This protocol presents the lowest cost and lowest tech methods for serving aging in sea elegans. An influential model organism in aging biology. The greatest advantage of these methods is the ease of implementation, using low cost equipment and material.
Making them amenable to any researcher regardless of experience or available funding. As an old aging studies there is natural variability in health within populations. Therefore, it's essential that experiments are performed on healthy, well fed and tightly synchronized animals.
To ensure rip disability Begin the preparation of one liter nematode growth media or NGM auger plate. Add 2.5 grams of pep tone, 3.0 grams of sodium chloride and 20 grams of auger into a one liter flask with a stir bar and add distilled water to a final volume of 970 milliliters. Sterilize the NGM auger solution in a one liter flask using a standard autoclave or media sterilizer.
And let the solution stir until it cools to 60 to 75 degrees Celsius. While the solution cools, heat a bead bath to 65 to 70 degrees Celsius. Once the NGM auger solution cools to 60 to 75 degrees Celsius.
Add the liquid additives and allow the solution to mix completely for five minutes. Then submerge the flask in a bead bath at 65 to 70 degrees Celsius to prevent the media from solidifying while pouring into the plate. Pipette nine to 11 milliliters of the solution into each 60 milliliter plate.
When done, place the pipette back in the heated solution to maintain the temperature and to prevent any media from solidifying in the pipette. Repeat the procedure for all the plates and allow NGM a plates to solidify overnight. Once solidified, use them for seeding plates with bacteria or store the plates in sealed containers for up to three months at four degrees Celsius to synchronize the worms via bleaching.
Collect the nematodes by pouring a small amount of M9 solution onto a plate containing the worms without overfilling the Petri dish. Then swirl the M nine solution gently to loosen worms off the bacterial lawns. Next, collect adult gravid worms into a 15 milliliter tube with a serological pipette.
Avoiding piercing the auger with the pipette tip. Pellet the animals by centr for 30 seconds at 1100 times G, and aspirate the supernatant. Add five milliliters of the freshly prepared bleaching solution to the worm pellet, and check the worms under a dissecting microscope every few minutes along with intermittent vigorous shaking until all the adult worm bodies are dissolved and only eggs are left in the mix.
Next, pellet the eggs by spinning down the egg bleach mix for 30 seconds at 1100 times G and aspirate the bleaching solution. Then wash the pelleted eggs by adding up to 15 milliliters of M9 solution and inverting the tube four or five times to disperse the eggs and solution. Again, centrifuge the tube for 30 seconds at 1100 times G to pellet the eggs and aspirate the M9 solution.
Perform the wash four times to eliminate any bleach from the egg mix. After the fourth wash centrifusion aspirate the M9 solution from 100 microliters to two milliliters depending on the total number of worms bleached. Shake the eggs thoroughly to break up the clumps and ensure that the eggs are fully resuspended in the M9 solution.
Alternatively, animals can be L one arrested for a tighter temporal synchronization by adding 10 to 12 milliliters of M9 solution to the egg pellet in a 15 milliliter chronicle tube. Let the worms spin in a rotator for up to 24 hours at 20 degrees Celsius. To approximate the egg or L one concentration pipette four microliters of the egg mixture onto an NGM plate seated with bacteria.
Then count and calculate the eggs present per microliter of media. Repeat the counting three or four times to improve the approximation, based on the approximation plate, the appropriate number of eggs or L one arrested animals onto the NGM auger plate seeded with bacteria of choice. To measure the locomotive behavior of the worms via thrashing.
Move a small number of day one adult worms from an NGM auger plate onto 10 to 20 microliters of M9 solution under a dissecting scope. Then focus on one worm at a time and count the number of times the specimen switches from a concave to convex formation in 15 seconds. Use a hand counter and a timer and repeat the procedure for 10 to 15 worms.
Age out the worms to desired age and repeat thrashing measurements at all desired ages. As visualized, older worms thrash at significantly slower speeds. For quantity and fertility measurements.
In 10 to 15 Caenorhabditis elegans worms, single out L four worms into a separate NGM auger plate seeded with bacteria of choice. Allow animals to grow overnight at 20 degrees Celsius and ensure that a newly seeded batch of plates is ready for the next day. To measure the amount of eggs laid by Caenorhabditis elegans transfer the adult worms into a fresh NGM auger blade seeded with that looted bacteria every 12 to 24 hours for seven to eight days or until the eggs are no longer visible and count the number of eggs laid on the plates.
To measure the brood size of Caenorhabditis elegans Transfer the adult worms onto a fresh NGM auger plate seeded with bacteria every 12 to 24 hours, or seven to eight days or until the progeny no longer visible. Keep all the plates containing eggs at 20 degrees Celsius. Two days after transferring worms.
Count the developed progeny on the plate. Count all the alive and developing worms at the L four stage or earlier to ensure that the F two generation does not confound results. Remove all the worms from the plate as they're counted.
Maintain the plates for an additional one to two days before scoring them again to ensure that any animals with delayed hatching or development are not missed. The effect of the sterilization method on the lifespans of the wild type N2 nematodes showed that changes in lifespans of the worms grown on the NGM auger plates were similar to those of worms grown on the NGM auger FUDR plates or the glp four BN2 mutant animals grown on the NGM auger plates. The short-lived HSF one knockdown animal showed a significant decline in the lifespan for all conditions.
A substantial decrease in the number of laid eggs and brood size was found in the HSF one over expression animals compared to the wild type controls. Some HSF one over expression animals exhibited full sterility indicating that reproductive health can be inversely correlated with longevity. The thrashing rate provided a reliable method for measuring health during aging as measured by a significant decrease in the thrashing in old animals compared to the young ones.
Survival to stress are reliable assays for measuring organismal health, Tunicamycin which causes reticulum stress resulted in a reduced lifespan regardless of concentration compared to DMSO control. The high levels of paraquat significantly shortened the lifespan while low concentrations of paraquat increased the lifespan due to hormetic effect. In the thermo tolerance assays, the over expression of HSF one increased the thermo tolerance at 37 degrees Celsius For all lifespan and aging experiments.
It is important to remember that sterilization techniques can have a pleiotropic effect on organismal health, which impacts results. Many additional and complimentary approaches exist for measuring health span if equipment and infrastructure are available including measurements of transcription, translation protein aggregation, organelle, morphology and metabolic functions.
