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

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.

1. 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.
2. Completely melt solid NGM by microwaving on high in 30 second pulses. Swirl media between pulses to prevent pressurized gas bubbles from building up.
3. Place liquid NGM in 55 °C water bath or on the bench to cool.
4. 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.
5. 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.
6. 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.
7. 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.
8. Place OP50 culture in 37 °C shaker and allow bacteria to grow overnight (culture should reach saturation).
9. Pellet OP50 bacteria by spinning at 3,500 g for 10 minutes.
10. Remove 90% of supernatant and resuspend bacteria to concentrate culture 10x.
11. 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.
12. Leave plates on the bench overnight to allow bacteria culture to dry on the solid NGM (typically takes about 24 hours).
13. 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:
    • Place plates in the Stratalinker and remove lids
    • Close the door and turn on the Stratalinker
    • Press ‘Energy’
    • Enter ‘9999’ using the keypad
    • Press ‘Start’
    • The plates will be exposed to UV for approximately 5 minutes
14. Plates with UV-killed bacteria can be stored upside down at 4 °C for up to 1 month.

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.

1. (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.
2. (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.
3. 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.
4. Leave the TEL plate at 25 °C for 6 hours in order to allow the worms time to lay eggs.
5. 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.
6. 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).

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.

1. 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
2. Place Amp/FUDR plates at 20 °C for 24 hours.
3. After 24 hours, visually assess worms, media, and bacteria. Transfer worms to fresh Amp/FUDR plates if any of the following is observed:
   • 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.
   • 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.
   • 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.
   • 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.
   • Using the 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 platinum transfer pick. Score the worm as dead if it does not respond by moving its head.
   • Dead worms can be removed from the plate.
4. Record the date and the number of worms that are alive and dead.
5. Return plates to 20 °C.
6. Repeat steps 3.3 through 3.6 every 2 to 3 days until all worms have died.

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

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)).

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).

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).

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...

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.