The authors would like to thank June Middleton for supplying E. faecalis strains and for guidance with bacteria culture. C. elegans were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

1) Preparation of Nematode Growth Media (NGM)
<ol>
	<li>To make 50 plates, add 1.5 g NaCl, 8.5 g Ultrapure Agar, and 1.25 g peptone to a 1 L flask.</li>
	<li>Add 487.5 ml of dH2O to flask. Swirl gently to mix and cover opening of flask with a piece of aluminum foil.</li>
	<li>Sterilize the solution by autoclaving at standard conditions (121 psi, 120 &#176;C, 20 min).</li>
	<li>Allow the solution to cool to 45 &#176;C in a water bath.</li>
	<li>To the solution, add the following in order: 500 &#181;l cholesterol from a 5 mg/ml stock solution (dissolved in ethanol), 500 &#181;l 1 M CaCl2, 500 &#181;l 1 M MgSO4, and 12.5 ml KPO4 buffer (54.15 g KH2PO4 and 17.8 g K2HPO4 in 500 ml of dH2O) as described in18.</li>
	<li>Using sterile pipettes, add 10 ml of agar solution to each 60 mm polystyrene Petri dish. Allow the media to solidify at room temperature overnight.</li>
</ol>
2) Preparation of B Broth E. coli&#160;Media
<ol>
	<li>To make five 100 ml cultures, add 5.0 g of Bacto Tryptone, 2.5 g yeast extract, 5.0 g NaCl to a beaker.</li>
	<li>Add sterile, distilled water to a final volume of 500 ml. Heat the solution on a hot plate, while stirring, until the solutes have dissolved.</li>
	<li>Pour 100 ml of B broth into five glass bottles (at least 200 ml volume).</li>
	<li>Sterilize the solutions by autoclaving at standard conditions (121 psi, 120 &#176;C, 20 min).</li>
	<li>Allow B broth to cool to room temperature before use.</li>
</ol>
3) Seeding C. elegans&#160;Maintenance Plates
<ol>
	<li>After allowing NGM plates to dry at room temperature for at least one week, inoculate one 100 ml B broth culture with a few colonies of E. coli (OP50).</li>
	<li>Grow E. coli culture at 37 &#176;C overnight.</li>
	<li>Pipette one-two drops (approximately 100 &#181;l) of OP50 culture onto the center of each NGM plate. Plates are typically stacked lid-side up. When pipetting (seeding), begin by pipetting the culture onto the plate at the bottom of the stack. Work your way up each stack. Try not to move the plates after seeding, because it is important that the culture not get too close to the edges of the plate.</li>
	<li>Allow the seeded plates to dry at room temperature for at least one week before using.</li>
	<li>Seeded plates are stored (inverted) in sealed plastic containers at room temperature.</li>
</ol>
4) Maintaining C. elegans&#160;Strains
<ol>
	<li>To maintain well-fed C. elegans stocks, use a worm pick to move three L4s or gravid adults to a new, seeded NGM plate every 3-4 days.</li>
	<li>Store plates (inverted) preferably inside an incubator at 15- 22 &#176;C. Cultures in the assays described were stored at 20 &#176;C. C. elegans development is temperature-dependent. As maintenance temperature increases, time between developmental stages decreases.</li>
</ol>
5) Preparation of Tryptic Soy Agar (TSA) E. faecalis&#160;Culture Media
<ol>
	<li>To make 50 plates, measure 500 ml of sterile, distilled water into a flask and bring to a boil, while stirring, on a hotplate.</li>
	<li>Add 20 g of powdered TSA agar to flask and allow solution to dissolve.</li>
	<li>Sterilize the solutions by autoclaving at standard conditions (121 psi, 120 &#176;C, 20 min).</li>
	<li>Allow the solution to cool to 45 &#176;C in a water bath.</li>
	<li>Using sterile pipettes, add 10 ml of agar solution to each 60 mm polystyrene Petri dish. Allow the media to solidify at room temperature overnight.</li>
</ol>
6) Preparation of Tryptic Soy Broth (TSB) E. faecalis&#160;Culture Media
<ol>
	<li>To make 250 culture tubes, measure 500 ml of sterile, distilled water into a flask and warm on a hotplate&#160;while stirring.</li>
	<li>Add 15 g of TSB agar powder to the flask and allow the powder to dissolve.</li>
	<li>Sterilize the solutions by autoclaving at standard conditions (121 psi, 120 &#176;C, 20 min).</li>
	<li>Allow solution to cool to room temperature, then pipette 2 ml into sterile, 5 ml test tubes.</li>
</ol>
7) Preparation of Brain Heart Infusion (BHI) Media, with Streptomycin, for E. faecalis&#160;Culture
<ol>
	<li>Measure 500 ml of sterile, distilled water into a flask and bring to a boil, while stirring, on a hotplate.</li>
	<li>Add 26 g of BHI medium to the flask and allow the solution to dissolve.</li>
	<li>Sterilize the solutions by autoclaving at standard conditions (121 psi, 120 &#176;C, 20 min).</li>
	<li>Allow solution to cool to 45 &#176;C in a water bath.</li>
	<li>Using sterile technique, add 0.5 ml of a 10 mg/ml streptomycin stock solution and swirl to mix.</li>
	<li>Add 10 ml of the solution to each 60 mm polystyrene Petri dish.</li>
	<li>Allow media to solidify at room temperature overnight. Store plates (inverted) at 4 &#176;C until a few hours before use.</li>
</ol>
8) Maintaining E. faecalis&#160;Strains
<ol>
	<li>To maintain E. faecalis stocks, use a sterile loop to streak individual colonies onto separate tryptic soy agar (TSA) plates.</li>
	<li>Grow cultures at 37 &#176;C overnight. Store plates (inverted) in a sealed bag or box at room temperature for several weeks.</li>
</ol>
9) Preparing E. faecalis&#160;Plates for EIW Assay
<ol>
	<li>Inoculate a 2 ml TSB culture with a colony of E. faecalis and incubate overnight at 37 &#176;C.</li>
	<li>Pipette 20 &#181;l of E. faecalis culture onto the center of a prepared BHI-streptomycin plate, and incubate overnight at 37 &#176;C.</li>
</ol>
10) Preparing E. coli&#160;Plates for EIW Assay (Control Plates)
<ol>
	<li>Inoculate a 100 ml B-broth culture with a colony of E. coli OP50 and incubate overnight at 37 &#176;C</li>
	<li>Pipette 20 &#181;l of the OP50 culture onto the center of an unseeded NGM plate and allow the seeded plates to dry at room temperature overnight.</li>
</ol>
11) Egg in Worm Assay
<ol>
	<li>Pick 15- 20 staged-L4 C. elegans (Figure 1) onto the center of a lawn of bacteria on a prepared BHI-Strep-E. faecalis plate. Be careful not to transfer a lot of OP50 to the BHI plate. Pick the same number of L4s to a control NGM-OP50 plate.</li>
	<li>Incubate plates for 40 hr at 20 &#176;C.</li>
	<li>Prepare 20% bleach solution. Mix a commercial bleach (6.0% sodium hypochlorite) with the appropriate volume of distilled water.</li>
	<li>Add 10 &#181;l drops of bleach solution to ten distinct locations on a plastic lid (of a worm or bacterial plate).</li>
	<li>Using a worm pick, transfer one worm into each bleach drop. Gently swirl the tip of the pick in the bleach droplet to make sure the worm has washed off the end of the pick (confirm by viewing the droplet under a dissecting microscope).</li>
	<li>Allow the cuticle to dissolve for approximately 10 min or until the worms burst open, expelling the eggs (Figure 2). Be careful not to transfer eggs from the plate.</li>
	<li>Using a dissecting microscope, count the eggs in each drop of bleach.</li>
</ol>

<ol><li>Horvitz, H. R., Chalfie, M., Trent, C., Sulston, J. E., Evans, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serotonin+and+octopamine+in+the+nematode+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Serotonin and octopamine in the nematode Caenorhabditis elegans.</a> Science. 216, 1012-1014 (1982).</li>
<li>Sawin, E. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Genetic+and+cellular+analysis+of+modulated+behaviors+in+Caenorhabditis+elegans+%5B%5D%22">Genetic and cellular analysis of modulated behaviors in Caenorhabditis elegans [dissertation]</a>. , Massachusetts Institute of Technology. Cambridge, MA. (1996).</li>
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<li>Chase, D. L., Koelle, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+analysis+of+RGS+protein+function+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Genetic analysis of RGS protein function in Caenorhabditis elegans.</a> Methods Enzymol. 389, 305-320 (2004).</li>
<li>Trent, C., Tsuing, N., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Egg-laying+defective+mutants+of+the+nematode+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Egg-laying defective mutants of the nematode Caenorhabditis elegans.</a> Genetics. 104, 619-647 (1983).</li>
<li>Weinshenker, D., Garriga, G., Thomas, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+and+pharmacological+analysis+of+neurotransmitters+controlling+egg+laying+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Genetic and pharmacological analysis of neurotransmitters controlling egg laying in Caenorhabditis elegans.</a> J. Neurosci. 15, 6975-6985 (1995).</li>
<li>Waggoner, L. E., Zhou, G. T., Schafer, R. W., Schafer, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+alternative+behavioral+states+by+serotonin+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Control of alternative behavioral states by serotonin in Caenorhabditis elegans.</a> Neuron. 21, 203-214 (1998).</li>
<li>Bany, A., Dong, M., Koelle, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+and+cellular+basis+for+acetylcholine+inhibition+of+Caenorhabditis+elegans+egg-laying+behavior%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Genetic and cellular basis for acetylcholine inhibition of Caenorhabditis elegans egg-laying behavior.</a> J. Neurosci. 23, 8060-8069 (2003).</li>
<li>Dempsey, C. M., Mackenzie, S. M., Gargus, A., Blanco, G., Sze, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serotonin+%285HT%29%2C+fluoxetine%2C+imipramine+and+dopamine+target+distinct+5HT+receptor+signaling+to+modulate+Caenorhabditis+elegans+egg-laying+behavior%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Serotonin (5HT), fluoxetine, imipramine and dopamine target distinct 5HT receptor signaling to modulate Caenorhabditis elegans egg-laying behavior.</a> Genetics. 169, 1425-1436 (2005).</li>
<li>Leung, M. C., Williams, P. L., Bennedetto, A., Au, C., Helmcke, K. J., Aschner, M., Meyer, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caenorhabditis+elegans%3A+an+emerging+model+in+biomedical+and+environmental+toxicology%5BTitle%5D)+AND+%22Toxicol.+Sci%22%5BJournal%5D)">Caenorhabditis elegans: an emerging model in biomedical and environmental toxicology.</a> Toxicol. Sci. 106, 5-28 (2008).</li>
<li>Mylonakis, E., Aballay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Worms+and+flies+as+genetically+tractable+animal+models+to+study+host-pathogen+interactions%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">Worms and flies as genetically tractable animal models to study host-pathogen interactions.</a> Infect. Immun. 73, 3833-3841 (2005).</li>
<li>Schafer, W. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSchafer%2C+WR+%22WormBook%22">WormBook</a>. , WormBook. (2005).</li>
<li>White, J., Southgate, E., Thomson, N., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structure+of+the+Caenorhabditis+elegans+nervous+system%5BTitle%5D)+AND+%22Philos.+Trans.+R.+Soc.+Lond.+B.+Biol.+Sci%22%5BJournal%5D)">The structure of the Caenorhabditis elegans nervous system.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 314, 1-340 (1986).</li>
<li>Desai, C., Garriga, G., McIntire, S. L., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genetic+pathway+for+the+development+of+the+Caenorhabditis+elegans+HSN+motor+neurons%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A genetic pathway for the development of the Caenorhabditis elegans HSN motor neurons.</a> Nature. 336, 638-646 (1988).</li>
<li>Garsin, D. A., Sifri, C. D., Mylonakis, E., Qin, X., Singh, K. V., Murray, B. E., Calderwood, S. B., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+model+host+for+identifying+Gram-positive+virulence+factors%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">A simple model host for identifying Gram-positive virulence factors.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 10892-10897 (2001).</li>
<li>Aballay, A., Yorgey, P., Ausubel, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Salmonella+typhmurium+proliferates+and+establishes+a+persistent+infection+in+the+intestine+of+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Curr.+Biol%22%5BJournal%5D)">Salmonella typhmurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans.</a> Curr. Biol. 10, 1539-1542 (2000).</li>
<li>O'Quinn, A. L., Wiegand, E. M., Jeddeloh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Burkholderia+pseudomallei+kills+the+nematode+Caenorhabditis+elegans+using+an+endotoxin-mediated+paralysis%5BTitle%5D)+AND+%22Cell.+Microbiol%22%5BJournal%5D)">Burkholderia pseudomallei kills the nematode Caenorhabditis elegans using an endotoxin-mediated paralysis.</a> Cell. Microbiol. 3, 381-393 (2001).</li>
<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetics+of+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li>
<li>Harvey, S. C., Shorto, A., Orbidans, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+genetic+analysis+of+life-+history+traits+of+Caenorhabditis+elegans+in+stressful+environments%5BTitle%5D)+AND+%22BMC+Evol.+Biol%22%5BJournal%5D)">Quantitative genetic analysis of life- history traits of Caenorhabditis elegans in stressful environments.</a> BMC Evol. Biol. 8, 15(2008).</li>
<li>Harvey, S. C., Orbidans, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((All+eggs+are+not+equal%3A+the+maternal+environment+affects+progeny+reproduction+and+developmental+fate+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">All eggs are not equal: the maternal environment affects progeny reproduction and developmental fate in Caenorhabditis elegans.</a> PLoS One. 6, e25840(2011).</li>
<li>Klass MR, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aging+in+the+nematode+Caenorhabditis+elegans%3A+major+biological+and+environmental+factors+influencing+life+span%5BTitle%5D)+AND+%22Mech.+Ageing.+Dev%22%5BJournal%5D)">Aging in the nematode Caenorhabditis elegans: major biological and environmental factors influencing life span.</a> Mech. Ageing. Dev. 6, 413-429 (1977).</li>
</ol>

The authors declare that they have no competing financial interests.

The most critical steps in successfully performing this assay are: 1) using well-fed stocks of C. elegans, 2) culturing single types of bacteria on the assay plate, 3) accurately identifying staged L4 worms for exposure to E. faecalis, 4) keeping exposure time to E. faecalis consistent across all trials and 5) bleaching time should not exceed ten minutes to prevent egg disintegration.
For this assay it is important to pick healthy, well-fed worms. Maternal starvation...

Caenorhabditis elegans, a microscopic, free-living roundworm, is a model organism traditionally used to study developmental and cell signaling processes because of its transparent anatomy, well-characterized development, fully-sequenced genome, short generation-time, and genetic homology to humans. More recently, C. elegans has become a model organism in the field of environmental toxicology and innate immunity10, 11.
	
	These self-fertilizing hermaphroditic worms become sexually mature within two to three days of hatching from the egg. During its life cycle, C. elegans passes through four larval stages (L1-L4), before reaching adulthood. One isolated hermaphrodite can produce, on average, 300 offspring within three days of peak fecundity. In reproductively mature C. elegans hermaphrodites, fertilized eggs are retained within the uterus for several hours before being laid. The normal number of eggs stored in the uterus at any one time (during peak fecundity) is between ten and fifteen12. The number of eggs in the uterus is a function of both the rate of egg production and the rate of egg laying. Fertilized eggs are expelled from the uterus by the contraction of sixteen vulval muscles arranged around the opening of the vulva13. Hermaphrodite-specific motorneurons (HSN's) and VC motorneurons synapse onto vulval muscles affecting muscle contraction and thus egg-laying behavior5,7,13,14. Expulsion of eggs from the uterus occurs due to coordinated activity of neurons and muscles.
	
	Lab cultures of C. elegans are typically raised on a diet of nonpathogenic Escherichia coli OP50. In the natural environment, C. elegans come into contact with a variety of food sources, such as pathogenic bacteria, that can be potentially harmful. When exposed to harmful substances in the environment, C. elegans retain eggs until the environment becomes more favorable. Presumably this egg retention is an effort to protect their progeny.
	
	In this egg in worm (EIW) assay, C. elegans are exposed to the potentially pathogenic bacteria, Enterococcus faecalis, which is found in the environment. Exposure to pathogenic forms of E. faecalis can cause persistent intestinal infection and even death in C. elegans15. Exposure to other forms of pathogenic bacteria have been shown to affect egg retention16,17, however the effect was not quantified. In addition, the effect of mildly pathogenic strains of E. faecalis, strains that are not immediately lethal, on egg-laying behavior has not been studied.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. Even though C. elegans are transparent, eggs accumulating in the uterus can be difficult to quantify in an intact animal. The EIW assay involves bleaching gravid adult C. elegans that were exposed to bacteria for a fixed period of time. The bleach solution dissolves the outer cuticle leaving the eggs behind. The eggs are refractive to the effects of bleach due to the presence of a protective eggshell. After bleaching, one is very easily able to count the number of eggs released from the uterus of the worms upon bleaching.
	
	The assay described is a simple, inexpensive, and quick method to quantify the number of eggs in the uterus at one time, and thus quantify the effects of E. faecalis on egg retention. This assay may be used to quantify the effect of other types of bacteria, environmental toxins or drugs on egg retention. This assay also has the potential to be used as a screen for bacterial pathogenicity.

<table><tbody><tr><td>Agar, ultrapure</td><td>Affymetrix</td><td>10906</td><td></td></tr><tr><td>Bacto Peptone</td><td>Becton Dickinson</td><td>211677</td><td></td></tr><tr><td>Bacto Tryptone</td><td>Becton Dickinson</td><td>211705</td><td></td></tr><tr><td>Brain Heart Infusion dehydrated medium</td><td>Carolina Biological Supply</td><td>781781</td><td></td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt;, N2 strain</td><td>Caenorhabditis Genetics Center</td><td></td><td>http://www.cbs.umn.edu/cgc</td></tr><tr><td>Cholesterol</td><td>Alfa Aesar</td><td>A11470</td><td></td></tr><tr><td>Culture plates for &lt;em&gt;C. elegans&lt;/em&gt;</td><td>Tritech Research Inc.</td><td>T3308</td><td></td></tr><tr><td>Culture plates for &lt;em&gt;E. faecalis&lt;/em&gt;</td><td>Fisher Scientific-Fisherbrand</td><td>875713</td><td></td></tr><tr><td>&lt;em&gt;E. coli &lt;/em&gt;(OP50)</td><td>Caenorhabditis Genetics Center</td><td></td><td>http://www.cbs.umn.edu/cgc</td></tr><tr><td>&lt;em&gt;E. faecalis&lt;/em&gt; strains</td><td></td><td></td><td>provided by J. Middleton. All isolates were confirmed as enterococci</td></tr><tr><td colspan="4">by observing growth on enterococcosel agar (BBL) and in 6% NaCl broth;</td></tr><tr><td colspan="4"> &lt;p&gt;all strains grew at 44.5 &amp;ordm;C and were catalase negative and hydrolyzed esculin. A simplified&amp;nbsp;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;dichotomous key based on pigmentation and fermentation reactions for six sugars&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;(arabinose, mannitol, methyl-&amp;alpha;-D-glucopyranoside (MGP), ribose, sorbose and sorbitol) allowed&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;presumptive identification of all &lt;em&gt;E&lt;/em&gt;. &lt;em&gt;faecalis&lt;/em&gt; strains (Efs lacks pigmentation and is arabinose, MGP&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;and sorbose negative and sorbitol, mannitol and ribose positive). All presumptive Efs strains&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;were confirmed using the API 20 STREP system (Biomerieux).&lt;/span&gt;&lt;/p&gt; </td></tr><tr><td>Microscope</td><td>Motic</td><td>SMZ 168B</td><td>any microscope with transmitted illumination and 50X magnification should be sufficient</td></tr><tr><td>Streptomycin sulfate</td><td>Fisher BioReagents</td><td>BP910-50</td><td></td></tr><tr><td>Tryptic Soy Agar (Soybean-Casein Digest Agar Medium), Difco</td><td>Becton Dickinson</td><td>236950</td><td></td></tr><tr><td>Trypticase Soy Broth (Soybean-Casein Digest Medium), BBL</td><td>Becton Dickinson</td><td>211768</td><td></td></tr><tr><td>Yeast extract</td><td>Acros</td><td>61180-1000</td><td></td></tr></tbody></table>

measuring the effects of bacteria on c. elegans behavior using an egg retention assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

This assay allows one to quantify the number of retained eggs within C. elegans after exposure to E. faecalis. L4 staged worms (characterized by transparent open space above their vulva, Figure 1) were exposed to E. faecalis through adulthood. After forty hours of exposure to E. faecalis, bleaching was performed. As the cuticle disintegrated in the bleach droplet, the eggs became more apparent (Figure 2). The number of retained eggs was easily quantifi...

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Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

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17.1K Views.  Fairleigh Dickinson University. Een ei-in-worm (EIW) test is een handige methode om eierleggende gedrag te kwantificeren. Veranderingen in eileg kan een gedragsmatige reactie van het modelorganisme zijn Caenorhabditis elegans Aan potentieel schadelijke stoffen uit het milieu, zoals die geproduceerd door pathogene bacteriën.

Video: Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Genetics

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 ...

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as self-fertilizing hermaphrodites, which produce large broods of progeny-when males are present, they can produce even larger broods of cross progeny. Errors in meiosis, fertilization, and embryogenesis can be rapidly assessed as phenotypes of sterility, reduced fertility, or embryonic lethality. This article describes a method to determine embryonic viability and brood size in C. elegans. We demonstrate how to set up this assay by picking a worm onto an individual Modified Youngren's, Only Bacto-peptone (MYOB) plate, establish the appropriate timeframe to count viable progeny and non-viable embryos, and explain how to accurately count live worm specimens. This technique can be used to determine viability in self-fertilizing hermaphrodites as well as cross-fertilization by mating pairs. These relatively simple experiments are easily adoptable for new researchers, such as undergraduate students and first-year graduate students.

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Here, we present a general method to determine the embryonic viability and total number of embryos produced (brood) using the model organism C. elegans.

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as ...

High-Throughput Assay to Measure Caenorhabditis elegans Food Intake and Lifespan

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans&#160;(C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Neuroscience

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

Medicine

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 ...

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

C. elegans Chemotaxis Assay: A Method to Test Chemosensation in Worms

Source: Campbell, J. C., et al. <a href="https://www.jove.com/video/55939/a-caenorhabditis-elegans-nutritional-status-based-copper-aversion">A Caenorhabditis elegans Nutritional-Status Based Copper Aversion Assay.</a> J. Vis. Exp. (2017).
This video introduces chemotaxis in the nematode C. elegans and shows a sample protocol testing chemotactic aversion to copper sulfate.

Source: Campbell, J. C., et al. A Caenorhabditis elegans Nutritional-Status Based Copper Aversion Assay. J. Vis. Exp. (2017).
This video introduces ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

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