ISO 4768:2023

INTERNATIONAL ISO
STANDARD 4768
First edition
2023-07
Measurement method of anti-biofilm
activity on plastic and other non-
porous surfaces
Méthode de mesure de l'activité anti-biofilm sur le plastique et autres
surfaces non poreuses
Reference number
ISO 4768:2023(E)
© ISO 2023

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ISO 4768:2023(E)
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© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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Published in Switzerland
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ISO 4768:2023(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Materials . 1
4.1 Bacterial strain to be used for the tests . 1
4.2 Reagents . 2
4.3 Culture media and solution . 2
4.3.1 General . 2
4.3.2 Nutrient agar (NA) . 2
4.3.3 Suspension medium-1/5 Tryptic soy broth (1/5TSB) . 2
4.3.4 Plate count agar . 2
4.3.5 Slant culture medium . . . 2
4.3.6 0,1 % Crystal violet aqueous solution . 2
4.3.7 1,0 % Sodium dodecyl sulfate aqueous solution . 3
5 Apparatus . 3
6 Sterilization of apparatus and storage of stock cultures . 4
6.1 Dry-heat sterilization . 4
6.2 High-pressure steam sterilization . 4
6.3 Preparation of glassware . 4
6.4 Maintenance of stock cultures. 4
7 Procedure .4
7.1 Biofilm formation on the surface of the test specimens . 4
7.1.1 Pre-culture of bacteria . 4
7.1.2 Preparation of test specimens . 4
7.1.3 Preparation of the inoculated incubating medium . 5
7.1.4 Inoculation of test specimens . 5
7.1.5 Incubation of the inoculated test specimens . 5
7.2 Determination of the amount of biofilm . 6
7.2.1 First washing of test specimens . 6
7.2.2 Staining of test specimens with crystal violet . 6
7.2.3 Second washing of test specimens . 6
7.2.4 Biofilm recovery from test specimens . 7
7.2.5 Absorbance measurement . 7
7.3 Determining the viable bacteria count . 7
8 Expression of results . 8
8.1 Conditions for valid test . 8
8.2 Calculation of anti-biofilm activity on the basis of absorption spectrum method
for anti-biofilm materials . 8
8.3 Effectiveness of the anti-biofilm agent . 8
9 Repeatability and reproducibility .8
10 Test report . 8
Annex A (informative) Repeatability and reproducibility .10
Bibliography .12
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ISO 4768:2023(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
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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).
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of (a) patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed
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www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 61, Plastics, Subcommittee SC 6, Ageing,
chemical and environmental resistance.
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.
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ISO 4768:2023(E)
Introduction
Biofilms are known to be compositions of substances such as polysaccharides, lipids, and nucleic acids
produced by microorganisms and the cells of microorganisms, and are widely found in our living
environment as visible harms such as slimes, where water is present.
ISO 22196 on antibacterial test and ISO 21702 on antiviral test are the test methods for evaluating
“invisible hygiene” on the surface of non-porous products. Similarly, ISO 20743 on antibacterial test and
ISO 18184 on antiviral test are the test methods for evaluating the same of porous products.
This document describes how to evaluate products that are treated to suppress biofilm formations on
the non-porous surfaces.
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INTERNATIONAL STANDARD ISO 4768:2023(E)
Measurement method of anti-biofilm activity on plastic
and other non-porous surfaces
1 Scope
This document specifies a test method to evaluate the anti-biofilm activity of anti-biofilm treated
plastics and other non-porous surfaces of products, including intermediate products. It is applicable
to products such as plastics, coating materials, ceramics, stainless steels and rubber. Textile and
photocatalytic materials are out of its scope.
This method is intended to be a screening test for material development, and it is not expected to
reflect effects observed in the actual environment in which materials will be deployed. A “Crystal violet
staining – Absorbance measurement assay” is used to quantify the amount of biofilm formation in this
document.
2 Normative references
There are no normative references in this document.
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
biofilm
microbial cells and their metabolites such as polysaccharides, proteins, lipids and nucleic acids, firmly
attached to the material surface of the product in water
Note 1 to entry: In this document, crystal violet is used to stain the biofilm.
3.2
anti-biofilm
controlling the formation of biofilm (3.1) attached to the material surface of the product
3.3
anti-biofilm activity
activity in suppressing formation of biofilm (3.1) on the material surface
4 Materials
4.1 Bacterial strain to be used for the tests
The test strain is Staphylococcus epidermidis ATCC35984.
Other species of bacteria may be used after appropriate validation, as the importance of the species
chosen can differ depending on the target application. If other species are used, the name of the species,
its strain number and the specific reason for their use shall be included in the test report.
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ISO 4768:2023(E)
4.2 Reagents
Water shall be distilled or deionized and shall have a conductivity of <1 μS/cm.
All reagents shall be of an analytical grade and/or of a grade appropriate for microbiological purposes.
4.3 Culture media and solution
4.3.1 General
The culture medium specified in 4.3.2, 4.3.3, 4.3.4 and 4.3.5 shall be used. The medium may be
obtained from commercial suppliers, where it shall be prepared in accordance with the manufacturer's
instructions.
The quantity of the culture medium can be changed according to the intended test volume, but the
composition of the culture medium should not be changed.
4.3.2 Nutrient agar (NA)
Prepare NA by dissolving 5,0 g of meat extract, 10,0 g of peptone, 5,0 g of sodium chloride and 15,0 g of
agar powder in 1 000 ml of distilled or deionized water. Heat, with stirring, on a hotplate or in a boiling-
water bath until the agar has dissolved. Adjust the pH to a value between 7,0 and 7,2 (at 25 °C) with
sodium hydroxide or hydrochloric acid. Sterilize by autoclaving (see 6.2). If it is not used immediately
after the preparation, store it at 5 °C to 10 °C. NA that has been kept for one month or longer after the
preparation shall not be used.
4.3.3 Suspension medium-1/5 Tryptic soy broth (1/5TSB)
Prepare 1/5 TSB by dissolving 17,0 g of a pancreatic digest of casein, 3,0 g of papaic digest of soybean,
5,0 g of sodium chloride, 2,5 g of dipotassium phosphate and 2,5 g of dextrose in 1 000 ml of distilled
or deionized water. Dilute the trypticase soy broth with distilled or deionized water 1:4 volumetrically
and adjust the pH to a value between 7,1 and 7,5 (at 25 °C) with sodium hydroxide or hydrochloric acid.
Sterilize by autoclaving (see 6.2). If it is not used immediately after the preparation, store it at 5 °C to
10 °C. 1/5 TSB that has been kept for one week or longer after the preparation shall not be used.
4.3.4 Plate count agar
Prepare a plate count agar by dissolving 2,5 g of a yeast extract, 5,0 g of tryptone, 1,0 g of glucose and
15,0 g of agar powder in 1 000 ml of distilled or deionized water. Heat, with stirring, on a hotplate or
in a boiling-water bath until the agar has dissolved. Adjust the pH to a value between 7,0 and 7,2 (at
25 °C) with sodium hydroxide or hydrochloric acid. Sterilize by autoclaving (see 6.2). If it is not used
immediately after the preparation, store it at 5 °C to
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