. Use these worms as quickly as possible after the population has reached the appropriate age. This minimizes the impact of egg hatching within the worms during the assay.</li>
	<li>If the strain being used is temperature-sterile (e.g., fer-15(b26); fem-1(hc17) or glp-4(bn2)), incubate worms at 15 &#176;C for ~16 h and then transfer to 25 &#176;C for 44 h to complete development and prevent embryogenesis.</li>
</ol>
4. Liquid Killing Assay Setup (Basic Protocol)
Note: This is a protocol for one bacterial strain and one source of worms.
<ol>
	<li>Using a cell scraper, remove the P. aeruginosa from an SK plate and resuspend in ~ 5 mL of S Basal. Scale up if needed. Measure the optical density of the bacterial suspension using a spectrophotometer (OD600)</li>
	<li>Prepare 24 mL of diluted stock of P. aeruginosa in S Basal at OD600 &#8776; 0.09 (3X final concentration). See 4.6.2 for final content per well, and scale volume of bacterial dilution accordingly.</li>
	<li>Add 21 mL of liquid Slow Kill media (3 g of NaCl, 3.5 g of peptone/L supplemented with CaCl2 and MgSO4 to a final concentration of 1 mM). Using a multichannel pipette, transfer 45 &#181;L of bacteria and media to each well of a 384-well plate.</li>
	<li>Wash worms from their source (i.e., Step 3.17.1) into a 50 mL conical tube and allow worms to settle under gravitational force. Aspirate supernatant to 5 mL. Resuspend in a total of 50 mL S basal.</li>
	<li>Repeat step 4.4 twice.</li>
	<li>Using a worm sorter, sort approximately 22 worms into each well of the 384-well plate. 
	Note: The setup drops each worm in ~1.1 &#181;L of liquid. 22 worms will amount to 25 &#181;L, bringing total assay volume to 70 &#181;L/well.
	<ol>
		<li>The final composition of each well consists of 70 &#181;L. 45 &#181;L of this volume is added as bacterial medium (0.03 OD600 bacteria in 24 &#181;L S Basal, and 21 &#181;L SK supplemented with CaCl2 and MgSO4). The other 25 &#181;L is added with the 22 worms.</li>
		<li>If the sorter used here (see Table of Materials) is unavailable, worms can be diluted to a final concentration of 1 worm/&#181;L in S Basal and pipetted 25 &#181;L/well using a multichannel pipette. However, results may exhibit increased variability. Alternatively, it is possible to use a peristaltic liquid dispenser, as described by Leung and colleagues19.</li>
	</ol>
	</li>
	<li>After worms have been sorted into the 384-well plate, seal the plate with a gas-permeable film and incubate plates at 25 &#176;C for 24 - 48 h.</li>
	<li>At the desired time, use a microplate washer to wash the 384-well plate with S Basal a total of 5 times.
	<ol>
		<li>After the second wash, aspirate most of the media, leaving ~20 &#181;L. Vigorously shake plates using a microplate vortexer for at least 30 seconds to loosen any debris from the bottom of the wells).</li>
	</ol>
	</li>
	<li>After the final wash, aspirate supernatant down to 20 &#181;L. Add 50 &#181;L of 0.98 &#181;M nucleic acid stain (see Table of Materials)/well of the 384-well plate, for a final concentration of 0.7 &#181;M.</li>
	<li>Incubate at room temperature for 12 - 16 h. This dye will only stain dead worms. After the desired incubation period, wash plates using the microplate washer to remove any excess stain (a minimum of 3 washes).</li>
	<li>For data acquisition, use a spectrophotometer or an automated microscope to image both transmitted light and fluorescence (531 nm excitation and 593 emission).
	<ol>
		<li>Use a low magnification objective to allow an image of the whole well to be captured.</li>
		<li>Alternatively, use flow vermimetry. This technique uses a large object flow cytometer as a worm fluorimeter, measuring the size and fluorescence of whole organism (i.e., C. elegans) in a fashion that is strongly similar to flow cytometry.</li>
	</ol>
	</li>
	<li>Use automated image analysis software (such as CellProfiler, a free image analysis studio based on MatLab <a href="http://cellprofiler.org/">http://cellprofiler.org/</a>) to calculate the fluorescent and total worm areas per well in an unbiased fashion. 
	Note: The quotient of these numbers represents the fraction death for each well. If one well had 7 stained worms out of 20 total, then the fraction death comprises 0.35 or 35%.
	<ol>
		<li>Prior to first use, the automated image processing pipeline must be validated by manual scoring. This is done by comparing results from the automated pipeline to scores obtained by visually inspecting images and recording fractions of dead worms for each of them. The validation process ensures that the parameters for the automated imaging processing are accurate and represent the data correctly.</li>
	</ol>
	</li>
</ol>
5. Adaptation for Screening Multiple C. elegans Strains or Knockdowns (RNAi Screen Setup)
Note: This is an RNAi screen described for a 24-well plate setup.
<ol>
	<li>Add 1 mL of S Basal per well of a 24-well plate (see step 3.17.3). Gently agitate worms by shaking the plate, then transfer worms to an empty, sterile 24 deep-well plate. Allow worms to settle gravitationally (~5 minutes).</li>
	<li>Aspirate supernatant, leaving approximately 1 mL. Add 7 mL of S Basal to each well.</li>
	<li>Repeat steps 5.1 - 5.2 a minimum of 2 more times. After final wash, aspirate supernatant, leaving approximately 400 &#181;L.</li>
	<li>Using the Resampler function of the worm sorter, sort 22 worms from the 24-well plate worm suspension into each well of a 384-well plate (each well of a 24-well plate yields 8 - 12 wells of a 384-well plate).
	<ol>
		<li>To avoid starvation, pipette bacteria into 384-well plates (either a strain that serves solely as food, such as OP50, or a pathogenic strain) prior to sorting. 
		Note: Pathogens can be added by following steps 2.3 - 2.5 or 6.4 - 6.5, and food source bacteria can be diluted and added by following the same scheme as for P. aeruginosa.</li>
	</ol>
	</li>
	<li>After worms have been sorted into the 384-well plate, add small molecules or other experiment-specific materials.</li>
</ol>
6. Adaptation for E. faecalis
Note: Only the differences from P. aeruginosa assay are described.
<ol>
	<li>Prepare a fresh source of E. faecalis by streaking bacteria from a frozen stock onto a BHI (Brain Heart Infusion) agar plate and incubating for 16 to 24 h at 37 &#176;C. 
	Note: Perform E. faecalis experiments inside of a BSL-2 biological safety cabinet to ensure sterility. Use appropriate safety precautions to minimize the chance of pathogenic infection or contamination.</li>
	<li>Plates can be stored at 4 &#176;C for up to one week. After this period, replace with a fresh plate.</li>
	<li>The night before sorting worms, inoculate 3 - 5 mL sterile BHI with a single colony of E. faecalis. Incubate 12 - 16 h at 37 &#176;C with agitation.</li>
	<li>Add bacteria to the wells as for P. aeruginosa (3x&#160;bacteria in S Basal, OD600&#8776;0.09). 
	Note: For E. faecalis, the final well composition is: E. faecalis (OD600&#8776;0.03), BHI = 10% v/v, with the remaining volume comprised of S basal. Remember that 25 &#181;L of S basal will be delivered with worms. 
	Note: E. faecalis produces thick biofilms that absorb the fluorescent stain, causing blurred images. Extensive washing may be insufficient to remove this biofilm. In this case, worms can be transferred to an empty plate using a multichannel pipette. To facilitate transfer, Tween 20 can be added to S Basal to a final concentration of 0.1% (v/v) Tween 20 in S Basal. This aids in removing the worms from the biofilm.</li>
</ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyoverdine,+a+siderophore+from+Pseudomonas+aeruginosa,+translocates+into+C.+elegans,+removes+iron,+and+activates+a+distinct+host+response.">Pyoverdine, a siderophore from Pseudomonas aeruginosa, translocates into C. elegans, removes iron, and activates a distinct host response.</a> Virulence. , 1-41 (2018).</li><li>Garsin, D. A., 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=Long-lived+C.+elegans+daf-2+mutants+are+resistant+to+bacterial+pathogens.">Long-lived C. elegans daf-2 mutants are resistant to bacterial pathogens.</a> Science. 300 (5627), 1921 (2003).</li><li>Estes, K. A., Dunbar, T. L., Powell, J. R., Ausubel, F. M., Troemel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bZIP+transcription+factor+zip-2+mediates+an+early+response+to+Pseudomonas+aeruginosa+infection+in+Caenorhabditis+elegans.">bZIP transcription factor zip-2 mediates an early response to Pseudomonas aeruginosa infection in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (5), 2153-2158 (2010).</li><li>Tjahjono, E., Kirienko, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+mitochondrial+surveillance+pathway+is+required+for+defense+against+Pseudomonas+aeruginosa.">A conserved mitochondrial surveillance pathway is required for defense against Pseudomonas aeruginosa.</a> PLoS Genetics. 13 (6), e1006876 (2017).</li><li>Moy, T. I., 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+novel+antimicrobials+using+a+live-animal+infection+model.">Identification of novel antimicrobials using a live-animal infection model.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (27), 10414-10419 (2006).</li><li>Henderson, S. T., Bonafe, M., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=daf-16+protects+the+nematode+Caenorhabditis+elegans+during+food+deprivation.">daf-16 protects the nematode Caenorhabditis elegans during food deprivation.</a> The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 61 (5), 444-460 (2006).</li><li>Beanan, M. J., Strome, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+germ-line+proliferation+mutation+in+C.+elegans.">Characterization of a germ-line proliferation mutation in C. elegans.</a> Development. 116 (3), 755-766 (1992).</li></ol>

The authors declare that they have no conflicts of interest to disclose.

This assay (or similar assays where other pathogens are substituted for P. aeruginosa or E. faecalis) is useful for a variety of purposes, including drug discovery. It is also useful for addressing fundamental biological questions, such as identifying virulence factors, the elucidation of host defense pathways, and determining the regulatory machinery involved in the host-pathogen interaction.
Although the P. aeruginosa Liquid Killing assay is robust, there are several points where careful attention should be paid to minimize the chance of failure. First, as noted, the bacterial source plate for P. aeruginosa inoculations must remain fresh, lest the bacteria lose virulence, despite overnight growth in LB. Second, it is key to make sure that calcium and magnesium are added to P. aeruginosa killing assays. Without it, virulence will be significantly compromised. Finally, it is worth noting that worms reared on HT115 (for RNAi-based assays) will die significantly later than worms reared on OP50 (N. Kirienko, personal communication). For this reason, if switching to RNAi-based studies, it is important to empirically determine the best time point for the assay.
For killing assays with either P. aeruginosa or E. faecalis, it is crucial to ensure that there is adequate food for the development of the worms from the L1 stage until they are ready to be used in the assay. Starvation, even in the short-term, is known to activate a number of host defense pathways, such as the DAF-2/DAF-16 insulin/IGF signaling network27. Our data suggest that DAF-16/FOXO is already slightly activated by being cultured in liquid20, and further activation is strongly undesirable, since DAF-16/FOXO promotes broad-spectrum pathogen resistance23.
Finally, it is crucial to use completely sterile worms in any assay that relies on mortality as a readout. If worms are fertile, being in liquid causes difficulties in egg laying. As a result, some of the embryos hatch within the parent, eventually causing its death. For this reason, we generally use a genetic lesion, such as glp-4(bn2)28, that demonstrates a completely penetrant sterile phenotype for these assays. The use of chemicals that prevent embryonic development but still allow egg laying, such as 5-fluorodeoxyuridine (FUdR), should be avoided as they may also interfere with bacterial growth and development.
In general, we have found it quite helpful to plan several time points for analysis. Although the assay is robust and the timing is generally consistent, stochastic and imperceptible changes can shift the timing of death in the assay within 2 - 4 hours. When troubleshooting is necessary, it is often helpful to consider first the simplest of answers; i.e., prepare fresh media, discard the most recent bacterial cultures, and re-streak bacterial strains from frozen stocks, etc. Only very rarely do these measures not restore the assay to functionality.
Due to the relative simplicity of the method, a wide variety of modifications can easily be performed. Changing the worms from a uniform glp-4(bn2) background for high-throughput RNAi screens, such as those we describe herein, is useful for the identification of host response pathways for a wide range of xenobiotics and toxic chemicals. For example, by adding the same chemical or toxin into each well of RNAi plates (e.g., Cry5B toxin, phenanthroline, etc.), pathways that alter the sensitivity to these toxins can be readily identified. These types of screens can also be used to identify defense networks against any of the panoply of pathogens that infect C. elegans.

The identification and development of effective, broad-spectrum antibiotics, now almost a century ago, led to a watershed moment in public health where there was a wide-spread belief that infectious disease would be a scourge of the past. Within a few short decades, this optimism began to wane, as pathogen after pathogen developed resistance mechanisms that limited these once miraculous treatments. For some time, the arms race between drug discovery efforts and the pathogens seemed balanced. However, the misuse of antimicrobials has recently culminated in the emergence of pan-drug resistant strains of Klebsiella pneumoniae, Acinetobacter baumanii, Serratia marcescens, and P. aeruginosa1,2,3,4.
P. aeruginosa is an opportunistic, gram negative, multi-host pathogen that is a severe threat to patients with severe burns, those who are immunocompromised, or have cystic fibrosis. It is also increasingly identified as a causative agent in severe nosocomial infections, particularly due to its ongoing acquisition of antimicrobial resistance. To begin to address this threat, we have used the well-documented C. elegans-P. aeruginosa infection system5. Our lab has leveraged this system to develop a liquid-based, high-throughput, high-content screening platform to identify novel compounds that limit the ability of the pathogen to kill the host6. Intriguingly, these compounds seem to belong to at least three general categories, including antimicrobials7 and virulence inhibitors8. Other high-content drug discovery assays in C. elegans have been reported for Mycobacterium tuberculosum, Chlamydia trachomatis, Yersinia pestis, Listeria monocytogenes, Francisella tularensis, Staphylococcus aureus, Candida albicans, and Enterococcus faecalis, among others9,10,11,12,13,14,15,16. These types of assays have several well-recognized advantages, such as limiting false positive hits that may be toxic to both the host and the pathogen, increased likelihood of bioavailability compared to a chemical screen, and the ability to identify hits beyond simply limiting microbial growth, such as anti-virulents, immune stimulatory molecules, or compounds that otherwise tilt the balance of the host-pathogen interaction in favor of the former. Additionally, the compounds discovered in these screens are often effective in mammalian hosts.
It is worth noting that at least two other assays17,18 are available to carry out high-throughput screens in C. elegans in liquid. However, each of these assays is a modification that allows the prototypical intestinal-colonization assay, known as slow-killing, to be performed in liquid, increasing throughput and allowing compounds to be more readily screened. Careful characterization has conclusively demonstrated that the mechanisms of bacterial virulence are different between these assays and our liquid-based screen7. Since both types of virulence are observed in mammalian systems, it is important to consider which virulence determinant is most relevant for the experimenter&#39;s interests prior to assay selection.
Here we demonstrate an optimized version of the liquid-based C. elegans-P. aeruginosa assay. We also report the adaptation of our liquid-based assay method to accommodate the gram-positive bacterial pathogen Enterococcus faecalis. Like P. aeruginosa, E. faecalis is increasingly identified as a serious nosocomial threat with a growing armament of antimicrobial resistance pathways1. Although a previous method for high-throughput screening of E. faecalis exists14, it requires preinfection with the pathogen, which complicates the procedure and increases the likelihood of contaminating equipment like the COPAS FlowSort. Our protocol eliminates the need for pre-infection, improving the safety profile. Finally, we report a means by which either of these assays can be combined with feeding RNAi, allowing the user to search for host factors that play a role in the establishment of, or resistance to, infection.

<table><tbody><tr><td>COPAS FP BioSorter</td><td>Union Biometrica</td><td></td><td>Large object flow cytometer/worm sorter</td></tr><tr><td>Cytation 5</td><td>BioTek</td><td></td><td></td></tr><tr><td>EL406 Washer Dispenser</td><td>BioTek</td><td></td><td></td></tr><tr><td>Multitron Pro</td><td>Infors HT</td><td></td><td></td></tr><tr><td>24 Deep-Well RB Block</td><td>Thermo Fisher Scientific</td><td>CS15124</td><td></td></tr><tr><td>384-Well plate</td><td>Greiner Bio-One</td><td>MPG-781091</td><td></td></tr><tr><td>Nematode Growth Media (NGM)</td><td></td><td></td><td>Amount per liter: 18 grams agar, 3 grams NaCl, 2.5 grams Peptone, 1 mL CaCl&lt;sub&gt;2&lt;/sub&gt; (1 M), 1 mL MgSO&lt;sub&gt;4&lt;/sub&gt; (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water</td></tr><tr><td>Slow Killing (SK) plates</td><td></td><td></td><td>Amount per liter: 18 grams agar, 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl&lt;sub&gt;2&lt;/sub&gt; (1 M), 1 mL MgSO&lt;sub&gt;4&lt;/sub&gt; (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water</td></tr><tr><td>Slow Killing (SK) media</td><td></td><td></td><td>Amount per liter:&amp;nbsp; 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl&lt;sub&gt;2 &lt;/sub&gt;(1 M), 1 mL MgSO&lt;sub&gt;4&lt;/sub&gt; (1 M), 25 mL Phosphate buffer, and 973 mL of milli-Q water</td></tr><tr><td>Lysogeny Broth (LB)</td><td>USBiological Life Sciences</td><td>L1520</td><td></td></tr><tr><td>Brian Heart Infusion broth (BHI)</td><td>Research Products International Corp</td><td>50-488-526</td><td></td></tr><tr><td>Worm Bleach Solution</td><td></td><td></td><td>Amount per 100 mL: 10 mL of 5 M NaOH solution, 20 mL of 5% Sodium Hypochlorite Solution, and 70 mL of sterile water</td></tr><tr><td>S Basal</td><td></td><td></td><td>Amount per liter: 5.85 grams NaCl, 6 grams KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 1 gram K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, and 1 Liter of milli-Q water</td></tr><tr><td>Agar</td><td>USBiological Life Sciences</td><td>A0930</td><td></td></tr><tr><td>NaCl</td><td>USBiological Life Sciences</td><td>S5000</td><td></td></tr><tr><td>Peptone</td><td>USBiological Life Sciences</td><td>P3300</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>USBiological Life Sciences</td><td></td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>M63-500</td><td></td></tr><tr><td>Phospate buffer</td><td></td><td></td><td>amount per liter: 132 mL of K2HPO4 (1M) and 868 mL of KH2PO4 (1M)</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Acros Organics</td><td>7778-77-0</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>USBiological Life Sciences</td><td>P5100</td><td></td></tr><tr><td>5% Sodium Hypochlorite Solution</td><td>BICCA</td><td>7495.5-32</td><td></td></tr><tr><td>NaOH solution</td><td>Fisher Scientific</td><td>SS255-1</td><td></td></tr><tr><td>Breathe-easy</td><td>Diversified Biotech</td><td>BEM-1</td><td></td></tr><tr><td>SYTOX Orange Nucleic Acid Stain</td><td>Fisher Scientific</td><td>S11368</td><td></td></tr><tr><td>&lt;strong&gt;Bacterial Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;P. aeruginosa &lt;/em&gt;(PA14)</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. faecalis&lt;/em&gt;(OG1RF)</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli &lt;/em&gt;superfood (OP50)</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli &lt;/em&gt;RNAi expressing bacteria (HT115)</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Worm Strains &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;glp-4&lt;/em&gt;(&lt;em&gt;bn2&lt;/em&gt;) (Beanan and Strome, 1992, PMID: 1289064)</td><td></td><td></td><td></td></tr><tr><td>PINK-1::GFP reporter (Kang et al., 2018, PMID: 29532717)</td><td></td><td></td><td></td></tr></tbody></table>

a high-throughput, high-content, liquid-based c. elegans pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

Important parameters for assay performance
A proper understanding of the biology underlying this assay is necessary for troubleshooting and optimizing the assay. To that end, we refer first to several key papers elucidating the mechanisms of pathogenesis of P. aeruginosa-mediated killing in liquid7,20. Provided that the steps outlined above are followed (see Figure 1 for a schematic of the assay protocol) a time-dependent killing of C. elegans will be observed only in the presence of the pathogen (Figure 2A). In contrast, in the absence of key nutritional supplements (e.g., if the peptone is left out of the media, and only S Basal is added), little to no killing will be observed (Figure 2B). Interestingly, the two-step incubation of P. aeruginosa (for 24 h at 37 &#176;C, then 24 h at 25 &#176;C) which is critical for conventional slow-killing assays, and was originally implemented in Liquid Killing21, is dispensable in this assay (Figure 2C). While it is possible to add P. aeruginosa straight from overnight LB culture, doing so significantly changes lethality kinetics and is not recommended.
Understanding assay biology also permits simplification of the assay, if appropriate and desirable. For example, the most important driver of host killing in this assay is the siderophore pyoverdine, and host toxicity caused by pyoverdine is contingent upon its ability to bind iron7. As such, the assay can be simplified by substituting pyoverdine-rich filtrate, purified pyoverdine, or even some synthetic iron-chelating chemicals (e.g., 1,10-phenanthroline) for live bacteria, as the transcriptional response of C. elegans to these treatments is very similar (Figure 3)22.
Several different lines of evidence speak to the robustness of the experimental setup. First, the setup tolerates a wide range of initial bacterial concentrations. Concentrations as low as OD600 = 0.0025 (approximately 10-fold lower than recommended) still exhibit time- and concentration-dependent killing, although the timing does shift (Figure 4A). Second, the reproducibility of the assay is such that as few as 4 wells is frequently sufficient to obtain statistically significant data (Figure 4B).
There are several changes that we do not recommend. For example, using initial bacteria inocula with concentrations much higher than those listed in the protocol; doing so results in thick, biofilm-like material that is difficult or impossible to effectively wash away, which complicates scoring lethality. Furthermore, high-concentration bacterial inocula also can compete for oxygen and trigger non-specific host killing7.
Another important note is to limit the period of time between stopping the assay and imaging the dead worms. Note that this time frame must include staining, so it is critical to be efficient. After death, the biological material within worms begins to extrude and/or be consumed by bacteria. Within a matter of 24 - 32 h, only the cuticle remains. The cuticle stains very poorly and is very light, making it easy to lose during washes. It is important to note that strains or strain/RNAi conditions with very different kinetics of killing can be complicated by this phenomenon (i.e., some worms may still be alive while others have already lost their content and are impossible to image). Figure 4A shows this phenomenon, as worms at the left side of the plate, inoculated with very low initial bacterial concentrations, are still largely alive while worms on the right side of the plate, which were exposed to much higher concentrations of bacteria, have been washed away.
A very important determinant of assay success is the method used to collect and analyze the data. In our lab, we have used at least three methods for collecting data from these assays: spectrophotometry, automated microscopy, and flow vermimetry (i.e., the adaptation of flow cytometry techniques to C. elegans; literally, the measurement of worms in a flowing solution). The first of these methods uses a spectophotometer to read the fluorescence of the dye or reporter of the worms in question. This method has the advantage of using a fairly ubiquitous piece of equipment (a standard spectrophotometer with a microplate reader) and is the fastest method to acquire data. However, it lacks the informational content available from automated microscopy and the statistical power of flow vermimetry.
Automated microscopy is another viable option. A number of microplate imaging systems are currently on the market, ranging in prices that are affordable to a single investigator (e.g., a Bio-Tek Cytation5) to larger, more expensive and higher-quality machines that are more commonly found in core facilities (e.g., a Molecular Devices ImageXpress Microscope). In practice, most of these solutions are amenable for scoring most screens and assays. Most automated imaging platforms can also be coupled to downstream software for image processing (e.g., MetaMorph, ImageJ, Gene5, or Cell Profiler) to further the yield of information. Examples of the utility of processing are shown in Figure 5 and Figure 6. When the signal-to-noise ratio is high (as in Figure 5),analysis is simple and discriminating between positive and negative conditions is trivial. In these cases, even weak hits can be readily identified. Many assays, however, have a weaker signal-to-noise ratio. For example, treatment of PINK-1::GFP22 worms (with constitutive mCherry expression in their pharynges) with 1,10-phenathroline increases the level of PINK-1::GFP. For data analysis, the inducible GFP reporter is normalized to the constitutive mCherry signal (Figure 6). In this case, the reporter has weaker expression and/or is not activated in the majority of the worms. Therefore, implementing additional image-processing tools, like Cell Profiler, that can reduce background and amplify signal may be advantageous.
Finally, if a COPAS FlowPilot is available, it can be used to acquire data via flow vermimetry. Much like the more familiar concept of flow cytometry, this instrument can be used to measure the fluorescence of C. elegans in at least two or three channels at a time. This method is very amenable to the acquisition of data with high statistical significance, but the throughput is very much diminished compared to automated microscopy. The most significant advantage of flow vermimetry is in the ability to analyze whole worm populations as a single group for a replicate, rather than splitting them into multiple wells and analyzing the average of the wells. Analyzing large groups in this fashion dramatically improves the statistical power and allows the detection of even small effects.
Modifications of the Liquid Killing Assay
Recent developments of the assay have extended its usefulness by including assaying activation of fluorescent reporters (Figure 6), using medium- to high-throughput RNAi techniques (Figure 3), and even the substitution of the original pathogen.
The most obvious reason to use RNAi set up is to test for host defense pathways against P. aeruginosa (or other pathogens). Examples of RNAi knockdown of genes relevant for survival in the assay are shown in Figure 3. Consistent with previous observations20,23,24,25, daf-2(RNAi) enhanced C. elegans survival during exposure to P. aeruginosa, while daf-16(RNAi), zip-2(RNAi) and cebp-1(RNAi) shortened it. As noted, RNAi is most simply included in the assay growing worms on multiwell plates where each well contains a different RNAi clone. Due to the high viscosity of the agar and small volumes involved, it is difficult to achieve uniform, bubble-free filling of 96-well plates without specialized equipment. In addition, drying the bacterial strains carrying the RNAi can also be an issue, as the outer wells dry faster than inner wells, leading to non-uniformity and significant potential for agar cracking. As noted above, this encourages worms to burrow, reducing the number of animals available for screening and risks clogging liquid-handling machinery. For both of these reasons, 24-well plates are easier to work with than 96-well plates. Although this reduces assay throughput, it improves reliability, and is suggested, particularly for initial screens.
Finally, the assay has been modified the assay to replace P. aeruginosa with E. faecalis. (In principle, substitution with other bacterial species should not provide undue difficulties, provided that appropriate culturing and exposure conditions are identified.) The most important factor to consider is the media requirements of the pathogen, both for growth and development and for the induction of virulence. For example, the Gram positive, opportunistic pathogen E. faecalis requires nutrient-rich media (like BHI) and incubation at higher temperatures compared to P. aeruginosa14,26. By taking these factors into account, adaptation of the the assay to use E. faecalis was straightforward. As noted for P. aeruginosa, we saw time-dependent killing (Figure 7). Interestingly, the timing of death was similar to previously published assays that used pre-infection on agar plates26, despite the absence of this step, which may suggest that the mechanisms involved in pathogenesis vary.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58068/58068fig1.jpg" /> 
Figure 1: Scheme for liquid-based C. elegans &#8211; P. aeruginosa infection assay. Steps involving nematode propagation and preparation are at left side, bacterial preparation are at right. Steps involving both are centered. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58068/58068fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58068/58068fig2.jpg" /> 
Figure 2: Killing of C. elegans by P. aeruginosa is specific and requires proper bacterial nutrition. (A) C. elegans death in the presence of E. coli and P. aeruginosa. (B) C. elegans death in the presence of P. aeruginosa with (right) and without (left) Slow Kill media added to mix (i.e., S basal only). S Basal is a minimal media and does not contain the nutrients required for bacterial growth and precludes the production of pyoverdine. (C) C. elegans death in the presence of differently prepared P. aeruginosa. *p &#60;0.01, based on Student&#39;s t-test. Error bars represent S.E.M. 10 wells were used per biological replicate per condition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58068/58068fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58068/58068fig3.jpg" /> 
Figure 3: C. elegans resistance to P. aeruginosa depends on host immune pathways. A panel of selected RNAi knockdowns was treated with either P. aeruginosa (A) or 1,10-phenanthroline (a chemical chelator that mimics exposure to P. aeruginosa in liquid) (B). *p &#60;0.01, #p &#60;0.05, based on Student&#39;s t-test. Error bars represent S.E.M. 10 wells were used per biological replicate per condition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58068/58068fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58068/58068fig4.jpg" /> 
Figure 4: C. elegans killing by P. aeruginosa is robust and depends on bacterial concentration. (A-B) Representative images (A) and quantification (B) of C. elegans death after exposure to varying concentrations of P. aeruginosa for 72 h. p-values were calculated based on Student&#39;s t-test. Scale bar in (A) is 1 mm. 16 wells were used per biological replicate per condition. BF: Bright Field, FL: Fluorescence. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58068/58068fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58068/58068fig5.jpg" /> 
Figure 5: Data acquisition and image processing of an assay with a strong signal. (A) Representative images of live (negative control) and dead (positive control) C. elegans. (B) Statistical analysis of negative and positive controls based on data acquisition method, processing mode, and number of wells analyzed. (C) Fluorescence was analyzed for worms from four individual wells of a 96-well plate (two positive and two negative control wells). Each well contained ~100 worms. Each dot on the graph represents a single worm, showing the time-of-flight (which correlates with but, due to the coiling of living worms, imperfectly represents size) and the measured fluorescence for worms that are stained with Sytox Orange. Positive control worms were pre-killed by heat exposure. Due to changes in tension and shape of dead worms (as can be seen in (A), dead worms appear more rod-like and have few body bends) compared to their living counterparts, dead worms have a longer TOF. p-values were calculated based on Student&#39;s t-test. Scale bar in (A) is 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58068/58068fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58068/58068fig6.jpg" /> 
Figure 6: Data acquisition and image processing of an assay with a moderate signal. (A) Representative images of C. elegans carrying an inducible PINK-1::GFP reporter and an mCherry constitutive marker (to normalize expression of genes from the extrachromosomal array). C. elegans exposed to DMSO (negative control) or 1,10-phenanthroline (positive control). (B) Statistical analysis of negative and positive controls based on data acquisition method, processing mode, and number of wells analyzed. (C) Four individual wells of a 96-well plate (two positive and two negative control wells) were analyzed using flow vermimetry. p-values were calculated based on Student&#39;s t-test. Scale bar in (A) is 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58068/58068fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58068/58068fig7.jpg" /> 
Figure 7: C. elegans killing by E. faecalis is robust and time dependent. (A-B) Representative images (A) and quantification (B) of C. elegans death after exposure to E. faecalis. *p &#60;0.01, based on Student&#39;s t-test. 10 wells were used per biological replicate per condition. Error bars represent S.E.M. Scale bar in (A) is 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58068/58068fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A High-throughput, High-content, Liquid-based C. elegans Pathosystem at JoVE.com

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

a-high-throughput-high-content-liquid-based-c-elegans-pathosystem

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

14.4K Views.  Rice University. Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

Video: A High-throughput, High-content, Liquid-based C. elegans Pathosystem

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Biochemistry

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Behavior

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...

Environment

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

Genetics

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Neuroscience

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Biology

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Summary

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Chapters in this video

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

A Caenorhabditis elegans Model System for Amylopathy Study

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

C. elegans Chemotaxis Assay

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health