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.