Methods: General Techniques

OEST 740042108042108

OutlineIntroductionTechniques for GrowthTechniques for Growth

Flow and steady state methodsMi iMicroscopic

Adherence FormationPhysiology and Structure

Chemical analysisQuantitative Analysis of BiofilmQuantitative Analysis of Biofilm formation

Introduction

Essential elements to understanding h f i f bi fil (B h lthe function of biofilms (Beech et al.

2002)determining factors influencing the processes of

bacterial adhesion and biofilm formationspatial arrangement of biofilms

Including distribution and composition of microorganisms within the matrix

h i i h i f h icharacterizing the properties of the matrix

IntroductionCommonly focuses on two primary stages of biofilm de elopmentof biofilm development

Bacterial Adhesion A di t d tit ti f ifi llA direct and quantitative means for specifically measuring the long-range interactions between bacteria and surfaces

can provide important information to direct the design of materials refractile to bacterial adhesion and for the control of biofilmadhesion and for the control of biofilm formations.

Biofilm FormationBiofilm Formation

IntroductionBiofilm Formation

Biofilm characteristicsBiofilm characteristicsStructure (thickness, differentiation, homogeneity and bacterial density)Biofilm accumulation and inhibition

Biofilm compositionChemical constituents (proteins, DNA, humic compounds and EPS)

IntroductionHistorical research on biofilm formation has relied onhas relied on

Various microscopic techniquesBacterial counts

Rely on indirect proxiesC ll d itCell density Biofilm thicknessCFUsCFUs

Robbins DeviceLow pressure flow applicationsSampling ports – removed and replaced asepticallyInoculation

Exponential phase culture injectionContinuous fermentation vessel

Robbin’s Device

Bacterial AdherenceBacterial AdherenceImmune response to biofilm bacteria

i i f i fiPhysiology and metabolism of biofilm bacteriaRegulation of bacterial genes and productsResistance to antimicrobial compoundsp

Laminar Flow Adhesion Cells

Multipurposegood reproducibilitygood reproducibility

Continuous MonitoringMonitoring Biofilm formation and succession

CDC Biofilm Reactor (CBR)

24 removable biofilm growth24 removable biofilm growth surfacesOperates under moderate toOperates under moderate to high shearDetecting biofilm formationDetecting biofilm formationCharacterizing structureEff t f ti i bi l tEffect of anti-microbial agents

Batch C i fl di iContinuous flow conditions

Transmitted Light MicroscopyTransmitted Light MicroscopyGeneral techniqueInitial colonization and film formation

Inference Reflection Microscopy (IRM)

Bacterial adhesion toBacterial adhesion to surface

Fil l tFilm or polymer generates an interference pattern in the reflected lightreflected lightSeparation distance of 0 –100 nm100 nmIntensity correlated to thickness

Atomic force MicroscopyInteractions between bacteria and s rfacesbacteria and surfacesRelies on cantilever tip to scan the surfacetip to scan the surface of the specimen. When tip is broughtWhen tip is brought near the specimens surface, forces deflect the tip in accordance with Hooke’s Law

F kF = -k x

v

AFM

Fluorescence Microscopy

Uses opaque surfaces d fland fluorescent

labels to visualize biofilmsbiofilms

effectively determine and localize biofilm

tcomponentsQuantify bacteria counts and densitiescou ts a d de s t es

Confocal laser scanning microscopyDirect, non-invasive optical sectioningprovides images which are free from out of focusprovides images which are free from out of focus blur

Can examine different layers of bacterial bi fil t l b t tbiofilms on natural substrata

Not sensitive to microtopographyUsed to study the effects of chemical treatmentsUsed to study the effects of chemical treatments on biofilm structure

Proves information on biofilm thickness and structurestructureCan also provide information of cell density

When combined with fluorescent probes can l b d t i li d tif bi filalso be used to visualize and quantify biofilm

components

Confocal laser scanning microscopymicroscopy

Confocal laser scanning microscopy

Lectins are commercially available with fl t l b lfluorescent labels

Working stock – 100 µg/ml – applied for 30-45 imins

Glycoconjugate distributionP b fl d h h i iProbes – fluorescent dyes that change emission spectra in response to intracellular activity ( ) t ( H)(e.g. enzymes) or parameter (e.g. pH).

Physiological status, metabolic activities, gene expression total cell densitiesexpression, total cell densities

Confocal laser scanning microscopy

K. MojicaK. Mojica

Scanning electron Microscopy (SEM)(SEM)

Good depth of focusClear images of organisms on opaque substrata across a wide range of magnificationsmagnifications

Only used to examine a film’s surface because lower layers become obscuredPreparation kills the constituent organisms and shrivels film

destroying the structuredestroying the structuremisrepresents some organisms

(SEM)

Chemical CompositionChemical CompositionExtraction of extracellular polymeric substances using p y gcation exchange resin

Especially suitable for humic substances and proteins but also useful for carbohydratesUsing DOWEX resin allow for the substitution of divalent cations with monovalent cations

Leads to loosening or weakening of biofilm matrix structure Physical extraction (EPS yield) sensitive to stirring intensity, amount of DOWEX, extraction time and extraction volume

After extraction the quantity of TOC, carbohydrates, uronic acids, DNA, protein and humic substances can be determined usingacids, DNA, protein and humic substances can be determined using specific individual methods

Ex. TOC can be quantified after acidification and purging of inorganic carbon with persulfate oxidation and evaluated using a analytical

b lcarbon analyzer

Chemical Composition

ExopolysaccharidesCan be analyzed using chemical or microscopic methodsBiochemical methods typically involves theBiochemical methods typically involves the isolation by extraction procedures that can result in the disruption of the biofilm matrixp

Goal: To contribute a technique to directly measure a component of the microbial EPSmeasure a component of the microbial EPS matrix of microbial biofilms in a manner that is amendable to high throughputthat is amendable to high throughput technologies.

SummarySummaryMicrobiologist are now appreciating that biofilms are important features of microbial growth.A large number of devices are currently g yemployed to grow biofilms under laboratory conditions.The future development of universal techniques that can control all aspects of features influencing biofilm formation and allow for across laboratory comparisons will continue to improve advances in biofilm research.

SummarySummary

The future of biofilm research is still in its infancy.Biofilm research will continue to makeBiofilm research will continue to make exciting discoveries as our techniques t h t i d d ib bi filto characterize and describe biofilm processes improves.