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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)