ISO-22196-2007.pdf

Reference number ISO 22196:2007(E) © ISO 2007 INTERNATIONAL STANDARD ISO 22196 First edition 2007-10-15 Plastics Measurement of antibacterial activity on plastics surfaces Plastiques Mesurage de l'action antibactérienne sur les surfaces en plastique -,-,- ISO 22196:2007(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat accepts no liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below. COPYRIGHT PROTECTED DOCUMENT © ISO 2007 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO's member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii © ISO 2007 All rights reserved -,-,- ISO 22196:2007(E) © ISO 2007 All rights reserved iii Contents Page Foreword iv 1 Scope 1 2 Normative references1 3 Terms and definitions .2 4 Materials .2 4.1 Bacteria to be used for the tests2 4.2 Reagents, culture media and solutions.3 5 Apparatus .5 6 Sterilization of apparatus and storage of stock cultures 5 6.1 Dry-heat sterilization .5 6.2 High-pressure steam sterilization6 6.3 Preparation of glassware6 6.4 Maintenance of stock cultures.6 7 Procedure .6 7.1 Pre-culture of bacteria.6 7.2 Preparation of test specimens .6 7.3 Preparation of test inoculum7 7.4 Inoculation of test specimens7 7.5 Incubation of the inoculated test specimens8 7.6 Recovery of bacteria from test specimens .8 7.7 Determining the viable bacteria count by the pour plate culture method.9 8 Expression of results 9 8.1 Determination of the number of viable bacteria.9 8.2 Conditions for a valid test.9 8.3 Calculation of the antibacterial activity.10 8.4 Effectiveness of the antibacterial agent10 9 Repeatability and reproducibility.10 10 Test report11 Annex A (normative) Quality of biological materials.12 Annex B (informative) Repeatability and reproducibility13 Bibliography16 -,-,- ISO 22196:2007(E) iv © ISO 2007 All rights reserved 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. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. 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. ISO 22196 was prepared by Technical Committee ISO/TC 61, Plastics, Subcommittee SC 6, Ageing, chemical and environmental resistance. -,-,- INTERNATIONAL STANDARD ISO 22196:2007(E) © ISO 2007 All rights reserved 1 Plastics Measurement of antibacterial activity on plastics surfaces 1 Scope WARNING Handling and manipulation of microorganisms which are potentially hazardous requires a high degree of technical competence and may be subject to current national legislation and regulations. Only personnel trained in microbiological techniques should carry out such tests. Appropriate practices for disinfection, sterilization and personal hygiene must be strictly observed. This International Standard specifies a method of evaluating the antibacterial activity of antibacterial-treated plastic products (including intermediate products). NOTE It may also be suitable for other non-porous materials. It is not intended to be used to evaluate the effects and propagation of bacteria on plastics without antibacterial treatments. ISO 846 6 describes tests to evaluate the effects and propagation of bacteria on plastics, which are different from those covered by this International Standard. Those who are interested are referred to ISO 846:1997, method C. Secondary effects of antibacterial treatments, such as the prevention of biodeterioration and odour, are not covered by this International Standard, which is not intended to be used or referenced as a method to document or claim biodegradability of plastics. For biodegradation, refer to ISO 14851, ISO 14852 and ISO 14855 (see the Bibliography) and related standards. This International Standard does not concern plastic building materials, such as PVC or composites, unless they act in the same way as treated articles. Any results obtained with this International Standard should always refer to this standard and the conditions used. Results obtained with this International Standard indicate antibacterial activity under the specified experimental conditions used herein, and do not reflect activity under other circumstances where a variety of factors, such as temperature, humidity, different bacterial species, nutrient conditions, etc., have to be considered. A minimum diffusion of the antibacterial agents/chemicals into the test inoculum is necessary with this procedure. It is recommended that workers consult ISO 7218. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations -,-,- ISO 22196:2007(E) 2 © ISO 2007 All rights reserved 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 antibacterial term describing a state where growth of bacteria on the surfaces of products is suppressed or describing the effect of an agent which suppresses the growth of bacteria on the surfaces of products 3.2 antibacterial agent agent that inhibits the growth of bacteria on the surfaces of products by the use of an antibacterial surface treatment or a compounded agent 3.3 antibacterial activity difference in the logarithm of the viable cell count found on an antibacterial-treated product and an untreated product after inoculation with and incubation of bacteria 3.4 antibacterial effectiveness ability of an antibacterial agent to inhibit the growth of bacteria on the surface of a plastic treated with the agent, as determined by the value of the antibacterial activity 4 Materials 4.1 Bacteria to be used for the tests Both of the following species of bacteria shall be used: a) Staphylococcus aureus; b) Escherichia coli. In addition to the above two species of bacteria, other species can be used, if required. If other species are used, the species and the reason for their use shall be included in the test report. The bacterial strains to be used are shown in Table 1. If bacterial strains obtained from culture collections other than those shown in Table 1 are used, they shall be obtained from a member agency of the World Federation for Culture Collections (WFCC) or of the Japan Society for Culture Collections (JSCC) and shall be the same strains as those shown in Table 1. Prepare stock cultures of these species in accordance with the suppliers directions. Table 1 Bacterial strains to be used Name Strain Staphylococcus aureus ATCC 6538P CIP 53.156 DSM 346 NBRC 12732 NCIB 8625 Escherichia coli ATCC 8739 CIP 53.126 DSM 1576 NBRC 3972 NCIB 8545 -,-,- ISO 22196:2007(E) © ISO 2007 All rights reserved 3 4.2 Reagents, culture media and solutions Any water used shall be distilled or deionized and have a conductivity of 1 µS/cm. All reagents shall be of analytical grade and/or of a grade appropriate for microbiological purposes. 4.2.1 Nonionic surfactant Use polyoxyethylene sorbitan monooleate. 4.2.2 Biological materials The following biological materials are required: lecithin; D-glucose; yeast extract; meat extract (see Annex A); peptone (see Annex A); casein peptone; soybean peptone; tryptone. 4.2.3 Culture medium 4.2.3.1 General The culture medium specified below shall be used. The medium may be obtained from commercial suppliers. If so, it shall be prepared for use in accordance with the manufacturers instructions. 4.2.3.2 Suspension medium 1/500 nutrient broth (1/500 NB) Prepare nutrient broth by dissolving 3,0 g of meat extract, 10,0 g of peptone and 5,0 g of sodium chloride in 1 000 ml of distilled or deionized water. Dilute the nutrient broth with distilled or deionized water to a 500-fold volume and adjust the pH to a value between 6,8 and 7,2 with sodium hydroxide or hydrochloric acid. Sterilize by autoclaving (see 6.2). If it is not used immediately after preparation, preserve it at 5 °C to 10 °C. Never use a 1/500 NB that has been kept for one week or longer after preparation. 4.2.3.3 Nutrient agar Prepare nutrient agar 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 preparation, then preserve it at 5 °C to 10 °C. Never use a nutrient agar that has been kept for one month or longer after preparation. -,-,- ISO 22196:2007(E) 4 © ISO 2007 All rights reserved 4.2.3.4 Plate count agar Prepare plate count agar by dissolving 2,5 g of 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 preparation, preserve it at 5 °C to 10 °C. Never use a plate count agar that has been kept for one month or longer after preparation. 4.2.3.5 Slant culture medium Pour 6 ml to 10 ml of nutrient agar, which has been warmed to dissolve, into a screw-capped test tube. Sterilize by autoclaving (see 6.2). After sterilization, place the test tube at a slant angle of about 15° to the horizontal and allow the contents to solidify. If it is not used immediately after preparation, preserve it at 5 °C to 10 °C. Never use a slant culture medium kept for one month or longer after preparation. 4.2.3.6 Soybean casein digest broth with lecithin and polyoxyethylene sorbitan monooleate (SCDLP broth) Prepare SCDLP broth by dissolving 17,0 g of casein peptone, 3,0 g of soybean peptone, 5,0 g of sodium chloride, 2,5 g of disodium hydrogen phosphate, 2,5 g of glucose and 1,0 g of lecithin in 1 000 ml of distilled or deionized water. Mix thoroughly and add 7,0 g of nonionic surfactant. Adjust the pH to a value between 6,8 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 preparation, preserve it at 5 °C to 10 °C. Never use an SCDLP broth kept for one month or longer after preparation. NOTE SCDLP is the default neutralizer in the majority of circumstances. Information about selection and evaluation of alternative antibacterial neutralizing agents can be found in ASTM E 1054 4 and EN 1040 5. 4.2.3.7 Phosphate buffer solution Prepare phosphate buffer solution by placing 34,0 g of potassium dihydrogen phosphate in a 1 000 ml volumetric flask. Add 500 ml of distilled or deionized water and mix to dissolve. Adjust the pH to a value between 6,8 and 7,2 (at 25 °C) with sodium hydroxide. Add distilled or deionized water to make up to 1 000 ml. Sterilize by autoclaving (see 6.2). Never use a phosphate buffer solution kept for one month or longer after preparation. 4.2.3.8 Phosphate-buffered physiological saline Prepare physiological saline by placing 8,5 g of sodium chloride in 1 000 ml of distilled or deionized water and mixing to dissolve. Dilute the phosphate buffer solution prepared in 4.2.3.7 with the physiological saline to an 800-fold volume. Sterilize the phosphate-buffered physiological saline solution by autoclaving (see 6.2). If this solution is not used immediately after preparation, preserve it at 5 °C to 10 °C. Never use a phosphate- buffered physiological saline kept for one month or longer after preparation. -,-,- ISO 22196:2007(E) © ISO 2007 All rights reserved 5 5 Apparatus Unless otherwise specified, use the following apparatus and materials: 5.1 Dry-heat sterilizer, capable of maintaining the temperature at a value between 160 °C and 180 °C within ± 2 °C of the set point at equilibrium conditions. 5.2 Autoclave, capable of maintaining a temperature of (121 ± 2) °C and a pressure of (103 ± 5) kPa. 5.3 Hotplate with stirrer, or hot-water bath. 5.4 pH-meter, capable of measuring to ± 0,2 units. 5.5 Balance, capable of weighing to ± 0,01 g. 5.6 Pipetters, sterile, with 1 000 µl tips. 5.7 Incubator, capable of maintaining the temperature within ± 1 °C of the se