University of Ottawa: Indicator Microorganism Enumeration Lab Report

This lab report details an experiment on indicator microorganism enumeration, focusing on the membrane filtration and defined substrate technology (DST) methods. The experiment, conducted at the University of Ottawa for the CVG3132 course, aimed to identify and quantify indicator microorganisms in a water sample. The report includes the theory behind the methods, materials used, and detailed procedures. Results from both techniques are presented, showing that the membrane filtration method found less than one colony in the 10% sample, while the DST method indicated 1.0 MPN/100ml of total coliforms and less than 1.0 MPN/100ml of E. coli. Calculations and a discussion section analyze the findings, comparing the two methods and addressing potential sources of error. The conclusion summarizes the experiment's outcomes, noting that the sample did not meet Canadian drinking water quality standards. The report highlights the DST method as more suitable for routine water quality testing and emphasizes the importance of accurate experimental procedures.

Lab 1 – Indicator Microorganism Enumeration
CVG3132 – Physical/Chemical Unit Operations of Water and Wastewater Treatment
Group 4 – B (Wednesday):
Saif El Husseini – 7023482
Ian Philion – 7023099
Jean-Sébastien Plouffe– 7044012
Experiment Performed: 2015 January 28th
Lab Report Due: 2015 February 5th
UNIVERSITY OF OTTAWA - CIVIL ENGINEERING

Table of Contents
List of Table and Figures............................................................................................................... ii
1.0 Theory......................................................................................................................................1
1.1 Membrane Filtration (MF) Technique..............................................................................1
1.2 Defined Substrate Technology (DST)...............................................................................2
1.3 Dilution Factor..................................................................................................................3
2.0 Objectives............................................................................................................................. 4
3.0 Materials............................................................................................................................... 4
3.1 Part A – Membrane Filtration Technique..........................................................................4
3.2 Part B – Defined Substrate Technology............................................................................4
4.0 Methods................................................................................................................................ 4
4.1 Part A – Membrane Filtration Technique..........................................................................4
4.2 Part B – Defined Substrate Technology............................................................................4
5.0 Results & Data......................................................................................................................5
5.1 Part A - Membrane Filtration Technique..........................................................................5
5.2 Part B - Defined Substrate Technology.............................................................................6
6.0 Calculations.......................................................................................................................... 7
6.1 Part A - Membrane Filtration Technique..........................................................................7
6.2 Part B - Defined Substrate Technology.............................................................................7
7.0 Discussion.............................................................................................................................9
8.0 Conclusion.......................................................................................................................... 10
9.0 References.......................................................................................................................... 11

List of Table and Figures
Table 1: Results of the Membrane Filtration Technique................................................................5
Table 2: Well counts of the Colilert Quanty-tray...........................................................................6
Table 3: The MPN for total coliform and E. coli bateria...............................................................8
Figure 1: Petri dish containing the 10% sample.............................................................................5
Figure 2: IDEXX Quanti-Tray®/2000 MPN Table.........................................................................7
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1.0 Theory
Indicator microorganisms are biological detectors help to target pathogens. In general,
pathogens are hard to isolate and are small in number. In order to identify pathogens that are
easier and safety detected (Lab Manual, 2015). There are certain requirements that need to be
met in order for the indicator microorganism to be most efficient. Firstly, the indicator must be
found in the fecal contamination as it occurs (Lab Manual, 2015).
Moreover, the population of the indicator must be greater than or equal to the population of
the pathogens, while having similar survival characteristics like the pathogens (Lab Manual,
2015). In addition, the indicator should not be able to reproduce outside of the host. Once these
samples have been collected, these samples should be stored in a cold and dark area (i.e. ice
chest or refrigerator between 1- 4 ℃ (Lab Manual, 2015). While performing this experiment, it
is crucial to constantly sterilize and clean your surroundings, especially when you are working
with samples that contain bacteria.
One method that can be used in this experiment that can sterilize equipment is through the
use of methanol and allows it to evaporate from the equipment. Make sure to indicate that
methanol is highly toxic to bacteria and can interfere with a sample (Lab Manual, 2015).
1.1 Membrane Filtration (MF) Technique
Firstly, a Membrane Filter Technique offers the advantage of isolating discrete colonies of
bacteria. Moreover, it allows testing of large sample volumes. Reduces the amount of preparation
time in comparison to a lot of traditional methods used in wastewater.
In the real world, the MF Technique is useful for monitoring bacteria in pharmaceuticals,
cosmetics, electronics, food, and beverage industries (Pall Corporation, 2014). Moreover, it

allows the removal of cidal or bacteriostatic agents that are mainly removed in Pour Plate,
Spread Plate or MPN Techniques (Pall Corporation, 2014).
In general, the MF Technique passes through a filter using a filter funnel and a vacuum
system. In addition, the filter and organisms are placed in a petri dish that includes a culture
medium (Lab Manual, 2015). Through the period of incubation, a bacterium reproduces on the
surface of the membrane and grows bacterial colonies that are countable.
There must be less than 200 bacterial colonies on the filter to consider this method accurate;
one can state that there are too many to count. Similarly, if there are not that many coliforms, one
can state that there is less than 1 per 100mL (Lab Manual, 2015). For Accuracy, make sure to
shake the sample prior to filtering, as this allows the separation of all bacteria found.
1.2 Defined Substrate Technology (DST)
Colilert is a commercially available enzyme-substrate that simultaneously detects the total
coliforms and E.Coli. (Lab Manual, 2015). It is found in presence/Absence format (PA) or most-
probable number (MPN).
In general, the defined substrate technology works simultaneously with two enzymes
substrates:
 Chromogen
 Fluorogen
Chromogen is an enzyme substrate that reacts with the enzyme that is found in total
coliforms. Similarly, Fluorogen is an enzyme substrate that reacts with an enzyme that is
commonly found in E.Coli.
Furthermore, placing these two enzymes in a 24-hour incubation at 35℃, the coliform-
positive would turns yellow in color. Meanwhile, an E.Coli-positive causes it to fluoresce from
ultra-violet light (Lab Manual, 2015).
Without a doubt, the colilert method is approved for drinking water since it helps to monitor
all kinds of water, especially those with high-suspended sedimentation. Lastly, the PA is mostly
appropriate for ground water or drinking-water studies (Lab Manual, 2015).
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Meanwhile, the MPN method helps to count from 1 to 2,400 MPN per 100mL of sample.
These tests help give a bigger range with the dilution of 1:100 or 1:10 ratios with the assistance
of sterile water (Lab Manual, 2015).
1.3 Dilution Factor
This formula of dilution, also known as the dilution factor, can be used where one solute
is present, this formula should be solved through stoichiometry if a reaction takes place (Quansys
Biosciences, 2013). The dilution formula can be written as follows:
V 1C 1=V 2C 2 (Eq. 1)
Where:V1 = Volume of starting solution needed to make new solution (mL)
C1 = Concentration of starting solution
V2 = Final volume of new solution (mL)
C2 = Final concentration of new solution
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2.0 Objectives
The objective of this experiment is to determine the membrane filtration technique and
defined substrate technology (DST).
3.0 Materials
3.1 Part A – Membrane Filtration Technique
Refer to page 15 of the CVG3132 lab manual.
3.2 Part B – Defined Substrate Technology
Refer to page 17 of the CVG3132 lab manual.
4.0 Methods
4.1 Part A – Membrane Filtration Technique
Refer to page 15 of the CVG3132 lab manual. The serial dilutions used in the experiment have
been changed to the following:
10% Sample – 10mL of sample and 90mL sterilized water
1% Sample – 10mL of 10% sample and 90mL water
0.1% Sample – 10mL of 1% sample, 90mL water
4.2 Part B – Defined Substrate Technology
Refer to page 17 of the CVG3132 lab manual.
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5.0 Results & Data
5.1 Part A - Membrane Filtration Technique
After letting the four sample incubate for 24 hours at 35°C, no colonies were clearly
observed on the filter. On the 10% sample some dark dot could be observe but it was not enough
to count as a colony. The distribution of the colonies is shown in the following table.
Table 1: Results of the Membrane Filtration Technique
Sample Colonies
Control
(Distilled water)
0
0.1% sample 0
1% sample 0
10% sample Less than one
Figure 1: Petri dish containing the 10% sample
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5.2 Part B - Defined Substrate Technology
For this part, the sample was incubated in the Colilert Quanti-tray for 24 hours at 35 ℃. The
sample was then compared to the color comparator for color change. The presence of total
coliform bacteria is shown when the sample turn to yellow. Only one large well of the colibert
Quanti-tray turned to yellow.
The second part of the experiment was to identify if E. coli was present in the sample.
The Colibert Quanti-tray was expose to the 365nm UV light and no fluorescent wells was
detected.
The number of wells that contain coliform bacteria and the result for the presence of E.
coli are shown in the table 2.
Table 2: Well counts of the Colilert Quanty-tray.
Positive Result Large Well Count Small Well Count
Total coliforms (yellow) 1 0
E. coli (fluorescence) 0 0
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6.0 Calculations
6.1 Part A - Membrane Filtration Technique
The calculation for the control sample, the 0.1% sample and the 1% sample are not possible
because the filter has no trace of bacteria on it. At the opposite, the 10% sample showed some
trace that can be defined as ''less than one'' colony. The result per 100ml of original sample is
calculated as follow.
Colonies per 100ml of original sample =
colonies per 100 ml of 10 % sample
10 % =less than 1
0.1 =less than 10 colonies .
6.2 Part B - Defined Substrate Technology
In this part, the most probable number (MPN) is use to identify the number of bacteria contained
in the sample. To determiner this number, the use of the IDEXX Quanti-Tray/2000 MPN Table
to determine the MPN for total coliform bacteria and E. coli bacteria. The table is shown in the
figure below.
Figure 2: IDEXX Quanti-Tray®/2000 MPN Table
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Using the data of the Table 2 and the IDEXX Quanti-Tray®/2000 MPN Table, the
amount of total coliform bacteria is 1.0 MPN/100ml and the density of the E. coli bacteria is less
than 1.0 MPN/100ml.The sample was not diluted for this analysis. Therefore, the density of
bacteria per 100ml of sample is given directly with the chart. The results are shown in the table
3.
Table 3: The MPN for total coliform and E. coli bacteria
Positive Result Large Well
Count
Small
Well
Count
MPN/100 mL
Total coliforms
(yellow)
1 0 1.0
E. coli (fluorescence) 0 0 Less than 1
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7.0 Discussion
The first part of experiment of the membrane filtration method ha four major samples
which were unable to show the presence of coliform. The samples included a control sample,
dilution sample of 10%, dilution sample of 1% and dilution sample of 0.1%. Some bacterial
colony were found to exist on the 10% dilution sample. Nevertheless, none of the were coliform
able to be differentiated by the red color and mettalic sheen. The use of non-dilited sample is
assumed to could have helped to find coliform on the sample. The experiment is able to show
that there was less than 10 coliform available per 100 ml.
Second part of the experiment was the DST method. At this part, no E-Coliform were
present. This meant that the most probable number of E-Coliform which can be found in a 100
ml is less than 1. In this part of the experiment, only one large positive to the total coliform was
found in our sample. According to the table, we can conclude that one MPN of the total coliform
per 100 ml is able to exist.
The major observation made while comparing the two tests is that they have the same
range and this proves that they are done in the right manner. The Canadian guidelines for
drinking water quality is that the samples aren't good for consummation. The guide means that
the concentration of the total coliform and E-Coli are not mostly detected in a sample of 100 ml.
Contamination is one of the source of error in this lab experiment. Nevertheless, this
source of error was not a major problem in this experiment due to the low results in the
experiment. In addition, another are which can be source of error in the experiment is the shaking
step which happens before the diluting and filtration. Lack of proper shaking means that the
bacteria will agglomerate together and form a big colony instead of multiple colonies as
required.
The need for high number of equipment and more disinfection step makes the membrane
filtration method more involving although its more precise. DST method is not much precise and
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