SIST-TS CEN/TS 17781:2023
(Main)Organic, organo-mineral and inorganic fertilizers - Detection of Escherichia coli
Organic, organo-mineral and inorganic fertilizers - Detection of Escherichia coli
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 colony-count technique at 44 °C on a solid medium containing a chromogenic ingredient for the detection of the enzyme ß-glucuronidase. The method is based on ISO 16649-2 [4].
Strains of Escherichia coli which do not grow at 44 °C and, in particular, those that are ß-glucuronidase negative, such as Escherichia coli O157, will not be detected. Detected microorganisms are presumptively determined β-glucuronidase-positive Escherichia coli, since some Enterobacteriaceae, in particular Shigella and Salmonella, can also show β-glucuronidase activity at 44 °C.
Organische, organisch-mineralische und mineralische Düngemittel - Nachweis von Escherichia coli
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 Koloniezählverfahren fest, das bei 44 °C auf einem festen Medium durchgeführt wird, das einen chromogenen Bestandteil zum Nachweis des Enzyms β Glucuronidase enthält. Das Verfahren basiert auf ISO 16649 2 [4].
Stämme von Escherichia coli, die bei 44 °C kein Wachstum aufweisen, und insbesondere Stämme, die β Glucuronidase-negativ sind, wie z. B. Escherichia coli O157, werden nicht nachgewiesen. Bei den nachgewiesenen Mikroorganismen handelt es sich um vermutlich β Glucuronidase-positive Escherichia coli, da einige Enterobacteriaceae, insbesondere Shigella und Salmonella, bei 44 °C ebenfalls β Glucuronidase-Aktivität zeigen können.
Engrais organiques, organo-minéraux et inorganiques - Recherche des Escherichia coli
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 technique de dénombrement des colonies à 44 °C sur un milieu solide contenant un ingrédient chromogène pour la recherche de l’enzyme β-glucuronidase. La méthode est basée sur l’ISO 16649-2 [4].
Les souches d’Escherichia coli qui ne poussent pas à 44 °C et, en particulier, celles qui sont β-glucuronidase négatives, telles que les Escherichia coli O157, ne peuvent pas être mises en évidence. Les microorganismes détectés sont des Escherichia coli β-glucuronidase positive déterminés par présomption, car certaines Enterobacteriaceæ, en particulier Shigella et Salmonella, peuvent également présenter une activité β-glucuronidase à 44 °C.
Organska, organsko-mineralna in anorganska gnojila - Ugotavljanje prisotnosti Escherichia coli
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 tehniko štetja kolonij pri 44 °C na trdnem gojišču, ki vsebuje kromogeno sestavino za določanje encima ß-glukuronidaze. Metoda temelji na standardu ISO 16649-2 [4].
Sevi Escherichia coli, ki ne rastejo pri 44 °C in zlasti tisti, ki so negativni na ß-glukuronidazo, kot je Escherichia coli O157, ne bodo zaznani. Odkriti mikroorganizmi so verjetno bakterije Escherichia coli, pozitivne na β-glukuronidazo, saj lahko nekatere bakterije Enterobacteriaceae, zlasti Shigella in Salmonella, kažejo aktivnost β-glukuronidaze tudi pri 44 °C.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN/TS 17781:2023
01-februar-2023
Organska, organsko-mineralna in anorganska gnojila - Ugotavljanje prisotnosti
Escherichia coli
Organic, organo-mineral and inorganic fertilizers - Detection of Escherichia coli
Organische, organisch-mineralische und mineralische Düngemittel - Nachweis von
Escherichia coli
Engrais organiques, organo-minéraux et inorganiques - Recherche des Escherichia coli
Ta slovenski standard je istoveten z: CEN/TS 17781:2022
ICS:
65.080 Gnojila Fertilizers
SIST-TS CEN/TS 17781:2023 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 17781:2023
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SIST-TS CEN/TS 17781:2023
CEN/TS 17781
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
April 2022
TECHNISCHE SPEZIFIKATION
ICS 65.080
English Version
Organic, organo-mineral and inorganic fertilizers -
Detection of Escherichia coli
Engrais organiques, organo-minéraux et inorganiques - Organische, organisch-mineralische und anorganische
Recherche des Escherichia coli Düngemittel - Nachweis von Escherichia coli
This Technical Specification (CEN/TS) was approved by CEN on 13 March 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 17781:2022 E
worldwide for CEN national Members.
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SIST-TS CEN/TS 17781:2023
CEN/TS 17781:2022 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 6
5 Diluents, culture media and reagents . 7
5.1 General. 7
5.2 Diluents . 7
5.3 Culture media . 7
6 Equipment and consumables . 8
7 Sampling . 8
8 Preparation of test sample . 9
9 Procedure (see Figure A.1 in Annex A (normative)) . 9
9.1 Preparation of the initial suspension and decimal dilutions . 9
9.2 Inoculation and incubation . 9
9.3 Enumeration of colonies . 10
9.4 Confirmation (optional) . 10
10 Expression of results . 11
11 Method validation . 12
12 Test report . 12
Annex A (normative) Diagram of the procedure . 13
Annex B (normative) Composition and preparation of culture media and reagents . 14
Bibliography . 18
2
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CEN/TS 17781:2022 (E)
European foreword
This document (CEN/TS 17781:2022) 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 organizations 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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CEN/TS 17781:2022 (E)
Introduction
This document describes a method for the detection and enumeration of Escherichia coli 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.
This methodology has been developed to detect and enumerate Escherichia coli in organic, organo-
mineral and inorganic fertilizers in order to be able to control certain hygienic requirements in the
Regulation (EU) 2019/1009 [1].
Escherichia coli is a 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 therefore cannot reflect
the behaviour of all pathogens in fertilizers.
Because of the large variety of fertilizers, this method might not be appropriate in every detail for certain
products. In this case, different methods which are specific to these products may be used if absolutely
necessary for justified technical reasons. Nevertheless, every attempt should be made to apply this
method as far as possible.
Mineral components in fertilizers can have a negative impact on the survivability of microorganisms
when they go into solution. In addition to an unfavourable shift in the pH value, the products can have a
strong osmotic effect or be toxic to cells themselves (e.g. copper). Therefore, it can be necessary to test
the inhibitory effect of the fertilizers to be investigated in a pre-test.
4
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SIST-TS CEN/TS 17781:2023
CEN/TS 17781:2022 (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 colony-count technique at 44 °C on a solid medium containing a chromogenic
ingredient for the detection of the enzyme β-glucuronidase. The method is based on ISO 16649-2 [4].
Strains of Escherichia coli which do not grow at 44 °C and, in particular, those that are β-glucuronidase
negative, such as Escherichia coli O157, will not be detected. Detected microorganisms are presumptively
determined β-glucuronidase-positive Escherichia coli, since some Enterobacteriaceae, in particular
Shigella and Salmonella, can also show β-glucuronidase activity at 44 °C.
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
laboratory sample
sample intended for laboratory inspection or testing
3.2
test sample
sample prepared from the laboratory sample (3.1) and from which test portions (3.3) will be taken
3.3
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
3.4
glucuronidase-positive presumptive Escherichia coli
bacteria which at 44 °C form typical blue colony on Tryptone-Bile-X-glucuronide (TBX) medium under
the conditions specified in this method
[SOURCE: ISO 16649-2:2001, 3.1, modified – The reference to ISO 16649-2 has been replaced with a
reference to this document and the term name has been modified.]
3.5
confirmed β-glucuronidase-positive Escherichia coli
β-glucuronidase-positive presumptive Escherichia coli (3.4) showing a positive indole reaction in
tryptophan broth under the conditions specified in this method
5
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CEN/TS 17781:2022 (E)
3.6
enumeration of β-glucuronidase-positive Escherichia coli
determination of the number of colony-forming units (CFU) of β-glucuronidase-positive Escherichia coli,
per milliliter or per gram of sample, when test, confirmation and calculations are carried out in
accordance with this method
[SOURCE: ISO 16649-2:2001, 3.2, modified – The reference to ISO 16649-2 has been replaced with a
reference to this document and the word “confirmation” has been added.]
3.7
initial suspension
primary dilution obtained after a weighed or measured quantity of the product under examination (or of
a test sample prepared from the product) has been mixed with, normally, a nine-fold quantity of diluent
Note 1 to entry: A closer ratio between the diluent and the quantity of product is often not recommended because
of possible inhibiting influences of the matrix.
3.8
further dilution
suspension or solution obtained by mixing a measured volume of the initial suspension (3.7) with an
x-fold volume of diluent and by repeating this operation with further dilutions until a dilution series,
suitable for the inoculation of culture media, is obtained.
Note 1 to entry: Ten-fold dilutions are normally used to produce a decimal dilution series, but other ratios may be
required for specific purposes.
[SOURCE: EN ISO 6887-1:2017, 3.7]
4 Principle
a) Preparation of sterile liquid Tryptone-Bile-X-glucuronide (TBX) medium tempered at 44 °C to 47 °C;
b) Drawing a representative test sample under aseptic conditions;
c) Preparation of the initial suspension with a tempered diluent to obtain a homogeneous distribution
of bacterial cells from the test portion;
d) Preparation of further dilutions of the initial suspension in order to reduce the number of
microorganisms per unit volume or to reduce the cell inhibitory properties of the initial suspension
to allow, after incubation, the counting of colonies;
e) Inoculation of blank plates with an aliquot of the optimum dilutions and pouring of the molten agar
medium into each plate, mixing and solidification;
f) Incubation of inverted plates at 44 °C ± 1 °C for 18 h to 24 h;
g) Counting of typical blue colonies, considering the specific properties of Escherichia coli
h) Biochemical verification of isolates during detection of indole if necessary;
i) Calculation of the number of colony-forming units (CFU) of β-glucuronidase-positive Escherichia coli
per gram or per millilitre of sample.
6
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CEN/TS 17781:2022 (E)
5 Diluents, culture media and reagents
5.1 General
For standard laboratory practice, EN ISO 7218 and EN ISO 11133 can be used.
Composition of culture media and reagents and their preparation are specified in Annex B (normative).
For uniformity of results, in the preparation of media, either use a dehydrated complete medium or use
constituents of uniform quality and chemicals of recognized analytical grade.
5.2 Diluents
5.2.1 General
Fertilizers with a high mineral content can significantly change the pH value of the initial suspension,
which can negatively affect the viability of the microorganisms under investigation. In general (organic
fertilizers), a basic phosphate buffer is sufficient to prepare the initial suspension. When testing organo-
mineral or inorganic fertilizers, the pH value of the substrate in solution should be determined in a
preliminary test. The general use of a double-buffered phosphate buffer is recommended. If the substrate
is simultaneously tested for the presence of Salmonella (CEN/TS 17780), the initial suspension for
enrichment can be used with buffered or double-buffered peptone water (with 10 g peptone). In this case,
rapid processing of the initial suspension is necessary.
5.2.2 basic phosphate buffer
See B.1.
5.2.3 double-buffered phosphate buffer
See B.2.
5.3 Culture media
5.3.1 Tryptone-Bile-X-glucuronide (TBX) medium
See B.3.
5.3.2 MacConkey Agar No. 3 (optional)
See B.4.
5.3.3 Medium and Reagent for indole reaction (optional)
5.3.3.1 Tryptone/tryptophan medium
See B.5.
5.3.3.2 Kovacs reagent
See B.6.
7
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CEN/TS 17781:2022 (E)
6 Equipment and consumables
6.1 General
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (EN ISO 7218 can be used) and, in particular, the following.
6.2 Equipment for dry sterilization (oven) and wet sterilization (autoclave).
EN ISO 7218 can be used.
6.3 Incubator.
Capable of maintaining a temperature of 44 °C ± 1 °C. Optionally also capable of maintaining a
temperature of 37 °C ± 1 °C and/or 35 °C ± 1 °C and/or 44 °C to 47 °C.
6.4 Blending equipment.
The following apparatus may be used:
— a peristaltic homogenizer with sterile bags (paddle homogenizer), possibly with the option to adjust
blending speed and time; or
— a laboratory shaker with sterile bags.
6.5 Mechanical stirrer.
A mech
...
SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17781:2021
01-december-2021
Organska, organsko-mineralna in anorganska gnojila - Ugotavljanje prisotnosti
Escherichia coli
Organic, organo-mineral and inorganic fertilizers - Detection of Escherichia coli
Organische, organisch-mineralische und mineralische Düngemittel - Nachweis von
Escherichia coli
Engrais organiques, organo-minéraux et inorganiques - Recherche des Escherichia coli
Ta slovenski standard je istoveten z: FprCEN/TS 17781
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17781:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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kSIST-TS FprCEN/TS 17781:2021
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kSIST-TS FprCEN/TS 17781:2021
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17781
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
November 2021
ICS 65.080
English Version
Organic, organo-mineral and inorganic fertilizers -
Detection of Escherichia coli
Engrais organiques, organo-minéraux et inorganiques – Organische, organisch-mineralische und anorganische
Recherche des Escherichia coli Düngemittel - Nachweis von Escherichia coli
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 NORMALISATIO N
EUROPÄISCHES KOMITEE FÜR NORMUN G
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 17781:2021 E
worldwide for CEN national Members.
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kSIST-TS FprCEN/TS 17781:2021
FprCEN/TS 17781:2021 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 6
5 Diluents, culture media and reagents . 7
5.1 General. 7
5.2 Diluents . 7
5.3 Culture media . 7
6 Equipment and consumables . 7
7 Sampling . 8
8 Preparation of test sample . 9
9 Procedure (see Figure A.1 in Annex A) . 9
9.1 Preparation of the initial suspension and decimal dilutions . 9
9.2 Inoculation and incubation . 9
9.3 Enumeration of colonies . 10
9.4 Confirmation (optional) . 10
10 Expression of results . 11
11 Method validation . 12
12 Test report . 12
Annex A (normative) Diagram of the procedure . 13
Annex B (normative) Composition and preparation of culture media and reagents . 14
Bibliography . 18
2
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kSIST-TS FprCEN/TS 17781:2021
FprCEN/TS 17781:2021 (E)
European foreword
This document (FprCEN/TS 17781:2021) 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 17781:2021
FprCEN/TS 17781:2021 (E)
Introduction
This document describes a method for the detection and enumeration of Escherichia coli 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.
This methodology has been developed to detect and enumerate Escherichia coli in organic, organo-
mineral and inorganic fertilizers in order to be able to control certain hygienic requirements in the
Regulation (EU) 2019/1009 [1].
Escherichia coli is a 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 therefore cannot reflect
the behaviour of all pathogens in fertilizers.
Because of the large variety of fertilizers, this method may not be appropriate in every detail for certain
products. In this case, different methods which are specific to these products may be used if absolutely
necessary for justified technical reasons. Nevertheless, every attempt should be made to apply this
method as far as possible.
Mineral components in fertilizers can have a negative impact on the survivability of microorganisms
when they go into solution. In addition to an unfavourable shift in the pH value, the products can have a
strong osmotic effect or be toxic to cells themselves (e.g. copper). Therefore, it may be necessary to test
the inhibitory effect of the fertilizers to be investigated in a pre-test.
4
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kSIST-TS FprCEN/TS 17781:2021
FprCEN/TS 17781:2021 (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 colony-count technique at 44 °C on a solid medium containing a chromogenic
ingredient for the detection of the enzyme ß-glucuronidase. The method is based on ISO 16649-2 [4].
Strains of Escherichia coli which do not grow at 44 °C and, in particular, those that are ß-glucuronidase
negative, such as Escherichia coli O157, will not be detected. Detected microorganisms are presumptively
determined β-glucuronidase-positive Escherichia coli, since some Enterobacteriaceae, in particular
Shigella and Salmonella, can also show β-glucuronidase activity at 44 °C.
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
laboratory sample
sample intended for laboratory inspection or testing
3.2
test sample
sample prepared from the laboratory sample (3.1) and from which test portions (3.3) will be taken
3.3
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
3.4
glucuronidase-positive presumptive Escherichia coli
bacteria which at 44 °C form typical blue colony on tryptone-bile-glucuronide medium (TBX) under the
conditions specified in this method
[SOURCE: ISO 16649-2:2001, 3.1, modified – The reference to ISO 16649-2 has been replaced with a
reference to this document]
3.5
confirmed ß-glucuronidase-positive Escherichia coli
ß-glucuronidase-positive presumptive Escherichia coli showing a positive indole reaction in tryptophan
broth under the conditions specified in this method
5
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kSIST-TS FprCEN/TS 17781:2021
FprCEN/TS 17781:2021 (E)
3.6
enumeration of ß-glucuronidase-positive Escherichia coli
determination of the number of colony-forming units (CFU) of ß-glucuronidase-positive Escherichia coli,
per milliliter or per gram of sample, when test, confirmation and calculations are carried out in
accordance with this method
[SOURCE: ISO 16649-2:2001, 3.2, modified – The reference to ISO 16649-2 has been replaced with a
reference to this document]
3.7
initial suspension
primary dilution obtained after a weighed or measured quantity of the product under examination (or of
a test sample prepared from the product) has been mixed with, normally, a nine-fold quantity of diluent
Note 1 to entry: A closer ratio between the diluent and the quantity of product is often not recommended because
of possible inhibiting influences of the matrix.
3.8
further dilution
suspension or solution obtained by mixing a measured volume of the initial suspension (3.7) with an
x-fold volume of diluent and by repeating this operation with further dilutions until a dilution series,
suitable for the inoculation of culture media, is obtained.
Note 1 to entry: Ten-fold dilutions are normally used to produce a decimal dilution series, but other ratios may be
required for specific purposes.
[SOURCE: EN ISO 6887-1:2017, 3.7]
4 Principle
a) Preparation of sterile liquid tryptone-bile-glucuronide medium (TBX) tempered at 44 °C to 47 °C;
b) Drawing a representative test sample under aseptic conditions;
c) Preparation of the initial suspension with a tempered diluent to obtain a homogeneous distribution
of bacterial cells from the test portion;
d) Preparation of further dilutions of the initial suspension in order to reduce the number of
microorganisms per unit volume or to reduce the cell inhibitory properties of the initial suspension.
To allow, after incubation, the counting of colonies;
e) Inoculation of blank plates with an aliquot of the optimum dilutions and pouring of the molten agar
medium into each plate, mixing and solidification;
f) Incubation of inverted plates at 44 °C ± 1 °C for 18 h to 24 h;
g) Counting of typical blue colonies, considering the specific properties of Escherichia coli
h) Biochemical verification of isolates during detection of indole if necessary;
i) Calculation of the number of colony-forming units (CFU) of ß-glucuronidase-positive Escherichia coli
per gram or per milliliter of sample.
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kSIST-TS FprCEN/TS 17781:2021
FprCEN/TS 17781:2021 (E)
5 Diluents, culture media and reagents
5.1 General
For standard laboratory practice, EN ISO 7218 and EN ISO 11133 can be used.
Composition of culture media and reagents and their preparation are specified in Annex B (normative).
For uniformity of results, in the preparation of media, either use a dehydrated complete medium or use
constituents of uniform quality and chemicals of recognized analytical grade.
5.2 Diluents
5.2.1 General
Fertilizers with a high mineral content can significantly change the pH value of the initial suspension,
which can negatively affect the viability of the microorganisms under investigation. In general (organic
fertilizers), a basic phosphate buffer is sufficient to prepare the initial suspension. When testing organo-
mineral or inorganic fertilizers, the pH value of the substrate in solution should be determined in a
preliminary test. The general use of a double-buffered phosphate buffer is recommended. If the substrate
is simultaneously tested for the presence of Salmonella (FprCEN/TS 17780), the initial suspension for
enrichment can be used with buffered or double-buffered peptone water (with 10 g peptone). In this case,
rapid processing of the initial suspension is necessary.
5.2.2 basic phosphate buffer
See B.1.
5.2.3 double-buffered phosphate buffer
See B.2.
5.3 Culture media
5.3.1 Tryptone-bile-glucuronic medium (TBX)
See B.3.
5.3.2 MacConkey Agar No. 3 (optional)
See B.4.
5.3.3 Medium and Reagent for indole reaction (optional)
5.3.3.1 Tryptone/tryptophan medium
See B.5.
5.3.3.2 Kovacs reagent
See B.6.
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (EN ISO 7218 can be used) and, in particular, the following.
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kSIST-TS FprCEN/TS 17781:2021
FprCEN/TS 17781:2021 (E)
6.1 Equipment for dry sterilization (oven) and wet sterilization (autoclave).
EN ISO 7218 can be used.
6.2 Incubator.
Capable of maintaining a temperature of 44 °C ± 1 °C. Optionally also capable of maintaining a
temperature of 37 °C ± 1 °C and/or 35 °C ± 1 °C and/or 44 °C to 47 °C.
6.3 Blending equipment.
The following apparatus may be used:
— a peristaltic homogenizer with sterile bags (paddle homogenizer), possibly with the option to adjust
blending speed and time; or
— a laboratory shaker with sterile bags.
6.4 Mechanical stirrer.
A mechanical stirrer e.g. Vortex Mixer facilitates the homogenous mixing of decimal dilutions.
6.5 Balance.
Balances of the required range and accuracy for the different products to be weighed.
6.6 Water bath.
Capable of maintaining temperatures of 44 °C to 47 °C
6.7 Cooling unit, adjustable at 5 °C ± 3°C.
6.8 pH-meter, capable of reading to the nearest 0,1 pH unit at 20 °C to 25 °C.
6.9 Sterile loops of approximate diameter, 3 mm (10 μl volume).
6.10 Sterile tubes, bottles, or flasks with caps of appropriate c
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