An automated method for the quantification of moving predators such as rotifers in biofilms by image analysis

In natural environments as well as in industrial processes, microorganisms form biofilms. Eukaryotic microorganisms, like metazoans and protozoans, can shape the microbial communities because of their grazing activity. However, their influence on biofilm structure is often neglected because of the lack of appropriate methods to quantify their presence. In the present work, a method has been developed to quantify moving population of rotifers within a biofilm. We developed an automated approach to characterize the rotifer population density. Two time lapse images are recorded per biofilm location at an interval of 1 s. By subtracting the two images from each other, rotifer displacements that occurred between the two images acquisition can be quantified. A comparison of the image analysis approach with manually counted rotifers showed a correlation of R2 = 0.90, validating the automated method. We verified our method with two biofilms of different superficial and community structures and measured rotifer densities of up to 1700 per cm2. The method can be adapted for other types of moving organisms in biofilms like nematodes and ciliates.