EN ISO 6888-2:2021
(Main)Microbiology of the food chain - Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) - Part 2: Method using rabbit plasma fibrinogen agar medium (ISO 6888-2:2021)
Microbiology of the food chain - Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) - Part 2: Method using rabbit plasma fibrinogen agar medium (ISO 6888-2:2021)
This document specifies a horizontal method for the enumeration of coagulase-positive staphylococci by counting the colonies obtained on a solid medium (rabbit plasma fibrinogen agar medium) after aerobic incubation at 34 °C to 38 °C (see Reference [10]).
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 and handling;
— 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 ISO 6888-1 and this document are given equivalent status.
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für die Zählung von koagulase-positiven Staphylokokken (Staphylococcus aureus und andere Spezies) - Teil 2: Verfahren mit Kaninchenplasma-Fibrinogen-Agar-Medium (ISO 6888-2:2021)
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 (Kaninchenplasma-Fibrinogen-Agar-Medium) erhalten wurden (siehe Literaturhinweis [10]).
Dieses Dokument ist anwendbar für:
- Erzeugnissen, die für den menschlichen Verzehr vorgesehen sind;
- Erzeugnissen, 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 zu einer Lebensmittelkette zählen, zu überprüfen.
Wegen der Vielzahl der Erzeugnisse in der Lebensmittelkette ist es möglich, dass dieses horizontale Verfahren für alle Erzeugnisse nicht 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; und
- Begleitflora, die die gesuchten Kolonien überdecken kann.
Trotzdem sind ISO 6888 1 und dieses Dokument gleichwertig.
WARNUNG - Zum Schutz der Gesundheit des Laborpersonals ist es wesentlich, die Prüfungen zur Zählung von Staphylokokken nur in angemessen ausgerüsteten Laboren durchzuführen, wobei die Prüfung unter der Leitung eines erfahrenen Mikrobiologen erfolgt, und dass bei der Entsorgung von 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, geeignete Sicherheits- und Gesundheitsmaßnahmen festzulegen.
Microbiologie de la chaîne alimentaire - Méthode horizontale pour le dénombrement des staphylocoques à coagulase positive (Staphylococcus aureus et autres espèces) - Partie 2 : Méthode utilisant le milieu gélosé au plasma de lapin et au fibrinogène (ISO 6888-2:2021)
Le présent document spécifie une méthode horizontale de dénombrement des staphylocoques à coagulase positive par comptage des colonies obtenues en milieu solide (milieu gélosé au plasma de lapin et au fibrinogène) après incubation en aérobiose entre 34 °C et 38 °C (voir la Référence [10]).
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 entièrement appropriée 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 (complètement) 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, l’ISO 6888-1 et le présent document bénéficient d’un statut équivalent.
Mikrobiologija v prehranski verigi - Horizontalna metoda za štetje koagulazno pozitivnih stafilokokov (Staphylococcus aureus in drugih vrst) - 2. del: Metoda uporabe agarja z zajčjo plazmo iz fibrinogenov (ISO 6888-2:2021)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 6888-2:2021
01-december-2021
Nadomešča:
SIST EN ISO 6888-2:1999
SIST EN ISO 6888-2:1999/A1:2003
Mikrobiologija v prehranski verigi - Horizontalna metoda za štetje koagulazno
pozitivnih stafilokokov (Staphylococcus aureus in drugih vrst) - 2. del: Metoda
uporabe agarja z zajčjo plazmo iz fibrinogenov (ISO 6888-2:2021)
Microbiology of the food chain - Horizontal method for the enumeration of coagulase-
positive staphylococci (Staphylococcus aureus and other species) - Part 2: Method using
rabbit plasma fibrinogen agar medium (ISO 6888-2:2021)Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für die Zählung von
koagulase-positiven Staphylokokken (Staphylococcus aureus und andere Spezies) - Teil
2: Verfahren mit Kaninchenplasma/Fibrinogen-Agar (ISO 6888-2:2021)Microbiologie de la chaîne alimentaire - Méthode horizontale pour le dénombrement des
staphylocoques à coagulase positive (Staphylococcus aureus et autres espèces) - Partie
2 : Méthode utilisant le milieu gélosé au plasma de lapin et au fibrinogène (ISO 6888-
2:2021)Ta slovenski standard je istoveten z: EN ISO 6888-2:2021
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 6888-2:2021 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------SIST EN ISO 6888-2:2021
---------------------- Page: 2 ----------------------
SIST EN ISO 6888-2:2021
EN ISO 6888-2
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2021
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes EN ISO 6888-2:1999, EN ISO 6888-
2:1999/A1:2003
English Version
Microbiology of the food chain - Horizontal method for the
enumeration of coagulase-positive staphylococci
(Staphylococcus aureus and other species) - Part 2:
Method using rabbit plasma fibrinogen agar medium (ISO
6888-2:2021)
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 2 : Méthode utilisant le milieu gélosé Spezies) - Teil 2: Verfahren mit
au plasma de lapin et au fibrinogène (ISO 6888- Kaninchenplasma/Fibrinogen-Agar (ISO 6888-2:2021)
2:2021)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 6888-2:2021 E
worldwide for CEN national Members.---------------------- Page: 3 ----------------------
SIST EN ISO 6888-2:2021
EN ISO 6888-2:2021 (E)
Contents Page
European foreword ....................................................................................................................................................... 3
---------------------- Page: 4 ----------------------SIST EN ISO 6888-2:2021
EN ISO 6888-2:2021 (E)
European foreword
This document (EN ISO 6888-2:2021) 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 6888-2:1999.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 6888-2:2021 has been approved by CEN as EN ISO 6888-2:2021 without any
modification.---------------------- Page: 5 ----------------------
SIST EN ISO 6888-2:2021
---------------------- Page: 6 ----------------------
SIST EN ISO 6888-2:2021
INTERNATIONAL ISO
STANDARD 6888-2
Second edition
2021-08
Microbiology of the food chain —
Horizontal method for the
enumeration of coagulase-positive
staphylococci (Staphylococcus aureus
and other species) —
Part 2:
Method using rabbit plasma
fibrinogen agar medium
Microbiologie de la chaîne alimentaire — Méthode horizontale
pour le dénombrement des staphylocoques à coagulase positive
(Staphylococcus aureus et autres espèces) —
Partie 2: Méthode utilisant le milieu gélosé au plasma de lapin et au
fibrinogène
Reference number
ISO 6888-2:2021(E)
ISO 2021
---------------------- Page: 7 ----------------------
SIST EN ISO 6888-2:2021
ISO 6888-2:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
---------------------- Page: 8 ----------------------
SIST EN ISO 6888-2:2021
ISO 6888-2:2021(E)
Contents Page
Foreword ........................................................................................................................................................................................................................................iv
Introduction ................................................................................................................................................................................................................................vi
1 Scope ................................................................................................................................................................................................................................. 1
2 Normative references ...................................................................................................................................................................................... 2
3 Terms and definitions ..................................................................................................................................................................................... 2
4 Principle ........................................................................................................................................................................................................................ 2
4.1 General ........................................................................................................................................................................................................... 2
4.2 Incubation ................................................................................................................................................................................................... 2
4.3 Enumeration ............................................................................................................................................................................................. 3
5 Culture media and reagents ...................................................................................................................................................................... 3
6 Equipment and consumables .................................................................................................................................................................. 3
7 Sampling ........................................................................................................................................................................................................................ 4
8 Preparation of the test sample ............................................................................................................................................................... 4
9 Procedure (see Figure A.1) ........................................................................................................................................................................ 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 RPFA medium ....................................................... 5
9.3.2 Colony counting procedure .................................................................................................................................... 5
10 Expression of results ........................................................................................................................................................................................ 5
11 Performance characteristics of the method ............................................................................................................................. 5
11.1 Interlaboratory study ........................................................................................................................................................................ 5
11.2 Repeatability limit ................................................................................................................................................................................ 5
11.3 Reproducibility limit .......................................................................................................................................................................... 6
12 Test report ................................................................................................................................................................................................................... 7
13 Quality assurance ................................................................................................................................................................................................ 7
Annex A (normative) Flow diagram of the procedure ........................................................................................................................ 8
Annex B (normative) Culture media and reagents ................................................................................................................................ 9
Annex C (informative) Results of the interlaboratory study .....................................................................................................12
Bibliography .............................................................................................................................................................................................................................14
© ISO 2021 – All rights reserved iii---------------------- Page: 9 ----------------------
SIST EN ISO 6888-2:2021
ISO 6888-2:2021(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 6888-2:1999), which has been technically
revised. It also incorporates the Amendment ISO 6888-2:1999/Amd 1:2003. 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 ISO 6888-1 and this document has been clarified;
— the document has been aligned with ISO 7218:2007, i.e. and 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;
— flow diagram procedure in Annex A has been updated;
— culture media and reagents with performance testing have been added and moved to Annex B;
— performance testing for rabbit plasma fibrinogen agar (RPFA) medium has been added;
— results of the interlaboratory study (from ISO 6888-2:1999/Amendment 1:2003 Precision data)
have been updated;iv © ISO 2021 – All rights reserved
---------------------- Page: 10 ----------------------
SIST EN ISO 6888-2:2021
ISO 6888-2:2021(E)
— 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 2021 – All rights reserved v
---------------------- Page: 11 ----------------------
SIST EN ISO 6888-2:2021
ISO 6888-2:2021(E)
Introduction
ISO 6888-1, this document 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 characterization of staphylococci 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 for
production of haemolysin, thermostable nuclease and acid from mannitol (see ISO 7218 and Reference
[13]).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 the method.The results of the interlaboratory study and samples tested are described in Annex C.
vi © ISO 2021 – All rights reserved---------------------- Page: 12 ----------------------
SIST EN ISO 6888-2:2021
INTERNATIONAL STANDARD ISO 6888-2:2021(E)
Microbiology of the food chain — Horizontal method
for the enumeration of coagulase-positive staphylococci
(Staphylococcus aureus and other species) —
Part 2:
Method using rabbit plasma fibrinogen agar medium
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests
for enumerating 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 ScopeThis document specifies a horizontal method for the enumeration of coagulase-positive staphylococci
by counting the colonies obtained on a solid medium (rabbit plasma fibrinogen agar medium) after
aerobic incubation at 34 °C to 38 °C (see Reference [10]).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 and handling;
— 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 ISO 6888-1 and this document are given equivalent status.
© ISO 2021 – All rights reserved 1
---------------------- Page: 13 ----------------------
SIST EN ISO 6888-2:2021
ISO 6888-2:2021(E)
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 6887 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examinationISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinationsISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media3 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 typical colonies in a selective culture medium (rabbit plasma fibrinogen agar
medium)Note 1 to entry: The typical colonies are described in 9.3.
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 Principle4.1 General
Preparation of poured plate of the rabbit plasma fibrinogen agar 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, follow the
procedure(s) in accordance with ISO 7218.NOTE The volume of rabbit plasma fibrinogen agar medium to be added to the inoculum is a critical point for
the coagulase method and reaction.4.2 Incubation
Aerobic incubation of the plates at 34 °C to 38 °C and examination after 24 h and, if necessary, after
48 h.2 © ISO 2021 – All rights reserved
---------------------- Page: 14 ----------------------
SIST EN ISO 6888-2:2021
ISO 6888-2:2021(E)
4.3 Enumeration
Calculation of the number of coagulase-positive staphylococci per gram of sample, per millilitre of
sample, per square centimetre or per sampling device from the number of typical colonies obtained on
plates at dilution levels chosen to give a significant result within the counting limits of the method and
in accordance with ISO 7218.NOTE See Annex A for a flow diagram.
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 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 Petri dishes, with a diameter of approximately 90 mm, made of glass or plastic.
6.5 Sterile graduated pipettes or automatic pipettes of nominal capacities 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.6.6 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 7218.6.7 Refrigerator, capable of operating at 5 °C ± 3 °C.
6.8 Sterile tubes, bottles or flasks with caps, of appropriate capacity. Bottles or flasks with non-toxic
metallic or plastic screw-caps may be used.6.9 Membranes, with 0,2 µm pore size.
© ISO 2021 – All rights reserved 3
---------------------- Page: 15 ----------------------
SIST EN ISO 6888-2:2021
ISO 6888-2:2021(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 receive 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 (see Figure A.1)
9.1 Test portion, initial suspension and dilutions
See the relevant part of the ISO 6887 series.
9.2 Inoculation and incubation
Transfer, by means of a sterile pipette (6.5), 1 ml of the test sample if liquid, or 1 ml of the initial
suspension (10 dilution) in the case of other products, to a Petri dish (see Annex B). For enumeration
techniques in microbiology of the food chain, the number of Petri dishes to be used, according to the
tested dilutions, is stated in ISO 7218. Repeat the procedure for further decimal dilutions if necessary.
Into each Petri dish (6.4), immediately pour 18 ml to 20 ml freshly prepared complete medium (B.2.3)
to obtain a depth of at least 3 mm.Carefully mix the inoculum with the culture medium and leave to solidify by placing the Petri dishes on
a horizontal surface.After complete solidification, invert the dishes prepared above and place them in the incubator (6.2) set
at 34 °C to 38 °C. After incubation for 24 h ± 2 h, mark on the bottom of the plates the positions of any
typical colonies present. If no colonies or no typical colonies are obtained at 24 h ± 2 h, re-incubate all
plates at 34 °C to 38 °C for a further 24 h ± 2 h (to a total of 48 h ± 4 h), and mark any typical colonies.
NOTE Even if there is an inhibitor of trypsin in the RPFA medium, colonies with typical appearance after
24 h ± 2 h incubation can lose typical appearance after 48 h ± 4 h incubation, due to enzymatic processes (trypsin)
[11]or due to overgrowth. Counting only at 48 h ± 4 h can lead to low counts or no counts.
4 © ISO 2021 – All rights reserved---------------------- Page: 16 ----------------------
SIST EN ISO 6888-2:2021
ISO 6888-2:2021(E)
9.3 Counting of colonies
9.3.1 General description of colonies growing on RPFA medium
Typical colonies are black or grey or even white, small and are surrounded by an opacity halo of
precipitation, indicating coagulase activity. Proteus colonies can appear to look like those of coagulase-
positive staphylococci early on in the incubation. However, they can be distinguished from staphylococci
after 24 h ± 2 h and 48 h ± 4 h of incubation, as their colonies become more or less brownish and start
to spread.9.3.2 Colony counting procedure
At the end of the incubation period (see 9.2), count the typical colonies in each dish.
At reading the plates after 24 h, mark on the bottom of the plates the positions of any typical colonies
present bef...
SLOVENSKI STANDARD
oSIST prEN ISO 6888-2:2020
01-maj-2020
Mikrobiologija v prehranski verigi - Horizontalna metoda za štetje koagulazno
pozitivnih stafilokokov (Staphylococcus aureus in drugih vrst) - 2. del: Tehnika
uporabe agarja z zajčjo plazmo iz fibrinogenov (ISO/DIS 6888-2:2020)
Microbiology of the food chain - Horizontal method for the enumeration of coagulase-
positive staphylococci (Staphylococcus aureus and other species) - Part 2: Technique
using rabbit plasma fibrinogen agar medium (ISO/DIS 6888-2:2020)Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für die Zählung von
koagulase-positiven Staphylokokken (Staphylococcus aureus und andere Spezies) - Teil
2: Verfahren mit Kaninchenplasma/Fibrinogen-Agar (ISO/DIS 6888-2:2020)Microbiologie des aliments - Méthode horizontale pour le dénombrement des
staphylocoques à coagulase positive (Staphylococcus aureus et autres espèces) - Partie
2: Technique utilisant le milieu gélosé au plasma de lapin et au fibrinogène (ISO/DIS
6888-2:2020)Ta slovenski standard je istoveten z: prEN ISO 6888-2
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 6888-2:2020 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------oSIST prEN ISO 6888-2:2020
---------------------- Page: 2 ----------------------
oSIST prEN ISO 6888-2:2020
DRAFT INTERNATIONAL STANDARD
ISO/DIS 6888-2
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2020-03-09 2020-06-01
Microbiology of the food chain — Horizontal method
for the enumeration of coagulase-positive staphylococci
(Staphylococcus aureus and other species) —
Part 2:
Technique using rabbit plasma fibrinogen agar medium
Microbiologie des aliments — Méthode horizontale pour le dénombrement des staphylocoques à coagulase
positive (Staphylococcus aureus et autres espèces) —Partie 2: Technique utilisant le milieu gélosé au plasma de lapin et au fibrinogène
ICS: 07.100.30THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 6888-2:2020(E)
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. ISO 2020
---------------------- Page: 3 ----------------------
oSIST prEN ISO 6888-2:2020
ISO/DIS 6888-2:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020
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
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 4 ----------------------
oSIST prEN ISO 6888-2:2020
ISO/DIS 6888-2:2020(E)
Contents Page
Foreword ........................................................................................................................................................................................................................................iv
Introduction ..................................................................................................................................................................................................................................v
1 Scope ................................................................................................................................................................................................................................. 1
2 Normative references ...................................................................................................................................................................................... 1
3 Terms and definitions ..................................................................................................................................................................................... 1
4 Principle ........................................................................................................................................................................................................................ 2
4.1 General ........................................................................................................................................................................................................... 2
4.2 Incubation ................................................................................................................................................................................................... 2
4.3 Enumeration ............................................................................................................................................................................................. 2
5 Culture media and reagents ...................................................................................................................................................................... 2
6 Equipment and consumables .................................................................................................................................................................. 3
7 Sampling ........................................................................................................................................................................................................................ 3
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 ........................................................................................................................................................................... 4
10 Expression of results ........................................................................................................................................................................................ 4
10.1 General case ............................................................................................................................................................................................... 5
10.2 Estimation of low numbers .......................................................................................................................................................... 5
11 Performance characteristics of the method ............................................................................................................................. 6
11.1 Interlaboratory study ........................................................................................................................................................................ 6
11.2 Repeatability limit ................................................................................................................................................................................ 6
11.3 Reproducibility limit .......................................................................................................................................................................... 7
12 Test report ................................................................................................................................................................................................................... 7
13 Quality assurance ................................................................................................................................................................................................ 8
Annex A (normative) Flow diagram of the procedure ........................................................................................................................ 9
Annex B (normative) Culture media and reagents .............................................................................................................................10
Annex C (informative) Results of the interlaboratory study .....................................................................................................13
Bibliography .............................................................................................................................................................................................................................15
© ISO 2020 – All rights reserved iii---------------------- Page: 5 ----------------------
oSIST prEN ISO 6888-2:2020
ISO/DIS 6888-2:2020(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 on 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 the following
URL: www .iso .org/ iso/ foreword .html.This document was prepared by Technical Committee ISO/TC 34, Food Products, Subcommittee SC 9,
Microbiology.This second edition cancels and replaces the first edition (ISO 6888-2:1999), which has been technically
revised.The main changes compared to the previous edition are as follows:
— Title relates to “Food chain”;
— Precision on the status of ISO 6888 part 1 and part 2;
— Consistency with ISO 7218 (2007, Amdt 1 2013): one plate per dilution and pour molten agar medium
at 44 °C to 47 °C;— All occurrences when appropriate; “35 °C or 37 °C” to “34 °C to 38 °C”;
— Normative reference ISO 11133;
— All available standards related to sampling techniques;
— Precision data;
— Flow diagram procedure in Annex A;
— Culture media and reagent with performance testing in Annex B;
— Performance testing for RPFA;
— Results of the interlaboratory study;
— Updated bibliography.
A list of all parts in the ISO 6888 series can be found on the ISO website.
iv © ISO 2020 – All rights reserved
---------------------- Page: 6 ----------------------
oSIST prEN ISO 6888-2:2020
ISO/DIS 6888-2:2020(E)
Introduction
1.1 Because of the large variety of food and feed products, this horizontal method may not be
appropriate in every detail for certain products. In this case, different methods, which are specific to
these products, may be used if absolutely necessary for justified technical reasons. Nevertheless, every
attempt should be made to apply this horizontal method as far as possible.When this part of ISO 6888 is next reviewed, account will be taken of all information then available
regarding the extent to which this horizontal method has been followed and the reasons for deviations
from this method in the case of particular products.The harmonization of test methods cannot be immediate and, for certain group of products,
International Standards and/or national standards may already exist that do not comply with this
horizontal method. It is hoped that when such standards are reviewed they will be changed to comply
with this part of ISO 6888 so that eventually the only remaining departures from this horizontal
method will be those necessary for well-established technical reasons.1.2 ISO 6888 describes three horizontal methods (part 1, part 2 and part 3) 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.“Both parts 1 and 2 of ISO 6888 are given equivalent status. Nevertheless, it is recommended to use the
procedure described in ISO 6888-2 for the foods (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 which can obscure the colonies being sought.
1.3 For the purposes of this part of ISO 6888, the characterisation of staphylococci 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 may be confused with other bacteria but they may
be distinguished from such other bacteria by the use of additional tests not included in this part of
ISO 6888, such as the sensitivity to lysostaphin, the production of haemolysin, thermostable nuclease
and acid from mannitol (see ISO 7218 and reference [1]).The main technical changes listed in the Foreword, introduced in this document compared to ISO 6888-2
(1999), are considered as minor (see ISO 17468).They have a minor impact on the performance characteristics of the method.
© ISO 2020 – All rights reserved v
---------------------- Page: 7 ----------------------
oSIST prEN ISO 6888-2:2020
---------------------- Page: 8 ----------------------
oSIST prEN ISO 6888-2:2020
DRAFT INTERNATIONAL STANDARD ISO/DIS 6888-2:2020(E)
Microbiology of the food chain — Horizontal method
for the enumeration of coagulase-positive staphylococci
(Staphylococcus aureus and other species) —
Part 2:
Technique using rabbit plasma fibrinogen 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 ScopeThis document specifies a horizontal method for the enumeration of coagulase-positive staphylococci
by counting of colonies obtained on a solid medium (rabbit plasma fibrinogen medium) after aerobic
incubation at 34 °C to 38 °C (see reference [2]).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.
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 6887 (all parts), Microbiology of the food chain — Rules for the preparation of the test sample, of initial
suspension and of decimal dilutions for microbiological examinationISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinationsISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture mediaISO 17468, Microbiology of the food chain — Technical requirements and guidance on establishment or
revision of a standardized reference method3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
© ISO 2020 – All rights reserved 1
---------------------- Page: 9 ----------------------
oSIST prEN ISO 6888-2:2020
ISO/DIS 6888-2:2020(E)
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at http:// www .iso .org/ obp— IEC Electropedia: available at http:// www .electropedia .org/
3.1
coagulase-positive staphylococci
bacteria which form typical colonies in or on a selective culture medium (rabbit plasma fibrinogen
agar medium).Note 1 to entry: : the typical colonies are described in clause 9.3.
3.2
enumeration of the coagulase-positive staphylococci
determination of the number of coagulase-positive staphylococci (3.1) per millilitre or per gram, per
square centimetre or per sampling device..4 Principle
4.1 General
Preparation of poured plate of the rabbit plasma fibrinogen agar 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 or of the initial
suspension, with one plate per dilution.4.2 Incubation
Aerobic incubation of the plates at 34 °C to 38 °C and examination after 24hand if necessary after 48 h.
4.3 EnumerationCalculation of the number of coagulase-positive staphylococci per gram, per millilitre, per square
centimetre or per sampling device of sample from the number of typical colonies obtained on plates at
dilution levels chosen to give a significant result.NOTE See Annex A for flow diagram
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, follow the procedures in accordance with ISO 11133 and
Annex B.For the diluent(s), see the relevant part of ISO 6887 series.
Commercially available media, in accordance with this document, can be used. Nevertheless,
considering the known variability of manufactured lots of the supplement, it is recommended that
each batch of bovine fibrinogen/rabbit plasma solution be tested before use, by running positive and
negative controls.2 © ISO 2020 – All rights reserved
---------------------- Page: 10 ----------------------
oSIST prEN ISO 6888-2:2020
ISO/DIS 6888-2:2020(E)
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 for 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 or 37
°C ± 1 °C.6.3 Water bath, or similar apparatus, capable of being maintained at 44 °C to 47 °C.
6.4 Petri dishes, with a diameter of approximately 90 mm, sterile, made of glass or plastic.
6.5 Sterile graduated pipettes or automatic pipettes of nominal capacities 1 ml, 2 ml and 10 ml,
graduated in 0,1 ml, 0,1 ml and 0,5 ml divisions, respectively.Graduated pipettes and pipettor tips should be fitted with a non-absorbent cotton wool plug to prevent
contamination when used to manipulate microbial cultures.6.6 pH-meter, it shall be 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.6.7 Refrigerator capable of operating at 5°C±3°C.
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, it is recommended that the parties concerned come to an
agreement on this subject.Recommended sampling techniques are given in the following documents:
[3]
— ISO/TS 17728 for food and animal feed ;
[4]
— ISO 707 for milk and milk products ;
[5]
— ISO 6887-3 for fish and fishery products ;
[6]
— ISO 13307 for primary production stage ;
[7]
— ISO 17604 for carcasses ;
[8]
— ISO 18593 for surfaces .
It is important that the laboratory receive a sample which is truly representative and has not been
damaged or changed during transport or storage.© ISO 2020 – All rights reserved 3
---------------------- Page: 11 ----------------------
oSIST prEN ISO 6888-2:2020
ISO/DIS 6888-2:2020(E)
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 ISO 6887 (all parts).
If there is no specific International Standard available, it is recommended that the parties concerned
come to an agreement on this subject.9 Procedure
9.1 Test portion, initial suspension and dilutions
See the relevant part of ISO 6887 series.
9.2 Inoculation and incubation
9.2.1 Transfer, by means of a sterile pipette (6.5), 1 ml of the test sample if liquid, or 1 ml of the initial
suspension (10 dilution) in the case of other products, to an agar Petri dish (Annex B). For enumeration
techniques in food microbiology, one Petri dish per dilution shall be used with at least two successive
dilutions. Repeat the procedure for the 10- dilution and for further decimal dilutions if necessary. If only
one dilution is plated, two plates have to be used.9.2.2 Into each Petri dish (9.2.1), immediately pour 18 – 20 ml freshly prepared complete medium
(B.2.3) (do not keep this in a liquid form) to obtain a depth of at least 3 mm.Carefully mix the inoculum with the culture medium and leave to solidify by placing the Petri dishes on
a cool horizontal surface.9.2.3 After complete solidification, invert the dishes and place them in the incubator (6.2) set at 34 °C
to 38 °C. Examine plates after 24 h ± 2 h and, if necessary, after 48 h ± 4 h incubation. If no colonies or no
typical colonies are obtained at 24 h ± 2 h, incubate until 48 h ± 4 h.NOTE Colonies with typical appearance after 24 h ± 2 h incubation can lose typical appearance after 48 h
± 4 h incubation, due to enzymatic processes (trypsin) of secondary growth or due to overgrowth by secondary
growth. Counting only at 24 h ± 2 h can lead to too low counts or no counts.9.3 Counting of colonies
After a sufficient incubation period (see 9.2.3), the staphylococci form black or grey or even white,
small colonies surrounded by a halo of precipitation, indicating coagulase activity. Proteus colonies may
appear to look like those of coagulase-positive staphylococci early on in the incubation. However, they
can be distinguished from staphylococci after 24 h and 48 h of incubation, as their colonies become
more or less brownish and start to spread.Count the typical colonies in each dish.
NOTE As the rabbit plasma fibrinogen agar is based on a coagulase reaction, it is not necessary to confirm
this activity.10 Expression of results
For calculation of the results, follow the procedure(s) in accordance with ISO 7218. Calculate and report
the results as the number of coagulase-positive staphylococci in cfu per gram, millilitre, per square
centimetre or per sampling device.In cases where no colonies of the target microorganism have been detected, follow ISO 7218 for the
expression of results for special cases.4 © ISO 2020 – All rights reserved
---------------------- Page: 12 ----------------------
oSIST prEN ISO 6888-2:2020
ISO/DIS 6888-2:2020(E)
10.1 General case
Select those dishes containing at the maximum 300 colonies, with 100 typical colonies per Petri dish.
One dish shall contain at least 10 typical colonies.Calculate the number N of coagulase-positive staphylococci present per millilitre or per gram of product
as a weighted mean from two successive dilutions using the following equation:Vn +01, nd
where
ΣC is the sum of the characteristic staphylococcal colonies on all the dishes selected;
V is the volume of inoculum on each dish, in millilitres;n is the number of dishes selected at the first dilution;
n is the number of dishes selected at the second dilution;
d is the dilution rate corresponding to the first dilution selected (the initial suspension is a
dilution).Round off the calculated results to two significant figures (see ISO 7218).
Take as the result the number of coagulase-positive staphylococci per millilitre (liquid products) or per
gram (other products), expressed as a number between 1,0 and 9,9 inclusive, multiplied by 10 where x
is the appropriate power of 10.EXAMPLE
A count of a product after inoculation with 1 ml of product gave the following results:
— for the first dilution selected (10 ): 66 typical colonies;— for the second dilution selected (10 ): 4 typical colonies.
66+4
N= =6364
11, ×10
The result, after rounding off, is 6,4 x 10 .
10.2 Estimation of low numbers
10.2.1 If the two dishes, corresponding to the test sample (liquid products) or the initial suspension
(other products) each contain less than 15 identified colonies, report the result as follows.
a) For liquid products, estimated number of coagulase-positive staphylococci per millitre (Ne):
N =V×2
where C is the sum of the colonies of coagulase-positive staphylococci counted (9.3) on the two
dishes selected;b) For other products, estimated number of coagulase-positive staphylococci per gram (Ne):
© ISO 2020 – All rights reserved 5---------------------- Page: 13 ----------------------
oSIST prEN ISO 6888-2:2020
ISO/DIS 6888-2:2020(E)
N =
V××2 d
where
C is the sum of the colonies of coagulase-positive staphylococci counted (9.3) on the two dishes
selected;V is the volume of inoculum on each dish, in millilitres;
d is the dilution rate of the initial suspension.
10.2.2 If the two dishes, corresponding to the test sample (liquid products) or the initial suspension
(other products) do not contain any coagulase-positive staphylococcal colony, report the result as
follows:— less than 1 coagulase-positive staphylococci per millilitre (liquid products);
— less than 1/d coagulase-positive staphylococci per gram (other products), where d is the dilution
rate of the initial suspension.11 Performance characteristics of the method
11.1 Interlaboratory study
Results of the interlaboratory study to determine the precision of the method are summarized in
Annex C. Repeatability and reproducibility limits were determined using five sample types (broiler
caecal material, frozen spinach, minced meat, raw milk, chicken skin) contaminated at various levels.
The values derived from the interlaboratory study may not be applicable to concentration ranges and
sample types other than those given in Annex C.11.2 Repeatability limit
The absolute difference between two single (log -transformed) test results (number of coagulase-
positive staphylococci per gram or per millilitre) or the ratio of the higher to the lower of the two test
results on the normal scale, obtained using the same method on identical test material in the same
laboratory by the same operator using the same equipment within the shortest feasible time interval
will, in no more than 5 % of cases, exceed the repeatability limit r.As a general indication of the repeatability limit r, the following values (med
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.