E Coli

ISO Methods for Detection of Coliforms/E.
coli
and Pseudomonas aeruginosa in Water
1 Copyright 2019 IDEXX Laboratories

IDEXX Laboratories, Inc.
o Headquarters in Westbrook,
Maine, USA
o Offices in 20 countries serving

customers in 175 countries
o Over 8,000 employees worldwide

IDEXX: A Company Dedicated to Water
Microbiology
o Global Leader in Microbiological Water Tests. Experts in water
microbiology.
o Products accepted and approved in more than 50 countries/organizations
throughout the globe;
o More than 20 million IDEXX tests for microbiology of water consumed
annually at a global level
o Helps in daily protection of water quality for more than 2.5 billion
people worldwide

Innovative Water Microbiology Tests
Coliform and
Enterococci P. aeruginosa L. pneumophila Quality Control
E. coli
Colilert® Enterolert® Pseudalert® Legiolert® Quanti-Cult®
Colilert®-18 Enterolert®-E Pseudalert® 250 IDEXX-QC
Colisure® Enterolert®-DW
Colilert ® 250 Enterolert® 250
Cryptosporidium/ Heterotrophic Plate

Quantification Accessories
Giardia Count
Filta-Max® SimPlate® for HPC Quanti-Tray® EZ-DPD™
Filta-Max xpress® HPC for Quanti-Tray® Quanti-Tray®/2000 Collection vessels
Invitrogen Quanti- Incubators
Dynabeads® for IMS Tray®/Legiolert Sealers

Approvals and Acceptance
Association of Official analytical Chemists International United States Environmental Protection Agency
Organizations U.S. Food and Drug Administration World Health Organization
Standard Methods for the Examination of Water and International Standard Organization (Method 9308-2)
Wastewater International Bottled Water Association
American Society for Testing and Materials European Bottled Water Association
US Department of Defense The United States Pharmacopeia
Coverage of Key Markets and Customer
Segments
Water Types Customers
o Drinking water o Public health laboratories
o Ambient water o Utilities
o Ground water o Private laboratories
o Source/surface water o Bottled water
o Wastewater o Industrial water producers
o Marine water o Cruise ships
o Dairy water o F&B / Pharmaceutical
o Hospital water companies
o Livestock companies

Standard Methods for the Examination of
Water & Wastewater
IDEXX Water Products are the only
commercial methods included
o Coliform/E. coli Method
• Colilert®, Colilert 18® and Colisure®
o Enterococcus Method
• Enterolert®
o Quantification Method
• Quanti-Tray® and Quanti-Tray ®/2000
o Heterotrophic Plate Count Method

• SimPlate®
*Pseudalert ®in pursue for peer review publications

Colilert-18/Quanti-Tray Method is an ISO Standard
Method and is the National Standards for EU
Member States
• ISO 9308-2:2012
– Water quality - enumeration of
Escherichia coli and coliform
bacteria
– Part 2: Most probable number
method
– IDEXX Colilert-18/Quanti-Tray

Pseudalert Accepted as Global ISO
Standard in 2018
ISO 16266-2:2018:
• Water quality – Detection and
enumeration of Pseudomonas
aeruginosa
• Part 2: Most probable number
method
• Water matrices included in ISO
designation are:
– Hospital waters
– Drinking water
– Swimming/spa pool waters

ISO 9308-2:2012
Water quality – Emuneration of E.coli
and coliform bacteria
Part 2: Most probable number method

ISO 9308-2:2012
Scope
Cancels and replaces the first edition ISO 9308-2:1990
Based on the growth of target organisms in a liquid medium
Determination of bacteria concentration by MPN
Method applicable to all types of water, including those with

suspended solids and high background counts of
heterotrophic bacteria
Detects E.coli based on the expression of the enzyme β-D-

glucuronidase

ISO 9308-2:2012
Definitions
Total Coliforms:
Original definition: members of the Enterobacteriaceae which ferment
lactose with acid and gas* production within 48
hours at 36±2C
Current definition: members of the Enterobacteriaceae which
demonstrate β-galactosidase activity
E. coli:
Original definition: coliforms which also produce indole from tryptophan
after 24 hours at 44°C
Current definition: members of the enterobacteriaceae which
demonstrate both β-D-galactosidase AND β-D-
glucuronidase activity

Total Coliforms
Pre 1994 Post 1994 (Report 71) 2006-
Acid & Gas from Lactose Acid from Lactose Enzymatic Definition
Escherichia Escherichia Escherichia
Klebsiella Klebsiella Klebsiella
Enterobacter Enterobacter Enterobacter
Citrobacter Citrobacter Citrobacter
Yersinia Yersinia
Serratia Serratia
Hafnia Hafnia
Pantoea Pantoea
Kluvyera Kluvyera
Cedecea
Ewingella
Moellerella
Leclercia
Rahnella
Yokonella
Buttiauxella
ISO 9308-2:2012
Principle
Based upon IDEXX’s Colilert-18 / Quanti-Tray system
Simultaneously detects coliforms & E.coli
Yellow colour indicates positive for coliforms, blue
fluorescence indicates positive for E.coli
No confirmation tests required
Can be used for both presence/absence and quantitative
Incubation at 36±2°C, results in 18-22 hours

Colilert-18 – Defined Substrate Technology


Differs from other methods where multiple nutrient sources

are available to bacteria as food sources
Using a defined nutrient as the primary carbon source is one

way to control the bacteria’s environment, which increases
the accuracy of the method

Colilert-18
ONPG Positive Reaction
Total Coliform and Fecal Coliform Reaction
ONPG provides the major source of carbon in Colilert -18

and can be metabolised by the Coliform enzyme ß-galactosidase

Colilert-18
MUG Positive Reaction
E.coli Reaction

Sample Inoculation for Presence/Absence Test
1 2
Add reagent to water sample Cap vessel and shake to Incubate vessel at
(100mL) dissolve 36±2°C for 18-22h
3 4
Read the color change in Read for fluorescence

visible light under UV light (365nm)
Presence/Absence Test Results
POSITIVE for E. Coli NEGATIVE POSITIVE for Coliforms

Sample is Yellow AND Sample remains Sample is Yellow
emits Fluorescence Colorless
Comparator
o Comparator is available for use when reading results

o Comparator provides the minimum yellow (and fluorescence)
for positive results

Sample Inoculation for Quantification Test
1 2 3
Add reagent to the Pour into Quanti-Tray or Seal using Quanti-Tray

sample Quanti-Tray/2000 Sealer PLUS
4 5
Incubate at 36±2°C for 18-22h Count coliform and E. coli

positive wells and refer to MPN
23 Copyright 2019 IDEXX Laboratories Table
Quanti-Tray & Quanti-Tray/2000
o Quanti-Tray: detects up to 200 MPN/100mL

o Quanti-Tray/2000: detects up to 2,419 MPN/100mL
Quanti-Tray®
Quanti-Tray®/2000

Reading Positive Coliform Counts on
Quanti-Tray/2000
o 16 positive large wells
o 3 positive small wells
o Result – 22.6 MPN/100mL

Interpreting Results with Quanti-Tray/2000
MPN Table

Advantages of Colilert-18
Easy
o 5-step method simplifies training
o Unit-dosed packaging eliminates media preparation
o No repeat testing due to clogged filters or heterotrophic

interference
o QC procedure can be done in 15 minutes per new lot of

product

Rapid
o Under 1 minute hands-on time
o Coliform and E. coli results in just 18 hours
o No confirmations necessary
o No colony counting

Accurate
o Identifies E. coli specifically
o β-galactosidase and β-glucuronidase activity detects slow or

non lactose fermenting coliforms
o Suppresses up to 2 million heterotrophs per 100ml
o Eliminates subjective interpretation
o Detects a single viable organism /100mL

ISO 16266-2:2018
Water quality – Detection and
emuneration of Pseudomonas
aeruginosa
Part 2: Most probable number method

ISO 16266-2:2018
Scope
Based on the growth of target organisms in a liquid medium
Determination of bacteria concentration by MPN
Method applicable to a range of different types of water

- hospital waters
- drinking water
- non-carbonated bottled water
- ground water
- swimming pool and spa pool waters
Detects Pseudomonas aeruginosa via a bacterial enzyme

technology
ISO 16266-2:2018
Definition
Pseudomonas aeruginosa:
species of microorganism capable of growing in a selective
broth and hydrolyze 7-amino-4-methylcoumarin aminopeptidase
substrate

ISO 16266-2:2018
Principle
o Based upon IDEXX’s Pseudalert
o Same platform as Colilert-18
o No color change; fluorescence indicates a positive
o No confirmation test required
o Can be used for both presence/absence and quantitative (100

and 250mL formats)
o Incubation temperature 38±0.5ºC, results in 24-28 hours

Pseudalert
Bacterial Enzyme Technology
o The microorganisms of P. aeruginosa rapidly grow and reproduce,

using the abundant supply of amino acids, vitamins, and other
nutrients present in the reagent.
o The strains of P. aeruginosa in active growth contain an enzyme that

hydrolyzes the 7-amino-4-methylcoumarin aminopeptidase substrate
to produce fluorescence in blue light

Sample Inoculation for Presence/Absence Test
Step 1 Step 2 Step 3
Add reagent and Incubate vessel at Read under 365 nm

antifoam to water 38±0.5°C for 24-28 UV light and record
sample and mix hours results
well
Results Interpretation
STRONG POSITIVE for P. WEAK POSITIVE for P. NEGATIVE

aeruginosa sample emits aeruginosa sample emits Sample remains
blue Fluorescence any Fluorescence Green

Sample Inoculation for Quantification Test
1 2 3
Add Pseudalert and Add sample to Seal the Quanti-Tray

antifoam to sample Quanti-Tray
4 5
Incubate at 38±0.5°C for Read under UV light

24-28 hours and refer to MPN table
Pseudalert
Advantages
o Easy to use, simplify training and testing
o Less than 1 minute hands-on time for sample preparation
o No media preparation required, contains no hazardous

materials
o Confirmed results in 24 hours, no additional confirmation

needed
o Eliminates subjective interpretation from traditional methods
o Can be run in both presence/absence or quantification tests

Limitations of
Conventional Methods

Multiple Tube Fermentation
Drinking Water
Waste Water
Liquid Culture @ 35±0.5°C

MPN Format
Nutrient Rich
Lactose Based (Acid & Gas)
Selectivity:
Detergents & Salts
E. coli Confirmation
Indole reaction (Kovac’s)
PRESUMPTIVE – 24-96h
CONFIRMED – 72-120h

Multiple Tube Fermentation
Brilliant Green
Lauryl Tryptose
Lactose Bile
Broth (LTB)
Broth (BGLB)
(24-48h @ 35C)
(48h @ 35C)
EC-Broth
(24h @ 44C)

TTC-Tergitol
Clean Water
Low Suspended Solids
MF Culture @ 36±2°C
CFU Format
Nutrient Rich
Lactose Based (Acid)
Selectivity:
Detergent & TTC
E. coli Confirmation
Indole reaction (Kovac’s)
PRESUMPTIVE – 18-24h

TTC-Tergitol
Non-target Target
The Reality
Potential Issues with Traditional MF Methods
o Bacterial growth can be dependent upon membrane type.
o Correct preparation and quality control is essential.
o Nutrient uptake on solid media can be variable:
• nutrient rich media benefit targets and non-targets – OVERGROWTH
• some targets may not be able to utilise a specific nutrient e.g. lactose
o Non-targets can cause:
• false positives if they appear similar to target organisms
• false negatives if they compete with target organisms
o Mechanisms used to reduce false positives/
negatives can be counter productive:
• detergents, salts, antibiotics, toxins, temp’
• may increase specificity at the expense
of sensitivity and vice versa
o May only be suitable for certain matrices*
o Technique, experience, time, resource! SENSITIVITY SPECIFICITY

Example 1: Lactose Fermentation
LACTOSE
β-GALACTOSIDASE
PERMEASE
GALACTOSE
LACTOSEEXT LACTOSEINT
+
Coliform
GLUCOSE
+
LACTIC ACID
Lactose Based Method FALSE NEGATIVE
β-GALACTOSIDASE
galactopyranoside
ONPGEXT ONPGINT
+ Coliform
O-nitrophenol
Enzyme/Substrate Method TRUE POSITIVE

Example 2: Membrane Performance
o Membrane type, manufacturer and batch have all been
demonstrated to affect method performance.
o Performance is also dependent on the method:

• different membranes favour different methods/flora
o Membranes MUST be considered when assessing performance of

MF methods.

Membrane Performance
Supplier Cat # Material Designation Colour Growth
Gelman 662/8 Mixed cellulose ester GN6 ++ ++
Schleicher & Schuell 406870 Mixed cellulose ester ME25/21 ++ ++
Whatman 141114 Cellulose nitrate na I I
Schleicher & Schuell 405370 Cellulose nitrate NC45 + +
Whatman 70604704 Polycarbonate Cyclopore + +
Gelman 61845 Polyethersulfone Supor 450 + +
Gelman 66408 Acrylopolymer (nylon substrate) Versapor 450 + +
Gelman 66223 Polysulfone HT 450 + +
Millipore HTTP04700 Polycarbonate na + +
Millipore HAWG04700 Mixed cellulose ester na + +
Millipore HAWG04700 Mixed cellulose ester (triton free) na + +
Whatman 7404004 Nylon na 0 +
Schleicher & Schuell 414112 Polyamide NL1 0 I
Sartorius 13906 Cellulose nitrate Type 139 0 +
Sartorius 11106 Cellulose acetate Type 111 0 +
Whatman 70000004 Cellulose acetate na 0 +
Asypor ZDMA047-045AZ Mixed cellulose ester na - +
Schleicher & Schuell 404012 Cellulose acetate OE67 0 -
Whatman 68095022 Alumina matrix Anodisc 47 - -
Reproduced from: OSSMER,R., SCHMIDT, W. and MENDE, U - TC & E.coli – CC Agar

Membrane Performance Variation
Defined suspension of an E. coli type strain on Chromogenic Coliform

Agar (media and membranes from various suppliers)

Example 3: Background Flora
o Most MF media are only recommended for water with low
bacterial counts
o Addition of antibiotics can reduce background, BUT:
• reduces sensitivity
• outside the scope of ISO 9308-1:2012

Pseudomonas Agar – ISO 16266-1
Multiple Matrices
MF Culture @ 44°C
CFU Format
Nutrient Rich
Selectivity:
Cetrimide, Naladixic Acid
Confirmation
Multiple Confirmations
PRESUMPTIVE – 48h

Pseudomonas Agar - supplemented (PACN)
o ISO 16266 Method
o relies upon membrane filtration
o nutrient rich media
o specificity achieved using cetrimide and sodium naladixate to inhibit other G-ve
(Klebsiella, Proteus, Providencia spp.)
o selectivity:
• blue-green colonies are confirmed P. aeruginosa – confirmed 48h
• Non-blue/green or cream, brown colonies require confirmation
o confirmation – ISO:
• non-blue/green but fluorescent – acetamide broth – confirmed 72h
• cream/brown – oxidase, acetamide, Kings-B – confirmed 72-168hr
o confirmation – UK MoDW
• milk cetrimide agar / 1:10-phenanthroline – confirmed 72hr

Pseudomonas Agar - supplemented (PACN)
PACN Oxidase Test Acetamide Broth
King’s B Agar Milk Cetrimide Agar 1:10 phenathroline

FAQs

FAQs
Question 1: IDEXX’s methods report results

in MPN. What is the relationship between
MPN and CFU?

What is Most Probable Number - MPN?
ESTIMATE of the Concentration of an Analyte
(bacteria in our case)
ASSUMPTIONS
2. Each positive 3. Analyst

1. Bacteria are
results from a correctly counts
evenly distributed
single bacterium positive results
Results in an ESTIMATED count

and a PROBABLE range
COULD BE HERE MPN COULD BE HERE

What is Colony Forming Unit - CFU?
ESTIMATE of the Concentration of an Analyte
(bacteria in our case)
ASSUMPTIONS
2. Each colony 3. Analyst

1. Bacteria are
results from a correctly counts
evenly distributed
single bacterium positive colonies
Results in an ESTIMATED count

and a PROBABLE range
COULD BE HERE CFU COULD BE HERE

The Journey of a Bacterium
Membrane Filtration IDEXX Methods
Bacterium Captured on Membrane Bacterium Captured in Well
Multiplies on Solid Media Multiplies in Liquid Media
Positive Colony
Positive Well (colored/fluorescent)
(colored/fluorescent)
Microbiologist Counts Positive Microbiologist Counts Positive

Colonies – Generates Count Wells – Generates Count
1 CFU 1 MPN
Another Common Objection…
“When I compare Colilert-18 to

my reference method I get
higher counts with Colilert-18.
So how can you say that MPN
and CFU are equivalent?”

Units are ~Equivalent! – Methods ARE NOT!
e.g. analysis of a relatively clean sample
Colilert-18 vs. m-ENDO

Colilert-18 Count Higher
V MPN vs. CFU
ENZYME vs. LACTOSE
CCA vs. m-ENDO

CCA Count Higher
V CFU vs. CFU
ENZYME vs. LACTOSE

In Summary
MICROBIOLOGICAL
METHODS
Make Assumptions
Estimate microbial
concentration
Approximate numbers
within a probable range
Ranges typically overlap
between methods
Methods (not units)
account for differences
Experts use these units
interchangeably
To Explain to an Auditor
o Liquid Media - MPN/100mL: is a maximum likelihood
estimate of the number of bacteria in a sample based upon
the distribution of bacteria within the sample (based upon the
premise that the bacteria are distributed homogeneously
(statistically speaking) and that each bacteria grows within the
well/tube).
o Solid Media - CFU/100mL: is a maximum likelihood estimate of

the number of bacteria in a sample based upon the number of
colonies which form on a solid growth medium (based upon the
premise that each bacterial cell forms a distinct colony and that all
bacteria in the sample grow and form a colony).

FAQs
Question 2: Is it necessary to analyse

microbiology samples in a biosafety cabinet
using IDEXX methods?

US CDC Biosafety Classification
Reference Standard Methods 9020B
Biosafety level 1 (BSL 1)
- BSL 1 is suitable for work involving well-characterized agents not
known to consistently cause disease in healthy adults and of
minimal potential hazard to laboratory personnel and the
environment
- Work is generally conducted on open bench tops using standard
microbiological practices
- Microbiological agents that should be handled under BSL 1 are:
• Total and thermotolerant coliforms
• E.coli
• Enterococci
• Iron and sulfur bacteria
• Actinomycetes
• Other non-pathogenic microorganisms
US CDC Biosafety Classification
Reference Standard Methods 9020B
Biosafety level 2 (BSL 2)
- Differs from BSL 1 in that
• Laboratory personnel have specific training in handling
pathogenic agents
• Access to laboratory is limited when work is in progress
• Extreme precautions are taken with contaminated sharp items
• Certain procedures in which infectious aerosols may be created
are conducted in biological safety cabinets
- Microbiological agents that should be handled under BSL 2 are:
• Pathogenic bacteria such as: Salmonella, Shigella, Diarrheagenic
E.coli, Campylobacter, Vibrio, Leptospira, Legionella, Yersinia
enterocolitica, Aeromonas, Mycobacterium
• Enteric viruses
• Fungi
• Pathogenic protozoa
IDEXX is green
Please project (don’t print) this presentation.

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ISO Methods for Detection of Coliforms/E.

1 Copyright 2019 IDEXX Laboratories

o Offices in 20 countries serving

2 Copyright 2019 IDEXX Laboratories

3 Copyright 2019 IDEXX Laboratories

Cryptosporidium/ Heterotrophic Plate

4 Copyright 2019 IDEXX Laboratories

6 Copyright 2019 IDEXX Laboratories

o Heterotrophic Plate Count Method

*Pseudalert ®in pursue for peer review publications

8 Copyright 2019 IDEXX Laboratories

9 Copyright 2019 IDEXX Laboratories

10 Copyright 2019 IDEXX Laboratories

Based on the growth of target organisms in a liquid medium

Determination of bacteria concentration by MPN

Method applicable to all types of water, including those with

Detects E.coli based on the expression of the enzyme β-D-

11 Copyright 2019 IDEXX Laboratories

12 Copyright 2019 IDEXX Laboratories

14 Copyright 2019 IDEXX Laboratories

15 Copyright 2019 IDEXX Laboratories

16 Copyright 2019 IDEXX Laboratories

Differs from other methods where multiple nutrient sources

Using a defined nutrient as the primary carbon source is one

17 Copyright 2019 IDEXX Laboratories

ONPG provides the major source of carbon in Colilert -18

18 Copyright 2019 IDEXX Laboratories

19 Copyright 2019 IDEXX Laboratories

Read the color change in Read for fluorescence

POSITIVE for E. Coli NEGATIVE POSITIVE for Coliforms

o Comparator is available for use when reading results

22 Copyright 2019 IDEXX Laboratories

Add reagent to the Pour into Quanti-Tray or Seal using Quanti-Tray

Incubate at 36±2°C for 18-22h Count coliform and E. coli

o Quanti-Tray: detects up to 200 MPN/100mL

24 Copyright 2019 IDEXX Laboratories

o 16 positive large wells

o 3 positive small wells

o Result – 22.6 MPN/100mL

25 Copyright 2019 IDEXX Laboratories

26 Copyright 2019 IDEXX Laboratories

o Unit-dosed packaging eliminates media preparation

o No repeat testing due to clogged filters or heterotrophic

o QC procedure can be done in 15 minutes per new lot of

27 Copyright 2019 IDEXX Laboratories

o Coliform and E. coli results in just 18 hours

28 Copyright 2019 IDEXX Laboratories

o β-galactosidase and β-glucuronidase activity detects slow or

o Suppresses up to 2 million heterotrophs per 100ml

o Eliminates subjective interpretation

o Detects a single viable organism /100mL

29 Copyright 2019 IDEXX Laboratories

30 Copyright 2019 IDEXX Laboratories

Determination of bacteria concentration by MPN

Method applicable to a range of different types of water

Detects Pseudomonas aeruginosa via a bacterial enzyme

32 Copyright 2019 IDEXX Laboratories

o Same platform as Colilert-18

o No color change; fluorescence indicates a positive

o No confirmation test required

o Can be used for both presence/absence and quantitative (100

33 Copyright 2019 IDEXX Laboratories

o The microorganisms of P. aeruginosa rapidly grow and reproduce,