Hi Elaine, The fact that nothing cultures after three weeks and yet you still have SYTO 9-only-positive cells may mean 1) All your microbes are in fact dead and therefore the advice that Howard gives will provide you with a better method of proving this 2) Not all of your bacteria are in fact dead. This is a messy hypothesis to prove and entails entertaining the idea of viable but non-culturable (VBNC) bacteria (or even freakier, "active but non-culturable" [ABNC] bacteria). Many microbiologist would bite your hand off for suggesting that VBNC bacteria exist. I prefer to talk of "not immediately culturable" microbes. These could be slightly damaged microbes that need time in a friendly, non-challenging environment in order to recover the capacity to reproduce and form colonies on agar. Perhaps the SYTO 9-only-positive cells need to be held in a recovery medium before they'll grow on solid media? Perhaps they need to be plated out therafter on a rich medium like blood agar (with nutrient agar as the base) for them to form colonies? If you have the option of sorting this SYTO 9-only-positive sub-population and performing a few experiments using various recovery media (peptone water, Ringers etc.) your curiousity might be salved. I hope the above has helped a little, Ultan Elaine Kunze wrote: > We want to ennumerate the number of live bacteria in a gel. The bugs > are Staph epidermidis. We are using the BacLite kit. We see a number > of green only bacteria under the microscope despite the fact that > nothing cultures after three weeks. We do have some PI reds, but > there are many green onlys. What am I missing? > > Elaine Kunze > Flow Cytometry and Imaging > Huck Institutes of the Life Sciences > 319 Life Sciences Building > Penn State University > University Park, PA 16802 > 814-863-2762 >Received on Thu Jun 8 14:18:00 2006
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