Total anaerob microflora in gastro-intesinal tract

gerne at my-dejanews.com gerne at my-dejanews.com
Fri Oct 23 13:21:44 EST 1998


For most accurate total counts I would avoid growth altogether as it is the
major reason for distortions. For total counts I would just use flow
cytometry as outlined in the attached article. However, I do not agree that
the cells will dye voluntarily on you just because they see oxygen. Regards
Gerhard

van der Waaij, L.A., Mesander, G., Limburg, P.C., and van der Waaij, D.
Direct flow cytometry of anaerobic bacteria in human feces. Cytometry
16:270-279, 1994.

Abstract : We describe a flow cytometry method for analysis of noncultured
anaerobic bacteria present in human fecal suspensions. Nonbacterial fecal
compounds, bacterial fragments, and large aggregates could be discriminated
from bacteria by staining with propidium iodide (PI) and setting a
discriminator on PI fluorescence and by exclusion of events with large
forward scatter. Since anaerobic bacteria, which account for over 99.9% of
all fecal bacteria, die during sample preparation, a fixation step was not
necessary. A second aim of this study was to investigate the technical
possibility of measurement of in vivo IgA coating of fecal anaerobic bacteria
as well as their bacterial size. Fecal samples of 22 healthy human volunteers
were analyzed. The fluorescence distribution of IgA-coated bacteria labeled
with fluorescein isothiocyanate (FITC)-anti-Hu-IgA had overlap with noncoated
bacteria. However, with match region subtraction, detection of low levels of
specific FITC fluorescence on IgA-coated bacteria was achieved. The median
bacterial two-dimensional surface area was 1.0 microns2. To validate flow
cytometry data, all samples were analyzed with an image analysis system as
well. With this new method, a rapid evaluation of fecal flora with high
sensitivity for specific FITC fluorescence is possible without culturing.





In article <362EE244.6B5C at hre.hydro.com>,
  Tom Granli <Tom.Granli at hre.hydro.com> wrote:
> I plan to do research on the normal microflora in gastro-intestinal
> tract (GIT) of various animals.  One aspect is to determine (as good as
> possible) the number of total anaerobic bacteria (preferably vial) in
> various parts of the GIT.
>
> In this regard I would appreciate suggestions for proper methods to use,
> both agar counts, chemical analyses or other methods.  Your experiences
> of the suggested methods is also very welcome.
>
> Regards,
> Tom Granli.
>

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