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 chromogene 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 a un traitement (tels que l’ajout de chaux, le séchage ou la pasteurisation) réduisant de façon significative les niveaux bactériens a un niveau inférieur a 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 chromogene 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 matiere seche inférieure a 20 %. Il convient d’utiliser le CEN/TR 15214-2 et le CEN/TR 15214-3 lorsque les matériaux ont une teneur en matiere seche supérieure a 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
30-Jun-2006
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
01-Jul-2006
Due Date
01-Jul-2006
Completion Date
01-Jul-2006

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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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SIST-TP CEN/TR 15214-1:2006
CEN/TR 15214-1:2006 (E)
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

2

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SIST-TP CEN/TR 15214-1:2006
CEN/TR 15214-1:2006 (E)
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.
3

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SIST-TP CEN/TR 15214-1:2006
CEN/TR 15214-1:2006 (E)
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".
4

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SIST-TP CEN/TR 15214-1:2006
CEN/TR 15214-1:2006 (E)
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
5

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SIST-TP CEN/TR 15214-1:2006
CEN/TR 15214-1:2006 (E)
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
-1
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
...

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