Name: Measuring Caenorhabditis elegans Life Span on Solid Media (Video) | JoVE Journal
Uploaded: 2009-05-12T00:00:00.000+00:00
Duration: 12 min 49 s
Description: Learn to measure C. elegans lifespan on solid media using synchronized populations and UV-killed bacteria. This protocol enables accurate aging research with systematic mortality tracking.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by a Glenn/AFAR Breakthroughs in Gerontology Award and NIH Grant 1R01AG031108-01 to M. K. G. S. is supported by NIH training grant P30AG013280. M. K. is an Ellison Medical Foundation New Scholar in Aging.

Part 1: Prepare nematode growth media (NGM) plates
This section describes the preparation of the solid NGM plates for use in the life span experiment. A basic life span experiment requires two types of plates: standard NGM plates, which contain no additives, and Amp/FUDR plates, which have both ampicillin (Amp) and fluorodeoxyuridine (FUDR) added to the NGM. Ampicillin is used to prevent foreign bacterial contamination. FUDR inhibits cell division, reduces egg production, and prevents eggs from hatching. The use of FUDR for longevity analysis does not affect adult life span and removes the need to transfer worms every few days in order to separate them from growing larva. Both types of plates are seeded with E. coli OP50 bacteria, which is subsequently killed by exposure to UV.
<ol>
	<li>Prepare NGM (see Section 5 for recipe and storage notes) and label petri plates. You will need 1 60 mm petri plate per 10 mL of NGM. If you are starting with previously prepared solid NGM, continue to step 1.2. If you are starting with freshly autoclaved NGM, skip to step 1.4.</li>
	<li>Completely melt solid NGM by microwaving on high in 30 second pulses. Swirl media between pulses to prevent pressurized gas bubbles from building up.</li>
	<li>Place liquid NGM in 55 °C water bath or on the bench to cool.</li>
	<li>Once NGM reaches 55 °C add 33 µL of 150 mM FUDR and 100 µL of 100 mg/mL Ampicillin per 100 mL NGM (for Amp/FUDR plates) and swirl to mix. For NGM plates without additional drugs proceed directly to step 1.5.</li>
	<li>Using sterile technique, pipette 10 mL NGM into each 60 mm petri plate. Avoid forming bubbles if possible. If bubbles do form, they can be popped using a pipette tip.</li>
	<li>Leave plates on the bench with lids on to allow NGM to solidify and dry. If possible, leave plates on bench for 2 days before adding bacteria, but 1 day will work if plates are needed sooner.</li>
	<li>On the day before the plates finish drying, seed liquid LB culture with a single colony from a fresh streak of E. coli OP50 bacteria on LB agar. You should prepare at least 1 mL of an overnight OP50 culture per 60 mm plate.</li>
	<li>Place OP50 culture in 37 °C shaker and allow bacteria to grow overnight (culture should reach saturation).</li>
	<li>Pellet OP50 bacteria by spinning at 3,500 g for 10 minutes.</li>
	<li>Remove 90% of supernatant and resuspend bacteria to concentrate culture 10x.</li>
	<li>Pipette 100 µL of 10x concentrated OP50 culture into the center of solidified NGM plates. Swirl plates gently to spread out bacteria culture if needed (ideally bacteria should cover the central area of the NGM without coming near the plate walls). Try to avoid touching the pipette tip to the surface of the NGM, as flaws in the surface of the media allow the worms to burrow into the NGM.</li>
	<li>Leave plates on the bench overnight to allow bacteria culture to dry on the solid NGM (typically takes about 24 hours).</li>
	<li>Once dry, expose the surface of the plates to a UV dose sufficient to arrest growth of the bacteria. If you are using a Stratagene UV Stratalinker 2400:
	<ul>
		<li>Place plates in the Stratalinker and remove lids</li>
		<li>Close the door and turn on the Stratalinker</li>
		<li>Press ‘Energy’</li>
		<li>Enter ‘9999’ using the keypad</li>
		<li>Press ‘Start’</li>
		<li>The plates will be exposed to UV for approximately 5 minutes</li>
	</ul>
	</li>
	<li>Plates with UV-killed bacteria can be stored upside down at 4 °C for up to 1 month.</li>
</ol>
Part 2: Perform a timed egg laying to acquire an age-synchronized population of animals
In this section we generate a population of worms with a common hatch-date. This is accomplished by allowing reproductively active adults to lay eggs on a plate for a defined period of time and allowing those eggs to develop.
<ol>
	<li>(Optional) Transfer approximately 20 young adult worms to a fresh NGM plate without FUDR. Leave plates at 25 °C in order to allow worms to propagate and eat through all of the bacterial food. After the bacterial food has been consumed newly hatched worms will enter the growth arrested dauer stage. You will start to see dauer larva around within a week, depending on how many worms you transfer to the plate initially. Dauer larva can be used for the next steps for up to 1 month.</li>
	<li>(Optional) Transfer 20 to 30 dauer larva to a fresh NGM plate without FUDR. In the presence of food dauer larva will become reproductively active adults within 2 days and remain reproductively active for a few days thereafter at 25 °C.</li>
	<li>Transfer 10 to 15 reproductively active adults to a fresh NGM plate without FUDR. This plate is referred to as the timed egg laying (TEL) plate.</li>
	<li>Leave the TEL plate at 25 °C for 6 hours in order to allow the worms time to lay eggs.</li>
	<li>Remove adult worms from TEL plate. Plate can be visually inspected for eggs before removing adults. If the worms have not laid a sufficient number of eggs the TEL plate can be left at 25 °C for up to a total of 24 hours before removing adult worms.</li>
	<li>Place TEL plate at 20 °C until the eggs have hatched and the worms have developed to the L4 larval stage (this usually takes 2 days for wild type C. elegans, but can take longer for strains with slow development phenotypes).</li>
</ol>
Part 3: Score animals for life span
In this section we follow the age-synchronized population of worms from Part 2 until they die. Worms are maintained on Amp/FUDR plates to prevent egg production and bacterial contamination and are considered dead when they fail to respond to external stimuli.
<ol>
	<li>Transfer L4 larva to seeded Amp/FUDR plates. For each strain or condition being tested, it is typical to set up 2-3 plates with 25-30 worms per plate. Images of the C. elegans life stages are provided for reference (Figure 1).1</li>
	<li>Place Amp/FUDR plates at 20 °C for 24 hours.</li>
	<li>After 24 hours, visually assess worms, media, and bacteria. Transfer worms to fresh Amp/FUDR plates if any of the following is observed:
	<ul>
		<li>Worms have eaten most of the bacterial food. Early in the life span experiment worms will have to be transferred 1 to 2 times a week to prevent the bacteria from being depleted.</li>
		<li>A significant number of larvae are present. This is usually an indication that the worms were transferred to the Amp/FUDR plates as young adults instead of L4s and were able to lay some eggs before the FUDR took effect. The FUDR will generally prevent the larvae from growing into full adults, but occasionally a few will grow into adults and become confused with the experimental animals.</li>
		<li>Live /growing bacteria are observed. Generally, the combination of Amp and UV-killing will ensure that no live bacteria contaminate the experiment, but occasionally it occurs.</li>
		<li>Fungal growth is observed on the media. If caught early enough, fungal growth can usually be cut out using a pipette tip or spatula. Once it grows to be larger than a few millimeters in diameter it is usually easier to transfer the worms to a new plate.</li>
		<li>Using the dissection scope, determine whether each worm is alive or dead.</li>
		<li>Gently tap the plate. The worm is alive if it moves in response to the tapping.</li>
		<li>If the worm does not respond to tapping the plate, zoom in on the head region.</li>
		<li>Gently tap the worm’s head with platinum transfer pick. Score the worm as dead if it does not respond by moving its head.</li>
		<li>Dead worms can be removed from the plate.</li>
	</ul>
	</li>
	<li>Record the date and the number of worms that are alive and dead.</li>
	<li>Return plates to 20 °C.</li>
	<li>Repeat steps 3.3 through 3.6 every 2 to 3 days until all worms have died.</li>
</ol>
Part 4: Representative Results.
The raw data produced by a nematode life span experiment is a list of dates with corresponding numbers of worms that are alive and dead for each strain tested. The number of worms that die on each day is typically inverted to calculate the proportion of worms alive on each day (Figure 2A), which is plotted graphically as a survival curve (Figure 2B; the day of the timed egg laying is considered day 0). The life span for each individual worm in the study can be calculated from the count of worms that die on each day and used to calculate mean and median life span for comparison between strains. The count of number of worms alive on each day is not used directly in life span analysis. Worms maintained on solid media will occasionally ‘flee’, or crawl either up the wall of the plate or down beneath the media. The number of worms alive on each day can be used to determine how many worms fled throughout the course of the experiment. Worms that flee are typically removed from analysis. As a benchmark, the typical median life span for N2, the C. elegans wild-type strain, maintained on UV-killed bacteria at 20 °C is approximately 25 days as measured from egg.
Part 5: Solutions
<table border="0">
	<tbody>
		<tr>
			<td colspan="2">Nematode Growth Media (NGM), 100 mL:</td>
		</tr>
		<tr>
			<td colspan="2">Combine:</td>
		</tr>
		<tr>
			<td>0.3 g</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>0.25 g</td>
			<td>Peptone</td>
		</tr>
		<tr>
			<td>2 g</td>
			<td>Agar</td>
		</tr>
		<tr>
			<td colspan="2">Autoclave for 40 minutes and let cool to 55 °C, then add:</td>
		</tr>
		<tr>
			<td>100 µL</td>
			<td>1 M MgSO4</td>
		</tr>
		<tr>
			<td>100 µL</td>
			<td>5 mg/mL Cholesterol</td>
		</tr>
		<tr>
			<td>100 µL</td>
			<td>1 M CaCl2</td>
		</tr>
		<tr>
			<td>1.625 mL</td>
			<td>1.5 M KPi pH 6.0</td>
		</tr>
		<tr>
			<td colspan="2">Liquid NGM can be used immediately to pour plates or allowed to solidify and stored long-term at room temperature</td>
		</tr>
	</tbody>
</table>
<table border="0">
	<tbody>
		<tr>
			<td colspan="2">Luria Broth (LB), 1L:</td>
		</tr>
		<tr>
			<td>10 g</td>
			<td>BactoTryptone</td>
		</tr>
		<tr>
			<td>5 g</td>
			<td>Yeast Extract</td>
		</tr>
		<tr>
			<td>10 g</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>10 mL</td>
			<td>1 M Tris pH 8.0</td>
		</tr>
		<tr>
			<td>1 L</td>
			<td>deionized water</td>
		</tr>
		<tr>
			<td colspan="2">Autoclave and store at room temperature.</td>
		</tr>
	</tbody>
</table>
<table border="0">
	<tbody>
		<tr>
			<td colspan="2">1 M MgSO4, 300 mL:</td>
		</tr>
		<tr>
			<td>73.95 g</td>
			<td>MgSO4</td>
		</tr>
		<tr>
			<td>300 mL</td>
			<td>deionized water</td>
		</tr>
		<tr>
			<td colspan="2">Autoclave and store at room temperature.</td>
		</tr>
	</tbody>
</table>
<table border="0">
	<tbody>
		<tr>
			<td colspan="2">5 mg/mL Cholesterol, 200 mL:</td>
		</tr>
		<tr>
			<td>1 g</td>
			<td>cholesterol</td>
		</tr>
		<tr>
			<td>200 mL</td>
			<td>100% ethanol</td>
		</tr>
		<tr>
			<td colspan="2">Filter sterilize and store at room temperature.</td>
		</tr>
	</tbody>
</table>
<table border="0">
	<tbody>
		<tr>
			<td colspan="2">1 M CaCl2, 500 mL:</td>
		</tr>
		<tr>
			<td>27.75 g</td>
			<td>CaCl2</td>
		</tr>
		<tr>
			<td>500 mL</td>
			<td>deionized water</td>
		</tr>
		<tr>
			<td colspan="2">Filter sterilize and store at room temperature.</td>
		</tr>
	</tbody>
</table>
<table border="0">
	<tbody>
		<tr>
			<td colspan="2">1.5 M KPi pH 6.0, 1L:</td>
		</tr>
		<tr>
			<td colspan="2">Combine:</td>
		</tr>
		<tr>
			<td>31.4 g</td>
			<td>KPi dibasic</td>
		</tr>
		<tr>
			<td>179.6 g</td>
			<td>KPi monobasic</td>
		</tr>
		<tr>
			<td>850 mL</td>
			<td>deionized water</td>
		</tr>
		<tr>
			<td colspan="2">Heat while mixing to allow KPi to dissolve into solution. Adjust pH to 6.0 with 10 N NaOH.</td>
		</tr>
		<tr>
			<td colspan="2">Add deionized water to 1 L.</td>
		</tr>
		<tr>
			<td colspan="2">Autoclave and store at room temperature.</td>
		</tr>
	</tbody>
</table>
<table border="0">
	<tbody>
		<tr>
			<td colspan="2">1 M Tris, pH 8.0:</td>
		</tr>
		<tr>
			<td>60.57 g</td>
			<td>Tris</td>
		</tr>
		<tr>
			<td>400 mL</td>
			<td>deionized water</td>
		</tr>
		<tr>
			<td colspan="2">Adjust pH to 8.0 with HCl.</td>
		</tr>
		<tr>
			<td colspan="2">Add deionized water to 500 mL.</td>
		</tr>
		<tr>
			<td colspan="2">Filter sterilize and store at room temperature.</td>
		</tr>
	</tbody>
</table>
<table border="0">
	<tbody>
		<tr>
			<td colspan="2">150 mM Fluorodeoxyuridine (FUDR), 10 mL:</td>
		</tr>
		<tr>
			<td>0.3693 g</td>
			<td>FUDR</td>
		</tr>
		<tr>
			<td>10 mL</td>
			<td>sterile deionized water</td>
		</tr>
		<tr>
			<td colspan="2">Store at -20 °C.</td>
		</tr>
	</tbody>
</table>
<table border="0">
	<tbody>
		<tr>
			<td colspan="2">100 mg/mL Ampicillin (Amp), 10 mL:</td>
		</tr>
		<tr>
			<td>1 g</td>
			<td>Ampicillin</td>
		</tr>
		<tr>
			<td>10 mL</td>
			<td>sterile deionized water</td>
		</tr>
		<tr>
			<td colspan="2">Store at -20 °C.</td>
		</tr>
	</tbody>
</table>
<table border="0">
	<tbody>
		<tr>
			<td colspan="2">50 mg/mL Carbenicillin (Carb), 10 mL:</td>
		</tr>
		<tr>
			<td>500 mg</td>
			<td>Carbenicillin</td>
		</tr>
		<tr>
			<td>10 mL</td>
			<td>sterile deionized water</td>
		</tr>
		<tr>
			<td colspan="2">Store at -20 °C.</td>
		</tr>
	</tbody>
</table>
<table border="0">
	<tbody>
		<tr>
			<td colspan="2">1 M Isopropyl β-d-Thiogalactopyranoside (IPTG), 10 mL:</td>
		</tr>
		<tr>
			<td>2.38 g</td>
			<td>IPTG</td>
		</tr>
		<tr>
			<td>10 mL</td>
			<td>sterile deionized water</td>
		</tr>
		<tr>
			<td colspan="2">Store at -20 °C.</td>
		</tr>
	</tbody>
</table>
<img alt="Nematode lifecycle diagram: egg to adult stages, including L1-L4 larvae and adult male development." src="/files/ftp_upload/1152/Figure%201.jpg" data-altupdatedat="1773047343000" data-alt="figure 1"> 
Figure 1. Bright field images of C. elegans life stages, including egg, the four larval stages (L1 – L4), and adult. All panels show hermaphrodites except the lower-right, which shows an adult male (Image from Wood (1988)).
<img alt="Life span analysis; survival outcome table and graph for Wild Type vs. daf-2 over time (days)." src="/files/ftp_upload/1152/Figure%202.jpg" data-altupdatedat="1773047343000" data-alt="figure 2"> 
Figure 2. Representative results from a C. elegans life span experiment comparing wild type strain N2 to a strain containing a mutation in the daf-2 gene. (A) A table showing collected data, including number of days since worms were eggs, number of dead worms observed on each day, and the percentage of the original sample remaining alive on each day (as calculated from the daily counts of dead worms). (B) Survival curves corresponding to the life span data provided in (A).
<img alt="C. elegans development stages, microscopy: bright field vs. DAPI fluorescence, Day 4 &amp; 8 comparison." src="/files/ftp_upload/1152/Figure%203.jpg" data-altupdatedat="1773047343000" data-alt="figure 3"> 
Figure 3. Representative comparison of lipofuscin between young and old adult C. elegans. Bright field images are shown on left and DAPI channel fluorescence images on the right. Top panels show a 4 day old worm and bottom panels show an 11 day old worm (as measured from egg).

<ol><li>Wood, W. B. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWood%2C+WB+%22The+Nematode+Caenorhabditis+elegans%22">The Nematode Caenorhabditis elegans</a>. Wood, W. B. , Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. 1-16 (1988).</li>
<li>Sutphin, G. L., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dietary+restriction+by+bacterial+deprivation+increases+life+span+in+wild-derived+nematodes%5BTitle%5D)+AND+%22Exp+Gerontol%22%5BJournal%5D)">Dietary restriction by bacterial deprivation increases life span in wild-derived nematodes.</a> Exp Gerontol. 43, 130-135 (2008).</li>
<li>Garigan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+analysis+of+tissue+aging+in+Caenorhabditis+elegans%3A+a+role+for+heat-shock+factor+and+bacterial+proliferation%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation.</a> Genetics. 161, 1101-1112 (2002).</li>
<li>Chen, D., Pan, K. Z., Palter, J. E., Kapahi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Longevity+determined+by+developmental+arrest+genes+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Aging+Cell%22%5BJournal%5D)">Longevity determined by developmental arrest genes in Caenorhabditis elegans.</a> Aging Cell. 6, 525-523 (2007).</li>
<li>Curran, S. P., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lifespan+regulation+by+evolutionarily+conserved+genes+essential+for+viability%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">Lifespan regulation by evolutionarily conserved genes essential for viability.</a> PLoS Genet. 3, e56-e56 (2007).</li>
<li>Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rates+of+behavior+and+aging+specified+by+mitochondrial+function+during+development%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Rates of behavior and aging specified by mitochondrial function during development.</a> Science. 298, 2398-2401 (2002).</li>
<li>Hamilton, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+systematic+RNAi+screen+for+longevity+genes+in+C.+elegans%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">A systematic RNAi screen for longevity genes in C. elegans.</a> Genes Dev. 19, 1544-1555 (2005).</li>
<li>Hansen, M., Hsu, A. L., Dillin, A., Kenyon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+genes+tied+to+endocrine%2C+metabolic%2C+and+dietary+regulation+of+lifespan+from+a+Caenorhabditis+elegans+genomic+RNAi+screen%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">New genes tied to endocrine, metabolic, and dietary regulation of lifespan from a Caenorhabditis elegans genomic RNAi screen.</a> PLoS Genet. 1, 119-128 (2005).</li>
<li>Lee, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+systematic+RNAi+screen+identifies+a+critical+role+for+mitochondria+in+C.+elegans+longevity%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity.</a> Nat Genet. 33, 40-48 (2003).</li>
<li>Smith, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-+and+calorie-independent+life+span+extension+from+dietary+restriction+by+bacterial+deprivation+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22BMC+Dev+Biol%22%5BJournal%5D)">Age- and calorie-independent life span extension from dietary restriction by bacterial deprivation in Caenorhabditis elegans.</a> BMC Dev Biol. 8, 49-49 (2008).</li>
<li>Hodgkin, J., Horvitz, H. R., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nondisjunction+Mutants+of+the+Nematode+CAENORHABDITIS+ELEGANS%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Nondisjunction Mutants of the Nematode CAENORHABDITIS ELEGANS.</a> Genetics. 91, 67-94 (1979).</li>
<li>Emmons, S. W., Sternberg, P. W. C. ELEGANS II. Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R. , Cold Spring Harbor Laboratory Press. Cold Springs Harbor, NY. (1997).</li>
<li>Lipton, J., Kleemann, G., Ghosh, R., Lints, R., Emmons, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mate+searching+in+Caenorhabditis+elegans%3A+a+genetic+model+for+sex+drive+in+a+simple+invertebrate%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Mate searching in Caenorhabditis elegans: a genetic model for sex drive in a simple invertebrate.</a> J Neurosci. 24, 7427-7434 (2004).</li>
<li>Davis, B. O., Anderson, G. L., Dusenbery, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Total+luminescence+spectroscopy+of+fluorescence+changes+during+aging+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Total luminescence spectroscopy of fluorescence changes during aging in Caenorhabditis elegans.</a> Biochemistry. 21, 4089-4095 (1982).</li>
<li>Klass, M. R. <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>
<li>Steinkraus, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dietary+restriction+suppresses+proteotoxicity+and+enhances+longevity+by+an+hsf-1-dependent+mechanism+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Aging+Cell%22%5BJournal%5D)">Dietary restriction suppresses proteotoxicity and enhances longevity by an hsf-1-dependent mechanism in Caenorhabditis elegans.</a> Aging Cell. 7, 394-404 (2008).</li>
</ol>

The genetic control of longevity has been studied extensively in C. elegans, largely due to the ease and rapidity with which life span can be determined. The protocol discussed in this article describes a basic framework for obtaining reproducible life span data in C. elegans and can also be applied to relatednematode species.2 By making some simple alterations these procedures can be adapted to measure life span under a variety of conditions. Here we will discuss several common variations, i...

<table><tbody><tr><td>Agar</td><td>Reagent</td><td>Fisher Scientific</td><td>DF0145-17-0 (214530)</td><td></td></tr><tr><td>Ampicillin</td><td>Reagent</td><td>MidSci</td><td>0339</td><td></td></tr><tr><td>BactoTryptone</td><td>Reagent</td><td>Fisher Scientific</td><td>DF0123-17-3 (211705)</td><td></td></tr><tr><td>BactoPeptone</td><td>Reagent</td><td>Fisher Scientific</td><td>DF0118-17-0 (211677)</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Reagent</td><td>Fisher Scientific</td><td>NC9699248 (1332-01)</td><td></td></tr><tr><td>Carbenicillin</td><td>Reagent</td><td>Gold Biotechnology</td><td>C0109</td><td></td></tr><tr><td>Cholesterol</td><td>Reagent</td><td>Sigma-Aldrich</td><td>C75209</td><td></td></tr><tr><td>Fluorodeoxyuridine (FUDR)</td><td>Reagent</td><td>Sigma-Aldrich</td><td>F0503</td><td></td></tr><tr><td>Isopropyl &amp;#946;-d-Thiogalactopyranoside (IPTG)</td><td>Reagent</td><td>Gold Biotechnology</td><td>I2481C5</td><td></td></tr><tr><td>KPi Dibasic</td><td>Reagent</td><td>Fisher Scientific</td><td>5087862 (3252-01)</td><td></td></tr><tr><td>KPi Monobasic</td><td>Reagent</td><td>Fisher Scientific</td><td>5087861 (3246-01)</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Reagent</td><td>Fisher Scientific</td><td>NC9561800 (2500-01)</td><td></td></tr><tr><td>NaCl</td><td>Reagent</td><td>Fisher Scientific</td><td>S251</td><td></td></tr><tr><td>Tris</td><td>Reagent</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Yeast Extract</td><td>Reagent</td><td>Fisher Scientific</td><td>DF0886-17-0 (288620)</td><td></td></tr></tbody></table>

measuring caenorhabditis elegans life span on solid media

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.

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 ...

In order to measure lifespan in C Elgan, an age synchronized population is generated using a timed egg lang reproductively. Active adult hermaphrodite worms are transferred onto nematode growth media plates seeded with UV killed e coli allowed to lay eggs for six to eight hours and then removed over the next two days, worms hatch from these eggs and grow to the L four larval stage. The L four worms are transferred to new NGM plates containing ampicillin to prevent live bacterial contamination and FUDR to prevent reproduction and allowed to develop into adults.From this point forward, the adult worms are observed every two to three days until all worms have died. At each time point, dead worms are removed from the plate and the number of live and dead worms recorded. I'm George Seton from the laboratory of Matt Capline in the Department of Pathology at the University of Washington.Today we're gonna show you how to measure lifespan and see our hepatitis elegance. We use this procedure in our laboratory to study the genetics of aging. So let's get started.A basic lifespan experiment requires two types of plates, standard NGM plates, which contain no additives, and AMP FUDR plates, which have both ampicillin and fluoro deoxy uridine added to the NGM. Both types of plates are seated with e coli OP 50 bacteria, which is subsequently killed by exposure to uv. Begin by preparing 10 milliliters of NGM solution.Per 60 millimeter Petri dish completely melt solid NGM by microwaving on high in 32nd pulses. Swirl media between pulses to prevent pressurized gas bubbles from building up place liquid NGM in a 55 degree Celsius water bath or on the bench to allow the media to cool. Once the media has cooled to 55 degrees Celsius, at which point you should be able to comfortably hold the glass container with bare hands, it is ready to have drugs added.And to pour the plates for plates with drugs, add 33 microliters of 150 millimolar FUDR and 100 microliters of 100 milligram per milliliter. Ampicillin per 100 milliliter NGM and swirl to mix ampicillin is used to prevent foreign bacterial contamination and FUDR to prevent eggs from hatching. So worms will not need to be transferred every few days in order to separate them from growing larvae.Now pour the media and allow the plates to dry on the bench for two days on the day before the plates finished drying seed liquid LB culture with a single colony from a fresh streak of e coli. OP 50 bacteria on LB auger. Prepare at least 0.5 milliliter culture per 60 millimeter plate.Place the OP 50 culture in a 37 degree Celsius shaker and allow bacteria to grow overnight. The next day pellet the OP 50 bacteria with 3, 500 g of centrifugal force for 10 minutes. Decant enough of the supernatant to resuspend the bacteria at a 10 x concentration.Now inoculate each plate with 100 microliters of the concentrated culture. Avoid touching the pipette tip to the surface of the NGM as flaws in the surface of the media will allow the worms to burrow swirl plates until the bacteria cover the central area of the NGM without coming near the plate walls and leave plates to dry on the bench for 24 hours. Once dry, expose the surface of the plates to a UV dose strong enough to stop the bacterial growth, we use a strata gene UV straddle linker 2, 400.Begin by loading the plates in the straddle linker without their lids on. Now press energy. Enter 9 9 9 9 on the keypad and press start.The plates will be exposed to UV for approximately five minutes. Plates with UV killed bacteria can be stored upside down at four degrees Celsius for up to a month. This section describes how to generate a population of worms with a common hatch date.Begin by transferring approximately 20 young adult worms to a fresh NGM plate without drugs. Leave the plate at 25 degrees Celsius for approximately a week to allow worms to propagate and eat through all of the bacterial food within a week. Newly hatched worms in the growth arrested dower stage should appear transferred 20 to 30 dower larvae to a fresh NGM plate without drugs and incubate them at 25 degrees Celsius in the presence of food.The dower larvae will become reproductively active adults within two days and remain reproductively active for a few days thereafter. Now transfer 10 to 15 of the reproductively active adults to a fresh NGM plate without drugs. This plate is referred to as the timed egg laying or TEL plate.Leave the TEL plate at 25 degrees Celsius so the worms can lay eggs. The animals that hatch from these eggs will be the experimental animals and have their lifespan scored after six hours. Inspect the TEL plate for eggs.If there are not enough eggs yet, leave the TEL plate at 25 degrees Celsius for up to a total of 24 hours. Checking periodically for eggs. When a sufficient number of eggs have been laid, remove the adults from the TEL plate.A typical lifespan experiment will require 30 to 90 eggs per experimental group, but several hundred eggs can be generated on a single TEL plate. Approximately 150 eggs will be laid by 10 to 15 reproductively active adults in six to eight hours. When comparing experimental groups, statistical significance can be achieved for large lifespan differences with as few as 30 animals.More animals should be used to distinguish more modest differences. Now place the TEL plate without adults at 20 degrees Celsius until the eggs of hatched and the worms have developed to the L four larval stage. This usually takes two days for wild type C elegance, but can take longer for strains with slow development phenotypes.This section describes how to follow the life of an age synchronized population. First, transfer the L four larva from the TEL plate to seated AMP FUDR plates. For each strain or condition being tested, it is typical to set up two to three plates with 25 to 30 worms per plate.Incubate the AMP FUDR plates at 20 degrees Celsius for 24 hours. And then visually assess the worms, media and bacteria transfer worms to fresh AMP FUDR plates. If the worms have eaten most of the bacterial food, this will typically occur once or twice a week.Early in the experiment, if a significant number of larvae are present, this usually indicates that the worms were transferred to the AMP FUDR plates as young adults instead of as L fours. Young adults can lay eggs before the FUDR takes effect, and occasionally larvae escape the effects of the FUDR and grow into adults. These larvae will confound the experiment in this situation, transfer the adult worms to fresh plates and discard the old plates containing the larvae.Worms should also be transferred to fresh plates if living growing bacteria are observed. Generally the combination of AMP and UV killing will ensure that no live bacteria contaminate the experiment, but occasionally it occurs. Also, transfer the worms.If fungal growth is observed on the media. If caught early enough, fungus can be cut out using a pipette tip or spatula. Once it grows to be larger than a few millimeters in diameter, it is usually easier to transfer the worms to a new plate.Score all worms for lifespan every two to three days. Using a dissection scope. Determine whether each worm is alive or dead.Gently tap the plate. The worm is alive if it moves in response to the tapping. If the worm does not respond to tapping the plate, zoom in on the head region, gently tap the worm's head with a platinum transfer pick.Score the worm as dead. If it does not respond by moving its head, remove the dead worm from the plate. After checking all the worms, record the date and the number of worms that are alive and dead.Worms maintained on solid media will occasionally escape the plate surface by burrowing or crawling up the wall. Worms that flee are completely omitted from the study. When finished, return the plates to the 20 degrees Celsius incubator.Wait another two to three days and then check the plates again. Continue this regime until every worm has died. The number of worms that die on each day is typically inverted to calculate the proportion of worms alive on each day, this data is plotted graphically as a survival curve with the day of the timed egg laying considered.Day zero. The typical median lifespan for N two. The Sea elgan wild type strain maintained on UV kill bacteria at 20 degrees Celsius is approximately 25 days as measured from egg.We've just shown you how to measure lifespan and see elegance. You can modify this basic procedure to test a variety of environmental conditions, including dietary restriction and RNA interference, which is discussed in the supporting text. So that's it.Thanks for watching and good luck with your experiment.

measuring-caenorhabditis-elegans-life-span-on-solid-media

Research

JoVE Journal

Biology

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

This video describes a traditional, plate-based method of measuring the lifespan of C. elegans. The example protocol measures the lifespan of worms treated with RNAi.

Lifespan Analysis: Measuring C. elegans Longevity

C. elegans egg-laying behavior can provide important information in developmental, reproductive, and anatomical studies.  This video introduces the important steps of an egg laying assay and shows a sample experiment in which a drug is tested for reproductive toxicity.

Egg Laying Assay: A Method to Quantify the Egg-Laying Behavior of C. elegans

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

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

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

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

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

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

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

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

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Learn to measure C. elegans lifespan on solid media using synchronized populations and UV-killed bacteria. This protocol enables accurate aging research with systematic mortality tracking.

Measuring Caenorhabditis elegans Life Span on Solid Media

Explore More Videos