mCherry10-expressing Mycobacterium tuberculosis (Mtb) was generated as described elsewhere [13 (link)]. Mtb H37Rv was obtained from American Type Culture Collection ((ATCC) 27294, Manassas, VA, USA). Strains were cultured as described in detail elsewhere [14 (link)]. In order to generate frozen stocks, mid-log-phase bacterial suspensions (OD600, 0.2 to 0.4) were stored at −80°C. After four weeks of storage, the number of bacteria (colony forming units, CFU) was determined by plating serial tenfold dilutions on 7H10 plates (0.05% Tween 80 / 10% heat-inactivated bovine serum in phosphate buffered saline (PBS)) and counting colonies after 3–4 weeks of incubation at 37°C.
For spiking experiments, frozen stocks of Mtb H37Rv were thawed, centrifuged (4000xg), bacteria re-suspended in PBS and a defined amount of this solution added to buffer or saliva samples. To address the impact of proteinase K plus DTT treatment on bacterial viability, Mtb H37Rv spiked PBS samples were incubated in the presence or absence of proteinase K (18U/ml, Carl Roth, Karlsruhe, Germany) and Dithiothreitol (DTT, 2mM, Sigma-Aldrich, St. Louis, USA) for 60 minutes at 37°C and subsequently subjected to CFU analysis as described.
For spiking experiments, frozen stocks of Mtb H37Rv were thawed, centrifuged (4000xg), bacteria re-suspended in PBS and a defined amount of this solution added to buffer or saliva samples. To address the impact of proteinase K plus DTT treatment on bacterial viability, Mtb H37Rv spiked PBS samples were incubated in the presence or absence of proteinase K (18U/ml, Carl Roth, Karlsruhe, Germany) and Dithiothreitol (DTT, 2mM, Sigma-Aldrich, St. Louis, USA) for 60 minutes at 37°C and subsequently subjected to CFU analysis as described.
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