ISO-Staphylococcus Aureus Parte 2
ISO-Staphylococcus Aureus Parte 2
ISO-Staphylococcus Aureus Parte 2
STANDARD 6888-2
Second edition
2021-08
Reference number
ISO 6888-2:2021(E)
© ISO 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 iTeh STANDARD PREVIEW
General description of colonies growing on RPFA medium........................................................ 5
9.3.2 Colony counting procedure..................................................................................................................................... 5
10
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Expression of results......................................................................................................................................................................................... 5
11 Performance characteristics of the ISO method6888-2:2021 .............................................................................................................................. 5
11.1 Interlaboratory study......................................................................................................................................................................... 5
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11.2 Repeatability limit.................................................................................................................................................................................
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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
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
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World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/
iso/foreword.html. (standards.iteh.ai)
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology, in collaboration with the European ISO 6888-2:2021
Committee for Standardization (CEN) Technical
Committee CEN/TC 463, Microbiology of the food chain, in accordance with the Agreement on technical
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cooperation between ISO and CEN (Vienna102782f5445b/iso-6888-2-2021
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;
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.
1 Scope
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
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aerobic incubation at 34 °C to 38 °C (see Reference [10]).
This document is applicable to: (standards.iteh.ai)
— products intended for human consumption;
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— products intended for animal feeding;
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— 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.
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 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
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, 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.
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.
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.
6.8 Sterile tubes, bottles or flasks with caps, of appropriate capacity. Bottles or flasks with non-toxic
metallic or plastic screw-caps may be used.
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.
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.
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 before further incubation.
If plates are re-incubated at 34 °C to 38 °C for 24 h ± 2 h, mark any new typical colonies.
For enumeration, only retain plates containing a maximum of 300 colonies, with 100 typical colonies.
One of the plates shall contain at least 10 colonies.
NOTE As the rabbit plasma fibrinogen agar medium is based on a coagulase reaction, it is not necessary to
confirm this activity.
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When the number of cfu is expected to be at or near the limit of determination, the use of duplicate
plates is preferable. If duplicate plates are used the minimum for the sum of colonies should be 10. In
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this case, the level of contamination is expected to be higher than 5 cfu/ml for liquid samples or higher
than 50 cfu/g for solid samples.
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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 (colony forming unit) 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.