International Baccalaureate Diploma Programme Sri KDU Smart School
Extended Essay -Biology-
In vitro study of the Synergism between Essential Oils, Cinnamon (Cinnamomum zeylanicum) and Clove (Syzygium aromaticum) in Inhibiting Growth of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923
Candidate Name: Michelle Tan Oon Ee Candidate Number: 002206-006 Word Count: 3992 words
Michelle Tan Oon Ee
002206-006 Abstract
Cinnamon (Cinnamomum zeylanicum) and clove (Syzygium aromaticum) are natural spices and remedies used to combat bacterial infection. The active ingredient that contains antimicrobial properties is primarily found in the spices’ essential oils. As spices are rarely used individually, the combination of essential oils of cinnamon and clove will produce a synergistic effect to inhibit bacteria growth. Thus, my research question is the In vitro study of the Synergism between Essential Oils, Cinnamon (Cinnamomum zeylanicum) and Clove (Syzygium aromaticum) in Inhibiting Growth of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923.
The Agar Disk Diffusion Method is used to determine antimicrobial effects of essential oils. The essential oils are obtained through steam distillation and extracted with dichloromethane. Different percentages of individual essential oil concentrations and different combinations of percentage volume per volume ratio concentrations are pipetted onto filter paper discs. The discs are positioned on agar plates and incubated for 24 hours at 37°C. The diameter of the clear area around the disc or inhibition zone is measured. The wider the diameter of inhibition zone, the higher the antimicrobial activity.
Analysis through ANOVA and Tukey’s HSD test show that cinnamon is a stronger antimicrobial agent against both bacteria strains. However, Staph. a is less resistant to the essential oils than E.coli. Higher percentage concentrations for cinnamon and clove produce larger inhibition zones. There are no synergistic effects identified as 100% individual cinnamon concentration gives the greatest antimicrobial activity. However, the most significantly different percentage cinnamon volume per clove volume ratio combination is (90% : 10%). Small amounts of cinnamon enhance antimicrobial activities of cloves but combined mixtures work optimally at higher concentrations of cinnamon essential oils. Thus, for best antimicrobial effects, larger quantities of cinnamon should be used in cooking. (290 words) Page | 2
Michelle Tan Oon Ee
002206-006 Acknowledgement
I wish to express my utmost gratitude to the individuals that have provided assistance and encouragement to me in the completion of this extended essay. Mr Lawrence Kok, my dedicated supervisor My wonderful parents and brother, Nicklaus Mr Masukor Sari My fellow EE mates: Ken and Eugene My dear seniors: Ju Anne and Jorrel
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Tables of Contents Abstract ................................................................................................................................................ 2 Acknowledgement ............................................................................................................................... 3 Tables of Contents ............................................................................................................................... 4 1.0
Introduction ............................................................................................................................... 6
1.1 Rationale of study ...................................................................................................................... 6 1.2 Cinnamon (Cinnamomum zeylanicum) ..................................................................................... 8 1.3 Clove (Syzygium aromaticum) .................................................................................................. 9 1.4 Two Types of Bacteria ............................................................................................................ 10 2.0
Hypotheses .............................................................................................................................. 11
2.1 Effects of Cinnamon and Clove on E.coli and Staph. a. ......................................................... 11 2.2 Synergistic effects of the combination of Cinnamon and Clove ............................................. 12 3.0
Variables .................................................................................................................................. 13
3.1 Manipulated Variables............................................................................................................. 13 3.2 Responding Variables .............................................................................................................. 13 3.3 Constant Variables................................................................................................................... 14 4.0
Materials and Methods ............................................................................................................ 15
4.1 Preparation of Mueller-Hinton Agar (MHA) .......................................................................... 15 4.2 Preparation of Sterilised Filter Paper Discs and Autoclaving Procedures .............................. 15 4.3 Preparation of Inoculums ........................................................................................................ 15 4.4 Extraction of Cinnamon Essential Oil by Steam Distillation .................................................. 16
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4.5 Extraction of Clove Essential Oil by Steam Distillation ......................................................... 17 4.6 Preparation of Cinnamon and Clove Percentage Ratios.......................................................... 18 4.7 Disk Diffusion Test (Kirby Bauer Method) ............................................................................ 19 4.8 Incubation of Agar Plates ....................................................................................................... 20 5.0
Data Collection ........................................................................................................................ 21
5.1 Data Processing ....................................................................................................................... 23 5.2 Data Analysis: ANOVA .......................................................................................................... 27 5.3 Tukey’s HSD Test ................................................................................................................... 28 6.0
Conclusion and Evaluations .................................................................................................... 30
6.1 Discussion................................................................................................................................ 30 6.1.1 Different Concentrations of Cinnamon and Clove Essential Oils. .................................. 30 6.1.2 Different Percentage Ratio of Combinations of Cinnamon and Clove Essential Oils ..... 30 6.1.3 Synergistic Effects of Cinnamon and Clove. ................................................................... 31 6. 2 Limitations ............................................................................................................................... 32 6.3 Ways to Improve Experiment .................................................................................................. 33 6.4 Areas of Further Investigations ............................................................................................... 34 6.5 Conclusion ............................................................................................................................... 35 7.0
References ............................................................................................................................... 36
8.0
Appendixes .............................................................................................................................. 39
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1.0
Introduction
1.1
Rationale of study
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In our technologically driven world, new improved antibiotics are constantly being produced to cure bacterial diseases. However, antibiotics are losing effectiveness as increasing numbers are used in animal feed [¹]. Countries like the EU and USA are phasing out antibiotics usage for animal growth, some of which are also human medicine. This overusage causes bacteria to undergo mutation or acquire antibiotic resistance genes from other bacteria. Today, more than 90% of Staphylococcus aureus strains are resistant to penicillin and other beta-lactam antibiotics [²]. Before modern drug therapy, natural antibiotics were common remedies for bacterial infections. Now, there is a renewed interest for natural approaches as many spice products have shown inhibition of pathogenic bacteria growth and can potentially replace synthetic antibiotics. These antimicrobial compounds have distinct structures and modes of action that differ from synthetic antibiotics, suggesting that if taken together, cross-resistance will be minimal. The selected spices are globally used as ingredients in cooking. Cinnamon and clove have strong inhibitory effectiveness to bacterial growth1 but the synergistic effects of a combination of both spices are not thoroughly established. In traditional Chinese medicine, cinnamon is commonly used for colds, flatulence, nausea and diarrhoea. Cinnamon, an ingredient in chai tea, aids with digestion of dairy products and fruits [³]. Clove heals digestive system ailments and promotes quick metabolism. Clove essential oil, an antiseptic, is useful for wounds, scabies, athlete’s foot and fungal infections [⁴]. Both spices also reduce glucose and cholesterol levels [⁵]. The spices give “Biryani” rice, a Malaysian dish, a rich aroma and enhance flavours in many local curries. Cinnamon is a marinating ingredient in Chinese stewed pork and clove is often used as garnish.
1
Refer to Appendix 1
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Most cuisines uses combination of spices and thus, there may be possible enhanced antimicrobial effects when cinnamon and clove are mixed. Synergism occurs when the effects of two spices is greater than the effect of each spice individually. The presence of one antimicrobial property will multiply the effects of the other and thus, known as a synergistic effect. The optimum combination ratio of percentage volume per volume concentration will produce the highest inhibition zone. Escherichia coli (E.coli) and Staphylococcus aureus (Staph. a) are common bacteria found in human intestines. Both cause diarrhoea, digestive diseases, food poisoning and skin infections. As remedies for such diseases, the spices should inhibit the growth of these bacteria. Thus, they are viable as affordable and abundant natural antibiotics. For this experiment, I have chosen the Agar Disk Diffusion Method from Performance Standards for Antimicrobial Disks Susceptibility Tests, Approved, 9th Edition by the Clinical and Laboratory Standards Institute [â ś]. Essential oils of cinnamon and clove are diluted in polysorbat-80 to allow diffusion in and out of the filter paper discs. The impregnated discs will be placed on the Mueller-Hinton agar (MHA)2 to produce an inhibition area. No bacteria colonies will form at the area where the concentration of active ingredients is equal or more than the effective concentration to inhibit the bacteria. A clear inhibition area which has no bacteria growth will be the measure of antimicrobial activity. Thus, a larger diameter of the zone will signify a higher antimicrobial activity. Thus, my research question will be In vitro study of the Synergism between Essential Oils, Cinnamon (Cinnamomum zeylanicum) and Clove (Syzygium aromaticum) in Inhibiting Growth of Escherichia coli and Staphylococcus aureus.
2
MH agar is a research microbiological growth medium used for antimicrobial susceptibility testing
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Cinnamon (Cinnamomum zeylanicum) Common varieties of cinnamon include Ceylon cinnamon (Cinnamon zeylanicum) and
Chinese cinnamon (C.cassia). Its dried barks are used for infusions, tinctures, and powders. The bark’s distillate produces an essential oil with a peppery smell. The stock essential oils to be used are extracted through steam distillation of Ceylon cinnamon barks imported by a local market from India. The essential oils of spices are known to contain active antimicrobial compounds. The proximate oil content for cinnamon is only 0.5-2.0% where a large component is cinnamaldehyde. Cinnamaldehyde or 3-phenylprop-2-enal, is an aromatic aldehyde with a mono-substituted benzene ring and an aldehyde group. Research shows that cinnamaldehyde inhibits cell division in Escherichia coli by decreasing the in-vitro assembling and bundling functions of FtsZ, a prokaryotic homolog of tubulin3. Cinnamaldehyde also reduces the frequency of Z-rings formed from FtsZ per unit cell length of E. coli. The polymerization of FtsZ is inhibited by cinnamaldehyde, disallowing the cell to regenerate
[7]
. Ceylon cinnamon also contains 4-10%
eugenol4. Its other components include ethyl cinnamate, beta-caryophyllene, linalool, and methyl chavicol, which are food additives and aromatic compounds [8].
Figure 1: Chemical Structure of Cinnamaldehyde Cinnamon bark is antispasmodic; it stimulates salivary glands and stomach mucous membranes. Scientists have discovered that both cinnamaldehyde and eugenol inhibit Helicobacter pylori[9], a peptic ulcer-causing bacteria.
3
A type of globular protein that makes up microtubules, a structural component involved in mitosis and cytokinesis.
4
An allyl chain-substituted phenol compound.
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Clove (Syzygium aromaticum) Cloves from the Myrtaceae family are aromatic dried flower buds. A native from Indonesia,
cloves are harvested around length 1.5-2cm. They consist of a long calyx branching out into four spreading sepals and four unopened petals in the middle resembling a small ball. Essential oils of cloves will be extracted through steam distillation of clove buds from Brazil. Approximately 16-18% of clove is essential oils. Clove oil consist 70% to 90% eugenol. Eugenol or 2-methoxy-4-allyl phenol is an allyl chain-substituted phenol 5 compound. It is a phytochemical6 that causes cytoplasm membrane coagulation and active transport unbalance
[10]
.
Eugenol, an analgesic, is known to combat inflammation and bacterial and fungal infection. Clove oil is the active ingredient in mouthwashes and over-the-counter pain-relief remedies for toothaches. It contains high tannin content of 10% to 19%, providing additional antimicrobial activity. Other constituents include acetyl eugenol, beta-caryophyllene and vanillin [11].
Figure 2: Chemical Structure of Eugenol Studies in Taiwan show that eugenol reduces blood clotting and is an effective antiinflammatory agent. Clove oil has shown considerable efficacy against several common hospitalacquired bacteria and yeasts including MRSA7.
[12]
Clove bud oil also significantly decreases the
production of alpha-toxin and entrotoxin A and B8 produced by Staph. a [13].
5
Comprises of an aromatic ring bonded directly to a hydroxyl group.
6
Small organic hydrophobic molecules and are naturally occurring antibiotics
7
Methicillin-resistant Staphylococcus aureus
8
Toxins that causes food poisoning by contaminating cooked meat, eggs, fish, milk and dairy products
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Two Types of Bacteria The two bacteria are chosen are Staphylococcus aureus, (Gram-positive bacterium) and
Escherichia coli, (Gram-negative bacterium). A clearer view on different susceptibilities of the two spectrums of bacteria towards cinnamon and cloves can be determined. In Figure 3(a), gram-positive bacteria have a thick layer of peptidoglycan enclosing the plasma membrane allowing it to retain the crystal violet stain from violet staining. Uniquely, the cell wall contains teichoic acids9 and lipoteichoic acids10, providing rigidity and a site for parasite attachment.
Figure 3(a)
Figure 3 (b) 11
Figure (b) shows gram-negative cells have thinner peptidoglycan layers with porins12 on an outer membrane. Gram-negative bacteria are generally more resistant than gram-positive bacteria as the outer membrane protects the bacteria from antibiotics, disinfectants, dyes and detergents. Thus, it is less permeable for antimicrobial agents to damage the peptidoglycan layer.
9
Tightly bounded bacterial polysaccharides
10
Teichoic acids which are anchored to the lipid bilayer via a glycerolipid
11
http://www.micro.cornell.edu/cals/micro/research/labs/angert-lab/low.cfm
12
Proteins that act as a pore to allow diffusion of molecules.
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Michelle Tan Oon Ee
2.0
Hypotheses
2.1
Effects of Cinnamon and Clove on E.coli and Staph. a.
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Antimicrobial agents attack specific cell sites to cause microbial death or damage. The four main targets are cell walls, cell membranes, biosynthesis pathways for DNA 13 and RNA 14 , or protein enzyme functions. Certain organic compounds can penetrate through or attach themselves to bacteria cell membranes, binding with bacteria structures and subsequently destroying it. Both cinnamon and clove essential oils contain phenol and aldehyde groups, which inhibit bacterial growth and thus, will show antibacterial activity against E.coli and Staph. a.
Hypotheses: i)
Cinnamon will produce larger zones of inhibition due to the presence of cinnamaldehyde. Cinnamaldehyde reduces the reproduction of E.coli by affecting nucleic acids synthesis [7] and
its aldehyde group inactivate proteins through polymerisation. For cloves with high eugenol content, its phenol group coagulates protein and disrupts cell walls and cell membranes.
ii)
There will be greater inhibition zones for Staph. a. as it is a gram-positive bacteria.
The absence of the additional outer membrane and porins on the external peptidoglycan layer makes Staph. a. less resistant to antimicrobial agents. Thus, cinnamaldehyde and eugenol have easier access to attack the peptidoglycan layer and cell wall.
13
Deoxyribonucleic acid is a double-stranded nucleic acid that contains genetic instruction for development and functioning of living organisms. 14
Ribonucleic acid is a single-stranded molecule consisting of a long chain of nucleotide units and is important for protein synthesis.
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Michelle Tan Oon Ee 2.2 iii)
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Synergistic effects of the combination of Cinnamon and Clove The combination of both spices will produced an enhanced effect with a larger zone of
inhibition. Although cinnamon and clove both have antimicrobial properties, the two spices are rarely used individually. Since both cinnamon and clove contain eugenol, the combination will increase the total amount of eugenol in the new derived antimicrobial agent. The aldehyde and phenol groups in the essential oils can work together to disrupt protein synthesis. However, through preliminary testing, cinnamon showed stronger inhibitory effects than clove. The antimicrobial effect of cinnamon, presumably from cinnamaldehyde, is significantly larger than that of clove’s eugenol.
iv)
A combination of increasing cinnamon volume concentration and decreasing clove volume
concentration will produce a larger zone of inhibition.
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3.0
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Variables
3.1 Manipulated Variables: Combination of cinnamon and clove essential oils at different percentage ratios. Volume of Cinnamon, Vcin/ μl
Volume of Clove, Vclove/ μl
5.0 9.0 15.0 21.0 45.0
45.0 21.0 15.0 9.0 5.0
Percentage ratio volume per volume, (%v/v) % Cinnamon: % Clove 10% : 90% 30% : 70% 50% : 50% 70% : 30% 90% : 10%
Table 1: Percentage ratios volume per volume (%v/v) of the combination of cinnamon and cloves. Volume to volume dilution is used to combine both extracts in a fixed percentage ratio. The small amounts of extract ranging from 5μl to 45μl are measured with a micropipette. All of the combined ratios are kept overnight in a micro centrifuge and spun in the centrifuge to obtain a homogenous mixture.
3.2
Responding Variables: Largest Inhibition Area The optimum ratio of combination of cinnamon and clove will produce the largest inhibition
zone. A zone of inhibition is the diameter of clear area around the impregnated filter paper disc on the MHA plate after 24 hours of incubation.
Zone of inhibition
Figure 4: Measuring zone of inhibition using a ruler Page | 13
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Constant Variables:
The bacteria used will be Escherichia coli ATCC 2592215 and Staphylococcus aureus ATCC 2592313. Initial trials shows both bacteria are susceptible to cinnamon and clove essential oils. The pathogenic strains are common contaminants of water sources and surfaces.
The concentration of bacteria is approximately 1x10⁸ cfu/ml according to 0.5 McFarland standard.
The amount of bacteria inoculated on every MHA plate is 40 μl 20 μl of bacteria is transferred twice with a micropipette onto centre of MHA surface.
All concentrations are obtained from extracted stock of cinnamon and clove essential oils. Initial trials showed that inhibition zones from 100% pure essential oils were too large. Thus, a four-fold dilution is carried out.
Incubation period of 24 hours at temperature 37.0°C These conditions ensure optimum rate of bacterial growth, sufficient to cover the entire MHA surface.
The diameter of filter paper discs is 6mm. The filter paper is punched with a hole puncher and only the flat discs are used. All discs are of same length so that the amount of extract absorbed is constant.
The soaking duration of filter paper in the extract is around 1 minute Due to limited quantities of extracts, 30 μl of combined extracts is transferred with a micropipette onto each filter paper and is left for a minute to allow complete absorption. 15
Non-pathogenic strains used for laboratory research studies
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Michelle Tan Oon Ee
4.0
Materials and Methods
4.1
Preparation of Mueller-Hinton Agar (MHA)16
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1.
7g of MHA is added to 250 ml of distilled water and autoclaved.
2.
The mixture is poured into 15 agar plates to a fixed height of 7mm and left to cool.
3.
After solidifying, the plates are covered and stored in the refrigerator upside-down.
4.2
Preparation of Sterilised Filter Paper Discs and Autoclaving Procedures
1.
6mm filter paper discs are prepared and any irregular-shaped discs are disposed.
2.
Paper discs, cotton buds and screw-cap jars are autoclaved in a pressure cooker. The forceps are sterilised by dipping it in ethanol before heating under a Bunsen flame.
4.3
Preparation of Inoculums
Escherichia coli ATCC 25922
Staphylococcus aureus ATCC 25923
A loopful of each bacteria strain grown overnight on a MHA slant17, is transferred individually to 5ml of Mueller-Hinton broth. After incubation, bacteria suspension’s turbidity is adjusted to match a 0.5 McFarland standard18 (1 x 10⁸ CFU/ml).
16
Refer to Appendix 2 for detailed procedure on the preparation of MHA.
17
The inoculum is prepared by my supervisor, Mr Lawrence Kok.
18
Refer to Appendix 3 for preparation of 0.5 McFarland standard
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Extraction of Cinnamon Essential Oil by Steam Distillation
1.
100g pounded cinnamon is transferred into a distilled water-filled distillate flask.
2.
Heat is continuously supplied by a blue Bunsen flame and steam is channelled through a distillation funnel into a conical flask to be condensed in a beaker of ice.
Figure 5: Set-up of Apparatus for Steam Distillation of Cinnamon. 3.
Dichloromethane19 (DCM) is added to the milky coloured distillate20. The flask is shaken to dissolve the essential oil in DCM and left aside.
4.
With a glass pipette, DCM is extracted from the bottom edge of flask into a boiling tube until it is quarter-filled.
5.
The boiling tube is heated in a water bath to evaporate DCM. Precautions must be taken as cinnamon extracts are very volatile at high temperatures.
6.
The remaining dark-yellowish substance is identified as cinnamon essential oil through its smell. It is kept in a micro centrifuge as stock of 100% pure cinnamon essential oil.
19
An organic compound CH2Cl2, widely used as an organic solvent.
20
Refer to Appendix 4 for more details on DCM extraction
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Extraction of Clove Essential Oil by Steam Distillation
1.
100g pounded cloves is transferred into a distilled water-filled distillate flask.
2.
Heat is continuously supplied by a blue Bunsen flame and steam is channelled through a distillation funnel into a conical flask to be condensed in a beaker of ice.
Figure 6: Set-up of Apparatus for Steam Distillation of Clove. 3.
DCM is added to the milky coloured distillate. The flask is shaken and left aside.
4.
With a glass pipette, DCM is extracted into a boiling tube until it is quarter-filled.
5.
The boiling tube is heated in a water bath to evaporate DCM. Precautions are taken even though cloves extracts are not as volatile.
6.
The remaining light-yellow substance is identified as clove essential oil through its distinctive smell. It is kept in a micro centrifuge as stock of 100% pure clove essential oil.
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Preparation of Cinnamon and Clove Percentage Ratios A four-fold dilution or 1:4 volume to volume dilution is carried out on pure 100% essential oils using polysorbat-8021, an organic solvent because oil is insoluble in water. For every 1ml of essential oil, 3 ml of polysorbat-80 is added.
The cinnamon and clove stocks are then diluted to concentrations 10%, 30%, 50%, 70% and 90%. A volume of essential oil is pipetted and added to a pipetted volume of polysorbat-80.22
Volume of Essential Oil, VEO / μl
Volume of Polysorbat-80, Vpolysorbat/ μl
50.0 90.0 150.0 210.0 450.0
450.0 210.0 150.0 90.0 50.0
Percentage Concentration of Essential Oil, (% vol/vol) 10% 30% 50% 70% 90%
Table 2: Percentage volume per volume (% vol/vol) concentrations of essential oils after volume to volume dilution.
Cinnamon and clove are combined in fixed ratios by adding the volumes of cinnamon and clove in quantities shown in table below.
Volume of Cinnamon, Vcin/ μl
Volume of Clove, Vclove/ μl
5 9 15 21 45
45 21 15 9 5
Percentage ratio volume per volume, (%v/v) % Cinnamon: % Clove 10% : 90% 30% : 70% 50% : 50% 70% : 30% 90% : 10%
Table 3: Percentage volume per volume (%vol/vol) ratio concentrations of cinnamon and clove essential oils.
Every micro centrifuge is shaken to thoroughly mix its contents. They are spun in a centrifuge for 2 minutes to produce a homogenous mixture.
21
Emulsion to enhance solubility and diffusion of essential oil. Refer to Appendix 5 for details on polysorbat-80
22
Refer to Appendix 6
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Disk Diffusion Test (Kirby Bauer Method)[6] 40 μl of Escherichia coli suspension is swabbed evenly over MHA with a cotton swab. The plate is continuously rotated by 60°.
2.
The plate is covered and left aside for 2 minutes.
3.
30 μl of 10% cinnamon oil is placed on a sterile disc for 1 minute.
4.
With a pair of sterile forceps, the disc is pressed down on MHA.
5.
Once in contact, the discs cannot be removed because of instantaneous antibiotic diffusion from disc to agar.
6.
Filter papers are positioned evenly spaced as shown below to avoid overlapping inhibition zones.
7.
Triplicates samples of each bacterium are prepared.
Positive control
Negative control
Figure 7: Position of filter papers on an agar plate.
95% alcohol is sprayed over the working bench before inoculation.
The negative control is polysorbat-80 and the positive control is Tetracycline 10μg.
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Incubation of Agar Plates 23
1.
Agar plates are incubated in inverted positions.
2.
Plates are stacked around 4 in a pile to ensure all plates reach 37째C within 24 hours.
3.
Without opening the lid, the diameters of the zones of complete inhibition are measured with a ruler.
23
Refer to Appendix 7 for set-up of the incubator
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Data Collection Essential Oil
Percentage volume per volume / % vol/vol
E. coli
Staph. A
Trial 1
Trial 2
Trial 3
Mean ± S.Da
Trial 1
Trial 2
Trial 3
Mean ± S.D
10
6.5
6.8
-b
6.65 ± 0.21
-
-
-
-
30
7.8
8.5
6.6
7.63 ± 0.96
8.2
8.5
7.5
8.07 ± 0.51
50
10.2
9.2
11.1
10.16 ± 0.95
10.0
9.5
12.5*
9.75 ± 0.35
70
15.0
16.1
15.5
15.53 ± 0.55
17.8
16.9
17.5
17.40 ± 0.46
90
18.3
19.5
19.9
19.23 ± 0.83
20.5
21.5
22.5
21.50 ± 1.00
100
22.1
22.5
23.4
22.67 ± 0.67
25.0
22.7*
24.2
24.60 ± 0.57
10
-
-
-
-
-
-
-
-
30
-
-
-
-
6.5
6.8
-
6.65 ± 0.21
50
6.3
6.1
-
6.20 ± 0.14
8.5
8.0
-
8.25 ± 0.35
70
6.8
7.0
6.7
6.83 ± 0.15
9.2
8.8
6.6*
9.00 ± 0.28
90
9.0
10.3
8.7
9.33 ± 0.85
11.0
12.2
8.5*
11.60 ± 0.85
100
13.6
13.0
14.0
13.53 ± 0.50
14.3
15.2
14.6
14.70 ± 0.46
Tetracyclinec
16.5
15.8
16.0
16.10 ± 0.36
20.1
19.8
19.5
19.8 ± 0.30
Polysorbat-80d
-
-
-
-
-
-
-
-
Cinnamon
Clove
Table 4: Inhibition zone (mm) produced by different percentages of cinnamon and clove essential oil on E.coli and Staph. a. a b
Mean zone (mm) ± standard deviation for triplicates No zone of inhibition
c d
Positive control Negative control
* Values are not taken into account due to inconsistencies.
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Essential Oil
E. coli
Staph. a
Trial 1
Trial 2
Trial 3
Mean ± S.D a
Trial 1
Trial 2
Trial 3
Mean ± S.D
10% : 90%
10.5
11.5
10.0
10.67 ± 0.76
11.5*
9.0
8.8
8.9 ± 0.14
30% : 70%
11.8
13.2*
11.0
11.4 ± 0.57
10.5
10.0
9.5
10.0 ± 0.50
50% : 50%
15.5*
13.0
12.8
12.9 ± 0.14
15.5*
12.5
11.5
12.0 ± 0.71
70% : 30%
14.5
13.8
13.0
13.77 ±0.75
12.8
12.5
11.3
12.2 ± 0.21
90% : 10%
15.0
16.2
15.3
15.5 ± 0.62
15.0
16.5
16.0
15.83 ± 0.77
Tetracycline b
16.5
15.8
16.0
16.1 ± 0.36
20.1
19.8
19.5
19.8 ± 0.30
Polysorbat-80 c
-d
-
-
-
-
-
-
-
Percentage Volume per Volume Ratios / % Vcin/Vclove
Table 5: Inhibition zone (mm) produced by different ratios of the combination of cinnamon and clove essential oil on E.coli and Staph. a. a
Mean zone (mm) ± standard deviation for triplicates Positive control c Negative control d No zone of inhibition * Values are not taken into account due to inconsistencies b
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Data Processing
Inhibition zone (mm) produced by different percentages of cinnamon and clove essential oil on E. coli and Staph. a. 26
Mean Inhibition Zone (mm)
24 22 20 18 16 14 12
E Coli
10
Staph. A
8 6
4 2 0 10
30
50
70
90 Cinnamon
100
10
30
50
70
90
100
Clove
Graph 1: Inhibition zone (mm) produced by different percentages of cinnamon and clove essential oil on E.coli and Staph. a. Page | 23
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Inhibition zone (mm) produced by different percentage ratios of combination for cinnamon and clove essential oils on E.coli and Staph. a. 18 16
Mean Inhibition Zone (mm)
14 12 10 E. Coli
8
Staph. A 6 4 2 0 10% : 90%
30% : 70%
50% : 50%
70% : 30%
90% : 10%
Percentage Volume per Volume Ratios / %Cinnamon : %Clove
Graph 2: Inhibition zone(mm) produced by different percentage ratios of combination for cinnamon and clove essential oil on E.coli and Staph. a.
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Inhibition Zone (mm) for Individual and Percentage Ratio Combinations of Cinnamon and Clove Essential Oils on E.coli. 24 Percentage Ratio Combination of Cinnamon and Clove
20 18 16 14
Cinnamon Essential Oil
12 10
100% Clove
100% Cinnamon
90% Cinnamon
90% : 10%
70% Cinnamon
70% : 30%
50% Clov
50% : 50%
70% Clove
30% Cinn
30% : 70%
2
90% Clove
4
10% Cinn
6
50% Cinnamon
8
10% : 90%
Minimmum inhbition zone (mm)
22
Clove Essential Oil
0 10% : 90%
30% : 70%
50% : 50%
70% : 30%
90% : 10%
Various Values of Cinnamon and Clove Essential Oils
Graph 3: Inhibition Zone by Individual and Percentage Ratio Combinations of Cinnamon and Clove Essential Oils on E.coli. Page | 25
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Inhibition Zone (mm) for Individual and Percentage Ratio Combinations of Cinnamon and Clove Essential Oils on Staph. a. 26
22 Percentage Ratio Combination of Cinnamon and Clove
20 18 16 14 12
Cinnamon Essential Oil
10
100% Clove
100% Cinnamon
90% Cinnamon
90% : 10%
30% Clov
70% Cinnamon
70% : 30%
50% Clove
50% : 50%
70% Clove
30% Cinn
2
30% : 70%
4
90% Clove
6
50% Cinnamon
8
10% : 90%
Minimmum inhbition zone (mm)
24
Clove Essential Oil
0 10% : 90%
30% : 70%
50% : 50%
70% : 30%
90% : 10%
Various Values of Cinnamon and Clove Essential Oils
Graph 4: Inhibition Zone by Individual and Percentage Ratio Combined Cinnamon and Clove Essential Oils on Staph. a. Page | 26
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Data Analysis: ANOVA [15] The ANOVA test (Analysis of Variance test) 24 compares two sources of variation: variation
between group means, đ?‘ đ?‘?2 and variation within each group, đ?‘ đ?‘¤2 . The results determine whether there is a significant difference in antimicrobial activity for different ratio combinations of cinnamon and clove essential oils. 
Our null hypothesis is đ??ť0 = đ?œ‡1 = đ?œ‡2 = đ?œ‡3 = đ?œ‡4 = đ?œ‡5 such that the mean of the five groups are equal.

The alternative hypothesis đ??ť1 is one of the means is not equal to the others or none of them are equal.
The đ??š ratio is calculated through the formula: đ??š đ?‘&#x;đ?‘Žđ?‘Ąđ?‘–đ?‘œ =
đ?‘€đ?‘’đ?‘Žđ?‘› đ?‘ đ?‘žđ?‘˘đ?‘Žđ?‘&#x;đ?‘’ đ?‘?đ?‘’đ?‘Ąđ?‘¤đ?‘’đ?‘’đ?‘› đ?‘”đ?‘&#x;đ?‘œđ?‘˘đ?‘?đ?‘ đ?‘€đ?‘’đ?‘Žđ?‘› đ?‘ đ?‘žđ?‘˘đ?‘Žđ?‘&#x;đ?‘’ đ?‘¤đ?‘–đ?‘Ąâ„Žđ?‘–đ?‘› đ?‘”đ?‘&#x;đ?‘œđ?‘˘đ?‘?
If any of the đ??š ratios is higher than đ??š critical at significant level of 0.05 (Îą = 0.05), đ??ť0 is rejected and thus, one of the means is significantly different from the others. Calculations are done though Microsoft Excel 2007. However, the đ??š-test does not indicates the location of the significant difference between means of groups. Variable
Percentage Ratio Combinations of Cinnamon and Clove Essential Oils
Bacteria Strain
F-value
F-critical
E.coli
24.59
3.84
Staph. a.
43.77
3.84
Conclusion The two F-values are larger than the F-critical values. Thus, the ANOVA test shows that one of the groups has a significant difference from the others in the group.
Table 6: Results on ANOVA for two bacterial strains to determine whether there is a significant difference between mean zone of inhibition when cinnamon and clove essential oils are combined at different ratios25.
24
See Appendix 7 for assumptions when carrying out an ANOVA
25
See Appendix 8 for more details on ANOVA and Appendix 10 for detailed ANOVA calculations
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Tukey’s HSD Test [15] A post hoc analysis is then carried out to identify the location of the differences. A Tukey’s
HSD26 test is a multiple comparison test that tests out the null hypothesis. The pair whose mean differences between groups exceed the HSD critical value is considered significantly different. These calculations are done manually.
Group Combination, % vol/vol (Mean Inhibition Zone of Percentage Ratio Combinations of Cinnamon and Clove Essential Oils on E. coli) 10% : 90% 30% : 70% 10% : 90% 50% : 50% 10% : 90% 70% : 30% 10% : 90% 90% : 10% 30% : 70% 50% : 50% 30% : 70% 70% : 30% 30% : 70% 90% : 10% 50% : 50% 70% : 30% 50% : 50% 90% : 10% 70% : 30% 90% : 10%
Mean Difference, mm
HSD critical value
Implication
0.73 2.23 3.16 4.83 1.50 2.37 4.10 0.87 2.60 1.73
1.85 1.85 1.85 1.85 1.85 1.85 1.85 1.85 1.85 1.85
No significant difference significant difference significant difference significant difference No significant difference significant difference significant difference No significant difference significant difference No significant difference
Table 7: Results of Tukey’s HSD test on mean inhibition zone of percentage ratio combinations of cinnamon and clove essential oils on E.coli to identify which the pairs that have significant difference
26
See Appendix 9 for Tukey’s HSD (honestly significant difference) formula
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Group Combination, % vol/vol (Mean Inhibition Zone of Percentage Ratio Combinations of Cinnamon and Clove Essential Oils on Staph. a.) 10% : 90% 30% : 70% 10% : 90% 50% : 50% 10% : 90% 70% : 30% 10% : 90% 90% : 10% 30% : 70% 50% : 50% 30% : 70% 70% : 30% 30% : 70% 90% : 10% 50% : 50% 70% : 30% 50% : 50% 90% : 10% 70% : 30% 90% : 10%
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Mean Difference, mm
HSD critical value
Implication
1.10 3.10 3.30 6.93 2.00 2.20 5.83 0.20 3.83 3.63
1.85 1.85 1.85 1.85 1.85 1.85 1.85 1.85 1.85 1.85
No significant difference significant difference significant difference significant difference significant difference significant difference significant difference No significant difference significant difference significant difference
Table 8: Results of Tukey’s HSD test on mean inhibition zone of percentage ratio combinations of cinnamon and clove essential oils on Staph. a. to identify which the pairs that have significant difference
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6.0
Conclusion and Evaluations
6.1
Discussion
6.1.1
Different Concentrations of Cinnamon and Clove Essential Oils on E.coli and Staph. a. Graph 1 show that cinnamon essential oils produce wider inhibition zones than clove essential
oils. Thus, the cinnamaldehyde in cinnamon is a stronger antimicrobial compound than eugenol in clove. Cinnamaldehyde interferes with DNA and RNA functions, either by disallowing DNA to replicate or by binding irreversibly to DNA, preventing translocation and transcription. Bacterial cells are inhibited from forming growth proteins and multiplying. The compound’s aldehyde group has a –CHO functional group on a terminal carbon which polymerises easily. The alkylating polymers react with amino groups on amino acids and cross-link to inactivate proteins [14]. The phenol group of eugenol denatures proteins, breaking bonds between secondary and tertiary protein structures and causes them to unfold and coil randomly. At higher essential oil concentrations, they act as cellular poisons, disrupting cell walls and membranes. In lower concentrations, they inactivate enzyme systems The inhibition zone for Staph. a. is larger than for E.coli except for 50% cinnamon concentration. Overall, Staph. a, the gram-positive bacteria, is more susceptible to both essential oils. There is no inhibition zone for 10% cinnamon concentration (Staph. a), 10% and 30% clove concentrations (E.coli) and 10% clove concentration (Staph. a). The content of antimicrobial agents in lower concentrations is not sufficient to inhibit bacteria growth.
6.1.2
Different Percentage Ratio of Combinations of Cinnamon and Clove Essential Oils Graph 2 shows different percentage ratio of combinations of cinnamon and clove essential
oil to be more effective against E.coli than Staph. a. This differs from individual essential oil effects on these bacteria. This is one possible enhanced synergistic effects of the combination of essential oils. A combination of cinnamon and clove would be more effective to combat gram-negative bacteria. Page | 30
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The ratio of cinnamon to clove that produces the largest inhibition zone is (90% : 10%). The Tukey’s HSD test shows that ratios (90% :10%) and (70% :10%) have significant differences with other ratios. With increasing percentage concentration of cinnamon essential oil in the combined mixture, the zone of inhibition enlarges. Thus, antimicrobial activity is proportional to the concentration of cinnamon in the combined mixture. However, with reference to table 7, there is no significant difference in antimicrobial activity for slight changes in percentage concentration of either essential oil.
6.1.3
Synergistic Effects of Cinnamon and Clove.
Bacteria E.coli Graph 3 show ratios (10% : 90%), (30% :70%) and (50% : 50%) present better antimicrobial activity with larger zones of inhibition than individual concentrations. However, the amount of antimicrobial agents in the individual concentrations is not 100% of the mixture. To determine whether there is a synergistic effect for cinnamon and cloves, both the combined and individual mixtures must contain 100% antimicrobial agents. Thus, ratios are compared with 100% essential oils. Clearly, 100% cinnamon’s antimicrobial activity is higher than the combined mixtures. Thus, cinnamon works better individually at high concentrations. For clove, results show that ratios (10% : 90%), (30% :70%) and (50% : 50%) produce enhanced effects. Therefore, there is no synergistic effect for all combined ratios of cinnamon and clove essential oils as none of the inhibition zones for ratios exceed that of 100% cinnamon. However, essential oils used have undergone 4-fold dilution to attain a smaller inhibition zones.
Bacteria Staph. a. In Graph 4, combined ratios (30% :70%) and (50% : 50%) produce larger zones of inhibition than individual concentrations. When compared with 100% cinnamon, all combined Page | 31
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ratios are significantly lower. Comparisons with 100% clove show that cinnamon enhances clove’s antimicrobial properties for ratios (10% : 90%), (30% :70%), (50% : 50%) and (70% : 30%). There is also no synergistic effect for all combined ratios of cinnamon and clove essential oils when tested on Staph. a.
6. 2 1.
Limitations The Kirby Bauer Method is designed for convenient and rapid production of clinical samples and simplified data handing. Methods of disk diffusion and microdilution were originally developed when antibiotic resistance was low. Thus, these principles have limited ability to detect resistance. Due to unstable antibiotic gradients in disk tests, zone sizes are directly influenced by inoculums and growth rates regardless of the antimicrobial agent’s susceptibility [16].
2.
Microdilution is restricted by volume and nutritive capacity, thus inclined to give false susceptible results. Since small inoculums are used, the odds of obtaining resistant subpopulations are minimal. The experiments were conducted in a small scale and may not be such an accurate representation of actual antimicrobial properties of essential oils.
3.
There are many potential sources for contaminations of agar or in essential oil preparations, producing inconsistent results.
4.
There is an uncertainty whether the essential oil obtained is purely 100% essential oil. The yellow colour of essential oil obtained is of different intensities. The assumption is the colour pigments have no effect on antimicrobial properties of essential oils and has no active ingredients.
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Polysorbat-80 has no antimicrobial properties and assists essential oils to penetrate bacteria cell wall and membrane. However, some studies show a decreasing effect on antimicrobial activity of essential oils[17].
6.3 1.
Ways to Improve Experiment Essential oils could be further refined through redistillation. This was not carried out due to time restrictions. Redistillation should be conducted in an equipped distillery, where refined methods are applied, depending on the oil’s nature. To prevent contamination and false susceptibility results, there is a need of a proper sterile chamber for microbial work with a laminar flow system.
2.
The determination of the essential oil’s minimum inhibitory concentration (MIC) could be carried out. MIC is the lowest concentration of an antimicrobial that visibly inhibits growth. Serial dilutions of the antimicrobial are carried out in tubes of broth where small uniform samples of bacteria are inoculated, incubated and examined for turbidity.
3.
A microatmosphere method could be used, allowing determination of antimicrobial activity of essential oils in vapour phase. The vaporised essential oil is diffused directly to agar in an inverted Petri dish. This reduces contamination and quickens inhibitory actions.
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Areas of Further Investigations Antibacterial activities of cinnamons and cloves may be less effective when cooked or
heated under intense temperatures. Aldehyde groups can oxidise to form carboxylic acids when exposed to air and under high temperatures. This will reduce the stability of cinnamaldehyde, which might decompose at high temperatures. The effects of temperature on cinnamons and cloves could be further investigated. Most organic compounds are also unstable at high pHs. Their antimicrobial activity could be hindered when digested in the stomach’s acidic medium. The optimum pH level at which cinnamon and clove antimicrobial activity is most efficient could be determined. The viability of cinnamon and clove as a potential antibacterial drug would require more research done on the evaluation of their in-vivo efficiency. This will include the effects of oxygen and light on antimicrobial activity of the essential oils. Further experimentation could be done with crude ethanolic extracts where spice materials are cut into small pieces and soaked in 95% ethanol. This could aid in commercial production of cinnamon remedies as their barks have small quantities of oils. However, inhibitory activity of essential oils is generally significantly greater than ethanolic extracts[18]. Spices’ antimicrobial activity decreases when added to food materials like protein, carbohydrate and fat. Thus, spices can be combined with preservatives such as acid, sugar, salt and vinegar. There may be synergistic interaction between the active components of spices with these factors at low or high temperatures to increase antimicrobial effects. To overcome the problem of antibiotics that can no longer combat bacteria, essential oils of cinnamon and cloves could possibly be modified into existing drugs. The synergistic effects can overcome bacteria resistance to these antibiotics.
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Conclusion Higher concentration of essential oils produces larger zones of inhibition and higher
antimicrobial activity. Cinnamon is a stronger antimicrobial agent than clove. Both spices can block bacterial protein syntheses without adversely affecting human cells. Staph. a has lower resistance to both essential oils. The best percentage volume per volume ratio is (90% : 10%), showing greatest antimicrobial activity against both bacteria. The combined mixture works best at a higher concentration of cinnamon essential oils. A larger amount of cinnamon should thus be used in cooking to attain the highest antimicrobial properties. Only cinnamon concentrations are more efficient in inhibiting growth individually. Smaller quantities of cinnamon can be added with clove spice to enhance antimicrobial activity for cloves. This may be applicable while cooking as large quantities of a single spice could produce a very strong smell. Although it seems that there is little or no synergistic effect between these essential oils, a combination of the two essential oils does inhibit bacteria growth.
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References
1. Antibiotic Uncovered [Online] 30 May 2003 [Cited: 20 March 2009.] http://www.foodqualitynews.com/Food-Alerts/Antibiotics-uncovered
2. American Society for Microbiology. Antibiotic Resistance - Current Status and Future Directions [Online] 6 July 1994 [Cited: 22 March 2009.] http://www.asm.org/ASM/files/CCPAGECONTENT/DOCFILENAME/0000005962/antibiot [1].pdf
3. Wong, Cathy. Health Benefits of Cinnamon [Online] 27 October 2007 [Cited: 7 July 2009.] http://altmedicine.about.com/od/cinnamon/a/cinnamon.htm
4. Health Benefits of Clove Oil [Online] 2006 [Cited: 7 July 2009.] http://www.organicfacts.net/organic-oils/natural-essential-oils/health-benefits-of-clove-oil.html
5. Wood, Shelley. Cinnamon and cloves: Benefits in diabetes probed [Online] 4 April 2006 [Cited: 7 July 2009] http://www.medscape.com/viewarticle/539016
6. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disks Susceptibility Tests, Approved standard, M2-A9, 9th Edition. CLSI. Wayne PA, 2006.
7. Domadia, Prerna; Swarup, Sanjay; Bhunia, Anirban; Sivaraman, J; Dasgupta, Debjani. Inhibition of bacterial cell division protein FtsZ by cinnamaldehyde. 11 June 2007 [Cited: 8 July 2009]
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8. Cinnamon. [Cited: 8 July 2009] http://en.wikipedia.org/wiki/Cinnamon
9. Ali, Shaik Mahaboob; Khan, Aleem A; Ahmed, Irshad; Musaddiq, M; Ahmed, Khaja S; Polasa, H; Rao, L Venkateswar; Habibullah, Chittoor M; Sechi, Leonardo A; Ahmed, Niyaz. Antimicrobial activities of Eugenol and Cinnamaldehyde against the human gastric pathogen Helicobacter pylori. 21 December 2009 [Cited: 8 July 2009]
10. Leite, Aristides Medeiros; Lima, Edeltrudes Oliveira; Souza, Evandro Leite; Diniz, Margareth de Fátima Formiga Melo; Trajano, Vinícius Nogueira; Medeiros, Isac Almeida. Inhibitory effect of β-pinene, α-pinene and eugenol on the growth of potential infectious endocarditis causing Gram-positive bacteria. 28 February 2007. [Cited: 9 July 2009]
11. Clove. [Cited: 9 July 2009] http://en.wikipedia.org/wiki/Clove
12. Warnke, Patrick H; Becker, Stephan T; Podschun, Rainer; Sivananthan, Sureshan; Springer, Ingo N; Russo, Paul A.J; Wiltfang, Joerg; Fickenscher, Helmut; Sherry, Eugene. The battle against multi-resistant strains: Renaissance of antimicrobial essential oils as a promising force to fight hospital-acquired infections. 28 March 2009. [Cited: 9 July 2009]
13. Sulieman, Abdel Moneim E; El Boshra, Iman M.O; El Khalifa, El Amin A. Nutritive Value of Cloves (Syzygium aromaticum) and Detection of Antimicrobial Effect of Its Bud Oil. 2007. [Cited: 9 July 2009]
14. Talaro, Kathleen Park. Foundations in Microbiology - Basic Principles , Sixth Edition. McGraw Hill, 2008.
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15. Kuzma, Jan W; Bohnenblust, Stephen E. Basic Statistics for the Health Sciences, Fourth Edition. McGraw-Hill International Edition, 2001
16. Schwalbe, Richard; Steele-Moore, Lynn; Goodwin, Avery C. Antimicrobial Susceptibility Testing Protocols. 2007. [Cited: 20 July 2009]
17. Suppakul, Panuwat; Miltz, Joseph; Sonneveld, Kees; Bigger, Stephen W. Antimicrobial Properties of Basil and Its Possible Applications in Food Packaging. 24 April 2003. [Cited: 25 July 2009]
18. Nanasombat, Suree; Lohasupthawee, Pana. Antibacterial Activity of Crude Ethanolic Extracts and Essential Oils of Spices against Salmonelle and Other Enterobacteria. 2005. [Cited: 25 July 2009]
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8.0 Appendixes Appendix 1: Table of Antimicrobial Effectiveness of Spices and Herbs Snyder, O. Peter. Antimicrobial effects of Spices and Herbs.1997 URL: http://www.hi-tm.com/Documents/Spices.html Spices and Herbs
Inhibitory Effect
Cinnamon, cloves, mustard
Strong
Allspice, bay leaf, caraway, coriander, cumin, oregano, rosemary, sage, thyme
Medium
Black pepper, red pepper, ginger
Weak
Table 9: Antimicrobial Effectiveness of Various Spices and Herbs Appendix 2: Preparation of Mueller-Hinton Agar 1.
7g MHA powder is weighed with an electronic balance. 250cm続 of distilled water is added to it in a beaker and the mixture is boiled under a blue Bunsen flame.
2.
The liquid agar is stirred continuously with a glass rod to avoid it from being charred.
3.
The mixture is transferred into a pressure bottle and is autoclaved in a pressure cooker. During the sterilisation process, the bottle cap is loosened to allow steam to escape and to prevent an occurrence of an explosion in the pressure cooker.
4.
After 15 minutes, the Mueller-Hinton agar is taken out of the pressure cooker and poured into 90mm agar plates up to 7mm in thickness.
5.
The agar is allowed to cool down and solidify. The plates are then covered to prevent contamination and stored upside-down in the refrigerator.
Figure 8: Agar plates left to cooled for 15 minutes Page | 39
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Appendix 3: Turbidity Standard Preparation A 0.5 McFarland standard was prepared by adding barium chloride (BaCl₂) to sulphuric acid (H₂SO₄) resulting in a barium sulphate (BaSO4) precipitation.
Appendix 4: Extraction of Essential Oils using Dichloromethane (DCM)
Figure 9: Apparatus used in DCM extraction 1.
3 drops of dichloromethane is added into the conical flask with a glass dropper.
2.
The conical flask is shaken vigorously but not too hard to prevent emulsion formation. The shaking will extract the essential oil into the organic layer.
3.
The flask is then tilted as shown in the figure below to allow the DCM to settle to one end.
Distillate
Dichloromethane layer with dissolved essential oils Figure 10: Conical flask of distillate with added DCM Page | 40
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A new glass dropper is used to slowly transfer out as much of the organic layer as possible into to a boiling tube. Steps 3 and 4 are repeated two more times.
5.
The boiling tube is placed in a heated water bath. The DCM evaporates, leaving behind the essential oil.
6.
The essential oil is transferred with a plastic dropper into a micro centrifuge to be stored.
7. All glass droppers, boiling tube and conical flasks are disposed properly as dichloromethane cannot be washed away with water.
Figure11: Evaporation of DCM through heating to obtain the essential oils
Appendix 5: Negative Control and Solvent used in Serial Dilution – Polysorbat-80
Figure 12: Polysorbat-80
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Polysorbat-80 is a viscous, water-soluble yellow liquid that is used in the dilution of the stock essential oil. The hydrophilic groups in this compound are polyethers also known as polyoxyethylene groups, which are polymers of ethylene oxide. In the nomenclature of polysorbates, the numeric designation following polysorbate refers to the lipophilic group, in this case the oleic acid.
Appendix 6: Preparation of Cinnamon and Clove Percentage Ratios
Appendix 7: Set-up of Incubator
Figure 13: Incubator
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The incubator comprises of a shelf in a closed cupboard with a single 100W bulb. It has removable black and white boards to reflect and absorb light, preventing overheating. The agar plates and inoculum suspensions are incubated here. Petri dishes are inverted to prevent condensation moisture from dripping onto the agar surface. A thermometer is placed inside the incubator to monitor its temperature. Before the carrying out of the experiments, the incubator is sprayed and cleaned with ethanol to avoid contamination.
Appendix 8: ANOVA Assumptions To perform an ANOVA, three assumptions must be made: 1. Observations are independent, i.e. the value of one observation is not correlated with the value of another. 2. Each group’s observations are normally distributed. 3. Variances of the various groups are homogenous i.e. the each group’s variance is equal to that of any other group.
Appendix 9: Further Details on ANOVA Some convential notations and their definitions used in ANOVA calculations: NOTATION
DEFINITION
MS within or đ?’”đ?&#x;?đ?’˜
Mean square within or Within-group variance
MS between or đ?’”đ?&#x;?đ?’ƒ
Mean square between or Between-group variance
đ?’…đ?’‡
Degree of freedom
đ?’Œ
Number of groups
đ?’?
Number of observations in a group
N
Total number of observation
Îą
Significant level
đ?‘şđ?‘şđ?’˜
Sum of squares within group
đ?‘şđ?‘şđ?’ƒ
Sum of squares between group Table 10: Convention notations and definitions Page | 43
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The one-way ANOVA table is shown below: Source of variation Between
Within
Total
Sum of squares
df
Mean Squares (s²)
đ?‘†đ?‘†đ?‘?
k−1
���
N−k
���
N−1
đ?‘€đ?‘†đ?‘? = đ?‘€đ?‘†đ?‘? =
F ratio
Critical F
P Value
đ?‘†đ?‘†đ?‘? đ?‘˜ − 1
���
đ?‘€đ?‘†đ?‘¤ đ?‘€đ?‘†đ?‘?
đ??šđ?‘˜âˆ’1,
đ?‘ −đ?‘˜
Computer generated
đ?‘ − đ?‘˜
Table 11: One-Way ANOVA

The critical F value is at significant level of 0.05 or 5%, thus, Îą = 0.05. The value can be obtained from Percentiles of F Distribution Table in Basic Statistic for the Health Sciences, Fourth Edition[15] (pg 300)
Appendix 10: Tukey’s HSD Test The formula for the Tukey’s HSD test is: đ??ťđ?‘†đ??ˇ = đ?‘ž(đ?›ź, đ?‘˜, đ?‘ − đ?‘˜)
��� �
such that � is obtained from Percentage Points of the Studentized Range for 2 Through 20 Treatments Table[15] (pg 304) ι = significant level of 0.05 k = total number of groups N – k = represents total number of results – total number of groups
Appendix 11: Detailed Calculation of ANOVA using Microsoft Excel 2007
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1. The effects of the combination of cinnamon and clove essential oils in different percentage ratios on their antimicrobial activity on inhibiting E. coli. Anova: Single Factor SUMMARY Groups (Percentage Volume per Volume Ratios / % Vcin/Vclove) Group 1 (10% : 90%) Group 2 (30% : 70%) Group 3 (50% : 50%) Group 4 (70% : 30%) Group 5 (90% : 10%)
Count
Sum 3 2 2 3 3
Average
Variance
32.0 22.8 25.8 41.3 46.5
10.67 11.40 12.90 13.77 15.50
0.58 0.32 0.02 0.56 0.39
4 8
MS 10.49 0.43
F 24.59
ANOVA Source of Variation Between Groups Within Groups
SS 41.96 3.41
Total
45.37
df
P-value 1.51E-04
F crit 3.84
12 0.43 = 1.85 3
đ??ťđ?‘†đ??ˇ = 4.89
2. The effects of the combination of cinnamon and clove essential oils in different percentage ratios on their antimicrobial activity on inhibiting Staph. a. Anova: Single Factor SUMMARY Groups (Percentage Volume per Volume Ratios / % Vcin/Vclove) Group 1 (10% : 90%) Group 2 (30% : 70%) Group 3 (50% : 50%) Group 4 (70% : 30%) Group 5 (90% : 10%)
Count
Sum 2 3 2 3 3
Average
17.80 30.00 24.00 36.60 47.50
Variance
8.90 10.00 12.00 12.20 15.83
0.02 0.25 0.50 0.63 0.58
MS 18.86 0.43
F 43.77
ANOVA Source of Variation Between Groups Within Groups
SS 75.42 3.45
Total
78.87 đ??ťđ?‘†đ??ˇ = 4.89
df 4 8
P-value 1.76E-05
F crit 3.84
12 0.43 = 1.85 3 Page | 45