FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 8784-3
ISO/TC 6/SC 2
Pulp, paper and board —
Secretariat: SIS
Microbiological examination —
Voting begins on:
2021-12-20
Part 3:
Voting terminates on:
Enumeration of yeast and mould
2022-02-14
based on disintegration
Pâtes, papiers et cartons — Analyse microbiologique —
Partie 3: Dénombrement des levures et des moisissures basé sur la
désintégration
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 SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/FDIS 8784-3:2021(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. © ISO 2021
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ISO/FDIS 8784-3:2021(E)
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© ISO 2021

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ISO/FDIS 8784-3:2021(E)
Contents Page

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

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

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

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

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

5 Culture media and diluents ......................................................................................................................................................................2

5.1 General ........................................................................................................................................................................................................... 2

5.2 Water ............................................................................................................................................................................................................... 2

5.3 Culture media for total yeast and mould count ......................................................................................................... 2

5.4 Diluents ......................................................................................................................................................................................................... 2

5.5 Non-ionic surfactant .......................................................................................................................................................................... 3

6 Apparatus and equipment ......................................................................................................................................................................... 3

7 Sampling ....................................................................................................................................................................................................................... 4

8 Preparation of the test material.......................................................................................................................................................... 4

8.1 General ........................................................................................................................................................................................................... 4

8.2 Weighing ...................................................................................................................................................................................................... 4

8.3 Disintegration ......................................................................................................................................................................................... 5

9 Determination of yeast and mould count .................................................................................................................................. 5

9.1 General ........................................................................................................................................................................................................... 5

9.2 Plating for yeast and mould count ......................................................................................................................................... 5

9.3 Incubation ................................................................................................................................................................................................... 5

10 Enumeration of the colonies ....................................................................................................................................................................6

11 Calculation and report ................................................................................................................................................................................... 6

11.1 Calculation .................................................................................................................................................................................................. 6

11.2 Interpretation .......................................................................................................................................................................................... 7

11.3 Report ............................................................................................................................................................................................................. 7

12 Test report .................................................................................................................................................................................................................. 7

Bibliography ................................................................................................................................................................................................................................ 9

iii
© ISO 2021 – All rights reserved
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ISO/FDIS 8784-3:2021(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 6, Paper, board and pulps, Subcommittee

SC 2, Test methods and quality specifications for paper and board.
A list of all parts in the ISO 8784 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
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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 8784-3:2021(E)
Pulp, paper and board — Microbiological examination —
Part 3:
Enumeration of yeast and mould based on disintegration
1 Scope

This document specifies a method for determining the total number of colony-forming units of yeast

and mould in dry market pulp, paper and paperboard after disintegration. The enumeration relates to

specific media.
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 186, Paper and board — Sampling to determine average quality

ISO 638-1, Paper, board, pulps and cellulosic nanomaterials - Determination of dry matter content by oven-

drying method - Part 1: Materials in solid form
ISO 7213, Pulps — Sampling for testing
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminology databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
yeast

mesophilic aerobic microorganism which, at 25 °C using mycological agar medium under the conditions

described in this document, develops matte or shiny round colonies on the surface of the medium,

usually having a regular outline and a more or less convex surface

Note 1 to entry: Yeasts within, rather than on, a medium develop round, lenticular, colonies.

3.2
mould

mesophilic aerobic filamentous microorganism which, on the surface of mycological agar medium

under the conditions described in this document, usually develops flat or fluffy spreading propagules/

germs or colonies often with coloured fruiting or sporing structures

Note 1 to entry: Moulds within, rather than on, a medium can develop round, lenticular, colonies.

3.3
yeast and mould count

number of colony-forming units (CFU) of yeast (3.1) and mould (3.1) formed after incubation in a

This pour plate method involves enumeration of colonies in a standard culture medium. A fibre

suspension, prepared from paper, paperboard, or pulp samples, is plated in agar.

The plates are incubated at 25 °C ± 1 °C for 5 days. The total numbers of yeast and mould are enumerated

by counting the colonies formed in the agar. The results are expressed as the number of CFU per gram

of sample.
5 Culture media and diluents
5.1 General

All substrates and diluents shall be appropriately sterilized. When preparing the culture medium, make

sure that the ingredients are completely dissolved by mixing while heating prior to dispensing into

suitable containers for sterilization. See ISO 11133 for quality assurance and guidelines on preparation

and production of culture media.
5.2 Water

When water is mentioned in a formula, use distilled water or purified water, see ISO 11133.

5.3 Culture media for total yeast and mould count

Culture medium shall be prepared as follows, or from commercially available dehydrated culture media

according to the manufacturer’s instructions. Ready-to-use medium may be used when its composition

is comparable to that given in this part of ISO 8784. To test the performance of the medium, see

ISO 11133.
Sabouraud dextrose chloramphenicol agar medium (SDCA) composition per litre:
— dextrose 40,0 g
— peptic digest of animal tissue 5,0 g
— pancreatic digest of casein 5,0 g
— chloramphenicol 0,050 g
— agar 15,0 g
— water 1 000 ml

Dissolve the components (including the chloramphenicol) or the dehydrated complete medium in the

water by heating. Dispense the medium into suitable containers. Sterilize in an autoclave at 121 °C for

15 min. After sterilization the pH shall be equivalent to 5,6 ± 0,2 when measured at room temperature.

5.4 Diluents
Ringer’s solution ¼ strength
Composition per litre:
— Sodium chloride (NaCl) 2,250 g;
— Potassium chloride (KCl) 0,105 g;
— Calcium chloride (CaCl ) 6 H O 0,120 g;
2 2
— Sodium hydrogen carbonate (NaHCO ) 0,050 g;
© ISO 2021 – All rights reserved
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ISO/FDIS 8784-3:2021(E)
— Water 1 000 ml.

NOTE 1 Ringer’s solution is preferred, although other isotonic solutions can be used. See ISO 6887-1.

NOTE 2 Ringer’s tablets are commercially available.
5.5 Non-ionic surfactant
Poly (oxyethylene)-sorbitan monooleate (Tween 80).

To facilitate the release of cells from the fibres, it is recommended to add 20 µl of Tween 80 per litre to

the Ringer’s solution prior to sterilization
...

ISO/FDIS 8784-3

Pulp, paper and board -- Microbiological examination

Pulp, paper and board -- Microbiological examination

Pâtes, papiers et cartons -- Analyse microbiologique

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Pulp, paper and board -- Microbiological examination

Pâtes, papiers et cartons -- Analyse microbiologique

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