I don't think you want to run your sample in acetone anyhow.
For details of a method try Susann's assay below
Have a healthy Christmas and 1998
Gerhard
Muller, S., Losche, A., Bley, T., and Scheper, T.
A flow cytometric approach for characterization and differentiation of bacteria
during microbial processes. Applied Microbiology And Biotechnology 43(1):93-101,
1995.
Abstract : The analysis of growing or resting bacterial populations by flow
cytometry offers several advantages over traditional methods for determining
mean-value parameters. This method has been applied here to measure both the
distribution of single- cell fluorescence intensity and the light-scatter
behaviour of the methylotrophical strains of Methylobacterium rhodesianum MB126
and Methylocystis GB25 as well as Pseudomonas fluorescens and a strain isolated
from the soil. The four different bacterial populations were analysed concerning
the DNA and the poly-3-hydroxybutyrate (PHB) content. A new cell- preservation
method is presented. Optimized staining methods for each strain were developed
in detail, in two cases DNA had to be dehybridized before staining with a
mixture of mithramycin/ethidium bromide. Nile red is used for detecting PHB.
Both stains were excited by an argon-ion laser at 488 nm; fluorescence emission
for mithramycin/ethidium bromide was measured from 520 nm and for Nile red from
600 nm onwards. It is shown that changes in the DNA content and in the forward-
lightscattering behaviour of the bacterial strains chosen were measurable. These
changes could be related to different cultivation conditions and correlated, in
the case of strains that accumulate PHB, with alterations of that biopolymer
content. In addition it was found that these methods provide a contribution to
the differentiation of mixed bacterial populations.
______________________________ Reply Separator _________________________________
Subject: Nile Red and Aquatic Organisms
Author: gebhard@aecom.yu.edu at INTERNET
Date: 17/12/97 16:47
Hello, I am posting this as a favor to a friend not currently on
line.....please address any responses to me and I'll forward them on
thanks..... Dave G.
We are currently involved in efforts to measure lipid content in
single-cell marine organisms. To achieve this, we would like to use Nile
Red, a stain that is soluble in acetone. Our question is this- Is there a
safe concentration of acetone that can be used to acheive a reasonable
signal in our biological sample that may be run on a FACScan? Are there
alternative solvents that would be more forgiving to the FACScan plumbing?
Any helpful comments would be greatly appreciated!!
>
Dave Gebhard--Director FACS Facility
Albert Einstein College of Medicine
Chanin Building 309
1300 Morris Park Avenue
Bronx, New York 10461
718-430-2724/ 3573
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