Total anaerob microflora in gastro-intesinal tract

Ralf Hartemink ralf.hartemink at
Fri Oct 23 09:17:39 EST 1998

The actual type of agar to use on total counts is not that important.
It should be a rich medium, such as Reinforced Clostridial Agar,
supplemented with either blood or faecal extract. In addition to this
we also add vitamin K and the salt solution described by Holdeman and
Moore in the VPI manual.
Do not use BHI-blood agar or Schaedler agar, as these give lower
counts. So does Brucella Blood agar.

More important is the way you handle the samples.
Be aware of the following points:
- do not use platic vials and containers for transport
- never freeze a sample (unpredictable and unreproducible changes)
- do not work on the bench, but in an anaerobic chamber
- treat the samples as fresh as possible
- do not use a vortex or ultra-turrax to homogenize the samples
outside an anaerobic chamber (inside is OK)
- pre-reduce your dilution medium (physiological salt + 0.5 g/l
cystein.HCl at pH 6.7)
- do not dilute the samples outside an anaerobic chamber

Finally, working with (slaughtered ?) animals : when you kill an
animal the mucous layer will loosen and the intestinal contents will
mix. So use anaesthisized animals.

We have submitted a manuscript on the effects of different media and
methods on total counts and also have a lot of experience with animal
samples. Don't hesitate to ask any further !

Ralf Hartemink
Division Food Science
Food Microbiology Dept
Intestinal microbiology group
Wageningen Agricultural University
PO Box 8129
6700 EV Wageningen
The Netherlands
ralf.hartemink at

On Thu, 22 Oct 1998 08:44:04 +0200, Tom Granli
<Tom.Granli at> 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.
>Tom Granli.

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