Hello everybody and Happy Holidays to all before I begin! I have a question which I hope someone can help me with. I have recently taken over as flow operator of a core analytical flow lab and I am somewhat new to flow in general so please forgive me if this is a trivial question. I have an investigator who would like to do bacterial counting (Porphrymonas Gingivalis (a gram neg. rod) using the Molecular Probes Kit (Bacterial Counting Kit #B-7277). According to the protocol, I should be able to get signals with log FS vs. lin SS. We ran a sample the other day and I was not able to destinguish between debris and the bacterial cells (i.e., I was getting the same type of signal with media alone). I tried different discriminator settings but could still not get a difference in settings. The media they use is BH1 media enriched for anaerobes (some contents= hemin, Vit. K, cysteine, resazurin). When I ran the beads (included in the kit for quantitation) in dH2O, I got exactly the pattern Molecular Probes info sheets says you should, however, when I ran beads in media only, the bead pattern disappears and I get alot of fluorescence (middle of Fluorescent intensity (log scale) and low on FS (log scale). I guess my question (in a round about way) is, do any of the items mentioned in the media show green fluorescence? We thought it might be the resazurin but we haven't tried that yet. Any suggestions or help would be greatly appreciated!! Merry Christmas to all, and to all a good night!!! Andy Oberyszyn The Ohio State University Analytical Cytometry Laboratory 416 Comprehensive Cancer Center 410 West 12th Avenue Columbus, Ohio 43210 Tel: 614/292-FLOW(3569) Fax: 614/292-7335 E-Mail: cytometry@osu.edu
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