FprEN ISO 6888-1

Microbiology of the food chain - Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) - Part 1: Mehod using Baird-Parker agar medium (ISO/FDIS 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/FDIS 6888-1:2021)

Dieses Dokument legt ein horizontales Verfahren für die Zählung von koagulase-positiven Staphylokokken in Lebensmitteln und Futtermitteln fest. In diesem Verfahren werden nach aerober Inkubation bei 34 °C bis 38 °C und Koagulase-Bestätigung auf einem festen Medium (Baird-Parker-Agar) [10] erhaltene Kolonien gezählt und bestätigt.
Dieses Dokument ist anzuwenden 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 zu einer Lebensmittelkette zählen, zu überprüfen.
Wegen der Vielzahl der Produkte in der Lebensmittelkette ist es möglich, dass dieses horizontale Verfahren nicht für alle Produkte bis ins Einzelne 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 Produkten oder sonstigen Produkten, die 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 atypische Kolonien bilden;
- Begleitflora, die die gesuchten Kolonien überdecken kann.
Trotzdem sind dieses Dokument und ISO 6888 2 gleichwertig.

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/FDIS 6888-1:2021)

Mikrobiologija v prehranski verigi - Horizontalna metoda za štetje koagulazno pozitivnih stafilokokov (Staphylococcus aureus in drugih vrst) - 1. del: Metoda uporabe Baird-Parkerjevega agarja (ISO/FDIS 6888-1:2021)

General Information

• Status: Not Published
• ICS: 07.100.30 - Food microbiology
• Technical Committee: CEN/TC 463 - Microbiology of the food chain
• Drafting Committee: CEN/TC 463 - Microbiology of the food chain
• Current Stage: 6055 - CEN Ratification completed (DOR) - Publishing
• Due Date: 27-Apr-2021
• Completion Date: 27-Apr-2021
• Ref Project: kSIST FprEN 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: Mehod using Baird-Parker agar medium (ISO/FDIS 6888-1:2021)

RELATIONS

• Revised: EN ISO 6888-1:1999 - Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) - Part 1: Technique using Baird-Parker agar medium (ISO 6888-1:1999)
• Effective Date: 26-Sep-2018
• Revised: EN ISO 6888-1:1999/A2:2018 - Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) - Part 1: Technique using Baird-Parker agar medium - Amendment 2: Inclusion of an alternative confirmation test using RPFA stab method (ISO 6888-1:1999/Amd 2:2018)
• Effective Date: 26-Sep-2018
• Revised: EN ISO 6888-1:1999/A1:2003 - Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) - Part 1: Technique using Baird-Parker agar medium - Amendment 1: Inclusion of precision data (ISO 6888-1:1999/Amd 1:2003)
• Effective Date: 26-Sep-2018

Standards Content (sample)

SLOVENSKI STANDARD
oSIST prEN ISO 6888-1:2020
01-maj-2020
Mikrobiologija v prehranski verigi - Horizontalna metoda za štetje koagulazno
pozitivnih stafilokokov (Staphylococcus aureus in drugih vrst) - 1. del: Tehnika
uporabe Baird-Parkerjevega agarja (ISO/DIS 6888-1:2020)

Microbiology of the food chain - Horizontal method for the enumeration of coagulase-

positive staphylococci (Staphylococcus aureus and other species) - Part 1: Technique

using Baird-Parker agar medium (ISO/DIS 6888-1:2020)
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/DIS 6888-1:2020)
Microbiologie des aliments - Méthode horizontale pour le dénombrement des

staphylocoques à coagulase positive (Staphylococcus aureus et autres espèces) - Partie

1: Technique utilisant le milieu gélosé de Baird-Parker (ISO/DIS 6888-1:2020)
Ta slovenski standard je istoveten z: prEN ISO 6888-1
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 6888-1:2020 en

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

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oSIST prEN ISO 6888-1:2020
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oSIST prEN ISO 6888-1:2020
DRAFT INTERNATIONAL STANDARD
ISO/DIS 6888-1
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 1:
Technique using Baird-Parker agar medium

Microbiologie des aliments — Méthode horizontale pour le dénombrement des staphylocoques à coagulase

positive (Staphylococcus aureus et autres espèces) —
Partie 1: Technique utilisant le milieu gélosé de Baird-Parker
ICS: 07.100.30
THIS 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 6888-1:2020(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
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oSIST prEN ISO 6888-1:2020
ISO/DIS 6888-1:2020(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

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oSIST prEN ISO 6888-1:2020
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Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Culture media and reagents ...................................................................................................................................................................... 2

6 Equipment and consumables .................................................................................................................................................................. 2

7 Sampling ........................................................................................................................................................................................................................ 3

8 Preparation of 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

9.4 Confirmation ............................................................................................................................................................................................. 5

9.4.1 General...................................................................................................................................................................................... 5

9.4.2 Tube test ...................................................................... ............................................................................................................ 5

9.4.3 Plate test using RPFA .................................................................................................................................................... 6

10 Expression of results ........................................................................................................................................................................................ 6

11 Performance characteristics of the method ............................................................................................................................. 7

11.1 Interlaboratory study ........................................................................................................................................................................ 7

11.2 Repeatability limit ................................................................................................................................................................................ 7

11.3 Reproducibility limit .......................................................................................................................................................................... 7

12 Test report ................................................................................................................................................................................................................... 8

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 .....................................................................................................16

Bibliography .............................................................................................................................................................................................................................19

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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 6888-1:1999), 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;
— 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;
— Description of typical and atypical colonies on BPA;

— RPFA as alternative to coagulase test for confirmation (from ISO 6888-1:1999/Amdendement2: 2018);

— Flow diagram procedure in Annex A;
— Culture media and reagent with performance testing in Annex B;

— Results of the interlaboratory study (from ISO 6888-1:1999/Amendement1: 2003 Precision data);

— Updated bibliography.
A list of all parts in the ISO 6888 series can be found on the ISO website.
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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 groups 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 (see reference [3]) 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 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 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 [9]).

The main technical changes listed in the Foreword, introduced in this document compared to ISO 6888-1

(1999) are considered as minor (see ISO 17468).They have a minor impact on the performance

characteristics of the method.
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oSIST prEN ISO 6888-1:2020
DRAFT INTERNATIONAL STANDARD ISO/DIS 6888-1:2020(E)
Microbiology of the food chain — Horizontal method
for the enumeration of coagulase-positive staphylococci
(Staphylococcus aureus and other species) —
Part 1:
Technique 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

by counting of colonies obtained on a solid medium (Baird-Parker medium) after aerobic incubation at

34 °C to 38 °C.
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 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 http:// www .iso .org/ obp
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— IEC Electropedia: available at http:// www .electropedia .org/
3.1
coagulase-positive staphylococci

bacteria which form typical and/or atypical colonies on the surface of a selective culture medium

(Baird-Parker agar medium) and which 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 clause 9.3.1
3.2
enumeration of the coagulase-positive staphylococci

determination of the number of coagulase-positive staphylococci (3.1) per gram, per millilitre, per

square centimetre, or per sampling device.
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 or of the initial

suspension.
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

4.4 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 typical and/or atypical colonies

obtained on plates at dilution levels chosen to give a significant result, and confirmed by a positive

coagulase test 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 Annex B and/or ISO 11133.
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.
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:

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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 Drying cabinet or oven, capable of operating between 25 °C and 50 °C.

6.4 Water bath, or similar apparatus, capable of being maintained at 44 °C to 47 °C.

6.5 Sterile tubes, bottles or flasks with caps, of appropriate capacity. Bottles or flasks with non-toxic

metallic or plastic screw-caps may be used.

6.6 Sterile Petri dishes, with a diameter of approximately 90 mm and (optional) large size (diameter

approximately 140 mm).
6.7 Straight wire, (see ISO 7218) or Pasteur pipette.

6.8 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.

Sterile Pasteur or graduated pipettes and pipettor tips should be fitted with a non-absorbent cotton

wool plug to prevent contamination when used to manipulate microbial cultures.
6.9 Spreaders, sterile, made of glass or plastic.

6.10 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.
6.11 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:
[7]
— ISO/TS 17728 for food and animal feed ;
[1]
— ISO 707 for milk and milk products ;
[17]
— ISO 6887-3 for fish and fishery products ;
[4]
— ISO 13307 for primary production stage ;
[6]
— ISO 17604 for carcasses ;
[8]
— ISO 18593 for surfaces .
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It is important that the laboratory receive a sample which is truly representative and has not been

damaged or changed during transport or storage.
8 Preparation of 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)

and if necessary ISO 18593. If there is no specific International Standard, 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 (6.8), 0,1 ml of the test sample if liquid, or 0,1 ml of the

initial suspension (10 dilution) in the case of other products, to an agar plate (B.2). For enumeration

techniques in food microbiology, one plate 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 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 (Ø 140mm) or on the

surface of three small agar plates (Ø 90 mm). Also prepare duplicate using an additional three small agar

plates or one large agar plates (see ISO 7218)

9.2.3 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 (6.9). Allow the plates to dry with their lids on for

about 15 min at laboratory temperature.
NOTE For inoculation using a spiral plater, see ISO 7218.

9.2.4 Invert the plates prepared according to the 9.2.3 and incubate them for 24 h ± 2 h then re-

incubate for a total of 48 h ± 4 h in the incubator (6.2) at 34 °C to 38 °C.

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

9.3.1 After incubation for 24 h ± 2 h, mark on the bottom of the plates the positions of any typical

colonies present (see note 1 below).

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 (see note 2 below).

For the enumeration, only retain plates containing a maximum of 300 colonies, including a maximum of

150 typical and/or atypical colonies, at two successive dilutions. ”One of the plates shall contain at least

10 colonies. Select for confirmation (9.4) a given number A (in general 5 typical colonies if there are

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only typical colonies, or 5 atypical colonies if there are only atypical colonies, or 5 typical and 5 atypical

colonies if both types are present, from each plate).”

If there are less than 10 typical and/or atypical colonies present on plates inoculated with undiluted

liquid product or the lowest dilution of other products, it is possible to make an estimated count as

described in 9.3.3 and ISO 7218.

NOTE 1 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 ± 2 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.

NOTE 2 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.

NOTE 3 Other colonies are all the remaining colonies possibly present on the plates that do not show the

typical or atypical appearance described in Notes 1 and 2, and are considered as the background flora.

NOTE 4 Bacteria belonging to genera other than staphylococci may 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 If a 1,0 ml inoculum was spread over three plates (see 9.2.2), treat these plates as one in all

subsequent counting and confirmation procedures.

9.3.3 To make an estimated count of lower numbers of coagulase-positive staphylococci, retain all

plates that contain any typical and atypical colonies. Select all such colonies for confirmation within the

limits set out above.
9.4 Confirmation
9.4.1 General

The confirmation of coagulase-positive staphylococci is undertaken by a tube test (9.4.2). Alternatively,

it can be undertaken by a plate test using RPFA (9.4.3) (see References [14],[15] and[16]).

NOTE An alternative procedure as mentioned in ISO 7218 can be used to confirm isolates as coagulase-

positive staphylococci, providing the suitability of the relevant procedure is verified.

9.4.2 Tube test

From the surface of each selected colony (9.3), remove an inoculum with a sterile wire (6.7) and transfer

it to a tube or bottle of brain-heart infusion broth (B.3).
Incubate at 34 °C to 38 °C for 24 h ± 2 h.

Aseptically add 0,1 ml of each culture to 0,3 ml of the rabbit plasma (B.4) (unless other amounts are

specified by the manufacturer) in sterile haemolysis tubes or bottles (6.5), and incubate at 34 °C to 38°C.

By tilting the tube, examine for clotting of the plasma after 4 h to 6 h of incubation and, if the test is

negative, re-examine after 24 h + 2h of incubation, or examine at the incubation times specified by the

manufacturer.
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Consider the coagulase test to be positive if the cultures yield at least 3+ coagulase reactions according

to the scoring guidance in Figure 1. Reactions from 1+ to 2+ are considered as intermediate.

As a negative control, for each batch of plasma, add 0,1 ml of sterile brain-heart infusion broth (B.3)

to the recommended quantity of rabbit plasma (B.4) and i
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