Thomas, maybe that your "lost" bacteria are under your (scatter?) discriminant. When doing two color viability stains with the elite a double discriminant on both fluorescences can be used, just to see only live and death cells (and ignore unstained cells or particles). If you are looking for unstained cells then you can try using a doble discriminant on FSC and SSC. When using scatter parameters with bacterial cells, is better to calibrate your instrument using small beads (we use the one micron from Polysciences or Mol Probes) to avoid counting background noise. It is surprising how little changes in the optics adjuntement or in the FSC mask position can change the amount of background (and bacterial cells) detected. Good luck, Jaume En/Na "R. Thomas Leonard" ha escrit: > I'm on a mission to determine bacterial viability as well as quantify > bacteria using Molecular Probes' BacLight viability kit. Using an EPICS > Elite, I have routinely found bacterial counts to be 10% of those > determined with fluorescence microscopy. The accuracy of the microscopy > counts have been verified by other labs. I recognize that the organism has > mechanisms to pump out fluorochrome, but to the extent that 90% of bacteria > go undetected? We're analyzing on the log scale. Our operators are > accustomed to analyzing mammalian cells, but claim they have done all the > "tweaking" possible to optimize bacterial analysis. > > Why such a disparity in counts? My conclusion is that the instrument simply > can't detect the smaller organisms in the population (of E. coli). > > Also, we can see distinct populations when analyzing live controls and dead > controls independently, but when we mix the live and dead populations we > are often unable to distinguish between live and dead populations. > Sometimes we can, other times we can't. It is not only strange but > frustrating. > > Has anyone had similar experiences? Any thoughts on what might be > contributing to these unexpected results? Thanks in advance. > > Tom Leonard -- Jaume Comas Citometria de Flux. Serveis CientíficoTècnics Universitat de Barcelona tel 34/93.403.46.54 fax 34/93.403.72.06 mailto:jaume@giga.sct.ub.es http://giga.sct.ub.es:800/
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