I anticipate that most of the lipophilic dyes such as DiO or DiI (structurally
the same as PKH 26) would work; however, FM 1-43 and FM 4-64 may be easier to
use due to their greater solubility in water. FM 1-43 and FM 4-64 are
nonfluorescent in water except when bound to membranes. They may get
internalized in cells, however. FM 1-43 may be preferred for flow cytometry
but both dyes are excitable at 488 nm.
I have not seen this specific experiment done, however.
Mol Microbiol 1999 Feb;31(4):1149-59
A vital stain for studying membrane dynamics in
bacteria: a novel mechanism controlling septation during Bacillus subtilis
sporulation.
Pogliano J, Osborne N, Sharp MD, Abanes-De Mello A,
Perez A, Sun YL, Pogliano K.
Department of Biology, University of California, San
Diego 92093-0349, USA.
At the onset of sporulation in Bacillus subtilis, two
potential division sites are assembled at each pole, one of which will be used
to synthesize the asymmetrically
positioned sporulation septum. Using the vital stain FM
4-64 to label the plasma membrane of living cells, we examined the fate of
these potential division sites in
wild-type cells and found that, immediately after the
formation of the sporulation septum, a partial septum was frequently
synthesized within the mother cell at the second
potential division site. Using time-lapse deconvolution
microscopy, we were able to watch these partial septa first appear and then
disappear during sporulation. Septal
dissolution was dependent on sigma E activity and was
partially inhibited in mutants lacking the sigma E-controlled proteins SpoIID,
SpoIIM and SpoIIP, which may play a
role in mediating the degradation of septal
peptidoglycan. Our results support a model in which sigma E inhibits division
at the second potential division site by two
distinct mechanisms: inhibition of septal biogenesis
and the degradation of partial septa formed before sigma E activation.
"Rochelle A. Diamond" wrote:
> Dear flow people,
>
> I have a client that needs to stain bacterial cell membranes. Is there
> anyone who does extracellular or intracellular staining of bacteria, but
> not cell-wall staining, or a reference where I could find information on
> this? We need to stain membrane proteins under the cell-wall and under the
> outer membrane to do flow cytometry.
>
> Thanks for any help on this issue,
> Rochelle Diamond
> Caltech Flow Cytometry Cell Sorting Facility
> diamond@its.caltech.edu
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