SIST-TP CEN/TR 16193:2013

Sludge, treated biowaste and soil - Detection and enumeration of Escherichia coli

This Technical Report specifies three methods for the detection and enumeration of Escherichia coli in sludge, treated biowaste and soil:
-   Method A - Membrane filtration method for quantification (see Clause 6);
-   Method B - Miniaturised method (Most Probable Number, MPN) by inoculation in liquid medium (see Clause 7);
-   Method C - Macromethod (Most Probable Number) in liquid medium (see Clause 8).

Schlamm, behandelter Bioabfall und Boden - Nachweis und Zählung von Escherichia coli

Dieser Fachbericht beschreibt drei Verfahren zur Bestimmung und Auszählung von Escherichia coli in Schlamm, behandeltem Bioabfall und Boden:
   Verfahren A - Membranfiltrationsverfahren zur quantitativen Bestimmung (siehe Abschnitt 6);
   Verfahren B - Miniaturisiertes Verfahren durch Animpfen in Flüssigmedium (MPN-Verfahren) (siehe Abschnitt 7);
   Verfahren C - Makroverfahren (MPN) in Flüssigmedium (siehe Abschnitt 8).

Boue, biodéchet traité et sol - Recherche et dénombrement des Escherichia coli

Le présent Rapport technique spécifie trois méthodes de recherche et de dénombrement des Escherichia coli
dans les boues, les biodéchets traités et les sols :
 Méthode A - Méthode par filtration sur membrane pour le dénombrement (voir Article 6) ;
 Méthode B - Méthode miniaturisée (nombre le plus probable, NPP) pour ensemencent en milieu liquide
(voir Article 7) ;
 Méthode C - Macrométhode (nombre le plus probable) en milieu liquide (voir Article 8).

Blato, obdelani biološki odpadki in tla - Ugotavljanje prisotnosti in števila Escherichia coli

To tehnično poročilo določa tri metode za ugotavljanje prisotnosti in števila Escherichia coli v blatu, obdelanih bioloških odpadkih in tleh:
– metoda A – metoda filtriranja z membrano za kvantifikacijo (glej točko 6);
– metoda B – miniaturizirana metoda (najbolj verjetno število (MPN)) z inokulacijo v tekoči medij (glej točko 7);
– metoda C – makrometoda (najbolj verjetno število) v tekočem mediju (glej točko 8).

General Information

• Status: Published
• Public Enquiry End Date: 31-Jan-2011
• Publication Date: 11-Jun-2013
• ICS: 13.030.20 - Liquid wastes. Sludge
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: KAT - Soil quality
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 04-Jun-2013
• Due Date: 09-Aug-2013
• Completion Date: 12-Jun-2013
• Directive: 86/278/EEC - Protection of the environment, and in particular of the soil, when sewage sludge is used in agriculture
• Directive: TP136 - Uredba o predelavi biološko razgradljivih odpadkov in uporabi komposta ali digestata
• Mandate: M/330 - Mandate given to CEN for the development of horizontal standards in the fields of sludge, biowaste and soil.
• Ref Project: CEN/TR 16193:2013 - Sludge, treated biowaste and soil - Detection and enumeration of Escherichia coli

Overview

CEN/TR 16193:2013 - "Sludge, treated biowaste and soil - Detection and enumeration of Escherichia coli" (CEN) is a Technical Report that consolidates validated laboratory procedures for measuring Escherichia coli in environmental matrices. Prepared by CEN/TC 400 under Mandate M/330, it is part of a modular, horizontal approach to analytical standards for sludge, biowaste and soil. The report brings together three complementary methods that have been evaluated in interlaboratory validation trials.

Key topics and technical requirements

• Methods included:
  • Method A - Membrane filtration (quantification): uses membrane filtration and chromogenic media for colony enumeration; includes sample preparation, dilution, resuscitation, confirmation and dry residue determination.
  • Method B - Miniaturised MPN (Most Probable Number): microtitre-plate liquid inoculation for MPN estimation; includes MPN statistical tables and guidance for medium manufacture and quality criteria.
  • Method C - Macromethod MPN: classical 3-tube (macrotube) MPN in liquid medium with associated MPN tables and performance metrics.
• Matrices tested: digested sewage sludge, pelletised and air‑dried sludge, presscake, composted sewage sludge, composted biowaste and green waste, anaerobic treated biowaste, and soils.
• Performance and validation: the report contains interlaboratory comparison results, precision (repeatability/reproducibility) assessments and MPN statistical tables.
• Sampling & sample handling: guidance on sampling, storage and handling for sludge, treated biowaste and soil to ensure representative and safe analysis.
• Quality assurance: procedures for media preparation, method performance checks and requirements for test reporting.
• Safety considerations: warnings about biological hazards, explosive gas risks in sealed samples and requirement for trained personnel and compliance with national biosafety rules.

Applications and users

This Technical Report is practical for:

• Environmental and microbiological laboratories performing E. coli detection and enumeration in sludge, treated biowaste and soil.
• Wastewater treatment operators, composting facilities and biowaste processors monitoring hygiene and treatment efficacy.
• Regulatory agencies and environmental consultants assessing compliance with hygiene targets and public‑health related parameters.
• Laboratories seeking a harmonised analytical approach as part of accreditation scopes covering multiple environmental matrices.

Related standards and context

• Part of CEN’s modular "Horizontal" project addressing analytical steps across matrices; complements standards on sampling and other hygienic, biological, inorganic and organic determinations.
• Intended as a Technical Report to support further method refinement toward eventual European Standard status.

Keywords: Escherichia coli detection, E. coli enumeration, sludge testing, treated biowaste analysis, soil microbiology, membrane filtration, MPN method, CEN/TR 16193:2013.

Standards Content (Sample)

SLOVENSKI STANDARD
01-julij-2013
Blato, obdelani biološki odpadki in tla - Ugotavljanje prisotnosti in števila
Escherichia coli
Sludge, treated biowaste and soil - Detection and enumeration of Escherichia coli
Schlamm, behandelter Bioabfall und Boden - Nachweis und Zählung von Escherichia coli
Boue, biodéchet traité et sol - Recherche et dénombrement des Escherichia coli
Ta slovenski standard je istoveten z: CEN/TR 16193:2013
ICS:
13.030.20 7HNRþLRGSDGNL%ODWR Liquid wastes. Sludge
13.080.30 Biološke lastnosti tal Biological properties of soils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

TECHNICAL REPORT
CEN/TR 16193
RAPPORT TECHNIQUE
TECHNISCHER BERICHT
May 2013
ICS 13.030.01
English Version
Sludge, treated biowaste and soil - Detection and enumeration
of Escherichia coli
Boue, biodéchet traité et sol - Recherche et dénombrement Schlamm, behandelter Bioabfall und Boden - Nachweis und
des Escherichia coli Zählung von Escherichia coli

This Technical Report was approved by CEN on 1 March 2011. It has been drawn up by the Technical Committee CEN/TC 400.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TR 16193:2013: E
worldwide for CEN national Members.

Contents Page
Foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Abbreviations . 7
5 Quality assurance . 7
6 Method A — Membrane filtration method for quantification . 7
6.1 Scope . 7
6.2 Principle. 8
6.3 Reagents, diluents and culture media . 8
6.4 Apparatus . 9
6.5 Sampling . 10
6.5.1 General . 10
6.5.2 Storage . 11
6.5.3 Handling . 11
6.6 Procedure . 11
6.6.1 Sample preparation . 11
6.6.2 Sample dilution . 12
6.6.3 Membrane filtration . 12
6.6.4 Resuscitation and enumeration of colonies on chromogenic agar . 13
6.6.5 Confirmation of colony identity . 13
6.6.6 Determination of the dry residue content . 13
6.7 Calculation and expression of results . 13
6.8 Performance data of the interlaboratory comparison — Method A . 14
6.8.1 Material used in the interlaboratory comparison study . 14
6.8.2 First assessment of the precision of the method . 15
6.8.3 Interlaboratory comparison results . 16
6.9 Pre-filtration and centrifugation — Comparison tests . 17
7 Method B — Miniaturised method (Most Probable Number) by inoculation in liquid
medium . 19
7.1 Scope . 19
7.2 Principle. 19
7.3 Reagents, diluents and culture media . 19
7.4 Apparatus . 21
7.5 Sampling . 22
7.5.1 General . 22
7.5.2 Storage . 22
7.5.3 Handling . 22
7.6 Procedure . 22
7.6.1 Sample preparation . 22
7.6.2 Analysis . 23
7.6.3 Determination of the dry residue content . 24
7.7 Expression of results . 24
7.7.1 Determination of the characteristic number . 24
7.7.2 Calculation of the MPN and its confidence interval . 25
7.8 Performance data . 27
7.8.1 MPN Statistical table . 27
7.8.2 Performance data of the interlaboratory comparison .3 5
7.8.3 First assessment of the precision of the method .3 6
7.8.4 Interlaboratory comparison results .3 7
7.9 Preparation of synthetic sea salt .3 9
7.9.1 Major ion composition of a convenient ocean synthetic sea salt . 39
7.9.2 Example for preparation from defined substances .3 9
7.10 Quality criteria for the manufacturing of the medium in microtitre plates (E. coli) . 40
8 Method C — Macromethod (Most Probable Number) in liquid medium . 41
8.1 Scope .4 1
8.2 Principle .4 1
8.3 Reagents, diluents and culture media .4 1
8.4 Apparatus .4 2
8.5 Sampling .4 3
8.5.1 General .4 3
8.5.2 Sample storage.4 3
8.5.3 Sample handling .4 3
8.6 Procedure .4 3
8.6.1 Sample preparation .4 3
8.6.2 Analysis .4 4
8.6.3 Determination of the dry residue content .4 4
8.7 Expression of the results .4 4
8.8 Performance data .4 6
8.8.1 MPN Statistical table for 3-tubes MPN procedure .4 6
8.8.2 Repeatability and reproducibility .4 7
8.8.3 First assessment of the precision of the method .4 7
8.8.4 Interlaboratory comparison results .4 8
9 Test report .5 0
Bibliography .5 1

Foreword
This document (CEN/TR 16193:2013) has been prepared by Technical Committee CEN/TC 400 “Project
Committee - Horizontal standards in in the fields of sludge, biowaste and soil”, the secretariat of which is held
by DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
This document is part of a modular horizontal approach in which this document belongs to the analytical step.
The preparation of this document by CEN is based on a mandate by the European Commission (Mandate
M/330). The mandate considers standards on sampling and analytical methods for hygienic and biological
parameters as well as inorganic and organic determinants. It was the aim of the mandate to develop
standards that are applicable to sludge, treated biowaste and soil and lead to equivalent results as far as this
is technically feasible.
Until now, test methods determining properties of materials within the environmental area were prepared in
Technical Committees (TCs) working on specific products/matrices (soil, waste, sludge etc). However, it is
recognised that many steps in test procedures can be used in test procedures for other products/matrices. By
careful determination of these steps and selection of specific questions within these steps, elements of the
test procedure can be described in a way that can be used for more matrices and materials with certain
specifications. This optimisation is in line with the development among end-users of standards. A majority of
routine environmental analyses are carried out by institutions and laboratories working under a scope which is
not limited to one single environmental matrix but covers a wide variety of matrices. Availability of standards
covering more matrices contributes to the optimisation of laboratory procedures and standard maintenance
costs, e.g. costs related to accreditation and recognition.
A horizontal modular approach was developed in the project 'Horizontal'. 'Modular' means that a test standard
developed in this approach concerns a specific step in assessing a property and not the whole "chain of
measurement” (from sampling to analyses). A beneficial feature of this approach is that “modules” can be
replaced by better ones without jeopardising the standard “chain”.
The results of the desk study as well as the evaluation and validation studies have been subject to
discussions with all parties concerned in the CEN structure during the development by project 'Horizontal'.
The results of these consultations with interested parties in the CEN structure have been presented to and
discussed in CEN/TC 400.
This Technical Report contains the most common detection and enumeration methods for the determination of
E. coli consolidated in one document. The individual methods are specified in the following clauses:
 Clause 6: Method A - Membrane filtration method for quantification;
 Clause 7: Method B - Miniaturised method (Most Probable Number) by inoculation in liquid medium;
 Clause 8: Method C - Macromethod (Most Probable Number) in liquid medium.
Introduction
Escherichia coli is a non-pathogenic, Gram negative bacterium with a faecal origin. Consequently, it can be
used as an indicator of faecal contamination. It can also be used to monitor the effectiveness of pasteurisation
or disinfection treatments but it is comparatively sensitive (to heat, high pH) and cannot therefore reflect the
behaviour of all pathogens in these materials.
This Technical Report contains three different methods for the detection and enumeration of Escherichia coli
which were included in a validation trial in 2007.
The results achieved in this validation trial have been judged differently by experts. Consequently, it was
decided by CEN/TC 400 to publish the methods as a Technical Report, aiming for further improvement of the
methods and a later publication as European Standard.
Table 1 — Matrices for which the methods described in this Technical Report are applicable and
tested in a validation trial
Matrix Method A Method B Method C
Mesophilic anaerobic Mesophilic anaerobic Mesophilic anaerobic
Sludge
digested sewage sludge digested sewage sludge digested sewage sludge
Pelletised air dried sludge Pelletised air dried sludge Pelletised air-dried sludge
Digested sewage sludge Digested sewage sludge Digested sewage sludge

presscake presscake presscake
Composted sewage sludge Composted sewage sludge Composted sewage sludge
Biowaste Composted biowaste Composted biowaste Anaerobic treated biowaste
Composted green waste Composted green waste Composted green waste
Anaerobic treated biowaste Anaerobic treated biowaste Composted biowaste
WARNING — Persons using this Technical Report should be familiar with normal laboratory practice.
This Technical Report does not purport to address all of the safety problems, if any, associated with
its use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
WARNING — Samples may contain hazardous and inflammable substances. They may contain
pathogens and be liable to biological action. Consequently, it is recommended that these samples be
handled with special care. The gases which can be produced by microbiological activity are
potentially inflammable and will pressurise sealed bottles. Exploding bottles are likely to result in
infectious shrapnel and/or pathogenic aerosols. Glass bottles should be avoided wherever possible.
National regulations should be followed with respect to microbiological hazards associated with this
method.
IMPORTANT — It is absolutely essential that tests conducted according to this Technical Report be
carried out by suitably trained staff.
1 Scope
This Technical Report specifies three methods for the detection and enumeration of Escherichia coli in sludge,
treated biowaste and soil:
 Method A - Membrane filtration method for quantification (see Clause 6);
 Method B - Miniaturised method (Most Probable Number, MPN) by inoculation in liquid medium (see
Clause 7);
 Method C - Macromethod (Most Probable Number) in liquid medium (see Clause 8).
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
EN 15934, Sludge, treated biowaste, soil and waste — Calculation of dry matter fraction after determination of
dry residue or water content
EN ISO 9308-3:1998, Water quality — Detection and enumeration of Escherichia coli and coliform bacteria in
surface and wastewater — Part 3: Miniaturized method (Most Probable Number) by inoculation in liquid
medium (ISO 9308-3:1998)
ISO 8199, Water quality — General guidance on the enumeration of micro-organisms by culture
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
Escherichia coli
E.coli
β-D-glucuronidase-positive microorganism growing at an incubation temperature of 44 °C in the specified
liquid medium containing 4-methylumbelliferyl-β-D-glucuronide (MUG)
[SOURCE: EN ISO 9308-3:1998]
Note 1 to entry: During growth, indole is produced from tryptophan and gas produced from lactose.
3.2
vegetative bacteria
bacteria which are capable of normal growth in broth or on agar media without pre-culture resuscitation
3.3
sub-lethally damaged bacteria
bacteria which have been stressed but not killed by storage or subsequent treatment by, e.g., mesophilic
anaerobic digestion, lime stabilisation or composting, and therefore may not be recovered
3.4
resuscitation
recovery to vegetative growth of sub-lethally damaged bacteria previously incapable of growth on agar media
3.5
quantitative resuscitation
recovery to vegetative growth of sub-lethally damaged bacteria isolated discretely on a membrane filter, prior
to transfer to chromogenic medium for growth of individual colonies
3.6
colony forming unit
cfu
growth of individual bacterial cells into visible colonies on agar media, including on membrane filters
overlaying the agar media
3.7
Most probable number
MPN
every well whose inoculum contains even one viable organism will produce detectable growth or change
Note 1 to entry: The individual wells of the sample are independent.
4 Abbreviations
BCIG: 5-bromo-4-chloro-3-indolyl-β-glucuronide
CN:  Characteristic number
DS:  Dry solid
E. coli:  Escherichia coli
MLGA:  Membrane Lactose Glucuronide Agar
MPN:  Most Probable Number
MUG: 4-methylumbelliferyl-β-D-glucuronide
SMD: Special Microplate Diluent
5 Quality assurance
Suitable quality control procedures, at least those described in ISO 8199, shall be applied.
6 Method A — Membrane filtration method for quantification
6.1 Scope
Method A specifies a membrane filtration procedure for the quantitative detection, by culture of individual
colonies on chromogenic agar media. It is not suitable for materials whose treatment will significantly reduce
bacterial levels to less than 10 viable E. coli per g wet weight, such as lime addition, drying or pasteurisation.
This membrane filtration method is not appropriate for enumeration and detection of other coliform bacteria
without modifications to the chromogenic agar medium.
It is suitable to evaluate the log reduction of E. coli through treatment, as well as the quality of the end
product.
-1
Method A has a limit of detection of approximately 27 E. coli cfu ∙ g wet weight according to ENV ISO 13843,
dependent on the solids content which at high concentrations (> 0,1 g/ml) may restrict filtration of the sample
volume through the membrane if not first diluted.
6.2 Principle
The homogenised diluted sample is filtered, the membrane filter recovered aseptically and incubated on
membrane lactose glucuronide agar (MLGA), initially at (30 ± 1) °C for (4,0 ± 0,5) h. Subsequently, the
temperature is increased to (44 ± 1) °C for (16 ± 2) h. The presence of E. coli is indicated by green colonies
resulting from the hydrolysis of BCIG.
6.3 Reagents, diluents and culture media
6.3.1 General instructions
To ensure reproducible results, prepare culture media and diluents using either constituents of uniform quality
and chemicals of recognised analytical grade, or a dehydrated diluent or complete medium prepared following
the manufacturer’s instructions. Prepare them with demineralised or distilled water free from substances
capable of inhibiting growth under the test conditions (see ISO 8199).
The use of chemicals of other grades is permissible provided that they are shown to be of equivalent
performance in the test.
6.3.2 Peptone saline solution
Bacteriological peptone 1,0 g
Sodium chloride 8,5 g
Distilled water 1 000 ml
Sodium hydroxide solution
Hydrochloric acid, 1mol/l
Dissolve the bacteriological peptone and sodium chloride into distilled water. Adjust the pH by adding sodium
hydroxide solution or hydrochloric acid so that, after sterilisation, it will correspond to (7,0 ± 0,5) at 25 °C.
Sterilise in the autoclave (6.4.1) at (121 ± 3) °C for (15 ± 1) min. Store at (5 ± 3) °C for a maximum of
3 months.
6.3.3 Membrane Lactose Glucuronide Agar (MLGA)
6.3.3.1 5-bromo-4-chloro-3-indolyl-β-glucuronide (BCIG) suspension
BCIG, monohexylammonium salt 0,2 g
Aqueous ethanol, 95 % 2,5 ml
Sodium hydroxide, 1 mol/l 0,5 ml
Dissolve 200 mg BCIG in a combined solution of 95 % aqueous ethanol and 1 mol/l sodium hydroxide.
6.3.3.2 MLGA
Peptone 40,0 g
Yeast extract 6,0 g
Lactose 30,0 g
Sodium lauryl sulphate 1,0 g
Phenol red 0,2 g
Sodium pyruvate 0,5 g
Bacteriological agar 10,0 g
Demineralised or distilled water 1 000 ml
Mix all ingredients and bring to the boil whilst stirring continuously.
Add the BCIG suspension to the molten base agar medium and mix thoroughly. Adjust the pH to (7,0 ± 0,5).
Sterilise by autoclaving at (121 ± 3) °C for (15 ± 1) min. Pour into 55 mm Petri dishes in volumes of
approximately 10 ml. Allow setting and store refrigerated at (5 ± 3) °C in the dark. Use within 7 days.
6.3.4 MacConkey Agar
Peptone 20,0 g
Lactose 10,0 g
Bile salts 5,0 g
Sodium chloride 5,0 g
Neutral red 0,075 g
Agar 12,0 g
Distilled water 1 000 ml
Suspend the ingredients in 1 000 ml of distilled water. Bring to the boil whilst stirring continuously to dissolve
all ingredients completely. Adjust the pH to (7,0 ± 0,5).
Sterilise in the autoclave (6.4.1) at (121 ± 3) °C for (15 ± 1) min. Store at (5 ± 3) °C for a maximum of 1 month.
Dry the surface of the agar before inoculation.
6.4 Apparatus
With the exception of equipment supplied sterile, the glassware shall be sterilised in accordance with the
instructions given in ISO 8199.
Usual microbiological laboratory equipment and in particular:
6.4.1 Apparatus for sterilisation – autoclave.
6.4.2 Thermostatic incubator(s) adjustable to (30 ± 1) °C and/or (44 ± 1) °C.
6.4.3 Homogeniser
1)
6.4.4 Centrifuge, capable of centrifuging 50 ml at 200 g to 300 g.
6.4.5 Membrane filters, 0,45 µm gridded, cellulose nitrate.
6.4.6 Glass fibre pre-filter discs, 47 mm diameter, pore size 2,7 µm.
6.4.7 Vacuum pump
6.4.8 Vacuum manifold – magnetic filter bases and cups.
6.4.9 Sterile homogeniser bags, 250 ml volume, with or without integrated mesh to exclude large
particulate matter.
6.4.10 Sterile Petri dishes, 50 mm in diameter, for holding MLGA medium.
6.4.11 Sterile universals of 20 ml volume, or containers with similar capacity.
6.4.12 Sterile pipettes, glass or disposable plastic ware, capable of dispensing 1 ml and 10 ml volumes.
6.4.13 Sterile conical centrifuge tubes, 50 ml volume, disposable plastic.
6.4.14 Tweezers, capable of sterilisation by immersion in ethanol and subsequent flaming.
6.4.15 Analytical balance
6.4.16 Refrigerator, capable of maintaining (5 ± 3) °C.
6.4.17 Vortex mixer
6.4.18 pH meter with an accuracy of ± 0,1.
6.4.19 Beakers or containers, 100 ml, 250 ml and 1 000 ml.
6.4.20 Laboratory spatula
6.4.21 Boiling bath
6.4.22 Bunsen burner
6.4.23 Sterile forceps
6.4.24 Filter funnels
6.5 Sampling
6.5.1 General
Take samples of at least 100 g wet weight and deliver them to the laboratory as quickly as possible (within
24 h). In order to prevent propagation or inactivation of E. coli during transport to the laboratory and
subsequent storage, refrigerate the sample at (5 ± 3) °C.
Samples are liable to ferment and may contain pathogenic micro-organisms. It is essential to keep them away
from any food or drink, and to protect any cuts. When transporting and handling samples, it is essential that
national and international regulations relating to bio-hazardous samples are followed.

-2
1) g = 9,81 m∙s
See also the Warning note in the introduction.
6.5.2 Storage
Do not store samples in the open laboratory. If samples are to be stored, store them at (5 ± 3) °C for no longer
than 72 h after receipt.
6.5.3 Handling
Cleanliness when working is essential. When handling sludge samples, it is necessary to wear gloves, face
and eye protection, and sufficient body protection to guard against bottles bursting. The gas evolved is usually
flammable, so all equipment used in the vicinity shall be flame proof to avoid any source of ignition.
6.6 Procedure
6.6.1 Sample preparation
6.6.1.1 General
Weigh a representative 10 g (wet weight) of the sample as received into a 250 ml container (6.4.19).
Add an appropriate volume of peptone saline solution (6.3.2) so that the final weight is 100 g and mix
thoroughly using a vortex mixer (6.4.17).
Place in homogeniser bag (6.4.9) and place in the homogeniser (6.4.3) and homogenise for 2 min to obtain
the sample suspension (dilution A). For samples with a dry solid content > 20 % a homogeniser bag with an
integrated mesh should be used. For samples with dry solid content < 20 % a homogeniser bag without
integrated mesh can be used.
6.6.1.2 For lime-treated materials
Adjust the pH to (7,0 ± 0,5) with 1 mol/l hydrochloric acid (6.3.2). The sample is mixed by shaking between
each addition of hydrochloric acid to ensure the correct pH is achieved. The sample is transferred to a sterile
250 ml container and tested using a pH meter (6.4.18).
If the pH drops below 4,5 during the neutralisation process, start a new analysis with a fresh test portion.
For other relevant treatment chemicals (e.g. peracetic acid), a suitable oxidant neutralisation procedure shall
be used (e.g. EN 1040).
Centrifugation and pre-filtration: The variation in the level of solid material contained within the matrices
applicable to Method A means that some samples will require centrifugation and pre-filtration before they can
be processed by membrane filtration without blocking the membrane. Not all samples will require these
optional steps; if necessary then centrifugation and pre-filtration should be applied.
6.6.1.3 Centrifugation (optional)
Transfer the homogeniser bag contents to two disposable centrifuge tubes (6.4.13) and centrifuge the two
50 ml aliquots at 200 g to 300 g for 3 min.
6.6.1.4 Pre-filtration (optional)
Decant the supernatant from the tubes in a beaker and filter through a glass-fibre pre-filter (6.6) using a filter
funnel with receiver (6.4.24) to remove fine debris.
The filter funnels should have been sterilised in a boiling bath (6.4.21) prior to analysis, the filter funnels are
removed from the boiling bath using sterile forceps (6.4.23) and attached to the vacuum pump (6.4.7).
The glass fibre filter (6.4.6) is placed on the filter funnel using sterile tweezers (6.4.14) before the filter funnel
cup is secured in position. The vacuum may now be used to draw the sample through the glass fibre filter; it is
recommended that the sample is not all introduced to the filter funnel cup at the same time because blockages
may occur.
The filter funnels should be returned to the boiling bath and be sterilised for a minimum of 5 min before being
used again.
6.6.2 Sample dilution
The number of dilutions to subsequently filter varies according to the presumed level of contamination of the
-1 -3
material to be tested. Typically, dilution A (the filtrate) should be serially diluted 10 to 10 with peptone
saline solution (6.3.2). This will permit the enumeration of up to 104 E. coli per g wet weight sample. Samples
-8
with greater concentrations or counts of bacteria will require additional dilutions of the filtrate to 10 (e.g.
8 9
untreated sludge may contain 10 to 10 E. coli per g wet weight).
Prepare the relevant number of sterile universals (6.4.11) according to the number of selected dilutions. Add
9 ml of sterile peptone saline solution (6.3.2) to each.
Using a sterile pipette (6.4.12), transfer 1 ml of the filtrate to the first universal containing 9 ml of peptone
saline solution (6.3.2) and mix thoroughly using a vortex mixer (6.4.17).
Using a fresh pipette (6.4.12), transfer 1 ml of the diluted sample to the second universal containing 9 ml of
peptone saline solution (6.4.2) and mix thoroughly using a vortex mixer (6.4.17).
Continue as above until all the dilutions have been prepared.
6.6.3 Membrane filtration
Transfer the magnetic filter base (6.4.8) from the boiling water bath (6.4.21) to the manifold using sterilised
forceps (6.4.23).
Membranes (6.4.5) are removed from their packets with sterilised tweezers (6.4.14) and placed grid side up
onto the magnetic filter base. Membranes that have torn are dropped or which touch any object shall be
discarded. Membranes should only be handled by the edge with tweezers designed for that purpose. The
tweezers shall be sterilised first by immersing them in ethanol which is subsequently flamed off using a
Bunsen burner (6.4.22), and then dipping directly into boiling water.
The magnetic filter cup is then removed from the boiling bath and attached to the magnetic base taking care
not to wrinkle the membrane. Magnetic filter cups should only be removed from the boiling bath with
disinfected forceps and placed directly onto magnetic bases. Hands may be used to transfer magnetic filter
cups back to the boiling bath. Neither forceps nor tweezers should be placed directly onto the bench. If the
filtration equipment is left for any significant length of time, the magnetic bases shall be returned to the boiling
bath. Magnetic bases left unused for short periods can be covered with the base or lid of a sterile Petri dish
until filtration recommences.
Add a sufficient volume of peptone saline solution (15 ± 5) ml into the filter cup, pipette 1 ml of the diluted
sample into the filter cup. Replace the top on the universal. Place the used universal back into the rack. The
universal shall not be placed on the filtration bench.
The sample may now be drawn through the filter by vacuum and only when filtration is complete should the
vacuum be turned off. The magnetic filter cup is lifted off, and returned to the boiling bath.
The membrane is carefully removed using sterile tweezers and transferred to the MLGA 55 mm Petri dish
(6.4.10). The membrane should be ‘rolled’ into the plate to prevent air bubbles becoming trapped between the
growth medium and the membrane, and the lid of the Petri dish is replaced. Bubbles should be excluded so
that the membranes are in intimate contact with the agar surface allowing unrestricted growth of viable
bacteria present on the membrane surface.
Any wrinkled or torn membranes discovered after filtration shall be discarded. The magnetic filter base shall
then be re-sterilised and the dilution filtered again.
Once filtration of samples is complete, the filter funnels are placed in to the boiling water bath for disinfection.
They shall be totally immersed in boiling water for at least 2 min before being removed to continue filtration.
2 3
A positive control suspension containing 10 to 10 target organisms is prepared using stock cultures (i.e.
reference material). The positive control sample should be analysed as the last sample in the analytical run. A
blank control suspension is prepared using peptone saline solution. The blank control should be analysed at
the beginning and as the penultimate sample of the analytical run.
6.6.4 Resuscitation and enumeration of colonies on chromogenic agar
Remove the filter from the housing using sterile tweezers (6.4.13) and transfer to the surface of a 55 mm
diameter MLGA plate (6.3.3). Incubate plates initially at (30 ± 1) °C for (4,0 ± 0,5) h. Subsequently, increase
the temperature to (44 ± 1) °C for (16 ± 2) h.
Enumerate typical green colonies by eye, only plates within the range 10 to 100 colonies should be
considered for the expression of results. If no counts are in this range it may be appropriate to consider counts
outside this range provided that an accurate enumeration is possible. The number of typical colonies that are
identified for confirmation is determined by the experience of the analyst.
When enumerating typical colonies, be aware that strains in environmental samples can give pale green
colonies on initial isolation and these should be considered for confirmation.
6.6.5 Confirmation of colony identity
The typical colonies are sub-cultured onto selective MacConkey agar (6.3.4). The MacConkey subculture
plates are incubated at (36 ± 2) °C for (21 ± 3) h.
It is important to subculture any green colonies suspected of being E. coli regardless of colour alone: a
minimum of two colonies per plate, per sample; and a maximum of each morphological type per plate and per
sample should be taken for subculture.
Typical green colonies on MLGA plates corresponding to typical red colonies observed by eye on MacConkey
plates should be considered as confirmed E. coli colonies for the expression of results.
For further confirmation additional biochemical tests can be performed.
6.6.6 Determination of the dry residue content
The numbers of E. coli may be calculated per wet weight or dry weight. For the latter, it is necessary to
determine the dry residue of the sample using the method specified in EN 15934. This shall be performed in
parallel with the microbiological analysis.
6.7 Calculation and expression of results
Calculation of the number of E. coli (present per g wet weight of the original sample) is by dividing the total
number of typical colonies (n) on the filter membrane of the selected plates (9.4) by the total volume filtered of
the initial sample. The result of the confirmation step shall be taken into account to estimate the total number
of typical colonies to calculate the final result (see ISO 8199).
n
c= (1)
V
where
c is the E. coli concentration per gram (g) wet weight of original sample;
n is the total number of typical E. coli colonies on the selected filter membranes: n = n + n + …;
1 2
V is the total volume filtered through the selected filter membranes (from 1 ml): V = V + V + …;
1 2
The dilution factor of the dilution A taken for filtration step should not be forgotten in the final calculation.
EXAMPLE If the volume of the test dilution used (V ) is 1 ml of dilution A and the following counts are obtained at the
i
respective dilutions:
Dilution Counts
10-2 81 colonies
10-3 15 colonies
Then:
n = 81 + 15 = 96
× × × ×
V = (0,1 1 0,01) + (0,1 1 0,001)
c =96/0,001 1 = 8,7 104 cfu/g ww
Numbers present per g dry weight of sample are calculated according to Formula (2):
n
(2)
c= ×100
Ve
where
e is the dry residue of the original wet sample in percent, %.
6.8 Performance data of the interlaboratory comparison — Method A
6.8.1 Material used in the interlaboratory comparison study
The interlaboratory comparison of the membrane filtration method for quantification of E. coli in soil, sludge
and treated biowaste took place from May to July 2007. It was carried out with 14 European laboratories on 7
different matrices. The matrices selected for the interlaboratory comparison were chosen to represent soil,
sludge and biowaste as broadly as possible, because Method A will find general application across different
types of soil and soil related materials (detailed information can be found in the final report on the
interlaboratory comparison study).
Table 2 provides a list of the matrices chosen for E. coli detection.
Table 2 — Matrices tested in the interlaboratory comparison trial
Matrix type Abbreviation
Mesophilic anaerobic digested sewage sludge MAD
Anaerobic treated biowaste ATB
Pelletised air-dried sludge PADS
Digested sewage sludge presscake DSSP
Composted sewage sludge CSS
Composted green waste CGW
Composted biowaste CBW
In the interlaboratory comparison study, the following starting points were used:
The laboratory samples were all taken from a large batch of the different matrices according to the normal
practice. The choice was made to analysed only spiked samples so as to obtain positive results. The spiking,
the mixing and the sub-sampling were carried out as needed to prepare representative laboratory samples of
approximately 150 g from the large batch sample. These were sent out by courier to each of the participating
laboratories.
The experimental plan requested information on the basis of each laboratory being given 3 laboratory samples
of each of the 2 batches of the seven matrices to be tested.
6.8.2 First assessment of the precision of the method
The statistical evaluation was conducted according to ENV ISO 13843. The limit of detection, the upper limit of
quantification, the range of quantification and the results of dispersion U were obtained (Table 3).
The limit of detection corresponds to the number of particles (germs per test portion) when the probability of a
negative result is 5 % (superior limit of the confidence interval of the null result).
Poisson distribution corresponds to the random distribution of the number of particles at the moment of
sampling a perfectly homogenised suspension.
The relative variance U corresponds to the relative standard deviation squared ratio of the standard deviation
2 2 2
squared and the mean squared as U = s / m .
NOTE 1 This statistic is commonly used to express dispersion or uncertainty of microbiological test results.
Table 3 — Summary of components of the E. coli Membrane filtration method precision
Limit of detection Upper limit of Range of Results of
quantification quantification dispersion U
5 % 5 % Log10 unit
E. coli/g wet weight E. coli/g wet weight
26,96 1,32 10 9,7 Less than 0,05

NOTE 2 In judging the results it is important to consider that they do not depend on the experimental data but only on
the design of the measurement protocol (random variation).
6.8.3 Interlaboratory comparison results
The statistical evaluation was conducted according to ISO 5725-2. The average values, the repeatability (r)
and the reproducibility (R) were obtained (Table 4).
The repeatability corresponds to the maximum difference that can be expected (with a 95 % statistical
confidence) between one test result and another, both test results being obtained under the following
conditions: the tests are performed in accordance with all the requirements of the method by the same
laboratory using its own facilities and testing laboratories samples obtained from the same primary field
sample and prepared under identical procedures.
The repeatability limit was calculated using the relationship: r = f ∙ √2 ∙ s with the critical range factor
test r.test
f = 2.
NOTE 1 The above relationship refers to the difference that may be found between two measurements r
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