Viability of intracellular bacteria - (Nov/18/2010 )

Hi all,

From my timelapse imaging, I always observe my bacteria trap in late endosomal compartments, even after overnight. Although the bacteria look fairly intact from the morphology, I would like to know if the bacteria are still viable.

Do you know if there is any way I can check it?

I came across the Live/Dead Baclight staining kit from Molecular Probes, but not sure if this kit is going to stain the intracellular bacteria specifically, or it will stain up my cells simultaneously.

I also thought of lysing the cells to release the intracellular bacteria and plate them on agar plates. However, this approach will only work if the gentamicin work at 100% efficiency to kill of the extracellular bacteria. Normally, I still get 1 or 2 colonies on my agar plate when I plate the medium containing gentamicin at the end of the experiment.

Any idea from all the genius out there?????

Many thanks in advance.

I would lyse the cells and plate out serial dilutions to work out viable CFU/ml. Is it the standard method for measuring bacterial invasion. One or two 'background' colonies shouldn't alter your result too much. From my experience, 100ug/ml gentamicin for 2 hours was sufficient to kill extracelluar bacteria.