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DAPI with soil bacteria ??

gerne at my-deja.com gerne at my-deja.com
Thu Jan 13 19:39:48 EST 2000


Hi Chris
You can chech for soil-interference by spiking your soil sample with
cultured bugs that give you bag's of signal. If they dont in a soil
sample you can tell that it is the soil.  Consider the possibilities of
loss of dye by non-specific binding as well as dye extrusion pumps. Dead
cells should be better in that context.
Some of the new dyes from molecular probes seem to show superior signal
to noise ratios/background values, but they do not give you a functional
reading from the cells. See
http://www.cyto.purdue.edu/flowcyt/research/micrflow/gerhard/gerhard.htm
for further info.
You might perhaps want to use either respiratory activity measurement
with tetrazoliun-chloride or esterase activity. There was a nice paper
by the group of Clive Edwards from Liverpool measuring soil bacteria by
flow cytometry.

Regards
Gerhard

In article <857tcv$4hl$1 at bnews.gigabell.net>,
  "chrisroesch" <chrisroesch at okay.net> wrote:
> Hello Newsgroup,
>
> I have to quantify forest soil bacteria. As a standard method I chose
the
> DAPI direct count method (DAPI is a DNA specific fluorescent dye) — my
phase
> contrast counts gave numbers about two magnitudes higher than the
accepted
> values for bacteria per gram soil.
>
> The problem is, that the bacteria so far resisted all of my tries to
get
> them stained with DAPI. It seems likely that remnants of humic acids
cause
> the disturbance (e.g. I get perfect signals for cultured cells).
>
> To reduce humic acids content, I already tried (di-)phosphate buffer
at pH
> 9, Crombach buffer, even NaOH at pH > 11 (which killed my bacteria off
but
> otherwise was of little use).
>
> Has anyone got similar problems with DAPI or could propose alternative
> methods??
> Would it be worth a try with acridine orange??
>
> Thanks,
> ChrisRoesch
>
>


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