bacterial viability assay - (Aug/05/2009 )

Hi,

I am going to look at bacterial viability after ingestion by macrophages. I will lyse the cells and initially plate out the lysates to count cfu's
However, I need to do this at a high thoroughput level, and therefore would like to use a fluorescent/colormetric assay in 96 well plates to determine bacterial viability.
I have heard conflicting results regarding the alamar blue (resazurin) assay, and wonder if anyone has used similar assays? or even looked at fluorescent assays such as calcein?
Thanks!

You can look into some DNA binding dye on fixed bacteria.

How do you propose to validate whatever technique you emply in this application?

I guess that's why I'm using this forum.

An obvious method would be correlation with cfu's, but am open to whatever has been tried and tested by others...

you can use for example SYTO-9 (live) and propidium iodide (dead) ...but i don't know how this interferse with your macrophages. I don't get it how you would do this in 96-well plates? How will you do the read out of such an assay? Maybe you can comment on that?

Regards,
p