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

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

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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)


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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

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 [⁶].

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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1.2 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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1.3 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 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 phenol5 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 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 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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1.4 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 acids

10, 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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2.0 Hypotheses

2.1 Effects of Cinnamon and Clove on E.coli and Staph. a.

Antimicrobial agents attack specific cell sites to cause microbial death or damage. The four

main targets are cell walls, cell membranes, biosynthesis pathways for DNA13

and RNA14

, 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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2.2 Synergistic effects of the combination of Cinnamon and Clove

iii) 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 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

Percentage ratio

volume per volume, (%v/v)

% 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. 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.

Figure 4: Measuring zone of inhibition using a ruler

Zone of inhibition


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3.3 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


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4.0 Materials and Methods

4.1 Preparation of Mueller-Hinton Agar (MHA)16

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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4.4 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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4.5 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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4.6 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

Volume of Essential Oil,

VEO / μl

Volume of Polysorbat-80,

Vpolysorbat/ μl

Percentage Concentration of

Essential Oil,

(% 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.

Volume of Cinnamon,

Vcin/ μl

Volume of Clove,

Vclove/ μl

Percentage ratio

volume per volume, (%v/v)

% 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-80

22 Refer to Appendix 6


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4.7 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.

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.

Positive control

Negative control


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4.8 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 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


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5.0 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

Cinnamon

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

Clove

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 -

- - - - - - -

Table 4: Inhibition zone (mm) produced by different percentages of cinnamon and clove essential oil on E.coli and Staph. a. a Mean zone (mm) ± standard deviation for triplicates

c Positive control * Values are not taken into account due to inconsistencies.

b No zone of inhibition

d Negative control


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Essential Oil

Percentage Volume

per Volume Ratios /

% Vcin/Vclove

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 - - - - - - -

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

b Positive control

c Negative control

d No zone of inhibition

* Values are not taken into account due to inconsistencies


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5.1 Data Processing

Graph 1: Inhibition zone (mm) produced by different percentages of cinnamon and clove essential oil on E.coli and Staph. a.

0

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Me

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Cinnamon Clove

Inhibition zone (mm) produced by different percentages of cinnamon and clove essential oil on E. coli and Staph. a.

E Coli

Staph. A


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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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mm

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Percentage Volume per Volume Ratios / %Cinnamon : %Clove

Inhibition zone (mm) produced by different percentage ratios of combination for cinnamon and clove essential oils on E.coli and Staph. a.

E. Coli

Staph. A


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Graph 3: Inhibition Zone by Individual and Percentage Ratio Combinations of Cinnamon and Clove Essential Oils on E.coli.

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inh

bit

ion

zo

ne

(m

m)

Various Values of Cinnamon and Clove Essential Oils

Inhibition Zone (mm) for Individual and Percentage Ratio Combinations of Cinnamon and Clove Essential Oils on E.coli.

Percentage Ratio Combination of Cinnamon and Clove

Cinnamon Essential Oil

Clove Essential Oil


Page | 26

Graph 4: Inhibition Zone by Individual and Percentage Ratio Combined Cinnamon and Clove Essential Oils on Staph. a.

10

% :

90

%

30

% :

70

%

50

% :

50

%

70

% :

30

%

90

% :

10

%

30

% C

inn

50

% C

inn

amo

n

70

% C

inn

amo

n

90

% C

inn

amo

n

10

0%

Cin

nam

on

90

% C

love

70

% C

love

50

% C

love

30

% C

lov

10

0%

Clo

ve

0

2

4

6

8

10

12

14

16

18

20

22

24

26

10% : 90% 30% : 70% 50% : 50% 70% : 30% 90% : 10%

Min

imm

um

inh

bit

ion

zo

ne

(m

m)

Various Values of Cinnamon and Clove Essential Oils

Inhibition Zone (mm) for Individual and Percentage Ratio Combinations of Cinnamon and Clove Essential Oils on Staph. a.

Percentage Ratio Combination of Cinnamon and Clove

Cinnamon Essential Oil

Clove Essential Oil


Page | 27

5.2 Data Analysis: ANOVA [15]

The ANOVA test (Analysis of Variance test) 24

compares two sources of variation: variation

between group means, 𝑠𝑏2 and variation within each group, 𝑠𝑤

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 𝐻0 = 𝜇1 = 𝜇2 = 𝜇3 = 𝜇4 = 𝜇5 such that the mean of the five groups are

equal.

The alternative hypothesis 𝐻1 is one of the means is not equal to the others or none of them are

equal.

The 𝐹 ratio is calculated through the formula:

𝐹 𝑟𝑎𝑡𝑖𝑜 =𝑀𝑒𝑎𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝑏𝑒𝑡𝑤𝑒𝑒𝑛 𝑔𝑟𝑜𝑢𝑝𝑠

𝑀𝑒𝑎𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝑤𝑖𝑡ℎ𝑖𝑛 𝑔𝑟𝑜𝑢𝑝

If any of the 𝐹 ratios is higher than 𝐹 critical at significant level of 0.05 (α = 0.05), 𝐻0 is

rejected and thus, one of the means is significantly different from the others. Calculations are done

though Microsoft Excel 2007. However, the 𝐹-test does not indicates the location of the significant

difference between means of groups.

Variable Bacteria Strain F-value F-critical Conclusion

Percentage Ratio

Combinations of

Cinnamon and

Clove Essential

Oils

E.coli 24.59 3.84

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.

Staph. a. 43.77 3.84

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


Page | 28

5.3 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)

Mean Difference, mm

HSD critical value

Implication

10% : 90% 30% : 70% 0.73 1.85 No significant difference

10% : 90% 50% : 50% 2.23 1.85 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


Page | 29

Group Combination, % vol/vol (Mean Inhibition Zone of Percentage Ratio Combinations of Cinnamon and

Clove Essential Oils on Staph. a.)

Mean Difference, mm

HSD critical value

Implication











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


Page | 30

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 | 31

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


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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 Limitations

1. 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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5. 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 Experiment

1. 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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6.4 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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6.5 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.


Page | 36

7.0 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. 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


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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.

Figure 10: Conical flask of distillate with added DCM

Distillate

Dichloromethane

layer with dissolved

essential oils


Page | 41

4. 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 𝒔𝒘𝟐 Mean square within or Within-group variance

MS between or 𝒔𝒃𝟐 Mean square between or Between-group variance

𝒅𝒇 Degree of freedom

𝒌 Number of groups

𝒏 Number of observations in a group

N Total number of observation

α Significant level

𝑺𝑺𝒘 Sum of squares within group

𝑺𝑺𝒃 Sum of squares between group

Table 10: Convention notations and definitions


Page | 44

The one-way ANOVA table is shown below:

Source of

variation

Sum of

squares

df Mean Squares

(s²)

F ratio Critical F P Value

Between 𝑆𝑆𝑏 k − 1 𝑀𝑆𝑏 =

𝑆𝑆𝑏𝑘 − 1

𝑀𝑆𝑤𝑀𝑆𝑏

𝐹𝑘−1, 𝑁−𝑘

Computer

generated Within 𝑆𝑆𝑤 N − k 𝑀𝑆𝑏 =

𝑆𝑆𝑤𝑁 − 𝑘

Total 𝑆𝑆𝑡 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 Test

The formula for the Tukey’s HSD test is:

𝐻𝑆𝐷 = 𝑞(𝛼,𝑘,𝑁 − 𝑘) 𝑀𝑆𝑤𝑛

such that 𝑞 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 groups

Appendix 11: Detailed Calculation of ANOVA using Microsoft Excel 2007


Page | 45

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)

Count Sum Average Variance

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

𝐻𝑆𝐷 = 4.89 0.43

3= 1.85

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)

Count Sum Average Variance

Group 1 (10% : 90%) 2 17.80 8.90 0.02 Group 2 (30% : 70%) 3 30.00 10.00 0.25 Group 3 (50% : 50%) 2 24.00 12.00 0.50 Group 4 (70% : 30%) 3 36.60 12.20 0.63 Group 5 (90% : 10%) 3 47.50 15.83 0.58

ANOVA Source of Variation SS df MS F P-value F crit

Between Groups 75.42 4 18.86 43.77 1.76E-05 3.84

Within Groups 3.45 8 0.43

Total 78.87 12

𝐻𝑆𝐷 = 4.89 0.43

3 = 1.85

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

Education

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