Because last week, my life span results found that the gas-1 mutant worm had lost its main phenotype, a short lifespan, my PI asked my to perform florescence tests on it to see if it still had chemical differences from the wild type. Since Fred, an undergrad, has been working with fluoresce for the past few months and perfecting the assay, he taught me what information fluorescence can uncover about the worms being studied. There are three different dyes that the worms are fed, Mito Sox, Mito Tracker Green (MTG), and TMRE. Mito Sox measures the oxidant burden in the animal's mitochondrion which should be higher in gas-1 mutant worms because the sicker animal experiences more oxidative stress in its cells. MTG measures the mito content in the animals cells and should be less in gas-1 worms because the mutant strain is born with less functioning mitochondria. TMRE measures the membrane potential in the animal's mitochondrial complex which should be less in gas-1 worms because their mitochondria are less capable of creating a concentration gradient with H+ ions.
To perform the fluorescence experiment, I first had to prepare enough plates. I had to spread them with OP50 e.coli and then with each dye. After placing about 60 young adults by hand on each plate, I incubated them for 24 hours so that they have time to ingest the e.coli with dye and the dye had time to adhere to the fatty cells in the animal. Then after 24 hours, I handpicked them again and transferred them onto a plate of fresh bacteria. This is done so that the worms can eat the e.coli without dye and clear their gut of the dye. We want only the dye that had penetrated into the cells to fluoresce not the food in the gut itself. After letting them clear their gut for 3-4 hours, I put levamisole (a drug that temporarily paralyzes them) on each plate making sure every worm is submerged in the drug. After giving the worms 30 minutes to fully paralyze, I took the plates to the microscope with a camera. I used different filters on the microscope for each dye, and zoomed into each individual worm and took pictures of each worm's pharyngal bulb.
Here's a good one:
About 2 hours of picture taking later I have to manually circle the pharyngal bulb using a software. This software then counts the pixels and measures the amount of light per pixel and calculates the necessary information. According to the fluorescence experiment, everything seems normal with the gas-1 strain. Nevertheless, my PI has ordered a fresh batch of worms for the lab to work with.
To perform the fluorescence experiment, I first had to prepare enough plates. I had to spread them with OP50 e.coli and then with each dye. After placing about 60 young adults by hand on each plate, I incubated them for 24 hours so that they have time to ingest the e.coli with dye and the dye had time to adhere to the fatty cells in the animal. Then after 24 hours, I handpicked them again and transferred them onto a plate of fresh bacteria. This is done so that the worms can eat the e.coli without dye and clear their gut of the dye. We want only the dye that had penetrated into the cells to fluoresce not the food in the gut itself. After letting them clear their gut for 3-4 hours, I put levamisole (a drug that temporarily paralyzes them) on each plate making sure every worm is submerged in the drug. After giving the worms 30 minutes to fully paralyze, I took the plates to the microscope with a camera. I used different filters on the microscope for each dye, and zoomed into each individual worm and took pictures of each worm's pharyngal bulb.
Here's a good one:
About 2 hours of picture taking later I have to manually circle the pharyngal bulb using a software. This software then counts the pixels and measures the amount of light per pixel and calculates the necessary information. According to the fluorescence experiment, everything seems normal with the gas-1 strain. Nevertheless, my PI has ordered a fresh batch of worms for the lab to work with.
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