Re: Laboratory Methods for E. coli and Enterococcus

["Muruleedhara Byappanahali" <byappan@usgs.gov>]

I had problems in sending in my reply before. Yes, our email is working
now.

The revised methods for E. coli and enterococci are available in the EPA
publication. The title of this publication is Improved Enumeration Methods
for the Recreational Water Quality Indicators: Enterococci and Escherichia
coli (EPA/821/R-97/004 March 2000). For E. coli, the revised procedure
involves only a slight modification to the original procedure in that a
chromogen (5-bromo-6-chloro-3-indoxyl-B-D-glucoronide), which gives a
magenta color to the colonies, is added to the medium. This eliminates the
additional confirmatory test (substrate test/urease activity) used in the
earlier method (9213 D, Standard Methods for the Examination of Water and
Wastewater). Rest of the procedures (incubation temperature, duration of
incubation) is pretty much the same.

As far as the enterococci analysis is concerned, the modified procedure
will provide the test results in 24 h. The difference between the mE (old
method) and the revised mEI method is the addition of an extra reagent (
indoxyl-B-D-glucoside) to the mE medium. The modified procedure for
enterococci reduces the total incubation time to 24 h as contrast to 48 h
in the old mE method. In addition, it is a single-step method for
enterococci isolation and identification in contrast to the old method,
which required an additional confirmatory test in the form esculin
hydrolysis (enterococci are positive for this reaction).

Although the modified procedures for E. coli and enterococci are not very
"new" and they can be handled with relative ease by most laboratories, a
major concern may be the cost of the chromogens. Both
5-bromo-6-chloro-3-indoxyl-B-D-glucoronide and indoxyl-B-D-glucoside are
quite expensive, and for 2 g these reagents cost about $500.00 & $285.00,
respectively.  You can buy these reagents from HACH, SIGMA and other
biotech companies.

Murulee Byappanahalli, Ph. D.
Research Microbiologist
U. S. Geological Survey
Lake Michigan Ecological Research Station




                                                                                                                 
                      "Terbush, Karen (ALB)"                                                                     
                      <Karen.Terbush@oprhp.s         To:      "Great lakes Beach Net" <beachnet@great-lakes.net> 
                      tate.ny.us>                    cc:                                                         
                      Sent by:                       Subject: Laboratory Methods for E. coli and Enterococcus    
                      owner-beachnet@great-l                                                                     
                      akes.net                                                                                   
                                                                                                                 
                                                                                                                 
                      07/29/02 10:06 AM                                                                          
                                                                                                                 
                                                                                                                 



Hello,
I am from New York State Parks and Recreation.  We operate 62 public
beaches all over New York State.  Some of them are on the great lakes, some
on the Atlantic Ocean and most on freshwater inland lakes.  For the past
couple of years we have been trying to provide guidance to our parks
regarding beach monitoring so that monitoring procedures are uniform all
over the state.
In New York state our bathing beach Standards are still for Total and Fecal
Coliform.  However, based on EPA's recommendations, we have asked all of
our beaches this year to also monitor for enterococcus or E. coli.
The problem is this:  Many of the labs have not even heard of enterococcus
and don't know what methods to use.  For E. coli, many are unfamiliar with
techniques to enumerate the organisms.  I have provided some of the labs
with the procedures recommended by EPA but some have run into problems
obtaining the reagents needed for these tests.
When I attended the Great Lakes Beach Conference in 2000, it seemed that we
were way behind the rest of you when it came to using these better
indicators.  Could folks please give me some advice on methods and
techniques being used for E. coli and Enterococcus (24 hour tests) and
where reagents can be obtained?
I'd appreciate any information you can provide.
Thank you.

Karen B. Terbush
Sr. Environmental Analyst
NYS Office of Parks,Recreation
and Historic Preservation
Environmental Management Bureau
Agency Bldg. 1, Empire State Plaza
Albany, NY 12238
(518) 474-0409
Fax 474-7013



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