I have not use the kit, but I remember that some time ago, we use to count live mycobacteria before the infection in a hemacytometer, using fluorescein diacetate.

The idea is that the fluorescein diacetate is permeable and once it is broken down by live mycobacteria, it is trapped inisde it. I guess, it should work for flow too.

Hola Mónica, I've used the kit for different bacteria species and, usually, works different in each specie. First, I used to dilute the kit because some times the dyes concentration is too high for certain bacteria. In order to help you better, please give me information about flow cytometer and optical density of your sample,

I have not used this for mycobacteria but can suggest that your cell suspension needed to be adjusted to ~10^9/ml at OD600 = 0.135-0..145. Use 0.5 microliter of Syto 9 and propidium iodide each.

I have tried to use the live/dead backlight for cytometry and the results were also not good. Both control and treated cells were stained with PI/Syto 9 . So I decided to evaluate the bacteria viability by confocal microscopy using the same kit with very satisfactory results