CEN/TR 15214-1:2006

Characterization of sludges - Detection and enumeration of Escherichia coli in sludges, soils, soil improvers, growing media and biowastes - Part 1: Membrane filtration method for quantification

This part of the CEN Technical Report specifies a membrane filtration procedure for the quantitative detection, by culture of individual colonies on chromogenic agar media, of Escherichia coli. in sludges, soils, soil improvers, growing media and biowastes. This part of the Technical Report 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. A liquid enrichment and most probable number estimation method may be suited for such purpose.
This membrane filtration method is not appropriate for enumeration and detection of other coliform bacteria without modifications to the chromogenic agar media.
It is suitable to evaluate the log reduction of E.coli through treatment, as well as the quality of the end product.
This method is for materials with dry residues less than 20 %. For materials with dry residues greater than 20 % and low numbers of E. coli,  CEN/TR 15214-2 and CEN/TR 15214-3  should be used.

Charakterisierung von Schlämmen - Quantitativer Nachweis von Escherichia coli in Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten sowie Bioabfällen - Teil 1: Membranfiltrationsverfahren zur quantitativen Bestimmung

Dieses Dokument legt ein Membranfiltrationsverfahren für den quantitativen Nachweis von Escherichia coli in Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten und Bioabfall durch Züchtung von einzelnen Kolonien auf chromogenen Agarmedien fest. Dieses Dokument ist nicht für Materialien geeignet, deren Behandlung zu einer wesentlichen Verringerung der Bakterienkonzentration auf weniger als 10 lebensfähige E. coli je Gramm Feuchtmasse führt, wie z. B. Hinzufügen von Kalk, Trocknen oder Pasteurisierung. In diesem Fall können die Anreicherung in einem flüssigen Medium und das Verfahren der wahrscheinlichsten Keimzahl (MPN-Schätzverfahren) geeignet sein.
Dieses Membranfiltrationsverfahren ist ohne Modifikation der chromogenen Agarmedien nicht für die Zählung und den Nachweis von anderen coliformen Bakterien geeignet.
Es eignet sich für die Bewertung der logarithmischen Abnahme von E. coli durch Behandlung sowie der Qualität des Endproduktes.
Das vorliegende Verfahren ist für Materialien mit einem Trockenrückstand von weniger als 20 % vorgesehen. Bei Materialien mit einem Trockenrückstand von mehr als 20 % und einer geringen Anzahl an E. coli sollten CEN/TR 15214-2 und CEN/TR 15214-3 Anwendung finden.

Caractérisation des boues - Détection et dénombrement des Escherichia coli dans les boues, les sols, les amendements du sol, les supports de culture et les biodéchets - Partie 1 : Méthode de quantification par filtration sur membrane

La présente partie du Rapport Technique CEN spécifie un mode opératoire de filtration sur membrane pour la détection quantitative par culture de colonies individuelles sur un milieu chromogène gélosé, d’Escherichia coli dans les boues, les sols, les amendements du sol, les supports de culture et les biodéchets. La présente partie du Rapport Technique ne convient pas aux matériaux soumis à un traitement (tels que l’ajout de chaux, le séchage ou la pasteurisation) réduisant de façon significative les niveaux bactériens à un niveau inférieur à 10 E. coli viables par gramme de masse humide. Pour cela, une fertilisation liquide et une estimation en utilisant la technique du nombre le plus probable peuvent convenir.
Cette méthode de filtration sur membrane ne convient pas pour la détection et le dénombrement d’autres bactéries coliformes si le milieu chromogène gélosé n’est pas modifié.
Elle convient en revanche pour évaluer la réduction log d’E. coli pendant le traitement ainsi que la qualité du produit fini.
Cette méthode s’applique aux matériaux ayant une teneur en matière sèche inférieure à 20 %. Il convient d’utiliser le CEN/TR 15214-2 et le CEN/TR 15214-3 lorsque les matériaux ont une teneur en matière sèche supérieure à 20 % et des nombres faibles de E. coli.

Karakterizacija blata – Ugotavljanje prisotnosti in števila Echerichia coli v blatu, zemljini, izboljševalcih tal, rastnih substratih in bio-odpadkih - 1. del: Metoda membranske filtracije za kvantifikacijo

General Information

• Status: Published
• Publication Date: 24-Jan-2006
• ICS: 07.100.99 - Other standards related to microbiology
• Technical Committee: CEN/TC 308 - Characterization of sludges
• Drafting Committee: CEN/TC 308/WG 1 - Characterization methods
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 25-Jan-2006
• Completion Date: 25-Jan-2006
• Ref Project: SIST-TP CEN/TR 15214-1:2006 - Characterization of sludges - Detection and enumeration of Escherichia coli in sludges, soils, soil improvers, growing media and biowastes - Part 1: Membrane filtration method for quantification

Standards Content (Sample)

SLOVENSKI STANDARD
SIST-TP CEN/TR 15214-1:2006
01-julij-2006

Karakterizacija blata – Ugotavljanje prisotnosti in števila Echerichia coli v blatu,

zemljini, izboljševalcih tal, rastnih substratih in bio-odpadkih - 1. del: Metoda

membranske filtracije za kvantifikacijo

Characterization of sludges - Detection and enumeration of Escherichia coli in sludges,

soils, soil improvers, growing media and biowastes - Part 1: Membrane filtration method

for quantification
Charakterisierung von Schlämmen - Quantitativer Nachweis von Escherichia coli in

Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten sowie Bioabfällen -

Teil 1: Membranfiltrationsverfahren zur quantitativen Bestimmung

Caractérisation des boues - Détection et dénombrement des Escherichia coli dans les

boues, les sols, les amendements du sol, les supports de culture et les biodéchets -

Partie 1 : Méthode de quantification par filtration sur membrane
Ta slovenski standard je istoveten z: CEN/TR 15214-1:2006
ICS:
13.030.20 7HNRþLRGSDGNL%ODWR Liquid wastes. Sludge
13.080.30 Biološke lastnosti tal Biological properties of soils
65.080 Gnojila Fertilizers
SIST-TP CEN/TR 15214-1:2006 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TP CEN/TR 15214-1:2006
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SIST-TP CEN/TR 15214-1:2006
TECHNICAL REPORT
CEN/TR 15214-1
RAPPORT TECHNIQUE
TECHNISCHER BERICHT
January 2006
ICS 07.100.99
English Version
Characterization of sludges - Detection and enumeration of
Escherichia coli in sludges, soils, soil improvers, growing media
and biowastes - Part 1: Membrane filtration method for
quantification

Caractérisation des boues - Détection et dénombrement Charakterisierung von Schlämmen - Quantitativer

des Escherichia coli dans les boues, les sols, les engrais, Nachweis von Escherichia coli in Schlämmen, Böden,

les amendements organiques et les biodéchets - Partie 1 : Düngemitteln und Bodenverbesserern, Kultursubstraten

Méthode de quantification par filtration sur membrane sowie Bioabfällen - Teil 1: Membranfiltrationsverfahren zur

quantitativen Bestimmung

This Technical Report was approved by CEN on 3 September 2005. It has been drawn up by the Technical Committee CEN/TC 308.

CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,

Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,

Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels

© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TR 15214-1:2006: E

worldwide for CEN national Members.
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Contents Page

Foreword ..........................................................................................................................................................3

Introduction......................................................................................................................................................4

1 Scope...................................................................................................................................................5

2 Normative references.........................................................................................................................5

3 Terms and definitions.........................................................................................................................5

4 Principle...............................................................................................................................................6

5 Apparatus............................................................................................................................................6

6 Sampling and hazards ........................................................................................................................7

7 Reagents, diluents and culture media...............................................................................................7

8 Procedure............................................................................................................................................8

9 Expression of results..........................................................................................................................9

10 Test report.........................................................................................................................................10

11 Performance data..............................................................................................................................10

Annex A (informative) Performance data of the interlaboratory comparison............................................11

Bibliography...................................................................................................................................................13

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Foreword

This Technical Report (CEN/TR 15214-1:2006) has been prepared by Technical Committee CEN/TC 308

“Characterization of sludges”, the secretariat of which is held by AFNOR.
This Technical Report does not replace any existing CEN method.

The standard is divided into three parts, part 1 gives a membrane filtration for quantification, part 2 gives a

miniaturised semi-quantitative MPN method and part 3 gives a semi-quantitative macromethod.

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Introduction

Sludges, soils, soil improvers, growing media and biowastes can contain pathogenic micro-organisms such as

Salmonella spp. which occur mainly in the intestinal tract of humans and animals and are transmitted through

faecal contamination. The use of such contaminated materials in agriculture may cause outbreaks of infection

due to the production of contaminated food and animal foodstocks. They may also be transmitted to wild

animals. There is a need to monitor the efficacy of the storage and treatment processes to control pathogens

such as Salmonella spp., and application rates to land.

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 pasteurization

or disinfection treatments but it is comparatively sensitive (to heat, high pH) and cannot therefore reflect the

behaviour of all pathogens in these materials.

Suitable quality control procedures, at least those described in ISO 8199, have to be applied.

WARNING — "Waste and sludge samples can contain hazardous and inflammable substances. They

can contain pathogens and be liable to biological action. Consequently, it is recommended that these

samples should 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".
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1 Scope

This part of the CEN Technical Report specifies a membrane filtration procedure for the quantitative detection,

by culture of individual colonies on chromogenic agar media, of Escherichia coli. in sludges, soils, soil

improvers, growing media and biowastes. This part of the Technical Report 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. A liquid enrichment and most probable number estimation method may be

suited for such purpose.

This membrane filtration method is not appropriate for enumeration and detection of other coliform bacteria

without modifications to the chromogenic agar media.

It is suitable to evaluate the log reduction of E.coli through treatment, as well as the quality of the end product.

This method is for materials with dry residues less than 20 %. For materials with dry residues greater

than 20 % and low numbers of E. coli, CEN/TR 15214-2 and CEN/TR 15214-3 should be used.

2 Normative references

The following referenced documents are indispensable for the application of this document. For dated

references, only the edition cited applies. For undated references, the latest edition of the referenced

document (including any amendments) applies.

EN 12880:2000, Characterisation of Sludges — Determination of dry residue and water content

ISO 8199, Water quality — General guidance on the enumeration of micro-organisms by culture

3 Terms and definitions

For the purposes of this Technical Report, the following terms and definitions apply.

3.1
Escherichia coli

Escherichia coli, belongs to the family of Enterobacteriaceae, is Gram-negative, non-sporulating, rod-shaped,

lactose positive and able to grow at 44 °C. Most E. coli strains are able to produce indole from tryptophan and

are β-glucuronidase-positive
3.2
method definition

β-glucuronidase-positive and able to hydrolyse 5-bromo-4-chloro-3-indolyl-β-glucuronide (BCIG) when

growing on an MLG agar medium at the temperature of 44 °C
3.3
cfu, colony forming unit

growth of individual bacterial cells into visible colonies on agar media, including on membrane filters

overlaying the agar media
3.4
vegetative bacteria

those bacteria which are capable of normal growth in broth or on agar media without pre-culture resuscitation

3.5
sub-lethally damaged bacteria

those bacteria which have been stressed but not killed by storage or subsequent treatment by, for example,

mesophilic anaerobic digestion, lime stabilisation or composting
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3.6
resuscitation

stimulation to vegetative growth of sub-lethally damaged bacteria previously incapable of growth on agar

media
3.7
quantitative resuscitation

stimulation to vegetative growth of sub-lethally damaged bacteria recovered discretely on a membrane filter,

prior to transfer to chromogenic medium for growth of individual colonies
3.8
dry residue

the dry mass portion of the material obtained after the specified drying process. It is expressed as percent or

in grams per kilogram [EN 12880:2000, 3.1]
4 Principle

The diluted sludge sample is filtered, the membrane filter recovered aseptically and incubated on membrane

lactose glucuronide agar (MLGA), initially at (30 ± 1) °C for (4 ± 0,5) h. Subsequently, the temperature is

increased to (44 ± 1) °C for (14 ± 2) hours. The presence of E. coli is indicated by green colonies resulting

from the hydrolysis of BCIG. The method has a limit of detection of approximately 10 cfu g wet weight sludge,

dependent on the solids content which at high concentrations (> 20 % (w/v)) may restrict filtration of the

sample volume through the membrane if not first diluted.
5 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:
5.1 Apparatus for sterilisation by dry heat (oven) or steam (autoclave).
5.2 Thermostatic incubator(s) regulated at (30 ± 1) °C and/or (44 ± 1) °C.
5.3 Homogeniser (e.g. Stomacher®, Seward
...