ISO 19045-2:2024

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
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
Email: copyright@iso.org
Website: www.iso.org
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,
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 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)
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 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
disposable Fuchs or modified Fuchs Rosenthal haemocytometer.
2 2
5.3.12 Sterile 75 cm and 175 cm flat polystyrene tissue culture flasks.
5.3.13 Orbital shaker.
5.3.14 Refrigerator, with a temperature of 2 °C to 8 °C.
5.4 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.
5.5 Culture maintenance
5.5.1 The strain should not be subcultured more than five passages as per American Type Culture
Collection (ATCC) protocols.
5.5.2 Maintenance of stock cultures and scaling up cultures for testing (see Annex C).
5.6 Growth and harvest of microbial challenge (trophozoite)
5.6.1 Grow trophozoites as described in Annex C using Acanthamoeba growth medium (Ac#6, Annex A).
Prepare a sufficient number of flasks based on the size of the experiment and the number of trophozoites
required.
5.6.2 After the 24 h scale up, dislodge the adherent trophozoites. Trophozoites may be dislodged by
vigorously shaking, by scraping the bottom of the flask with a cell scraper or by striking the flask with
moderate force.
5.6.3 Decant trophozoites into 50 ml polypropylene centrifuge tubes and centrifuge at 500 × g for 5 min at
room temperature.
5.6.4 Resuspend one tube pellet in 10 ml of ¼ strength Ringer’s solution as specified in Annex B. If more
inoculum is required, resuspend additional pellets using this same method.
5.6.5 Wash 3 times with 10 ml of ¼ strength Ringer’s solution by centrifugation at 500 × g for 2 min at
room temperature.
5.6.6 Resuspend pellet by vortexing in 1 ml to 2 ml of ¼ strength Ringer’s solution.
5.7 Preparation of Acanthamoeba stock solution
5.7.1 Enumerate trophozoite numbers in the stock solution using a cell counting chamber (make a 1:10 to
1:100 dilution in ¼ strength Ringer’s solution or appropriate diluent to assist) and record number cells/ml.
5.7.2 Adjust the Acanthamoeba stock concentration in ¼ strength Ringer’s Solution to 5 × 10 cells/ml
to 5 × 10 cells/ml based on the value obtained using the haemocytometer; this solution shall be called the
standardized Acanthamoeba stock solution.
Inoculate 10 ml of 1/4 strength Ringer’s solution with 0,1 ml of the standardized Acanthamoeba stock
4 5
solution to result in 5 × 10 cells/ml and 5 × 10 cells/ml for the inoculum control solution.
5.8 Stand-alone procedure – inoculation
5.8.1 If the product is sensitive to light, protect it from light during the period of the test.
5.8.2 Prepare a set of three round-bottomed tubes (for each lot tested) with each tube containing 10 ml of
test product solution per challenge organism. Tubes that are compatible with the test solution shall be used.
5.8.3 Inoculate the sample tube of the product to be tested with 0,1 ml of a suspension of the standardized
4 5
Acanthamoeba stock solution providing the cell concentration range (4 × 10 cells/ml to 6 × 10 cells/ml)
specified in 5.7.2 Ensure that the volume of inoculum does not exceed 1 % of the sample volume.
5.8.4 Mix contents of tubes using a vortex mixer (until a vortex forms). Ensure complete dispersion of the
inoculum by adequate mixing.
5.8.5 Store the inoculated product at room temperature. The temperature shall be monitored using a
calibrated device and the temperature documented.
5.9 Recovery procedures
Use at least four replicates in any recovery procedure. All recovery wells shall be observed at 14 days. Please
see Annex J for representative photographic images of positive and negative wells.
5.9.1 Stand-alone procedure – recovery method one (12 well plate method)
5.9.1.1 Take 1,0 ml aliquots of the inoculated product for determination of viable count at the disinfecting
time of interest following mixing using vortex mixer until a vortex forms. Recommended time points include:

25 % and 100 % of the minimum recommended disinfecting time for all organisms. If overnight contact lens
disinfection is recommended, use a soaking time of 8 h.
5.9.1.2 At the specified time intervals remove 1,0 ml aliquot from the test article and add to 9,0 ml of
-1
validated neutralising broth (see Annex G) (10 dilution). Mix the suspension well using the vortex mixer
until vortex forms. Allow to sit for appropriate time to allow neutralisation to be completed.
5.9.1.3 Perform a further five (5) 10-fold serial dilutions in ¼ strength Ringer’s solution (see Annex B)
-2 -3 -4 -5 -6
(dilutions 10 , 10 , 10 , 10 , 10 ).
5.9.1.4 Determine the viable count of organisms in appropriate dilutions by removing 1 ml of each dilution
and placing it into the corresponding well of a 12-well tissue culture plate containing NNA as specified in
Annex D with a lawn of E. coli (see Annex F). Plate each dilution in quadruplicate.
5.9.1.5 Incubate plates at 28 ± 2 °C and inspect microscopically for growth. All recovery wells shall be
observed at 14 days. Please see Annex J for representative photographic images of positive and negative wells.
5.9.1.6 The absence of growth per well shall be documented, e.g. by recording a “-” (no recovery), the
observance of growth per well shall be documented, e.g. by recording a “+” (recovery).
5.9.1.7 Determine log reduction values by using the most-probable number method using the Reed and
Muench computation as specified in Annex H or the Spearman-Karber computation specified in Annex I. For
recovery method one, the Reed and Muench spreadsheet will indicate 1 ml per well.
5.9.2 Stand-alone procedure – recovery method two (96 well plate method)
5.9.2.1 Take 20 μl aliquots of the inoculated product for determination of viable count at the disinfecting
time of interest following mixing using vortex mixer until a vortex forms. Recommended time points include:
25 % and 100 % of the minimum recommended disinfecting time for all organisms. If overnight contact lens
disinfection is recommended, use a soaking time of 8 h.
5.9.2.2 At the specified time intervals remove 20 μl from the test article and add to at least four outer
wells of a 96-well microtitre plate (A1 to A4) containing 180 μl of validated neutraliser broth (see Annex G)
-1
(10 dilution). Allow to sit for appropriate time to allow neutralization to be completed. Refer to Figure 1 for
an example of a 96 well microtiter plate layout.
5.9.2.3 Mix the contents of the outer wells by pipetting gently up and down six times and make five serial
10-fold dilutions across the microtitre plate by transferring 20 µl to the next well, mixing and transferring
another 20 µl, etc. (wells B1-B4, C1-C4, D1-D4, E1-E4 and F1-F4). Discard the final 20 µl. The following
-2 -3 -4 -5 -6
dilutions will therefore be prepared in this step: 10 , 10 , 10 , 10 , 10 .
For recovery method two, the Reed and Muench spreadsheet will indicate 0,2 ml per well.
5.9.2.4 Add 50 µl of E. coli (see Annex E) to each well.
5.9.2.5 Cover and incubate the plates at 28 ± 2 °C and inspect microscopically for growth. All recovery wells
must be observed at 14 days. Please see Annex J for representative photographic images of positive and negative
wells. Trophozoites may undergo encystment and so the wells may contain immature and mature cysts
5.9.2.6 The absence of growth per well shall be documented, e.g. by recording a “-” (no recovery), the
observance of growth per well shall be documented, e.g. by recording a “+” (recovery).

5.9.2.7 Determine log reduction values by using the most-probable number method using the Reed and
Muench computation (see Annex H) or the Spearman-Karber computation (see Annex I).
Figure 1 — Layout of the 96-well Plate for Method 2
Divide 96-well flat bottomed microtitre plates as shown in Figure 1:
Add 180 µl of validated neutralising broth (Annex G) to outer wells (column A) and 180 µl of ¼ strength
Ringer’s solution to the rest of the wells (columns B-F).
6 Controls
6.1 Inoculum control
6.1.1 The inoculum control shall be conducted at each trial using the same materials and methods
employed in the assay substituting ¼ Ringer’s for the test solution. Prepare an inoculum control by
dispersing 0,1 ml of the standardized Acanthamoeba stock solution (5.7.2) into 10 ml of the ¼ Ringer’s as
used in 5.8.3. Execute 5.8.4 and 5.8.5 and either 5.9.1 or 5.9.2 depending upon the recovery method to be
used for the product evaluation. The inoculum concentration shall be confirmed by haemocytometer count
of the cells/ml in the inoculated ¼ Ringer’s solution and the value recorded. For the purpose of determining
log reductions, the inoculum concentration and cell concentrations challenged in the test solution shall be
measured using the Reed and Muench spreadsheet or the Spearman-Karber spreadsheet.
6.2 Recovery medium control
6.2.1 Mix a 1/10 dilution (1 ml into 9 ml) of the disinfecting product in validated neutraliser broth using a
vortex mixer and let it stand for the appropriate time to allow neutralisation to be completed. Inoculate the
tube using 0,1 ml of the standardised Acanthamoeba stock solution (5.7.2) into the neutralised disinfection
product. Execute 5.8.4 and 5.8.5 and either 5.9.1 or 5.9.2 depending upon the recovery method to be used for
the product evaluation.
6.2.2 Ensure that the recovery from the neutraliser broth is at least 50 % of the inoculum control.
7 Performance criteria
If the average concentration of the cells on the inoculum control plates is below 1,0 × 10 cells/ml or above
5,0 × 10 cells/ml, the experiment is considered invalid and the test must be repeated.

Annex A
(normative)
Preparation of Acanthamoeba growth medium (Ac#6)
A.1 Intended use
Acanthamoeba growth medium (Ac#6) is used for axenic culture of Acanthamoeba trophozoites.
A.2 Composition
The composition of Ac#6 growth medium is given in Table A.1.
Table A.1 — Composition of Ac#6 growth medium
Material Amount
Biosate (e.g. BBL: BD-211862) 20,0 g
Glucose (e.g. Sigma, G7021) 5,0 g
KH PO (anhydrous: e.g. Fluka, 60219 or EMD, PX1565-1) 0,3 g
2 4
a
Vitamin B12 stock solution (100 μg/ml: e.g. Sigma, B4051 or EMD,1.11988.0100) 100 μl
b
L-Methionine stock solution (5 mg/ml: e.g. Fluka, 64319 or Calbiochem, 4500) 3 ml
Deionised or Nanopure™ water to 1 000 ml
a
Preparation of vitamin B12 stock solution (100 μg/ml):
Dissolve 10 mg vitamin B12 in 100 ml of deionised or Nanopure H O, aliquot into 10 ml
...