Antimicrobial effect of garlic against E Coli and Stap.A

Extended Essay – Biology LIM JU ANNE 002206‐018

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EXTENDED ESSAY

BIOLOGY

Optimal Condition for Antimicrobial Activity:

In Vitro study on the effects of temperature and pH on Antimicrobial Activity of Aqueous Garlic Extract against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923.

Submitted by: Lim Ju Anne

Candidate Number: 002206 – 018

Word Count: 3995 only


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Table of Contents

Figures ........................................................................................................................................................... 3

Tables ............................................................................................................................................................ 4

Abstract ......................................................................................................................................................... 6

1.0 Introduction ........................................................................................................................................ 7

1.1 Rationale of Study ............................................................................................................................... 7

1.2 Aim ...................................................................................................................................................... 8

1.3 Garlic (Allium Sativum) ........................................................................................................................ 9

1.4 Bacteria ............................................................................................................................................. 11

2.0 Variables ................................................................................................................................................ 13

2.1 Independent variable ........................................................................................................................ 13

2.2 Dependent variable ........................................................................................................................... 13

2.3 Fixed variable .................................................................................................................................... 13

3.0 Procedures ............................................................................................................................................ 14

3.1 Preparation before experiment ........................................................................................................ 14

3.2 Preparation of Aqueous Garlic Extract for different temperature and pH treatment ..................... 15

3.3 Method for Agar Disk Diffusion Method .......................................................................................... 17

4.0 Data Collection ................................................................................................................................. 21

4.1 Raw Data: Antimicrobial Activity of Aqueous Garlic Extract Incubated at Different Temperature . 21

4.2 Raw Data: Antimicrobial Activity of Aqueous Garlic Extract Incubated at Different pH .................. 23

4.3 Data Processing: Comparing Mean Inhibition Zone of E. Coli and Staph. a ..................................... 25

4.4 Data Processing: ANOVA and Tukey’s HSD test................................................................................ 26

5.0 Conclusion ........................................................................................................................................ 30

5.1 Part 1: Temperature ......................................................................................................................... 30

5.2 Part 2: pH .......................................................................................................................................... 32

6.0 Evaluation and Suggestion ................................................................................................................ 34

7.0 References ........................................................................................................................................ 36

8.0 Appendixes ....................................................................................................................................... 37


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Figures Figure 1: Generation of Allicin in Garlic Clove (2) ......................................................................................... 9 Figure 3: Cell Wall of Gram‐positive Bacteria (7) ........................................................................................ 11 Figure 2: Cell wall of Gram‐negative Bacteria (10) ..................................................................................... 11 Figure 4: Swabbing direction on the nutrient agar plate ............................................................................ 18 Figure 5: Labelling the Nutrient Agar Plate (left figure: for testing of different incubation pH; right figure: for testing of different incubation temperature) ....................................................................................... 19 Figure 6: Measuring Inhibition Zone of Aqueous garlic Extract Treated at Different pH ........................... 20


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Tables

Table 1: Microcentrifuge labelling and its corresponding temperature ..................................................... 15 Table 2: Microcentrifuge labelling and its corresponding pH ..................................................................... 17 Table 3: Labelling legend on Nutrient agar plate ........................................................................................ 19 Table 4: Inhibition Zone (mm) of aqueous garlic extract incubated at various temperatures on E. coli ... 21 Table 5: Inhibition Zone (mm) of aqueous garlic extract incubated at various temperatures on Staph. a 22 Table 6: Inhibition Zone (mm) of aqueous garlic extract incubated at various pH on E. coli ..................... 23 Table 7: Inhibition Zone (mm) of aqueous garlic extract incubated at various pH on Staph. a ................. 24 Table 8: Results of ANOVA on 4 sets of data to determine whether there is significant difference between mean inhibition zone of aqueous garlic extract incubated at different temperature and pH on E. Coli and Staph. a ..................................................................................................................................... 27 Table 9: Results of Tukey’s HSD test on mean inhibition zone of aqueous garlic extract incubated at different temperature on E. Coli to determine which group is significantly different than the other....... 28 Table 10: Results of Tukey’s HSD test on mean inhibition zone of aqueous garlic extract incubated at different temperature on Staph. a to determine which group is significantly different than the other ... 28 Table 11: Results of Tukey’s HSD test on mean inhibition zone of aqueous garlic extract incubated at different pH on E. Coli to determine which group is significantly different than the other ....................... 29 Table 12: Results of Tukey’s HSD test on mean inhibition zone of aqueous garlic extract incubated at different pH on Staph. a to determine which group is significantly different than the other ................... 29 Table 13: Conventional notations and its meaning .................................................................................... 41 Table 14: One‐way ANOVA table ................................................................................................................ 42 Table 15: Summary ..................................................................................................................................... 43 Table 16: ANOVA table ............................................................................................................................... 43 Table 17: Summary ..................................................................................................................................... 44


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Table 18: ANOVA table ............................................................................................................................... 44 Table 19: Summary ..................................................................................................................................... 45 Table 20: ANOVA table ............................................................................................................................... 45 Table 21: Summary ..................................................................................................................................... 46 Table 22: ANOVA Table ............................................................................................................................... 46


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Abstract

Garlic (Allium Sativum) is known to have antimicrobial properties. This study investigates the effects of

pH and temperature on the antimicrobial activity of aqueous garlic extract. The aqueous garlic extract is

tested on non‐pathogenic Escherichia coli ATCC 25922 and Staphylococus aureus ATCC 25923 in an in

vitro environment. The method chosen is Agar Disc Diffusion Method. Filter paper discs are dipped into

the aqueous garlic extract which are pre‐treated at different pH and temperature and then put onto

inoculated agar plates. The agar plates are incubated at approximately 37oC for 24 hours. The diameter

of inhibition zone (clear zone around the filter paper disc) is measured; the larger the diameter of zone

of inhibition, the higher the antimicrobial activity of the aqueous garlic extract. ANOVA (calculated at

significance level of 0.05) and Tukey’s HSD test is used to analyse the data. For E. coli and Staph. a,

inhibition decreases as incubation temperature of aqueous garlic extract increases; there is no inhibition

at 100oC. Inhibition is greatest for both bacteria strains when aqueous garlic extract is incubated at

room temperature (25oC). Antimicrobial activity of aqueous garlic extract incubated at 75oC and 80oC is

significantly lesser than aqueous garlic extract incubated at 25oC and 40oC. There is inhibition in Staph.

a and E. coli for aqueous garlic extract incubated in pH 1 and pH 7 medium. However, antimicrobial

activity on E. coli is significantly higher when aqueous garlic extract is incubated in pH 1 compared to pH

7. There is no antimicrobial activity on Staph. a and E. coli when aqueous garlic extract is incubated in

medium of pH 12 and pH 14. The results of my study suggest that optimum incubation temperature for

antimicrobial activity of aqueous garlic extract is between 25oC to 40oC and optimum pH is between

acidic (pH 1) to neutral medium (pH 7).

(298 words only)


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1.0 Introduction

1.1 Rationale of Study

Overuse of antibiotics caused increasing antibiotic‐resistant bacteria; the perception that antibiotic is

the ultimate cure had pressured physicians into prescribing antibiotics for every infection, even if it is a

viral infection. Not finishing the entire course of antibiotic prescribed leaves behind weakened bacteria

in the host. Animals are sometimes given low dosage of antibiotics for long duration (to prevent

bacterial infection) to increase the rate of weight gain (1). The unnecessary administration of antibiotics

leaves weakened bacteria which then mutate and gain antibiotic resistant gene. Antibiotic like

penicillin1 is ineffective now.

Studies suggest that allicin works by inhibiting certain thiol‐containing enzymes in the microorganisms

by rapid reaction of thiosulfinates with thiol groups of the enzyme (2). It is difficult for bacteria to gain

resistance because alteration of enzyme’s structure is not an alternative; according to the lock‐and‐key

model, the action of enzyme is specific because the geometric shape of enzyme’s active site and its

substrate is complementary. Alteration of enzyme’s shape causes substrate to be unable to fit into

enzyme’s active‐site. The bacteria cannot survive because bio‐chemical enzyme‐catalysed reaction such

as respiration cannot be carried out.

Lastly, as Escherichia coli (E. coli) and Staphylococcus aureus (Staph. a) are common bacteria in our

daily lives which have the potential to turn pathogenic and garlic is a common spice used in most

culture, I feel that it is worthwhile studying the possibility of using aqueous garlic extract as natural

antibiotic.

1 See Appendix 1


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1.2 Aim

The aim of my paper is to study the effects of pH and temperature on the antimicrobial activity of

aqueous garlic extract.

Hence, my research question is Optimal Condition for Antimicrobial Activity: In Vitro study on the

effects of temperature and pH on Antimicrobial Activity of Aqueous Garlic Extract against Escherichia

coli ATCC 25922 and Staphylococcus aureus ATCC 25923.

The Agar Disk Diffusion Method is chosen for this experiment (3). This method is adapted2 from

Performance Standards for Antimicrobial Disks Susceptibility Tests; Approved – Ninth Edition published

by Clinical and Laboratory Standards. I chose this method because of the hydrophilic nature of aqueous

garlic extract. Filter paper discs are impregnated with aqueous garlic extract and placed onto inoculated

nutrient agar. The active ingredient will diffuse through the nutrient agar’s surface. No colonies will

grow near the area where the concentration is equal or more than the effective concentration to kill or

inhibit the bacteria. The size of the clear zone (no bacteria growth) is a measure of the antimicrobial

activity. The larger the diameter of the clear zone, the higher the antimicrobial activity.

2 See Appendix 2


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1.3 Garlic (Allium Sativum)

Garlic (Allium Sativum) is one of the oldest spices that had been noted for its medicinal value across

many cultures (Egyptians, Greeks, Romans, Chinese, Islamic and Indians). For example, Herodotus

wrote that garlic is given to labourer building the pyramids to increase their stamina; Hippocrates

thought that garlic is good for many ailments; Mohammed, the prophet claimed garlic applied directly to

a sting wound would relieve its pain (4).

The earliest documentation garlic’s antimicrobial property of garlic was done by Louis Pasteur in 1858

(4). Due to its antimicrobial property, garlic poultice is used to dress wound during World War I. In

World War II, the Russian army also turned to garlic when they ran out of penicillin and thus, garlic was

named the ‘Russian Penicillin’ (5).

Allicin, produced when crushed, is attributed by many researches to the antimicrobial activity of garlic.

It is responsible for the typical garlic odour. Whole garlic bulbs contain odourless, sulphur containing

amino acid derivative called alliin and an enzyme called allinase. Alliin is contained in the mesophyll

cells while allinase in the bundle sheath cells of the whole garlic. When garlic is crushed, alliin and

allinase will interact (2). Allinase converts alliin into allicin as illustrated in Figure 1.

Figure 1: Generation of Allicin in Garlic Clove (2)


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Though, theoretically, allicin is deemed responsible for garlic’s antimicrobial activity, many researches

show that allicin is a volatile compound which decomposes into other sulphurous compound such as

diallys sulphide, diallyl disulphide, and ajoene after its formation (6). Due to ambiguity of information

presented, limitation of my knowledge at this level of education and limitation of school lab instrument,

I could not possibly verify allicin as the compound responsible for garlic’s antimicrobial activity.

Therefore, compound responsible for the antimicrobial activity of aqueous garlic extract will be referred

collectively as inhibitory components in this study.


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1.4 Bacteria

I have chosen E. coli and Staph. a because they represent the two spectrums of bacteria: E. coli

represent the gram‐negative bacteria while Staph. a represent the gram‐positive bacteria. This will

therefore give a general idea of the different susceptibility level of gram‐positive and gram‐negative

bacteria towards aqueous garlic extract.

Figure 3: Cell Wall of Gram‐positive Bacteria (7)

Figure 2: Cell wall of Gram‐negative Bacteria (10)


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As shown in Figure 2, the outer membrane of the gram‐negative cell wall has a type of protein called

porin which controls what enters and leaves the cell – this gives the quality of selective permeability to

bile, disinfectants and drugs. In Figure 3, cell wall of the gram‐positive bacteria has peptidoglycan which

contains tightly bound acidic polysaccharides (teichoic acid). The presence of outer membrane in gram‐

negative bacteria provides extra barrier, making it less permeable to antimicrobial substances compared

to gram‐positive bacteria. Therefore, it is generally easier to inhibit or destroy gram‐positive bacteria

due to difference in the cell wall structure(8).


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2.0 Variables

2.1 Independent variable

Aqueous garlic extract is subjected to different temperature using the water bath for 15 minutes. The

temperatures tested are 100oC, 80 oC, 75 oC, 40 oC and room temperature.

0.5 cm3 of aqueous garlic extract is added into 0.5 cm3 either hydrochloric acid or sodium hydroxide

adjusted to pH 1, pH7, pH12 and pH14 using pH meter (±0.01).

2.2 Dependent variable

The optimal pH and temperature is indicated by aqueous garlic extracts which produces the largest

inhibition zone after being subjected to a particular pH and temperature. The inhibition zone is the

diameter of the clear zone around impregnated filter paper disc on the nutrient agar plate after 24

hours of incubation.

2.3 Fixed variable

The fixed variables are the amount of bacteria inoculated on the nutrient agar plate; concentration of

aqueous garlic extract; concentration, pH (7.2) and volume of nutrient agar used; the duration of filter

paper disc being soaked in the extract; diameter of filter paper disc (6mm).


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3.0 Procedures

3.1 Preparation before experiment

3.1.1 Apparatus preparation

Filter paper discs are cut out with hole puncher of 6mm diameter. Cheese cloth, mortar and

pestle, filter paper discs, forceps, and cotton bud are sterilised using an autoclave.

7g of Gene Chemicals nutrient agar powder is added to 250cm3 of distilled water. The mixture

is heated while stirring until it boils. Then, it is poured into a glass bottle and sterilised in the pressure

cooker. During sterilization, glass bottle cap is loosened to allow steam to escape and prevent explosion

in the pressure cooker.

The nutrient agar solution is then poured into 90 mm nutrient agar plate3 up to 7 mm

thickness and allowed to set on a flat surface. After it had cooled down, the nutrient agar plate is

covered to prevent contamination.

3.1.2 Turbidity standard preparation

McFarland 0.5 standard is prepared by mixing 0.05 cm3 of 0.048 mol of barium chloride (BaCl2)

and 9.95 cm3 of 0.18 mol of sulphuric acid (H2SO4) (5) in a screw‐cap tube used for preparing the

bacteria inoculums.

3 See Appendix 3


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3.1.3 Inoculum preparation

The bacteria inoculums are prepared by Mr Lawrence Kok4. The approximate cell density of

McFarland 0.5 standard is 1.5 x 108 CFU/mL. The turbidity of bacteria is visually compared to the

standard5 to make sure bacteria turbidity is similar to turbidity of McFarland 0.5 standard.

3.2 Preparation of Aqueous Garlic Extract for different temperature and pH treatment

3.2.1 Preparation of aqueous garlic extract

100g of garlic is weighed using electronic weighing machine and then pounded into pulp using

the mortar and pestle. Pulp is pressed into a beaker using two layers of cheese cloth to obtain the

aqueous garlic extract.

3.2.2 Preparing aqueous garlic extract at different temperature

5 microcentrifuges are filled with 1cm3 of aqueous garlic extract using the 1000µL micro pipette.

The microcentrifuges are labelled as shown in table 1.

4 See Appendix 4 5 See Appendix 5

Microcentrifuge label

Temperature (±0.01)oC

1 100.00 2 80.00 3 75.00 4 40.00 5 Room temperature

Table 1: Microcentrifuge labelling and its corresponding temperature


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As there are only 2 water bath machine in my school lab, I devised other ways to incubate the

aqueous garlic extract. To incubate in temperature 100oC, microcentrifuge 1 is put through a

polystyrene piece (which acts as a float)6 in a beaker with boiling water.

Incubation at temperature 80oC and 75oC are done in the water bath machine. The machine is

set to the required temperature and allowed to heat up. When the thermometer shows that the

temperature had risen to the required temperature, microcentrifuges 2 and 3 are put into its respective

water bath with its float.

As for temperature at 40oC, a hot plate is used to heat up the water in the beaker. As the heat

adjustment tuner on the heat plate does not state the temperature, the desired water temperature

(40oC) is attained by adjustment of the heat adjustment tuner through trial‐and‐error. The

microcentrifuge with its float is put into the water.

The temperature of the aqueous garlic extract is made sure to be at the desired temperature by

measuring the content in microcentrifuge using a temperature probe (±0.01)oC by Texas Instrument.

Each microcentrifuge is incubated for 15 minutes. After the microcentrifuges are taken out of its water

bath, some coagulated white matter is observed. Therefore, it is spun in a microcentrifuge at 4000 rpm

for 6 minutes to spin down the residue. 6 filter paper discs are added into each microcentrifuge (for

triplicates when testing each strain of bacteria) using sterilised forceps and left for 30 minutes.

6 See Appendix 6


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3.2.3 Preparing aqueous garlic extract at different pH

4 microcentrifuges are filled with 0.5 cm3 of aqueous garlic extract using the micropipette. The

4 microcentricuges are labelled as shown in table 2.

Aqueous solution of pH 1 is prepared by adding distilled water7 to 1 M HCl. Distilled water is

used to represent medium of pH 7. Aqueous solution of pH 12 and pH 14 are prepared by adding

distilled water to 1M NaOH. pH of aqueous solution is gauged using pH meter(±0.01). 0.5 cm3 of pH 1

HCl, distilled water, pH 12 NaOH, pH 14 NaOH is added into microcentrifuge 6, 7, 8, and 9 respectively

using micropipette. Content in the microcentrifuge is shaken for thorough mixing and left for 15

minutes. As some white coagulated matter is observed in the microcentrifuge, it is spun in the

centrifuge at 4000 rpm for 6 minutes to bring the residue to the bottom. 6 filter paper discs are added

into each microcentrifuge (for triplicates when testing each strain of bacteria) using sterilised forceps

and left for 30 minutes.

3.3 Method for Agar Disk Diffusion Method

3.3.1 Inoculation of nutrient agar plate

Before inoculation, the working bench is wiped with 95% alcohol and the fan is turned off to

prevent contamination. 100µL of nutrient broth containing E. coli is transferred using a micro pipette

with clean tip onto the nutrient agar. A sterile cotton swab is used to swab the surface of nutrient agar 7 See Appendix 7

Microcentrifuge label

pH (±0.01)

6 1 7 78 12 9 14

Table 2: Microcentrifuge labelling and its corresponding pH


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in one direction. The plate is swabbed twice more, turning the plate 60o every time. Then, swab around

the rim of the agar as shown by figure 4 .

This step is repeated with Staph. a. Each bacteria strain is inoculated in 6 nutrient agar plates (3 plates

to be tested for effects of temperature while another 3 plates to be tested for the effects of pH). Fresh

micro pipette tips are used for each transfer of bacteria onto nutrient agar surface. The inoculated

nutrient agar plates are left for 5 minutes to dry to ensure an even bacteria lawn growth on the surface.

3.3.2 Application of filter paper discs onto agar plates

When the inoculated nutrient agar surface is dry, filter paper discs are transferred onto the

nutrient agar individually using forceps. The forceps is sterilised by dipping it into 95% alcohol and

burning it over the Bunsen burner. The impregnated filter paper discs are taken out from their

Figure 4: Swabbing direction on the nutrient agar plate


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microcentrifuges. Any excess liquid on the filter paper disc is dabbed off the wall of the microcentrifuge.

The filter paper disc is then transferred onto their respective position on the agar.

Figure 5: Labelling the Nutrient Agar Plate (left: for testing of different incubation pH; right: for testing of different incubation temperature)

Label Content of filter paper disc 1

Aqueous garlic extract incubated at pH 1, 7, 12 and 14 respectively.

7 12 14 C1 Aqueous solution of pH 1,

7, 12 and 14 respectively as negative control.

C7 C12

C14

100 Aqueous garlic extract

incubated at 100oC, 80oC, 75 oC, 40 oC and 25 oC

respectively.

80 75 40 25

C Distilled water as negative control

C Antibiotic discs as positive

control Table 3: Labelling legend on Nutrient agar plate

H2O


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The filter paper discs are pressed gently onto the surface of the nutrient agar to ensure good

contact. The filter paper discs are not relocated once it had been placed because some antimicrobial

agents are known to act instantly. The forceps are sterilised with the same method for transfer of each

filter paper disc.

3.3.3 Incubation and Data Collection

The incubator8 is a shelf with two 100W bulbs in a shelf. After the transfer of filter paper discs

onto nutrient agar, all the agar plates placed inverted into the incubator. The temperature of the

incubator is adjusted to 37 (± 2) oC. All agar plates are incubated for 24 hours. The zone of inhibition is

measured with centimetre ruler ((±1.0)mm) as shown in figure 6.

Figure 6: Measuring Inhibition Zone of Aqueous garlic Extract Treated at Different pH

8 See Appendix 8.

12mm


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4.0 Data Collection

4.1 Raw Data: Antimicrobial Activity of Aqueous Garlic Extract Incubated at Different Temperature

Substance tested

Temperature/ oC (±0.01 oC)

Diameter of Inhibition Zone (mm) 1 2 3 Mean±S.D

Aqueous garlic extract

100.0

‐ ‐ ‐ ‐

80.0

8 7 7 7.3±0.6

75.0

10 9 9 9.3±0.6

40.0

16 15 17 16.0±1.0

25.0 (room temperature)

19 16 18 17.7±1.5

Distilled Watera N/A

‐ ‐ ‐ ‐

Nalidixic acidb#

N/A 26 26 26 26.0±0.0

Table 4: Inhibition Zone (mm) of aqueous garlic extract incubated at various temperatures on E. coli

(‐) = no activity (N/A) = not applicable (S.D) = Standard Deviation aNegative control bPositive control # Disc content: 30µg


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Substance tested

Temperature/ oC (±0.01 oC)

Diameter of Inhibition Zone (mm) 1 2 3 Mean±S.D

Aqueous garlic extract

100.0

‐ ‐ ‐ ‐

80.0

13 12 12 12.3±0.6

75.0

15 14 16 15.0±1.0

40.0

20 20 19 19.7±0.6

25.0 (room temperature)

22 18 17 19.0±2.6

Distilled Watera N/A

‐ ‐ ‐ ‐

Methicillinb#

N/A 17 18 17 17.3±0.6

Table 5: Inhibition Zone (mm) of aqueous garlic extract incubated at various temperatures on Staph. a



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4.2 Raw Data: Antimicrobial Activity of Aqueous Garlic Extract Incubated at Different pH

Substance tested pH of medium

Diameter of Inhibition Zone (mm) 1 2 3 mean±S.D

Aqueous garlic extract + hydrochloric acid

1 10 11 10 10.3±0.6

Aqueous garlic extract + distilled water

7

12 14 14 13.3±1.2

Aqueous garlic extract +

Sodium Hydroxide

12

‐ ‐ ‐ ‐

14

‐ ‐ ‐ ‐

Hydrochloric acida 1

7 8 7 7.3±0.6

Distilled Watera 7

7 8 ‐ 7.5±0.7

Sodium Hydrodixea 12

8 8 ‐ 8.0±0.0

14

7 7 ‐ 7.0±0.0

Nalidixic acidb#

N/A 26 27 26 26.3±0.6

Table 6: Inhibition Zone (mm) of aqueous garlic extract incubated at various pH on E. coli



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Substance tested pH of medium

Diameter of Inhibition Zone (mm) 1 2 3 mean±S.D

Aqueous garlic extract + hydrochloric acid

1

20 17 17 18.0±1.7

Aqueous garlic extract + distilled water

7

22 18 20 20.0±2.0

Aqueous garlic extract +

Sodium Hydroxide

12

‐ ‐ ‐ ‐

14

‐ ‐ ‐ ‐

Hydrochloric acida 1

‐ ‐ ‐ ‐

Distilled Watera 7

‐ ‐ ‐ ‐

Sodium Hydrodixea 12

‐ ‐ ‐ ‐

14

‐ ‐ ‐ ‐

Methicillinb# N/A 20 19 19 19.3±0.6

Table 7: Inhibition Zone (mm) of aqueous garlic extract incubated at various pH on Staph. a

(‐) = no activity (N/A) = not applicable (S.D) = Standard Deviation aNegative control bPositive control #Disc content: 5µg

4.3

Graph 1: M

Graph 2: M

Data Proc

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4.4 Data Processing: ANOVA and Tukey’s HSD test

Further data analysis is carried out using Analysis of Variance (ANOVA) and Tukey’s HSD (honestly

significant difference) (9). ANOVA partitions the variance of all observations into variations between

group and variations within each group. The result of ANOVA indicates whether the manipulated

variable (pH and temperature) causes significance difference in the antimicrobial activity. If yes, a post

hoc analysis called Tukey’s HSD test is carried out. Tukey’s HSD test is a multiple comparison test used

to test the hypothesis that all possible pairs of means are equal. It determines which group is

significantly different than the others.

For ANOVA to be carried out, three assumptions are made:

1. Observations are independent (the value of one observation is not correlated with the value of

another observation).

2. Observations in each group are normally distributed.

3. Homogeneity of variances (variance of each group is equal to that of any other group).

The null hypothesis is first assumed – there is no difference between means of different groups. Then,

the statistic test is carried out to find the F ratio.

If the computed F ratio is greater than the F critical value at the significance level of 0.05 (α=0.05), the

null hypothesis is rejected. There is one group that is significantly different from others.


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For Tukey’s HSD test, a critical value, HSD is calculated. If the mean difference between groups is

greater than HSD critical value, there is significant difference between these pairs.

The ANOVA is carried out using Microsoft Excel 2007 while Tukey HSD9 is calculated manually. Below

are the results of my tests:

Variable Bacteria strain F‐value F‐critical Indication

Temperature

E. Coli 151.73 3.48 ANOVA test on the four sets of data shows that there is a group which is significantly different from others in their own respective set of data.

i.e.: mean inhibition zone of aqueous garlic extract incubated at a certain temperature or pH is significantly

larger than those incubated at other temperature or pH.

Staph. a 109.77 3.38

pH E. Coli 346.87 4.07

Staph. a 207.43 4.07

Table 8: Results of ANOVA on 4 sets of data to determine whether there is significant difference between mean inhibition zone of aqueous garlic extract incubated at different temperature and pH on E. Coli and Staph. a

9 For detailed calculations, see Appendix 9


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One group is significantly different from the others in their respective set of data, thus Tukey’s HSD test is carried out and the results are as shown in below:

Group Combination, oC (Mean Inhibition Zone of aqueous garlic extract incubated at different temperature on E.coli)

Mean difference,

mm

HSD critical value

Implication

100 80 7.3 2.40 significant difference100 75 9.3 2.40 significant difference 100 40 16.0 2.40 significant difference 100 25 17.7 2.40 significant difference 80 75 2.0 2.40 No significant difference 80 40 8.7 2.40 significant difference 80 25 10.3 2.40 significant difference 75 40 6.7 2.40 significant difference 75 25 8.3 2.40 significant difference 40 25 1.7 2.40 No significant difference

Table 9: Results of Tukey’s HSD test on mean inhibition zone of aqueous garlic extract incubated at different temperature on E. Coli to determine which group is significantly different than the other

Group Combination, oC (Mean Inhibition Zone of aqueous garlic extract incubated at different temperature on Staph. a)

Mean difference,

mm

HSD critical value

Implication

100 80 12.33 3.53 significant difference 100 75 15.00 3.53 significant difference 100 40 19.67 3.53 significant difference 100 25 19.00 3.53 significant difference 80 75 3.33 3.53 No significant difference 80 40 7.30 3.53 significant difference 80 25 6.67 3.53 significant difference 75 40 4.67 3.53 significant difference 75 25 4.00 3.53 significant difference 40 25 0.67 3.53 No significant difference

Table 10: Results of Tukey’s HSD test on mean inhibition zone of aqueous garlic extract incubated at different temperature on Staph. a to determine which group is significantly different than the other


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Group Combination, pH (Mean Inhibition Zone of aqueous garlic extract

incubated at different pH on E. Coli)

Mean difference,

mm

HSD critical value

Implication

1 7 3.33 1.69 significant difference 1 12 10.33 1.69 significant difference 1 14 10.33 1.69 significant difference 7 12 13.33 1.69 significant difference 7 14 13.33 1.69 significant difference 12 14 0.00 1.69 No significant difference

Table 11: Results of Tukey’s HSD test on mean inhibition zone of aqueous garlic extract incubated at different pH on E. Coli to determine which group is significantly different than the other

Group Combination, pH (Mean Inhibition Zone of aqueous garlic extract

incubated at different pH on Staph. a)

Mean difference,

mm

HSD critical value

Implication

1 7 2.00 3.46 No significant difference 1 12 18.00 3.46 significant difference 1 14 18.00 3.46 significant difference 7 12 20.00 3.46 significant difference 7 14 20.00 3.46 significant difference12 14 0.00 3.46 No significant difference

Table 12: Results of Tukey’s HSD test on mean inhibition zone of aqueous garlic extract incubated at different pH on Staph. a to determine which group is significantly different than the other


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5.0 Conclusion

5.1 Part 1: Temperature

Graph 1 and 2 shows that Staph. a (gram‐positive) is more susceptible to antimicrobial effects of

garlic regardless of how aqueous garlic extract is pre‐treated compared to E. coli (gram‐negative) as the

inhibition zone of Staph. a is always larger than E. coli in this study. However, as this study does not

concern the susceptibility of different bacteria, no further analysis is carried out to verify whether there

is significant difference between susceptibility of E. coli and Staph. a.

Graph 1 indicates the effects of temperature on the antimicrobial activity of aqueous garlic

extract on E. coli and Staph. a:

1. Aqueous garlic extract incubated at room temperature, 25 oC yield the highest antimicrobial

activity while aqueous garlic extract incubated at 100 oC has no antimicrobial activity

2. Antimicrobial activity has an inverse relationship with the incubation temperature of aqueous

garlic extract: as temperature decreases from 100 oC to 25 oC, the inhibition zone increases.

The Tukey’s HSD test is used to determine whether the antimicrobial activity of aqueous garlic

extract incubated at 25 oC is significantly higher than being incubated at other temperature. All

calculations are based on significance level of 0.05, α=0.05. Table 8 and table 9 (involving E. coli and

Staph. a) show that there is no significant difference between antimicrobial activity of aqueous garlic

extract incubated at 80 oC and 75 oC. There is also no significant difference between antimicrobial

activity of aqueous garlic extract incubated at 40 oC and 25 oC.

As aqueous garlic extract incubated at 100 oC does not show any antimicrobial activity, there is

significant difference in antimicrobial activity when compared to aqueous garlic extract incubated at


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other temperatures (80 oC, 75 oC, 40 oC, 25 oC). This is shown by results in the first four rows in table 8

and 9.

Aqueous garlic extract incubated at 75 oC and 80 oC shows significantly lower antimicrobial

activity when compared to aqueous garlic extract incubated at 40 oC and 25 oC. This is shown by results

in table 8 and 9, suggesting that the optimum incubation temperature for aqueous garlic extract’s

antimicrobial activity is between 40 oC and 25 oC.

Though there is no concrete evidence why aqueous garlic extract loses its antimicrobial activities

when incubated at high temperature, there are two plausible explanations.

Increase in temperature increases the kinetic energy of atoms in molecules; atoms vibrate more

violently. If kinetic energy of atoms in molecule overcomes bond enthalpy, bonds between atoms in a

molecule are broken. The 3‐D structure of inhibitory components is altered and thus is rendered

dysfunctional. If the inhibitory component is an enzyme, it is denatured, i.e.: loss of structure and

function.

Lastly, it is possible that the inhibitory components are volatile at high temperatures. The

increased in kinetic energy causes molecules of inhibitory components to have enough energy to

overcome the attractive forces between the molecules. Inhibitory components become gaseous

molecules and escape into the atmosphere.


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5.2 Part 2: pH

Graph 2 shows that aqueous garlic extract incubated at pH 7 yields higher antimicrobial activity

compared to pH 1 for both E. coli and Staph. a.. Aqueous garlic extract incubated at pH 12 and 14 does

not show any antimicrobial activity in both strains of bacteria.

For both strains of bacteria, the results of the Tukey’s HSD test (calculated at significance level

of 0.05) in table 10 and 11 show that aqueous garlic extract incubated at pH 1 and pH 7 has significant

difference in antimicrobial activity when compared to that of aqueous garlic extract incubated at pH 12

and 14.

Table 10 also indicates significant difference between antimicrobial activity of aqueous garlic

extract incubated at pH 1 and pH 7 on E. coli. It appears that antimicrobial activity of aqueous garlic

extract on E. coli is significantly higher when aqueous garlic extract is incubated at pH 7 compared to pH

1. However, there is no significant difference between antimicrobial activity of aqueous garlic extract

incubated at pH 1 and pH 7 on Staph. a. as indicated in table 11.

Table 5 shows distilled water (control) causes inhibition zone of 7‐8 mm in E. coli. This indicates

that random errors (possibly contaminated distilled water) occurred during this part of the experiment

because distilled water should not show any antimicrobial activity. Thus, inhibition zone of 7‐8 mm

caused by hydrochloric acid and sodium hydroxide can be disregarded, i.e.: antimicrobial activity on E.

coli is solely caused by aqueous garlic extract.

This part of the experiment suggests that the optimum incubation pH for aqueous garlic extract

is from pH 1 to pH 7 (acidic to neutral medium).


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One possible explanation to aqueous garlic extract’s lost of antimicrobial activity when

incubated in alkaline solution is that the hydroxide ions (OH‐) may have affected the polar bonds which

exist between atoms in molecule of the inhibitory components. Polar bonds exist between one atom

which is partially positive and another which is partially negative. The OH‐ may have neutralised or alter

the bond polarity between atoms, causing bonds between atoms to break. Broken bonds in a molecule

causes structural change of inhibitory components, leading to loss of function.

Lastly, if the formation of inhibitory component involves enzymatic reaction, loss of

antimicrobial activity may be due to denaturation of enzyme. Enzymes are pH sensitive; it functions

optimally at certain pH. Besides the breaking of bonds between atoms in an enzyme which causes

structural change, OH‐ could affect the polarity of the enzyme’s active site. OH‐ would get attached to

positively charged active site, preventing negatively charged substrate from sitting on the active site.

Enzymatic reaction cannot take place, causing the loss of antimicrobial activity.


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6.0 Evaluation and Suggestion

Possible Errors

As this experiment is very time consuming, I was only able to do three repetitions for each

bacteria strain and at the same time control all the fixed variables. As there are only three repetitions,

my results may not be conclusive due to the high possibility of random errors.

Realising the possibility of anomalous results due to experimental techniques, I have included

positive control in my experiment. Nalidixic acid (30µg) and Methycillin (5µg) is used as a positive

control for E. coli and Staph. a respectively. According to M100‐S2 – Performance Standards for

Antimicrobial Susceptibility Testing; Second International Supplement, control limits for inhibitory

diameter zones of E. coli and Staph. a when tested with antibiotic discs are 22‐28 mm and 17‐22 mm

respectively. The recorded inhibition zones for Staph. a when tested with methycillin (5µg) is 17‐19 mm

while for E. coli when tested with Nalidixic acid (30µg) is 26‐27 mm. The inhibition zone of both bacteria

strain is well within the control limits, indicating that my methodology is valid despite possibility of

random error.

Limitations

My study only involves one strain of E. coli and Staph. a; thus antimicrobial activity of aqueous

garlic extract on E. coli, Staph. a, gram‐positive and gram‐negative bacteria cannot be generalised. In

order to generalise the antimicrobial activity of differently pre‐treated aqueous garlic extract on

different bacteria, different strains of E. coli and Staph. a, as well as other common gram‐positive and

gram‐negative bacteria should be tested. Testing aqueous garlic extract with a wider range of bacteria

will enable us to establish its status as an antibiotic.


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Due to limitation of school lab facilities, aqueous garlic extract was only incubated at 5 different

temperatures; thus I could only approximate the temperature at which antimicrobial activity of aqueous

garlic extract decreases. For further investigation, I would incubate aqueous garlic extract at

temperature ranging between 80oC to 100oC (because this study shows that antimicrobial activity of

aqueous garlic extract starts to decrease when incubated at temperature greater than 75 oC) to

determine the exact temperature at which there is no antimicrobial activity.

Further Research

It would be interesting to observe how multiple antibiotic resistant strains of E. coli and Staph. a

reacts to aqueous garlic extract. Should the growth of multiple antibiotic resistant E. coli and Staph. a

be inhibited, it could lead to discovery of a new generation of antibiotics.

There is possibility of synergistic effect between aqueous garlic extract and acidic food

substance such as lime, lemon and vinegar. Though this study shows lower antimicrobial activity when

aqueous garlic extract is incubated at pH 1, there is possibility of synergistic effect because acidic

medium is known to be able to kill bacteria (just like the acidic medium in the stomach which kills

ingested bacteria). Besides, there is no significant difference between antimicrobial activities of

aqueous garlic extract incubated at pH 1 and pH 7 when tested against Staph. a..

Lastly, some unanswered questions are how exactly alkaline solution (pH) and temperature

affects the antimicrobial activity of aqueous garlic extract. It is also unknown which inhibitory

component inhibits the bacterial growth.


Page 36 of 48

7.0 References

1. Lewis, Ricki. U.S Food and Drug Administration. The Rise of Antibiotic‐Resistant Infection. [Online] September 1995. [Cited: June 27, 2007.] http://www.fda.gov/Fdac/features/795_antibio.html.

2. Ankri, Serge and Mirelman, David. Antimicrobial Properties of Garlic. Microbes and Infection. Elesvier, Paris : s.n., 1999.

3. Performance Standards for Antimicrobial Disks Susceptibility Tests; Approved Standard ‐ Ninth Edition. Clinical and Labaratory Standards Institute. 2006, pp. M2‐A9.

4. Heinrich, Michael, Pieroni, Michael and Bremner, Paul. Plants as Medicine. [book auth.] Ghillean Prance and Mark Nesbitt. Cultural History of Plants. New York : Routledge, 2005, pp. 219‐221.

5. Schou, Chad. Economic Botany Leaflets. [Online] may 09, 2000. [Cited: February 24, 2008.] http://www.siu.edu/~ebl/leaflets/garlic2.htm.

6. Inhibition of Microbial growth by Ajoene, a Sulphur‐Containing Compound Derived from Garlic. Nagavana, Rie, et al. 11, New York : American Society for Microbiology, 1996, Vol. 62. 0099‐2240.

7. Introduction to Biotic. Antibiotic Attack. [Online] [Cited: December 12, 2007.] http://maflib.mtandao‐afrika.net/TQA01074/english/bio.htm.

8. Talaro, Kathleen Park. An Introduction to Cell and Procaryotic Cell Structure and Function. Foundations in Microbiology: Basic Principles, Sixth Edition. New York : Mc Graw Hill, pp. 99‐101.

9. McGraw‐Hill. ANOVA. [book auth.] Jan W Kuzma and Stephen E Bohnenblust. Basic Statistic for the Health Sciences‐4th edition. Singapore : Mayfield Publishing Company, 2001.

10. Chapter 2 ‐ Cell structure and organization. Microbiology and Bacteriology :: The World of Microbes. [Online] [Cited: December 12, 2007.] http://www.bact.wisc.edu/Microtextbook/index.php?module=Book&func=displaychapter&chap_id=35&theme=printer.


Page 37 of 48

8.0 Appendixes

Appendix 1: Excerpt from ‘The Rise of Antibiotic‐Resistant Infection’.

URL: http://www.fda.gov/Fdac/features/795_antibio.html

Disease‐causing microbes thwart antibiotics by interfering with their mechanism of action. For example, penicillin kills bacteria by attaching to their cell walls, then destroying a key part of the wall. The wall falls apart, and the bacterium dies. Resistant microbes, however, either alter their cell walls so penicillin can't bind or produce enzymes that dismantle the antibiotic.

In another scenario, erythromycin attacks ribosomes, structures within a cell that enable it to make proteins. Resistant bacteria have slightly altered ribosomes to which the drug cannot bind. The ribosomal route is also how bacteria become resistant to the antibiotics tetracycline, streptomycin and gentamicin.

Appendix 2: Why were the adaptations made

Mueller‐Hinton agar was not used in my study because the school lab did not have this particular type of agar at the time of my experiment. Therefore, I used common nutrient agar from Gene Chemicals. I believe that this would not be much of a limitation to my experiment because E. Coli and Staph. a are common bacteria; therefore, it is able to survive in most environments. The nutrient agar from Gene Chemicals has a softer texture. Therefore, the thickness of nutrient agar in this experiment is 7mm instead of 4mm; the increased thickness is to compensate for the fragility of the nutrient agar.


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Appendix 3: Nutrient Agar Plate

Appendix 4: Method of Preparing Bacteria Culture from Mr Lawrence Kok

Screw‐cap bottles that are needed to contain bacteria culture and pre‐prepared nutrient broth are sterilized in the autoclave. Pure Escherichia Coli ATCC 25922 and Staphylococcus aureus ATCC 25923 is purchased in dry lypholised form. Two bottles are filled half‐way with sterilized nutrient broth. Pure strains of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 are put into the nutrient broth. Inoculated nutrient broth is incubated at 37oC for 24 hours.

90 mm


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Appendix 5: Comparison of inoculums turbidity with 0.5 McFarland Standard.

Appendix 6: Polystyrene as a float in the water bath

Escherichia Coli Staphylococcus aureus 0.5 McFarland Standard

microcentrifuge

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Page 40 of 48

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Page 41 of 48

Appendix 9: Detailed Calculation of ANOVA from Microsoft Excel 2007

ANOVA first starts with null hypotheses, , where mean of groups are hypothesised to be the same.

:

ANOVA partitions the variance of all observations into two sources of variation: variation between the group means and variation within the group means. The sampling distribution used for testing is called the F distribution (in honour of R. A. Fisher, who developed F statistic). Between‐group variance measure the treatment effect, which in this study is the antimicrobial effects of aqueous garlic extract when incubated at different temperature and pH. Below is the conventional notation of ANOVA and its explanation:

NOTATION MEANING

or Within‐group variance or

mean square within

or Between‐group variance or

mean square between

Degree of freedom

Number of groups

Number of observation in each group

Total number of observation

Significance level

Sum of squares between group

Sum of squares within group

Table 13: Conventional notations and its meaning


Page 42 of 48

If the between group variance is greater than the within group variance, , then there is

treatment effect. If , then there is no treatment effect.

Test of Hypothesis is performed by comparing the ratio of the two variance estimates, .

has 1 degree of freedom; labelled as

has degree of freedom; labelled as .

Source of

variation

Sum of

Squares df

Mean

Squares, F ratio Critical F* P value

Between 1 1

, Computer generated

Within

Total 1

Table 14: One‐way ANOVA table

*the critical F value is at significance level of 0.05, 0.05and the value is obtained from the Percentiles of F Distribution table in APPENDIX 10‐A below.

There are differences between at least one pair of means when F ratio is greater than critical F. In order to find out exactly where the differences are, Tukey’s HSD (honestly significant difference) is carried out to make multiple comparisons.

The formula for computing HSD is:

, ,

Where is obtained from Percentage Points of the Studentized Range for 2 Through 20 treatments table in APPENDIX 10‐B below.

There is significant difference between groups of a certain pair if the mean difference between pair is greater than HSD value.


Page 43 of 48

For this experiment, I used Microsoft 2007 to generate ANOVA tables. Below are the tables generated:

1. The effects of temperature on the antimicrobial activity of aqueous garlic extract on inhibition zone of E. coli.

Groups (temperature, 0C) Count Sum Average Variance

Group 1 (100 oC) 3 0 0.00 0.00 Group 2 (80 oC) 3 22 7.33 0.33 Group 3 (75 oC) 3 28 9.33 0.33 Group 4 (40 oC) 3 48 16.00 1.00Group 5 (25 oC) 3 53 17.67 2.33

Table 15: Summary

Source of Variation SS df MS F P‐value F crit

Between Groups 606.93 4 151.73 189.67 2.2117E‐09 3.48

Within Groups 8.00 10 0.80

Total 614.93 14 Table 16: ANOVA table

4.650.83

2.40


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2. The effects of temperature on the antimicrobial activity of aqueous garlic extract on inhibition zone of Staph. a.

Groups(temperature, 0C) Count Sum Average Variance

Column 1 (100 oC) 3 0 0.00 0.00

Column 2 (80 oC) 3 37 12.33 0.33

Column 3 (75 oC) 3 45 15.00 1.00

Column 4 (40 oC) 3 59 19.67 0.33

Column 5 (25 oC) 3 57 19.00 7.00 Table 17: Summary


Between Groups 761.07 4 190.27 109.77 3.22E‐08 3.48



4.651.733

3.53


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3. The effects of pH on the antimicrobial activity of aqueous garlic extract on inhibition zone of E. coli.

Groups(pH) Count Sum Average Variance

Groups 1 (pH 1) 3 31 10.33 0.33

Groups 2 (pH 7) 3 40 13.33 1.33

Groups 3 (pH 12) 3 0 0.00 0.00

Groups 4 (pH 14) 3 0 0.00 0.00 Table 19: Summary


Between Groups 433.58 3 144.53 346.87 8.31E‐09 4.07



4.530.423

1.69


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4. The effects of pH on the antimicrobial activity of aqueous garlic extract on inhibition zone of Staph. a.

Groups(pH) Count Sum Average Variance

Group 1 (pH 1) 3 54 18.00 3.00

Group 2 (pH 7) 3 60 20.00 4.00

Group 3 (pH 12) 3 0 0.00 0.00

Group 4 (pH 14) 3 0 0.00 0.00 Table 21: Summary


Between Groups 1089.00 3 363.00 207.43 6.35E‐08 4.07


Total 1103.00 11 Table 22: ANOVA Table

4.531.753

3.46


Page 47 of 48

APPENDIX 10 – A: Percentiles of F Distribution (9)


Page 48 of 48

APPENDIX 10 – B: Points of the Studentized Range for 2 Through 20 treatments (9)

Education

Antimicrobial effect of garlic against E Coli and Stap.A