Since March 9th, I had conducted a series of serial dilutions to test the survival of viable BCG cells in the presence of GSH over about two weeks to collect data from Trial 1 due to previous immiscible data. I inoculated 4 different cultures onto four separate agar plates every day for twelve days. All of the plates have been incubating for some time now and have been hopefully forming colonies. However, due to unforeseen circumstances of the Covid-19 virus pandemic I was unable to collect the data from the plates. Hopefully by the time the quarantine is lifted and we are allowed access back into the labs the plates have still upheld and the agar has not dried out and the colonies are still countable.
In regards to the NADH assays, another member of my lab group had finished her ATP trials before I was able to finish mine and she was able to take over the experiment. Unfortunately, she was unable to conduct a trial because of the pandemic as well. Being that this experiment was passed onto another colleague in my lab group the next experiment I will hopefully be able to attempt is a GSH assay. This assay detects the amount of free thiol groups present in the cytoplasm of an organism. Although it detects GSH it can also detect MSH, cysteine, cys-gly and any other free thiol. The more free thiol there is the more reductive stress is present.
From this experience I have learned a multitude of things being both technical and mental. I have learned to be very patient throughout my time working on this project. I expected to be finished with it by the end of the first semester but between running into obstacles and cramming for exams for classes it took a lot longer than I anticipated. I have also learned that science is very much so a trial and error process and things may never go the way you plan them to. I have been working on this project for some time now and I was so close to finishing it but because of things that I or anyone else can control it must be put on hold. This has made me put everything into perspective to be very cautious in the lab thinking through each step I need to do and making sure I am using sterile technique as much as I can.
From the data that I have collected so far it is determined that by Day 3 GSH is not killing BCG that was grown in a cholesterol-based media.
Communication with my faculty mentor was definitely key in carrying out this project. When I first tried the serial dilutions, I seemed to have carried out the protocol correctly but had an over exceeding amount of colony forming units. To figure out how I could reduce the number of colonies I had to get some insight from Dr. Kelly and we figured that if we did another dilution within the protocol it would lessen the number of bacteria and CFUs. Learning new techniques was needed for this project as well and Dr. Kelly was the best person to learn from.
Overall, I had an awesome time learning and conducting research and I hope to continue doing so next year and possibly this summer.