> "chrisroesch" <chrisroesch at okay.net> wrote:
> 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) ...
> Has anyone got similar problems with DAPI or could propose alternative
> methods?? Would it be worth a try with acridine orange??
Non-specific fluorescence is a problem with soil samples
stained with DAPI or AO (see Kepner and Pratt 1994).
This problem can be largely circumvented (see Weinbauer
et al. 1998). by use of the high-affinity green fluorescent nucleic
acid dye, SYBR II (Molecular Probes), with aluminum oxide
membrane filters (0.2 micron Anodisk, Whatman).
Another rapid and inexpensive method uses DTAF (a
fluorescein-based fluorochrome) and CTC (a reducible
tetrazolium dye) for quantifying total microbial biomass
while evaluating levels of microbial metabolic activity in
complex soil samples without enrichment or cell elution.
(see Bhupathiraju 1999).
Hope this is of help,
Bhupathiraju V.K. et al. New direct microscopy based method
for evaluating in-situ bioremediation. J. Hazard. Mater.
1999 Jun 30;67(3):299-312
Kepner R. L., Jr., and J. R. Pratt. Use of fluorochromes for
direct enumeration of total bacteria in environmental samples:
Past and present. Microbiol. Rev. 1994 58:603-615
Weinbauer M.G. et al. Utility of green fluorescent nucleic
acid dyes and aluminum oxide membrane filters for rapid
epifluorescence enumeration of soil and sediment bacteria.
Appl. Environ. Microbiol. 1998 Dec;64(12):5000-3
John Hegarty, M.Sc.
johnh at psu.edu
Penn State University - Harrisburg
Environmental Engineering & Microbiology