Dear flowers, my question to the flow community was: Hi, a collegue attempts to count bacteria (Bacillus subtilis)before and after they are gone through the Ribolyser procedure to determine/confirm the percentage of undestroyed cells. I was looking for a suitable dye and found the LIVE/DEAD BacLight Bacterial viability and counting kit from INVITROGEN. Has anyone general experiences with this kit, and do you think it is possible to use it for out purpose? Thanks! Petra I appreciate very much that so many of you took the time to answer! Here are some of the replies: Hi there, I have used this kit and other syto dyes to stain bugs and it works very well. We found that the syto dye requires titration since each species ability to uptake the dye differed. Also, for our experimental needs to compare the counts and percentages of viable cells, we typically acquired sample based on time so that the relative volume of sample was approximately the same from sample to sample. Over time, we stop getting the kit and bought the syto dye and PI separately, more cost effective. We tried sytox on a Beckman Coulter XL and the cytometer could not see the bacteria. Had to go to a BD aria. That worked! I am not sure what the "Ribolyser" procedure entails. However I would guess that after this treatment all the bacteria are dead, and that your colleague wants to determine the number of particles that contain nucleic acid (by fluorescence) vs. particles that do not contain nucleic acid. If this is the case, the Live/Dead stain will work and and you need only examine PI fluorescence because all your cells are dead to begin with. Viability is not the issue; the questions are about nucleic acid content and cell lysis. If you haven't bought the the Live/Dead kit and have no need to examine bacterial viability (I doubt that is an issue for "ribolysis"), then DAPI or acridine orange would be cheaper to simply reveal particles that have DNA inside them. Both are mutagenic so care should be taken not to inhale the powders and to avoid contact of the aqueous solutions with the skin. I am not sure about the Live/Dead components, but I would bet that any nucleic-acid-binding xenobiotic can be shown to be a mutagen. The kit works well with Bacillus subtilis in our hands - we use it routinely for "live /dead" analysis. I'm not sure what the Ribolyser procedure is - but as long as it leaves your cells intact, so that they retain their DNA and can trigger the electronics by scattering light - you should be able to distinguish between cells with intact and compromised cell membranes. Whether this works for your application depends on what the difference between your "undestroyed" cells versus your destroyed cellsis, i.e. the extent of membrane permeability. Petra >> Dr. Petra Hildebrandt >> Ernst Moritz Arndt-Universität Greifswald >> Medizinische Fakultät >> AG Funktionelle Genomforschung >> Friedrich-Ludwig-Jahnstr. 15 A >> D-17489 Greifswald >> Germany >> >> Phone: +49-3834-865874, 864074, 865881 >> Fax: +49-3834-8680005 Petra.Hildebrandt@uni-greifswald.deReceived on Tue Jul 1 15:18:00 2008
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