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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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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+model+host+for+identifying+Gram-positive+virulence+factors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+typhmurium+proliferates+and+establishes+a+persistent+infection+in+the+intestine+of+Caenorhabditis+elegans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burkholderia+pseudomallei+kills+the+nematode+Caenorhabditis+elegans+using+an+endotoxin-mediated+paralysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+Caenorhabditis+elegans.">Genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li><li>Harvey, S. C., Shorto, A., Orbidans, H. 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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...

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 border="0" cellpadding="0" cellspacing="0" width="2472">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Agar, ultrapure</td>
			<td style="width:191px;">Affymetrix</td>
			<td style="width:119px;">10906</td>
			<td style="width:1791px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Bacto Peptone</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Bacto Tryptone</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Brain Heart Infusion dehydrated medium</td>
			<td style="width:191px;">Carolina Biological Supply</td>
			<td style="width:119px;">781781</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:371px;">C. elegans, N2 strain</td>
			<td style="width:191px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td>http://www.cbs.umn.edu/cgc</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:371px;">Cholesterol</td>
			<td style="width:191px;">Alfa Aesar</td>
			<td style="width:119px;">A11470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Culture plates for C. elegans</td>
			<td style="width:191px;">Tritech Research Inc.</td>
			<td style="width:119px;">T3308</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Culture plates for E. faecalis</td>
			<td style="width:191px;">Fisher Scientific-Fisherbrand</td>
			<td style="width:119px;">875713</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">E. coli (OP50)</td>
			<td style="width:191px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td>http://www.cbs.umn.edu/cgc</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">E. faecalis strains</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>provided by J. Middleton. All isolates were confirmed as enterococci</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>by observing growth on enterococcosel agar (BBL) and in 6% NaCl broth;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>
			all strains grew at 44.5 &#186;C and were catalase negative and hydrolyzed esculin. A simplified&#160;dichotomous key based on pigmentation and fermentation reactions for six sugars&#160;(arabinose, mannitol, methyl-&#945;-D-glucopyranoside (MGP), ribose, sorbose and sorbitol) allowed&#160;presumptive identification of all E. faecalis strains (Efs lacks pigmentation and is arabinose, MGP&#160;and sorbose negative and sorbitol, mannitol and ribose positive). All presumptive Efs strains&#160;were confirmed using the API 20 STREP system (Biomerieux).
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Microscope</td>
			<td style="width:191px;">Motic</td>
			<td style="width:119px;">SMZ 168B</td>
			<td>any microscope with transmitted illumination and 50X magnification should be sufficient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Streptomycin sulfate</td>
			<td style="width:191px;">Fisher BioReagents</td>
			<td style="width:119px;">BP910-50</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Tryptic Soy Agar (Soybean-Casein Digest Agar Medium), Difco</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">236950</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Trypticase Soy Broth (Soybean-Casein Digest Medium), BBL</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211768</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Yeast extract</td>
			<td style="width:191px;">Acros</td>
			<td style="width:119px;">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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16.9K 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

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of 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 ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...