Bacterial identification & colony counting from a mixture of 5 different bacteria

ash at umich.edu ash at umich.edu
Tue Sep 17 18:11:34 EST 2002

"Haider, Amir A" <ahaider at iupui.edu> wrote: : 1-I want to mix and grow the
following bacteria for one of my projects: : Streptococcus mutans,
Lactobacillus caseii, S. salivarius, S. parasanguis, A. naeslundii . :
2-After growing them, I need to identify and count the colonies, so that,
I can compare the growth rates under the conditions of my experiment. :
Now the question is , what type of agar plates should I use so that I can
separate and count them? : Thanks. : A.Haider : ahaider at iupui.edu

Excellent experiment idea.  Certain strains
produce bacteriocins which hinder the survival
of competitors.  Other pairings may synergize
with one another.  

BHI Agar should suffice for all these organisms.
To distinguish which strain is which in a pair
train one of the pair to resistance to an antibiotic:
say, like streptomycin, or rifampin, or erythromycin.

Measure the absorbance of the cultures independently
and when mixed.  Streak your organisms back
onto BHI plates with the appropriate antibiotics
to do the colony counts.  Recover the organisms 
in air or 5% co2; probably 5% co2 is the better 
environment for survival.

SDS-PAGE profiles might be interesting too.

Ratios of initial inocculation might have an effect.

Also, you might want to wash your cells before innoculation
to remove any soluble growth factors or inhibitors which may
have accumulated during the overnight growth.

Consider that with the chained microbes you are actually
counting colonies which have been formed from many cells.
Your cocci per chain number may vary, so you might
want to include microscope observations or endeavor
to learn how to break the chains up into individual
yet viable cocci.  It might be easier to get your growth
rate data from the absorbances and determine the
ratio of organism A to organism B at various time 
points on the agar plates.

regards, Andrew S Heath
	Research Associate
	Infectious Diseases
	University of Michigan

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