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>

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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 can affect fecundity and growth of progeny19,20, therefore it is important that worms have been well fed for several generations before performing this assay. A well-fed stock of worms is easily maintained by simply transferring a few adult worms to a new seeded NGM plate every 2-3 days. Two days before the assay, adult worms should be placed on a fresh lawn of OP50 in order to have sufficient L4 progeny available for the assay.
It is also important that EIW assay plates promote the growth of only a single type of bacteria. E. faecalis strains were grown on BHI plates. Streptomycin was added to the plates to select against E. coli OP50, which is transferred with the L4 worms as they are transferred from maintenance NGM plates to the assay plates. OP50 also grows on BHI plates. If not selected against, C. elegans would feed on two types of bacteria (E. coli and E. faecalis) while on the BHI plates. The strains of E. faecalis used in this assay are resistant to streptomycin. If different types of bacteria were used for this assay, a different antibiotic, and perhaps different culture media, should be used to select for the bacteria of choice and against E. coli OP50.
Accurately selecting staged worms for this assay is crucial for several reasons. First, it is important to count egg retention during the time of egg production/laying in the self- fertilizing C. elegans. Egg production occurs only during the first five days of C. elegans adulthood (peaking around 40 hours post-L4) and quickly declines thereafter4,21. Adult worms live for an average of two to three weeks, so many adult worms live for at least a week while not producing fertilized eggs. Therefore, it is important to make sure that the EIW assay is not performed on unstaged adult worms of unknown age.
The second reason it is important to use staged animals in the EIW assay is so that the time in development at which worms have been exposed to the bacteria, toxin or drug is tightly controlled. Cultures of C. elegans are typically synchronized at the egg, L1 or L4 stages. The L4 stage was used for this assay because of the concern that longer exposure to E. faecalis would result in lethality before worms became gravid adults.
The duration of time worms are left in the bleach solution is also important to monitor. Worms should typically dissolve in under ten minutes, although this time does vary from worm to worm. Fertilized eggs are eventually dissolved by bleach, but the process is slower than for the cuticle due to the presence of a protective eggshell. The eggshell will begin to be dissolved by the bleach if left in the solution for longer than 10-15 min.
Egg retention reflects the balance between egg production and egg laying. The EIW assay alone does not distinguish whether the number of eggs retained in the uterus is due to alterations in egg production or egg laying. This is especially a concern if worms treated with a bacterial strain or toxin retain fewer eggs than those in the control group. In these cases especially, a follow-up brood size assay should be done. Brood size assays quantify the number of eggs laid over the worm&#39;s reproductive lifetime4. If the brood size of worms in the experimental and control groups are the same, then differences in egg retention can be attributed to differences in egg-laying behavior. Because it is unlikely that mildly pathogenic bacterial strains like E. faecalis would act to increase egg production, it is reasonable to assume that an increase in egg retention after exposure to E. faecalis (as seen in Figure 3) is a result of decreased egg-laying behavior.
The EIW assay also does not determine the mechanism by which bacteria may alter egg retention. It is possible that C. elegans may retain eggs because the pathogenic bacteria affects feeding by colonizing in and blocking the mouth opening or because it is a poor food source. It is also possible that the bacteria may colonize and block the vulval opening, or affect the function of cells in the egg-laying system. Further analysis is required to determine the mechanism by which egg retention is altered.
This EIW assay can be easily modified to determine the effects of many types of compounds on egg retention. In addition, this assay is simple enough that it could be incorporated into a screen to identify genes, in either the host (C. elegans) or pathogen (E. faecalis), required for the effect of the pathogen. The EIW assay can also be used to screen for genes required for regulating egg-laying itself. C. elegans egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9, and requires the coordinated activity of several neurons and muscle groups12. EIW assays have been and continue to be used to identify genes important for neurotransmitter signaling and/or muscle activity. EIW assays provide a quantitative, rather than qualitative, analysis of an important C. elegans behavior.

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 quantified by counting while viewing under dissecting microscope.
More eggs were retained in the presence of E. faecalis strains tested, as compared to E. coli OP50 controls (Figure 3). There is some variation in the effect of any one strain of E. faecalis on individual worms, as seen from the error bars. However, the effect of strains of E. faecalis on egg retention within a population of worms does vary significantly from the controls. The degree of egg retention elicited by different strains of E. faecalis tested may also vary. This varied effect may be due to the expression of different virulence factors in each strain.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/51203/51203fig1highres.jpg" src="/files/ftp_upload/51203/51203fig1.jpg" /> 
Figure 1. Identifying L4 staged C. elegans. A. L4 stage C. elegans larvae are the largest larvae present on plates- only slightly shorter in length than adult worms. L4s are thinner than adults as well, due to the absence of retained eggs (which make adult worms slightly wider). B. After identifying those larvae that are slightly shorter than adults, L4-staged C. elegans are further identified by the lighter half-moon spot located in the middle of their body (black arrowheads). This spot is visible in smaller larvae (L2 and L3 stages), but is absent in the adult. All images were taken of worms on NGM plates, with a camera mounted to a dissecting microscope (40X and 50X magnification for A and B ,respectively). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51203/51203fig1highres.jpg">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/51203/51203fig2highres.jpg" src="/files/ftp_upload/51203/51203fig2.jpg" /> 
Figure 2. Bleach solution dissolves the C. elegans cuticle over time. A staged adult worm is placed in a droplet of bleach solution (A). Over five to ten minutes, the cuticle of the worm begins to dissolve (B-F). As most of the cuticle is dissolved, the eggs become visible (F black arrows). Fat stores inside the worm also dissolve slowly in bleach, and remain visible (F, gray arrows). All images were taken of worms on NGM plates, with a camera mounted to a dissecting microscope (40X). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51203/51203fig2highres.jpg">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/51203/51203fig3.jpg" src="/files/ftp_upload/51203/51203fig3.jpg" /> 
Figure 3. Some E. faecalis strains cause increased egg retention after 40 hours of exposure. Worms exposed to two of the six E. faecalis strains (orange bars) showed an increased level of egg retention compared to worms fed the E. coli (OP50) control strain (blue bars). Ten worms were fed each bacterial strain in each trial. Trials were done in triplicate on different days. * indicates p&#60;0.05 (Student&#39;s t-test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51203/51203fig3.jpg">Click here to view larger image</a>.

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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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17.0K Views.  Fairleigh Dickinson University. 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.

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 ...

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

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

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 ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Bacterial Metabolic Inactivation in Caenorhabditis elegans Using Paraformaldehyde

Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a diet of live bacteria, the metabolic activity of their food source can confound experiments looking for the direct effects of various interventions on the worm. To avoid the confounding effects of bacterial metabolism, C. elegans researchers have used multiple methods to metabolically inactivate bacteria, including ultraviolet (UV)-irradiation, heat-killing, and antibiotics. UV treatment is relatively low-throughput and cannot be used in liquid culture because each plate must be examined for successful bacterial killing. A second treatment method, heat-killing, negatively affects the texture and nutritional quality of the bacteria, leading to the developmental arrest of C. elegans. Finally, antibiotic treatment can directly alter C. elegans physiology in addition to preventing bacterial growth. This manuscript describes an alternative method to metabolically inactivate bacteria using paraformaldehyde (PFA). PFA treatment cross-links proteins within bacterial cells to prevent metabolic activity while preserving cellular structure and nutritional content. This method is high-throughput and can be used in liquid culture or solid plates, as testing one plate of PFA-treated bacteria for growth validates the whole batch. Metabolic inactivation through PFA treatment can be used to eliminate the confounding effects of bacterial metabolism on studies of drug or metabolite supplementation, stress resistance, metabolomics, and behavior in C. elegans.

Author Spotlight: Advancing Caenorhabditis elegans Research Using Paraformaldehyde-Treated Bacteria 

The food source for Caenorhabditis elegans in the lab is live Escherichia coli. Since bacteria are metabolically active, they present a confounding variable in metabolic and drug studies in C. elegans. A detailed protocol to metabolically inactivate bacteria using paraformaldehyde is described here.

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a ...

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

Source: Campbell, J. C., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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

Summary

Explore More Videos

Chapters in this video

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

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

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

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

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

C. elegans Chemotaxis Assay

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

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