Inhibition of E Coli and Staph A of essential oils, cinnamon and cloves
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Inhibition of E Coli and Staph A of essential oils, cinnamon and cloves

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Research work done by my IB student Michelle Tan. Please cite and provide proper referencing to her on her work if you use this material.

Research work done by my IB student Michelle Tan. Please cite and provide proper referencing to her on her work if you use this material.

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Inhibition of E Coli and Staph A of essential oils, cinnamon and cloves Inhibition of E Coli and Staph A of essential oils, cinnamon and cloves Document Transcript

  • 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 AbstractCinnamon (Cinnamomum zeylanicum) and clove (Syzygium aromaticum) are natural spices andremedies used to combat bacterial infection. The active ingredient that contains antimicrobialproperties is primarily found in the spices’ essential oils. As spices are rarely used individually, thecombination of essential oils of cinnamon and clove will produce a synergistic effect to inhibitbacteria growth. Thus, my research question is the In vitro study of the Synergism betweenEssential Oils, Cinnamon (Cinnamomum zeylanicum) and Clove (Syzygium aromaticum) inInhibiting 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. Theessential oils are obtained through steam distillation and extracted with dichloromethane. Differentpercentages of individual essential oil concentrations and different combinations of percentagevolume per volume ratio concentrations are pipetted onto filter paper discs. The discs are positionedon agar plates and incubated for 24 hours at 37°C. The diameter of the clear area around the disc orinhibition zone is measured. The wider the diameter of inhibition zone, the higher the antimicrobialactivity.Analysis through ANOVA and Tukey’s HSD test show that cinnamon is a stronger antimicrobialagent against both bacteria strains. However, Staph. a is less resistant to the essential oils thanE.coli. Higher percentage concentrations for cinnamon and clove produce larger inhibition zones.There are no synergistic effects identified as 100% individual cinnamon concentration gives thegreatest antimicrobial activity. However, the most significantly different percentage cinnamonvolume per clove volume ratio combination is (90% : 10%). Small amounts of cinnamon enhanceantimicrobial activities of cloves but combined mixtures work optimally at higher concentrations ofcinnamon essential oils. Thus, for best antimicrobial effects, larger quantities of cinnamon shouldbe used in cooking. (290 words) Page | 2
  • Michelle Tan Oon Ee 002206-006 AcknowledgementI wish to express my utmost gratitude to the individuals that have provided assistance andencouragement to me in the completion of this extended essay.Mr Lawrence Kok, my dedicated supervisorMy wonderful parents and brother, NicklausMr Masukor SariMy fellow EE mates: Ken and EugeneMy dear seniors: Ju Anne and Jorrel Page | 3
  • Michelle Tan Oon Ee 002206-006Tables of ContentsAbstract ................................................................................................................................................ 2Acknowledgement ............................................................................................................................... 3Tables of Contents ............................................................................................................................... 41.0 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 ............................................................................................................ 102.0 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 ............................................. 123.0 Variables .................................................................................................................................. 13 3.1 Manipulated Variables............................................................................................................. 13 3.2 Responding Variables .............................................................................................................. 13 3.3 Constant Variables................................................................................................................... 144.0 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 Page | 4
  • Michelle Tan Oon Ee 002206-006 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 ....................................................................................................... 205.0 Data Collection ........................................................................................................................ 21 5.1 Data Processing ....................................................................................................................... 23 5.2 Data Analysis: ANOVA .......................................................................................................... 27 5.3 Tukey’s HSD Test ................................................................................................................... 286.0 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 ............................................................................................................................... 357.0 References ............................................................................................................................... 368.0 Appendixes .............................................................................................................................. 39 Page | 5
  • Michelle Tan Oon Ee 002206-0061.0 Introduction1.1 Rationale of study In our technologically driven world, new improved antibiotics are constantly being producedto cure bacterial diseases. However, antibiotics are losing effectiveness as increasing numbers areused in animal feed [¹]. Countries like the EU and USA are phasing out antibiotics usage for animalgrowth, some of which are also human medicine. This overusage causes bacteria to undergomutation or acquire antibiotic resistance genes from other bacteria. Today, more than 90% ofStaphylococcus aureus strains are resistant to penicillin and other beta-lactam antibiotics [²]. Before modern drug therapy, natural antibiotics were common remedies for bacterialinfections. Now, there is a renewed interest for natural approaches as many spice products haveshown inhibition of pathogenic bacteria growth and can potentially replace synthetic antibiotics.These antimicrobial compounds have distinct structures and modes of action that differ fromsynthetic antibiotics, suggesting that if taken together, cross-resistance will be minimal. The selected spices are globally used as ingredients in cooking. Cinnamon and clove havestrong inhibitory effectiveness to bacterial growth1 but the synergistic effects of a combination ofboth spices are not thoroughly established. In traditional Chinese medicine, cinnamon is commonlyused for colds, flatulence, nausea and diarrhoea. Cinnamon, an ingredient in chai tea, aids withdigestion of dairy products and fruits [³]. Clove heals digestive system ailments and promotes quickmetabolism. Clove essential oil, an antiseptic, is useful for wounds, scabies, athlete’s foot andfungal 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 Page | 6
  • Michelle Tan Oon Ee 002206-006 Most cuisines uses combination of spices and thus, there may be possible enhancedantimicrobial effects when cinnamon and clove are mixed. Synergism occurs when the effects oftwo spices is greater than the effect of each spice individually. The presence of one antimicrobialproperty will multiply the effects of the other and thus, known as a synergistic effect. The optimumcombination ratio of percentage volume per volume concentration will produce the highestinhibition zone. Escherichia coli (E.coli) and Staphylococcus aureus (Staph. a) are common bacteria foundin 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 areviable as affordable and abundant natural antibiotics. For this experiment, I have chosen the Agar Disk Diffusion Method from PerformanceStandards for Antimicrobial Disks Susceptibility Tests, Approved, 9th Edition by the Clinicaland Laboratory Standards Institute [⁶]. Essential oils of cinnamon and clove are diluted inpolysorbat-80 to allow diffusion in and out of the filter paper discs. The impregnated discs will beplaced on the Mueller-Hinton agar (MHA)2 to produce an inhibition area. No bacteria colonies willform at the area where the concentration of active ingredients is equal or more than the effectiveconcentration to inhibit the bacteria. A clear inhibition area which has no bacteria growth will bethe measure of antimicrobial activity. Thus, a larger diameter of the zone will signify a higherantimicrobial activity. Thus, my research question will be In vitro study of the Synergism between EssentialOils, Cinnamon (Cinnamomum zeylanicum) and Clove (Syzygium aromaticum) in InhibitingGrowth of Escherichia coli and Staphylococcus aureus.2 MH agar is a research microbiological growth medium used for antimicrobial susceptibility testing Page | 7
  • Michelle Tan Oon Ee 002206-0061.2 Cinnamon (Cinnamomum zeylanicum) Common varieties of cinnamon include Ceylon cinnamon (Cinnamon zeylanicum) andChinese cinnamon (C.cassia). Its dried barks are used for infusions, tinctures, and powders. Thebark’s distillate produces an essential oil with a peppery smell. The stock essential oils to be usedare extracted through steam distillation of Ceylon cinnamon barks imported by a local market fromIndia. The essential oils of spices are known to contain active antimicrobial compounds. Theproximate 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 benzenering and an aldehyde group. Research shows that cinnamaldehyde inhibits cell division inEscherichia coli by decreasing the in-vitro assembling and bundling functions of FtsZ, aprokaryotic homolog of tubulin3. Cinnamaldehyde also reduces the frequency of Z-rings formedfrom FtsZ per unit cell length of E. coli. The polymerization of FtsZ is inhibited by [7]cinnamaldehyde, disallowing the cell to regenerate . Ceylon cinnamon also contains 4-10%eugenol4. Its other components include ethyl cinnamate, beta-caryophyllene, linalool, and methylchavicol, which are food additives and aromatic compounds [8]. Figure 1: Chemical Structure of Cinnamaldehyde Cinnamon bark is antispasmodic; it stimulates salivary glands and stomach mucousmembranes. Scientists have discovered that both cinnamaldehyde and eugenol inhibit Helicobacterpylori[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. Page | 8
  • Michelle Tan Oon Ee 002206-0061.3 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 fourspreading sepals and four unopened petals in the middle resembling a small ball. Essential oils ofcloves 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 aphytochemical6 that causes cytoplasm membrane coagulation and active transport unbalance [10] .Eugenol, an analgesic, is known to combat inflammation and bacterial and fungal infection. Cloveoil is the active ingredient in mouthwashes and over-the-counter pain-relief remedies fortoothaches. It contains high tannin content of 10% to 19%, providing additional antimicrobialactivity. 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 anti-inflammatory agent. Clove oil has shown considerable efficacy against several common hospital-acquired bacteria and yeasts including MRSA7. [12] Clove bud oil also significantly decreases theproduction 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 antibiotics7 Methicillin-resistant Staphylococcus aureus8 Toxins that causes food poisoning by contaminating cooked meat, eggs, fish, milk and dairy products Page | 9
  • Michelle Tan Oon Ee 002206-0061.4 Two Types of Bacteria The two bacteria are chosen are Staphylococcus aureus, (Gram-positive bacterium) andEscherichia coli, (Gram-negative bacterium). A clearer view on different susceptibilities of the twospectrums of bacteria towards cinnamon and cloves can be determined. In Figure 3(a), gram-positive bacteria have a thick layer of peptidoglycan enclosing theplasma membrane allowing it to retain the crystal violet stain from violet staining. Uniquely, thecell wall contains teichoic acids9 and lipoteichoic acids10, providing rigidity and a site for parasiteattachment. Figure 3(a) Figure 3 (b) 11 Figure (b) shows gram-negative cells have thinner peptidoglycan layers with porins12 on anouter membrane. Gram-negative bacteria are generally more resistant than gram-positive bacteria asthe 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 polysaccharides10 Teichoic acids which are anchored to the lipid bilayer via a glycerolipid11 http://www.micro.cornell.edu/cals/micro/research/labs/angert-lab/low.cfm12 Proteins that act as a pore to allow diffusion of molecules. Page | 10
  • Michelle Tan Oon Ee 002206-0062.0 Hypotheses2.1 Effects of Cinnamon and Clove on E.coli and Staph. a. Antimicrobial agents attack specific cell sites to cause microbial death or damage. The fourmain targets are cell walls, cell membranes, biosynthesis pathways for DNA 13 and RNA 14 , orprotein enzyme functions. Certain organic compounds can penetrate through or attach themselves tobacteria cell membranes, binding with bacteria structures and subsequently destroying it. Bothcinnamon and clove essential oils contain phenol and aldehyde groups, which inhibit bacterialgrowth 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] andits aldehyde group inactivate proteins through polymerisation. For cloves with high eugenolcontent, 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 peptidoglycanlayer makes Staph. a. less resistant to antimicrobial agents. Thus, cinnamaldehyde and eugenol haveeasier access to attack the peptidoglycan layer and cell wall.13 Deoxyribonucleic acid is a double-stranded nucleic acid that contains genetic instruction for development andfunctioning of living organisms.14 Ribonucleic acid is a single-stranded molecule consisting of a long chain of nucleotide units and is important forprotein synthesis. Page | 11
  • Michelle Tan Oon Ee 002206-0062.2 Synergistic effects of the combination of Cinnamon and Cloveiii) The combination of both spices will produced an enhanced effect with a larger zone ofinhibition. Although cinnamon and clove both have antimicrobial properties, the two spices are rarelyused individually. Since both cinnamon and clove contain eugenol, the combination will increasethe total amount of eugenol in the new derived antimicrobial agent. The aldehyde and phenolgroups in the essential oils can work together to disrupt protein synthesis. However, throughpreliminary testing, cinnamon showed stronger inhibitory effects than clove. The antimicrobialeffect of cinnamon, presumably from cinnamaldehyde, is significantly larger than that of clove’seugenol.iv) A combination of increasing cinnamon volume concentration and decreasing clove volumeconcentration will produce a larger zone of inhibition. Page | 12
  • Michelle Tan Oon Ee 002206-0063.0 Variables3.1 Manipulated Variables: Combination of cinnamon and clove essential oils at differentpercentage ratios. Percentage ratio Volume of Cinnamon, Volume of Clove, volume per volume, (%v/v) Vcin/ μl Vclove/ μl % Cinnamon: % Clove 5.0 45.0 10% : 90% 9.0 21.0 30% : 70% 15.0 15.0 50% : 50% 21.0 9.0 70% : 30% 45.0 5.0 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. Thesmall amounts of extract ranging from 5μl to 45μl are measured with a micropipette. All of thecombined ratios are kept overnight in a micro centrifuge and spun in the centrifuge to obtain ahomogenous mixture.3.2 Responding Variables: Largest Inhibition Area The optimum ratio of combination of cinnamon and clove will produce the largest inhibitionzone. A zone of inhibition is the diameter of clear area around the impregnated filter paper disc onthe MHA plate after 24 hours of incubation. Zone of inhibition Figure 4: Measuring zone of inhibition using a ruler Page | 13
  • Michelle Tan Oon Ee 002206-0063.3 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 Page | 14
  • Michelle Tan Oon Ee 002206-0064.0 Materials and Methods4.1 Preparation of Mueller-Hinton Agar (MHA)161. 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 Procedures1. 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 25923A loopful of each bacteria strain grown overnight on a MHA slant17, is transferred individually to5ml of Mueller-Hinton broth. After incubation, bacteria suspension’s turbidity is adjusted to matcha 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 Page | 15
  • Michelle Tan Oon Ee 002206-0064.4 Extraction of Cinnamon Essential Oil by Steam Distillation1. 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 Page | 16
  • Michelle Tan Oon Ee 002206-0064.5 Extraction of Clove Essential Oil by Steam Distillation1. 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. Page | 17
  • Michelle Tan Oon Ee 002206-0064.6 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 Percentage Concentration of Volume of Essential Oil, Volume of Polysorbat-80, Essential Oil, VEO / μl Vpolysorbat/ μl (% vol/vol) 50.0 450.0 10% 90.0 210.0 30% 150.0 150.0 50% 210.0 90.0 70% 450.0 50.0 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. Percentage ratio Volume of Cinnamon, Volume of Clove, volume per volume, (%v/v) Vcin/ μl Vclove/ μl % Cinnamon: % Clove 5 45 10% : 90% 9 21 30% : 70% 15 15 50% : 50% 21 9 70% : 30% 45 5 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-8022 Refer to Appendix 6 Page | 18
  • Michelle Tan Oon Ee 002206-0064.7 Disk Diffusion Test (Kirby Bauer Method)[6]1. 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. Page | 19
  • Michelle Tan Oon Ee 002206-0064.8 Incubation of Agar Plates 231. Agar plates are incubated in inverted positions.2. Plates are stacked around 4 in a pile to ensure all plates reach 37°C 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 Page | 20
  • Michelle Tan Oon Ee 002206-0065.0 Data Collection Essential Oil E. coli Staph. A Percentage volume per Trial 1 Trial 2 Trial 3 Mean ± S.Da Trial 1 Trial 2 Trial 3 Mean ± S.D volume / % vol/vol 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 ± 0.51Cinnamon 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 Clove 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 - - - - - - - -Table 4: Inhibition zone (mm) produced by different percentages of cinnamon and clove essential oil on E.coli and Staph. a.a c Mean zone (mm) ± standard deviation for triplicates Positive control * Values are not taken into account due to inconsistencies.b d No zone of inhibition Negative control Page | 21
  • Michelle Tan Oon Ee 002206-006 Essential Oil E. coli Staph. a Percentage Volume Trial 1 Trial 2 Trial 3 Mean ± S.D a Trial 1 Trial 2 Trial 3 Mean ± S.D per Volume Ratios / % Vcin/Vclove 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 - - - - - - - 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 triplicatesb Positive controlc Negative controld No zone of inhibition* Values are not taken into account due to inconsistencies Page | 22
  • Michelle Tan Oon Ee 002206-0065.1 Data Processing Inhibition zone (mm) produced by different percentages of cinnamon and clove essential oil on E. coli and Staph. a. 26 24 22 Mean Inhibition Zone (mm) 20 18 16 14 12 E Coli 10 Staph. A 8 6 4 2 0 10 30 50 70 90 100 10 30 50 70 90 100 Cinnamon Clove Graph 1: Inhibition zone (mm) produced by different percentages of cinnamon and clove essential oil on E.coli and Staph. a. Page | 23
  • Michelle Tan Oon Ee 002206-006 Inhibition zone (mm) produced by different percentage ratios of combination for cinnamon and clove essential oils on E.coli and Staph. a. 18 16 14 Mean Inhibition Zone (mm) 12 10 8 E. Coli Staph. A 6 4 2 0 10% : 90% 30% : 70% 50% : 50% 70% : 30% 90% : 10% Percentage Volume per Volume Ratios / %Cinnamon : %CloveGraph 2: Inhibition zone(mm) produced by different percentage ratios of combination for cinnamon and clove essential oil on E.coli and Staph. a. Page | 24
  • Michelle Tan Oon Ee 002206-006 Inhibition Zone (mm) for Individual and Percentage Ratio Combinations of Cinnamon and Clove Essential Oils on E.coli. 24 22 Percentage Ratio 20 Combination of Minimmum inhbition zone (mm) Cinnamon and Clove 18 16 14 Cinnamon Essential 12 Oil 10 8 100% Cinnamon 6 50% Cinnamon 70% Cinnamon 90% Cinnamon Clove Essential Oil 100% Clove 90% Clove 70% Clove 10% : 90% 30% : 70% 50% : 50% 70% : 30% 90% : 10% 4 10% Cinn 30% Cinn 50% Clov 2 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
  • Michelle Tan Oon Ee 002206-006 Inhibition Zone (mm) for Individual and Percentage Ratio Combinations of Cinnamon and Clove Essential Oils on Staph. a. 26 24 22 Minimmum inhbition zone (mm) Percentage Ratio 20 Combination of 18 Cinnamon and Clove 16 14 12 Cinnamon Essential Oil 10 8 100% Cinnamon 50% Cinnamon 70% Cinnamon 90% Cinnamon 6 100% Clove 90% Clove 70% Clove 50% Clove 10% : 90% 30% : 70% 50% : 50% 70% : 30% 90% : 10% 4 30% Cinn 30% Clov 2 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
  • Michelle Tan Oon Ee 002206-0065.2 Data Analysis: ANOVA [15] The ANOVA test (Analysis of Variance test) 24 compares two sources of variation: variation 2 2between group means, and variation within each group, . The results determine whether thereis a significant difference in antimicrobial activity for different ratio combinations of cinnamon andclove essential oils. Our null hypothesis is 0 = 1 = 2 = 3 = 4 = 5 such that the mean of the five groups are equal. The alternative hypothesis 1 is one of the means is not equal to the others or none of them are equal.The ratio is calculated through the formula: = ℎ If any of the ratios is higher than critical at significant level of 0.05 (α = 0.05), 0 isrejected and thus, one of the means is significantly different from the others. Calculations are donethough Microsoft Excel 2007. However, the -test does not indicates the location of the significantdifference between means of groups. Variable Bacteria Strain F-value F-critical Conclusion The two F-values are E.coli 24.59 3.84 larger than the F-criticalPercentage Ratio values. Thus, theCombinations of ANOVA test shows that Cinnamon and one of the groups has a Clove Essential significant difference Oils Staph. a. 43.77 3.84 from the others in the group. Table 6: Results on ANOVA for two bacterial strains to determine whether there is a significantdifference 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 ANOVA25 See Appendix 8 for more details on ANOVA and Appendix 10 for detailed ANOVA calculations Page | 27
  • Michelle Tan Oon Ee 002206-0065.3 Tukey’s HSD Test [15] A post hoc analysis is then carried out to identify the location of the differences. A Tukey’sHSD26 test is a multiple comparison test that tests out the null hypothesis. The pair whose meandifferences 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 Mean HSD critical ImplicationRatio Combinations of Cinnamon and Difference, mm value Clove Essential Oils on E. coli) 10% : 90% 30% : 70% 0.73 1.85 No significant difference 10% : 90% 50% : 50% 2.23 1.85 significant difference 10% : 90% 70% : 30% 3.16 1.85 significant difference 10% : 90% 90% : 10% 4.83 1.85 significant difference 30% : 70% 50% : 50% 1.50 1.85 No significant difference 30% : 70% 70% : 30% 2.37 1.85 significant difference 30% : 70% 90% : 10% 4.10 1.85 significant difference 50% : 50% 70% : 30% 0.87 1.85 No significant difference 50% : 50% 90% : 10% 2.60 1.85 significant difference 70% : 30% 90% : 10% 1.73 1.85 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 difference26 See Appendix 9 for Tukey’s HSD (honestly significant difference) formula Page | 28
  • Michelle Tan Oon Ee 002206-006 Group Combination, % vol/vol(Mean Inhibition Zone of Percentage Mean HSD critical ImplicationRatio Combinations of Cinnamon and Difference, mm value Clove Essential Oils on Staph. a.) 10% : 90% 30% : 70% 1.10 1.85 No significant difference 10% : 90% 50% : 50% 3.10 1.85 significant difference 10% : 90% 70% : 30% 3.30 1.85 significant difference 10% : 90% 90% : 10% 6.93 1.85 significant difference 30% : 70% 50% : 50% 2.00 1.85 significant difference 30% : 70% 70% : 30% 2.20 1.85 significant difference 30% : 70% 90% : 10% 5.83 1.85 significant difference 50% : 50% 70% : 30% 0.20 1.85 No significant difference 50% : 50% 90% : 10% 3.83 1.85 significant difference 70% : 30% 90% : 10% 3.63 1.85 significant differenceTable 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 Page | 29
  • Michelle Tan Oon Ee 002206-0066.0 Conclusion and Evaluations6.1 Discussion6.1.1 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 essentialoils. Thus, the cinnamaldehyde in cinnamon is a stronger antimicrobial compound than eugenol inclove. Cinnamaldehyde interferes with DNA and RNA functions, either by disallowing DNA toreplicate or by binding irreversibly to DNA, preventing translocation and transcription. Bacterialcells are inhibited from forming growth proteins and multiplying. The compound’s aldehyde grouphas a –CHO functional group on a terminal carbon which polymerises easily. The alkylatingpolymers 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 andtertiary protein structures and causes them to unfold and coil randomly. At higher essential oilconcentrations, they act as cellular poisons, disrupting cell walls and membranes. In lowerconcentrations, they inactivate enzyme systems The inhibition zone for Staph. a. is larger than for E.coli except for 50% cinnamonconcentration. Overall, Staph. a, the gram-positive bacteria, is more susceptible to both essentialoils. There is no inhibition zone for 10% cinnamon concentration (Staph. a), 10% and 30% cloveconcentrations (E.coli) and 10% clove concentration (Staph. a). The content of antimicrobial agentsin 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 essentialoil to be more effective against E.coli than Staph. a. This differs from individual essential oil effectson these bacteria. This is one possible enhanced synergistic effects of the combination of essentialoils. A combination of cinnamon and clove would be more effective to combat gram-negativebacteria. Page | 30
  • Michelle Tan Oon Ee 002206-006 The ratio of cinnamon to clove that produces the largest inhibition zone is (90% : 10%). TheTukey’s HSD test shows that ratios (90% :10%) and (70% :10%) have significant differences withother ratios. With increasing percentage concentration of cinnamon essential oil in the combinedmixture, the zone of inhibition enlarges. Thus, antimicrobial activity is proportional to theconcentration of cinnamon in the combined mixture. However, with reference to table 7, there is no significant difference in antimicrobialactivity 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 antimicrobialactivity with larger zones of inhibition than individual concentrations. However, the amount ofantimicrobial agents in the individual concentrations is not 100% of the mixture. To determine whether there is a synergistic effect for cinnamon and cloves, both thecombined and individual mixtures must contain 100% antimicrobial agents. Thus, ratios arecompared with 100% essential oils. Clearly, 100% cinnamon’s antimicrobial activity is higher thanthe 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 cloveessential 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 ofinhibition than individual concentrations. When compared with 100% cinnamon, all combined Page | 31
  • Michelle Tan Oon Ee 002206-006ratios are significantly lower. Comparisons with 100% clove show that cinnamon enhances clove’santimicrobial properties for ratios (10% : 90%), (30% :70%), (50% : 50%) and (70% : 30%). Thereis also no synergistic effect for all combined ratios of cinnamon and clove essential oils when testedon Staph. a.6. 2 Limitations1. 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. Page | 32
  • Michelle Tan Oon Ee 002206-0065. 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 Ways to Improve Experiment1. 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. Page | 33
  • Michelle Tan Oon Ee 002206-0066.4 Areas of Further Investigations Antibacterial activities of cinnamons and cloves may be less effective when cooked orheated under intense temperatures. Aldehyde groups can oxidise to form carboxylic acids whenexposed to air and under high temperatures. This will reduce the stability of cinnamaldehyde, whichmight decompose at high temperatures. The effects of temperature on cinnamons and cloves couldbe further investigated. Most organic compounds are also unstable at high pHs. Their antimicrobial activity could behindered when digested in the stomach’s acidic medium. The optimum pH level at which cinnamonand clove antimicrobial activity is most efficient could be determined. The viability of cinnamon and clove as a potential antibacterial drug would require moreresearch done on the evaluation of their in-vivo efficiency. This will include the effects of oxygenand light on antimicrobial activity of the essential oils. Further experimentation could be done with crude ethanolic extracts where spice materialsare cut into small pieces and soaked in 95% ethanol. This could aid in commercial production ofcinnamon remedies as their barks have small quantities of oils. However, inhibitory activity ofessential 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 andvinegar. There may be synergistic interaction between the active components of spices with thesefactors at low or high temperatures to increase antimicrobial effects. To overcome the problem of antibiotics that can no longer combat bacteria, essential oils ofcinnamon and cloves could possibly be modified into existing drugs. The synergistic effects canovercome bacteria resistance to these antibiotics. Page | 34
  • Michelle Tan Oon Ee 002206-0066.5 Conclusion Higher concentration of essential oils produces larger zones of inhibition and higherantimicrobial activity. Cinnamon is a stronger antimicrobial agent than clove. Both spices can blockbacterial protein syntheses without adversely affecting human cells. Staph. a has lower resistance toboth essential oils. The best percentage volume per volume ratio is (90% : 10%), showing greatestantimicrobial activity against both bacteria. The combined mixture works best at a higherconcentration of cinnamon essential oils. A larger amount of cinnamon should thus be used incooking to attain the highest antimicrobial properties. Only cinnamon concentrations are moreefficient in inhibiting growth individually. Smaller quantities of cinnamon can be added with clove spice to enhance antimicrobialactivity for cloves. This may be applicable while cooking as large quantities of a single spice couldproduce a very strong smell. Although it seems that there is little or no synergistic effect betweenthese essential oils, a combination of the two essential oils does inhibit bacteria growth. Page | 35
  • Michelle Tan Oon Ee 002206-0067.0 References1. Antibiotic Uncovered [Online] 30 May 2003 [Cited: 20 March 2009.]http://www.foodqualitynews.com/Food-Alerts/Antibiotics-uncovered2. American Society for Microbiology. Antibiotic Resistance - Current Status and FutureDirections [Online] 6 July 1994 [Cited: 22 March 2009.]http://www.asm.org/ASM/files/CCPAGECONTENT/DOCFILENAME/0000005962/antibiot[1].pdf3. Wong, Cathy. Health Benefits of Cinnamon [Online] 27 October 2007 [Cited: 7 July 2009.]http://altmedicine.about.com/od/cinnamon/a/cinnamon.htm4. 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.html5. Wood, Shelley. Cinnamon and cloves: Benefits in diabetes probed [Online] 4 April 2006[Cited: 7 July 2009] http://www.medscape.com/viewarticle/5390166. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial DisksSusceptibility 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 July2009] Page | 36
  • Michelle Tan Oon Ee 002206-0068. Cinnamon. [Cited: 8 July 2009] http://en.wikipedia.org/wiki/Cinnamon9. 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 pathogenHelicobacter 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 infectiousendocarditis causing Gram-positive bacteria. 28 February 2007. [Cited: 9 July 2009]11. Clove. [Cited: 9 July 2009] http://en.wikipedia.org/wiki/Clove12. 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 promisingforce 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 ItsBud Oil. 2007. [Cited: 9 July 2009]14. Talaro, Kathleen Park. Foundations in Microbiology - Basic Principles , Sixth Edition.McGraw Hill, 2008. Page | 37
  • Michelle Tan Oon Ee 002206-00615. Kuzma, Jan W; Bohnenblust, Stephen E. Basic Statistics for the Health Sciences, FourthEdition. McGraw-Hill International Edition, 200116. Schwalbe, Richard; Steele-Moore, Lynn; Goodwin, Avery C. Antimicrobial SusceptibilityTesting 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 Salmonelleand Other Enterobacteria. 2005. [Cited: 25 July 2009] Page | 38
  • Michelle Tan Oon Ee 002206-0068.0 AppendixesAppendix 1: Table of Antimicrobial Effectiveness of Spices and HerbsSnyder, O. Peter. Antimicrobial effects of Spices and Herbs.1997URL: http://www.hi-tm.com/Documents/Spices.html Spices and Herbs Inhibitory Effect Cinnamon, cloves, mustard Strong Allspice, bay leaf, caraway, coriander, Medium cumin, oregano, rosemary, sage, thyme Black pepper, red pepper, ginger Weak Table 9: Antimicrobial Effectiveness of Various Spices and HerbsAppendix 2: Preparation of Mueller-Hinton Agar1. 7g MHA powder is weighed with an electronic balance. 250cm³ 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
  • Michelle Tan Oon Ee 002206-006Appendix 3: Turbidity Standard PreparationA 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 extraction1. 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
  • Michelle Tan Oon Ee 002206-0064. 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 oilsAppendix 5: Negative Control and Solvent used in Serial Dilution – Polysorbat-80 Figure 12: Polysorbat-80 Page | 41
  • Michelle Tan Oon Ee 002206-006Polysorbat-80 is a viscous, water-soluble yellow liquid that is used in the dilution of the stockessential oil. The hydrophilic groups in this compound are polyethers also known aspolyoxyethylene groups, which are polymers of ethylene oxide. In the nomenclature ofpolysorbates, the numeric designation following polysorbate refers to the lipophilic group, in thiscase the oleic acid.Appendix 6: Preparation of Cinnamon and Clove Percentage RatiosAppendix 7: Set-up of Incubator Figure 13: Incubator Page | 42
  • Michelle Tan Oon Ee 002206-006The incubator comprises of a shelf in a closed cupboard with a single 100W bulb. It has removableblack and white boards to reflect and absorb light, preventing overheating. The agar plates andinoculum suspensions are incubated here. Petri dishes are inverted to prevent condensation moisturefrom dripping onto the agar surface. A thermometer is placed inside the incubator to monitor itstemperature. Before the carrying out of the experiments, the incubator is sprayed and cleaned withethanol to avoid contamination.Appendix 8: ANOVA AssumptionsTo 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 ofanother.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 ofany other group.Appendix 9: Further Details on ANOVASome convential notations and their definitions used in ANOVA calculations: NOTATION DEFINITION MS within or Mean square within or Within-group variance MS between or Mean square between or Between-group variance Degree of freedom Number of groups Number of observations in a group N Total number of observation α Significant level Sum of squares within group Sum of squares between group Table 10: Convention notations and definitions Page | 43
  • Michelle Tan Oon Ee 002206-006The one-way ANOVA table is shown below: Source of Sum of df Mean Squares F ratio Critical F P Value variation squares (s²)Between k−1 = − 1 −1, − ComputerWithin N−k generated = − Total N−1 Table 11: One-Way ANOVA  The critical F value is at significant level of 0.05 or 5%, thus, α = 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 TestThe formula for the Tukey’s HSD test is: = (, , − ) such that is obtained from Percentage Points of the Studentized Range for 2 Through 20 Treatments Table[15] (pg 304) α = significant level of 0.05 k = total number of groups N – k = represents total number of results – total number of groupsAppendix 11: Detailed Calculation of ANOVA using Microsoft Excel 2007 Page | 44
  • Michelle Tan Oon Ee 002206-0061. 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 Count Sum Average Variance Ratios / % Vcin/Vclove) Group 1 (10% : 90%) 3 32.0 10.67 0.58 Group 2 (30% : 70%) 2 22.8 11.40 0.32 Group 3 (50% : 50%) 2 25.8 12.90 0.02 Group 4 (70% : 30%) 3 41.3 13.77 0.56 Group 5 (90% : 10%) 3 46.5 15.50 0.39 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 41.96 4 10.49 24.59 1.51E-04 3.84 Within Groups 3.41 8 0.43 Total 45.37 12 0.43 = 4.89 = 1.85 32. 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 FactorSUMMARY Groups(Percentage Volume per Volume Count Sum Average Variance Ratios / % Vcin/Vclove)Group 1 (10% : 90%) 2 17.80 8.90 0.02Group 2 (30% : 70%) 3 30.00 10.00 0.25Group 3 (50% : 50%) 2 24.00 12.00 0.50Group 4 (70% : 30%) 3 36.60 12.20 0.63Group 5 (90% : 10%) 3 47.50 15.83 0.58ANOVA Source of Variation SS df MS F P-value F critBetween Groups 75.42 4 18.86 43.77 1.76E-05 3.84Within Groups 3.45 8 0.43Total 78.87 12 0.43 = 4.89 = 1.85 3 Page | 45