DETECTION AND ENUMERATION OF THE MOST PROBABLE

NUMBER (MPN) OF COLIFORM IN WATER

K.S.L. PERERA

GS/MSC/FOOD/3631/08

2008/2010

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4.0 Introduction

MPN is a procedure to estimate the population density of viable microorganisms in a test sample.
It is based upon the application of the theory of probability to the numbers of observed positive
growth responses to a standard dilution series of sample inoculum placed into a set number of
culture media tubes. Positive growth response after incubation may be indicated by such
observations as gas production in fermentation tubes or visible turbidity in broth tubes, depending
upon the type of media employed. The sample should be diluted in such a manner that higher
dilutions of the sample will result in fewer positive culture tubes in the series. The number of
sample dilutions to be prepared is generally based on the expected population contained within
the sample. If particularly high microbial populations are expected, the sample must be diluted to
a range where the MPN can be obtained. Most reliable results occur when all tubes at the lower
dilution are positive and all tubes at the higher dilution are negative. Generally tenfold serial
dilutions are used in either a 3, 5 or 10 tube MPN series. When a higher number of tubes are
inoculated in the series, the confidence limits of the MPN are narrowed. For particularly high
microbial populations, the values obtained by MPN are generally not considered to be as precise
as population numbers derived from direct plating methods; however, it should be emphasized
that MPN values are only estimates while plate counts are direct counts of living organisms
expressed in cfu/ml. MPN values are, however, particularly useful when low concentrations of
organisms (<100/g) are encountered in such materials as milk, food, water and soil where
particulate matter of the matrix may interfere with obtaining accurate colony counts.

Most probable number (MPN) test

Most probable number (MPN) test tubes of lactose containing Macconkey broth are inoculated
with the samples of interest (usually water) measuring 10 ml, 1 ml, and 0.1 ml. During incubation,
coliform organisms produce gas. Depending upon which tubes from which water samples display
gas, an MPN table is consulted and a statistical range of the number of coliform bacteria is
determined. The MPN test is very easy to perform and interpret, but it does not determine the
exact number of bacteria as the standard plate count does.

4.1.1 Materials :

Beaker (1L), Pipettes (1ml), Pipettes (10 ml), Tubes containing Macconkey Broth (sterilized)

4.1.2 Procedure :

100 ml of tap water and pond water was measured. Two set of series consisting of 5 tubes filled
with 10.0 ml of double strength Macconkey broth and another ten tubes (two sets) filled with 10.0

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ml of single strength Macconkey broth, with inverted Durham tubes was prepared. (it was
ensured-that Durham tubes do not contain air bubbles.)

5 tubes with double strength Macconkey broth was inoculated with 10.0 ml aliquots of the
sample to be tested and another 5 tubes with single strength medium with 1.0 ml aliquots of the
sample and the other 5 tubes with 0.1 ml of the sample using sterile pipettes. They were then
mixed well and kept at 370C for 24 hours and observed the tubes at the end of 24 hours for acid
and gas production. Negative tubes were re incubated for additional 24 hours. Positive tubes were
recorded. These are considered as positive for presumptive coliforms and estimate the microbial
content using the MPN table.
"Note: For the first five tubes (tube with double strength Macconkey broth), inoculum can be
prepared through a serial dilution as direct inoculums may contain immeasurable load of
microorganisms. This is applicable if the sample is contaminated water but not samples like milk.

4.1.3 Results

Type of Sample Tap water Pond water

Quantity of water put up in each tube 10 ml 1 ml 0.1 ml 10 ml 1 ml 0.1 ml
Number of tube used 5 5 5 5 5 5
Number of tubes giving positive 0 0 0 5 2 2
reaction

4.1.4 Conclusion

According to the MPN Table tap water has not been contaminated by coliforms and pond water
has been contaminated by micro organism. MPN of coliforms 100 ml of the pond water 95.

MPN Table - for Coliforms / E.coli /100 ml water.

Quantity of water put up in each 10 ml 1 ml 0.1 ml Most probable number of

tube coliforms organisms in 100 ml
Number of tubes used 5 5 5 of the original water
0 0 0 0*
0 0 1 2
0 0 2 4

Number of tubes giving positive 5 2 0 50

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reaction 5 2 1 70
5 2 2 95 *
5 2 3 120
5 2 4 150
5 2 5 175

5 3 0 80
5 3 1 110
5 3 2 150
5 3 3 175
5 3 4 200

5 3 5 250
5 4 0 130
5 4 1 170
5 4 2 225
5 4 3 275

5 4 4 350
5 4 5 425
5 5 0 250
5 5 1 350
5 5 2 550

5 5 3 900
5 5 4 1 600
5 5 5 1 800

4.2 Conformation of coliform

Conformation of the results is necessary since positive presumptive tests may be the result of
organisms of non coliform origin that are not recognized as indicators of faecal pollution. The
confirmed test requires that selective and differential media such as eosin methylene blue (EMB)
streaked from a positive lactose broth tube obtained from the presumptive media. Eosin
methylene blue contains the dye methylene blue, which inhibits the growth of gram - positive
organisms. In the presence of an acid environment EMB forms a complex that precipitates out on
the coliform colonies producing dark centers and a green metallic sheen. This reaction is
characteristic for Eschirichia coli, the major indicator of faecal pollution.

4.2.1 Materials:

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EMB agar plates, Inoculating loops, lamps

4.2.2 Procedure :

Labeled EMB agar plates was inoculated by streaking Using lactose broth culture which gave
positive results in 24 hours and Incubated the plates in an inverted position for 24 hours at 37 0C
Examined the colony characters.

4.2.3 Results & Conclusion

Type of Sample Results Conclusion

Tape water no dark cerves and agreen metallic no faecal pollution in tape
sheen water
Pond Preducing dark centres and a green There is a facal pollution in
metallic sheen pond water

4.3 Completed test :

The completed test is the final analysis of the sample. It is used to examine the coliform colonies
that papered on the EMB plates used in the confirmatory test. An isolated colony is picked from
the confirmatory test plate and inoculated into a tube of lactose broth and streaked on a nutrient
agar slant to perform Gram stain. The test tubes that shows acid and gas in the lactose broth and
the presence of gram negative bacilli on microscopic examination after inoculation and incubation
are further confirmation of the presence of E.coli and they are indicative of a positive completed
test.

4.3.1 Result

When examined, slide which prepared from the EMB Culture plate (belongs to sample of pond
water) by a microscope it contained pale to dark red coloured rod shaped bacteria, when spore
stain was done it got clared that these are non sporing rods.

Therefore this sample is bacteriologically unsatisfactory andit is contaminated by faecal bacteria

(E-Coli)

4.4 Discussion

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In application of probability theory to the determination of MPN values, it should be kept in mind
that the following assumptions are generally considered to be accepted: (a) the organisms are
randomly and evenly distributed throughout the sample, (b) the organisms exist as single entities,
not as chains, pairs or clusters and they do not repel one another, (c) the proper growth medium,
temperature and incubation conditions have been selected to allow even a single viable cell in an
inoculum to produce detectable growth and (d) the population does not contain viable, sub-
lethally injured organisms that are incapable of growth in the culture medium used.

On occasions when more than three dilutions of a sample are used in a decimal series of dilutions
of a 3-tube MPN determination; the following guidelines should be followed. Results from only
three consecutive dilutions are used to determine the MPN. If one or more dilutions have all tubes
positive, select the highest dilution (smallest sample quantity) with positive results in all tubes and
the next two higher dilutions, as shown in examples a and b below. When none of the dilutions
yield all tubes positive, select the three lowest dilutions for which the middle dilution contains the
positive result, as shown in example c below. If a positive result occurs in a higher unselected
dilution, add the number of positive tubes in this dilution to the results of the highest dilution of
the three selected, as shown in example d below. When all dilutions tested yield all tubes positive,
select the three highest dilutions.

Disadvantages of MPN Techniques

1. MPN procedure takes very long time for the confirmed test result.
2. In MPN the results are probability calculations and cannot be accurate.
3. MPN requires more glass wares and media.
4. False positive results are of common occurrence.

Advantages of MPN Techniques

1. Interpretation of the results requires minimal experience and training as results can be got by
simply observing for the presence of gas or no gas.
2. Water samples with high turbidity can be analyzed, since there is no apparent deleterious
effect.
3.Because of the dilutions used in the range of 1:0 or 1:100, toxic substances present in the
sample can be diluted out.
4. MPN technique is the effective method for analyzing samples such as muds, sludges, sediments
etc.

References :

01. Food and Agriculture organization of the united nations 1992 Mannal of food quality
control 14/4 microbiological analysis FAO, Rome I taly.
02. Sri Lanka standard 614 Part 2 (1983) specification for potable water (Part 2)
Bacteriological Requirements.

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