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TEDANKARACOLLEGEFOUNDATIONHIGHSCHOOL

EXTENDEDESSAY

ResearchQuestion: Is thereasignificantmeandifferenceamongantisepticsused inmedical

institutions,intermsoftheirbactericidaleffectsonStaphylococousepidermidisinlaboratory

conditionsevaluatedbytheutilizationoftheQuantitativeSuspensionTestMethod?

Subject:Biology

CandidateName:KeremNazlıel

CandidateNumber:1129‐0085

Tutor:Demetİzgü

ExamSession:May2014

WordCount:3998

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AbstractTheaimofthisextendedessaywastoevaluatethebactericidaleffectsofdifferentbrandsofantisepticsonthebacteriumStaphylococcusepidermidis.Myresearchquestionwas:“Isthereasignificantmeandifferenceamongantisepticsusedinmedical institutions, in termsof theirbactericidaleffectsonStaphylococousepidermidisinlaboratoryconditionsevaluatedbytheuseofQuantitativeSuspensionTestMethod?”Itwashypothesizedthattherewouldbeameandifferenceinbetweenantisepticpropertiesofvariousagents.Sincetheantisepticsincludedifferentchemicals,theymayhavedifferentbactericidalpropertiesintermsofdestroyingmicroorganisms.Inordertotestthehypothesisandtoanswertheresearchquestion,QuantiativeSuspensionTestMethodwasused.S.epidermidiscolonieswerecultivatedontoMuellerHintonPlate.Inorder to compare the data, a control trial was needed. In my research, S. epidermidissuspensions were prepared, by using dilutions and thenmixed with different brands ofantiseptics. After 60 seconds of preparation the solution was neutralized. Then thesuspensionswere spreaded on a TSA and incubated for 24 hours at 37°C. Following theincubation the colonies were taken out and counted by using a colored marker andmultipliedbythedilutionratioinordertodeterminetheactualnumber.

The results achieved turnedout to prove that thehypothesiswas right. Thepvaluewas0.042onsingle factorANOVA.Thisvalueshows that therewasameandifferenceamongthe bactericidal effects of antiseptics.Monorapid turned out to be themost effective onewhile Aniosrub was the least. My study demonstrated that, different antiseptics havedifferentbactericidal effects. It ispossible thatwith time, bacteriamaygain resistance tocommercial antiseptics. I believe in order to prevent the spread of these infectionsespecially inhospitalsettings it ismandatory to test theantisepticpropertiesofdifferentantisepticsinregulartimeintervals.Wordcount:303

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TableofContents

I. Introduction3

II. Hypothesis6

III. MethodDevelopmentandPlanning7

IV. Method12

V. DataAnalysis15

VI. Evaluation19

VII. Conclusion23

VIII. Bibliography25

IX. Appendices

Appendix127

Appendix228

Appendix329

Appendix431

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I.Introduction:

Myparentsarebothphysicians.Iregularlygoto theirhospitaltovisitthem.Duringmy

visits I have seen hand antiseptics that are placed in the corner of each room and on

corridors. I saw that my parents were frequently using them, especially following the

contactwithapatient.Afterseveralvisits,Inoticedthepresenceofadifferentbrandofan

antiseptic.Then I askedmyparents “Whichantiseptic is themosteffectiveone? “Bothof

them said “Actually we did not study the real effects of them against bacteria, but the

producer companies say that each of them effectively destroy the microorganisms.

MeanwhileIheardnewsontheTVreportingthatthehandantisepticshadpowerfuleffects

on hospital infections as well as on other infections. The news was focusing on the

importanceofkeepinghandscleaninordertoavoidhavinginfluenzainfectionsandtohalt

thespreadingof hospitalinfections.Itwasmentionedthatthemaincauseofspreadwas

due tocrosscontaminationof themicroorganisms frompatient topatientviadoctorand

nurses. That made me wonder which antiseptics have the best bactericidal effect on

bacteria.

Keepinghandscleanisoneofthemostimportantstepswecanusetoavoidgetting

sick and spreading microorganisms to people around us. Many diseases and conditions

spreadbecauseofnotwashinghandswithsoapandavailablewater.Ifsoapandwaterare

unavailable,itisthebestfortheindividualstouseantiseptics.

Agents applied to living tissue to destroy or inhibit the growth of infectious

microorganismareknownasantiseptics.Antisepticscomeinavariety:ointment,liquid,gel

and spray.When using a hand‐antiseptic; antiseptic should be placed to the palm of the

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handandhandsshouldberubbedtogethersotheliquidcoverallthesurfacesofthehands

andfingersandrubbeduntiltheyaredry.1Ittakesapproximately30secondsforhandsto

getdry.

Companies who make commercial hand antiseptics use different chemicals and

formulas. Among the major families of antiseptics, there are alcohol, phenols, chlorine,

iodine compounds.2Alcohol based sanitizers aremore effective at killing organisms than

soaps and do not dry out hands asmuch.3Most commonly used are ethanol (60‐90%),I

propanol (60‐70%) and 2‐propanol/isopropanol (70‐80%) ormixtures of these alcohols

(70‐80%).Onestudyshowedthatalcoholbasedhandsanitizersaremoreeffectiveatkilling

microorganismsthansoapsanddonotdryouthandasmuch.4

So Iwanted to evaluate the efficiency of different types of hand antiseptics that

contain various compounds in different percentages, which are mainly used in the

hospitalsinordertodestroymicroorganisms.

The aim of the present study was to compare the bactericidal effects of hand

antiseptics used in medical institutions. The term bactericidal refers to any agent that

directlyinducesthedeathofbacterium,throughdisruptingitsenzymemechanismsorelse.

AsthetestorganismIdecidedtouseStaphylococcusepidermidis,(whichisagram‐positive

bacteria)inordertolimitthesubjectofmyextendedessay.Thisbacteriumisgrampositive

and is one the most common skin bacterium found on the human skin. Because it is a

1Tentative Final Monograph for Health-Care Antiseptic Drug Products; Proposed Rule74 (56). United States Federal Food and Drug Administration. March 2009. pp. 12613–12617.2http://www.ehow.com/info_7865502_chemicals‐antiseptic‐soap.html3http://www.uoguelph.ca/foodsafetynetwork/alcohol‐based‐hand‐sanitizers4http://www.uoguelph.ca/foodsafetynetwork/alcohol‐based‐hand‐sanitizers

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common microorganism; it is usually selected for research purposes in laboratory

conditions.Myresearchquestionis,

Isthereasignificantmeandifferenceamongantisepticsusedinmedicalinstitutions,

in terms of their bactericidal effects on Staphylococous epidermidis in laboratory

conditions evaluated by the utilization of the Quantitative Suspension Test

Method?

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II.Hypothesis:

Antiseptics, agents applied to living tissue to destroy or inhibit the growth of infectious

microorganism are known as antiseptics. Antiseptics are usually used in hospitals or on

otherfacilities.Inordertohaltthespreadingofinfectionsantisepticsarewidelyusedin

hospitals as well as in our daily lives. However, with time the chemicals can lose their

bactericidalpotencyduetotimeorheat,andneedtobecheckedperiodicallytoascertain

their potency and efficiency. Antiseptics usually contain alcohol, phenols, and chlorine,

iodine compounds to inhibit the growth of bacteria.5Commercial companies usually

produce ethanol based andpropanol based antiseptics. Tobe licensed as an antiseptic;

antisepticshouldbekilling99.9%ofthebacteriaonhands,30secondsafterapplicationand

99.999%to99.999%inoneminute.6Sinceallthecommercialantisepticsarelicensed,we

do not expect to see a huge difference in between the antiseptics. Since the chemical

propertiesofantisepticscanvary,itcanbeforeseenthattheremaybeadifferenceamong

theeffectsofantiseptics.InastudyconductedbyJokarandMohebbi, itwasreportedthat

isopropanolbasedantisepticshadahigherdisinfectanteffect thanethanolbasedoneson

instruments aswell as on skin surface.7According to this data, itwas hypothesized that

therewillbeasignificantmeandifferenceintermsofdestroyingbacteriain60secondsin

between different brands of hand antiseptics by the utilization of the Quantitative

SuspensionTestMethod.

5http://www.ehow.com/info_7865502_chemicals‐antiseptic‐soap.html6http://www.cdc.gov/handwashing/7http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/(Z)

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III.MethodDevelopment/Planning

While I was trying to find a logical and an appropriate method for my research

question, which was, Is there a significant mean difference among antiseptics used in

medical institutions, in terms of their bactericidal effects on S.epidermidis in laboratory

conditions?Iconfrontedwithsomeproblems.

A.BacteriumtoUse

Firstofall,myfirstaimwastodeterminetheappropriatebacteriumtobeusedin

the experiment. I needed a bacterium,which can be easily found in the human skin and

easily obtained. I carried out an evaluation and realized that S.epidermidismay be the

ideal bacterium for me to use in my research. First, it was one of the most common

bacteriumfoundonthehumanepitheliaandalsoitisnotpathogenic8(inthesafetyclassof

IatATCC).Thismeansthatitissafeformetoworkonit.DuringmyresearchIdecidedto

use the 2nd subculture of the bacteria, in order for the bacteria to be more active.

Additionally,thebacteriathatI’llbeusingwillbebetterthanthepreviousgenerationsince

theoffspring’softhebacteriahasthechancetosurvivelonger.Forpreparationprocedure

of2ndsubcultureseeappendix2.

B.AntisepticstoUse

8Levinson,W.(2010).ReviewofMedicalMicrobiologyandImmunology(11thed.).pp.94–99.

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Secondly, my aim was to determine which antiseptics should I be using in my

experiment?Imadeanevaluationandsawthattheethanolbasedantisepticsandpropanol

based antiseptics were themost commonly preferred ones Therefore I decided to use

“Monochol”,”Monorapid”,”Steriderm”and“Aniosrub”brands,whichareusedfrequentlyin

hospital settings as well as on other laboratory conditions. The chemicals that the

antisepticsaremadeofcanbeseeninappendix1.

Afterdecidingontheantisepticstoworkon,Ifacedupwiththeprobleminchoosing

themethodofmyresearch.Ineededamethodthatwouldletmetogetaprecisedatawith

less random errors. Because my research question is based on the comparison of

bactericidal effects of different antiseptics; the method I choose should be able to

demonstratethebactericidalpropertiesofdifferentagents.Fortheprocedure,Ineededa

mediumwhereIwouldbeabletocultivatethebacteria.Imadeanevaluationandsawthat

MuellerHintonagarwouldbethebestonebecauseitisaculturethatenablesdifferentkind

ofbacteriatogrowon.9

C.TheControlTrial

SincemyaimwastoevaluatethebactericidalpropertiesofsolutionsfirstIneedto

findouthowthebacterialcoloniesreproduceinapropermediumwithoutthepresenceof

any antiseptic. I need a medium where I can obtain the 2nd subculture. Therefore all

coloniescultivatedherewouldhaveahighchanceofgrowth.Inordertocounttheamount

ofbacteriafollowingtheprocedureadilutionisneededinordertobeaccurate.Without

the dilution procedure, there may be a large number of bacteria which would made it

impossibletocount.

9Atlas, R.M. (2004). Handbook of Microbiological Media. London: CRC Press. p. 1226. ISBN 0-8493-1818-1.

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I used an easy method to find the number of colonies I needed while I was

cultivating the bacterium. I decided that the dilution method would be appropriate. If I

dilute the bacterial solution I would have the chance of counting the bacterial colonies

easily.Consequently;multiplyingitwiththerateofdilutionIwouldbeabletogettheactual

numberofbacteriathatispresentintheTSA.

SinceI’llbeplanningtodilutethebacterialsolution,Ineededtochooseadiluentthat

I can use in my research. According to European Union (EU) countries protocol (prEN

13727:April2009)inordertodeterminetheeffectofantiseptic;aspecificdiluentneedsto

beused.Thematerialsthatmakeupthediluentispresentedintheappendix3.ThenIhad

todecideontheamountofbacteriathat I’llbeusingontrials.AccordingtotheEuropean

Union countries protocols the number of bacterium in a suspension should be between

1.5*10^8‐ 5.0*10^8 cfu/ml. To determine this value a spectrophotometer should be

present.InordertogetanaveragevalueIdecidedtoadjustthevalueto+0.03OD.Bythis

wayIwouldbeabletodeterminetheamountofbacteriawhichIwillbeusingineachtrial.

Todothis,I’llneedtomakeasolutionthatbothcontainthediluentandthebacteria.

Throughoutmy trials I’ll benaming this solutionas the test suspension.Since Ineededa

smallamountofbacteriatofindanappropriatenumberofcolonies,Ineededtohaveatest

solutionthatissmallinvolume.Idecidedtouse2mlofdiluentandsomebacteriainorder

toreachthevalueof+0.03OD.I’llbeplacingthediluentandthebacteriaintoatesttubeso

thattheycanmixhomogeneously.TodilutethetestsolutionIdecidedtotake100μlof2ml

testsolution.BythiswayI’llbediluting thesolution in1/50=0.02. I’llbeadding100μlof

test solution into4900μl. I chose theamountof solutionas4900μlbecausewhen I’ll be

oncetaking100μlfromthesolutionI’llhavethechancetodiluteitintheratioof1/50.SoI

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decidedtorepeatthedilutionprocessforthreetimes.InthiswayIwouldbeabletodilute

thesolutioninratioof1/125000.

Then Ineededagrowthmediumwhere I cangrowthebacterial coloniesafter the

test. This medium has to have enough nutrients for bacterial growth. So, I chose

TSA10becauseitcontainssoybeanwhichenablesthegrowthofbacteriaeasily.

FollowingthedilutionprocessIshouldplacethetestsolutioninaTSA*withtheaid

of a glass loop; in order to distribute the test solution homogeneously. In order to

determine how the bacteria colonies reproduce; an ideal environment was needed. I

decidedtoplace them intoan incubator,which isset to37°C ,whichwould enable the

enzymesof bacteria towork properly. In order for thebacterial colonies to grow in an

appropriateamounttheyhavetobestoredintheincubatoratleastfor24hours.

Following the incubationprocess Iwouldhave the chance to count thenumberof

colonies formed. To facilitate the process I decided to count the colonieswith a colored

markerbystartingfromthebottomuptothetop.ThenIwouldbeabletodeterminethe

numberofcoloniesthatarepresentinthedilutedtestsolution.Inordertoobtaintheactual

numberofcolonies,Ishouldbemultiplyingthenumberofbacteriawiththedilutionratio

whichinthiscaseis1.25*10^6.

Afterdecidingonthemethodtodeterminethenumberofbacteriapresentinthetest

solution,Ineededtofindamethod,whichwouldevaluatetheeffectoftheantisepticonthe

bacteria.SinceIwillbeusingtheantisepticIshouldbeusingthetestsolutiononcemore.

D.MixingAntisepticsWithTestSuspension

*TrypticSoyAgar

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Sincebacteriahavethechancetokillof%99.999to%99.99999withinfiveminutes

11Ineedtolookattheeffectinaspecifictimeperiod,thereforeIdecidedtotakethetime

spanas60seconds.

When I placed the antiseptic in the test tube with the test solution I need a

neutralizer, which will halt the effects on the antiseptics. According to the Quantitative

SuspensionTestMethodthereisadeterminedneutralizerforuse.Hardwaterneedstobe

usedinordertoachieveneutralization.(seeappendix2)SoIthoughtthatthevolumesIwill

beusingshouldbesimilartothecontroltrialanddecidedtotake100μloftestsuspension

andplaceditinaglasstube.I added900μlantisepticoverit,andwaitedforaminute.

Then100μlofthepreparedsolutionwasplacedtothetubethatcontains800μlneutralizer

and100μlhardwater.Aminuteisneededfortheneutralizertobeeffective.Then,likein

thecontroltrialIshouldtake100μlandadditonaTSAandplaceitintheincubatorfor24

hours.

AfterwardsIshouldcountthenumberofbacteriaasinthecontroltrial.InthistrialI

diluted in a 0.01 ratio, so I should bemultiplying the numberwith 100. In order to get

accurateresultsIshouldrepeatthetrialforfivetimesforeachantiseptictodeterminethe

number of bacteria present in the culture without the presence of any antiseptic

interaction.

After deciding on the appropriate method I needed a laboratory where I would

conductmyresearch.ImadeanevaluationandfoundthatGaziUniversityMedicalFaculty

Hospital’s Microbiology Department have the technical capability and experience to

conductmystudy.

11http://www.cdc.gov/handwashing/

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.IV.Method

Quantitative Suspension Test Method

Quantitativesuspensiontestmethod(prEN13727.April2009)isadesignatedprotocolusedinEuropeanUnioncountriestodeterminethebactericidalactivityofachemicaldisinfectantoranantisepticagainstStaphylococcusepidermidis.A) PreparationoftheTestSuspension

1.Put on the gloves, and disinfect the table so that no bacteria would contaminate the

medium.

2.TaketheMuellerHiltonAgarthatcontainsthe2ndbacteriasubculture.(seeappendix3)

3.Byusingthelooptake2coloniesandplaceitinatesttube.

4.Thenwithautomaticpipettetake2mlofdiluentandplaceitintothetesttube.

5.Place the tube in the spectrophotometer, andmake sure the value is +0.03 OD. If not

adjustittothisvaluebyeitheraddingdiluentoraddingsomebacteria.

6.Thiswaythesolution’sabsorptionvaluewillbeintherange   cfu/ml.(ColonyFormingUnit/ml)

6.Thetestsuspensionisprepared.

B) EstimatingTheNumberofBacteriaPresent(CFU)inTheTestSuspension

1.100μloftestsuspensionis takenfromthe2mlsuspensionbyusinga100μlautomatic

pipette.

2.Thenitisplacedinatesttubewhichcontains4900μlofdiluent.

3.Bythisstep,thetestsolutionis1/50diluted.

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4.Thestepsbetween2‐4arerepeatedfortwomoretimes.

5.Thiswaythetestsuspensionwouldbedilutedfor3times.

6.Aftertheserialdilution100μloftestsuspensionistakenandpouredonaTSA

7.By thehelpof a glass spreader the suspension is spreadedonto theTSA in clockwise

directionfortwotimes.

8.AfterwardstheTSAisplacedinanincubator,whichissetto37°C±0.5°C

9.Thebacterialcultureiskeptintheincubatorfor24hours.

10.Aftertheincubationprocess,acoloredmarkeristakenandthewhitebacterialcolonies

arecountedstartingfrombottomgoingtothetop.

11.Thenumberofbacteriaaliveinthedilutedsuspensionisdetermined.

12.Inordertoobtainthenumberofbacteriainthetestsuspensionthenumberofbacteria

ismultipliedwiththedilutionratioof

13.Thenumberofbacteriapresentinthetestsolutionisdetermined.

C) TestingofDifferentAntisepticsByQuantitativeSuspensionTestMethod

1) 100μloftestsuspensionispreparedinthesamestepsasinA.

2) Itisplacedinatesttube.

3) 900μl of antiseptics is added to the test tube in order to mix it with the test

suspension.

4) Waitfor60secondsfortheantisepticandthetestsuspensiontointeractataroom

temperatureof24°C

5) Followingtheinteraction,100μlofthemixsolutionistakenby100μlpipette.

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6) Itisplacedinatesttubewhere800μlneutralizerand100μlhardwaterarepresent.

7) Wait for 5 minutes for the neutralizer and the hard water to react against the

antiseptic.

8) 100μlofthesolutionistakenbyusinga100μlpipetteandplacedonaTSA

9) By using a glass spreader the suspension is spreaded in the TSA in a clockwise

directionfor2times.

10) Thebacterialcultureisobtained

11) Then the culture is placed in an incubatorwhich is set to 37°C and kept for 24

hours.

12) After the incubationprocess, thenumberofbacteriaon theagar is counted from

bottomtothetopbyusingacoloredmarker.

13) Thenumberofbacteriapresentinthedilutedsolutionisdetermined.

14) Tocalculatethenumberofbacteriainthesolutionthenumberofbacteriapresent

inthedilutedsolutionismultipliedwithdilutionratioof10^2.

15) Thesteps1‐14arerepeatedforeachantiseptic.

16) Thesteps1‐15arerepeatedforfivetimes.

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V.DataAnalysis

Experiment   Trials  Number of Colonies Formed After Incubation

Control  1  287 2  283 3  282 

4  286 5  293 

Table1.1:TableShowing theNumberofColoniesFormedonTrypticSoyAgar inControlExperimentin24Hoursat37°C

Whenthedatafromtable1.1usedandthenumberofcolonies ismultipliedby CFU/mlisobtainedforeachtrialTable1.2:Numberof totalcoloniesobtained in thecontrolexperiment,DilutionFactor is1.25*10^6

Table 2.1: Raw Data Table Showing The Number of Colonies Formed After PerformingQuantitative Suspension Test Method in the Diluted Solution following the mixture ofantisepticswithTestSuspension

Trials  Number  of  Colonies  Formed  By  Diluted  Suspension  in  1 Minute  

  Monochol  Steriderm  Aniosrub  Monorapid 1  1  2  2  0 2  2  3  3  0 3  1  2  1 2

4  0  1  2  1 5  1  1  4  1 

Experiment   Trials  CFU/ml  Mean (CFU/ml) 

Control  1  3.58  *10^8 3.53*10^8

2  3.53  *10^8

3  3.52 *10^8

4  3.57 *10^8 

5  3.66 * 10^8

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DescriptiveStatistics1)Mean

:Numberofcolonies

n:numberoftrials2)Variance:

n:Numberoftrialsxi:NumberofcoloniesVar(x):Varianceμ:Mean3)StandardDeviation:

:Mean

xi:Numberofcoloniesσ:StandardDeviationn:Numberoftrials4)StandardError:

σ:StandardDeviation:NumberofTrials

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  Monochol  Steriderm Aniosrub Monorapid 

Mean  1.0  1.8 2.4 0.8 Standard Error  0.3  0.4 0.5 0.4 Standard Deviation  0.7  0.8 1.1 0.8 Count  5 5 5 5

Confidence  Level (95,0%) 

0.9  1.0 1.4 1.0 

Variance  0.5  0.7 1.3 0.7 

Table 2.2: Calculated Data Table Showing Descriptive Statistics of Number of ColoniesFormedBydifferentantiseptics

Mean  Number  of  Colonies  Formed  in  the Antiseptic‐BacteriaSuspension  (CFU/ml) 

Monocohol   Monorapid  Steriderm Aniosrub100  80  180  240 

Table2.3:CalculatedDataTableShowingtheNumberofColoniesFormedintheAntiseptic‐BacteriaSuspension(CFU/ml)TheevaluationmethodofCFUcanbeseenonappendix4.ANOVA:Source of Variation  SS  df  MS  F P‐value F crit 

Between Groups  8.2 3  2.733333333 3.416666667 0.042965426 3.238871517 Within Groups  12.8 16  0.8                    Total  21 19          

Table2.4: ANOVAResultsofCalculatedValuesofNumberofcolonies formedfromTable2.1

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Bargraph:

Figure 1Graph ShowingMeanNumber of S.epidermidis colonies mixedwithdifferent brands of antisepticsfollowing24°CincubationonTSAplatesat37°C

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VI. Evaluation

Theaimofthisstudywastofindananswertomyresearchquestion:Isthereasignificant

mean difference among antiseptics used inmedical institutions, in terms of their

bactericidal effects on Staphylococous epidermidisin laboratory conditions using

QuantitativeSuspensionTestMethod? It was hypothesized that therewould not be a

mean difference among antiseptics because the commercial antiseptics should have a

specificstandardinordertobelicensed.

To beginwith,my control trial had themean CFU/ml as 3.53*10^8. According to

Quantitative Suspension TestMethod the valuemust be between 1.5*10^8‐5.0*10^8. So

whenthemeanvalueiscomparedtothedeterminedintervalitcanbeseenthatthetrials

including the number of colonies formed without the presence of an antiseptic were

successful.Thismeansthattherewasnotanycontaminationofdifferentbacteriainthetest

suspensionotherthanS.epidermidis.

Attheendofthetrials, forMonorapidthemeannumberofcoloniesformedinthe

dilutedsolutionwas0.7,forMonochol,1.0,forSteriderm1.8andforAniosrub2.4.When

comparing the data with the hypothesis, it can be concluded that the hypothesis was

correct. It canbe seen that there is a difference in themeannumberof colonies formed.

SincethenumberofcoloniesformedbyMonorapidislessthantheerest,itcanbeaccepted

asthemosteffectiveonewhiletheAniosrubisastheleast.

Whenconsideredaccording tothecriteriaofQuantitativeSuspensionTestMethod

all antiseptics were accepted as effective at the end. According to the Quantitative

SuspensionTestMethodevaluationcriteria,theremustbeadifferenceof10^5.Whenwe

comparethetable2.4andthe1.2thereisadifferenceofmorethan10^5.Alltheantiseptics

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tested on the trials were successful according to QSTM criteria. So, the antiseptics are

appropriateforeverydayuse.

WhentheTable2.3isanalyzedthoroughlyitcanbeseenthatonthestandarderror

section, the standard error values are smaller than themean values. However, Standard

Errorvaluesaresignificantbecausethevaluesarerelativelyclosetothemeanvalues.This

maymean that theremayhavebeen some errors thatmayhave changed the results. As

seeninFigure1;theerrorbarshavehighintervalswhencomparedtothemeannumberof

coloniesformedinthedilutedsolution.Whenthestandarddeviationvaluesareanalyzed;it

is obvious that there is a significant difference especially in between Monocohol and

Monorapid. Since themean values and the standard deviation are close relation to each

otheritcanbeconcludedthatonceagainthattheremustbesomeproblemsthatcouldhave

affectedtheresultsoftheexperiment.

Accordingtothenullhypothesis,therewouldnotbeanydifferenceamongthedata

iftheαvaluewerelessthan0.05.Theαwassmallerthan0.05,somynullhypothesiswas

rejected. This shows that there is a significant difference in between each and every

antisepticgroup.

When ANOVA table is analyzed we see that some standard errors are present.

StandardErorrvaluesofeachantisepticaresimilar,thereforetheerrorsintheexperiment

mayhaveeffectedtheoutcomeoftheexperiment.

Thevariouscompositionsofchemicalsineachantisepticmaybethereasonforthis

discrepancy. The most effective antiseptic according to our results was Monorapid.

Monorapidcontains%70ofispropanolwhileMonocoholcontains%45ofisopropanol.The

percentageofisopropanolpresentonthesolutioncanhaveanaffectonitsantibacterial

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properties.SteridermandAniosrubbothcontain%70ofethanol.However,Steridermhas

a stronger antiseptic activity, because it contains additional agents other than alcohol

whichmaymake it tobemore effective. Antiseptics that contain isopropanol as their

main component are more effective than the ones who have ethanol as their main

component.

Duringmyresearch,therewerenounexpectedincidentsthatmayhaveaffectedthe

outcomeofmyexperiment.However,whengoingthroughoverthedatathatIobtained;I

realizedthattheremightbesomeerrorsthatmayhaveaffectedtheresults.

1. Theremay have been contamination of themedium. In order to solve this

problemmoreattentionshouldbepaidtothesurroundingsthatthestudy

isconductedon.

2. The bacteria thatwere usedwere 2nd subculture. Theremight be a chance

thattheywerenotstrongenough.So,ifIhaveused3rdsubculture,Iwould

havethechancetoincreasethepossibilityofhavingmoreactivebacteria.

3. Onlyonetypeofbacteriawasused.Therearedifferenttypesofbacteriathat

inhabitonthehumanskin.Ifdifferenttypesofbacteriawereexaminedwe

would have the chance to evaluate the effects of antiseptics on different

microorganism.

4. Different types of measuring methods such as spectrophotometric method

mayhavebeenused.

5. Tomakeabettercomparison;differentantisepticswithdifferentproperties

shouldbeused.

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6. In order to obtain a more precise number of bacteria; the dilution ratio

shouldbedecreased.

7. Differenttypesofbacteriashouldbeusedtoevaluatetheeffectsofantiseptics

onhumanskin.

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VII.Conclusion

Myresearchquestion“Isthereasignificantmeandifferenceamongantisepticsusedin

medical institutions, in terms of their bactericidal effects on Staphylococous

epidermidis in laboratory conditions usingQuantitative Suspension TestMethod?”is

answeredwiththeresultsthatIhaveobtainedfrommytrials.Thereisasignificant

meandifference in betweenbactericidalpropertiesofdifferentantiseptics. InmystudyI

haveseenthattheantisepticsthataremoreconcentratedwithisopropanolarelikelytobe

more effective than the antiseptics that include ethanol as their main component. Since

eachantisepticbactericidalpropertyinQuantitativeSuspensionTestMethodinterval,they

allcanberegardedassuccessful.Therefore,Iconsideredmystudyasbeingaccurate.

Themain reasonwhy I have chosen to domy extended essay particularly on this

topic was to determine the difference in between bactericidal properties of different

antiseptics used in medical instutions. However, the topic goes beyond my scope and

capabilities. Some studies have been conducted before,whichwere similar tomine. The

studies I have come across were usually drawing attention to the comparison of

disinfectantsratherthantheantiseptics.

Peopleallaroundtheworld useantisepticseverydayinorder togetridoff the

bacteriaon theirhands. Individualshavedifferent choices for antiseptics.Theantiseptic

marketisgrowingdaybydayandbecauseeachantiseptichasadifferentcompositionis

hardtoconcludewhichoneissuperiorandrecommendedforgeneraluse.

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VII.Bibliography

1. Atlas, R.M. (2004). Handbook of Microbiological Media. London: CRC Press. p. 1226.  ISBN 0‐

8493‐1818‐1 

2. Author’s name not stated December 2013 

 Handwashing: Clean Hands Saves Lives 

http://www.cdc.gov/handwashing/ 

3. Levinson, W. (2010). Review of Medical Microbiology and Immunology (11th ed.). pp. 94–99. 

4. Author’s name not stated. Time and date not stated. 

 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/(Z)  

5. Rao,Sridhar Date and time not stated. 

Testing of Disinfectants 

http://www.microrao.com/micronotes/pg/testing_of_disinfectants.pdf  

6. Edmonds, Sarah L‐Macinga,David ‐Mays‐Suko, Patricia 

Date and time not stated.

Comparative Efficacy of Commercially Available Alcohol‐Based Hand rubs and WHO‐Recommended Hand rubs: Which Is More Critical, Alcohol Content or Product Formulation? 

http://www.gojo.com/~/media/GOJO/Countries/USA/Markets/Healthcare/_Shared/Files/Resources/ResearchReport_CompEfficacy%20ABHR%20winner%20of%20Rutala%20award.pd 

7.Author’s name not stated. Time and date not stated. 

 EN 1276 Quantitative Suspension Test of Bactericidal Activity of Chemical Disinfectants  

http://www.ats‐labs.com/testing‐services/antimicrobial‐test‐library/european‐standards‐en‐

test‐methods 

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8. P. D.; Olson, M. E. (2010). "Current concepts in biofilm formation of Staphylococcus

epidermidis". Future Microbiology 5 (6): 917–933.

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IX.Appendices

A)Appendix1

Materials

*1000mlManorapid(Antisepticachem.pharm.ProduckteGmbH–Germany)

70%(v/v)iso‐propanol,1,3Butanediol,PEG‐75,

Lanoline,water,parfume

*1000mlManochol(GülBiolojiLabaratuarlarıSanayiTic.Limited–Turkey)

Ethylalcoholl96%(64‐17‐5)45%,Isopropylalcohol(67‐30‐0)25%,odor

Component,moisturizer,de‐ioniatedwater

*1000mlAnioscrub(ANIOSLaboratoires‐France)

%70Ethanol(700mg/gi.e755ml/L‐CASNo64‐17‐5)

*1000mlSteriderm(KimpaİlaçLabveTic.LTD.Şt‐Turkey.)

70%h/hethanol(CAS:64‐17‐5),Chlorhexidinedigluconate(CAS:18472‐51‐0),

1‐3butandiol

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B)AppendixII

SolutionsUsedInQuantitativeSuspensionTestDiluents:‐Tryptone1.0g‐Sodiumcloride8.5g‐Distilledwater1000mlNeutralizer:‐Tween8030g‐Saponin30g‐L‐histidine1g‐Lecithin3g‐Nathiosulfate5g‐Diluents1000mlHardWaterSolutionAMgCl219.84g

CaCl246.24g.

Distilledwater1000mlSterilizedinautoclave,andcanbestoredinrefrigeratorforamonth.SolutionBNaHCO335.02gDistilledwater1000mlSterilizedwithfiltrationandcanbestoredinrefrigeratorforaweek.HardWaterWorkingSolutionSolutionA6mlSolutionB8mlDistilledwater1000mlpHshouldbe7.Shouldbefreshlypreparedforeverytestandconsumedwithin1‐2hourofpreparation.

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C)AppendixIIIPreparationofTrypticsoyagarContentsofthemedium:Caseinpeptone15g/LSoybeanpeptone5g/LSodiumchloride5g/LAgar15g/LpH7.3±0.240grofpowderedmediumand960mlofdistilledwateraresterilized inautoclaveat1atmosphericpressureon121°Cfor15minutes..Sampleseachcontaining25mlofmixture are pouredtoasterilePetridish so that thethicknessis4mmwhenbecamehard,andstoredat4ºConrefrigeratoruntilconsumed.PreparationofMuellerHintonAgarContentsofthemediumCattleinfusion300g/LCaseinacidhydrolysate17.5g/LStarch1.50g/LAgar17.00g/LpH7.3±0.238grofpowderedmediumand 960mlofdistilledwateraresterilized inautoclaveat1atmosphericpressureon121°Cfor15minutes..Samples each containing 25 ml of mixture , poured to a sterile Petri dish so that thethicknessis4mmwhenbecamehard,andstoredat4°Conrefrigeratoruntilconsumed.MandatoryContactIntervalMandatorycontactintervalforsurfacedisinfectionis5or60minutes,and60secondsforhygienicand5minutesforsurgicalhandwashing.Ifcontaminatedmaterialofthepatientis presenton the surface ; proposed contact interval for disinfection is5minutes. If the

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productwouldbeusedincleanconditions; inthosecircumstances60minuteswouldbeusedasamandatorycontactintervalWaterUsedInExperimentsWatershouldbecleandistilledwater,notdemineralizedwater.ProductTestSolutionsThesedisinfectantsareusedaccordingtotheconcentrationproposedbythemanufacturer.BacterialSuspensionsTestsuspension;bacterialsuspensionisthesuspensionusedtodeterminethebactericidalactivityofdisinfectants.StockcultureswereobtainedbythemicroorganismspassagedtoMuellerHintonplaqueswhichwerestoredat‐20°C.Secondsubcultureswereobtainedfromthisstockculturesfollowingthere‐passageofculturestoMuellerHintonplaques.2ndsubculturesofthebacteriawereusedinourexperiment..

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D)Appendix4.ColonyFormingUnit(FCU)EvaluationInthetestmethodbacterialsuspension(N)iscalculatedas1,5x108‐5x108CFU/mlandthissuspensionisusedintheexperiment.Accordingtothetestprocedure100µlofliquidfromthesuspensionisprocessedwith900µlofdisinfectant.Duringthisstagenumberofbacteriainthesuspensionisdecreasedbyaratiooftenbecauseofdilution;whiletheColonyFormingUnit(CFU)inthesuspensionisbetween1,5x107‐5x107.When100µlofthismixtureisaddedto800µlneutralizerand100µlhardwater,itwouldbediluted10timesmore.100µlfromthisfinalsolutionwheninoculatedtotheplaquesitisre‐dilutedas10.Thenumbersofcoloniesthataregrownintheplaquesaremultipliedbythedilutionratioof100.Thebactericidalactivityofadisinfectantisconsideredaseffective;ifthereisadecreaseof10‐5ormoreonthenumberofcolonyformingunit(CFU)