A standardized pre-treatment method of biofilm flocs for fluorescence microscopic characterization

Journal of Microbiological Methods
Oxana NosykFritz H Frimmel

Abstract

The availability of epifluorescence microscopy opens the door for the visualization of biofilm flocs via the fluorescence staining technique. However, one of the main problems in the application of the method is a missing standardized protocol for sample preparation. In this study, different pre-treatment methods for the simultaneous staining of different sludge flocs components e.g. extracellular polymeric substances (EPS) and microorganisms (MO) were compared. A method was chosen, which remains the shape of sludge flocs from a municipal wastewater treatment plant and allows to stain major parts of the flocs thoroughly: For this the surrounding wastewater was removed by centrifugation. Then the flocs were resuspended in sterile tap water, immobilized by drying on a diagnostic slide and fixated with formaldehyde. The staining of the different compounds was performed with tetramethylrhodamine isothiocyanate-labeled lectin Concanavalin A (TRITC-Con A; glycoconjugates), fluoresceinisothiocyanate isomer I (FITC; amino groups) and 4,6-diamidino-2-phenylindoldihydrochloride (DAPI; nucleic acids). This staining method was successfully applied for protein- and carbohydrate-quantification using gelatin mixtures of dextran and bovine ser...Continue Reading

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Citations

May 17, 2012·Journal of Microbiological Methods·Fiona J BairdJane E Hill
Dec 10, 2015·Cell Adhesion & Migration·Robert SwierczewskiChris P F Redfern
Aug 14, 2018·Journal of Fish Biology·Hiroshi Ueda
Jun 4, 2019·Canadian Journal of Microbiology·Da-Seul Shin, Yong-Bin Eom
Nov 20, 2016·Folia Microbiologica·Seung-Gyun WooYong-Bin Eom
Sep 28, 2020·Journal of Applied Microbiology·H-R Kim, Y-B Eom

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