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.
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.
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.
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
Nematode Growth Media (NGM), 100 mL: | |
Combine: | |
0.3 g | NaCl |
0.25 g | Peptone |
2 g | Agar |
Autoclave for 40 minutes and let cool to 55 °C, then add: | |
100 µL | 1 M MgSO4 |
100 µL | 5 mg/mL Cholesterol |
100 µL | 1 M CaCl2 |
1.625 mL | 1.5 M KPi pH 6.0 |
Liquid NGM can be used immediately to pour plates or allowed to solidify and stored long-term at room temperature |
Luria Broth (LB), 1L: | |
10 g | BactoTryptone |
5 g | Yeast Extract |
10 g | NaCl |
10 mL | 1 M Tris pH 8.0 |
1 L | deionized water |
Autoclave and store at room temperature. |
1 M MgSO4, 300 mL: | |
73.95 g | MgSO4 |
300 mL | deionized water |
Autoclave and store at room temperature. |
5 mg/mL Cholesterol, 200 mL: | |
1 g | cholesterol |
200 mL | 100% ethanol |
Filter sterilize and store at room temperature. |
1 M CaCl2, 500 mL: | |
27.75 g | CaCl2 |
500 mL | deionized water |
Filter sterilize and store at room temperature. |
1.5 M KPi pH 6.0, 1L: | |
Combine: | |
31.4 g | KPi dibasic |
179.6 g | KPi monobasic |
850 mL | deionized water |
Heat while mixing to allow KPi to dissolve into solution. Adjust pH to 6.0 with 10 N NaOH. | |
Add deionized water to 1 L. | |
Autoclave and store at room temperature. |
1 M Tris, pH 8.0: | |
60.57 g | Tris |
400 mL | deionized water |
Adjust pH to 8.0 with HCl. | |
Add deionized water to 500 mL. | |
Filter sterilize and store at room temperature. |
150 mM Fluorodeoxyuridine (FUDR), 10 mL: | |
0.3693 g | FUDR |
10 mL | sterile deionized water |
Store at -20 °C. |
100 mg/mL Ampicillin (Amp), 10 mL: | |
1 g | Ampicillin |
10 mL | sterile deionized water |
Store at -20 °C. |
50 mg/mL Carbenicillin (Carb), 10 mL: | |
500 mg | Carbenicillin |
10 mL | sterile deionized water |
Store at -20 °C. |
1 M Isopropyl β-d-Thiogalactopyranoside (IPTG), 10 mL: | |
2.38 g | IPTG |
10 mL | sterile deionized water |
Store at -20 °C. |
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, including live bacteria, RNA interference (RNAi), dietary restriction by bacterial deprivation, and drug-free NGM.
Probably the most common variation from this protocol is the maintenance of worms on live bacteria. This can be accomplished by making a few minor changes. First, change the procedure for seeding the plates with bacteria (steps 1.7 through 1.13). Instead of growing OP50 cultures to saturation, grow to mid-log phase and pipette 200 µL onto the plates directly from the liquid culture. Allow the bacteria to grow up on the plates over night and omit exposure to UV. Ampicillin should also be excluded from the plates with FUDR. An advantage to using live bacteria is that worms do not have to be transferred as often to new plates, since bacterial food is alive and growing. The disadvantage of live bacterial food is that OP50 is pathogenic to C. elegans.3 Worms grown on live bacteria have a shorter live span than worms grown on UV-killed bacteria,3 which could potentially mask life span phenotypes associated with aging. Median life span on live bacteria is approximately 20 days.
Gene knock down by RNAi is easily accomplished in C. elegans by modifying their bacterial food so that it produces double-stranded RNA corresponding to the gene to be knocked down. Two RNAi bacterial libraries are available that cover more than 90% of the open reading frames in the C. elegans genome.4-9 To utilize RNAi in the context of life span, replace the OP50 bacteria with the RNAi clone of interest and follow the modifications discussed in the previous paragraph for measuring life span on live bacteria. The RNAi libraries use a plasmid based expression system. The plasmid is selected for using a carbenicillin resistance cassette and expression of the double stranded RNA is induced by isopropyl β-d-thiogalactopyranoside (IPTG). Both carbenicillin and IPTG need to be included in the NGM plates. Modify step 1.4 by adding 100 µL 1 M IPTG and 50 µL 50 mg/mL carbenicillin per 100 mL NGM to both types of plates. Ampicillin does not need to be added to either type of plate, since carbenicillin fulfills the same role.
Dietary restriction is the most widely studied intervention for slowing aging across evolutionarily divergent species. In C. elegans, maximum life span extension on solid media is observed when the bacterial food source is completely removed during adulthood, a form of dietary restriction termed bacterial deprivation (BD) .9, 10 To measure life span in the context of BD, make two modifications to the above protocol. First, prepare a third type of plate. These plates should be identical to the Amp/FUDR plates except lacking the bacterial food source. Second, modify Part 3 to include an additional step between step 3.2 and step 3.3. On the 4th day of adulthood (4 days after transferring the L4 larvae to Amp/FUDR) transfer BD worms to Amp/FUDR plates lacking bacteria. One complication associated with this form of dietary restriction is the worms’ tendency to flee. In the absence of food the worms will not stay near the center of the plate, but will increase their area of exploration in search of food. As a result, a larger fraction of the worms will crawl up the walls of the plate and desiccate. We often see 50% to 70% of the animals flee after being transferred to BD plates. To address this issue, start with three times as many worms in order to have a significant number remaining on the plates post-flight. BD can also be combined with live bacterial food with no additional modifications, or with RNAi with one additional modification. For BD with RNAi, an additional antibiotic must be added to the plates without bacteria to prevent bacteria transferred with the worms from growing and providing an unwanted food source. Two examples are tetracycline and kanamycin, either of which can be added to the FUDR plates without bacterial food during step 1.4. BD can also be initiated as early as 2 days of adulthood or as late as 14 days of adulthood, with no significant impact on life span.10
The final modification that we will discuss is measuring life span on NGM plates without additional drugs (e.g. ampicillin or FUDR). This can be accomplished by simply not adding the drugs to the NGM during step 4 and adding one additional step in Part 3. Without FUDR the worms will continue laying eggs and producing larva. During their reproductive phase (approximately the first week of adulthood) all experimental worms will have to be transferred to fresh plates every 2 days to separate them from their offspring.
C. elegans are primarily hermaphroditic with rare occurrence of males. Life span is typically measured for hermaphrodites only, but can also be measured for males. There are two challenges associated with working with male C. elegans. The first is acquiring a large quantity of male worms, as hermaphrodite self-fertilization produces a very small fraction of male offspring (0.1%).11 Once a population containing male worms is attained, male/hermaphrodite mating produces approximately equal numbers of males and hermaphrodites as long as worms are maintained in the presence of food.12 Male stocks can be ordered from the Caenorhabditis Genetics Center or generated by visually screening for founding males produced from hermaphrodite self-fertilization. The second challenge is male scavenging behavior. In the absence of either food or potential mates (hermaphrodites), male worms enter a searching behavioral mode that involves a wide range of movement.13 When maintained on plates this behavior results in a large fraction of the worms fleeing up the plate walls and desiccating. The general method for dealing with this difficulty is simply to start with enough males that a substantial number remain after most have fled.
Apart from life span, a common age-associated phenotype is lipofuscin accumulation. Lipofuscin is complex cellular waste that cannot be degraded that builds up in cells with age and is used as a biomarker of aging in C. elegans.14, 15 Lipofuscin fluoresces and can be easily visualized in C. elegans using the DAPI channel of a fluorescence microscope (Figure 3). Lipofuscin accumulation can be visualized in worms being scored for life span directly on the NGM plates, allowing collection of a useful secondary phenotype in parallel with life span.
In addition to life span, the protocol described in this article can also be used to score phenotypic progression of age-associated paralysis in C. elegans models of proteotoxicity disease.16 When a worm becomes paralyzed it becomes unable to crawl across the plate, but can still move its head. A worm is scored as paralyzed if it fails to move relative to the NGM in response to plate tapping or prodding with a platinum transfer pick, but does move its head. Worms that die typically retain the paralysis score (paralyzed or not paralyzed) that they were given during the most recent live observation. Importantly, even wild type C. elegans become paralyzed with advanced age. For this reason paralysis is typically not scored for worms older than approximately 20 days, as beyond this point it becomes difficult to distinguish between paralysis caused by normal aging and paralysis caused by progression of the proteotoxicity disease.
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.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Agar | Reagent | Fisher Scientific (BD Diagnostic Systems) | DF0145-17-0 (214530) | |
Ampicillin | Reagent | MidSci | 0339 | |
BactoTryptone | Reagent | Fisher Scientific (BD Diagnostic Systems) | DF0123-17-3 (211705) | |
BactoPeptone | Reagent | Fisher Scientific (BD Diagnostic Systems) | DF0118-17-0 (211677) | |
CaCl2 | Reagent | Fisher Scientific (JT Baker) | NC9699248 (1332-01) | |
Carbenicillin | Reagent | Gold BioTechnology (GBT) | C0109 | |
Cholesterol | Reagent | Sigma-Aldrich | C75209 | |
Fluorodeoxyuridine (FUDR) | Reagent | Sigma-Aldrich | F0503 | |
Isopropyl β-d-Thiogalactopyranoside (IPTG) | Reagent | Gold BioTechnology (GBT) | I2481C5 | |
KPi Dibasic | Reagent | Fisher Scientific (JT Baker) | 5087862 (3252-01) | |
KPi Monobasic | Reagent | Fisher Scientific (JT Baker) | 5087861 (3246-01) | |
MgSO4 | Reagent | Fisher Scientific (JT Baker) | NC9561800 (2500-01) | |
NaCl | Reagent | Fisher Scientific | S251 | |
Tris | Reagent | Sigma-Aldrich | T1503 | |
Yeast Extract | Reagent | Fisher Scientific (BD Diagnostic Systems) | DF0886-17-0 (288620) |