Due to the fastidious nature of Mycobacterium bovis-BCG and the minimal media that I am attempting to culture it in, I have run into multiple different problems. Once I was finally able to establish a media that was suitable for my bacterium and experiment, I began growth trials. Initially, I was dealing with a contamination problem, so I was unsure if my media was successful, or if another organism was using it for itself. After weeks of waiting, my research team was able to culture pure, uncontaminated BCG, that we are now able to use for all our experiments.
Normally when growth trials are conducted, we dilute our cultures down to an optical density of 0.1, where after a week we are able to see a steady growth curve. Following this procedure, I conducted two more growth trials with the new BCG. I noticed that all my samples were remaining at an optical density of 0.1 for more than a week, which is abnormal. Assuming that the bacteria needed more time to adjust to the minimal, nutrient depleted media, I allowed it to continue to grow for another week. After two weeks the optical density remained the same. With both of my growth trials following this pattern, I started to think my media was unsuccessful. Then I thought maybe the bacteria weren’t dying after all. Maybe they were protecting themselves from their harsh environment by entering non replicative persistence.
I am now conducting experiments to determine if the BCG has become dormant. I am currently conducting viability trials, where I allow the bacteria to grow in the cholesterol media for a week (hopefully they will have entered NRP), and plate them on 7H11 plates. After two weeks of incubation, I can count their colonies and see if the bacteria are still alive, or if I need to alter my 7H12 media.