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Reconsideration of the derivation of Most Probable Numbers, their standard

deviations, confidence bounds and rarity values

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 Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Reconsideration of the derivation of Most Probable

Numbers, their standard deviations, confidence bounds
and rarity values
B. Jarvis1, C. Wilrich2 and P.-T. Wilrich3
1 Ross Biosciences Ltd., Upton Bishop, Ross-on-Wye, UK
2 Bundesanstalt für Materialforschung und –prüfung, Berlin, Germany
3 Institut für Statistik und Ökonometrie, Freie Universität Berlin, Berlin, Germany

Keywords
confidence bounds, microbiological analyses,
most probable number, MPN, rarity value.
Correspondence
Basil Jarvis, Ross Biosciences Ltd, Upton
Bishop, Ross-on-Wye HR9 7UR, UK.
E-mail: basil.jarvis@btconnect.com
2010 ⁄ 0180: received 31 January 2010,
revised 3 June 2010 and accepted 4 June
2010
doi:10.1111/j.1365-2672.2010.04792.x
Abstract
Aims: The purpose of this work was to derive a simple Excel spreadsheet and a
set of standard tables of most probable number (MPN) values that can be
applied by users of International Standard Methods to obtain the same output
values for MPN, SD of the MPN, 95% confidence limits and test validity. With
respect to the latter, it is considered that the Blodgett concept of ‘rarity’ is
more valuable than the frequently used approach of improbability (vide de
Man).
Methods and Results: The paper describes the statistical procedures used in the
work and the reasons for introducing a new set of conceptual and practical
approaches to the determination of MPNs and their parameters. Examples of
MPNs derived using these procedures are provided. The Excel spreadsheet can
be downloaded from http://www.wiwiss.fu-berlin.de/institute/iso/mitarbeiter/
wilrich/index.html.
Conclusions: The application of the revised approach to the determination of
MPN parameters permits those who wish to use tabulated values, and those
who require access to a simple spreadsheet to determine values for nonstandard
test protocols, to obtain the same output values for any specific set of multiple
test results. The concept of ‘rarity’ is a more easily understood parameter to
describe test result combinations that are not statistically valid. Provision of
the SD of the log MPN value permits derivation of uncertainty parameters that
have not previously been possible.
Significance and Impact of the Study: A consistent approach for the derivation
of MPNs and their parameters is essential for coherence between International
Standard Methods. It is intended that future microbiology standard methods
will be based on the procedures described in this paper.

recording the number of tubes showing growth at each

Introduction
The most probable number (MPN) procedure is used
widely to estimate microbial densities in many matrices
including foods and water. The procedure, derived from
the original work of McCrady (1915), consists of adding
a volume of each of several serial dilutions of a sample to
a number of replicate tubes of culture medium and,
following incubation under appropriate conditions,
level of inoculum. The estimate of density is based on the
application of the theory of probability based on certain
assumptions. The first primary assumption is that the
inoculum contains a random distribution of microbial
cells. This implies that each dilution is thoroughly mixed
and clumps or aggregates of cells do not occur or, if they
do, that they are not disrupted during further dilution
stages – an assumption that may not always be correct.

ª 2010 The Authors

1660 Journal of Applied Microbiology 109, 1660–1667 ª 2010 The Society for Applied Microbiology
B. Jarvis et al.
Secondly, that each volume of inoculum containing at
least one viable organism will exhibit growth when incu-
bated in the culture medium. If these assumptions are
not met, then the MPN procedure may underestimate
true microbial cell density.
Over time, different approaches have been made to
derive MPN values. Seminal papers include those by
Halvorson and Ziegler (1933), Barkworth and Irwin
(1938), Haldane (1939), Finney (1947), Cochran (1950)
and de Man (1975, 1977, 1983). Although much early
work was concerned with testing replicate tubes at a
single dilution level, the concept of repeating the tests
with replicate tubes at multiple dilution levels soon
became normal practice. Haldane (1939) and Cochran
(1950) describe the statistical theory behind single and
multiple dilution tests and explain some of the problems
associated with the calculations of MPN values for
multiple tube tests. Cochran (1950) also stressed the need
to ensure that the level of inoculum should lie within
certain limits in order properly to cover the expected level
of organisms in the original sample and provide a means
of assessing the standard deviation of the log10 of the cell
density for different combinations of dilution ratios and
replicate tests at each level.
Since that time, several authors have published tables
of MPN values for various combinations of replicate
tubes and dilution levels. Pre-eminent are those of de
Man (1983) that provide confidence bounds for the MPN
estimates together with a measure of the improbability of
the estimate. In addition, various workers have developed
computer software to enable the calculation of MPN esti-
mates – these include Hurley and Roscoe (1983), Klee
(1993), Curiale (2000), Garthright and Blodgett (2003),
US FDA (2006) and La Budde (2008).
Garthright and Blodgett (2003) describe the FDA’s pre-
ferred MPN methods for standard tests (e.g. five tubes at
three dilution levels) and also for large and unusual com-
binations of tests. Such test systems may use 25 or more
replicate tests at each dilution level (Moruzzi et al. 2000)
or may use well trays with automatic pipetting for serial
dilution assays (Irwin et al. 2000; Walser 2000). One of
the benefits of the US FDA (2006) spreadsheet system is
that it can accommodate situations where one or more
tube in a series has been lost or where overgrowth makes
a tube reading unreliable. However, use of the US FDA
(2006) spreadsheet system results in determination of
confidence bounds that differ from those cited in de
Man’s and other standard tables, including the FDA
tables, thus risking confusion amongst practitioners. The
reason for these differences is that different approxima-
tions have been used to derive the confidence bounds.
Blodgett (personal communication) says that the FDA
spreadsheet approach uses the normal approximation
ª 2010 The Authors
Journal of Applied Microbiology 109, 1660–1667 ª 2010 The Society for Applied Microbiology
Derivation of MPNs and their parameters
described originally by Haldane (1939), whereas the tables
are based on a modification of the method devised by de
Man (1983). For a more detailed consideration of the dif-
ferent approaches that have been made to determination
of MPN confidence intervals, see Garthright and Blodgett
(2003).
Another important aspect of deriving MPN estimates is
that of improbable outcomes. Such outcomes may occur,
for instance, as a consequence of laboratory errors. Sup-
pose three sets of results based on five tubes at each of
three successive tenfold serial dilutions give outcomes of
0-0-5. The calculated estimate of this MPN is 9Æ05 per ml
if the inocula are 0Æ1, 0Æ01 and 0Æ001 ml of original sam-
ple. But the statistical likelihood for the outcome is only
0Æ000025. In de Man’s (1983) MPN tables, and also in
those of the US FDA (2006), an improbability index pro-
vides guidance to practitioners as to the acceptability or
otherwise of a set of results. However, Blodgett (2002)
suggested an alternative approach to the assessment of
improbability that he describes as the ‘rarity’ value
(Blodgett 2008). Improbability is the sum of the probabil-
ities of all possible outcomes as likely, or less likely, than
the actual outcome. In contrast, the ‘rarity’ value mea-
sures the probability of the actual outcome divided by the
probability of the most likely outcome. In this work, we
have developed a rarity score that is used to indicate
whether a particular outcome is likely or not.
With the plethora of publications on MPN methods, it
is pertinent to ask why we have considered it necessary to
‘reinvent the wheel’ with yet another publication. In
2008, a number of mistakes were identified in part of a
revised International Standard (Anon 2007) dealing inter
alia with MPN techniques. The statistics working group
(SWG) of ISO TC34 ⁄ SC9 was asked to recommend
amendments to this standard. In so doing, we reviewed
all international standards on food, dairy and water
microbiology and determined that of 15 published stan-
dards, only five standards (Anon 2003, 2004, 2005,
2006a,b) cross-refer to Anon (2007), or its predecessor,
whilst others use different sets of MPN tables and ⁄ or
refer the user to one of several different software systems
for estimation of MPN values. The SWG recommended
that all relevant ISO microbiological standards should be
revised to include reference to a revised edition of Anon
(2007), which would include both tables of MPNs for
standard combinations of tests and reference to a specific,
generally available software that could be used for any
combination of test systems. We evaluated the ‘free’ soft-
ware available and concluded that new approaches would
be desirable. We recommended that for an international
standard, it was essential that both the tabulated parame-
ters and those determined by use of a spreadsheet should
give the same results. We concluded, also, that it would
1661
Derivation of MPNs and their parameters
be sensible to replace the concept of improbability with
the ‘rarity’ approach of Blodgett (2002, 2008) and to
provide also an estimate of standard deviation that can
be used to derive the measurement uncertainty of MPN
values. This paper describes the statistical approach used
and provides examples of the outputs.
Methods
MPN estimations are essentially ‘presence or absence’
tests performed using serial dilutions to estimate the
density l [as colony forming units (CFU) per g or ml] of
a target micro-organism in a test matrix. Samples drawn
from the matrix are diluted to several different levels, and
at each dilution level several tubes of culture medium are
inoculated with a portion (typically 1 ml) of that dilution.
After incubation under defined conditions, the number of
tubes that show the presence of the micro-organism at
each separate inoculum level is counted. These counts are
the basis of the calculation of the MPN as an estimate of
the concentration l.
Figure 1 Screenshot of Excel spreadsheet for determination of Most Probable Number estimates and their parameters.
1662 Journal of Applied Microbiology 109, 1660–1667 ª 2010 The Society for Applied Microbiology
B. Jarvis et al.
We assume that the target micro-organisms are
randomly distributed within the matrix, that they are
separate, not clustered together, and that they do not
repel each other. The numbers of micro-organisms in
each quantum of inoculum are independent. We
assume also that every tube whose inoculum contains
at least one viable target micro-organism will show the
presence of the micro-organism after incubation. Under
these assumptions, it is reasonable to assume a Poisson
distribution of the number of micro-organisms in the
tubes.
Let k be the number of dilutions, di the relative
dilution level per tube (i.e. for a tenfold serial dilution
from 10)1 to 10)3, di = 0Æ1, 0Æ01 and 0Æ001, respec-
tively), wi the volume (or weight) of the inoculum at
dilution level i, ni the number of tubes and xi the
number of positive tubes (tubes that show the presence
of the micro-organism) at dilution i; i ¼ 1; 2; :::; k.
Hence, a serial dilution test with k dilutions and its
results can be described by the k quadruples (di, wi, -
ni, xi), as illustrated in Fig. 1.
ª 2010 The Authors
B. Jarvis et al. Derivation of MPNs and their parameters

log (f(x)) of a function has its maximum at the same value

Determination of the MPN
as the function f(x), we use the Loglikelihood function
At each dilution, i the number Y of micro-organisms in
the tubes follows the Poisson distribution with expecta- ln L
tion diwil. The probability of y ¼ 0; 1; 2; ::: micro- Xk    
ni
organisms in a tube is given by ¼ ln þ xi lnð1  expðdi wi lÞÞ  ðni  xi Þdi wi l
i¼1 xi

ðdi wi lÞy and calculate the MPN as the value l ^ at which the first
PðY ¼ yÞ ¼ expðdi wi lÞ; y ¼ 0; 1; 2; :::
y! derivative of the Loglikelihood function with respect to l,

Particularly, the probability of no micro-organism in a k  

@ ln L X xi di wi expðdi wi lÞ
tube is ¼  ðni  xi Þdi wi
@l i¼1
1  expðdi wi lÞ
PðY ¼ 0Þ ¼ expðdi wi lÞ
is 0:
and the probability of at least one micro-organism in a
Xk  
tube is xi di wi expðdi wi l ^Þ xi di wi ð1expðdi wi l
^ÞÞ
þ ni di wi
i¼1
1expðdi wi l ^Þ ^Þ
1expðdi wi l
pi ¼ PðY>0Þ ¼ 1  PðY ¼ 0Þ ¼ 1  expðdi wi lÞ: X k  
xi di wi
¼ ni di wi ¼0:
i¼1
1expðd ^Þ
i wi l
The number Xi of positive tubes at dilution step i follows
the binomial distribution with parameters pi and ni. The
probability of xi ¼ 0; 1; :::; ni positive tubes is given by An estimate V^arð^ lÞ of the variance Varð^ lÞ of l
^ is
obtained from the second derivative of the Loglikelihood
  function,
ni xi
PðXi ¼ xi Þ ¼ p ð1  pi Þðni xi Þ " #
xi i @ 2 ln L Xk
xi ðdi wi Þ2 expðdi wi lÞ
  ¼ ;
ni @l2 ð1  expðdi wi lÞÞ2
¼ ð1  expðdi wi lÞÞxi ðexpðdi wi lÞÞðni xi Þ : i¼1
xi
as

The probability of observing (x1, x2, ..., xk) positive tubes 1 1

lÞ ¼ 
V^arð^  ¼ " #:
2  Pk ðd Þ2
^Þ
at the k dilutions is @ ln L  x i i i expðdi wi l
w
@l2  l ¼ l
^
^ÞÞ2
ð1  expðdi wi l
i¼1
PðX1 ¼ x1 ; :::; Xk ¼ xk Þ
Yk  
ni
¼ ð1  expðdi wi lÞÞxi ðexpðdi wi lÞÞðni xi Þ : The standard deviation of the estimate l
^ is given by
i¼1 xi
pffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Varð^ lÞ:
Given the results (x1, x2, ..., xk) of the serial dilution test,
this is the likelihood function of l, If only negative test results have been observed, xi = 0 for
i = 1, ..., k, the MPN is l ^ ¼ 0; and if only positive test
L ¼ Lðl; di ; wi ; xi ; i ¼ 1; :::; kÞ
results have been observed, xi = ni for i = 1, ..., k, the
Yk  
ni MPN is infinity.
¼ ð1  expðdi wi lÞÞxi ðexpðdi wi lÞÞðni xi Þ :
i¼1 x i

A confidence interval for the concentration

The likelihood function L(l) gives, for each possible
concentration l, the probability of the result of the serial
dilution test that has been observed. As an estimated
MPN of the concentration l, we use the value of l that
maximizes the likelihood function. As the logarithm1
1
For convenience we use natural logarithms ln(x) = loge(x).
There are various ways to construct a confidence interval
for the concentration l. The calculation of an ‘exact’
1 ) a = 95% confidence interval [lL, lU] for the concen-
tration l is tedious. It is described in detail by de Man
(1983). Its limits additionally depend on the rule by
which the a = 5% improbable concentrations are divided
into those below lL and those above lU and hence,

ª 2010 The Authors

Journal of Applied Microbiology 109, 1660–1667 ª 2010 The Society for Applied Microbiology 1663
Derivation of MPNs and their parameters B. Jarvis et al.

different authors end up with different confidence inter- positive tubes we find MPN = l ^ ¼ 006 CFU ml)1. How-
vals. We propose to use an easy approximation. As a ever, the result x1 ¼ 0; x2 ¼ 5; x3 ¼ 10 strongly contra-
maximum likelihood estimator of ln l, the (natural) loga- dicts our expectation that, with increasing dilution levels,
rithm ln l
^ of l
^ follows an approximately normal distribu- the numbers of positive results should decrease. Hence,
tion with estimated variance this result violates our assumptions underlying the MPN
determination. As the MPN calculation works irrespective
^2ln l^ ¼ V^arðln l
r ^Þ ¼ V^arð^ l2 :
lÞ=^ of the unlikeliness of the result of the serial dilution test,
we need a measure of this unlikeliness to decide whether
Hence, the interval to trust the result of the test or not.
 The rarity index (r), introduced by Blodgett (2002,
^  2^
ln l ^ þ 2^
rln l^ ; ln l rln l^ 2008), is the ratio of two likelihood values:

is an approximate 95% confidence interval for ln l and Lð^lÞ

r¼ ;
 L0 ð^
lÞ
l rln l^ Þ; l
^  expð2^ ^  expð2^
rln l^ Þ
In the numerator, we have the likelihood
is an approximate 95% confidence interval for the X k   
ni
concentration l. lÞ ¼ exp
Lð^ ln þ xi lnð1  expðdi wi l
^ÞÞ
If only negative test results have been observed, xi = 0 for i¼1 xi
i = 1, ..., k, the lower confidence limit lL is 0, and the !
upper confidence limit l U is the value l for which the ðni  xi Þdi wi l
^
likelihood function Lðl U Þ ¼ 0  025; where 0Æ025 =
(1 ) 0Æ95) ⁄ 2 : of the result (x1, x2, ..., xk) of the serial dilution test, i.e.
Y
k the value that we find if we insert l ^ into the likelihood
expðdi wi l U Þni ¼ 0  025: function L(l).
i¼1 In the denominator, we have the maximum of the likeli-
hood Lð^lÞwith respect to (x1, ..., xk),
( !)
Xk   
max max ni
L0 ð^
lÞ ¼ lÞg ¼
fLð^ exp ln þ xi lnð1  expðdi wi l
^ÞÞ  ðni  xi Þdi wi l
^ ;
ðx1 ; :::; xk Þ ðx1 ; :::; xk Þ i¼1 xi

This gives i.e. the value of the likelihood if the result of the serial
dilution test was most likely under a concentration l
ln 0025 ln 40 equal to the estimate l ^ of the concentration; this maxi-
lU ¼  ¼ k :
Pk P mum is achieved if
di wi ni di wi ni
i¼1 i¼1

If only positive test results have been observed, xi = ni for
i = 1, ..., k, the upper confidence limit lU is infinity, and
the lower confidence limit lL is the value for which the
likelihood function L(lL) = 0Æ025:
Y
k
ð1  expðdi wi lL ÞÞni ¼ 0025:
i¼1
This is a nonlinear equation for lL.
The rarity index
If we perform a serial dilution test with k = 3 dilutions,
dilution factors d1 ¼ 1; d2 ¼ 01; d3 ¼ 001, inocula
volumes w1 = w2 = w3 and numbers of tubes
n1 = n2 = n3 = 10 and observe x1 ¼ 0; x2 ¼ 5; x3 ¼ 10
xi ¼ ½ðni þ 1Þð1  expðdi wi l
^Þ; i ¼ 1; :::; k
where [x] denotes the largest integer not larger than x.
The rarity index is a value between 0 and 1. It is 1 if
the result of the serial dilution test is most likely a
concentration equal to the estimated MPN. If it is in the
neighbourhood of 0, the result of the serial dilution test
is very unlikely for a concentration equal to the estimated
MPN. Following the approach of de Man (1975, 1983),
we use three categories of rarity:
Category 1: The MPN value would be very likely to
occur if its rarity value falls within the range 0Æ05–1Æ00
(0Æ05 £ r £ 1Æ00).
Category 2: The MPN value would be expected to occur
only rarely if its rarity value falls within the range 0Æ01–
0Æ05 (0Æ01 £ r < 0Æ05).

ª 2010 The Authors

1664 Journal of Applied Microbiology 109, 1660–1667 ª 2010 The Society for Applied Microbiology
B. Jarvis et al. Derivation of MPNs and their parameters

Category 3: The MPN value would be expected to occur
extremely rarely if its rarity value falls within the range 0–
0Æ01 (0 < r < 0Æ01).
If only negative results or only positive results have
been observed, the rarity index is r = 1 and hence, the
category is also 1.
Software
An Excel spreadsheet for estimation of the MPN and its
parameters has been developed that is freely available
from http://www.wiwiss.fu-berlin.de/institute/iso/mitarbe
iter/wilrich/index.html.
Results
Table 1 illustrates the MPN parameters for a three-tube
assay with tenfold dilutions. The MPN values, derived
using the procedures described by Arndt et al. (1981), are
essentially identical to those found in the tables of de
Man (1983) and on the BAM website (Garthright and
Blodgett 2003; US FDA, 2006). The data columns in the
table are as follows:
Columns 1–3 show the numbers of positive test results
for inoculum quanta of 1Æ0, 0Æ1 and 0Æ01 g or ml of sam-
ple, respectively.
Column 4 shows the derived MPN estimates referenced
to the primary level of inoculum (i.e. 1Æ0 g or ml)
rounded to two significant figures. Respectively, MPN
values for larger or smaller quantities of primary inocula
should be divided or multiplied by the additional factor
involved, e.g. if the series contains 10, 1 and 0Æ1 g inocu-
lum, the listed MPN value per g should be divided by 10;
if the series contains 0Æ01, 0Æ001 and 0Æ0001 g inoculum,
the MPN value per g should be multiplied by 100.
Columns 5 and 6 show the log MPN and the standard
deviation, respectively, of the log MPN. Thus, it is possi-
ble to derive an estimate of the expanded microbiological
uncertainty for the calculated MPN value and combine it
with uncertainties stemming from other sources.
Columns 7 and 8 provide approximate bounds of the
95% confidence interval for each MPN value.
Column 9 lists the calculated ‘rarity value’ for the MPN
result, based on the procedure of Blodgett (2008) that is
used to determine the acceptability category of potential
MPN results (Column 10).

Table 1 Most Probable Number (MPN) table for a 3 · 3 design (i.e. three sequential tenfold dilution levels and a reference quantum of 1Æ0 g) for
outcomes with rarity index category 1 and 2

Number of positive results for 95% confidence

inoculum volume (ml or g) MPN SD of limits
per ml or log10 log10 Rarity
1Æ00 0Æ10 0Æ01 per g MPN MPN Lower Upper index Category

0 0 0 0 NA* NA 0 1Æ1 1Æ000 1

0 1 0 0Æ30 )0Æ52 0Æ43 0Æ041 2Æ3 0Æ087 1
1 0 0 0Æ36 )0Æ45 0Æ44 0Æ048 2Æ7 1Æ000 1
1 0 1 0Æ72 )0Æ14 0Æ31 0Æ17 3Æ0 0Æ021 2
1 1 0 0Æ74 )0Æ13 0Æ31 0Æ18 3Æ1 0Æ211 1
1 2 0 1Æ1 0Æ056 0Æ26 0Æ35 3Æ7 0Æ021 2
2 0 0 0Æ92 )0Æ037 0Æ32 0Æ21 4Æ0 1Æ000 1
2 0 1 1Æ4 0Æ16 0Æ26 0Æ42 4Æ8 0Æ041 2
2 1 0 1Æ5 0Æ17 0Æ27 0Æ43 5Æ0 0Æ426 1
2 1 1 2Æ0 0Æ31 0Æ23 0Æ69 6Æ0 0Æ019 2
2 2 0 2Æ1 0Æ32 0Æ24 0Æ71 6Æ2 0Æ069 1
3 0 0 2Æ3 0Æ36 0Æ31 0Æ55 9Æ7 1Æ000 1
3 0 1 3Æ8 0Æ59 0Æ31 0Æ93 16 0Æ084 1
3 1 0 4Æ3 0Æ63 0Æ33 0,95 19 1Æ000 1
3 1 1 7Æ5 0Æ87 0Æ30 1Æ9 30 0Æ209 1
3 1 2 12 1Æ1 0Æ26 3Æ6 37 0Æ021 2
3 2 0 9Æ3 0Æ97 0Æ32 2Æ2 40 1Æ000 1
3 2 1 15 1Æ2 0Æ27 4Æ4 51 0Æ420 1
3 2 2 21 1Æ3 0Æ24 7Æ2 64 0Æ068 1
3 3 0 24 1Æ4 0Æ32 5Æ6 100 1Æ000 1
3 3 1 46 1Æ7 0Æ34 9Æ6 220 1Æ000 1
3 3 2 110 2Æ0 0Æ32 25 480 1Æ000 1
3 3 3 ¥ NA NA 36 ¥ 1Æ000 1

*NA, not available.

ª 2010 The Authors

Journal of Applied Microbiology 109, 1660–1667 ª 2010 The Society for Applied Microbiology 1665
Derivation of MPNs and their parameters B. Jarvis et al.

Table 2 Examples of Most Probable Number (MPN) estimates for large and unusual combinations of tests

95%
confidence
limits
Weight of MPN per log10 log10 SD Rarity Rarity
Inocula (ml) sample* (g) No. tubes No. positives ml or per g MPN MPN Lower Upper index category

1Æ0, 0Æ1, 0Æ01 1Æ0 20, 20, 20 20, 14, 3 13 1Æ1 0Æ11 7Æ6 21 0Æ794 1
1Æ0, 0Æ1, 0Æ01 1Æ0 50, 50, 50 50, 35, 7 13 1Æ1 0Æ071 9Æ0 17 0Æ806 1
1Æ0, 0Æ1, 0Æ01 2Æ0 50, 50, 50 50, 35, 7 6Æ3 0Æ80 0Æ071 4Æ5 8Æ7 0Æ806 1
1Æ0, 0Æ1, 0Æ01 2Æ0 50, 49, 49 50, 34, 7 6Æ2 0Æ79 0Æ071 4Æ5 8Æ6 0Æ746 1
10Æ0, 1Æ0, 0Æ1, 0Æ01 1Æ0 1, 10, 10, 10 1, 9, 4, 1 3Æ3 0Æ51 0Æ15 1Æ7 6Æ4 0Æ089 1
10Æ0, 1Æ0, 0Æ1, 0Æ01 1Æ0 1, 10, 10, 10 1, 4, 2, 1 0Æ80 )0Æ096 0Æ17 0Æ37 1Æ7 0Æ017 2
10Æ0, 1Æ0, 0Æ1, 0Æ01 1Æ0 1, 10, 10, 10 0, 5, 1, 0 0Æ33 )0Æ49 0Æ18 0Æ14 0Æ74 0Æ004 3
5Æ0, 1Æ0, 0Æ5, 0Æ1, 0Æ05 1Æ0 1, 5, 5, 5, 5 1, 5, 3, 1, 1 2Æ7 0Æ42 0Æ16 1Æ3 5Æ5 0Æ512 1

*Quantity of sample in 1 ml of the initial homogenate (wi).

In Table 1, and also in the output of the computer Acknowledgements

programme, we show that if results at all test levels are
negative, then the MPN is 0; this value is supplemented
by the 95% confidence interval (with the lower confi-
dence limit 0). Most published MPN tables give a value
<x where x is the MPN for the next set of results with
the same number of tubes and only one positive result.
For instance, if in the design (3 · 1Æ0, 3 · 0Æ1,
3 · 0Æ01 ml) all results are negative, we report
MPN = 0 with a 95% confidence interval [0, 1Æ1],
whereas for this design some tables (e.g. de Man 1983)
and computer programmes (e.g. Curiale 2000) show
MPN < 0Æ30, with no confidence limits. We believe that
our statement is much more informative: i.e. 0 is the
most probable concentration, but a concentration up to
1Æ1 is possible with 95% confidence. Similarly, if all test
results are positive the MPN is infinity (¥) with 95%
confidence interval [36, ¥], whereas de Man (1983),
Curiale (2000) and others give MPN > 110 with no
confidence limits.
Table 2 illustrates results for some different combina-
tions of dilution factor, inoculum level and number of
replicate tests undertaken. These values were derived
using the Excel spreadsheet version of the programme,
for which a partial screenshot is presented as Fig. 1.
Discussion
The procedure described here provides a means of
obtaining MPN estimates, and their parameters, for both
standard and nonstandard assay combinations using
either derived tables of values or a freely available spread-
sheet, both of which provide identical outputs for the
same inputs. The system is to be included in the revised
ISO 7218 Standard to which other international standards
will cross-refer.
The authors are grateful to their colleagues on the SWG
of ISO TC34 ⁄ SC9 and the anonymous reviewers for help-
ful comments and discussion.
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