1、BRITISH STANDARD BS ISO 14461-1:2005 Milk and milk products Quality control in microbiological laboratories Part 1: Analyst performance assessment for colony counts ICS 07.100.30 BS ISO 14461-1:2005 This British Standard was published under the authority of the Standards Policy and Strategy Committe
2、e on 13 September 2005 BSI 13 September 2005 ISBN 0 580 46093 2 National foreword This British Standard reproduces verbatim ISO 14461-1:2005 and implements it as the UK national standard. The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology, which has the r
3、esponsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international publications referred to in this document may be found in the BSI Catalogue under the section entitled “Internatio
4、nal Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. 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
5、 Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep UK interests informed; monitor related international and Europ
6、ean developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii to vi, pages 1 to 34, an inside back cover and a back cover. The BSI copyright notice displayed in this document indicates when the document was
7、 last issued. Amendments issued since publication Amd. No. Date Comments Reference numbers ISO 14461-1:2005(E) IDF 169-1:2005(E)INTERNATIONAL STANDARD ISO 14461-1 IDF 169-1 First edition 2005-05-01 Milk and milk products Quality control in microbiological laboratories Part 1: Analyst performance ass
8、essment for colony counts Lait et produits laitiers Contrle de qualit en laboratoires microbiologiques Partie 1: valuation de la performance des analystes effectuant les comptages de colonies BS ISO 14461-1:2005 ii BS ISO 14461-1:2005 iii Contents Page Foreword iv Introduction . vi 1 Scope 1 2 Norma
9、tive references . 1 3 Terms and definitions. 1 4 Principle (see Figure 1) 2 5 Diluent, culture media and reagents . 3 6 Apparatus and glassware. 5 7 Sampling 5 8 Preparation of test sample . 6 8.1 Milk . 6 8.2 Dried milk . 6 9 Procedure. 6 9.1 General . 6 9.2 Number of decimal dilution steps . 6 9.3
10、 Preparation of first decimal dilution . 6 9.4 Preparation of further decimal dilutions. 7 9.5 Melting the medium 7 9.6 Preparation of binary dilutions and inoculation of the medium 7 9.7 Incubation 7 9.8 Randomization and counting of colonies. 8 10 Statistical evaluation 10 10.1 Adequacy of the dat
11、a set 10 10.2 Evaluation of the complete data set (see Figure 2) 10 Annex A (informative) Weighted mean and homogeneity testing of colony counts 27 Annex B (Informative) BASIC programme for calculating the likelihood ratio index G 2 33 Bibliography . 34 iv Foreword ISO (the International Organizatio
12、n 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
13、 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. Inter
14、national 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. Publicat
15、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 document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent righ
16、ts. ISO 14461-1IDF 169-1 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International.
17、 ISO 14461IDF 169 consists of the following parts, under the general title Milk and milk products Quality control in microbiological laboratories: Part 1: Analyst performance assessment for colony counts Part 2: Determination of the reliability of colony counts of parallel plates and subsequent dilu
18、tion steps BS ISO 14461-1:2005 v Foreword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing Committees carrying out the technical work.
19、 IDF collaborates with ISO and AOAC International in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Publication as an Inte
20、rnational Standard requires approval by at least 50 % of the National Committees casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. This
21、 part of International Standard ISO 14461-1IDF 169 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separat
22、ely by AOAC International. All work was carried out by the Joint ISO/IDF/AOAC Action Team, Statistics of analytical data, of the Standing Committee on Quality assurance, statistics of analytical data and sampling, under the aegis of its project leaders, Dr. H. Glaeser (EU) and Prof. Dr. H. Weiss (DE
23、). This edition of ISO 14461-1IDF 169-1, together with ISO 14461-2IDF 169-2, cancels and replaces IDF 169:1994, which has been technically revised. ISO 14461IDF 169 consists of the following parts, under the general title Milk and milk products Quality control in microbiological laboratories: Part 1
24、: Analyst performance assessment for colony counts Part 2: Determination of the reliability of colony counts of parallel plates and subsequent dilution steps BS ISO 14461-1:2005 vi Introduction Every microbiological method consists of several steps that are followed in a specific sequence (sub-sampl
25、ing, diluting, plating and counting). The final result has a margin of uncertainty that is determined by the variability of all the steps involved. In order to obtain results with a margin of uncertainty not much larger than what can be expected from the correct application of the method, it is nece
26、ssary to follow the rules of Good Laboratory Practice (GLP). The three most important factors in obtaining a correct plate count are the homogeneity of the sample material, the exactness with which the dilutions are performed, and the technique of inoculation and/or counting of the plates. By homoge
27、nizing a sample material very well, making multiple dilution series, and inoculating several plates from the same dilution, it is possible to assess how well a laboratory can perform the colony-count technique, taking into account the expected variability of the method. A too large variability indic
28、ates that at least one of the steps in the performance of the method is out of control. The identification of those steps is done by comparison of the replicate inoculations, the different dilution levels and the dilution series. When the steps with excessive variability have been identified, the ne
29、cessary measures should be taken to bring these steps under control. BS ISO 14461-1:2005 1 Milk and milk products Quality control in microbiological laboratories Part 1: Analyst performance assessment for colony counts 1 Scope This part of ISO 14461IDF 169 describes a procedure for testing the perfo
30、rmance of the colony-count technique within a laboratory by establishing the within-laboratory variability of its technique and identifying those steps that are associated with excessive variability. The procedure is also suitable for checking the proper observance of Good Laboratory Practice (GLP),
31、 which may be a prerequisite for participation in interlaboratory tests of colony-count methods. EXAMPLE Appropriate test samples are raw milk, pasteurized milk and dried milk. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated
32、 references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 648:1977, Laboratory glassware One-mark pipettes ISO 835-4, Laboratory glassware Graduated pipettes Part 4: Blow-out pipettes ISO 4788, Laborator
33、y glassware Graduated measuring cylinders ISO 7218, Microbiology of food and animal feedings stuffs General rules for microbiological examinations ISO 8261IDF 122, Milk and milk products Preparation of samples and dilutions for microbiological examination 3 Terms and definitions For the purposes of
34、this document, the following terms and definitions apply. 3.1 colony-count technique counting of the number of microorganisms as determined by the procedure specified in this part of ISO 14461IDF 169 NOTE The number of microorganisms is expressed per gram or per millilitre of test sample. BS ISO 144
35、61-1:2005 2 4 Principle (see Figure 1) Figure 1 Quality assurance in the microbiological laboratory: Design of pilot studies for plate count 4.1 A test sample is homogenized then diluted to a suitable working density. (e.g. 500 CFU to 10 000 CFU per millilitre). A suspension is prepared. 4.2 From th
36、is first dilution, four dilution series are prepared, each consisting of 12 binary dilution steps. NOTE Binary (two-fold) dilution steps are used, not decimal (10-fold) dilutions as is the usual practice. With binary dilutions it is possible to count colonies on plates originating from five to six d
37、ilutions, and this large number of counts improves considerably the testing of the dilution steps. BS ISO 14461-1:2005 3 4.3 Three parallel plates are poured from each dilution of each series. 4.4 The plates are incubated. 4.5 The sequence of the plates is randomized and the colonies on each plate a
38、re counted. 4.6 The counts are tabulated and the “statistical homogeneity” of the counts in two steps is calculated. 4.7 If the values obtained are statistically homogeneous, then the quality of the application of the method is satisfactory and no further evaluation is needed. 4.8 If the results are
39、 not statistically homogeneous, an analysis of variance (ANOVA) is performed in order to identify the variation of the results with one or more of the factors that were varied (i.e. dilution series, dilution levels, plating). Further investigations are carried out and the factor(s) identified are ad
40、justed. NOTE Users will designate the particularly important sources of error in the performance of the method. 5 Diluent, culture media and reagents The operations described in detail in this clause and in Clause 9 shall either be carried out by one person alone or be divided over a group with clea
41、rly defined tasks for each participant. Use only reagents of recognized analytical grade and distilled water or water of at least equivalent purity, unless otherwise specified. The reagents and the water shall be free from substances that may adversely influence the growth of microorganisms under th
42、e test conditions. The culture medium shall be of recognized bacteriological quality. Any dehydrated medium shall be prepared according to the manufacturers instructions. 5.1 Sodium hydroxide solution or hydrochloric acid (approx. 0,1 mol/l), to adjust the pH of the diluent and the culture medium. 5
43、.2 Culture medium: Tryptone-glucose-yeast extract agar, with addition of skimmed milk powder. 5.2.1 Composition Yeast extract 2,5 g Tryptic digest of casein (tryptone) 5,0 g Glucose monohydrate (C 6 H 12 O 6 H 2 O) 1,0 g Skimmed milk powder 1,0 g Agar 10 g to 15 g aWater 1 000 ml aDepending on the g
44、el strength of the agar. In all cases it is necessary to add the skimmed milk powder, even if the dehydrated complete medium is purchased and even if the suppliers consider such an addition unnecessary. 5.2.2 Preparation For the experiment 2 litres of medium of the same lot will be needed. If a comm
45、ercial dehydrated complete medium is used, follow the manufacturers instructions but add the skimmed milk powder. Adjust the pH so that after sterilization it is 7,0 0,2 at about 45 C. BS ISO 14461-1:2005 4 If the medium is prepared from dehydrated basic components, then dissolve and disperse in pre
46、heated water, in the following order: yeast extract, tryptone, glucose and, finally, the skimmed milk powder. Heating the water will assist in the dissolving and dispersion procedure. Add the agar and heat to boiling, while stirring frequently, until the agar is completely dissolved. Alternatively,
47、steam the mixture for about 30 min. Filter the medium through filter paper, if necessary. Adjust the pH so that after sterilization it is 7,0 0,2 at about 45 C. Dispense the culture medium in amounts of 250 ml into bottles (6.10). Sterilize all the bottles at one time in the autoclave (6.1) set at 1
48、21 C for 15 min. Store the prepared medium in the dark at a temperature between 0 C and 5 C for no longer than 1 month. 5.3 Diluents: Peptone/salt solution or quarter-strength Ringers solution, from a single lot. 5.3.1 Peptone/salt solution This is the diluent selected for general use. 5.3.1.1 Compo
49、sition Peptone 1,0 g Sodium chloride (NaCl) 8,5 g Water up to 1 000 ml 5.3.1.2 Preparation Dissolve the components in the water, by heating if necessary. Adjust the pH so that after sterilization it is 7,0 0,2 at 25 C. 5.3.2 Quarter-strength Ringers solution 5.3.2.1 Composition Sodium chloride (NaCl) 2,25 g Potassium chloride (KCl) 0,105 g Calcium chloride, anhydrous (CaCl 2 ) 0,06 g Sodium hydrogen ca
