1、BRITISH STANDARDBS ISO 14461-2:2005Milk and milk products Quality control in microbiologicallaboratories Part 2: Determination of the reliability of colony counts of parallel plates and subsequent dilution stepsICS 07.100.30g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g4
2、8g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS ISO 14461-2:2005This British Standard was published under the authority of the Standards Policy and Strategy Committee on 4 August 2005 BSI 4 August 2005ISBN 0 580 46093 2Na
3、tional forewordThis British Standard reproduces verbatim ISO 14461-2:2005 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 responsibility to: A list of organizations represented on this commit
4、tee can be obtained on request to its secretary.Cross-referencesThe British Standards which implement international publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility
5、 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 Standard does not of itself confer immunity from legal obligations. ai
6、d 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 European developments and promulgate them in the UK.Summary of pagesThis doc
7、ument comprises a front cover, an inside front cover, the ISO title page, pages ii to vi, pages 1 to 18, an inside back cover and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments issued since publicationAmd. No. Date CommentsRefe
8、rence numbersISO 14461-2:2005(E)IDF 169-2:2005(E)INTERNATIONAL STANDARD ISO14461-2IDF169-2First edition2005-04-01Milk and milk products Quality control in microbiological laboratories Part 2: Determination of the reliability of colony counts of parallel plates and subsequent dilution steps Lait et p
9、roduits laitiers Contrle de qualit en laboratoire microbiologique Partie 2: Dtermination de la fiabilit des comptages de colonies en botes parallles et des dilutions dcimales suivantes BS ISO 14461-2:2005iiBS ISO 14461-2:2005iiiContents PageForeword ivIntroduction . vi1 Scope 12 Normative references
10、 . 13 Terms and definitions. 14 Principle . 25 Procedure. 25.1 General. 25.2 Counting results of two parallel plates. 25.3 Sum of counting results of subsequent dilution steps. 26 Evaluation 36.1 Tables of results 36.2 Examples of testing parallel plates . 76.3 Examples of testing sums of two subseq
11、uent dilution steps 77 Calculation formulae and examples 157.1 In Table 1 157.2 In Table 2 167.3 Examples 16BS ISO 14461-2:2005ivForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing Interna
12、tional 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 the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with
13、ISO, also take part in the work. ISO collaborates 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
14、committees is to prepare International Standards. 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 t
15、he 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 rights. ISO 14461-2IDF 169-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products
16、, 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. ISO 14461IDF 169 consists of the following parts, under the general title Milk and milk products Quality control in microbiolog
17、ical laboratories: Part 1: Analyst performance assessment for colony counts Part 2: Determination of the reliability of colony counts of parallel plates and subsequent dilution steps BS ISO 14461-2:2005vForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector w
18、ith 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. IDF collaborates with ISO and AOAC International in the development of standard methods of analysis and sampling for milk and mil
19、k products. Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Publication as an International Standard requires approval by at least 50 % of the National Committees casting a vote. Attention is drawn to the possibi
20、lity 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. ISO 14461-2IDF 169-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the
21、International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately 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 assur
22、ance, statistics of analytical data and sampling, under the aegis of its project leaders, Dr. H. Glaeser (EU) and Prof. Dr. H. Weiss (DE). This edition of ISO 14461-2IDF 169-2, together with ISO 14461-1IDF 169-1, cancels and replaces IDF 169:1994, which has been technically revised. ISO 14461IDF 169
23、 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 dilution steps BS ISO
24、 14461-2:2005viIntroduction Every microbiological method consists of several steps that are followed in a specific sequence (sub-sampling, 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
25、 results with a margin of uncertainty not much larger than what can be expected from the correct application of the method, it is necessary 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 mat
26、erial, the exactness with which the dilutions are performed, and the technique of inoculation and/or counting of the plates. By homogenizing 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 labora
27、tory can perform the colony-count technique, taking into account the expected variability of the method. Too large a variability indicates that at least one of the steps in the performance of the method is out of control. The identification of those steps is carried out by comparison of the replicat
28、e inoculations, the different dilution levels and the dilution series. When the steps with excessive variability have been identified, necessary measures should be taken to bring these steps under control. BS ISO 14461-2:20051Milk and milk products Quality control in microbiological laboratories Par
29、t 2: Determination of the reliability of colony counts of parallel plates and subsequent dilution steps 1 Scope This part of ISO 14461IDF 169 describes a routine procedure for the evaluation of results of the enumeration of microorganisms using colony-count methods with subsequent 10-fold dilution s
30、teps and one plate or two parallel plates within each dilution step. This routine procedure is applied regularly in each laboratory performing colony counts. It provides criteria for the acceptability of differences between the results from parallel plates and subsequent dilution steps, as follows.
31、a) The results (colony counts) obtained from parallel plates are compared with tabulated limits for given colony counts. If these limits are exceeded, a technical problem when performing the parallel determinations may be indicated. b) The results (sums of colony counts) of two parallel plates of tw
32、o subsequent 10-fold dilution steps are compared with tabulated limits for given sums of colony counts. If these limits are exceeded, a technical problem when performing the dilutions may be indicated. c) If the limits mentioned above are exceeded in more cases than expected, this indicates that the
33、 test procedure lacks reliability. NOTE The formulae for calculating the values in Table 1 and 2 are given and explained in Clause 7. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies
34、. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 14461-1IDF 169-1, Milk and milk products Quality control in microbiological laboratories Part 1: Analyst performance assessment for colony counts3 Terms and definitions For the purposes of
35、 this document, the following terms and definitions apply 3.1colony count number of microorganisms found, as determined by the method specified in ISO 14461-1|IDF 169-1 NOTE The number of microorganisms is expressed per gram or per millilitre of test sample. BS ISO 14461-2:200524 Principle The count
36、ing results obtained are compared with tabulated limits for given colony counts. Decisions are based on the way the limits are exceeded. The tabled values are calculated and explained. 5 Procedure 5.1 General The procedure shall be applied routinely in laboratories carrying out colony counts. A stan
37、dardized method for performing colony counts must be applied in any case. If the applied method is not in accordance with an International Standard or another accepted standard, a detailed description of the method shall be available and followed precisely. In the case that a method is followed with
38、 only one plate per dilution step, the procedure described in 5.2 shall be carried out with a certain minimum frequency (e.g. once per hundred sample units tested). 5.2 Counting results of two parallel plates Compare the results (colony counts) of two parallel plates with the limits tabulated in Tab
39、le 1. Compare the upper and lower colony counts of an observed pair of results with the corresponding colony counts given in Table 1. Use the upper colony count given in Table 1 as basis for the comparison. Then compare the lower colony count given in Table 1 with the observed lower count. A lower o
40、bserved count below the lower colony count of Table 1 indicates that the difference between the colony counts obtained with the two parallel plates is unacceptably high. (See the results of the first dilution step in Examples 1 and 2 in Clause 7.) A lower observed count, which is at least equal to t
41、he lower colony count, indicates that the difference is acceptable. (See the results of the second dilution step in Examples 1 and 2 in Clause 7.) 5.3 Sum of counting results of subsequent dilution steps 5.3.1 Use for the following test the colony counts from the two sets of parallel plates that pas
42、sed the test in 5.2. Compare the sums of colony counts from parallel plates over two 10-fold dilution steps with the tabulated limits in Table 2. For an observed sum with dilution step 10x, compare the sum obtained with dilution step 10(x+1)with the tabulated lower limit for the sum. Observed sums f
43、or the dilution step 10(x+1)within the ranges given in Table 2 are acceptable. Observed sums outside these limits indicate that the ratio of the colony counts obtained over two 10-fold dilution steps deviates significantly from the expected ratio. 5.3.2 Use for the following test the colony counts f
44、rom the parallel plates that did not pass the test in 5.2 or those colony counts from one plate per dilution step. Compare the colony counts from plates over two subsequent 10-fold dilution steps with the limits tabulated in Table 2. For an observed colony count with dilution step 10x, compare the c
45、olony count obtained with dilution step 10(x+1)with the tabulated lower limit for the count. Observed colony counts for dilution step 10(x+1)within the ranges given in Table 2 are acceptable. Observed colony counts outside these limits indicate that the ratio of the colony counts obtained over two 1
46、0-fold dilution steps deviates significantly from the expected ratio. (Two comparisons of results are given in Examples 1 and 2 in Clause 7). BS ISO 14461-2:20053See Figure 1 for a flowchart of the procedure. NOTE A minus sign () indicates an acceptable result within limits. A plus sign (+) indicate
47、s a result that is out of limits, and is an indication of technical problems. Figure 1 Flowchart describing the testing of colony-counting procedure and its evaluation 6 Evaluation 6.1 Tables of results For each test described in 5.3.1 and 5.3.2, the results outside the limits specified in Table 1 o
48、r Table 2 shall not occur more often than once in a 100 cases. If results outside these limits occur more often, the microbiological test procedure shall be scrutinized. BS ISO 14461-2:20054Table 1 Limits of agreement for colony counts of two parallel Petri dishes (with a probability of 99 % per com
49、parison) Colony count Upper Lower Sum 10 2 1211 3 1412 3 1513 4 1714 4 1815 5 2016 5 2117 6 2318 6 2419 7 2620 7 2721 8 2922 9 3123 9 3224 10 3425 11 3626 11 3727 12 3928 12 4029 13 4230 14 4431 14 4532 15 4733 16 4934 16 5035 17 5236 18 5437 19 5638 19 5739 20 5940 21 6141 21 6242 22 6443 23 6644 24 6845 24 6946 25 7147 26 7348 27 7549 27 7650 28 7851 29 8052 29 8153 30 83Colony count Upper Lower Sum 54 31 8555 32 8756 32 8857 33 9058 34 9259 35 9460 36 9661 36 9762 37 9963 38 10164 39 10365 39 10466
