In Situ Techniques and Digital Image Analysis Methods for Quantifying Spatial Localization Patterns of Nitrifiers and Other Microorganisms in Biofilm and Flocs

The spatial localization patterns of microorganisms in multispecies biofilms reflect numerous phenomena that influence sessile microbial life, such as substrate concentration gradients within the biofilm and biological interactions with other biofilm populations. Quantitative and population-specific in situ analyses of spatial patterns have a high potential to provide novel insights into the biology of biofilm organisms, including yet uncultured microbes, but such approaches have been developed and used in a few studies only. Here, we outline digital image analysis methods to quantify the coaggregation, mutual avoidance, or random distribution of microbial populations in biofilm and flocs. A protocol is provided for fluorescence in situ hybridization with rRNA-targeted probes, which preserves the three-dimensional biofilm architecture for confocal microscopy and image analysis, and the combined use of these approaches is demonstrated by spatial analyses of nitrifying bacteria in complex biofilm samples.