SIST-TP CEN/TR 15214-3:2006

SLOVENSKI STANDARD
01-julij-2006
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Characterization of sludges - Detection and enumeration of Escherichia coli in sludges,
soils, soil improvers, growing media and biowastes - Part 3: Macromethod (Most
Probable Number) in liquid medium
Charakterisierung von Schlämmen - Quantitativer Nachweis von Escherichia coli in
Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten sowie Bioabfällen -
Teil 3: Makroverfahren in Flüssigmedium (MPN-Verfahren)
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 3 : Macrométhode (nombre le plus probable) par ensemencement en milieu
liquide
Ta slovenski standard je istoveten z: CEN/TR 15214-3:2006
ICS:
13.030.20
13.080.30
65.080
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

TECHNICAL REPORT
CEN/TR 15214-3
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 3: Macromethod (Most Probable Number)
in liquid medium
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 3 : Düngemitteln und Bodenverbesserern, Kultursubstraten
Macrométhode (nombre le plus probable) par sowie Bioabfällen - Teil 3: Makroverfahren in
ensemencement en milieu liquide Flüssigmedium (MPN-Verfahren)
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 15214-3:2006: E
worldwide for CEN national Members.

Contents Page
Foreword .3
Introduction.4
1 Scope .5
2 Normative references .5
3 Terms and definitions.5
4 Principle.5
5 Apparatus .6
6 Sampling and hazards .6
7 Reagents, diluents and culture media.7
8 Procedure .8
9 Expression of the results .9
10 Test report .9
11 Performance data.10

Annex A (informative) Performance of method.11
Bibliography.12

Foreword
This Technical Report (CEN/TR 15214-3:2006) 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 macro method
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 feedstocks. It 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 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".
1 Scope
This part of the CEN Technical Report specifies a most probable number (MPN) method for the quantitative
detection of Escherichia coli in sludges, soils, soil improvers, growing media and biowastes. It allows further
differentiation within the test than part 2 of this standard. It is suitable to evaluate the log reduction of E.coli
through treatment as well as the quality of the end product.
The method is for material with dry residues of more than 10 %.
For materials with dry residues less than 10 %, the procedure given in CEN/TR 15214-1 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 guide to 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, which belongs to the family of Enterobacteriaceae, are Gram-negative, non-sporulating, rod-
shaped, lactose positive bacteria able to grow at 44 °C. Most E.coli strains are able to produce indole from
tryptophane and are β-glucuronidase-positive
3.2
method definition
for the purpose of the present method, the following E. coli definition shall apply: - β-glucuronidase-positive
and able to hydrolyse 4-methylumbelliferyl-B-D-glucoronide (MUG) when at an incubation temperature of
44 °C in the specified liquid medium. In addition, indole shall be produced from tryptophan and gas produced
from lactose
3.3
dry residue
the dry mass portion of the sample obtained after the specified drying process. It is expressed as percent or in
grams per kilogram
[EN 12880:2000, 3.1].
4 Principle
This method is based on that described to Schindler 1991 (Fluorcult™ or equivalent).
a) Suspension of the sample in 0.9 % m/V sodium chloride;
b) Serial dilutions of this suspension in the same diluent;
c) Transfer 1ml of these diluted suspensions into 3 tubes containing 9 ml MUG fluorocult lauryl sulfate broth;
d) Incubation at (44 ± 1) °C for (40 ± 4) h;
e) Detection of gas production, indole formation and fluorescence;
f) Quantification by the MPN technique.
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 Wide-mouth glass flasks or beakers for exampl
...