Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Overview: The protocol is split into four parts. Part 1 describes how to prepare the feeding bacteria. Part 2 describes how to prepare the worm culture. Part 3 describes how to score lifespan. Part 4 shows some representative results, and Part 5 describes how to prepare the required solutions. Lifespan experiments take several weeks to complete. For each step of the protocol week, day, and time of the day is included to facilitate planning. The L4 stage (Day 0) is used as the reference point for the entire protocol.

1. Preparation of Feeding Bacteria

This section describes the preparation of the feeding bacteria. The specific E. coli strain used to feed C.elegans is called OP50. Prior to this protocol, to prevent cross contamination of the worm culture with other bacteria, the OP50 strain has been made Carbenicillin/Ampicillin resistant²¹. Prepare the OP50 four to five days in advance. All materials coming in contact with OP50 must be sterile.

Day -7: Thursday (week 1): Inoculate 5 mL of TB containing 100 μg/ mL Ampicillin and 0.1 μg/ mL Amphotericin B with a single OP50 colony and incubate over night at 37°C in a bacterial shaker.

Day -6: Friday (week 1).

Morning 8:30. Inoculate early to allow enough time for the culture to reach saturation

1. Dilute the overnight culture of OP50 1:2000 in 300 mL TB containing 50 μg/ mL Ampicillin. Incubate the culture in a bacterial shaker for 8-12 hours at 37°C until saturation is reached. Do not allow the culture to grow longer than 14 hours.
2. Transfer the OP50 into a sterile, pre-weighted centrifugation tube. Pellet the OP50 by centrifugation for 10 min at 3500 rpm (2200 x g) in a table top centrifuge.
3. Discard the supernatant and re-suspend the OP50 pellet in sterile water and re-pellet by centrifugation. Repeat this wash twice.
4. After the second wash, carefully remove all the remaining water. No water should be left in the tube. Weigh the centrifugation tube containing the pellet and subtract the weight of the empty centrifugation tube in order to determine the weight of the pellet.
5. Thoroughly re-suspend the pellet in S-complete to a concentration of 100 mg/ mL. No clumps should be left.
6. The concentration of 100 mg/ mL OP50 should correspond to 2 x10¹⁰ bacteria/ mL. Use a photo spectrometer to determine the number of bacteria per mL if the relationship between the optical density and number of bacteria per mL is known. If necessary, adjust the concentration of the OP50 feeding solution to 2 x10¹⁰ bacteria/ mL.
7. Store the OP50solution at 4°C until it is used for the worm culture.

2. Preparation of a Synchronous Worm Culture

This section describes the preparation of the worm culture. Its goal is to generate an age-synchronous population of worms. All materials coming in contact with C.elegans after the bleach treatment in step 5 must be sterile. Plates are kept at 20°C unless otherwise indicated.

Day -6: Friday (week 1), 4:00 p.m.: Transfer the animals to a fresh plate

1. Take a 5-10 day old NGM plate on which the majority of the worm population consists of starved L1 larvae.
2. Sterilize a metal spatula by shortly heating it over a Bunsen burner. Let it cool. Use the cooled spatula to cut out agar chunks from the plate containing the starved worms. Transfer several of these chunks onto a fresh 10 cm NGM plate seeded with OP50. Incubate for approx. 65 hours at 20 °C until the majority of the worm population consists of gravid adults. The time it takes for the starved L1 animals to grow into gravid adults may vary from strain to strain.

Day -3: Monday (week 2) 10:00 a.m.: Establish a synchronous population

3. Collect worms from the 10 cm plate by washing them off the plate with 5-10 mL sterile water. Transfer the worm/water solution into a 15 mL conical tube.
4. Wash the worms by centrifuging 2 min in a tabletop centrifuge at 1200 rpm (280 x g). Discard supernatant and add 10 mL water. Repeat.
5. Remove supernatant and add 5 mL of freshly prepared bleach/NaOH solution (2.5 mL household bleach, 1 mL 10N NaOH, 6.5 mL H₂O). Incubate for 5 minutes at RT until the worms break open. Be sure to vortex gently every minute. Monitor the progress of the reaction under the dissecting microscope.
6. As soon as all the adults dissolve, add 5 mL of M9 buffer to neutralize the reaction.
7. Wash the eggs three times with 10 mL M9 buffer by centrifuging 2 min at 2500 rpm (1100 x g).
8. Wash the eggs once with 10 mL S-complete by centrifuging 2 min at 2500 rpm (1100 x g).
9. Remove the supernatant, add 10 mL S-complete and transfer the solution to a fresh 50 mL conical tube. Add 30 mL of S-complete to a final volume of 40 mL. Gently shake the tube overnight at room temperature on a nutator or similar device.

Day -2: Tuesday (week 2), 12:00 noon: Seed the animals into plates

10. Under the dissecting scope, check whether the worms hatched during the night. Determine the concentration of worms in the S-complete solution by counting the number of worms in 10 μL drops using a dissecting scope. Count at least 10 drops for each sample.
11. Re-suspend the worms at a concentration of 80-100 worms/ mL in S-complete. Add Carbenicillin (stock 100 mg/ mL) to a final concentration of 50 μg/ mL and Amphotericin B (stock 250 μg/ mL) to a final concentration of 0.1 μg/ mL. Shake on a nutator. If preparing larger quantities of worms, use a 600 mL nunclon flask with filter cap.
12. At 2:30 p.m. add the OP50 prepared in Part 1 to a final concentration of 6 mg/ mL (= 1.2 x10⁹ bacteria/ mL). Return the OP50 to 4°C.
13. Transfer 120 μL of the worm/OP50 solution into each well of a 96 well plate. Use 96 well plates with a transparent bottom. Make sure to keep the worms in suspension while pipetting.
14. Seal the plate using a tape sealer to avoid contamination and evaporation. Shake the plate on a microtiter plate shaker for 2 min and incubate for 2 days at 20°C until the animals reach the L4 stage.

Day 0: Thursday (week 2) before noon: Sterilize animals by adding Fluorodeoxyuridine (FUDR)

15. To sterilize the animals add 30 μL of a 0.6 mM FUDR stock solution to each well. This step brings the final volume in each well to 150 μL and reduces the final concentration of OP50 from 6mg/ mL to 5 mg/ mL (1×10⁹ bacteria/ mL). Seal the plate using tape sealers and shake it for 2-3 min on a microtiter plate shaker. If the OP50 was added at 2:30 on day -2, it is important that the FUDR is added before noon. Return plates to the 20°C incubator

Day 1: Friday (week 2): Add drugs to culture

16. By 9:00 a.m. most of the animals should be gravid and contain several eggs each. Add the drugs whose effect on lifespan is to be tested at the desired concentration. If dissolving the drugs in DMSO the final concentrations of DMSO should not exceed 0.6%, as DMSO concentrations higher than 0.6% shorten C.elegans lifespan. After addition of the drug, seal the plates with tape sealer and shake it for 2-3 min on a microtiter plate shaker. Return plates to the 20°C incubator
17. Adding the drugs can occasionally kill a few animals per plate, especially if using a solvent other than water. Use an inverted microscope to check for dead animals. Generally, there should be less than 10 dead animals per 96 well plate. Return plates to the 20°C incubator.

Day 4: Monday (week 3): Change sealers

18. To allow fresh oxygen to enter the culture remove the sealer, wait 1minute and reseal the plate. Shake the plate for 2-3 minutes on a microtiter plate shaker. Repeat once every week.

Day 5: Tuesday (week 3): Add new OP50 to prevent starvation

19. Add 5 μL of the 100 mg/ mL OP50 solution that was prepared in part 1 to each of the 96 wells to prevent starvation.

3. Scoring of Lifespan.

This section describes how survival of the synchronized worm population of Part 2 is monitored until the animals died. To observe the animals in the 96 well plates, use an inverted microscope with a 2x or 2.5x objective. Record survival data three times a week; Monday, Wednesday and Friday. Use movement to determine whether the animals are alive or dead. Strong light, especially blue light, induces the animals to move. Do not remove dead animals from the assay. Occasionally, animals that did not move and were determined dead in the preceding count might move later on.

1. On day 0, at the beginning of the experiment count the total number of worms in each well. Censor wells that contain more than 18 animals from the analysis as animals in these wells will not have enough OP50 and will show effects of dietary restriction.
2. In order to increase the chance that the animals move shake the 96 well plate on a microtiter plate shaker for 2 min. before counting.
3. In each counting session, record date and number of animals which move as live animals. Movement in liquid is much easier than on solid media and can be induced by strong lights. The use of a higher magnification may help to spot very subtle movements like those of the tip of the pharynx. Such subtle movements are often the only movement observed in very old animals.
4. Return plates to the 20°C incubator
5. Repeat step 2-4 every two to three days until all the animals are dead.

4. Representative Results.

This section shows an example of how to keep records of the lifespan data generated by this assay and some representative results.

Figure 1A shows an example of how to record lifespan data during this assay. An excel sheet is used to keep track of the survival the populations in each well. For each well it’s coordinate in the plate, strain, drug, concentration of the drug and the total number of animals alive on day 0 (X0) is recorded at the beginning of the experiment. Record date, as well as the number of living and dead animals three times a week in order to follow the survival of the various populations in each well. To graph the results, calculate the fraction of animals alive for each day and plot it as a function of time in days. P-values should be calculated using statistical packages like STATA or similar software.

The medium lifespan of C.elegans is temperature dependent. Figure 1B shows that temperature dependent changes in lifespan are accurately reproduced by the microtiter based lifespan assay²². Similarly, the microtiter plate assay reproduces changes in lifespan from mutants reported to have lifespans that differ from wild type N2 animals^{23-24 25}(Fig 1C).

The assay also can be used to make more quantitative statements. In Fig1D mean lifespan is plotted as a function of Mirtazepine concentration⁷. Each data point represents the mean lifespan of 7-12 population, each living in a different well. Even though the number of animals per well is relatively low (5-15 animals) the well-to-well variation is relatively small as can be seen from the error bars.

Figure 1. The microtiter plate based lifespan assay accurately reproduces changes in lifespan reported in the literature.
(A) Sample data collected in an excel spreadsheet. During each counting session date, coordinate of the well and the number of live and dead animal was recorded. At the beginning of the experiment, the total number of animals in each well was determined. (B) Survival curve of wild type (N2) animals grown at 20°C and 25°C. (C) Survival curves of strains carrying mutations that affect lifespan. All four strains were assayed in parallel at 20°C. (D) Dose response curve of Mirtazepine treated N2 animals. Mean lifespan is plotted as a function of Mirtazepine concentration. Error bars indicate the S.E.M of 8 wells per condition.

5. Materials.

M9 buffer, 1000 mL

• 6 g Na₂HPO₄
• 3 g KH₂PO₄
• 5 g NaCl
• 0.25 g MgSO₄∙7H₂O
• Add deionized water to 1000 mL
• Autoclave

Potassiumphosphate buffer pH 6.0, 1000 mL

• 136 g KH₂PO₄
• Add deionized water to 900 mL
• Adjust pH to 6.0 with 5M KOH
• Add deionized water to 1000 mL
• Autoclave

Trace metal solution

• 1.86 g Na₂EDTA
• 0.69 g FeSO₄ ∙7H₂O
• 0.20 g MnCl₂ ∙ 4H₂O
• 0.29 g ZnSO₄ ∙7H₂O
• 0.016 g CuSO₄
• 1000 mL deionized water
• Autoclave and store in the dark.

S-basal medium, 1000 mL

• 5.9 g NaCl
• 50 mL of 1M potassium phosphate, pH 6.0
• 1000 mL deionized water
• Autoclave
• Let the solution cool and then add 1 mL of 5mg/ mL cholesterol (dissolved in Et-OH).

Potassium citrate 1M, 1000 mL

• 268.8 g tripotassium citrate
• 26.3 citric acid monohydrate
• Add 900 mL deionized water
• Adjust pH to 6.0 with 5M KOH
• Add deionized water to 1000 mL
• Autoclave

S-complete medium, 1000 mL

• 977 mL S-basal
• 10 mL 1M potassium citrate pH 6 (sterile)
• 10 mL Trace metals solution (sterile)
• 3 ml 1M CaCl₂ (sterile)
• 3 mL 1M MgSO₄ (sterile)

NGM agar

• 3.0g NaCl
• 2.5g Pepton (from casein, pancreatic digest)
• 17g Agar
• Add deionized water to 975 mL and a stirring bar
• Autoclave
• After autoclaving cool down to 55°C and add the following components:
  • 0.5 mL of 1M CaCl₂ (sterile)
  • 1 mL of 5mg/ mL Cholesterol in ethanol
  • 1 mL of 1M MgSO₄ (sterile)
  • 25 mL Potassiumphosphate buffer, pH 6.0 (sterile)

TB, 1000 mL

• 12g Bacto Tryptone
• 24 g Yeast Extract
• 4 mL Glycerol
• Add 900 mL deionized water
• Autoclave
• After autoclaving cool down to 55°C and add the following component:
  • add 100 mL of 0.17M KH₂PO₄/0.72M K₂HPO₄

0.6 mM Fluorodeoxyuridine (FUDR, sigma cat# F0503), 1000 mL

• 100 mg FUDR
• Dissolve in 670 mL sterile S-complete, make 10 mL or 45 mL aliquots.
• Store at -20 °C.

100 mg/ mL Carbenicillin, 10 mL

• 1 g Carbenicillin
• 10 mL sterile deionized water
• Sterile filtrate and aliquot
• Store at -20 °C.
• Use at a final concentration of 50 μg/ mL

250ug/ mL Amphotericin B, 4 mL

• 1mg Amphotericin B
• 4 mL Et-OH
• Store at -20 °C
• Use at a final concentration of 0.1 μg / mL