Methods: General Techniques OEST 740

Transcription

1 Methods: General Techniques OEST

2 Outline Introduction Techniques for Growth Flow and steady state methods Microscopici Adherence Formation Physiology and Structure Chemical analysis Quantitative Analysis of Biofilm formation

3 Introduction Essential elements to understanding the function of biofilms (Beech et al. 2002) determining factors influencing the processes of bacterial adhesion and biofilm formation spatial arrangement of biofilms Including distribution and composition of microorganisms within the matrix characterizing i the properties of the matrix

4 Introduction Commonly focuses on two primary stages of biofilm development elopment Bacterial Adhesion A 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 biofilm formations. Biofilm Formation

5 Biofilm Formation Biofilm characteristics Introduction Structure (thickness, differentiation, homogeneity and bacterial density) Biofilm accumulation and inhibition Biofilm composition Chemical constituents (proteins, DNA, humic compounds and EPS)

6 Introduction Historical research on biofilm formation has relied on Various microscopic techniques Bacterial counts Rely on indirect proxies Clld Cell density Biofilm thickness CFUs

7 Robbins Device Low pressure flow applications Sampling ports removed and replaced aseptically Inoculation Exponential phase culture injection Continuous fermentation vessel

8 Robbin s Device Bacterial Adherence Immune response to biofilm bacteria Physiology and metabolism of biofilm bacteria Regulation of bacterial genes and products Resistance to antimicrobial compounds

9 Laminar Flow Adhesion Cells Multipurpose good reproducibility Continuous Monitoring Biofilm formation and succession

10 CDC Biofilm Reactor (CBR) 24 removable biofilm growth surfaces Operates under moderate to high shear Detecting biofilm formation Characterizing structure Effect of anti-microbial i agents Batch Continuous flow conditions i

11 Transmitted Light Microscopy General technique Initial colonization and film formation

12 Inference Reflection Microscopy Bacterial adhesion to surface (IRM) Film or polymer generates an interference pattern in the reflected light Separation distance of nm Intensity correlated to thickness

13 Atomic force Microscopy Interactions between bacteria and surfaces Relies on cantilever tip to scan the surface of the specimen. When tip is brought near the specimens surface, forces deflect the tip in accordance with Hooke s Law F = -k x

14 v AFM

15 Fluorescence Microscopy Uses opaque surfaces and dfluorescent labels to visualize biofilms effectively determine and localize biofilm components Quantify bacteria counts and densities es

16 Confocal laser scanning microscopy Direct, non-invasive optical sectioning provides images which are free from out of focus blur Can examine different layers of bacterial biofilms on natural substrata t Not sensitive to microtopography Used to study the effects of chemical treatments on biofilm structure Proves information on biofilm thickness and structure Can also provide information of cell density When combined with fluorescent probes can also be used dto visualize and quantify biofilm components

17 Confocal laser scanning microscopy

18 Confocal laser scanning microscopy Lectins are commercially available with fluorescent labels l Working stock 100 µg/ml applied for mins Glycoconjugate distribution Probes fluorescent dyes that change emission i spectra in response to intracellular activity (e.g. enzymes) or parameter (e.g. ph). Physiological status, metabolic activities, gene expression, total cell densities

19 Confocal laser scanning microscopy K. Mojica

20 Scanning electron Microscopy (SEM) Good depth of focus Clear images of organisms on opaque substrata across a wide range of magnifications Only used to examine a film s surface because lower layers become obscured Preparation kills the constituent organisms and shrivels film destroying the structure misrepresents some organisms

21 (SEM)

22 Chemical Composition Extraction of extracellular polymeric substances using cation exchange resin Especially suitable for humic substances and proteins but also useful for carbohydrates Using 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 using specific individual methods Ex. TOC can be quantified after acidification and purging of inorganic carbon with persulfate oxidation and evaluated using a analytical carbon analyzer

23 Chemical Composition Exopolysaccharides Can be analyzed using chemical or microscopic methods Biochemical methods typically involves the isolation by extraction procedures that can result in the disruption of the biofilm matrix Goal: To contribute a technique to directly measure a component of the microbial EPS matrix of microbial biofilms in a manner that is amendable to high throughput technologies.

24 Summary Microbiologist are now appreciating that biofilms are important features of microbial growth. A large number of devices are currently employed 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.

25 Summary The future of biofilm research is still in its infancy. Biofilm research will continue to make exciting discoveries as our techniques to characterize and describe biofilm processes improves.