1、BS ISO21527-1:2008ICS 07.100.30NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDMicrobiology of foodand animal feedingstuffs Horizontalmethod for theenumeration of yeastsand mouldsPart 1: Colony count technique inproducts with water activity greaterthan 0,95This
2、British Standardwas published under theauthority of the StandardsPolicy and StrategyCommittee on 31 August2008 BSI 2008ISBN 978 0 580 57107 7Amendments/corrigenda issued since publicationDate CommentsBS ISO 21527-1:2008National forewordThis British Standard is the UK implementation of ISO 21527-1:20
3、08.The UK participation in its preparation was entrusted to TechnicalCommittee AW/9, Microbiology.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are respons
4、ible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.BS ISO 21527-1:2008Reference numberISO 21527-1:2008(E)ISO 2008INTERNATIONAL STANDARD ISO21527-1First edition2008-07-01Microbiology of food and animal feeding stuffs Horizontal method for
5、the enumeration of yeasts and moulds Part 1: Colony count technique in products with water activity greater than 0,95 Microbiologie des aliments Mthode horizontale pour le dnombrement des levures et moisissures Partie 1: Technique par comptage des colonies dans les produits activit deau suprieure 0,
6、95 BS ISO 21527-1:2008ISO 21527-1:2008(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes 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 performi
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9、etariat at the address given below. COPYRIGHT PROTECTED DOCUMENT ISO 2008 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 writ
10、ing 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. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2008 All rights reservedBS ISO 215
11、27-1:2008ISO 21527-1:2008(E) ISO 2008 All rights reserved iiiForeword 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 commi
12、ttees. 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 I
13、nternational 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 Internationa
14、l 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
15、 subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 21527-1 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology. ISO 21527 consists of the following parts, under the general title Microbiology of fo
16、od and animal feedings stuffs Horizontal method for the enumeration of yeasts and moulds: Part 1: Colony count technique in products with water activity greater than 0,95 Part 2: Colony count technique in products with water activity less than or equal to 0,95 This part of ISO 21527, together with I
17、SO 21527-2, cancel and replace ISO 7698:1990, ISO 7954:1987 and ISO 13681:1995. BS ISO 21527-1:2008ISO 21527-1:2008(E) iv ISO 2008 All rights reservedIntroduction Because of the large variety of food and feed products, the applications of the horizontal method specified in ISO 21527 (all parts) may
18、not be appropriate for certain products. In this case, different methods, which are specific to these products, may be used if absolutely necessary for justified technical reasons. Nevertheless, every attempt shall be made to apply the horizontal method as specified in ISO 21527 (all parts) as far a
19、s possible. When ISO 21527 (all parts) is next reviewed, account will be taken of all information then available regarding the extent to which the horizontal method has been followed and the reasons for deviations from this method in the case of particular products. The harmonization of test methods
20、 cannot be immediate, and for certain groups of products International Standards and/or national standards may already exist that do not comply with the horizontal method as specified in ISO 21527 (all parts). It is hoped that when such standards are reviewed they will be changed to comply with ISO
21、21527 (all parts) so that eventually the only remaining departures from this horizontal method will be those necessary for well-established technical reasons. BS ISO 21527-1:2008INTERNATIONAL STANDARD ISO 21527-1:2008(E) ISO 2008 All rights reserved 1Microbiology of food and animal feeding stuffs Ho
22、rizontal method for the enumeration of yeasts and moulds Part 1: Colony count technique in products with water activity greater than 0,95 WARNING It is essential that enumeration of moulds is carried out with the greatest care to protect the operator and to prevent contamination of the atmosphere wi
23、th mould spores. 1 Scope This part of ISO 21527 specifies a horizontal method for the enumeration of viable yeasts and moulds in products intended for human consumption or feeding of animals that have a water activity greater than 0,95 eggs, meat, dairy products (except milk powder), fruits, vegetab
24、les, fresh pastes, etc., by means of the colony count technique at 25 C 1 C (References 1, 2). This part of ISO 21527 does not allow the enumeration of mould spores. Neither the identification of fungal flora nor the examination of foods for mycotoxins lie within the scope of this part of ISO 21527.
25、 The method specified in this part of ISO 21527 is not suitable for enumeration of heat-resistant fungi, such as Byssochlamys fulva or Byssochlamys nivea, in canned or bottled fruit and vegetables. 2 Normative references The following referenced documents are indispensable for the application of thi
26、s document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 6887 (all parts), Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal
27、 dilutions for microbiological examination ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations ISO 8261, Milk and milk products General guidance for the preparation of test samples, initial suspensions and decimal dilutions for
28、microbiological examination ISO/TS 11133 (all parts), Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. NOTE There are some intermediate forms
29、and the distinction between a yeast (3.1) and a mould (3.2) can be arbitrary. BS ISO 21527-1:2008ISO 21527-1:2008(E) 2 ISO 2008 All rights reserved3.1 yeast mesophilic aerobic microorganism which, at 25 C using mycological agar medium under the conditions described in this part of ISO 21527, develop
30、s matt or shiny round colonies (3.4) on the surface of the medium, usually having a regular outline and a more or less convex surface NOTE Yeasts within, rather than on, a medium develop round, lenticular, colonies. 3.2 mould mesophilic aerobic filamentous microorganism which, on the surface of myco
31、logical agar medium under the conditions described in this part of ISO 21527, usually develops flat or fluffy spreading propagules/germs (3.3) or colonies (3.4) often with coloured fruiting or sporing structures. NOTE Moulds within, rather than on, a medium can develop round, lenticular, colonies. 3
32、.3 propagule germ viable entity capable of growth in a nutrient medium EXAMPLE Vegetative cell, group of cells, spore, spore cluster, or a piece of fungal mycelium. ISO 6107-6:2004, 65 3.4 colony localized visible accumulation of microbial mass developed on or in a solid nutrient medium from a viabl
33、e particle ISO 6107-6:2004, 15 4 Principle 4.1 Surface-inoculated plates are prepared using a specified selective culture medium. Depending on the expected number of colonies, a specified quantity of the sample (if the product is liquid), or of an initial suspension (in the case of other products),
34、or decimal dilutions of the sample/suspension are used. Additional plates can be prepared under the same conditions, using decimal dilutions of the test sample or of the initial suspension. 4.2 The plates are then aerobically incubated at 25 C 1 C for 5 d. If necessary, the agar plates are left to s
35、tand in diffuse daylight for 1 d to 2 d. 4.3 Colonies/propagules are then counted and, if required (to distinguish yeast colonies from bacterial colonies), the identity of any doubtful colonies is confirmed by examination with a binocular magnifier or microscope. 4.4 The number of yeasts and moulds
36、per gram or per millilitre of sample is calculated from the number of colonies/propagules/germs obtained on plates chosen at dilution levels producing countable colonies. Moulds and yeasts are counted separately, if necessary. BS ISO 21527-1:2008ISO 21527-1:2008(E) ISO 2008 All rights reserved 35 Di
37、luent and culture medium For current laboratory practice, see ISO 6887 (all parts) and ISO 8261. 5.1 Diluent 5.1.1 General See ISO 6887 (all parts), ISO 8261 and the specific International Standard dealing with the product concerned. NOTE It is possible to add surface-active agents such as sodium po
38、ly(oxyethylene)sorbatitanmonooleate 1)0,05 % (mass concentration) to diluents to reduce clumping of mould spores and conidia (Reference 2). Except for specific preparation of the test sample, the use of 0,1 % (mass concentration) peptone water broth as diluent is recommended. 5.1.2 Composition of 0,
39、1 % (mass concentration) peptone water broth Enzymatic digest of animal or vegetal tissues 1,0 g Water 1 000 ml 5.1.3 Preparation of 0,1 % (mass concentration) peptone water broth Dissolve the components in the water, by heating if necessary. If necessary, adjust the pH so that, after sterilization,
40、 it is 7,0 0,2 at 25 C. 5.2 Culture medium 5.2.1 Dichloran-rose bengal chloramphenicol agar (DRBC) (References 3, 4) 5.2.1.1 Composition Enzymatic digest of animal and plant tissues 5,0 g D-Glucose (C6H12O6) 10,0 g Potassium dihydrogenphosphate (KH2PO4) 1,0 g Magnesium sulfate (MgSO4 H2O) 0,5 g Dich
41、loran (2,6-dichloro-4-nitroaniline) 0,002 g Rose bengal 0,025 g Agar 12 g to 15 g aChloramphenicol 0,1 g Water, distilled or deionized 1 000 ml aDepending on the gel strength of the agar. 1) Tween 80 is an example of a suitable product available commercially. This information is given for the conven
42、ience of users of this International Standard and does not constitute an endorsement by ISO of this product. BS ISO 21527-1:2008ISO 21527-1:2008(E) 4 ISO 2008 All rights reserved5.2.1.2 Preparation 5.2.1.2.1 General Suspend all the ingredients except chloramphenicol in the water and bring to the boi
43、l to dissolve completely. If necessary, adjust the pH (6.4) so that after sterilization it is 5,6 0,2 at 25 C. Add 10 ml of a 1 % (mass concentration) solution of chloramphenicol in ethanol and mix. Dispense the medium in quantities into suitable containers (6.5) of suitable capacity. Sterilize by a
44、utoclaving at 121 C for 15 min. Immediately, cool the medium in a water bath (6.3) maintained at a temperature of 44 C to 47 C. Cool to below 50 C and dispense 15 ml amounts into sterile Petri dishes (6.6). Allow the medium to solidify, and dry, if necessary, the surface of the plates as described i
45、n ISO 7218 and ISO/TS 11133 (all parts). Use immediately, or store in the dark, according to ISO/TS 11133 (all parts) until required. CAUTION Avoid exposure of the medium to light, since cytotoxic breakdown products can result in underestimation of mycoflora in samples. 5.2.1.2.2 Optional addition o
46、f chlortetracycline hydrochloride Where bacterial overgrowth may be a problem (e.g. raw meats), chloramphenicol (50 mg/l) and chlortetracycline (50 mg/l) are recommended. In this case, prepare the basic medium as described above, with only chloramphenicol 50 mg, dispense it in quantities of 100 ml a
47、nd sterilize. Prepare also a 0,1 % (mass concentration) solution of chlortetracycline hydrochloride in water (relatively unstable in solution, it must be freshly prepared) and sterilize by filtration. Just prior to use, add 5 ml of this solution aseptically to 100 ml of the basic medium, and pour pl
48、ates. Gentamicin is not recommended, as it has been reported to cause inhibition of some yeast species. 5.2.1.2.3 Optional addition of trace elements In order for moulds to exhibit their full morphology, particularly any pigments they normally produce, they need trace elements that may not be presen
49、t in DRBC. To identify moulds on this medium, add the following trace element solution at 1 ml per litre of the medium, prior to autoclaving: ZnSO4 7H2O 1g; CuSO4 5H2O 0,5 g; water, distilled or deionized 100 ml (Reference 1). 5.2.1.2.4 Optional addition of Tergitol 2)In order to avoid overgrowth of Mucoraceae on agar plates, the addition of Tergitol 2)(1 ml/l) to the culture medium is recommended. 5.2.1.3 Performance testing for the quality assurance of the culture medium 5
