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

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 serum albumin (BSA). The pre-treatment and staining methods were appropriate to compare the composition of different biofilm flocs such as nitrifying activated sludge from a municipal wastewater treatment plant and a pure culture of the bacterial strain Pseudomonas putida (ATCC 17514) as revealed by the results of conventional analytical methods.