SIST EN 15788:2022

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

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.

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

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;
-1
— Replacement of the required laboratory mixer with a rotation speed of 18 000 min to
-1
22 000 min by homogenization devices, for example according to EN ISO 7218, with a maximal
-1
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.
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.
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.
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;
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.
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.
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
a
Agar
8 g to 18 g
Water, distilled or deionized 1 000 ml
a
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
a
Agar
8 g to 18 g
Water, distilled or deionized 1 000 ml
a
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.
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.
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.
...