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.