Iso 16140-3

DRAFT INTERNATIONAL STANDARD
ISO/DIS 16140-3
ISO/TC 34/SC 9 Secretariat: AFNOR

Voting begins on: Voting terminates on:
2017-12-15 2018-03-09
Microbiology of the food chain — Method validation —

Part 3:
Protocol for the verification of reference and validated
alternative methods implemented in a single laboratory
Microbiologie de la chaîne alimentaire — Validation des méthodes —
Partie 3: Protocole pour la vérification de méthodes de référence et alternatives validées appliquées dans
un laboratoire
ICS: 07.100.30
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ISO/DIS 16140-3:2017(E)
Contents
Foreword ..........................................................................................................................................................................5
Introduction.....................................................................................................................................................................6
1 Scope ...............................................................................................................................................................................8
2 Normative references ...............................................................................................................................................8
3 Terms and definitions ..............................................................................................................................................8
4 Principle ..................................................................................................................................................................... 11
4.1 General ...................................................................................................................................................................... 11
4.2 Implementation verification ............................................................................................................................ 11
4.3 (Food) item verification ..................................................................................................................................... 12
4.4 Implementation verification and (food) item verification .................................................................. 12
4.5 Performance characteristics ............................................................................................................................ 15
5 Qualitative methods — Technical protocol for verification .................................................................... 15
5.1 Estimated LOD50 (eLOD50) determination .................................................................................................. 15
5.2 Experimental design ........................................................................................................................................... 15
5.3 Selection of (food) items.................................................................................................................................... 16
5.4 Artificial contamination ..................................................................................................................................... 16
5.4.1 Selection of strains ............................................................................................................................................... 16
5.4.2 Inoculation of the test portions ...................................................................................................................... 16
5.5 Results ....................................................................................................................................................................... 17
5.6 Acceptance criteria .............................................................................................................................................. 19
5.7 Root cause analysis .............................................................................................................................................. 19
6 Quantitative methods — Technical protocol for verification ............................................................ 20
6.1 Precision (intralaboratory reproducibility) determination ............................................................... 20
6.1.1 General ...................................................................................................................................................................... 20
6.1.2 Experimental design ........................................................................................................................................... 20
6.1.3 Selection of (food) item...................................................................................................................................... 22
6.1.4 Natural contamination ....................................................................................................................................... 22
6.1.5 Artificial contamination ..................................................................................................................................... 22
6.1.6 Results ....................................................................................................................................................................... 23
6.1.7 Acceptance criteria .............................................................................................................................................. 24
6.1.8 Root cause analysis .............................................................................................................................................. 25
6.2 Estimated bias determination ......................................................................................................................... 26
6.2.1 General ...................................................................................................................................................................... 26
6.2.2 Experimental design ........................................................................................................................................... 26
6.2.3 Selection of (food) items.................................................................................................................................... 26
6.2.4 Artificial contamination ..................................................................................................................................... 26
6.2.5 Results ....................................................................................................................................................................... 28
6.2.6 Acceptance criteria .............................................................................................................................................. 28
6.2.7 Root cause analysis .............................................................................................................................................. 28
7 Summary of acceptance criteria ................................................................................................................... 29
Annex A (informative) — Classification of (food) categories and suggested target combinations
for verification studies.............................................................................................................................................. 30
Annex B (normative) —Requirements and guidance on how to choose challenging (food) item(s)
for (food) item verification ..................................................................................................................................... 47
B.1 General ......................................................................................................................................................................
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B.2 Matrix effects to consider Single user..................................................................................................................................
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B.2.1 Microbial characteristics ................................................................................................................................... 47

B.2.2 Physical and chemical characteristics ......................................................................................................... 47
B.2.3 Food process characteristics ........................................................................................................................... 48
B.3 Selection of (food) items for verification .................................................................................................... 48
Annex C (informative) — Protocol for QUALITATIVE method verification ........................................... 49
Annex D (informative) — Protocol for QUANTITATIVE method verification ....................................... 52
D.1 Protocol for precision (intralaboratory reproducibility) determination...................................... 52
D.2 Protocol for estimated bias (eBias) determination ................................................................................ 53
Bibliography ................................................................................................................................................................. 56

4 © ISO 2017 – All rights reserved

ISO/DIS 16140-3:2017(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national
standards bodies (ISO member bodies). The work of preparing International Standards is normally
carried out through ISO technical committees. Each member body interested in a subject for which a
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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.
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 ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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 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 www.iso.org/patents).
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 ISO 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: www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology (Working Group WG 3, Method validation).
A list of all parts of the ISO 16140 series can be found on the ISO website.


ISO/DIS 16140-3:2017(E)
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: Protocol 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) method validation
 Part 5: Protocol for factorial interlaboratory validation for non-proprietary methods
 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[2], 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 ISO 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.
NOTE 1 Standardized reference methods (with and without published validation data) only require verification
before implementation in the laboratory.
NOTE 2 In this part of ISO 16140, the words ‘category’, ‘type’ and ‘item’ are sometimes combined with ‘food’ to
improve the readability of this document. However, the word ‘food’ is interchangeable with ‘feed’ and the other
areas of the food chain as mentioned in the Scope of ISO 16140-3.
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.

ISO/DIS 16140-3:2017(E)
The flow chart in 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 reference 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.
Figure 1 — Flow chart for application of the different parts of the ISO 16140 series
Part 6 of ISO 16140, is somewhat different from the other parts in the ISO 16140 series in that it 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 (e.g. serotyping of Salmonella) is included in part 6 of ISO 16140.


ISO/DIS 16140-3:2017(E)
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 (micro)organisms 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.
ISO 6887 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 16140-1:2016, Microbiology of the food chain — Method validation — Part 1: Vocabulary
ISO 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: availableISO
at http://www.electropedia.org/
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ISO/DIS 16140-3:2017(E)
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
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]
3.2
category
group of (food) types of the same origin
EXAMPLE Heat-processed milk and dairy products.
Note 1 to entry: The (food) categories are listed in Annex A of this document.
[SOURCE: ISO 16140-1:2016, 2.11, modified]
3.3
estimated bias (eBias)
determination of the bias based on the experimental design described in this document
Note 1 to entry: An accurate determination of the bias is not possible as the number of samples tested is small.
Therefore, the term estimated bias (eBias) is used in this document.
3.4
estimated LOD50 (eLOD50)
determination of the LOD50 (level of detection at 50 % probability of detection) based on the
experimental design described in this document
Note 1 to entry: An accurate determination of the LOD50 is not possible as the number of samples tested is small.
Therefore, the term estimated LOD50 (eLOD50) is used.
Note 2 to entry: LOD50 is defined in ISO 16140-1:2016, 2.35.
3.5
item
single specified food, feed, environmental, or primary production matrix
EXAMPLE Food category: heat-processed milk and dairy products; food type: pasteurised dairy product;
food item: crème brûlée.
[SOURCE: ISO 16140-1:2016, 2.34]
3.6
laboratory sample
sample prepared for sending to the laboratory and intended for inspection or testing
[SOURCE: ISO 6887-1:2017, 3.1]
3.7
matrix (product)
all the components of thetosample
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[SOURCE: ISO 16140-1:2016, 2.38]
3.8
reference material
material, sufficiently homogeneous and stable with respect to one or more specified properties, which
has been established to be fit for its intended use in a measurement process
Note 1 to entry: Properties can be quantitative or qualitative, e.g. identity of substances or species.
Note 2 to entry: Uses may include the calibration of a measurement system, assessment of a measurement
procedure, assigning values to other materials, and quality control.
[SOURCE: ISO Guide 30:2015, 2.1.1, modified]
3.9
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 a broader range (scope) of analytes, matrices and
concentrations than the scope that will be claimed by a user laboratory. The scope of laboratory application
is ≤ 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, 2.70]
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 entry: 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.
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.
3.13
type
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
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3.14
user laboratory
laboratory which implements a validated alternative method and/or a reference method
3.15
verification
demonstration that a validated method performs in the user’s hands according to the method’s
specifications determined in the validation study and is fit for its intended purpose
4 Principle
4.1 General
Before performing method verification, it is crucial to refer to the published validation data for the
method in order to confirm the scope of validation and to select the appropriate (food) items.
The verification is undertaken in two parts:
 implementation verification;
 (food) item verification.
User laboratories intending to verify methods that have published validation data for comparison (this
includes all validated alternative methods) shall perform both the implementation and the (food) item
verification. Some reference methods may not have published validation data. For those methods, the
user laboratory will only perform (food) item verification.
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;
 select one (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.

ISO/DIS 16140-3:2017(E)
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 Alternative method Reference method Alternative method
Implementation   Not applicable Not applicable a
verification
(Food) item    Not applicable a
verification
aNot applicable: the method shall first be validated to ISO 16140-2 or ISO 16140-5 (see Figure 1) after which validation data
are available.
Figure 2a to Figure 2d show the number of (food) items required for implementation verification and
(food) item verification under different circumstances.
Figure 2a — (Food) items required when verifying a reference method with published validation
dataLicensed
or a validated alternative
to Biomerieux Colombia SASmethod
/ Carolina for a “broad
Espitia range of foods” scope
(carolina.espitia@biomerieux.com)

ISO/DIS 16140-3:2017(E)
Figure 2b — (Food) items required when verifying a reference method with published validation
data or a validated alternative method for a “limited range of foods” scope
Figure 2c — Licensed
(Food)toitems required
Biomerieux Colombiawhen verifying
SAS / Carolina a (carolina.espitia@biomerieux.com)
Espitia reference method with no published
validation data
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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 Total minimum
validation validation data? verification verification number
Broad scope Yes 1 ≥5 6
(≥ 5 categories) No a 0 ≥5 5
Limited scope Yes 1 N N+1
(N categories) No a 0 N N
a Not applicable for validated alternative methods as these will always have validation data.
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 received by the user laboratory, and should, as far as
possible, be items withto components
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/ Carolina antimicrobial
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ISO/DIS 16140-3:2017(E)
probiotics, etc., as described in Annex B that may interfere with the detection of the target
microorganism.
If feed and pet food, environmental samples and primary production stage samples have been included
as additional (non-food) categories in the validation study, and if these are claimed by the user
laboratory, then one item from each of these categories claimed, shall also be included in the (food)
item verification.
4.5 Performance characteristics
Table 3 describes the required performance characteristics for method verification.
Table 3 — Required performance characteristics to be determined for verification

Method Performance characteristic Implementation verification (Food) item verification
Qualitative Estimated LOD50 (eLOD50)  
Quantitative Intralaboratory reproducibility (SIR) 
Estimated bias (eBias) 
NOTE Regarding intralaboratory reproducibility (SIR), the relationship with ISO/CD 19036 is further explained in 6.1.
5 Qualitative methods — Technical protocol for verification

5.1 Estimated LOD50 (eLOD50) determination
The estimated LOD50 (eLOD50) determination is required for both the implementation and the (food)
item verification.
 The user laboratory first follows the technical protocol outlined below, in its entirety to complete
the implementation verification, demonstrating the ability to conduct the validated method.
 The user laboratory then applies this same technical protocol 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, although
the number of colonies for confirmation may be reduced to one.
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.
 At a 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 of the test portions: 2 at high level, 2 at intermediate level and 2 at low level. Leave the
remaining 2 test portions uninoculated. Choose a (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 If the level of the

Licensed to culture used
Biomerieux for inoculation
Colombia is not
SAS / Carolina known,
Espitia additional test portions can be inoculated
with extra dilutions to ensure thatISO
theStore
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 Determine the level of contamination by plating the high-level inoculum, according to ISO 7218,
using a non-selective medium (e.g. Plate Count Agar); at the same time as the test portions are
inoculated. The contamination levels of the other inocula are calculated using the counts obtained
for the high-level inoculum and taking into account the dilution factors used.
 The test portions shall be tested according to the full protocol of the method being verified.
 The eLOD50 is calculated based on the positive and negative results (see 5.5).
5.3 Selection of (food) items
For the implementation verification, one (food) item is required. The (food) item selected shall be one
included in the validation study and preferably is tested by the user laboratory.
For the (food) item verification, the user laboratory shall test a minimum number 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) item being verified.
They can be from:
 culture collections;
 user laboratory collection;
 reference material (including commercial reference material, e.g. 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.
5.4.2 Inoculation of the test portions
Use the LOD50 data from the validation study of the method to determine the range of contamination
(between 1 to 10 times the LOD50) that will be used to inoculate the test portion. If the LOD50 is not
known (e.g. 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
Licenseddilutions to Colombia
to Biomerieux cover the targeted
SAS / Carolina range of contamination. This step is not required
Espitia (carolina.espitia@biomerieux.com)

ISO/DIS 16140-3:2017(E)
if the conditions of growth of the strain is well known by the user laboratory (e.g. stability after storage
at 4 °C overnight).
If the user laboratory works with ready to use target strains with known levels (e.g. reference material),
the steps described above are not required.
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:
 High inoculation level: this should be at a maximum of ten times the expected LOD50. When the
LOD50 is not known, a recommended level of 10 cfu/test portion is used.
 Intermediate inoculation level: from the high inoculation level, perform a 1:2 dilution to achieve the
intermediate level. The level of contamination for unknown LOD50 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 LOD50 should be 1 cfu/test portion.
NOTE The abbreviation of colony forming units is cfu.
To determine the inoculum level, enumerate the high-level inoculum according to ISO 7218 (using a
non-selective medium [e.g. Plate Count Agar]); 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 a Salmonella method.

 Based on the LOD50 (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 prepared and assumed to
contain 5×108 cfu/ml based on the preliminary enumeration.
 As the actual count of the new overnight culture 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:
 1 ml and 0,5 ml of 10-7 dilution (to cover unforeseen lower concentration);
 1 ml and 0,5 ml of 10-7 dilution, diluted 1:2;
 1 ml and 0,5 ml of 10-8 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 calculation.
 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 ml of dilution 10-7), 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
dilution 10-8) is 1,8 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
eLOD50.
The blank level shall not produce positive results. If positive results are obtained, the experiments shall
be repeated for all levels. Single user licence only, copying and networking prohibited.

ISO/DIS 16140-3:2017(E)
The high-level inoculum (10×LOD50) shall produce only positive results. If negative results are obtained,
the experiments shall be repeated for all levels.
Table 4 — Estimation of eLOD50 based on the number of positive results per level of
contamination
High inoculation Intermediate Low inoculation Blank level eLOD50
level inoculation level level (cfu/test portion)
(targeted 10×LOD50 (targeted 5×LOD50 (targeted 1×LOD50
/test portion) /test portion) /test portion)
2/2 2/2 2/2 0/2 < 1×LLI a
2/2 2/2 1/2 0/2 = 1×LLI
2/2 2/2 0/2 0/2 = 2×LLI
2/2 1/2 2/2 0/2 = 2×LLI
2/2 1/2 1/2 0/2 = 3×LLI
2/2 1/2 0/2 0/2 = 4×LLI
2/2 0/2 2/2 0/2 Unreliable result b
2/2 0/2 1/2 0/2 = 5×LLI
2/2 0/2 0/2 0/2 = 8×LLI
NOTE The MPN calculator used can be found at: http://standards.iso.org/iso/7218.
a LLI = low level of inoculation.
b This combination of results is very unlikely to occur with the levels of contamination tested and therefore leads to an
unreliable result according to the MPN calculator. The test shall therefore be repeated.
Based on the actual inoculum level, determined as described in 5.4.2, Table 4 can be modified – see
Table 5. In this example a high-level contamination of 18 cfu/test portion is used with the
corresponding, intermediate level contamination of 9 cfu/test portion and low-level contamination of
1,8 cfu/test portion.
Table 5 — Example for the calculation of the eLOD50 using a known inoculum level
High inoculation Intermediate Low inoculation level Blank level eLOD50
level inoculation level (= 1,8 cfu/test portion) (cfu/test portion)
(= 18 cfu/test portion) (= 9 cfu/test portion)
2/2 2/2 2/2 0/2 < 1,8
2/2 2/2 1/2 0/2 1,8
2/2 2/2 0/2 0/2 3,6 (2 × 1,8)
2/2 1/2 2/2 0/2 3,6 (2 × 1,8)
2/2 1/2 1/2 0/2 5,4 (3 × 1,8)
2/2 1/2 0/2 0/2 7,2 (4 × 1,8)
2/2 0/2 2/2 0/2 Unreliable result a
2/2 0/2 1/2 0/2 9,0 (5 × 1,8)
2/2 0/2 0/2 0/2 14,4 (8 × 1,8)
a This combination of results is very unlikely
to occur with the levels of contamination tested and therefore leads to an
unreliable result according to the MPN calculator. The test shall therefore be repeated.


ISO/DIS 16140-3:2017(E)
5.6 Acceptance criteria
The eLOD50, determined according to Table 4, shall be compared to the LOD50 from the validation study.
The eLOD50 shall not be > 3×LOD50 observed in the validation study. If the LOD50 is not known from the
validation study data, e.g. reference methods with no published validation data, the eLOD 50 shall be
equal or lower than 3 cfu/test portion. The value 3 cfu/test portion is derived from an acceptable
theoretical LOD50 value of 1 cfu/test portion (plus a margin of 3 × higher).
NOTE Clause 7 provides a summary for the acceptance criteria.
EXAMPLE
 The LOD50 observed in the validation study can be expressed as cfu/ml or cfu/test portion. The validation
LOD50 will need to be expressed in cfu/test portion to be able to compare the result with the result of the
verification study.
 In the case where the LOD50 observed in the validation study is given as cfu/g or cfu/ml, then the LOD50
needs to be multiplied by the size of the test portion used. Therefore, an LOD 50 of 0,1 cfu/g or cfu/ml will
give an LOD50 of 2,5 when a 25 g or ml test portion is used.
 In the case where the LOD50 from the validation study is, for example, 2,5 cfu/test portion the maximum
acceptable value for the eLOD50 will be 7,5 cfu/test portion (maximum of 3×LOD50).
 Based on the example given in 5.4.2, the last three combinations in Table 5 are not acceptable as either
the eLOD50 is unreliable or too high (eLOD50 of 9,0 or 14,4 cfu/test portion).
5.7 Root cause analysis
When the result does not meet the acceptance criterion (if the eLOD50 is > 3×LOD50 observed in the
validation study), 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 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, etc.);
 (food) item specificity, e.g. 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 LOD50 of 1 cfu/ test portion (e.g. Campylobacter);
 etc.
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.

ISO/DIS 16140-3:2017(E)
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
the reproducibility is considered. As this is performed in a single laboratory, the reproducibility is
expressed as the intralaboratory reproducibility (SIR). The intralaboratory reproducibility (SIR)
determination is based on 4.2.1 ‘Reproducibility standard deviation derived from intralaboratory
experiments’ of ISO/CD 19036.
NOTE The determination of the intralaboratory reproducibility (SIR) in the scope of implementation
verification is only required for one (food) item. However, user laboratories implementing ISO 19036 are required
to determine the measurement uncertainty for other (food) items as well – see ISO/CD 19036 for further
information.
The (food) item used in the implementation verification is any (food) item relevant to the user
laboratory.
During the implementation verification, run 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 some samples due to
practical errors/mishaps during testing.
The contamination levels used shall be representative of the range of the natural contamination found
in 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 (e.g. 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).


ISO/DIS 16140-3:2017(E)
Figure 3 — Experimental protocol to estimate the standard deviation of the

(intralaboratory) reproducibility (SIR)
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,
ISO Store pipettes,
Order: etc.).
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ISO/DIS 16140-3:2017(E)
Test conditions 1 and 2 are considered to cause the 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. If the 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 (SIR) will be compared
to the interlaboratory reproducibility (SR) obtained from the interlaboratory study. The interlaboratory
reproducibility (SR) represents the technical uncertainty as defined by ISO/CD 19036 and this value is
therefore independent from the (food) item.
6.1.4 Natural contamination
Whenever possible, use naturally contaminated items. For the (food) item chosen, the individual test
portions evaluated shall have contamination levels representative of the range of the natural
contamination found in the laboratory samples analysed in the user laboratory.
If the expected level of natural contamination is less than 10 cfu/g in the test portion, artificial
contamination is used (see 6.1.5) to cover the range of use of the method.
6.1.5 Artificial contamination
6.1.5.1 Selection of the strain
They can be from:
 reference material (including commercial reference material e.g. freeze-dried strain with known
concentration).
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 onBiomerieux

Licensed to non-selective
Colombia media to determine
SAS / Carolina the concentration of the strain. It is
assumed that this level will be consistently achieved when the same culture conditions are used.

ISO/DIS 16140-3:2017(E)
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 (e.g. stability after storage at 4 °C
overnight).
If the user laboratory works with ready to use target strains with known levels (e.g. reference material),
the steps described above are not required.
EXAMPLE A user laboratory wants to verify the ISO 21528-2 Enterobacteriaceae enumeration method.
The validation of this method was performed using one (food) item within each of five (food) categories:
1. (Food) category: raw meat and ready to cook meat products (except poultry); (food) type: fresh meats
(unprocessed); (food) item: raw pork chop.
2. (Food) category: eggs and egg products (derivatives); (food) type: eggs (unprocessed); (food) item: shell
eggs.
3. (Food) category: pet food and animal feed; (food) type: animal origin ingredient; (food) item: meat and
bone meal.
4. (Food) category: heat processed milk and dairy products; (food) type: pasteurised milk-based products;
(food) item: pasteurised milk.
5. (Food) category: chocolate, bakery products and confectionary; (food) type: pastries; (food) item:
tiramisu.
For implementation verification, the food item ‘tiramisu’ [a (food) item tested in the validation study] was chosen
and E. coli was chosen as the strain for the artificial inoculation.
 An overnight E. coli culture gives approximately 109 cfu/ml after when enumerated on non-selective
media. This concentration will be used as a starting point for the dilutions to inoculate the test portion.
 Based on the range of contamination used in the validation study, tiramisu will be inoculated between
30 (1,5 log10) cfu/g to 30 000 (4,5 log10) cfu/g.
 A minimum of ten different (brands, lots) laboratory samples of tiramisu will be prepared and each
divided into two test portions - A and B (see Figure 3).
Both test portions, A and B, originating from the same laboratory sample (see Figure 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 initial suspensions.
 In order to do this, an overnight culture is prepared and assumed to contain 10 9 cfu/ml (based on results
of previous enumerations – see Annex D).
 To contaminate at the level of 30 cfu/g, 6 serial decimal dilutions of the overnight culture are prepared, to
reduce the initial level from 109 cfu/ml to 103 cfu/ml.
 The initial suspensions of the two (10 g) test portions per laboratory sample are prepared.
 0,3 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/g can be obtained with different
dilutions and/or inoculation volume.
6.1.6 Results
The intralaboratory reproducibility (SIR) is calculated, based on a minimum of 10 laboratory samples,

according to the formula given below.
1
𝑆𝐼𝑅 = √ ∑(𝑦𝑖𝐴 − 𝑦𝑖𝐵 )2
2𝑝
where
SIR is to
Licensed the intralaboratory
Biomerieux Colombia SASreproducibility;
/ Carolina Espitia (carolina.espitia@biomerieux.com)
i is the index of the
ISO Store laboratory
Order: OP-345435sample, i = 1 2019-02-01
/ Downloaded: to p (p ≥ 10);

ISO/DIS 16140-3:2017(E)
p is the number of samples;

𝑦𝑖𝐴 , yiB are the log-transformed data, in log10 (cfu/g) or log10 (cfu/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
results) cannot be used for the calculation of SIR. The criteria for low counts are given in ISO 7218.
6.1.7 Acceptance criteria
The intralaboratory reproducibility (SIR) of the verified method shall be ≤ the interlaboratory
reproducibility (SR) of the most homogeneous (food) item used in the validation study. The most
homogeneous (food) item is defined as the (food) item with the lowest SR mean value of the inoculation
levels.
EXAMPLE
 The user laboratory has evaluated 12 laboratory samples of tiramisu for the level of Enterobacteriaceae
(using ISO 21528-1) following the experimental design given in Figure 3. The following results (calculated
as cfu/g of the laboratory sample) in Table 6 were obtained.
Table 6 — Test results

Laboratory Expected Result A (xiA) Result B (xiB) Log result A Log result B
sample contamination level (cfu/g) (cfu/g) yiA = log10(xiA) yiB = log10(xiB)
number (cfu/g)
1 30 10 30 1 1,477121
2 300 110 182 2,041393 2,260071
3 300 410 620 2,612784 2,792392
4 600 640 330 2,80618 2,518514
5 600 690 570 2,838849 2,755875
6 600 780 640 2,892095 2,80618
7 600 620 1 300 2,792392 3,113943
8 600 870 1 500 2,939519 3,176091
9 6000 8 600 6 400 3,934498 3,80618
10 6000 16 000 5 000 4,20412 3,69897
11 6000 > 15 000 13 400 > 4,176091 4,127105
12 30 000 20 000 32 000 4,30103 4,50515
 The results of laboratory samples 1 and 11 cannot be used because one of the counts was either too high
(‘>’ result) or too low (below permitted counting range according to ISO 7218). Results of 10 laboratory
samples remain for the calculation.
 Based on the 10 remaining laboratory samples, the SIR can be calculated as shown in Table 7.
Table 7 — Calculation of SIR

Laboratory sample Log result A Log result B Absolute difference Squared difference
number yiA = log10(xiA) yiB = log10(xiB) |yiA – yiB| |yiA – yiB|2
1 1,000000 1,477121 Not used Not used

2 2,041393 2,260071 0,218678 0,04782
3 2,612784 2,792392 0,179608 0,032259
4 Licensed to Biomerieux2,80618
Colombia SAS / Carolina2,518514 0,287666
Espitia (carolina.espitia@biomerieux.com) 0,082752
5 ISO2,838849
Store Order: OP-345435 / Downloaded:
2,755875 2019-02-01 0,082974 0,006885

ISO/DIS 16140-3:2017(E)
Laboratory sample Log result A Log result B Absolute difference Squared difference
number yiA = log10(xiA) yiB = log10(xiB) |yiA – yiB| |yiA – yiB|2
6 2,892095 2,80618 0,085915 0,007381

7 2,792392 3,113943 0,321551 0,103396
8 2,939519 3,176091 0,236572 0,055966
9 3,934498 3,80618 0,128318 0,016466
10 4,20412 3,69897 0,50515 0,255177
11 > 4,176091 4,127105 Not used Not used
12 4,30103 4,50515 0,20412 0,041665
Sum 0,649766
Sum/(2×10) 0,032488
SIR = √(0,032488) 0,18
 The obtained SIR of 0,18 is compared to the results of the validation study (data taken over from
ISO 21528-2). Table 8 provides the SR values obtained from that validation study.
Table 8 — Summary of SR values from the validation study for ISO 21528-2
SR values from the validation study
(Food) item Low inoculation Intermediate High inoculation Mean value of
level inoculation level level three inoculation levels
Liquid pasteurized egg 0,32 0,50 0,48 0,43
Minced meat 0,28 0,36 0,57 0,40
(Animal) feed 0,18 0,17 0,20 0,18
Pasteurised milk 0,24 0,18 0,19 0,20
Tiramisu 0,22 0,28 0,13 0,21
 The design of the test is to not consider the effect of the (food) item. The SIR obtained is compared to the
SR of a homogenous (food) item. In this example, the homogenous (food) item was (animal) feed (mean of
the 3 inoculation levels was 0,18).
 The SIR found in the verification study (0,18) is compared to SR of 0,18 from the validation study.
 As the SIR of the verification study (0,18) is equal to 0,18; the conclusion is that the precision of the
method carried out in this user laboratory is acceptable.
6.1.8 Root cause analysis
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 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,

Licensed e.g. root
to Biomerieux Colombia SAScause analysis)
/ Carolina can be given in the study report to provide
an explanation of the findings. ISO Store Order: OP-345435 / Downloaded: 2019-02-01

ISO/DIS 16140-3:2017(E)
If a (food) item is not verified, it is recommended that the user laboratory contacts a relevant
6.2 Estimated bias determination
6.2.1 General
The 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
The 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 B for
each level).
 Run in parallel the method to be verified with the artificially contaminated (food) item and the
same method with the inoculum 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
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.
6.2.4 Artificial contamination
6.2.4.1 Selection of strains
They can be from:
 reference material (including commercial reference material, e.g. freeze-dried strain with known
concentration).
use a different strain for each of the (food) items to be tested.


ISO/DIS 16140-3:2017(E)
6.2.4.2 Inoculation of the test portions
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 each inoculum suspension on non-selective 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 (e.g. stability after storage
at 4 °C overnight).
If the user laboratory works with ready to use strains with known levels (e.g. reference material), the
steps described above are not required.
6.2.5.3 Preparation of the test portions
The inoculation levels shall be selected and justified based on the levels expected in routine analyses.
Three inoculum levels are required for this work. Choose the lower and upper levels to represent the
lower and upper levels a user laboratory would expect to find during routine use of the method.
Set up more than three inoculum levels (e.g. five or six) so that the user laboratory is more likely to
obtain the three levels required for comparison studies.
EXAMPLE A user laboratory has decided that it would usually expect to find between 10 2 and 106 cfu/g in
submitted samples. This study required inoculation of the initial suspension to the levels of: 10 1 cfu/ml, 103 cfu/ml
and 105 cfu/ml (as the initial suspension is a 10-fold dilution of the test portion, this is equivalent to 10 2 cfu/g,
104 cfu/g and 106 cfu/g of the test portion). It is assumed that the test portion is 10 g and the final volume of the
initial suspension is 100 ml.
 A fresh overnight culture of the required microorganism (see 6.1.5.1) was prepared and assumed through
previous measurement/experience to have 107 cfu/ml. Appropriate dilutions were made covering the
assumed range of 101 cfu/ml to 106 cfu/ml.
 One ml of each dilution of the culture was transferred into duplicate initial suspensions, giving assumed
concentrations in the duplicate initial suspensions of: 100 cfu/ml; 101 cfu/ml; 102 cfu/ml; 103 cfu/ml;
104 cfu/ml; 105 cfu/ml (this was equivalent to 10 1 cfu/g to 106 cfu/g in the test portions).
An uninoculated test portion was included.
 The inoculated initial suspensions were then each enumerated using the method to be verified.
 The dilutions used to inoculate the initial suspensions were also plated out on a non-selective medium to
allow the actual concentration of microorganisms to be determined.
 After incubation and counting of the inoculum culture it was determined that the overnight culture
actually contained 108 cfu/ml (i.e. 1 log10 > the assumed level). Therefore, the actual levels in the initial
suspensions were 101 to 107 cfu/ml, and the calculated levels in the test portions were 10 2 cfu/g to
108 cfu/g.
 The user laboratory was then able to select the results relating to 10 2 cfu/g; 104 cfu/g and 106 cfu/g of the
test portion (101 cfu/ml; 103 cfu/ml and 105 cfu/ml of the initial suspensions) and compare actual
counted results for the method being verified, with the counts, of the inoculum, on non-selective medium.

ISO/DIS 16140-3:2017(E)
6.2.5 Results
The results shall be expressed in log10 cfu/g or log10 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 log10 cfu/g.
EXAMPLE A user laboratory wants to verify the eBias of the method for the Enterobacteriaceae count by a
validated alternative method using the (food) item ‘boiled pasta’. The expected range of contamination is between
102 cfu/g and 104 cfu/g. The results of the tests are given in Table 9.
Table 9 — Test results

Results Mean result per batch Difference between
(log10 cfu/g) (log10 cfu/g) method to be verified
Method to be Method to be Method to be Method to be with artificially
verified with verified with verified with verified with contaminated (food)
artificially inoculum artificially inoculum item and the inoculum
contaminated suspension contaminated suspension suspension
(food) item [without (food) (food) item [without (food)
item] item]
Laboratory sample 1
(from batch 1), test 1,87 2,15
portion 1
2,06 2,17 -0,11
Laboratory sample 1
portion 2
Laboratory sample 2
portion 1
3,99 4,29 -0,30
Laboratory sample 2
portion 2
Laboratory sample 3
portion 1
3,68 3,62 0,06
Laboratory sample 3
portion 2
The results indicate that at each level of contamination the absolute difference between the two methods is less
than 0,5 log10, so 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
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ISO/DIS 16140-3:2017(E)
 identify analytical error in protocol application (e.g. no correct inoculation level, etc.);
 (food) item specificity e.g. very challenging (food) items that required higher dilution factor in the
initial suspension;
 etc.
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 when the eBias is > 0,5 log10 cfu/g.
If a (food) item is not verified, it is recommended that the user laboratory contacts a relevant
7 Summary of acceptance criteria

Table 10 summarises the acceptance criteria that are used for method verification.
Table 10 — Acceptance criteria in verification

Method Acceptance criteria
eLOD50 (when LOD50 available in the validation study)
Qualitative eLOD50 ≤ 3 × LOD50
eLOD50 (when no LOD50 available)
eLOD50 ≤ 3 cfu/test portion
SIR (for methods with validation data)
SIR ≤ SR of the most homogeneous (food) item used in the validation study
Quantitative (lowest SR mean value of the inoculation levels)
eBias
≤ 0,5 log10 cfu/g for all inoculation levels


ISO/DIS 16140-3:2017(E)
Annex A (informative) — Classification of (food) categories and suggested target

combinations for verification studies
Table A.1 outlines the classification of mainly foods intended to guide user laboratories in the selection
of (food) items in its corresponding (food) categories when performing method verification. The
intrinsic properties of foods such as levels of indigenous microbiota, fat content, pH, salt content, water
activity and the presence of antimicrobial compounds can have a substantial influence on the outcome
of a method. Therefore, the intrinsic properties of foods have been considered to the extent possible in
the classification of foods, but the wide variety of foods available makes this consideration difficult to
apply past the level of (food) type.


ISO/DIS 16140-3:2017(E)
Table A.1 — Classification of samples and their relevance for testing for various microorganisms
Shiga (Patho- Bacillus Clostridium Clostridium
Items Total Lactic Yeasts Entero- Esche- Coagulase Salmo L. mono- toxin- Crono- genic) cereus perfringens botulinum
Listeria Campylo- Vibrio
Categories Types (some viable Acid and bacteri- richia positive nella cyto- producing bacter Yersinia (vegetative (vegetative (vegetative
spp. bacter spp.
examples) count Bacteria moulds aceae coli staphylococci spp. genes E. coli spp. entero- cells or cells or cells or
(STEC) litica spores) spores) spores)
Raw milks Raw milk Y Y Y Y Y Y Y Y Y

and/or
fermented/ Raw fermented/ Y Y Y Y Y Y Y
acidified acidified, raw
milks (not milk yoghurts,
heat raw dairy-based
treated) drinks
Raw butters Y Y Y Y Y Y Y
Raw milk and Raw creams Y Y Y Y Y Y Y

dairy products Raw milk- Hard and semi- Y Y Y Y Y Y Y
based
hard cheeses
products,
(e.g. Comté,
with high
Beaufort)
fat content
and/or high Blue cheeses Y Y Y Y Y Y Y
background (Roquefort)
microbiota
Soft cheeses Y Y Y Y Y Y Y
(e.g. Brie,
Munster)


ISO/DIS 16140-3:2017(E)

spp. bacter spp.
Pasteurized Milk-based Y Y Y Y Y Y Y Y Y Y
dairy desserts, ice
products creams, drinks,
creams
Sterilized or UHT milks, Y

UHT dairy canned milks or
products creams
Fermented/ Y Y Y Y Y Y Y Y
acidified
pasteurized
milk, yoghurts,
dairy-based
products
Pasteurized Y Y Y Y Y Y Y Y
milks
Heat-processed Butters Y Y Y Y Y Y Y
milk and dairy
Pasteurized
products Creams Y Y Y Y Y Y Y
milk-based
products Hard and semi- Y Y Y Y Y Y
hard cheeses
(heat processed)
(e.g. Comté,
Emmental,
Gouda)
Blue cheeses Y Y Y Y Y
(Bleu de Bresse)
Soft cheeses (e.g. Y Y Y Y Y Y

Brie, Munster)
Dry Milk powders Y Y Y Y Y Y Y Y
Powder for Y Y Y Y Y Y Y Y
milk-based
desserts ISO Store Order: OP-345435 / Downloaded: 2019-02-01

ISO/DIS 16140-3:2017(E)

spp. bacter spp.
Carcasses, meat Y Y Y Y Y Y Y Y Y Y
cuts, Carpaccio's
Minced meat, Y Y Y Y Y Y Y Y Y Y
Fresh meats
meat
Raw meat and (unprocesse
preparations,
ready-to-cook d)
Carpaccio's
meat products
(except Carcasses, Y Y Y Y Y Y Y Y Y Y
poultry) swabs, rinsates
Ready-to- Frozen burger Y Y Y Y Y Y Y Y Y Y

cook patties,
(processed) marinated beef
shish-kabobs
Cooked Cooked ham, Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

meat pâté
products
Fermented Salami Y Y Y Y Y Y Y Y Y Y Y Y Y
or dried
meat
products
Ready-to-eat,
Raw cured Filet de sax, lard Y Y Y Y Y Y Y Y Y Y Y Y Y
ready-to-
(smoked)
reheat meat
(aw >0,92)
products
Raw cured Cobourg ham, Y Y Y Y Y Y Y Y
(smoked) dry cured ham
(aw <0,92)
Canned Corned beef Y Y Y

meat
(ambient
stable)


ISO/DIS 16140-3:2017(E)

spp. bacter spp.
Carcasses, Y Y Y Y Y Y Y Y
meats, cuts
Fresh meats Carcasses, Y Y Y Y Y Y Y Y

(unprocesse swabs, rinsates
Raw poultry d)
and ready-to- Minced meat, Y Y Y Y Y Y Y Y
cook poultry meat
products preparations
Ready-to- Seasoned Y Y Y Y Y Y Y Y
cook chicken breasts
products
(processed)
Cooked Cooked turkey Y Y Y Y Y Y Y Y Y Y Y Y Y

meat filet
products
Fermented Chicken sausage Y Y Y Y Y Y Y Y Y Y Y

or dried
meat
products
Raw cured Smoked turkey Y Y Y Y Y Y Y Y Y Y Y

Ready-to-eat,
(smoked) filet
ready-to-
(aw >0,92)
reheat meat
poultry Canned Canned poultry Y Y Y
products (ambient meat, canned
stable) duck pâté


ISO/DIS 16140-3:2017(E)

spp. bacter spp.
Eggs Shell eggs Y Y Y Y Y Y Y Y

(unprocesse
d)
Egg Egg yolk, egg Y Y Y Y Y Y Y Y Y Y Y Y

products white, whole
(heat liquid egg
processed)
with
Eggs and egg additives
products (salt or
(derivates) sugar >2 %)
Egg Egg yolk, egg Y Y Y Y Y Y Y Y Y Y Y Y

products white, whole
(heat liquid egg
processed)
without
additives
Dry Egg powder Y Y Y Y Y Y Y Y Y Y
Fish Fish Y Y Y Y Y Y Y Y Y Y
(unprocesse
d)
Shellfish Oyster, clam, Y Y Y Y Y Y Y Y Y Y

(unprocesse scallop, mussel
Raw and ready- d)
to-cook fish Crustaceans Shrimp, crab Y Y Y Y Y Y Y Y Y Y
and seafoods (unprocesse and crab meat,
(unprocessed) d) lobster
Ready-to- Frozen fish Y Y

cook fish sticks
and
seafoods
(processed) Licensed to Biomerieux Colombia SAS / Carolina Espitia (carolina.espitia@biomerieux.com)

ISO/DIS 16140-3:2017(E)

spp. bacter spp.
Cooked Shelled and Y Y Y Y Y Y Y Y Y Y Y Y Y

fishery shucked
products products of
cooked
crustaceans, fish
and seafood
terrines
Acidified Roll herring, Y Y Y Y Y Y Y Y Y Y

and anchovy
marinated
fishery
products
Smoked or Smoked fish Y Y Y Y Y Y Y Y Y Y Y Y

cured, and
other
Ready-to-eat, processed
ready-to- products
reheat fishery (aw >0,92)
products
Smoked or Smoked fish, Y Y Y Y Y Y Y Y Y Y Y
cured, and dried (salted)
other fish
processed
products
(aw <0,92)
Canned Canned fish, Y Y Y

(ambient canned crab
stable fish)


ISO/DIS 16140-3:2017(E)

spp. bacter spp.
Cut ready- Fruit mixes Y Y Y Y Y Y Y Y Y Y

to-eat fruits
Cut ready- Bagged pre-cut Y Y Y Y Y Y Y Y Y Y Y

to-eat leafy vegetables,
vegetables salads,
shredded carrot
Produce Potatoes, yams, Y Y Y Y Y Y Y Y

grown in or sweet potatoes,
in contact cassava, dahlia,
with the carrots,
ground cruciferous
vegetables
Sprouts Soy, fenugreek, Y Y Y Y Y Y Y Y Y Y Y

alfalfa, mung
Raw Freshly Y Y Y Y Y Y Y Y Y Y Y Y
Fresh produce fruit/vegeta squeezed
and fruits ble juices strawberry
(unpasteuri juice, smoothies,
zed) carrot juice
Leafy Basil, cilantro, Y Y Y Y Y Y Y Y Y Y

greens green onions,
lettuce and
parsley
Vegetables Crops Y Y Y Y Y Y Y Y Y Y
and fruits
(unprocesse
d) not
described
above


ISO/DIS 16140-3:2017(E)

spp. bacter spp.
Heat- Pasteurized Y Y Y Y Y Y Y
processed apple juice
fruit/vegeta
bles juices
Canned Canned Y Y Y
fruits and pineapples
vegetables
Processed (ambient
fruits and stable)
vegetables
Heat- Blanched Y Y Y Y Y Y Y Y Y Y
processed spinach, frozen
vegetables vegetables
and fruits blanched
Fermented/ Fermented Y Y Y Y Y Y Y Y Y Y
acidified cabbage, pickle
vegetables
Low and Syrups, Y Y Y Y Y

IMF fruits concentrates,
(aw < 0,85) jams, semi-dried
prunes
Seasonings Spices, herbs, Y Y Y Y Y

peppers
Dried cereals,
Nuts and Nuts, nut meats, Y Y Y Y Y Y Y
fruits, nuts,
seeds nut butters,
seeds and
seeds
vegetables
Dried fruits Freeze-dried Y Y Y Y Y Y
&vegetables vegetables
(aw < 0,60)
Dried Corn, oat, Y Y Y Y Y Y

cereals breakfast
cereals

ISO/DIS 16140-3:2017(E)

spp. bacter spp.
Flours Wheat, Y Y Y Y Y Y
buckwheat, oat
Probiotic Pre-blend, spray Y Y Y Y Y Y Y Y

ingredients dried, culture
powders
Non- Dehydrated Y Y Y Y Y Y Y Y
probiotic milk,
ingredients dehydrated
yoghurt,
dehydrated
berries
Non- Whey-based Y Y Y Y Y Y Y Y
probiotic (dairy), soy-
infant based
formula (vegetables)
fortification
Infant formula formulation
and infant
Probiotic Whey-based Y Y Y Y Y Y Y Y
cereals
infant (dairy), soy-
formula based
(vegetables)
fortification
formulation
Non- Infant cereals Y Y Y Y Y Y Y Y

probiotic
infant
cereals
Probiotic Probiotic infant Y Y Y Y Y Y

infant cereals
cereals


ISO/DIS 16140-3:2017(E)

spp. bacter spp.
Pastries Bakery products Y Y Y Y Y Y Y Y Y Y

with custard,
confectionaries
Dry Cake mixes Y Y Y Y Y Y Y

powdered
Low Crackers, Y Y Y Y Y
Chocolate, moisture breads, cookies
bakery
products and Dry and Cake, pralines, Y Y Y Y Y
confectionary sugared low marzipan
moisture
(aw <0,85)
Dry and Biscuits, Y Y Y Y Y

sugared low chocolate,
moisture confectionary,
(aw <0,65) honey, sugar,
candy syrups


ISO/DIS 16140-3:2017(E)

spp. bacter spp.
Composite Refrigerated Y Y Y Y Y Y Y Y Y Y Y Y
foods with pasta salads,
substantial sandwiches,
raw chocolate
ingredients mousse,
(excluding bavarois
patisserie)
Composite Hot meals Y Y Y Y Y Y Y Y Y Y Y

processed
foods
(cooked)
Ready to Cooked chilled Y Y Y Y Y Y Y Y Y Y Y Y

(re)heat foods, boiled
food: rice or pasta,
refrigerated vol-au-vent in
Multi- vacuum
component
Ready to Frozen fries, Y Y Y Y Y Y Y Y Y Y Y
foods or meal
(re)heat pizza, stuffed
components
food: frozen croissants
Ready to Vol-au-vent in Y Y Y
(re)heat food: glass bottles
ambient
stable
(canned)
Ready to Dehydrated Y Y Y Y Y Y Y
(re)heat (instant) soups
food: dry
Mayon- Raw vegetable Y Y Y Y Y Y Y Y Y Y Y Y

naise-based salads with
delisalads dressing
(acid) with
raw
ingredients

ISO/DIS 16140-3:2017(E)

spp. bacter spp.
Mayon- Sandwich Y Y Y Y Y Y Y Y Y Y Y
naise-based spreads
delisalads
(acid) with
processed
ingredients
Ambient Ketchup, sauces, Y Y Y Y Y Y Y Y Y Y Y

stable acid dressings,
foods mayonnaises,
(pH <4,8) mustard
Animal Meat and bone Y Y Y Y Y Y Y Y

origin meal, chicken
ingredients and feather
meal, fish meal,
animal digest
Plant origin Corn meal, Y Y Y Y Y Y Y

ingredients soybean meal,
vegetables
Other Microbial products Y Y Y Y Y Y Y

ingredients such as yeast
extracts,
Pet food and probiotics
animal feed
Dry food Pellets, treats Y Y Y Y Y Y Y
(aw ≤0,7)
Wet food Fresh meat, Y Y Y Y Y Y Y Y Y Y

(aw >0,7) sausages,
croquettes
Canned Meat, fish Y
Animal Cereals, flours Y Y Y Y Y Y Y

feeds
(bovine, Licensed to Biomerieux Colombia SAS / Carolina Espitia (carolina.espitia@biomerieux.com)
ovine, pig) ISO Store Order: OP-345435 / Downloaded: 2019-02-01

ISO/DIS 16140-3:2017(E)

spp. bacter spp.

feeds
(poultry)

feeds (fish)
Equipment Swabs, dusts Y Y Y Y Y Y Y Y Y Y Y Y Y Y

or
production
Environmental environmen
samples (food t
or feed
production) Waters (Recycled) Y Y Y Y Y Y Y Y Y Y Y Y Y Y
used in the washing water,
manufacturi process water
ng process
Animal Swab samples Y Y Y Y

faeces (boot socks),
faeces rectal
Primary Environmen Dust samples, Y Y Y Y
production tal samples hygiene swabs,
samples (PPS) and non- water from
faeces drinkers, litters,
hatchery
samples


ISO/DIS 16140-3:2017(E)
NOTE 1 Minced meat preparations include portioned, cut, or minced meat (<1 % NaCl or spices) intended to
undergo a heat treatment before consumption presented as seasoned, marinated, coated, with herbs and spices, or
other ingredients are added to improve sensory properties or texture.
NOTE 2 Poultry meat preparations include marinated and spiced meat cuts, chicken fillets, and chicken wing,
i.e. intact structure either with or without skin.
NOTE 3 Seafoods include live bivalve molluscs and by analogy marine gastropods, echinoderms, and tunicates.
NOTE 4 Ready-to-eat (RTE) foods: Food intended by the producer or the manufacturer for direct human
consumption without the need for cooking or other processing effective to eliminate or reduce to an acceptable
level of microorganisms of concern.
NOTE 5 Ready-to-cook (RTC) foods: Food designed by the producer or the manufacturer as requiring cooking
or other processing effective to eliminate or reduce to an acceptable level microorganisms of concern.
NOTE 6 Ready-to-reheat (RTRH) foods: Food designed by the producer or the manufacturer as suitable for
direct human consumption without the need for cooking, but which may benefit in organoleptic quality from some
warming prior to consumption.
NOTE 7 For definitions of feeding stuff, refer to REGULATION (EC) No 79/373/EEC.
NOTE 8 Water mentioned in Table A.1 is water used in the manufacturing process or for PPS. In these cases,
filtration of samples is not needed.
NOTE 9 If the verification targets spore-formers, both vegetative cells and spores are included.


ISO/DIS 16140-3:2017(E)
Annex B (normative) —Requirements and guidance on how to choose challenging

(food) item(s) for (food) item verification
B.1 General
It is important to select (food) items that are representative of those encountered in the user
laboratory. This annex is specifically applicable to (food) item verification.
The (food) item can affect the outcome of an analysis. The composition of the food, its background
microbiota and other contaminants can interfere with the test method and invalidate the result. It is
therefore expected that the user laboratory will ensure that the method is fit-for-purpose for the (food)
items of interest. Even if a method is validated for a broad range of foods, not all (food) items have
undergone validation; therefore, the user laboratory shall demonstrate that the method is applicable to
the (food) items tested in the laboratory. As only one (food) item is required per (food) category, it is
then important 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 been sterilised, for example canned food, (food) items are naturally contaminated
by microorganisms, which can be categorised as:
 Technological microbiota such as microbial cultures and probiotics, e.g. fermented and cured foods,
probiotic food products inoculated with a level of microorganisms from 106 cfu/g to 109 cfu/g, etc.
 High background microbiota samples, e.g. poultry minced meat, faecal samples, raw milk, etc.
 Spoilage microorganisms: the presence of this native microbiota can influence the recovery and
growth of the target microorganism.
The effect of the background microbiota on the test method should be evaluated when the difference in
their counts and that of the target microorganism is greater than 3 log10. For example, high
Enterobacteriaceae counts may affect isolation of Salmonella spp. and the presence of Gram-positive
starter cultures may interfere with the recovery of Listeria monocytogenes.
B.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, e.g. high fat content, lecithin, thickener, nutrient content;
 pH, e.g. pH < 4-5 (beverages, sauces, etc.);
 oxidation reduction potential;
 water activity, e.g. aw < 0,85 (flour, low moisture foods);
 antimicrobial constituents and growth inhibitors, e.g. polyphenols, enzymes, molecular inhibitors;
 physical structure of the food, e.g. viscosity, solubility;

ISO/DIS 16140-3:2017(E)
 colour, e.g. food dyes.
B.2.3 Food process characteristics
The manufacturing process of the considered matrix can often have a treatment step (heating, high
pressure process, etc.) that may result in injuring microbial cells. This affects the culturability of the
cells and therefore affects the recovery of the microorganism of concern.
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.
When selecting a challenging (food) item per category, the user laboratory shall choose, among the
(food) items tested in its laboratory, an item which shows one of the challenging characteristics. It is
best to select a (food) item presenting a combination of the characteristics (e.g. pH + aw) as this
represents the worst-case scenario.
For a broad range of foods scope, a minimum of five (food) items selected from five (food) categories is
required (4.4). When possible, each of the five (food) items shall have a different characteristic or a
combination of characteristics in order to cover different cases. Table B.1 provides an example.
Table B.1 – Example of (food) items and its characteristics

Category Item Characteristic
1 1 pH
2 2 Viscosity
3 3 Fat content
4 4 High background microbiota and pH
5 5 Polyphenol
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 of competitive Physical characteristics Chemical compound
(micro)organisms
Technological High background pH aw Solubility/ Colour Vanillin, Enzyme Polyphenol Molecular
microbiota microbiota, viscosity salt, … inhibitor
spoilage
Cultural method x x x x x x x x x
Immuno-enzymatic x x x x x x x x x
Molecular test x x x x x x x x x x
Flow cytometry x x x x x x x
ATP x x x x x x x

ISO/DIS 16140-3:2017(E)
Annex C (informative) — Protocol for QUALITATIVE method verification

Preparation for verification: a preliminary enumeration of the microorganism that will be used for
the method verification is performed to provide an estimate of the concentration of the inoculum that
will be used.
1) Prepare a culture of the microorganism under appropriate conditions (medium, temperature,

time of incubation). Follow the procedures specified in ISO 11133.
2) Perform the enumeration of the culture on a non-selective medium to determine the cell
concentration. For decimal dilution and enumeration details, follow ISO 7218 and ISO 6887-1.
Check the purity of the strain.
Figure C.1 — Example of preliminary determination of the inoculum level
The result of the enumeration will be used as the starting point for the dilutions to inoculate the
test portions. In this example, the initial concentration was determined to be 5×108 cfu/ml
(see Figure C.1).
NOTE Overnight culture is intended to obtain microorganisms in stationary phase of growth. Modify the
culture conditions if the target microorganism requires longer incubation times.
Verification: Using the previously determined concentration (5×108 cfu/ml for this example), prepare
dilutions to cover target contamination levels (see Figure C.2). The dilutions selected for inoculation are
based on the validation LOD50 (assumed to be 2,5 cfu/test portion in this example).


ISO/DIS 16140-3:2017(E)
Figure C.2 — Example of the preparation of the inoculum
1) For each contamination level, perform 2 replicates. In theory, 3 contamination levels (high,
intermediate and low) and 1 blank 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 1 ml into the
initial suspension of the individual test portion.
Figure C.3 — Example of the inoculation of the test portions
In the example shown in Figure C.3, the expected high-level inoculum is prepared using the 10-6
dilution, in case the new overnight culture has a lower than expected count.
2) Perform the method to ISO
be verified.
Store Order: OP-345435 / Downloaded: 2019-02-01

ISO/DIS 16140-3:2017(E)
3) Record the positive and negative results.
4) Calculate the actual contamination based on the actual enumerated result (refer to ISO 7218).
In the example shown in Figure C.4, the new overnight culture count result is 1,8×10 7 cfu/ml
and not 5×108 cfu/ml. Therefore, test portions with positive results are most likely to appear at
the 10-6 and 10-7 dilutions instead of 10-7 and 10-8 dilutions.
Figure C.4 — Example of the enumeration of the actual inoculum level


ISO/DIS 16140-3:2017(E)
Annex D (informative) — Protocol for QUANTITATIVE method verification

D.1 Protocol for precision (intralaboratory reproducibility) determination
The contamination levels used shall be representative of the range of the natural contamination found
in the samples tested in the user laboratory.
Preparation of the laboratory samples and test portions (see Figure D.1):
1) For one (food) item e.g. tiramisu, a minimum of 10 laboratory samples are collected. Each
laboratory sample is homogenised and divided into 2 test portions.
2) If no (food) item with natural contamination is available (or contamination < 10 cfu/g),
inoculate the initial suspension with a selected strain. If artificial contamination is used,
enumerate, in parallel, the inoculum suspension (used to prepare the initial suspension) using a
non-selective medium.
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.


ISO/DIS 16140-3:2017(E)
Figure D.2 — Suggestions for variations for precision determination
4) From the obtained results, calculate the intralaboratory reproducibility SIR (see 6.1.6 and 6.1.7).
D.2 Protocol for estimated bias (eBias) determination
Preparation for verification: a preliminary enumeration of the microorganism to be used for the
method verification is performed to provide an estimate of the concentration (see Figure D.3).
Prepare a culture of the microorganism under appropriate conditions (medium, temperature, time of
incubation). Follow the procedures specified in ISO 11133. Check the purity of the strain.

Figure D.3 — Example of the preliminary determination of the inoculum
level

ISO/DIS 16140-3:2017(E)
Verification:
1) Repeat the culture taking into account the concentration previously determined. For this study,
the inoculation of the initial suspension with 101 cfu/ml, 103 cfu/ml and 105 cfu/ml is required.
2) Based on the results of the previous enumeration test, appropriate dilutions are made covering
the assumed range of 101 cfu/ml to 106 cfu/ml (see Figure D.4).
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:
100 cfu/ml; 101 cfu/ml; 102 cfu/ml; 103 cfu/ml; 104 cfu/ml; 105 cfu/ml (see Figure D.5).

Figure D.5 — Example of the inoculation of the test portions

ISO/DIS 16140-3:2017(E)
4) Enumerate in duplicate as follows (see 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-selective
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 results of the inoculum suspension tested with the same method.


ISO/DIS 16140-3:2017(E)
Bibliography
[1] ISO 16140 (all parts), Microbiology of the food chain — Method validation
[2] ISO 17468:2016, Microbiology of the food chain — Technical requirements and guidance on
establishment or revision of a standardized reference method
[3] ISO/CD 19036, Microbiology of the food chain — Estimation of measurement uncertainty for
quantitative determinations
[4] ISO 21528-1, Microbiology of the food chain — Horizontal method for the detection and
enumeration of Enterobacteriaceae — Part 1: Detection of Enterobacteriaceae
[5] ISO 21528-2, Microbiology of the food chain — Horizontal method for the detection and
enumeration of Enterobacteriaceae — Part 2: Colony-count technique


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DRAFT INTERNATIONAL STANDARD

ISO/TC 34/SC 9 Secretariat: AFNOR

Microbiology of the food chain — Method validation —

THIS DOCUMENT IS A DRAFT CIRCULATED

COPYRIGHT PROTECTED DOCUMENT

ii ﻿ © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 3

B.2.1 Microbial characteristics ................................................................................................................................... 47

Licensed to Biomerieux Colombia SAS / Carolina Espitia (carolina.espitia@biomerieux.com)

4 © ISO 2017 – All rights reserved

Licensed to Biomerieux Colombia SAS / Carolina Espitia (carolina.espitia@biomerieux.com)

© ISO 2017 – All rights reserved 5

 Part 4: Protocol for single-laboratory (in-house) method validation

 Part 5: Protocol for factorial interlaboratory validation for non-proprietary methods

6 © ISO 2017 – All rights reserved

Licensed to Biomerieux Colombia SAS / Carolina Espitia (carolina.espitia@biomerieux.com)

© ISO 2017 – All rights reserved 7

Microbiology of the food chain — Method validation — Part 3:

 products intended for human consumption,

 products intended for animal feeding,

 samples from the primary production stage.

3 Terms and definitions

8 © ISO 2017 – All rights reserved

— ISO Online browsing platform: available at http://www.iso.org/obp

[SOURCE: ISO 16140-1:2016, 2.9]

EXAMPLE Heat-processed milk and dairy products.

[SOURCE: ISO 16140-1:2016, 2.11, modified]

Note 2 to entry: LOD50 is defined in ISO 16140-1:2016, 2.35.

[SOURCE: ISO 16140-1:2016, 2.34]

[SOURCE: ISO 6887-1:2017, 3.1]

© ISO 2017 – All rights reserved 9

[SOURCE: ISO 16140-1:2016, 2.38]

[SOURCE: ISO Guide 30:2015, 2.1.1, modified]

[SOURCE: ISO 16140-1:2016, 2.70]

[SOURCE: ISO 6887-1:2017, 3.5, modified]

[SOURCE: ISO 6887-1:2017, 3.4, modified]

10 © ISO 2017 – All rights reserved

[SOURCE: ISO 16140-1:2016, 2.78, modified]

The verification is undertaken in two parts:

 (food) item verification.

4.2 Implementation verification

 reference methods; and

 validated alternative methods.

The user laboratory shall:

 review the published validation data for the method;

© ISO 2017 – All rights reserved 11

4.3 (Food) item verification

4.4 Implementation verification and (food) item verification

Table 1 — Implementation verification and (food) item verification

12 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 13

14 © ISO 2017 – All rights reserved

4.5 Performance characteristics

Table 3 describes the required performance characteristics for method verification.

Table 3 — Required performance characteristics to be determined for verification

5 Qualitative methods — Technical protocol for verification

5.2 Experimental design

NOTE If the level of the

ii © ISO 2017 – All rights reserved