Thanks to everyone for your helpful replies to my earlier request (see text below). Accordingly, I feel compelled to share our results. Based on your suggestions, we've (the operator and I) concluded that the discriminator setting makes all the difference when attempting to quantify bacteria. While we obtained very poor quantitative results triggering on forward scatter, triggering on log side scatter worked beautifully in terms of accurately quantifying bacteria. Even triggering on PI yielded acceptable results. As for viability discrimination, we found wonderful separation between independent live and dead controls, yet when the two populations were mixed the separation appeared to become more of a gradient. Nonetheless, when "live" and "dead" regions established from the controls were actually sorted, about 80% of the "live" cells were live and less than 5% of the "dead" sorted cells were live. Can't complain about that (or at least I won't). Thanks Folks! Tom Leonard 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
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