CEN/TR 15214-1:2006

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

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.

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

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.
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".
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
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 Laboratories or equivalent).
5.4 Membrane filters (0,45 µm gridded, cellulose nitrate, 47 mm diameter or equivalent).
5.5 Glass fibre filter discs (47 mm diameter, e.g. Sartorius 13430-0475 or equivalent nominal pore size).
5.6 Vacuum pump.
5.7 Vacuum manifold.
5.8 Sterile homogeniser bags, 250 ml volume, with or without integrated mesh to exclude large particulate
matter (e.g. Stomacher®, Seward Laboratories 6041, 6041/STR or equivalent).
5.9 Sterile Petri dishes, 50 mm in diameter, for holding MLGA medium.
5.10 Sterile test tubes of 20 ml volume, or flasks with similar capacity.
5.11 Automatic pipettes, capable of dispensing 1,0-5,0 ml volumes.
5.12 Sterile pipettes, glass or disposable plastic ware, capable of dispensing 1 ml and 10 ml volumes.
5.13 Sterile tips for automatic pipettes.
5.14 Sterile conical centrifuge tubes, 50 ml volume, disposable plastic.
5.15 Tweezers, capable of sterilisation by immersion in ethanol and subsequent flaming.
6 Sampling and hazards
6.1 Introduction
Take samples of at least 100 g wet weight and deliver them to the laboratory as quickly as possible, preferably
chilled at (5 ± 3) °C.
6.2 General
Samples are liable to ferment, particularly if untreated, and may contain pathogenic micro-organisms. It is
essential to keep them away from any food or drink, and to protect any cuts. Bursting glass bottles containing
sludge can produce micro-organism contaminated shrapnel. Plastic bottles can also burst and produce a
hazardous spray and aerosol.
See also the Warning note in the introduction.
6.3 Storage
It is not advisable to store samples in the open laboratory. If samples are to be stored, store them at (5 ± 3) °C.
6.4 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.
See also the Warning note in the introduction.
7 Reagents, diluents and culture media
7.1 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. If the media are not used immediately, preserve them in
the dark at (5±3) °C for up to one month in conditions avoiding any alterations in their composition.
NOTE The
...