Eventually, after proving myself to be a somewhat competent microbiologist and able to follow the lab safety rules, it was agreed that we could start trying to grow TB according to Friedrich's instructions. My work with BCG had persauded me that the freshly made growing medium according to Friedrich's instructions was the best, but also I had quite a lot of problems with contamination. This problem followed me into the CL3 (containment level 3 - extra security) lab and a number of our initial attempts to grow TB had to be discarded.
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| Oxoid Nutrient Broth |
While waiting to access one of the additional ingredients I required to make the growing medium - Peptone - I had used some ready made medium called Nutrient Broth and made by Oxoid. This had proved to be very good in growing BCG so we agreed to give it a try for the TB and at the same time reduce the likelihood of contamination by changing our methods of adding the additional ingredients.
Despite the TB growing well it failed at first to form biofilms - films of TB colonies on the surface of the liquid which is what we needed. With a little experimentation we found that one issue seemed to be the amount of TB we added to our growing medium. 7ml of recently flagged (using a MGIT) in 100 ml of medium proved to be successful and working with the most studied strain of TB - H37rv - we were rewarded with a nice biofilm after three weeks of growth in the incubator.
This flask shows the growth of the early biofilm after four weeks growth. and a close up pic of the biofilm
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| Close up of the biofilm |
It is interesting to note that most reports of TB biofilm growth only cover a period of 5 weeks growth as for example this study by Ojha et al in 2012. A big issue in TB treatment and other diseases is the antibiotic tolerance which is often associated with the formation of biofilms. Biofilms provide an important reservoir of cells that can repopulate colonized sites upon removal of drug treatment. The biofilms are also linked to drug tolerance as in this study so it is clearly important to study the underlying mechanisms.
As the biofilms in vivo are long lasting, it is curious that the work with biofilms is over such a short period. The issue appears to be that in addition to the fact that biofilms have not in the past been extensively studied, when the biofilms are grown in vitro in the lab after a couple of months the biofilm appears to sink under its own weight to the bottom and the biofilm is lost. As I explain in the next blog, Friedrich's methodology has circumvented this problem.
