The most common discrepancy between CFU's and cytometric counts originates from identifying non-bacterial events as bacteria e.g. counting noise or bacteria originating from buffers etc but not from the sample. The use of DNA stains can help to identify what is bacteria and what is noise it can be misleading if the bacteria possess transport systems to chuck out the DNA stains. Other problems are aggregation and coincidence of events that need to be considered. Once those potential interferences are sorted out things should not be too bad. However there are systematic problems originating from the term "viability". A staining based on DNA stains with differential membrane permeability can only indicate the permeability of the membrane to the respective probes, so you measure membrane integrity. Whilst in general cells taking up PI would be classified as dead permeabilisation can be transient as under conditions of electroporation, chemoporation etc. Nonpermeabilised cells have at least the potential to form gradients but do not necessarily do so. Thus a discrepancy between reproductive viable cells and intact cells has to be expected. In fact if you irradiate bacteria of treat them with strong oxidative stresses you can generate bacteria that show membrane integrity, even metabolic activity but still won't reproduce. If you look at perfectly happy marine samples it is generally accepted that even if the cells show metabolic activity, the number of cells growing in culture is still only a small fraction of the cells detectable as they are very fastidious. Similarly if you look at stressed or injured cells most culture based recovery methods fail. The discrepancy between different recovery methods can give you up to 5 log difference. In the light of those problems even the differentiation system described in http://www1.elsevier.com/homepage/sah/mimet/speciss/1378.pdf does not help to consolidate the data. Overall you should expect the numbers of intact cells to exceed the number of reproductive viable cells if measurements are done correctly, as the reproductive growing cells are a subpopulation of the metabolic active cells which in turn are a subpopulation of the intact cells. regards Gerhard -----Original Message----- From: Therese M. McGinn [mailto:tmcginn@benchmarkbiolabs.com] Sent: 28 November 2005 16:16 To: Cytometry Mailing List Subject: Bacterial enumeration: beads versus CFU Hello All, 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 Therese M. McGinn Ph.D. Senior Research Scientist Benchmark Biolabs, Inc. Phone: 402-475-8104 Fax: 402-475-8511 www.benchmarkbiolabs.com ********************************************************************** This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you have received this email in error please notify the system manager. This footnote also confirms that this email message has been swept by MIMEsweeper for the presence of computer viruses. www.mimesweeper.com **********************************************************************Received on Fri Dec 2 15:58:00 2005
This archive was generated by hypermail 2.1.8 : Sat Jan 14 2006 - 22:04:00 EST
