Hi Louise, I am not sure if this can solve your problem, but perhaps you should try and increase linear amplification on FSC up to 32 which could bring your bacteria and beads on the same scale. You should use FINE GAIN control bellow the oscilloscope on Vantage by pressing 1 (as parameter 1 - FSC) then FINE GAIN button and then up-arrow button to increase amplification up to 32 (setting it first on 16 in CellQuest Det/Amp window). I am using 2.5 µm beads for daily alignment and I expect to see them on the middle of oscilloscope screen/plot with FSC lin 16 amplification and ND filter out. It also depends on the size and position of FSC obscuration bar. If you are using normal pressures (11-15 PSI) you should have small, standard obscuration bar. Tweaking the position of the bar is going to diminish FSC or to go up to the pure laser light noise. Hope this helps, Sasha. On Mon, 25 Oct 1999 14:04:09 -0500 "Barnett, Louise " <BarnettL@missouri.edu> wrote: > > 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 > ---------------------- Dr Sasha Sreckovic Dept Path & Micro University of Bristol University Walk Bristol, BS8 1TD, UK Sasa.Sreckovic@bristol.ac.uk 0117-928-8606
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