Aliquots of 0.5 mL of each actively growing M. avium isolate were transferred to 1.5-mL polypropylene screw-cap microtubes (Sarstedt). The tubes were then inoculated with 0.5 mL of: 64, 32, 16, 8, 4, 2, 1, 0.2, 0.02 or 0.002 mg of amikacin/L; 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.1, 0.02 or 0.002 mg of ciprofloxacin/L; 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.1, 0.02 or 0.002 mg of clarithromycin/L; and 16, 8, 4, 2, 1, 0.5, 0.24, 0.1, 0.02 or 0.002 mg of rifabutin/L to produce final concentrations of half of these amounts. Drug-free controls of M. avium were also prepared by inoculating them with 0.5 mL of 7H9 broth (Difco). Thereafter, the suspensions were incubated for 24 h at 37 °C in an environment of 7–8% CO2. After incubation, 0.2 mL of each assay suspension was removed and placed in a sterile 1.5-mL screw-cap microtube containing 57 μL of 8% paraformaldehyde (pH 7.4) and 0.2 mL of sterile phosphate-buffered saline (final paraformaldehyde concentration was 1%). Samples were mixed and held at room temperature for 45 min before being analyzed with a Bryte HS flow cytometer with WinBryte software (Bio-Rad Laboratories, Hercules, CA, USA). Samples fixed with paraformaldehyde were periodically tested for viable cells by plating 0.01 mL of the suspension on 7H10 agar medium (Difco). Subsequently, the plates were incubated at 37 °C for 14 days. No viable M. avium cells were detected. After treatment with paraformaldehyde, M. avium cells were detected and differentiated from non-M. avium particles in 7H9 medium by flow cytometry using forward and side-angle light scatter signals. Electronic noise and background particles in the 7H9 medium were excluded from analysis by adjusting the threshold monitor listed on the WinBryte software program. Forward and side-angle light scatter were then used to analyze M. avium cells that were incubated with or without antimycobacterial agents. For each sample acquired, the flow cytometer provided a histogram profile showing the number of M. avium organisms in each of 2048 logarithmic channels of increasing light scatter. Five thousand events were acquired for each sample at a flow rate of 2 μL/min. In addition, 2.5-μm polystyrene beads (Molecular Probes, Eugene, OR, USA) were used daily for calibration of the instrument.