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EXTENDEDESSAY
BIOLOGY
OptimalConditionforAntimicrobialActivity:
InVitrostudyontheeffectsoftemperatureandpHonAntimicrobialActivityofAqueousGarlicExtract
againstEscherichiacoliATCC25922andStaphylococcusaureusATCC25923.
Submittedby:LimJuAnne
CandidateNumber:002206018
WordCount:3995only
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TableofContentsFigures........................................................................................................................................................... 3
Tables............................................................................................................................................................ 4
Abstract......................................................................................................................................................... 6
1.0 Introduction........................................................................................................................................ 7
1.1 RationaleofStudy............................................................................................................................... 7
1.2Aim...................................................................................................................................................... 8
1.3Garlic(AlliumSativum)........................................................................................................................ 9
1.4Bacteria............................................................................................................................................. 11
2.0Variables................................................................................................................................................ 13
2.1Independentvariable........................................................................................................................ 13
2.2Dependentvariable........................................................................................................................... 13
2.3Fixedvariable.................................................................................................................................... 13
3.0Procedures............................................................................................................................................ 14
3.1 Preparationbeforeexperiment........................................................................................................ 14
3.2
Preparation
of
Aqueous
Garlic
Extract
for
different
temperature
and
pH
treatment
.....................
15
3.3 MethodforAgarDiskDiffusionMethod.......................................................................................... 17
4.0 DataCollection................................................................................................................................. 21
4.1 RawData:AntimicrobialActivityofAqueousGarlicExtractIncubatedatDifferentTemperature.21
4.2 RawData:AntimicrobialActivityofAqueousGarlicExtractIncubatedatDifferentpH..................23
4.3 DataProcessing:ComparingMeanInhibitionZoneofE.ColiandStaph.a..................................... 25
4.4 DataProcessing:ANOVAandTukeysHSDtest................................................................................ 26
5.0 Conclusion........................................................................................................................................ 30
5.1 Part1:Temperature......................................................................................................................... 30
5.2 Part2:pH.......................................................................................................................................... 32
6.0 EvaluationandSuggestion................................................................................................................ 34
7.0 References........................................................................................................................................ 36
8.0 Appendixes....................................................................................................................................... 37
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FiguresFigure1:GenerationofAllicininGarlicClove(2)......................................................................................... 9
Figure3:CellWallofGrampositiveBacteria(7)........................................................................................ 11
Figure2:CellwallofGramnegativeBacteria(10)..................................................................................... 11
Figure4:Swabbingdirectiononthenutrientagarplate............................................................................ 18
Figure5:LabellingtheNutrientAgarPlate(leftfigure:fortestingofdifferentincubationpH;rightfigure:
fortestingofdifferentincubationtemperature)....................................................................................... 19
Figure6:MeasuringInhibitionZoneofAqueousgarlicExtractTreatedatDifferentpH........................... 20
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TablesTable
1:
Microcentrifuge
labelling
and
its
corresponding
temperature
.....................................................
15
Table2:MicrocentrifugelabellinganditscorrespondingpH..................................................................... 17
Table3:LabellinglegendonNutrientagarplate........................................................................................ 19
Table4:InhibitionZone(mm)ofaqueousgarlicextractincubatedatvarioustemperaturesonE.coli...21
Table5:InhibitionZone(mm)ofaqueousgarlicextractincubatedatvarioustemperaturesonStaph.a22
Table6:InhibitionZone(mm)ofaqueousgarlicextractincubatedatvariouspHonE.coli..................... 23
Table7:InhibitionZone(mm)ofaqueousgarlicextractincubatedatvariouspHonStaph.a.................24
Table8:ResultsofANOVAon4setsofdatatodeterminewhetherthereissignificantdifference
betweenmeaninhibitionzoneofaqueousgarlicextractincubatedatdifferenttemperatureandpHon
E.ColiandStaph.a..................................................................................................................................... 27
Table9:ResultsofTukeysHSDtestonmeaninhibitionzoneofaqueousgarlicextractincubatedat
differenttemperatureonE.Colitodeterminewhichgroupissignificantlydifferentthantheother.......28
Table10:ResultsofTukeysHSDtestonmeaninhibitionzoneofaqueousgarlicextractincubatedat
differenttemperatureonStaph.atodeterminewhichgroupissignificantlydifferentthantheother...28
Table11:ResultsofTukeysHSDtestonmeaninhibitionzoneofaqueousgarlicextractincubatedat
differentpHonE.Colitodeterminewhichgroupissignificantlydifferentthantheother....................... 29
Table12:ResultsofTukeysHSDtestonmeaninhibitionzoneofaqueousgarlicextractincubatedat
differentpHonStaph.atodeterminewhichgroupissignificantlydifferentthantheother................... 29
Table13:Conventionalnotationsanditsmeaning.................................................................................... 41
Table14:OnewayANOVAtable................................................................................................................ 42
Table15:Summary..................................................................................................................................... 43
Table16:ANOVAtable............................................................................................................................... 43
Table17:Summary..................................................................................................................................... 44
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Table18:ANOVAtable............................................................................................................................... 44
Table19:Summary..................................................................................................................................... 45
Table20:ANOVAtable............................................................................................................................... 45
Table21:Summary..................................................................................................................................... 46
Table22:ANOVATable............................................................................................................................... 46
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AbstractGarlic(AlliumSativum)isknowntohaveantimicrobialproperties. Thisstudyinvestigatestheeffectsof
pHandtemperatureontheantimicrobialactivityofaqueousgarlicextract. Theaqueousgarlicextractis
tested on nonpathogenicEscherichiacoli ATCC 25922 andStaphylococusaureus ATCC 25923 in an invitroenvironment. ThemethodchosenisAgarDiscDiffusionMethod. Filterpaperdiscsaredippedinto
the aqueous garlic extract which are pretreated at different pH and temperature and then put onto
inoculatedagarplates. Theagarplatesareincubatedatapproximately37
o
Cfor24hours. Thediameter
ofinhibitionzone(clearzonearoundthefilterpaperdisc)ismeasured;thelargerthediameterofzone
of inhibition, the higher the antimicrobial activity of the aqueous garlic extract. ANOVA (calculated at
significance level of 0.05) and Tukeys HSD test is used to analyse the data. ForE.coli andStaph.a,inhibitiondecreasesasincubationtemperatureofaqueousgarlicextractincreases;thereisnoinhibition
at 100oC. Inhibition is greatest for both bacteria strains when aqueous garlic extract is incubated at
roomtemperature(25oC). Antimicrobialactivityofaqueousgarlicextractincubatedat75oCand80oCis
significantlylesserthanaqueousgarlicextractincubatedat25oCand40oC. ThereisinhibitioninStaph.aandE.coli for aqueous garlic extract incubated in pH 1 and pH 7 medium. However, antimicrobialactivityonE.coliissignificantlyhigherwhenaqueousgarlicextractisincubatedinpH1comparedtopH7. ThereisnoantimicrobialactivityonStaph.aandE.coliwhenaqueousgarlicextractisincubatedinmediumofpH12andpH14. Theresultsofmystudysuggestthatoptimumincubationtemperaturefor
antimicrobial activity of aqueous garlic extract is between 25oC to 40
oC and optimum pH is between
acidic(pH1)toneutralmedium(pH7).
(298wordsonly)
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1.0 Introduction1.1 RationaleofStudyOveruse of antibiotics caused increasing antibioticresistant bacteria; the perception that antibiotic is
theultimatecurehadpressuredphysiciansintoprescribingantibioticsforeveryinfection,evenifitisa
viralinfection. Notfinishingtheentirecourseofantibioticprescribedleavesbehindweakenedbacteria
in the host. Animals are sometimes given low dosage of antibiotics for long duration (to prevent
bacterialinfection)toincreasetherateofweightgain(1). Theunnecessaryadministrationofantibiotics
leaves weakened bacteria which then mutate and gain antibiotic resistant gene. Antibiotic like
penicillin1isineffectivenow.
Studies suggest that allicin works by inhibiting certain thiolcontaining enzymes in the microorganisms
byrapidreactionofthiosulfinateswiththiolgroupsoftheenzyme(2). Itisdifficultforbacteriatogain
resistancebecausealterationofenzymesstructureisnotanalternative;accordingtothelockandkey
model, the action of enzyme is specific because the geometric shape of enzymes active site and its
substrate is complementary. Alteration of enzymes shape causes substrate to be unable to fit into
enzymesactivesite. Thebacteriacannotsurvivebecausebiochemicalenzymecatalysedreactionsuch
asrespirationcannotbecarriedout.
Lastly, asEscherichia coli (E. coli) andStaphylococcusaureus (Staph.a) are common bacteria in ourdaily 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.
1SeeAppendix1
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1.2 AimThe aim of my paper is to study the effects of pH and temperature on the antimicrobial activity of
aqueousgarlicextract.
Hence, my research question isOptimal Condition forAntimicrobialActivity: InVitro studyon the
effectsoftemperatureandpHonAntimicrobialActivityofAqueousGarlicExtractagainstEscherichiacoliATCC25922andStaphylococcusaureusATCC25923.
The Agar Disk Diffusion Method is chosen for this experiment (3). This method is adapted2 from
PerformanceStandardsforAntimicrobialDisksSusceptibilityTests;ApprovedNinthEdition published
byClinicalandLaboratoryStandards. Ichosethismethodbecauseofthehydrophilicnatureofaqueous
garlicextract. Filterpaperdiscsareimpregnatedwithaqueousgarlicextractandplacedontoinoculated
nutrient agar. The active ingredient will diffuse through the nutrient agars surface. No colonies will
growneartheareawheretheconcentrationisequalormorethantheeffectiveconcentrationtokillor
inhibit the bacteria. The size of the clear zone (no bacteria growth) is a measure of the antimicrobial
activity. Thelargerthediameteroftheclearzone,thehighertheantimicrobialactivity.
2SeeAppendix2
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1.3 Garlic(AlliumSativum)Garlic (AlliumSativum) 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
thoughtthatgarlicisgoodformanyailments;Mohammed,theprophetclaimedgarlicapplieddirectlyto
astingwoundwouldrelieveitspain(4).
The earliest documentation garlics 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
WorldWarII,theRussianarmyalsoturnedtogarlicwhentheyranoutofpenicillinandthus,garlicwas
namedtheRussianPenicillin(5).
Allicin,producedwhencrushed,isattributedbymanyresearchestotheantimicrobialactivityofgarlic.
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
allinasewillinteract(2). AllinaseconvertsalliinintoallicinasillustratedinFigure1.
Figure1:GenerationofAllicininGarlicClove(2)
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Though, theoretically, allicin is deemed responsible for garlics antimicrobial activity, many researches
show that allicin is a volatile compound which decomposes into other sulphurous compound such as
diallys sulphide,diallyldisulphide, and ajoeneafter its formation (6). Due to ambiguity of information
presented,limitationofmyknowledgeatthislevelofeducationandlimitationofschoollabinstrument,
I could not possibly verify allicin as the compound responsible for garlics antimicrobial activity.
Therefore,compoundresponsiblefortheantimicrobialactivityofaqueousgarlicextractwillbereferred
collectivelyasinhibitorycomponentsinthisstudy.
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1.4 Bacteria
I have chosen E. coli and Staph. a because they represent the two spectrums of bacteria: E. colirepresent the gramnegative bacteria while Staph. a represent the grampositive bacteria. This willtherefore give a general idea of the different susceptibility level of grampositive and gramnegative
bacteriatowardsaqueousgarlicextract.
Figure3:CellWallofGrampositiveBacteria(7)
Figure2:CellwallofGramnegativeBacteria(10)
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As shown in Figure 2, the outer membrane of the gramnegative cell wall has a type of protein called
porinwhichcontrolswhatentersandleavesthecellthisgivesthequalityofselectivepermeabilityto
bile,disinfectantsanddrugs. InFigure3,cellwallofthegrampositivebacteriahaspeptidoglycanwhich
containstightlyboundacidicpolysaccharides(teichoicacid). Thepresenceofoutermembraneingram
negativebacteriaprovidesextrabarrier,makingitlesspermeabletoantimicrobialsubstancescompared
to grampositive bacteria. Therefore, it is generally easier to inhibit or destroy grampositive bacteria
duetodifferenceinthecellwallstructure(8).
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2.0 Variables2.1 IndependentvariableAqueousgarlicextractissubjectedtodifferenttemperatureusingthewaterbathfor15minutes. The
temperaturestestedare100oC,80oC,75
oC,40oCandroomtemperature.
0.5 cm3
of aqueous garlic extract is added into 0.5 cm3
either hydrochloric acid or sodium hydroxide
adjustedtopH1,pH7,pH12andpH14usingpHmeter(0.01).
2.2 DependentvariableThe 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
hoursofincubation.
2.3 FixedvariableThe fixed variables are the amount of bacteria inoculated on the nutrient agar plate; concentration of
aqueousgarlic extract;concentration,pH(7.2)andvolumeofnutrientagarused; thedurationoffilter
paperdiscbeingsoakedintheextract;diameteroffilterpaperdisc(6mm).
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3.0 Procedures3.1 Preparationbeforeexperiment3.1.1 Apparatuspreparation
Filterpaperdiscsarecutout withholepuncherof6mmdiameter. Cheesecloth,mortarand
pestle,filterpaperdiscs,forceps,andcottonbudaresterilisedusinganautoclave.
7gofGeneChemicalsnutrientagarpowderisaddedto250cm3
ofdistilledwater. Themixture
isheatedwhilestirringuntilitboils. Then,itispouredintoaglassbottleandsterilisedinthepressure
cooker. Duringsterilization,glassbottlecapisloosenedtoallowsteamtoescapeandpreventexplosion
inthepressurecooker.
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
coveredtopreventcontamination.
3.1.2 Turbiditystandardpreparation
McFarland0.5standardispreparedbymixing0.05cm3of0.048molofbariumchloride(BaCl2)
and 9.95 cm3 of 0.18 mol of sulphuric acid (H2SO4) (5) in a screwcap tube used for preparing the
bacteriainoculums.
3SeeAppendix3
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3.1.3 Inoculumpreparation
ThebacteriainoculumsarepreparedbyMrLawrenceKok4. Theapproximatecelldensityof
McFarland0.5standardis1.5x108CFU/mL. Theturbidityofbacteriaisvisuallycomparedtothe
standard5tomakesurebacteriaturbidityissimilartoturbidityofMcFarland0.5standard.
3.2 PreparationofAqueousGarlicExtractfordifferenttemperatureandpHtreatment
3.2.1 Preparationofaqueousgarlicextract
100g ofgarlicisweighedusing electronic weighing machine and then poundedintopulpusing
the mortar and pestle. Pulp is pressed into a beaker using two layers of cheese cloth to obtain the
aqueousgarlicextract.
3.2.2 Preparingaqueousgarlicextractatdifferenttemperature
5microcentrifugesarefilledwith1cm3ofaqueousgarlicextractusingthe1000Lmicropipette.
Themicrocentrifugesarelabelledasshownintable1.
4SeeAppendix4
5SeeAppendix5
Microcentrifuge
label
Temperature
(0.01)oC
1 100.00
2 80.00
3 75.00
4 40.00
5 Roomtemperature
Table1:Microcentrifugelabellinganditscorrespondingtemperature
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3.2.3 PreparingaqueousgarlicextractatdifferentpH
4microcentrifugesarefilledwith0.5cm3
ofaqueousgarlicextractusingthemicropipette. The
4microcentricugesarelabelledasshownintable2.
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
distilledwaterto1MNaOH. pHofaqueoussolutionisgaugedusingpHmeter(0.01). 0.5cm3
ofpH1
HCl,distilledwater,pH12NaOH,pH14NaOHisaddedintomicrocentrifuge6,7,8,and9respectively
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 thecentrifugeat4000rpmfor6minutestobringtheresiduetothebottom. 6filterpaperdiscsareadded
into each microcentrifuge (for triplicates when testing each strain of bacteria) using sterilised forceps
andleftfor30minutes.
3.3 MethodforAgarDiskDiffusionMethod3.3.1 Inoculationofnutrientagarplate
Before inoculation, the working bench is wiped with 95% alcohol and the fan is turned off to
prevent contamination. 100L of nutrient broth containingE.coli is transferred using a micro pipettewithcleantipontothenutrientagar. Asterilecottonswabisusedtoswabthesurfaceofnutrientagar
7SeeAppendix7
Microcentrifuge
label
pH
(0.01)
6 1
7 7
8 12
9 14
Table2:MicrocentrifugelabellinganditscorrespondingpH
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inonedirection. Theplateisswabbedtwicemore,turningtheplate60oeverytime. Then,swabaround
therimoftheagarasshownbyfigure4.
ThisstepisrepeatedwithStaph.a. Eachbacteriastrainisinoculatedin6nutrientagarplates(3platestobetestedforeffectsoftemperaturewhileanother3platestobetestedfortheeffectsofpH). Fresh
micro pipette tips are used for each transfer of bacteria onto nutrient agar surface. The inoculated
nutrientagarplatesareleftfor5minutestodrytoensureanevenbacterialawngrowthonthesurface.
3.3.2 Applicationoffilterpaperdiscsontoagarplates
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
Figure4:
Swabbing
direction
on
the
nutrient
agar
plate
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microcentrifuges. Anyexcessliquidonthefilterpaperdiscisdabbedoffthewallofthemicrocentrifuge.
Thefilterpaperdiscisthentransferredontotheirrespectivepositionontheagar.
Figure5:LabellingtheNutrientAgarPlate(left:fortestingofdifferentincubationpH;right:fortestingofdifferent
incubationtemperature)
Label Contentoffilterpaperdisc
1Aqueousgarlicextract
incubatedatpH1,7,12and14respectively.
7
12
14
C1AqueoussolutionofpH1,
7,12and14respectivelyas
negativecontrol.
C7
C12C14100
Aqueousgarlicextract
incubatedat100oC,80oC,
75oC,40oCand25oC
respectively.
80
75
40
25
CDistilledwaterasnegative
control
CAntibioticdiscsaspositive
controlTable3:LabellinglegendonNutrientagarplate
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
agentsareknowntoactinstantly. Theforcepsaresterilisedwiththesamemethodfortransferofeach
filterpaperdisc.
3.3.3 IncubationandDataCollection
Theincubator8
isashelfwithtwo100Wbulbsinashelf. Afterthetransferoffilterpaperdiscs
onto nutrient agar, all the agar plates placed inverted into the incubator. The temperature of the
incubatorisadjustedto37(2)oC. Allagarplatesareincubatedfor24hours. Thezoneofinhibitionis
measuredwithcentimetreruler((1.0)mm)asshowninfigure6.
Figure6:MeasuringInhibitionZoneofAqueousgarlicExtractTreatedatDifferentpH
8SeeAppendix8.
12mm
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4.0 DataCollection4.1 RawData:AntimicrobialActivityofAqueousGarlicExtractIncubatedatDifferentTemperature
Substance
tested
Temperature/oC
(0.01oC)
DiameterofInhibitionZone(mm)
1 2 3 MeanS.D
Aqueousgarlic
extract
100.0
80.0 8 7 7 7.30.6
75.0 10 9 9 9.30.6
40.0 16 15 17 16.01.0
25.0(room
temperature)
19 16 18 17.71.5
DistilledWatera
N/A
Nalidixicacidb# N/A 26 26 26 26.00.0
Table4:InhibitionZone(mm)ofaqueousgarlicextractincubatedatvarioustemperaturesonE.coli()=noactivity
(N/A)=notapplicable(S.D)=StandardDeviationaNegativecontrol
bPositivecontrol
#Disccontent:30g
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Substancetested
Temperature/o
C(0.01
oC)
Diameterof
Inhibition
Zone
(mm)
1 2 3 MeanS.D
Aqueousgarlic
extract
100.0
80.0 13 12 12 12.30.6
75.0 15 14 16 15.01.0
40.0 20 20 19 19.70.6
25.0(room
temperature)
22 18 17 19.02.6
DistilledWatera N/A
Methicillinb# N/A 17 18 17 17.30.6
Table5:InhibitionZone(mm)ofaqueousgarlicextractincubatedatvarioustemperaturesonStaph.a()=noactivity
(N/A)=notapplicable
(S.D)=StandardDeviationaNegativecontrolbPositivecontrol#
Disccontent:5g
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4.2 RawData:AntimicrobialActivityofAqueousGarlicExtractIncubatedatDifferentpH
Substancetested pHof
medium
DiameterofInhibitionZone(mm)
1 2 3 meanS.D
Aqueousgarlicextract
+hydrochloricacid
1 10 11 10 10.30.6
Aqueousgarlicextract
+distilledwater
7 12 14 14 13.31.2
Aqueousgarlicextract
+
SodiumHydroxide
12
14
Hydrochloricacida
1 7 8 7 7.30.6
DistilledWatera 7 7 8 7.50.7
SodiumHydrodixea
12 8 8 8.00.0
14 7 7 7.00.0
Nalidixicacidb# N/A 26 27 26 26.30.6
Table6:InhibitionZone(mm)ofaqueousgarlicextractincubatedatvariouspHonE.coli()=noactivity
(N/A)=notapplicable
(S.D)=StandardDeviationaNegativecontrol
bPositivecontrol
#Disccontent:30g
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Substancetested
pH
of
mediumDiameter
of
Inhibition
Zone
(mm)
1 2 3 meanS.D
Aqueousgarlicextract
+hydrochloricacid
1 20 17 17 18.01.7
Aqueousgarlicextract
+distilledwater
7 22 18 20 20.02.0
Aqueousgarlicextract
+
SodiumHydroxide
12
14
Hydrochloricacida 1
DistilledWatera 7
SodiumHydrodixea
12
14
Methicillinb# N/A 20 19 19 19.30.6
Table7:InhibitionZone(mm)ofaqueousgarlicextractincubatedatvariouspHonStaph.a()=noactivity
(N/A)
=
not
applicable
(S.D)=StandardDeviationaNegativecontrol
bPositivecontrol
#Disccontent:5g
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4.3
Graph1:
Graph2:
DataPro
eanInhibitio
eanInhibitio
0
5
10
15
20
25
MeanInhibitio
nZone(mm)
0
5
10
15
20
25
MeanInhibitionZone(mm)
g
essing:
nZone(mm)o
nZone(mm)o
100
eanIgarli
temp
1
pHat
MeanIrlicext
omparin
faqueousgarl
faqueousgarl
80
T
hibitioextracrature
7
whichaqueo
hibitio
ractinccoli
Extend
gMeanI
icextractincu
icextractincu
75
emperature,
nZonetincubonE.
usgarlicextr
nZoneubatedandSt
ed Ess
hibition
atedatvario
atedatvario
40
oC
(mm)tedatoliand
12
ctisincubat
(mm)atvariph.a
y Bio
Zoneof
stemperature
spHonE.coli
25
faquevarious
Staph.
14
ed(pH)
faqueuspH
logyLI
00
P
.Coliand
sonE.colian
andStaph.a
usa
E.c
Sta
usnE.
E.
Sta
JUANNE
2206018
age25of4
Staph.a
Staph.a
oli
ph.A
oli
ph.A
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For Tukeys HSD test, a critical value, HSD is calculated. If the mean difference between groups is
greaterthanHSDcriticalvalue,thereissignificantdifferencebetweenthesepairs.
The ANOVA is carried out using Microsoft Excel 2007 while Tukey HSD9 is calculated manually. Below
aretheresultsofmytests:
Variable Bacteriastrain Fvalue Fcritical Indication
Temperature
E.Coli 151.73 3.48 ANOVAtestonthefoursetsofdatashowsthatthereisagroupwhichis
significantlydifferentfromothersin
theirownrespectivesetofdata.
i.e.:meaninhibitionzoneofaqueous
garlicextractincubatedatacertain
temperatureorpHissignificantly
largerthanthoseincubatedatother
temperatureorpH.
Staph.a 109.77 3.38
pH
E.Coli 346.87 4.07
Staph.a 207.43 4.07Table8:ResultsofANOVAon4setsofdatatodeterminewhetherthereissignificantdifferencebetweenmeaninhibition
zoneofaqueousgarlicextractincubatedatdifferenttemperatureandpHonE.ColiandStaph.a
9Fordetailedcalculations,seeAppendix9
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Onegroupissignificantlydifferentfromtheothersintheirrespectivesetofdata,thusTukeysHSDtest
iscarriedoutandtheresultsareasshowninbelow:
GroupCombination,oC
(MeanInhibitionZoneofaqueousgarlicextract
incubatedatdifferenttemperatureonE.coli)
Mean
difference,
mm
HSD
critical
value
Implication
100 80 7.3 2.40 significantdifference
100 75 9.3 2.40 significantdifference
100 40 16.0 2.40 significantdifference
100 25 17.7 2.40 significantdifference
80 75 2.0 2.40 Nosignificantdifference
80 40 8.7 2.40 significantdifference
80 25 10.3 2.40 significantdifference
75 40 6.7 2.40 significantdifference
75 25 8.3 2.40 significantdifference
40 25 1.7 2.40 Nosignificantdifference
Table9:ResultsofTukeysHSDtestonmeaninhibitionzoneofaqueousgarlicextractincubatedatdifferenttemperatureon
E.Colitodeterminewhichgroupissignificantlydifferentthantheother
GroupCombination,oC
(MeanInhibitionZoneofaqueousgarlicextract
incubatedatdifferenttemperatureonStaph.a)Mean
difference,
mm
HSD
critical
value
Implication
100 80 12.33 3.53 significantdifference
100 75 15.00 3.53 significantdifference
100 40 19.67 3.53 significantdifference
100 25 19.00 3.53 significantdifference
80 75 3.33 3.53 Nosignificantdifference
80 40 7.30 3.53 significantdifference
80 25 6.67 3.53 significantdifference75 40 4.67 3.53 significantdifference
75 25 4.00 3.53 significantdifference
40 25 0.67 3.53 Nosignificantdifference
Table10:ResultsofTukeysHSDtestonmeaninhibitionzoneofaqueousgarlicextractincubatedatdifferenttemperature
onStaph.atodeterminewhichgroupissignificantlydifferentthantheother
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GroupCombination,pH
(MeanInhibitionZoneofaqueousgarlicextract
incubatedatdifferentpHonE.Coli)Mean
difference,
mm
HSD
critical
value
Implication
1 7 3.33 1.69 significantdifference
1 12 10.33 1.69 significantdifference
1 14 10.33 1.69 significantdifference
7 12 13.33 1.69 significantdifference
7 14 13.33 1.69 significantdifference
12 14 0.00 1.69 Nosignificantdifference
Table11:ResultsofTukeysHSDtestonmeaninhibitionzoneofaqueousgarlicextractincubatedatdifferentpHonE.Colitodetermine
which
group
is
significantly
different
than
the
other
GroupCombination,pH
(MeanInhibitionZoneofaqueousgarlicextract
incubatedatdifferentpHonStaph.a)Mean
difference,
mm
HSD
critical
value
Implication
1 7 2.00 3.46 Nosignificantdifference
1 12 18.00 3.46 significantdifference
1 14 18.00 3.46 significantdifference
7 12 20.00 3.46 significantdifference
7 14 20.00 3.46 significantdifference
12 14 0.00 3.46 Nosignificantdifference
Table12:ResultsofTukeysHSDtestonmeaninhibitionzoneofaqueousgarlicextractincubatedatdifferentpHonStaph.atodeterminewhichgroupissignificantlydifferentthantheother
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5.0 Conclusion5.1 Part1:Temperature
Graph1and2showsthatStaph.a(grampositive)ismoresusceptibletoantimicrobialeffectsofgarlicregardlessofhowaqueousgarlicextractispretreatedcomparedtoE.coli(gramnegative)astheinhibition zone ofStaph.a is always larger thanE.coli in this study. However, as this study does notconcernthesusceptibilityofdifferentbacteria,nofurtheranalysisiscarriedouttoverifywhetherthere
issignificantdifferencebetweensusceptibilityofE.coliandStaph.a.Graph 1 indicates the effects of temperature on the antimicrobial activity of aqueous garlic
extractonE.coliandStaph.a:
1. Aqueous garlic extract incubated at room temperature, 25 oC yield the highest antimicrobialactivitywhileaqueousgarlicextractincubatedat100
oChasnoantimicrobialactivity
2. Antimicrobial activity has an inverse relationship with the incubation temperature of aqueousgarlicextract:astemperaturedecreasesfrom100oCto25oC,theinhibitionzoneincreases.
TheTukeysHSDtestisusedtodeterminewhethertheantimicrobialactivityofaqueousgarlic
extract incubated at 25oC is significantly higher than being incubated at other temperature. All
calculations are based on significance level of 0.05, =0.05. Table 8 and table 9 (involvingE.coli andStaph.a) show that there is no significant difference between antimicrobial activity of aqueous garlicextract incubated at 80
oC and 75oC. There is also no significant difference between antimicrobial
activityofaqueousgarlicextractincubatedat40oCand25oC.
Asaqueousgarlicextractincubatedat100oCdoesnotshowanyantimicrobialactivity,thereis
significant difference in antimicrobial activity when compared to aqueous garlic extract incubated at
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othertemperatures(80oC,75oC,40oC,25oC). Thisisshownbyresultsinthefirstfourrowsintable8
and9.
Aqueous garlic extract incubated at 75 oC and 80 oC shows significantly lower antimicrobial
activitywhencomparedtoaqueousgarlicextractincubatedat40oCand25oC. Thisisshownbyresults
in table 8 and 9, suggesting that theoptimum incubation temperature for aqueous garlic extracts
antimicrobialactivityisbetween40oCand25oC.
Thoughthereisnoconcreteevidencewhyaqueousgarlicextractlosesitsantimicrobialactivities
whenincubatedathightemperature,therearetwoplausibleexplanations.
Increaseintemperatureincreasesthekineticenergyofatomsinmolecules;atomsvibratemore
violently. Ifkineticenergyofatomsinmoleculeovercomesbondenthalpy,bondsbetweenatomsina
molecule are broken. The 3D 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
moleculesandescapeintotheatmosphere.
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5.2 Part2:pHGraph2showsthataqueousgarlicextractincubatedatpH7yieldshigherantimicrobialactivity
comparedtopH1forbothE.coliandStaph.a.. AqueousgarlicextractincubatedatpH12and14doesnotshowanyantimicrobialactivityinbothstrainsofbacteria.
Forbothstrainsofbacteria,theresultsoftheTukeysHSDtest(calculatedatsignificancelevel
of0.05)intable10and11showthataqueousgarlicextractincubatedatpH1andpH7hassignificant
differenceinantimicrobialactivitywhencomparedtothatofaqueousgarlicextractincubatedatpH12
and14.
Table 10 also indicates significant difference between antimicrobial activity of aqueous garlic
extract incubated at pH 1 and pH 7 onE.coli. It appears that antimicrobial activity of aqueous garlicextractonE.coliissignificantlyhigherwhenaqueousgarlicextractisincubatedatpH7comparedtopH1. However, there is no significant difference between antimicrobial activity of aqueous garlic extract
incubatedatpH1andpH7onStaph.a.asindicatedintable11.
Table5showsdistilledwater(control)causesinhibitionzoneof78mminE.coli.Thisindicatesthatrandomerrors(possiblycontaminateddistilledwater)occurredduringthispartoftheexperiment
because distilled water should not show any antimicrobial activity. Thus, inhibition zone of 78 mm
causedbyhydrochloric acid andsodium hydroxide can bedisregarded, i.e.:antimicrobial activity onE.coliissolelycausedbyaqueousgarlicextract.
ThispartoftheexperimentsuggeststhattheoptimumincubationpHforaqueousgarlicextract
isfrompH1topH7(acidictoneutralmedium).
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One possible explanation to aqueous garlic extracts lost of antimicrobial activity when
incubatedinalkalinesolutionisthatthehydroxideions(OH
)mayhaveaffectedthepolarbondswhich
exist between atoms in molecule of the inhibitory components. Polar bonds exist between one atom
whichispartiallypositiveandanotherwhichispartiallynegative. TheOH
mayhaveneutralisedoralter
thebondpolaritybetweenatoms,causingbondsbetweenatomstobreak. Brokenbondsinamolecule
causesstructuralchangeofinhibitorycomponents,leadingtolossoffunction.
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
structuralchange,OH
couldaffectthepolarityoftheenzymesactivesite. OH
wouldgetattachedto
positively charged active site, preventing negatively charged substrate from sitting on the active site.
Enzymaticreactioncannottakeplace,causingthelossofantimicrobialactivity.
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6.0 EvaluationandSuggestionPossibleErrors
As this experiment is very time consuming, I was only able to do three repetitions for each
bacteriastrainandatthesametimecontrolallthefixedvariables. Asthereareonlythreerepetitions,
myresultsmaynotbeconclusiveduetothehighpossibilityofrandomerrors.
Realising the possibility of anomalous results due to experimental techniques, I have included
positive control in my experiment. Nalidixic acid (30g) and Methycillin (5g) is used as a positive
control for E. coli and Staph. a respectively. According to M100S2 Performance Standards forAntimicrobial Susceptibility Testing; Second International Supplement, control limits for inhibitory
diameter zones ofE.coli andStaph.a when tested with antibiotic discs are 2228mm and 1722 mmrespectively. TherecordedinhibitionzonesforStaph.awhentestedwithmethycillin(5g)is1719mmwhileforE.coliwhentestedwithNalidixicacid(30g)is2627mm. Theinhibitionzoneofbothbacteriastrain is well within the control limits, indicating that my methodology is valid despite possibility of
randomerror.
Limitations
MystudyonlyinvolvesonestrainofE.coliandStaph.a;thusantimicrobialactivityofaqueousgarlic extract onE.coli,Staph.a, grampositive and gramnegative bacteria cannot be generalised. Inorder to generalise the antimicrobial activity of differently pretreated aqueous garlic extract on
differentbacteria,differentstrainsofE.coliandStaph.a,aswellasother commongrampositiveandgramnegativebacteriashouldbetested. Testingaqueousgarlicextractwithawiderrangeofbacteria
willenableustoestablishitsstatusasanantibiotic.
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Duetolimitationofschoollabfacilities,aqueousgarlicextractwasonlyincubatedat5different
temperatures;thusIcouldonlyapproximatethetemperatureatwhichantimicrobialactivityofaqueous
garlic extract decreases. For further investigation, I would incubate aqueous garlic extract at
temperature ranging between 80oC to 100
oC (because this study shows that antimicrobial activity of
aqueous garlic extract starts to decrease when incubated at temperature greater than 75 oC) to
determinetheexacttemperatureatwhichthereisnoantimicrobialactivity.
FurtherResearch
ItwouldbeinterestingtoobservehowmultipleantibioticresistantstrainsofE.coliandStaph.areactstoaqueousgarlicextract. ShouldthegrowthofmultipleantibioticresistantE.coliandStaph.abeinhibited,itcouldleadtodiscoveryofanewgenerationofantibiotics.
There is possibility of synergistic effect between aqueous garlic extract and acidic food
substancesuchaslime,lemonandvinegar. Thoughthisstudyshowslowerantimicrobialactivitywhen
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
aqueousgarlicextractincubatedatpH1andpH7whentestedagainstStaph.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
componentinhibitsthebacterialgrowth.
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7.0 References1.Lewis,Ricki.U.SFoodandDrugAdministration.TheRiseofAntibioticResistantInfection.[Online]
September1995.[Cited:June27,2007.]http://www.fda.gov/Fdac/features/795_antibio.html.
2.Ankri,SergeandMirelman,David.AntimicrobialPropertiesofGarlic.MicrobesandInfection.
Elesvier,Paris:s.n.,1999.
3.PerformanceStandardsforAntimicrobialDisksSusceptibilityTests;ApprovedStandard NinthEdition.
ClinicalandLabaratoryStandardsInstitute.2006,pp.M2A9.
4.Heinrich,Michael,Pieroni,MichaelandBremner,Paul.PlantsasMedicine.[bookauth.]Ghillean
PranceandMarkNesbitt.CulturalHistory
of
Plants.
NewYork:Routledge,2005,pp.219221.
5.Schou,Chad.EconomicBotanyLeaflets.[Online]may09,2000.[Cited:February24,2008.]
http://www.siu.edu/~ebl/leaflets/garlic2.htm.
6.InhibitionofMicrobialgrowthbyAjoene,aSulphurContainingCompoundDerivedfromGarlic.
Nagavana,Rie,etal.11,NewYork:AmericanSocietyforMicrobiology,1996,Vol.62.00992240.
7.IntroductiontoBiotic.AntibioticAttack.[Online][Cited:December12,2007.]http://maflib.mtandao
afrika.net/TQA01074/english/bio.htm.
8.
Talaro,
Kathleen
Park.
An
Introduction
to
Cell
and
Procaryotic
Cell
Structure
and
Function.
FoundationsinMicrobiology:BasicPrinciples,SixthEdition.NewYork:McGrawHill,pp.99101.
9.McGrawHill.ANOVA.[bookauth.]JanWKuzmaandStephenEBohnenblust.BasicStatisticforthe
HealthSciences4thedition.Singapore:MayfieldPublishingCompany,2001.
10.Chapter2 Cellstructureandorganization.MicrobiologyandBacteriology::TheWorldofMicrobes.
[Online][Cited:December12,2007.]
http://www.bact.wisc.edu/Microtextbook/index.php?module=Book&func=displaychapter&chap_id=35
&theme=printer.
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8.0 AppendixesAppendix1:ExcerptfromTheRiseofAntibioticResistantInfection.
URL:http://www.fda.gov/Fdac/features/795_antibio.html
Diseasecausingmicrobesthwartantibioticsbyinterferingwiththeirmechanismofaction.Forexample,
penicillin kills bacteria by attaching to their cell walls, then destroying a key part of the wall. The wall
fallsapart,andthebacteriumdies.Resistantmicrobes,however,eitheraltertheircellwallssopenicillin
can'tbindorproduceenzymesthatdismantletheantibiotic.
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
ribosomalrouteisalsohowbacteriabecomeresistanttotheantibioticstetracycline,streptomycinand
gentamicin.
Appendix2:Whyweretheadaptationsmade
MuellerHintonagarwasnotusedinmystudybecausetheschoollabdidnothavethisparticulartypeof
agar at the time of my experiment. Therefore, I used common nutrient agar from Gene Chemicals. I
believethatthiswouldnot bemuchof a limitationto my experiment becauseE.ColiandStaph.aare
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
insteadof4mm;theincreasedthicknessistocompensateforthefragilityofthenutrientagar.
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Appendix3:NutrientAgarPlate
Appendix4:MethodofPreparingBacteriaCulturefromMrLawrenceKok
Screwcap bottles that are needed to contain bacteria culture and preprepared nutrient broth are
sterilizedintheautoclave. PureEscherichiaColiATCC25922andStaphylococcusaureusATCC25923ispurchased in dry lypholised form. Two bottles are filled halfway with sterilized nutrient broth. Pure
strainsofEscherichiacoliATCC25922andStaphylococcusaureusATCC25923areputintothenutrientbroth. Inoculatednutrientbrothisincubatedat37
oCfor24hours.
90mm
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Appendix5:Comparisonofinoculumsturbiditywith0.5McFarlandStandard.
Appendix6:Polystyreneasafloatinthewaterbath
EscherichiaColiStaphylococcusaureus0.5McFarlandStandard
microcentrifuge
Polystyrene
Waterbath
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Appendix9:DetailedCalculationofANOVAfromMicrosoftExcel2007
ANOVAfirststartswithnullhypotheses,,wheremeanofgroupsarehypothesisedtobethesame.: ANOVApartitionsthevarianceofallobservationsintotwosourcesofvariation:variationbetweenthe
groupmeansandvariationwithinthegroupmeans. Thesamplingdistributionusedfortestingiscalled
theFdistribution(inhonourofR.A.Fisher,whodevelopedFstatistic). Betweengroupvariance
measurethetreatmenteffect,whichinthisstudyistheantimicrobialeffectsofaqueousgarlicextract
whenincubatedatdifferenttemperatureandpH. BelowistheconventionalnotationofANOVAandits
explanation:
NOTATION MEANING
or Withingroupvarianceormeansquarewithin
or Betweengroupvarianceormeansquarebetween
Degreeoffreedom Numberofgroups Numberofobservationineachgroup Totalnumberofobservation Significancelevel
Sumofsquaresbetweengroup
SumofsquareswithingroupTable13:Conventionalnotationsanditsmeaning
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Ifthebetweengroupvarianceisgreaterthanthewithingroupvariance, ,thenthereistreatmenteffect. If
,thenthereisnotreatmenteffect.
TestofHypothesisisperformedbycomparingtheratioofthetwovarianceestimates, .
has 1degreeoffreedom;labelledas has degreeoffreedom;labelledas.
Sourceof
variation
Sumof
Squaresdf
Mean
Squares,
Fratio CriticalF* Pvalue
Between 1 1 , ComputergeneratedWithin Total 1Table14:OnewayANOVAtable
*thecriticalFvalueisatsignificancelevelof0.05, 0.05andthevalueisobtainedfromthePercentilesofFDistributiontableinAPPENDIX10Abelow.
TherearedifferencesbetweenatleastonepairofmeanswhenFratioisgreaterthancriticalF. In
ordertofindoutexactlywherethedifferencesare,TukeysHSD(honestlysignificantdifference)is
carriedouttomakemultiplecomparisons.
Theformulaforcomputing HSDis:
,, Where
isobtainedfromPercentagePointsoftheStudentizedRangefor2Through20treatmentstable
inAPPENDIX10Bbelow.
Thereissignificantdifferencebetweengroupsofacertainpairifthemeandifferencebetweenpairis
greaterthanHSDvalue.
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Forthisexperiment,IusedMicrosoft2007togenerateANOVAtables. Belowarethetablesgenerated:
1.
TheeffectsoftemperatureontheantimicrobialactivityofaqueousgarlicextractoninhibitionzoneofE.coli.
Groups (temperature,0C) Count Sum Average Variance
Group1(100oC) 3 0 0.00 0.00
Group2(80oC) 3 22 7.33 0.33
Group3(75oC) 3 28 9.33 0.33
Group4(40oC) 3 48 16.00 1.00
Group5(25oC) 3 53 17.67 2.33
Table15:Summary
SourceofVariation SS df MS F Pvalue Fcrit
BetweenGroups 606.93 4 151.73 189.67 2.2117E09 3.48
WithinGroups 8.00 10 0.80
Total 614.93 14
Table16:ANOVAtable
4.650.83 2.40
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2. TheeffectsoftemperatureontheantimicrobialactivityofaqueousgarlicextractoninhibitionzoneofStaph.a.
Groups(temperature,0C) Count Sum Average Variance
Column1(100oC) 3 0 0.00 0.00
Column2(80oC) 3 37 12.33 0.33
Column3(75oC) 3 45 15.00 1.00
Column4(40oC) 3 59 19.67 0.33
Column5(25oC) 3 57 19.00 7.00
Table17:Summary
SourceofVariation SS df MS F Pvalue Fcrit
BetweenGroups 761.07 4 190.27 109.77 3.22E08 3.48
WithinGroups 17.33 10 1.73
Total 778.40 14
Table18:ANOVAtable
4.651.733 3.53
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3. TheeffectsofpHontheantimicrobialactivityofaqueousgarlicextractoninhibitionzoneofE.coli.
Groups(pH) Count Sum Average Variance
Groups1(pH1) 3 31 10.33 0.33
Groups2(pH7) 3 40 13.33 1.33
Groups3(pH12) 3 0 0.00 0.00
Groups4(pH14) 3 0 0.00 0.00
Table19:Summary
SourceofVariation SS df MS F Pvalue Fcrit
BetweenGroups 433.58 3 144.53 346.87 8.31E09 4.07
WithinGroups 3.33 8 0.42
Total 436.92 11
Table20:ANOVAtable
4.530.423 1.69
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4.
TheeffectsofpHontheantimicrobialactivityofaqueousgarlicextractoninhibitionzoneofStaph.a.
Groups(pH) Count Sum Average Variance
Group1(pH1) 3 54 18.00 3.00
Group2(pH7) 3 60 20.00 4.00
Group3(pH12) 3 0 0.00 0.00
Group4(pH14) 3 0 0.00 0.00
Table21:Summary
SourceofVariation SS df MS F Pvalue Fcrit
BetweenGroups 1089.00 3 363.00 207.43 6.35E08 4.07
WithinGroups 14.00 8 1.75
Total 1103.00 11
Table22:ANOVATable
4.531.753 3.46
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APPENDIX10A:PercentilesofFDistribution(9)
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APPENDIX10B:PointsoftheStudentizedRangefor2Through20treatments(9)