Bacterial counting

Andy Oberyszyn oberyszyn.2 at osu.edu
Tue Dec 23 15:18:26 EST 1997


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 at osu.edu




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