Capsaicin Survival Assay for Streptococci

Survival in presence of capsaicin was tested by the live/dead assay using SYBR Green I (Invitrogen, Eugene, OR, USA) and propidium iodide (Sigma–Aldrich), two nucleic acid dyes differing in their ability to penetrate bacterial cells (Magi et al., 2015 (link)). Briefly, overnight-grown streptococci were suspended in 1 mL capsaicin-supplemented BHI broth [∼1 × 10⁸ colony forming units (CFU)/mL] and incubated for 15, 30, or 60 min at 37°C in 5% CO₂. After staining with 1 × SYBR Green I and 40 μg/mL propidium iodide, cells were incubated at room temperature for 25 min in the dark, harvested on GTBP filters (Ø = 0.2 μm, Millipore, Billerica, MA, USA), and examined under an epifluorescence microscope (Axioskop2, Zeiss, Milano, Italy).