In the beginning of this year, I aimed to understand the effects of the human immune response on persistent Mycobacterium tuberculosis. Using Mycobacterium bovis-BCG as a model organism, I planned to conduct growth trials for active and persistent culture grown in a cholesterol rich 7H12T media and treat them with glutathione to determine if active culture could undergo a metabolic shift similar to that of NRP BCG. If the active BCG was able to shift towards an NRP metabolism, it would indicate that the active BCG was able to induce a persistent state solely through the presence of cholesterol. I also planned to conduct a NAD+/NADH, H+ Glo Assay to understand how cholesterol is impacting the cell.
Over the past year our laboratory has been suffering from contamination issues, preventing the growth of our BCG and the ability to obtain accurate data from pure culture. Luckily, we were able to order new BCG and grow it up into new pure culture. Creating bacterial frozen stocks from a dry pellet takes about four weeks, putting my work slightly behind schedule, but I was able to start growth trials within the last week. Being in the first two days of the trial, I have not obtained any statistically significant data, but the bacteria appears to be growing in the harsh environment I have provided for it. I plan to continue this growth trial for about a week and determine my next steps from there. If the bacteria show a normal growth curve, I will treat new cultures with glutathione and hopefully observe their persistence. If they do not grow during this trial I will reassess my media recipe and attempt to create a more nutritious environment for the bacteria.
From the first half of my research I have learned that science does not always behave on a schedule! It is very difficult to try and force bacteria to grow, especially when you have deadlines to meet! I also learned a lot about controlling contamination and how to continue keeping the lab as pure as possible.