The procedure performs dietary restriction on the standard abased plate in sea elegance to study its effect on longevity, reproduction, and metabolism. This is accomplished by first accurately measuring the concentration of the bacteria that will be used as a food source. The second step of the procedure is to properly dilute the bacteria culture into suitable concentrations.
Then prepare abased SDR plates with killed bacteria. Finally set up SDR experiments with an age synchronized population of worms. Results of progeny production and lifespan measurements can show delayed and reduced progeny production as well as increased mean lifespan.
The main advantage of this technique over 60 methods like Dian in AZA medium, is that all the experiment will be done on a standard solid AGA based play that is used by most warm laboratories For dietary restriction experiments and see elegance establish separate calibration curves for each bacteria strain used as a food source. Pick a single colony of e coli OP 50 bacteria from a fresh streak on lb agar and inoculate three milliliters of liquid lb broth culture in a 37 degree Celsius shaker overnight. Prepare serial twofold dilution of the overnight culture.
Then measure the absorbence of each dilution at 600 nanometers by spectrophotometer in triplicate. Further dute each bacterial suspension, 1 million fold evenly seed 0.4 milliliters of bacterial suspension of each dilution on a 100 millimeter lb agri plate and incubate at 37 degrees Celsius for 18 to 24 hours. Count the number of bacterial colonies grown on each plate in order to calculate colony forming units per milliliter for each sample.
Now plot OD 600 as a function of bacterial concentration and perform a linear regression analysis to establish the relationship between measured OD 600 values and OP 50 bacteria concentrations. For this method, a bacteria concentration of one times 10 to the 11th CFUs per milliliter is used in the ad lido feeding condition and one times 10 to the eighth CFU per milliliter as the optimal DR feeding condition. However, four to six different bacteria concentrations may be needed to establish a complete dose dependent relationship.
For SDR and its responses. Prepare an inoculum with a single colony of e coli OP 50 bacteria in three milliliters of liquid LB broth, cultured in a 37 degree Celsius shaker for six to eight hours. Transfer the OP 50 culture to a new flask containing 500 milliliters of LB broth and grow overnight for the bacteria to reach saturation if necessary.
Prepare appropriate dilution of the OP 50 culture for the measurement of absorbence at OD 600. To determine the bacteria concentration, pellet the bacteria by centrifugation at 1500 Gs for 20 minutes. Remove the supernatant and resuspend the bacteria to a concentration of one times 10 at of the 12th CFUs per milliliter.
Now, prepare 10 milliliter bacteria cultures in six different concentrations, ranging from one times 10 of the 12th to one times 10 of the seventh CFU per milliliter pipette. Point two milliliters of bacteria culture into the center of a 60 millimeter solidified N GM plate. Avoid touching the surface of the plate with the pipette tip as nicks on the surface allow worms to burrow into the aerates.
Place the plates in a 37 degree Celsius incubator for one hour. Then apply 10 microliters of 100 milligrams per milliliter carbonic soin and 10 microliters of 50 milligrams per milliliter can mycin to each plate to a rest growth of the bacteria. Store plates with different concentrations of antibiotic killed bacteria at four degrees Celsius for future use for up to one month.
It is important to prepare and store enough plates, preferably the same batch for the entire plant. Experiment at least one to two days ahead to verify the viability of the bacteria. Scrape some bacteria out of the SDR plates and spread it on an empty N GM plate with carbonic, sein and antimycin.
Place the plate in a 37 degree Celsius incubator for six to eight hours and confirm the absence of bacterial growth. For most of the SDR related studies, such as lifespan analysis and progeny production profiling, preparation of an age synchronized population of worms is essential. Use a platinum worm picker to transfer 20 to 30 reproductively active adults onto several regular NGM plates.
With OP 50. Depending on the number of eggs required for the experiments, more adults may be needed. Leave the plates at 20 degrees Celsius for four hours to allow egg laying.
Remove the adults from the plate and visually inspect the plates for eggs. Continue to incubate the plates at 20 degrees Celsius to allow the progeny to develop into the late L four day one adult stage. Usually this takes three days for N two wild type worms at 20 degrees Celsius.
For lifespan analysis. Transfer 10 to 12 day one adult worms onto an SDR plate to initiate the dietary restriction. To obtain sufficient statistic power, use a total of 60 to 100 worms for each SDR condition.
Score the viability of each worm every two to three days until all the worms have died. Importantly, transfer the worms to fresh SDR plates every other day to avoid starvation. This calibration curve plots OP 50 concentration in relation to OD 600.
The equation generated by linear regression analysis can be used for all future calculations of OP 50 bacteria concentrations. This study assays the effect of chronic SDR treatments on mean lifespan of different worm strains compared to a normal dose dependent response of wild type worms to SDR. The lifespan effect of SDR is partially suppressed by DR two overexpression while DAF 16 MU 86 mutation has no effect on SDR induced lifespan extension.
After watching this video, you should have a good understanding of how to perform Dian on the standard AGA based play in sea elegance.
