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.