FACSing Bacteria

Gerhard Nebe-von-Caron Gerhard.Nebe-von-Caron at unilever.com
Fri Nov 8 04:48:05 EST 1996



          Hi Scott

          See attached message below.The instrument setting principle 
          described works for nearly all machines. According to my 
          message log I must have put that on the mail server, but 
          perhaps it only got to Dyane.
          Regards
          Gerhard.Nebe-von-caron at unilever.com





______________________________ Reply Separator _________________________________
Subject: FACSing Bacteria
Author:  ssimpson at bu.edu at INTERNET
Date:    08/11/96 04:14


To all Flow'ers

     I'm trying to quantify the amount of complement factor H deposited on
Neisseria gonorrhoeae by FACS. I'm using a BD FACScan machine. When
acquiring the data I have the FSC set at E-1 (PMT is at 868) and I'm
noticing that I'm getting non-specific background noise that will not go
away even when water is running thru the machine.  Do you have any
suggestions about what settings (FSC/SSC) I should be using to analyze
bugs with this machine and any thoughts on the background noise.

     Thanks in advance for any help.

     Scott Simpson


______________________________ Forward Header __________________________________
Subject: Re: FACSCalibur parameter settings for analysis of baacteria
Author:  Gerhard Nebe-von-Caron at 1890CHPE
Date:    28/10/96 12:44


          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 at 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 at 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 at wsunix.wsu.edu



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