UGR Grant Final Report
In the beginning of the summer, I aimed to understand the intracellular response of Mycobacterium tuberculosis when attacked by the detoxification molecule, Glutathione. Using Mycobacterium bovis-BCG (BCG) as our model organism, our previous researched showed that non replicative persistent (NRP) BCG was resistant to glutathione induced reductive stress killing, while normal BCG succumbed to detoxification. This shows us that NRP BCG has an extra line of defense against the usual way the body would try to kill the bacteria. This data, combined with research from other laboratories stating that the presence of cholesterol in the granuloma can act as an electron sink helping to overcome reductive stress, gave way to my hypothesis.
I hypothesized that a cholesterol induced metabolism will protect M. bovis-BCG from GSH induced reductive stress killing similar to how NRP BCG is able to resist GSH. In order to test this, I planned to use an NAD/NADH-GloTM Assay in hopes of seeing BCG accumulating more NAD+/NADP when associated with cholesterol. These markers would indicate a metabolic shift towards an oxidative environment in the bacterial cytoplasm and would prevent glutathione induced reductive stress killing.
Before I could use the NAD/NADH-GloTM Assay, I had to create a media that contains enough cholesterol to induce a metabolic shift while also ensuring that the organism is not nutrient deprived. A recipe was created for 7H12 media using information from other labs who also worked with MTB and cholesterol. This recipe created our first obstacle because in order to fully dissolve the cholesterol, we had to heat 200 proof ethanol to 80C; which is highly dangerous. Ultimately, with much trial and error, we were able to create a media with the dissolved cholesterol without heating ethanol to 80C, called 7H12T. Additionally, a detergent to prevent the clumping of these naturally sticky organisms was needed. Originally, we attempted Tyloxapol, which was very hard to use due its thick consistency and a large amount was necessary to be effective. This made the media too bubbly when we had to filter sterilize it. Instead we found that we were able to use Tween80, a detergent that we use in our regular 7H9 media when we culture BCG without cholesterol.
Once we finished creating the media, we conducted growth trials to ensure that we would be able to culture BCG with it. When conducting growth trials, 1 mL of frozen BCG suspended in glycerol, then added to 4mL 7H9 media and was allowed to incubate for 24 hours. Then, 1 mL was added to 24mL of 7H12T media and incubated for three days. The growth was monitored each day by measuring optical density using a spectrophotometer. After the first day, we suspected contamination because of the doubling time. BCG usually takes three days to reach mid-log stage with an optical density of 0.6-0.8 nm. But, this culture reached 1.1 nm by day two. With other contamination issues being observed in the lab, by using a crystal violet stain we were able to observe three different types of contamination including yeast spores. The contamination was traced backed to our original frozen stock, forcing us to purchase fresh BCG.
The need for new BCG was a huge setback for the project, because we could not continue without it. Being a biosafety level 2 organism, it was difficult to obtain, and the shipping process took some time. After a few weeks, we finally received our new BCG and are currently working on culturing it to create new frozen stocks. Although I was not able to complete the goals that I had set, I still benefitted from this process. I was able to learn a lot about contamination; when to be suspicious, how to find it and how to get rid of it. A large part of the process included staining samples and observing them under microscopes, which was very interesting. Although the bacteria was damaging to my research it was fascinating to see all the other microbes living around us and how easily something could become infected. I also think that the many setbacks in creating the media was helpful to me as a researcher and scientist. With guidance from my advisor, I was able to learn how to go about making media that is suitable for a specific experiment and it really expanded my ability to comprehend scientific literature.
For the remainder of the summer and into the first semester, once the new BCG has been cultured; I will continue working on obtaining a successful growth curve which will lead to performing a NAD/NADH-GloTM Assay. Although scientific research does not always go as planned, I am incredibly grateful for the opportunity to work with BCG and further my knowledge in the field of microbiology.