Bacterial enumeration: beads versus CFU

Gray, Diane Diane.Gray at invitrogen.com
Tue Nov 29 19:28:44 EST 2005


 
Terry-
I, too, have noticed a discrepancy between counts generated by flow
cytometry vs.CFU. I have accounted this observation to the following:
 
-individual cells can be stained "live" that may not be "vital" enough
to grow on plates
-cells that stain "dead" (slightly to severely permeabilized membranes)
may not actually be dead, but can "recover" if given optimal conditions;
the assumption with this flow assay is that a permeabilized cell is not
"alive"
-precision and accuracy of counting bead solution concentration
-flow rate of the instrument or rate of events/second
-ability to discriminate "real cells" from debris or noise
-the assumption that a single colony comes from from a single bacterium
 
With these considerations, I am not convinced plate counts can verify
flow counts or vise versa, and I'm not sure if validation of a
particular method should operate under that assumption.
 
Best regards,
 
Diane R. Gray
Microbiologist
Functional Biology/Flow Cytometry
Molecular Probes Labeling and Detection Technologies
Invitrogen Corporation
29851 Willow Creek Road
Eugene, OR 97402-9132
p. 541.465.8300 x59754
f. 541.335.0177
Diane.Gray at invitrogen.com
http://probes.invitrogen.com

 

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.  

Thank you for your consideration,

Terry McGinn

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