SHAYLENE MOONILAL
GENETICS PRACTICAL
(GNE3704)
YEAR: 2018
STUDENT NUMBER: 55202063
PORTFOLIO
,Shaylene Moonilal
55202063
GNE3704
Experiment 1: Water Quality Testing II: PCR –Based Testing of Water Contaminants
Introduction:
Water does not only make up 50-65% of our body mass, rather it is needed for surviving and living
not only for humans, but all living organisms. Water is a solvent that transports many essential
molecules and other particles around the body, some of which include nutrients and waste products
from the body’s metabolic processes.
Hence, the impending impact of waterborne pathogens on human health has become a growing
concern. Studies show that for 2011-2012, 32 drinking water-associated outbreaks were reported,
accounting for at least 431 cases of illness, 102 hospitalizations, and 14 deaths in the US. (Beer et al,
2015). This is also the case in South Africa, especially because there is no water systems in many
rural parts of the country as well as no proper sanitation. South Africa has large areas that lack
adequate supplies of potable water and sanitation. (Singh et al, 2013).
According to Altintas et al, 2015, a range of viruses can contaminate drinking water sources, causing
illnesses such as diarrhoea, pneumonia and gastroenteritis which can result in death. These
contaminants enter the supply through point source which is a single known source such as from a
chemical industry. They can also enter through non-point pollution which is from several unknown
sites such as over flow of septic tanks or water run-offs from farms.
Various water quality tests are available to detect the number and types of micro-organisms in
waters and to assist many communities in keeping microbial content of water supplies at a low level.
Indicator bacteria can be detected to give an estimate of pathogens. The most common indicator
organisms in water bacteriology are the coliform bacteria. These are Gram-negative rods normally
found in the intestine and typified by Escherichia coli. A fecal dwelling coliform bacteria is a specific
microorganism that can be used as an indicator of good water quality. So the presence of
gastrointestinal bacterial such as E.coli in water will mean that it is fecal contaminated.
In this experiment, we look at Polymerase Chain Reaction (PCR) and a Standard Plate Count.
PCR can be used to monitor water contamination because an assay based on Polymerase Chain
Reaction (PCR) amplifies the DNA of microbes present, thus determining the DNA recovery efficiency
of the genetically engineered microbe and calculating the number of cells of the test microbe in the
sample. PCR can be used to detect and quantify pathogenic bacteria, but these reactions may be
inhibited by humic acids or substances commonly present in soil and sediment, resulting in
inaccurate results. Furthermore, some portions of DNA and/or microbial cells can be lost during
testing, resulting in reduced DNA recovery efficiencies. This technology standardizes the DNA
extraction efficiencies for pathogen detection and yields accurate measurements of pathogenic
microbe concentrations.
It is generally impractical to test for all pathogenic organisms, but the total number of bacteria can
be calculated. In the Standard Plate Count, samples of water are diluted in jars containing 9ml sterile
water or broth, and samples are placed in Petri dishes with nutrient agar or other nutritious
medium. After incubation, the colony count is taken and multiplied by the dilution factor to obtain
the total number of bacteria per ml of sample.
, Shaylene Moonilal
55202063
GNE3704
Experimental Objectives:
To monitor water quality through detection of coliform bacteria.
To extract DNA from water samples.
To detect the presence of E.coli using PCR.
To analyse the amplified / PCR products using Gel Electrophoresis.
To be able to do Serial dilution and a Standard Plate Count as well as work out Colony
forming units (CFU) per ml.
Materials and Methods:
The experiment was done based on the protocol, our group used the Lab contaminated water to test
for the presence of E.coli. DNA from the water was extracted, amplified, run through the gel and
finally visualized under UV trans-illuminator. We also did a Standard Plate Count (SPC). Each group
member tested a different water, but 1 of the 3 waters was used to validate PCR results. That was
Kimberly’s SPC with contaminated water.
Results and Discussion:
Figure 1: The results after running the PCR on a gel and visualizing under a BioRad UV trans-illuminator.
We observed that our sample had a band that matched with the positive control, however it was
more like a smear than a clear band as seen in Figure 1 above.
GENETICS PRACTICAL
(GNE3704)
YEAR: 2018
STUDENT NUMBER: 55202063
PORTFOLIO
,Shaylene Moonilal
55202063
GNE3704
Experiment 1: Water Quality Testing II: PCR –Based Testing of Water Contaminants
Introduction:
Water does not only make up 50-65% of our body mass, rather it is needed for surviving and living
not only for humans, but all living organisms. Water is a solvent that transports many essential
molecules and other particles around the body, some of which include nutrients and waste products
from the body’s metabolic processes.
Hence, the impending impact of waterborne pathogens on human health has become a growing
concern. Studies show that for 2011-2012, 32 drinking water-associated outbreaks were reported,
accounting for at least 431 cases of illness, 102 hospitalizations, and 14 deaths in the US. (Beer et al,
2015). This is also the case in South Africa, especially because there is no water systems in many
rural parts of the country as well as no proper sanitation. South Africa has large areas that lack
adequate supplies of potable water and sanitation. (Singh et al, 2013).
According to Altintas et al, 2015, a range of viruses can contaminate drinking water sources, causing
illnesses such as diarrhoea, pneumonia and gastroenteritis which can result in death. These
contaminants enter the supply through point source which is a single known source such as from a
chemical industry. They can also enter through non-point pollution which is from several unknown
sites such as over flow of septic tanks or water run-offs from farms.
Various water quality tests are available to detect the number and types of micro-organisms in
waters and to assist many communities in keeping microbial content of water supplies at a low level.
Indicator bacteria can be detected to give an estimate of pathogens. The most common indicator
organisms in water bacteriology are the coliform bacteria. These are Gram-negative rods normally
found in the intestine and typified by Escherichia coli. A fecal dwelling coliform bacteria is a specific
microorganism that can be used as an indicator of good water quality. So the presence of
gastrointestinal bacterial such as E.coli in water will mean that it is fecal contaminated.
In this experiment, we look at Polymerase Chain Reaction (PCR) and a Standard Plate Count.
PCR can be used to monitor water contamination because an assay based on Polymerase Chain
Reaction (PCR) amplifies the DNA of microbes present, thus determining the DNA recovery efficiency
of the genetically engineered microbe and calculating the number of cells of the test microbe in the
sample. PCR can be used to detect and quantify pathogenic bacteria, but these reactions may be
inhibited by humic acids or substances commonly present in soil and sediment, resulting in
inaccurate results. Furthermore, some portions of DNA and/or microbial cells can be lost during
testing, resulting in reduced DNA recovery efficiencies. This technology standardizes the DNA
extraction efficiencies for pathogen detection and yields accurate measurements of pathogenic
microbe concentrations.
It is generally impractical to test for all pathogenic organisms, but the total number of bacteria can
be calculated. In the Standard Plate Count, samples of water are diluted in jars containing 9ml sterile
water or broth, and samples are placed in Petri dishes with nutrient agar or other nutritious
medium. After incubation, the colony count is taken and multiplied by the dilution factor to obtain
the total number of bacteria per ml of sample.
, Shaylene Moonilal
55202063
GNE3704
Experimental Objectives:
To monitor water quality through detection of coliform bacteria.
To extract DNA from water samples.
To detect the presence of E.coli using PCR.
To analyse the amplified / PCR products using Gel Electrophoresis.
To be able to do Serial dilution and a Standard Plate Count as well as work out Colony
forming units (CFU) per ml.
Materials and Methods:
The experiment was done based on the protocol, our group used the Lab contaminated water to test
for the presence of E.coli. DNA from the water was extracted, amplified, run through the gel and
finally visualized under UV trans-illuminator. We also did a Standard Plate Count (SPC). Each group
member tested a different water, but 1 of the 3 waters was used to validate PCR results. That was
Kimberly’s SPC with contaminated water.
Results and Discussion:
Figure 1: The results after running the PCR on a gel and visualizing under a BioRad UV trans-illuminator.
We observed that our sample had a band that matched with the positive control, however it was
more like a smear than a clear band as seen in Figure 1 above.
