1、BSI Standards PublicationBS ISO 19045:2015Ophthalmic optics Contactlens care products Methodfor evaluating Acanthamoebaencystment by contact lenscare productsBS ISO 19045:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 19045:2015.The UK participation in it
2、s preparation was entrusted to TechnicalCommittee CH/172/9, Contact lenses and contact lens care products.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are
3、 responsible for its correctapplication. The British Standards Institution 2015. Published by BSI StandardsLimited 2015ISBN 978 0 580 85629 7ICS 11.040.70Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStan
4、dards Policy and Strategy Committee on 30 April 2015.Amendments issued since publicationDate Text affectedBS ISO 19045:2015 ISO 2015Ophthalmic optics Contact lens care products Method for evaluating Acanthamoeba encystment by contact lens care productsOptique ophtalmique Produits dentretien de lenti
5、lles de contact Mthode dvaluation de lenkystement de Acanthamoeba au contact des produits dentretien des lentilles de contactINTERNATIONAL STANDARDISO19045First edition2015-04-01Reference numberISO 19045:2015(E)BS ISO 19045:2015ISO 19045:2015(E)ii ISO 2015 All rights reservedCOPYRIGHT PROTECTED DOCU
6、MENT ISO 2015All rights reserved. Unless otherwise specified, 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
7、 be requested from either ISO at the address below or ISOs member body in the country of the requester.ISO copyright officeCase postale 56 CH-1211 Geneva 20Tel. + 41 22 749 01 11Fax + 41 22 749 09 47E-mail copyrightiso.orgWeb www.iso.orgPublished in SwitzerlandBS ISO 19045:2015ISO 19045:2015(E)Forew
8、ord ivIntroduction v1 Scope . 12 Terms and definitions . 13 Principle 23.1 General . 23.2 Encystment test 24 Encystment test method . 24.1 General . 24.2 Test organism . 24.3 Culture media and reagents . 24.4 Test materials . 24.5 Test samples 34.6 Culture maintenance 34.7 Preparation of microbial c
9、hallenge . 34.8 Encystment procedure . 44.8.1 General 44.8.2 Control plate 44.8.3 Test samples. 54.9 Encystment calculation 75 Controls 7Annex A (normative) Acanthamoeba growth medium (Ac#6) . 9Annex B (informative) 1/4 strength Ringers Solution .10Annex C (informative) Sarkosyl-Calcofluor White sol
10、ution .11Annex D (informative) Preparation of encystment control solutions 12Annex E (informative) Maintenance of Acanthamoeba trophozoites and preparation for testing .14Bibliography .15 ISO 2015 All rights reserved iiiContents PageBS ISO 19045:2015ISO 19045:2015(E)ForewordISO (the International Or
11、ganization 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 establ
12、ished 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 standardizati
13、on.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
14、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 pat
15、ent 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
16、an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT), see the following URL: Foreword Supplementary Information .The committee responsible for t
17、his document is ISO/TC 172, Optics and photonics, Subcommittee SC 7, Ophthalmic optics and instruments.iv ISO 2015 All rights reservedBS ISO 19045:2015ISO 19045:2015(E)IntroductionAcanthamoeba is a genus of small free-living amoeba common to most soil and aquatic habitats.1,2The organism is characte
18、rized by a life cycle of a feeding and dividing trophozoite which, in response to adversity, can transform into a resistant cyst stage.1,2Acanthamoeba cysts have been shown to resist extremes of temperature, pH, desiccation, and most chemical disinfectants at normal concentration for use.1,2,3,4Rece
19、ntly, it has been observed that a contact lens disinfecting solution associated with a significant number of Acanthamoeba keratitis cases was able to induce trophozoite encystment.5,6,7Such a phenomenon is of important concern as Acanthamoeba cysts can be resistant to contact lens disinfection syste
20、ms and this can increase the risk of acquiring Acanthamoeba keratitis.3,4,7,8,9This 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 th
21、e efficacy of a contact lens disinfecting product against Acanthamoeba spp. ISO 2015 All rights reserved vBS ISO 19045:2015BS ISO 19045:2015Ophthalmic optics Contact lens care products Method for evaluating Acanthamoeba encystment by contact lens care products1 ScopeThis International Standard speci
22、fies 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 disinfe
23、ction efficacy of contact lens disinfecting products.2 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.2.1trophozoitemotile, feeding amoeboid form of Acanthamoeba2.2encystmentphase in the life cycle of Acanthamoeba where the trophozoite stage transfo
24、rms into the cyst stage2.3mature cystdormant 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.2.4immature cystcyst comprised only of the inner c
25、ell wall2.5room temperaturetemperature defined as 18 C to 25 C2.6passagetransfer or transplantation of cells, with or without dilution, from one culture vessel to anotherNote 1 to entry: It is understood that any time cells are transferred from one vessel to another, a certain portion of the cells m
26、ay be lost and, therefore, dilution of cells, whether deliberate or not, can occur.Note 2 to entry: This term is synonymous with the term “subculture”.102.7passage numbernumber of times cells in the culture have been subcultured or passaged (2.6)10INTERNATIONAL STANDARD ISO 19045:2015(E) ISO 2015 Al
27、l rights reserved 1BS ISO 19045:2015ISO 19045:2015(E)3 Principle3.1 GeneralThe 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 testThe encystment test is used to me
28、asure a disinfecting solutions 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 l
29、ens 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
30、method4.1 GeneralPrior 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 Te
31、st organism4.2.1 A. castellanii (ATCC 50370).4.3 Culture media and reagents4.3.1 Ac#6 axenic semi-defined Acanthamoeba growth medium (see Annex A).4.3.2 1/4 strength Ringers solution (see Annex B).4.3.3 Sarkosyl-Calcofluor White (see Annex C).4.3.4 Encystment positive and negative control solutions
32、(see Annex D).4.4 Test materials4.4.1 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: 1
33、0 ml disposable, 20 l, 100 l, and 1 000 l.2 ISO 2015 All rights reservedBS ISO 19045:2015ISO 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 nm380 nm,
34、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 cel
35、l scraper.4.4.12 Sterile 75 cm2and 150 cm2/180 cm2flat polystyrene tissue culture flasks.4.5 Test samplesAliquots 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. Thr
36、ee lots of product shall be tested. Each lot of product shall be tested with a separate inoculum preparation.4.6 Culture maintenance4.6.1 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).
37、4.6.3 Scaling up cultures for testing (24 h prior to test) (see E.2).4.7 Preparation of microbial challenge4.7.1 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 s
38、hake 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 pe
39、llet in 10 ml of 1/4 strength Ringers 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 Ringers solution by centrifugation at 500 g for 2 min at room temperature. ISO 2015 All rights reserved 3BS ISO 19045:2015ISO
40、 19045:2015(E)4.7.6 Resuspend pellet by vortexing in 1 ml to 2 ml of 1/4 strength Ringers solution.4.7.7 Enumerate trophozoite numbers using a cell counting chamber (make a 1:10 to 1:100 dilution in 1/4 strength Ringers solution to assist) and record number/ml. A volume of 20 l is used for cell coun
41、ting using the hemocytometer.NOTE A 1:100 dilution can be prepared by two 1:10 serial dilutions of 100 l into 900 l.4.7.8 Adjust the stock concentration to 1,0 107/ml to 1,5 107/ml in 1/4 strength Ringers solution and use immediately for testing.4.8 Encystment procedure4.8.1 GeneralThe encystment pr
42、ocedure 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 plate4.8.2.1 Dispense the encystment positive control solution into three wells and dispense the negative control solution (see Annex D) into s
43、ix wells each of a 12 well microtitre plate as shown in Figure 1.Key1 positive control2 negative control T243 negative control T0Figure 1 12 well microtitre plate for controls4.8.2.2 Add 30 l of 1,0 107/ml trophozoites in 1/4 strength Ringers solution to all wells. This is designed to yield a final
44、concentration of 1,0 105/ml in the test and control wells. If the stock trophozoite concentration is found to be 1,0 107/ml, then add an appropriate volume to the wells to yield the required inoculum concentration of 1 105trophozoites/ml (e.g. 20 l if the stock suspension was counted as 1,5 107/ml).
45、NOTE Test solutions can be assayed on separate plates if the same trophozoite inoculum preparation is used and one plate has the appropriate encystment positive and negative controls.4 ISO 2015 All rights reservedBS ISO 19045:2015ISO 19045:2015(E)4.8.2.3 Mix contents of wells by gently pipetting up
46、and down three times with a 1 000 l pipettor set to deliver 500 l or a 3 ml disposable pipette.4.8.2.4 Immediately perform cell chamber counts using the hemocytometer on each of the three T0encystment negative control wells and record the individual and averaged total cell count/ml in Table 1.4.8.2.
47、5 Measure the background cyst count in the inoculum by adding 50 l of the Sarkosyl-Calcofluor White solution (see Annex C) to each of the three T0encystment negative control wells pipetting vigorously to mix and leaving at room temperature for 5 min.NOTE It has been observed that 5 min is critical f
48、or optimal assessment. Longer exposure times can cause the cysts to lyse and become difficult to recognize.4.8.2.6 Again, mix vigorously by pipetting up and down using a 1 000 l pipettor set to deliver 500 l or a 3 ml disposable pipette.4.8.2.7 Immediately perform cell chamber counts using the hemoc
49、ytometer on the Sarkosyl-Calcofluor White treated cells (under UV fluorescence with appropriate filter for Calcofluor white detection) to determine the background cyst level. Switch between UV fluorescence and white light to confirm observation of cysts. Count the number of refractile and fluorescent cells/ml and record in Table 1. These represent immature and mature cyst forms and give the background levels for the experiment (see Figu
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