Lab 4:
Most Probable Number
Method (MPN)
Most Probable Number Method (MPN)
Objective
• What is the MPN method?
• How to determine the amount of
bacteria from the MPN method?
Most Probable Number Method (MPN)
 The most probable number (MPN) method is familiar to quality
control (QC) microbiologists as part of the microbial limits tests.
• Its usefulness goes far beyond this one test, however.
• The theory behind the MPN method is central to the commonly
used D-value determination by fraction-negative method, and a
variant of this method has been suggested for trending of
environmental monitoring data from the aseptic core.
• MPN can be adjusted to provide a sensitive method
to determine differences between two qualitative
microbiological methods. As such, it can be used as
a tool in validation of rapid microbiological methods
and for growth promotion testing of broth media.
Most Probable Number Method (MPN)
 The most probable number (MPN) is particularly useful for low
concentrations of organisms (<100/g), especially in milk and
water, and for those foods whose particulate matter may
interfere with accurate colony counts.
 Only viable organisms are enumerated by the MPN
determination.
 If, in the microbiologist's experience, the bacteria in
the prepared sample in question can be found attached
in chains that are not separated by the preparation and
dilution, the MPN should be judged as an estimate of
growth units (GUs) or colony-forming units (CFUs)
instead of individual bacteria.
Most Probable Number Method (MPN)
For simplicity, however, here we will speak of
these GUs or CFUs as individual bacteria.
The following assumptions are necessary to
support the MPN method.
The sample is prepared in such a way that the
bacteria are distributed randomly within it.
The bacteria are separate, not clustered
together, and they do not repel each other.
 The growth medium and conditions of incubation have been
chosen so that every inoculum that contains even one viable
organism will produce detectable growth.
 The essence of the MPN method is the dilution of a sample to
such a degree that inoculate will sometimes but not always
contain viable organisms.
 The "outcome", i.e., the numbers of inoculate producing
growth at each dilution, will imply an estimate of the
original, undiluted concentration of bacteria in the sample.
 In order to obtain estimates over a broad range of
possible concentrations, microbiologists use serial
dilutions, incubating several tubes (or plates, etc.) at
each dilution.
Confidence Intervals
 The 95 percent confidence intervals in the tables have the
following meaning.
 Before the tubes are inoculated, the chance is at least 95
percent that the confidence interval associated with the
eventual result will enclose the actual concentration.
Selecting Three Dilutions for Table Reference
 An MPN can be computed for any numbers of tubes at any
numbers of dilutions.
 MPN values based on 3 decimal dilutions, however, are very close
approximations to those based on 4 or more dilutions.
 When more than three dilutions are used in a decimal series of
dilutions, refer to the 3 dilution table according to the following
two cases, illustrated by the table of examples below (with 5
tubes at each dilution).
Inconclusive Tubes
 In special cases where tubes or plates cannot be
judged either positive or negative (e.g., plates
overgrown by competing microflora at low dilutions),
these tubes or plates should be excluded from the
results.
 The entire dilutions at or below those in which
exclusion occurs may be excluded.
 If it is not desired to exclude the remaining tubes at or
below the dilution of the excluded tubes, the results
will now have an unequal number of tubes at several
dilutions.
Table 1
Example
1.0 g
0.1 g
0.01 g
0.001 g
0.0001 g
Combination
of Positives
MPN/g
a
5
5
1
0
0
5-1-0
33
b
4
5
1
0
0
5-1-0
33
c
5
4
4
1
0
4-4-1
40
d
5
4
4
0
1
4-4-1
40
e
5
5
5
5
2
5-5-2
5400
f
0
0
1
0
0
0-0-1
0.20
g
4
4
1
1
0
4-2-2
4.7
Example: The following inoculated tubes give a positive
reading:
1. 5 tubes with 10 ml of 1:10 dilution of test material - all 5 are
positive
2.
5 tubes with 1 ml of 1:10 dilution of test material - 1 is
positive
3. 5 tubes with 1 ml of 1:100 dilution of test material –
none are positive
 The quantities in each of the five tubes of the three dilution
series represent 1, 0.1 and 0.01 g (ml), respectively of the test
material.
 According to Table I, a reading of 5-1-0 gives a value of 33 when
10, 1 and 0.1 g (ml) respectively are used. However, since only 1/10
of these amounts were actually used in the analysis, the value of 33
obtained from Table II must be multiplied by 10
giving 33 x 10 = 330 organisms per 100 g (ml) of test material
 If the results need to be expressed per g (ml), the
MPN value is 330 ÷ 100 = 3.3. When higher dilutions
are used, the same procedure is followed, but the
multiplier (dilution factor) is enlarged to relate the
amount of test material actually present to the values
given for 10, 1.0 and 0.1 g (ml) in Table I.
MPN/100 ml=No .microorganisms x dilution factor of
middle set of tubes (Table I)
Most Probable Number
Advantages
 Relatively simple and
sensitive
 Can count a specific
type in the presence of
others
 Can use large sample
volumes
Disadvantages
 Time consuming and
labor intensive
 Requires large
volumes of glassware
 Doesn’t give the “real”
value
 Doesn’t give isolated
colonies
SUMMARY
 The basic concept for the MPN method is to dilute the sample to
such a degree that inocula in the tubes will sometimes (but not
always) contain viable organisms.
 By replicates, and dilution series, this will result in a fairly accurate
estimate of the most probable number of cells in the sample.
 While this method is most commonly used in the personal
products, medical device, and pharmaceutical QC microbiology
labs for water testing or the compendial bioburden test, it has
significant potential for other applications.
 These possible applications of MPN include D-value
determination, environmental monitoring, media
growth promotion studies, and aspects of the
validation of rapid microbiological methods.
END OF LECTURE