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DAPI with soil bacteria ??

chrisroesch chrisroesch at okay.net
Sat Jan 15 16:27:08 EST 2000


Hello Gerhard,

thank you very much for answering my question!


gerne at my-deja.com <gerne at my-deja.com> schrieb in Nachricht
<85lr8i$s70$1 at nnrp1.deja.com>...
> 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.

Yes, I did such a seeding experiment already – the result was, that
pre-stained cultured cells gave still signals when mixed with independently
„stained“ soil isolates (which alone gave no signals). Staining soil samples
mixed previously with cultured cells gave no signals at all. Therefore my
assumption that humic acids might be interfering.

> 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.

The dye seemed to have vanished as I couldn’t detect it anymore after trying
to stain soil isolates.
After that I fixed the cells in paraformaldehyd to prevent enzymatic break
down but it was of no help.

> 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.

After your answer (and two other’s) I’ll try using SybrGreen-I which should
give me the best results.

> 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.

Thank you for this hint, but this seems to be unnecessary for my work, as I
need the total bacterial cell number of soil for reference only (but I’m
well aware that the viable cell number might be significantly different from
the total number of cells).

Regards, Chris







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