WATER ENGINEERING LABORATORY ECW341

OCTOBER 2022 – FEBRUARY 2023

TITLE OF EXPERIMENT:
ENUMERATION OF BACTERIA

LEVEL OF OPENNESS: LEVEL 2

GROUP MEMBERS:
CLASS: EC1105
TITLE OF EXPERIMENT

Enumeration of Bacteria

INTRODUCTION
It is often necessary to count the number of organisms present in a sample of a
material, such as water, food, or a bacterial culture. Coliform bacteria are Gram-negative,
non-spore-forming microorganisms that can ferment lactose to create gas and acid. Fecal
coliforms, a subgroup of these bacteria, are prevalent in both human and animal intestines at
high concentrations. Since they are rarely observed thriving in nature when there is no fecal
contamination, fecal coliform bacteria like Escherichia coli (E. coli) are frequently used as
indicator species. E. coli reveals the presence of feces, which means that dangerous bacteria
like Salmonella species and Campylobacter species may also be present.

OBJECTIVES
1. To quantify the presence of E. Coli in water samples
2. To compare the results obtained from the experiments with the national drinking
water standard.
3. To compare the results obtained from the experiments with the IDEXX
Quanti-Tray/2000 MPN Table.
THEORTICAL BACKGROUND
Water pollution caused by fecal contamination is a serious problem due to the
potential for contracting diseases from pathogens. The presence of pathogens is determined
with indirect evidence by testing for an "indicator" organism such as coliform bacteria. The
most basic test for bacterial contamination of a water supply is the test for total coliform
bacteria.
Total coliforms include bacteria that are found in the soil and in water. Fecal
coliforms are the group of the total coliforms that are considered to be present specifically in
the feces of warm-blooded animals and human. Escherichia coli (E. coli) is the major species
in the fecal coliform group and is considered to be the species of coliform bacteria that is the
best indicator of fecal pollution and the possible presence of pathogens.
According to National Primary Drinking Water Regulations USEPA. Not more than
5.0% samples should be total coliform-positive (TC-positive) in a month. Every sample that
has total coliform must be analyzed for either fecal coliforms or E. coli. If two consecutive
TC-positive samples, and one is also positive for E. coli fecal coliforms, system has an acute
MCL violation.
Quanti-Tray is a method that uses a tray that consist of many small boxes which is
called wells conduct the Enumeration of Bacteria lab experiment. The standard 15-tube serial
dilution is based on the same statistical model as the Quanti-Tray/2000. When the Quanti-
Tray Sealer PLUS seals the Quanti-Tray/2000, the sample is automatically divided into the
appropriate sections. No Durham tubes, test tubes, or dilutions are needed when using the
Quanti-Tray system. In comparison to a 15-tube serial dilution, the Quanti-Tray/2000
delivers a counting range of 1-2,419 with a far superior 95% confidence limit by
automatically dividing the material into 97 wells of two distinct sizes. The Quanti-Tray
System delivers semiautomated quantification methods based on the Most Probable Number
(MPN) model in Standard Methods for the Examination of Water and Wastewater.
Quanti-Tray is easy, rapid, accurate and cost-effective as it does not involve the use of
pipette unlike the traditional enumeration of bacteria method which is standard/viable
plate/colony count. Quanti-Tray enumeration method detects down to one organism per 100
mL and provides result in less than 24 hours.
A 100 mL sample is split into 51 wells using Quanti-Tray. The number of bacteria in
the first sample is then calculated using the Most Probable Number (MPN) approach
described in Standard Methods. Automation reduces labour requirements and increases
output (up to 4 samples per minute). A high counting range of 1-200 (without dilutions) and
very close 95% confidence limits are made possible by the comparatively large number of
wells.
The Quanti-Tray system's newest sealer is the Quanti-Tray Sealer PLUS. The Quanti-
Tray and Quanti-Tray/2000 sealer allows for quantification while reducing time and
enhancing laboratory productivity. Features include an access panel that makes cleaning
simple, a warm-up duration of 2-3 minutes, quicker tray sealing, and energy economy.

PROBLEM STATEMENTS
According to Drinking Water Quality Standard by MOH Malaysia, acceptable value
for raw water quality is 5000 MPN / 100 m. Based on this requirement, each group is
required to investigate the presence of E-Coli in one (1) water samples gather from around
UiTM to be tested in the lab. Each group is required to prepare the required apparatus and
procedure to carry out the experimental works.

APPARATUS

i. Gloves
ii. Water sample
iii. Beaker
iv. Sterile vessel
v. Colilert
vi. Quanti-Tray
vii. Quanti-Tray mould
viii. Quanti-Tray Sealer
ix. Ultra-violet light
x. Incubator
PROCEDURE
i. Glove was put on before experiment is conducted.
ii. Quanti-Tray Sealer PLUS was turned on to warm up for sealing.
iii. A 100 ml water sample was prepared using beaker.
iv. The water was poured into a sterile vessel.
v. One capsule of Colilert was opened and put into the sterile vessel containing water
sample.
vi. The sterile vessel was sealed tight to prevent spilling.
vii. The sterile vessel was shaken until the Colilert has dissolved and fully incorporated
with the water sample.
viii. The water sample is placed into the Quanti-tray aseptically.
ix. The Quanti-Tray was put into the Quanti-Tray mould, then was sealed using Quanti-
Tray sealer.
x. The Quanti-Tray was put into incubator for 24 hours at 35 degree Celsius.
xi. Quanti-Tray sample is taken out. Ultra-violet light were used to determine the
presence of E. Coli in the aqueous material by observing whether the large and small
wells lights fluorescent blue or not. If it is luminant, E. Coli is present and should be
avoided. Small and large fluorescing wells are counted.
xii. Most Probable Number (MPN) is referred from the IDEXX Quanti-Tray MPN table.

RESULT AND DATA ANALYSIS

DISCUSSION
The water sample is collected from a drainage system in UiTM Kampus Samarahan 2,
which is the waste water that is created by humans. Every warm-blooded animal, including
humans, excretes coliform bacteria into its environment. Coliform bacteria are unlikely to
cause illness. However, the discovery that they are present in drinking water raises the
possibility that there are pathogenic organisms (pathogens), including Shigellosis (Shigella)
and Salmonellosis (Salmonella) in the water system. Most illnesses that could pollute water
sources start in either human or animal waste. It is challenging, time-consuming, and
expensive to test drinking water for every possible pathogen. However, with the presence of
Quanti-Tray 2000, it is easy and inexpensive, and accurate to test for the presence of coliform
bacteria. If testing reveals the presence of coliform bacteria in a water sample, water systems
search for the source of contamination and restore safe drinking water by treatment of the
water.
Coliform bacteria can be divided into three categories. Each one of the categories
indicates the quality of drinking water and carries a different level of risk. The large
collection of diverse bacterial species known as total coliform. Feces contain a group of
coliforms known as fecal coliforms. E. coli is a member of the subgroup of fecal coliforms.
Labs analyze drinking water samples for total coliform. If total coliform is detected, the
laboratory also looks for E. coli in the sample.
Coliform bacteria are widespread and often found in the environment such as in soils
or in plants. Coliform bacteria are generally safe as fecal contamination is rare when a lab
discovered the total coliform bacteria that is found in drinking water. But the drinking water
as a whole is not safe because if coliform exist in the water, other pathogens that will carry
water-borne disease have a high chance to be present in it. Hence why it is critical to identify
and address the contamination’s source as soon as possible.
CONCLUSION
Presence of E. Coli in a water sample has been verified and the method of testing has
been proved effective, efficient and accurate. Students may learn and check about the
presence of E. Coli in a water sample and compare to National Drinking Water Standard.
There are several factors that contribute to the contamination of raw water, which
increases the risk of drinking water. However, it is important to state that water is essential to
the survival of humanity. Hence, it is critical to find the contamination source when a water is
polluted so that it person in charge can act accordingly and swiftly to treat the water source.
According to the IDEXX Quanti-Tray MPN table, the total MPN is over 2419.6,
which means that the water is not safe for drinking as it could exceed over 5000 MPN/100 m
raw water quality according to Drinking Water Quality Standard by MOH Malaysia. In a
nutshell, water source that comes from the drainage system are not suitable for the water
treatment plant and it is the wastewater treatment plant’s responsibility to take care of the
wastewater.

REFERENCES
https://www.idexx.com/en/about-idexx/investors/company-overview/
(IDEXX, 2010)

http://www.ohiowea.org/docs/E_Coli_QuanitTray_.pdf
(Staley, n.d.)

https://bio.libretexts.org/Courses/North_Carolina_State_University/
MB352_General_Microbiology_Laboratory_2021_(Lee)/
05%3A_Enumeration_of_Bacteria/5.01%3A_Introduction_to_Enumeration_of_Bacteria?
readerView
(Biology, n.d.)
Appendix 1: IDEXX Quanti-Tray/2000 MPN Table