1、BRITISH STANDARDCosmetics Microbiology Enumeration and detection of aerobic mesophilic bacteriaICS 07.100.99; 71.100.70g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g
2、42g43g55g3g47g36g58BS EN ISO 21149:2009National forewordThis British Standard is the UK implementation of EN ISO 21149:2009. It is identical to ISO 21149:2006. It supersedes BS ISO 21149:2006 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee CW/217, Cosm
3、etics.A list of organizations represented on this committee can be obtained on request to its secretary. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard cannot confer immuni
4、ty from legal obligations.BS EN ISO 21149:2009This British Standard was published under the authority of the Standards Policy and Strategy Committee on 28 April 2006 BSI 2010Amendments/corrigenda issued since publicationDate Comments 31 January 2010 This corrigendum renumbers BS ISO 21149:2006 as IS
5、BN 978 0 580 66832 6BS EN ISO 21149:2009EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 21149June 2009ICS 07.100.99; 71.100.70English VersionCosmetics - Microbiology - Enumeration and detection ofaerobic mesophilic bacteria (ISO 21149:2006)Cosmtiques - Microbiologie - Dnombrement et dtectionde
6、s bactries arobies msophiles (ISO 21149:2006)Kosmetik - Mikrobiologie - Zhlung und Nachweis vonaeroben mesophilen Bakterien (ISO 21149:2006)This European Standard was approved by CEN on 23 May 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditio
7、ns for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three offi
8、cial versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, B
9、elgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR
10、 STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 21149:2009: EForeword The text of ISO 21149:200
11、6 has been prepared by Technical Committee ISO/TC 217 “Cosmetics” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 21149:2009 by Technical Committee CEN/SS H99 “Products for household and leisure use - Undetermined” the secretariat of which is held by CMC
12、. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by December 2009, and conflicting national standards shall be withdrawn at the latest by December 2009. Attention is drawn to the possibility that so
13、me of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to imp
14、lement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerlan
15、d and the United Kingdom. Endorsement notice The text of ISO 21149:2006 has been approved by CEN as a EN ISO 21149:2009 without any modification. iiBS EN ISO 21149:2009iiiContents Page 1 Scope . 1 2 Normative references . 1 3 Terms and definitions. 1 4 Principle. 2 4.1 General. 2 4.2 Plate count. 2
16、4.3 Membrane filtration. 2 4.4 Detection 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
17、 Strains of microorganisms 7 8 Handling 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 Detectio
18、n of growth (enrichment method) . 9 12 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 inoc
19、ulum 14 13.3 Validation of counting methods 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 cultu
20、re media . 20 Annex D (informative) Neutralizers of antimicrobial activity of preservatives and rinsing liquids . 23 Bibliography . 24 BS EN ISO 21149:2009This page deliberately set blank1Cosmetics Microbiology Enumeration and detection of aerobic mesophilic bacteria 1 Scope This International Stand
21、ard 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 products within
22、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 has been otherw
23、ise 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 microbiological risk anal
24、ysis, 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 following referenc
25、ed 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 instructions for microbi
26、ological 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 conditions, other typ
27、es of microorganisms (e.g. yeast, mould) can be detected. 3.2 product portion of an identified cosmetic product received in the laboratory for testing BS EN ISO 21149:20092 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 initia
28、l suspension suspension (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 medi
29、um or by the presence 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
30、 product shall be checked 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. Ae
31、robic incubation of the 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 s
32、uitable amount of the 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 sp
33、ecified in ISO 21148. 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. BS EN ISO 21149:200934.4 Detection of bacteria by enrichment D
34、etection of bacteria by enrichment consists of the following 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
35、suspension on non-selective solid agar medium. Aerobic incubation 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 specification
36、s are given in ISO 21148. When water is used in a formula, 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
37、 may be used if they have been demonstrated to be suitable 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 demo
38、nstrated before the determination of the count (see Clause 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
39、lecithin 5,0 g Polysorbate 20 40,0 ml Water 960,0 ml 5.2.2.1.2 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
40、suitable containers. Sterilize in the autoclave at 121 C for 15 min. After sterilization, the pH shall be equivalent to 7,3 0,2 when measured at room temperature. BS EN ISO 21149:20094 5.2.2.2 Other neutralizing diluents Other neutralizing diluents may be used as appropriate (see Annex A and Annex D
41、). 5.2.3 Diluent 5.2.3.1 Fluid A 5.2.3.1.1 Composition Peptic digest of animal tissue 1,0 g Water 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 steril
42、ization, the pH shall be equivalent to 7,1 0,2 when measured at room temperature. 5.2.3.2 Other 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
43、 Sodium chloride 8,5 g Water 1 000 ml 5.3.2 Preparation Dissolve the components in the water by 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 C
44、ulture media 5.4.1 General Culture media may be prepared as follows, or from dehydrated culture 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 medi
45、a for counting 5.4.2.1 Soybeancasein digest agar medium (SCDA) or tryptic soy agar (TSA) 5.4.2.1.1 Composition Pancreatic digest of casein 15,0 g Papaic digest of soybean meal 5,0 g BS EN ISO 21149:20095Sodium chloride 5,0 g Agar 15,0 g Water 1 000 ml 5.4.2.1.2 Preparation Dissolve the components or
46、 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 0,2 when measured at room temperature. 5.4.2.2 Other media for c
47、ounting Other media may be used as appropriate (see Annex C). 5.4.3 Culture media for detection 5.4.3.1 General When chosen, an enrichment broth and an agar medium shall be used for bacterial detection. The enrichment broth is used to disperse the sample and to increase the initial microbial populat
48、ion. It may contain neutralizers if the specimen to be tested has antimicrobial properties. 5.4.3.2 Enrichment broth: Eugon LT 100 broth 5.4.3.2.1 General This medium contains ingredients which neutralize inhibitory substances present in the sample: lecithin and polysorbate 80, dispersing agent: oct
49、oxynol 9. 5.4.3.2.2 Composition Pancreatic digest of casein 15,0 g Papaic digest of soybean meal 5,0 g L-cystine 0,7 g Sodium chloride 4,0 g Sodium sulfite 0,2 g Glucose 5,5 g Egg lecithin 1,0 g Polysorbate 80 5,0 g Octoxynol 9 1,0 g Water 1 000 ml BS EN ISO 21149:20096 5.4.3.2.3 Preparation Dissolve successively polysorbate 80, octoxynol 9 and egg lecithin into boiling water until their complete dissolution. Dissolve the other components by mixing while heating. Dispense the medium into
