Animal feeding stuffs: Methods of sampling and analysis - Detection and enumeration of Enterococcus (E. faecium) spp. used as feed additive

This European Standard defines general rules for the enumeration of enterococci in feed samples (additives, premixtures and feeding stuffs) that contain enterococci (E. faecium) as a single microorganism component or in a mixture with other microorganisms. Applying the method to feeds with a high copper content (> 400 mg/kg) demands a special procedure (see Annex A).This standard is not applicable to mineral feeds which are defined as complementary feeding stuffs composed mainly of minerals and containing at least 40 % crude ash (Council Directive 79/373/EEC).
There are different categories of feed samples:
a)   Additives containing about 10+10 colony forming units (CFU)/g;
b)   Premixtures containing 10+8 CFU/g;
c)   Feeds, meal or pellets which contain about 10+6 CFU/g and include complete feeding stuffs,and milk replacers.
The detection limit is as defined in EN ISO 7218.

Futtermittel: Probenahme- und Untersuchungs-verfahren - Nachweis und Zählung von Enterococcus spp. (E. faecium) als Futtermittelzusatzstoff

Dieses Dokument legt allgemeine Regeln für die Zählung von Enterokokken (E. faecium) in Futtermitteln (Zusatzstoffe, Vormischungen und Mischfuttermittel mit Ausnahme von mineralischen Futtermitteln) fest, die Enterokokken als einzelnen mikrobiellen Bestandteil oder in einem Gemisch mit anderen Mikroorganis-men enthalten. Die Anwendung des Verfahrens auf Mischfuttermittel mit kritischen Kupfermengen erfordert ein besonderes Verfahren (siehe Anhang A). Dieses Dokument ist nicht anwendbar auf mineralische Futter-mittel, die als Ergänzungsfuttermittel definiert sind, welche hauptsächlich aus Mineralstoffen zusammen-gesetzt sind und mindestens 40 % Rohasche enthalten (Verordnung R767/2009) [4].
Es gibt unterschiedliche Kategorien von Futtermittelproben:
a) Zusatzstoffe, die etwa 1010 koloniebildende Einheiten (KbE/g) enthalten;
b) Vormischungen, die etwa 1011 KbE/kg enthalten;
c) Mischfuttermittel, Mehl oder Pellets, die etwa 109 KbE/kg enthalten.
Die Nachweisgrenze entspricht der in EN ISO 7218 festgelegten.

Aliments des animaux: Méthodes d’échantillonnage et d’analyse - Détection et dénombrement des souches de Enterococcus (E. faecium) spp. utilisées comme additifs pour l’alimentation animale

Le présent document définit des règles générales pour le dénombrement d’entérocoques (E. Faecium) présents dans les aliments pour animaux (additifs, prémélanges et aliments composés, à l’exception des aliments minéraux) qui contiennent des entérocoques comme seul micro-organisme constitutif ou en mélange avec d’autres micro-organismes. L’application de la méthode aux aliments composés pour animaux présentant une teneur critique en cuivre nécessite de mettre en oeuvre un mode opératoire spécial (voir Annexe A). Le présent document ne s’applique pas aux aliments minéraux, qui se définissent comme des aliments complémentaires constitués principalement de minéraux et contenant au moins 40 % de cendre brute (Règlement R767/2009) [4].
Il existe différentes catégories d’échantillons d’aliments pour animaux :
a) les additifs contenant environ 1010 UFC/g (UFC : unités formant colonie) ;
b) les prémélanges contenant 1011 UFC/kg ;
c) les aliments composés, farines ou granulés qui contiennent environ 109 UFC/kg.
La limite de détection est définie dans l’EN ISO 7218.

Krma: metode vzorčenja in analize - Določanje in štetje prisotnih Enterococcus (E. faecium) spp., uporabljenih kot krmni dodatek

Ta evropski standard določa splošna pravila za štetje enterokokov v vzorcih krme (dodatki, premiksi in krma), ki vsebujejo enterokoke (E. faecium) kot eno komponento mikroorganizma ali v kombinaciji z drugimi mikroorganizmi. Uporaba metode za krmo z visoko vsebnostjo bakra (> 400 mg/kg) zahteva poseben postopek (glej dodatek A). Ta standard se ne uporablja za mineralno krmo, ki je opredeljena kot dopolnilna krma, sestavljena predvsem iz mineralov, in vsebuje najmanj 40 % surovega pepela (Direktiva Sveta 79/373/EGS).
Obstajajo različne kategorije vzorcev krme:
a)   dodatki, ki vsebujejo približno 10+10 enot, ki tvorijo kolonije (CFU)/g;
b)   premiksi, ki vsebujejo 10+8 CFU/g;
c)   krma, moka ali peleti, ki vsebujejo približno 10+6 CFU/g ter vključujejo popolno krmo in mlečne nadomestke.
Meja zaznavanja je opredeljena v standardu EN ISO 7218.

General Information

Status
Published
Public Enquiry End Date
19-Mar-2020
Publication Date
13-Jan-2022
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
28-Dec-2021
Due Date
04-Mar-2022
Completion Date
14-Jan-2022

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Standards Content (sample)

SLOVENSKI STANDARD
SIST EN 15788:2022
01-februar-2022
Nadomešča:
SIST EN 15788:2009

Krma: metode vzorčenja in analize - Določanje in štetje prisotnih Enterococcus (E.

faecium) spp., uporabljenih kot krmni dodatek

Animal feeding stuffs: Methods of sampling and analysis - Detection and enumeration of

Enterococcus (E. faecium) spp. used as feed additive
Futtermittel: Probenahme- und Untersuchungs-verfahren - Nachweis und Zählung von
Enterococcus spp. (E. faecium) als Futtermittelzusatzstoff
Aliments des animaux: Méthodes d’échantillonnage et d’analyse - Détection et

dénombrement des souches de Enterococcus (E. faecium) spp. utilisées comme additifs

pour l’alimentation animale
Ta slovenski standard je istoveten z: EN 15788:2021
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN 15788:2022 en,fr,de

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

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SIST EN 15788:2022
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SIST EN 15788:2022
EN 15788
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2021
EUROPÄISCHE NORM
ICS 65.120 Supersedes EN 15788:2009
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Detection and enumeration of Enterococcus (E. faecium)
spp. used as feed additive

Aliments des animaux: Méthodes d'échantillonnage et Futtermittel: Probenahme- und Untersuchungs-

d'analyse - Détection et dénombrement des souches de verfahren - Nachweis und Zählung von Enterococcus

Enterococcus (E. faecium) spp. utilisées comme spp. (E. faecium) als Futtermittelzusatzstoff

additifs pour l'alimentation animale
This European Standard was approved by CEN on 2 August 2021.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

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 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. EN 15788:2021 E

worldwide for CEN national Members.
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SIST EN 15788:2022
EN 15788:2021
Contents Page

European foreword ....................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 5

1 Scope .................................................................................................................................................................... 6

2 Normative references .................................................................................................................................... 6

3 Terms and definitions ................................................................................................................................... 6

4 Principle ............................................................................................................................................................. 6

5 Diluents and culture media ......................................................................................................................... 7

5.1 Diluents ............................................................................................................................................................... 7

5.2 Culture media ................................................................................................................................................... 8

6 Apparatus ........................................................................................................................................................ 10

7 Sampling .......................................................................................................................................................... 11

8 Preparation of the test sample ................................................................................................................ 11

9 Procedure........................................................................................................................................................ 11

9.1 Preparation of poured agar plates for spread plate method ....................................................... 11

9.2 Preparation of culture media for pour plate method ..................................................................... 12

9.3 Preparation of the initial suspension and decimal dilutions ....................................................... 12

9.4 Inoculation and incubation of the plates ............................................................................................. 13

9.5 Enumeration of colonies ............................................................................................................................ 14

9.6 Confirmation .................................................................................................................................................. 14

10 Expression of results ................................................................................................................................... 15

11 Precision .......................................................................................................................................................... 15

11.1 General ............................................................................................................................................................. 15

11.2 Interlaboratory studies .............................................................................................................................. 15

11.3 Repeatability .................................................................................................................................................. 16

11.4 Reproducibility ............................................................................................................................................. 16

12 Test report ...................................................................................................................................................... 16

Annex A (informative) Notes on the procedure ............................................................................................... 17

A.1 General ............................................................................................................................................................. 17

A.2 Critical copper concentration .................................................................................................................. 17

Annex B (informative) Results of the interlaboratory studies ................................................................... 18

B.1 Interlaboratory study based on bile aesculin azide agar .............................................................. 18

B.2 Interlaboratory study based on Slanetz and Bartley agar ............................................................ 18

Bibliography ................................................................................................................................................................. 20

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EN 15788:2021
European foreword

This document (EN 15788:2021) has been prepared by Technical Committee CEN/TC 327 “Animal

feeding stuffs - Methods of sampling and analysis”, the secretariat of which is held by NEN.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by May 2022, and conflicting national standards shall be

withdrawn at the latest by May 2022.

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 supersedes EN 15788:2009.
The main changes compared to the previous edition are as follows:
— Amendment of the title;

— Extension of the scope of application to all Enterococci used as feed additive;

— Updating of normative cross references;
— Supplement of phosphate buffered saline with Tween 80;

— Addition of the option to use Tween 80 supplemented phosphate buffered saline for the

preparation of the initial suspension as well as diluent for serial dilutions;
— Addition of Slanetz and Bartley agar as selective detection medium;

— Replacement of the required laboratory mixer with a rotation speed of 18 000 min to

22 000 min by homogenization devices, for example according to EN ISO 7218, with a maximal

requested rotation speed of 10 000 min ;

— Unification of the homogenization time for the preparation of initial suspensions to five minutes for

all feed matrices;
— Addition of the option to use a spiral plater for plating;
— Preparation of initial suspensions generally conducted with tempered tPBS;
— Addition of the pour plate method as an alternative cultivation technique;

— Addition of a procedure for the investigation of feeding stuffs containing high amounts of copper in

the informative Annex A;

— Addition of validation data derived from VDLUFA ring trials of different feeding stuff matrices using

Slanetz and Bartley agar as enumeration media;

— Adjustment of the range of accepted colony numbers for counting from '≥ 30 to ≤ 350' to '≥ 10

to ≤ 200' colonies per plate.

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.
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EN 15788:2021

According to the CEN-CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to implement this European Standard: 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.
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Introduction

This methodology has been developed to enumerate enterococci (E. faecium) as feed additives to enable

the European Commission to control proper labelling of animal feeding products. It was compiled first

during the EU project SMT4-CT98-2235 “Methods for the official control of probiotics used as feed

additives” [1]. It was amended with a second medium from VDLUFA method 28.2.3 “Enumeration of

Enterococcus faecium” [2]. The method is based on an extensive screening of 12 pre-selected,

commercially available media for the detection and enumeration of enterococci. The specified

methodology was validated in interlaboratory studies for E. faecium ([1], [2], [3]). It can be assumed that

the method is also suitable for other Enterococcus spp.

This method is not selective for enterococci (E. faecium) used as feed additives, but can be applied to

enumerate Enterococcus spp. in additives, premixtures and compound feeds assuming that the added

enterococci (E. faecium) are present in far higher numbers than any other enterococci.

This method is not applicable for the detection of any ubiquitous or faecal contaminants of Enterococcus

spp. in food and animal feeding stuffs.
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EN 15788:2021
1 Scope

This document specifies general rules for the enumeration of enterococci (E. faecium) in feeding stuffs

(additives, premixtures and compound feeds excluding mineral feeds) that contain enterococci as a single

microorganism component or in a mixture with other microorganisms. Applying the method to

premixtures and compound feeds with critical amounts of copper demands a special procedure (see A.2).

The document is not applicable to mineral feeds which are defined as complementary feeding stuffs

composed mainly of minerals and containing at least 40 % crude ash (Regulation (EC) 767/2009) [4].

There are different categories of feed samples:
a) Additives containing about 10 colony forming units (CFU)/g;
b) Premixtures containing 10 CFU/kg;
c) Compound feeds, meal or pellets which contain about 10 CFU/kg.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements 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 ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)

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:

— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
3.1
Enterococci

Gram-positive, catalase negative cocci, which usually occurs in pairs or short chains

Note 1 to entry: This description is based on their characteristics as used for this document.

Note 2 to entry: Enterococci are classified as aerotolerant anaerobes with the ability to reduce 2,3,5-triphenyl

tetrazolium chloride to formazan and capable of hydrolyzing aesculin at 44 °C ± 0,5 °C. Enterococci form colonies

fitting the description of this species on the specified culture media after incubation at a temperature of 37 °C under

aerobic conditions for 24 h resp. 48 h (see 9.6).
4 Principle

a) Preparation of sterile and dry poured agar plates or preparation of sterile liquid culture media

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;
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d) Preparation of further decimal dilutions of the initial suspension in order to reduce the number of

microorganisms per unit volume to allow, after incubation, the counting of colonies;

e) Inoculation of the prepared poured plates with an aliquot of the optimum dilutions and dispersion

of the inoculum using a sterile spreader or 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 for 24 h resp. 48 h at 37 °C ± 1 °C under aerobic conditions;

g) Counting of typical colonies, considering the specific properties of enterococci;

h) Morphological verification of isolates though the use of microscopy or biochemical confirmation if

necessary;

i) Calculation of the colony forming units of enterococci per gram or kilogram of feed sample.

5 Diluents and culture media
5.1 Diluents
5.1.1 Diluents for initial suspension

The diluent is used for the preparation of the initial suspension and may also be used for the preparation

of further decimal dilutions. The composition of the diluent is given in Table 1.

® 1
Table 1 — Phosphate buffered saline with Polysorbate 80 (Tween 80) (tPBS)
Sodium chloride NaCl 8,00 g
Potassium chloride KCl 0,20 g
Na HPO
Disodium hydrogen phosphate anhydrous 1,15 g
2 4
KH PO
Potassium dihydrogen phosphate anhydrous 0,20 g
2 4
C H O
Polyoxyethylene (20) sorbitan monooleate 1 ml
64 124 26
® 1
(Tween 80)
Water, distilled or deionized H O 1 000 ml

Dissolve the components (see Table 1) in water. If necessary, adjust to a final pH of 7,3 ± 0,2 at 25 °C after

sterilization. Fill the solution into appropriate containers (e.g. bottles, flasks, or test tubes) and sterilize

at 121 °C ± 3 °C for 15 min. To avoid loss during autoclaving, screw cap bottles are recommended.

For immediate use, hold at 40 °C ± 1 °C in a water bath or incubator.

NOTE When using commercially available PBS buffer tablets, please take note that variations in composition

and pH can occur between products from different manufacturers and could therefore give results different from

the ones obtained with the buffer as specified in this document.
1 ®

Tween 80 is an example of a suitable product available commercially. This information is given for the

convenience of users of this document and does not constitute an endorsement by CEN of this product.

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EN 15788:2021
5.1.2 Diluents for serial dilutions

For serial dilutions, the diluent for initial suspension (5.1.1) or alternatively a peptone salt solution (PSS)

according to EN ISO 6887-1 [5] can be used. The composition of PSS is given in Table 2.

Table 2 — Peptone salt solution (PSS) according to EN ISO 6887-1
Enzymatic digest casein 1,0 g
Sodium chloride NaCl 8,5 g
Water, distilled or deionized H O 1 000 ml

Dissolve the components (see Table 2) in water in flasks or bottles. Adjust the pH if necessary so that,

after sterilization, it is 7,0 ± 0,2 at 25 °C. For decimal dilutions, prepare test tubes containing

9,0 ml ± 0,1 ml after sterilization or use screw cap bottles to avoid volume loss during autoclaving.

Sterilize at 121 °C ± 3 °C for 15 min. Bring the diluent to room temperature before use.

NOTE Commercially available, ready-to-use PSS tubes of 9 ml are suitable.
5.2 Culture media
5.2.1 General
Two different culture media are proposed:
a) Bile aesculin azide agar;

b) Enterococcus selective medium according to Slanetz and Bartley (Slanetz and Bartley agar).

NOTE Both media can be used for the spread plate method as well as for the pour plate method.

WARNING — The culture media described contain sodium azide. As this substance is highly toxic and

mutagenic, precautions shall be taken to avoid contact with it, especially by inhalation of fine dust during

the preparation of commercially available dehydrated complete media.

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.
5.2.2 Composition
5.2.2.1 Bile aesculin azide agar

The composition of the bile aesculin azide agar is given in Table 3. The resulting pH value at 25 °C is

7,1 ± 0,2.
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EN 15788:2021
Table 3 — Composition of the bile aesculin azide agar
Casein peptone (tryptone) 17,0 g
Yeast extract 5,0 g
Peptone 3,0 g
Ox bile, dried 10,0 g
Sodium chloride 5,0 g
Aesculin 1,0 g
Ammonium iron(III) citrate 0,5 g
Sodium azide 0,15 g
Agar
8 g to 18 g
Water, distilled or deionized 1 000 ml
Depending on the gel strength of the agar.
5.2.2.2 Slanetz and Bartley agar

The composition of the Slanetz and Bartley agar is given in Table 4. The resulting pH value at 25 °C is

7,2 ± 0,2.
Table 4 — Composition of the Slanetz and Bartley agar
Tryptose 20,0 g
Yeast extract 5,0 g
D-(+)-glucose 2,0 g
Di-potassium hydrogen phosphate 4,0 g
Sodium azide 0,4 g
2,3,5-triphenyl tetrazolium chloride (TTC) 0,1 g
Agar
8 g to 18 g
Water, distilled or deionized 1 000 ml
Depending on the gel strength of the agar.
5.2.3 Preparation
5.2.3.1 Bile aesculin azide agar

Dispense the medium (Table 3) into suitable containers, e.g. bottles or flasks with non-toxic metal screw-

caps. Dissolve all components specified in Table 3 in water by boiling. If necessary, adjust to a final pH of

7,1 ± 0,2 at 25 °C. Sterilize at 121 °C ± 3 °C for 15 min.
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5.2.3.2 Slanetz and Bartley agar

Dispense the medium (Table 4) into suitable containers, e.g. bottles or flasks with non-toxic metal screw-

caps. Dissolve all components specified in Table 4 in water by boiling in a steamer or on a heatable

magnetic stirrer. Excessive heating shall be avoided. If necessary, adjust to a final pH of 7,2 ± 0,2 at 25 °C.

According to the manufacturer’s instructions, this medium may not be autoclaved. It should not be re-

melted.
6 Apparatus
Usual microbiological laboratory equipment and, in particular, the following:

6.1 Equipment for dry sterilization (oven) and wet sterilization (autoclave), for example

according to EN ISO 7218 [6].

6.2 Incubator, capable of maintaining a temperature of 37 °C ± 1 °C. Optionally also capable of

maintaining a temperature of 40 °C ± 1 °C and/or between 44 °C and 47 °C.

6.3 Water bath, capable of maintaining a temperature of 40 °C ± 1 °C and between 44 °C and 47 °C.

6.4 Blending equipment.
The following apparatus may be used according to EN ISO 7218 [6]:
-1 -1

— a rotary homogenizer (blender) with a notional variable speed of 3 000 min to 10 000 min , as

well as aseptic glass or metals bowls equipped with covers; or

— a peristaltic homogenizer with sterile bags (paddle homogenizer), possibly with the option to adjust

blending speed and time; or
— a vibrational mixer with sterile bags; or

— any other homogenizing system with equivalent efficiency (e.g. a hand blender with aseptic beaker).

6.5 Mechanical stirrer.

A mechanical stirrer (e.g. Vortex Mixer) facilitates the homogenous mixing of decimal dilutions, as

described in e.g. EN ISO 7218 [6].

6.6 Balances, of the required range and accuracy, for example according to EN ISO 7218 [6], for the

different products to be weighed.
6.7 Flasks or screw-cap bottles, of appropriate capacities.
6.8 Test tubes, of appropriate capacities.
6.9 Pipettes or pipettor and sterile tips, to dispense 0,1 ml to 1 ml.

6.10 Sterile pipettes, to dispense 5 ml, for full outlet with wide (approx. 3 mm) tips (e.g. serological

pipette.
NOTE As alternative, 5 ml graduated pipettes without tips can be used.
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6.11 Spreading spatula, sterile L- or triangular-shaped spreaders from glass or metal or sterile

disposable plastic spreaders.

NOTE As alternatives, a spiral plater with a sanitized dispensing system or disposable one-way micro syringes

can be used.
6.12 Sterile Petri dishes, with triple vents (plates), 90 mm in diameter.
6.13 Laminar flow cabinet.

6.14 Microscope, capable of phase-contrast microscopy at a magnification of 600× to 1 000×.

6.15 pH meter, having an accuracy of calibration of ± 0,1 pH unit at 20 °C to 25 °C.

6.16 Steamer or heatable magnetic stirrer, capable to generate the boiling temperature.

7 Sampling

Carry out the sampling procedure in accordance with the specific standard appropriate to the product

concerned. If such a specific standard is not available, it is recommended that agreement be reached on

this subject among the parties concerned. Apply community rules [1] for official control sampling of

animal feeds.

NOTE Sampling can be done according to EN ISO 6497 [7]. Although EN ISO 6497 is not specifically applicable

for microorganisms, due to the lack of other references it seems to be the most suitable protocol to be taken into

account for microorganisms as feed additives.

Take precautions to avoid potential cross-contamination of samples with microorganisms, particularly

after sampling additives and premixtures supplemented with microorganisms. Where required, clean

and disinfect the sampling equipment between each sample, particularly after sampling additives and

premixtures.
Put the sample in a sterile container.
8 Preparation of the test sample

The test sample preparation shall be done in accordance with EN ISO 6498 and the congruent product

standard.
NOTE EN ISO 6498 gives general guidelines on test sample preparation.
9 Procedure
9.1 Preparation of poured agar plates for spread plate method

The culture media are prepared as specified in 5.2.3 according to the manufacturer’s directions. Cool after

autoclaving (for bile aesculin azide agar) or cooking (Slanetz and Bartley agar) in a water bath to a

temperature between 44 °C and 47 °C. Pour portions of approximately 15 ml into each plate (6.12) under

sterile conditions and spread to give a homogeneous layer.

Sodium azide and TTC are heat sensitive ingredients, therefore do not re-melt solidified agar.

When the medium has solidified, pile quantities of four inverted plates on each other and dry at room

temperature or in an incubator at 37 °C ± 1 °C for approximately 12 h or overnight. Alternatively, spread

the plates out in a laminar flow cabinet and dry the agar surface with the lids partially removed for about

30 min.
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Check the dried plates for sterility. If correctly protected against dehydration, dried plates may be stored

in a refrigerator at 5 °C ± 3 °C for up to two weeks. Bring the plates to room temperature before use.

9.2 Preparation of culture media for pour plate method

After sterilization or cooking, hold the medium at a temperature between 44 °C and 47 °C in a water bath

or incubator until the preparation of the pour plates. The prepared medium shall be used at the same day

and the homogeneity of the ingredients shall be ensured.

Sodium azide and TTC are heat sensitive ingredients, therefore do not re-melt solidified agar.

9.3 Preparation of the initial suspension and decimal dilutions

Table 5 gives recommended sample quantities and corresponding volumes of tempered diluent for the

preparation of initial suspensions for various feeds and treatment of the initial suspension.

Table 5 — Recommended sample quantities and corresponding volumes of tempered diluent for

the preparation of initial suspensions for various feeds and treatment of initial suspension

Nature of Critical Sample Diluent for Dilution Treatment of
sample copper quantity suspension factor of initial
content (5.1.1) initial suspension
g or ml
suspension
mg/kg
Additives n.a.
5,0 ± 0,25 (495 ± 9,9) ml 1 : 100
Premixtures > 2 000
Mixer or paddle
blender 5 min
Compound feeds > 200
50,0 ± 2,5 (450 ± 9) ml 1 : 10
Milk replacers n.a.
(90 ± 1,8) ml +
Paste-like and Paddle blender
5 g of
> 400 5,0 ± 0,25 1 : 20
oleaginous feed 5 min
Tween 80 [8]

Weigh the recommended quantity of the sample according to Table 5 into an aseptic blender bowl, beaker

or plastic bag. Add the corresponding amount of tempered tPBS (5.1.1) according to Table 5. If

encapsulated enterococci are analysed, an adequate amount of sample material depending on the size of

the capsules shall be used for the preparation of the initial suspension. The number of capsules should be

greater than 25, to obtain a standard deviation in repeated analysis of less than 20 % for a good repeat of

analysis.

It is recommended to examine the copper content of the initial suspension in a pre-test. Copper contents

exceeding 20 mg/l in the initial suspension require the application of a chelating agent e.g. iminodiacetic

acid in an appropriate concentration with regard to the pH value (see A.2).

If a significantly lower value than expected is obtained in a first analysis, it is recommended to repeat the

analysis with a wider ratio of sample mass to diluent volume (5.1.1) for initial suspension or to blend the

sample with low-germ grain bran or any other neutral carrier to reduce the influence of interfering

substances.

For compound feed, it is strongly recommended to perform the analysis in duplicate to minimize

variation. The result will be the average of both samples. For pelleted compound feeds, a low speed dry

grinding for 30 s is recommended to obtain an unrefined powder. Avoid excessive heating that can

damage the microorganisms.

According to specific procedures for dehydrated products and products with low water activity in

EN ISO 6887-4 [8] it is recommended to weigh out the sample accurately into a pre-dispensed volume of

tempered tPBS (5.1.1) to minimize osmotic shock to the microflora.
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It is recommended to check the pH of the initial suspension and to correct it to a pH between 7,1 and 8,1.

-1 -1
Blend the samples for 5 min in a mixer at a speed of 3 000 min to 5 00
...

SLOVENSKI STANDARD
oSIST prEN 15788:2020
01-marec-2020
Krma: metode vzorčenja in analize - Izolacija in štetje domnevno prisotnih
Enterococcus (E. faecium) spp

Animal feeding stuffs: Methods of sampling and analysis - Isolation and enumeration of

Enterococcus (E. faecium) spp.
Futtermittel: Probenahme- und Untersuchungsverfahren - Trennung und Zählung von
Enterococcus spp. (E. faecium)
Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Isolement et
dénombrement de l’entérocoque (E. faecium) spp.
Ta slovenski standard je istoveten z: prEN 15788
ICS:
65.120 Krmila Animal feeding stuffs
oSIST prEN 15788:2020 en,fr,de

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

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oSIST prEN 15788:2020
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oSIST prEN 15788:2020
DRAFT
EUROPEAN STANDARD
prEN 15788
NORME EUROPÉENNE
EUROPÄISCHE NORM
January 2020
ICS 65.120 Will supersede EN 15788:2009
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Isolation and enumeration of Enterococcus (E. faecium)
spp.

Aliments des animaux - Méthodes d'échantillonnage et Futtermittel: Probenahme- und

d'analyse - Isolement et dénombrement de Untersuchungsverfahren - Trennung und Zählung von

l'entérocoque (E. faecium) spp. Enterococcus spp. (E. faecium)

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

CEN/TC 327.

If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations

which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other

language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC

Management Centre has the same status as the official versions.

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 European Standard. It is distributed for review and comments. It is subject to change without

notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 15788:2020 E

worldwide for CEN national Members.
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Contents Page

European foreword ....................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Principle ............................................................................................................................................................. 6

5 Diluents and selective media ...................................................................................................................... 6

6 Apparatus and glassware ............................................................................................................................. 9

7 Sampling .......................................................................................................................................................... 10

8 Preparation of test sample ....................................................................................................................... 10

9 Procedure........................................................................................................................................................ 10

9.1 Preparation of poured agar plates ......................................................................................................... 10

9.2 Preparation of selective media for pour plate method .................................................................. 11

9.3 Preparation of the initial suspension and decimal dilutions ....................................................... 11

9.4 Inoculation and incubation of the plates ............................................................................................. 12

9.5 Counting of colonies .................................................................................................................................... 13

9.6 Confirmation .................................................................................................................................................. 13

10 Expression of results ................................................................................................................................... 14

11 Precision .......................................................................................................................................................... 15

11.1 General ............................................................................................................................................................. 15

11.2 Interlaboratory studies .............................................................................................................................. 15

11.3 Repeatability .................................................................................................................................................. 15

11.4 Reproducibility ............................................................................................................................................. 15

12 Test report ...................................................................................................................................................... 15

Annex A (informative) Notes on procedure ..................................................................................................... 16

Annex B (informative) Results of the interlaboratory studies ................................................................. 17

Bibliography ................................................................................................................................................................. 20

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European foreword

This document (prEN 15788:2019) has been prepared by Technical Committee CEN/TC 327 “Animal

feeding stuffs: Methods of sampling and analysis”, the secretariat of which is held by NEN.

This document is currently submitted to the CEN Enquiry.
This document will supersede EN 15788:2009.
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Introduction

This methodology has been developed to enumerate enterococci (E. faecium) as feed additives to enable

the European Commission to control proper label ling of animal feeding products (EU project SMT4-

CT98-2235 “Methods for the official control of probiotics (microorganisms) used as animal feeds”) [1]. It

was amended by a second medium and validation data from VDLUFA method 28.2.3 “Enumeration of

Enterococcus faecium” [7]. The method is based on an extensive screening of 12 pre-selected,

commercially available media for the detection and enumeration of enterococci. The described

methodology was validated in interlaboratory studies for Enterococcus faecium. It may be assumed that

the method is also suitable for other Enterococcus spp.

This method is not selective for enterococci (E. faecium) as feed additives, but can be applied to

enumerate enterococci in additives, premixtures and compound feeds assuming that the added

enterococci (E. faecium) are present in far higher numbers than any other enterococci.

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1 Scope

This document defines general rules for the enumeration of enterococci (E. faecium) in feedingstuffs

(additives, premixtures and compound feeds excluding mineral feeds) that contain enterococci as a single

microorganism component or in a mixture with other microorganisms. Applying the method to

compound feeds with critical amounts of copper demands a special procedure (see Annex A). The

document is not applicable to mineral feeds which are defined as complementary feeding stuffs

composed mainly of minerals and containing at least 40 % crude ash (Regulation R767/2009) [4].

There are different categories of feed samples:
a) Additives containing about 10 colony forming units (CFU)/g;
b) Premixtures containing 10 CFU/kg;
c) Compound feeds, meal or pellets which contain about 10 CFU/kg;
The detection limit is defined in EN ISO 7218.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements 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 ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)

EN ISO 7218, Microbiology of food and animal feeding stuffs – General requirements and guidance for

microbiological examinations (ISO 7218)

EN ISO 6887-1, Microbiology of the food chain - Preparation of test samples, initial suspension and decimal

dilutions for microbiological examination - Part 1: General rules for the preparation of the initial suspension

and decimal dilutions (ISO 6887-1)
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 http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
3.1
enterococcus faecium

gram-positive, catalase negative coccus, which usually occurs in pairs or short chains

Note1 to entry This description is based on their characteristics as used for this standard

Note 2 to entry Enterococcus faecium is classified as aerotolerant anaerobe with the ability to reduce 2,3,5-

triphenyl tetrazolium chloride to formazan and capable of hydrolyzing aesculin at 44 °C ± 0,5 °C. It forms colonies

fitting the description of this species on the specified selective media after incubation at a temperature of 37 °C

under aerobic conditions for 24 h resp. 48 h (see 9.6).
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4 Principle

a) Preparation of sterile and dry poured agar plates or preparation of sterile liquid selective medium

tempered at 44 °C to 47 °C.
b) A representative test sample is taken under aseptic conditions.

c) An initial suspension is prepared with a tempered diluent to obtain a homogeneous distribution of

enterococci cells from the test portion.

d) The number of microorganisms per unit volume is reduced by the preparation of further decimal

dilutions from the initial suspension to obtain a countable number of colonies on the selective

enumeration media.

e) Inoculation of prepared poured plates with an aliquot of the optimum dilutions and dispersion of the

inoculum using a sterile spreader or inoculation of blank petri dishes with an aliquot of the optimum

dilutions and pouring of the molten agar medium into each Petri dish, mixing and solidification.

f) The inoculated plates are incubated for 24 h resp. 48 h at 37 °C ± 1 °C under aerobic conditions.

g) Counting of typical colonies, considering the specific properties of Enterococcus faecium as listed in

3.1.

h) Morphological verification of isolates by use of microscopy or biochemical confirmation if necessary.

i) Calculation of the colony forming units of Entercoccus faecium per g or kg of feed sample.

5 Diluents and selective media
5.1 Diluents
5.1.1 Diluent for initial suspension

This diluent is used for the preparation of the initial suspension and may also be used for the preparation

of further decimal dilutions.
Table 1 — Phosphate buffered saline supplemented with Tween 80 (tPBS)
Sodium chloride NaCl 8,00 g
Potassium chloride KCl 0,20 g
Disodium hydrogen phosphate anhydrous Na HPO 1,15 g
2 4
Potassium dihydrogen phosphate anhydrous KH PO 0,20 g
2 4
Polyoxyethylensorbitanmonooleate (Tween 80) 1 ml
Water, distilled or deionized 1 000 ml

Dissolve the components in water. If necessary, adjust to a final pH of 7,3 ± 0,2 at 25 °C after sterilization.

The solution is filled into appropriate containers (e.g. bottles or flasks, test tubes) and sterilized at

121 °C ± 1 °C for 15 min. To avoid loss during autoclaving, screw cap bottles are recommended.

Tween® is an example of a suitable product available commercially. This information is given for the convenience

of users of this document and does not constitute an endorsement by CEN of this product.

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Temper to 40 ± 1°C in a water bath or incubator immediately before usage.

NOTE The use of commercially available PBS buffer tablets is acceptable. However, please take note that

variations in composition and pH can occur between products from different manufacturers and could therefore

give results different from the ones obtained with the medium as specified in this International Standard.

5.1.2 Diluents for serial dilutions

For serial dilutions, the diluent for initial suspension (5.1.1) or alternatively peptone salt solution (PSS)

according to EN ISO 6887-1 can be used.
Table 2 — Peptone salt solution PSS according to EN ISO 6887-1
Enzymatic digest casein 1,0g
Sodium chloride (NaCl) 8,5g
Water, distilled or deionized 1 000 ml

Dissolve the components in the water in flasks, bottles or test tubes. Adjust the pH if necessary so that,

after sterilization, it is 7,0 ± 0,2 at 25 °C. For decimal dilutions, prepare test tubes containing

9,0 ml ± 0,1 ml after sterilization or use screw cap bottles to avoid weight loss during autoclaving.

Sterilize at 121 °C ± 1 °C for 15 min. Bring the diluent to room temperature before use.

NOTE Commercially available, ready-to-use PSS tubes of 9 ml are suitable.
5.2 Enumeration media
5.2.1 General
Two different media are proposed:
a) Bile Aesculin Azide Agar;
b) Enterococcus selective medium according to Slanetz and Bartley.

NOTE Both media can be used for the spread plate method as well as for the pour plate method.

WARNING — The selective media described contain sodium azide. As this substance is highly toxic and

mutagenic, precautions shall be taken to avoid contact with it, especially by inhalation of fine dust during

the preparation of commercially available dehydrated complete media.

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.
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5.2.2 Composition
5.2.2.1 Bile Aesculing Azide Agar
Table 3 — Composition of the agar
Casein peptone (tryptone) 17,0 g
Yeast extract 5,0 g
Peptone 3,0 g
Ox bile, dried 10,0 g
Sodium chloride NaCl 5,0 g
Aesculin 1,0 g
Ammonium iron – III-citrate C H O FeNH 0,5 g
6 8 7 3
Sodium azide NaN 0,15 g
Agar agar 8 g to 18 g
Water, distilled or deionized 1 000 ml
pH 7,1 ± 0,1 at 25 °C
a Depending on the gel strength of the agar.
5.2.2.2 Slanetz and Bartley medium
Table 4 — Composition of the agar
Tryptose 20,0 g
Yeast extract 5,0 g
D-(+)-Glucose 2,0 g
Di-Potassiumhydrogenphosphate K HPO 4,0 g
2 4
Sodium azide NaN 0,4 g
2,3,5-Triphenyltetrazolium chloride 0,1 g
Agar agar 8 g to 18 g
Water, distilled or deionized 1 000ml
pH 7,2 ± 0,2 at 25 °C
a Depending on the gel strength of the agar.
5.2.3 Preparation
5.2.3.1 Bile Aesculin Azide Agar

Dispense the agar medium into suitable containers (bottles or flasks with non-toxic metal screw-caps

may be used). Dissolve all components described in 5.2.2.1in water by boiling. If necessary adjust the pH

so that after sterilization it is pH 7,1 ± 0,1 at 25 °C. Sterilize at 121 °C ± 1 °C for 15 min.

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5.2.3.2 Slanetz and Bartley medium

Dissolve all components described in 5.2.2.2 in water by boiling in a steamer or on a heatable magnetic

stirrer. If necessary, adjust the pH so that after heating it is pH 7,2 ± 1 °C at 25 °C. Excessive heating must

be avoided. According to the manufacturer’s instructions, this medium may not be autoclaved. It should

not be remelted.
6 Apparatus and glassware
Usual microbiological laboratory equipment and, in particular, the following:
6.1 Equipment for dry sterilization (oven) and wet sterilization (autoclave)
According to EN ISO 7218.
6.2 Incubator

Capable of maintaining a temperature of 30 °C ± 1 °C. Optionally also capable of maintaining a

temperature of 40 ± 1°C and/or 44 °C to 47 °C.
6.3 Water bath
Capable of maintaining a temperature of 44 °C to 47 °C and 40 °C ± 1 °C.
6.4 Blending equipment
The following apparatus may be used (EN ISO 7218):

— a rotary homogenizer (blender) with a notional variable speed of 3 000 rpm to 10 000 rpm, as well

as aseptic glass or metals bowls equipped with covers; or

— a peristaltic blender with sterile bags (paddle homogenizer), possibly with the option to adjust

blending speed and time; or
— a vibrational mixer with sterile bags; or

— any other homogenizing system with equivalent efficiency (e.g. a hand blender with aseptic beaker).

6.5 Mechanical stirrer
A mechanical stirrer e.g. Vortex Mixer (see EN ISO 7218), or equivalent.
6.6 Balance

Balances of the required range and accuracy according EN ISO 7218 for the different products to be

weighed.
6.7 Flasks or screw-cap bottles of appropriate capacities
6.8 Test tubes of appropriate capacities
6.9 Pipettes or Pipettor and sterile tips to dispense 0.1 ml to 1 ml
6.10 Sterile 5 ml graduated pipettes

For full outlet with wide (approx. 3 mm) tips (e.g. serological pipette; alternative: 5 ml-graduated pipettes

without tips).
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6.11 Bacterial Cell spreaders

Sterile L- or triangular-shaped spreaders from glass or metal or sterile disposable plastic spreaders.

Alternatively a spiral plater with a sanitized dispensing system or disposable one–way microsyringes can

be used.
6.12 Sterile Petri dishes, triple vent, 90 mm in diameter
6.13 Laminar flow cabinet
6.14 Microscope
Capable of phase-contrast microscopy at a magnification of 600x to 1 000x.
6.15 pH meter
Having an accuracy of calibration of ± 0,1 pH unit at 20 °C to 25 °C.
6.16 Steamer or heatable magnetic stirrer
Capable of maintaining a temperature of 100 °C.
7 Sampling

Carry out the sampling procedure in accordance with the specific standard appropriate to the product

concerned. If such a specific standard is not available, it is recommended that agreement be reached on

this subject among the parties concerned. Apply community rules [1] for official control sampling of

animal feeds.

NOTE Sampling can be done according to EN ISO 6497 [3]. Although EN ISO 6497 is not applicable for

microorganisms, due to the lack of other references, it seems to be the most suitable protocol to be taken into

account for microorganisms as feed additives.

WARNING — Take precautions to avoid potential cross-contamination of samples with microorganisms.

Particularly after sampling additives and premixtures supplemented with microorganisms. Where

required, clean and disinfect sampling equipment between each sample, particularly after sampling

additives and premixtures containing microorganisms. Put the sample in a sterile container.

8 Preparation of test sample

The test sample preparation shall be done in accordance with EN ISO 6498 and the congruent product

standard. EN ISO 6498 gives general guidelines on test sample preparation.
9 Procedure
9.1 Preparation of poured agar plates

The media are prepared as described in 5.2.3 or according to the manufacturer’s directions. Cool after

autoclaving (Bile Aesculin Acide agar) or cooking (Slanetz and Bartley medium) in a water b

...

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