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Jaoac 1089
Jaoac 1089
5, 2003 1089
Testing laboratories wishing to comply with the re- be used but certified reference materials for running as con-
quirements of ISO/IEC 17025:1999 need to estimate trols alongside tests are not generally available and where
uncertainty of measurement for their quantitative these are available, it will be unlikely that they will be matrix
methods. Many microbiological laboratories have matched ...” (2).
Table 1. Calculation of intermediate precision for Heterotrophic Plate Counts of clean watersa
a
Data provided by Palmerston North City Council (New Zealand).
95% confidence level, based on the Poisson distribution, are where S is the standard deviation, C is the colony count, and u
included in the publications described above. is the overdispersion factor, calculated from the slope of the
In simple equations, the uncertainty associated with a count line relating the variance-to-mean ratio to concentration. In
depends primarily on the total colony count, dilutions, and the the ISO/TR 13843 example, u = 0.088.
number of replicate plates. All contributions to uncertainty are At a 95% confidence interval, the count
therefore not necessarily included in the estimation. Various
publications have recognized that when samples are analyzed = C ± 2 ´ C + u 2C 2
in replicate, variability is greater than fully random (in the Pois-
son sense), i.e., overdispersion may be observed (6, 7). Pure
cultures of bacteria can be expected to follow a Poisson series, If C = 105, the 95% confidence interval
but mixed cultures may deviate from Poisson, especially when
sublethal cell damage has occurred (9). = 105 ± 2´ 105 + (0.00766 ´ 1052 )
ISO/TR 13843 includes a worked example in which the
overdispersion factor is estimated (6; Annex B). In this exam-
ple, results from 12 laboratories are used to demonstrate the = 105 ± 28, i.e., 77 – 133
calculations involved. Each laboratory analyzed a sample of
the same type, but of its own choice, and performed quadrupli- Other approaches are available for estimating u (7).
cate parallel counts on its homogenized sample suspensions. A laboratory may use the ISO/TR 13843 procedure to esti-
All laboratories used the same method. The results obtained mate the uncertainty or the confidence interval for each of its
were used to calculate the overdispersion factor by a number methods and sample types by undertaking a series of replicate
of statistical techniques including linear regression. determinations. ISO/TR 13843 suggests replicates be in ex-
The data from ISO/TR 13843 can be developed further and cess of quadruplicate for each sample and that more than 12
used as an example of the additional calculations needed to samples may be desirable for reliable results.
determine the confidence interval for microbiological counts Many laboratories lack the resources of staff numbers and
on a particular type of sample at a 95% confidence level using the time required to accumulate sufficient data for the
an overdispersion model. In this calculation: ISO/TR 13843 approach. They will, however, be conversant
with the concept of precision criterion, and a similar approach
for estimating uncertainty from duplicate data could therefore
S = C + u 2C 2 be more readily adopted.
FORSTER: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 1091
Table 2. Transformation of ISO/TR 13843 data into logarithms10 and estimation of confidence intervals
The EURACHEM/CITAC Approach is not meaningful to consider correction for bias intrinsic to
these methods (10).
In 1995, the 1st Ed. of the EURACHEM/CITAC publica- It may well be that the majority of quantitative microbio-
tion Quantifying Uncertainty in Analytical Measurement was logical methods can be considered to be empirical methods,
published; the 2nd Ed. was published in 2000. This protocol where results generated are dependent on the media used,
establishes general rules for the evaluation and expression of times and temperatures of incubation, and inclusion or exclu-
uncertainty in quantitative chemical analysis, based on the ap- sion of resuscitative steps in the methods. Variations in the re-
proach laid down in the ISO Guide to the Expression of Un- covery of organisms resulting from the above factors have
certainty in Measurement. In the evaluation of the measure- been well documented over the years (5, 9).
ment uncertainty of a method, the EURACHEM guide Each of the above steps specified in the EURACHEM
requires the analyst to look closely at all the possible sources guide for estimating uncertainty can be considered in turn.
of uncertainty within a method and states that “in practice, a Specification
preliminary study will quickly identify the most significant
sources of uncertainty” which will be the dominating influ- In this step, what is being measured is clearly defined. The
ences in the total uncertainty of the method. equation used to calculate the value of the measurand at the
Many of the following concepts and procedures in this end of the method process is a good starting point. In microbi-
EURACHEM guide apply equally well to microbiological ology, very simple equations are usually involved in the calcu-
testing: lation of colony-forming units (CFUs) or specific organisms
in a sample. These equations normally take into account the
(a) Specifying clearly what is being measured or specify-
average of duplicate results, the dilution used, and the volume
ing the measurand
of the inoculum.
(b) Identifying contributions to uncertainty in the method
Specification can also include an overview or flowchart of
concerned
the steps undertaken in the performance of the method. Ini-
(c) Estimating the size of each identified contribution to tially, a laboratory may wish to consider contributions to un-
uncertainty as a standard deviation certainty from the subsampling stage, which is normally a
(d) If necessary, combining the values obtained for uncer- characteristic of a test method.
tainties
Identification of Sources of Uncertainty
(e) Calculating the expanded uncertainty
An important point is made in the EURACHEM guide re- On the whole, general quantitative microbiological analy-
garding empirical methods. In such methods, the analytical re- ses are very straightforward, most being based on the same
sults obtained are dependent on the procedures used in the general principles, i.e., subsampling, dilution, plating, incuba-
analysis. The method accordingly defines the measurand or, tion, and counting (with, on occasion, confirmation of the
in other words, the “right” answer is not a property of the sam- identity of organisms).
ple or of the target organisms, but of the method. Where such a The EURACHEM guide recommends the use of “cause
method is in use within its defined field of application, the bias and effect” diagrams for identifying contributions to uncer-
associated with the method is defined as being zero. That is, it tainty. A good starting point in the construction of a cause and
1092 FORSTER: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003
Table 3. Fifteen replicate results for fecal coliforms (MPN) and estimation of the confidence intervala
a
Data supplied by Watercare Laboratory Services (Auckland, New Zealand).
effect diagram is the equation used to calculate the measurand where (yi1 – yi2) is the difference between individual duplicate
ì t 2 ü
1/ 2 It can be expected that all major contributions to uncer-
ïï å ( y i 1 - y i 2 ) ïï tainty are accounted for in calculating intermediate precision.
S = í i=1 ý On occasion, minor contributions to uncertainty may be quan-
ï 2t ï tified separately as standard deviations and, if relevant, com-
ïî ïþ bined with the intermediate precision value.
FORSTER: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 1093
Conclusions
S = (3 ´ 0.092033)/(48 – 12), S = 0.0876, 2S = 0.1752
2