BAe) (oli Reference number 16140-32017) 21502017 Scanned with CamScannerScanned with CamScannerISO/DIS 16: Contents Foreword. Introduction.. 1 Scope... 2 Normative references, 3 Terms and definitions 4 Principle 41 Gen é 4.2 Implementation verification Ese ee u 4.3 (Food) item verification. Lit Lin sates ae 12 44 Implementation verification and (food) item verification et aia 4.5 Performance characteristics... 15 5 Qualitative methods — Technical protocol for verificstio 5.1 Estimated LODso(eL0Ds:) determination... 5.2 Experimental design — 5.3 Selection of (food) items. 5.4 Artificial contamination. 5.4.1 Selection of strains enn. 5.4.2 Inoculation of the test portions -ae-ee---—— 5 Results... 7 Acceptance crit Root cauise analysis 6 Quantitative methods — Technical protocal for verification 61 Precision (intralaboratory reproducibility) determination .. 611° Gene 612 Experimental design an mnee nomen 6.1.3 Selection of (food) item. 614 Natural contamination... 6.15 Artificial contamination 616 Results 61.7 Acceptance eriteria 6.1.8 Root cause analysis... 62 Estimated bias determination... 621 General... 622 Experimental design 6.23. Selection of (food) items. 624 Artificial contamination. 625 Results... 62.6 Acceptance criteria. 627 Root cause analysis.. 7 Summary of acceptance criteria ~~ : Anmex A (informative) — Classification of (food) categories and suggested target combinations for verification studies.. ‘Annex B (normative) Requirements and guidance on how to choose challenging (food) item(s) for (food) item verification . 47 Bi B2 © 150.2017 — Allrights reserved 3 Scanned with CamScannerScanned with CamScanner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 1S0 technical committees. Each member body Interested in a subject for which @ technical committee has been established has the right to be represented on that committee International organizations, governmental and non-governmental, in liaison with 1SO, also take part in the work ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the 1S0/IEC Directives, Part 2 (see www {so.org/directives) Attention is drawn to the possibility that some of the elements of this document may be the stibject of patent rights. 1SO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see suv iso ore /natents) Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement. For an explanation on the voluntary nature of standards, the meaning of 1SO specific terms and expressions related to conformity assessment, as well as information about ISO's adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: wowwisoore/iso/foreword btm! This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology (Working Group WG 3, Method validation). list ofall parts of the ISO 16140 series can be found on the ISO website, ©1S0 2017 ~ Allrights reserved Scanned with CamScanneriSO/DIS 164 Introduction ‘The ISO 16140 series has been elaborated in response to the need for various ways to validate or verify test methods. It is the successor of ISO 16140:2003, Microbiology of food and animal feeding stuffs — Protocol for the validation of alternative methods. ISO 16140 series consists of several parts with the general title, Microbiology of the food chain — Method validation: — Part 1: Vocabulary — Part 2: Protocal for the validation of alternative (proprietary) methods against a reference method — Part 3: Protocol for the verification of reference and validated alternative methods implemented in a single laboratory — Part 4. Protocol for single-laboratory (in-house) metitod validation — Part 5: Protocol for factorial interlaboratory validation for non-proprietary metiiods — Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation and typing procedures ISO 17468, Microbiology of the food chain — Technical requirements and guidance on establishment or revision of a standardized reference method, is a closely linked International Standard This International Standard, which establishes technical rules for the development and validation of standardized methods, is intended for the development of standardized methods by ISO/TC 34, Food products, Subcommittee SC 9, Microbiology and CEN/TC 275/WG 6, Microbiology of the food chain. {In general, two stages are needed before a method can be used in a laboratory: — The first stage is the validation of the method. This is either conducted in several laboratories (parts 2 and 5 of ISO 16140) or in one laboratory (part 4 of ISO 16140) — The second stage is method verification, where a laboratory demonstrates that it can satisfactorily perform a validated method. This is described in part 3 of 1S0 16140 (method verification). In part 3, a separation is made between verification of (food) items that are included in the validation study and (food) items that are not tested in the validation study but belong within the scope of validation. NOTE1 — Standardized reference methods (with and without published validation data) only require verification before implementation in the laboratory. NOTE2 In this partof!SO 16140, the words ‘category, type’ and ‘tem’ are sometimes combined with ‘food’ to improve the readability ofthis document. However, the word Tood’ is interchangeable with ‘feed’ and the other areas of the food chain as mentioned in the Scope of SO 16140-2. Part 4 of ISO 16140 addresses validation within a single laboratory. The results are therefore only valid in the laboratory which conducted the study. In this case, verification (part 3 of ISO 16140) is not required. Part 5 of ISO 16140 describes protocols for situations where a more rapid validation is required or ‘when the method to be validated is highly specialised, and, the number of participating laboratories required by ISO 16140-2 cannot be reached. 6 © 150 2017 - All rights reserved Scanned with CamScannerISO/DIS 11 The flow chartin Figure 1 gives an overview of the links between the different parts mentioned above. It also guides the users in selecting the right part of the ISO 16140 series, taking into account the purpose of the study and the remarks given above. For this, It is important to distinguish between ‘reference method’ and ‘standardized feference method’. A reference method is an internationally recognized and widely’ accepted method (term 2.59 of ISO 16140-1:2016) and a standardized reference method is a reference method described in a standard (term 3.5 of ISO 17468:2016). In the ISO 16140 series, reference method includes standardized reference method. The flow diagram acknowledges that published validation data may not be available for some standardized reference methods. “prpete vacuo cehrence Pees Fears Ae iT Ha ED AOE (Fass are mecinpane pe mame pened ie ee ecccrr ayes note 5 ee Seen Figure 1 — Flow chart for application of the different parts of the [SO 16140 series Part 6 of ISO 16140, is somewhat different from the other parts in the ISO 16140 series in thatiit relates to a very specific situation where only the confirmation procedure of a method is validated. The confirmation procedure advances a suspected (presumptive) result to a confirmed positive result. The ‘typing of pure strains (eg serotyping of Salmonella) is included in part 6 of ISO 16140. © 180 2017 — allrights reserved 2 Scanned with CamScannerISO/DIS 161.49-3:2017(8) Microbiology of the food chain — Method validation — Part 3: Protocol for the verification of reference and validated alternative methods implemented in a single laboratory 1 Scope This part of ISO 16140 describes the protocol for the verification of reference methods, standardized reference methods and validated alternative methods for implementation in the user laboratory. Method verification does not apply to non-validated alternative methods. This part of ISO 16140 is applicable to the verification of methods used for the analysis (detection ‘and/or quantification) of microorganisms in — products intended for human consumption, — products intended for animal feeding, — environmental samples in the area of food and feed production, handling, and — samples from the primary production stage. ‘This part of ISO 16140 is, in particular, applicable to bacteria and fungi. Some clauses can be applicable to other (microorganisms or their metabolites, to be determined on a case-by-case basis. ‘The verification focuses on those (food) items that are within the scope of validation and are tested in the user laboratory. 2 Normative references ‘The following documents are referred to in the text in such a way that some or all of their content constitutes requirements 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. 180 6887 (all parts), Microbiology of the food chain — Preparation of test samples, intial suspension and decimal dilutions for microbiological examination 1S0 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for microbiological examinations 10 16140-1:2016, Microbiology of the food chain — Method validation — Part 1: Vocabulary 1s0 16140-2:2016, Microbiology of the food chain — Method validation — Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method 3 Terms and definitions For the purposes of this document, the terms and definitions given in ISO 16140-1 and the following apply. ISO and IEC maintain terminological databases for use in standardization at the following addresses: — IEC Electropedia: available at http://www-electropediaore/ © 180 2017 - Allrights reserved Scanned with CamScanner— 150 Online browsing platform: available at http://www iso ore/oby 34 bias measurement bias estimate of a systematic measurement error, or the systematic difference between the quantitative assigned value and the average of measurement replicate results [SOURCE: ISO 16140-1:2016, 2.9] 32 category group of (food) types of the same origin Note I to entry: The (food) categories are listed in Annex A of tis document (SOURCE: 1S0 16140-1:2016, 2.11, modified] 33 estimated bias (eBias) determination of the bias based on the experimental design described in this document 1¢ determination of the bias is not possible as the number of samples tested is small. ‘used in this document. Note 1 to entry: An accu! ‘Therefore, the term estimated bias 34 estimated LODso (eLODsa) determination of the LODs: (level of detection at 50 % probability of detection) based on the experimental design described in this document ination of the LOD: 's not possible as the number of samples tested is small » (@LOD::) is used 6,235 item single specified food, feed, environmental, or primary production matrix EXAMPLE Food category: heat-processed mili and dairy products; food type: pasteurised dairy produc; food item: créme bralée. [SOURCE: 1S0 16140-1:2016, 234) 36 laboratory sample sample prepared for sending to the laboratory and intended for inspection or testing [SOURCE: 1SO 6887-1:2017, 3.1] 37 matrix (product) all the components of the sample © 150 2017 - allrights reserved Scanned with CamScanner[SOURCE: ISO 16140-1:2016, 238] 38 reference material material, sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit forits intended use in a measurement process Nore 1 10 en y: Properties can be quantitative or qualitative, eg. identity of substances or species. Nove 2 to entry: Uses may include the callbration of a measurement system. assessment of @ measurement procedure, assigning values to other materials, ard quality controt [SOURCE: ISO Guide 30-2015, 2.1.1, modified] 39 scope of laboratory application analytes, matrices, and concentrations for an analytical method that a user laboratory claims to be capable of satisfactorily testing in its laboratory Note 1 to entry: A method may have been validated to 2 broader range (scope) of analytes, matrices and concentrations than the scope that will be claimed by 2 user laboratory. The scope of laboratory application 1s the scope of validation, 3.10 scope of validation analytes, matrices and concentrations for which a validated method of analysis can be used satisfactorily [SOURCE: ISO 16140-1:2016, 3.11 test portion measured (volume or mass) representative sample taken from the laboratory sample for use in the preparation of the initial suspension Note 1 to entey: Sometimes preparation of a test sample from the laboratory sample is required before the test portion is taken, but this is infrequently used in microbiological examinations. [SOURCE: 150 6887-1:2017, 35, modified] 3.12 test sample sample prepared from the laboratory sample according to the procedure specified in the test method and from which test portions are taken Note 1 to entry: Preparation of the laboratory sample before the test portion is taken is infrequently used in ‘microbiological examinations. (SOURCE: 1S0 6887-1:2017, 3.4, modified] 3.13 type ae for a given category, a group of (food) items processed in a similar way, with similar intrinsic characteristics and a similar microbial ecology EXAMPLE Food category: heat-processed milk and dairy products; food type: pasteurised dalry product. 10 © 1802017 ~ Allrights reserved Scanned with CamScannerISO/DIS 16140- {SOURCE: ISO 16140-1:2016, 2.78, modified] 3.14 aser laboratory laboratory which implements a validated alternative method and/or a reference method 3.15 verification demonscration that a validated method performs in the user's hands according to the method's sped smined in the validation study and is ft for its intended purpose ation: 4 Principle 4.1 General lished validation data for the method verification, it is crucial to refer to the publ the scope of validation and to select the appropriate (food) items, Before performi: method in order to conf The veri ication is undertaken in two parts: sation verification; — imple — (food) item verification. User laboratories intending to verify methods that wublished validation data for comparison (this includes all validated alternative methods) shall perform both the implementation and the (food) item validation data. For those methods, the cation. Some reference methods may not have publish: il] only perform (food) item verification. user laboratory Alternative methods that have not been validated shall first be validated, before a user laboratory can verify its use In their laboratory (see Figure 1). 4.2. Implementation verification Implementation verification aims to demonstrate the competence of the user laboratory to perform the validated method. It compares user laboratory performances to those obtained during the validation. Implementation verification applies only to the following methods with published validation data: — reference methods; and — validated alternative methods. ‘The user laboratory shall: — review the published validation data for the method: — selectone (food) item tested during the validation study that belongs within the scope of laboratory application of the user laboratory, if possible, and; — use this (food) item and the sample size used in the validation study to perform implementation verification. © 180 2017 - All rights reserved . Scanned with CamScanner4.3 (Food) item verification (Food) item verification applies to reference methods, with and without published validation data, and validated alternative methods with validation data. The (food) item verification sets out to demonstrate the competence of the user laboratory to perform the validated method with (food) items that are tested in the user laboratory. 4.4 Implementation verification and (food) item verification Table 1 provides guidance on when to use implementation verification and when to use (food) item verification. Table 1 — Implementation verification and (food) item verification Method with published validation data _| Method without published validation data Reference method | AKermative method | Reference method | Alkernative method Implementation 7 Y Nocapplicable | Nocapplicable rerifcation | (Food) item 7 1 E 7 | Notapplicable* verification | Nex appliatie be Gai be valeaed wo BO 161402 or 0 361305 (He 7 wich aidan ca Figure 2a to Figure 2d show the number of (food) items required for implementation verification and (food) item verification under different circumstances. laboratory anplicatinn dation Scope of Category 1 > Types > Htems Category 2 3 Types 3 hems Category 39 Types > hems wary 4-9 Types 3 hems Categury $3 Types > lems Category 15 Figure 2a — (Food) items required when verifying a reference method with published validation data or a validated alternative method for a “broad range of foods” scope 12 © 150 2017 ~ all rights reserved Scanned with CamScannerScope of Fulldanion [we | Scope of tavoratery app Figure 26 — (Food) items required when verifying a reference method with published validation data ora validated alternative method for a “limited range of foods” scope Scope of validation Reference method with no published. Category 1 > Types > Items Category 29 Types > ems Category 39 Types > Items Category 4 > Types > Items Category 5 > Types > Items Category 6 Category 15 Figure 2c — (Food) items required when verifying a Scope of laboratory application Implementation verification (Yoo) tes each iForent (load reference method with no published validation data for a “broad range of foods” scope © 150 2017 - all rights reserved B/DIS 16140-3:2017(E) Scope of validation* Scope of laboratory application Reference method with no published validation data + of foods” scope Implementation verification ‘Limited rang Category 1 > Types > Items a5 hea Category 2 > Types > Items > Types > hems Figure 2d — (Food) items required when verifying a reference method with no published validation data for a “limited range of foods” scope ‘Table 2 summarises the minimum number of (food) items required for the different scenarios. ‘Table 2 — Summary of the minimum number of (food) items required for verification. Number of samples Scope of Published | ~ implementation (Food) item otal minimum validation | validation data? verification verification number Broad scope Yes I 25 é (2 Scaregores) Now 0 25 5 Limited scope Yes 1 N Nel (categories) ae 7 F 7 | Notapplcabi for validated akernaive methods as these wll abrays have ralaon 3 ‘Table A.1 of Annex A provides the list of (food) categories and corresponding (food) items and Annex B provides requirements and further guidance on selecting a challenging (food) item from each (food) category for (food) item verification. The (food) items chosen from ‘each (food) category shall be items that reflect the range of the laboratory samples sived by the user laboratory, and should, as far as possible, be items with components such as natural antimicrobial properties, vitamins, flavours, rc © 1S0 2017 ~ all rights reserved Scanned with CamScannerthe number of colonies for confirmation may be redu with the ribed in Annex B that may in probiotics, roorgan mic ism. tal samples and primary production stage samples‘have been included lidation study, and if these are claimed by the user fortes claimed, shall also be included in the (food) ed and pet food, environn 3s additional (non-food) cat ory, then one item from each of these cat sgories in the va -m verification 5 Performance characteristics -actaristics for method verification. Table 3 describes the required performance char Table 3 — Required performance characteristics to be determined for verification Method | _Perlrmance characterise Implementation verification] (Food) item verification [Qualitanive _[Esumared LODs:(eL.0Ds3) | v: t = [Quanniave linvalsborscore 7 JNOT= __ Regarding invalabarston reproduc 5 Qualitative methods — Technical protocol for verification 5.1 Estimated LODso(2LODso) determination tion and the (food) d for both the implement [eLOD:3] determination is reaui The estimated LO item verification. In its entirety to complete — The user laboratory first follows the technical protocol outlined below. validated method. the implementation verification, demonstrating the ability to conduct — The user laboratory then applies this same technical protocol for (food) item verification. During the method verification, run the full protocol of the method a3 described, including the confirmation procedure (if there is one). Each individual test portion needs to be confirmed, although 5.2 Experimental design ‘The protocol shall be performed as follows (see also Annex C for additional guidance and examples): — Prepare cultures of the target microorganisms for inoculating the (food) items. — _Ata minimum. prepare 8 test portions of the same (food) item, Choose a (food) item which should ‘not be naturally contaminated by the target microorganism. — Inoculate 6 ofthe test portions: 2 at high evel.2 at intermediate level and 2 at low level Leave the remaining 2 test portions uninoculated, Choose 2 (food) item which is demonstrated to be not naturally contaminated by the target microorganism. These are the blank test portions which shall give negative results, NOTE Ifthe level of the culture used for inoculation is not known, additional test portions can be inoculated ‘with extra dilusons to ensure thatthe targetlevels are included inthe verification. © 180 2017 - All rights reserved a5) Oo Scanned with CamScannerIS0/DIS 16440-3:2017(B} inoculum, according to 150 7218, same time as the test portions are unts obtained — Detennine the level of contamination by plating the high-lev using a non-selective medium (eg. Plate Count Agar); at the same | noculated. The contamination levels of the other inocula are calculated using th ¢ high-level inoculum and taking into account the dilution factors used. + portions shall be tested according to the full protocol of the method being for’ fled. — Thetes — The eLODsois calculated based om the positive and negative results (see 5. 5.3. Selection of (food) items = : 3 (For the implement Gicatighl one (ood) item is required. The (food) item selected shall be one included in the validation study and preferably is tested by the user laboratory. (FOrTKE (food) item verification the user laboratory shall test a minimum aumber of one (food) item: preferably a challenging one, from the (food) categories claimed in the scope of laboratory application (see 4.4). Annex B provides guidance on how to choose challenging (food) items. 5.4 Artificial contamination 5.4.1. Selection of strains Preferably, strains used in the verification are from sources relevant to the (food) tem being verified. ‘They can be from: — culture collections: — userlaboratory collection; — reference material (including commercial reference material, e.g. freeze-dried strain with known concentration). 218. Preferably, Strain characterisation and selection shall follow the requirements specified in 1S0 use a different strain for each of the (food) items to be tested. 5.4.2 Inoculation of the test portions Use the LODs: data from the validation study of the method to determine the range of contamination (between 1 to 10 times the LODs) that will be used to inoculate the test portion. Ifthe LODs» is not known (eg. reference methods with no published validation data), the value will be assumed to be equal to or lower than 1 cfu/test portion. ‘The following guidance is given as an example of procedures suitable for producing inocula. The selected strain shall be grown in a culture medium under conditions that enable optimal growth of the strain (e.g. overnight culture); follow the procedures specified in ISO 11133. Enumerate the culture on a non-selective medium to determine the concentration of the strain. It is ‘assumed that this level will be consistently achieved when the same culture conditions are used. Repeat growing the culture under the same conditions taking into account the concentration previously determined to prepare dilutions to cover the targeted range of contamination. This step is not required 16 © 180 2017 - All rights reserved Scanned with CamScannerISO/DIS 16140-3:2017(E) conditions of growth of the strain is well know: +*C overnight) oratory (eg, stability afte! storage Ifthe user laboratory works with ready to use the steps described above are not re t strains with known levels (e.g reference material) cd The prepared inoculum is introduced directly into the initial suspension of the individual test portions Mix well after inoculation to ensure uniform suspension of the inoculum. Use of stressed cultures is recommended, but not required. A minimum of 3 inoculum levels per (Food) item shall be used. ‘The 3 levels are: |: this should be at a maximum of ten times the expected LODss. Whi |. arecomm vel of 10 cfu/test portion is used. — High inoculation le: LODse is not know — Intermediate inoculation level: from the high inoculation level, perform @ 1:2 dilution to achieve the intermediate level. The level of contamination for unknown LODs: should be 5 cfu/test portion. — Low inoculation level: from the high inoculation level, perform 1:10 dilution to achieve the low level. The contamination level for unknown LODs: should be 1 cfu/test portion. NOTE The abbreviation of colony form: To determine the inoculum level, enumerate the high-level inoculum according to 150.7216 (using a non-selective medium (eg. Plate Count Agarl); at the same time as the test portions are inoculated. The count of the low and intermediate levels will be calculated using the counts obtained for the high-level inoculum and taking into account the dilution factors used. EXAMPLE A user laboratory wants to verify 2 Scimoneila method. — Based on the LODss (2,5 cfu/test portion) determined in the validation study, the range of contamination for (food) item A will, theoretically, be 25 cfu/test portion, 12,5 cfu/test portion and 2,5 cfu/test portion. In order to determine the range of contamination, an overnight culture is orepared and assumed to contain 5x10! cfu/ml based on the preliminary enumeration. ‘As the actual count of the new overnight culrure is unknown, the user laboratory can use several dilutions to cover the three target levels, using each dilution on two test portions. In this case: — 1mland 0.5 ml of 10" dilution (to cover unforeseen lowe: concentration); — 1 mland0.5 ml of 107 dilucion, dured 1 — ml and 0,5 mi of 10° dilution (to cover unforeseen higher concentration). In total, 12 inoculated test portions will be examined plus 2 blank test portions but only the three relevant levels and the blank level will be retained for the calevlation. — In this example, the counts on duplicate plates of one of the dilutions set up for enumeration of the inoculum, are 14 and 22, making the final average contamination level for this dilution 18 cfu/ml. _— Using this selected dilution for the high inoculation level (1 mi of dilusion 10°), then the intermediate level (1:2 dilution of the high inoculum) is considered to be 9 cfu/test portion and the low level (1 ml of Gilution 10) is 18 cfu/test portion. See also Annex C. 5.5. Results Record the number of positive results obtained at each inoculum level and use Table 4 to determine the eLODso. ‘The blank level shall not produce positive results. If positive results are obtained, the experiments shall be repeated for all levels. © 150 2017 ~ allrights reserved = Scanned with CamScannerISO/DIS 16140-3:2017(E) ‘The high-level inoculum (10xLODs«) shall produce only positive results, If negative results are obtained the experiments shall be repeated for al levels. ‘Table 4— Estimation of eLODso based on the number of positive results per level of contamination Tigh inoculation | tatermediate | Lowinoculation | Blanklevel 3xLODss observed inthe validation study. 1f the LODss is not known from the validation study data, eg. reference methods with no published validation data, the eLODs» shall be equal or lower than 3 cfu/test portion. The value 3 cfu/test portion is derived from an accepiable ODse value of 1 cfu/test portion (plus a margin of 3 « higher). Clause ? provides 2 summary for the acceptance cri 1 LODss observed in che validation stady can be expressed as cfu/ml or cfu/test porvion. T will need to be expressed in cfu,/'test portion ro be able xo compare the result with th sfa/g oF cfu/ml then the LODs: ere the LODsy observed in the validation seu LODs: of 0.1 cfu/g or cfu/ml will size ofthe test rortion used. Taet when 2 25. or mi test portion is im the validation smady is, for example, 2.5 cfu/test portion the maximum ven in 5.42, the last th 9,0 o: 13.4 cutest portion}. Based on the exam} the eLODsp is unreliable or too high (2L0I 5.7 Root cause analysis When the result does not meet the acceptance criterion (if the eLODss is > 3xLODse observed in the validation study), perform 2 root cause analysis in order to provide an explanation for the observed sults, ‘The root cause analysis could be conducted as follows: — identify analytical error due to human factors: — identify analytical error due to the equipment: — identify analytical error due to lack of good laboratory practice: — identify analytical error in protocol application (e.g. no correct inoculation level, et (food) item specificity, eg very challenging (food) items that required higher dilution factor in the initial suspension; — identify if the method is capable of achieving the acceptability limit of 1 cfu/ test portion. Some methods may not be able to achieve an LODs« of 1 cfu/ test portion (e.2, Campylobacter); = ete, ‘Once the problems are identified, Implement corrective actions and repeat the test. Information (based on investigation, e.g. root cause analysis) can be given in the study report to provide ‘an explanation of the findings. If a (food) item cannot be verified, it is recommended that the user laboratory contacts a relevant organization depending of the method, e.g. standardization body, supplier, certification body. © 150 2027 ~ Allrights reserved am —<$—$— Scanned with CamScanner6 Quantitative methods — Technical protocol for verification 6.1. Precision (intralaboratory reproducibility) determination 6.1.1 General The precision (Intralaboratory reproducibility) determination only applies to implementation verification, Precision is comprised of repeatability and reproducibility. For the implementation verification, only he reproducibility is considered, As this is performed in a single laboratory, the reproducibility is pressed as the intralaboratory reproducibility (Ss). The intralaboratory reproducibility (52) determination is based on 42.1 ‘Reproducibility standard deviation derived from intralaboratory experiments’ of [S0/CD 19036. pe of implementation ing ISO 19036 are required see 1S0/CD 19036 for further ion of the intralaboratory: reproducibly (Sz) in the ‘or one (food) item. However, user laboratories ipl wainty for other (food) items as w The (food) item used in the implementation verification is any (food) item relevant to the user laboratory. During the implementation verification, rum the full protocol of the method as described. including the confirmation procedure, Each individual test portion need to be confirmed. 6.1.2 Experimental design ‘The protocol shall be performed as follows (see also Annex D.1 for additional guidance and examples). At least 10 laboratory samples, originating from different batches but belonging to the same (food) item, are required. More samples may be tested to cover the possible loss of sonie samples due to practical errors/mishaps during testing. ‘The contamination levels used shall be representative of the range of the natural contamination found im the samples tested in the user laboratory. Each laboratory sample (or test sample) shall be homogenised before two test portions are taken (see Figure 3). Homogenisation is essential to ensure that microorganisms are uniformly distributed. For liquid products, the homogenisation shall be performed by shaking the laboratory sample (or test sample) by hand (eg, 25 times through an arc of 25 cm). For solid products, the homogenisation may be performed by mechanical means which could include stomachers and blenders. For details, follow the procedure in the ISO 6887 series. If artificial contamination is used, inoculate the initial suspension of each test portion with a known level of the strain. Naturally contaminated test portions can be analysed directly after homogenisation of the laboratory sample (or test sample). 150 2017 - Allrights reserved 20 Scanned with CamScanner180/DIS 16140-3:2017(E) Laboratory sample Test sampl Homogenise Test portion portion A | Initial Initial suspension A suspension B T | | Analysis Analysis T ae Result A Different conditions: A.B Figure 3 — Experimental protocol to estimate the standard deviation of the (intralaboratory) reproducibility (Si) The test sample, as shown in Figure 3, is infrequently used in microbiological examinations. In that case, the laboratory sample is directly used for homogenisation. ‘Test conditions used in the analysis of test portion A and B shall be different in as many ways as possible within the scope of validation. These shall include but not be limited to: 1) technicians; 2). batches of culture media and reagents (optional: when relevant, different strains may also be used to inoculate different initial suspensions) and; 3) apparatus (Incubators, vortex mixer, pipettes, etc). © 180 2017 — All rights reserved ay Scanned with CamScannerTest conditions 1 and 2 are considered to cause tite most variability in the results of a method and shall be varied unless the user laboratory can justify otherwise. Test condition 3 shall be varied based on the availability of the apparatus in the user laboratory. Ifthe contamination of the (food) item can be shown to be sufficiently stable, the analysis may be conducted on different days. Results shall be analysed according to 6.1.6. 6.1.3 Selection of (food) item One (food) item is required for the implementation verification. Any (food) item, relevant to the user laboratory can be selected. The reason is that the intralaboratory reproducibility (Sz) will be compared to the interlaboratory reproducibility (5,) obtained fron laboratory study. The interlaboratory reproducibility ( sents the technical uncertainty as defined by ISO/CD 19036 and this value is therefore independent from the 9) rer. 6.1.4 Natural contamination Whenever possible, use naturally contaminated items. For the (food) item chosen, the individual test portions evaluated shall have contamination {et resentative of the range of the natural contamination found in the laboratory samples analysed in the user laboratory. jected level of natural contamination is less than 10 cftt/g in the test portion, artificial contamination is used (see 6.1.5) to co of use of the method. 6.15 Artificial contamination 6.1.5.1 Selection of the strain Preferably, strains used in the verification are from sources relevant to the (food) item being verified. They can be from: — culture collectioi — user laboratory collection; — reference material (Including commercial reference material eg. freeze-dried strain with known concentration). Strain characterisation and selection shall follow the requirements specified in ISO 7218. Preferably, use one strain per (food) item. 6.1.5.2 Inoculation of the test portion The following guidance is given as an example of procedures suitable for producing inocula. When inoculating the test portion, the contamination levels used shall be representative of the range of the natural contamination found in the laboratory samples analysed in the user laboratory. ‘The selected strain shall be grown in a culture medium under conditions that enable optimal growth of the strain (e.g, overnight culture); follow the procedures specified in ISO 11133. Enumerate the culture on non-selective media to determine the concentration of the strain. It is ‘assumed that this level will be consistently achieved when the same culture conditions are used. 2 © 150 2017 - All rights reserved Scanned with CamScanner§sO/DIS 16140-3:2017(3) Repeat the overnight culture and take into account the concentration previously determined to prepare dilutions to cover the representative range of natural contamination. This step is not required if the conditions of the strain growth is well known by the user laboratory (eg. stability after storage at 4 °C overnight) Ifthe user laboratory works with ready to use target strains with known levels (eg reference material), the steps described above are not required. The prepated inoculum is introduced directly into the initial suspension of the individual test portions. Mix well after inoculation to ensure uniform suspension of the inoculum. Use of stressed cultures is recommended, but not required EXAMPLE A user laboratory wants «0 veri The validation of this method was performed using o: L (Food) category: raw mest and ready to cook (unprocessed): (food) item: rave pork chop 2. (Food) category: eggs and egg products (Gerivatives): (food) ne: exes eration method. 10d) categories ‘it Boul); (food) type: fresh meats eat products (ex -aprocessed); (food) item: shell esas. 3. (Food) pet food and 4: (food imal origin ingredient: (food) item: meat and one meal 4, (Food) category: heat processed milk and dairy products: (food) 1772 ed milk-based products; eurised milk. chocolate, bakery products and (food) sype: pastries: (food) ikem: (food) isem tested in the validation smady] was chosen ial moculation. coli was chosen as the E coli culture gives approximat concer: all be used as a star — Based on the range of comamination used 30 (15 log:s) cfu/g to 30 000 (4,5 logs:) cfu/g — A minimum of ran different (brands, lois) laboratory samples of tiramisu divided into two test portions - A and B (see Figure 3). Both test portions. A and B, originating {rom the same laborat igure 3) will be inoculated at the same level. Each set of the 10 or more laboratory samples will be inoculated at different levels: ‘between 30 and 30.000 cfu/g. The culture will be inoculated into the intial suspensions. — inorder to do this, an overnight culture is prepared and assumed to contain 10° cfu/ml (based on results of previous enumeration - see Annex D). — To contaminate at the level of 30 cfu/s, 6 serial decimal dilutions ofthe over reduce the initial ievel from 10° cfu/ml to 10* cfu/ml. — The initial suspensions of the two (10 g) test portions per laboratory sample are prepared. = 03 ml of the culture suspension are inoculated into each of two initial suspensions. Different contamination levels covering the range of 30 cfu/g and 30 000 cfu/s can be obtained with different ‘ddutions and/or inoculation volume. lutions ro inoculate he test portion. wl be inoculated between ll be prepared and each ight culture are prepared, ro 6.1.6 Results ‘The intralaboratory reproducibility (Sz) is calculated, based on a minimum of 10 laboratory samples, according to the formula given below. Sa isthe intralaboratory reproducibility; i isthe index of the laboratory sample, i= 1 to p (p> 10); 23 (© 150 2017 — Allrights reserved Scanned with CamScannerp is the number of samples; Yu Yoo are the log-transformed data, in logis (cfu/g) of logs (cft/ml), from conditions A and B respectively. An example of a manual calculation is given in Table 8. Results of tests with low counts (e.g. Indicative sults) cannot be used for the calculation of Sz. The criteria for low counts are given in 150 7218. 6.1.7 Acceptance criteria The intralaboratory reproducibility (Sz) of the verified method shall be < the interlaboratory S;) of the most homogeneous (food) item used in the validation study. The most homogeneous (food) item is ‘ood) item with the lowest 5; mean value of the inoculation I NOTE Clause 7 provides 2 summary for the acceptance criteria EXAMPLE — The user laboratory has evaluated 12 laboratory samples of tiramisu for the level of Er (esing ISO 21528-1) following the experimental design given in Figure 3. The following results (calculated ble 6 were obtained. srobacceriacece Table 6 — Test results Taborstory | __Ewpeced _] femitae) | RemiNBGs) | legresikA | Logrenikp sample | contamization evel | Ge og) sumber_| | ane En FT) 7 7a | Swannalaearts 300| 382 226007; 3 T 300] 620) 2.792392] atl 200) 530) 13] Saal 600) 230) 570) 72838845] 2.755875| 8 200) ad 230) 20920951 20siel a 600) 620) 1300) 2.792392) 3.113943] ‘< 600) 870) 1500) 2.939529) 376094] : Zoee] 3609) 6400) Saas] 3.80634 og 5000) 76000) $000) 208221 369097 Fry 5000 315,001 73400) 3476094) 3227205 2 30 004 20000) 32000) -430203] 150545] _— The results of laboratory samples 1 and 11 cannot be used because one of the counts was either too high (2 result) or too low (below permitted counting range according to 1S0 7218). Results of 10 laboratory samples remain for the calculation. — Based on the 10 remaining laboratory samples, the Sa can be calculated as shown in Table 7. Table 7 — Calculation of Sz Taboratorysample | __LogresuleA TogresukB | Absolute difference | Squared difference umber pat lga) a= logetaa) ba-ral anya? 7 7,p00009 ana Norased Norse] z 2.041394] 2.28007 02186 opa7er| 3 2612784 2.792892 0.179609) 03225 4 50618] 2510514 0.287666] 002753] s z3s8609 2755875] 0.082975] 1.006805 2 © 150 2017 - All rights reserved Scanned with CamScannerISO/DIS 16140-3:21 Taboratory ample | _LegresultA TogrenikB | Absolute diference Timber | se loga(t) pas logics) bars | TESS 3007384 z I 9.103394] 8 I ct 0.085988] z T 00-6464 20 | 9255177 I Norused| Not weal i 920422) 0051685] sem Fam/{2=10)] 05789 a= v(0.032488)) aq — The obtained Sx of 0,18 is compared to esults of the validation ined from that validation study. (data taken ov 150 21526-2) Table 8 provides the Ssvalues 25 Table 8 — Summary of Sr values from the validation study for IS0 21528-2 Sivalues from the validation study Food) tem | towineculation | Intermediate | High inoculation | __Mean value of ‘ eal inoculate oe evel three inoculation levels O33 os os o2% os on 3 020 the testis to not conside: St of s homogenous (Food) icem. In this examale, the 3 inoculation levels was 0,18) — The Si found in the verification study (0.18) is compared to Ss of 0,18 from the validation study. (0.18) 1s equal to 0,18; the conclusion is that the precision of the — As the Sin of the verification stu method carried out in this user laboratory is acceptable. 61,8 Root cause analysis btained is com red to the sm was (animal) feed (mean of When the result does not meet the acceptance criterion, perform a root cause analysis in order to provide an explanation for the observed results. The root cause analysis could be conducted as follows: — identify analytical error due to humian factors: — identify analytical error due to the equipment: — identify analytical error due to lack of good laboratory practice; — identify analytical error in protocol application (e.g. no correct inoculation level, etc.) Once identified the gap, implement corrective action and repeat the test. Information (based on investigation, eg. root cause analysis) can be given in the study report to provide an explanation of the findings, © 150 2017 - Allrights reserved Scanned with CamScanner 25a (food) item is not verified, it is recommended that the user laboratory contacts a relevant organization depending of the method, eg. standardization body, supplier, certification body. 6.2 Estimated bias determination 6.2.1 General estimated bias (eBias) determination is only applied for (food) item verification, During the method verification, run the full protocol of the method as described, including the confirmation procedure (if there is one). Each individual test portion needs to be confirmed. 6.2.2 Experimental design Protocol shall be performed as follows (see also Annex D.2 for additional guidance and examples) — Select the (food) item(s) for testing. — Artificially contaminate the (food) item(s) at three inoculation levels. The artificial contamination is done in the initial suspension. Each level is performed in duplicate (Initial suspension A and 8 for each level), — Run in parallel the method to be verified with the artificially contaminated (food) item and the same method with the inoculura suspension used to contaminate the (Food) item. — Test the uninoculated test portion, in duplicate, to determine the background contamination level. 6.2.3. Selection of (food) items For the (food) item verification, the user laboratory shall test a minimum of one (food) item, preferably 4 challenging one, from the (food) categories claimed in the scope of laboratory application (see 4.4) Annex B provides guidance on how to choose challenging (food) items. 6.24 Artificial contamination 6.2.4.1 Selection of strains Preferably, strains used in the verification are from sources relevant to the (food) item being verified. ‘They can be from: — culture collections; — user laboratory collection; — teference material (including commercial reference material, eg. freeze-dried strain with known concentration), Strain characterisation and selection shall follow the requirements specified in ISO 7218. Preferably, use a different strain for each of the (food) items to be tested. 26 © 150 2017 ~ allrights reserved Scanned with CamScanner6 culation of the test portions The following guidance is given as an example of procedures suitable for producing selected strain shall be grown in a culture medium under conditions that enable optimal growth of the strain (e.g. overnight culture), Follow the procedures specified iz ISO 11133, numerate each inoculum suspension on non-s media to confirm the levels used. The inoculation levels are expressed as cfu/g or cfu/ml. Repeat the culture under the same conditions and take into account the concentration previously determined, to prepare dilutions to cover the targeted range of contamination. This step is not required if the conditions of growth of the strain is well known by the user laboratory (+g. stability after storage at 4*C overnight) If the user laborator steps described abo: Y works with ready reference material), the re not required to use strains with The prepared inoculum is is Mix well after inoculation recommended, but not requi individual test port Use of stressed cultu! roduced directly into t ensure uniform suspension of the inocuh ns. s Is 6.2.5.3 Preparation of the test portions The inoculation levels shall be selected and justified based on the levels = the assumed level). Therefore. the actual levels in the intial suspensions were 10 to 10° cfu/ml, and the calculated levels in the test portions were 10? cfu/s to 10° efu/g — The user laboratory was then able to select the results relating to 102 cfu/g; 10* cfu/g and 10cfu/g of the test portion (10: cfu/ml; 10 cfu/ml and 10° cfu/ml of the initial suspensions) and compare actual counted results for the method being verified, with the counts, ofthe inoculum, on non-selective medium. ‘© 180 2017 ~ All rights reserved a Scanned with CamScannerIs0/DIS 16 017(E) 6.25 Results ‘The results shall be expressed in log:s cfui/g or log:» cfu/ml. Compare the results of the method to be verified with artificially contaminated (food) item to the results of the inoculum suspension tested with the same method. 6.2.6 Acceptance criteria It is expected that, at each level, the difference between the results of the method to be verified with artificially contaminated (food) item and that of the inoculum suspension tested with the same method is equal to or less than 0.5 logis cfu/g. vides a summa’ NOTE Clause 7 pr for the acceptance criteria. las of the method for the Enterobacteriaceae count by 2 of contamination is between. EXAMPLE A.user laboratory w validated alternativ 10% cfu/g and 10° cfu, ts to veri Table 9 — Test results ] Dilference between method to be verified | with ardBiclally | contaminated (food) | tem and the inoculum suspension. Resales ‘Mean result per batch Tangle sample t test lLaboratory sample 2 i \irom batch 2), 050 393 be | a2, orton t lrsboratory sample 2 a ae 030 |(from batch 2) test 404 435 fportion 2 [taboratory sample 3 (om batch 3) test 373 359 [portion l-aboratory sample 3 (irom batch 3), est 363 345 orion 2 362 0.08 ‘The results indicate that at each level of contamination the absolute difference berween the two methods is less than 0.5 logzs, o the method to be verified works correctly in the user laboratory. 6.2.7 Root cause analysis When the result does not meet the acceptance criteria, perform a root cause analysis in order to provide an explanation for the observed results, ‘The root cause analysis could be conducted as follows: 28 © 1502017 - All rights reserved Scanned with CamScannerOnce the problems are identified, imp! Information (based on investigation, 2 180/DIS 16140-3:2017(6) Identify analytical error due to human factors: nntify analytical error due to the equipment; Identify analytical error due to lack of good laboratory practice: identify analytical error in protocol application (eg. no correct inoculation level, etc.) igher dilution factor in the (food) item specificity =g. very challenging (food) items that r initial suspension; et. tthe t e actions and t @ analysis) can be given in the study report to provide an explanation of the findings when the eBiasis > 0.5 logse cfu/z, If a (food) item is not verified, it is recommende organization depending ofthe m 7 Table 10 summarises the ac © 180 2017 ~ Allrights reserved t the user hori, eg. standardization body, supplier, certification body oratory contacts 2 relevant Summary of acceptance criteria ance crite! Table 10 — Acceptan: Method] Teceptance cnteria ToDsavalableinthe Qualitenve ODse3 10D GOD fvhen no LOD avaiable) LOD £3 cytes persion ‘Saf athods with validation data) ES seo te cree fear eacous (00d) hem used fe he valcadion rudy Quantitative owest mean salve ofthe inoculation levels) Bias 205 legs dug for all inoculaton levels 29 Scanned with CamScannerScanned with CamScannerte poniosou suit nV ~ £102 081 ‘ fe [A | Ase Ae ee [ae x AR |e | a a | Tal aro opera, Scanned with CamScannerte ‘un pnersosehieny souotou Scanned with CamScannerfe V~ £102 0510 ‘ ‘ ‘ Tear) (paren shears (se fee face [ae uo , Goo eea toot A A A ape cae [eae | eas [een aes ras os rl enone oo 4 a ‘ ie ae fee | tes [ee | le es spina 4 4 4 eo enero Acs ena seats | wpa H/OSL Scanned with CamScannerve road, | sapotono, (1) 2107:£-0V 091 S1d/0S1 Scanned with CamScanner14 V-210z 0s opr shes ane] esto: rssceyen sponyene co ape mn soy, | soptoney ae Lor sta/ost Scanned with CamScanner90-00 sopse3a409 (12102-00191 SIG/OSI su niacalel Scanned with CamScannerut HIV ~ £102 0s1@ M noe sayin Scanned with CamScanner19 oe sin twearl soc, | operon ‘OPL9E SIG/OSL | eet Scanned with CamScannerov IV=£102, 081 (a0>%0] “Asewononpio} — ineon “yms0yp| op Ae |e A as ‘ewronnl peu uw ‘owieoip| wok, (a)210z*£-0VF9F sta/ost Scanned with CamScanneroe = 2102 0510 sopotaie Scanned with CamScannerWIV~Z102 081 souk, | sou ay Scanned with CamScannerv= c107, wear] sinpastl wa 4 sv (a)z102:8-0r191 sia/ost Scanned with CamScannera ponssau suit ny ~ £02 OSI (sua) extu oxnepoid © ales 4 4 ‘a Alben 4 4 captains rng] imutey 4 Aen i sae a A | rou amon 1 ] so rsa Scanned with CamScannerScanned with CamScannerx B (norm: (food) item | | —Requirements and guidance on how to choose challenging s) for (food) item verification Bl General It is important to select (food) items that are representative of those encountered in the user applicable to (food) item veriti ‘This annex is specifica The (food) item can affect the outcome of an analysis. The composition of the food, its background crobiota and other contaminants can interfere with the test method and invalidate the result. It is re expected that the user laboratory will ensure that the method is fit-for-purpose for the (food) if a method is validated for a broad range of foods, not all (food) items have dation; therefore, the user laboratory shall demonstrate that the method is applicable to (food) items tested in the laboratory. as only one (food) item is required per (food) category. itis iportant to perform the (food) item verification with the most challenging one. B.2_ Matrix effects to consider B.2.1 Microbial characteristics Unless, the food has be: ~e naturally contaminated sterilised, for example canned food, (food) it by microorganisms, which can be categorised as: — Technological microbiota such as microbial cultures and probiotics, eg. fermented and cured foods, Probiotic food products inoculated with a level of microorganisms from 10 cfy/g to 10° cfu/g, etc — High background microbiota samples, eg. poultry minced meat, fzecal samples, raw milk, ete. — Spoilage microorganisms; the presence of this native microbiota can influence the recovery and growth of the target microorganism. ‘The effect ofthe background microbiota on the test method should be evaluated when the difference in ir counts and that of the target microorganism is greater than 3 loge. For example. high ‘obacteriaceae counts may affect isolation of Salmonella spp. and the presence of Gram-positive rter cultures may interfere with the recovery of Listeria monocytogenes. 8.2.2 Physical and chemical characteristics The following physical and chemical parameters are known to affect the recovery of microorganisms and/or on the method performance (see ISO 6887 series): — composition, eg high fat content, lecithin, thickener, nutrient content — pl.eg pH < 4-5 (beverages, sauces, etc); — oxidation reduction potential; — water activity, eg. ax < 0,85 (flour, low moisture foods); — antimicrobial constituents and growth inhibitors, eg. polyphenols, enzymes, molecular inhibitors; — Physical structure of the food, eg. viscosity, solubility; ©1S0 2017 - Allrights reserved aa Scanned with CamScannerI — colour, e.g. food dyes B.2.3 Food process characteristics considered matrix can often have a treatment step (heating. high result in injuring microbial cells. This affects the culturability of the 2 The manufactur pressure process, i thereto: ° 9f the microorganism of con B.3. Selection of (food) items for verification The microbial, physical, chemical and process characteristics recommended above can be found in (food) items amongst all (food) categories described in Annex A. ting a challenging (food) item per category, the user laboratory shall choose, among the. tested in its laboratory, an item which shows one of the challenging characteristics. It is food) item presenting a combination of the characteristics (¢2 pH ~ 2.) as this the worst-case scenario. When (food) i best to s represent of foods scope, a minimum of five (food) items selected from five (ood) categories is possible, each of the five (food) items shall have a different characteristic or a order to cover different cases. Table B.1 provides an example Fora broad ran: d (4.4). combination of characteristic Table B.1- Example of (food) items and its characteristics Giegory | item Characteristic i Far conte High background microbiots and pil Palyphensl (a ‘The selection of (food) items also depends on the method principle which can orientate the user laboratory in the selection of the (food) items. Table B.2 indicates examples of principles of methods whose performance can be affected by the food characteristics. ‘Table B.2 - Examples of (food) items characteristics that may affect performance of different method principles Method] High number fcompetitive | Physicalcharacterisics Chemical compound [© (esterojorganisms | Faciaolgical] High background | pH | a- [Sohiblig/| Coeur Vanilin nayme] Polyphenol | Molec | microbioea | microbiota viscosity Iimhibitor i spoilage Carimesed | =| [BS [ee] | = muno-emare]| | era = ea ae E Moenbres |= | = er [| ana |e | = 5 Row enomeny | =. = 5 [iss ae are joey | Seca | rll b= 48 © 180 2017 -Allrights reserved Scanned with CamScannerPreparation for verification: a preliminary eni the method verification is performed to provide an willbe used. 1) Prepare a culture of the nucroorganism_ ‘time of incubation). Follow the pro pa 2) Perform the enumeration of the « Scanned with CamScanner10 ditraon 10 aileron Expected 50 cum! Expected Ste/id sin pacalel wit the test porions Figure C.2 — Example of the preparation of the inoculum 1) For each contamination level, pesform 2 replicates. In theory, 3 contamination levels (high. intermediate and low) and 1 biaak level are required. However, as the actual count is not known at the time of inoculation, it is recommended to perform a “range” of serial dilutions that would include the 3 target contamination levels (see Figure C.3). Inoculate 0,5 ml and 1m] into the initial suspension of the individual 10? ansion Expert 505 /e Expecred Sad ae sa vise 1 1 1 1 1 1 1 L ahtewt Intermeate Io Lov lot Blank eer Target 25 fuytes porvon Tange 23 ce portion Tepe 25 clues porten 0 du/tex parton Figure C3 — Example of the inoculation of the test portions Inthe example shown in Figure C3, the expected high-level inoculum is prepared using the 10¢ dilution, in case the new overnight culture has a lower than expected count. 2) Perform the method to be verified. 50 ©180 2017 Allright reservedee ee 3) Record the positive and negative results. 4) Calculate the actual contamination based on the actual enumerated result (refer to 1SO 7218). In the example shown in Figure C4, the new overnight culture count result is 1,810" cfu/ml and not 3105 cfu/ml. Therefore, fest portions with positive results are most likely to appear at the 104 and 10° dilutions instead of 10” and 10* dilutions. rin ame — =] U 10 diluion 107 dilutien 10? dilunion Expected 5x10? cfu/m! Expected 50 chi/ml Expected 5 cfu/ml Scanned with CamScanneromnative) — Protocol for QUANTITATIVE method verification D.1. Protocol for precision (intralaboratory reproducibility) determination ge of the natural contamination found ‘The contamination levels used shall be representative of the ran in the samples tested in the user laboratory. Preparation of the laboratory samples and test portions (see Figure D.1) minimum of 10 laboratory samples are collected. Each nd divided into 2 test portions. 2 (food) item eg tramisu, laboratory sample is homogenised ar al contamination is available (or contamination <10 cfu/g), ion with a selected strain. If artificial contamination is used, noculum suspension (used to prepare the initial suspension) using a ed) item with nai initial suspen enumerate, in parallel, the i non-selective medium Test samples infrequent used ia mcrbclopral examinations, In tha cate, the Laboratory samples recy used for homagentsation. Inia suspension Init suspension a Figure D.1— Preparation of samples for precision determination 3) Each initial suspension is then analysed following the method protocol, Test conditions used in the analysis of the test portion A and B shall be different in as many ways as possible within the scope of validation (e.g. technicians, batches of culture media and reagents, when relevant ~ ‘strains used for artificial contamination, apparatus and days - if the contamination of the laboratory sample can be shown to be sufficiently stable) See Figure D.2. 5 © 130 2017 ~ All rights reserved Scanned with CamScannerined results, calculat 4) From the obt: D.2 Protocol for estimated bias (eBias) determination ation of the microorganism to be used for concentration (see Figure D.3) Preparation for verification: 3 pre! method verification is performed to pr propriate conditions (medium, temperature, tim Prepare a culture of the microorga incubation). Follow the procedures specified in ISO 11133. Check the purity of the strain. ce al DDprmeetrumare © 10ideton 10a Exered the ae ied C=) () ae Figure D.3 — Example of the preliminary determination of the inoculum level 53 © 150 2017 - all rights reserved Scanned with CamScannerVerification: 1) Repeat the culture taking into account the concentration previous! mined, For this study, ‘cfu/ml is required. the inoculation of the initial suspension with L0® cfu/ml, 10% cfu/ml and 1 Based on the results of the previous enuineration test, appropriate dilutions are made covering the assumed range of LO: cfu/ml to 10¢ cfu/ml (see Figure D.4). 1:10 diduton eg Lmlin® | © dituent UO u New ovemight culture 10 dilaton 10 dilution 10 "dilution Expected 5108 cfu/ml Expected 5+105 cfu/ml Expected 5«10° cfu/ Figure D.4 — Example of the preparation of the inoculum: 3) Inoculate 1 ml of each dilution into duplicate initial suspensions to give concentrations of: 110° cfu/ml; 10% cfu/ml: 10° cfu/ml; 10° cfu/ml; 10° cf/ml; 10° cfu/ml (see Figure D.5) aa 10 dluten eg Lestin ml i iiaeat | | \ Newovernight culture 1D dion 10! dhanon, Expected 510% cu/mt Expected 5410! cfu/ml ‘a | Initial nuspension A val suspension B {Un10 eucion 610 (110 dikrton of 10 3) ee koe eee Figure D.5 — Example of the inoculation of the test portions © 1502017 ~ All rights reserved 54180/DIS 16140-3:2037(z) 4) Enumerate in duplic: Figure D.6) — the inoculated initial suspensions and an uninoculated (blank) test portion using the method to be verified, and — the inoculum suspension (used to prepare the initial suspension) using a non-s medium, iy x cfu/ml \ Inoculation in the initial suspension Method to be verified without (food) item eae Initial suspension A Initial suspension 8 Method to be verified with (food) item = = : 7 = oe? anon! ea eet vetiet) ou ete EUs see oo ieee tate eee ae QO Ore eae) Same method (plate, medium) Figure D.6 — Example of quantitative method verification using artificial contamination 5) Compare the results of the method to be verified with artificially contaminated (food) item to the resultsof the inoculum suspension tested with the same method. ©1SO 2017 - All rights reserved oF Scanned with CamScannerScanned with CamScanner