Methods: Quantitative Techniques - University of...
Transcript of Methods: Quantitative Techniques - University of...
![Page 1: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/1.jpg)
Methods: Quantitative QTechniques
OEST 740050608050608
![Page 2: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/2.jpg)
Conventional Techniques
Dry weightTetrazolium Reduction Assay
Tetrazolium salts – colorimetric determinates measured spectrophometricallyReduced by mitochondrial ydehydrogenases
MTT – violet – DMSOXTT – brown – easier –water soluble product
Biofilm formation Hawser & Douglas 1994
![Page 3: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/3.jpg)
Conventional TechniquesRadiolabling
I i f [3H] L iIncorporation of [3H] LeucinePrecipitated by TCA and radioactivity measured Used to assess the effects of antibiotics, biofilm formation Dix et al. 1988
E. coli, Candida spp.
Hawser & Douglas 1994
![Page 4: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/4.jpg)
Conventional Techniques
Imaging(ex. Confocal Laser Scanning Microscopy)
Thickness, cell density, composition
Cell countsDirect viable cell counts (DVC)
To asses disinfection efficiency
Plate countsEstimate survival populations after disinfection
![Page 5: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/5.jpg)
MBEC AssayRelies on CFU’s as a proxy for the presence
f i bi l bi filof a microbial biofilmAssumes bacteria are only present if they formed a biofilmPro: high-throughput, commercially g g p , yavailableCon: does not delineateCon: does not delineate other effects
![Page 6: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/6.jpg)
MBEC AssayMBEC AssayPrimarily utilized to analyze the effects of chemical treatment on biofilm formation.
Harrison et al. 2005
![Page 7: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/7.jpg)
MBEC Assayy
Harrison et al 2005Harrison et al. 2005
![Page 8: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/8.jpg)
MBEC Assay
Relies on the recovery of attached cells from substratum exposed to chemical treatment.
Harrison et al. 2005
![Page 9: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/9.jpg)
MBEC Assay
Provides 3 comparison measurements in each experiment: MIC, MBC and MBEC
Harrison et al. 2005
![Page 10: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/10.jpg)
MBEC Assayy
Growth of bacterial biofilms in MBEC assay have been found to be very reproducible
Harrison et al. 2005
![Page 11: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/11.jpg)
Electrochemical Impedancemetry
Electrochemical Impedance SpectroscopyBacterial adhesion and biofilm proliferationUtilizes a one-compartment, 3-electrode cellp ,
Working electrodesGrids - colonizedCoupons – non- colonized
Can account for non-specific adsorption of substancesof substances Dheilly et al. 2008
![Page 12: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/12.jpg)
Electrochemical ImpedancemetryImpedance could be correlated to biofilm gro th time and confirmed b CLSMgrowth time and confirmed by CLSM
Dheilly et al. 2008
![Page 13: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/13.jpg)
Electrochemical ImpedancemetryCan provide real-time monitoring of both biofilm formation and gro thbiofilm formation and growth.
P. aeruginosa
Dheilly et al. 2008
![Page 14: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/14.jpg)
Crystal violetApplied to pre-formed biofilms tobiofilms to assess biofilmformation.Dissolvedwith ethanol MeasuredMeasured spectrophoto-metrically
O’Toole
![Page 15: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/15.jpg)
Crystal violet
Biofilm formation of avirulent and WTBiofilm formation of avirulent and WT strain of B. pertussis.
Setta et al. 2007
![Page 16: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/16.jpg)
Fourier Transform Infrared SSpectroscopy
Setta et al. 2007
![Page 17: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/17.jpg)
Fourier Transform Infrared SpectroscopySpectroscopy
Setta et al. 2007
![Page 18: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/18.jpg)
Alamar Blue
Redox indicatorAntibiotic susceptibility testingSimple and amendable to highSimple and amendable to high-throughput technologies.
Pettit et al. 2005
![Page 19: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/19.jpg)
Alamar Blue
Method verified using XTT reduction and CFU/ml
Pettit et al. 2005
![Page 20: Methods: Quantitative Techniques - University of Hawaiimcooney/oest740/Biofilms_Methods_3_ppt.pdfSummary The importance of biofilm formation and the roles it plays inand the roles](https://reader033.fdocuments.us/reader033/viewer/2022052711/5ad59dda7f8b9a6d708d1b70/html5/thumbnails/20.jpg)
SummaryThe importance of biofilm formation and the roles it plays in biocorrosionand the roles it plays in biocorrosionand antibiotic resistance has lead to the advancement of several quantitativeadvancement of several quantitative methods.
detect the formation of biofilms susceptibility to different chemical agents.
Despite advances conventional biofilm techniques remain invaluable to thetechniques remain invaluable to the verification of new methods.