Viability Assay for B. burgdorferi

To estimate the viability of B. burgdorferi, the SYBR Green I/propidium iodide (PI) assay was performed as described by Feng et al. [35 (link)]. Briefly, SYBR Green I 5 µL (100 × stock, Invitrogen, Waltham, MA, USA) and 5 µL propidium iodide (0.5 mM, Sigma, St. Louis, MO, USA) were added to each well and mixed thoroughly. The plates were incubated in the dark for 15 min at room temperature, with λ_ex at 450 nm and λ_em at 535 nm (green emission) and 635 nm (red emission) for each well of the screening plate using a TECAN Genios Pro microplate reader (Männedorf, Switzerland). Meanwhile, B. burgdorferi suspensions (live and 70% isopropyl alcohol killed) at five different proportions of live: dead cells (0:10, 2:8, 5:5, 8:2, 10:0) were mixed and added to the wells of the 96-well plate. Then, the SYBR Green I/PI reagent was added to each of the five samples, and the green/red fluorescence ratios for each proportion of live/dead B. burgdorferi were measured using the same parameters on the TECAN Genios Pro microplate reader as above. Using least-square fitting analysis, the regression equation and regression curve of the relationship between the percentage of live bacteria and the ratios of green/red fluorescence were obtained. The regression equation was used to calculate the percentage of live cells in each well of the screening plate.