Bacterial and Viral Particle Quantification

Swab samples were thawed at room temperature immediately prior to sample preparation. Once thawed, swab tips were cut off into 1 ml of sterile (0.2 µm filtered and UV treated) TE buffer (10 mM Tris, 1 mM EDTA, pH 7.4, Sigma). Samples were then vortexed for 3 minutes to elute the bacteria and viruses from the swab tip.
Eluted swab samples were diluted (1:100) in 0.2 µm filtered TE buffer for optimal visualisation of bacterial and virus-like particle (VLP) populations. Diluted samples were then stained with SYBR-I Green (1:20,000 final dilution; Molecular Probes) and incubated for 10 minutes in the dark at 80°C as per previously established and optimised methods [17 (link),18 (link),21 ]. Control samples of sterile rayon swabs eluted in sterile TE buffer were prepared in the same manner as the participant swab samples. These samples were used to eliminate any background artefacts introduced during sample preparation or from the rayon swabs themselves (S1 Fig). Triplicates of each swab sample were prepared for analysis (S1-S4 Tables). Fluorescent beads (1 µm, Molecular Probes) were added to each sample at a concentration of 10⁵ beads ml⁻¹ [22 ]. Using the bead fluorescence and concentration as a control, flow cytometric parameters were normalised [22 ].