SIST-TP CEN/TR 15215-1:2006

Characterization of sludges - Detection and enumeration of Salmonella spp. in sludges, soils, soil improvers, growing media and biowastes - Part 1: Membrane filtration method for quantitative resuscitation of sub-lethally stressed bacteria (to confirm efficacy of log drop treatment procedures)

This part of the CEN Technical Report specifies a membrane filtration procedure for the quantitative resuscitation and enumeration, by culture of individual colonies on chromogenic agar media, of Salmonella spp. including potentially sub-lethally damaged Salmonella spp. in sewage sludges. It may be suitable for other sludges, soils, soil improvers, growing media and biowastes but the user shall validate the method using these materials.  The fully defined scope will be determined after the proposed validation trials have been agreed and carried out.
NOTE 1   The objective is to cover untreated and treated sludges, soils, soil improvers, growing media and biowastes.
The method is particularly suited to determining the efficiency of treatment procedures for the elimination of pathogens in sewage sludge as outlined in the Revision of Directive 86/278/EEC (3rd Draft, CEN/TC 308 – doc 525). Treatment type A processes are initially to be validated through a to be defined Log10 reduction with a test organism such as Salmonella senftenberg W775.
The method has a limit of detection of approximately 1 cfu/g wet weight sludge, dependent on the solids content which at high concentrations (> 20 % (w/v)) can restrict filtration of the sample volume through the membrane if not first diluted.
NOTE 2   Salmonella spp. can be present in biosolids including untreated and treated sewage sludge as both vegetative and sub-lethally damaged cells; the latter require resuscitation to enable colony growth for accurate enumeration on agar media.
NOTE 3   This method is not suitable for treated sludges containing less than 1 viable Salmonella spp. per 1 g wet weight.
NOTE 4   This method is not suitable for untreated sludges containing low levels of Salmonella.

Charakterisierung von Schlämmen - Quantitativer Nachweis von Salmonella spp. in Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten sowie Bioabfällen - Teil 1: Membranfiltrationsverfahren zur quantitativen Miterfassung vorgeschädigter Bakterien (zur Bestätigung der logarithmischen Verminderung durch ein Behandlungsverfahren)

Dieses Dokument legt ein Membranfiltrationsverfahren für die Quantifizierung von Salmonella ssp., einschließlich möglicherweise subletal geschädigter Salmonella ssp., in Klärschlamm durch Kultivieren von einzelnen Kolonien auf chromogenen Agarmedien fest. Das Verfahren kann auch für andere Schlämme, Böden, Bodenverbesserungsmittel, Kultursubstrate und Bioabfall geeignet sein, der Anwender muss jedoch das Verfahren unter Verwendung dieser Materialien validieren. Der vollständig definierte Anwendungsbereich wird bestimmt, nachdem die vorgeschlagenen Validierungsuntersuchungen vereinbart und durchgeführt sind.
ANMERKUNG 1   Der vorgesehene Anwendungsbereich umfasst die Untersuchung von unbehandelten und behandelten Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten und Bioabfall umfasst.
Das Verfahren ist besonders geeignet zur Bestimmung der Wirksamkeit von Behandlungsverfahren zur Beseitigung von Krankheitserregern im Klärschlamm, die in der Überarbeitung der Richtlinie 86/278/EWG (3. Entwurf, CEN/TC 308 – doc 525), Verfahren zur Behandlung vom Typ A, kurz beschrieben sind und die zunächst durch eine festzulegende log10-Reduzierung mit Prüfkeimen, wie z. B. Salmonella Senft
Das Verfahren hat eine Nachweisgrenze von etwa 1 KBE/g Feuchtschlammmasse, in Abhängigkeit vom Feststoffanteil, der bei hohen Konzentrationen (> 20 % (Massenvolumenkonzentration)) und ohne vorherige Verdünnung die Filtration des Probenvolumens durch die Membran behindern kann.
ANMERKUNG 2   Salmonella spp. kann in Biofeststoffen, einschließlich unbehandelten und behandelten Klärschlamms, sowohl in Form von vegetativen als auch von subletal geschädigten Zellen vorliegen; letzteres erfordert eine Wiederbelebung, um zur genauen Zählung auf Agarmedien ein Koloniewachstum zu ermöglichen.
ANMERKUNG 3   Dieses Verfahren ist nicht für behandelte Schlämme geeignet, die weniger als 1 lebensfähige Salmonella spp. je Gramm Feuchtmasse enthalten.

Caractérisation des boues - Détection et dénombrement de Salmonella spp. dans les boues, les sols, les amendements du sol, les supports de culture et les biodéchets - Partie 1 : Méthode par filtration sur membrane permettant la ressuscitation quantitative des bactéries stressées de maniere sub-léthale (pour confirmer l'efficacité de l'abattement de logs lors des procédés de traitement)

La présente partie du Rapport Technique CEN définit une méthode par filtration sur membrane permettant la revivification quantitative et le dénombrement, au moyen de la culture de colonies individuelles sur milieu chromogene gélosé, de Salmonella spp., y compris des salmonelles potentiellement endommagées, mais de façon non létale, dans des boues d’épuration. Cette méthode peut s’avérer convenir a d’autres boues, sols, amendements du sol, supports de culture et biodéchets mais l’utilisateur doit valider la méthode au moyen de ce matériel. Le domaine d’application pleinement défini ne sera établi qu’une fois les essais de validation proposés pris en compte et réalisés.
NOTE 1   L’objectif est de couvrir le domaine des boues, sols, amendements du sol, supports de culture et biodéchets bruts et traités.
Cette méthode permet surtout de déterminer l’efficacité des procédés de traitement pour l’élimination des pathogenes dans les boues d’épuration comme souligné dans la Révision de la Directive 86/278/CEE (3eme version, CEN/TC 308 – doc. 525). Les procédés de traitement de type A doivent etre initialement validés au moyen d’un abattement de log10 a définir, avec un organisme d’essai de type Salmonella senftenberg W775.
La méthode a une limite de détection d’environ 1 ufc/g de masse de boue humide, dépendant de la teneur en solides qui peut, a forte concentration (> 20 % (m/v)), limiter la filtration d’un volume d’échantillon a travers la membrane si celui-ci n’est pas préalablement dilué.
NOTE 2   Salmonella spp. peut etre présente dans les biosolides comprenant des boues d’épuration brutes et traitées, que ce soit sous la forme de cellules endommagées végétatives et endommagées de façon non létale, ces dernieres nécessitant une revivification pour permettre le développement de la colonie afin d’obtenir un dénombrement précis sur milieu gélosé.
NOTE 3   Cette méthode n’est pas adaptée aux boues traitées contenant moins d’une cellule viable de Salmonella spp. par gramme de

Karakterizacija blata – Ugotavljanje prisotnosti in števila Salmonela sp. v blatu, zemljini, izboljševalcih tal, rastnih substratih in bio-odpadkih - 1. del: Metoda membranske filtracije za kvantitativno obnovo (ponovno oživljanje) bakterij, ki so pod subletalnim stresom (za potrjevanje učinkovitosti postopka logaritemskega dodajanja po kapljicah)

General Information

• Status: Published
• Publication Date: 30-Jun-2006
• ICS: 13.030.20 - Liquid wastes. Sludge
• ICS: 13.080.30 - Biological properties of soils
• ICS: 65.080 - Fertilizers
• Technical Committee: KAT - Soil quality
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 01-Jul-2006
• Due Date: 01-Jul-2006
• Completion Date: 01-Jul-2006
• Ref Project: CEN/TR 15215-1:2006 - Characterization of sludges - Detection and enumeration of Salmonella spp. in sludges, soils, soil improvers, growing media and biowastes - Part 1: Membrane filtration method for quantitative resuscitation of sub-lethally stressed bacteria (to confirm efficacy of log drop treatment procedures)

Standards Content (Sample)

SLOVENSKI STANDARD
SIST-TP CEN/TR 15215-1:2006
01-julij-2006
.DUDNWHUL]DFLMDEODWD±8JRWDYOMDQMHSULVRWQRVWLLQãWHYLOD6DOPRQHODVSYEODWX
]HPOMLQLL]EROMãHYDOFLKWDOUDVWQLKVXEVWUDWLKLQELRRGSDGNLKGHO0HWRGD
PHPEUDQVNHILOWUDFLMH]DNYDQWLWDWLYQRREQRYRSRQRYQRRåLYOMDQMHEDNWHULMNLVR
SRGVXEOHWDOQLPVWUHVRP]DSRWUMHYDQMHXþLQNRYLWRVWLSRVWRSNDORJDULWHPVNHJD
GRGDMDQMDSRNDSOMLFDK
Characterization of sludges - Detection and enumeration of Salmonella spp. in sludges,
soils, soil improvers, growing media and biowastes - Part 1: Membrane filtration method
for quantitative resuscitation of sub-lethally stressed bacteria (to confirm efficacy of log
drop treatment procedures)
Charakterisierung von Schlämmen - Quantitativer Nachweis von Salmonella spp. in
Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten sowie Bioabfällen -
Teil 1: Membranfiltrationsverfahren zur quantitativen Miterfassung vorgeschädigter
Bakterien (zur Bestätigung der logarithmischen Verminderung durch ein
Behandlungsverfahren)
Caractérisation des boues - Détection et dénombrement de Salmonella spp. dans les
boues, les sols, les amendements du sol, les supports de culture et les biodéchets -
Partie 1 : Méthode par filtration sur membrane permettant la ressuscitation quantitative
des bactéries stressées de maniere sub-léthale (pour confirmer l'efficacité de
l'abattement de logs lors des procédés de traitement)
Ta slovenski standard je istoveten z: CEN/TR 15215-1:2006
ICS:
13.030.20
13.080.30
65.080
SIST-TP CEN/TR 15215-1:2006 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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TECHNICAL REPORT
CEN/TR 15215-1
RAPPORT TECHNIQUE
TECHNISCHER BERICHT
January 2006
ICS 07.100.99

English Version
Characterization of sludges - Detection and enumeration of
Salmonella spp. in sludges, soils, soil improvers, growing media
and biowastes - Part 1: Membrane filtration method for
quantitative resuscitation of sub-lethally stressed bacteria (to
confirm efficacy of log drop treatment procedures)
Caractérisation des boues - Détection et dénombrement de Quantitativer Nachweis von Salmonella spp. in
Salmonella spp. dans les boues, les sols, les engrais, les Schlämmen, Böden, Düngemitteln und Bodenverbesserern,
amendements organiques et les biodéchets - Partie 1 : Kultursubstraten sowie Bioabfällen - Teil 1:
Méthode par filtration sur membrane permettant la Membranfiltrationsverfahren zur quantitativen Miterfassung
ressuscitation quantitative des bactéries stressées de vorgeschädigter Bakterien (zur Bestätigung des
manière sub-léthale (pour confirmer l'efficacité de logarithmisch-tropfenweisen Behandlungsverfahrens)
l'abattement de logs lors des procédés de traitement)
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 15215-1:2006: E
worldwide for CEN national Members.

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CEN/TR 15215-1:2006 (E)
Contents Page
Foreword .3
Introduction.4
1 Scope .5
2 Normative references .5
3 Terms and definitions.5
4 Principle.6
5 Apparatus .7
6 Sampling and hazards .8
7 Reagents, diluents and culture media.8
8 Procedure .10
9 Expression of results.12
10 Test report .12
11 Performance data.12

Annex A (informative) Performance data of the interlaboratory comparison.13
Bibliography.15

2

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CEN/TR 15215-1:2006 (E)
Foreword
This Technical Report (CEN/TR 15215-1:2006) has been prepared by Technical Committee CEN/TC 308
“Characterization of sludges”, the secretariat of which is held by AFNOR.
This document does not replace any existing CEN method.
This standard is divided into three parts:
 part 1 gives a membrane filtration method
 part 2 is a liquid enrichment method and determination by MPN and
 part 3 is a presence / absence method by liquid enrichment.
3

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CEN/TR 15215-1:2006 (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 pathogen-contaminated materials in agriculture can cause outbreaks of
infection due to the production of contaminated food or animal feedstocks and may also be transmitted to wild
animals, consequently, there is a need to monitor rates to land. See CEN/TR 15215-2.
Examination for Salmonellae should only be carried out in laboratories competent for carrying out work
involving pathogens. 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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CEN/TR 15215-1:2006 (E)
1 Scope
This part of the CEN Technical Report specifies a membrane filtration procedure for the quantitative
resuscitation and enumeration, by culture of individual colonies on chromogenic agar media, of Salmonella
spp. including potentially sub-lethally damaged Salmonella spp. in sewage sludges. It may be suitable for
other sludges, soils, soil improvers, growing media and biowastes but the user shall validate the method using
these materials. The fully defined scope will be determined after the proposed validation trials have been
agreed and carried out.
NOTE 1 The objective is to cover untreated and treated sludges, soils, soil improvers, growing media and biowastes.
The method is particularly suited to determining the efficiency of treatment procedures for the elimination of
rd
pathogens in sewage sludge as outlined in the Revision of Directive 86/278/EEC (3 Draft, CEN/TC 308 –
doc 525). Treatment type A processes are initially to be validated through a to be defined Log reduction with
10
a test organism such as Salmonella senftenberg W775.
The method has a limit of detection of approximately 1 cfu/g wet weight sludge, dependent on the solids
content which at high concentrations (> 20 % (w/v)) can restrict filtration of the sample volume through the
membrane if not first diluted.
NOTE 2 Salmonella spp. can be present in biosolids including untreated and treated sewage sludge as both vegetative
and sub-lethally damaged cells; the latter require resuscitation to enable colony growth for accurate enumeration on agar
media.
NOTE 3 This method is not suitable for treated sludges containing less than 1 viable Salmonella spp. per 1 g wet
weight.
NOTE 4 This method is not suitable for untreated sludges containing low levels of Salmonella.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applied. 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
Salmonella spp.
member of the family of Enterobacteriaceae, these are Gram-negative, non-sporulating, rod-shaped bacteria,
most of which are motile. They can be distinguished from other genera of the Enterobacteriaceae family by
biochemical methods and serologically identified by their somatic or flagellar antigens (O and H-antigens)
3.2
method definition
Salmonella spp. capable of being resuscitated on Tetrathionate broth at (36 ± 2) °C followed by fermentation
of propylene glycol and acid production on Rambach agar at (36 ± 2) °C. Most serovars are unable to ferment
lactose and are β-galactosidase negative, but capable of fermenting propylene glycol and producing acid on
Rambach agar when incubated at (36 ± 2) °C (See also 8.5)
5

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CEN/TR 15215-1:2006 (E)
NOTE Some Salmonella (e.g. S. typhi and S. paratyphi) will not be detected
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 in treatment processes or storage
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
presumptive positives
isolates which are believed to be Salmonella spp., but not yet confirmed
3.9
dry residue
the dry mass portion of the sludge obtained after the specified drying process. It is expressed as percent or in
grams per kilogram
[EN 12880:2000, 3.1]
4 Principle
The homogenised diluted sludge sample is centrifuged and filtered, the membrane filter recovered aseptically
and incubated at (36 ± 2) °C on a sterile glass fibre disk soaked with resuscitation medium (Tetrathionate
broth). After 24 h the membrane is recovered aseptically and incubated at (36 ± 2) °C on chromogenic
medium (Rambach agar). The membranes are examined after 24 h and 48 h (the latter to detect more
fastidious S. dublin) and positive colonies are quantified. The presence of Salmonella spp. is indicated by
bright red colonies resulting from fermentation of propylene glycol; other coliforms appear blue, green, violet
or colourless due to their inability to ferment propylene glycol while some produce β-galactosidase which
hydrolyses colourless X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) in the medium to produce a
blue chromophore.
6

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CEN/TR 15215-1:2006 (E)
To distinguish Salmonella spp. from occasional Citrobacter spp., spray an aerosolised solution of
4-methylumbelliferyl caprylate (1 mg/ml) in ethanol directly onto the filters on the Rambach agar. The
presence of Salmonella spp. is indicated by fluorescence of the colonies under UV light at 366 nm, resulting
from the production of C esterase activity. The colonies may also be confirmed using biochemical test strips
8
or serological identification of their somatic and flagellar antigens (O- and H- antigens).
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 regulated at (36 ± 2) °C.
5.3 Homogeniser (e.g. Stomacher®, Seward Laboratories or equivalent).
5.4 Centrifuge capable of centrifuging 50 ml at 200 × g to 300 × g for 1 min.
5.5 Disposable filter units (0,45 µm gridded, cellulose nitrate, 47 mm diameter and 0,2 µm cellulose
nitrate, 25 mm diameter).
5.6 Coarse glass fibre filter discs (47 mm diameter) (e.g. Whatman GF/D pore size 2,7 µ or equivalent).
5.7 Vacuum pump (e.g. Neuberger Model N726-3FT-18 or equivalent).
5.8 Vacuum manifold (e.g. Millipore or equivalent) to hold filter units.
5.9 Stereo microscope fitted with × 10 eyepieces; use × 6 magnification).
5.10 Cold light source, fitted with a Schott KG 1,45 × 45 filter (blue) or equivalent. Illuminate membrane
filters with dual fibre-optic light guides.
5.11 UV observation lamp or chamber (366 nm).
WARNING — UV light cause
...