Name: Solid Plate-based Dietary Restriction in Caenorhabditis elegans (Video) | JoVE Journal
Uploaded: 2011-05-28T14:00:00.000+00:00
Duration: 6 min 13 s
Description: Learn solid plate-based dietary restriction in C. elegans using killed bacteria to study longevity, reproduction, and metabolism. This protocol enables precise control of food intake for aging research.

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5 minutes

This work was supported by a pilot award from the Nathan Shock Center (P30 AG13283) and a R01 grant (R01 AG028516) from the National Institute on Aging (NIA) to A.-L.H.

1. Establishing a calibration curve for the estimation of bacteria concentrations
This section describes a method for the estimation of bacteria concentration for any given bacteria strain (e.g. OP50, HT115) that will be used as a food source for Dietary restriction experiments in C. elegans. Separate calibration curves should be established for each bacteria strain used. 
<ol>
 <li>Inoculate a single colony of E. coli OP50 bacteria picked from a fresh streak of bacteria on LB agar in 3 mL liquid LB broth.</li>
 <li>Place OP50 culture in 37 °C shaker and allow bacteria to grow overnight. </li>
 <li>Prepare serial 2-fold dilutions of the bacterial suspension from the overnight culture (2x, 4x, 8x, 16x, 32x).</li>
 <li>Measure the absorbance (optical density) of each dilution at 600 nm by spectrophotometer in triplicate.</li>
 <li>Further dilute each bacterial suspension 1,000,000-fold (1000-fold dilution twice).</li>
 <li>Spread 0.4 mL of bacterial suspension of each dilution evenly on a 100 mm LB agar plate.</li>
 <li>Place LB plates with OP50 in a 37 °C incubator for 18-24 hours.</li>
 <li>Count the number of bacterial colonies grown on each plate. The colony counts are used to calculate cfu (colony forming unit) per mL for each sample.</li>
 <li>The relationship between measured OD600 values and OP50 bacteria concentrations can be established by plotting OD600 as a function of bacterial concentration (cfu/mL) and performing a linear regression analysis (Figure 1).</li>
</ol>
 Solutions:
<table border="1">
 <tbody>
 <tr>
 <td colspan="2">Luria Broth (LB), 1L:</td>
 </tr>
 <tr>
 <td>10 g</td>
 <td>Bacto-tryptone</td>
 </tr>
 <tr>
 <td>5 g </td>
 <td>Yeast Extract</td>
 </tr>
 <tr>
 <td>10 g</td>
 <td>NaCl</td>
 </tr>
 </tbody>
</table>
Add deionized water to 1L. 
 Autoclave and store at room temperature. 
2. Preparation of solid plate-based dietary restriction (sDR) plates
This section describes the preparation of plates for use in solid-plate based dietary restriction (sDR) experiments. Normally, we consider a bacteria concentration of 1 x 1011 cfu/mL as the ad libitum feeding condition and 1 x 108 cfu/mL as the optimal DR feeding condition for this method. However, 4-6 different bacteria concentrations may be needed to establish a complete dose-dependent relationship for sDR and its responses.
<ol>
 <li>Inoculate a single colony of E. Coli OP50 bacteria in 3 mL liquid LB broth.</li>
 <li>Place the OP50 culture in 37 °C shaker and allow the bacteria to grow for 6-8 hours.</li>
 <li>Transfer the OP50 culture to a new flask containing 500 mL LB broth, and place the OP50 culture back in 37 °C shaker overnight. </li>
 <li>Measure the OD600 of the OP50 culture to determine the bacteria concentration (the concentration is calculated using the calibration curve generated in Part 1). Appropriate dilutions may be needed for an accurate measurement of absorbance. </li>
 <li>Pellet OP50 bacteria by centrifugation at 1,500 g for 20 minutes.</li>
 <li>Resuspend bacteria to a concentration of 1 x 1012 cfu/mL.</li>
 <li>Prepare 10 mL bacteria culture in six different concentrations: 1 x 1012, 1011, 1010, 109, 108, 107 cfu/mL.</li>
 <li>Pipette 0.2 mL of bacteria culture into the center of a 60 mm solidified NGM plate. Avoid touching the surface of the plate with pipette tip, as nicks on the surface allow worms to burrow into the agar plates.</li>
 <li>Place plates in 37 °C incubator for 1 hour.</li>
 <li>Apply 10μL of 100mg/mL carbenicillin and 10 μL of 50mg/mL kanamycin to each plate to arrest growth of the bacteria. </li>
 <li>Store plates with different concentrations of antibiotic-killed bacteria at 4 °C for future use (up to one month). It is important to prepare and store enough plates for the entire planned experiment at least 1-2 days ahead. Using the same batch of plates for the entire experiments is preferable.</li>
 <li>To verify the viability of the bacteria, scrape some bacteria out of the sDR plates and spread it on an empty NGM plate with carbenicillin and kanamycin. Place the plate in 37 °C incubator for 6-8 hours and then check for growth of the bacteria (no bacteria growth should be observed).</li>
</ol>
Solutions
<table border="1">
 <tbody>
 	<tr>
 <td colspan="2">Nematode Growth Media (NGM), 1L:</td>
 </tr>
 <tr>
 <td>3 g </td>
 <td>NaCl</td>
 </tr>
 <tr>
 <td>2.5 g</td>
 <td>Bacto-Peptone </td>
 </tr>
 <tr>
 <td>20g</td>
 <td>Agar</td>
 </tr>
 </tbody>
</table>
Autoclave for 40 minutes and let cool to 65 °C, then add:
<table border="1">
 <tbody>
 <tr>
 <td>1 mL </td>
 <td>1 M MgSO4</td>
 </tr>
 <tr>
 <td>1 mL</td>
 <td>1 M CaCl2</td>
 </tr>
 <tr>
 <td>1 mL</td>
 <td>5 mg/mL Cholesterol</td>
 </tr>
 <tr>
 <td>25 mL</td>
 <td>1M Potassium Phosphate, pH 6.0</td>
 </tr>
 </tbody>
</table>
Immediately pour liquid NGM onto 60 mm x 15 mm petri dishes in 10 mL aliquots
3. Setting up sDR experiments
For most of the sDR-related studies, such as life span analysis and fertility profiling, preparation of an age-synchronized population of worms is essential. For other studies, simply transfer worms from a regular OP50 plate to an sDR plate to initiate the dietary restriction. In this section, a protocol to set up a lifespan analysis for sDR animals is described as an example. 
<ol>
 <li>Use a platinum worm picker to transfer 20-30 reproductively active adults onto several regular NGM plates with OP50. Depending on the number of eggs required for the experiments, more adults may be needed.</li>
 <li>Leave the plates at 20°C for 4 hours to allow egg-laying.</li>
 <li>Remove the adults from the plate. Plates should be visually inspected for eggs.</li>
 <li>Leave the plates at 20°C and allow the progeny to develop into the Late-L4/day 1 adult stage. Usually, this takes 3 days for N2 wild types worms at 20 °C.</li>
 <li>Transfer a suitable number of day 1 adults onto sDR plate to initiate the dietary restriction. </li>
 <li>For a lifespan analysis, set up a total of 6-8 plates with 10-20 worms per plate for each sDR condition. </li>
 <li>Score the viability of each worm every 2 days until all the worms have died. It is critical that worms be transferred to fresh sDR plates every 12-48 hours to avoid complete deprivation of food. </li>
</ol>
4. Representative Results:
Shown in Figure 1 is a plot of a representative calibration curve for the estimation of OP50 concentration in relation to OD600. The equation generated by liner regression analysis can be used for all future calculations of OP50 bacteria concentrations. Shown in Figure 2 is an example of assaying the effect of chronic sDR treatments on mean lifespan of different worm strains. The blue line represents a normal dose-dependent response of wild-type worms to sDR. The red line indicates that the lifespan effect of sDR is partially suppressed by drr-2 o.e., while daf-16(mu86) has no effect on sDR-induced lifespan extension. Detail method for performing lifespan analysis in C. elegans is described in previous literature 1-3.
<img src="/files/ftp_upload/2701/2701fig1.jpg" alt="Bacteria concentration vs. optical density graph, linear regression, R²=0.99505, OD600 analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Representative result of a calibration curve for the estimation of E. Coli OP50 concentration. OP50 concentration is plotted as a function of measured OD600 values. Error bars = S.D. Overnight bacterial culture was diluted 1.5x, 2x, 4x, 8x, 16x, 32x before absorbance measurement, and further diluted 1,000x twice before cfu measurement.
<img src="/files/ftp_upload/2701/2701fig2.jpg" alt="Bacteria concentration vs. mean lifespan graph; N2, daf-16(mu86), drr-2 o.e. analysis." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Representative result of a C. elegans lifespan experiment comparing wild type N2 animals (blue) with two different mutants (green and red) in response to sDR (Figure from Ching et al. 4). Error bars = S.E.M.

<ol><li>Hsu, A. L., Murphy, C. T., Kenyon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+aging+and+age-related+disease+by+DAF-16+and+heat-shock+factor%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Regulation of aging and age-related disease by DAF-16 and heat-shock factor.</a> Science. 300, 1142-1145 (2003).</li>
<li>Kenyon, C., Chang, J., Gensch, E., Rudner, A., Tabtiang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+C.+elegans+mutant+that+lives+twice+as+long+as+wild+type%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A C. elegans mutant that lives twice as long as wild type.</a> Nature. 366, 461-464 (1993).</li>
<li>Apfeld, J., Kenyon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+lifespan+by+sensory+perception+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Regulation of lifespan by sensory perception in Caenorhabditis elegans.</a> Nature. 402, 804-809 (1999).</li>
<li>Ching, T. T., Paal, A. B., Mehta, A., Zhong, L., Hsu, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((drr-2+encodes+an+eIF4H+that+acts+downstream+of+TOR+in+diet-restriction-induced+longevity+of+C.+elegans%5BTitle%5D)+AND+%22Aging+Cell%22%5BJournal%5D)">drr-2 encodes an eIF4H that acts downstream of TOR in diet-restriction-induced longevity of C. elegans.</a> Aging Cell. 9, 545-557 (2010).</li>
<li>Weindruch, R., Walford, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWeindruch%2C+R+%22The+Retardation+of+Aging+and+Disease+by+Dietary+Restriction%22">The Retardation of Aging and Disease by Dietary Restriction</a>. , Thomas. (1988).</li>
<li>Masoro, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subfield+history%3A+caloric+restriction%2C+slowing+aging%2C+and+extending+life%5BTitle%5D)+AND+%22Sci+Aging+Knowledge+Environ%22%5BJournal%5D)">Subfield history: caloric restriction, slowing aging, and extending life.</a> Sci Aging Knowledge Environ. , RE2-RE2 (2003).</li>
<li>Masoro, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overview+of+caloric+restriction+and+ageing%5BTitle%5D)+AND+%22Mech+Ageing+Dev%22%5BJournal%5D)">Overview of caloric restriction and ageing.</a> Mech Ageing Dev. 126, 913-922 (2005).</li>
<li>Houthoofd, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Axenic+growth+up-regulates+mass-specific+metabolic+rate%2C+stress+resistance%2C+and+extends+life+span+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Exp+Gerontol%22%5BJournal%5D)">Axenic growth up-regulates mass-specific metabolic rate, stress resistance, and extends life span in Caenorhabditis elegans.</a> Exp Gerontol. 37, 1371-1378 (2002).</li>
<li>Houthoofd, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((No+reduction+of+metabolic+rate+in+food+restricted+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Exp+Gerontol%22%5BJournal%5D)">No reduction of metabolic rate in food restricted Caenorhabditis elegans.</a> Exp Gerontol. 37, 1359-1369 (2002).</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>Bishop, N. A., Guarente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+neurons+mediate+diet-restriction-induced+longevity+in+C.+elegans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Two neurons mediate diet-restriction-induced longevity in C. elegans.</a> Nature. 447, 545-549 (2007).</li>
<li>Panowski, S. H., Wolff, S., Aguilaniu, H., Durieux, J., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PHA-4%2FFoxa+mediates+diet-restriction-induced+longevity+of+C.+elegans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">PHA-4/Foxa mediates diet-restriction-induced longevity of C. elegans.</a> Nature. 447, 550-555 (2007).</li>
<li>Lakowski, B., Hekimi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+genetics+of+caloric+restriction+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">The genetics of caloric restriction in Caenorhabditis elegans.</a> Proc Natl Acad Sci U S A. 95, 13091-13096 (1998).</li>
<li>Kaeberlein, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lifespan+extension+in+Caenorhabditis+elegans+by+complete+removal+of+food%5BTitle%5D)+AND+%22Aging+Cell%22%5BJournal%5D)">Lifespan extension in Caenorhabditis elegans by complete removal of food.</a> Aging Cell. 5, 487-494 (2006).</li>
<li>Greer, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+AMPK-FOXO+pathway+mediates+longevity+induced+by+a+novel+method+of+dietary+restriction+in+C.+elegans%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C. elegans.</a> Curr Biol. 17, 1646-1656 (2007).</li>
</ol>

No conflicts of interest declared.

Dietary restriction (DR) is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals 5-7. Both environmental and genetic manipulations have been used to model DR in C. elegans, including culturing worms in a semi-defined liquid medium 8,9, dilution of the bacterial food source in liquid medium 10-12, and use of behavior mutants that feed at a slower rate 13. However, genetic mimics of DR may produce ...

<table><tbody><tr><td>Carbenicillin</td><td>Duchefa Biochemie</td><td>C0109.25</td><td>Dissolved in water.</td></tr><tr><td>Kanamycin</td><td>Roche Group</td><td>10106801001</td><td>Dissolved in water.</td></tr><tr><td>Bacto-peptone</td><td>BD Biosciences</td><td>DF0118072</td><td></td></tr><tr><td>Bacto-tryptone</td><td>BD Biosciences</td><td>DF0123075</td><td></td></tr><tr><td>Bacto-yeast extract</td><td>BD Biosciences</td><td>DF0127071</td><td></td></tr><tr><td>Agar</td><td>BD Biosciences</td><td>DF00145170</td><td></td></tr><tr><td>Sterile culture tube</td><td>USA Scientific, Inc.</td><td>1485-0810</td><td></td></tr><tr><td>Spectrophotometer</td><td>Amersham</td><td>Gene Quant pro</td><td>Amersham is now part of GE Healthcare.</td></tr><tr><td>Dissecting Microscope</td><td>Carl Zeiss, Inc.</td><td>Stemi 2000</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter Inc.</td><td>Accuspin FR</td><td></td></tr></tbody></table>

solid plate-based dietary restriction in caenorhabditis elegans

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

The procedure performs dietary restriction on the standard abased plate in sea elegance to study its effect on longevity, reproduction, and metabolism. This is accomplished by first accurately measuring the concentration of the bacteria that will be used as a food source. The second step of the procedure is to properly dilute the bacteria culture into suitable concentrations.Then prepare abased SDR plates with killed bacteria. Finally set up SDR experiments with an age synchronized population of worms. Results of progeny production and lifespan measurements can show delayed and reduced progeny production as well as increased mean lifespan.The main advantage of this technique over 60 methods like Dian in AZA medium, is that all the experiment will be done on a standard solid AGA based play that is used by most warm laboratories For dietary restriction experiments and see elegance establish separate calibration curves for each bacteria strain used as a food source. Pick a single colony of e coli OP 50 bacteria from a fresh streak on lb agar and inoculate three milliliters of liquid lb broth culture in a 37 degree Celsius shaker overnight. Prepare serial twofold dilution of the overnight culture.Then measure the absorbence of each dilution at 600 nanometers by spectrophotometer in triplicate. Further dute each bacterial suspension, 1 million fold evenly seed 0.4 milliliters of bacterial suspension of each dilution on a 100 millimeter lb agri plate and incubate at 37 degrees Celsius for 18 to 24 hours. Count the number of bacterial colonies grown on each plate in order to calculate colony forming units per milliliter for each sample.Now plot OD 600 as a function of bacterial concentration and perform a linear regression analysis to establish the relationship between measured OD 600 values and OP 50 bacteria concentrations. For this method, a bacteria concentration of one times 10 to the 11th CFUs per milliliter is used in the ad lido feeding condition and one times 10 to the eighth CFU per milliliter as the optimal DR feeding condition. However, four to six different bacteria concentrations may be needed to establish a complete dose dependent relationship.For SDR and its responses. Prepare an inoculum with a single colony of e coli OP 50 bacteria in three milliliters of liquid LB broth, cultured in a 37 degree Celsius shaker for six to eight hours. Transfer the OP 50 culture to a new flask containing 500 milliliters of LB broth and grow overnight for the bacteria to reach saturation if necessary.Prepare appropriate dilution of the OP 50 culture for the measurement of absorbence at OD 600. To determine the bacteria concentration, pellet the bacteria by centrifugation at 1500 Gs for 20 minutes. Remove the supernatant and resuspend the bacteria to a concentration of one times 10 at of the 12th CFUs per milliliter.Now, prepare 10 milliliter bacteria cultures in six different concentrations, ranging from one times 10 of the 12th to one times 10 of the seventh CFU per milliliter pipette. Point two milliliters of bacteria culture into the center of a 60 millimeter solidified N GM plate. Avoid touching the surface of the plate with the pipette tip as nicks on the surface allow worms to burrow into the aerates.Place the plates in a 37 degree Celsius incubator for one hour. Then apply 10 microliters of 100 milligrams per milliliter carbonic soin and 10 microliters of 50 milligrams per milliliter can mycin to each plate to a rest growth of the bacteria. Store plates with different concentrations of antibiotic killed bacteria at four degrees Celsius for future use for up to one month.It is important to prepare and store enough plates, preferably the same batch for the entire plant. Experiment at least one to two days ahead to verify the viability of the bacteria. Scrape some bacteria out of the SDR plates and spread it on an empty N GM plate with carbonic, sein and antimycin.Place the plate in a 37 degree Celsius incubator for six to eight hours and confirm the absence of bacterial growth. For most of the SDR related studies, such as lifespan analysis and progeny production profiling, preparation of an age synchronized population of worms is essential. Use a platinum worm picker to transfer 20 to 30 reproductively active adults onto several regular NGM plates.With OP 50. Depending on the number of eggs required for the experiments, more adults may be needed. Leave the plates at 20 degrees Celsius for four hours to allow egg laying.Remove the adults from the plate and visually inspect the plates for eggs. Continue to incubate the plates at 20 degrees Celsius to allow the progeny to develop into the late L four day one adult stage. Usually this takes three days for N two wild type worms at 20 degrees Celsius.For lifespan analysis. Transfer 10 to 12 day one adult worms onto an SDR plate to initiate the dietary restriction. To obtain sufficient statistic power, use a total of 60 to 100 worms for each SDR condition.Score the viability of each worm every two to three days until all the worms have died. Importantly, transfer the worms to fresh SDR plates every other day to avoid starvation. This calibration curve plots OP 50 concentration in relation to OD 600.The equation generated by linear regression analysis can be used for all future calculations of OP 50 bacteria concentrations. This study assays the effect of chronic SDR treatments on mean lifespan of different worm strains compared to a normal dose dependent response of wild type worms to SDR. The lifespan effect of SDR is partially suppressed by DR two overexpression while DAF 16 MU 86 mutation has no effect on SDR induced lifespan extension.After watching this video, you should have a good understanding of how to perform Dian on the standard AGA based play in sea elegance.

solid-plate-based-dietary-restriction-in-caenorhabditis-elegans

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Learn solid plate-based dietary restriction in C. elegans using killed bacteria to study longevity, reproduction, and metabolism. This protocol enables precise control of food intake for aging research.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

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