Accred Qual Assur (2012) 17:597–601
DOI 10.1007/s00769-012-0933-z
PRACTITIONER’S REPORT
Alternative calculation of measurement uncertainty with global
approach in food microbiology
Emrah Torlak
Received: 7 July 2012 / Accepted: 5 October 2012 / Published online: 19 October 2012
Ó Springer-Verlag Berlin Heidelberg 2012
Abstract Calculation of measurement uncertainty is a
requirement for all laboratories accredited to ISO/IEC
17025 including those carrying out microbiological anal-
yses. Today, calculation of measurement uncertainty in
microbiological analyses using precision data according to
global approach principles is widely recognized by the
microbiologists due to difficulties in quantification of
individual uncertainty sources. In food microbiology, pre-
cision data obtained from different samples usually show
over-dispersion, and the use of over-dispersed data may
result in large variance. The current ISO standard on
measurement uncertainty in food microbiology proposes
the use of log-transformed precision data to overcome this
problem. This paper proposes an alternative procedure to
calculate the measurement uncertainty in food microbiol-
ogy using non-logarithmic precision data. The calculations
used in this procedure based on relative range of duplicate
analyses can be applied to intra-laboratory reproducibility
data obtained from microbiological analyses of which
duplicate results show relatively low variation.
Keywords Measurement uncertainty 
Food microbiology  Global approach  Precision
Introduction
Measurement uncertainty, one of the technical requirements
of ISO/IEC 17025 [1], provides quantitative estimates of the
level of confidence for test results and is therefore an essential
E. Torlak (&)
Department of Biology, Faculty of Science,
Necmettin Erbakan University, 42050 Konya, Turkey
e-mail: torlakemrah@yahoo.com
component of a quality system for food microbiology labo-
ratories. Measurement uncertainty is an important factor in
comparing the results with reference values or criteria given
in product standards, microbiological guidelines or legisla-
tive limits. It has also importance in comparing the results
obtained from different laboratories.
Calculation of measurement uncertainty has been com-
monplace in physical and chemical laboratories for many
years, and procedures for measurement uncertainty are well
established. But, it is only relatively recently that the subject
has been addressed by microbiologists [2]. In food micro-
biology, protocols and formulations given in the guidance
documents for measurement uncertainty are applicable to
enumeration methods using a colony-count technique. For
most probable number (MPN) methods, the theoretical
95 % confidence intervals given in McCrady’s tables can be
used as expanded uncertainty range if contributory com-
ponents can be shown to be under control [3–5].
The current ISO standard on measurement uncertainty in
food microbiology is based on the log-transformed repro-
ducibility data [5]. However, most of the microbiological
criteria for foods in the EU and Turkish regulations [6, 7]
have been provided non-logarithmically. Due to nature of
logarithm, ISO protocol yield uncertainties with asym-
metric confidence limits in natural (back-transformed)
values. Asymmetrical confidence limits cannot be expres-
sed as plus or minus half the confidence interval width
divided by the estimated value of the variable (e.g. ±10 %)
and can be only expressed as confidence interval.
In the field of food microbiology, unlike in other testing
fields (e.g. chemical analyses), expression of uncertainty in
test reports together with the results as logarithmic values
or an interval in natural values may cause difficulties in the
interpretation of results for customers and authorities. For
this reason, food microbiology laboratories seek an
123
598
alternative calculation protocol for measurement uncer-
tainty using non-logarithmic data. This contribution
describes a procedure for the estimation of measurement
uncertainty in food microbiology using non-logarithmic
precision data.
Different approaches to measurement uncertainty
Currently, there are two approaches to estimate uncer-
tainty. They are referred to as the bottom-up approach and
the top-down or global approach [3, 5, 8]. The bottom-up
approach of which the basic principles were defined in the
JCGM reference document entitled ‘‘Guide to the Expres-
sion of Uncertainty in Measurement’’ requires the
identification and estimation of each separate source of
uncertainty and their combination to arrive at an overall
estimate of the uncertainty [9, 10]. This approach may be
considered to be unsuitable for microbiological testing
methods, because they have many sources of uncertainty
and some of these sources of uncertainty (e.g. incubation
temperature fluctuations and media quality) are not effec-
tively quantifiable for any individual experiment [11]. For
such reasons, international consensus supports the use of
the global approach to evaluate microbiological measure-
ment uncertainty as opposed to calculation of the combined
contributions of individual components of uncertainty [3].
The bottom-up approach can always be used in calibration
laboratories, but in testing laboratories, it should only be
used in cases where there is insufficient data to establish a
reliable estimate of its long-term reproducibility.
Global approach
The global approach which is based upon a statistical
evaluation of the test results from samples that have
undergone the complete measurement procedure [3, 5, 10].
This approach has the advantage that most testing labora-
tories are already familiar with precision experiments and a
suitably devised reproducibility experiment will include
the effects of all of the major contributors to uncertainty.
The global approach has two major uncertainty con-
tributions: trueness and precision [8, 12]. Trueness is
expressed in terms of the bias, which is difference
between the mean test results and true value. However,
due to empirical nature of quantitative microbiological
methods, it is not possible determine a true value which is
required to determine bias. Results of microbial enumer-
ations are dependent on method-defined conditions such as
the strain and condition of the test organism, the tem-
perature, incubation period and culture media. Therefore,
in practice, only precision is taken into account for the
123
Accred Qual Assur (2012) 17:597–601
uncertainty estimation in food microbiology. Laboratories
can only assess their bias by reviewing of inter-laboratory
studies and proficiency trials results or testing certified
reference materials. Proficiency trials can also provide
useful confirmation of realistic uncertainty estimates
[4, 5].
Measurement uncertainty associated with the precision
can be realistically evaluated with reproducibility data
[13]. Estimates of reproducibility may be derived from
inter-laboratory studies and proficiency trials, which have
the benefit of providing estimates of laboratory bias, or
through the determination of intra-laboratory reproduc-
ibility. There are some drawbacks of using precision values
from inter-laboratory studies and proficiency trials, because
these precision values are generally obtained under limited
precisely defined combinations of matrix, strain of micro-
organism, contamination level, and background microflora.
Additionally, natural variations in samples and variations
from sub-sampling have not been taken into account since
the analyses are performed on certain amount (e.g. 25 g) of
homogenized samples. Thus, use of precision values from
inter-laboratory studies and proficiency trials causes an
under-estimation of the uncertainty. For these reasons,
laboratories should always give priority to intra-laboratory
reproducibility data for measurement uncertainty in food
microbiology although these data do not reflect laboratory
bias [5].
Intra-laboratory reproducibility
Precision is closeness of the test results obtained from the
same sample and can be evaluated under three different
conditions: repeatability, intra-laboratory reproducibility and
reproducibility. Repeatability condition refers to stable fac-
tors such as same laboratory, same equipment, same analyst,
and short intervals of time. Reproducibility condition refers
to different laboratories [14]. When the term reproducibility
is adopted under changed factors in a single laboratory, it is
named as intra-laboratory reproducibility (within lab repro-
ducibility, Rw). According to ISO 5725-3 [15], the intra-
laboratory reproducibility condition is achieved by changing
one or more of the factors from the repeatability conditions.
Among these factors, considerable change of time cannot be
applied to food microbiology due to its nature.
Intra-lab reproducibility, sometimes called as interme-
diate precision, can be satisfactorily evaluated for
quantitative analyses in food microbiology by duplicate
analyses performed within an acceptable time interval.
Duplicate samples are tested by different analysts to allow
for monitoring of the effect of different staff as well as the
variability in different portions of the same sample. In
addition to changing analysts, duplicate analyses should be
Accred Qual Assur (2012) 17:597–601
performed under conditions as different as possible and
should ideally include as many variables as may be
encountered within a laboratory, in terms of batches of
culture media and reagents, equipment and time of analy-
sis. Thus, results of duplicate analyses can realistically
reflect the intra-laboratory imprecision [3, 16]. Variation of
influence quantities, such as equipment for homogenization
of the initial suspension, position and elapsed time inside
the incubator, and batches of nutrient media and reagents,
can only be approximated by a random choice of variables.
Samples used in duplicate analyses should represent the
scope of the method. The routine sample composition of
laboratories and matrix heterogeneity caused by the het-
erogeneous distribution of microorganisms should be taken
into account for the selection of samples. Ideally, duplicate
analyses should be performed starting from the sub-sam-
pling stage with naturally contaminated samples.
Physiological conditions of microorganisms may vary
considerably for different samples depending on their
processing and storage conditions. Artificially contami-
nated samples may be insufficient to represent these
variations for certain types of food [5]. As well as opera-
tional variability, results of duplicate analyses starting from
sub-sampling stage represent unpredictable variation that is
due to distribution of microorganisms [17].
According to the technical requirements of ISO/IEC
17025, the precision of test results must be investigated by
quality control programs. Duplicate testing of a certain
number of samples is a part of internal quality program
performed by most of food testing laboratories. Therefore,
the use of data obtained from an internal quality control
program offers a practical solution for laboratories to
determine measurement uncertainty without the need for
additional data.
Standard deviation of intra-laboratory reproducibility
Analyses results may vary considerably for different sam-
ples depending on their contamination levels in food
microbiology. The use of data of duplicate analyses to
calculate reproducibility standard deviation over a wide
data range may give a large standard deviation value. To
overcome this difficulty, the current practice in food
microbiology is to transform the results logarithmically or
classify the results with respect to their contamination
levels calculating the reproducibility standard deviation for
each contamination level [5]. Alternatively, if relative
differences between results of duplicate analyses (Relative
ranges) are used instead of absolute differences, distribu-
tion of range of data can be decreased considerably. Thus,
obtained data are approximately normally distributed,
which allow the use of the average of relative ranges based
599
directly on the natural values for calculation of the relative
standard deviation of intra-laboratory reproducibility.
In colony-count technique, unpredictable variation
increases rapidly as the number of colonies on plate
decreases and global design is not suitable for low counts.
Enumeration results based on less than 10 counted colonies
should be excluded from calculations [18, 19].
The relative standard deviation of intra-laboratory
reproducibility (RSDRw) is calculated by [19–21]:
R
RSDRw ¼
d2
where R is the mean relative range and d2 is the conversion
factor. For duplicate counts, d2 is taken as 1.128 [22].
The mean relative range (R) for n duplicate determina-
tions is calculated by [19]:
P
Ri
R ¼
n
where Ri is the relative range.
Relative range (Ri) for per duplicate determination is
calculated by [19]:
j Ai  Bi j
Ri ¼
mi
based on the results of two analysts (Ai and Bi) and their
arithmetic mean (mi = (Ai ? Bi)/2).
Expanded uncertainty
The intra-laboratory reproducibility standard deviation can
be accepted as the combined standard uncertainty (u) for
microbiological analyses according to global approach
principles. An interval expected to include a large fraction
of the distribution of values reasonably attributable to the
measurand is covered by expanded uncertainty (U) which
is calculated as U = ku with an appropriate coverage factor
k [9]. Routinely, when 30 or more sets of duplicate anal-
yses are performed, a coverage factor of 2 is used to give
distribution limits (confidence interval) with an approxi-
mation of 95 % around the normally distributed mean
value [19].
Suppose that the relative intra-laboratory reproducibility
standard deviation (RSDRw) is calculated as 30 %; then
expanded uncertainty (U) is obtained as 60 %. If testing
result of a sample is 2000 cfu/g, then result with uncer-
tainty can be expressed as (2000 ± 1200) cfu/g in test
report.
An example of the uncertainty calculations for enu-
meration of Staphylococcus aureus in cheese by the
relative range-based protocol proposed in this paper and
the ISO protocol are given in Table 1. In order to compare
123
600 Accred Qual Assur (2012) 17:597–601

Table 1 Calculations of measurement uncertainty by relative range-based and ISO protocols using results of duplicate analyses
Sample Results R Logarithmic results
A (cfu/g) B (cfu/g) log10{A} log10{B} (log10{A} - log10{B})2

1 210 280 0.29 2.32 2.45 0.016

2 3100 2200 0.34 3.49 3.34 0.022
3 18000 24000 0.29 4.26 4.38 0.016
4 960 1300 0.30 2.98 3.11 0.017
5 16000 13000 0.21 4.20 4.11 0.008
6 1600 2100 0.27 3.20 3.32 0.014
7 650 310 0.71 2.81 2.49 0.103
8 460 580 0.23 2.66 2.76 0.010
9 6700 8100 0.19 3.83 3.91 0.007
10 190 110 0.53 2.28 2.04 0.056
11 540 790 0.38 2.73 2.90 0.027
12 1200 870 0.32 3.08 2.94 0.020
13 1300 1600 0.21 3.11 3.20 0.008
14 540 350 0.43 2.73 2.54 0.035
15 6600 4400 0.40 3.82 3.64 0.031
Relative range-based protocol ISO protocol
P 
R 0.34 ðlog10 fAg  log10 fBgÞ2 =2n 0.013
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 ffi
P
R=d2 ¼ RSDRw 0.30 ðlog10 fAg  log10 fBgÞ2 =2n ¼ SDRw 0.114

U (%) 60.0 U (log10) 0.23

Data were kindly provided by Konya Food Control Laboratory, Turkey
R, SDRw denote the relative range and standard deviation of intra-laboratory reproducibility, respectively
{A}, {B} denote the numerical values of the results A and B, respectively

the confidence intervals of relative range-based protocol
and the ISO protocol, some examples based on the uncer-
tainty values from Table 1 are given in Table 2. These
examples show that using the relative range-based protocol
instead of ISO protocol for uncertainty calculation in
quantitative food microbiology does not make considerable
difference in the confidence intervals. However, differ-
ences between confidence intervals obtained by two
protocol increase as the uncertainty values increase.
Moreover, expanded uncertainties may be calculated equal
or higher than 100 % by relative range-based protocol for
microbiological quantifications, especially for the ones
requiring additional confirmation steps and these uncer-
tainty values produces unacceptable confidence intervals
which overlap zero. Therefore, ISO protocol is more suit-
able for microbiological analyses which can yield results
with the high variation, due to its asymmetrical upper and
lower limits of the confidence interval (e.g. -45 %,
?70 %) in natural values. It can be proposed that the
microbiological data yielded uncertainty values up to 75 %

Table 2 Examples of confidence intervals obtained by relative range-based and ISO protocols
Results/(cfu/g) Lower/upper limits of confidence intervala
Relative range-based protocol (U = 60 %) ISO protocol (U(log10) = 0.23)
Linear Logarithmic cfu/g Logarithmic values cfu/g

200 2.30 80b 320c 2.07b 2.53c 120b 340c

2000 3.30 800 3200 3.07 3.53 1200 3400
20000 4.30 8000 32000 4.07 4.53 12000 34000
200000 5.30 80000 320000 5.07 5.53 120000 340000
a
Limit values in non-logarithmic form were rounded to two significant numbers
b
Lower limit
c
Upper limit

123
Accred Qual Assur (2012) 17:597–601
can be treated by relative range-based protocol. The ISO
protocol should be followed when the calculated uncer-
tainty value is higher than 75 %.
Conclusions
The calculation protocol described in this paper is based on
relative differences between results of duplicate analyses; it
is therefore not necessary to have logarithmic results or to
calculate the reproducibility standard deviations for dif-
ferent contamination levels. This alternative protocol may
be useful especially for small scale food laboratories of
which uncertainties for many microbiological analyses
may be calculated as lower than 75 %, due to their rela-
tively low operational variations. Thus, measurement
uncertainty for results of these microbiological analyses
can be expressed the same as chemical test results
expressed in test reports.
References
1. ISO/IEC 17025 (2005) General requirements for the competence
of testing and calibration laboratories. International Organization
for Standardization, Geneva
2. Torlak E (2008) Measurement uncertainty in testing for antimi-
crobial activity on textile materials. Accred Qual Assur 13:563–566
3. HPA QSOP 4 (2005) Uncertainty of measurement in testing.
National Standard Methods. Health Protection Agency, London
4. IANZ AS TG5 (2004) Uncertainty of measurement, precision and
limits of detection in chemical and microbiological testing lab-
oratories. International Accreditation New Zealand, Auckland
5. ISO/TS 19036/Amd 1 (2009) Microbiology of food and animal
feeding stuffs—guide on measurement of uncertainty for quantita-
tive determinations. International Organization for Standardization,
Geneva
6. EC Commission Regulation 2073/2005 (2005) Microbiological
criteria for foodstuffs. European Commission, Brussels
7. TFC Regulation (2011) Microbiological criteria for foodstuffs.
Turkish Food Codex, Ankara
601
8. EUROLAB Technical Report No. 1/2007 (2007) Measurement
uncertainty revisited: alternative approaches to uncertainty test
results and the uncertainty of evaluation. www.eurolab.org
9. EURACHEM (2000) Quantifying uncertainty in analytical mea-
surement. www.eurachem.org
10. BIPM, IEC, IFCC, ILAC, IUPAC, IUPAP, ISO, OIML (2008)
Evaluation of measurement data—guide for the expression of
uncertainty in measurement. JCGM 100, http://www.bipm.org/en/
publications/guides/gum.html
11. Corry JEL, Jarvis B, Passmore SM, Hedges AJ (2007) A critical
review of measurement uncertainty in the enumeration of food
microorganisms. Food Microbiol 24:230–253
12. Barwick VJ, Ellison SLR (2000) Protocol for uncertainty evalu-
ation from validation data. LGC/VAM/1998/088
13. ISO 21748 (2010) Guidance for the use of repeatability, reproduc-
ibility and trueness estimates in measurement uncertainty
estimation. International Organization for Standardization, Geneva
14. BIPM, IEC, IFCC, ILAC, IUPAC, IUPAP, ISO, OIML (2008)
International vocabulary of metrology—basic and general con-
cepts and associated terms (VIM) 3rd edn, JCGM 200;
http://www.bipm.org/vim
15. ISO 5725-3 (1994) Accuracy (trueness and precision) of mea-
surement methods and results—part 3: intermediate measures of
the precision of a standard measurement method. International
Organization for Standardization, Geneva
16. HPA QSOP 18 (2005) Recommended minimum internal quality
control in food microbiology testing laboratories. National
Standard Methods, Health Protection Agency, London
17. ISO 29201 (2012) Water quality—the variability of enumeration
methods measurement of microbiological enumeration methods.
International Organization for Standardization, Geneva
18. ISO 7218 (2007) Microbiology of food and animal feeding
stuffs—general requirements and guidance for microbiological
examinations. International Organization for Standardization,
Geneva
19. FDA Ora-Lab.5.9 (2003) Assuring quality of test results. Food
and Drug Administration, Silver Spring
20. ISO/TS 13530 (2009) Water quality—guidance on analytical
quality control for chemical and physicochemical water analysis.
International Organization for Standardization, Geneva
21. NORDTEST Report TR 569 (2011) Internal quality control
handbook for chemical laboratories. www.nordtest.info
22. ASTM MNL 7A (2002) Manual on presentation of data and
control chart analysis. American Society for Testing and Mate-
rials, West Conshohocken
123