The strength of activated sludge flocs is important for the flocculation, settling and dewatering properties of activated sludge and thus the performance of wastewater treatment plants. Little is known about how different bacteria affect the floc properties, so in this study it was investigated whether the strength and other characteristics of large microcolonies within activated sludge flocs from a full-scale nutrient removal plant varied significantly between different phylogenetic groups of bacteria. The investigation was carried out by using a shear method for deflocculation of activated sludge flocs, combined with different chemical manipulations under defined conditions. The identification and quantification of the microcolony-forming bacteria were conducted with group-specific gene probes and fluorescence in situ hybridization. The focus was on the microcolonies and not on the entire sludge flocs. In general, the results showed large difference in the strength and colloid-chemical properties of the different probe-defined microcolonies. By applying extensive shear to the system, less than 12% of the microcolony biovolume of the Beta-, Gamma- and Deltaproteobacteria and Actinobacteria could be disrupted, thus forming strong microcolonies. Alphaproteobacteria and Firmicutes formed weaker microcolonies (42-61% could be disrupted by shear). For most groups, several intermolecular forces determined the strength of the microcolonies: hydrophobic interactions, cross-linking by multivalent cations and perhaps entanglements of extracellular polymeric substances. However, the dominant force varied between the various phylogenetic groups. The large difference between the different phylogenetic groups indicated that only a few species were present within each group, rather than many different bacterial species within each phylogenetic group had similar floc properties.