A Reference number
ISO 6888-2:1999(E)
INTERNATIONAL
STANDARD
ISO
6888-2
First edition
1999-02-15
Microbiology of food and animal feeding
stuffs — 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
ISO 6888-2:1999(E)
© ISO 1999
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic
or mechanical, including photocopying and microfilm, without permission in writing from the publisher.
International Organization for Standardization
Case postale 56 • CH-1211 Genève 20 • Switzerland
Internet iso@iso.ch
Printed in Switzerland
ii
Contents
1 Scope ........................................................................................................................................................................1
2 Normative references ..............................................................................................................................................1
3 Terms and definitions .............................................................................................................................................1
4 Principle....................................................................................................................................................................2
5 Diluent and culture media.......................................................................................................................................2
6 Apparatus and glassware .......................................................................................................................................3
7 Sampling...................................................................................................................................................................4
8 Preparation of test sample......................................................................................................................................4
9 Procedure .................................................................................................................................................................4
10 Expression of results ............................................................................................................................................5
11 Precision.................................................................................................................................................................6
12 Test report ..............................................................................................................................................................6
Bibliography................................................................................................................................................................7
© ISO ISO 6888-2:1999(E)
iii
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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3.
Draft International Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote.
International Standard ISO 6888-1 was prepared by Technical Committee ISO/TC 34, Agricultural food products,
Subcommittee SC 9, Microbiology.
This first edition of ISO 6888-2, together with ISO 6888-1, cancels and replaces ISO 6888:1983, which has been
technically revised.
ISO 6888 consists of the following parts, under the general title 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
Part 2: Technique using rabbit plasma fibrinogen agar medium
ISO 6888-2:1999(E) © ISO
iv
0 Introduction
0.1 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.
0.2 ISO 6888 describes two horizontal methods (part 1 and part 2) for the 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.
In the general case, use part 1 of ISO 6888. However, it is preferable to use the procedure described in part 2 only
for foodstuffs (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.
0.3 For the purposes of this part of ISO 6888, 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 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, of thermostable nuclease and of acid from mannitol (see reference [1]).
INTERNATIONAL STANDARD © ISO ISO 6888-2:1999(E)
1
Microbiology of food and animal feeding stuffs — Horizontal
method for the enumeration of coagulase-positive staphylococci
(Staphylococcus aureus and other species) —
Part 2:
Technique using rabbit plasma fibrinogen agar medium
1 Scope
This part of ISO 6888 describes a horizontal method for the enumeration of coagulase-positive staphylococci in
products intended for human consumption or feeding of animals by counting of colonies obtained on a solid medium
(rabbit plasma fibrinogen medium) after aerobic incubation at 35 °C or 37 °C (see reference [2]).
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this part of ISO 6888. For dated references, subsequent amendments to, or revisions of, any of these publications
do not apply. However, parties to agreements based on this part of ISO 6888 are encouraged to investigate the
possibility of applying the most recent editions of the normative documents indicated below. For undated
references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain
registers of currently valid International Standards.
ISO 6887-1, Microbiology of food and animal feeding stuffs — Rules for the preparation of the test samples, of initial
suspension and of decimal dilutions for microbiological examination — Part 1: General rules for the preparation of
the initial suspension and of decimal dilutions.
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 7218, Microbiology of food and animal feeding stuffs — General rules for microbiological examinations.
3 Terms and definitions
For the purposes of this part of ISO 6888, the following terms and definitions apply.
3.1
coagulase-positive staphylococci
bacteria which form typical colonies in a rabbit plasma fibrinogen selective agar medium when the test is carried out
according to the method specified in this part of ISO 6888
3.2
enumeration of the coagulase-positive staphylococci
determination of the number of coagulase-positive staphylococci found per millilitre or per gram of sample when the
test is carried out according to the method specified in this part of ISO 6888
ISO 6888-2:1999(E) © ISO
2
4 Principle
4.1 Preparation of duplicate poured plates 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
two plates per dilution.
4.2 Incubation of the plates at 35 °C or 37 °C 1) for 18 h to 24 h, and a further 24 h if necessary.
4.3 From the number of typical colonies per Petri dish, calculation of the number of coagulase-positive
staphylococci per millilitre or per gram of test sample.
5 Diluent and culture media
5.1 General
For current laboratory practice, see ISO 7218.
5.2 Diluent
See ISO 6887-1 and the specific standard dealing with the product to be examined.
5.3 Rabbit plasma fibrinogen agar medium (see references [3] and [4]).
NOTE Commercially available media, in accordance with this part of ISO 6888, can be used. Nevertheless, considering the
experienced 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.
5.3.1 Base medium
Prepare the base medium as stated in ISO 6888-1:1999, 5.3.1, with the exception of the distribution of the base
medium, in quantities of 90 ml per flask or bottle.
5.3.2 Solutions
5.3.2.1 Potassium tellurite solution
Prepare the potassium tellurite solution as indicated in ISO 6888-1:1999, 5.3.2.1.
5.3.2.2 Bovine fibrinogen solution
5.3.2.2.1 Composition
Bovine fibrinogen 5 g to 7 g 1)
Sterile water 100 ml
1) Depending on the purity of the bovine fibrinogen.
5.3.2.2.2 Preparation
Under aseptic conditions, dissolve the bovine fibrinogen in the water just prior to use.
1) The temperature is agreed between the interested parties and is indicated in the test report.
© ISO ISO 6888-2:1999(E)
3
5.3.2.3 Rabbit plasma and trypsin inhibitor solution
5.3.2.3.1 Composition
Rabbit plasma with EDTA for coagulase (EDTA
coagulase plasma)
30 ml
Trypsin inhibitor 30 mg
5.3.2.3.2 Preparation
Operating under aseptic conditions, dissolve the components in the water, just prior to use.
5.3.3 Complete medium
5.3.3.1 Composition
Base medium (5.3.1) 90 ml
Potassium tellurite solution (5.3.2.1) 0,25 ml
Bovine fibrinogen solution (5.3.2.2) 7,5 ml
Rabbit plasma and trypsin inhibitor solution (5.3.2.3) 2,5 ml
5.3.3.2 Preparation
Melt the base medium, then let it cool down to 48 °C ± 1 °C in a water bath (6.3).
Under aseptic conditions, add the three solutions previously warmed to 48 °C ± 1 °C in a water bath. Mix thoroughly
after each addition by rotation to minimize foaming.
Use the complete medium immediately after its preparation, in order to avoid any precipitation of the plasma.
WARNING If a commercially available solution of bovine fibrinogen/rabbit plasma is used, follow with
great care the manufacturer's instructions for the preparation of this solution and of the complete medium
(in particular the temperature of the base medium). Otherwise, the medium can completely lose its activity.
5.4 Preparation of agar plates
See ISO 6888-1:1999, 5.3.4.
6 Apparatus and glassware
NOTE Disposable apparatus 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, for maintaining the inoculated media, plates and tubes within the temperature range 35 °C ± 1 °C or
37 °C ± 1 °C.
6.3 Water bath, or similiar apparatus, capable of being maintained at 48 °C ± 1 °C.
6.4 Petri dishes, sterile, made of glass or plastic.
6.5 Total-delivery graduated 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.
ISO 6888-2:1999(E) © ISO
4
7 Sampling
Sampling is not part of the method specified in this part of ISO 6888. If there is no specific International Standard
dealing with sampling of the product concerned, it is recommended that the parties concerned come to an
agreement on this subject.
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 in accordance with the specific International Standard appropriate to the product
concerned. 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 dilution
See ISO 6887-1 and the specific standard appropriate to the product concerned.
9.2 Inoculation and incubation
9.2.1 Take two sterile Petri dishes (6.4). Transfer, by means of a sterile pipette (6.5), 1 ml of the test sample if the
product is liquid, or 1 ml of the initial suspension in the case of other products, to each of the dishes. Take two other
sterile Petri dishes and transfer 1 ml of the first decimal dilution to each of the dishes.
Repeat these operations with successive dilutions using a new sterile pipette for each decimal dilution.
9.2.2 Into each Petri dish (9.2.1), immediately pour freshly prepared complete medium (5.3.3) (do not keep this in
a liquid form) to a depth of approximately 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 thus prepared dishes and place them in the incubator (6.2) set at
35 °C or 37 °C 2) for 18 h to 24 h. If necessary, re-incubate for 18 h to 24 h.
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 show, at the beginning of
incubation, an appearance similar to coagulase-positive staphylococci colonies. However, after 24 h or 48 h of
incubation, they may have the appearance of a spreading culture, more or less brownish, which allows them to be
distinguished from staphylococci.
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.
2) The temperature is agreed between the interested parties and is indicated in the test report.
© ISO ISO 6888-2:1999(E)
5
10 Expression of results
10.1 General case
Select those dishes containing at the maximum 300 colonies, with 100 typical colonies at two successive dilutions.
One dish shall contain at least 15 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:
( )N
C
V n n d
=
+
∑
1 201,
where
C∑ is the sum of the characteristic staphylococcal colonies on all the dishes selected;
V is the volume of inoculum on each dish, in millilitres;
n1 is the number of dishes selected at the first dilution;
n2 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 10x where x is the appropriate power
of 10.
EXAMPLE
A count of a product after inoculation with 0,1 ml of product gave the following results:
for the first dilution selected (10–2): 66 typical colonies and 54 typical colonies;
for the second dilution selected (10–3): 4 typical colonies and 7 typical colonies.
N = + + +
×
=
−
66 54 4 7
2 2 10
5 9552
,
The result, after rounding off, is 6,0 · 103.
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 colonies, report the result as follows.
a) For liquid products, estimated number of coagulase-positive staphylococci per millilitre:
N Ce = 2
where C is the sum of the colonies of coagulase-positive staphylococci counted (9.3) on the two dishes selected;
ISO 6888-2:1999(E) © ISO
6
b) For other products, estimated number of coagulase-positive staphylococci per gram:
N C
de
=
×2
where
C is the sum of the colonies of coagulase-positive staphylococci counted (9.3) on the two dishes selected;
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 Precision
See ISO 7218.
12 Test report
The test report shall specify:
all information necessary for the complete identification of the sample;
the sampling method used, if known;
the test method used, with reference to this part of ISO 6888;
the incubation temperature used;
all operating details not specified in this part of ISO 6888, or regarded as optional, together with details of any
incidents which may have influenced the test results;
the results obtained.
© ISO ISO 6888-2:1999(E)
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Bibliography
[1] KLOOS W.E. Systematics and the natural history of staphylococci. In: Staphylococci, J. Appl. Bacteriol. Symp.
Suppl., 69, 1990, pp. 25 s - 37 s; and Bergey’s Manual of Determinative Bacteriology, 9th edn., 1994.
[2] IDF 145A:1997, Milk and milk products — Enumeration of coagul