[Microbiology] Re: enumeration of non-cultureable bacteria

myrehfuss at ucdavis.edu myrehfuss at ucdavis.edu
Thu Aug 17 13:12:09 EST 2006

biovirus04 at gmail.com wrote:
> gholamreza ahmadian wrote:
> > Hello all!
> > Does anybody know how we can enumerate non-cultureable but viable bacteria (E.Coli) in the sea water
> >  to check environmental contamination? your answer will be really useful for me and I appreciate it.
> > Reza
> Reza,
> You could plate your sample (or dilutions of it) on EMB (eosin
> methylene blue) agar.  This particular agar is specialized for
> detecting E. coli.  On these plates, E. coli takes on a green, metallic
> sheen.  So, you'd count the colonies that appeared this way, and ignore
> anything else that might grow up.
> Any other ideas out there?
> --Alex
> **************************
> Alex B. Berezow, Grad Student
> Dept. of Microbiology
> University of Washington School of Medicine
> Seattle, WA  98195

Greetings.  Since they're apparently non culturable, EMB or any
selective media wouldn't work (assuming this E coli is truly VBNC)

If you had a real time PCR machine, this could be done in a fairly easy
way via quantitative PCR, although it would take a few steps.   Amplify
a region of 16S specific for E coli using specific primers.  Purify,
clone into your favorite cloning vector.  Transform E coli with your
vector.  Grow up more calls, extract more plasmid.  Quantify your
plasmid,  then set up a standard curve of say, 30 through 3x10e6 copies
of the plasmid.  Use this formula:

m= (n) 1.096 x 10e-21 g / bp

    m=mass of one plasmid molecule
    n=size of vector  + insert (in basepairs)

You'll get grams of plasmid per copy from that.  Set up your standard
curve from that, of course accounting for how much you typically run
per reaction in your setup.  Run DNA extracted from a known volume of
seawater along with your standard curve.

Plot your Ct (cycle threshold) of your samples on the standard curve
Cts (which haveknown copy number values), and you'll get copies of 16S
rDNA specific to E coli.  There are 7 copies of the 16s rRNA gene in E
coli, so divide your copy number by 7 to get the number representing
one copy per one cell.  Multiply that by the mL in your original
seawater sample that you extracted the DNA from, and you'll # E coli
per mL.   I think that would work.

Of course it's all moot if you don't have a real time PCR machine e.g.
Lightcycler or ICycler)  You could do competitive PCR with a regular
PCR machine, but that's a PITA and I'm not sure it be accurate enough.

That being said, I bet these E coli are not truly VBNC and you probably
could enumerate via culture methods.


Marc Rehfuss, PhD
Departments of Medical Microbiology & Immunology
Center for Comparative Medicine
University of California, Davis

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