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1、 Reference number ISO 21149:2006(E) ISO 2006 INTERNATIONAL STANDARD ISO 21149 First edition 2006-03-01 Cosmetics Microbiology Enumeration and detection of aerobic mesophilic bacteria Cosmtiques Microbiologie Dnombrement et dtection des bactries arobies msophiles Copyright International Organization
2、for Standardization Provided by IHS under license with ISO Licensee=Aramco HQ/9980755100 Not for Resale, 04/16/2007 09:06:13 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 21149:2006(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Ado
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5、e 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. ISO 2006 All rights reserved. Unless otherwise specified, no part of this publication may
6、 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 ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel.
7、+ 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2006 All rights reserved Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Aramco HQ/9980755100 Not for Resale, 04/16/2007 09:06:13 M
8、DTNo reproduction or networking permitted without license from IHS -,-,- ISO 21149:2006(E) ISO 2006 All rights reserved iii Contents Page Foreword iv 1 Scope . 1 2 Normative references. 1 3 Terms and definitions. 1 4 Principle. 2 4.1 General. 2 4.2 Plate count. 2 4.3 Membrane filtration. 2 4.4 Detec
9、tion of bacteria by enrichment. 3 5 Diluents, neutralizers and culture media 3 5.1 General. 3 5.2 Neutralizing diluents and diluents 3 5.3 Diluent for the bacterial suspension (tryptone sodium chloride solution). 4 5.4 Culture media 4 6 Apparatus and glassware 6 7 Strains of microorganisms 7 8 Handl
10、ing of cosmetic products and laboratory samples . 7 9 Procedure 7 9.1 General recommendation 7 9.2 Preparation of the initial suspension 7 9.3 Counting methods 8 9.4 Enrichment 9 10 Counting of colonies (plate counts and membrane filtration methods). 9 11 Detection of growth (enrichment method). 9 1
11、2 Expression of results . 10 12.1 Method of calculation for plate count. 10 12.2 Interpretation. 10 12.3 Examples . 11 12.4 Detection after enrichment 13 13 Neutralization of the antimicrobial properties of the product 13 13.1 General. 13 13.2 Preparation of inoculum 14 13.3 Validation of counting m
12、ethods 14 13.4 Validation of the detection method by enrichment. 15 13.5 Interpretation of validation results 15 14 Test report . 16 Annex A (informative) Other neutralizing diluents . 17 Annex B (informative) Other diluents. 19 Annex C (informative) Other culture media. 20 Annex D (informative) Neu
13、tralizers of antimicrobial activity of preservatives and rinsing liquids. 23 Bibliography. 24 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Aramco HQ/9980755100 Not for Resale, 04/16/2007 09:06:13 MDTNo reproduction or networking permitted w
14、ithout license from IHS -,-,- ISO 21149:2006(E) iv ISO 2006 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
15、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
16、 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.
17、 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
18、document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 21149 was prepared by Technical Committee ISO/TC 217, Cosmetics. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee
19、=Aramco HQ/9980755100 Not for Resale, 04/16/2007 09:06:13 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARD ISO 21149:2006(E) ISO 2006 All rights reserved 1 Cosmetics Microbiology Enumeration and detection of aerobic mesophilic bacteria 1 Scope This Int
20、ernational Standard gives general guidelines for enumeration and detection of mesophilic aerobic bacteria present in cosmetics, by counting the colonies on agar medium after aerobic incubation, or by checking the absence of bacterial growth after enrichment. Because of the large variety of cosmetic
21、products within this field of application, this method may not be appropriate for some products in every detail (e.g. certain water immiscible products). Other methods (e.g. automated) may be substituted for the tests presented here provided that their equivalence has been demonstrated or the method
22、 has been otherwise validated. If needed, microorganisms enumerated or detected may be identified using suitable identification tests described in the standards given in the Bibliography. In order to ensure product quality and safety for consumers, it is advisable to perform an appropriate microbiol
23、ogical risk analysis, so as to determine the types of cosmetic products to which this International Standard is applicable. Products considered to present a low microbiological risk include those with low water activity, hydro-alcoholic products, extreme pH values, etc. 2 Normative references The fo
24、llowing 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 21148:2005, Cosmetics Microbiology General instruct
25、ions for microbiological examination 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 aerobic mesophilic bacteria mesophilic bacteria growing aerobically under the conditions specified in this International Standard NOTE In the described condi
26、tions, other types of microorganisms (e.g. yeast, mould) can be detected. 3.2 product portion of an identified cosmetic product received in the laboratory for testing Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Aramco HQ/9980755100 Not for
27、 Resale, 04/16/2007 09:06:13 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 21149:2006(E) 2 ISO 2006 All rights reserved 3.3 sample portion of the product (at least 1 g or 1 ml) which is used in the test to prepare the initial suspension 3.4 initial suspension suspensi
28、on (or solution) of a sample in a defined volume of an appropriate liquid (diluent, neutralizer, broth or combination of them) 3.5 sample dilution dilution of the initial suspension 4 Principle 4.1 General This method involves enumeration of colonies on a non-selective agar medium or by the presence
29、 or absence of bacterial growth after enrichment. The possible inhibition of microbial growth by the sample shall be neutralized to allow the detection of viable microorganism 1. In all cases and whatever the methodology, the neutralization of the antimicrobial properties of the product shall be che
30、cked and validated 2 3 4. 4.2 Plate count Plate count consists of the following steps. Preparation of poured plates or spread plates, using a specified culture medium, and inoculation of the plates using a defined quantity of the initial suspension or dilution of the product. Aerobic incubation of t
31、he plates at 32,5 C 2,5 C for 72 h 6 h. Counting the number of colony forming units (CFU) and calculation of the number of aerobic mesophilic bacteria per millilitre or per gram of product. 4.3 Membrane filtration Membrane filtration consists of the following steps. Transfer a suitable amount of the
32、 sample prepared as validated in the filtration apparatus wetted with a small volume of an appropriate sterile diluent, filter immediately and wash according to the validated procedure (see 13.3.4). Transfer the membrane filter onto the surface of the specified agar medium as specified in ISO 21148.
33、 Aerobic incubation of the membranes at 32,5 C 2,5 C for 72 h 6 h. Counting the number of colony forming units (CFU) and calculation of the number of aerobic mesophilic bacteria per millilitre or per gram of product. Copyright International Organization for Standardization Provided by IHS under lice
34、nse with ISO Licensee=Aramco HQ/9980755100 Not for Resale, 04/16/2007 09:06:13 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 21149:2006(E) ISO 2006 All rights reserved 3 4.4 Detection of bacteria by enrichment Detection of bacteria by enrichment consists of the follow
35、ing steps: Incubation at 32,5 C 2,5 C for at least 20 h of a defined quantity of the initial suspension in a non-selective liquid medium containing suitable neutralizers and/or dispersing agents. Transfer of a defined quantity of the previous suspension on non-selective solid agar medium. Aerobic in
36、cubation at 32,5 C 2,5 C for 48 h to 72 h. Detection of growth and expression of results as “presence/absence” of aerobic mesophilic bacteria per sample S of product. 5 Diluents, neutralizers and culture media 5.1 General General specifications are given in ISO 21148. When water is used in a formula
37、, use distilled water or purified water as specified in ISO 21148. The following diluents, neutralizers and culture media are suitable for enumeration and detection of aerobic mesophilic bacteria. Other diluents, neutralizers and culture media may be used if they have been demonstrated to be suitabl
38、e for use. 5.2 Neutralizing diluents and diluents 5.2.1 General The diluent is used to disperse the sample. It may contain neutralizers if the specimen to be tested has antimicrobial properties. The efficacy of the neutralization shall be demonstrated before the determination of the count (see Claus
39、e 13). Information relative to suitable neutralizers is given in Annex D. 5.2.2 Neutralizing diluents 5.2.2.1 Fluid casein digest soy lecithin polysorbate 20 medium (SCDLP 20 broth) 5.2.2.1.1 Composition Pancreatic digest of casein 20,0 g Soy lecithin 5,0 g Polysorbate 20 40,0 ml Water 960,0 ml 5.2.
40、2.1.2 Preparation Dissolve the polysorbate 20 in 960 ml of water by mixing while heating in a water bath at 49 C 2 C. Add pancreatic digest of casein and soy lecithin. Heat for about 30 min to obtain solution. Mix and dispense the medium into suitable containers. Sterilize in the autoclave at 121 C
41、for 15 min. After sterilization, the pH shall be equivalent to 7,3 0,2 when measured at room temperature. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Aramco HQ/9980755100 Not for Resale, 04/16/2007 09:06:13 MDTNo reproduction or networking
42、 permitted without license from IHS -,-,- ISO 21149:2006(E) 4 ISO 2006 All rights reserved 5.2.2.2 Other neutralizing diluents Other neutralizing diluents may be used as appropriate (see Annex A and Annex D). 5.2.3 Diluent 5.2.3.1 Fluid A 5.2.3.1.1 Composition Peptic digest of animal tissue 1,0 g Wa
43、ter 1 000 ml 5.2.3.1.2 Preparation Dissolve 1 g of peptone in water to make 1 l. Heat with frequent agitation. Dispense into suitable containers. Sterilize in the autoclave at 121 C for 15 min. After sterilization, the pH shall be equivalent to 7,1 0,2 when measured at room temperature. 5.2.3.2 Othe
44、r diluents Other diluents may be used as appropriate (see Annex B). 5.3 Diluent for the bacterial suspension (tryptone sodium chloride solution) 5.3.1 Composition Tryptone, pancreatic digest of casein 1,0 g Sodium chloride 8,5 g Water 1 000 ml 5.3.2 Preparation Dissolve the components in the water b
45、y mixing while heating. Dispense into suitable containers. Sterilize in the autoclave at 121 C for 15 min. After sterilization, the pH shall be equivalent to 7,0 0,2 when measured at room temperature. 5.4 Culture media 5.4.1 General Culture media may be prepared as follows, or from dehydrated cultur
46、e media according to the instructions of the manufacturer. Ready-to-use media may be used when their composition and/or growth yields are comparable to those of the formulas given herein. 5.4.2 Culture media for counting 5.4.2.1 Soybeancasein digest agar medium (SCDA) or tryptic soy agar (TSA) 5.4.2
47、.1.1 Composition Pancreatic digest of casein 15,0 g Papaic digest of soybean meal 5,0 g Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Aramco HQ/9980755100 Not for Resale, 04/16/2007 09:06:13 MDTNo reproduction or networking permitted without
48、 license from IHS -,-,- ISO 21149:2006(E) ISO 2006 All rights reserved 5 Sodium chloride 5,0 g Agar 15,0 g Water 1 000 ml 5.4.2.1.2 Preparation Dissolve the components or the dehydrated complete medium in the water by mixing while heating. Dispense the medium into suitable containers. Sterilize in the autoclave at 121 C for 15 min. After sterilization and cooling down, the pH shall be equivalent to 7,3