ISO 19045:2015
(Main)Ophthalmic optics — Contact lens care products — Method for evaluating Acanthamoeba encystment by contact lens care products
Ophthalmic optics — Contact lens care products — Method for evaluating Acanthamoeba encystment by contact lens care products
ISO 19045:2015 specifies a method for evaluating the potential of products for contact lens disinfection to induce encystment of Acanthamoeba species. This method excludes the evaluation of oxidative systems that require a special lens case for use. This International Standard does not address the evaluation of disinfection efficacy of contact lens disinfecting products.
Optique ophtalmique — Produits d'entretien de lentilles de contact — Méthode d’évaluation de l’enkystement de Acanthamoeba au contact des produits d’entretien des lentilles de contact
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Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 19045
First edition
2015-04-01
Ophthalmic optics — Contact lens care
products — Method for evaluating
Acanthamoeba encystment by contact
lens care products
Optique ophtalmique — Produits d’entretien de lentilles de contact —
Méthode d’évaluation de l’enkystement de Acanthamoeba au contact
des produits d’entretien des lentilles de contact
Reference number
ISO 19045:2015(E)
©
ISO 2015
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ISO 19045:2015(E)
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ii © ISO 2015 – All rights reserved
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ISO 19045:2015(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Terms and definitions . 1
3 Principle . 2
3.1 General . 2
3.2 Encystment test . 2
4 Encystment test method . 2
4.1 General . 2
4.2 Test organism . 2
4.3 Culture media and reagents . 2
4.4 Test materials . 2
4.5 Test samples . 3
4.6 Culture maintenance . 3
4.7 Preparation of microbial challenge . 3
4.8 Encystment procedure . 4
4.8.1 General. 4
4.8.2 Control plate . 4
4.8.3 Test samples. 5
4.9 Encystment calculation. 7
5 Controls . 7
Annex A (normative) Acanthamoeba growth medium (Ac#6) . 9
Annex B (informative) 1/4 strength Ringer’s Solution .10
Annex C (informative) Sarkosyl-Calcofluor White solution .11
Annex D (informative) Preparation of encystment control solutions .12
Annex E (informative) Maintenance of Acanthamoeba trophozoites and preparation for testing .14
Bibliography .15
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ISO 19045:2015(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 on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers
to Trade (TBT), see the following URL: Foreword — Supplementary Information.
The committee responsible for this document is ISO/TC 172, Optics and photonics, Subcommittee SC 7,
Ophthalmic optics and instruments.
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ISO 19045:2015(E)
Introduction
[1],[2]
Acanthamoeba is a genus of small free-living amoeba common to most soil and aquatic habitats.
The organism is characterized by a life cycle of a feeding and dividing trophozoite which, in response to
[1],[2]
adversity, can transform into a resistant cyst stage. Acanthamoeba cysts have been shown to resist
extremes of temperature, pH, desiccation, and most chemical disinfectants at normal concentration for
[1],[2],[3],[4]
use.
Recently, it has been observed that a contact lens disinfecting solution associated with a significant
[5],[6],[7]
number of Acanthamoeba keratitis cases was able to induce trophozoite encystment. Such a
phenomenon is of important concern as Acanthamoeba cysts can be resistant to contact lens disinfection
[3],[4],[7],[8],[9]
systems and this can increase the risk of acquiring Acanthamoeba keratitis.
This International Standard provides a methodology for assessing the capability of a contact lens
disinfecting solution to induce Acanthamoeba trophozoite encystment. This method does not describe
methodology to assess the efficacy of a contact lens disinfecting product against Acanthamoeba spp.
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INTERNATIONAL STANDARD ISO 19045:2015(E)
Ophthalmic optics — Contact lens care products — Method
for evaluating Acanthamoeba encystment by contact lens
care products
1 Scope
This International Standard specifies a method for evaluating the potential of products for contact lens
disinfection to induce encystment of Acanthamoeba species. This method excludes the evaluation of
oxidative systems that require a special lens case for use. This International Standard does not address
the evaluation of disinfection efficacy of contact lens disinfecting products.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
trophozoite
motile, feeding amoeboid form of Acanthamoeba
2.2
encystment
phase in the life cycle of Acanthamoeba where the trophozoite stage transforms into the cyst stage
2.3
mature cyst
dormant form of Acanthamoeba composed of an inner and outer cell wall, typically more resistant to a
range of challenges than trophozoites (2.1)
Note 1 to entry: Challenges include heat, dehydration, chemical, etc.
2.4
immature cyst
cyst comprised only of the inner cell wall
2.5
room temperature
temperature defined as 18 °C to 25 °C
2.6
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, can occur.
[10]
Note 2 to entry: This term is synonymous with the term “subculture”.
2.7
passage number
[10]
number of times cells in the culture have been subcultured or passaged (2.6)
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ISO 19045:2015(E)
3 Principle
3.1 General
The assay tests the capability for a solution to induce Acanthamoeba trophozoite encystment as this
physiological event can afford the organism protection from disinfection.
3.2 Encystment test
The encystment test is used to measure a disinfecting solution’s potential for inducing trophozoite
encystment to either the immature or mature cyst form. Assessment of this phenomenon is considered
important as Acanthamoeba cysts can be resistant to many disinfecting systems at operating conditions.
In the encystment test, contact lens disinfecting solutions are exposed to Acanthamoeba trophozoites.
Following detergent treatment and calcofluor white staining to lyse remaining trophozoites and stain
the inner cell wall, the organisms are observed microscopically for the production of immature and
mature cysts.
4 Encystment test method
4.1 General
Prior to conducting encystment studies, personnel should be trained and experienced in the following:
a) culturing and manipulating Acanthamoeba;
b) recognizing immature and mature cyst forms;
c) calculating the level of cyst formation as described in this International Standard.
4.2 Test organism
4.2.1 A. castellanii (ATCC 50370).
4.3 Culture media and reagents
4.3.1 Ac#6 axenic semi-defined Acanthamoeba growth medium (see Annex A).
4.3.2 1/4 strength Ringer’s solution (see Annex B).
4.3.3 Sarkosyl-Calcofluor White (see Annex C).
4.3.4 Encystment positive and negative control solutions (see Annex D).
4.4 Test materials
4.4.1 Sterile 50 ml and 14 ml/15 ml polypropylene centrifuge tubes.
4.4.2 Sterile 12 well flat bottomed plasma treated microtitre plates of material compatible with the
test material.
4.4.3 Calibrated pipettes (fixed and adjustable volume and multichannel) to deliver: 10 ml disposable,
20 µl, 100 µl, and 1 000 µl.
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ISO 19045:2015(E)
4.4.4 3 ml sterile disposable plastic Pasteur pipettes.
4.4.5 Fluorescence microscope with ×10, ×20, and ×40 phase contrast and fluorescence objectives with
a UV-2A filter, excitation 330 nm–380 nm, and emission greater than 420 nm.
4.4.6 An inverted microscope with ×10, ×20, and ×40 objectives.
4.4.7 28 °C ± 2 °C and 32,5 °C ± 2,5 °C incubators.
4.4.8 Centrifuge.
4.4.9 Vortex mixer.
4.4.10 Cell counting chamber (e.g. Modified Fuchs Rosenthal INCYTO disposable hemocytometer).
4.4.11 Optional: Pivoting blade cell scraper.
2 2 2
4.4.12 Sterile 75 cm and 150 cm /180 cm flat polystyrene tissue culture flasks.
4.5 Test samples
Aliquots of the product to be tested shall be representative of the product to be marketed. The product
should be taken directly from the final product container immediately prior to testing. Three lots of
product shall be tested. Each lot of product shall be tested with a separate inoculum preparation.
4.6 Culture maintenance
4.6.1 The strain should not be subcultured more than five passes as per American Type Culture
Collection (ATCC) protocols.
4.6.2 Maintenance of stock cultures (see E.1).
4.6.3 Scaling up cultures for testing (24 h prior to test) (see E.2).
4.7 Preparation of microbial challenge
4.7.1 Grow trophozoites as described in 4.6.2 and 4.6.3.
NOTE Prepare a sufficient number of flasks based on the size of the experiment and the number of
trophozoites required.
4.7.2 Vigorously shake flasks to dislodge adherent trophozoites (rinse with a pipette if necessary).
NOTE Scrape the bottom of the flask with a cell scraper if necessary.
4.7.3 Decant trophozoites into 50 ml polypropylene centrifuge tubes and centrifuge at 500 × g for 5 min
at room temperature.
4.7.4 Resuspend one tube pellet in 10 ml of 1/4 strength Ringer’s solution (see Annex B) and use to
resuspend the other pellets if additional inoculum is required.
4.7.5 Wash ×3 with 10 ml of 1/4 strength Ringer’s solution by centrifugation at 500 × g for 2 min at
room temperature.
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ISO 19045:2015(E)
4.7.6 Resuspend pellet by vortexing in 1 ml to 2 ml of 1/4 strength Ringer’s solution.
4.7.7 Enumerate trophozoite numbers using a cell counting chamber (make a 1:10 to 1:100 dilution
in 1/4 strength Ringer’s solution to assist) and record number/ml. A volume of 20 µl is used for cell
counting using the hemocytometer.
NOTE A 1:100 dilution can be prepared by two 1:10 serial dilutions of 100 μl into 900 μl.
7 7
4.7.8 Adjust the stock concentration to 1,0 × 10 /ml to 1,5 × 10 /ml in 1/4 strength Ringer’s solution
and use immediately for testing.
4.8 Encystment procedure
4.8.1 General
The encystment procedure consists of pipetting 3,0 ml ± 0,1 ml or weighing 3,0 g ± 0,1 g of control
and/or test solutions into a well of a 12 well microtitre plate.
4.8.2 Control plate
4.8.2.1 Dispense the encystment positive control solution into three wells and dispense the negative
control solution (see Annex D) into six wells each of a 12 well microtitre plate as shown in Figure 1.
Key
1 p
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