Comparison of methods for determining the effectiveness of antibacterial functionalized textiles

16 Pages·2017·4.24 MB·English

RESEARCHARTICLE
Comparison of methods for determining the
effectiveness of antibacterial functionalized
textiles
HajoHaase1*,LisaJordan1,LauraKeitel1,ClaudiaKeil1,BorisMahltig2
1 DepartmentofFoodChemistryandToxicology,InstituteforFoodTechnologyandFoodChemistry,
TechnischeUniversita¨tBerlin,Berlin,Germany,2 FacultyofTextileandClothingTechnology,Hochschule
Niederrhein,UniversityofAppliedScience,Mo¨nchengladbach,Germany
*[email protected]
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a1111111111 Abstract
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Antimicrobialfunctionalizationoftextilesisimportantforvariousapplications,suchaspro-
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tectionoftextilematerialsfromdecomposition,generationofmoreeffectivewounddress-
ings,andthepreventionofinfectionsormalodorsresultingfrombacterialgrowth.Inorderto
testtheefficacyofnewproducts,theirantibacterialactivityneedstobeevaluated.Atpres-
ent,severaldifferentproceduresarebeingusedforthispurpose,hinderingcomparisons
OPENACCESS amongdifferentstudies.Thepresentpapercomparesfiveoftheseassaysusingasample
Citation:HaaseH,JordanL,KeitelL,KeilC, panelofdifferenttextilesfunctionalizedwithcopper(Cu)andsilver(Ag)asantibacterial
MahltigB(2017)Comparisonofmethodsfor
agents,anddiscussesthesuitabilityofthesemethodsfordifferentanalyticalrequirements.
determiningtheeffectivenessofantibacterial
Bacterialviabilitywasdeterminedbymeasuringtheopticaldensityat600nm,acolorimetric
functionalizedtextiles.PLoSONE12(11):
e0188304.https://doi.org/10.1371/journal. assaybasedonMTT(3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazoliumbromide)con-
pone.0188304 version,anagardiffusionassay,andcolonyformation,eitherafterculturinginmediacon-
Editor:YogendraKumarMishra,Instituteof tainingtextilesamples,orafterrecoveryfromtextilessoakedwithbacterialsuspension.All
MaterialsScience,GERMANY experimentswereperformedwithaGram-negative(Escherichiacoli)andaGram-positive
Received:July21,2017 (Staphylococcuswarneri)modelorganism.Ingeneral,theresultsyieldedbythedifferent
methodswereofgoodcomparability.Toidentifythemostsuitabletestsystemforthepartic-
Accepted:November4,2017
ulartypeofantibacterialcoating,severalfactorsneedtobetakenintoaccount,suchas
Published:November21,2017
choosingappropriateendpointsforanalyzingpassiveoractiveantibacterialeffects,selec-
Copyright:©2017Haaseetal.Thisisanopen tionofrelevantmicroorganisms,correctingforpotentialinterferencebyleachingofcolored
accessarticledistributedunderthetermsofthe
textilecoatings,requiredhandsontime,andthenecessarysensitivity.
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation
Introduction
files.
Antimicrobialfunctionalizationoftextileshasmultipleapplications.Itcanbeusedtoprotect
Funding:WeacknowledgesupportbytheGerman
ResearchFoundationandtheOpenAccess textilesfrommicrobialgrowthanddegradation,isusedinmedicalapplicationssuchaswound
PublicationFundsofTechnischeUniversita¨tBerlin. dressingsandtopreventsecondaryinfectionsoftheskinunderconditionssuchasatopicder-
matitis,andmaybeappliedtopreventodorformationresultingfrombacterialmetaboliteson
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. clothesortextilesusedforcleaningpurposes[1–3].Tothisend,textilesaretreatedwithagents
PLOSONE|https://doi.org/10.1371/journal.pone.0188304 November21,2017 1/16
Determiningtheeffectivenessofantibacterialfunctionalizedtextiles
containingvarioussubstanceswithantibacterialproperties.Theseinclude,amongstmanyoth-
ers,antibioticssuchasCiprofloxacin,disinfectantssuchasTriclosan,polysaccharidessuchas
chitosan,andmetal-basedantibacterialcoatings,inparticularonescontainingsilver,zincor
copper[1,4,5].
Antibacterialsubstancescaneitheractactively,havingadirectbactericidaleffectorhinder-
inggrowthwithoutkillingthebacteria,ortheyactpassivelybyimpedingbacteriatocolonize
thetextilefibers,e.g.,byobstructingbacterialadhesion[2].Toevaluateandcomparetheeffec-
tivenessofdifferenttextiletreatments,theirantibacterialactivityneedstobeexperimentally
confirmed,asthereisconsiderablevariationintheactivityofdifferentproducts[6].Atpres-
ent,variousdifferentmethodsarebeingusedfortestingtheeffectivenessofthedifferent
coatings:
ThecolorimetricMTTassayisbasedonthereductionofthetetrazole3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)toitsrespectiveformazan.Theresulting
purpledyeisthenquantifiedat570nminaphotometerormultiplatereader.Thistestiscom-
monlyusedformeasuringproliferationandviabilityofmammaliancells,butMTTandother
relatedsubstances,suchas2,3,5-triphenyl-tetrazoliumchloride,canalsobeutilizedformea-
suringbacterialviability[7].Infact,theuseofMTTfortestingbactericidalactivityoffunctio-
nalizedtextileshasalreadybeenreportedseveraltimes[8–10].
Thediskdiffusionmethodhadinitiallybeenappliedfortestingtheefficacyofantibiotics
[11].Bacteriaareincubatedonanagarplateonwhichafilterisplaced,whichhasbeenloaded
withthetestcompound(s).Antibacterialactivityisthendeterminedbytheabsenceofbacterial
growthinazonearoundthefilter.Modifiedversionsofthismethodhavebeenusedfortesting
thetoxicityofnanoparticlesolutionsinawellintheagar[12],orbysubstitutingthefilterfora
textilesample,sothatitsantimicrobialeffectivenesscanbeevaluatedinacomparablemanner
[8,13,14].
Anothermethodtoquantifybacterialgrowthisbymeasuringtheopticaldensityat600
nm(OD ),whichisbasedonturbidityresultingfromlightscatteringbybacteria[15].
600
Thisismainlyusedasaquickandaffordablemethodtomonitorgrowthofbacteriaduring
theircultureinliquidmedia,buthasalsobeenappliedfortestingantibacterialpropertiesof
nanostructures[7,16],andmightbeasuitablemethodforevaluatingantibacterialeffectsof
textilesamples,aswell.
Countingthenumberofviablebacteriabyformationofvisiblecoloniesonagarplates,
knownascolonyformation,isanothermethodfordeterminingbacterialviability.Itcaneither
beperformedusingbacterialsolutionsinwhichtextilesampleshadbeenincubated,oronbac-
teriathatwereelutedfromabacteria-soakedtextilesample.Thelatteristhebasicprinciple
underlyingtheprotocol100–2004recommendedbytheAmericanAssociationofTextile
ChemistsandColorists(AATCC)toassesstheefficacyofantibacterialfinishes,andofthecor-
respondingnormENISO20743bytheInternationalOrganizationforStandardization.Both
protocols,orvariousmodificationsthereof,arefrequentlyusedforinvestigatingantibacterial
propertiesoffabrics[17–21].
Oneimportantobstaclethatiscurrentlyhinderingthedevelopmentofeffectiveantibacte-
rialtextilesisthelackofcomparabilitybetweendifferentstudies[22].Sofar,onlyfewpublica-
tionsusemorethanonemethodtoassessantibacterialactivity,leadingtoextremelylimited
informationonpotentialdifferencesbetweentheresultsofcommonlyusedtestmethods.
Therefore,thepresentstudyusesasamplesetofantibacterialfurnishedtextiles,basedoncop-
perorsilverasactiveingredients,aimingtoevaluatethecomparabilityoftheresultsobtained
withfivedifferenttestmethodsandtodiscussthemethods’suitabilityforevaluatingtheanti-
bacterialpropertiesoftextiles.
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Determiningtheeffectivenessofantibacterialfunctionalizedtextiles
Materialsandmethods
Textilesamples
Apanelof28differenttextilesampleswaspreparedfollowingacoatingprocedurealready
describedindetail[23].Briefly,apolyesterfilamentfabricwithaspecificweightof180g/cm2
wasusedasasubstrate.Itwascoatedwithlayersof50μm,100μmor200μmthickness,con-
sistingofacrylateoracrylate/polyanilinecoatings,whichweresupplementedwithupto30%
copperorsilverpigments.Forthepresentexperiments,circleswith5mmdiameterwerepre-
paredusingaconventionalholepuncher.
Bacterialculture
E.coli(strainBL21(D3))wereculturedinLuria–Bertani(LB)medium.S.warneri(straindsm-
20316)weregrowninTrypticaseSoyYeastExtract(TYSE)medium.Glycerolstockswere
expandedinovernightculturesat37˚Cand150rpm.Fortheexperiments,cellswerediluted
1:250intheirrespectiveculturemediaandtreatedasdescribedbelow.Agrowthcurveindi-
catesthatbothbacterialstrainsareinaphaseoflogarithmicgrowthafter3h(S1Fig).Conse-
quently,thishasbeenusedastheincubationtimeforsubsequentexperiments.
MTTassay
AntimicrobialtestingbasedonthereductionofMTTwasdoneasdescribedelsewhere[8].
Briefly,200μlbacterialsuspensionpercavityweregrowninsterile96-multiwellcellculture
platesfromTPP(TechnoPlasticProductsAG,Trasadingen,Switzerland)intheirrespective
culturemediaandinthepresenceoftextilesamplesfor3hat37˚C,rotatingat250rpmina
PST-60-HL-4orbitalshaker(BioSan,Riga,Latvia).Subsequently,cellswereincubatedinthe
presenceof0.1mg/lMTTinculturemediumfor5min,followedbylysisinisopropanolfor
further30minanddeterminationoftheabsorptionat570nmwithareferencewavelengthof
630nmonanELx800absorbancemicroplatereader(BioTekInstruments,Winooski,USA).
Thecontrolviabilityof100%wasmeasuredinatestarrangementintheabsenceofanytextile
fabric,butotherwiseidenticaltotheothersamples;0%wasdeterminedinthesamesetupin
theabsenceofbacteria.
OD assay
600
Cellsweregrownin96microwellplatesinthepresenceoftextiledisksasdescribedforthe
MTTassay.Aftertheincubations,100μlofthebacterialsuspensionsweretransferredtoanew
plateandabsorptionwasmeasuredat600nmonanInfiniteM200microplatereader(Tecan,
Crailsheim,Germany).100%viabilitywasdefinedasbacterialgrownintheabsenceoftextile
samples,0%viabilityasmediumblanks.
Diskdiffusionassay
Thediskdiffusionassaywasperformedaspreviouslydescribed[8].Inbrief,textilediskswere
placedonLBorTSYEagarplatesonwhichbacterialsuspensionshadbeenseededwithaDri-
galskispatula.Afterincubationfor24hat37˚C,acalipergaugewasusedtodeterminethe
widthofthebacteria-freeareola.Resultsaregivenastheareaofthezoneofgrowthinhibition
inmm2.
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Determiningtheeffectivenessofantibacterialfunctionalizedtextiles
Table1. ConditionsforAAS.
Element Wavelength SlitWidth Dilution(v/v) Standardcurve
[nm] [nm] [mg/l]
Ag 328.1 0.7 1:5 0.1–2.5
Cu 324.8 0.7 1:50 0.5–5
https://doi.org/10.1371/journal.pone.0188304.t001
Colonyformationwithincubationinsolution(Colony(Sol))
Cellsweregrownon96microwellplatesinthepresenceoftextiledisksasdescribedforthe
MTTassay.Afterwardsaseriesof10-folddilutionswasprepared,ofwhich10μlaliquotswere
placedonLBorTSYEagarplatesintriplicatesandincubatedfor24hat37˚C.Colonieswere
countedforthefirstdilutionatwhichdistinguishablecolonieswereobservableandusedto
calculatethenumberofviablebacteriaintheoriginalsolution.
Colonyformationonsoakedtextiledisks(Colony(Soak))
Textiledisksweresoakedwith20μlbacterialsuspensionandincubatedfor3hat37˚Cina
humidifiedatmosphere.Afterwards,bacteriawereelutedin100μl0.85%NaClsolutionfor1
min.Serialdilutionandcolonyformationwereperformedasdescribedabove.Dataareshown
asthepercentageofviablecellsrecoveredfromthetextilesampleat3h,relativetorecovery
fromtherespectivesampleat0h.Thequantitativeretrievalofbacteriafromthetextileswas
efficient;theaveragerecoveryfromalltextilesamplesimmediatelyaftersoakingcomparedto
theamountofbacteriaoriginallyplacedontothetextileswas86.4%forE.coliand114.5%for
S.warneri,respectively.
Measuringmetalionreleasebyatomicabsorptionspectrometry(AAS)
Toanalyzemetalrelease,textilediskswereplacedinto200μlculturemediain96wellplates
andincubatedfor3hat37˚C,rotatingat250rpm.Afterwards,mediaweredilutedin0.65%
HNO andanalyzedbyflameAASonanAAnalyst800instrument(PerkinElmer,Rodgau,
3
Germany),usingtheconditionssummarizedinTable1.Acetyleneflowwassetto2.0l/min
andoxidant(air)to17.0l/min;allsampleswereanalyzedintriplicates.Reagentsforatomic
absorptionmeasurementswereofappropriatequalityfortraceelementanalysis(TraceSelect,
Fluka,Germany).
Statisticalanalyses
Allexperimentswereperformedindependentlyatleastthreetimesandresultsareshownas
meanswiththestandarderrorofmeans(S.E.M.)aserrorbars.Statisticalsignificanceofthe
experimentalresults(significancelevelofp<0.05)wascalculatedbyGraphPadprismsoftware
(version5.0,GraphPadSoftware,SanDiegoUSA)usingone-wayANOVAandTukey´spost-
hoctest.Tocomparetheresultsobtainedwiththedifferenttestmethods,datawereanalyzed
bylinearcorrelationanalysiscalculatingthePearsonproduct-momentcorrelationcoefficient.
Results
AnMTTassayofbacteriaculturedinthepresenceoftextilesamplesshowsthatadditionof
copperorsilverpigmentstotextilecoatingsappliedontoapolyesterfabricreducestheviability
ofE.coli(Fig1A)andS.warneri(Fig1B).Forsilver,whichhasbeenappliedinfourdifferent
concentrations(rangingfrom5to30%)inapolyacrylatecoating,aconcentration-dependent
effectisobserved.Thiscorrelateswithaconcentration-dependentsilverreleaseintothe
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Determiningtheeffectivenessofantibacterialfunctionalizedtextiles
Fig1.MTTassay.ViabilityofE.coli(A)orS.warneri(B)after3hcultureinthepresenceoftextilesampleswasanalyzedbytheMTTassay.A
viabilityof100%correspondstothesignalobtainedwithbacteriaintheabsenceofanytextilesamples.Dataareshownasmeans+S.E.M.of
n=6independentexperiments.*Meansignificantlydifferentfromuntreatedtextile;One-wayANOVAwithTukey´spost-hoctest(p<0.05).n.d.:
notdetected.
https://doi.org/10.1371/journal.pone.0188304.g001
culturemedia,asshowninFig2Aforthecoatingof200μmthickness.Analogousresultsare
alsoobservedforallothersilver-containingcoatings(datanotshown).Remarkably,inthe
MTTassaytextilesfurnishedwithcoppershowhighertoxicitythanthecorrespondingsilver-
treatedsamples.Incontrast,silverions(Ag+)insolutionaremoretoxicthancopperions(Cu2
+),showinglowerEC valuesforbothtypesofbacteria(S2Fig).Theseobservationsarerecon-
50
ciledbymeasurementsofmetalreleaseintotheculturemediabyAAS.Theresultingconcen-
trationsofcopperaremillimolar,andherebymorethanoneorderofmagnitudehigherthan
thoseofsilver(Fig2B).
DeterminationoftheOD confirms,inprinciple,theresultsoftheMTTassayforsilver-
600
furnishedtextiles(Fig3).Incontrast,nolossofviabilityisobservedforcopper-containing
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Determiningtheeffectivenessofantibacterialfunctionalizedtextiles
Fig2.Metalrelease.Tomonitormetalreleaseduringincubationofthebacteria,textilesampleswerekeptfor3hat37˚CinLBorTYSEmediaona
rotatingincubator.Subsequently,thetotalamountsofAgreleasedfrompolyacrylatecoating,thickness200μm(A)orCureleasedfrompolyaniline
coatingcontaining5%Cu(B)inthemediawerequantifiedbyAAS.Dataareshownasmeans+S.E.M.ofn=3independentexperiments.
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coatings.Notably,copperreleaseleadstodiscolorationoftheculturemedia.Measuringthe
absorptionofmediaincubatedwithcopper-treatedtextileintheabsenceofbacteriaindi-
catesasignificantabsorptionat600nm.Ifthesevaluesaresubtractedasbackground,a
reductionofviabilityisobservedforcopperaswell(Fig4).AstheOD isanon-destruc-
600
tiveassay,itallowsfortime-resolvedmeasurementsinsteadofasingleendpointmeasure-
ment.AsshowninFig5,cultureinthepresenceof10and25μMAg+bothinhibitthe
growthofE.coliduringthestandardassaydurationof3h.However,at10μMAg+bacterial
growthisonlydelayedby3hours,asshownbytheirentryintothephaseofexponential
growth4hafterthestartoftheexperiment.
Inthediskdiffusionassay(Fig6),highervaluesindicateantibacterialefficiency,astheyrep-
resenttheareaofgrowthinhibitionaroundthetextilesampleonanagarplate.Uncoatedpoly-
esterfabricandsampleswithoutadditionofsilverorcopperpigmentstothecoatingare
ineffective.Notably,allcopper-containingcoatingsdonotshowanybactericidalactivity,as
well.Incontrast,silver-containingcoatingsabrogatebacterialgrowthinthevicinityofthetex-
tilesamples.Ingeneral,theseeffectscorrelatewiththeamountofsilverpigmentaddedtothe
coating,exceptforthecaseofpolyaniline-coatedsamplesappliedtoE.coli.
Inthenextexperiment,bacteriaareculturedinthepresenceoftextilesamplesasforMTT
andOD assays,followedbycountingthenumbersofcoloniesafterplatingdilutedaliquots
600
ontoagarplates(Fig7).Theeffectsonbacterialviabilitycorrespondwellwiththeresultsfrom
bothothermethods,exceptforasomewhathighertoxicityofsilver-coatedtextilesonE.coli.
Notably,thismethodinvolvingserialdilutionallowsforamoreprecisedeterminationoflow
numbersofviablebacteria.WhentheE.colidatafromFig7,whichshownoapparentremain-
ingviabilityformanysilver-containingsamples,aredepictedonalogarithmicscale,this
allowsthesensitivequantificationoflowbacterialnumbersoverseverallogsteps(S3Fig).
Inthefinalsetofexperiments,textilesamplesaresoakedinbacterialsolution,bacteria
recoveredbyelutionwithsodiumchloridesolutionanddilutedintheirrespectiveculture
media,followedbydeterminingtheremainingbacterialviabilitybycolonyformationasinthe
previousexperiment.Theresultingviabilityisshownasthepercentageofcoloniesrecovered
after3hincubation,comparedtotheimmediaterecoverydirectlyaftersoaking.WhileE.coli
givesresultsthatareingoodagreementwiththeothermethods,nonotabledifferences
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Determiningtheeffectivenessofantibacterialfunctionalizedtextiles
Fig3.OD assay.ViabilityofE.coli(A)orS.warneri(B)after3hcultureinthepresenceoftextilesampleswasanalyzedbymeasuringthe
600
opticaldensityat600nm.Aviabilityof100%correspondstothesignalobtainedwithbacteriaintheabsenceofanytextilesamples.Dataare
shownasmeans+S.E.M.ofn=3independentexperiments.*Meansignificantlydifferentfromuntreatedtextile;One-wayANOVAwithTukey´s
post-hoctest(p<0.05).
https://doi.org/10.1371/journal.pone.0188304.g003
betweenmetal-freecontrolsandsilver-containingpolyacrylatecoatingsareobservedforS.
warneri.
Tocomparetheresultsofthedifferentmethods,thePearsonproduct-momentcorrelation
coefficientwasdetermined(Table2).ForE.coli,statisticallysignificantlinearcorrelations
(p<0.05)wereobservedbetweenallmethods.However,thecorrelationcoefficientsindicate
thatnotallcorrelationscanbeconsideredtobestrong,indicatingacertaindegreeofvariation
betweenthedifferenttests.Thenegativecorrelationcoefficientsfordiskdiffusionarenotan
indicationofpoorcorrelation,butresultfromthefactthatinthiscasedatacannotbe
expressedaspercentviability,butareindicatedasareawithoutbacterialgrowthinstead,which
isinverselyproportionaltoviability.ForS.warneri,theColony(Soak)assayshowedno
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Determiningtheeffectivenessofantibacterialfunctionalizedtextiles
Fig4.OD assaywithbackgroundcorrection.InacomplementaryexperimenttoFig2,theeffectofleachingofcoatingfromthetextile
600
samplesafterincubationinbacteria-freemediaforE.coli(LB-medium,A)orS.warneri(TYSE-medium,B)wasmeasuredat600nm.Originaldata
(monochromaticbars)anddataaftercorrectionbysubtractingtheabsorptionat600nmcausedbyleaching(checkeredbars)areshownasmeans
+S.E.M.ofn=3independentexperiments.
https://doi.org/10.1371/journal.pone.0188304.g004
statisticallysignificantcorrelationwiththediskdiffusionassay,whichwastheonlycorrelation
withapvaluegreater0.05.
Discussion
Variousprotocolsareroutinelybeingemployedtodeterminetheantibacterialeffectivenessof
textiletreatments[2].Comparisonoftheresultsishinderedbythelackofinformationregard-
ingthereproducibilityandcomparabilitybetweenthedifferentassays.Thepresentstudywas
initiatedtoprovideafirstoverviewonthecomparabilityofsomecommonlyusedmethodsby
usingonesetofsamplesbasedoncoatingscontainingcopperorsilverastheanti-bacterial
agent,whicharetestedinparallelwithfivedifferentassays.
Fig5.OD assayatdifferenttimepoints.ViabilityofE.coliculturedinthepresenceoftheindicated
600
concentrationsofAgNO .Dataareshownasmeans+S.E.M.ofn=3independentexperiments.
3
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Determiningtheeffectivenessofantibacterialfunctionalizedtextiles
Fig6.Diskdiffusionassay.Theareaofthebacteria-freezonearoundtextiledisksplacedonagarplatescoatedwithE.coli(A)orS.warneri
(B)wasmeasured.Dataareshownasmeans+S.E.M.ofn=4independentexperiments.*Meansignificantlydifferentfromuntreatedtextile;
One-wayANOVAwithTukey´spost-hoctest(p<0.05).
https://doi.org/10.1371/journal.pone.0188304.g006
Ingeneral,theresultsfromallfivemethodsareingoodagreement,althoughdifferentend-
pointsareinvestigated.MetabolicactivityisusedfortheMTTassay[24].Itwillnotprovide
informationonthenumberofviablebacteria,asitcannotdistinguishbetweenareduction
resultingfromlowernumbersofbacteriawithunalteredmetabolicactivity,andequaltotal
numbersofbacteriathathavedifferentialmetabolicactivity.Incontrast,theOD ispropor-
600
tionaltothetotalnumberofbacteria,butdoesnotprovideanyinformationregardingmeta-
bolicactivityortheirviability.Fordiskdiffusionandthemethodsdependingoncolony
formation,onlyviable,dividingbacteriaarerelevant,whereasdeadbacteriaorevenbacteriain
atemporarygrowtharrestwillremainundetected.
Severallimitationsexistforthedifferentmethods.Diskdiffusionstrictlydependsonthe
diffusionandsolubilityoftheantibacterialsubstancefromthetextileintothesurrounding
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Fig7.Colony(Sol)assay.ViabilityofE.coli(A)orS.warneri(B)after3hcultureinthepresenceoftextilesampleswasanalyzedbycolony
formation.Aviabilityof100%correspondstothesignalobtainedwithbacteriaintheabsenceofanytextilesamples.Dataareshownasmeans
+S.E.M.ofn=3independentexperiments.*Meansignificantlydifferentfromuntreatedtextile;One-wayANOVAwithTukey´spost-hoctest
(p<0.05).
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agar[11].Evenextraordinarilyefficientantibacterialagentswillshownoeffect,iftheystay
boundtothetextileorareunabletodiffuseintheagar,e.g.,duetoprecipitation.Thelatter
maybethereasonwhythecoppercoatedtextiles,whichshowconsiderabletoxicityinother
assays,havenoeffectinthediskdiffusionassay.
TheOD methodallowsforasimpletime-resolvedmonitoringofbacterialgrowth,
600
becausebacteriaarenotlysedandcanbemeasuredrepeatedly.However,theapplicationfor
antimicrobialtestingislimitedtotextileswithoutleachingfromcoatingswithabsorptionat
thiswavelength,ornecessitatesrunningparallelexperimentsforbackgroundcorrection,asin
Fig4.Alternatively,lightscatteringbybacteriaisnotlimitedtoaparticularwavelength.This
opensthepossibilitytoperformtheassayatanydifferentwavelength,ifitisundisturbedby
thespecificsamples.LeachingmightalsoaffecttheabsorptionintheMTTassaymeasuredat
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