Unfortunately formazan formation is equally flawed as there are
numerous limitations to it's use. The discussion of bacterial
viability, let alone the detection of life would take too long.
FDA works best if you look at the accumulation of the dye within the
bacteria looking through a fluorescent microscope. Unfortunatelly it
is pumped out actively by a lot of species it they posess proton
antiport pumps. Other limitations are pH sensitivity, leading to
significant loss of fluorescence below pH7 and slow breakdown of the
non-fluorescent into the fluorescent component above pH 7.5.
Di-chloro-carboxyfluorescein-diacetate is at least less sensitive to
pH quenching below down to pH4.5, the use of its succinimidyl ester
aids covalent binding to proteins inside the bacterium.
I am not sure what 55oC will do to your esterases, but most of your
bugs won't like it.
With kind regards
"Bruce A. Caldwell" <caldwelb at fsl.orst.edu> wrote in message news:<3B5DEE30.A0D65703 at fsl.orst.edu>...
> HI --
> FDA is a non-specific esterase substrate and not a direct indicator of
> life. Many cell-free soil enzymes (e.g., phosphatases, lipases) can
> react with this substrate.
>> A better approach would be to use one of several chromogenic terminal
> electron acceptors (TTC, INT) which actually response to microbial
> metabolism. There are many papers on this (dehydrogenase) assay.
> Basically, you incubate the soil with substrate and then extract the
> reduced "formazan" with methanol. Microbial respiratiopn turns the
> colorless INT or TTC to red-purple "formazan".
>> Good luck,
> Bruce Caldwell
> Dept of Forest Science
> 321 Richardson Hall
> Oregon State University
> Corvallis OR 97331-5752
>> martin weiss wrote:
>> > We have developed an activity for visitors to detect life in soil
> > using fluorescein diacetate (in DMSO). However, the reaction proceeds
> > very slowly reaching a peak after a day or so.
> > Does anyone have any suggestions to speed up the reaction?. We've
> > tried heat (55 C) and SDS (2% final) to no avail.
> > Cheers,
> > Martin
> > --
> > Martin Weiss, Ph.D.
> > Director of Science
> > New York Hall of Science
> > 47-01 111 th Street
> > Corona, New York 11368
> > phone: 718 699 0005 x 356
> > facsimile:718 699 1341
> > mweiss at nyhallsci.org> > http://www.nyhallsci.org> >
> > ---