The Most Probable Number Method and Its

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Microbiology Topics.

Scott Sutton ]
The Most Probable Number
Method and Its Uses in
Enumeration, Qualification,
and Validation
Scott Sutton

“Microbiology Topics” discusses various topics in INTRODUCTION


microbiology of practical use in validation and com- The “most probable number” (MPN) method is a useful,
pliance. We intend this column to be a useful if underutilized, tool for the microbiologist. It is part of
resource for daily work applications. the harmonized compendial chapter on bacterial enu-
Reader comments, questions, and suggestions are meration (1) and has been part of the “Microbial Limits
needed to help us fulfill our objective for this col- Test” chapter in the United States Pharmacopeia since the
umn. Please send your comments and suggestions chapter inception in USP XVIII (2). The test is a method
to column coordinator Scott Sutton at scott.sutton@ to estimate the concentration of viable microorganisms
microbiol.org or journal coordinating editor Susan in a sample by means of replicate liquid broth growth in
Haigney at shaigney@advanstar.com. ten-fold dilutions and is particularly useful with samples
that contain particulate material that interferes with
KEY POINTS plate count enumeration methods.
The following key points are discussed: The basic concept to the MPN method is similar to
• The most probable number (MPN) method is useful the fraction negative method of D-value determination.
for estimating quantitative bioburden if plating for Nutrient broth will support growth of organisms and
colony forming units is not advised. turn turbid. The basic pattern of growth vs. no-growth
• This method is described in United States Pharma- can provide information as it is a reflection of sampling
copeia chapter <61> and by the US Food and Drug error. For example, if one replicate tube of media receives
Administration in the Bacteriological Analytical Man- a dilution of the sample that contains a bacertial cell, the
ual. Details of the method are discussed. tube will turn turbid. Its neighbor, an “identical” replicate,
• The MPN method has direct application in quali- may not receive any bacteria in its sample due to pipetting
fication studies for media and for alternate (rapid) or sampling and would not turn turbid. This information
microbiological methods. It has also been suggested is particularly useful at low numbers of organisms. How-
as a consideration for an alternate method to trend ever, this accuracy can be greatly increased by diluting the
environmental monitoring studies. inoculum and then comparing the recoveries of all tubes
in the dilution series. This is the basis of the MPN method

[
For more Author ABOUT THE AUTHOR
information, Scott Sutton, Ph.D., is owner and operator of The Microbiology Network (www.microbiol.org),
go to which provides services to microbiology-related user’s groups. Dr. Sutton may be reached by e-mail at
gxpandjvt.com/bios scott.sutton@microbiol.org.

gxpandjv t.com JOURNAL OF VALIDATION T ECHNOLOGY [SUMMER 2010] 35


Microbiology Topics.

Table: MPN table for a three-replicate design from FDA’s Bacterial Analytical Manual.
Positive Tubes Positive Tubes
0.1 0.01 0.001 MPN 95% Confidence Range 0.1 0.01 0.001 MPN 95% Confidence Range
0 0 0 <3.0 0-9.5 2 2 0 21 4.5-42
0 0 1 3 0.15-9.6 2 2 1 28 8.7-94
0 1 0 3 0.15-11 2 2 2 35 8.7-94
0 1 1 6.1 1.2-18 2 3 0 29 8.7-94
0 2 0 6.2 1.2-18 2 3 1 36 8.7-94
0 3 0 9.4 3.6-38 3 0 0 23 4.6-94
1 0 0 3.6 0.17-18 3 0 1 38 8.7-110
1 0 1 7.2 1.3-18 3 0 2 64 17-180
1 0 2 11 3.6-38 3 1 0 43 9-180
1 1 0 7.4 1.3-20 3 1 1 75 17-200
1 1 1 11 3.6-38 3 1 2 120 37-420
1 2 0 11 3.6-42 3 1 3 160 40-420
1 2 1 15 4.5-42 3 2 0 93 18-420
1 3 0 16 4.5-42 3 2 1 150 37-420
2 0 0 9.2 1.4-38 3 2 2 210 40-430
2 0 1 14 3.6-42 3 2 3 290 90-1000
2 0 2 20 4.5-42 3 3 0 240 42-1000
2 1 0 15 3.7-42 3 3 1 460 90-2000
2 1 1 20 4.5-42 3 3 2 1100 180-4100
2 1 2 27 8.7-94 3 3 3 >1100 420-4000

(also known and multiple tube, dilution tube, or dilution dilution series (although varying numbers of replicates
tube methods). The method offers real opportunities as and different dilution series may also be used). In this
an enumeration tool. It can also be employed for semi- design, if all tubes showed growth, then the results will
quantitative estimation of growth-promotion capability of be noted as 333. If only one tube in each replicate shows
liquid media and in estimation of precision for alternate growth it would be denoted as 111. The pattern of growth
microbiological methods with a simple modification. is then read from the table to provide the most probable
number and 95% confidence interval. By this, the result
THE METHOD of 210 would reflect an MPN of 21, and a result of 322
In the compendial version of the MPN test, the sample would be interpreted as an MPN of 210.
to be tested is prepared in 10-fold dilution series, and Figures 1 and 2 show this in graphic depiction. As the
then 1mL samples of each dilution are inoculated into incubated tubes would be read 321, the MPN would be
triplicate broth culture tubes for incubation. As the dilu- recorded as 150.
tions increase, the possibility that the broth tubes will fail The MPN table normally only presents results for three
to be inoculated with any microorganism increases. At dilutions in sequence (e.g., 10-1, 10-2, 10-3), but the dilution
some point therefore, very few of the replicate tubes will series tested might have been from the 10-2 to 10-4 tubes
be inoculated with viable microorganisms. (see the FDA BAM discussion on how to select appro-
Following incubation, all tubes are examined for tur- priate tubes to read). The worker will need to take the
bidity and the pattern of growth in the tubes is scored dilution factors in the table and in the actual experiment
against a table of such values (3). The MPN table from the into account to derive the most probable number from
US Food and Drug Administration’s Bacterial Analytical this study. The results of this test should be expressed as
Manual (BAM) (http://www.fda.gov) is provided above. A “MPN” rather than CFU (colony forming unit) to reflect
typical design uses three replicates with a three-log10 unit the capabilities of the method.
36 JOURNAL OF VALIDATION T ECHNOLOGY [SUMMER 2010] iv thome.com
Scott Sutton, Coordinator.

Figure 1: Three-tube design for Figure 2: Three-tube design for


MPN (unincubated). MPN (incubated).

The method assumes a random distribution of micro- the count is approaching three. These control levels are in
organisms in the sample and an accurate dilution of the truth of little value despite their popularity in regulatory
sample through the dilution series. It also assumes that circles (6-8). One approach suggested to deal with this
the microorganisms are separate and do not affect each mismatch between regulatory expectations and plate
other (i.e., attract or repel). In addition, it must be assumed count capabilities has been to explore the possibility of
that every tube (or plate, etc.) whose inoculum has a single looking at a frequency distribution models to establish
viable organism will result in visible growth. control levels in these areas (9) or incident models (10).
Although the compendial version utilizes three replicates A recent publication by Sun et al. (11) pointed out the
and a ten-fold dilution series, there is no theoretical reason possibilities in using MPN methods for evaluation of clean
for these parameters. In fact, it is well known that the accu- room monitoring data. The basic idea is to use the fun-
racy of the method increases dramatically when increasing damental statistics as if only a single dilution were being
the number of replicates and decreasing the interval of the considered. In this approach, the application is not dis-
dilution series (five-fold or two-fold) (4, 5). The FDA BAM similar to fraction negative studies of biological indicators
website referenced provides an Excel spreadsheet to assist for sterilization studies. Preliminary studies presented by
in creating different MPN tables as needed. this group look promising, and this is an approach that
could be pursued with existent data for evaluation.
APPLICATIONS OF MPN IN
ENVIRONMENTAL MONITORING DATA MPN IN QUALIFICATION OF BROTH MEDIA
Environmental monitoring data is a problem for micro- The compendial chapters of the USP on microbial limits
biology. We are urged to “qualify” our control levels and tests and sterility tests (revised in 2009) both place great
our sample sites. However, we are using a technology emphasis on media growth promotion studies as a requi-
(plate count) that is exceedingly imprecise at numbers of site quality control activity. This has been reinforced by
CFU below 25. The aseptic core of a modern facility will the USP chapter <1117> (revised in 2010) (12) discussion
commonly yield counts of zero, with concern expressed if of the importance of media control in the lab. While
gxpandjv t.com JOURNAL OF VALIDATION T ECHNOLOGY [SUMMER 2010] 37
Microbiology Topics.

the methods at hand to compare bacterial growth on compendial bioburden test, it has significant potential
solid media are quantitative in nature (recovery within to the quality control microbiology lab. These possible
50% or within 70% by CFU), the tools described in the applications of MPN include environmental monitor-
compendia for broth growth promotion are qualitative at ing, media growth promotion studies and aspects of the
best. “Liquid media are suitable if clearly visible growth validation of rapid microbiological methods.
of the microorganisms comparable to that previously
obtained with a previously tested and approved batch REFERENCES
of medium occurs” (12). 1. USP, Chapter <61> “Microbiological Examination of Non-
Weenk (13) reviewed the MPN method in his extensive sterile Products: Microbial Enumeration Tests,” USP 32.
review of methods used to qualify media and is recom- vol. 1 pp 71-75, 2009.
mended for its detailed description of how to improve 2. USP, “Microbial Limit Tests,” USP XVIII, p. 846, 1970.
the precision of the method. The basic approach rec- 3. FDA, Bacterial Analytical Manual, Appendix 2 available on-
ommended is to approach the MPN method from the line at http://www.fda.gov/Food/ScienceResearch/Labo-
opposite direction than that of the bioburden MPN. In ratoryMethods/BacteriologicalAnalyticalManualBAM/
the bioburden test, we have a sample with an unknown ucm109656.htm.
bioburden and we are trying to deduce the most probable 4. Eisenhart, C. and W. Perry, “Statistical Methods and Control
number of cells. In the recommended growth promotion In Bacteriology,” Bacterial Rev. 7:57-137, 1943.
test for liquid media, we have two batches dispensed in 5. Cochran, W., “Estimation of Bacterial Densities by Means of
tubes. The “sample” is a known inoculum in a known the Most Probable Number,” Biometrics. 6:105-116, 1950.
dilution series. The inoculum dilutions are seeded into 6. Hussong, D. and RE Madsen, “Analysis of Environmental
the two media, and then incubated. If the media exhibit Microbiology Data From Cleanroom Samples,” Pharm Tech-
identical growth promoting properties, then the 95% con- nol Aseptic Proc:10-15, 2004.
fidence intervals of the two MPN determinations should 7. Farrington, JK., “Environmental Monitoring in Pharmaceuti-
overlap. In this manner, a (semi)-quantitative growth cal Manufacturing-A Product Risk Issue,” Amer Pharm Rev
promotion study may be performed for liquid media. 8(4):26-30, 2005.
8. Agalloco, J. and Akers, J., “The ‘Myth’ Called Sterility,” Pharm
MPN IN QUALIFICATION OF ALTERNATE Tech, Bioprocess Sterile Manufact:s44-s45, 2010.
(RAPID) MICROBIOLOGICAL METHODS 9. Caputo, RA and A. Huffman, “Environmental Monitoring:
It should be obvious to the reader that the previous discus- Data Trending Using a Frequency Model,” PDA J Pharm Sci
sion on the use of MPN in growth promotion studies has Tech. 58(5):254-260, 2004.
immediate application for determination of the relative 10. USP, Chapter <1116> “Microbiological Control and Monitor-
limit of detection for two microbiological methods, for ing Environments Used for the Manufacture of Healthcare
example a “traditional” method and an “alternate” method. Products,” Pharm Forum 33(3), 2007.
This is in fact referenced in USP chapter <1223> (14). 11. Sun, X et al., “The Expanded Application of Most Probable
The USP chapter recommends this approach even for Number to the Quantitative Evaluation of Extremely Low
quantitative methods. Although this might at first seem Microbial Count,” PDA J Pharm Sci Tech 60(2):124-134,
counter-intuitive, the MPN method (when used with a 2006.
dilution series) can actually be more accurate than plate 12. USP, Chapter <1117> “Microbiological Best Laboratory Prac-
counts at low numbers. The only modification that needs tices,” USP 33, Reissue, Second Supplement, pp. R-1100-R-
to be made is to ignore the counts and treat every plate or 1105, 2010.
membrane as a separate “tube”—the MPN method fits 13. Weenk, G.H., “Microbiological Assessment of Culture
right into the experimental design. Media: Comparison and Statistical Evaluation of Methods,”
Intl J Food Microbiol., 17:159-181, 1992.
SUMMARY 14. USP, Chapter <1223> “Validation of Alternative Microbio-
The basic concept for the MPN method is to dilute the logical Methods,” USP 32, vol. 1 pp730-3, 2009. JVT
sample to such a degree that inocula in the tubes will
sometimes (but not always) contain viable organisms. ARTICLE ACRONYM LISTING
By replicates, and dilution series, this will result in a fairly BAM Bacterial Analytical Manual
accurate estimate of the most probable number of cells in CFU Colony Forming Unit
the sample. While this method is most commonly used MPN Most Probable Number
in the pharmaceutical industry for water testing or the USP United States Pharmacopeia

38 JOURNAL OF VALIDATION T ECHNOLOGY [SUMMER 2010] iv thome.com

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