HELP!!! Anaerobic Bacteria Project

Ralf Hartemink ngw at teleoffice.nl
Mon Sep 23 18:47:32 EST 1996


Lesley Ollada wrote:
> 
> I'm thinking of doing a science project on anaerobic bacteria.  OK,
> Specifically, my topic would be "Testing the Survival of Anaerobic
> Bacteria in Various Thioglycolate and Chopped meat Broth Formulations."
> So, I'd apprec
> iate feedback on these or others...
> 
> 1) Do you think this is a good idea?  If not, what needs to happen to
> make this a successful project?

I think it is an interesting project, but what kind of different
formulations are you going to try ? Just some published formulations, or
are you going to make your own formulations ?

> 2) Do you have any procedural suggestions?



> 3) Do you have any suggestions as to what Anaerobic Bacteria to use?
> Which are the best to use in this type of experiment?

You obviously need several bacteria with different growth requirements
and from different genera. I suggest typical proteolytic species
(Clostridium histolyticum or Peptostreptococcus anaerobius will do), as
well as typical saccharolytic species (bifidobacteria or lactobacilli).
Also the redox potential will be different in the different
formulations, so you need strains which are aerobic (E. coli),
aerotolerant (lactobacilli), microaerophilic (Clostridium perfringens,
Cl. tertium, Campylobacter), anaerobic (Bacteroides fragilis) and
stricktly anaerobic (Cl. novyi, Eubacterium species)

> 4) Are there too many variables involved in working with this type of
> experiment?  Can they be minimized?

You can always think of more variables, just see what you can handle !

> 5) Any suggestions as far as how to measure the survival, besides
> whether they are present or not?

Survival is best tested by plating them. Optical density measurements is
of no use with chopped meat. pH measurements are not conclusive with
proteolytic species. Gas formation is not useful for all species. Other
methods are often very expensive (ATP-measurements etc). However, for
properly plating and counting anaerobes you need anaerobic media
(prepared PRAS) and an anaerobic chamber. Working on a bench will
severely affect your results, especially for the strict anaerobes. Also,
you need a proper medium to count them.

If you don't have an anaerobic chamber, it is possible to determine
survival using test tubes. You prepare a dilution series (2 ml into 8
ml) in reduced physiological salt solution (i.e. with 0.5 g/l
cysteine.HCl, pH 6.7 and resazurin as an indicator). From these you
inoculate test tubes with a rich medium, which you know supports growth
(f.e. reinforced clostridial medium, PY medium). By adding different
amounts of the dilutions and from different dilutions, you can determine
approximately the number of bacteria which are still alive, simply by
looking for growth after 48-72h incubation at 37 degrees centigrade.

> 6) Any general comments/constuctive criticism welcome also.

Working with anaerobes is not as easy as we often think ! So be sure you
have the right equipment, otherwise you don't know what you are
measuring !
 

Ralf Hartemink
Intestinal microbiology group
Dept. Food Microbiology
Wageningen Agricultural University
The Netherlands

Ralf.Hartemink at algemeen.lenm.wau.nl



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