In Vitro Study on the Effects of Temperature and pH on Antimicrobial Activity of Aqueous Garlic Extract

8/2/2019 In Vitro Study on the Effects of Temperature and pH on Antimicrobial Activity of Aqueous Garlic Extract

1/48

Extended Essay Biology LIMJUANNE002206018

Page1of48

EXTENDEDESSAY

BIOLOGY

OptimalConditionforAntimicrobialActivity:

InVitrostudyontheeffectsoftemperatureandpHonAntimicrobialActivityofAqueousGarlicExtract

againstEscherichiacoliATCC25922andStaphylococcusaureusATCC25923.

Submittedby:LimJuAnne

CandidateNumber:002206018

WordCount:3995only


2/48


Page2of48

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


3/48


Page3of48

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


4/48


Page4of48

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


differenttemperatureonStaph.atodeterminewhichgroupissignificantlydifferentthantheother...28


differentpHonE.Colitodeterminewhichgroupissignificantlydifferentthantheother....................... 29


differentpHonStaph.atodeterminewhichgroupissignificantlydifferentthantheother................... 29

Table13:Conventionalnotationsanditsmeaning.................................................................................... 41

Table14:OnewayANOVAtable................................................................................................................ 42

Table15:Summary..................................................................................................................................... 43

Table16:ANOVAtable............................................................................................................................... 43

Table17:Summary..................................................................................................................................... 44


5/48


Page5of48


Table19:Summary..................................................................................................................................... 45


Table21:Summary..................................................................................................................................... 46

Table22:ANOVATable............................................................................................................................... 46


6/48


Page6of48

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)


7/48


Page7of48

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


8/48


Page8of48

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


9/48


Page9of48

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)


10/48


Page10of48

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.


11/48


Page11of48

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)


12/48


Page12of48

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).


13/48


Page13of48

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).


14/48


Page14of48

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


15/48


Page15of48

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


16/48


17/48


Page17of48

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


18/48


Page18of48

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


19/48


Page19of48

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


20/48


Page20of48

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


21/48


Page21of48

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


22/48


Page22of48

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


23/48


Page23of48

4.2 RawData:AntimicrobialActivityofAqueousGarlicExtractIncubatedatDifferentpH

Substancetested pHof

medium

DiameterofInhibitionZone(mm)

1 2 3 meanS.D


+hydrochloricacid

1 10 11 10 10.30.6


+distilledwater

7 12 14 14 13.31.2


+

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


24/48


Page24of48

Substancetested

pH

of

mediumDiameter

of

Inhibition

Zone

(mm)

1 2 3 meanS.D


+hydrochloricacid

1 20 17 17 18.01.7


+distilledwater

7 22 18 20 20.02.0


+

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


25/48

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


26/48


27/48


Page27of48

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


28/48


Page28of48

Onegroupissignificantlydifferentfromtheothersintheirrespectivesetofdata,thusTukeysHSDtest

iscarriedoutandtheresultsareasshowninbelow:

GroupCombination,oC

(MeanInhibitionZoneofaqueousgarlicextract

incubatedatdifferenttemperatureonE.coli)

Mean

difference,

mm

HSD

critical

value

Implication

100 80 7.3 2.40 significantdifference




80 75 2.0 2.40 Nosignificantdifference






Table9:ResultsofTukeysHSDtestonmeaninhibitionzoneofaqueousgarlicextractincubatedatdifferenttemperatureon

E.Colitodeterminewhichgroupissignificantlydifferentthantheother

GroupCombination,oC


incubatedatdifferenttemperatureonStaph.a)Mean

difference,

mm

HSD

critical

value

Implication







80 25 6.67 3.53 significantdifference75 40 4.67 3.53 significantdifference



Table10:ResultsofTukeysHSDtestonmeaninhibitionzoneofaqueousgarlicextractincubatedatdifferenttemperature

onStaph.atodeterminewhichgroupissignificantlydifferentthantheother


29/48


Page29of48

GroupCombination,pH


incubatedatdifferentpHonE.Coli)Mean

difference,

mm

HSD

critical

value

Implication







Table11:ResultsofTukeysHSDtestonmeaninhibitionzoneofaqueousgarlicextractincubatedatdifferentpHonE.Colitodetermine

which

group

is

significantly

different

than

the

other

GroupCombination,pH


incubatedatdifferentpHonStaph.a)Mean

difference,

mm

HSD

critical

value

Implication







Table12:ResultsofTukeysHSDtestonmeaninhibitionzoneofaqueousgarlicextractincubatedatdifferentpHonStaph.atodeterminewhichgroupissignificantlydifferentthantheother


30/48


Page30of48

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


31/48


Page31of48

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.


32/48


Page32of48

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).


33/48


Page33of48

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.


34/48


Page34of48

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.


35/48


Page35of48

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.


36/48


Page36of48

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.


37/48


Page37of48

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.


38/48


Page38of48

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


39/48


Page39of48

Appendix5:Comparisonofinoculumsturbiditywith0.5McFarlandStandard.

Appendix6:Polystyreneasafloatinthewaterbath

EscherichiaColiStaphylococcusaureus0.5McFarlandStandard

microcentrifuge

Polystyrene

Waterbath


40/48


41/48


Page41of48

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


42/48


Page42of48

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.


43/48


Page43of48

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


44/48


Page44of48

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


BetweenGroups 761.07 4 190.27 109.77 3.22E08 3.48


Total 778.40 14

Table18:ANOVAtable

4.651.733 3.53


45/48


Page45of48

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


BetweenGroups 433.58 3 144.53 346.87 8.31E09 4.07


Total 436.92 11

Table20:ANOVAtable

4.530.423 1.69


46/48


Page46of48

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


BetweenGroups 1089.00 3 363.00 207.43 6.35E08 4.07


Total 1103.00 11

Table22:ANOVATable

4.531.753 3.46


47/48


Page47of48

APPENDIX10A:PercentilesofFDistribution(9)


48/48


APPENDIX10B:PointsoftheStudentizedRangefor2Through20treatments(9)

In Vitro Study on the Effects of Temperature and pH on Antimicrobial Activity of Aqueous Garlic Extract

Documents