cell cycle in bacteria using PI

Hi Luciana,

One thing you need to do is make sure you are only collecting the 
fluorescence from single cells and not doublets or chains.  Mild 
sonication (circa 10 W for 10-20 sec) should work in breaking up chains 
and doublets and may have the added bonus of permeabilising the cells in 
a more satisfactory way than treatment with chemicals.	If your 
instrument allows it you should use pulse width to differentiate single 
cells from doublets or chains.	For some machines you can tell the 
difference between single cells and all the rest using FSC width vs SSC 
width or FSC area vs SSC area (or a mixture of these!). If you can 
resolve single cells in this way, gate	these cells into a histogram of 
PI fluorescence.  Also, the slower you run your samples the better the 
resolution.  As an aside, you may get better results with cyanine 
dimers, considered to be very high affinity nucleic acid stains e.g. 
YOYO-1.  Finally, as suggested recently, incuding an RNase in your 
staining buffer may give you sharper peaks as bacteria have a high ratio 
of RNA to DNA at certain points of their life cycles.

Ultan Cronin