DETECTION AND

ENUMERATION OF THE MOST

PROBABLE NUMBER (MPN)
OF COLIFORM BACTERIA IN A
SAMPLE OF WATER

GS/M.Sc./FOOD/3608/08

B.K.K.K.Jinadasa
Introduction

Coliforms are normally present on raw produce, and their presence does not necessarily signify
that pathogens are present or that the produce has come in contact with feces.

The coliform group includes species from the genera Citrobacter, Klebsiella, Enterobacter, and
E.coli. Other than E. coli, no foodborne outbreaks have been associated with coliforms.
Coliforms were historically used as "indicator microorganisms" to serve as a measure of fecal
contamination, and thus potentially of the presence of enteric pathogens. Although some
coliforms are found in the intestinal tract of man, most are found throughout the environment and
have little sanitary significance. The presence of indicator organisms such as E. coli and
coliforms can be an indication of faecal contamination arising from within the slaughter facility
or post slaughter, inadequate processing, post-processing contamination or a combination of any
or all of the above.

Coliform counts are inadequate to differentiate between faecal and non-faecal contamination. E.
coli is considered to be more closely associated with faecal contamination from warm-blooded
vertebrates than are other members of the coliforms. Hence, the presence of E. coli in food or
water became accepted as indicative of recent faecal contamination and the possible presence of
frank pathogens.

In an effort to indicate ‘quality’ or ‘safety’ of such things as dairy products, fruits, vegetables,
drinking water, composted manure, and treated sewage, microbiologists developed the positive
association of groups of bacteria to fecal contamination and dubbed this group of bacteria ''faecal
coliform" . The term "thermotolerant coliforms" has recently been used to describe these
organisms and is more appropriate. These general terms for a large and diverse class of bacteria
are useful and remain relevant in specific food, wastewater management, and water quality
applications. The ability to rapidly and accurately detect E. coli and coliform bacteria is
important to any food quality and safety program.

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Most Probable Number (MPN)

The most probable number (MPN) technique is an important technique in estimating microbial
populations in soils, waters, and agricultural products. Many soils are heterogeneous, therefore
exact cell numbers of an individual organism can be impossible to determine. The MPN
technique is used to estimate microbial population sizes in situations like this. The technique
does not rely on quantitative assessment of individual cells; instead it relies on specific
qualitative attributes of the microorganism being counted. The important aspect of MPN
methodology is the ability to estimate a microbial population size based on a process-related
attribute.

This test method is a statistical, multi-step assay consisting of presumptive, confirmed and
completed phases. In the assay, serial dilutions of a sample are inoculated into broth media.
Analysts score the number of gas positive (fermentation of lactose) tubes, from which the other 2
phases of the assay are performed and then uses the combinations of positive results to consult a
statistical tables to estimate the number of organisms present. Typically only the first 2 phases
are performed in coliform and fecal coliform analysis, while all 3 phases are done for E. coli.
The 3-tube MPN test is used for testing most foods. The 5-tube MPN is used for water,

4.1. Presumptive coliform test

The presumptive coliform test is used to detect and estimate coliforms population of a water
sample. In this test, a known volume of water is added to lactose fermentation tubes and
production of acid and gas from the fermentation of lactose is a positive test for coliform
bacteria. The lactose broth used in this test is selective for the isolation of coliforms. A pH
indicator such as bromocresol purple is added to lactose broth for the detection of acid. The
colour of pH indicator changes to yellow with the fermentation of acid from lactose.

Materials

Pond water sample

McConkey broth of double and single with Durham tubes (5 tubes from double strength and 10
from single strength)

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Procedure

10 ml of pond water was added into each of the double strength McConkey broth.

1 ml of pond water was added to each of the 5 tubes of single strength tubes.

0.1 ml of pond water was added to another set of single strength tubes.

These samples were incubated at 37ºC for 48 hours for acid and gas production.

Results

Double strength Single strength Single strength

Mcconkey 10ml 10ml 10ml 10ml 10ml 10ml 10ml 10ml 10ml
Broth

Pond Water 10ml 10ml 10ml 1.0ml 1.0ml 1.0ml 0.1ml 0.1ml 0.1ml
sample

Durham tubes √ √ √ √ √ √ √ √ √

Observation- *** *** *** ** ** ** * * *

Gas
Production

Discussion

Production of acid and gas after 48 hours incubation indicates a doubtful presumptive test for
coliform bacteria and suggests that the water sample is polluted.

Positive and doubtful tubes were retained to use in the confirmed test.

4.2. Confirmed test for coliform bacteria

This test is used to confirm the presence of coliforms in water samples showing positive or
doubtful presumptive test. In the confirmed test, the samples showing positive presumptive
lactose broth tubes are streaked onto a selective differential eosin-methylene blue (EMB)
medium for coliforms. EMB medium is selective in nature because of the presence of the dye

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methylene blue which inhibits the growth of Gram positive bacteria and allowing the growth of
Gram negative bacteria. EMB medium is differential in nature I that lactose fermenting bacteria
gives coloured colonies due to the formation of a complex in EMB that precipitates out onto the
coliform colonies while non-lactose fermenters produce colourless colonies on EMB agar.

4.2.1. Brilliant Green Bile Broth Test

Brilliant Green Bile Broth contains the bile salt which can inhibit the growth of coliforms other
than faecal inhabitant. Since E coli living in faecal matter they can tolerate bile salt and other
cannot withstand bile salt. Here they only producing gas and inverted derham tubes kept in the
medium to collect the gas.

Materials

Inoculating loops, Spirits lamps, 10ml Pipettes, Tubes containing Brilliant Green Bile broth
(Sterilized)

Procedure

Brilliant Green Bile broth containing (10ml) tubes were labeled and all tubes were inserted with
Durham tubes in invert position. Each tube was enumerated with one loopful of amount from
positive MacConkey tubes. The tubes were then incubated for 24 hour and the broth was
observed for the colony development and colour changes.

4.2.2. Eosin Methylene Blue Test

Eosin Methylene Blue (EMB) agar contains the dye methylene blue, which inhibits the growth of
gram positive organisms. In the present of an acid environment EMB agar forms a complex that
precipitates out on the coliform colonies producing dark centers and a green metallic sheen. This
reaction is characteristic for Eschiria coli, the major indicator of faecal contamination.

Materials

EMB agar plates, Inoculating loops, Spirits lamps

Procedure

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EMB agar plates were labeled. Using the positive 24 hour lactose broth culture, surface of EMB
agar plates were streaked. The plates were incubated in an inverted position for 24 hours at
370C. The colonies formed green metallic sheen were examined.

4.3. Completed test

The completed test is the final analysis of the sample. It is used to examine the coliform colonies
that appear on the EMB plates used tube in the confirmatory test. An isolated colony was 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. Following inoculation and incubation tubes showing
acid and gas in the lactose broth and the presence of gram negative bacilli on microscopic
examination are further confirmation on the presence of E. coli and they are indicative of a
positive completed test.

4.4. Results and Observations

The observations were recorded for the each tests and interpreted for the identification of
coliform type in the sample. be maintained at 37 ºC for this test.