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Ronaldo Chacha Proposal
Ronaldo Chacha Proposal
RONALDO CHACHA
A Research proposal submitted in partial fulfilment of the requirements for the degree of
Bachelor of Science in Industrial Biotechnology of Jomo Kenyatta University of
Agriculture and Technology.
1
SEPTEMBER 2019
DECLARATION
I hereby declare that this research proposal is my original work and has not been presented
elsewhere for a degree award
Signature……………………… Date…………………….
Declaration by Supervisor
This Research Proposal has been submitted for examination with my approval as supervisor.
Signature……………………… Date………………….
2
TABLE OF CONTENTS
DECLARATION.......................................................................................................................................2
TABLE OF CONTENTS..........................................................................................................................3
LIST OF DIAGRAMS..............................................................................................................................4
LIST OF ABBREVIATIONS AND ACRONYMS..................................................................................5
ABSTRACT...............................................................................................................................................6
CHAPTER ONE........................................................................................................................................7
1.0 INTRODUCTION...........................................................................................................................7
1.1 Background Information................................................................................................................7
1.3 Justification......................................................................................................................................8
1.4 Research questions..........................................................................................................................9
1.5 Hypothesis........................................................................................................................................9
1.6 Study objectives...............................................................................................................................9
1.6.1 General objectives.....................................................................................................................9
To determine antibacterial property of ear wax against pathogenic bacteria isolated within Juja town
.............................................................................................................................................................9
1.6.2 Specific objectives.....................................................................................................................9
2.0 LITERATURE REVIEW.............................................................................................................10
2.1 Ear wax/ cerumen..........................................................................................................................10
2.2 Composition of cerumen...............................................................................................................10
2.4 Antibacterial properties................................................................................................................12
2.5 Forms of cerumen..........................................................................................................................12
2.7 Mechanism of action of pathogenic bacteria...............................................................................14
2.8 Bacteria isolation...........................................................................................................................14
3.2 Study area and sampling...............................................................................................................15
3.4 Study design...................................................................................................................................16
3.5 Methods used.................................................................................................................................16
3.5.1 Bacteria isolation,media preparation and culturing............................................................16
3.5.2 Bacterial inhibition test using cerumen.................................................................................16
3.7 Data analysis..................................................................................................................................17
WORK PLAN......................................................................................................................................18
REFERENCES..........................................................................................................................................20
3
4
LIST OF DIAGRAMS
Figure 2.2 showing dry earwax.................................................................................................................14
Figure 2.1 showing wet earwax.................................................................................................................14
5
LIST OF ABBREVIATIONS AND ACRONYMS
6
ABSTRACT
Ear wax is a product of the ear that protects the skin of the ear from water and infection, its
normally made of a yellowish waxy substance called cerumen secreted by apocrine ceruminous
glands in the outer canal. It can be used as an antibacterial agent. The main objective of the study
is to test for the antibacterial property of human earwax against pathogenic bacteria within Juja
town which will be isolated from meat, water and sardine samples. Different composition of ear
wax has been tested against different ear pathogenic strains and has proven to possess
antibacterial activity. The present work is aimed to test the antibacterial property and inhibitory
property of ear wax Bacteria isolated from three different sources within Juja town will be
cultured to different plates and serial dilution carried out to reduce a dense culture of cells to a
more usable concentration. The grown cultures will then be grown on various selective media to
obtain a sizeable number of pathogenic microbes to work with. Inhibitory property of ear wax
will be tested against the bacteria at different concentration at various percentages after carrying
out serial dilutions. Minimum inhibitory concentration will be determined on basis of our
concentration. Diameter of zones of inhibition will be measured to determine the extent of
antibacterial property of ear wax. Data obtained will then be collected into excel worksheets and
analysed by ONE WAY Analysis OF VARIANCE and results presented in pictures and tables.
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CHAPTER ONE
1.0 INTRODUCTION
1.1 Background Information
Ear wax also referred to as cerumen, is a rich biological fluid that has unique advantage as a bio
monitoring medium of a high diagnostic potential. Cerumen is formed at the ear canal by
sebaceous glands, ceruminous glands and apocrine glands present at the outer one third of the
human external auditory canal and creates an acidic coat which aids in prevention of infection of
external auditory canal (Dien et al., 2011)
Although mortality from infectious disease has declined globally, bacterial infections still impact
the world’s most vulnerable populations. Bacterial infections including pneumonia and diarrhea
are major killers in young children. Tuberculosis and viral hepatitis are among the leading causes
of deaths due to infectious disease in adults living in low- and middle-income countries
(Singleton, 2012). Treatment for these devastating diseases exists but their use and effectiveness
face numerous challenges including timely diagnosis, drug resistance and access to healthcare.
Antibacterial property entails the ability of a certain compound to act against a certain bacterium.
It may involve inhibiting of viability or growth or germination of certain bacteria, for our case
we are testing the antibacterial property of human ear wax against pathogenic bacteria existing
within Juja town. Pathogenic bacteria strains are harmful and have been associated with the
release of toxins causing food poisoning and other infections like diarrhoea kidney failure, in
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susceptible individuals, respiratory illness, and pneumonia, typhoid, gut infections (WHO,2012;
Kumar et al.,2007).
Problems associated with infection by pathogenic bacteria within Juja town has been of great
concern. Pathogenic bacteria which have been studied to cause illness in human including food
poisoning, urinary tract infections, respiratory illness, pneumonia, meningitis, haemolytic
uraemic syndrome in young children, diarrhoea. It is associated with symptoms like fever,
vomiting, abdominal pains, and anaemic conditions. Food poisoning, diarrhoea and gut related
infections has posed great challenge to the Juja community and Kiambu county at large. (The
main sources of the pathogenic bacteria have been observed to be food and water.). In
susceptible individuals, pathogenic bacteria have been observed to cause kidney failures thus
these bacteria are more life threatening in infants and people with weakened immune systems.
(Todar, 2009; Lim et al., 2010). In addition, antibiotic resistance has also become a big threat to
global health, it has risen in all parts of the world making it hard to treat infectious diseases.
(Parovic and Schutz, 2016.). Many cases of antibiotic resistance have also been experienced
within Juja area and Nairobi regions according to WHO, 2015 article and approximately 700,000
lives worldwide are lost annually due to disease resistance (Neill, 2016).
1.3 Justification
Due to the fact that absence of ear wax in the human ear has resulted to various ear microbial
infection such as outer ear canal, otitis external ear itching and some other properties ear wax
possesses i.e. acidic nature, presence of antibodies, hormones, lipids, enzymes (lysozyme). Ear
wax is thus considered to have great antibacterial property. It prevents entry of any microbial
agent into the ear, it has a lubricating and cleansing property, and it repels water, traps that
prevent entry of any insect of any microbial agent. This antibacterial property of ear wax could
help come up with a long-term solution against bacterial infections and resistance and especially
those caused by pathogenic bacteria strains. (Schwab et al., 2011).
The ability of ear wax to inhibit microbial growth or viability of bacterial growth at different
concentrations could be the first step for the remedy against infections caused by the pathogenic
bacteria, further tests could be carried out and included in vaccine production and antibiotics that
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will act against them. This will help save infant deaths since the pathogenic bacteria is more life
threatening to infants. (Todar, 2009).
Antibiotic resistance has been experienced in treatment of some pathogenic bacteria stains
(WHO, 2015; Neill, 2016), therefore addition of some active components maybe the ear wax
may help solve the menace and maintain the various drugs pharmacodynamics. Determination
of inhibition zones will help to show the various extents of antibacterial property.
Does ear wax have a greater or lower antibacterial activity than ciprofloxacin against pathogenic
bacteria isolated within Juja Town?
What are the common pathogenic bacteria found within food samples in Juja?
1.5 Hypothesis
HO: Ear wax lacks antibacterial properties against pathogenic bacteria isolated within Juja town.
H1: Ear wax has antibacterial properties against pathogenic bacteria isolated in Juja town.
2. To identify the common pathogenic bacteria found in food samples within Juja town To
determine and compare the antibacterial activity of ear wax and ciprofloxacin against
pathogenic bacteria at different concentrations.
3. To determine and compare the antibacterial activity of ear wax and ciprofloxacin against
pathogenic bacteria at different concentrations.
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CHAPTER TWO
Ear wax is slightly acidic which discourages fungal growth in moist and dark environment of ear
canal. Without ear wax it would be almost impossible to avoid ear infections. Individuals vary in
how much wax their ears produce, some produce relatively large amounts while others produce
relatively smaller amounts. Factors like emotional stress, fear, pain, use of certain drugs, anxiety
tends to increase production of ear wax. Swimming has also been studied to show an increase in
production of ear wax. (Lum et al., 2009; Katauria A, and Katauria K 2013.)
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2.2 Composition of cerumen
Cerumen is a mixture of keratinocyte from the outer part of external auditory canal and
secretions form sebaceous glands along with apocrine sweat glands. It contains 60% keratin, 6-
9% cholesterol, saturated and unsaturated long chained fatty acids, and alcohols 12-20%.
Glandular secretions coming from hair follicles of external auditory canal also mix with cerumen
and make it a sticky substance. Cerumen is present in 10% of all children and up to 57% of older
persons. (Lum et al., 2009; Roeser and Ballachanda 2007). It is also composed of lipids,
proteins, carbohydrates, amino acids, hormones, anti-bodies, immunoglobulin glycopeptides. It
also possesses some enzymes like lysozyme which is an antibacterial enzyme capable of
destroying bacterial cell walls. It is composed of squalene whose concentration differs in gender
and age. Its acidic which discourages bacterial or fungal attack. It contains esters which have
long aliphatic four chains of carbon molecules which ensures there are insoluble in water.
(Yassin et al; 2012 ).
The common proteins present in the ear wax include antimicrobial peptides, e.g. hBD1-3, and
lactoferrin, LL37, BP1 and HNP1-3. These antibacterial peptides prevent bacteria and fungi
causing infections in the ear canal. (Schwaab et al, .2011). The human beta defensins (hBD1-3)
has a stronger antibacterial property on gram negative bacteria. The hBD2 has a strong
antibacterial property against E. coli, P aeruginosa an C. albicans. All hBD have proven to have
antibacterial effects. (Schneider et al.,2005; Harder et al.,2013). The human LL37 is a 37 amino
acid long C terminus with active antimicrobial component. It is expressed on leucocytes like
neutrophils, monocytes, epithelial cells like skin, respiratory tract, gastrointestinal tract. It has
antibacterial property against both gram negative and positive bacteria (Wang et al,2004;
Haussen et al.,2008). The Human lactoferrin is present in saliva, tears, milk, nasal mucosa,
granulocytes. It has proven to have antibacterial property against vibrio cholera, C. albicans and
Streptococcus mutants and E. coli (Stenfors et al.,2002; Schwaab et al 2011), The Human
bactericidal permeability increasing protein (BPI): - it is a single chain cationic protein divided
by proteolysis into two segments. It’s mainly seen in granules of neutrophils dermal fibro blasts.
It has a bacterial effect on gram negative bacteria (Cassels, 2012;Gupta et al; 2012).
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The ear wax provides protection against bacteria, fungi, insects. It contains a sticky wax that acts
like a stopping point or traps bacteria, fungus. It helps in shedding dead tissues, cleanse the ear to
ensure vibrations can pass through easily to be transformed into sound (Diep 2014; Shapiro and
Clarke, 2002). Lubrication is another function as it prevents desiccation of skin within the ear
canal, its lubricative property is due to its high lipid content of sebum. Ear wax cleaning is done
or removed during jaw movement especially during chewing. It dislodges the unwanted particles
attached to walls of ear canal increasing likelihood of its removal ( Saxby et al.,2013).
A specific gene determines whether people have wet or dry ear wax, the gene is referred to as
ATP binding cassette C11 gene. Dry type individual is homozygous for adenine while wet types
require at least one guanine. (Yoshiura et al.,2006; Guest et al.,2004; Diep,2014).
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Figure 2.1 showing wet earwax Figure 2.2 showing dry earwax
(http:www.en.wikipedia.org).’Cerumen’
2.6 Bacteria.
Bacteria are prokaryotic microorganisms a few micrometres in length, their cell wall is made of
peptidoglycan and they exist in various shapes like sphere, rod and spirals. Main sources of
bacteria include soil water, food sources, air. they also have symbiotic and parasitic relationship
with plant and animals. (Greenwood et al., 2012). Bacteria may be harmful i.e. pathogenic or
non-pathogenic. All animal life on earth is dependent on bacteria for their survival and some
possess genes and enzymes important in synthesising vitamin B12, that is soluble in water and is
involved in metabolism of every cell in human body. It is a co factor in DNA synthesis fatty acid
amino acid metabolism and is also key in normal functioning of nervous system (Fang et al
2017; Moore and Warren 2012). Bacteria may also be important in nutrient cycle by recycling
nutrients such as nitrogen fixation in the environment. They may also provide nutrients needed to
sustain life by converting dissolved compound like methane to energy (Choi 2013; Glud et al
2013). In addition, the large amount of bacteria exists in gut flora and in the skin. Majority of
bacteria in the body are harmless due to the protective ability of immune system though many
are beneficial especially in the gut flora. (Sears, 2005;Fan et al; 2017) .
Pathogenic bacteria are harmful and trigger an infection. Main sources of these bacterial include
air, water, soil. Example of these pathogenic bacteria from these sources include E.coli, coliform,
Vibrio cholerae, Salmonella typhi, Staphylococcus aureus, Mycobacteria tuberculosis,
Helicobacter pylori, Listeria monocytogens, Bacillus subtilis. These pathogenic bacteria cause
infectious diseases including; cholera, typhoid, diarrhoea, respiratory infection, gut infection,
pneumonia, tetanus (WHO, 2011; Kumar et al., 2007;Choi et al.,2013).
Pathogenic bacteria can be treated with antibiotics classified as bactericidal (if they kill the
bacteria) or bacteriostatic (if they prevent bacterial growth). (Yonath and Bashan 2004; Cassels
2012).
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2.7 Mechanism of action of pathogenic bacteria
Pathogenic bacteria act or cause damages directly or indirectly. They cause infection indirectly
as a result of inappropriate immune response triggered by an infection which may damage the
host cell. They act directly where by the pathogen attached to the host cell causes direct damage
as pathogen will use the host cell for nutrient and produce products, in addition they produce
toxins that cause must of direct damage e.g. endotoxins are release when bacteria lysis which is
after antibiotic treatment (Greenwood et al 2012; Nash 2015).
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CHAPTER THREE
An informed consent and clearance from Institutional clearance Ethics comittee will be obtained
on the use ear wax samples for research. It will be obtained from the Research ,Production and
Extension office in JKUAT.
The isolates will be collected from meat samples, sardine and water samples from various points
in Juja town. Meat samples will be collected in sterile bags, water sample collected in sterile
bottle while sardine will be collected in sterile bags for transportation to biochemistry lab in
JKUAT for further preparation. Ear wax will be collected using sterile swabs under proper
hygienic conditions and will be transferred to the laboratory.
The necessary sample size was determined using the following sstatististical test;
The Z score represents the confidence level which is 90%, The standard deviation is 0.5, The
margin of error expected in the study is 10%. Therefore:
=68
Earwax samples will be collected from 68 healthy individuals both male and female from
within the institution (JKUAT; College of Health Sciences), out of a population of 2400 students
using stratified systematic random sampling with our stratum group being two; male and female
and will use the following formula;
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Total population= 2400
sample size=68
female population=1100
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3.5.2 Bacterial inhibition test using cerumen.
Approximately 1g of Cerumen obtained will be dissolved in sterile buffer containing 5% sodium
bicarbonate and 30% glycerol after which it will be serial diluted with saline buffer up to 10-2 and
used for bacterial inhibition test. Agar plate diffusion method will be used to introduce the
antibacterial agent where by, filter papers will be punched with a punching machine of about
6mm diameter. They will be then soaked in the prepared cerumen of different concentrations.
The soaked discs at desired concentrations will be placed on the agar surface on the different
plates as labelled according to their concentrations. The plates will be incubated for 7 days at 37
degrees Celsius. The diameter of zones of inhibition will be measured to determine minimum
inhibitory concentration. The antibacterial agent will be expected to diffuse into agar and inhibit
growth of the test microorganism for positive test.
Data will be collected by observation and measurement of diameter of the zones of inhibition
shown by earwax on the pathogenic bacteria to be studied. Diameter of zones of inhibition
shown by the standard (ciprofloxacin) against the pathogenic bacteria being tested will also be
measured and a comparison made on basis of the extent of inhibition.
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WORK PLAN
The research work will be carried out from September 2019 to April 2020. The breakdown is as
follows:
2019
Proposal writing
Submission,,oral
presentation of
the proposal
Sample
collection
Progress report
2020
In vitro studies-
start of the lab
work.
Finalising lab
work
Project report
compilation
Project report
submission,
presentation
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BUDGET.
12 Transport - 500
13 Stationery - 200
Total 8500/-
-Refrigerator, autoclave, weigh balance, biosafety cabinet, and microbial test organism will be
provided by the institution’s biochemistry laboratory.
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