Fluorescent in situ hybridization (FISH) and confocal scanning laser microscopy (CSLM) are the key techniques used to investigate bacterial community structure at wastewater treatment plants. An optimum nitrifying bacterial population is necessary for nitrification, which plays a significant ecological role in regulating the overall quality of water. Nitrifying bacteria mainly appear as dense aggregates within activated sludge flocs. The impacts of five different pre-treatment methods (physical, chemical, enzymatic and combinations) on floc dispersion from two different wastewater treatment plants were determined. The effect of pre-treatment on the enumeration of the nitrifying bacterial population was also investigated. This study on floc dispersion using CSLM images showed sonication was the superior method for all the samples tested, irrespective of the sludge type. For samples from industrial wastewater plants, an optimized sonication level of 8 W for 8 min could reduce the floc size to 10 microm, whereas for domestic wastewater samples, the floc size was reduced to 10 microm at 8 W for 5 min. The maximum number of nitrifying bacterial cells was observed at this optimized level for different samples. A decrease in the number of cells was observed beyond this optimized level for both the plants. The results presented here highlight the importance of optimizing pre-treatment methods for different types of wastewater for accurate bacterial community analysis using FISH-CSLM.