Organic, organo-mineral and inorganic fertilizers - Detection of Salmonella spp.

This document is applicable to fertilizing products, which are classified as PFC 1(A) and PFC 1(B) or the PFC 1(A) and PFC 1(B) component in PFC 7 of Regulation (EU) 2019/1009 [1]. However, the present method was not validated for blends.
This document specifies a method for the detection of Salmonella spp. in organic, organo-mineral and inorganic fertilizers. The method is based on EN ISO 6579-1 and its validated alternative methods for the detection of Salmonella spp. in food and feeding stuff.
It requires three successive steps: A selective enrichment, an isolation on a chromogenic agar, and if positive a confirmation with a serological test (and if required, a selective media).

Organische, organisch-mineralische und mineralische Düngemittel - Nachweis von Salmonella spp

Dieses Dokument ist für Düngeprodukte anzuwenden, die den Kategorien PFC 1(A) und PFC 1(B) zugeordnet oder als Komponente der Kategorien PFC 1(A) und PFC 1(B) in PFC 7 der Verordnung (EU) 2019/1009 [1] eingestuft sind. Das vorliegende Verfahren wurde jedoch nicht für Mischungen validiert.
Dieses Dokument legt ein Verfahren zum Nachweis von Salmonella spp. in organischen, organisch-mineralischen und anorganischen Düngemitteln fest. Das Verfahren basiert auf EN ISO 6579 1 und ihren validierten alternativen Verfahren zum Nachweis von Salmonella spp. in Lebensmitteln und Futtermitteln.
Es erfordert drei aufeinanderfolgende Schritte: Eine selektive Anreicherung, eine Isolierung auf einem chromogenen Agar und bei positivem Befund eine Bestätigung mit einer serologischen Untersuchung (und, falls erforderlich, einem Selektivmedium).

Engrais organiques, organo-minéraux et inorganiques - Recherche des Salmonella spp.

Le présent document s’applique aux fertilisants, classés PFC 1(A) et PFC 1(B) ou aux PFC 1(A) et PFC 1(B) entrant dans la composition de PFC 7 du règlement (UE) 2019/1009 [1]. Toutefois, la présente méthode n’a pas été validée pour les mélanges.
Le présent document spécifie une méthode pour la recherche des Salmonella spp. dans les engrais organiques, organo-minéraux et inorganiques. La méthode est basée sur l’EN ISO 6579-1 et ses méthodes alternatives validées pour la recherche des Salmonella spp. dans les aliments et les aliments pour animaux.
Elle nécessite trois phases successives : un enrichissement sélectif, un isolement sur un milieu gélosé chromogène, et, en cas de résultat positif, une confirmation par un essai sérologique (et, si nécessaire, un milieu sélectif).

Organska, organsko-mineralna in anorganska gnojila - Ugotavljanje prisotnosti salmonele (Salmonella spp.)

Ta dokument se uporablja za sredstva za gnojenje, ki so razvrščena v funkcijske kategorije PFC 1(A) in PFC 1(B) ali PFC 1(A) in PFC 1(B) v PFC 7 v skladu z Uredbo (EU) 2019/1009 [1]. Vendar ta metoda ni bila potrjena za mešanice.
Ta dokument določa metodo za ugotavljanje prisotnosti Salmonella spp. v organskih, organsko-mineralnih in anorganskih gnojilih. Metoda temelji na standardu EN ISO 6579-1 in njegovih potrjenih alternativnih metodah za odkrivanje Salmonella spp. v živilih in krmi.
Metoda poteka v treh zaporednih korakih: selektivna obogatitev, izolacija na kromogenem agarju in, če je rezultat pozitiven, potrditev s serološkim preskusom (in po potrebi s selektivnim gojiščem).   
7

General Information

Status
Published
Public Enquiry End Date
02-Jan-2022
Publication Date
09-Jan-2023
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
06-Jan-2023
Due Date
13-Mar-2023
Completion Date
10-Jan-2023

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SLOVENSKI STANDARD
SIST-TS CEN/TS 17780:2023
01-februar-2023
Organska, organsko-mineralna in anorganska gnojila - Ugotavljanje prisotnosti
salmonele (Salmonella spp.)
Organic, organo-mineral and inorganic fertilizers - Detection of Salmonella spp.
Organische, organisch-mineralische und mineralische Düngemittel - Nachweis von
Salmonella spp
Engrais organiques, organo-minéraux et inorganiques - Recherche des Salmonella spp.
Ta slovenski standard je istoveten z: CEN/TS 17780:2022
ICS:
65.080 Gnojila Fertilizers
SIST-TS CEN/TS 17780:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN/TS 17780:2023

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SIST-TS CEN/TS 17780:2023


CEN/TS 17780
TECHNICAL SPECIFICATION

SPÉCIFICATION TECHNIQUE

April 2022
TECHNISCHE SPEZIFIKATION
ICS 65.080
English Version

Organic, organo-mineral and inorganic fertilizers -
Detection of Salmonella spp.
Engrais organiques, organo-minéraux et inorganiques - Organische, organisch-mineralische und mineralische
Recherche des Salmonella spp. Düngemittel - Nachweis von Salmonella spp.
This Technical Specification (CEN/TS) was approved by CEN on 21 February 2022 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS
available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in
parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N

EUROPÄISCHES KOMITEE FÜR NORMUN G

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17780:2022 E
worldwide for CEN national Members.

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SIST-TS CEN/TS 17780:2023
CEN/TS 17780:2022 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 6
4.1 General. 6
4.2 Enrichment in selective liquid medium . 6
4.3 Plating out on selective solid media. 6
4.4 Confirmation . 6
5 Culture media and reagents . 6
5.1 General. 6
5.2 Isolation chromogenic agar. 6
5.3 Non-selective agar . 6
5.4 Confirmation selective agar . 7
6 Equipment and consumables . 7
7 Sampling . 7
8 Preparation of test sample . 7
9 Procedure (see Figure A.1) . 8
9.1 Test portion and initial suspension . 8
9.2 Selective pre-enrichment . 8
9.3 Isolation . 8
9.4 Confirmation . 8
10 Expression of results . 10
11 Performance assays . 11
12 Test report . 11
Annex A (normative) Diagrams of the procedures . 12
Annex B (normative) Culture media and reagents . 13
Annex C (informative) Examples of selective plating-out media . 17
Bibliography . 19

2

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SIST-TS CEN/TS 17780:2023
CEN/TS 17780:2022 (E)

European foreword
This document (CEN/TS 17780:2022) has been prepared by Technical Committee CEN/TC 260
“Fertilizers and liming materials”, the secretariat of which is held by DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN/CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United
Kingdom.
3

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SIST-TS CEN/TS 17780:2023
CEN/TS 17780:2022 (E)

Introduction
This document describes a method for the detection of Salmonella spp. in fertilizers of the following
Product Function Categories (PFCs) of EU fertilizing products, as described in the Regulation (EU)
2019/1009 [1]:
— PFC 1(A): Organic fertilizer;
— PFC 1(B): Organo-mineral fertilizer;
— PFC 1(C): Inorganic fertilizer, which contains more than 1 % by mass of organic carbon, other than
organic carbon from chelating or complexing agents, nitrification inhibitors, denitrification
inhibitors or urease inhibitors, coating agents, urea or calcium cyanamide. The present method was
validated on products known as present on the market in April 2021 and conform to Regulation (EU)
2019/1009 [1] that are inorganic fertilizers with more than 1 % of organic carbon such as struvite
with low level of organic matter. In case that other products would be developed having other
physical and chemical characteristics, it might become necessary to develop different methods to
correctly account for pathogenic microorganisms they might contain.
4

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SIST-TS CEN/TS 17780:2023
CEN/TS 17780:2022 (E)

1 Scope
This document is applicable to fertilizing products, which are classified as PFC 1(A) and PFC 1(B) or the
PFC 1(A) and PFC 1(B) component in PFC 7 of Regulation (EU) 2019/1009 [1]. However, the present
method was not validated for blends.
This document specifies a method for the detection of Salmonella spp. in organic, organo-mineral and
inorganic fertilizers. The method is based on EN ISO 6579-1 and its validated alternative methods for the
detection of Salmonella spp. in food and feeding stuff.
It requires three successive steps: A selective enrichment, an isolation on a chromogenic agar, and if
positive a confirmation with a serological test (and if required, a selective media).
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at https://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
3.1
Salmonella spp.
microorganism which forms typical colonies on solid selective media described and which displays the
morphological, physiological and biochemical characteristics described when the analysis is carried out
in accordance with this document
3.2
detection of Salmonella
determination of the detection or not detection of Salmonella spp. (3.1), in 25 g or 25 ml of product, when
tests are carried out in accordance with this document
3.3
laboratory sample
sample intended for laboratory inspection or testing
3.4
test sample
sample prepared from the laboratory sample (3.3) and from which test portions (3.5) will be taken
3.5
test portion
quantity of material taken from the test sample (or if both are the same, from the laboratory sample) and
on which the test is carried out
5

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SIST-TS CEN/TS 17780:2023
CEN/TS 17780:2022 (E)

4 Principle
4.1 General
The detection of Salmonella spp. requires three successive steps as specified in normative Annex A. The
three steps are the selective enrichment, the isolation on a chromogenic agar, and the confirmation with
a serological test (and if required, a selective media).
NOTE Salmonella can be present in small numbers and are often accompanied by considerably larger numbers
of other bacteria such as Enterobacteriaceae or of other families. Enrichment is used to allow the detection of low
numbers of Salmonella or stressed Salmonella.
Stressed microorganisms are defined in this document as those present in the environment that might be injured
or that might have developed in harsh environments. Such organisms can be difficult to detect because they struggle
to grow on selective media. However, under suitable conditions, they can repair the cellular damage and recover
their normal properties.
Alternative methods based on molecular biology can be used if giving the same results as those given in this
document (see EN ISO 7218).
4.2 Enrichment in selective liquid medium
Buffered peptone water (BPW) containing 10 mg/l novobiocin at room temperature is inoculated with
the test portion (refer to 9.1), then incubated between 34 °C and 38 °C for 18 h ± 2 h.
For large quantities (e.g. 1 l or more), it is recommended to pre-warm the BPW to 34 °C to 38 °C before
mixing it with the test portion.
4.3 Plating out on selective solid media
From the enrichment obtained in 4.2, the chromogenic solid media (5.2) is inoculated.
This selective agar is incubated between 34 °C and 38 °C for 24 h ± 3 h (or according to the
manufacturer’s instructions if explicitly recommended).
4.4 Confirmation
Colonies of presumptive Salmonella are confirmed by means of appropriate serological test. If the
serological test gives a negative result, the inoculation of a selective agar (Table C.2) is required. If the
test gives a negative result, up to 4 other presumptive colonies will be tested (if possible and up to
5 colonies in total).
5 Culture media and reagents
5.1 General
For current laboratory practice, see EN ISO 7218 and EN ISO 11133.
Composition of culture media and reagents and their preparation are described in Annex B.
5.2 Isolation chromogenic agar
This isolation medium is chosen by the testing laboratory and shall highlight the C8-esterase enzymatic
activity. For examples of isolation media, see Table C.1.
5.3 Non-selective agar
General purpose agar supporting the growth of a wide range of non-fastidious strains is chosen by the
testing laboratory. For example, for non-selective agar, see B.4.
6

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SIST-TS CEN/TS 17780:2023
CEN/TS 17780:2022 (E)

5.4 Confirmation selective agar
The confirmation medium is chosen by the testing laboratory and shall highlight the production of
hydrogen sulphide (H S) by the strains (see B.5). For examples of confirmation media, see Table C.2.
2
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (see EN ISO 7218) and, in particular, the following.
6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave), see EN ISO 7218.
6.2 Drying cabinet or oven, capable of operating between 25 °C and 50 °C.
6.3 Incubator(s), capable of operating in the range 34 °C to 38 °C or at 37 °C ± 1 °C.
6.4 Water bath, capable of operating at 47 °C to 50 °C, containing an antibacterial agent.
6.5 Cooling unit, adjustable at 5 °C ± 3 °C.
6.6 Freezer, capable of operating at -20 °C ± 5 °C.
6.7 Sterile loops, of approximate diameter, 3 mm (10 μl volume).
6.8 pH-meter, with a reading to the nearest 0,1 pH unit from 20 °C to 25 °C.
6.9 Sterile tubes, bottles, or flasks, with caps of appropriate capacity.
6.10 Sterile filter, with a pore size of 0,2 µm.
6.11 Sterile Petri dishes, with a diameter of approximately 90 mm and (optional) large size (diameter
approximately 140 mm).
7 Sampling
Sampling should be performed carefully, following the principles described in EN 1482 (all parts) with
appropriate adaptations, required to account for specificities of organic and organo-mineral fertilizers
and to microbiological quality of the samples.
It is important that the laboratory receives a laboratory sample (3.3) which is representative and has not
been damaged or changed during transport or storage.
8 Preparation of test sample
The sample is prepared in accordance to the European or International Standard that is specific to the
concerned matrix [see EN ISO 6887 (all parts)]. If there is no specific European or International Standard,
it is advised that the different parties agree on this subject.
7

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SIST-TS CEN/TS 17780:2023
CEN/TS 17780:2022 (E)

9 Procedure (see Figure A.1)
9.1 Test portion and initial suspension
For preparation of the initial suspension, use as diluent the enrichment medium specified in Annex B, B.2
(buffered peptone water). Pre-warm the BPW supplemented with Novobiocin (nBPW) to room
temperature before use.
A test portion (3.5) of 25 g or 25 ml is weighed and nBPW (refer to 4.2) is added to yield a tenfold dilution
(mass or volume) (see EN ISO 6887-1).
NOTE 1 If the samples are suspected to exhibit extreme pH values, double buffered peptone water
(supplemented with novobiocin) can be used as diluent for the initial suspension.
NOTE 2 If the initial suspension is used for other microbiological tests, novobiocin can be added subsequently.
9.2 Selective pre-enrichment
Incubate the initial suspension (9.1) between 34 °C and 38 °C (6.3) for 18 h ± 2 h.
It is permissible to store the pre-enrichment culture at 5 °C (6.5) for a maximum of 72 h after the
incubation.
9.3 Isolation
From the selective pre-enrichment (9.2), inoculate the surface of the chromogenic isolation agar (5.2) by
means of a 10 µl loop (6.7) so that well-isolated colonies will be obtained.
Allow the plates to equilibrate at room temperature if they were stored at a lower temperature. If
necessary, dry the surface of the plates before use (see EN ISO 11133).
Incubate the selective plating-out
...

SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17780:2021
01-december-2021
Organska, organsko-mineralna in anorganska gnojila - Ugotavljanje prisotnosti
vrst Salmonella (Salmonella spp.)
Organic, organo-mineral and inorganic fertilizers - Detection of Salmonella spp.
Organische, organisch-mineralische und mineralische Düngemittel - Nachweis von
Salmonella spp
Engrais organiques, organo-minéraux et inorganiques - Recherche des Salmonella spp.
Ta slovenski standard je istoveten z: FprCEN/TS 17780
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17780:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
kSIST-TS FprCEN/TS 17780:2021

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kSIST-TS FprCEN/TS 17780:2021


FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17780
SPÉCIFICATION TECHNIQUE

TECHNISCHE SPEZIFIKATION

October 2021
ICS 65.080
English Version

Organic, organo-mineral and inorganic fertilizers -
Detection of Salmonella spp.
Engrais organiques, organo-minéraux et inorganiques - Organische, organisch-mineralische und mineralische
Recherche des Salmonella spp. Düngemittel - Nachweis von Salmonella spp


This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee
CEN/TC 260.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change
without notice and shall not be referred to as a Technical Specification.


EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17780:2021 E
worldwide for CEN national Members.

---------------------- Page: 3 ----------------------
kSIST-TS FprCEN/TS 17780:2021
FprCEN/TS 17780:2021 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 6
4.1 General. 6
4.2 Enrichment in selective liquid medium . 6
4.3 Plating out on selective solid media. 6
4.4 Confirmation . 6
5 Culture media and reagents . 6
5.1 General. 6
5.2 Isolation chromogenic agar. 6
5.3 Non-selective agar . 7
5.4 Confirmation selective agar . 7
6 Equipment and consumables . 7
7 Sampling . 7
8 Preparation of test sample . 7
9 Procedure (see Figure A.1) . 8
9.1 Test portion and initial suspension . 8
9.2 Selective pre-enrichment . 8
9.3 Isolation . 8
9.4 Confirmation . 8
10 Expression of results . 10
11 Performance assays . 11
12 Test report . 11
Annex A (normative) Diagrams of the procedures . 12
Annex B (normative) Culture media and reagents . 13
Annex C (informative) Examples of selective plating-out media . 17
Bibliography . 19

2

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kSIST-TS FprCEN/TS 17780:2021
FprCEN/TS 17780:2021 (E)

European foreword
This document (FprCEN/TS 17780:2021) has been prepared by Technical Committee CEN/TC 260
“Fertilizers and liming materials”, the secretariat of which is held by DIN.
This document is currently submitted to the Vote on TS.
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association.
3

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kSIST-TS FprCEN/TS 17780:2021
FprCEN/TS 17780:2021 (E)

Introduction
This document describes a method for the detection of Salmonella spp. in fertilizers of the following
Product Function Categories (PFCs) of EU fertilizing products, as described in the Regulation (EU)
2019/1009 [1]:
— PFC 1(A): Organic fertilizer;
— PFC 1(B): Organo-mineral fertilizer;
— PFC 1(C): Inorganic fertilizer, which contains more than 1 % by mass of organic carbon, other than
organic carbon from chelating or complexing agents, nitrification inhibitors, denitrification
inhibitors or urease inhibitors, coating agents, urea or calcium cyanamide. The present method was
validated on products known as present on the market in April 2021 and conform to Regulation (EU)
2019/1009 [1] that are inorganic fertilizers with more than 1 % of organic carbon such as struvite
with low level of organic matter. In case that other products would be developed having other
physical and chemical characteristics, it might become necessary to develop different methods to
correctly account for pathogenic microorganisms they might contain.
4

---------------------- Page: 6 ----------------------
kSIST-TS FprCEN/TS 17780:2021
FprCEN/TS 17780:2021 (E)

1 Scope
This document is applicable to fertilizing products, which are classified as PFC 1(A) and PFC 1(B) or the
PFC 1(A) and PFC 1(B) component in PFC 7 of Regulation (EU) 2019/1009 [1]. However, the present
method was not validated for blends.
This document specifies a method for the detection of Salmonella spp. in organic, organo-mineral and
inorganic fertilizers. The method is based on EN ISO 6579-1 and its validated alternative methods for the
detection of Salmonella spp. in food and feeding stuff.
It requires three successive steps: A selective enrichment, an isolation on a chromogenic agar, and if
positive a confirmation with a serological test (and if required, a selective media).
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at https://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
3.1
Salmonella spp.
microorganism which forms typical colonies on solid selective media described and which displays the
morphological, physiological and biochemical characteristics described when the analysis is carried out
in accordance with this document
3.2
detection of Salmonella
determination of the detection or not detection of Salmonella (3.1), in 25 g or 25 mL of product, when
tests are carried out in accordance with this document
3.3
laboratory sample
sample intended for laboratory inspection or testing
3.4
test sample
sample prepared from the laboratory sample (3.3) and from which test portions (3.5) will be taken
3.5
test portion
quantity of material taken from the test sample (or if both are the same, from the laboratory sample) and
on which the test is carried out
5

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kSIST-TS FprCEN/TS 17780:2021
FprCEN/TS 17780:2021 (E)

4 Principle
4.1 General
The detection of Salmonella spp. requires three successive steps as specified in normative Annex A. The
three steps are the selective enrichment, the isolation on a chromogenic agar, and the confirmation with
a serological test (and if required, a selective media).
NOTE 1 Salmonella can be present in small numbers and are often accompanied by considerably larger numbers
of other bacteria such as Enterobacteriaceae or of other families. Enrichment is used to allow the detection of low
numbers of Salmonella or stressed Salmonella.
Stressed microorganisms are defined in this document as those present in the environment that may be injured or
that may have developed in harsh environments. Such organisms may be difficult to detect because they struggle to
grow on selective media. However, under suitable conditions, they may repair the cellular damages and recover
their normal properties.
Alternative methods based on molecular biology can be used if giving the same results as those given in this
document (see EN ISO 7218).
4.2 Enrichment in selective liquid medium
Buffered peptone water (BPW) containing 10 mg/l novobiocin at room temperature is inoculated with
the test portion (refer to 9.1), then incubated between 34 °C and 38 °C for 18 h ± 2 h.
For large quantities (e.g. 1 l or more), it is recommended to pre-warm the BPW to 34 °C to 38 °C before
mixing it with the test portion.
4.3 Plating out on selective solid media
From the enrichment obtained in 4.2, the chromogenic solid media (5.2) is inoculated.
This selective agar is incubated between 34 °C and 38 °C for 24 h ± 3 h (or according to the
manufacturer’s instructions if explicitly recommended).
4.4 Confirmation
Colonies of presumptive Salmonella are confirmed by means of appropriate serological test. If the
serological test gives a negative result, the inoculation of a selective agar (Table C.2) is required. If the
test gives a negative result, up to 4 other presumptive colonies will be tested (if possible and up to
5 colonies in total).
5 Culture media and reagents
5.1 General
For current laboratory practice, see EN ISO 7218 and EN ISO 11133.
Composition of culture media and reagents and their preparation are described in Annex B.
5.2 Isolation chromogenic agar
This isolation medium is chosen by the testing laboratory and shall highlight the C8-esterase enzymatic
activity. For examples of isolation media, see Table C.1.
6

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kSIST-TS FprCEN/TS 17780:2021
FprCEN/TS 17780:2021 (E)

5.3 Non-selective agar
General purpose agar supporting the growth of a wide range of non-fastidious strains is chosen by the
testing laboratory. For example, for non-selective agar, see B.4.
5.4 Confirmation selective agar
The confirmation medium is chosen by the testing laboratory and shall highlight the production of
hydrogen sulphide (H S) by the strains (see B.5). For examples of isolation media, see Table C.2.
2
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (see EN ISO 7218) and, in particular, the following.
6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave), see EN ISO 7218.
6.2 Drying cabinet or oven, capable of operating between 25 °C and 50 °C.
6.3 Incubator(s), capable of operating in the range 34 °C to 38 °C or at 37 °C ± 1 °C.
6.4 Water bath, capable of operating at 47 °C to 50 °C, containing an antibacterial agent.
6.5 Cooling unit, adjustable at 5 °C ± 3 °C.
6.6 Freezer, capable of operating at -20 °C ± 5 °C.
6.7 Sterile loops, of approximate diameter, 3 mm (10 μl volume).
6.8 pH-meter, with a reading to the nearest 0,1 pH unit from 20 °C to 25 °C.
6.9 Sterile tubes, bottles, or flasks, with caps of appropriate capacity.
6.10 Sterile filter, with a 0,2 µm porosity.
6.11 Sterile Petri dishes, with a diameter of approximately 90 mm and (optional) large size (diameter
approximately 140 mm).
7 Sampling
Sampling should be performed carefully, following the principles described in EN 1482 (all parts) with
appropriate adaptations, required to account for specificities of organic and organo-mineral fertilizers
and to microbiological quality of the samples.
It is important that the laboratory receives a laboratory sample (3.3) which is representative and has not
been damaged or changed during transport or storage.
8 Preparation of test sample
The sample is prepared in accordance to the European or International Standard that is specific to the
concerned matrix [see EN ISO 6887 (all parts)]. If there is no specific European or International Standard,
it is advised that the different parties agree on this subject.
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kSIST-TS FprCEN/TS 17780:2021
FprCEN/TS 17780:2021 (E)

9 Procedure (see Figure A.1)
9.1 Test portion and initial suspension
For preparation of the initial suspension, use as diluent the enrichment medium specified in Annex B, B.2
(buffered peptone water). Pre-warm the BPW supplemented with Novobiocin (nBPW) to room
temperature before use.
A test portion (3.5) of 25 grams or 25 millilitres is weighed and nBPW (refer to 4.2) is added to yield a
tenfold dilution (mass or volume) (see EN ISO 6887-1).
NOTE 1 If the samples are suspected to exhibit extreme pH values, double buffered peptone water
(supplemented with novobiocin) can be used as diluent for the initial suspension.
NOTE 2 If the initial suspension is used for other microbiological tests, novobiocin can be added subsequently.
9.2 Selective pre-enrichment
Incubate the initial suspension (9.1.) between 34 °C and 38 °C (6.3.) for 18 h ± 2 h.
It is permissible to store the pre-enrichment culture at 5 °C (6.5.) for a maximum of 72 h after the
incubation.
9.3 Isolation
From the selective enrichment (9.2.), inoculate the surface of the chromogenic isolation agar (5.2) by
means of a 10 µl loop (6.7.) so that well-isolated colonies will be obtained.
Allow the plates to equilibrate at room temperature if they were stored at a lower temperature. If
necessary, dry the surface of the plates before use (see EN ISO 11133).
Incubate the selective plating-out medium between 34 °C and 38 °C (6.3.) for 24 h ± 3 h (or according to
the manufacturer’s instructions if explicitly recommended).
Typical colonies exhibiting the C8-esterase activity are smooth (or rarely rough), round and magenta.
After incubation, check the chromogenic medium for the presence of colonies, which, from their
characteristics, are considered to be suspect colonies.
9.4 Confirmation
9.4.1 General
The combination of enzymatic activity and serological test results
...

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