Research ArticleBiofilm Localization in the Vertical Wall of Shaking96-Well Plates

Luciana C Gomes Joana M R Moreira Manuel SimotildeesLuiacutes F Melo and Filipe J Mergulhatildeo

LEPABE Department of Chemical Engineering Faculty of Engineering University of Porto Rua Dr Roberto Frias4200-465 Porto Portugal

Correspondence should be addressed to Filipe J Mergulhao filipemfeuppt

Received 20 November 2013 Accepted 19 December 2013 Published 13 April 2014

Academic Editors M Rodrigues and A Sellam

Copyright copy 2014 Luciana C Gomes et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Microtiter plates with 96 wells are being increasingly used for biofilm studies due to their high throughput low cost easy handlingand easy application of several analyticalmethods to evaluate different biofilmparametersThesemethods provide bulk informationabout the biofilm formed in each well but lack in detail namely regarding the spatial location of the biofilms This location canbe obtained by microscopy observation using optical and electron microscopes but these techniques have lower throughput andhigher cost and are subjected to equipment availability This work describes a differential crystal violet (CV) staining method thatenabled the determination of the spatial location of Escherichia coli biofilms formed in the vertical wall of shaking 96-well platesIt was shown that the biofilms were unevenly distributed on the wall with denser cell accumulation near the air-liquid interfaceThe results were corroborated by scanning electronmicroscopy and a correlation was found between biofilm accumulation and thewall shear strain rates determined by computational fluid dynamics The developed method is quicker and less expensive and hasa higher throughput than the existing methods available for spatial location of biofilms in microtiter plates

1 Introduction

Biofilms are defined as structured microbial communitiesthat are attached to a surface and encapsulated within a self-produced matrix [1 2] They constitute a serious problem forpublic health because of the increased resistance of biofilm-associated microorganisms to antimicrobial agents and theirpotential to cause infections in patients with indwellingmedical devices [1 3]

Intensive studies on themechanisms of biofilm formationand resistance have encouraged the development of differentin vitro platforms such as microtiter plates (MTPs) whichare one of the most widely used biofilmmodel systems [4 5]In these systems biofilms are formed on the bottom and onthe wall [6] of the microtiter plate wells (most commonly a96-well plate) or they are grown on the surface of a couponplaced in the wells of the MTP (most commonly a 6- 12-or 24-well plate) The large number of advantages offered bythese straightforward and user-friendly systems explain their

widespread use (Table 1) In addition several standard assaysare available for the determination of different parametersrelated to the biofilm in MTPs [7] They can be categorizedinto biofilm biomass assays (quantitation of matrix and bothliving and dead cells) viability assays (determination ofviable cells) and matrix quantitation assays (through specificstaining of matrix components) (Table 1) Microtiter plateshave been intensively used in clinical research for screeningof antimicrobial compounds [8 9] and for studying biofilmformation [10 11] and inhibition [12 13] The most widelyused method for following biofilm formation in MTPs isthe crystal violet (CV) staining derived from the originalChristensen et al [14] method which only measured biofilmbiomass at the bottom of the well CV is a basic dye that stainsboth living and dead cells by binding to negatively chargedsurface molecules and polysaccharides in the extracellularmatrix of biofilms [15] Later the CV assay was modifiedto increase its accuracy and to allow for biofilm biomassquantitation in the entire well by the solubilization of the

Hindawi Publishing CorporationScientificaVolume 2014 Article ID 231083 6 pageshttpdxdoiorg1011552014231083

2 Scientifica

Table 1 Advantages of MTPs as biofilm reactors and standardmethods for quantitation of biofilm parameters in MTPs

Advantages References Quantitation assays ReferencesHigh throughput [4 40] Biofilm biomass [1 16]Small volumes ofreagents [4 40] Microbial

physiological activity [7 41]

Automation [42 43] Microbial cells in thebiofilm [7 41]

Multiplexing [4] Biofilm matrix [44]

dye [16 17] This method can therefore be considered a bulkmethod which provides information about the total amountof biofilms produced without revealing any informationabout biofilm localization It has been shown that the biofilmdistribution in the wall of a 96-well MTPmay not be uniformwhen dynamic conditions are used (when the MTP is shakenwith an orbital motion) [6]This biofilm heterogeneity can beanalyzed for instance by microscopy either using standardoptical microscopy [18] confocal laser scanning microscopy(CLSM) [19] or scanning electron microscopy (SEM) [20]The relative advantages and limitations of these two latertechniques are presented in Table 2 It is interesting to pointout that for the most part these microscopy analyses aremade at the bottom of the well disregarding the biofilm thatis formed on the vertical wall Both techniques enable spatiallocalization of the biofilms but are inherently low throughputtechniques with a high cost and are subjected to equipmentavailabilityThe aimof this workwas to develop a low cost andhigh throughput method that would enable the quantitationof the total amount of biofilm produced inside a well butproviding the same information about the spatial location ofthe formed biofilm A differential CV staining method washere developed by combining the high throughput featuresof the usual CV staining but enabling spatial localization ofthe biofilm without the use of expensive equipment

2 Materials and Methods

21 Computational Fluid Dynamic (CFD) SimulationsNumerical simulations were made in Ansys Fluent CFDpackage (version 130) as previously described [6] Acylindrical well (diameter of 66mm and height of 117mm)was built in Design Modeller 130 and discretized into a gridof 18876 hexahedral cells by Meshing 130 Simulation wasmade for a shaking diameter of 50mm and frequency of150 rpm The location of the interface was determined byAnsys Fluent as well as the magnitude of the shear strainrate The time averaged shear strain rate was obtained byaveraging the steady state shear strain rate of the liquid sideduring a complete orbit

22 Biofilm Formation in 96-Well Microtiter PlatesEscherichia coli JM109(DE3) from Promega (USA) wasused to form biofilms in 96-well microtiter plates becausethis strain has shown a good biofilm forming ability inboth turbulent [21] and laminar [6] flow conditions Its

Its genotype is endA1 recA1 gyrA96 thi hsdR17 (rkminus

mk+) relA1 supE44 120582minus Δ(lac-proAB) (F1015840traD36 proAB119897119886119888IqZΔM15) and 120582(DE3) Cells from an overnight culturewere prepared as previously described [6] and biofilms wereproduced by pipetting 20 120583L of these cells into six wellsof sterile 96-well flat-bottomed microtiter plates (OrangeScientific USA) filled with 180120583L of nutrient media Themedia used was identical to the one described in [22]except for glucose A glucose concentration of 1 g Lminus1 wasused in this work since it has been demonstrated that thisconcentration originated the maximum biofilm amount at24 h in different shaking conditions [6] Microtiter plateswere placed for 24 h at 30∘C in an orbital shaking incubatorwith 50mm of diameter at 150 rpm (CERTOMAT BS-1Sartorius AG Germany)

23 BiofilmQuantitation by Crystal Violet (CV) Staining Theamount of biofilm formed was measured using the CV dyein a differential form this means that the well has beendivided into four different sections (Figure 1(d)) and thecorresponding vertical wall section was stained sequentiallyup to a maximum volume of 200 120583L Biofilm quantitation onthe bottom of the well was performed by using 25 120583L of CVfor staining (as detailed below) Section 1 corresponded toa volume between 25 and 50 120583L (and a height of 077mm)Section 2 corresponded to a volume between 50 and 100120583Lsection 3 corresponded to a volume between 100 and 150 120583Land section 4 corresponded to a volume between 150 and200120583L Sections 2 3 and 4 had an equivalent height of154mm

Prior to CV staining the contents of the microtiter plateswere discarded and the wells were washed with 200120583L ofsterile water to remove non-adherent bacteria [16] Then thebiofilms were fixed with 250120583L of 96 ethanol [23] Thefirst section of the well to be quantified was the bottom (notrepresented in Figure 1(d)) which corresponds to a volumeof 25120583L and a height of 077mm The cells adhered to thisregion were stained for 5min with the correspondent volumeof 1 (vv) crystal violet (Merck Germany) and the dyebound to this specific region was solubilized with 200120583Lof 33 (vv) acetic acid (VWR Portugal) The absorbancewas measured at 570 nm using a microtiter plate reader(SpectraMax M2E Molecular Devices UK) The biofilmsformed in the successive sections of the well (sections 1 to 4 ofFigure 1(d)) were quantified using the described procedurespecifically using the CV volume equivalent to the maximumlevel of each section (section 1mdash50120583L section 2mdash100 120583Lsection 3mdash150120583L section 4mdash200120583L) The absorbance cor-responding to each of the defined sections and presentedon Figure 1(d) was calculated by subtracting the absorbancesfrom the previous sections and considering the dilutionfactor To quantify the biofilm formed in each of the wellsections six replica wells were used per experiment and threeindependent experiments were performed

24 Scanning Electron Microscopy (SEM) Microtiter platewells containing 24 h old biofilms were imaged by SEM aspreviously described [24] Briefly biofilms were fixed in 3

Scientifica 3

Table 2 Advantages and limitations of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) on biofilmanalysis

Technique Advantages Limitations References

Scanning electronmicroscopy

High resolutionWide range of magnificationsGood comparative informationAbility to image complex shapes

Not real timeRequires additional sample preparationLimited quantification

[45ndash49]

Confocal laser scanningmicroscopy

Living fully hydrated samplesNon-invasiveQuantitative evaluationReflection and fluorescence mode

Low resolutionNarrow range of magnificationsNot applicable to thick biofilms

[35 47 50ndash52]

1

2

3

4

Increased wetted areaStatic fill volume

0253

0028

0

0066

Absorbance (570 nm)

(a) (b) (d) (c) (e)

20 40 60 80 100 120 140 160 180

Strain rate (sminus1)

Figure 1 Biofilm localization in shaking 96-well microtiter plates placed in a 50mm incubator at 150 rpm (a) Photograph of a well stainedwith crystal violet (b) schematic representation of a well where the dark grey area corresponds to the wetted area without shaking the lightgrey area represents the area increase upon shaking and the dotted line depicts the inclination of the air-liquid interface (c) time averagedshear strain rates (values below 20 sminus1 are not represented) (d) illustration of the biofilm distribution on the vertical wall assayed by thedifferential CV staining (e) representative scanning electron micrographs of the wall sections defined in image (d) (5000x magnification bar= 10120583m)

(ww) glutaraldehyde in cacodylate buffer dehydrated withethanol and hexamethyldisilazane (HMDS) and sputter-coated with a palladium-gold thin filmThe different sectionsalong the vertical wall of three independent wells wereobserved with a SEMEDS system (FEI Quanta 400FEGESEMEDAX Genesis X4M FEI Company USA)

3 Results and Discussion

The effect of orbital shaking on biofilm formation in 96-well microtiter plates was firstly assessed by the conventionalprocedure of crystal violet staining [6 16] Through directobservation of the stained wells (prior to solubilization ofthe dye) it was possible to observe that biofilms weremainly formed on the wall and not on the bottom of thewell Moreover since the amount of produced biofilms isproportional to the amount of CV adsorbed it seemed thatthe biofilm was unevenly distributed in the cylindrical wall

and higher amounts were formed closer to the air-liquidinterface (Figure 1(a))

It is widely known that hydrodynamics affects biofilmformation [25ndash27] as a result of the shear forces that canmodulate cell adhesion to a given surface [28ndash31] Eventhough microtiter plates are broadly accepted as biofilmformation reactors by the scientific community there is stilla lack of information on the impact of hydrodynamics onbiofilm formation in this system In this work the flowinside the wells was simulated using computational fluiddynamics (Figures 1(b) and 1(c)) in order to find out ifthe hydrodynamic conditions were related to the unevenbiofilm distribution that was visible to the naked eye after CVstaining (Figure 1(a)) Figure 1(b) represents a scale modelof a microwell with the wetted area without orbital motionthe area increase upon shaking and the inclination of theair-liquid interface For the shaking conditions chosen forthis work (50mm of orbital diameter and 150 rpm) an areagain of 85 and a maximum angle of 78∘ were obtained

4 Scientifica

Similar to what was observed in the photograph of thestained well (Figure 1(a)) the shear strain rate distributionwas not uniform along the wall being much higher in theliquid side near the interface (Figure 1(c)) In this regionCFD simulations revealed some spots corresponding to amaximum shear strain rate of 180 sminus1 which are related toregions of unstable vortices [6]

Observing the results of the differential CV staining(Figure 1(d)) the amount of biofilm detected along the differ-ent sections of the wall was not constantThe highest value ofabsorbance (corresponding to the highest amount of biofilmbiomass) was measured in the region located immediatelybelow the liquid level (section 4) In the intermediate sectionsof the well the absorbance values were about 10 times lowerthan near the interface (section 3) or almost zero (section 2)increasing slightly in the section closer to the bottom of thewell (section 1)

In order to validate the results obtained by the differentialCV staining SEM analysis was performed on the vertical wallof the wells Figure 1(e) shows scanning electronmicrographsrepresentative of the four sections defined in the applicationof the differential CV staining (Figure 1(d)) SEM analysisshowed that E coli adhesion varied across the wall andthat higher attachment occurred closer to the air-liquidinterface (section 4) compared to the intermediate (sections2 and 3) and near bottom (section 1) regions of the wellBiofilms consisting of bacterial aggregates were observednear the interface while in the lower regions of the wellthere were only few cells randomly distributed on the surface(Figure 1(e))

Most likely the spatial location of the biofilms (assayed bydifferential staining and SEM analysis) is conditioned by thenon-homogeneous distribution of shear strain calculated byCFD during shaking The higher cell density of the biofilmsformed closer to the interface is probably due to the higheroxygen and substrate mass transfer from the bulk solution tothe biofilm [32] resulting from amore efficient liquid mixingin this region These results are in agreement with previousstudies [29 33 34] showing that higher shear forces promotethe formation of denser biofilms

Both methods used for biofilm analysis in this work(differential CV staining and SEM) enabled a higher detailin analysis of biofilms grown in 96-well microtiter plateswhich is not achieved by the spectrophotometric methodscommonly used in MTPs (Table 1) Indeed methods such asthe traditional CV assay provide bulk data from a biofilmand are classified as macroscale methods whereas SEM is amicroscale technique [24] It is interesting to notice that thedifferential CV staining presented in this work correspondsto an intermediate scale between the traditional CV assay andSEM analysis The traditional CV assay quantifies the biofilmformed on the wall and bottom of each well of a microtiterplate which corresponds to an area of about 146mm2 (fora 96-well plate) while the method proposed here evaluateswall sections of about 14 and 28mm2 In comparison thearea covered by a SEM image taken at 5000x magnificationwas of 2 times10minus3mm2 and this technique enables detailedanalysis of individual cells within the biofilm rather than

simply determining their localization SEM offers highermagnification (ranging from 20x to approximately 30000x)and resolution (from 50 to 100 nm) together with the abilityof imaging complex shapes (Table 2) It is also highly rec-ommended for the visualization of cellular morphology cell-to-cell interactions and matrix components within biofilms[35] However one must bear in mind that most laboratoriesare not equipped with an electron microscope and thistechnique has a considerably lower throughput than themethods described in Table 1

Observation of biofilms formed on the bottom of thewells of microtiter plates is very common using opticalmicroscopes at magnifications of 100 to 200x which typicallycover areas of 03mm2 [36ndash39] These observations haveprovided some information about the architecture of thebiofilms formed on the bottom of the wells (particularlywhen CLSM is used) but usually disregard the biofilm thatforms on the vertical wall It has been shown that in dynamicconditions the amount of biofilm formed on the vertical wallcan be higher than the one formed on the bottom of the well[6] and therefore a method was developed in this work todetermine the spatial localization of these biofilms in a highthroughput manner using common laboratory equipmentThe area under analysis in each of the four sections definedfor the differential staining is equivalent to the area analyzedby optical microscopy using a 15x magnification This areacould be further reduced (thus increasing the precision of themethod) by dividing the well in sections of smaller height

The novel approach presented in this work demonstratesthat the CV dye can be extremely useful in locating theadherent cells in microtiter plates when used in a differentialway The method is slightly more laborious and slowerthan the traditional staining procedure but it requires fewerresources and has higher throughput than other techniquesthat are used to determine the spatial location of biofilms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support providedby Operational Programme for Competitiveness Factors(COMPETE) European Fund for Regional Development-FEDER and the Portuguese Foundation for Scienceand Technology (FCT) through Projects PTDCEBB-BIO1028632008 and PTDCEBB-BIO1049402008Luciana Gomes acknowledges the receipt of a PhD Grantfrom FCT (SFRHBD804002011) Joao Miranda and JoseAraujo (Transport Phenomena Research Center FEUP) areacknowledged for the numerical simulations

References

[1] J W Costerton P S Stewart and E P Greenberg ldquoBacterialbiofilms a common cause of persistent infectionsrdquo Science vol284 no 5418 pp 1318ndash1322 1999

Scientifica 5

[2] P Stoodley K Sauer D G Davies and J W CostertonldquoBiofilms as complex differentiated communitiesrdquo AnnualReview of Microbiology vol 56 pp 187ndash209 2002

[3] V Hancock L Ferrieres and P Klemm ldquoBiofilm formation byasymptomatic and virulent urinary tract infectious Escherichiacoli strainsrdquo FEMSMicrobiology Letters vol 267 no 1 pp 30ndash372007

[4] T Coenye and H J Nelis ldquoIn vitro and in vivo model systemsto studymicrobial biofilm formationrdquo Journal ofMicrobiologicalMethods vol 83 no 2 pp 89ndash105 2010

[5] J S Teodosio M Simoes L F Melo and F J Mergulhao ldquoPlat-forms for in vitro biofilm studiesrdquo in Biofilms in BioengineeringM Simoes and F Mergulhao Eds pp 45ndash62 Nova ScienceNew York NY USA 2013

[6] J M R Moreira L C Gomes J D P Araujo et al ldquoThe effectof glucose concentration and shaking conditions on Escherichiacoli biofilm formation in microtiter platesrdquo Chemical Engineer-ing Science vol 94 pp 192ndash199 2013

[7] E Peeters H J Nelis and T Coenye ldquoComparison of multiplemethods for quantification of microbial biofilms grown inmicrotiter platesrdquo Journal of Microbiological Methods vol 72no 2 pp 157ndash165 2008

[8] M Erriu F M G Pili E Tuveri et al ldquoOil essentialmouthwashes antibacterial activity againstAggregatibacter acti-nomycetemcomitans a comparison between antibiofilm andantiplanktonic effectsrdquo International Journal of Dentistry vol2013 Article ID 164267 5 pages 2013

[9] D Minardi O Cirioni R Ghiselli et al ldquoEfficacy of tigecyclineand rifampin alone and in combination against Enterococcusfaecalis biofilm infection in a ratmodel of ureteral stentrdquo Journalof Surgical Research vol 176 no 1 pp 1ndash6 2012

[10] C Sanchez K Mende M Beckius et al ldquoBiofilm formationby clinical isolates and the implications in chronic infectionsrdquoBMC Infectious Diseases vol 13 article 47 2013

[11] E Hell C Giske K Hultenby et al ldquoAttachment and biofilmforming capabilities of Staphylococcus epidermidis strains iso-lated from preterm infantsrdquo Current Microbiology vol 67 no 6pp 712ndash717 2013

[12] M-H Lin F-R Chang M-Y Hua Y-C Wu and S-T Liu ldquoInhibitory effects of 12346-penta-O-galloyl-120573-D-glucopyranose on biofilm formation by Staphylococcus aureusrdquoAntimicrobial Agents and Chemotherapy vol 55 no 3 pp 1021ndash1027 2011

[13] C De La Fuente-Nunez V Korolik M Bains et al ldquoInhibitionof bacterial biofilm formation and swarming motility by asmall synthetic cationic peptiderdquo Antimicrobial Agents andChemotherapy vol 56 no 5 pp 2696ndash2704 2012

[14] G D ChristensenW A Simpson and J J Younger ldquoAdherenceof coagulase-negative staphylococci to plastic tissue cultureplates a quantitative model for the adherence of staphylococcito medical devicesrdquo Journal of Clinical Microbiology vol 22 no6 pp 996ndash1006 1985

[15] X Li Z Yan and J Xu ldquoQuantitative variation of biofilmsamong strains in natural populations of Candida albicansrdquoMicrobiology vol 149 no 2 pp 353ndash362 2003

[16] S Stepanovic D Vukovic I Dakic B Savic and M Svabic-Vlahovic ldquoA modified microtiter-plate test for quantificationof staphylococcal biofilm formationrdquo Journal of MicrobiologicalMethods vol 40 no 2 pp 175ndash179 2000

[17] L Cremet S Corvec E Batard et al ldquoComparison of threemethods to study biofilm formation by clinical strains of

Escherichia colirdquoDiagnosticMicrobiology and Infectious Diseasevol 75 no 3 pp 252ndash255 2013

[18] L R Martinez and A Casadevall ldquoSpecific antibody canprevent fungal biofilm formation and this effect correlates withprotective efficacyrdquo Infection and Immunity vol 73 no 10 pp6350ndash6362 2005

[19] A Bridier D Le Coq F Dubois-Brissonnet V Thomas SAymerich and R Briandet ldquoThe spatial architecture of Bacillussubtilis biofilms deciphered using a surface-associated modeland in situ imagingrdquo PLoS ONE vol 6 no 1 Article ID e161772011

[20] V Kostenko M M Salek P Sattari and R J MartinuzzildquoStaphylococcus aureus biofilm formation and tolerance toantibiotics in response to oscillatory shear stresses of physiolog-ical levelsrdquo FEMS Immunology and Medical Microbiology vol59 no 3 pp 421ndash431 2010

[21] J S Teodosio M Simoes and F J Mergulhao ldquoThe influence ofnonconjugative Escherichia coli plasmids on biofilm formationand resistancerdquo Journal of Applied Microbiology vol 113 no 2pp 373ndash382 2012

[22] J S Teodosio M Simoes L F Melo and F J MergulhaoldquoFlow cell hydrodynamics and their effects on E coli biofilmformation under different nutrient conditions and turbulentflowrdquo Biofouling vol 27 no 1 pp 1ndash11 2011

[23] S Shakeri R K Kermanshahi M M Moghaddam and GEmtiazi ldquoAssessment of biofilm cell removal and killing andbiocide efficacy using the microtiter plate testrdquo Biofouling vol23 no 2 pp 79ndash86 2007

[24] L Gomes J M R Moreira J M Miranda et al ldquoMacroscaleversusmicroscalemethods for physiological analysis of biofilmsformed in 96-well microtiter platesrdquo Journal of MicrobiologicalMethods vol 95 no 3 pp 342ndash349 2013

[25] Y Liu and J-H Tay ldquoThe essential role of hydrodynamic shearforce in the formation of biofilm and granular sludgerdquo WaterResearch vol 36 no 7 pp 1653ndash1665 2002

[26] P Stoodley S Wilson L Hall-Stoodley J D Boyle H MLappin-Scott and J W Costerton ldquoGrowth and detachment ofcell clusters from mature mixed-species biofilmsrdquo Applied andEnvironmentalMicrobiology vol 67 no 12 pp 5608ndash5613 2001

[27] S Wasche H Horn and D C Hempel ldquoInfluence of growthconditions on biofilm development and mass transfer at thebulkbiofilm interfacerdquoWater Research vol 36 no 19 pp 4775ndash4784 2002

[28] H J Busscher and H C van der Mei ldquoMicrobial adhesion inflow displacement systemsrdquo Clinical Microbiology Reviews vol19 no 1 pp 127ndash141 2006

[29] M Simoes M O Pereira S Sillankorva J Azeredo andM J Vieira ldquoThe effect of hydrodynamic conditions on thephenotype of Pseudomonas fluorescens biofilmsrdquoBiofouling vol23 no 4 pp 249ndash258 2007

[30] J S Teodosio F C Silva J M R Moreira et al ldquoFlow cellsas quasi ideal systems for biofouling simulation of industrialpiping systemsrdquo Biofouling vol 29 no 8 pp 953ndash966 2013

[31] M C M Van Loosdrecht D Eikelboom A Gjaltema AMulder L Tijhuis and J J Heijnen ldquoBiofilm structuresrdquoWaterScience and Technology vol 32 no 8 pp 35ndash43 1995

[32] J M R Moreira J S Teodosio F C Silva et al ldquoInfluenceof flow rate variation on the development of Escherichia colibiofilmsrdquo Bioprocess and Biosystems Engineering vol 36 no 11pp 1787ndash1796 2013

6 Scientifica

[33] WKKwokC Picioreanu S LOng et al ldquoInfluence of biomassproduction and detachment forces on biofilm structures in abiofilm airlift suspension reactorrdquo Biotechnology and Bioengi-neering vol 58 no 4 pp 400ndash407 1998

[34] A Rochex J-J Godon N Bernet and R Escudie ldquoRole ofshear stress on composition diversity and dynamics of biofilmbacterial communitiesrdquo Water Research vol 42 no 20 pp4915ndash4922 2008

[35] A Bridier T Meylheuc and R Briandet ldquoRealistic represen-tation of Bacillus subtilis biofilms architecture using combinedmicroscopy (CLSM ESEM and FESEM)rdquo Micron vol 48 pp65ndash69 2013

[36] M G White S Piccirillo V Dusevich D J Law T Kaprosand S M Honigberg ldquoFlo11p adhesin required for meioticdifferentiation in Saccharomyces cerevisiaeminicolonies grownon plastic surfacesrdquo FEMS Yeast Research vol 11 no 2 pp 223ndash232 2011

[37] C L Quave M Estevez-Carmona C M Compadre et alldquoEllagic acid derivatives from Rubus ulmifolius inhibit Staphy-lococcus aureus biofilm formation and improve response toantibioticsrdquo PLoS ONE vol 7 no 1 Article ID e28737 2012

[38] A Lizcano T Chin K Sauer E I Tuomanen and C JOrihuela ldquoEarly biofilm formation on microtiter plates is notcorrelated with the invasive disease potential of Streptococcuspneumoniaerdquo Microbial Pathogenesis vol 48 no 3-4 pp 124ndash130 2010

[39] A S Melo A L Colombo and B A Arthington-Skaggs ldquoPara-doxical growth effect of caspofungin observed on biofilms andplanktonic cells of five differentCandida speciesrdquoAntimicrobialAgents and Chemotherapy vol 51 no 9 pp 3081ndash3088 2007

[40] W A Duetz ldquoMicrotiter plates as mini-bioreactors miniatur-ization of fermentation methodsrdquo Trends in Microbiology vol15 no 10 pp 469ndash475 2007

[41] K Honraet E Goetghebeur and H J Nelis ldquoComparisonof three assays for the quantification of Candida biomassin suspension and CDC reactor grown biofilmsrdquo Journal ofMicrobiological Methods vol 63 no 3 pp 287ndash295 2005

[42] S Kumar C Wittmann and E Heinzle ldquoReview minibioreac-torsrdquo Biotechnology Letters vol 26 no 1 pp 1ndash10 2004

[43] N F Azevedo S P Lopes C W Keevil M O Pereira and MJ Vieira ldquoTime to ldquogo largerdquo on biofilm research advantages ofan omics approachrdquoBiotechnology Letters vol 31 no 4 pp 477ndash485 2009

[44] K Tote D V Berghe L Maes and P Cos ldquoA new colorimetricmicrotitre model for the detection of Staphylococcus aureusbiofilmsrdquo Letters in AppliedMicrobiology vol 46 no 2 pp 249ndash254 2008

[45] T A Norton R C Thompson J Pope et al ldquoUsing confocallaser scanning microscopy scanning electron microscopy andphase contrast light microscopy to examine marine biofilmsrdquoAquatic Microbial Ecology vol 16 no 2 pp 199ndash204 1998

[46] D O Serra A M Richter G Klauck et al ldquoMicroanatomy atcellular resolution and spatial order of physiological differen-tiation in a bacterial biofilmrdquo mBio vol 4 no 2 pp e100103ndashe100113 2013

[47] P S Stewart R Murga R Srinivasan and D De Beer ldquoBiofilmstructural heterogeneity visualized by three microscopic meth-odsrdquoWater Research vol 29 no 8 pp 2006ndash2009 1995

[48] S B Surman J T Walker D T Goddard et al ldquoComparison ofmicroscope techniques for the examination of biofilmsrdquo Journalof Microbiological Methods vol 25 no 1 pp 57ndash70 1996

[49] C Hannig M Follo E Hellwig and A Al-Ahmad ldquoVisualiza-tion of adherent micro-organisms using different techniquesrdquoJournal of Medical Microbiology vol 59 no 1 pp 1ndash7 2010

[50] A Bridier F Dubois-Brissonnet A Boubetra V Thomas andR Briandet ldquoThe biofilm architecture of sixty opportunisticpathogens deciphered using a high throughput CLSMmethodrdquoJournal of Microbiological Methods vol 82 no 1 pp 64ndash702010

[51] M Fedel P Caciagli V Chiste et al ldquoMicrobial biofilm imagingESEM vs HVSEMrdquo Imaging ampMicroscopy vol 9 no 2 pp 44ndash47 2007

[52] R J Palmer Jr J J Haagensen T Neu and C Sternberg ldquoCon-focal microscopy of biofilmsmdashspatiotemporal approachesrdquo inHandbook of Biological Confocal Microscopy J B Pawley Edpp 870ndash888 Springer New York NY USA 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 Scientifica

Table 1 Advantages of MTPs as biofilm reactors and standardmethods for quantitation of biofilm parameters in MTPs

Advantages References Quantitation assays ReferencesHigh throughput [4 40] Biofilm biomass [1 16]Small volumes ofreagents [4 40] Microbial

physiological activity [7 41]

Automation [42 43] Microbial cells in thebiofilm [7 41]

Multiplexing [4] Biofilm matrix [44]

dye [16 17] This method can therefore be considered a bulkmethod which provides information about the total amountof biofilms produced without revealing any informationabout biofilm localization It has been shown that the biofilmdistribution in the wall of a 96-well MTPmay not be uniformwhen dynamic conditions are used (when the MTP is shakenwith an orbital motion) [6]This biofilm heterogeneity can beanalyzed for instance by microscopy either using standardoptical microscopy [18] confocal laser scanning microscopy(CLSM) [19] or scanning electron microscopy (SEM) [20]The relative advantages and limitations of these two latertechniques are presented in Table 2 It is interesting to pointout that for the most part these microscopy analyses aremade at the bottom of the well disregarding the biofilm thatis formed on the vertical wall Both techniques enable spatiallocalization of the biofilms but are inherently low throughputtechniques with a high cost and are subjected to equipmentavailabilityThe aimof this workwas to develop a low cost andhigh throughput method that would enable the quantitationof the total amount of biofilm produced inside a well butproviding the same information about the spatial location ofthe formed biofilm A differential CV staining method washere developed by combining the high throughput featuresof the usual CV staining but enabling spatial localization ofthe biofilm without the use of expensive equipment

2 Materials and Methods

21 Computational Fluid Dynamic (CFD) SimulationsNumerical simulations were made in Ansys Fluent CFDpackage (version 130) as previously described [6] Acylindrical well (diameter of 66mm and height of 117mm)was built in Design Modeller 130 and discretized into a gridof 18876 hexahedral cells by Meshing 130 Simulation wasmade for a shaking diameter of 50mm and frequency of150 rpm The location of the interface was determined byAnsys Fluent as well as the magnitude of the shear strainrate The time averaged shear strain rate was obtained byaveraging the steady state shear strain rate of the liquid sideduring a complete orbit

22 Biofilm Formation in 96-Well Microtiter PlatesEscherichia coli JM109(DE3) from Promega (USA) wasused to form biofilms in 96-well microtiter plates becausethis strain has shown a good biofilm forming ability inboth turbulent [21] and laminar [6] flow conditions Its

Its genotype is endA1 recA1 gyrA96 thi hsdR17 (rkminus

mk+) relA1 supE44 120582minus Δ(lac-proAB) (F1015840traD36 proAB119897119886119888IqZΔM15) and 120582(DE3) Cells from an overnight culturewere prepared as previously described [6] and biofilms wereproduced by pipetting 20 120583L of these cells into six wellsof sterile 96-well flat-bottomed microtiter plates (OrangeScientific USA) filled with 180120583L of nutrient media Themedia used was identical to the one described in [22]except for glucose A glucose concentration of 1 g Lminus1 wasused in this work since it has been demonstrated that thisconcentration originated the maximum biofilm amount at24 h in different shaking conditions [6] Microtiter plateswere placed for 24 h at 30∘C in an orbital shaking incubatorwith 50mm of diameter at 150 rpm (CERTOMAT BS-1Sartorius AG Germany)

23 BiofilmQuantitation by Crystal Violet (CV) Staining Theamount of biofilm formed was measured using the CV dyein a differential form this means that the well has beendivided into four different sections (Figure 1(d)) and thecorresponding vertical wall section was stained sequentiallyup to a maximum volume of 200 120583L Biofilm quantitation onthe bottom of the well was performed by using 25 120583L of CVfor staining (as detailed below) Section 1 corresponded toa volume between 25 and 50 120583L (and a height of 077mm)Section 2 corresponded to a volume between 50 and 100120583Lsection 3 corresponded to a volume between 100 and 150 120583Land section 4 corresponded to a volume between 150 and200120583L Sections 2 3 and 4 had an equivalent height of154mm

Prior to CV staining the contents of the microtiter plateswere discarded and the wells were washed with 200120583L ofsterile water to remove non-adherent bacteria [16] Then thebiofilms were fixed with 250120583L of 96 ethanol [23] Thefirst section of the well to be quantified was the bottom (notrepresented in Figure 1(d)) which corresponds to a volumeof 25120583L and a height of 077mm The cells adhered to thisregion were stained for 5min with the correspondent volumeof 1 (vv) crystal violet (Merck Germany) and the dyebound to this specific region was solubilized with 200120583Lof 33 (vv) acetic acid (VWR Portugal) The absorbancewas measured at 570 nm using a microtiter plate reader(SpectraMax M2E Molecular Devices UK) The biofilmsformed in the successive sections of the well (sections 1 to 4 ofFigure 1(d)) were quantified using the described procedurespecifically using the CV volume equivalent to the maximumlevel of each section (section 1mdash50120583L section 2mdash100 120583Lsection 3mdash150120583L section 4mdash200120583L) The absorbance cor-responding to each of the defined sections and presentedon Figure 1(d) was calculated by subtracting the absorbancesfrom the previous sections and considering the dilutionfactor To quantify the biofilm formed in each of the wellsections six replica wells were used per experiment and threeindependent experiments were performed

24 Scanning Electron Microscopy (SEM) Microtiter platewells containing 24 h old biofilms were imaged by SEM aspreviously described [24] Briefly biofilms were fixed in 3

Scientifica 3

Table 2 Advantages and limitations of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) on biofilmanalysis

Technique Advantages Limitations References

Scanning electronmicroscopy

High resolutionWide range of magnificationsGood comparative informationAbility to image complex shapes

Not real timeRequires additional sample preparationLimited quantification

[45ndash49]

Confocal laser scanningmicroscopy

Living fully hydrated samplesNon-invasiveQuantitative evaluationReflection and fluorescence mode

Low resolutionNarrow range of magnificationsNot applicable to thick biofilms

[35 47 50ndash52]

1

2

3

4

Increased wetted areaStatic fill volume

0253

0028

0

0066

Absorbance (570 nm)

(a) (b) (d) (c) (e)

20 40 60 80 100 120 140 160 180

Strain rate (sminus1)

Figure 1 Biofilm localization in shaking 96-well microtiter plates placed in a 50mm incubator at 150 rpm (a) Photograph of a well stainedwith crystal violet (b) schematic representation of a well where the dark grey area corresponds to the wetted area without shaking the lightgrey area represents the area increase upon shaking and the dotted line depicts the inclination of the air-liquid interface (c) time averagedshear strain rates (values below 20 sminus1 are not represented) (d) illustration of the biofilm distribution on the vertical wall assayed by thedifferential CV staining (e) representative scanning electron micrographs of the wall sections defined in image (d) (5000x magnification bar= 10120583m)

(ww) glutaraldehyde in cacodylate buffer dehydrated withethanol and hexamethyldisilazane (HMDS) and sputter-coated with a palladium-gold thin filmThe different sectionsalong the vertical wall of three independent wells wereobserved with a SEMEDS system (FEI Quanta 400FEGESEMEDAX Genesis X4M FEI Company USA)

3 Results and Discussion

The effect of orbital shaking on biofilm formation in 96-well microtiter plates was firstly assessed by the conventionalprocedure of crystal violet staining [6 16] Through directobservation of the stained wells (prior to solubilization ofthe dye) it was possible to observe that biofilms weremainly formed on the wall and not on the bottom of thewell Moreover since the amount of produced biofilms isproportional to the amount of CV adsorbed it seemed thatthe biofilm was unevenly distributed in the cylindrical wall

and higher amounts were formed closer to the air-liquidinterface (Figure 1(a))

It is widely known that hydrodynamics affects biofilmformation [25ndash27] as a result of the shear forces that canmodulate cell adhesion to a given surface [28ndash31] Eventhough microtiter plates are broadly accepted as biofilmformation reactors by the scientific community there is stilla lack of information on the impact of hydrodynamics onbiofilm formation in this system In this work the flowinside the wells was simulated using computational fluiddynamics (Figures 1(b) and 1(c)) in order to find out ifthe hydrodynamic conditions were related to the unevenbiofilm distribution that was visible to the naked eye after CVstaining (Figure 1(a)) Figure 1(b) represents a scale modelof a microwell with the wetted area without orbital motionthe area increase upon shaking and the inclination of theair-liquid interface For the shaking conditions chosen forthis work (50mm of orbital diameter and 150 rpm) an areagain of 85 and a maximum angle of 78∘ were obtained

4 Scientifica

Similar to what was observed in the photograph of thestained well (Figure 1(a)) the shear strain rate distributionwas not uniform along the wall being much higher in theliquid side near the interface (Figure 1(c)) In this regionCFD simulations revealed some spots corresponding to amaximum shear strain rate of 180 sminus1 which are related toregions of unstable vortices [6]

Observing the results of the differential CV staining(Figure 1(d)) the amount of biofilm detected along the differ-ent sections of the wall was not constantThe highest value ofabsorbance (corresponding to the highest amount of biofilmbiomass) was measured in the region located immediatelybelow the liquid level (section 4) In the intermediate sectionsof the well the absorbance values were about 10 times lowerthan near the interface (section 3) or almost zero (section 2)increasing slightly in the section closer to the bottom of thewell (section 1)

In order to validate the results obtained by the differentialCV staining SEM analysis was performed on the vertical wallof the wells Figure 1(e) shows scanning electronmicrographsrepresentative of the four sections defined in the applicationof the differential CV staining (Figure 1(d)) SEM analysisshowed that E coli adhesion varied across the wall andthat higher attachment occurred closer to the air-liquidinterface (section 4) compared to the intermediate (sections2 and 3) and near bottom (section 1) regions of the wellBiofilms consisting of bacterial aggregates were observednear the interface while in the lower regions of the wellthere were only few cells randomly distributed on the surface(Figure 1(e))

Most likely the spatial location of the biofilms (assayed bydifferential staining and SEM analysis) is conditioned by thenon-homogeneous distribution of shear strain calculated byCFD during shaking The higher cell density of the biofilmsformed closer to the interface is probably due to the higheroxygen and substrate mass transfer from the bulk solution tothe biofilm [32] resulting from amore efficient liquid mixingin this region These results are in agreement with previousstudies [29 33 34] showing that higher shear forces promotethe formation of denser biofilms

Both methods used for biofilm analysis in this work(differential CV staining and SEM) enabled a higher detailin analysis of biofilms grown in 96-well microtiter plateswhich is not achieved by the spectrophotometric methodscommonly used in MTPs (Table 1) Indeed methods such asthe traditional CV assay provide bulk data from a biofilmand are classified as macroscale methods whereas SEM is amicroscale technique [24] It is interesting to notice that thedifferential CV staining presented in this work correspondsto an intermediate scale between the traditional CV assay andSEM analysis The traditional CV assay quantifies the biofilmformed on the wall and bottom of each well of a microtiterplate which corresponds to an area of about 146mm2 (fora 96-well plate) while the method proposed here evaluateswall sections of about 14 and 28mm2 In comparison thearea covered by a SEM image taken at 5000x magnificationwas of 2 times10minus3mm2 and this technique enables detailedanalysis of individual cells within the biofilm rather than

simply determining their localization SEM offers highermagnification (ranging from 20x to approximately 30000x)and resolution (from 50 to 100 nm) together with the abilityof imaging complex shapes (Table 2) It is also highly rec-ommended for the visualization of cellular morphology cell-to-cell interactions and matrix components within biofilms[35] However one must bear in mind that most laboratoriesare not equipped with an electron microscope and thistechnique has a considerably lower throughput than themethods described in Table 1

Observation of biofilms formed on the bottom of thewells of microtiter plates is very common using opticalmicroscopes at magnifications of 100 to 200x which typicallycover areas of 03mm2 [36ndash39] These observations haveprovided some information about the architecture of thebiofilms formed on the bottom of the wells (particularlywhen CLSM is used) but usually disregard the biofilm thatforms on the vertical wall It has been shown that in dynamicconditions the amount of biofilm formed on the vertical wallcan be higher than the one formed on the bottom of the well[6] and therefore a method was developed in this work todetermine the spatial localization of these biofilms in a highthroughput manner using common laboratory equipmentThe area under analysis in each of the four sections definedfor the differential staining is equivalent to the area analyzedby optical microscopy using a 15x magnification This areacould be further reduced (thus increasing the precision of themethod) by dividing the well in sections of smaller height

The novel approach presented in this work demonstratesthat the CV dye can be extremely useful in locating theadherent cells in microtiter plates when used in a differentialway The method is slightly more laborious and slowerthan the traditional staining procedure but it requires fewerresources and has higher throughput than other techniquesthat are used to determine the spatial location of biofilms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support providedby Operational Programme for Competitiveness Factors(COMPETE) European Fund for Regional Development-FEDER and the Portuguese Foundation for Scienceand Technology (FCT) through Projects PTDCEBB-BIO1028632008 and PTDCEBB-BIO1049402008Luciana Gomes acknowledges the receipt of a PhD Grantfrom FCT (SFRHBD804002011) Joao Miranda and JoseAraujo (Transport Phenomena Research Center FEUP) areacknowledged for the numerical simulations

References

[1] J W Costerton P S Stewart and E P Greenberg ldquoBacterialbiofilms a common cause of persistent infectionsrdquo Science vol284 no 5418 pp 1318ndash1322 1999

Scientifica 5

[2] P Stoodley K Sauer D G Davies and J W CostertonldquoBiofilms as complex differentiated communitiesrdquo AnnualReview of Microbiology vol 56 pp 187ndash209 2002

[3] V Hancock L Ferrieres and P Klemm ldquoBiofilm formation byasymptomatic and virulent urinary tract infectious Escherichiacoli strainsrdquo FEMSMicrobiology Letters vol 267 no 1 pp 30ndash372007

[4] T Coenye and H J Nelis ldquoIn vitro and in vivo model systemsto studymicrobial biofilm formationrdquo Journal ofMicrobiologicalMethods vol 83 no 2 pp 89ndash105 2010

[5] J S Teodosio M Simoes L F Melo and F J Mergulhao ldquoPlat-forms for in vitro biofilm studiesrdquo in Biofilms in BioengineeringM Simoes and F Mergulhao Eds pp 45ndash62 Nova ScienceNew York NY USA 2013

[6] J M R Moreira L C Gomes J D P Araujo et al ldquoThe effectof glucose concentration and shaking conditions on Escherichiacoli biofilm formation in microtiter platesrdquo Chemical Engineer-ing Science vol 94 pp 192ndash199 2013

[7] E Peeters H J Nelis and T Coenye ldquoComparison of multiplemethods for quantification of microbial biofilms grown inmicrotiter platesrdquo Journal of Microbiological Methods vol 72no 2 pp 157ndash165 2008

[8] M Erriu F M G Pili E Tuveri et al ldquoOil essentialmouthwashes antibacterial activity againstAggregatibacter acti-nomycetemcomitans a comparison between antibiofilm andantiplanktonic effectsrdquo International Journal of Dentistry vol2013 Article ID 164267 5 pages 2013

[9] D Minardi O Cirioni R Ghiselli et al ldquoEfficacy of tigecyclineand rifampin alone and in combination against Enterococcusfaecalis biofilm infection in a ratmodel of ureteral stentrdquo Journalof Surgical Research vol 176 no 1 pp 1ndash6 2012

[10] C Sanchez K Mende M Beckius et al ldquoBiofilm formationby clinical isolates and the implications in chronic infectionsrdquoBMC Infectious Diseases vol 13 article 47 2013

[11] E Hell C Giske K Hultenby et al ldquoAttachment and biofilmforming capabilities of Staphylococcus epidermidis strains iso-lated from preterm infantsrdquo Current Microbiology vol 67 no 6pp 712ndash717 2013

[12] M-H Lin F-R Chang M-Y Hua Y-C Wu and S-T Liu ldquoInhibitory effects of 12346-penta-O-galloyl-120573-D-glucopyranose on biofilm formation by Staphylococcus aureusrdquoAntimicrobial Agents and Chemotherapy vol 55 no 3 pp 1021ndash1027 2011

[13] C De La Fuente-Nunez V Korolik M Bains et al ldquoInhibitionof bacterial biofilm formation and swarming motility by asmall synthetic cationic peptiderdquo Antimicrobial Agents andChemotherapy vol 56 no 5 pp 2696ndash2704 2012

[14] G D ChristensenW A Simpson and J J Younger ldquoAdherenceof coagulase-negative staphylococci to plastic tissue cultureplates a quantitative model for the adherence of staphylococcito medical devicesrdquo Journal of Clinical Microbiology vol 22 no6 pp 996ndash1006 1985

[15] X Li Z Yan and J Xu ldquoQuantitative variation of biofilmsamong strains in natural populations of Candida albicansrdquoMicrobiology vol 149 no 2 pp 353ndash362 2003

[16] S Stepanovic D Vukovic I Dakic B Savic and M Svabic-Vlahovic ldquoA modified microtiter-plate test for quantificationof staphylococcal biofilm formationrdquo Journal of MicrobiologicalMethods vol 40 no 2 pp 175ndash179 2000

[17] L Cremet S Corvec E Batard et al ldquoComparison of threemethods to study biofilm formation by clinical strains of

Escherichia colirdquoDiagnosticMicrobiology and Infectious Diseasevol 75 no 3 pp 252ndash255 2013

[18] L R Martinez and A Casadevall ldquoSpecific antibody canprevent fungal biofilm formation and this effect correlates withprotective efficacyrdquo Infection and Immunity vol 73 no 10 pp6350ndash6362 2005

[19] A Bridier D Le Coq F Dubois-Brissonnet V Thomas SAymerich and R Briandet ldquoThe spatial architecture of Bacillussubtilis biofilms deciphered using a surface-associated modeland in situ imagingrdquo PLoS ONE vol 6 no 1 Article ID e161772011

[20] V Kostenko M M Salek P Sattari and R J MartinuzzildquoStaphylococcus aureus biofilm formation and tolerance toantibiotics in response to oscillatory shear stresses of physiolog-ical levelsrdquo FEMS Immunology and Medical Microbiology vol59 no 3 pp 421ndash431 2010

[21] J S Teodosio M Simoes and F J Mergulhao ldquoThe influence ofnonconjugative Escherichia coli plasmids on biofilm formationand resistancerdquo Journal of Applied Microbiology vol 113 no 2pp 373ndash382 2012

[22] J S Teodosio M Simoes L F Melo and F J MergulhaoldquoFlow cell hydrodynamics and their effects on E coli biofilmformation under different nutrient conditions and turbulentflowrdquo Biofouling vol 27 no 1 pp 1ndash11 2011

[23] S Shakeri R K Kermanshahi M M Moghaddam and GEmtiazi ldquoAssessment of biofilm cell removal and killing andbiocide efficacy using the microtiter plate testrdquo Biofouling vol23 no 2 pp 79ndash86 2007

[24] L Gomes J M R Moreira J M Miranda et al ldquoMacroscaleversusmicroscalemethods for physiological analysis of biofilmsformed in 96-well microtiter platesrdquo Journal of MicrobiologicalMethods vol 95 no 3 pp 342ndash349 2013

[25] Y Liu and J-H Tay ldquoThe essential role of hydrodynamic shearforce in the formation of biofilm and granular sludgerdquo WaterResearch vol 36 no 7 pp 1653ndash1665 2002

[26] P Stoodley S Wilson L Hall-Stoodley J D Boyle H MLappin-Scott and J W Costerton ldquoGrowth and detachment ofcell clusters from mature mixed-species biofilmsrdquo Applied andEnvironmentalMicrobiology vol 67 no 12 pp 5608ndash5613 2001

[27] S Wasche H Horn and D C Hempel ldquoInfluence of growthconditions on biofilm development and mass transfer at thebulkbiofilm interfacerdquoWater Research vol 36 no 19 pp 4775ndash4784 2002

[28] H J Busscher and H C van der Mei ldquoMicrobial adhesion inflow displacement systemsrdquo Clinical Microbiology Reviews vol19 no 1 pp 127ndash141 2006

[29] M Simoes M O Pereira S Sillankorva J Azeredo andM J Vieira ldquoThe effect of hydrodynamic conditions on thephenotype of Pseudomonas fluorescens biofilmsrdquoBiofouling vol23 no 4 pp 249ndash258 2007

[30] J S Teodosio F C Silva J M R Moreira et al ldquoFlow cellsas quasi ideal systems for biofouling simulation of industrialpiping systemsrdquo Biofouling vol 29 no 8 pp 953ndash966 2013

[31] M C M Van Loosdrecht D Eikelboom A Gjaltema AMulder L Tijhuis and J J Heijnen ldquoBiofilm structuresrdquoWaterScience and Technology vol 32 no 8 pp 35ndash43 1995

[32] J M R Moreira J S Teodosio F C Silva et al ldquoInfluenceof flow rate variation on the development of Escherichia colibiofilmsrdquo Bioprocess and Biosystems Engineering vol 36 no 11pp 1787ndash1796 2013

6 Scientifica

[33] WKKwokC Picioreanu S LOng et al ldquoInfluence of biomassproduction and detachment forces on biofilm structures in abiofilm airlift suspension reactorrdquo Biotechnology and Bioengi-neering vol 58 no 4 pp 400ndash407 1998

[34] A Rochex J-J Godon N Bernet and R Escudie ldquoRole ofshear stress on composition diversity and dynamics of biofilmbacterial communitiesrdquo Water Research vol 42 no 20 pp4915ndash4922 2008

[35] A Bridier T Meylheuc and R Briandet ldquoRealistic represen-tation of Bacillus subtilis biofilms architecture using combinedmicroscopy (CLSM ESEM and FESEM)rdquo Micron vol 48 pp65ndash69 2013

[36] M G White S Piccirillo V Dusevich D J Law T Kaprosand S M Honigberg ldquoFlo11p adhesin required for meioticdifferentiation in Saccharomyces cerevisiaeminicolonies grownon plastic surfacesrdquo FEMS Yeast Research vol 11 no 2 pp 223ndash232 2011

[37] C L Quave M Estevez-Carmona C M Compadre et alldquoEllagic acid derivatives from Rubus ulmifolius inhibit Staphy-lococcus aureus biofilm formation and improve response toantibioticsrdquo PLoS ONE vol 7 no 1 Article ID e28737 2012

[38] A Lizcano T Chin K Sauer E I Tuomanen and C JOrihuela ldquoEarly biofilm formation on microtiter plates is notcorrelated with the invasive disease potential of Streptococcuspneumoniaerdquo Microbial Pathogenesis vol 48 no 3-4 pp 124ndash130 2010

[39] A S Melo A L Colombo and B A Arthington-Skaggs ldquoPara-doxical growth effect of caspofungin observed on biofilms andplanktonic cells of five differentCandida speciesrdquoAntimicrobialAgents and Chemotherapy vol 51 no 9 pp 3081ndash3088 2007

[40] W A Duetz ldquoMicrotiter plates as mini-bioreactors miniatur-ization of fermentation methodsrdquo Trends in Microbiology vol15 no 10 pp 469ndash475 2007

[41] K Honraet E Goetghebeur and H J Nelis ldquoComparisonof three assays for the quantification of Candida biomassin suspension and CDC reactor grown biofilmsrdquo Journal ofMicrobiological Methods vol 63 no 3 pp 287ndash295 2005

[42] S Kumar C Wittmann and E Heinzle ldquoReview minibioreac-torsrdquo Biotechnology Letters vol 26 no 1 pp 1ndash10 2004

[43] N F Azevedo S P Lopes C W Keevil M O Pereira and MJ Vieira ldquoTime to ldquogo largerdquo on biofilm research advantages ofan omics approachrdquoBiotechnology Letters vol 31 no 4 pp 477ndash485 2009

[44] K Tote D V Berghe L Maes and P Cos ldquoA new colorimetricmicrotitre model for the detection of Staphylococcus aureusbiofilmsrdquo Letters in AppliedMicrobiology vol 46 no 2 pp 249ndash254 2008

[45] T A Norton R C Thompson J Pope et al ldquoUsing confocallaser scanning microscopy scanning electron microscopy andphase contrast light microscopy to examine marine biofilmsrdquoAquatic Microbial Ecology vol 16 no 2 pp 199ndash204 1998

[46] D O Serra A M Richter G Klauck et al ldquoMicroanatomy atcellular resolution and spatial order of physiological differen-tiation in a bacterial biofilmrdquo mBio vol 4 no 2 pp e100103ndashe100113 2013

[47] P S Stewart R Murga R Srinivasan and D De Beer ldquoBiofilmstructural heterogeneity visualized by three microscopic meth-odsrdquoWater Research vol 29 no 8 pp 2006ndash2009 1995

[48] S B Surman J T Walker D T Goddard et al ldquoComparison ofmicroscope techniques for the examination of biofilmsrdquo Journalof Microbiological Methods vol 25 no 1 pp 57ndash70 1996

[49] C Hannig M Follo E Hellwig and A Al-Ahmad ldquoVisualiza-tion of adherent micro-organisms using different techniquesrdquoJournal of Medical Microbiology vol 59 no 1 pp 1ndash7 2010

[50] A Bridier F Dubois-Brissonnet A Boubetra V Thomas andR Briandet ldquoThe biofilm architecture of sixty opportunisticpathogens deciphered using a high throughput CLSMmethodrdquoJournal of Microbiological Methods vol 82 no 1 pp 64ndash702010

[51] M Fedel P Caciagli V Chiste et al ldquoMicrobial biofilm imagingESEM vs HVSEMrdquo Imaging ampMicroscopy vol 9 no 2 pp 44ndash47 2007

[52] R J Palmer Jr J J Haagensen T Neu and C Sternberg ldquoCon-focal microscopy of biofilmsmdashspatiotemporal approachesrdquo inHandbook of Biological Confocal Microscopy J B Pawley Edpp 870ndash888 Springer New York NY USA 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Scientifica 3

Table 2 Advantages and limitations of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) on biofilmanalysis

Technique Advantages Limitations References

Scanning electronmicroscopy

High resolutionWide range of magnificationsGood comparative informationAbility to image complex shapes

Not real timeRequires additional sample preparationLimited quantification

[45ndash49]

Confocal laser scanningmicroscopy

Living fully hydrated samplesNon-invasiveQuantitative evaluationReflection and fluorescence mode

Low resolutionNarrow range of magnificationsNot applicable to thick biofilms

[35 47 50ndash52]

1

2

3

4

Increased wetted areaStatic fill volume

0253

0028

0

0066

Absorbance (570 nm)

(a) (b) (d) (c) (e)

20 40 60 80 100 120 140 160 180

Strain rate (sminus1)

Figure 1 Biofilm localization in shaking 96-well microtiter plates placed in a 50mm incubator at 150 rpm (a) Photograph of a well stainedwith crystal violet (b) schematic representation of a well where the dark grey area corresponds to the wetted area without shaking the lightgrey area represents the area increase upon shaking and the dotted line depicts the inclination of the air-liquid interface (c) time averagedshear strain rates (values below 20 sminus1 are not represented) (d) illustration of the biofilm distribution on the vertical wall assayed by thedifferential CV staining (e) representative scanning electron micrographs of the wall sections defined in image (d) (5000x magnification bar= 10120583m)

(ww) glutaraldehyde in cacodylate buffer dehydrated withethanol and hexamethyldisilazane (HMDS) and sputter-coated with a palladium-gold thin filmThe different sectionsalong the vertical wall of three independent wells wereobserved with a SEMEDS system (FEI Quanta 400FEGESEMEDAX Genesis X4M FEI Company USA)

3 Results and Discussion

The effect of orbital shaking on biofilm formation in 96-well microtiter plates was firstly assessed by the conventionalprocedure of crystal violet staining [6 16] Through directobservation of the stained wells (prior to solubilization ofthe dye) it was possible to observe that biofilms weremainly formed on the wall and not on the bottom of thewell Moreover since the amount of produced biofilms isproportional to the amount of CV adsorbed it seemed thatthe biofilm was unevenly distributed in the cylindrical wall

and higher amounts were formed closer to the air-liquidinterface (Figure 1(a))

It is widely known that hydrodynamics affects biofilmformation [25ndash27] as a result of the shear forces that canmodulate cell adhesion to a given surface [28ndash31] Eventhough microtiter plates are broadly accepted as biofilmformation reactors by the scientific community there is stilla lack of information on the impact of hydrodynamics onbiofilm formation in this system In this work the flowinside the wells was simulated using computational fluiddynamics (Figures 1(b) and 1(c)) in order to find out ifthe hydrodynamic conditions were related to the unevenbiofilm distribution that was visible to the naked eye after CVstaining (Figure 1(a)) Figure 1(b) represents a scale modelof a microwell with the wetted area without orbital motionthe area increase upon shaking and the inclination of theair-liquid interface For the shaking conditions chosen forthis work (50mm of orbital diameter and 150 rpm) an areagain of 85 and a maximum angle of 78∘ were obtained

4 Scientifica

Similar to what was observed in the photograph of thestained well (Figure 1(a)) the shear strain rate distributionwas not uniform along the wall being much higher in theliquid side near the interface (Figure 1(c)) In this regionCFD simulations revealed some spots corresponding to amaximum shear strain rate of 180 sminus1 which are related toregions of unstable vortices [6]

Observing the results of the differential CV staining(Figure 1(d)) the amount of biofilm detected along the differ-ent sections of the wall was not constantThe highest value ofabsorbance (corresponding to the highest amount of biofilmbiomass) was measured in the region located immediatelybelow the liquid level (section 4) In the intermediate sectionsof the well the absorbance values were about 10 times lowerthan near the interface (section 3) or almost zero (section 2)increasing slightly in the section closer to the bottom of thewell (section 1)

In order to validate the results obtained by the differentialCV staining SEM analysis was performed on the vertical wallof the wells Figure 1(e) shows scanning electronmicrographsrepresentative of the four sections defined in the applicationof the differential CV staining (Figure 1(d)) SEM analysisshowed that E coli adhesion varied across the wall andthat higher attachment occurred closer to the air-liquidinterface (section 4) compared to the intermediate (sections2 and 3) and near bottom (section 1) regions of the wellBiofilms consisting of bacterial aggregates were observednear the interface while in the lower regions of the wellthere were only few cells randomly distributed on the surface(Figure 1(e))

Most likely the spatial location of the biofilms (assayed bydifferential staining and SEM analysis) is conditioned by thenon-homogeneous distribution of shear strain calculated byCFD during shaking The higher cell density of the biofilmsformed closer to the interface is probably due to the higheroxygen and substrate mass transfer from the bulk solution tothe biofilm [32] resulting from amore efficient liquid mixingin this region These results are in agreement with previousstudies [29 33 34] showing that higher shear forces promotethe formation of denser biofilms

Both methods used for biofilm analysis in this work(differential CV staining and SEM) enabled a higher detailin analysis of biofilms grown in 96-well microtiter plateswhich is not achieved by the spectrophotometric methodscommonly used in MTPs (Table 1) Indeed methods such asthe traditional CV assay provide bulk data from a biofilmand are classified as macroscale methods whereas SEM is amicroscale technique [24] It is interesting to notice that thedifferential CV staining presented in this work correspondsto an intermediate scale between the traditional CV assay andSEM analysis The traditional CV assay quantifies the biofilmformed on the wall and bottom of each well of a microtiterplate which corresponds to an area of about 146mm2 (fora 96-well plate) while the method proposed here evaluateswall sections of about 14 and 28mm2 In comparison thearea covered by a SEM image taken at 5000x magnificationwas of 2 times10minus3mm2 and this technique enables detailedanalysis of individual cells within the biofilm rather than

simply determining their localization SEM offers highermagnification (ranging from 20x to approximately 30000x)and resolution (from 50 to 100 nm) together with the abilityof imaging complex shapes (Table 2) It is also highly rec-ommended for the visualization of cellular morphology cell-to-cell interactions and matrix components within biofilms[35] However one must bear in mind that most laboratoriesare not equipped with an electron microscope and thistechnique has a considerably lower throughput than themethods described in Table 1

Observation of biofilms formed on the bottom of thewells of microtiter plates is very common using opticalmicroscopes at magnifications of 100 to 200x which typicallycover areas of 03mm2 [36ndash39] These observations haveprovided some information about the architecture of thebiofilms formed on the bottom of the wells (particularlywhen CLSM is used) but usually disregard the biofilm thatforms on the vertical wall It has been shown that in dynamicconditions the amount of biofilm formed on the vertical wallcan be higher than the one formed on the bottom of the well[6] and therefore a method was developed in this work todetermine the spatial localization of these biofilms in a highthroughput manner using common laboratory equipmentThe area under analysis in each of the four sections definedfor the differential staining is equivalent to the area analyzedby optical microscopy using a 15x magnification This areacould be further reduced (thus increasing the precision of themethod) by dividing the well in sections of smaller height

The novel approach presented in this work demonstratesthat the CV dye can be extremely useful in locating theadherent cells in microtiter plates when used in a differentialway The method is slightly more laborious and slowerthan the traditional staining procedure but it requires fewerresources and has higher throughput than other techniquesthat are used to determine the spatial location of biofilms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support providedby Operational Programme for Competitiveness Factors(COMPETE) European Fund for Regional Development-FEDER and the Portuguese Foundation for Scienceand Technology (FCT) through Projects PTDCEBB-BIO1028632008 and PTDCEBB-BIO1049402008Luciana Gomes acknowledges the receipt of a PhD Grantfrom FCT (SFRHBD804002011) Joao Miranda and JoseAraujo (Transport Phenomena Research Center FEUP) areacknowledged for the numerical simulations

References

[1] J W Costerton P S Stewart and E P Greenberg ldquoBacterialbiofilms a common cause of persistent infectionsrdquo Science vol284 no 5418 pp 1318ndash1322 1999

Scientifica 5

[2] P Stoodley K Sauer D G Davies and J W CostertonldquoBiofilms as complex differentiated communitiesrdquo AnnualReview of Microbiology vol 56 pp 187ndash209 2002

[3] V Hancock L Ferrieres and P Klemm ldquoBiofilm formation byasymptomatic and virulent urinary tract infectious Escherichiacoli strainsrdquo FEMSMicrobiology Letters vol 267 no 1 pp 30ndash372007

[4] T Coenye and H J Nelis ldquoIn vitro and in vivo model systemsto studymicrobial biofilm formationrdquo Journal ofMicrobiologicalMethods vol 83 no 2 pp 89ndash105 2010

[5] J S Teodosio M Simoes L F Melo and F J Mergulhao ldquoPlat-forms for in vitro biofilm studiesrdquo in Biofilms in BioengineeringM Simoes and F Mergulhao Eds pp 45ndash62 Nova ScienceNew York NY USA 2013

[6] J M R Moreira L C Gomes J D P Araujo et al ldquoThe effectof glucose concentration and shaking conditions on Escherichiacoli biofilm formation in microtiter platesrdquo Chemical Engineer-ing Science vol 94 pp 192ndash199 2013

[7] E Peeters H J Nelis and T Coenye ldquoComparison of multiplemethods for quantification of microbial biofilms grown inmicrotiter platesrdquo Journal of Microbiological Methods vol 72no 2 pp 157ndash165 2008

[8] M Erriu F M G Pili E Tuveri et al ldquoOil essentialmouthwashes antibacterial activity againstAggregatibacter acti-nomycetemcomitans a comparison between antibiofilm andantiplanktonic effectsrdquo International Journal of Dentistry vol2013 Article ID 164267 5 pages 2013

[9] D Minardi O Cirioni R Ghiselli et al ldquoEfficacy of tigecyclineand rifampin alone and in combination against Enterococcusfaecalis biofilm infection in a ratmodel of ureteral stentrdquo Journalof Surgical Research vol 176 no 1 pp 1ndash6 2012

[10] C Sanchez K Mende M Beckius et al ldquoBiofilm formationby clinical isolates and the implications in chronic infectionsrdquoBMC Infectious Diseases vol 13 article 47 2013

[11] E Hell C Giske K Hultenby et al ldquoAttachment and biofilmforming capabilities of Staphylococcus epidermidis strains iso-lated from preterm infantsrdquo Current Microbiology vol 67 no 6pp 712ndash717 2013

[12] M-H Lin F-R Chang M-Y Hua Y-C Wu and S-T Liu ldquoInhibitory effects of 12346-penta-O-galloyl-120573-D-glucopyranose on biofilm formation by Staphylococcus aureusrdquoAntimicrobial Agents and Chemotherapy vol 55 no 3 pp 1021ndash1027 2011

[13] C De La Fuente-Nunez V Korolik M Bains et al ldquoInhibitionof bacterial biofilm formation and swarming motility by asmall synthetic cationic peptiderdquo Antimicrobial Agents andChemotherapy vol 56 no 5 pp 2696ndash2704 2012

[14] G D ChristensenW A Simpson and J J Younger ldquoAdherenceof coagulase-negative staphylococci to plastic tissue cultureplates a quantitative model for the adherence of staphylococcito medical devicesrdquo Journal of Clinical Microbiology vol 22 no6 pp 996ndash1006 1985

[15] X Li Z Yan and J Xu ldquoQuantitative variation of biofilmsamong strains in natural populations of Candida albicansrdquoMicrobiology vol 149 no 2 pp 353ndash362 2003

[16] S Stepanovic D Vukovic I Dakic B Savic and M Svabic-Vlahovic ldquoA modified microtiter-plate test for quantificationof staphylococcal biofilm formationrdquo Journal of MicrobiologicalMethods vol 40 no 2 pp 175ndash179 2000

[17] L Cremet S Corvec E Batard et al ldquoComparison of threemethods to study biofilm formation by clinical strains of

Escherichia colirdquoDiagnosticMicrobiology and Infectious Diseasevol 75 no 3 pp 252ndash255 2013

[18] L R Martinez and A Casadevall ldquoSpecific antibody canprevent fungal biofilm formation and this effect correlates withprotective efficacyrdquo Infection and Immunity vol 73 no 10 pp6350ndash6362 2005

[19] A Bridier D Le Coq F Dubois-Brissonnet V Thomas SAymerich and R Briandet ldquoThe spatial architecture of Bacillussubtilis biofilms deciphered using a surface-associated modeland in situ imagingrdquo PLoS ONE vol 6 no 1 Article ID e161772011

[20] V Kostenko M M Salek P Sattari and R J MartinuzzildquoStaphylococcus aureus biofilm formation and tolerance toantibiotics in response to oscillatory shear stresses of physiolog-ical levelsrdquo FEMS Immunology and Medical Microbiology vol59 no 3 pp 421ndash431 2010

[21] J S Teodosio M Simoes and F J Mergulhao ldquoThe influence ofnonconjugative Escherichia coli plasmids on biofilm formationand resistancerdquo Journal of Applied Microbiology vol 113 no 2pp 373ndash382 2012

[22] J S Teodosio M Simoes L F Melo and F J MergulhaoldquoFlow cell hydrodynamics and their effects on E coli biofilmformation under different nutrient conditions and turbulentflowrdquo Biofouling vol 27 no 1 pp 1ndash11 2011

[23] S Shakeri R K Kermanshahi M M Moghaddam and GEmtiazi ldquoAssessment of biofilm cell removal and killing andbiocide efficacy using the microtiter plate testrdquo Biofouling vol23 no 2 pp 79ndash86 2007

[24] L Gomes J M R Moreira J M Miranda et al ldquoMacroscaleversusmicroscalemethods for physiological analysis of biofilmsformed in 96-well microtiter platesrdquo Journal of MicrobiologicalMethods vol 95 no 3 pp 342ndash349 2013

[25] Y Liu and J-H Tay ldquoThe essential role of hydrodynamic shearforce in the formation of biofilm and granular sludgerdquo WaterResearch vol 36 no 7 pp 1653ndash1665 2002

[26] P Stoodley S Wilson L Hall-Stoodley J D Boyle H MLappin-Scott and J W Costerton ldquoGrowth and detachment ofcell clusters from mature mixed-species biofilmsrdquo Applied andEnvironmentalMicrobiology vol 67 no 12 pp 5608ndash5613 2001

[27] S Wasche H Horn and D C Hempel ldquoInfluence of growthconditions on biofilm development and mass transfer at thebulkbiofilm interfacerdquoWater Research vol 36 no 19 pp 4775ndash4784 2002

[28] H J Busscher and H C van der Mei ldquoMicrobial adhesion inflow displacement systemsrdquo Clinical Microbiology Reviews vol19 no 1 pp 127ndash141 2006

[29] M Simoes M O Pereira S Sillankorva J Azeredo andM J Vieira ldquoThe effect of hydrodynamic conditions on thephenotype of Pseudomonas fluorescens biofilmsrdquoBiofouling vol23 no 4 pp 249ndash258 2007

[30] J S Teodosio F C Silva J M R Moreira et al ldquoFlow cellsas quasi ideal systems for biofouling simulation of industrialpiping systemsrdquo Biofouling vol 29 no 8 pp 953ndash966 2013

[31] M C M Van Loosdrecht D Eikelboom A Gjaltema AMulder L Tijhuis and J J Heijnen ldquoBiofilm structuresrdquoWaterScience and Technology vol 32 no 8 pp 35ndash43 1995

[32] J M R Moreira J S Teodosio F C Silva et al ldquoInfluenceof flow rate variation on the development of Escherichia colibiofilmsrdquo Bioprocess and Biosystems Engineering vol 36 no 11pp 1787ndash1796 2013

6 Scientifica

[33] WKKwokC Picioreanu S LOng et al ldquoInfluence of biomassproduction and detachment forces on biofilm structures in abiofilm airlift suspension reactorrdquo Biotechnology and Bioengi-neering vol 58 no 4 pp 400ndash407 1998

[34] A Rochex J-J Godon N Bernet and R Escudie ldquoRole ofshear stress on composition diversity and dynamics of biofilmbacterial communitiesrdquo Water Research vol 42 no 20 pp4915ndash4922 2008

[35] A Bridier T Meylheuc and R Briandet ldquoRealistic represen-tation of Bacillus subtilis biofilms architecture using combinedmicroscopy (CLSM ESEM and FESEM)rdquo Micron vol 48 pp65ndash69 2013

[36] M G White S Piccirillo V Dusevich D J Law T Kaprosand S M Honigberg ldquoFlo11p adhesin required for meioticdifferentiation in Saccharomyces cerevisiaeminicolonies grownon plastic surfacesrdquo FEMS Yeast Research vol 11 no 2 pp 223ndash232 2011

[37] C L Quave M Estevez-Carmona C M Compadre et alldquoEllagic acid derivatives from Rubus ulmifolius inhibit Staphy-lococcus aureus biofilm formation and improve response toantibioticsrdquo PLoS ONE vol 7 no 1 Article ID e28737 2012

[38] A Lizcano T Chin K Sauer E I Tuomanen and C JOrihuela ldquoEarly biofilm formation on microtiter plates is notcorrelated with the invasive disease potential of Streptococcuspneumoniaerdquo Microbial Pathogenesis vol 48 no 3-4 pp 124ndash130 2010

[39] A S Melo A L Colombo and B A Arthington-Skaggs ldquoPara-doxical growth effect of caspofungin observed on biofilms andplanktonic cells of five differentCandida speciesrdquoAntimicrobialAgents and Chemotherapy vol 51 no 9 pp 3081ndash3088 2007

[40] W A Duetz ldquoMicrotiter plates as mini-bioreactors miniatur-ization of fermentation methodsrdquo Trends in Microbiology vol15 no 10 pp 469ndash475 2007

[41] K Honraet E Goetghebeur and H J Nelis ldquoComparisonof three assays for the quantification of Candida biomassin suspension and CDC reactor grown biofilmsrdquo Journal ofMicrobiological Methods vol 63 no 3 pp 287ndash295 2005

[42] S Kumar C Wittmann and E Heinzle ldquoReview minibioreac-torsrdquo Biotechnology Letters vol 26 no 1 pp 1ndash10 2004

[43] N F Azevedo S P Lopes C W Keevil M O Pereira and MJ Vieira ldquoTime to ldquogo largerdquo on biofilm research advantages ofan omics approachrdquoBiotechnology Letters vol 31 no 4 pp 477ndash485 2009

[44] K Tote D V Berghe L Maes and P Cos ldquoA new colorimetricmicrotitre model for the detection of Staphylococcus aureusbiofilmsrdquo Letters in AppliedMicrobiology vol 46 no 2 pp 249ndash254 2008

[45] T A Norton R C Thompson J Pope et al ldquoUsing confocallaser scanning microscopy scanning electron microscopy andphase contrast light microscopy to examine marine biofilmsrdquoAquatic Microbial Ecology vol 16 no 2 pp 199ndash204 1998

[46] D O Serra A M Richter G Klauck et al ldquoMicroanatomy atcellular resolution and spatial order of physiological differen-tiation in a bacterial biofilmrdquo mBio vol 4 no 2 pp e100103ndashe100113 2013

[47] P S Stewart R Murga R Srinivasan and D De Beer ldquoBiofilmstructural heterogeneity visualized by three microscopic meth-odsrdquoWater Research vol 29 no 8 pp 2006ndash2009 1995

[48] S B Surman J T Walker D T Goddard et al ldquoComparison ofmicroscope techniques for the examination of biofilmsrdquo Journalof Microbiological Methods vol 25 no 1 pp 57ndash70 1996

[49] C Hannig M Follo E Hellwig and A Al-Ahmad ldquoVisualiza-tion of adherent micro-organisms using different techniquesrdquoJournal of Medical Microbiology vol 59 no 1 pp 1ndash7 2010

[50] A Bridier F Dubois-Brissonnet A Boubetra V Thomas andR Briandet ldquoThe biofilm architecture of sixty opportunisticpathogens deciphered using a high throughput CLSMmethodrdquoJournal of Microbiological Methods vol 82 no 1 pp 64ndash702010

[51] M Fedel P Caciagli V Chiste et al ldquoMicrobial biofilm imagingESEM vs HVSEMrdquo Imaging ampMicroscopy vol 9 no 2 pp 44ndash47 2007

[52] R J Palmer Jr J J Haagensen T Neu and C Sternberg ldquoCon-focal microscopy of biofilmsmdashspatiotemporal approachesrdquo inHandbook of Biological Confocal Microscopy J B Pawley Edpp 870ndash888 Springer New York NY USA 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 Scientifica

Similar to what was observed in the photograph of thestained well (Figure 1(a)) the shear strain rate distributionwas not uniform along the wall being much higher in theliquid side near the interface (Figure 1(c)) In this regionCFD simulations revealed some spots corresponding to amaximum shear strain rate of 180 sminus1 which are related toregions of unstable vortices [6]

Observing the results of the differential CV staining(Figure 1(d)) the amount of biofilm detected along the differ-ent sections of the wall was not constantThe highest value ofabsorbance (corresponding to the highest amount of biofilmbiomass) was measured in the region located immediatelybelow the liquid level (section 4) In the intermediate sectionsof the well the absorbance values were about 10 times lowerthan near the interface (section 3) or almost zero (section 2)increasing slightly in the section closer to the bottom of thewell (section 1)

In order to validate the results obtained by the differentialCV staining SEM analysis was performed on the vertical wallof the wells Figure 1(e) shows scanning electronmicrographsrepresentative of the four sections defined in the applicationof the differential CV staining (Figure 1(d)) SEM analysisshowed that E coli adhesion varied across the wall andthat higher attachment occurred closer to the air-liquidinterface (section 4) compared to the intermediate (sections2 and 3) and near bottom (section 1) regions of the wellBiofilms consisting of bacterial aggregates were observednear the interface while in the lower regions of the wellthere were only few cells randomly distributed on the surface(Figure 1(e))

Most likely the spatial location of the biofilms (assayed bydifferential staining and SEM analysis) is conditioned by thenon-homogeneous distribution of shear strain calculated byCFD during shaking The higher cell density of the biofilmsformed closer to the interface is probably due to the higheroxygen and substrate mass transfer from the bulk solution tothe biofilm [32] resulting from amore efficient liquid mixingin this region These results are in agreement with previousstudies [29 33 34] showing that higher shear forces promotethe formation of denser biofilms

Both methods used for biofilm analysis in this work(differential CV staining and SEM) enabled a higher detailin analysis of biofilms grown in 96-well microtiter plateswhich is not achieved by the spectrophotometric methodscommonly used in MTPs (Table 1) Indeed methods such asthe traditional CV assay provide bulk data from a biofilmand are classified as macroscale methods whereas SEM is amicroscale technique [24] It is interesting to notice that thedifferential CV staining presented in this work correspondsto an intermediate scale between the traditional CV assay andSEM analysis The traditional CV assay quantifies the biofilmformed on the wall and bottom of each well of a microtiterplate which corresponds to an area of about 146mm2 (fora 96-well plate) while the method proposed here evaluateswall sections of about 14 and 28mm2 In comparison thearea covered by a SEM image taken at 5000x magnificationwas of 2 times10minus3mm2 and this technique enables detailedanalysis of individual cells within the biofilm rather than

simply determining their localization SEM offers highermagnification (ranging from 20x to approximately 30000x)and resolution (from 50 to 100 nm) together with the abilityof imaging complex shapes (Table 2) It is also highly rec-ommended for the visualization of cellular morphology cell-to-cell interactions and matrix components within biofilms[35] However one must bear in mind that most laboratoriesare not equipped with an electron microscope and thistechnique has a considerably lower throughput than themethods described in Table 1

Observation of biofilms formed on the bottom of thewells of microtiter plates is very common using opticalmicroscopes at magnifications of 100 to 200x which typicallycover areas of 03mm2 [36ndash39] These observations haveprovided some information about the architecture of thebiofilms formed on the bottom of the wells (particularlywhen CLSM is used) but usually disregard the biofilm thatforms on the vertical wall It has been shown that in dynamicconditions the amount of biofilm formed on the vertical wallcan be higher than the one formed on the bottom of the well[6] and therefore a method was developed in this work todetermine the spatial localization of these biofilms in a highthroughput manner using common laboratory equipmentThe area under analysis in each of the four sections definedfor the differential staining is equivalent to the area analyzedby optical microscopy using a 15x magnification This areacould be further reduced (thus increasing the precision of themethod) by dividing the well in sections of smaller height

The novel approach presented in this work demonstratesthat the CV dye can be extremely useful in locating theadherent cells in microtiter plates when used in a differentialway The method is slightly more laborious and slowerthan the traditional staining procedure but it requires fewerresources and has higher throughput than other techniquesthat are used to determine the spatial location of biofilms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support providedby Operational Programme for Competitiveness Factors(COMPETE) European Fund for Regional Development-FEDER and the Portuguese Foundation for Scienceand Technology (FCT) through Projects PTDCEBB-BIO1028632008 and PTDCEBB-BIO1049402008Luciana Gomes acknowledges the receipt of a PhD Grantfrom FCT (SFRHBD804002011) Joao Miranda and JoseAraujo (Transport Phenomena Research Center FEUP) areacknowledged for the numerical simulations

References

[1] J W Costerton P S Stewart and E P Greenberg ldquoBacterialbiofilms a common cause of persistent infectionsrdquo Science vol284 no 5418 pp 1318ndash1322 1999

Scientifica 5

[2] P Stoodley K Sauer D G Davies and J W CostertonldquoBiofilms as complex differentiated communitiesrdquo AnnualReview of Microbiology vol 56 pp 187ndash209 2002

[3] V Hancock L Ferrieres and P Klemm ldquoBiofilm formation byasymptomatic and virulent urinary tract infectious Escherichiacoli strainsrdquo FEMSMicrobiology Letters vol 267 no 1 pp 30ndash372007

[4] T Coenye and H J Nelis ldquoIn vitro and in vivo model systemsto studymicrobial biofilm formationrdquo Journal ofMicrobiologicalMethods vol 83 no 2 pp 89ndash105 2010

[5] J S Teodosio M Simoes L F Melo and F J Mergulhao ldquoPlat-forms for in vitro biofilm studiesrdquo in Biofilms in BioengineeringM Simoes and F Mergulhao Eds pp 45ndash62 Nova ScienceNew York NY USA 2013

[6] J M R Moreira L C Gomes J D P Araujo et al ldquoThe effectof glucose concentration and shaking conditions on Escherichiacoli biofilm formation in microtiter platesrdquo Chemical Engineer-ing Science vol 94 pp 192ndash199 2013

[7] E Peeters H J Nelis and T Coenye ldquoComparison of multiplemethods for quantification of microbial biofilms grown inmicrotiter platesrdquo Journal of Microbiological Methods vol 72no 2 pp 157ndash165 2008

[8] M Erriu F M G Pili E Tuveri et al ldquoOil essentialmouthwashes antibacterial activity againstAggregatibacter acti-nomycetemcomitans a comparison between antibiofilm andantiplanktonic effectsrdquo International Journal of Dentistry vol2013 Article ID 164267 5 pages 2013

[9] D Minardi O Cirioni R Ghiselli et al ldquoEfficacy of tigecyclineand rifampin alone and in combination against Enterococcusfaecalis biofilm infection in a ratmodel of ureteral stentrdquo Journalof Surgical Research vol 176 no 1 pp 1ndash6 2012

[10] C Sanchez K Mende M Beckius et al ldquoBiofilm formationby clinical isolates and the implications in chronic infectionsrdquoBMC Infectious Diseases vol 13 article 47 2013

[11] E Hell C Giske K Hultenby et al ldquoAttachment and biofilmforming capabilities of Staphylococcus epidermidis strains iso-lated from preterm infantsrdquo Current Microbiology vol 67 no 6pp 712ndash717 2013

[12] M-H Lin F-R Chang M-Y Hua Y-C Wu and S-T Liu ldquoInhibitory effects of 12346-penta-O-galloyl-120573-D-glucopyranose on biofilm formation by Staphylococcus aureusrdquoAntimicrobial Agents and Chemotherapy vol 55 no 3 pp 1021ndash1027 2011

[13] C De La Fuente-Nunez V Korolik M Bains et al ldquoInhibitionof bacterial biofilm formation and swarming motility by asmall synthetic cationic peptiderdquo Antimicrobial Agents andChemotherapy vol 56 no 5 pp 2696ndash2704 2012

[14] G D ChristensenW A Simpson and J J Younger ldquoAdherenceof coagulase-negative staphylococci to plastic tissue cultureplates a quantitative model for the adherence of staphylococcito medical devicesrdquo Journal of Clinical Microbiology vol 22 no6 pp 996ndash1006 1985

[15] X Li Z Yan and J Xu ldquoQuantitative variation of biofilmsamong strains in natural populations of Candida albicansrdquoMicrobiology vol 149 no 2 pp 353ndash362 2003

[16] S Stepanovic D Vukovic I Dakic B Savic and M Svabic-Vlahovic ldquoA modified microtiter-plate test for quantificationof staphylococcal biofilm formationrdquo Journal of MicrobiologicalMethods vol 40 no 2 pp 175ndash179 2000

[17] L Cremet S Corvec E Batard et al ldquoComparison of threemethods to study biofilm formation by clinical strains of

Escherichia colirdquoDiagnosticMicrobiology and Infectious Diseasevol 75 no 3 pp 252ndash255 2013

[18] L R Martinez and A Casadevall ldquoSpecific antibody canprevent fungal biofilm formation and this effect correlates withprotective efficacyrdquo Infection and Immunity vol 73 no 10 pp6350ndash6362 2005

[19] A Bridier D Le Coq F Dubois-Brissonnet V Thomas SAymerich and R Briandet ldquoThe spatial architecture of Bacillussubtilis biofilms deciphered using a surface-associated modeland in situ imagingrdquo PLoS ONE vol 6 no 1 Article ID e161772011

[20] V Kostenko M M Salek P Sattari and R J MartinuzzildquoStaphylococcus aureus biofilm formation and tolerance toantibiotics in response to oscillatory shear stresses of physiolog-ical levelsrdquo FEMS Immunology and Medical Microbiology vol59 no 3 pp 421ndash431 2010

[21] J S Teodosio M Simoes and F J Mergulhao ldquoThe influence ofnonconjugative Escherichia coli plasmids on biofilm formationand resistancerdquo Journal of Applied Microbiology vol 113 no 2pp 373ndash382 2012

[22] J S Teodosio M Simoes L F Melo and F J MergulhaoldquoFlow cell hydrodynamics and their effects on E coli biofilmformation under different nutrient conditions and turbulentflowrdquo Biofouling vol 27 no 1 pp 1ndash11 2011

[23] S Shakeri R K Kermanshahi M M Moghaddam and GEmtiazi ldquoAssessment of biofilm cell removal and killing andbiocide efficacy using the microtiter plate testrdquo Biofouling vol23 no 2 pp 79ndash86 2007

[24] L Gomes J M R Moreira J M Miranda et al ldquoMacroscaleversusmicroscalemethods for physiological analysis of biofilmsformed in 96-well microtiter platesrdquo Journal of MicrobiologicalMethods vol 95 no 3 pp 342ndash349 2013

[25] Y Liu and J-H Tay ldquoThe essential role of hydrodynamic shearforce in the formation of biofilm and granular sludgerdquo WaterResearch vol 36 no 7 pp 1653ndash1665 2002

[26] P Stoodley S Wilson L Hall-Stoodley J D Boyle H MLappin-Scott and J W Costerton ldquoGrowth and detachment ofcell clusters from mature mixed-species biofilmsrdquo Applied andEnvironmentalMicrobiology vol 67 no 12 pp 5608ndash5613 2001

[27] S Wasche H Horn and D C Hempel ldquoInfluence of growthconditions on biofilm development and mass transfer at thebulkbiofilm interfacerdquoWater Research vol 36 no 19 pp 4775ndash4784 2002

[28] H J Busscher and H C van der Mei ldquoMicrobial adhesion inflow displacement systemsrdquo Clinical Microbiology Reviews vol19 no 1 pp 127ndash141 2006

[29] M Simoes M O Pereira S Sillankorva J Azeredo andM J Vieira ldquoThe effect of hydrodynamic conditions on thephenotype of Pseudomonas fluorescens biofilmsrdquoBiofouling vol23 no 4 pp 249ndash258 2007

[30] J S Teodosio F C Silva J M R Moreira et al ldquoFlow cellsas quasi ideal systems for biofouling simulation of industrialpiping systemsrdquo Biofouling vol 29 no 8 pp 953ndash966 2013

[31] M C M Van Loosdrecht D Eikelboom A Gjaltema AMulder L Tijhuis and J J Heijnen ldquoBiofilm structuresrdquoWaterScience and Technology vol 32 no 8 pp 35ndash43 1995

[32] J M R Moreira J S Teodosio F C Silva et al ldquoInfluenceof flow rate variation on the development of Escherichia colibiofilmsrdquo Bioprocess and Biosystems Engineering vol 36 no 11pp 1787ndash1796 2013

6 Scientifica

[33] WKKwokC Picioreanu S LOng et al ldquoInfluence of biomassproduction and detachment forces on biofilm structures in abiofilm airlift suspension reactorrdquo Biotechnology and Bioengi-neering vol 58 no 4 pp 400ndash407 1998

[34] A Rochex J-J Godon N Bernet and R Escudie ldquoRole ofshear stress on composition diversity and dynamics of biofilmbacterial communitiesrdquo Water Research vol 42 no 20 pp4915ndash4922 2008

[35] A Bridier T Meylheuc and R Briandet ldquoRealistic represen-tation of Bacillus subtilis biofilms architecture using combinedmicroscopy (CLSM ESEM and FESEM)rdquo Micron vol 48 pp65ndash69 2013

[36] M G White S Piccirillo V Dusevich D J Law T Kaprosand S M Honigberg ldquoFlo11p adhesin required for meioticdifferentiation in Saccharomyces cerevisiaeminicolonies grownon plastic surfacesrdquo FEMS Yeast Research vol 11 no 2 pp 223ndash232 2011

[37] C L Quave M Estevez-Carmona C M Compadre et alldquoEllagic acid derivatives from Rubus ulmifolius inhibit Staphy-lococcus aureus biofilm formation and improve response toantibioticsrdquo PLoS ONE vol 7 no 1 Article ID e28737 2012

[38] A Lizcano T Chin K Sauer E I Tuomanen and C JOrihuela ldquoEarly biofilm formation on microtiter plates is notcorrelated with the invasive disease potential of Streptococcuspneumoniaerdquo Microbial Pathogenesis vol 48 no 3-4 pp 124ndash130 2010

[39] A S Melo A L Colombo and B A Arthington-Skaggs ldquoPara-doxical growth effect of caspofungin observed on biofilms andplanktonic cells of five differentCandida speciesrdquoAntimicrobialAgents and Chemotherapy vol 51 no 9 pp 3081ndash3088 2007

[40] W A Duetz ldquoMicrotiter plates as mini-bioreactors miniatur-ization of fermentation methodsrdquo Trends in Microbiology vol15 no 10 pp 469ndash475 2007

[41] K Honraet E Goetghebeur and H J Nelis ldquoComparisonof three assays for the quantification of Candida biomassin suspension and CDC reactor grown biofilmsrdquo Journal ofMicrobiological Methods vol 63 no 3 pp 287ndash295 2005

[42] S Kumar C Wittmann and E Heinzle ldquoReview minibioreac-torsrdquo Biotechnology Letters vol 26 no 1 pp 1ndash10 2004

[43] N F Azevedo S P Lopes C W Keevil M O Pereira and MJ Vieira ldquoTime to ldquogo largerdquo on biofilm research advantages ofan omics approachrdquoBiotechnology Letters vol 31 no 4 pp 477ndash485 2009

[44] K Tote D V Berghe L Maes and P Cos ldquoA new colorimetricmicrotitre model for the detection of Staphylococcus aureusbiofilmsrdquo Letters in AppliedMicrobiology vol 46 no 2 pp 249ndash254 2008

[45] T A Norton R C Thompson J Pope et al ldquoUsing confocallaser scanning microscopy scanning electron microscopy andphase contrast light microscopy to examine marine biofilmsrdquoAquatic Microbial Ecology vol 16 no 2 pp 199ndash204 1998

[46] D O Serra A M Richter G Klauck et al ldquoMicroanatomy atcellular resolution and spatial order of physiological differen-tiation in a bacterial biofilmrdquo mBio vol 4 no 2 pp e100103ndashe100113 2013

[47] P S Stewart R Murga R Srinivasan and D De Beer ldquoBiofilmstructural heterogeneity visualized by three microscopic meth-odsrdquoWater Research vol 29 no 8 pp 2006ndash2009 1995

[48] S B Surman J T Walker D T Goddard et al ldquoComparison ofmicroscope techniques for the examination of biofilmsrdquo Journalof Microbiological Methods vol 25 no 1 pp 57ndash70 1996

[49] C Hannig M Follo E Hellwig and A Al-Ahmad ldquoVisualiza-tion of adherent micro-organisms using different techniquesrdquoJournal of Medical Microbiology vol 59 no 1 pp 1ndash7 2010

[50] A Bridier F Dubois-Brissonnet A Boubetra V Thomas andR Briandet ldquoThe biofilm architecture of sixty opportunisticpathogens deciphered using a high throughput CLSMmethodrdquoJournal of Microbiological Methods vol 82 no 1 pp 64ndash702010

[51] M Fedel P Caciagli V Chiste et al ldquoMicrobial biofilm imagingESEM vs HVSEMrdquo Imaging ampMicroscopy vol 9 no 2 pp 44ndash47 2007

[52] R J Palmer Jr J J Haagensen T Neu and C Sternberg ldquoCon-focal microscopy of biofilmsmdashspatiotemporal approachesrdquo inHandbook of Biological Confocal Microscopy J B Pawley Edpp 870ndash888 Springer New York NY USA 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Scientifica 5

[2] P Stoodley K Sauer D G Davies and J W CostertonldquoBiofilms as complex differentiated communitiesrdquo AnnualReview of Microbiology vol 56 pp 187ndash209 2002

[3] V Hancock L Ferrieres and P Klemm ldquoBiofilm formation byasymptomatic and virulent urinary tract infectious Escherichiacoli strainsrdquo FEMSMicrobiology Letters vol 267 no 1 pp 30ndash372007

[4] T Coenye and H J Nelis ldquoIn vitro and in vivo model systemsto studymicrobial biofilm formationrdquo Journal ofMicrobiologicalMethods vol 83 no 2 pp 89ndash105 2010

[5] J S Teodosio M Simoes L F Melo and F J Mergulhao ldquoPlat-forms for in vitro biofilm studiesrdquo in Biofilms in BioengineeringM Simoes and F Mergulhao Eds pp 45ndash62 Nova ScienceNew York NY USA 2013

[6] J M R Moreira L C Gomes J D P Araujo et al ldquoThe effectof glucose concentration and shaking conditions on Escherichiacoli biofilm formation in microtiter platesrdquo Chemical Engineer-ing Science vol 94 pp 192ndash199 2013

[7] E Peeters H J Nelis and T Coenye ldquoComparison of multiplemethods for quantification of microbial biofilms grown inmicrotiter platesrdquo Journal of Microbiological Methods vol 72no 2 pp 157ndash165 2008

[8] M Erriu F M G Pili E Tuveri et al ldquoOil essentialmouthwashes antibacterial activity againstAggregatibacter acti-nomycetemcomitans a comparison between antibiofilm andantiplanktonic effectsrdquo International Journal of Dentistry vol2013 Article ID 164267 5 pages 2013

[9] D Minardi O Cirioni R Ghiselli et al ldquoEfficacy of tigecyclineand rifampin alone and in combination against Enterococcusfaecalis biofilm infection in a ratmodel of ureteral stentrdquo Journalof Surgical Research vol 176 no 1 pp 1ndash6 2012

[10] C Sanchez K Mende M Beckius et al ldquoBiofilm formationby clinical isolates and the implications in chronic infectionsrdquoBMC Infectious Diseases vol 13 article 47 2013

[11] E Hell C Giske K Hultenby et al ldquoAttachment and biofilmforming capabilities of Staphylococcus epidermidis strains iso-lated from preterm infantsrdquo Current Microbiology vol 67 no 6pp 712ndash717 2013

[12] M-H Lin F-R Chang M-Y Hua Y-C Wu and S-T Liu ldquoInhibitory effects of 12346-penta-O-galloyl-120573-D-glucopyranose on biofilm formation by Staphylococcus aureusrdquoAntimicrobial Agents and Chemotherapy vol 55 no 3 pp 1021ndash1027 2011

[13] C De La Fuente-Nunez V Korolik M Bains et al ldquoInhibitionof bacterial biofilm formation and swarming motility by asmall synthetic cationic peptiderdquo Antimicrobial Agents andChemotherapy vol 56 no 5 pp 2696ndash2704 2012

[14] G D ChristensenW A Simpson and J J Younger ldquoAdherenceof coagulase-negative staphylococci to plastic tissue cultureplates a quantitative model for the adherence of staphylococcito medical devicesrdquo Journal of Clinical Microbiology vol 22 no6 pp 996ndash1006 1985

[15] X Li Z Yan and J Xu ldquoQuantitative variation of biofilmsamong strains in natural populations of Candida albicansrdquoMicrobiology vol 149 no 2 pp 353ndash362 2003

[16] S Stepanovic D Vukovic I Dakic B Savic and M Svabic-Vlahovic ldquoA modified microtiter-plate test for quantificationof staphylococcal biofilm formationrdquo Journal of MicrobiologicalMethods vol 40 no 2 pp 175ndash179 2000

[17] L Cremet S Corvec E Batard et al ldquoComparison of threemethods to study biofilm formation by clinical strains of

Escherichia colirdquoDiagnosticMicrobiology and Infectious Diseasevol 75 no 3 pp 252ndash255 2013

[18] L R Martinez and A Casadevall ldquoSpecific antibody canprevent fungal biofilm formation and this effect correlates withprotective efficacyrdquo Infection and Immunity vol 73 no 10 pp6350ndash6362 2005

[19] A Bridier D Le Coq F Dubois-Brissonnet V Thomas SAymerich and R Briandet ldquoThe spatial architecture of Bacillussubtilis biofilms deciphered using a surface-associated modeland in situ imagingrdquo PLoS ONE vol 6 no 1 Article ID e161772011

[20] V Kostenko M M Salek P Sattari and R J MartinuzzildquoStaphylococcus aureus biofilm formation and tolerance toantibiotics in response to oscillatory shear stresses of physiolog-ical levelsrdquo FEMS Immunology and Medical Microbiology vol59 no 3 pp 421ndash431 2010

[21] J S Teodosio M Simoes and F J Mergulhao ldquoThe influence ofnonconjugative Escherichia coli plasmids on biofilm formationand resistancerdquo Journal of Applied Microbiology vol 113 no 2pp 373ndash382 2012

[22] J S Teodosio M Simoes L F Melo and F J MergulhaoldquoFlow cell hydrodynamics and their effects on E coli biofilmformation under different nutrient conditions and turbulentflowrdquo Biofouling vol 27 no 1 pp 1ndash11 2011

[23] S Shakeri R K Kermanshahi M M Moghaddam and GEmtiazi ldquoAssessment of biofilm cell removal and killing andbiocide efficacy using the microtiter plate testrdquo Biofouling vol23 no 2 pp 79ndash86 2007

[24] L Gomes J M R Moreira J M Miranda et al ldquoMacroscaleversusmicroscalemethods for physiological analysis of biofilmsformed in 96-well microtiter platesrdquo Journal of MicrobiologicalMethods vol 95 no 3 pp 342ndash349 2013

[25] Y Liu and J-H Tay ldquoThe essential role of hydrodynamic shearforce in the formation of biofilm and granular sludgerdquo WaterResearch vol 36 no 7 pp 1653ndash1665 2002

[26] P Stoodley S Wilson L Hall-Stoodley J D Boyle H MLappin-Scott and J W Costerton ldquoGrowth and detachment ofcell clusters from mature mixed-species biofilmsrdquo Applied andEnvironmentalMicrobiology vol 67 no 12 pp 5608ndash5613 2001

[27] S Wasche H Horn and D C Hempel ldquoInfluence of growthconditions on biofilm development and mass transfer at thebulkbiofilm interfacerdquoWater Research vol 36 no 19 pp 4775ndash4784 2002

[28] H J Busscher and H C van der Mei ldquoMicrobial adhesion inflow displacement systemsrdquo Clinical Microbiology Reviews vol19 no 1 pp 127ndash141 2006

[29] M Simoes M O Pereira S Sillankorva J Azeredo andM J Vieira ldquoThe effect of hydrodynamic conditions on thephenotype of Pseudomonas fluorescens biofilmsrdquoBiofouling vol23 no 4 pp 249ndash258 2007

[30] J S Teodosio F C Silva J M R Moreira et al ldquoFlow cellsas quasi ideal systems for biofouling simulation of industrialpiping systemsrdquo Biofouling vol 29 no 8 pp 953ndash966 2013

[31] M C M Van Loosdrecht D Eikelboom A Gjaltema AMulder L Tijhuis and J J Heijnen ldquoBiofilm structuresrdquoWaterScience and Technology vol 32 no 8 pp 35ndash43 1995

[32] J M R Moreira J S Teodosio F C Silva et al ldquoInfluenceof flow rate variation on the development of Escherichia colibiofilmsrdquo Bioprocess and Biosystems Engineering vol 36 no 11pp 1787ndash1796 2013

6 Scientifica

[33] WKKwokC Picioreanu S LOng et al ldquoInfluence of biomassproduction and detachment forces on biofilm structures in abiofilm airlift suspension reactorrdquo Biotechnology and Bioengi-neering vol 58 no 4 pp 400ndash407 1998

[34] A Rochex J-J Godon N Bernet and R Escudie ldquoRole ofshear stress on composition diversity and dynamics of biofilmbacterial communitiesrdquo Water Research vol 42 no 20 pp4915ndash4922 2008

[35] A Bridier T Meylheuc and R Briandet ldquoRealistic represen-tation of Bacillus subtilis biofilms architecture using combinedmicroscopy (CLSM ESEM and FESEM)rdquo Micron vol 48 pp65ndash69 2013

[36] M G White S Piccirillo V Dusevich D J Law T Kaprosand S M Honigberg ldquoFlo11p adhesin required for meioticdifferentiation in Saccharomyces cerevisiaeminicolonies grownon plastic surfacesrdquo FEMS Yeast Research vol 11 no 2 pp 223ndash232 2011

[37] C L Quave M Estevez-Carmona C M Compadre et alldquoEllagic acid derivatives from Rubus ulmifolius inhibit Staphy-lococcus aureus biofilm formation and improve response toantibioticsrdquo PLoS ONE vol 7 no 1 Article ID e28737 2012

[38] A Lizcano T Chin K Sauer E I Tuomanen and C JOrihuela ldquoEarly biofilm formation on microtiter plates is notcorrelated with the invasive disease potential of Streptococcuspneumoniaerdquo Microbial Pathogenesis vol 48 no 3-4 pp 124ndash130 2010

[39] A S Melo A L Colombo and B A Arthington-Skaggs ldquoPara-doxical growth effect of caspofungin observed on biofilms andplanktonic cells of five differentCandida speciesrdquoAntimicrobialAgents and Chemotherapy vol 51 no 9 pp 3081ndash3088 2007

[40] W A Duetz ldquoMicrotiter plates as mini-bioreactors miniatur-ization of fermentation methodsrdquo Trends in Microbiology vol15 no 10 pp 469ndash475 2007

[41] K Honraet E Goetghebeur and H J Nelis ldquoComparisonof three assays for the quantification of Candida biomassin suspension and CDC reactor grown biofilmsrdquo Journal ofMicrobiological Methods vol 63 no 3 pp 287ndash295 2005

[42] S Kumar C Wittmann and E Heinzle ldquoReview minibioreac-torsrdquo Biotechnology Letters vol 26 no 1 pp 1ndash10 2004

[43] N F Azevedo S P Lopes C W Keevil M O Pereira and MJ Vieira ldquoTime to ldquogo largerdquo on biofilm research advantages ofan omics approachrdquoBiotechnology Letters vol 31 no 4 pp 477ndash485 2009

[44] K Tote D V Berghe L Maes and P Cos ldquoA new colorimetricmicrotitre model for the detection of Staphylococcus aureusbiofilmsrdquo Letters in AppliedMicrobiology vol 46 no 2 pp 249ndash254 2008

[45] T A Norton R C Thompson J Pope et al ldquoUsing confocallaser scanning microscopy scanning electron microscopy andphase contrast light microscopy to examine marine biofilmsrdquoAquatic Microbial Ecology vol 16 no 2 pp 199ndash204 1998

[46] D O Serra A M Richter G Klauck et al ldquoMicroanatomy atcellular resolution and spatial order of physiological differen-tiation in a bacterial biofilmrdquo mBio vol 4 no 2 pp e100103ndashe100113 2013

[47] P S Stewart R Murga R Srinivasan and D De Beer ldquoBiofilmstructural heterogeneity visualized by three microscopic meth-odsrdquoWater Research vol 29 no 8 pp 2006ndash2009 1995

[48] S B Surman J T Walker D T Goddard et al ldquoComparison ofmicroscope techniques for the examination of biofilmsrdquo Journalof Microbiological Methods vol 25 no 1 pp 57ndash70 1996

[49] C Hannig M Follo E Hellwig and A Al-Ahmad ldquoVisualiza-tion of adherent micro-organisms using different techniquesrdquoJournal of Medical Microbiology vol 59 no 1 pp 1ndash7 2010

[50] A Bridier F Dubois-Brissonnet A Boubetra V Thomas andR Briandet ldquoThe biofilm architecture of sixty opportunisticpathogens deciphered using a high throughput CLSMmethodrdquoJournal of Microbiological Methods vol 82 no 1 pp 64ndash702010

[51] M Fedel P Caciagli V Chiste et al ldquoMicrobial biofilm imagingESEM vs HVSEMrdquo Imaging ampMicroscopy vol 9 no 2 pp 44ndash47 2007

[52] R J Palmer Jr J J Haagensen T Neu and C Sternberg ldquoCon-focal microscopy of biofilmsmdashspatiotemporal approachesrdquo inHandbook of Biological Confocal Microscopy J B Pawley Edpp 870ndash888 Springer New York NY USA 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 Scientifica

[33] WKKwokC Picioreanu S LOng et al ldquoInfluence of biomassproduction and detachment forces on biofilm structures in abiofilm airlift suspension reactorrdquo Biotechnology and Bioengi-neering vol 58 no 4 pp 400ndash407 1998

[34] A Rochex J-J Godon N Bernet and R Escudie ldquoRole ofshear stress on composition diversity and dynamics of biofilmbacterial communitiesrdquo Water Research vol 42 no 20 pp4915ndash4922 2008

[35] A Bridier T Meylheuc and R Briandet ldquoRealistic represen-tation of Bacillus subtilis biofilms architecture using combinedmicroscopy (CLSM ESEM and FESEM)rdquo Micron vol 48 pp65ndash69 2013

[36] M G White S Piccirillo V Dusevich D J Law T Kaprosand S M Honigberg ldquoFlo11p adhesin required for meioticdifferentiation in Saccharomyces cerevisiaeminicolonies grownon plastic surfacesrdquo FEMS Yeast Research vol 11 no 2 pp 223ndash232 2011

[37] C L Quave M Estevez-Carmona C M Compadre et alldquoEllagic acid derivatives from Rubus ulmifolius inhibit Staphy-lococcus aureus biofilm formation and improve response toantibioticsrdquo PLoS ONE vol 7 no 1 Article ID e28737 2012

[38] A Lizcano T Chin K Sauer E I Tuomanen and C JOrihuela ldquoEarly biofilm formation on microtiter plates is notcorrelated with the invasive disease potential of Streptococcuspneumoniaerdquo Microbial Pathogenesis vol 48 no 3-4 pp 124ndash130 2010

[39] A S Melo A L Colombo and B A Arthington-Skaggs ldquoPara-doxical growth effect of caspofungin observed on biofilms andplanktonic cells of five differentCandida speciesrdquoAntimicrobialAgents and Chemotherapy vol 51 no 9 pp 3081ndash3088 2007

[40] W A Duetz ldquoMicrotiter plates as mini-bioreactors miniatur-ization of fermentation methodsrdquo Trends in Microbiology vol15 no 10 pp 469ndash475 2007

[41] K Honraet E Goetghebeur and H J Nelis ldquoComparisonof three assays for the quantification of Candida biomassin suspension and CDC reactor grown biofilmsrdquo Journal ofMicrobiological Methods vol 63 no 3 pp 287ndash295 2005

[42] S Kumar C Wittmann and E Heinzle ldquoReview minibioreac-torsrdquo Biotechnology Letters vol 26 no 1 pp 1ndash10 2004

[43] N F Azevedo S P Lopes C W Keevil M O Pereira and MJ Vieira ldquoTime to ldquogo largerdquo on biofilm research advantages ofan omics approachrdquoBiotechnology Letters vol 31 no 4 pp 477ndash485 2009

[44] K Tote D V Berghe L Maes and P Cos ldquoA new colorimetricmicrotitre model for the detection of Staphylococcus aureusbiofilmsrdquo Letters in AppliedMicrobiology vol 46 no 2 pp 249ndash254 2008

[45] T A Norton R C Thompson J Pope et al ldquoUsing confocallaser scanning microscopy scanning electron microscopy andphase contrast light microscopy to examine marine biofilmsrdquoAquatic Microbial Ecology vol 16 no 2 pp 199ndash204 1998

[46] D O Serra A M Richter G Klauck et al ldquoMicroanatomy atcellular resolution and spatial order of physiological differen-tiation in a bacterial biofilmrdquo mBio vol 4 no 2 pp e100103ndashe100113 2013

[47] P S Stewart R Murga R Srinivasan and D De Beer ldquoBiofilmstructural heterogeneity visualized by three microscopic meth-odsrdquoWater Research vol 29 no 8 pp 2006ndash2009 1995

[48] S B Surman J T Walker D T Goddard et al ldquoComparison ofmicroscope techniques for the examination of biofilmsrdquo Journalof Microbiological Methods vol 25 no 1 pp 57ndash70 1996

[49] C Hannig M Follo E Hellwig and A Al-Ahmad ldquoVisualiza-tion of adherent micro-organisms using different techniquesrdquoJournal of Medical Microbiology vol 59 no 1 pp 1ndash7 2010

[50] A Bridier F Dubois-Brissonnet A Boubetra V Thomas andR Briandet ldquoThe biofilm architecture of sixty opportunisticpathogens deciphered using a high throughput CLSMmethodrdquoJournal of Microbiological Methods vol 82 no 1 pp 64ndash702010

[51] M Fedel P Caciagli V Chiste et al ldquoMicrobial biofilm imagingESEM vs HVSEMrdquo Imaging ampMicroscopy vol 9 no 2 pp 44ndash47 2007

[52] R J Palmer Jr J J Haagensen T Neu and C Sternberg ldquoCon-focal microscopy of biofilmsmdashspatiotemporal approachesrdquo inHandbook of Biological Confocal Microscopy J B Pawley Edpp 870ndash888 Springer New York NY USA 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology