S11494

Virus-like particles (VLP) were quantified using epifluorescence microscopy after staining with nucleic acid dye [68, 69 (link)]. Briefly, viral inoculum were first fixed with glutaraldehyde (Sigma-Aldrich G6257, final concentration 0.5 %) and diluted in 1 % potassium citrate solution (pH 7) to prevent aggregation of viral particles. Inoculum were vacuum filtrated to capture viruses on a 0.02 µm aluminium oxide filter (Whatman 6809–6002 Anodisc 25 inorganic membrane filter), before subsequent staining (SYBR Gold nucleic acid gel stain Invitrogen S11494, diluted at 0.2 % in Tris-EDTA) and washing by 4 ml of 1 % potassium citrate solution. Filters were then mounted on glass slides and VLP were observed and enumerated under epifluorescence microscopy (Olympus BH2-RFCA).

Primary stock dilutions of antibiotic powders were made in 100% DMSO and frozen at − 20 °C protected from light. Fresh working dilutions were prepared in PBS prior to each experiment, 0.22 nm filtered, and stored wrapped in tin foil at 2–5 °C refrigeration. Final concentrations of antimicrobials used in M. bovis BCG time-kill experiments are reported in multiples of the MIC as indicated. The MIC were: rifampicin, 0.01 μg/ml; isoniazid 0.125 μg/ml; kanamycin 1.0 μg/ml; ethambutol 1.0 μg/ml.
Calcein-AM 50 μg vials (ThermoFisher, C3100MP) were reconstituted in 50μL of DMSO on the day of each experiment, further diluted to 200μL with PBS for “working stock”. For cell staining, 5μL of working stock was added per 100μL of sample, with pipette mixing, then incubated in the dark at room temperature for 45–60 min before resuspension of bacilli with pipetting. SYBR-gold propriety stock (ThermoFisher, S-11494) was diluted 1000-fold in PBS, aliquoted and frozen at − 20 °C until use. After thawing aliquot, a further tenfold dilution (to 10^–4) in PBS was performed, and 5μL of this working stock added per 100μL of sample to be stained, with pipette mixing, followed by incubation in dark at room temperature for 45–60 min before resuspension of bacilli with pipetting.