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Candidate name: Tan Hui Min, Rachel

Candidate session number: 004130-0113


_____________________________________________________________________

Which antimicrobial substance: honey, Manuka honey, lemon

juice (C. Limon), honey-lemon juice or Manuka honey-lemon

juice inhibits the largest amount of gram-negative bacterial

growth and how does varying the concentration of that substance

affect the inhibition of gram-negative bacterial growth?

International Baccalaureate Diploma Programme

Session: November 2015

Biology Extended Essay

Candidate Name: Tan Hui Min, Rachel

Candidate Session Number: 004130-0113

Personal code: fnp532

Supervisor: Ms Vivienne Loh

Word Count: 4000 words

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
CONTENTS

I. Cover page…………………………………………………………………………..1
II. Contents page………………………………………………………………………2
III. Abstract……………………………………………………………………………3

1. Introduction………………………………………………………………………4
1.1 Research question……………………………………………………………...4
1.2 Introduction……………………………………………………………………4
1.3 Hypothesis……………………………………………………………...…….12

2. Materials and procedure………………………………………………………13


2.1 Variables……………………………………………………………………..13
2.2 Apparatus…………………………………………………………….………15
2.3 Materials……………………………………………………………………..15
2.4 Procedure Part A…………………………………………………………….16
2.5 Procedure Part B………………………….………………………………….26

3. Data collection and processing………………………………………………21


3.1 Results Part A….…………………………………………………………….21
3.2 Results Part B…….………………………………………………………….27
3.3 Discussion of results………………………………………………………...31

4. Conclusion………………………………………………………………………35

5. Evaluation…………………………….…………………………………………35
5.1 Possible extensions…………………………………………………………..35

6. Bibliography…………………………………………………………………….36

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
Abstract

Honey-lemon juice is a popular home remedy in many households worldwide. It is commonly

used to treat sore throat. Lemon juice and honey is mixed together and consumed. However,

the efficacy and mechanisms of honey-lemon juice have not

been fully elucidated. The purpose of this experiment is to investigate and elucidate

the antibacterial effects of honey-lemon juice, its separate components as well as Manuka

honey. The research question is: Which antimicrobial substance: honey, Manuka honey,

lemon juice(C. limon), honey-lemon juice or Manuka honey-lemon juice inhibits the

largest amount of gram-negative bacterial growth and how does varying the

concentration of that substance affect the inhibition of gram-negative bacterial growth?

In this experiment, the five antimicrobial substances were diluted to concentrations

commonly used by the majority of a population. Gram-negative bacteria from the mouth and

throat were inoculated and cultured and made into a broth. A Kirby-Bauer test was performed

to obtain a representation of the inhibition of bacterial growth.

Results show that Manuka honey-lemon juice inhibited the largest amount of bacterial

growth and there is a positive correlation between increasing the concentration of Manuka

honey and the area of the zone of inhibition seen. Therefore, the higher the concentration of

Manuka honey in Manuka honey-lemon juice, the larger the amount of bacterial growth

inhibited. The results show that Manuka honey-lemon juice can potentially be an affordable

alternative form of treatment for sore throat. (229 words)

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
1.1 Research Question

Which antimicrobial substance: honey, Manuka honey, lemon juice(C. limon), honey-lemon

juice or Manuka honey-lemon juice inhibits the largest amount of gram-negative bacterial

growth and how does varying the concentration of that substance affect the inhibition of

gram-negative bacterial growth?

1.2 Introduction

Diseases and ailments are caused by numerous factors such as virus, bacteria, etc. These

diseases affect many and it can cause increased health issues. One common ailment is sore

throat, which is also known as pharyngitis. Recently, the prevalence of sore throat incidences

has increased and in the United States alone, 15 million people see the doctor for sore throat

every year and 70% receive antibiotics. 1 Of these people, approximately 40% have strep

throat, which is an infection caused by the streptococcus bacteria.1 This infection is treated

with penicillin or amoxicillin that have antibacterial properties that can effectively inhibit the

bacterial growth and cure the infection.

However, due to the over usage of such drugs, bacterial strains have developed greater

resistance.2 Bacteria can change their structure or mutate their genetic material and eliminate

the effectiveness of drugs. 3 The surviving bacteria will multiply and the offspring will

receive the antimicrobial resistant properties of its parent. This poses a significant problem

for those affected by sore throat. Therefore, there is renewed scientific interest in finding

alternative solutions that are not bacteria resistant and possess the ability to inhibit bacterial

growth.

1. "Bad Sore Throat? It's Probably Not Strep, Most Likely Viral." IDSA :. N.p., n.d. Web. 19 June 2015.
<http://www.idsociety.org/2012_Strep_Throat_Guideline/>.
2. "Why Are Bacteria Becoming Resistant to Antibiotics? - RxList." RxList. N.p., n.d. Web. 19 June 2015.
<http://www.rxlist.com/antibiotic_resistance-page3/drugs-condition.htm>.
3. "Effects of Biocides on Antibiotic Resistance." Biocides: 4. How Can Bacteria Become Resistant to Biocides or Antibiotics? N.p., n.d.
Web. 19 June 2015. <http://ec.europa.eu/health/opinions/en/biocides-antibiotic-resistance/l-2/4-mechanisms.htm>.

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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In many households, honey-lemon juice is a home remedy for sore throat. Honey-lemon juice

is an inexpensive beverage that is believed to treat a host of ailments, such as preventing

dehydration.4 There is evidence of the antibacterial properties of honey-lemon juice like its

ability to treat sore throat. 5 However, honey is not the sole substance that has caught the

attention of scientists. Recently, Manuka honey has also been discovered to have

antibacterial properties.

However, the extent of bacterial growth inhibitory effects by these antimicrobial substances

has not been directly quantified. Should these substances have significant antibacterial effects

on cultured bacteria, further investigations can be conducted to determine if they are a

suitable alternative treatment for sore throat.

Therefore, the aim of this investigation is to find out which antimicrobial substance; Honey,

Manuka honey, lemon juice, honey-lemon juice or Manuka honey-lemon juice (MHLJ) can

inhibit the largest amount of gram-negative bacterial growth, proving to be the best inhibitor

of gram-negative bacterial growth. The diluted concentrations of these five substances are

chosen to be the same as what is commonly used by the majority of people. Also, 100.0% of

each individual substance is used as a control. This is to test for any synergistic effects

between the mixtures. Once the best inhibitor is discovered, the concentrations of the

substance will be varied to find the optimum concentration for the substance to inhibit

bacterial growth.

4. "Common Cold." Cold Remedies: What Works, What Doesn't, What Can't Hurt. N.p., n.d. Web. 19 June 2015.
<http://www.mayoclinic.org/diseases-conditions/common-cold/in-depth/cold-remedies/art-20046403>.
5. "Manuka Honey and Lemon: The Superfood Duo with 5 Unique Superpowers." The Alternative Daily. N.p., n.d. Web. 19 June 2015.
<http://www.thealternativedaily.com/manuka-honey-lemon-superfood-duo-5-unique-superpowers/>.

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
The approach taken in this investigation is to use the Kirby-Bauer test on self-cultured

bacteria taken from the mouth and throat area. The test employs the usage of agar plates and

filter paper discs to show the extent of the inhibition of bacterial growth. Filter paper discs

with the substances will be placed onto the agar plate with bacteria and after 24 hours, a zone

of inhibition will appear around the disc. This zone marks the area where there was no

bacterial growth and shows the amount of inhibition of bacterial growth.

The area of bacterial growth inhibited will be calculated using the formula of an ellipse to

accommodate zones of inhibition with two different radii. Once the best inhibitor of bacterial

growth is discovered, it will be diluted to five different concentrations to find out the

optimum concentration for it to inhibit bacterial growth.

This investigation is split into part A and B, where the former answers the first independent

variable – which substance inhibits the largest amount of bacterial growth, and the latter

answers the second independent variable – how does varying the concentration of that

substance affect the inhibition of bacterial growth.

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
Literature review

Bacteria

Figure 1 Gram-positive and gram-negative bacteria. Image taken from ars.els-cdn.com

Bacteria are prokaryotic cells, which do not contain membrane-bound organelles.6 There are

gram-negative and gram-positive bacteria, which can be differentiated by the presence of a

lipopolysaccharide outer membrane in gram-negative bacteria.

6. "Bacteria." Microbiology Online. N.p., n.d. Web. 19 June 2015. <http://www.microbiologyonline.org.uk/about-


microbiology/introducing-microbes/bacteria>.

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
Lemon (Citrus limon)

Figure 2 Citrus limon. Image taken from sahultrading.com

Lemon is a citrus fruit that contains nutrients required by the body. Preliminary research has

shown that lemon has several health benefits associated with it such as reducing high blood

pressure.7 Its primary active compound is limonene, which is found in both the rind and juice

of lemons.8

Limonene (1-methyl-4-prop-1-en-2-ylcyclohexene)

Figure 3 showing the structure of limonene. Image taken from gfd-dennou.org

7. "Health Benefits of Lemon | Organic Facts." Organic Facts. N.p., 18 Dec. 2008. Web. 19 June 2015.
<https://www.organicfacts.net/health-benefits/fruit/health-benefits-of-lemon.html>.
8. "Role of Citrus Fruit Juices and Distinctive Components in the Modulation of Degenerative Processes: Genotoxicity,
Antigenotoxicity, Cytotoxicity, and Longevity in Drosophila." National Center for Biotechnology Information. U.S. National Library
of Medicine, n.d. Web. 19 June 2015. <http://www.ncbi.nlm.nih.gov/pubmed/21707429>.
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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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Limonene is a liquid hydrogen, which possesses a lemon odor. 9 Limonene’s antibacterial

property comes from its negative apoptosis regulation ability. Apoptosis, known as

“programmed cell death”, is the process that eliminates unwanted or dangerous cells by

inducing the cell to break down.10 Limonene targets and binds to the cell wall and penetrate

into the phospholipid bilayer. Limonene proceeds to bind to proteins and alters their shape,

preventing them from functioning normally.11 For example, the proteins: GroEL and GroES.

These proteins are involved in promoting protein folding by binding to short, extended

peptides in an ATP-dependent cycle.11 Unfolded substrate proteins bind to the hydrophobic

binding site of GroEL, forming a binary complex with chaperonin proteins that promote

favourable conditions for protein folding. The binding of limonene to the cell wall can cause

outer membrane proteins to become mis-folded. Limonene induces bacterial envelope stress

due to the accumulation of mis-folded proteins and subsequently causes the cell membrane

and the cell to break down.11 Limonene has greater efficacy on gram-positive bacteria.11

9. "LIMONENE." LIMONENE. N.p., n.d. Web. 19 June 2015.


<http://pubchem.ncbi.nlm.nih.gov/compound/Dipentene#section=Experimental-Properties>.
10. APOPTOSIS IN ONCOLOGY." What Is Apoptosis? N.p., n.d. Web. 19 June 2015.
<http://www.abbvie.com/oncology/home/apoptosis.html>.
11. Nazzaro, Filomena, Florinda Fratianni, Laura De Martino, Raffaele Coppola, and Vincenzo De Feo. "Effect of Essential Oils on
Pathogenic Bacteria." Pharmaceuticals. MDPI, n.d. Web. 19 June 2015. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873673/>.

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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Honey

Figure 4 Honey. Image taken from getnativ.com

Honey is a liquid that is made by bees using the nectar of flowers.12 The nectar is converted

to honey through regurgitation and evaporation. After the final regurgitation, the aqueous

solution undergoes evaporation to reduce the remaining amount of water present. 13 Honey’s

primary active compound is hydrogen peroxide.14

Hydrogen peroxide

Figure 5 structure of hydrogen peroxide. Image taken from chemeddl.org

12. Palermo, By Elizabeth. "What Is Honey?" LiveScience. TechMedia Network, 20 June 2013. Web. 19 June 2015.
<http://www.livescience.com/37611-what-is-honey-honeybees.html>.
13. Mandal, Manisha Deb, and Shyamapada Mandal. "Honey: Its Medicinal Property and Antibacterial Activity." Asian Pacific Journal of
Tropical Biomedicine. Asian Pacific Tropical Medicine Press, n.d. Web. 19 June 2015.
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609166/>.
14. Elvira Mavric. (2008). Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka
(Leptospermum scoparium) honeys from New Zealand. Molecular nutrition. 52 (4), 483-489.

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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Hydrogen peroxide is a chemical compound with the formula H2O2. Hydrogen peroxide is

able to oxidize the electron carriers of the electron transport chain (ETC). Hydrogen peroxide

removes an electron from the ETC. As a result the electron cannot move down the chain and

release energy for the cell to sustain its metabolic processes, causing it to die eventually.

Manuka honey

Manuka honey is a type of honey that is only made using nectar from Manuka flowers. It

differs from regular honey as it contains large amounts of methylglyoxal (MGO), which is

the dominant antibacterial constituent of Manuka honey.15

Methylglyoxal (MGO)

Figure 6 structure of methylglyoxal. Image taken from mpbio.com

In Manuka honey, MGO is converted from dihydroxyacetone, a substance that is found in

high concentration in the nectar of Manuka flowers. 16 MGO gives Manuka honey its

antibacterial properties. There is a greater concentration of MGO in Manuka honey than

regular honey.17 MGO is a by-product of glycolysis. MGO can recognize and target specific

15. "Methylglyoxal—A Potential Risk Factor of Manuka Honey in Healing of Diabetic Ulcers." Methylglyoxal—A Potential Risk Factor
of Manuka Honey in Healing of Diabetic Ulcers. N.p., n.d. Web. 19 June 2015.
<http://www.hindawi.com/journals/ecam/2011/295494/>.

16. "Manuka Honey Information ." Manuka Honey Information. N.p., n.d. Web. 15 June 2015.
<http://honeycentre.com/Manuka_Honey_Info.php>.
17. KRYMKIEWICZ, Norberto. "Properties and Mode of Action of a Bactericidal Compound (= Methylglyoxal) Produced by a Mutant of
Escherichia Coli." Properties and Mode of Action of a Bactericidal Compound (= Methylglyoxal) Produced by a Mutant of
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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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bacterial cells and inhibit protein synthesis by the bacterial cell.18 MGO inhibits penicillin

binding proteins (PBPs), namely PBP2A. PBP2A is responsible for the synthesis of

peptidoglycan; which is the main component of bacterial cell walls. 19 Peptidoglycan

synthesis is essential to cell growth and the maintenance of the cell wall structure. By

inhibiting PBP2A, peptidoglycan cannot be synthesized and this leads to irregularities in the

structure of the cell wall such as loss of selective permeability and eventually cell death. By

preventing the cell from producing the products required, the cell cannot sustain its metabolic

processes, causing it to die.

1.3 Hypothesis

As prior research has shown that MHLJ inhibits a large amount of bacterial growth, it is

hypothesized that MHLJ will inhibit the largest amount of bacterial growth, and as the

concentration of Manuka honey in MHLJ increases, the amount of bacterial growth inhibited

increases.

Escherichia Coli (n.d.): n. pag. Ncbi.nlm.nih.gov. Web. 19 June 2015.


<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC247224/pdf/jbacter00367-0404.pdf>.
18. Liu, Michael, Jing Lu, Patrick Müller, Lynne Turnbull, Catherine M. Burke, Ralf C. Schlothauer, Dee A. Carter, Cynthia B.
Whitchurch, and Elizabeth J. Harry. "Antibiotic-specific Differences in the Response of Staphylococcus Aureus to Treatment with
Antimicrobials Combined with Manuka Honey." Frontiers in Microbiology. Frontiers Media S.A., 27 Jan. 2015. Web. 19 June 2015.
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4307217/>.
19. Bata, Mari Msheila. "COMPARATIVE STUDY OF ANTIBACTERIAL EFFICACY OF HONEY, LEMON JUICE AND
STANDARD ANTIBIOTIC FORMULATIONS AGAINST STAPHYLOCOCCUS AUREUS ISOLATES FROM RESPIRATORY
TRACT INFECTIONS." Academia.edu. Academia.edu, n.d. Web. 19 June 2015.
<http://www.academia.edu/8852654/COMPARATIVE_STUDY_OF_ANTIBACTERIAL_EFFICACY_OF_HONEY_LEMON_JUIC
E_AND_STANDARD_ANTIBIOTIC_FORMULATIONS_AGAINST_STAPHYLOCOCCUS
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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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2.1 Variables

Independent variables

The first independent variable is the different types of antimicrobial substances, namely

honey, Manuka honey, lemon juice, honey-lemon juice and MHLJ.

The second independent variable is the concentration of the substance that inhibits the largest

amount of bacterial growth.

Dependent variable

The dependent variable is the amount of inhibited bacterial growth on the agar plates, which

is measured by the area of the zone of inhibition.

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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Controlled variables

Table 1 showing the controlled variables

Controlled variables How it is controlled


The location that the bacteria was Inside of mouth and throat was
swabbed from swabbed
The type of bacteria used Rod shaped, gram negative
bacteria from the same
inoculated colony was used for
all trials
The temperature that bacteria was Kept to its optimum temperature
cultured and inoculated in at 37°C for all trials
The pH value of the nutrient agar Kept to pH 6.2 for all trials and
monitored using a pH probe
The Unique Manuka factor of Kept to UMF 12+ for all trials
Manuka honey used
The brand of honey used Only the “Honey world” brand
for both Manuka honey and
honey was used
Volume of nutrient agar used per petri 5.00 cm3 was used for all trials
dish
Volume of sterile nutrient broth used 2.00 cm3 was used for all trials
Volume of gram negative bacteria 0.15 cm3 was used for all trials
used per petri dish
Volume of antimicrobial substance 0.02 cm2 was used for all trials
used per petri dish
Volume of Gram Crystal violet 10.00 cm2 was used
solution used when gram staining
Volume of Gram Lugol’s Iodine 10.00 cm2 was used
solution used when gram staining
Volume of Gram Decolorizer solution 10.00 cm2 was used
used when gram staining
Volume of Gram Safranin solution 10.00 cm2 was used
used when gram staining
The total duration that the bacteria Each was cultured for 48 hours
was left to be cultured

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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2.2 Apparatus

1 Laminar flow cabinet

20 Sterile petri dishes

5 Sterile inoculating loops

1 Microscope

1 Vernier caliper

1 pair of tongs

1 pair of tweezers

1 Bunsen burner

1 Lighter

3 Stopwatches

5 1ml syringes

5 5ml syringes

5 10ml syringes

2.3 Materials

1000.00 cm3 of Nutrient agar

5 Sterile cotton swabs

20 strips of Parafilm

1 pair of gloves

1 Microscope slide

1 Coverslip

5 2.00 cm3 Micro centrifuge tubes

100 pieces of 6mm filter paper

500.00 cm3 of distilled water

10.00 cm3 of Ethanol


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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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10.00 cm3 of Gram Crystal violet solution

10.00 cm3 of Gram Lugol’s Iodine solution

10.00 cm3 of Gram Decolorizer solution

10.00 cm3 of Gram Safranin solution

10.00 cm3 of Sterile nutrient broth

150.00 cm3 Honey

150.00 cm3 Manuka Honey

150.00 cm3 Fresh lemon juice

2.4 Procedure (PART A)

I. Preparation of agar plates

A petri dish was filled with 15.00 cm3 of nutrient agar and covered with a lid. The lid was

removed and the agar plate was left in the hood to dry and harden. The petri dish was covered

and sealed with Parafilm. This was repeated 25 times to produce 25 agar plates.

II. Preparation of bacterial cultures

A sterile swab was used to swab the inside of the mouth and throat for bacteria. The swab

was moved over an agar plate in a zigzag manner. The petri dish was incubated for 48 hours

at 37°C. Once bacterial colonies were visible, the petri dish was refrigerated.

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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III. Inoculation of bacteria

A visible colony was chosen from the agar plate in II that was distinct from the rest. A

sample of the bacterial colony was collected with a sterile inoculating loop. The sample was

smeared on a new agar plate. The inoculating loop

was used to streak 4 lines, extending from the initial

smear site. (See 1) Another 4 lines were streaked

from the previous streaks. (See 2) This was repeated

twice, giving a total of 16 lines. This was done to

spread out the bacteria on the agar plate. (See 3 + 4)

The agar plate was incubated for 48 hours at 37°C.

Once the bacteria colonies were visible, the agar


Figure 7 showing streaking of agar plates
plates were refrigerated.

IV. Gram staining of bacterial colony

A drop of water was placed on a glass slide. An inoculating loop was used to collect a sample

from the bacterial colony that was inoculated in III. The sample was smeared on the glass

slide and mixed with the water. The slide held over the Bunsen burner for 2-3 seconds before

being removed from the flame. The slide was left to cool for 3 minutes. 10.00 cm3 of Gram

crystal violet solution was poured over the area that the bacteria were smeared on. After 30

seconds, the solution was washed off with distilled water. 10.00 cm3 of Gram Lugol’s Iodine

solution was poured over the slide. After 90 seconds, the solution was washed off with

distilled water. 10.00 cm3 of Gram Decolorize solution was poured over the slide. After 5

seconds, the slide was washed with distilled water. 10.00 cm3 of Gram Safranin solution was

poured over the slide. After 30 seconds, the slide was washed with distilled water. The slide

was pat dry with a paper towel. A drop of water was placed over the area with bacteria and

covered with a coverslip. The bacteria were identified under the microscope.
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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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V. Preparation of bacterial broth

2.00 cm3 of sterile nutrient broth was measured into a micro centrifuge tube with a syringe.

An inoculating loop was used to introduce another sample of the bacteria that was cultured in

III into the nutrient broth. The broth was left in the laminar flow for 24 hours until the

solution appeared cloudy.

VI. Dilution of antimicrobial substances

Table 2 showing the dilution of the antimicrobial substances

Antimicrobial Volume of 100.0% Volume of distilled Final concentration


substance stock substance water /cm3 of antimicrobial
/cm3 substance /%
Honey 12.00 8.00 60.0
Manuka honey 12.00 8.00 60.0
Lemon juice 8.00 12.00 40.0

The antimicrobial substances were diluted according to the table above.

When making the honey-lemon juice mixture, a 10.00 cm3 solution with 6.00 cm3 of 100.0%

honey and 4.00 cm3 of 100.0% lemon juice was made. This was repeated for the MHLJ

mixture. Note that in a mixture, the former value is the concentration of honey and the latter

value is the concentration of lemon juice. For example, 60.0%-40.0% honey lemon juice is

60.0% honey and 40.0% lemon juice.

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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VII. Kirby-Bauer Test

0.02 cm3 of honey was added onto a piece of circular 6 mm filter paper and left to dry. This

was repeated five times to produce 5 pieces of filter paper with honey. 0.15 cm3 of the cloudy

bacterial broth from V was introduced onto a new agar plate. The broth was left to set for 15

minutes. The glass rod was sterilized each time after spreading by soaking in ethanol and

heating it. The 5 dry pieces of filter paper were placed on each agar plate. The filter papers

were equidistant from one another.

This was repeated to produce 2 agar

plates with a total of 10 pieces of

filter paper containing honey. (Refer

to figure 8) All the steps above were

repeated for the remaining

antimicrobial substances; lemon

juice, Manuka honey, honey-lemon Figure 8 showing arrangement of filter paper discs

juice and MHLJ. (See figure 9 in next page) All the agar plates were left in the laminar flow

for 24 hours. The smallest and largest radius of the zone of inhibition that appeared around

each filter paper was measured using a Vernier caliper. (See figure 10 in next page) The area

of the inhibition of bacterial growth was calculated by multiplying the smallest and largest

radius of the zone of inhibition with π. For the negative control, a piece of filter paper that

contained distilled water was used. The smallest and largest radius of the zone of inhibition

for the negative control was measured as well. A graph of the average area of the zone of

inhibition was plotted against each antimicrobial substance.

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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Figure 9 showing the set-up for 1 replicate of the Kirby-Bauer test

Honey Manuka honey Lemon juice

Honey-lemon juice Manuka honey-lemon juice Distilled water (Control)

Figure 10 showing the method of measuring the radii of the zone of inhibition

Red line: Measures the smallest radius


Blue line: Measures the largest radius

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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3.1 Results (PART A)
Table 3 showing the smallest and largest radius of the zone of inhibition for the five different antimicrobial substances.

Antimicrobial substance
Manuka Manuka Lemon Lemon Honey-lemon Manuka honey-
Honey Honey
Honey Honey Juice Juice juice (60- lemon juice (60.0-
(60.0%) (100.0%)
(60.0%) (100.0%) (40.0%) (100.0%) 40.0%) 40.0%)
Trial Smallest radius 0.46 0.47 0.48 0.55 0.26 0.40 0.41 0.59
1 Largest radius 0.50 0.47 0.51 0.55 0.31 0.40 0.42 0.62
Trial Smallest radius 0.48 0.50 0.51 0.56 0.30 0.31 0.39 0.50
2 Largest radius 0.49 0.49 0.53 0.58 0.32 0.35 0.43 0.55
Trial Smallest radius 0.50 0.51 0.44 0.49 0.30 0.29 0.40 0.62
3 Largest radius 0.51 0.39 0.45 0.53 0.33 0.33 0.45 0.64
Trial Smallest radius 0.39 0.42 0.40 0.53 0.26 0.31 0.40 0.43
The radius 4 Largest radius 0.40 0.45 0.46 0.55 0.30 0.34 0.41 0.49
of the Smallest radius 0.43 0.46 0.50 0.49 0.30 0.32 0.39 0.55
Trial
zone of 5 Largest radius 0.50 0.48 0.54 0.50 0.33 0.35 0.40 0.56
inhibition
Trial Smallest radius 0.38 0.37 0.51 0.51 0.27 0.41 0.43 0.55
/cm
6 Largest radius 0.41 0.40 0.51 0.53 0.30 0.44 0.45 0.58
(±0.01
cm) Trial Smallest radius 0.40 0.45 0.47 0.58 0.25 0.36 0.52 0.60
7 Largest radius 0.41 0.49 0.48 0.59 0.29 0.38 0.55 0.61
Trial Smallest radius 0.40 0.50 0.50 0.45 0.26 0.40 0.51 0.62
8 Largest radius 0.44 0.50 0.52 0.47 0.30 0.41 0.53 0.65
Trial Smallest radius 0.37 0.53 0.45 0.49 0.31 0.39 0.43 0.55
9 Largest radius 0.41 0.54 0.47 0.53 0.32 0.42 0.47 0.58
Trial Smallest radius 0.38 0.43 0.50 0.47 0.30 0.34 0.42 0.52
10 Largest radius 0.45 0.44 0.53 0.50 0.33 0.35 0.45 0.57

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
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Table 4 showing the area of the zone of inhibition for the five different antimicrobial substances.

Antimicrobial substance
Manuka Manuka Lemon Lemon Honey-lemon Manuka honey-
Honey Honey
Honey Honey Juice Juice juice (60- lemon juice (60.0-
(60.0%) (100.0%)
(60.0%) (100.0%) (40.0%) (100.0%) 40.0%) 40.0%)
Trial 1 0.723 0.694 0.769 0.950 0.253 0.503 0.541 1.149

Trial 2 0.739 0.770 0.849 1.021 0.302 0.341 0.527 0.864

Trial 3 0.801 0.625 0.622 0.816 0.311 0.301 0.566 1.247

Trial 4 0.490 0.594 0.578 0.916 0.245 0.331 0.515 0.662

Trial 5 0.676 0.694 0.848 0.770 0.311 0.352 0.490 0.968


The area
of the Trial 6 0.490 0.465 0.817 0.849 0.255 0.567 0.608 1.002
zone of
inhibitio
Trial 7 0.515 0.693 0.709 1.075 0.228 0.430 0.899 1.150
n/ cm2
Trial 8 0.553 0.786 0.817 0.665 0.245 0.515 0.849 1.266

Trial 9 0.477 0.899 0.665 0.816 0.312 0.515 0.635 1.002

Trial 10 0.537 0.594 0.833 0.738 0.311 0.374 0.594 0.931


Average 0.600 0.681 0.751 0.862 0.277 0.423 0.622 1.024
Standard
0.122 0.121 0.100 0.128 0.035 0.095 0.140 0.185
deviation

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________

Sample calculations:

Area of zone of inhibition = Area of ellipse = π x smallest radius x largest radius

Using trial 1 of 60.0% honey as an example,

Area of zone of inhibition = π x 0.46 x 0.50

= 0.723 cm2

Using the results from 60.0% honey as an example,

Average of the area of zone of inhibition

= (0.723 + 0.739 + 0.801 + 0.490 + 0.676 + 0.490 + 0.515 + 0.553 + 0.477 +0.537) / 10

= 0.600 cm2

Standard deviation was calculated using Microsoft Excel.

Figure 11 showing the self-cultured bacteria

From the absence of staining, it can be seen


that it is gram-negative bacteria with a rod
shape. It is assumed to resemble
Escherichia coli. (E. coli)

23
Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
Graph 1 showing the average area of the zone of inhibition in cm2 against different antimicrobial substances as a display of the amount of bacterial growth

inhibited.

Graph 1 showing the average area of the zone of inhibition against different antimicrobial substances
1.2

1
average area of the zone of inhibition /cm2

0.8

0.6

0.4

0.2

0.495 0.614 0.680 0.758 0.246 0.400 0.595 0.910


0
Honey (60.0%) Honey (100.0%) Manuka Honey Manuka Honey Lemon Juice Lemon Juice Honey-lemon juice Manuka honey-
(60.0%) (100.0%) (40.0%) (100.0%) (60-40.0%) lemon juice (60.0-
40.0%)
Antimicrobial substances

24
Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
From the results obtained in tables 3 and 4 and graph 1, it can be seen that MHLJ has the

largest average area of zone of inhibition at 0.910 cm2 and thus inhibits the largest amount of

bacterial growth. This shows that MHLJ is the best inhibitor of bacterial growth as compared

to the other antimicrobial substances. This substance will proceed to be diluted to different

concentrations in the second part of the investigation procedure.

Table 5 showing the concentrations of Manuka honey-lemon juice.

Concentration
Manuka Honey /% Lemon Juice/%
40.0 60.0
50.0 50.0
60.0 40.0
70.0 30.0
80.0 20.0

Negative control

After incubating for 24 hours, there was no zone of inhibition present in the negative control.

This shows that the bacterial growth and lack thereof is attributed only by the antimicrobial

substances tested and not by any external factors.

25
Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
2.4 Procedure (PART B)

I. Dilution of Manuka honey-lemon juice

Table 6 showing dilution of Manuka honey-lemon juice

Antimicrobial Volume of 100.0% Volume of distilled Final concentration


substance stock substance water /cm3 of antimicrobial
/cm3 substance /%
Manuka honey 8.00 12.00 40.0
Manuka honey 10.00 10.00 50.0
Manuka honey 12.00 8.00 60.0
Manuka honey 14.00 6.00 70.0
Manuka honey 16.00 4.00 80.0
Lemon juice 4.00 16.00 20.0
Lemon juice 6.00 14.00 30.0
Lemon juice 8.00 12.00 40.0
Lemon juice 10.00 10.00 50.0
Lemon juice 12.00 8.00 60.0

Dilute the antimicrobial substances according to the table above.

II. Kirby-Bauer test

0.02 cm3 of 40.0%-60.0% MHLJ was added onto a piece of circular filter paper and left it to

dry. 0.15 cm3 of the cloudy bacterial broth from IV in procedure part A was introduced onto a

new agar plate and spread out. 5 dry pieces of filter paper were placed on the agar plate with

tweezers. This was repeated to produce 2 agar plates with a total of 10 pieces of filter paper

containing honey. All the steps above were repeated for the remaining concentrations of

MHLJ at 50.0%-50.0%, 60.0%-40.0%, 70.0%-30.0% and 80.0%-20.0%. All the agar plates

were left in the laminar flow for 24 hours. The smallest and largest radius of the zone of

inhibition was measured with a Vernier caliper. (See figure 6) The area of the inhibition of

bacterial growth was calculated by multiplying the smallest and largest radius of the zone of

inhibition with π. A graph of the average area of the zone of inhibition was plotted against

each concentration of MHLJ.

26
Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
3.2 Results (PART B)

Table 7 showing the first 5 trials of the smallest and largest radius of the zone of inhibition for the different concentrations of Manuka honey-lemon juice.

Concentration of Manuka honey-lemon juice /%


40.0%-60.0% 50.0%-50.0% 60.0%-40.0% 70.0%-30.0% 80.0%-20.0%
Smallest radius 0.17 0.37 0.59 0.60 0.65
Trial 1
Largest radius 0.20 0.39 0.62 0.62 0.68
Smallest radius 0.19 0.40 0.50 0.57 0.66
Trial 2
Largest radius 0.19 0.41 0.55 0.60 0.69
Smallest radius 0.32 0.40 0.62 0.56 0.69
Trial 3
Largest radius 0.38 0.42 0.64 0.57 0.70
Smallest radius 0.37 0.44 0.43 0.59 0.66
Trial 4
Largest radius 0.41 0.45 0.49 0.62 0.68
Smallest radius 0.30 0.43 0.55 0.62 0.70
Trial 5
Largest radius 0.32 0.45 0.56 0.65 0.71
The radius of the zone of inhibition /cm (±0.01 cm) Smallest radius 0.24 0.46 0.55 0.57 0.60
Trial 6
Largest radius 0.28 0.47 0.58 0.60 0.62
Smallest radius 0.25 0.40 0.60 0.64 0.66
Trial 7
Largest radius 0.30 0.41 0.61 0.66 0.66
Smallest radius 0.22 0.40 0.62 0.60 0.67
Trial 8
Largest radius 0.24 0.42 0.65 0.60 0.70
Smallest radius 0.28 0.48 0.55 0.57 0.68
Trial 9
Largest radius 0.31 0.50 0.58 0.59 0.70
Smallest radius 0.27 0.46 0.52 0.60 0.70
Trial 10
Largest radius 0.29 0.47 0.57 0.61 0.71

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
Table 8 showing the area of the zone of inhibition for the five different concentrations of Manuka honey-lemon juice.

Concentration of Manuka honey-lemon juice /%


40.0%-60.0% 50.0%-50.0% 60.0%-40.0% 70.0%-30.0% 80.0%-20.0%
Trial 1 0.107 0.453 1.149 1.169 1.389

Trial 2 0.113 0.515 0.864 1.075 1.431

Trial 3 0.382 0.528 1.247 1.003 1.518

Trial 4 0.477 0.622 0.662 1.149 1.410

Trial 5 0.302 0.608 0.968 1.266 1.562

The area of the zone of inhibition/ cm2 Trial 6 0.211 0.679 1.002 1.075 1.169

Trial 7 0.236 0.515 1.150 1.327 1.369

Trial 8 0.166 0.528 1.266 1.131 1.474

Trial 9 0.273 0.754 1.002 1.057 1.496

Trial 10 0.246 0.679 0.931 1.150 1.562

Average 0.251 0.588 1.024 1.140 1.438


Standard deviation 0.115 0.095 0.185 0.098 0.116

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
Table 9 showing the p-values of T-Tests of the area of the zone of inhibition recorded after being

exposed to the different concentrations of Manuka honey-lemon juice.

Concentration of 40.0-60.0 50.0-50.0 60.0-40.0 70.0-30.0 80.0-20.0


Manuka honey-
lemon juice /%
40.0-60.0 0.00 0.00 0.00 0.00
50.0-50.0 0.00 0.00 0.00
60.0-40.0 0.06 0.00
70.0-30.0 0.00
80.0-20.0

The T-test was calculated using Microsoft Excel using tails 2 and type 3.

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
Graph 2 showing the average area of the zone of inhibition in cm2 against the different concentrations of Manuka honey-lemon juice.

Graph 2 showing the average area of zone of inhibition against the different concentrations of Manuka
honey-lemon juice
1.600
R² = 0.976
1.400

1.273
1.200
Average area of zone of inhibition /cm2

1.000
1.030
0.900

0.800

0.600
0.556

0.400

0.225
0.200

0.000
40.0%-60.0% 50.0%-50.0% 60.0%-40.0% 70.0%-30.0% 80.0%-20.0%

Concentration of Manuka honey-lemon juice /%

30
Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
3.3 Discussion of results

From the results in Part A, it can be seen that 60.0%-40.0% MHLJ inhibited the largest amount

of bacterial growth than the other four substances. The average area of the zone of inhibition

by MHLJ is 0.910 cm2. The standard deviations of the substances were relatively small

below 0.185 cm2. This is 18.1% of the average value for the area. Thus, the results collected

are relatively close together and are considered quite precise and reliable.

40.0% Lemon juice inhibited the least amount of bacterial growth, with an average area of

only 0.246 cm2. This is almost 3 times lesser than the area inhibited by MHLJ. This is due to

its active compound, limonene. From the literature review, it is known that limonene can

cause cell death by binding and altering the conformation of proteins like GroEL and GroES,

therefore preventing them from functioning. Thus resulting in the mis-folding of outer

membrane proteins. As their binding sites are mis-shaped, the proteins cannot facilitate signal

transduction. Should the cell receive a signal to induce the synthesis of products vital to the

survival of the cell, it cannot respond. This could lead to cell death. However, limonene has

lower efficacy on gram-negative bacteria, which is the bacteria used in this investigation. In a

gram-negative bacterial cell, the presence of a selectively permeable outer membrane of

lipopolysaccharide restricts the movement of substances in and out of the cell. Limonene

cannot penetrate into the cell and induce apoptosis. Thus, the bacteria used in the

investigation were less susceptible to the effects of limonene. Hence, lemon juice inhibited

the least amount of bacterial growth.

Honey had an average area of zone of inhibition 0.680 cm2 and Manuka honey had an average

area of zone of inhibition 0.758 cm2. This is attributed to the honeys’ hydrogen peroxide

activity. H2O2 can oxidize the electron carriers on the ETC and remove an electron from the

ETC of the cell, and cause it to die. By removing the electron, energy cannot be released as it
31
Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
progresses down the chain. There will be no energy for the metabolic processes of the cell and

hence it cannot sustain itself.

Manuka honey inhibited the second largest amount of bacterial growth, with an average area

of 0.758 cm2. Honey inhibited a slightly lesser amount of bacterial growth, with an average

area of 0.680 cm2. Although these two substances are honeys, the abundance of the active

compound MGO in Manuka honey causes Manuka honey to inhibit a larger amount of

bacterial growth than honey. From the literature review, Manuka honey has larger

concentration of MGO than regular honey. Even though both honeys were diluted to 60.0%,

Manuka honey still contains a greater concentration of MGO than honey. At higher

concentrations, there are more MGO molecules available to recognize and bind to more

bacterial cells. There will be more PBP2A proteins being modified irreversibly resulting in

fewer remaining normal PBP2A proteins. This results in more bacterial cells with non-

functional proteins that cannot synthesize peptidoglycan needed for the cell wall. This would

lead to a larger amount of bacterial cells with irregular and weaker cell walls. These cell walls

are more vulnerable to lysis, which causes the break down of the cell and eventual cell death.

Therefore higher concentrations of Manuka honey inhibited a larger amount of bacterial

growth than the lower concentrations. Thus, Manuka honey had a larger area of the zone of

inhibition than honey.

According to the research done, lemon juice is more effective against gram-positive bacteria

while the honeys are effective against both gram-positive and gram-negative bacteria. As

gram-negative bacteria were used in this investigation, this explains why the honeys inhibited

more bacterial growth than lemon juice.

32
Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
Figure 12 showing the results from Part B.

40.0%-60.0% 50.0%-50.0% 60.0%-40.0% 70.0%-30.0% 80.0%-20.0%


Manuka honey- Manuka Manuka honey- Manuka honey- Manuka honey-
lemon juice honey-lemon lemon juice lemon juice lemon juice
juice

From the results in Part B, it can be seen that as the concentration of Manuka honey in a

MHLJ mixture increases from 40.0% to 80.0%, the average area of the zone of inhibition

increases from 0.225 cm2 to 1.273 cm2. From figure 12, it can be seen that the zones of

inhibition grow larger in radius as the concentration of Manuka honey in MHLJ increases.

There is a positive correlation between the concentration of Manuka honey in MHLJ and the

average area of the zone of inhibition. This is evident from the R2 value at 0.976. The value is

approaching 1, showing that the data is strongly positively correlated. The data collected for

each concentration is statistically significant (p < 0.05) between one another. However, data

collected for each concentration is statistically insignificant (p > 0.05) between 70.0%-30.0%

and 60.0%-40.0%. This suggests that the increase in concentration of MHLJ causes the

increase in the average area of the zone of inhibition.

The standard deviations of the substances were relatively small below 0.185 cm2. This is

18.1% of the average value for the area. Thus, the results collected are relatively close

together and are considered quite precise and reliable.

Limonene in lemon juice is not very effective against gram-negative bacteria, thus it can be

seen that the decrease in the concentration of lemon juice in MHLJ does not affect the average
33
Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
area of the zone of inhibition. This shows that when combined, Manuka honey has a greater

effect over the amount of inhibition of bacterial growth than lemon juice. As the

concentration of Manuka honey in the mixture increases, the amount of inhibition of bacterial

growth increases. Doubling the concentration of Manuka honey caused the average area of the

zone of inhibition to increase by five times. As the concentration of Manuka honey in the

mixture increases, the amount of MGO molecules increases. There are more MGO molecules

available to recognize and bind to more bacterial cells. There will be more proteins being

modified irreversibly resulting in fewer remaining normal proteins. This results in more

bacterial cells with non-functional proteins that cannot produce the products needed for the

cell to survive. The results collected reflect the synergistic effects of Manuka honey and

lemon juice when used together. The average area of the zone of inhibition for 80.0%-20.0%

MHLJ at 1.273 cm2 is higher than 100.0% Manuka honey and 100.0% lemon juice at 0.758

cm2 and 0.400 cm2 respectively. The effects of the two individual substances are not as

effective as compared to when they are combined, showing their synergistic effects. Therefore

as the concentration of Manuka honey increases, the area of the zone of inhibition increases.

Existing research conducted on honey shows that it has antibacterial properties on

staphylococcus aureus (S. aureus), which is gram-negative and the same class –

Gammaproteobacteria – as the bacteria used in this investigation, which is assumed to be E.

coli.21 Being the same class, it is assumed that they will react to the honeys and lemon juice

similarly. This supports the data collected in the experiment and is concurrent with the

literature reviews.

34
Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
4. Conclusion

The hypothesis proposed for the experiment is proven to be true. MHLJ inhibited the largest

amount of bacterial growth and as the concentration of Manuka honey in MHLJ increased, the

average area of the zone of inhibition increases.

5. Evaluation

One limitation of this investigation is that bacteria inoculated from the saliva found in the

mouth and throat was used. This bacterium might not be a causal factor of sore throat. To

improve on this use a pure strain of Streptococcus pyogenes, the bacteria that causes strep

throat.

Another limitation is that the Manuka honey was left in the open for long periods of time

during the experiments. Manuka honey might lose efficacy after some period of time in the

sun and the results reflected might not show its optimum abilities. An improvement on this

would be conducting the experiment in a dark room to minimize sunlight.

5.2 Further extensions

Further studies with pure isolates of the active compounds can be performed to ascertain if

similar effects were observed as compared to this experiment. This is done to exclude any

other cofounders present in the substance.

Further studies with a different preparation method of lemon juice can be performed as some

people use the rind of the lemon peel as well. This can broaden the understanding of lemon’s

inhibitory effects.

Further studies with lemons can be performed to determine if they have anti- proliferative

effects on cells using the data collected as a baseline.

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Candidate name: Tan Hui Min, Rachel
Candidate session number: 004130-0113
_____________________________________________________________________
6. Bibliography

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Candidate name: Tan Hui Min, Rachel
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 "Gram Stain Technique." Value@Amrita. Amrita Vishwa Vidyapeetham University,

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specific Differences in the Response of Staphylococcus Aureus to Treatment with

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Candidate name: Tan Hui Min, Rachel
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Degenerative Processes: Genotoxicity, Antigenotoxicity, Cytotoxicity, and Longevity in


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Candidate name: Tan Hui Min, Rachel
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Drosophila." National Center for Biotechnology Information. U.S. National Library of

Medicine, n.d. Web. 19 June 2015. <http://www.ncbi.nlm.nih.gov/pubmed/21707429>.

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