Blog #4: The Final Report (Conclusions, Future Experiments, Setbacks)

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.

Blog #3- More Progress and More Setbacks

I have been conducting a series of serial dilutions to test the survival of viable BCG cells in the presence of GSH over about a week now to collect data from Trial 1 due to previous immiscible data. Almost all of the plates have been incubating for some time to form colonies now. I inoculated 4 different cultures onto four separate agar plates every day for twelve days. I have two more days of plating and then I will enter the waiting phase of the experiment. I however seem to have run out of agar plates meaning that I need to make more which may set me back a day because it takes time for the plates to solidify. However, if all works out, in five days, I will be recording colony data and comparing it to data from Trials 2&3. I did come across one obstacle when conducting this trial and that was that culture B (7H9, BCG, GSH) became lysed at day 5 meaning that the culture is no longer able to be utilized because all of the cells are not viable. I then disposed of that culture and luckily, I had made a new culture of 7H9 and BCG at the start of the whole trial in case something like this happened. I had to grow the bacteria back to around the same optical density as the previous culture to indicate day 5. I then treated that culture with Glutathione every day before I could inoculate culture B on any plates.

In regards to the NADH assays, I have been prepping to begin the trial after data for the plating experiment is collected. I prepared all of the buffers and solutions needed for the experiment as well as record how much product we had left from the previous trial that was carried out. The previous trial that was done was carried out incorrectly and not all of the NADH kit was utilized. I am currently waiting for the rest of the reagents to conduct the assay.

I have learned that not everything will come easily and or on the first try. Everything takes time, patience, and practice. I have also learned that sterile technique is very imperative when it comes to biohazard safety level 2 experiments.

Blog #2: Progress and Setbacks

I have been conduction a series of serial dilutions to test the survival of viable BCG cells in the presence of GSH over a time period of two weeks for quite some time now. I have retrieved data for Trials 2 &3. It is very apparent that Mycobacterium bovis-BCG (BCG) grown in a cholesterol-based media was not killed at as early as day 3, when exposed to glutathione (GSH). Data for Trial 1 was immiscible done improperly due to lack of technique and contamination due to use of wrong lab equipment (use of wrong sized filter for GSH). This resulted in too many colonies to count and  everything must be done in triple kit in order to be sustainable data. This means that I have to redo trial 1. Before retrying Trial 1 I had to make agar plates to inoculate the bacteria onto. This is when I ran into some trouble starting off with the agar plates not setting this may have been because of improper technique such as missing a step or wrong measurements,the ultraviolet light within the hood, or the agar powder itself (This was later debunked because the same powder was used to make a successful batch later on).Once the plates were successfully made our lab found out that our BCG bacteria cultures were not growing properly. When making cultures frozen stock must have an optical density of 0.8-0.1, the cultures that were being produced in the lab remained at an optical density of around 0.5 with no signs of growth. It took our lab weeks and weeks to figure out what went wrong. There were a multitude of possibilities such as the bacteria itself, the conditions in which the cultures were being made, or even the media that the bacteria were being grown in. Our lab had to scrap all cultures that were being grown as well as all experiments that were underway. We soon pin pointed that it was indeed the media that the bacteria were being grown in that was the problem. We have made fresh media and have just begun starting to make frozen stock and cultures. Experiments will soon be on their way.

I have also begun thinking about the next project I will take on being that it will be in the imminent future. I am happy to be continuing a project that my fellow lab mate/lab mentor began working on before he graduated. I will be performing NADH assays, which will determine the relative NADH concentrations of the BCG cultures.

Blog #1: The effects of a cholesterol-based metabolism by Mycobacterium bovis-BCG on resistance to glutathione

The title of my research project is “The effects of a cholesterol-based metabolism by Mycobacterium bovis-BCGon resistance to glutathione”. The purpose of this research project is to help further our knowledge of the unknown in order to answer questions that have not been previously answered regarding tuberculosis. The bacteria that is dealt within this research project, (Mycobacterium bovis-BCG) mirrors the bacteria of which is found in tuberculosis, an ongoing upper respiratory disease that many doctors are able to treat however not treat in its entirety. This research is to help understand the bacteria and the way the human immune response reacts, in hopes of one day finding a permanent cure.

At the conclusion of this research project I hope to obtain and learn proper technique and use of appropriate equipment and reagents needed to work in a professional laboratory. I hope to achieve a paper publication as well as present data collected from this research at Eastern Colleges Science Conference (Undergraduate regional conference) and American Society for Microbiology Microbe 2020 in Chicago, Il (Professional scientific conference).

I hypothesize that Mycobacterium bovis-BCG(BCG ) grown in a cholesterol-based media will not be killed when exposed to glutathione (GSH).This imitates the bacteria in its latent NRP state. The methods used to determine this will be through a series of serial dilutions using proper laboratory equipment and technique. This will test the survival of viable BCG cells in the presence of GSH over a time period of two weeks. This hopefully will help us conclude that cholesterol plays a role helping persistent NRP BCG remain alive when exposed to GSH because M. tuberculosis utilizes cholesterol as a primary carbon source as its metabolic processes is decreased.