Re: FACSCalibur parameter settings for analysis of baacteria

Gerhard Nebe-von-Caron (Gerhard.Nebe-von-Caron@unilever.com)
28 Oct 1996 13:52:07 Z

In principle most of the modern cytometers are capable of
analysing bacteria to a certain degree. We recently had a go
at that at the flow cytometry course in Mannheim/Germany. To
find your instrument settings you can use 0.5micron yellow
green beads. We actually used the green fluorescent bacteria
from the Phagotest kit (Becton Dickinson / Orpegen). First
you set the instrument on green discrimination and increase
the voltage until you get a cluster forming. Green
fluorescence is fairly intense. Once you have that cluster
you set it to the top of the log green axis and the side
scatter (forget about forward scatter) to the middle of the
log scale. Then you can change the settings to side scatter
discrimination. If you loose your cells at that point you
start realising the first law of microbial cytometry:
Sterile is not particle free!
Sterile filter solutions into disposable labware as you will
never be able to obtain clean glassware out of a dishwasher
stuffed with autoclave tape...
I better stop her before I get sarcastic, but with the
bacteria from that Orpegen testkit we could also then set up
the machine for a simple double staining of FITC versus PI
(1 to 5 ug/ml) and subsequently test some resuspended
cultures for their viability, measuring side scatter versus
PI.

Gerhard Nebe-v.Caron
Unilever Research, Colworth,
Sharnbrook, Bedfordshire
GB - MK44 1LQ
Tel: +44(0)1234-222066
FAX: +44(0)1234-222344
gerhard.nebe-von-caron@unilever.com

P.S. If you dilute the BAClight samples / dye, the supravital staining does not
work propperly.

______________________________ Reply Separator _________________________________
Subject: FACSCalibur parameter settings for analysis of baacteria
Author: dsonier@wsunix.wsu.edu at INTERNET
Date: 25/10/96 23:34

I am a graduate student working on my MS. I would like to use flow
cytometry to quantify viable cells in various applications. I am
currently working on using flow cytometry to analyze isopropanol killed
(or fixed) E. coli cells stained with: Molecular Probes' BacLight Bacteria
Viability Staining Kit (adapted for flow cytometry), Ethidium Bromide, or
Propidium Iodide. I have had great difficulty acquiring events from
stained cell samples that do not look the same as a stain + water only
sample. I have used FACSComp to set the machine settings, and am using
FSC=E02 Lin. After varying parameter settings, looking for a stained
bacterial population, I was/am still unable to "find" bacteria with or
without gating. I would greatly appreciate any suggestions for instrument
or parameter settings that would help me acquire data on bacteria, or any
comments on others' use of the BacLight Kit. Thank you for your time,

Dyane N. Sonier
Program in Environmental Science and Regional Planning
Washington State University
Pullman, WA 99164
(509)335-1051
e-mail: dsonier@wsunix.wsu.edu

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