SIST-TP CEN/TR 15215-2:2006

Characterization of sludges - Detection and enumeration of Salmonella spp. in sludges, soils, soil improvers, growing media and biowastes - Part 2: Liquid enrichment method in selenite-cystine medium followed by Rapport-Vassiliadis for semi-quantitative Most Probable Number (MPN) determination

This part of the CEN Technical Report method describes a method to detect and semi-quantitatively determine Salmonellae in sludges, soils, soil improvers, growing media and biowastes in accordance with the requirements of the European Sewage Sludge Regulation Revision of Directive 86/278/EEC (3rd Draft, CEN/TC 308 - doc525).
The fully defined scope will be determined after the proposed validation trials have been agreed and carried out. The method has a limit of detection of approximately 1cfu/g wet weight sample.
NOTE   The objective is to cover untreated and treated sludges, soils, soil improvers, growing media, biowastes and associated materials.

Charakterisierung von Schlämmen - Quantitativer Nachweis von Salmonella spp. in Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten sowie Bioabfällen - Teil 2: Flüssiganreicherungsverfahren in Selenit-Cystein-Medium in Kombination mit Rappaport-Vassiliadis zur semiquantitativen Bestimmung der höchstwahrscheinlichen Keimzahl (MPN)

Dieses Dokument legt in Übereinstimmung mit den Anforderungen der europäischen Bestimmung über Klärschlamm, Überarbeitung der Richtlinie 86/278/EWG (3. Entwurf, CEN/TC 308 – doc 525) ein Verfahren zum Nachweis und zur halbquantitativen Bestimmung von Salmonellen in Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten und Bioabfällen fest.
Der vollständig definierte Anwendungsbereich wird bestimmt, nachdem die vorgeschlagenen Validierungsuntersuchungen vereinbart und durchgeführt sind. Das Verfahren hat eine Nachweisgrenze von etwa 1 KBE/g Feuchtprobenmasse.
ANMERKUNG   Der vorgesehene Anwendungsbereich umfasst die Untersuchung von unbehandelten und behandelten Schlämmen, Böden, Bodenverbesserern, Kultursubstraten und Bioabfall umfasst.

Caractérisation des boues - Détection et dénombrement de Salmonella spp. dans les boues, les sols, les amendements du sol, les supports de culture et biodéchets - Partie 2 : Méthode par enrichissement en milieu liquide sélénite-cystine puis en milieu de Rapport-Vassiliadis pour la détermination semi-quantitative par la méthode du Nombre le Plus Probable (NPP)

La présente partie du Rapport Technique CEN décrit une méthode permettant la détection des Salmonella et leur détermination semi-quantitative dans les boues, sols, amendements du sol, supports de culture et biodéchets, conformément aux exigences contenues dans la révision de la Directive 86/278/CEE (3eme version, CEN/TC 308 – doc. 525), portant sur la Réglementation européenne relative aux boues d’épuration.
Le domaine d’application exact ne sera établi qu’une fois les essais de validation proposés pris en compte et réalisés. La méthode a une limite de détection d’environ 1 ufc/g de masse d’échantillon de boue humide.
NOTE   L’objectif est de couvrir le domaine des boues traitées et non traitées, sols, amendements du sol, supports de culture, biodéchets et produits assimilés.

Karakterizacija blata – Ugotavljanje prisotnosti in števila Salmonela sp. v blatu, zemljini, izboljševalcih tal, rastnih substratih in bio-odpadkih - 2. del: Metoda s tekočo obogatitvijo v selenit-cisteinskem mediju/gojišču, ki mu sledi metoda po Vassiliadisu za semikvantitativno določevanje najverjetnejšega števila (MPN)

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 15215-2:2006 - Characterization of sludges - Detection and enumeration of Salmonella spp. in sludges, soils, soil improvers, growing media and biowastes - Part 2: Liquid enrichment method in selenite-cystine medium followed by Rapport-Vassiliadis for semi-quantitative Most Probable Number (MPN) determination

Standards Content (Sample)

SLOVENSKI STANDARD
SIST-TP CEN/TR 15215-2:2006
01-julij-2006
.DUDNWHUL]DFLMDEODWD±8JRWDYOMDQMHSULVRWQRVWLLQãWHYLOD6DOPRQHODVSYEODWX
]HPOMLQLL]EROMãHYDOFLKWDOUDVWQLKVXEVWUDWLKLQELRRGSDGNLKGHO0HWRGDV
WHNRþRRERJDWLWYLMRYVHOHQLWFLVWHLQVNHPPHGLMXJRMLãþXNLPXVOHGLPHWRGDSR
9DVVLOLDGLVX]DVHPLNYDQWLWDWLYQRGRORþHYDQMHQDMYHUMHWQHMãHJDãWHYLOD031
Characterization of sludges - Detection and enumeration of Salmonella spp. in sludges,
soils, soil improvers, growing media and biowastes - Part 2: Liquid enrichment method in
selenite-cystine medium followed by Rapport-Vassiliadis for semi-quantitative Most
Probable Number (MPN) determination
Charakterisierung von Schlämmen - Quantitativer Nachweis von Salmonella spp. in
Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten sowie Bioabfällen -
Teil 2: Flüssiganreicherungsverfahren in Selenit-Cystein-Medium in Kombination mit
Rappaport-Vassiliadis zur semiquantitativen Bestimmung der höchstwahrscheinlichen
Keimzahl (MPN)
Caractérisation des boues - Détection et dénombrement de Salmonella spp. dans les
boues, les sols, les amendements du sol, les supports de culture et biodéchets - Partie
2 : Méthode par enrichissement en milieu liquide sélénite-cystine puis en milieu de
Rapport-Vassiliadis pour la détermination semi-quantitative par la méthode du Nombre le
Plus Probable (NPP)
Ta slovenski standard je istoveten z: CEN/TR 15215-2:2006
ICS:
13.030.20
13.080.30
65.080
SIST-TP CEN/TR 15215-2:2006 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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TECHNICAL REPORT
CEN/TR 15215-2
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 2: Liquid enrichment method in selenite-
cystine medium followed by Rapport-Vassiliadis for semi-
quantitative Most Probable Number (MPN) determination
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 biodéchets - Partie 2 : Kultursubstraten sowie Bioabfällen - Teil 2:
Méthode par enrichissement en milieu liquide sélénite- Flüssiganreicherungsverfahren in Selenit-Cystein-Medium
cystine puis en milieu de Rapport-Vassiliadis pour la gefolgt durch Rapport-Vassiliadis zur semiquantitativen
détermination semi-quantitative par la méthode du Nombre Bestimmung der höchstwahrscheinlichen Keimzahl (MPN)
le Plus Probable (NPP)
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 15215-2:2006: E
worldwide for CEN national Members.

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CEN/TR 15215-2:2006 (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.8
8 Procedure .12
9 Expression of results.14
10 Test Report.14
11 Performance data.15
Annex A (normative) Most Probable Number in 100 ml of non-diluted suspension (8.1.2) = 10 g
dry residue (combination 10 ml – 1 ml – 0,1 ml) .16
Annex B (informative) Method performance Characterisation of Salmonella spp. enumeration
method.17
Bibliography.20

2

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CEN/TR 15215-2:2006 (E)
Foreword
This Technical Report (CEN/TR 15215-2:2006) 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.
This Standard is divided in three parts:
- part 1 gives a membrane filtration method
- part 2 is a liquid enrichment method and determination MPN and
- part 3 is a presence/absence method by liquid enrichment.
3

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CEN/TR 15215-2:2006 (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.
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 15215-2:2006 (E)
1 Scope
This part of the CEN Technical Report method describes a method to detect and semi-quantitatively
determine Salmonellae in sludges, soils, soil improvers, growing media and biowastes in accordance with the
rd
requirements of the European Sewage Sludge Regulation Revision of Directive 86/278/EEC (3 Draft,
CEN/TC 308 – doc525).
The fully defined scope will be determined after the proposed validation trials have been agreed and carried
out. The method has a limit of detection of approximately 1cfu/g wet weight sample.
NOTE The objective is to cover untreated and treated sludges, soils, soil improvers, growing media, biowastes and
associated materials.
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
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 enriched in selenite cystine broth at (36 ± 2) °C followed by growth in
Rappaport-Vassiliadis medium at (42 ± 1) °C followed by characteristic growth on SMID/Rambach agar or
XLD agar at (36 ± 2) °C (see also 4 and 8.5)
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
5

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CEN/TR 15215-2:2006 (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
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 (see EN 12880:2000, 3.1)
4 Principle
The steps involved in this method have been made as close as possible to those involved in the
ISO CD 6340-2:2000. The main differences are the following:
 sample preparation suitable for a solid matrix;
 a selective pre-enrichment step according to the possible high contamination of the sludge with interfering
bacteria.
Three series of three tubes containing serial dilutions of the sludge suspension should be used for the Most
Probable Number enumeration method.
The detection of Salmonella spp. requires four stages:
a) culturing of bacteria in a primary selective medium;
b) enrichment in a secondary selective medium which inhibits the growth of other micro-organisms but
promotes that of Salmonellae (selective enrichment);
c) preparation of pure cultures by inoculating special solid media with subcultures;
d) biochemical and serological identification tests.
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 example, 125 ml, 200 ml, 500 ml and 2 000 ml
5.2 Thermostatic incubator regulated at (36 ± 2) °C and (42 ± 1) °C
5.3 Autoclave (steam sterilizer)
6

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CEN/TR 15215-2:2006 (E)
5.4 Refrigerator
5.5 Sterile plastics culture dishes, with lid of about 90 mm in diameter
5.6 Sterile graduated pipettes, glass or disposable plastic ware, capable of dispersing 0,1 ml, 1 ml and
10 ml
5.7 Inoculating loop (10µl) (e.g. platinum-iridium wire), loop diameter approximately 3 mm
5.8 Apparatus for shaking the culture tubes
5.9 Culture tubes, 25 ml capacity, or equivalent containers
5.10 Vortex mixer suitable for 25 ml culture tubes or equivalent containers
5.11 pH meter, with temperature compensation and pH-measuring cell
5.12 Homogeniser (e.g. Stomacher ®, Seward Laboratories or equivalent)
5.13 Filter membrane, for media sterilisation (0,2 µm cellulose nitrate 47 mm diameter)
5.14 Boiling water bath
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 (within
24 hours). In order to prevent propagation or inactivation of Salmonella during transport to the laboratory and
subsequent storage, the necessary precautions depending upon the matrix shall be taken.
NOTE Generally chilling the sample to (5 ± 3) °C is recommended.
6.2 General
Samples are liable to ferment and can contain pathogenic micro-organisms. It is essential to keep them away
from any food or drink, and to protect any cuts. When transporting and handling samples, it is essential that
national and international regulations relating to biohazardous samples are followed.
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
for a maximum period of 36 hours.
6.4 Handling
Cleanliness when working is essential. When handling sludge samples, it is necessary to wear gloves, a face
and eye protection, and ensure adequate protection against bottles bursting. The gas evolved is flammable.
See also the Warning note in the introduction.
7

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CEN/TR 15215-2:2006 (E)
6.5 Toxic chemicals
Extreme care must be taken when handling sodium selenite and its solutions due to their high toxicity.
7 Reagents, diluents and culture media
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 fit for purpose demineralised or distilled water free from
substances capable of inhibiting growth under the test conditions. (ISO 8199). 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 1 The use of chemicals of other grades is permissible provided that they are shown to be of equivalent
performance in the test.
NOTE 2 Ready to use media may also be used for the examination provided their compositions are equivalent to those
specified in this sub clause.
7.1 Saline solution
Dissolve 0,85 g of sodium chloride in 100 ml of water and adjust the pH value of the solution to (7,0 ± 0,1) with
sodium hydroxide or hydrochloric acid (0,1 mol/l). Pour the solution into suitable glass containers as required
and sterilise in an autoclave (5.3) at (121 ± 3) °C for (15 ± 1)min.
7.2 Bromocresol purple solution
Dissolve 1 g of bromocresol purple C H Br O S in 100 ml of water.
21 16 2 5
7.3 Kovac’s reagent (indole reagent)
4-dimethylaminobenzaldehyde, C HNO 5 g
9 11
Isoamyl alcohol, C HO 75 ml
5 12
Hydrochloric acid (0,1 mol/ l) 25 ml
Dissolve the 4-dimethylaminobenzaldehyde, C H NO, in 75 ml of isoamyl alcohol, C H O, and heat in a
9 11 5 12
water bath at 60 °C for 5 min. Then add 25 ml of hydrochloric acid (0,1 mol/l). The reagent will be ready for
use after about 6 h to 7 h (indicated by a yellow colour).
Commercially available Kovac’s reagent can be used according to the manufacturer’s instructions.
7.4 Magnesium chloride solution
Dissolve 36 g of magnesium chloride hexahydrate. MgCl . 6H O, in 100 ml of water.
2 2
7.5 Malachite green solution
Dissolve 0,72 g of malachite green oxalate, C H CIN . C O , in 100 ml of water.
23 25 2 2 4
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CEN/TR 15215-2:2006 (E)
7.6 Phenol red solution
Dissolve 1g of phenol red in 1,25 ml of sodium hydroxide solution (0,1 mol/l) and make up to 250 ml with
water.
7.7 Lactose/peptone solution
Dissolve 20 g of tryptone and 5 g of sodium chloride in 1 000 ml of water in a 2 000 ml flat bottom flask, while
heating in a boiling water bath. Adjust the pH value of the solution to (7,2 ± 0,1) using sodium hydroxide
solution (0,1 mol/l), the
...