INTERNATIONAL ISO

STANDARD 6611
IDF
94
Second edition
2004-10-15

Milk and milk products — Enumeration

of colony-forming units of yeasts
and/or moulds — Colony-count
technique at 25 °C
Lait et produits laitiers — Dénombrement des unités formant colonie de
levures et/ou moisissures — Comptage des colonies à 25 °C
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Reference numbers
ISO 6611:2004(E)
IDF 94:2004(E)

© ISO and IDF 2004

ISO 6611:2004(E)
IDF 94:2004(E)

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 ISO 6611:2004(E)
IDF 94:2004(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.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. 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.

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.

ISO 6611IDF 94 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5,
Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International.
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It is being published jointly by ISO and IDF and separately by AOAC International.
(standards.iteh.ai)
This edition of ISO 6611IDF 94 cancels and replaces ISO 6611:1992, of which it constitutes a minor revision.

ISO 6611:2004
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ISO 6611:2004(E)
IDF 94:2004(E)

Foreword
IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National
Committee in every member country. Every National Committee has the right to be represented on the IDF
Standing Committees carrying out the technical work. IDF collaborates with ISO and AOAC International in
the development of standard methods of analysis and sampling for milk and milk products.

Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the
National Committees for voting. Publication as an International Standard requires approval by at least 50 % of
the National Committees casting a vote.

ISO 6611IDF 94 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5,
Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International.
It is being published jointly by ISO and IDF and separately by AOAC International.

All work was carried out by the Joint ISO/IDF/AOAC Group of Experts, Enumeration of yeasts and moulds in
dairy products (E34), under the aegis of its chairman, Mr J.J. Devoyod (FR).

This edition of ISO 6611IDF 94 cancels and replaces IDF 94B:1990, of which it constitutes a minor revision.
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 ISO 6611:2004(E)
INTERNATIONAL STANDARD
IDF 94:2004(E)

Milk and milk products — Enumeration of colony-forming units

of yeasts and/or moulds — Colony-count technique at 25 °C

1 Scope
This International Standard specifies a method for the detection and enumeration of colony-forming units
(CFU) of viable yeasts and/or moulds in milk and milk products by means of the colony-count technique at
25 °C.

The method is applicable to

 milk, liquid milk products,

 dried milk, dried sweet whey, dried buttermilk, lactose,

 cheese, iTeh STANDARD PREVIEW

 (standards.iteh.ai)
acid casein, lactic casein, rennet casein,

 caseinate, acid whey powder, ISO 6611:2004

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 butter, bc5dcc5ce7a9/iso-6611-2004
 frozen milk products (including edible ices),

 custard, desserts, fermented milk and cream.

NOTE This method is not suitable for a large number of thermolabile yeasts (in fresh cheese). In such cases the
agar-surface-plating method is preferred.

2 Normative references
The following referenced documents are indispensable for the application 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 6887-1, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial
suspension and decimal dilutions

ISO 7218, Microbiology of food and animal feeding stuffs — General rules for microbiological examinations

ISO 8261IDF 122:2001, Milk and milk products — General guidance for the preparation of test samples,
initial suspensions and decimal dilutions for microbiological examination

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ISO 6611:2004(E)
IDF 94:2004(E)

3 Terms and definitions

For the purposes of this document, the following terms and definitions apply.

3.1
yeasts and moulds
microorganisms which at 25 °C form colonies in a selective medium under the conditions specified in this
International Standard

4 Principle
4.1 Poured plates are prepared using a specified selective culture medium and a specified quantity of the
test sample if the initial product is liquid, or of an initial suspension in the case of other products.

Other plates are prepared, under the same conditions, using decimal dilutions of the test sample or of the
initial suspension.

4.2 The plates are aerobically incubated at 25 °C for 5 days.

4.3 The number of colony-forming units (CFU) of yeasts and/or moulds per gram or per millilitre of product
is calculated from the number of colonies obtained on plates chosen at dilution levels so as to give a
significant result.

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5 Diluents and culture medium(standards.iteh.ai)
For general guidance, see ISO 7218.
ISO 6611:2004
5.1 Basic materials
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See ISO 8261IDF 122.

5.1.1 Diluents

For diluents for general use and diluents for special purposes, see ISO 8261IDF 122.

5.1.2 Distribution, sterilization and storage of diluents

See ISO 8261IDF 122.

5.2 Yeast extract/dextrose/oxytetracycline/agar medium

5.2.1 Basic medium

5.2.1.1 Components

Yeast extract powder 5,0 g

Dextrose (C6H12O6) 20,0 g
Agar 10 g to 15 ga
Water 900 ml
a Depending on the gel strength of the agar.

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 ISO 6611:2004(E)
IDF 94:2004(E)

5.2.1.2 Preparation

Dissolve the components or dehydrated complete medium in the water, by heating if necessary.

Adjust the pH, if necessary, so that after sterilization it is 6,6 at 25 °C.

Sterilize in an autoclave (6.1) at 121 °C ± 1 °C for 15 min.

5.2.2 Oxytetracycline hydrochloride solution

5.2.2.1 Components

Oxytetracycline hydrochloride (C22H30O11⋅HCl) 50 mg

Water 50 ml

5.2.2.2 Preparation

Dissolve the oxytetracycline hydrochloride in the water. The solution shall be freshly prepared before use.
Sterilize the solution by means of filtration.

5.2.3 Complete medium

5.2.3.1 Components
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Oxytetracycline hydrochloride solution 10 ml
Basic medium 90 ml
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Cool the sterilized basic medium (5.2.1) to 45 °C. Just before use, bring the oxytetracycline hydrochloride
solution (5.2.2) to 45 °C and add 10 ml of this solution aseptically to 90 ml of the basic medium.

5.3 Yeast extract/dextrose/chloramphenicol/agar medium

5.3.1 Components

Yeast extract powder 5,0 g

Dextrose (C6H12O6) 20,0 g
Chloramphenicol (C11H12Cl2N2O5) 0,1 ga
Agar 12 g to 15 gb
Water 1 000 ml
a In order to obtain a final concentration of 100 µg/ml of medium.
b Depending on the gel strength of the agar.

5.3.2 Preparation

Dissolve the components in the water by heating, if necessary.

Adjust the pH, if necessary, so that after sterilization it is 6,6 at 25 °C.

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ISO 6611:2004(E)
IDF 94:2004(E)

Dispense the agar medium into suitable containers (6.8).

Sterilize in an autoclave (6.1) at 121 °C ± 1 °C for 15 min.

6 Apparatus and glassware

CAUTION — Sterilize all apparatus that will come into contact with the test sample, the diluents, the
IDF 122:2001, 6.1.
dilutions or the culture medium in accordance with ISO 8261

Disposable apparatus is an acceptable alternative to reusable glassware if it has suitable specifications.

Usual microbiological laboratory equipment, the apparatus required for the preparation of test samples and
dilutions as specified in ISO 8261IDF 122 and, in particular, the following.

6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave).

See ISO 7218.

6.2 Incubator, capable of operating at 25 °C ± 1 °C.

6.3 Petri dishes, of 90 mm to 100 mm diameter.

6.4 Graduated pipettes, plugged with cotton wool, calibrated to deliver 1 ml ± 0,02 ml, or 10 ml ± 0,2 ml or
11 ml ± 0,2 ml. iTeh STANDARD PREVIEW
6.5 45 °C ± 1 °C.
Water bath, capable of operating at(standards.iteh.ai)

6.6 Colony-counting equipment, consisting of an illuminated base with a dark background, fitted with a
ISO
magnifying lens to be used at a magnification of ×1,5, 6611:2004
and a mechanical or electronic digital counter.
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6.7 pH-meter, temperature-compensated, accurate to ± 0,1 pH units at 25 °C.
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6.8 Culture bottles or flasks.

Bottles or flasks with non-toxic metal screwcaps may be used.

7 Sampling
A representative sample should have been sent to the laboratory. It should not have been damaged or
changed during transport or storage.

Sampling is not part of the method specified in this International Standard. A recommended sampling method
is given in ISO 707.

In cheeses that are matured with a yeast or mould coat, it may be desirable to exclude the coat from the
sample for analysis. In these instances, the coat may be removed using a sterile scalpel or knife before
sampling is commenced.

8 Procedure

8.1 General
In order to improve the precision of the method, the preparation of dilutions should be carefully standardized.
Factors that affect precision are as follows:

 type of blending equipment;

 blending time;

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 ISO 6611:2004(E)
IDF 94:2004(E)

 diluent;

 time allowed for large particles to settle;

 mixing time allowed in the preparation of decimal dilutions.

CAUTION — Usual aseptic precautions shall be taken. The operations described in 8.2 and 8.3 shall
not be carried out in sunlight.

8.2 Preparation of the test sample and primary dilution

See ISO 8261IDF 122.

8.3 Further decimal dilutions

See ISO 8261IDF 122.

8.4 Duration of the procedure

See ISO 6887-1.

8.5 Inoculation and incubation

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8.5.1 Take two sterile Petri dishes (6.3). Transfer to each dish, by means of a sterile pipette (6.4), 1 ml of
the test sample, if liquid, or 1 ml of the initial suspension in the case of other products.

8.5.2
(standards.iteh.ai)
Take two further sterile Petri dishes. Transfer to each dish, by means of another sterile pipette, 1 ml of
the 10−1 dilution (liquid product) or 1 ml of theISO −2 dilution (other products).
106611:2004
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8.5.3 If necessary, repeat this operation using further decimal dilutions.
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8.5.4 Pour about 15 ml of the medium containing oxytetracycline hydrochloride (5.2) or the medium
containing chloramphenicol (5.3), previously melted and maintained at 45 °C in the water bath (6.5), into each
Petri dish.

8.5.5 Carefully mix the inoculum with the medium by rotating the Petri dishes, and allow the mixture to
solidify by leaving the Petri dishes to stand on a cool horizontal surface.

8.5.6 The time taken between the preparation of the first dilution and the mixing of the inoculum with the
medium shall not exceed 15 min.

8.5.7 Prepare a sufficient number of control plates to check the sterility.

8.5.8 After inverting the prepared dishes (8.5.5), place them (while keeping in an upright position) in the
incubator (6.2) set at 25 °C for 5 days.

To prevent spreading, some precautions should be taken, such as

 the addition of an overlayer of culture medium after resolidifying, or

 the addition of a drop of glycerol on filter paper in the lid of the dish.

8.5.9 Do not stack the dishes more than six high. Separate the stacks of dishes from one another and from
the walls and top of the incubator.

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