HL BIO IB Extended Essay

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

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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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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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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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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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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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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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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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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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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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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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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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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.
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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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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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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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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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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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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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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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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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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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
2 Largest radius 0.49 0.49 0.53 0.58 0.32 0.35 0.43 0.55
3 Largest radius 0.51 0.39 0.45 0.53 0.33 0.33 0.45 0.64
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
/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
8 Largest radius 0.44 0.50 0.52 0.47 0.30 0.41 0.53 0.65
9 Largest radius 0.41 0.54 0.47 0.53 0.32 0.42 0.47 0.58
10 Largest radius 0.45 0.44 0.53 0.50 0.33 0.35 0.45 0.57
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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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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)
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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
_____________________________________________________________________
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
_____________________________________________________________________
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
_____________________________________________________________________
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
27
_____________________________________________________________________
Table 8 showing the area of the zone of inhibition for the five different concentrations 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
28
_____________________________________________________________________
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.
29
_____________________________________________________________________
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%
30
_____________________________________________________________________
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
_____________________________________________________________________
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
_____________________________________________________________________
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
_____________________________________________________________________
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
_____________________________________________________________________
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.
35
_____________________________________________________________________
6. Bibliography
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<http://www.abbvie.com/oncology/home/apoptosis.html>.
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<http://www.microbiologyonline.org.uk/about-microbiology/introducing-
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 "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/>.
 Bata, Mari Msheila. "COMPARATIVE STUDY OF ANTIBACTERIAL EFFICACY OF
HONEY, LEMON JUICE AND STANDARD ANTIBIOTIC FORMULATIONS
AGAINST STAPHYLOCOCCUS AUREUS ISOLATES FROM RESPIRATORY
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<http://www.academia.edu/8852654/COMPARATIVE_STUDY_OF_ANTIBACTERIAL
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 "Common Cold." Cold Remedies: What Works, What Doesn't, What Can't Hurt. N.p., n.d.
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 "Effects of Biocides on Antibiotic Resistance." Biocides: 4. How Can Bacteria Become
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<http://ec.europa.eu/health/opinions/en/biocides-antibiotic-resistance/l-2/4-
mechanisms.htm>.
 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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 "Gram Stain Technique." Value@Amrita. Amrita Vishwa Vidyapeetham University,
n.d. Web. 19 June 2015. <
http://vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=2>.
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home/learn-about-honey/>.
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June 2015. <https://www.organicfacts.net/health-benefits/fruit/health-benefits-of-
lemon.html>.
 Hudzicki, Jan. "Kirby-Bauer Disk Diffusion Susceptibility Test Protocol." ASM
MicrobeLibrary. N.p., 8 Dec. 2009. Web. 19 June 2015. <
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bauer-disk-diffusion-susceptibility-test-protocol>.
 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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<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC247224/pdf/jbacter00367-0404.pdf>.
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<http://pubchem.ncbi.nlm.nih.gov/compound/Dipentene#section=Experimental-
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specific Differences in the Response of Staphylococcus Aureus to Treatment with
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Antimicrobials Combined with Manuka Honey." Frontiers in Microbiology. Frontiers
Media S.A., 27 Jan. 2015. Web. 19 June 2015.
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Alternative Daily. N.p., n.d. Web. 19 June 2015.
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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

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

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

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

the efficacy and mechanisms of honey-lemon juice have not

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

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

to obtain a representation of the inhibition of bacterial growth.

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

gram-negative bacterial growth?

bacterial growth and cure the infection.

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

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

suitable alternative treatment for sore throat.

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

optimum concentration for it to inhibit bacterial growth.

substance affect the inhibition of bacterial growth.

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

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-

Figure 2 Citrus limon. Image taken from sahultrading.com

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

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

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

Figure 4 Honey. Image taken from getnativ.com

primary active compound is hydrogen peroxide.14

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

the dominant antibacterial constituent of Manuka honey.15

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

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

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

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

processes, causing it to die.

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

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

amount of bacterial growth.

is measured by the area of the zone of inhibition.

Table 1 showing the controlled variables

Controlled variables How it is controlled

1 Laminar flow cabinet

20 Sterile petri dishes

5 Sterile inoculating loops

1000.00 cm3 of Nutrient agar

5 Sterile cotton swabs

5 2.00 cm3 Micro centrifuge tubes

100 pieces of 6mm filter paper

500.00 cm3 of distilled water