Hi, I'm having trouble with a bacteria counting kit (B-7277) from Molecular Probes which I bought because I don't have a coulter counter. The kit consists of SYTO BC bacteria stain (fluorescein channel) and unlabeled 6 um beads which are supplied at a concentration of 1 x 10(8) beads/ml. The procedure stains 1 ml of bacteria, adds 10 ul of the beads, and is then counted on a FL1 vs FSC plot. Since you spike the sample with a known concentration of beads, you draw a region around each population and can calculate the number of bacteria. I have no trouble seeing unstained and stained Haemophilus using log FSC vs log SSC on the FACSVantage ( I remove the ND filter in front of the FSC detector and use the wide obscuration bar) but cannot see the 6 um beads. They're evident by the rate counter and on the oscilloscope but are not piling up on the end of the histogram. If I switch to linear FSC my beads obviously appear but I lose my bacteria. I have talked to both BD and Molecular Probes about this. Has anyone with a FACSVantage used this kit? Could you also give me some pointers on how to see bacteria and 6 um beads on a log FSC vs log SSC plot? Another lab has been able to see them on an analyzer. Any help would be greatly appreciated. Louise Barnett University of Missouri barnettl@missouri.edu (573)884-7320
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