CEN/TR 15214-2:2006

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 2: Miniaturised method (Most
Probable Number) by inoculation in liquid medium
Charakterisierung von Schlämmen - Quantitativer Nachweis von Escherichia coli in
Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten sowie Bioabfällen -
Teil 2: Miniaturisiertes Verfahren durch Animpfen 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 2 : Méthode miniaturisée (nombre le plus probable) par ensemencement en milieu
liquide
Ta slovenski standard je istoveten z: CEN/TR 15214-2:2006
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 15214-2
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 2: Miniaturised method (Most Probable
Number) by inoculation 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 2 : Düngemitteln und Bodenverbesserern, Kultursubstraten
Méthode miniaturisée (nombre le plus probable) par sowie Bioabfällen - Teil 2: Miniaturisiertes Verfahren durch
ensemencement en milieu liquide Animpfen in 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 15214-2:2006: 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.6
5 Apparatus .6
6 Sampling and hazards .7
6.1 Introduction.7
6.2 General .7
6.3 Storage .7
6.4 Handling.7

7 Reagents, diluents and culture media.7
7.1 Tryptone salt diluent.8
7.2 Special diluent (SD) .8
7.3 Demineralized or distilled water .8
7.4 Culture medium: MUG/EC medium.8
8 Procedure .9
8.1 Sample preparation.9
8.2 Analysis .9
9 Expression of results.10
9.1 Determination of the characteristic number .10
9.2 Calculation of the MPN and its confidence interval .11
10 Test report .12
11 Performance data.12
Annex A (informative) MPN Statistical Table.13
6 dilutions (1/20 to 1/2000000) / 16 wells seeded per dilution .13
Annex B (informative) Synthetic sea salt.22
Annex C (informative) Performance data of the method .24
Annex D (normative) Quality criteria for the manufacturing of the medium in microtitre plates
(E.coli).27
Bibliography.28

Foreword
This Technical Report (CEN/TR 15214-2:2006) 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 standard.
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 pathogen-contaminated materials in agriculture may cause outbreaks
of infection due to the production of contaminated food and animal feedstocks and may also be transmitted to
wild animals, consequently, there is a need to monitor rates to land.
Escherichia coli is a non-pathogenic, Gram negative bacterium with an exclusive faecal origin. Consequently,
it can be used as an indicator of faecal contamination. It can also be used to monitor the effectiveness of
disinfection treatments but it is comparatively sensitive (to heat, high pH) and cannot therefore reflect the
behaviour of all pathogens in sewage sludge. Suitable quality control procedures, at least those described in
ISO 8199, have to be applied.
WARNING — "Waste and sludge samples may contain hazardous and inflammable substances. They
may contain pathogens and be liable to biological action. Consequently it is recommended that these
samples should be handled with special care. The gases which may 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 describes a miniaturized most probable number (MPN) method for the
quantitative detection of Escherichia coli in sludges, soils, soil improvers, growing media and biowastes. 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 convenient for material with dry residue of more than 10 %. For materials with dry residue less
than 20 %, the procedure specified in CEN/TR 15214-1 will 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
EN ISO 9308-3, Water quality - Detection and enumeration of Escherichia coli and coliform bacteria in surface
and waste water - Part 3: Miniaturized method (Most Probable Number) by inoculation in liquid medium (ISO
9308-3:1998)
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 belongs to the family of Enterobacteriaceae, is 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
β-glucuronidase-positive micro-organism able to hydrolyse 4-.methylumbelliferyl-β-D-glucuronide (MUG) when
growing at an incubation temperature of 44 °C in the specified liquid medium
3.3
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
This method is based on EN ISO 9308-3: “Water quality - Detection and enumeration of Escherichia coli and
coliform bacteria in surface and waste water – Part 3: Miniaturized method (Most Probable Number) by
inoculation in liquid medium”. The following text describes sample preparation in order to prepare a liquid
suspension, after which the analysis is performed following EN ISO 9308-3, reaching Most Probable Number
results in 100 ml. The number of E. coli is then calculated to express the number of E. coli per gram dry
residue of sludge. In order to perform the analysis of E. coli in sludges, soils, soil improvers, growing media
and biowastes, this document describes the whole procedure corresponding to the one of EN ISO 9308-3.
The detection and enumeration of E. coli from biological wastes and soils requires the following stages:
a) sample preparation: suspension of sludge in tryptone salt diluent;
b) inoculation of the diluted sample in a row of microtitre plate wells containing dehydrated culture medium;
c) examination of the microtitre plates under ultraviolet light at 366 nm in the dark after an incubation period
of 36 h minimum and 72 h maximum at (44 ± 1) °C. The presence of E. coli is indicated by a blue
fluorescence resulting from hydrolysis of MUG;
d) results are given as most probable number per 10 g sludge dry residue.
5 Apparatus
With the exception of equipment supplied sterile, the glassware shall be sterilized 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 regulated at (44 ± 1) °C.
5.3 Tunnel drier or vertical laminar airflow cabinet (preferably class II).
R
5.4 Homogeniser (e.g. Stomacher , Seward Laboratories or equivalent).
5.5 Sterile homogeniser bags, 250 ml volume, with or without integrated mesh to exclude large particulate
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matter (e.g. Stomacher , Seward Laboratories 6041, 6041/STR or equivalent).
5.6 Ultraviolet observation chamber (Wood’s Lamp , 366 nm).
WARNING — UV light causes irritation of eye and skin. Use protective glasses and gloves.
5.7 Portable refractometer (optional).
5.8 pH meter with an accuracy of ± 0.1.
5.9 Sterile test tubes of 25 ml volume, or flasks with similar capacity.
5.10 Sterile flasks, of nominal capacities e.g. 250 ml.
5.11 Sterile graduated pipettes, glass or disposable plastic ware, capable of dispersing 2 ml and 18 ml.
5.12 Adjustable or pre-set 8-channel multi-pipette or any other suitable system used for measuring
and distributing 200 µl per well.
5.13 Sterile tips for multi-pipette.
5.14 Equipm
...