ANNEX 1

m-MTEC Method for E. coli

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Water Resources--Office of Water Quality

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7.1.3
IDENTIFICATION AND ENUMERATION METHODS
The membrane filtration (MF) and most probable number (MPN) methods are used for
the presumptive identification, confirmation, and enumeration of indicator bacteria. For
general use, the MF method is preferable to the MPN method. The MPN method is
preferred if toxic substances are present in the sample or if, after filtration, a residue
heavy enough to block the micropores of the membrane filter is visible. The MPN
method is described in Standard Methods for the Examination of Water and Wastewater,
18th edition (American Public Health Association and others, 1992, p. 9-45 to 9-53) and
in Britton and Greeson (1989). Procedures for analyzing water samples by use of MF
methods are described below.

Indicator bacteria for presumptive identification and enumeration are cultured on

selective media after filtration of several different sample volumes onto gridded
membrane filters. Detailed confirmation, identification, and enumeration of these bacteria
require additional culturing and biochemical testing, the details of which are beyond the
scope of this manual. However, additional confirmation procedures are needed under
certain circumstances, such as use of the data in support of environmental regulation and
enforcement.

The fecal indicator bacteria are operationally defined by the method employed for
identification and enumeration, as follows:

The total coliform bacteria are defined as the organisms that produce red
colonies with a golden-green metallic sheen within 24 ± 2 hours when
incubated at 35.0 ± 0.5°C on m-Endo medium.
The fecal coliform bacteria are defined as the organisms that produce blue
colonies in whole or part within 24 ± 2 hours when incubated at 44.5 ± 0.2°C
on m-FC medium.
E. coli are defined as the organisms that produce yellow or yellow-brown
colonies that remain so when placed on a filter pad saturated with urea
substrate broth for 15 minutes after rescusitation at 35.0 ± 0.5°C for 2 hours
and incubation for 22 to 24 hours at 44.5 ± 0.2°C on m-TEC medium.
E. coli are defined as the organisms that produce a blue fluorescent margin
around a darker colony center within 4 hours when incubated at 35 ± 0.5°C

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on NA-MUG medium after primary culturing as total coliform bacteria on m-
Endo medium.
The fecal streptococci are defined as the organisms that produce red or pink
colonies within 48 ± 2 hours when incubated at 35.0 ± 0.5°C on KF medium.
Enterococci are defined as the organisms that produce pink to red colonies
with a black or reddish-brown precipitate after primary culture for 48 to 50
hours at 41.0 ± 0.5°C on m-E medium followed by incubation for 20 minutes
at 41.0°C on EIA medium.

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 Analytical Methods
mTEC agar method for Escherichia coli: U.S.
Environmental Protection Agency Method 1103.1.
Updated October 2007

The mTEC agar method is a two-step membrane-filtration

method for detection of Escherichia coli (E. coli). This method
can be done in the field or laboratory.

THEORY:
mTEC agar plates are incubated at 35°C for 2 hours followed by
incubation at 44.5°C for 22-24 hours. The mTEC agar contains
selective and differential agents. Sodium lauryl sulfate and
sodium desoxycholate inhibit Gram + cocci and endospore-
forming bacteria. Brom-cresol purple and brom-phenol red
inhibit nontarget bacteria and cause a color change from purple
to yellow when lactose is utilized. Lactose is utilized on mTEC
by E. coli and other thermotolerant coliforms.

The mTEC method includes a second step—transfer of the membrane to another

substrate for identification of E. coli. After incubation, filter membranes
containing yellow colonies are transferred to pads saturated with urea-phenol
solution. After 15-20 minutes of incubation on the urea substrate, colonies that
are not E. coli turn from yellow to purple. This color change is caused by the
breakdown of urea by the enzyme urease and a subsequent increase in pH. E.
coli is urease negative, and colonies of E. coli remain yellow, yellow-brown, or
yellow-green.

USE:
The mTEC method is recommended for use in monitoring fresh,
estuarine, and marine surface waters. It was specifically
developed to be used as a measure of recreational water
quality.

MEDIA:
The mTEC medium is available commercially in the dehydrated
form from Hardy Diagnostics (800/266-2222, Cat C7741 (500
g)). To prepare urea-phenol substrate, ingredients can be
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purchased from Fisher Scientific (800/766-7000): phenol red
(Cat AC41724-0050 (5 g)) and urea (Cat U15-500 (500 g)).
(See preparation instructions (Appendix F)).

Pre-poured plates can be purchased in lots of 20 plates from Fisher Scientific

(Cat BD215126) or VWR by special order. Other quantities and plate sizes are
available from these manufacturers by special order.

Use phosphate buffered dilution water and 0.45 mm membrane

filters. Buffer can be purchased from Hardy Diagnostics (Cat
D699 (99mL) or Cat U193 (500mL)). (See buffer preparation
(Appendix M)).

REFERENCES:
U.S. Environmental Protection Agency, 2000, Improved
enumeration methods for the recreational water quality
indicators: Enterococci and Escherichia coli: Washington, D.C.,
EPA 821-R-97-004, 53 p.

U.S. Environmental Protection Agency, 2006, Method 1103.1—E. coli in water

by membrane filtration using membrane-thermotolerant Escherichia coli agar
(mTEC): Washington, D.C., EPA 821-R-06-010, 45 p.
NWIS PARAMETER CODES:
31633 E. coli on mTEC agar, colonies per 100 mL

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 ANNEX 2

MI Agar - a New
Chromogenic/Fluorogenic Medium

For Total Coliform and E.coli

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8
 MI Agar - a New Chromogenic/Fluorogenic Medium from BD

BD Diagnostic Systems, Sparks,

MD, has announced the release of
BD MI Agar, a new
chromogenic/fluorogenic medium
formulated to simultaneously
detect total coliforms and
BD MI Agar Escherichia coli in drinking water
by membrane filtration.

BD MI Agar conforms to the

USEPA 1604 approved procedure
for monitoring drinking water
under the Total Coliform Rule and
source water under the Surface
Water Treatment Rule. A
significant enhancement to the
membrane filtration (MF) test
method, MI Agar increases the
analytical quality, while reducing
analysis time when compared to
conventional techniques.
Final results are available in 24 hours or less.

The benefit for testing facilities is a more efficient, cost-

effective, sensitive and specific tool for the detection of
total coliforms and E. coli in drinking water. BD MI Agar
offers precise, quantifiable results as compared to the most
probable numbers method which provides only a statistical
estimate that is more costly and time consuming.

While BD MI Agar is approved for use by certified drinking

water laboratories for microbial analysis of potable water,
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it's also ideal for a wider range of applications.

BD MI Agar can be used to test recreational, surface or

marine water, bottled water, groundwater, well water,
treatment plant effluents, water from drinking water
distribution lines, drinking water source water and possibly
foods.1

MI Agar was developed by the USEPA for testing drinking

water as an enhancement to the MF technique. As a single-
step MF technique, MI Agar can be used to simultaneously
detect and enumerate both total coliforms and E. coli in
water samples in 24 hours or less on the basis of their
specific enzyme activities.MI Agar detects the presence of
the bacterial enzymes ß- galactosidase and ß-glucuronidase
produced by total coliforms and E. coli, respectively.

Typically found in fecally polluted water, coliform bacteria

are species that inhabit the intestines of warm-blooded
animals or occur naturally in soil, vegetation and water.
They are often associated with disease outbreaks. Although
these bacteria are not usually pathogenic themselves, their
presence in drinking water indicates the possible presence
of other pathogens. E. coli is one species in this group of
coliform bacteria. Since it is always found in feces, it is a
more direct indicator of fecal contamination and the
possible presence of enteric pathogens.

For more information on BD MI Agar, please call 1-800-

638-8663 in the US, or to send a request for more details

1. U.S. Environmental Protection Agency. 2002. Method

1604: Total coliforms and Escherichia coli in water by

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membrane filtration using a simultaneous detection
technique (MI medium), September 2002, Publication EPA-
821-R-02-024, USEPA Office of Water (4203T),
Washington, DC

BD is a trademark of Becton, Dickinson and Company.

©2002 BD December 2002

11
MI Medium - U.S. EPA Method 1604
Science

Method 1604 (EPA 821-R-02-024) is a sensitive and

differential membrane filter (MF) method that uses MI agar
or MI broth for the simultaneous detection of both total
coliforms (TC) and Escherichia coli (E. coli) in water
samples in 24 hours or less. Identification of the two groups
of bacteria is based on their specific enzyme activities. Two
enzyme substrates are included in the medium to detect the
enzymes that are produced by TC and E. coli.

Total coliforms include species that may inhabit the

intestines of warm-blooded animals or occur naturally in
soil, vegetation, and water. They usually are found in fecally-polluted water and often are
associated with disease outbreaks. Although they usually are not pathogenic themselves,
their presence in drinking water indicates the possible presence of pathogens. E. coli, one
species of the coliform group, is always found in feces and is, therefore, a more direct
indicator of fecal contamination and the possible presence of enteric pathogens. In addition,
some strains of E. coli are pathogenic.

Applications

Method 1604 has been approved for use in monitoring drinking water, source water, and
ambient water (including fresh recreational water). It also has been proposed for use with
ground water, and may be useful for analyzing other types of samples, such as treatment
plant effluents, foods, pharmaceuticals, and clinical specimens (human or veterinary). The
method is sensitive, selective, and specific with low false-positive and false-negative rates.
Use of this method simplifies compliance monitoring of drinking water by eliminating the
additional time, labor, and expense of repeat or serial analyses that can delay the detection
of contaminated water. The method can be found on the EPA NERL-Cincinnati
Microbiology Home Page at www.epa.gov/microbes/ (click on "Method 1604" under "E.
coli" or "Total Coliforms").

Current Licensees

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Becton, Dickinson and Company
Whatman Inc.

EPA Contact Information

Kristen P. Brenner, Ph.D.

U.S. EPA
26 W. M.L. King Drive - ML 314
Cincinnati, OH 45268
Phone: (513) 569-7317
Fax: (513) 569-7170
E-mail: [email protected]

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14
 ANNEX 3

CHROMOCULT FOR COLIFORMS

ENZYMATIC METHODS
RAPID METHODS

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16
Chromocult® Coliform Agar

Cat. No. 1.10426.0100/500

(100 g, 500 g)
Selective agar for the simultaneous detection of total coliforms and E. coli in drinking water and processed
food samples.

The approval by US-EPA is pending.

Mode of Action Quality Control

Typical Composition (g/litre) Additives/Reagents
Preparation Picture/Image
Experimental Procedure and Evaluation Literature

ChemDAT®

Mode of Action
In the first instance, the interaction of selected peptones, pyruvate,
sorbitol and phosphate buffer guarantees rapid colony growth, even
for sublethally injured coliforms. The growth of Gram-positive bacteria
as well as some Gram-negative bacteria is largely inhibited by the
content of Tergitol® 7 which has no negative effect on the growth of
the coliform bacteria.
For the second stage, Merck has developed a new combination of
two chromogenic substrates which allow for the simultaneous
detection of total coliforms and E. coli.

Coliform identification
The characteristic enzyme for coliforms, ß-D-galactosidase cleaves
the Salmon-GAL substrate and causes a salmon to red colour of the
coliform colonies.

E. coli identification
The substrate X-glucuronide is used for the identification of ß-D-
glucuronidase, which is characteristic for E. coli
E. coli cleaves both Salmon-GAL and X-glucuronide, so that positive

17
colonies take on a dark-blue to violet colour. These are easily
distinguished from other coliform colonies which have a salmon to red
colour. As part of an additional confirmation of E. coli, the inclusion of
tryptophane improves the indole reaction, thereby increasing
detection reliability when it is used in combination with the Salmon-
GAL and X-glucuronide reaction.

Typical Composition (g/litre)

Peptones 3.0; sodium chloride 5.0; sodium dihydrogen phosphate
2.2; di-sodium hydrogen phosphate 2.7; sodium pyruvate 1.0;
tryptophane 1.0; agar-agar 10.0; Sorbitol 1.0; Tergitol ® 7 0.15;
chromogenic mixture 0.4.

Preparation
Suspend 26.5 g in 1 litre of demin. water by heating in a boiling water
bath or in free flowing steam. Stir the content to assist dissolution
(approx. 35 mn). Some turbidity may occur, but this does not effect
the performance!

 Do not autoclave! Do not overheat!

pH: 6.8 ± 0.2 at 25 °C.
Note: After autoclaving add E. coli / Coliform Supplement to the
medium cooled to 45-50 °C if the sample material contains high
gram-positive bacteria resp. Pseudomonas and Aeromonas spp.
The plates are opalescent to turbid and yellowish. Store in a
refrigerator and protect from light. To prevent plates from drying, seal
in plastic pouches or bags.
Shelf-life under these conditions: 6 months.

Experimental Procedure and Evaluation

Inoculate the medium by the pour plate method or by spreading the
sample material on the surface of the plates. In addition the
membrane-filter-technique can also be used.
Incubation: 24 hours at 35-37 °C.

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E. coli: dark-blue to violet colonies (Salmon-GAL and X-glucuronide
reaction).
Total coliforms: salmon to red colonies (Salmon-GAL reaction) and
dark-blue to violet colonies (E. coli).
Other Gram-negatives: colourless colonies, except for some
organisms which possess ß-D-glucuronidase activity. These
colonies appear light-blue to turquoise.
In order to confirm E. coli, coat the dark-blue to violet colonies with a
drop of KOVACS' indole reagent. If the reagent turns to a cherry-red
colour after some seconds, a positive indole formation confirms the
presence of E. coli.

Membrane-filter method:
The simultaneous detection of total coliforms and E. coli using
Chromocult® Coliform Agar (CCA) relies on the production of specific colony
colours. OSSMER et. al (1999) reported on the effect of the type and brand of membrane
filters on the growth and colour formation of coliforms and E. coli on CCA. The best
performance was obtained when using filters of cellulose-mixed-ester material, s. a.
Gelman GN6 or Schleicher and Schüll ME25. For the validation of membrane filters it
is advised to use one of these filters as reference.

Quality control
Test strains Growth Colony colour Salmon-GAL X-Glucuronide Indole
Escherichia coli ATCC 11775 good/very good dark-blue to + + +
violet
Citrobacter freundii ATCC 8090 good/very good salmon to red + - -
Escherichia coli DSM 502 good/very good blue to violet + - +
Salmonella enteritidis ATCC fair/very good colourless - - -
13076
Enterococcus faecalis ATCC none
19433

Additives
Product Pack Size
Merck Cat.No.
1.00898.0001 E. Coli/Coliform Selective-Supplement 16 vials
1.09293.0100 KOVACS Indole Reagent 100 ml
1.10156.0001 Chromoplate® Coliform Agar 20 plates
1.11350.0001 Bactident® Indole (dropper bottle) 1 x 30 ml
Schüll 406870 Cellulose-mixed-ester-ME 25/21
Gelman 66278 Cellulose-mixed-ester-GNG membrane filters

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 E.coli, Citrobacter freunii, Salmonella enteritidis

Literature

FRAMPTON, E.W., RESTAINO, L. a. BLASZKO, L.: Evaluation of

ß-glucuronidase substrate 5-bromo-4-chloro-3-indol-ß-D-
glucuronide (X-GLUC) in a 24 hour direct plating method for
Escherichia coli. – J. Food Protection, 51; 402-404 (1988).
KILIAN, M. a. BÃœLOW, P.: Rapid diagnosis of Enterobacteriaceae.
I. Detection of bacterial glycosidases. – Acta Pathol. Microbiol.
Scand. Sect. B 84; 245-251 (1976).
LE MINOR, L. a. HAMIDA, F. BEN: Advantages de la recherche de la
ß–galactosidase sur celle de la fermentation du lactose en milieu
complexe dans le diagnostic bactériologique, en particulier des
Enterobacteriaceae. – Ann. Inst. Pasteur (Paris), 102; 267-277
(1962).
MANAFI, M. a. KNEIFEL, W.: A combined chromogenic-fluorogenic
medium for the simultaneous detection of total coliforms and E. coli in
water. – Zentralabl. Hyg. 189; 225-234 (1989).
OSSMER, R., SCHMIDT, W., MENDE, U.: Chromocult® Coliform Agar
– Influence of Membrane Filter Quality on Performance. – XVII
Congresso de la Sociedad, Granada (1999).
New Zealand Dairy Industry: Microbiological Methods Manual,
Section 48: Product Test Methods – Enteric Indicator Organisms.
– NZTM 2; 48.5.1-48.5.10 (1998).

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© 2002 Merck KGaA, Darmstadt, Germany

21
Methods for detecting pathogenic viruses and bacteria
from water samples

Viruses
Concentration of viruses is an important facet of their detection and
identification. The most common method involves passing very large
volumes of water through filters that are electronegative or
electropositive in nature. The virus particles are adsorbed to the
surface of the filter by electrostatic charges and are then eluted by
passing smaller volumes of a protein-containing liquid (e.g. beef
extract).

Detection is often achieved through a tissue culture of the viruses in

human or primate tissue samples. More recently,
immunofluorescence (antibodies linked with fluorescent dyes such as
FITC - see Biology 446, Chapter on Methodology) , enzyme-linked
immunosorbent assays (ELISA both direct and indirect), nucleic acid

22
probes, the Polymerase Chain Reaction (PCR) and
radioimmunofocus assays (RIFA) have become more common.

Bacteria
The most common indicator of potential pathogen contamination is
still the coliform test in its various guises. The Total coliform test has
many forms; the most common so far has been the membrane filter
method in which a known volume of water is filtered through a 0.45
&muM or 0.22 &muM filter and the filter incubated on M-endo or LES-
Endo agar. Red colonies with a metallic sheen are considered
coliforms. A slower method, the Most Probable Number method is
also used and involves serial decimal dilution of the water sample
followed by adding 5 aliquots of the dilutions to 5 tubes of nutrient
media. If the dilutions are chosen correctly, some of the serial decimal
dilutions will have some tubes with growth and some without. A
statistical procedure based on these sets gives the number of
bacteria in the original sample. This test is sometimes referred to as
"dilution to extinction" - an unfortunate phrase !

More recently, with changes in many countries drinking water

regulations with regard to coliforms, no coliforms at all are allowed in
100 mL of sample. This permits a simple presence/absence test to be
used where 100 mL of sample is placed in nutrient medium at
suitable concentrations and incubated; any growth means the water
has coliforms and so fails the regulations.

A remaining problem is the time taken to assay for coliform number or

presence/absence; up to 3 days is required for some of the tests. The
quickest tests (P/A and membrane filter) take 24 hours. Very often,
quicker results reporting is essential or desirable. Rapid detection
tests have been developed for these situations. They vary in their
acceptance by the regulatory bodies, but at least one (Defined
Substrate Systems) has achieved broad use and regulatory standing.
In this test (various commercial versions are available), the water
sample is filtered through 0.45 &muM membrane filters, removed and
and incubated at 35C on a medium containing 4-methylumbelliferone-
beta-D-galactoside. If coliforms are present, they break the bond
between the methylumbelliferone and the galactoside, releasing the
fluorescent umbelliferone derivative. The degree of fluorescence is

23
measured with a fluorimeter and shows the degree of contamination
by coliforms.

A variant of this test (the Colilert method) uses ortho-nitrophenyl-beta-

D-galactosidase (ONPG) and 4-methyl umbelliferyl-beta-D-
glucuronide (MUG) for detecting total coliforms and Escherichia coli
in a single solution. The coliforms break down ONPG with their beta-
galactosidase enzymes releasing the yellow coloured indicator
portion of the molecule. If E. coli is also present, the enzyme
glucuronidase hydrolyzes the MUG to glucuronide and the indicator
portion 4-methyl-umbelliferone that fluoresces under ultraviolet light.
This permits separate and independent estimates of total coliform
and E. coli counts in the same sample. Levels as low as 1 CFU/100
mL have been reliably detected with this method. This test can also
be used with the MPN or P/A tests to improve resolution and
sensitivity.

For a complete assessment of the coliform bacteria and

methodologies to determine their numbers, etc. see the
Canadian Water Quality Guidelines article.

Gene probe and PCR technology could also be used to produce

more sensitive and specific test for coliforms or E. coli (from Kimball's
Biology Pages)

24
Animations from http://www.iupui.edu/
%7Ewellsctr/MMIA/htm/animations.htm (Click on PCR in side
menu) - From "Molecular Medicine in Action"

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Bernhard, A.E., and Field, K.G., 2000b, A PCR assay to discriminate

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Dombek, P.E., Johnson, L.K., Zimmerley, S.T., and Sadowsky, M.J.,

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Farnleitner, A.H., Kreuzinger, N., Kavka, G.G., Grillenberger, S., Rath,

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Hagedorn, C., Robinson, S.L., Filtz, J.R., Grubbs, S.M., Angier, T.A.,
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Kariuki, S., Gilks, C., Kimari, J., Obanda, A., Muyodi, J., Waiyaki, P.,
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Rapid Detection Kits For Coliform/E.coli

The IDEXX Colilert: Coliform/E.coli test kit simultaneously detects and confirms
coliforms and E.coli in water samples in 24 hours or less. Simply add the Colilert
reagent to the sample to incubate for 24 hours, and read results. Colilert is easy to read,
as positive coliform samples turn yellow, and when E.coli is present, samples fluoresce
under UV light. Colilert is easy to use, involving less than one minute hands-on time.
Colilert is used by over 90% of state labs in the United States and it is the only USEPA-
approved, 24-hour test for drinking and source waters.

28
Rapid Assessment Methodology for the Detection of Microbiological
Indicators.
To assess beach water quality, the Department of Environmental
Services (NHDES) monitors fecal indicator bacteria levels at coastal
beaches on a routine basis.
Unfortunately, sample analysis can take anywhere from 24 to 48 hours.
Because it takes at least 24 hours to receive results, beach managers and
the public are not informed of water quality problems until after the public
may have been exposed. This is an issue facing beach officials throughout
the world, and is a top priority of the US Environmental Protection Agency
(EPA). The EPA, universities and private entities are researching rapid
assessment methods to enumerate bacteria and viruses. These methods will
allow beach officials to post advisories on the same day water quality is
impaired, thus, better protecting public health. There are three different rapid
assessment method technologies available: Molecular surface recognition,
nucleic acid detection and enzyme/substrate based methods. All rapid
assessment methods will take less than two hours to obtain results.
Molecular surface recognition methods capture and/or label the target
bacterium by binding to molecular structures on the exterior surface or in its
genetic material. Analyses of coastal beach water samples currently employ
culture-based methods for the detection of Enterococci bacteria, an indicator
for fecal pollution in marine water. The quickest culture-based method takes

29
up to 24 hours to provide results. Now, a new method is being developed to
enumerate Enterococci. This new method uses Transcription-Mediated
Amplification (TMA) with a fluorescently-labeled probe to amplify a
specific region of Enterococci ribosomal RNA (rRNA).
The TMA rapid assessment method is currently being tested in Southern
California. Development of methods is moving quickly and will likely come
to execution within five years. One reason why the new technology is not
currently being used is that it is more expensive. Once this procedure is
widely and routinely used, the expenses will lower.
This rapid assessment method is very beneficial because it will allow beach
managers to take action towards protecting the public from exposure to
waterborne pathogens on the same day that the water is sampled.
Another rapid assessment method that is being developed for the detection
of fecal indicators is called Quantitative Polymerase Chain Reaction
(QPCR). QPCR is a nucleic acid detection method that targets genetic
material of bacteria, viruses or protozoan indicators. QPCR is being used to
test for both E. coli and Enterococci. Results while using this method can be
obtained on an average of two hours after sampled. These collections have
demonstrated 85-90 percent agreement with existing routine methods. This
method can be used to detect other water quality indicators such as
Bacteroides thetaiotamicron and human enterovirus. Studies indicate that
ratios of B. thetaiotamicron may provide useful information as to the source
of fecal contamination in samples collected.
The final rapid assessment technology methods available are the
enzyme/substrate based methods. These methods pair chromogenic or
fluorogenic substrate methods already widely used with advanced optical or
electrical detectors. These methods are directed at reducing the incubation
periods of current membrane filtration methods. Some of these methods
measure excitation and absorbance of the fluorescent metabolite of
Enterococci
using a fluorometer which will speed up the rate of detection. A popular type
of enzyme/substrate method is called the Dual-Wavelength Fluorimetry
(DWF). These rapid assessments methods are currently being tested for its
accuracy, sensitivity and efficiency. Research indicates that these new
methods will be made available within the next five years. Once these
technologies are made available and laboratories adopt the methods, beach
management will be able to better protect public health. With assistance
from EPA Beach Grants, New Hampshire is hoping to employ these methods
once they become available.

30
 ANNEX 4

BIORAD DETECTION METHOD

FOR E. COLI

31
32
Bio-Rad Laboratories, S.A.
Email: mailto:[email protected]?
Subject=Bio-Rad’s RAPID’E.coli 2™ Agar Granted
Performance Tested Method Status by AOAC RI [Ref:
rapidmicrobiology news - 1027h13]
Tel : +[33] 1 47 95 60 00
Fax: +[33] 1 47 41 91 33

Bio-Rad’s RAPID’E.coli 2™ Agar Granted Performance

Tested Method Status by AOAC RI
RAPID’E.coli 2 agar, manufactured by Bio-Rad Laboratories,
was granted Performance Tested Method status by the
AOAC Research Institute (certificate # 050601).

RAPID’E.coli 2 is a chromogenic medium for detection and

enumeration of E. coli and other coliform bacteria in food in
24 hours. It is a rapid method producing accurate and easy-
to-read results. Current methods for enumeration of E. coli
and coliform bacteria can be costly and laborious.

The use of chromogenic substrates in media has lead to

development of faster and easier methods for detection,
differentiation and enumeration of target bacteria.

RAPID’E.coli 2 is validated for enumeration of E. coli and

other coliform bacteria in raw ground beef, raw boneless
pork, fermented sausage, processed ham, processed turkey,
frozen turkey breast, raw ground chicken, cottage cheese,
processed ricotta cheese, unpasteurized raw milk, and dry
infant formula. It is validated at two incubation
temperatures, 37°C and 44°C (cottage cheese and processed
ricotta cheese are only validated at 37°C only).

33
The principle of RAPID’E.coli 2 medium relies on
simultaneous detection of two enzymatic activities, Beta-D-
Glucuronidase (GLUC) and Beta-DGalactosidase (GAL). The
medium contains two chromogenic substrates. One substrate
is specific to GAL and results in blue green coloration of
colonies positive for this enzyme and one substrate is
specific to GLUC and results in violet coloration of colonies
positive for this enzyme.

Coliforms, other than E. coli, (GAL+/GLUC-) form blue to

green colonies while, specifically, E. coli (GAL+/GLUC+) form
violet colonies. A count of total coliforms can be obtained by
adding the number of blue colonies and the number of violet
colonies. Differentiation of coliforms and specifically E. coli is
carried out by observing a simple color change reaction.
Observation of gas bubbles for differentiation is not
necessary.

RAPID’E.coli 2 is available in two formats, dehydrated media

(Item # 356- 4024) or ready-to-use bottled media (Item #
355-5299).

34
 ANNEX 5

LOW COST BACTERIA MONITORING

COLISCAN EASYGEL

35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
During 2004 and 2005, volunteers in Indiana and Iowa were trained to assess
E. coli using both field/home lab test methods and to collect samples that
were analyzed in a state certified laboratory in Iowa. Six E. coli tests were
assessed by these volunteer monitors. Following that sampling season, two
test methods were recommended for use in all 6 participating states based on
volunteer preference surveys, costs of the methods, and comparability of
results between the methods and laboratory methods. In 2005 volunteers in
MI, MN, OH, and WI initiated use of 3M Petrifilm and Coliscan Easgygel.
Volunteers in all 6 participating states continue to test these two methods in
2006.

A brief description of each of the 6 test methods follows, with links to the
distributors' websites and to the National Environmental Methods Index, so
that users can find more detailed information about each method.

Coliscan Easygel (not incubated)

This test method, distributed by Micrology Laboratories, was assessed by

volunteers in Indiana and Iowa. To use the kit for analysis, a small amount
(0.5-5 mL) of water sample is poured into a bottle of Coliscan Easygel and
mixed by swirling the bottle. The mixture is then poured into a labeled Petri
dish/plate, covered, and swirled to uniformly distribute the sample. After
sitting undisturbed for approximately 45 minutes, the Petri dishes are
inverted. The dishes are then left at room temperature for 48 hours, at which
time, purple/blue colonies (the E. coli colonies) on the plates are counted. E.
coli numbers are represented in colony forming units (CFU) / 100 ml of
water. For more information about this method visit the National
Environmental Methods Index.

Coliscan Easygel (incubated)

This test method, distributed by Micrology Laboratories, is being assessed in

all participating states. To use the kit for analysis, a small amount (0.5-5 mL)
of water sample is poured into a bottle of Coliscan Easygel and mixed by
swirling the bottle. Then, the mixture is poured into a labeled petri dish,
covered, and swirled to uniformly distribute the sample. After sitting
undisturbed for approximately 45 minutes, the Petri dishes are inverted and
incubated at 35 degrees C for 48 hours. Then, like with the non-incubated
Easygel method, purple/blue colonies (the E. coli colonies) on the plates are
counted. E. coli numbers are represented in colony forming units (CFU) / 100
ml of water. For more information about this method visit the National
55
Detection of Waterborne Coliforms and E. coli with
Coliscan Easygel

Introduction:

The Coliscan Easygel medium is a patented formulation for water

testing. It contains a sugar linked to a dye which, when acted on by
the enzyme ß-galactosidase (produced by coliforms including E. coli),
turns the colony a pink color. Similarly, there is a second sugar linked
to a different dye which produces a blue-green color when acted on
by the enzyme ß-glucuronidase. Because E. coli produces both ß-
galactosidase and ß-glucuronidase, E. coli colonies grow with a
purple color (pink + blue). The combination of these two dyes makes
possible the unique ability to use one test to differentiate and quantify
coliforms and E. coli. (Because E. coli is a member of the coliform
group, add the number of purple colonies to the number of pink
colonies when counting total coliforms.)

Instructions:

Either collect your water sample in a sterile container and transport

the water back to the test site, or take a measured water sample
directly from the source and place directly into the bottle of Coliscan
Easygel. Water samples kept longer than one (1) hour prior to
plating, or any Coliscan Easygel bottle that has had a sample
placed into it for transport longer than ten (10) minutes, should be
kept on ice or in a refrigerator until plated.

1. Label the petri dishes with the appropriate sample information.

A permanent marker or wax pencil will work.

2. Sterilely transfer water from the sample containers into the

bottles of Coliscan Easygel (Consult the following table for rough
guidelines for inoculum amount). Swirl the bottles to distribute the
inoculum and then pour the medium/inoculum mixtures into the
correctly labeled petri dishes. Place the lids back on to the petri
dishes. Gently swirl the poured dish until the entire dish is covered
with liquid (but be careful not to splash over the side or on the lid).

56
 Inoculation of Coliscan Easygel
Water Sources Inoculum Amount
Environmental: 1.0 to 5.0 mL
river, lake, pond,
stream, ditch
Drinking water: 5.0 ml
well, municipal, bottled

3. The dishes may be placed right-side-up directly into a level

incubator or warm level spot in the room while still liquid.
Solidification will occur in approximately 40 minutes.

4. Incubate at 35° C (95° F) for 24 hours, or at room temperature

for 48 hours. (See comments on incubation)
5. Inspect the dishes
a. Count all the purple colonies on the Coliscan dish
(disregard any light blue, blue-green or white colonies) , and report
the results in terms of E. coli per ml of water. NOTE: To report in
terms of E. coli per 100 ml of water, first find the number to multiply
by. To do this: first, divide 100 by the number of ml that you used for
your sample. Then, multiply the count in your plate by the result
obtained from #1. For example, a 3 ml sample, 100 / 3 = 33.3. So, 4
E. coli colonies multiplied by 33.3 will equal 133.2 E. coli per 100 ml
of water.
b. Count all the pink and purple colonies on the Coliscan
dish (disregard any light blue, blue-green or white colonies) and
report the results in terms of coliforms per ml of water.
6. Do one of the following prior to disposal in normal trash:
a. Place dishes and Coliscan bottles in a pressure cooker
and cook at 15 lbs. for 15 minutes. This is the best method.
b. Place dishes and Coliscan bottles in an ovenproof bag,
seal it, and heat in an oven at 300° F for 45 minutes.
c. Places dishes and Coliscan bottles in a large pan, cover
with water and boil for 45 minutes.

57
d. Place 5 ml (about 1 teaspoon) of straight bleach onto the
surface of the medium of each plate. Allow to sit at least 5 minutes.
Place in a watertight bag and discard in trash.

Comments on Incubation:
Micrology Laboratories, LLC in-house studies indicate that Coliscan
can effectively differentiate general coliforms from E. coli when
incubated at either room temperatures or at elevated temperatures
(such as 90-98° F). However, some further explanation may be
helpful.

There is no one standard to define room temperature. Most would

consider normal room temperature to vary from 68-74° F, but even
within this range the growth of bacteria will be varied. Members of
the bacterial family Enterobacteriaceae (which includes coliforms
and E. coli*) are generally hardy growers that prefer higher than
room temperatures, but which will grow at those temperatures. They
tend to grow at a faster rate than most other bacterial types when
conditions are favorable. It is therefore logical to try to place
inoculated dishes in a "warm" place in a room for incubation if a
controlled temperature incubator is not available. It is a very easy
task to make an adequate incubator from a box with a 40-60 watt
bulb in it to provide heat at an even rate. One can also use a heat
tape such as it is used to prevent the freezing of pipes in the winter
as your heat source.

Our general instructions indicate that incubation times for coliforms

(including E. coli) are generally 24-48 hours at elevated
temperatures (90-98° F) and 48 or more hours at room
temperatures. At elevated temperatures, no counts should be made
after 48 hours as any coliforms present will be quite evident by that
time and if new colonies form after 48 hours as any coliforms present
will be quite evident by that time and if new colonies form after 48
hours they are most likely not coliforms, but some other type of slow
growing organisms that should not be included in your data. At room
temperatures, the best procedure is to watch the plates by checking
them at 10-12 hour intervals until you observe some pink or purple
colonies starting to form and then allowing another 24-30 hours for
the maturation of those colonies. Since the coliforms (including E.
coli) are generally the faster growing organisms, these will be the

58
first to grow and be counted. Colonies that may show up at a later
time are likely to not be coliforms. As you can see, there are
advantages to incubating your dishes at elevated temperatures.
First, you can count the results earlier. At 95° F, it is often possible to
do accurate counts at 18-20 hours of incubation. There is also less
probability of variation from batch to batch when the incubation
temperatures are kept at one uniform level. And a higher incubation
temperature will tend to inhibit the growth of non-coliforms that may
prefer lower temperatures.

*E. coli is the primary fecal coliform, however, Klebsiella is

sometimes of fecal origin. Other general coliform genera include
Enterobacter and Citrobacter.

Interpretation of Results
This test method utilizes well established, widely accepted criteria for
the recognition of coliforms and E. coli and proper application of the
method will result in accurate results. Therefore, if you suspect that
your water is dangerously contaminated based on the results you get
using Coliscan Easygel, you should contact your local health
department and ask for their help in performing an official
assessment of water.

Non-fecal coliforms are widely distributed in nature, being found both

as naturally occurring soil organisms, and in the intestines of warm-
blooded animals and humans. Fecal coliforms are coliforms found
naturally only in the intestines of warm-blooded animals and
humans. Fecal coliform contamination is therefore the result of some
form of fecal contamination. Sources may be either animal or
human.

General Notes on Differentiating Coliforms and E. coli

Generally, water containing E. coli (the fecal contamination indicator
organism) should not be used for drinking water unless it is sanitized
in some manner. Contact your local health department for guidelines
regarding E. coli and coliforms in recreational waters. Inform them if
you suspect that contamination may be occurring from a specific
source.

59
Colonies which have the blue-green color are not exhibited any ß-
galactosidase activity (which is evidenced by the pink color).
Because of this, they are not considered to be either coliforms or E.
coli and therefore should be ignored when counting your coliform or
E. coli colonies. Similarly, colonies which are white are exhibiting
neither color-causing enzyme, and should also be ignored.

Colonies on the surface of the plate are exposed to the medium on

only the underside of the colony. This causes these colonies to
appear with much less of the indicator color. E. coli colonies may
only have a slight purple tinge to them, and it may appear only in the
center of the colony with the remainder of the colony being white.
Similarly, coliforms on the surface may be light pink or white with a
pink center.

60
 Detection of Waterborne Coliforms and Fecal
Coliforms
with Coliscan® Easygel®

Introduction

The Coliscan Easygel medium is a patented formulation for

water testing. It contains a sugar linked to a dye which, when
acted on by the enzyme p-galactosidase (produced by
coliforms including E. coli), turns the colony a pink color.
Similarly, there is a second sugar linked to a different dye
which produces a blue-green color when acted on by the
enzyme p-glucuronidase. Because E. coli produces both p-
galactosidase and p-glucuronidase, E.Coli colonies grow with
a purple color (pink + blue). The combination of these two
dyes makes possible the unique ability to use one test to
differentiate and quantify coliforms and E. coli. (Because E.
coli is a member of the coliform group, add the number of
purple colonies to the number of pink colonies when
counting total coliforms.)
® Coliscan and Easygel are registered trademarks of
Micrology Laboratories, LLC.
To order, call Voigt Global Distribution Inc
[email protected] www.VGDLLC.com
In the USA, call Toll Free: 1-877-484-3552
Instructions
1. Either collect your water sample in a sterile container and
transport the water back to the test site, or take a measured
water sample directly from the source and place directly into
the bottle of Coliscan Easygel. Water samples kept longer
than 1 hour prior to plating, or any Coliscan Easygel bottle
that has had sample placed into it for transport longer than
10 minutes, should be kept on ice or in a refrigerator until
plated.

61
2. Label the petri dishes with the appropriate sample
information. A permanent marker or wax pencil will work.
3. In a sterile manner, transfer water from the sample
containers into the bottles of Coliscan Easygel (Consult the
following
table for rough guidelines for inoculum amount). Swirl the
bottles to distribute the inoculum and then pour the
medium/inoculum mixtures into the correctly labeled petri
dishes. Place the lids back on to the Petri dishes. Gently swirl
the poured dish until the entire dish is covered with liquid
(but be careful not to splash over the side or on the lid).
Inoculation of Coliscan Easygel

Water Sources Inoculum Amount

Environmental: River, lake, pond, stream, ditch 1.0 ~ 5.0mL
Drinking water: Well, municipal, bottled 5.0mL
4. The dishes may be placed right-side-up directly into a
level incubator or warm level spot in the room while still
liquid. Solidification will occur in approximately 45 minutes.
5. Incubate at 35° C (95° F) for 24 hours, or at room
temperature for 48 hours, (see Comments on incubation)
6. Inspect the dishes.
a. Count all the purple colonies on the Coliscan dish
(disregard any light blue, blue-green or white colonies), and
report the results in terms of E. coli or Fecal Coliform per mL
of water.
Note: To report in terms of E. coli or Fecal Coliform per 100
mL of water, first find the number to multiply by:
1. Divide 100 by the number of mL that you used for your
sample.
2. Multiply the count in your plate by the result obtained
from #1.
e.g. For a 3 mL sample, 100/3 =33.3. So, 4 E. coli colonies
multiplied by 33.3 will be equal to 133.2 E. coli per 100 mL
of water.
b. Count all the pink and purple colonies on the Coliscan dish
(disregard any light blue, blue- green or white colonies) and
report the results in terms of coliforms per mL of water.

62
7. Do one of the following prior to disposal in normal trash:
a. Place dishes and Coliscan bottles in a pressure cooker and
cook at 15 Lbs. for 15 minutes.(This is the best method.)
b. Place dishes and Coliscan bottles in an oven-proof bag,
seal it, and heat in an oven at 300° F for 45 minutes.
c. Place dishes and Coliscan bottles in a large pan, cover
with water and boil for 45 minutes.
d. Place 5 mL (about 1 teaspoon) of straight bleach onto the
surface of the medium of each plate.
Allow to sit at least 5 minutes. Place in a water-tight bag and
discard in trash.
Comments on Incubation
Micrology Laboratories, LLC. in-house studies indicate that
Coliscan can effectively differentiate general coliforms from
E. coli when incubated at either room temperatures or at
elevated temperatures (such as 90-98° F). However, some
further explanation may be helpful.
There is no one standard to define room temperature. Most
would consider normal room temperature to vary from 68-
74° F, but even within this range the growth of bacteria will
be varied. Members of the bacterial family
Enterobacteriaceae (which includes coliforms and E. coli )
are generally hardy growers that prefer higher than room
temperatures, but which will grow at those temperatures.
They tend to grow at a faster rate than most other bacterial
types when conditions are favorable. It is therefore logical to
try to place inoculated dishes in a "warm" place in a room for
incubation if a controlled temperature incubator is not
available. It is a very easy task to make an adequate
incubator from a box with a 40-60 watt bulb in it to provide
heat at an even rate. One can also use a heat tape such as is
used to prevent the freezing of pipes in the winter-as your
heat source.
Our general instructions indicate that incubation times for
coliforms (including E. coli*) are generally 24-48 hours at
elevated temperatures (90-98° F) and 48 or more hours at
room temperatures. At elevated temperatures, no counts
should be made after 48 hours as any coliforms present will

63
be quite evident by that time and if new colonies form after
48 hours they are most likely not coliforms, but some other
type of slow growing organism that should not be included in
your data. At room temperatures, the best procedure is to
watch the plates by checking them at 10-12 hour intervals
until you observe some pink or purple colonies starting to
form and then allowing another 24-30 hours for the
maturation of those colonies. Since the coliforms (including
£. coif) are generally the fastest growing organisms, these
will be the first to grow and be counted. Colonies that may
show up at a later time are likely to not be coliforms. As you
can see, there are advantages to incubating your dishes at
elevated temperatures. First, you can count the results
earlier. At 95° F, it is often possible to do accurate counts at
18-20 hours of incubation. There is also less probability of
variation from batch to batch when the incubation
temperatures are kept at one uniform level. And a higher
incubation temperature will tend to inhibit the growth of non-
coliforms that may prefer lower temperatures.
*E.coli is the primary fecal coliform, however, Klebsiella is
sometimes of fecal origin. Other general coliform genera
include
Enterobacter and Citrobacter.
Interpretation of Results
This test method utilizes well established, widely accepted
criteria for the recognition of coliforms and E. coli and proper
application of the method will result in accurate results
Therefore, if you suspect that your water is dangerously
contaminated based on the results you get using Coliscan
Easygel, you should contact your local health department
and ask for their help in performing an official assessment of
the water.
Non-fecal coiiforms are widely distributed in nature, being
found both as naturally occurring soil organisms, and in the
intestines of warm-blooded animals and humans. Fecal
coliforms are coliforms found naturally only in the intestines
of warm-blooded animals and humans. Fecal coliform
contamination is therefore the result of some form of fecal
contamination. Sources may be either animal or human.

64
General Notes on Differentiating Coliforms and E.Coli
Generally, water containing E coli (the fecal contamination
indicator organism) should not be used for drinking water
unless it is sanitized in some manner. Contact your local
health department for guidelines regarding E. coli and
coliforms in recreational waters. Inform them if you suspect
that contamination may be occurring from a specific source.
Colonies which have the blue-green color are not exhibiting
any p-galactosidase activity (which is evidenced by the pink
color). Because of this, they are not considered to be either
coliforms or E. coli and therefore should be ignored when
counting your coliform or E. coli colonies. Similarly, colonies
which are white are exhibiting neither color-causing enzyme,
and should also be ignored.
Colonies on the surface of the plate are exposed to the
medium on only the underside of the colony. This causes
these colonies to appear with much less of the indicator
color. E. coli colonies may only have a slight purple tinge to
them, and it may appear only in the center of the colony
with the remainder of the colony being white. Similarly,
coliforms on the surface may be light pink or white with a
pink center.

Background information on Coliscan® Easygel®

Coliform bacteria are members of the family
Enterobacteriaceae and are defined as gram negative, non-
spore-forming rods
which ferment the sugar lactose with the evolution of gas
and acids. Many coliforms are normally found in soil and
water and
do not necessarily indicate the presence of fecal
contamination, but Escherichia coli (E. coli) is a primary
bacterium in the
human and animal intestinal tract and its presence in food or
water indicates fecal contamination. Therefore, E. coli is the
coliform that is used as an indicator for fecal contamination.
Other coliform genera include Citrobacter, Enterobacter and

65
Klebsiella. The US EPA acknowledges that E. coli is the best
indicator of health risk in fresh water and is currently
recommending testing for E. coli instead of fecal coliforms.
The term "fecal coliform" indicates coliforms which will grow
at a temperature of 44.5° C. This is not an accurate
designation as there are coliforms of non-fecal origin that will
grow at 44.5° C and there are strains of E. coli that will not
grow at 44.5° C.
Traditional tests for coliforms and E. coli or fecal coliforms
require the inoculation of media containing lactose,
incubation under carefully controlled temperatures, and
examination for the presence of gas from lactose
fermentation. Additional special media must then be
inoculated and incubated at elevated, carefully controlled
temperatures to confirm the presence of E. coli or fecal
coliforms. All these require extra equipment and careful
regulation of time and temperature. This approach is not
only expensive and time consuming, but can be less than
precise in indicating the numbers of specific organisms
present.

As a result of the difficulties and lack of precision inherent in

the older technology, new approaches have been developed
and are being used very successfully. One of the best
approaches is based on the fact that in order for coliforms to
ferment lactose, they must produce certain enzymes which
can be identified and used to verify the presence of the
coliforms. General coliforms produce the enzyme
galactosidase in lactose fermentation and E. coli produces
the enzyme glucuronidase in addition to galactosidase.
Coliscan Easygel utilizes this information to produce a
simple, accurate and quantitative method of identifying and
differentiating coliforms and E. coli (true fecal coliform) from
other bacteria in water and other types of samples. This
patented method incorporates two special chromogenic
substrates which are acted upon by the enzymes
galactosidase and glucuronidase to produce pigments of
different colors. To identify the presence and numbers of
coliforms and E. coli, Just add a test sample to the medium,

66
pour it into a pretreated petri dish and incubate it at room
temperature or at a higher controlled temperature (35° C. is
suggested). General coliforms will produce the enzyme
galactosidase and the colonies that grow will be a pink color.
E. coli will produce galactosidase and glucuronidase, and will
grow as blue/purple colonies. It is a simple task to count the
blue/purple colonies as E coli and the pink colonies as other
coliforms. The sum of the E. coli and the other coliforms is
the total coliform number.
Any non-colored colonies that grow in/on the medium are not
coliforms or E. coli, but may be members of the family
enterobacteriaceae such as Salmonella, Shigella, Proteus,
etc. Likewise, it is possible that samples may contain some
unusual types or strains of related organisms which produce
only the enzyme glucuronidase and not the enzyme
galactosidase, and these will grow as teal green colonies.
Any non-colored or teal green colonies that grow on/in
Coliscan Easygel should not be counted as E. coli or
coliforms without further biochemical testing. It is possible
that they are very unusual forms of coliform or E coli, or
significant other bacterial types which should not be ignored,
especially if they occur in large numbers.
Since Coliscan contains inhibitors, most other bacterial types
do not grow in/on it. Coliscan is best incubated at 30-37° C.
(85-99° F) as coliforms grow faster at this temperature range
than at lower room temperatures. At this range, counts can
normally be made at 24 hours, while at lower room
temperatures, counts may be best at 36-48 hours.
The beauty of the Coliscan method is that it uses proven and
accepted technology to allow anyone to do effective
coliform/E.
coli testing. For water testing, you can add up to a 5 ml_
sample of water to the bottle of medium that makes one
petri plate. This will detect as small a number of coliforms or
E. coli as one living bacterium in five milliliters of water. The
method is also easily adapted for large samples with
membrane filter use. Beware of copycat methods by other
manufacturers who claim similar red and blue colors for
coliforms and fecal coliforms, but whose results are

67
unreliable due to inferior technology. They cannot legally
copy the patented Coliscan technology.

68
Coliscan® Easygel® Colony Color Guide

The left half of the photo (#1-4) consists of colonies of E coli

(#1A, 1B) and Enterobacter aerogenes (#2, 3, 4) growing
in/on Coliscan® Easygel® medium. The right half of the

69
photo (#5, 6, 7) represents the appearance of organisms
other than E. coli or coliforms.
1A- Two E. coli CFU (colony forming units) showing purple
color with obvious pink diffused halos. Fecal coliform.
(Glucuronidase +, Galactosidase +)
1B- E. coli CFUs blue/purple color with minimal pink halos.
Fecal coliform. (Glucuronidase +, Galactosidase +)
2 - Enterobacter aerogenes CFUs as dark, solid pink color.
Non-fecal coliform, (Contain no blue/purple) (Glucuronidase
-, Galactosidase +)
3 - Enterobacter aerogenes CFUs as light pink color. Non-
fecal coliform (Glucuronidase -, Galactosidase +)
4 - Enterobacter aerogenes CFUs spread on surface of
medium.(2 original colonies) Non-fecal coliform.
(Glucuronidase -, Galactosidase +)
5 - Teal green CFU growing on surface of medium.
(Glucuronidase +) This colony type should not be counted as
E. coli or coliform
6 - Teal green CFU growing in the medium. (Glucuronidase +)
This colony type should not be counted as E. coli or coliform.
7 - Colorless CFU (indicates no Glucuronidase or
Galactosidase activity). This colony tvpe should not be
counted as E. coli or coliform.
® Coliscan and Easygel are registered trademarks of
Micrology Laboratories, LLC.
To order, call Voigt Global Distribution Inc Tel: 877-484-3552
[email protected] www.VGDLLC.com

70
 INTRODUCING THE NEW
Coliscan Easygel ® Plus* & Coliscan ® MF Plus*
®

for the
Identification and Differentiation
of
E. coli and Other Coliforms

The new Coliscan® Easygel® Plus does everything that the original
Coliscan® Easygel® does, plus it allows those who may be unsure of
the difference in color of the colonies growing in the medium to very
easily verify that the colony is or is not glucuronidase positive. (That
is, does it have blue color verifying it as E. coli or is it just a deep
magenta and therefore a general coliform?) All that is needed is a
long wave UV light source (available from Micrology Labs or other
sources) and a dark room for viewing.

Your Coliscan® Easygel dishes and colonies will look identical to the
original Coliscan® Easygel dishes with Blue/purple E. coli and
pink/magenta other coliforms visible in ambient day or room light, but
when you turn off the lights and shine the long wave UV on the
bottom of the dish, any E. coli will be surrounded by a bright bluish
fluorescent zone.

This application of two enzyme substrates testing for the same

enzyme means that you have double verification for the presence of
E. coli. It also means that the presence of E. coli may be detected and
quantified as early as 12 hours incubation time.

71
72
 ANNEX 6

OTHER LOW COST METHODS

73
74
July 27, 2006

Purdue creates new low-cost system to detect

bacteria
WEST LAFAYETTE, Ind. — Researchers at Purdue
University have developed a new low-cost system that
analyzes scattered laser light to quickly identify bacteria
for applications in medicine, food processing and
homeland security at one-tenth the cost of conventional
technologies.

The technique — Bacteria Rapid Detection Using

Optical Scattering Technology — works by shining a
laser through a petri dish containing bacterial colonies
growing in a nutrient medium.

"Unlike conventional methods, we don't have to do any

biochemical staining, DNA analysis or other types of
manipulation," said Bartek Rajwa, a staff scientist at the
Bindley Bioscience Center in Purdue's Discovery Park, the
university's hub for interdisciplinary research.

Particles of light, called photons, bounce off of the

colony, and the pattern of scattered light is projected
onto a screen behind the petri dish. This "light-scatter
pattern" is recorded with a digital camera and analyzed
with sophisticated software to identify the types of
bacteria growing in colonies.

"There are potentially thousands of applications for this

new technology, from identifying stem cells to drug-
resistant staph infections to pathogens on the
battlefield." said J. Paul Robinson, a researcher at the
Bindley Center and a professor in the Weldon School of
Biomedical Engineering and the School of Veterinary
Medicine.

75
The work was initiated by Arun Bhunia, a professor of
food microbiology in the Department of Food Science;
and E. Daniel Hirleman, a professor and William E. and
Florence E. Perry Head of Purdue's School of Mechanical
Engineering. Findings are detailed in a research paper
appearing this month in the Journal of Biomedical Optics.

Hirleman has specialized in research to develop new

types of sensors that work by analyzing light scattering
off objects for applications such as detecting impurities
on silicon wafers in computer chip manufacturing and
measuring the size and speed of fuel droplets in jet
engines.

"We adapted some ideas from that research to build a

scatterometer for food safety, and now we're using the
second generation of that instrument," Hirleman said.

A major motivation for the research is to reduce the time

it takes for industry to identify harmful organisms in food
processing. Scientists in food-processing plants routinely
grow cultures to test for dangerous pathogens.

"The dairy industry, for example, grows bacteria on petri

dishes to make sure products are safe, but industry is
trying to develop technologies that will very quickly
identify organisms," Robinson said. "The same sort of
thing holds true for clinical microbiology and other
laboratories. With our light-scattering method, it takes
less than five minutes to identify harmful organisms after
they have grown in a petri dish. The analysis is faster
than any other methods in existence, and it’s simple."

The technique might be used to identify staph infections

that are resistant to antibiotics.

76
"This is an extremely dangerous infection, and you want
to catch it as early as possible," Robinson said.

A mass-produced system based on the technology would

consist of inexpensive, off-the-shelf hardware, such as
red lasers and low-resolution digital cameras available at
consumer electronics stores, and likely would cost less
than $1,000, Hirleman said.

A critical part of the technique was made possible by

adapting a mathematical method created in 1934 by
Dutch physicist Fritz Zernike, who created a set of
mathematical "descriptors" subsequently called radial
Zernike polynomials. These descriptors can be used to
analyze how light-wave patterns are distorted after
passing through lenses having complex flaws or
aberrations.

Individual bacterial colonies growing in a petri dish also

distort light passing through them, just as a lens changes
light-wave patterns.

"Therefore, we can treat the colonies as lenses and use

Zernike polynomials," Rajwa said.

Factors such as the shape of bacteria, their refractive

indexes — or how much they bend light — the types of
substances secreted by a particular bacterium and the
distance between individual bacteria in a colony, all
contribute to how a colony distorts light. The procedure
identifies a bacterial colony by comparing an image of its
scatter pattern against a template that contains 120
features described by Zernike polynomials.

"A good analogy is the method used by law enforcement

to identify a person's face using specialized recognition
software," Rajwa said. "You could describe the face as
being made up of a combination of geometric shapes,
77
like ovals, squares and triangles, but each face has a
unique blend of these shapes. We did something similar.
We reduced complicated scatter patterns to 120 numbers
based on Zernike polynomials."

This reduced collection of numbers describes how well

the colony fits the template, and then pattern recognition
software is used to classify the bacteria.

"One of the most important developments is being able

to convert images to numbers, which makes it possible
to classify the patterns," Rajwa said. "We are able to
take images and convert them to numbers that uniquely
describe every picture."

The researchers used the new system to classify six

species of listeria, only one of which is a dangerous food-
borne pathogen for humans.

"If you have a mixture of different listeria, you would like

to know which is the one that can kill you," Rajwa said.
"We took pictures of the scatter patterns from different
listeria, and we were able to classify all of them
accurately."

The system also was able to accurately identify other

types of bacterial colonies, including salmonella, vibrio,
E. coli and bacillus.

"We were able to classify bacterial colonies with greater

than a 90 percent probability of being correct, which is as
good as you could do with equipment costing more than
$100,000, " Rajwa said. "And, unlike conventional
systems, our method is 100 percent non-invasive, which
means we can carry out the procedure without staining,
manipulating or killing the biological samples.

"The power of this technology is that it does not require

78
complicated lab equipment, and it could be designed so
that it wouldn't require someone with a doctoral degree
to operate. The whole beauty of the system is you don't
invade the biological environment that you want to
measure," Rajwa said. "If you are working with stem
cells, you don't want to stain them to see if they are
stem cells. You want to be able to look at colonies on a
petri dish without touching the colonies, without staining
or destroying the colonies."

The research has recently received funding from the U.S.

Department of Agriculture through Purdue's Center for
Food Safety Engineering.

Further work will include research to develop a graphical

user interface.

"Now it requires a qualified, trained person to do all the

recognition," Rajwa said. "We want a system where you
can actually put a petri dish or some other container into
the system, you press enter and the computer says, ‘This
is salmonella of this type and this strain, ' and it does
this quickly in real time. There is absolutely no
fundamental reason why we wouldn't be able to do this,
and we are pretty close to having an actual prototype of
a product that could be commercialized."

A provisional patent has been filed for the data-

processing technique, and a full patent application has
been filed on the underlying light-scattering technology.

The paper published in the Journal of Biomedical Optics

was written by Bulent Bayraktar, a postdoctoral
researcher working with Robinson; Padmapriya P.
Banada, a postdoctoral researcher in the Department of
Food Science; Hirleman, Bhunia, Robinson and Rajwa.

79
Writer: Emil Venere, (765) 494-4709,
[email protected]

Sources: J. Paul Robinson, (765) 494-0757,

[email protected]

Bartek Rajwa, (765) 494 0757,

[email protected]

Arun Bhunia, (765) 494-5443, [email protected]

E. Daniel Hirleman, (765) 494-5688,

[email protected]

Purdue News Service: (765) 494-2096;

[email protected]

Note to Journalists: An electronic or hard copy of the

research paper is available from Emil Venere, (765) 494-
4709, [email protected].

PHOTO CAPTION:
Arun Bhunia (standing) and Padmapriya Banada use a
laser and a computer monitor to observe scatter patterns
in a petri dish in their Purdue University lab. The
technique may provide cost-cutting applications for
medicine, food processing and homeland security. Bhunia
is a professor of food microbiology, and Banada is a
postdoctoral researcher. (Purdue University photo/Tom

80
Campbell.)

A publication-quality photo is available at

http://news.uns.purdue.edu/images/+2006/bhunia-
scatter.jpg

ABSTRACT

Feature extraction from light-scatter patterns of

Listeria colonies for identification and classification

Bulent Bayraktar1, Padmapriya P. Banada2, E. Daniel

Hirleman3, Arun K. Bhunia2, J. Paul Robinson4, Bartek
Rajwa5
1
Purdue University, Bindley Bioscience Center, Purdue
University Cytometry Laboratories, Department of
Electrical and Computer Engineering, West Lafayette,
Indiana 47907
2
Purdue University, Molecular Food Microbiology
Laboratory
3
Purdue University, School of Mechanical Engineering
4
Purdue University, Bindley Bioscience Center, Purdue
University Cytometry Laboratories, Department of Basic
Medical Sciences, School of Veterinary Medicine and
Weldon School of Biomedical Engineering
5
Bindley Bioscience Center, Purdue University Cytometry
Laboratories, Department of Basic Medical Sciences

Bacterial contamination by Listeria monocytogenes not

only puts the public at risk, but also is costly for the
food-processing industry. Traditional biochemical
81
methods for pathogen identification require complicated
sample preparation for reliable results. Optical scattering
technology has been used for identification of bacterial
cells in suspension, but with only limited success.
Therefore, to improve the efficacy of the identification
process using our novel imaging approach, we analyze
bacterial colonies grown on solid surfaces. The work
presented here demonstrates an application of
computervision and pattern-recognition techniques to
classify scatter patterns formed by Listeria colonies.
Bacterial colonies are analyzed with a laser
scatterometer. Features of circular scatter patterns
formed by bacterial colonies illuminated by laser light are
characterized using Zernike moment invariants. Principal
component analysis and hierarchical clustering are
performed on the results of feature extraction.
Classification using linear discriminant analysis, partial
least squares, and neural networks is capable of
separating different strains of Listeria with a low error
rate. The demonstrated system is also able to determine
automatically the pathogenicity of bacteria on the basis
of colony scatter patterns. We conclude that the obtained
results are encouraging, and strongly suggest the
feasibility of image-based biodetection systems.

82
 ANNEX 7

PETRI FILM

TOTAL COUNT – COLIFORM –E.COLI

COLIFAST

83
84
WATER ANALYSIS

Water supplies have to be constantly monitored for a variety of

materials----bacteria, nitrates, pesticides, metals, etc. In this lab, you
will analyze water for bacteria, and, in particular, an indicator group of
bacteria called the coliforms. Although you will be looking at the
total counts of aerobic bacteria in the water sample that YOU BRING
TO LAB, the coliform bacteria are the critical test organisms which
are found in improperly treated water or water that has fecal
contamination. They have been used as such for most of the 20th
century and still today.

Coliforms are bacteria that are naturally occurring in animals and in

the environment: they are indicators of other potentially harmful
microorganisms in drinking water. They are all gram negative
bacteria that ferment lactose, and are non-sporeforming. FECAL
coliforms, exemplified by E. coli, indicate water contaminated with
animal or human waste, i.e. feces. Microbes in the fecal-
contaminated water may cause food-borne illness that has short-term
symptoms—nausea, diarrhea, vomiting—or if severe enough may
cause death. This is a real problem in the immunocompromised,
immunodepressed, and babies and children.

METHODS OF EVALUATING FOR BACTERIA

Membrane filter technique: Filtering 100 ml of water through a
millipore filter with holes smaller than the bacteria causes the bacteria
to be trapped on top of the filter. The filter pad is then placed on
special coliform media which allows a colfirm count to be done.

Most probable number: The water sample is diluted and inoculated

into a variety of specialized media tubes. The MPN is determined
with the help of a standard chart, based on the number of tests that
have turned positive.

The results are given as number of coliforms per 100 ml of water. If

coliforms are present, the lab will generally recommend that a second
sample be analyzed. If the number of coliforms was over 30, a
second sample is essentially useless.
85
Possible results:
One or more coliform bacteria/100 ml = "does not meet the
bacteriological standard purity"
specific counts
too numerous to count (TNTC)
confluent growth

This lab incorporates a newer method of performing counts on

bacteria, called Petrifilms from 3M corporation. There is more
information—-interpretation and photos—-on these materials at the
3M website. Since the aerobic counts include all coliforms, the water
sample will be diluted out. When the water sample is added to the
dehydrated med, the water-soluble gel will rehydrate, forming a thin
media plate of sorts.

OBJECTIVES:
Identify coliform bacteria using 3M Petrifilms.
Differentiate between coliforms and fecal coliforms.
Analyze water samples for bacterial counts.

MATERIALS NEEDED: per table

water sample
1 coliform/E. coli count petrifilm
2 aerobic count petrifilms
1 ml pipettes
scissors
2-99 ml phosphate dilution containers
humidified container to incubate petrifilm plates
86
THE PROCEDURE:
1. Bring water sample to class, preferably a rather dirty sample—not
tap water.
2. Prepare a dilution of the water sample by taking a 1ml aliquot and
placing it into a 99ml phosphate buffer solution. This is the 1/100
dilution.
3. Using a fresh pipette, prepare a 1/10,000 dilution by taking a 1ml
aliquot and placing it into another 99ml phosphate buffer solution.
4. Be sure the containers are shaken well.
5. Place the Petrifilm flat on the table. Lift top film.
6. THE AEROBIC COUNT PLATE:
Place 1 ml of each dilution in the center of 2 petrifilm plates (already
labeled with 1/100 and 1/10,000).
7. THE COLIFORM COUNT PLATE
Place 1 ml of the original water sample in the center of the petrifilm
plate.
8. Release the top film and allow it to drop.
9. Using the small plastic spreader, ridge side DOWN, place it over
the inoculum. Gently apply pressure on spreader to distribute the
sample over the circular area. Do not rotate or twist the spreader.
10. Remove the spreader and wait at least 1 minute for the gel to
form.
11. Incubate the plates with clear film side up in a humidifed
container (1 for the entire class). You may stack the petrifilms.
12. Incubate the petrifilms at 30 degrees C for 48 hours.

INTERPRETATION:
1. Count the colonies on a Quebec colony counter or other magnified
light source (with clear films down).
2. Refer to the INTERPRETATION GUIDE in print form or at the 3M
website given above.
3. After counting the clear film covers can be lifted, and the colonies
can be used for testing or staining.

87
 AEROBIC COUNT PLATE:
There is a red indicator dye in the media gel that
colors the colonies.
Count all red colonies of any size or intensity of red.

COLIFORM COUNT PLATE:

The pH indicator violet red is incorporated into the
gel, along with bile which inhibits gram positive
bacteria. In addition, there is an indicator of the
enzyme glucuronidase which will turn blue if the
bacterium makes the enzyme. You may also see
carbon dioxide gas bubbles between the film and
the bottom of the petrifilm. Most E. coli make
both the glucuronidase and CO2, and those colonies will be blue or
blue-red. Non-fecal coliforms will be red colonies.

QUESTIONS:

1. What criteria are used to define the coliform group?

2. Why is there a dye added to the coliform petrifilms?

3. Why are coliforms used as indicator organisms for

water impurity?

88
3M™ Petrifilm™ E. coli count
( Arrow Scientific )

The top film of the Petrifilm plate traps gas (seen as bubbles)
produced by coliforms, providing “confirmed” counts. In addition, a
glucoronidase indicator forms a blue precipitate around any E. coli
colonies that may be present. Results in 24 hours. No extra
confirmation steps required.

Fast, Accurate, Results in either 24 or 48 hours with three easy

steps:

1) Inoculate – 3M™ Petrifilm™ Plates are easy to inoculate. Lift the

top film and add sample

2) Incubate – The space saving design maximizes incubator space and

3) Enumerate – Confirmed coliforms are red and blue colonies with

associated gas bubbles. Confirmed E. coli coliforms are blue colonies
with associated gas bubbles. A built-in grid facilitates counting
colonies, giving you fast, precise and accurate results.

This Petrifilm Plate can also be read in 4 seconds using the 3M™
Petrifilm™ Plate Reader.

Increased Productivity. Reduced Costs.

Quick & easy confirmed test results provide you with better
information to make the right decisions. 3M™ Petrifilm™ E.
coli/Coliform Count Plates provide the most cost-effective,
convenient and reliable confirmation method for testing equipment,
raw materials, food products and the manufacturing environment
enabling you to monitor critical control points more frequently. A
survey of 274 plants who converted to Petrifilm Plates from
traditional agar methods found an average labor savings of 45% and
an average increased technician efficiency of 80%. The end result is
higher quality product, better process control and an overall

89
reduction in operational costs.

Note that E. coli O157:H7 is glucoronidase negative and will not

produce a blue precipitate.

Note: Most E. coli (about 97%) produce beta-glucoronidase which

produces a blue precipitate associated with the colony.

The top film of the Petrifilm plate traps gas produced by the lactose
fermenting coliforms and E. coli. About 95% of E. coli produce gas,
indicated by blue to red-blue colonies associated with entrapped gas
on the Petrifilm EC plate (within approximately one colony
diameter). AOAC international and U.S. FDA Bacteriological
Analytical Manual (BAM) define coliforms as gram-negative rods
which produce acid and gas from lactose during
fermentation. Coliform colonies growing on the Petrifilm EC plate
produce acid which causes the pH indicator to make the gel colour
darker red. Gas trapped around red coliform colonies on the
Petrifilm plate indicates confirmed coliforms.

Click here to download a brochure for more information.

Click here to download instructions & interpretation guide.

Video of Tips on how to use Petrifilm plates with a foam dam.

90
91
APPLICATIONS

Assessing microbial water quality is a central function in total water

quality management.
The Colifast Analyser is a semi-automated early warning system for
rapid and systematic water quality monitoring.
Tests for thermotolerant coliforms /E.coli, total coliforms, Total Viable
Organisms and P. aeruginosa, are available. The Colifast Analyser can
also be used for detecting coliforms in pasteurised milk.
• Emergency testing
- Flood water management
- Effluent line cross-contamination
- Mains breakages
• Water monitoring
- Source water quality
- Surface and groundwater
- Non-disinfected waters
- Marine and recreational waters
• Operational testing
- Systems maintenance testing
• Beverage and bottled water production
• Industrial process water in food and pharmaceutical industries
• Research application work
• Pasteurised milk

THE COLIFAST ANALYSER PROCEDURE

Photo 1. Add sample directly to Colifast medium, or Photo 2. Filter
sample to concentrate and aseptically place membrane filter in vial
containing liquid medium. Photo 3. Register samples, place vials in
incubator blocks and press "Start". Photo 4. A result report is
automatically generated.

92
id

BENEFITS & FEATURES

• Rapid results, e.g., Coliform results are obtained within 2-12 hours
(18-24 h for traditional methods).
• Automated analysis ➟ Easy-to-use. After addition of the test sample
to the Colifast medium and registration, only hit "Start", and the Colfast
Analyser performs the analysis automatically.
• Remote warning system. The Colifast Analyser´s remote warning
system gives automatic and early warning of positive sample status
allowing earlier operational decisions by e-mail.
• More frequent sub-sampling = faster results. Automatic sub-sampling
allows more frequent analyses without increasing labour. Faster results
ensure better control of water and safety for
the public.
• Time saving = cost saving. Rapid results for rapid decisions reduces
release time of product.
• Multiple sample loading for all day testing. Multiple sample batches
can be loaded and tested simultaneously throughout a work-shift
according to the day’s testing requirements.
• Customised test runs for specific testing regimes. Test run
parameters can be customised for specific testing
matrices/requirements. Test confidence levels can be adjusted for

93
earlier reporting of positive results, according to experience with local
water sources and test regimes.

Perfomance for detection of thermotolerant coliforms / E.coli

DETECTION PRINCIPLE

The patented Colifast technology combines a selective growth medium

with an automated analyser. The growth medium for coliforms and
thermotolerant coliforms includes the substrate 4-methylumbelliferyl-ß-
D-galactoside, which fluoresces after hydrolysis by the ß-galactosidase
enzyme present in coliform bacteria. The reaction is illustrated in the
figure below.
Differential incubation temperatures of 44-44.5 °C and 35-37 °C are
used to distinguish between thermotolerant coliforms and total
coliforms respectively. Inhibitors suppress the growth of nontarget
organisms. Media for TVO and P. aeruginosa, containing other
fluorogenic substrates, are also available.
The fluorescence is monitored by the Colifast Analyser. Advanced
software controls the testing process and subsequent data handling
and reporting.

94
TECHNICAL SPECIFICATIONS
Power
• Electrical input: 110-240 VAC 50/60 Hz
Environmental
• Temperature: 10-30 °C
• Relative humidity: <90%
Computer requirements
• Type: IBM compatible
• Speed: Pentium 133 or greater
• Memory: 32 MB or greater
• Hard Drive Capacity: 1 GB Hard Drive
• Operating system: Windows 95/98 /NT
• Mouse: Bus Mouse
• Graphics: VGA graphic card 1 MB RAM on-board
• Com Port: 1 free high speed Com port
Please contact Colifast® for further information
and technical notes:
Design:

95