Bacterial enumeration: beads versus CFU

Howard Shapiro hms at shapirolab.com
Mon Nov 28 20:49:36 EST 2005


Terry McGinn wrote-

>I have been using the Becton Dickinson Cell Viability Kit with 
>Counting Beads to determine numbers of live bacteria in 
>cultures.  This method involves staining with propidium iodide and 
>thiazole orange to discriminate live from dead cells.	Numbers of 
>live cells are determined by a simple calculation relating the 
>number of events in the live gate to the number of beads.
>
>We have been comparing the numbers of live cells by this method to 
>CFU/ml as determined by traditional enumeration assay.  The 
>flow-based method shows much higher numbers of live cells than the 
>enumeration assay.  I would appreciate any feedback or suggestions 
>on how to reconcile this discrepancy.

The "viability" measured by a kit such as this is not reproductive 
viability, which is what you measure when you look for colonies on 
plates. When you do plate bacteria, you see a colony, and you don't 
know whether the "CFU" that gave rise to that colony was one cell, or 
two, or ten, or a hundred, etc. So, one possible cause of a 
discrepancy between "viable cells" by flow and a CFU assay is that 
aggregates of viable cells could be broken up by shear stress in the 
flow system, so you would measure individual cells in the flow 
cytometer, and get many times more "viable" cells than you would if 
you looked for CFU. I wouldn't bet on this being the actual cause of 
the discrepancy, but it's possible.

Conventional wisdom has it that cells - bacterial, mammalian, etc. - 
only take up propidium when their membranes are damaged. There are a 
lot of ways in which cells can be made reproductively nonviable 
without immediately becoming permeable to propidium, treatment with 
radiation being one example. As far as thiazole orange uptake goes, 
it does not require active metabolism; it will get into cells with 
intact membranes and cells with damaged membranes, and, unless that 
particular type of cell has an efflux pump that will get rid of it, 
it will stay there. In other words, a cell that you call "live" by 
virtue of its uptake of thiazole orange and not propidium may not be 
alive; it may just not have been dead long enough for the membrane to 
let in propidium.

As if all of that weren't bad enough, it now appears that, under 
stressful conditions, some cells that are not dead may transiently 
become permeable to propidium. Cytometry is easy. Biology is hard.

-Howard





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