EN ISO 6888-1:2021
(Main)Microbiology of the food chain - Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) - Part 1: Method using Baird-Parker agar medium (ISO 6888-1:2021)
Microbiology of the food chain - Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) - Part 1: Method using Baird-Parker agar medium (ISO 6888-1:2021)
This document specifies a horizontal method for the enumeration of coagulase-positive staphylococci by counting the colonies obtained on a solid medium (Baird-Parker medium)[10] after aerobic incubation at 34 °C to 38 °C and coagulase confirmation.
This document is applicable to:
— products intended for human consumption;
— products intended for animal feeding;
— environmental samples in the area of food and feed production, handling, and
— samples from the primary production stage.
This horizontal method was originally developed for the examination of all samples belonging to the food chain.
Because of the large variety of products in the food chain, it is possible that this horizontal method is not appropriate in every detail for all products. Nevertheless, it is expected that the required modifications are minimized so that they do not result in a significant deviation from this horizontal method.
Based on the information available at the time of publication of this document, this method is not considered to be (fully) suited to the examination of fermented products or other products containing technological flora based on Staphylococcus spp (e.g. S. xylosus) (such as cheeses made from raw milk and certain raw meat products) likely to be contaminated by:
— staphylococci forming atypical colonies on a Baird-Parker agar medium;
— background flora that can obscure the colonies being sought.
Nevertheless, both this document and ISO 6888-2 are given equivalent status.
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für die Zählung von koagulase-positiven Staphylokokken (Staphylococcus aureus und andere Spezies) - Teil 1: Verfahren mit Baird-Parker-Agar-Medium (ISO 6888-1:2021)
Dieses Dokument legt ein horizontales Verfahren für die Zählung von koagulase-positiven Staphylokokken durch die Zählung von Kolonien fest, die nach aerober Bebrütung bei 34 °C bis 38 °C und Koagulase-Bestätigung auf einem festen Medium (Baird-Parker-Agar) [10] erhaltenen wurden.
Dieses Dokument ist anwendbar für:
- Erzeugnisse, die für den menschlichen Verzehr vorgesehen sind;
- Erzeugnisse, die als Futtermittel vorgesehen sind;
- Umgebungsproben im Bereich der Herstellung und Handhabung von Lebensmitteln und Futtermitteln; und
- Proben aus dem Bereich der Primärproduktion.
Dieses horizontale Verfahren wurde ursprünglich dazu entwickelt, alle Proben, die zur Lebensmittelkette zählen, zu überprüfen.
Wegen der Vielzahl der Erzeugnisse in der Lebensmittelkette ist es möglich, dass dieses horizontale Verfahren nicht für alle Erzeugnisse bis ins Detail geeignet ist. Dennoch wird erwartet, dass die erforderlichen Modifikationen derart gering gehalten werden, dass sie nicht zu einer erheblichen Abweichung von diesem horizontalen Verfahren führen.
Entsprechend den zum Zeitpunkt der Veröffentlichung dieses Dokuments verfügbaren Informationen gilt dieses Verfahren nicht als (vollständig) geeignet für die Untersuchung von fermentierten Erzeugnissen oder sonstigen Erzeugnissen, die eine auf Staphylococcus spp basierende technologische Flora enthalten (z. B. S. xylosus) (wie z. B. Rohmilchkäse und bestimmte rohe Fleischerzeugnisse), die wahrscheinlich kontaminiert sind durch:
- Staphylokokken, die auf Baird-Parker-Agar-Medium atypische Kolonien bilden;
- Begleitflora, die die gesuchten Kolonien überdecken kann.
Trotzdem sind dieses Dokument und ISO 6888 2 gleichwertig.
WARNUNG - Zum Schutz der Gesundheit des Laborpersonals ist es wesentlich, die Prüfungen zum Nachweis von Staphylokokken nur in angemessen ausgerüsteten Laboren durchzuführen, wobei die Prüfung unter der Leitung eines erfahrenen Mikrobiologen erfolgt, und dass bei der Entsorgung aller bebrüteten Materialien mit großer Sorgfalt vorgegangen wird. Anwender dieses Dokuments sollten mit der üblichen Laborpraxis vertraut sein. Dieses Dokument gibt nicht vor, alle unter Umständen mit der Anwendung des Verfahrens verbundenen Sicherheitsaspekte anzusprechen. Es liegt in der Verantwortlichkeit des Anwenders, angemessene Sicherheits- und Gesundheits¬maßnahmen festzulegen.
Microbiologie de la chaîne alimentaire - Méthode horizontale pour le dénombrement des staphylocoques à coagulase positive (Staphylococcus aureus et autres espèces) - Partie 1: Méthode utilisant le milieu gélosé de Baird-Parker (ISO 6888-1:2021)
Le présent document spécifie une méthode horizontale de dénombrement des staphylocoques à coagulase positive par comptage des colonies obtenues en milieu solide (milieu de Baird-Parker)[10] après incubation en aérobiose entre 34 °C et 38 °C et confirmation par coagulase.
Le présent document s’applique aux:
— produits destinés à la consommation humaine;
— produits destinés à l’alimentation des animaux;
— échantillons environnementaux prélevés dans les secteurs de la production et de la distribution des aliments; et
— échantillons prélevés au stade de production primaire.
Cette méthode horizontale a été élaborée à l’origine pour l’examen de tous les échantillons appartenant à la chaîne alimentaire.
En raison de la grande diversité des produits de la chaîne alimentaire, il est possible que cette méthode horizontale ne soit pas appropriée dans ses moindres détails à tous les produits. Néanmoins, il est attendu que les modifications requises soient réduites le plus possible afin de ne pas s’écarter de manière significative de cette méthode horizontale.
D’après les informations disponibles au moment de la publication du présent document, cette méthode n’est pas considérée comme étant (parfaitement) adaptée à l’examen des produits fermentés ou d’autres produits contenant une flore technologique fondée sur Staphylococcus spp (par exemple, S. xylosus) (tels que les fromages à base de lait cru et certains produits à base de viande crue) susceptibles d’être contaminés par:
— des staphylocoques formant des colonies non caractéristiques sur un milieu gélosé de Baird-Parker;
— une flore annexe pouvant masquer les colonies recherchées.
Néanmoins, le présent document et l’ISO 6888-2 bénéficient d’un statut équivalent.
Mikrobiologija v prehranski verigi - Horizontalna metoda za štetje koagulazno pozitivnih stafilokokov (Staphylococcus aureus in drugih vrst) - 1. del: Metoda uporabe Baird-Parkerjevega agarja (ISO 6888-1:2021)
Ta dokument določa horizontalno metodo za štetje koagulazno pozitivnih stafilokokov
s štetjem kolonij, pridobljenih z uporabo trdnega medija (Baird-Parkerjevega medija)[10] po aerobni inkubaciji pri temperaturi med 34 °C in 38 °C in potrditvi koagulaze.
Ta dokument se uporablja za:
– proizvode, namenjene za prehrano ljudi;
– proizvode, namenjene za živalsko krmo;
– okoljske vzorce na področju proizvodnje hrane in krme; ter
– vzorce iz faze primarne proizvodnje.
Ta horizontalna metoda je bila prvotno razvita za preiskovanje vseh vzorcev, ki pripadajo prehranski verigi.
Zaradi velike raznolikosti proizvodov v prehranski verigi morda ta horizontalna metoda v vseh podrobnostih ni v celoti primerna za vse proizvode. Vseeno gre pričakovati, da je število zahtevanih sprememb manjše, s tem pa ni bistvenih odstopanj od te horizontalne metode.
Na podlagi informacij, ki so bile na voljo v času objave tega dokumenta, ta metoda ni (povsem) primerna za preiskovanje fermentiranih proizvodov ali drugih proizvodov, ki vsebujejo
tehnološko floro na osnovi bakterije Staphylococcus spp (npr. S. xylosus) (npr. sir iz surovega mleka in določeni izdelki iz surovega mesa) in so lahko kontaminirani s/z:
– bakterijo staphylococci, ki razvije atipične kolonije na Baird-Parkerjevem agarju;
– zaledno floro, ki lahko skrije iskane kolonije.
Vseeno imata ta dokument in standard ISO 6888-2 enakovreden status.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-december-2021
Nadomešča:
SIST EN ISO 6888-1:1999
SIST EN ISO 6888-1:1999/A1:2003
SIST EN ISO 6888-1:1999/A2:2018
Mikrobiologija v prehranski verigi - Horizontalna metoda za štetje koagulazno
pozitivnih stafilokokov (Staphylococcus aureus in drugih vrst) - 1. del: Metoda
uporabe Baird-Parkerjevega agarja (ISO 6888-1:2021)
Microbiology of the food chain - Horizontal method for the enumeration of coagulase-
positive staphylococci (Staphylococcus aureus and other species) - Part 1: Method using
Baird-Parker agar medium (ISO 6888-1:2021)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für die Zählung von
koagulase-positiven Staphylokokken (Staphylococcus aureus und andere Spezies) - Teil
1: Verfahren mit Baird-Parker-Agar (ISO 6888-1:2021)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour le dénombrement des
staphylocoques à coagulase positive (Staphylococcus aureus et autres espèces) - Partie
1: Méthode utilisant le milieu gélosé de Baird-Parker (ISO 6888-1:2021)
Ta slovenski standard je istoveten z: EN ISO 6888-1:2021
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN ISO 6888-1
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2021
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes EN ISO 6888-1:1999, EN ISO 6888-
1:1999/A1:2003, EN ISO 6888-1:1999/A2:2018
English Version
Microbiology of the food chain - Horizontal method for the
enumeration of coagulase-positive staphylococci
(Staphylococcus aureus and other species) - Part 1:
Method using Baird-Parker agar medium (ISO 6888-
1:2021)
Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales
horizontale pour le dénombrement des staphylocoques Verfahren für die Zählung von koagulase-positiven
à coagulase positive (Staphylococcus aureus et autres Staphylokokken (Staphylococcus aureus und andere
espèces) - Partie 1: Méthode utilisant le milieu gélosé Spezies) - Teil 1: Verfahren mit Baird-Parker-Agar (ISO
de Baird-Parker (ISO 6888-1:2021) 66888-1:2021)
This European Standard was approved by CEN on 27 April 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 ISO 6888-1:2021 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
This document (EN ISO 6888-1:2021) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 463 “Microbiology of the food chain” the
secretariat of which is held by AFNOR.
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 March 2022, and conflicting national standards shall
be withdrawn at the latest by March 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 ISO 6888-1:1999.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN websites.
According to the CEN-CENELEC Internal Regulations, the national standards organizations 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.
Endorsement notice
The text of ISO 6888-1:2021 has been approved by CEN as EN ISO 6888-1:2021 without any
modification.
INTERNATIONAL ISO
STANDARD 6888-1
Second edition
2021-08
Microbiology of the food chain —
Horizontal method for the
enumeration of coagulase-positive
staphylococci (Staphylococcus aureus
and other species) —
Part 1:
Method using Baird-Parker agar
medium
Microbiologie de la chaîne alimentaire — Méthode horizontale
pour le dénombrement des staphylocoques à coagulase positive
(Staphylococcus aureus et autres espèces) —
Partie 1: Méthode utilisant le milieu gélosé de Baird-Parker
Reference number
ISO 6888-1:2021(E)
©
ISO 2021
ISO 6888-1:2021(E)
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
ISO 6888-1:2021(E)
Contents Page
Foreword .iv
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 2
4.1 General . 2
4.2 Incubation . 2
4.3 Enumeration and confirmation . 2
5 Culture media and reagents . 3
6 Equipment and consumables . 3
7 Sampling . 4
8 Preparation of the test sample . 4
9 Procedure. 4
9.1 Test portion, initial suspension and dilutions . 4
9.2 Inoculation and incubation . 4
9.3 Counting of colonies . 5
9.3.1 General description of colonies growing on BPA medium . 5
9.3.2 Colony counting procedure . 5
9.4 Confirmation . 6
9.4.1 General. 6
9.4.2 Tube test . . 6
9.4.3 Plate test using RPFA medium . 7
10 Expression of results . 7
11 Performance characteristics of the method . 8
11.1 Interlaboratory study . 8
11.2 Repeatability limit . 8
11.3 Reproducibility limit . 8
12 Test report . 9
13 Quality assurance . 9
Annex A (normative) Flow diagram of the procedure .10
Annex B (normative) Culture media and reagents .11
Annex C (informative) Results of the interlaboratory study .18
Bibliography .20
ISO 6888-1:2021(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 463, Microbiology of the food chain, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
This second edition cancels and replaces the first edition (ISO 6888-1:1999), which has been technically
revised. It also incorporates the amendments ISO 6888-1:1999/Amd 1:2003 and ISO 6888-1:1999/
Amd 2:2018. The main changes compared with the previous edition are as follows:
— the title has been changed to relate to the “Food chain”;
— the status of this document and ISO 6888-2 has been clarified;
— the document has been aligned with ISO 7218:2007, i.e. pour molten agar medium at 44 °C to 47 °C;
— all occurrences, when appropriate, have been changed from “35 °C or 37 °C” to “34 °C to 38 °C”;
— all occurrences of incubation time, when appropriate, have been changed from “18 h to 24 h” to
“24 h ± 2 h”;
— requirements have been added to use ISO 11133;
— all available standards related to sampling techniques have been updated;
— a description of typical and atypical colonies on Baird-Parker agar (BPA) medium has been updated;
— the rabbit plasma fibrinogen agar (RPFA) medium has been added as an alternative to the coagulase
test for confirmation;
— the flow diagram procedure in Annex A has been updated;
— culture media and reagents with performance testing in Annex B have been added;
iv © ISO 2021 – All rights reserved
ISO 6888-1:2021(E)
— results of the interlaboratory study (from ISO 6888-1:1999/Amd 1:2003, Precision data) has been
updated;
— the Bibliography has been updated.
A list of all parts in the ISO 6888 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
ISO 6888-1:2021(E)
Introduction
This document, ISO 6888-2 and ISO 6888-3 describe three horizontal methods for the detection
and enumeration of coagulase-positive staphylococci among which enterotoxinogenic strains are
encountered. It is mainly concerned with Staphylococcus aureus, but also with S. intermedius and certain
strains of S. hyicus.
For the purposes of this document, the confirmation of typical and atypical colonies is based on a
positive coagulase reaction, but it is recognized that some strains of Staphylococcus aureus give weakly
positive coagulase reactions. These latter strains can be confused with other bacteria but they can be
distinguished by the use of additional tests not included in this document, such as tests for sensitivity
to lysostaphin, and production of haemolysin, thermostable nuclease and acid from mannitol (see
ISO 7218 and Reference [15]).
The main technical changes listed in the Foreword, introduced in this document compared with the
previous edition are considered as minor (see ISO 17468). They have a minor impact on the performance
characteristics of this method.
Results of the interlaboratory study and samples tested are described in Annex C.
vi © ISO 2021 – All rights reserved
INTERNATIONAL STANDARD ISO 6888-1:2021(E)
Microbiology of the food chain — Horizontal method
for the enumeration of coagulase-positive staphylococci
(Staphylococcus aureus and other species) —
Part 1:
Method using Baird-Parker agar medium
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests
for detecting staphylococci are only undertaken in properly equipped laboratories, under the
control of a skilled microbiologist, and that great care is taken in the disposal of all incubated
materials. Persons using this document should be familiar with normal laboratory practice.
This document does not purport to address all of the safety aspects, if any, associated with its
use. It is the responsibility of the user to establish appropriate safety and health practices.
1 Scope
This document specifies a horizontal method for the enumeration of coagulase-positive staphylococci
[10]
by counting the colonies obtained on a solid medium (Baird-Parker medium) after aerobic incubation
at 34 °C to 38 °C and coagulase confirmation.
This document is applicable to:
— products intended for human consumption;
— products intended for animal feeding;
— environmental samples in the area of food and feed production, handling, and
— samples from the primary production stage.
This horizontal method was originally developed for the examination of all samples belonging to the
food chain.
Because of the large variety of products in the food chain, it is possible that this horizontal method is not
appropriate in every detail for all products. Nevertheless, it is expected that the required modifications
are minimized so that they do not result in a significant deviation from this horizontal method.
Based on the information available at the time of publication of this document, this method is not
considered to be (fully) suited to the examination of fermented products or other products containing
technological flora based on Staphylococcus spp (e.g. S. xylosus) (such as cheeses made from raw milk
and certain raw meat products) likely to be contaminated by:
— staphylococci forming atypical colonies on a Baird-Parker agar medium;
— background flora that can obscure the colonies being sought.
Nevertheless, both this document and ISO 6888-2 are given equivalent status.
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.
ISO 6888-1:2021(E)
ISO 6887 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
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 http:// www .electropedia .org/
3.1
coagulase-positive staphylococci
bacteria that form either typical or atypical colonies, or both, on the surface of a selective culture
medium (Baird-Parker agar medium) and that show a positive coagulase reaction in a tube test or on
rabbit plasma fibrinogen agar
Note 1 to entry: The typical and atypical colonies are described in 9.3.1.
3.2
enumeration of coagulase-positive staphylococci
determination of the number of coagulase-positive staphylococci (3.1) per gram, per millilitre, per square
centimetre or per sampling device/sampled area
Note 1 to entry: A sampled area is an area not defined by a numerical size, for example, a hot tap, a door handle.
4 Principle
4.1 General
Inoculation of the surface of a solid selective culture medium, with a specified quantity of the test
sample if the product is liquid, or with a specified quantity of the initial suspension in the case of other
products.
Inoculation, under the same conditions, using decimal dilutions of the test sample.
4.2 Incubation
Aerobic incubation of the plates at 34 °C to 38 °C and examination after both 24 h and 48 h.
4.3 Enumeration and confirmation
Calculation of the number of coagulase-positive staphylococci per gram, per millilitre, per square
centimetre or per sampling device of sample from the number of either typical or atypical colonies, or
both, obtained on plates at dilution levels chosen to give a significant result, and confirmed by a positive
coagulase test result, within the counting limits of the method and in accordance with ISO 7218.
NOTE See Annex A for a flow diagram.
2 © ISO 2021 – All rights reserved
ISO 6888-1:2021(E)
5 Culture media and reagents
Follow current laboratory practices in accordance with ISO 7218.
The composition of culture media and reagents and their preparation are specified in Annex B.
For performance testing of culture media and reagents, follow the procedures in accordance with either
Annex B or ISO 11133, or both.
For the diluent(s), see the relevant part of the ISO 6887 series.
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following.
6.1 Apparatus for dry sterilization (oven) and wet sterilization (autoclave).
See ISO 7218.
6.2 Incubator, capable of maintaining the inoculated media within the temperature range 34 °C to
38 °C.
NOTE The range 34 °C to 38 °C for incubation of media includes the use of incubators set at 35 °C ± 1 °C,
36 °C ± 2 °C or 37 °C ± 1 °C.
6.3 Water bath, or similar apparatus, capable of being maintained at 44 °C to 47 °C.
6.4 Sterile tubes, bottles or flasks with caps, of appropriate capacity. Bottles or flasks with non-toxic
metallic or plastic screw-caps may be used.
6.5 Sterile Petri dishes, with a diameter of approximately 90 mm or a larger size (optional; diameter
of approximately 140 mm), made of glass or plastic.
6.6 Straight wire (see ISO 7218) or Pasteur pipette.
Loops (of diameter approximately 3 mm) and wires, made of platinum/iridium or nickel/chromium, or
glass rods, or equivalent sterile disposable loops or inoculating needles.
6.7 Sterile graduated pipettes or automatic pipettes of nominal capacities of 1 ml, 2 ml and 10 ml,
graduated in 0,1 ml, 0,1 ml and 0,5 ml divisions, respectively. See ISO 7218.
Graduated pipettes and pipettor tips should be fitted with a non-absorbent cotton wool plug to prevent
contamination when used to manipulate microbial cultures.
6.8 Spreaders, sterile, made of glass or plastic.
6.9 pH-meter, capable of being read to the nearest 0,01 pH unit, enabling measurements to be made
with a tolerance of ±0,1 pH unit. The pH meter shall be equipped with either manual or automatic
temperature compensation. See ISO 7218.
6.10 Refrigerator, capable of operating at 5 °C ± 3 °C.
6.11 Membranes, with a 0,2 µm pore size.
ISO 6888-1:2021(E)
7 Sampling
Sampling is not part of the method specified in this document. Follow the specific International
Standard dealing with the product concerned. If there is no specific International Standard dealing
with the sampling of the product concerned, the parties concerned should come to an agreement on this
subject.
Recommended sampling techniques are given in the following documents:
— ISO/TS 17728 for food and animal feed;
— ISO 707 for milk and milk products;
— ISO 6887-3 for fish and fishery products;
— ISO 13307 for the primary production stage;
— ISO 17604 for carcasses;
— ISO 18593 for surfaces.
It is important that the laboratory receives a sample that is representative. The sample should not have
been damaged or changed during transport or storage.
8 Preparation of the test sample
Prepare the test sample from the laboratory sample in accordance with the specific International
Standard dealing with the product concerned: follow the procedures specified in the ISO 6887 series
and, if necessary, ISO 18593. If there is no specific International Standard available, the parties
concerned should come to an agreement on this subject.
9 Procedure
9.1 Test portion, initial suspension and dilutions
Refer to the relevant part of the ISO 6887 series.
9.2 Inoculation and incubation
Transfer, by means of a sterile pipette (6.7), 0,1 ml of the test sample if liquid, or 0,1 ml of the initial
−1
suspension (10 dilution) in the case of other products, to a Baird-Parker agar (BPA) plate (see B.2).
For enumeration techniques in microbiology of the food chain, the number of Petri dishes to be used
according to the tested dilutions is stated into ISO 7218. Repeat the procedure for further decimal
dilutions if necessary.
If, for certain products, it is desirable to count low numbers of coagulase-positive staphylococci, the
level of detection can be raised by a factor of 10 by inoculating 1,0 ml of the test sample liquid, or 1,0 ml
of the initial suspension for other products, either on the surface of one plate (d = 140 mm) or on the
surface of three small agar plates (d = 90 mm). The number of Petri dishes to be used according to the
dilutions tested is stated in ISO 7218.
Carefully spread the inoculum as quickly as possible over the surface of the agar plate, trying not to
touch the sides of the Petri dish, using the spreader (6.8). Allow the plates to dry with their lids on for
about 15 min at laboratory temperature.
NOTE 1 For inoculation using a spiral plater, see ISO 7218.
4 © ISO 2021 – All rights reserved
ISO 6888-1:2021(E)
Invert the dishes prepared above and place them for 24 h ± 2 h in the incubator (6.2) set at 34 °C to
38 °C. Then re-incubate for a total of 48 h ± 4 h.
NOTE 2 Colonies with typical appearance after 24 h ± 2 h incubation can lose their typical appearance
after 48 h ± 4 h incubation, due to overgrowth with enlargement of the clear zone during the second phase of
incubation. Counting only at 48h ± 4h can lead to low counts or no counts.
9.3 Counting of colonies
9.3.1 General description of colonies growing on BPA medium
9.3.1.1 Colonies presumed to be coagulase-positive staphylococci
Typical colonies are black or grey, shining and convex (1 mm to 1,5 mm in diameter after incubation
for 24 h ± 2 h, and 1,5 mm to 2,5 mm in diameter after incubation for 48 h ± 4 h) and are surrounded
by a clear zone, which can be partially opaque. After incubation for at least 24 h, an opalescent ring
immediately in contact with the colonies can appear in this clear zone.
Atypical colonies have the same size as typical colonies and can present one of the following
morphologies:
— shining black colonies with or without a narrow white edge; the clear zone is absent or barely visible
and the opalescent ring is absent or hardly visible;
— grey colonies free of clear zone.
Atypical colonies are formed mainly by strains of coagulase-positive staphylococci contaminating,
for example, dairy products, shrimps and giblets. They are less often formed by strains of coagulase-
positive staphylococci contaminating other products.
9.3.1.2 Colonies not presumed to be coagulase-positive staphylococci
Other colonies are all the remaining colonies possibly present on the plates that do not show the typical
or atypical appearance described in 9.3.1.1 and are considered as the background flora.
NOTE Bacteria belonging to genera other than staphylococci can give colonies with an appearance similar to
staphylococci. Microscopic examination of Gram stain, before confirmation, will enable the distinction of other
genera from staphylococci.
9.3.2 Colony counting procedure
After incubation for 24 h ± 2 h, mark on the bottom of the plates the positions of any typical colonies
present.
Re-incubate all plates at 34 °C to 38 °C for a further 24 h ± 2 h and mark any new typical colonies. Also
mark any atypical colonies present.
For enumeration, only retain plates containing a maximum of 300 colonies in total (typical, atypical,
background flora), and including a maximum of either 150 typical or atypical colonies, or both, at two
successive dilutions.
EXAMPLE 1
0 typical colonies, 150 atypical colonies and 150 background flora.
EXAMPLE 2
150 t
...
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