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1 Introduction

Level 2 laboratory activities refer to the condition where only the problem is guided and
given. Students are required to find the ways & means and provide the answers to the given
assignment using the group creativity and innovativeness. The activity will enable the
students to appreciate independent learning and prepare them for a much harder task of
open ended laboratory activities. In this partially open laboratory activity the students are
required to carry out tests to quantify the presence of E-Coli and Total Coliform in water

1.2 Objective

To make students understand and implement suitable method to prove the existing of E-Coli
and Total Coliform in water bodies.

1.3 Learning Outcome

At the end of the laboratory activities, students would be able to:

1. To identify E-Coli and Total Coliform.
2. To explain the procedure to conduct the E-Coli and Total Coliform test.
3. Analyse test data and present the solution in proper technical format.
4. Work in a group to undertake the task and produce the relevant technical

1.3 Theoretical Background

IDEXX Colilert reagent Is used around the world for the detection of coliform and E.Coli in
water . Colilert is used for the simultaneous detection , specific identification, and
confirmation of total coliform and E.Coli in water. Colilert based on the Defined Substrate
Technology . DST utilizer indicator nutrients which cause target microbes contained in the
sample and incubated in the DST reagent system to produce a colour change ( or another
signal , fluorescent ) both indicating and confirming their presence . The indicator nutrient is
cleaved by the target microbe with metabolize the nutrient and frees the indicator to express
a specific colour . Total coliform and E. coli are specifically and simultaneously detected and
identified the 1CFU/100ml of sample, in 24 hours or less, by inoculating the reagent with the
sample and incubating it .

2 Problem Statement

The presence of coliform bacteria in the water indicates that the water may have been
contaminated with sewage and is not safe to drink. Regulations require that coliform bacteria
be completely absent from 95% of the water samples. Each group has to conduct an
experiment to identify the amount of E-Coli and Total Coliform in different water samples.
The experiment result should include pictures and calculation to prove the E-Coli and Total
Coliform existence.

3.1 Apparatus
Sterile bottle
Colilert Reagent
Quantity Tray
Quantity Tray Sealer
Comparator sample
Rubber Glove

3.2 Procedures

1. Contents of one pack to a 100ml water sample was added to a sterile vessel.
2. The vessel was cap and shake until dissolved.
3. The reagent mixture was poured into a Quantity tray and sealed in an INDEXX
Quantity Tray Sealer.
4. Next , the sealed tray was placed in 35 incubator for 24 hours .
5. Then, the result was read according to the result interpretation table .
6. If no yellow colour is observed, the test is negative.
7. It the sample has yellow colour equal to or greater than the comparator , the
presence of total coliform is confirmed.

8. The number of positive well was counted and MPN table provided was referred .

3.3 Data Acquisition

Result Indicator
No Colour of Less Yellow Than
No Coliform
Yellow Coliform
Fluorescent Blue E. Coli

Number of Box MPN of Bacteria

Type of Bacteria
Large Small ( MPN table)
Coliform 49 49 2419.6
E Coli 49 49 2419.6


Coliform bacteria and E.Coli are the type of bacteria that are part of the
enterobacteriaceae family . Coliform bacteria are a commonly used bacterial indicator of
sanitary quality of foods and water. They are defined as rod-shaped Gram-negative non-
spore forming and motile or non-motile bacteria which can ferment lactose with the
production of acid and gas when incubated at 3537C.Coliforms can be found in the
aquatic environment, in soil and on vegetation; they are universally present in large numbers
in the feces of warm-blooded animals. While coliforms themselves are not normally causes
of serious illness, they are easy to culture, and their presence is used to indicate that other
pathogenic organisms of fecal origin may be present. Such pathogens include disease-
causing bacteria, viruses, or protozoa and many multicellular parasites. Coliform procedures
are performed in aerobic or reduced oxygen conditions

Escherichia coli (also known as E. coli) is a Gram-negative, facultatively anaerobic,

rod-shaped bacterium of the genus Escherichia that is commonly found in the lower intestine
of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some
serotypes can cause serious food poisoning in their hosts, and are occasionally responsible
for product recalls due to food contamination.The harmless strains are part of the normal
flora of the gut, and can benefit their hosts by producing vitamin K2, and preventing
colonization of the intestine with pathogenic bacteria. The bacterium can be grown and
cultured easily and inexpensively in a laboratory setting, and has been intensively
investigated for over 60 years.
The most probable number method, otherwise known as the method of Poisson
zeroes, is a method of getting quantitative data on concentrations of discrete items from
positive/negative (incidence) data. The MPN method involves taking the original solution or
sample, and subdividing it by orders of magnitude and assessing presence/absence in
multiple subdivisions

The major weakness of MPN methods is the need for large numbers of replicates at
the appropriate dilution to narrow the confidence intervals. However, it is a very important
method for counts when the appropriate order of magnitude is unknown a priori and
sampling is necessarily destructive.
The use of UV light is to detect the presence of bacteria . Because bacteria tend to
congregate on surface where high level of flavin are present. Flavin is a material that emits a
fluorescent glow when exposed to UV light.