EN ISO 21322:2021
(Main)Cosmetics - Microbiology - Testing of impregnated or coated wipes and masks (ISO 21322:2020)
Cosmetics - Microbiology - Testing of impregnated or coated wipes and masks (ISO 21322:2020)
This document gives guidance for the enumeration and/or detection of microorganisms present in a cosmetic product that is impregnated or coated onto a substrate (i.e. wipes and masks) where sampling and microbiological influence of the manufactured product presents particular challenges in terms of microbiological sampling and testing.
The principle of this document can also be applied to test similar products (e.g. cushion, impregnated sponge, etc.) or applicators (e.g. brush, puff, sponge, etc.) with modification of the procedure as appropriate.
Kosmetische Mittel - Mikrobiologie - Prüfung von getränkten oder beschichteten Reinigungstüchern und Masken (ISO 21322:2020)
Dieses Dokument enthält eine Anleitung für die Auszählung und/oder zum Nachweis von Mikroorganismen, die in kosmetischen Mitteln vorhanden sind, die sich auf einem damit getränkten oder beschichteten Träger befinden (z. B. Reinigungstücher und Masken), wenn die Probenahme und der mikrobiologische Einfluss des hergestellten Produktes im Hinblick auf die mikrobiologische Probenahme und Prüfung eine besondere Herausforderung darstellen.
Der Grundsatz dieses Dokumentes kann auch auf ähnliche Produkte (z. B. Kissen, getränkte Schwämme usw.) oder Applikatoren (z. B. Pinsel, Quasten, Schwämme usw.) angewendet werden, ggf. mit einer Veränderung des Verfahrens.
Cosmétiques - Microbiologie - Essais sur lingettes et masques imprégnés ou enduits (ISO 21322:2020)
Le présent document fournit des recommandations pour le dénombrement et/ou la détection des micro-organismes présents dans les produits cosmétiques imprégnés ou enduits sur un substrat (lingettes et masques), pour lesquels l'échantillonnage et le contexte microbiologique du produit fabriqué posent des problèmes particuliers lorsqu'il s'agit de réaliser un échantillonnage et des essais microbiologiques.
Le principe du présent document peut également être appliqué à des produits similaires (par exemple, cushion, éponges imprégnées, etc.) ou des applicateurs (par exemple, pinceau, houpette, éponge, etc.), en modifiant le mode opératoire comme approprié.
Kozmetika - Mikrobiologija - Preskušanje impregniranih izdelkov ali izdelkov, obdelanih s premazi - Robčki in maske (ISO 21322:2020)
Ta dokument vsebuje napotke za štetje in/ali odkrivanje mikroorganizmov, ki so prisotni v kozmetičnem izdelku, uporabljenem za impregnacijo ali nanos na podlago (tj. robčki in maske), pri čemer z vidika mikrobiološkega vzorčenja in preskušanja vzorčenje oziroma ugotavljanje mikrobiološkega vpliva proizvedenega izdelka predstavljata še poseben izziv. Načelo tega dokumenta se lahko uporabi tudi za preskušanje podobnih izdelkov (npr. blazina, impregnirana gobica itd.) ali aplikatorjev (npr. čopič, pudrna blazinica, gobica itd.), pri čemer se postopek po potrebi ustrezno prilagodi.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-julij-2022
Kozmetika - Mikrobiologija - Preskušanje impregniranih izdelkov ali izdelkov,
obdelanih s premazi - Robčki in maske (ISO 21322:2020)
Cosmetics - Microbiology - Testing of impregnated or coated wipes and masks (ISO
21322:2020)
Kosmetische Mittel - Mikrobiologie - Prüfung von getränkten oder beschichteten
Feuchttüchern und Masken (ISO 21322:2020)
Cosmétiques - Microbiologie - Essais sur lingettes et masques imprégnés ou enduits
(ISO 21322:2020)
Ta slovenski standard je istoveten z: EN ISO 21322:2021
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
71.100.70 Kozmetika. Toaletni Cosmetics. Toiletries
pripomočki
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN ISO 21322
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2021
EUROPÄISCHE NORM
ICS 07.100.40
English Version
Cosmetics - Microbiology - Testing of impregnated or
coated wipes and masks (ISO 21322:2020)
Cosmétiques - Microbiologie - Essais sur lingettes et Kosmetische Mittel - Mikrobiologie - Prüfung von
masques imprégnés ou enduits (ISO 21322:2020) getränkten oder beschichteten Reinigungstüchern und
Masken (ISO 21322:2020)
This European Standard was approved by CEN on 5 December 2021.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21322:2021 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
The text of ISO 21322:2020 has been prepared by Technical Committee ISO/TC 217 "Cosmetics” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 21322:2021 by
Technical Committee CEN/TC 392 “Cosmetics” the secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by June 2022, and conflicting national standards shall be
withdrawn at the latest by June 2022.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 21322:2020 has been approved by CEN as EN ISO 21322:2021 without any modification.
INTERNATIONAL ISO
STANDARD 21322
First edition
2020-06
Cosmetics — Microbiology — Testing
of impregnated or coated wipes and
masks
Cosmétiques — Microbiologie — Essais sur lingettes et masques
imprégnés ou enduits
Reference number
ISO 21322:2020(E)
©
ISO 2020
ISO 21322:2020(E)
© ISO 2020
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
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Published in Switzerland
ii © ISO 2020 – All rights reserved
ISO 21322:2020(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
4.1 General information . 2
4.2 Selection of the test sample . 2
4.3 Selection of the method . 3
4.4 Recovery of microorganisms from the test sample . 3
4.5 Enumeration of aerobic mesophilic microorganisms . 3
4.5.1 General. 3
4.5.2 Plate count method overview . 3
4.5.3 Membrane filtration method overview . 3
4.6 Detection of specified microorganisms by enrichment method . 4
5 Diluents, neutralizers and culture media . 4
5.1 General . 4
5.2 Diluents and neutralizers . 4
5.3 Culture media . 4
5.3.1 Media for enumeration and detection . 4
5.3.2 Media for preparation of spores of Bacillus subtilis . 4
6 Apparatus and glassware . 4
7 Strains of microorganisms . 4
8 Handling of cosmetic products and laboratory samples . 5
9 Procedure. 5
9.1 General recommendation . 5
9.2 Selection and preparation of the test sample . 5
9.2.1 Selection of the test sample . 5
9.2.2 Preparation of the initial suspension . 5
9.3 Recovery of microorganisms . 6
9.3.1 General. 6
9.3.2 Stomaching . 6
9.3.3 Shaking/Stirring . . 6
9.4 Enumeration of aerobic mesophilic microorganisms . 6
9.4.1 General. 6
9.4.2 Pour plate method . 6
9.4.3 Surface spread method . 7
9.4.4 Membrane filtration method . 7
9.4.5 Incubation . 7
9.4.6 Counting of colonies. 7
9.5 Detection of specified microorganisms by enrichment method . 8
9.5.1 General. 8
9.5.2 Test for specified microorganisms . 8
10 Expression of results . 9
10.1 Enumeration of aerobic mesophilic microorganisms . 9
10.2 Detection of specified microorganisms . 9
11 Suitability test . 9
12 Test report . 9
ISO 21322:2020(E)
Annex A (normative) Guidance on methods for microbiological testing of impregnated or
coated products — Wipes and masks .10
Annex B (informative) Expression and interpretation of results .14
Annex C (normative) Suitability test method .21
Bibliography .26
iv © ISO 2020 – All rights reserved
ISO 21322:2020(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 217, Cosmetics.
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 21322:2020(E)
Introduction
For technical reasons, current standards in cosmetics microbiology may not be applicable to
impregnated or coated cosmetic products, such as wipes and masks, in which there is no direct access
to the formulation.
Based on their product form or delivery there is a need to adapt these standards to assess the
microbiological quality of these products.
vi © ISO 2020 – All rights reserved
INTERNATIONAL STANDARD ISO 21322:2020(E)
Cosmetics — Microbiology — Testing of impregnated or
coated wipes and masks
1 Scope
This document gives guidance for the enumeration and/or detection of microorganisms present in a
cosmetic product that is impregnated or coated onto a substrate (i.e. wipes and masks) where sampling
and microbiological influence of the manufactured product presents particular challenges in terms of
microbiological sampling and testing.
The principle of this document can also be applied to test similar products (e.g. cushion, impregnated
sponge, etc.) or applicators (e.g. brush, puff, sponge, etc.) with modification of the procedure as
appropriate.
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 11930, Cosmetics — Microbiology — Evaluation of the antimicrobial protection of a cosmetic product
ISO 16212, Cosmetics — Microbiology — Enumeration of yeast and mould
ISO 18416, Cosmetics — Microbiology — Detection of Candida albicans
ISO 21148, Cosmetics — Microbiology — General instructions for microbiological examination
ISO 21149, Cosmetics — Microbiology — Enumeration and detection of aerobic mesophilic bacteria
ISO 21150, Cosmetics — Microbiology — Detection of Escherichia coli
ISO 22717, Cosmetics — Microbiology — Detection of Pseudomonas aeruginosa
ISO 22718, Cosmetics — Microbiology — Detection of Staphylococcus aureus
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 https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
cosmetic formulation
preparation of raw materials with a qualitatively and quantitatively defined composition
3.2
cosmetic product
cosmetic formulation (3.1) that has undergone all stages of production, including packaging in its final
container, for shipment
ISO 21322:2020(E)
3.3
impregnated product
product absorbed on the support
3.4
coated product
product adsorbed on the support
3.5
test sample
representative unit of the entire cosmetic product (3.2) for testing
4 Principle
4.1 General information
The method determines the population of viable microorganisms by enumeration of colonies on a
non-selective agar medium and by the presence or absence of specified microorganisms growth after
enrichment.
The method involves the following steps:
— selection of the test sample;
— selection of an appropriate method;
— recovery of microorganisms;
— enumeration of the population of viable microorganisms by filtration or plate count method;
— tests for specified microorganisms by enrichment method.
The experimental conditions shall be evaluated to ensure that the method should not affect the viability
of microorganisms and the recovery of bioburden from the sample and should include the verification
of the efficacy of the neutralization (see Clause 11).
In order to ensure product quality and safety for the consumer, an appropriate microbiological risk
assessment should be performed to determine the types of cosmetic products to which this document
is applicable (see ISO 29621:2017, Table 2).
Other methods may be substituted provided that their equivalence has been demonstrated.
4.2 Selection of the test sample
— Whenever practical, the entire unit should be used for testing with a minimum weight of 1 g. If for
technical reasons the entire unit cannot be tested, a defined Unit Item Portion (UIP) is used for
testing. A “UIP” is a microbiologically-representative subunit of the test sample and is referenced
throughout the document.
— If the unit is < 1 g per unit then the appropriate number of units should be sampled to achieve the
appropriate weight or volume.
— The weight of the test sample shall be recorded even if the results are expressed by unit.
Selection of the test sample shall be according to A.1.
2 © ISO 2020 – All rights reserved
ISO 21322:2020(E)
4.3 Selection of the method
The method should be conducted according to an appropriate procedure based on the specifics of the
product (size, volume, single unit/multi–unit package, level of bioburden. etc.) and should ensure that a
representative sample is evaluated.
Selection of the method shall be according to A.1 and A.2.
4.4 Recovery of microorganisms from the test sample
The degree to which microorganisms adhere to the test sample surface is dependent on the wipe or
mask in which the liquid portion of the formulation has been either impregnated or coated. Preliminary
treatments may be necessary to separate microorganisms from the test sample.
Regardless of the treatment, the verification of recovery method should be performed in order to
demonstrate that the method can release microorganisms from the test sample without having an
adverse effect on their viability (see Clause 11).
4.5 Enumeration of aerobic mesophilic microorganisms
4.5.1 General
The enumeration of aerobic mesophilic microorganisms includes bacteria, yeasts and moulds.
Based on the nature of the test sample, the volume of diluent used to immerse the test sample and the
expected level of bioburden, two types of counting methods may be used:
— plate count method;
— membrane filtration method.
4.5.2 Plate count method overview
Plate count method consists of either using a pour plate or spread plate method.
Each method consists of the following steps.
— Prepare the agar plates and diluent using a non-selective agar medium for plating the diluent in
which the sample was immersed.
— Incubate the plates for enumeration and/or detection.
— Count the number of colonies forming units (CFU) based on the number of aerobic mesophilic
microorganisms recovered per unit or g.
4.5.3 Membrane filtration method overview
Membrane filtration consists of the following steps.
— Transfer the diluent or a defined quantity of diluent in which the test sample was immersed to a
filtration apparatus wetted with a small volume of an appropriate sterile diluent.
— After filtration and rinsing, transfer the membrane filter onto the surface of plates with non-
selective agar medium.
— Aerobic incubation of the plates.
— Count the number of colony forming units (CFU) and calculate of the number of aerobic mesophilic
microorganisms per g or unit.
ISO 21322:2020(E)
4.6 Detection of specified microorganisms by enrichment method
The objective of the enrichment method is to incubate a test sample in a non-selective broth medium to
increase the number of microorganisms that are present in a test sample.
— The first step of an enrichment method is to incubate the test sample in a non-selective broth
medium to increase the number of microorganisms present in the test sample.
— The second step of an enrichment method is to isolate specified microorganisms that may be present
on a test sample through the use of selective agar media followed by confirmatory identification
tests for characteristic colonies. See ISO 18416, ISO 21150, ISO 22717 and ISO 22718.
5 Diluents, neutralizers and culture media
5.1 General
The diluents, neutralizers and culture media suitable for enumeration and detection of aerobic
mesophilic microorganisms are described in ISO 11930, ISO 16212 and ISO 21149. Other diluents,
neutralizers and culture media may be used if they have been demonstrated to be suitable for use.
Use the general instructions given in ISO 21148. When water is mentioned in this document, use
distilled water or purified water as specified in ISO 21148.
5.2 Diluents and neutralizers
The diluent is used to disperse the sample. It is required that it contain neutralizers if the sample to be
tested has antimicrobial properties or contains a preservative. The efficacy of the neutralization shall
be demonstrated before the determination of the count (see Clause 11). Diluents and neutralizers shall
be in accordance with ISO 11930, ISO 16212, ISO 18416, ISO 21149, ISO 21150, ISO 22717 and ISO 22718.
5.3 Culture media
5.3.1 Media for enumeration and detection
Culture media for enumeration and/or detection shall be in accordance with ISO 11930, ISO 16212,
ISO 18416, ISO 21149, ISO 21150, ISO 22717 and ISO 22718.
5.3.2 Media for preparation of spores of Bacillus subtilis
See C.1.3.1.
6 Apparatus and glassware
The laboratory equipment, apparatus and glassware are described in ISO 21148.
7 Strains of microorganisms
The culture should be reconstituted according to the procedures provided by the supplier of the
reference strain. The strains may be stored in the laboratory conforming to EN 12353 or according to
another suitable method.
For testing the recovery of microorganisms on the test samples, spores of Bacillus subtilis ATCC 6633
(equivalent strain CIP 52.62 or NCIMB 8054 or NBRC 3134 or other equivalent national collection
strain) are used.
4 © ISO 2020 – All rights reserved
ISO 21322:2020(E)
For testing the efficacy of neutralizers, two strains representative of both Gram negative and Gram
positive bacteria and a yeast are used:
— Staphylococcus aureus ATCC 6538 (equivalent strain: CIP 4.83 or NCIMB 9518 or NBRC 13276 or
KCTC 1916 or other equivalent national collection strain);
— Pseudomonas aeruginosa ATCC 9027 (equivalent strain: CIP 82.118 or NCIMB 8626 or NBRC 13275
or KCTC 2513 or other equivalent national collection strain).
An alternative Gram negative strain may be Escherichia coli ATCC 8739 (equivalent strain: CIP 53.126 or
NCIMB 8545 or NBRC 3972 or KCTC 2571 or other equivalent national collection strain).
— Candida albicans ATCC 10231 (equivalent strain: IP 48.72 or NCPF 3179 or NBRC 1594 or KCTC 17205,
or other equivalent national collection strain).
The strains may be kept in the laboratory according to the EN 12353.
8 Handling of cosmetic products and laboratory samples
If storage is necessary, keep the products to be tested at room temperature. Do not incubate, refrigerate
or freeze products and samples before or after analysis. Sampling and test procedures should follow
the guidelines specified in ISO 21148 and in accordance with the procedure outlined in Clause 9.
9 Procedure
9.1 General recommendation
Use sterile equipment and aseptic technique whenever preparing the test sample and diluent.
For the preparation of an initial suspension, the time which elapses between the end of the preparation
of the test sample and the moment the diluent of the initial suspension comes into contact with the
culture medium shall not exceed (30 ± 15) min, unless specifically mentioned in the established
protocols or documents.
The method should follow the procedure developed during the suitability test, to ensure neutralization
of potential inhibitory effects (see Clause 11) and to guarantee the recovery of microorganisms.
9.2 Selection and preparation of the test sample
9.2.1 Selection of the test sample
The test sample shall weigh at least 1 g.
The test sample can be either the entire unit, or multiple units if the weight of one unit is less than 1 g,
or the UIP (see A.1).
Record the exact weight of the test sample, S, and, the number of units, n.
If a UIP is used for testing, record the UIP value of the test sample (see 4.2).
9.2.2 Preparation of the initial suspension
Place the test sample (see 9.2.1) into an appropriate container, with a known volume of diluent, defined
in the suitability test (see Clause 11). The test sample should be completely immersed in the diluent.
Record the value for “V”, the volume of diluent used.
ISO 21322:2020(E)
9.3 Recovery of microorganisms
9.3.1 General
After immersion, the following treatments may be used to remove microorganisms from the test sample:
— stomaching;
— shaking/stirring;
— vortexing (see A.3).
NOTE If necessary, record the volume of the diluent after stomaching or shaking of test sample.
9.3.2 Stomaching
Prepare the initial suspension utilizing a sterile stomacher bag and then place it into the stomacher.
Proceed according to the parameters (time and speed) outlined in the suitability test (see Clause 11).
Record the time and the speed at which the stomaching took place.
9.3.3 Shaking/Stirring
Prepare the initial suspension in the appropriate closed container and mix according to the parameters
(duration and frequency) applied in the suitability test (see Clause 11).
Sterile glass beads may be added to enhance product mixing and organism recovery.
Record the time, frequency and the speed of shaking/stirring (if applicable) and whether glass beads
are added or not.
9.4 Enumeration of aerobic mesophilic microorganisms
9.4.1 General
The enumeration of aerobic mesophilic microorganisms includes bacteria, yeasts and moulds.
The choice of the method depends on the volume of diluent used for the preparation of the initial
suspension (see A.2).
Based on the test sample size, the level of bioburden and the sensitivity of the method, all of the diluent
in which the test sample is immersed (V) or a fraction of V (Vd) is used for enumeration.
Usually, the volume V or Vd of the initial suspension diluent is the dilution used for enumeration. No
further diluting of the initial preparation is required.
The minimal volume of diluent shall be equivalent to at least 1 g of the test sample.
9.4.2 Pour plate method
Use the appropriate number of Petri dishes to properly evaluate the volume of diluent needed to
properly immerse and transfer the product to be plated (V or Vd).
The diluent (V) is divided into two work streams: one half (V/2) is for the enumeration of bacteria and
the other half (V/2) for enumeration of yeasts and moulds.
If the enumeration is conducted on a fraction of the total diluent (Vd), two equal fractions (Vd/2) shall
be plated: one for bacteria and the other for yeasts and moulds.
6 © ISO 2020 – All rights reserved
ISO 21322:2020(E)
If Petri dishes, 85 mm to 100 mm in diameter are used, add 1 ml of the diluent and pour 15 ml to
20 ml of the melted agar medium (as specified in ISO 21149 and ISO 16212) kept in a water bath not to
exceed 48 °C.
If larger Petri dishes (140 mm in diameter) are used, add no more than 10 ml of diluent in each plate and
the appropriate amount of agar medium based on the volume of diluent dispensed.
Slowly mix the transferred diluent with the medium, carefully rotating or tilting the plates to
sufficiently disperse the agar. Allow the mixture in the Petri dishes to solidify on a horizontal surface at
room temperature. Record the volume of diluent plated for each medium V/2 or Vd/2.
9.4.3 Surface spread method
Use the appropriate number of Petri dishes to properly evaluate the volume of diluent needed to
properly immerse and transfer the product to be plated (V or Vd).
In Petri dishes 85 mm to 100 mm in diameter, put 15 ml to 20 ml of the melted agar medium kept in
a water bath at no more than 48 °C. If larger Petri dishes are used, the volume of the agar is increased
accordingly. Allow plates to cool and solidify on a horizontal surface at room temperature.
The diluent (V) is divided into two work streams: one half (V/2) is for the enumeration of bacteria and
the other half (V/2) for enumeration of yeasts and moulds.
If the enumeration is conducted on a fraction of the total diluent (Vd), two equal fractions (Vd/2) shall
be plated: one for bacteria and the other for yeasts and moulds.
Spread over the surface of the medium a measured volume (V or Vd) of not less than 0,1 ml of the initial
suspension and/or sample dilution prepared as described in Clause 11.
Record the volume of diluent plated for each medium V/2 or Vd/2.
9.4.4 Membrane filtration method
Use membranes having a nominal pore size no greater than 0,45 μm.
If all of the diluent (V) is filtered, half (V/2) is for the enumeration of bacteria and the other half (V/2)
for enumeration of yeasts and moulds.
If the enumeration is conducted on a fraction of the diluent (Vd), two equal fractions (Vd/2) shall be
filtered: one for bacteria and the other for yeasts and moulds.
Filter the volume V/2 of diluent in which the test sample was immersed or the fraction Vd/2 through
each of two sterile filter membranes and wash according to the results of the suitability test (See
Clause 11).
Transfer the membrane filters onto the surface of the agar media as specified in ISO 21149 and
ISO 16212.
Record the volume of diluent filtered for each medium V/2 or Vd/2.
9.4.5 Incubation
Unless otherwise stated, place the inoculated dishes in the incubator set at 32,5 °C ± 2,5 °C for 72 h ± 6 h
(bacteria) or at 25 °C ± 2,5 °C for 3 d to 5 d (yeasts and moulds) if antibiotic is added to the medium. If a
culture medium without antibiotic is used, incubate at 22,5 °C ± 2,5 °C for 5 days to 7 days.
9.4.6 Counting of colonies
After incubation, count the colonies on each culture medium (N for bacteria or N for yeasts and
b ym
moulds) within the recommended countable ranges (see Annex B).
ISO 21322:2020(E)
If several plates have been used, take the sum of the colonies counted on each plate for each culture
medium.
Calculate the number of bacteria, N and the number of yeasts and moulds, N present in the volume
b, ym,
of tested diluent plated or filtered for each medium (V/2 or Vd/2) as follows.
N or N is equal to the sum of the number of colonies obtained from the plates (plate-count method)
b ym
or to the number of colonies counted on a single plate (filtration method).
Check that the counts are obtained from experimental conditions applied during the suitability test
procedure (see Clause 11).
9.5 Detection of specified microorganisms by enrichment method
9.5.1 General
The test for specified microorganisms includes the detection of P. aeruginosa, S. aureus, E. coli, and C.
albicans.
The principle of the test is the enrichment in a non-selective broth medium, followed by an isolation on
selective agar media and the identification of characteristics colonies.
The test may be conducted on the test sample or on 1 g of the test sample.
9.5.2 Test for specified microorganisms
9.5.2.1 Preparation of the initial suspension in the enrichment broth
The initial suspension is prepared by transferring the test sample (see 9.2.) or 1 g of the test sample
in a closed container with an appropriate volume of enrichment broth chosen following the procedure
developed during the suitability test (see Clause 11). The test sample shall be totally covered and the
minimal quantity to be tested should be equivalent to at least 1 g of the test sample. Record the volume
of enrichment broth, V, the exact weight of the test sample, S, and the number of units tested, n, if
applicable.
9.5.2.2 Incubation of the initial suspension
After recovery of microorganisms (9.3), incubate the initial suspension prepared in broth (see 9.5.2.1)
at 32,5 °C ± 2,5 °C for a minimum of 20 h and a maximum of 72 h max.
9.5.2.3 Isolation
After incubation, using a sterile loop streak an aliquot of the incubated enrichment broth onto the
surface of suitable detection agar media.
Invert the Petri dishes and incubate at 32,5 °C ± 2,5 °C for 48 h to 72 h in order to obtain isolated
colonies, according to ISO 21150, ISO 22717, ISO 22718 and ISO 18416.
9.5.2.4 Detection of specified microorganisms
Growth of characteristic colonies indicates the possible presence of specified microorganisms to
confirm by identification tests according to ISO 21150, ISO 22717, ISO 22718 and ISO 18416.
8 © ISO 2020 – All rights reserved
ISO 21322:2020(E)
10 Expression of results
10.1 Enumeration of aerobic mesophilic microorganisms
Calculate NS the total number of bacteria and NS the total number of yeasts and moulds present in
b ym
the unit. Expression of the results is given in Annex B.
The total number of aerobic mesophilic microorganisms can be expressed in CFU per unit (NS + NS )
b ym
or in CFU per g (NS + NS )/S.
b ym
10.2 Detection of specified microorganisms
If the identification of the colonies confirms the presence of specified microorganism, express the
result as:
— “Presence of P. aeruginosa, S. aureus, E. coli or C. albicans in the test sample S or 1 g of the test
sample”.
If no growth after enrichment is observed and/or if the identification of the colonies does not confirm
the presence of this species, express the result as:
— “Absence of P. aeruginosa, S. aureus, E. coli or C. albicans in the test sample S or in 1 g of the test
sample”.
11 Suitability test
The suitability of the test method consists of demonstrating that the method allows for the detection of
viable microorganisms and requires:
— The demonstration of the ability of the method for removing the microorganisms from the test sample.
— The evaluation of the effect of the test sample processing on microorganisms.
— The verification of the efficacy of the neutralization.
The method shall be as described in Annex C.
12 Test report
The test report shall specify the following:
a) all information necessary for the complete identification of the product;
b) description of the method used, suitability test included or standard operating procedure reference;
c) results obtained;
d) any point not specified in this document, or regarded as optional, together with details of any
incidents that may have influenced the results.
ISO 21322:2020(E)
Annex A
(normative)
Guidance on methods for microbiological testing of impregnated
or coated products — Wipes and masks
A.1 Selection of the test sample
The choice of an adequate test sample may depend on many factors such as size, volume, nature (wipes
or masks) packaging (unitary units or identical units packaged together).
For wipes or masks in a single package, the test sample is the entire unit.
For wipes or masks in a multi-use package, the test sample is one identical unit.
A minimum of 1 unit (wipe or mask) shall be tested except for the following cases.
— If for technical reasons the entire unit cannot be used, a defined Unit Item Portion (UIP) may be
used for testing. A UIP is a microbiologically representative subunit of the test sample. In this case
attention should be paid to the representativeness of the UIP.
— In the case where the unit is less than 1 g, then a sufficient number of units representing at least 1 g
shall be tested.
For example:
— in the case of a wipes pack, the unit is one wipe;
— for a single dose face mask, the unit is the whole mask;
— when the unit is 0,4 g, a minimum of 3 units shall be tested.
A.2 Selection of the enumeration method for aerobic mesophilic microorganisms
The choice of an adequate enumeration method may depend on the volume of diluent necessary to
immerse the test sample and the level of expected bioburden.
For a small volume of diluent (not more than 20 ml) use the plate count method and for larger volume of
diluent, the membrane filtration method is preferable (see decision tree).
If high levels of bioburden are expected, the enumeration may be conducted on a fraction of the
diluent (Vd).
The sensitivity of the method should provide sufficient margin below the threshold of the product
specification.
See Figure A.1.
10 © ISO 2020 – All rights reserved
ISO 21322:2020(E)
NOTE In case the test volume is Vd, the dilution factor, the threshold of the method and the specifications
are taken into account.
Figure A.1 — Flow chart for the choice of plate count method
A.3 Recovery of microorganisms
Considering that the degree of adhesion of microorganisms may depend on the nature of the product,
preliminary treatments may be necessary to remove microorganisms. Treatments should not affect the
viability of microorganisms.
Treatment may consist of stomaching, shaking, stirring and/or vortexing.
— Stomaching
Stomaching consists of the action of twin reciprocating paddles on the sample contained inside a
sterile bag forcing the eluent throughout the sample to ensure proper mixing.
Stomaching is suitable for soft, fibrous and/or absorbent materials such as wipes, facial masks,
sponges, puffs, etc. It would be unsuitable for any materials which would puncture the bag.
ISO 21322:2020(E)
The duration and the speed of stomaching shall be sufficient to allow the separation of
microorganisms from the substrate (see C.4).
— Shaking/stirring
Shaking or stirring, consists of an initial suspension mixed with a mechanical shaker by
reciprocating or orbital action. Vortex mixing may also be used for small samples with regular
surfaces.
Whatever the treatment, the recovery verification should be performed, demonstrating that the
method can remo
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