Figure 8. Fluoromycobacteriophage M. tuberculosis susceptibility testing using flow cytometry.

A. M. tuberculosis mc²6230 strains grown in the absence of Tween detergent were infected with phAE87::hsp60-EGFP with or without antibiotic treatment, fixed with paraformaldehyde, and analyzed by flow cytometry. In panels a, mock-infected control cells and b, phage infected cells, the intensity of fluorescence is plotted against light side scatter. The signal observed in mock-infected cells corresponds to autofluorescence of M. tuberculosis and the shift towards greater fluorescence in phage-infected cells shows that a high proportion of cells are infected and expressed GFP. In panels c–i the level of fluorescence is plotted against the number of events counted. In each experiment mock-infected cells are shown in red, phage infected cells in green, and antibiotic-treated phage infected cells in blue. When treated with rifampicin (Rif) or streptomycin (Str) antibiotics were added simultaneously with phage (φ), incubated for 16 hours, fixed and analyzed; for INH treatment cells were pre-incubated with antibiotic for 48 hours, incubated with phage for 16 hours, fixed, and analyzed. B. M. tuberculosis mc²6230 strains were grown in the presence of Tween, washed, preincubated with isoniazid for 24 hours, infected with phAE87::hsp60-EGFP, fixed, and analyzed by flow cytometry. Panels a–e correspond to panels a–c, f, and i of Figure 8A respectively.