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.
