1、BRITISH STANDARD BS ISO 17410:2001 Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of psychrotrophic micro-organisms ICS 07.100.30 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBS ISO 17410:2001 This British Standard, having been prepared
2、under the direction of the Consumer Products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 July 2001 BSI 07-2001 ISBN 0 580 37544 7 National foreword This British Standard reproduces verbatim ISO 17410:2001 and implements it a
3、s the UK national standard. It supersedes BS 4285-3:Section 3.1:1986 which is withdrawn. The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology, which has the responsibility to: A list of organizations represented on this committee can be obtained on request
4、to its secretary. Cross-references The British Standards which implement international publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards
5、 Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to u
6、nderstand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comp
7、rises a front cover, an inside front cover, the ISO title page, pages ii and iii, a blank page, pages 1 to 7 and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsReference number ISO
8、 17410:2001(E) INTERNATIONAL STANDARD ISO 17410 First edition 2001-05-15 Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of psychrotrophic microorganisms Microbiologie des aliments Mthode horizontale pour le dnombrement des micro-organismes psychrotrophesISO 1741
9、0:2001(E) ii ISO 17410:2001(E)iii 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 int
10、erested 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 Electrotechnic
11、al Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publicat
12、ion 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 International Standard may be the subject of patent rights. ISO shall not be held responsible for identifying any or all su
13、ch patent rights. International Standard ISO 17410 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology.INTERNATIONAL STANDARD ISO 17410:2001(E)1 Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of psychrotrophic microorgan
14、isms 1 Scope This International Standard specifies a method for the enumeration of psychrotrophic microorganisms by means of the colony-count technique at 6,5 C. The method is applicable to products intended for human consumption or for animal feeding stuffs. 2 Normative references The following nor
15、mative documents contain provisions which, through reference in this text, constitute provisions of this International Standard. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on this International Standar
16、d are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid International Standa
17、rds. ISO 6887-1, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions. 1) ISO 7218, Microbiology of food and anima
18、l feeding stuffs General rules for microbiological examinations. ISO 8261, Milk and milk products Preparation of samples and dilutions for microbiological examination. 3 Term and definition For the purposes of this International Standard, the following term and definition applies. 3.1 psychrotrophic
19、 microorganism bacteria, yeasts and moulds forming countable colonies under the conditions specified in this International Standard 4P r i n c i p l e 4.1 Two agar plates are prepared using a solid non-selective culture medium, and using a specified quantity of the test sample if the initial product
20、 is liquid, or using a specified quantity of an initial suspension in the case of other products. In order to prevent heat stress, surface inoculation is used. Other pairs of agar plates are prepared, under the same conditions, using decimal dilutions of the test sample or the initial suspension. 1)
21、 Parts 2 to 5 are in preparation, see biliography.ISO 17410:2001(E) 2 4.2 The plates are subjected to aerobic incubation at 6,5 C for 10 days. 4.3 The number of microorganisms per millilitre or per gram of sample is calculated from the number of colonies obtained on the plates chosen. 5 Culture medi
22、a and dilution fluid For current laboratory practice, see ISO 7218. Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or water of equivalent purity. If the prepared culture media and reagents are not used immediately, store them, unles
23、s otherwise stated, in the dark at a temperature of 3 C 2 C for no longer than 1 month, under conditions which do not produce any change in their composition. 5.1 Dilution fluid See ISO 6887-1. 5.2 Plate count agar (PCA) 5.2.1 Composition Enzymatic digest of casein 5,0 g Yeast extract 2,5 g Glucose
24、1,0 g Agar 9gto1 8g a Water 1 000 ml a Depending on the gel strength of the agar. When dairy products are examined, it is recommended to add 1,0 g of skimmed milk powder per litre of the culture medium. The skimmed milk powder shall be free from inhibitory substances. 5.2.2 Preparation Dissolve the
25、components or the dehydrated complete medium in the water, by heating if necessary. Mix thoroughly and leave to stand for several minutes. Adjust the pH (6.9), if necessary, so that after sterilization it is 7,0 0,2 at 25 C. Dispense the medium into flasks or bottles (6.10) in appropriate quantities
26、. Sterilize for 15 min in the autoclave (6.1) set at 121 C. If the medium is to be used immediately, cool it before use in the water bath (6.7) set at 44 Ct o4 7C. If not, allow the medium to solidify in the flask or bottle. Before use, melt the medium completely in a boiling water bath, then cool i
27、t in the water bath (6.7) set at 44 Cto4 7C. Place in each of an appropriate number of Petri dishes (6.5) about 15 ml of the freshly prepared, or remelted, complete medium. Allow to solidify. Immediately before use, dry the agar plates carefully (preferably with the lids off and the agar surface dow
28、nwards) in an incubator (6.3) set between 37 C and 55 C until the surface of the agar is dry. The agar plates may also be dried in a laminar-flow safety cabinet for 30 min with half-open lids, or overnight with the lids in place (see ISO 7218).ISO 17410:2001(E)3 6 Apparatus and glassware For general
29、 requirements, see ISO 7218. Disposable apparatus is an acceptable alternative to reusable glassware if it has similar specifications. Usual microbiological laboratory equipment and, in particular, the following. 6.1 Autoclave, capable of operating at a temperature of 121 C. 6.2 Oven for dry sterili
30、zation, capable of operating at a temperature between 170 C and 175 Cfo r1h . 6.3 Incubator, capable of operating at a temperature of 37 Cto5 5C. 6.4 Incubator, capable of operating at a temperature of 6,5 C 1 C. 6.5 Petri dishes, made of glass or plastic, of approximately 90 mm to 100 mm diameter.
31、6.6 Pipettes, calibrated for bacteriological use, having nominal capacities of 0,1 ml, 1 ml and 10 ml. 6.7 Water baths, or similar apparatus, one capable of operating at a temperature of 44 Ct o4 7C and another capable of boiling water. 6.8 Colony-counting equipment, consisting of an illuminated bas
32、e and, optionally, a mechanical or electronic digital counter. 6.9 pH-meter, capable of being read to the nearest 0,01 pH unit at 25 C, enabling measurements to be made which are accurate to 0,1 pH unit. 6.10 Bottles or flasks, of appropriate capacity, for preparation, sterilization and, if necessar
33、y, storage of culture media. 6.11 Glass or plastic spreaders, sterile, for spreading inoculation material on the surface of the culture medium. 7 Sampling It is important that the laboratory receive a sample which is truly representative of the product and has not been damaged or changed during tran
34、sport or storage. Sampling is not part of the method specified in this International Standard. See the specific International Standard dealing with the product concerned. If there is no specific International Standard, it is recommended that the parties concerned come to an agreement on this subject
35、. 8 Preparation of test sample Prepare the test sample in accordance with ISO 6887-1 or, for dairy products, ISO 8261. 9 Procedure 9.1 Test portion, initial suspension and dilutions Prepare the test portion, initial suspension (primary dilution) and further dilutions in accordance with ISO 6887-1 or
36、, for dairy products, ISO 8261.ISO 17410:2001(E) 4 9.2 Inoculation and incubation 9.2.1 Prepare two dishes from the test sample (if liquid) or from the primary initial solution of other products and from each dilution chosen. Transfer using a sterile pipette (6.6) 0,1 ml of liquid product, the initi
37、al suspension (primary dilution) or the appropriate dilutions to the centre of each labelled Petri dish containing the PCA medium (5.3). 9.2.2 Carefully spread the inoculum uniformly and as quickly as possible over the surface of the agar plate, without touching the sides of the dish, with a spreade
38、r (6.11) until there is no longer any liquid visible on the agar surface. A control plate without inoculum should be included for checking sterility. Use a fresh spreader for each plate. It is possible to use the same spreader for all dilutions from one sample beginning with the highest dilution and
39、 progressing in order to the dilution having the greatest amount of test material. 9.2.3 Invert the dishes prepared in 9.2.2 and place them in the incubator (6.4) set at 6,5 C. Incubate them for 10 days. 9.3 Enumeration After the specified period of incubation (see 9.2.3), count the colonies in each
40、 Petri dish containing not more than 150 colonies, using the colony-counting equipment (6.8). It is important that pinpoint colonies be included in the count but it is essential that the operator avoid mistaking particles of undissolved or precipitated matter in dishes for pinpoint colonies. Spreadi
41、ng colonies shall be considered as single colonies. If less than one-quarter of the dish is overgrown by spreading colonies, count the colonies on the unaffected part of the dish and calculate the corresponding number for the entire dish. If more than one-quarter of the dish is overgrown by spreadin
42、g colonies, discard the count for that dish. 10 Calculation and expression of results 10.1 Calculation See ISO 7218. For a result to be valid, in general it is considered that it is necessary to count the colonies on at least one dish containing as a minimum 15 colonies. Calculate the number, N, of
43、colony-forming units (CFU) of psychrotrophic microorganisms per gram or per millilitre of sample, using equation (1): 12 01 C N V(n , n )d (1) where C is the sum of all the colonies counted on all the dishes retained from two successive dilutions, one of which contains at least 15 colonies; V is the
44、 volume, in millilitres, of inoculum applied to each dish; n 1 is the number of dishes retained at the first dilution; n 2 is the number of dishes retained at the second dilution; d is the dilution factor corresponding to the first dilution retained.ISO 17410:2001(E)5 NOTE The lowest dilution is the
45、 dilution with the highest content of test sample. 10.2 Expression of results 10.2.1 General case: Dishes containing between 15 and 150 colonies Round off the results to two significant figures. In order to do this, if the third figure is less than 5, do not modify the preceding figure; if the third
46、 figure is greater than or equal to 5, increase the preceding figure by one unit. For example, 28 500 is rounded to 29 000 and 11 500 is rounded to 12 000. Take as the result the number of CFU of psychrotrophic microorganisms per millilitre (liquid products) or per gram (other products), expressed p
47、referably as a number between 1,0 and 9,9 multiplied by the appropriate power of 10, or a whole number with two significant figures. 10.2.2 Estimation of low numbers If the two dishes corresponding to the test sample (liquid products) or the initial suspension (other products) contain less than 15 c
48、olonies, calculate the arithmetic mean of the colonies counted on both dishes. Express the result as follows: estimated number of psychrotrophic microorganisms, N E , per millilitre (liquid products) or per gram (other products) using equation (2): E C N Vnd (2) where C is the sum of the colonies co
49、unted on the two dishes; V is the volume, in millilitres, of inoculum applied to each dish; n is the number of retained dishes (in this case n=2); d is the dilution factor of the initial suspension or of the dilution inoculated. 10.2.3 No colonies present If the two dishes corresponding to the test sample (liquid products) or the initial suspension (other products) contain no colonies, report the result as follows: less than 1/(V d) psychrotrophic microorganisms per millilitre (liquid products) or per gram (oth
