A Comparison between Bright Field and Phase-Contrast Image Analysis Techniques in Activated Sludge Morphological Characterization D.P. Mesquita, 1 O. Dias, 1 A.L. Amaral, 1,2 and E.C. Ferreira 1, * 1 IBB—Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2 Instituto Superior de Engenharia de Coimbra, Instituto Politécnico de Coimbra, Rua Pedro Nunes, Quinta da Nora, 3030-199 Coimbra, Portugal Abstract: Different approaches using microscopy image analysis procedures were employed for characterization of activated sludge systems. The approaches varied mainly on the type of visualization and acquisition method used for collection of data. In this context, this study focused on the comparison of the two most common acquisition methods: bright field and phase-contrast microscopy. Images were acquired from seven different wastewater treatment plants for a combined period of two years. Advantages and disadvantages of each acquisition technique and the results are discussed. Bright field microscopy proved to be more simple and inexpensive and provided the best overall results. Key words: activated sludge, image analysis, bright field, phase contrast, aggregates, filaments I NTRODUCTION An activated sludge system includes a complex ecosystem composed of different types of microorganisms such as protozoa, metazoa, and filamentous or zoogleal bacteria. A good balance between the different microorganisms is essen- tial to guarantee good settling properties and a clear super- natant ~ Jenkins et al., 2003!. Generally, the evaluation of aerated tanks may be performed by visual inspection under an optical microscope coupled to automated image analysis methods. Activated sludge processes have been increasingly monitored through microscopy observations for aggregate contents and morphology and determination of protruding filamentous bacteria content ~da Motta et al., 2001b; Jenné et al., 2006!. Subsequently, the gathered image analysis information is correlated with the sludge settling abilities ~Ganczarczyk, 1994; Grijspeerdt & Verstraete, 1997; Ba- nadda et al., 2005! to assess biomass morphology changes ~ Jenné et al., 2003! and to monitor bulking events in pilot plants ~da Motta et al., 2001a, 2001b; Jenné et al., 2004, 2007; Amaral & Ferreira, 2005!. Use of automated image analysis applications in analy- sis of activated sludge has increased in recent years, with two image acquisition methods standing out: phase-contrast microscopy as proposed in the works of Cenens et al. ~2002! and Jenné et al. ~2006, 2007! among others, and bright field microscopy as in the works of da Motta et al. ~2001a, 2001b!, Amaral and Ferreira ~2005!, and Mesquita et al. ~2009a, 2009b!. In comparison, bright field microscopy is the cheapest and simplest method to examine activated sludge, whereas phase-contrast microscopy requires more expensive equipment and a more skilled operator. Further- more, the nature of phase-contrast microscopy causes the aggregate borders to become ill-defined as the object’s halo hinders the assessment of their boundaries. However, this method presents, at least in theory, the advantage of a more precise determination of the protruding filamentous bacte- ria content. The high transparency of the filamentous bac- teria poses a contrast problem in bright field microscopy acquisition, which is opposite of the clear filament/dark background distinction in phase-contrast microscopy. There- fore, studies have been performed using bright field acquisi- tion methods to survey the aggregated biomass and phase- contrast acquisition for assessment of filamentous bacteria ~Amaral, 2003; Abreu et al., 2007; Costa et al., 2007!. Based on the reported advantages and disadvantages of both methodologies, the present work aims to survey the aggregates and protruding filamentous bacteria contents of activated sludge using image analysis procedures for bright field and phase-contrast microscopy. The best acquisition method for activated sludge characterization was deter- mined by monitoring the activated sludge of seven different wastewater treatment plants for a period of two years. Received April 8, 2009; accepted December 15, 2009 *Corresponding author. E-mail: ecferreira@deb.uminho.pt Microsc. Microanal. 16, 166–174, 2010 doi:10.1017/S1431927609991358 Microscopy AND Microanalysis © MICROSCOPY SOCIETY OF AMERICA 2010