DETECTION AND ENUMERATION OF THE MOST PROBABLE

NUMBER (MPN) OF COLIFORM IN WATER

T.L.V. PEIRIS

GS/MSC/FOOD/3630/08

2008/2010

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4.0 Introduction

The MPN procedure involves a multiple tube fermentation technique where three or more decimal
dilutions of the sample are inoculated into tubes of broth medium and incubated at a specific
temperature and for a specific time. The method is progressive; i.e., first determining the presence
of coliforms in the tubes, then determining if these tubes also contain faecal coliforms, and then
confirming whether E. coli is present. Based on the number of tubes indicating the presence /
absence of the three groups of organisms, the most probable number present can be estimated
from a standard statistical MPN table.
This method has been shown to produce satisfactory results with naturally-contaminated and
artifically-contaminated water in sealed containers (including mineral and spring water) and
prepackaged ice.
The presence of coliforms, faecal coliforms and aerogenic E. coli in water may be determined by
means of the MPN procedure. Briefly, this method involves serially diluting out the target
organisms in the sample, in 5-replicate aliquots, to extinction .The probable level of the target
organisms is then statistically estimated from an MPN table.
Gas production is used as an indication of ability to ferment lactose from LST broth (presumptive
coliform test); gas production from BGLB broth is considered confirmation of coliform presence;
gas production at 45o C from EC broth is used as confirmation of faecal coliform presence; and
appearance of typical nucleated, dark-centred colonies with or without metallic sheen when
positive EC broths are streaked onto L-EMB agar are indicative of E. coli. The typical colonies on
L-EMB agar must be confirmed by further biochemical tests to prove the presence of E. coli.

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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The test methods described in this Practical provide guidelines for detection and enumeration of
bacteria of food and water origin. Then we can prevent food borne infections and diseases
occurring more commonly in Sri Lanka.
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.