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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
(‐) = no activity (N/A) = not applicable (S.D) = Standard Deviation aNegative control bPositive control # Disc content: 5µg
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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
(‐) = no activity (N/A) = not applicable (S.D) = Standard Deviation aNegative control bPositive control # Disc content: 30µg
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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
Mean Inhibitio
Mean Inhibitio
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10
15
20
25
Mean Inhibition
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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.
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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.
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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
Polystyrene
Water bath
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M JU ANNE 2206‐018
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to show its ble to g only oxygen and ve hydrogen om of
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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
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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.
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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
Source of Variation SS df MS F P‐value F crit
Between Groups 761.07 4 190.27 109.77 3.22E‐08 3.48
Within Groups 17.33 10 1.73
Total 778.40 14 Table 18: ANOVA table
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
Source of Variation SS df MS F P‐value F crit
Between Groups 433.58 3 144.53 346.87 8.31E‐09 4.07
Within Groups 3.33 8 0.42
Total 436.92 11 Table 20: ANOVA table
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
Source of Variation SS df MS F P‐value F crit
Between Groups 1089.00 3 363.00 207.43 6.35E‐08 4.07
Within Groups 14.00 8 1.75
Total 1103.00 11 Table 22: ANOVA Table
4.531.753
3.46
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APPENDIX 10 – A: Percentiles of F Distribution (9)
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APPENDIX 10 – B: Points of the Studentized Range for 2 Through 20 treatments (9)