ISO 19045-2:2024
(Main)Ophthalmic optics — Contact lens care products — Part 2: Method for evaluating disinfecting efficacy by contact lens care products using trophozoites of Acanthamoeba species as the challenge organisms
Ophthalmic optics — Contact lens care products — Part 2: Method for evaluating disinfecting efficacy by contact lens care products using trophozoites of Acanthamoeba species as the challenge organisms
This document specifies a test method to be used in evaluating the antimicrobial activity of products for contact lens disinfection by chemical methods using the trophozoite form of Acanthamoeba species as the challenge organism. This document is not applicable to the evaluation of oxidative systems that require a special lens case for use.
Optique ophtalmique — Produits d'entretien de lentilles de contact — Partie 2: Méthode d'évaluation de l'efficacité désinfectante des produits d'entretien des lentilles de contact utilisant des trophozoïtes de l'espèce Acanthamoeba comme organismes pour l'épreuve microbienne
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International
Standard
ISO 19045-2
First edition
Ophthalmic optics — Contact lens
2024-11
care products —
Part 2:
Method for evaluating disinfecting
efficacy by contact lens care
products using trophozoites of
Acanthamoeba species as the
challenge organisms
Optique ophtalmique — Produits d'entretien de lentilles de
contact —
Partie 2: Méthode d'évaluation de l'efficacité désinfectante
des produits d'entretien des lentilles de contact utilisant des
trophozoïtes de l'espèce Acanthamoeba comme organismes pour
l'épreuve microbienne
Reference number
© ISO 2024
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Published in Switzerland
ii
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Acanthamoeba trophozoite disinfecting test method . 2
5.1 Organisms .2
5.2 Culture media and reagents.2
5.3 Test materials .3
5.4 Test samples .3
5.5 Culture maintenance .3
5.6 Growth and harvest of microbial challenge (trophozoite) .3
5.7 Preparation of Acanthamoeba stock solution .4
5.8 Stand-alone procedure – inoculation .4
5.9 Recovery procedures .4
5.9.1 Stand-alone procedure – recovery method one (12 well plate method) .4
5.9.2 Stand-alone procedure – recovery method two (96 well plate method) .5
6 Controls . . 6
6.1 Inoculum control .6
6.2 Recovery medium control .6
7 Performance criteria . 7
Annex A (normative) Preparation of Acanthamoeba growth medium (Ac#6) . 8
Annex B (normative) Preparation of ¼ strength Ringer’s solution . 9
Annex C (normative) Maintenance of Acanthamoeba trophozoites and preparation for testing .10
Annex D (normative) Preparation of non-nutrient agar (NNA) .11
Annex E (informative) Preparation of E. coli suspension .13
Annex F (informative) Preparation of non-nutrient agar (NNA) plates with E. coli . 14
Annex G (informative) Preparation of neutraliser broth for recovery methods .15
Annex H (normative) Reed and Muench computation method for calculation of the 50 %
endpoint titre . 16
Annex I (normative) Spearman-Karber computation method for calculation of the 50 %
endpoint titre .20
Annex J (informative) Micrographs of cultures using the 96 well plate method .22
Bibliography .26
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,
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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 document 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)
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www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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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 172, Optics and photonics, Subcommittee SC 7,
Ophthalmic optics and instruments.
A list of all parts in the ISO 19045-2 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
International Standard ISO 19045-2:2024(en)
Ophthalmic optics — Contact lens care products —
Part 2:
Method for evaluating disinfecting efficacy by contact lens
care products using trophozoites of Acanthamoeba species as
the challenge organisms
1 Scope
This document specifies a test method to be used in evaluating the antimicrobial activity of products for
contact lens disinfection by chemical methods using the trophozoite form of Acanthamoeba species as the
challenge organism.
This document is not applicable to the evaluation of oxidative systems that require a special lens case for use.
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
contact lens disinfection
chemical or physical process to reduce the number of viable microorganisms
Note 1 to entry: This is specified in the performance requirement clauses of ISO 14729 or ISO 18369-1.
3.2
trophozoite
motile, feeding amoeboid form of Acanthamoeba
[SOURCE: ISO 19045:2015, 2.1]
3.3
encystment
phase in the life cycle of Acanthamoeba where the trophozoite stage transforms into the cyst stage
3.4
mature cyst
dormant form of Acanthamoeba, composed of an inner and outer cell wall, typically more resistant to a range
of challenges than trophozoites (3.2)
Note 1 to entry: Challenges include heat, dehydration, chemical, etc.
3.5
immature cyst
cyst comprised only of the inner cell wall
3.6
room temperature
temperature between 18 °C to 25 °C
3.7
refrigerator temperature
temperature between 2 °C to 8 °C
3.8
passage
transfer or transplantation of cells, with or without dilution, from one culture vessel to another
Note 1 to entry: It is understood that any time cells are transferred from one vessel to another, a certain portion of the
cells may be lost and, therefore, dilution of cells, whether deliberate or not, may occur.
Note 2 to entry: This term is synonymous with the term “subculture”.
3.9
passage number
number of times cells in the culture have been subcultured or passaged
4 Principle
This assay challenges a contact lens disinfecting product with a standard inoculum of trophozoites of the
specified Acanthamoeba species and establishes the extent of their viability at pre-determined time intervals
comparable with those during which the product may be used.
5 Acanthamoeba trophozoite disinfecting test method
5.1 Organisms
5.1.1 A. castellanii (ATCC 50370), A. polyphaga (ATCC 30461).
5.1.2 Do not use Acanthamoeba trophozoites beyond passage number 5.
5.1.3 Escherichia coli (ATCC 8739).
NOTE E. coli is used for preparation of agar overlays for recovery of challenge organisms for recovery method one
(5.9.1) and for inoculation of microtitre wells for recovery of challenge organisms for recovery method two (5.9.2).
5.2 Culture media and reagents
5.2.1 Ac#6 axenic semi-defined Acanthamoeba growth medium (in accordance with Annex A).
5.2.2 ¼ strength Ringer’s solution (see Annex B).
5.2.3 Page’s saline non-nutrient agar (see Annex D) – recovery method one (see 5.9.1).
5.2.4 Trypticase soy broth (TSB) – (for use in Annex E).
5.2.5 Neutralising Broth for both recovery methods (see Annex G).
5.3 Test materials
5.3.1 Sterile 50 ml polypropylene centrifuge tubes.
5.3.2 Sterile 15 ml round-bottomed tubes (polystyrene, polypropylene or glass, depending on the
formulations to be tested).
5.3.3 Sterile 12-well flat bottom opto-mechanical- or plasma-treated microtitre plates.
5.3.4 Sterile 96-well flat bottom opto-mechanical- or plasma-treated microtitre plates.
5.3.5 Calibrated pipettes (fixed and adjustable volume and multichannel) to deliver: 20 µl, 50 µl, 100 µl,
180 µl, 200 µl and 1 000 µl.
5.3.6 Sterile, disposable transfer pipets, capable of pipetting 3 ml and 10 ml.
5.3.7 Inverted microscope, with ×10, ×20 and ×40 phase contrast objectives.
5.3.8 (28 ± 2) °C incubator.
5.3.9 Centrifuge.
5.3.10 Vortex mixer.
5.3.11 Cell counting chamber (haemocytometer), with a depth of 0,2 mm; e.g. an appropriate reusable or
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