ISO/FDIS 21322

• Abstract:

  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.

• Status: Not Published
• ICS: 07.100.40 - Cosmetics microbiology
• Technical Committee: ISO/TC 217 - Cosmetics
• Drafting Committee: ISO/TC 217/WG 1 - Microbiological standards and limits
• Current Stage: 5020 - FDIS ballot initiated: 2 months. Proof sent to secretariat
• Start Date: 02-Sep-2026
• Completion Date: 02-Sep-2026

• Revises: ISO 21322:2020 - Cosmetics — Microbiology — Testing of impregnated or coated wipes and masks
• Effective Date: 27-Dec-2025

Overview

ISO/FDIS 21322: Cosmetics - Microbiology - Testing of Impregnated or Coated Wipes and Masks is an international standard published by the International Organization for Standardization (ISO). This document provides essential guidance for the enumeration and/or detection of microorganisms present in cosmetic products that are impregnated or coated onto substrates. It specifically addresses challenges in microbiological testing related to wipes and masks, acknowledging the sampling complexity unique to these types of products. The standard’s principles may also be adapted for similar items such as impregnated cushions, sponges, or cosmetic applicators.

The standard is managed by ISO Technical Committee ISO/TC 217 (Cosmetics) and offers an updated approach to ensure cosmetic safety and quality by providing standardized procedures for microbiological examination.

Key Topics

• Microbiological Sampling and Testing
  ISO/FDIS 21322 outlines recommendations for collecting microbiologically representative test samples from impregnated or coated wipes and masks, ensuring reliable detection and enumeration of microorganisms.

• Test Sample Selection
  The standard details how to select the appropriate test amount (minimum 1 g per sample or corresponding multiple units), and addresses special cases like small or composite samples by using Unit Item Portions (UIP).

• Enumeration and Detection Methods
  Guidance is provided on the use of plate count and membrane filtration methods for enumerating aerobic mesophilic microorganisms, including bacteria, yeasts, and molds. The enrichment method for detection of specified organisms like Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans is also explained.

• Materials and Handling
  The use and quality control of diluents, neutralizers, and culture media suitable for cosmetic testing is specified, referencing other ISO standards for best practices.

• Suitability Testing
  A critical focus is given to suitability tests, ensuring that the chosen method effectively recovers and identifies microorganisms without adverse impacts from the product matrix or preservative content.

• Test Reporting
  The standard specifies essential reporting requirements, including complete product identification, description of methods used, and explicit results for regulatory compliance and quality assurance.

Applications

• Cosmetic Product Safety Assurance
  By implementing ISO/FDIS 21322, manufacturers of cosmetic wipes, facial masks, and coated products can reliably assess microbiological quality, protecting consumers from potential health hazards arising from contamination.

• Quality Control for New Product Forms
  The guidance helps cosmetic laboratories and production managers adapt microbiological quality control methods for novel and evolving product forms (e.g., untraditional substrates like cushions or applicators) by providing modification guidance.

• Regulatory Compliance
  The standardized approach facilitates compliance with international regulations and customer requirements in the cosmetic industry, supporting both local and global market access.

• Risk Assessment
  Performing appropriate microbiological risk assessments, as advised, helps in determining which products require comprehensive testing, optimizing resources, and ensuring high standards of hygiene.

Related Standards

ISO/FDIS 21322 is part of a broader framework of international standards for cosmetics microbiology. Relevant related standards include:

• ISO 21149: Cosmetics - Microbiology - Enumeration and detection of aerobic mesophilic bacteria
• ISO 18416: Cosmetics - Microbiology - Detection of Candida albicans
• ISO 21150: Cosmetics - Microbiology - Detection of Escherichia coli
• ISO 22717: Cosmetics - Microbiology - Detection of Pseudomonas aeruginosa
• ISO 22718: Cosmetics - Microbiology - Detection of Staphylococcus aureus
• ISO 11930: Cosmetics - Microbiology - Evaluation of the antimicrobial protection of a cosmetic product
• ISO 16212: Cosmetics - Microbiology - Enumeration of yeast and mould
• ISO 21148: Cosmetics - Microbiology - General instructions for microbiological examination
• ISO 4973: Cosmetics - Microbiology - Quality control of culture media and diluents

Together, these standards support a robust microbiological quality management system in the cosmetics sector, with ISO/FDIS 21322 providing targeted protocols for wipes, masks, and other coated cosmetic products.

Keywords: ISO 21322, Cosmetics microbiology, Testing wipes, Microbiological analysis, Cosmetic product safety, Quality control, Cosmetic masks, Microbial enumeration, Microbiological detection, Cosmetic standards.

FINAL DRAFT
International
Standard
ISO/TC 217
Cosmetics — Microbiology —
Secretariat: INSO
Testing of impregnated or coated
Voting begins on:
wipes and masks
2026-09-02
Cosmétiques — Microbiologie — Essais sur lingettes et masques
Voting terminates on:
imprégnés ou enduits
2026-11-25
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
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/CEN PARALLEL PROCESSING 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.
Reference number
FINAL DRAFT
International
Standard
ISO/TC 217
Cosmetics — Microbiology —
Secretariat: INSO
Testing of impregnated or coated
Voting begins on:
wipes and masks
Cosmétiques — Microbiologie — Essais sur lingettes et masques
Voting terminates on:
imprégnés ou enduits
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.
© ISO 2026
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/CEN PARALLEL PROCESSING
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
or ISO’s member body in the country of the requester.
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland Reference number
ii
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 .2
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.3
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 pizizenii (formerly known as Bacillus
subtilis and Bacillus subtilis subsp. spizizenii) .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 . 8
10.1 Enumeration of aerobic mesophilic microorganisms .8
10.2 Detection of specified microorganisms .9
11 Suitability test . 9
12 Test report . 9

iii
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 .13
Annex C (normative) Suitability test method .20
Bibliography .25

iv
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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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.
This second edition cancels and replaces the first edition (ISO 21322:2020), of which it constitutes a minor
revision.
The changes are as follows:
— the normative references (Clause 2) has been updated.
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.

v
Introduction
For technical reasons, current standards in cosmetics microbiology might 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
FINAL DRAFT International Standard ISO/FDIS 21322:2026(en)
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 4973, Cosmetics — Microbiology — Quality control of culture media and diluents used in cosmetics standards
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 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 http:// www .electropedia .org/
3.1
cosmetic formulation, noun
preparation of raw materials with a qualitatively and quantitatively defined composition
3.2
cosmetic product, noun
cosmetic formulation (3.1) that has undergone all stages of production, including packaging in its final
container, for shipment
3.3
impregnated product, noun
product absorbed on the support
3.4
coated product, noun
product adsorbed on the support
3.5
test sample, noun
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
[1]
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 Clause A.1.
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 Clause A.1 and Clause 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.
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
[2] [3] [4] [5]
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
[6] [7] [8]
microorganisms are described inISO 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, including
quality control, shall be in accordance with ISO 11930, ISO 16212, ISO 18416, ISO 21149,ISO 21150, ISO 22717
ISO 22718 and ISO 4973.
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, ISO 22718 and ISO 4973.
5.3.2 Media for preparation of spores of Bacillus pizizenii (formerly known as Bacillus subtilis and
Bacillus subtilis subsp. spizizenii)
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
[9]
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 pizizenii (formerly known as
® 1) ® TM1)
Bacillus subtilis and Bacillus subtilis subsp. spizizenii) ATCC 6633TM (equivalent strain: WDCM 00003
or CIP 52.62 or NCIMB 8054 or NBRC 3134 or other equivalent national collection strain) are used.
For testing the efficacy of neutralizers, two strains representative of both Gram negative and Gram positive
bacteria and a yeast are used:
® TM1) ® TM1)
— Staphylococcus aureus ATCC 6538 (equivalent strain: WDCM 00032 or CIP 4.83 or NCIMB 9518
or NBRC 13276 or KCTC 1916 or other equivalent national collection strain);
1) This information is given for the convenience of users of this document and does not constitute an endorsement by
ISO.
® TM1)
— Pseudomonas paraeruginosa (formerly Pseudomonas aeruginosa) ATCC 9027 (equivalent strain:
® TM1)
WDCM 00026 or CIP 82.118 or NCIMB 8626 or NBRC 13275 or KCTC 2513 or other equivalent
national collection strain).
® TM1)
An alternative Gram negative strain may be Escherichia coli ATCC 8739 (equivalent strain:
® TM1)
WDCM 00012 or CIP 53.126 or NCIMB 8545 or NBRC 3972 or KCTC 2571 or other equivalent national
collection strain).
® TM1) ® TM1)
— Candida albicans ATCC 10231 (equivalent strain: WDCM 00054 or CIP 48.72 or NCPF 3179 or
NBRC 1594 or KCTC 17205, or other equivalent national collection strain).
[9]
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
[10]
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 Clause 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.

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 Clause 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 Clause 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.

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
...

ISO/DISFDIS 21322
ISO/TC 217
Secretariat: INSO
Date: 2026-08-0318
Cosmetics — Microbiology — Testing of impregnated or coated wipes
and masks
Cosmétiques — Microbiologie — Essais sur lingettes et masques imprégnés ou enduits
DISFDIS stage
TThhiiss d drraftaft i iss s suubbmmiitttteded t too a pa pararallel vallel vootte e iinn I ISSOO,, C CEENN.

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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
E-mail: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
Contents
Foreword . iv
Introduction . v
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
4.6 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
6 Apparatus and glassware . 4
7 Strains of microorganisms . 5
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 . 6
9.3 Recovery of microorganisms . 6
9.4 Enumeration of aerobic mesophilic microorganisms . 6
9.5 Detection of specified microorganisms by enrichment method. 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 . 10
Annex A (normative) Guidance on methods for microbiological testing of impregnated or
coated products — Wipes and masks . 11
Annex B (informative) Expression and interpretation of results . 17
Annex C (normative) Suitability test method . 24
Bibliography . 29

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.
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 drawnISO draws attention to the possibility that some of the elementsimplementation of this
document may beinvolve the subjectuse of (a) patent(s). ISO takes no position concerning the evidence,
validity or applicability of any claimed patent rights in respect thereof. As of the date of publication of this
document, ISO had not received notice of (a) patent(s) which may be required to implement this document.
However, implementers are cautioned that this may not represent the latest information, which may be
obtained from the patent database available at www.iso.org/patents. 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
).
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.
This second edition cancels and replaces the first edition (ISO 21322:2020), of which it constitutes a minor
revision.
The changes are as follows:
— the normative references (Clause 2) has been updated.
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.
iv
Introduction
For technical reasons, current standards in cosmetics microbiology maymight 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.
v
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 4973, Cosmetics — Microbiology — Quality control of culture media and diluents used in cosmetics standards
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 terminologicalterminology 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/http://www.electropedia.org/
3.1
cosmetic formulation, noun
preparation of raw materials with a qualitatively and quantitatively defined composition
3.2
cosmetic product, noun
cosmetic formulation (3.1) that has undergone all stages of production, including packaging in its final
container, for shipment
3.3
impregnated product, noun
product absorbed on the support
3.4
coated product, noun
product adsorbed on the support
3.5
test sample, noun
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
[1]
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 Clause A.1.
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 Clause A.1 and Clause 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.
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
[2] [3] [4]
, ISO 21150 ISO 18416 , ISO 21150, ISO 22717, ISO 22717 and
characteristic colonies. See ISO 18416
[5]
ISO 22718 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
[6] [7] [8]
microorganisms are described inISO 11930 inISO 11930, ISO 16212, ISO 16212 and ISO 21149 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, including
quality control, shall be in accordance with ISO 11930, ISO 16212, ISO 18416, ISO 21149,ISO 21150, ISO
22717ISO 22718 and ISO 4973.
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, ISO 22718 and ISO 22718. Quality control of diluents shall be in
accordance with ISO 4973.
5.3.2 Media for preparation of spores of Bacillus pizizenii (formerly known as Bacillus subtilis and
Bacillus subtilis subsp. spizizenii)
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
[9]
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 pizizenii (formerly known
® 1)
as Bacillus subtilis and Bacillus subtilis subsp. spizizenii) ATCC 6633TM (equivalent strain:
® TM TM1)
WDCM 00003 00003 or CIP 52.62 or NCIMB 8054 or NBRC 3134 or other equivalent national collection
strain) are used.
For testing the efficacy of neutralizers, two strains representative of both Gram negative and Gram positive
bacteria and a yeast are used:
® TM TM1) ® TM TM1)
— Staphylococcus aureus ATCC 6538 6538 (equivalent strain: WDCM 00032 00032 or CIP 4.83
or NCIMB 9518 or NBRC 13276 or KCTC 1916 or other equivalent national collection strain);
® TM TM1)
— Pseudomonas paraeruginosa (formerly Pseudomonas aeruginosa) ATCC 9027 9027 (equivalent
® TM TM1)
strain: WDCM 00026 00026 or CIP 82.118 or NCIMB 8626 or NBRC 13275 or KCTC 2513 or other
equivalent national collection strain).
® TM TM1)
An alternative Gram negative strain may be Escherichia coli ATCC 8739 8739 (equivalent strain:
® TM TM1)
WDCM 00012 00012 or CIP 53.126 or NCIMB 8545 or NBRC 3972 or KCTC 2571 or other equivalent
national collection strain).
® TM TM1) ® TM TM1)
— Candida albicans ATCC 10231 10231 (equivalent strain: WDCM 00054 00054 or CIP 48.72 or
NCPF 3179 or NBRC 1594 or KCTC 17205, or other equivalent national collection strain).
[9]
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
[11]
guidelines specified in ISO 21148 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.

1)
This information is given for the convenience of users of this document and does not constitute an endorsement by ISO.

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 Clause 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.
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 Clause 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 Clause 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.
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 accordance with 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 moulds)
b ym
within the recommended countable ranges (see Annex B).
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 of tested
b, ym,
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) or to
b ym
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. paraeruginosa, 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
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