You might be interested to come to the microbiology workshop
at the ISAC meeting where such things will be addressed (see
abstract below). Bacterial growth media are full of green
fluorescent stuff even without adding dyes. One of the
major components hit by the 488nm line are riboflavins
which come form yeast extracts. I am not sure about your
additives. Resazurin should react more in the UV where it is
used in the commercial Almar Blue Assay. Other oxidation r
eactions occur in the autoclaving process which also give r
ise to precipitates that screw up light scatter
discrimination, so it is worthwhile to filter all solutions
in use. Best bet is to try starting with a stationary
culture broth (approx 10^8 cells/ml or more) and dilute
1:100 in buffered peptone water to sort out your instrument
settings.
I would suggest log/log scatter settings anyhow, and try to
set up the instrument on yellow/green 0.5um beads (first on
green fluorescence threshold) which give scatter similar to
most bugs. Alternatively you can use heatfixed bacteria in
the presence of 2 to 5 ug/ml PI or EB and trigger on red
fluorescence. Once you established a clear scatter cluster
you can try switching to scatter gating. In most
instruments side scatter is the better option.
Good luck
Gerhard
The Venue:
ISAC XIX congress 28/Feb/1998 Colorado Springs
(see http://nucleus.immunol.washington.edu/ISAC.html)
Tutorial on Microbial Flow Cytometry,
The Program:
The aim of the tutorial is to give flow cytometry users the confidence
and the technical background to tackle the measurement of bacteria. To
achieve that there will be a theoretical part and presentations of
practical applications, accompanied by protocols and reference
literature. It is envisaged to organise some practical demonstration
with regards to instrument set-up within the week.
1. Background information : (Gerhard Nebe-von-Caron)
1.1 Technical background
Setting the environment : Requirement on labware and reagents.
Setting up the instrument : Calibration standards
Signal processing: Triggering / threshold settings, Bacterial
discrimination, back-gating
Light Scatter measurements : Opportunities and limitations
Sorting bacteria : Instrument preparations for sterile sorting
1.2 Functional and differential labelling of bacteria
Bacterial enumeration: Sample handling, disaggregation and counting
methods.
The viability concept : Measurement of reproductive viability,
metabolic activity and membrane integrity.
Bacterial differentiation : Antibody staining in of "environmental"
samples.
2. Practical applications :
2.1 Microbial Flow Cytometry in Biotechnology (Susann Muller)
The measurements of physiological changes in biological processes and
their influence on process control will be presented. This will cover
the measurement of cell proliferation by DNA synthesis and changes in
the cell wall structure and the prediction of "survival capacity" by
assessment of cell constituents such as stored energy and cell membrane
functionality.
2.2 Antibiotic susceptibility of Mycobacterium tuberculosis and Candida
albicans (Ronald Shell)
This presentation describes a method to test the antibiotic
susceptibility of clinical isolates of the above species based on
metabolic activity. Materials and sample preparation, instrument
set-up, analysis and data interpretation will be discussed.
The team:
Dr. Susann Muller, University of Leipzig, Fakultaet fuer Biowissenschaften
Dr. R.F.Schell and S.M.Kirk, University of Wisconsin, State Laboratory
of Hygiene
Mr G.Nebe-von-Caron, Unilever Research Laboratory Colworth
______________________________ Reply Separator _________________________________
Subject: Bacterial counting
Author: oberyszyn.2@osu.edu at INTERNET
Date: 24/12/97 12:41
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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