[Microbiology] Re: Bacterial staining for Confocal analysis

shiraz14 via microbio%40net.bio.net (by shiraz14 from singnet.com.sg)
Sun Jul 11 11:42:53 EST 2010


On Jul 11, 12:53=A0am, RUTH SIELE <ruthsi... from yahoo.com> wrote:
> =A0Hi all,I have a modified RO (reverse osmosis) membrane and I want to c=
heck biofilm growth on the membrane surface. For this purpose, I'm using tw=
o bacterial strains (E.coli and Pseudomonas aeruginosa) in my system. I wan=
t to see and analyze (using confocal microscope) which strain is causing mo=
re biofilm on the membrane surface when grown in mixed culture. But, since =
I'm not microbiologist I don't really know which stains I have to use.
> - =A0Do I have to stain each of the bacteria before mixing and growing th=
em in the system (e.g. =A0 =A0 =A0 =A0red for E.coli=A0and green for P.aeru=
ginosa)?=A0
> - Are the stains (e.g. SYTO 59) permanent stains?
> Thanks a lot for your help!
> Ruth

Hi,

Firstly, I trust that you are using wild-type E. coli and P.
aeruginosa for your experimental purposes =96 please verify that this is
so (else please take note of the organism strain you are using [e.g.
E. coli BL21, etc) and potential mutations in the organism/(s) which
may cause variations in results obtained due to altered binding
efficiencies of the fluorochromes, etc].

For your experimental purposes [i.e. imaging under confocal laser
scanning microscopy (CLSM)], you=92d need to do a dual channel
acquisition using red and green channels. And yes, you should be able
to use SYTO 59 to stain the cells (although this is a nucleic acid
binding stain, so beware any potential inherent mutations which may
arise in the cellular DNA as a result of this venture). Please also be
aware though that both E. coli and P. aeruginosa will be stained with
the stain, causing them to both fluoresce red under CLSM. You would
then need to switch to the green channel (using the FITC filter set) =96
wild-type P. aeruginosa [which natively produces fluorescein (a yellow-
green pigment forming the principal component for the aromatic
synthesis of FITC)] will fluoresce green, while E. coli will not.
Superpositioning (i.e. merger) of the two acquisition channels should
then produce red fluorescent E. coli and yellow colonies corresponding
to P. aeruginosa (red + green =3D yellow), so this makes it easier for
you to perform any further quantitative analysis (e.g. cell counting,
statistical analysis, etc) on these organisms. (*N.B. please however
verify first that the P. aeruginosa that you are using is able to
produce fluorescein under native conditions, else you may have to re-
look at the strain =96 it is likely that that there is a mutation
somewhere in the host cells=92 genome).

As for your second query on permanence of SYTO 59, please note that
SYTO 59 (being a fluorochrome) is subjected to the same setbacks of
all known fluorochromes (despite their widespread usage) i.e. an
exponential decay in detectable fluorescence emission with time, the
need to store fluorescently-labelled slides at 20 degrees Celsius for
a period of 6 months at most (to maintain optimal fluorescence of
labelled slides, etc), unlike conventional stains (e.g. Gram=92s stain,
etc), so I=92d say that SYTO 59 (although likely to remain in the cell
nucleus for a long time after having stained the cell, may potentially
be degraded with time, or else diminish in its fluorescence with time
so that it is no longer detectable).

Based on your queries, I trust that you are a fourth-year
undergraduate (likely to be in your Honours year) so it may be the
first time you are getting to use a CLSM, in which case it would
definitely be advisable for you to do it (i.e. fluorochrome staining
of cells and all further techniques including time-lapse imaging, Z-
stack acquisition, etc) under supervision by a more senior member of
your lab (preferably a PhD student, if not a post-doctoral student).

Have fun using the CLSM and enjoy learning!

Best Regards,
Shiraz


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