Tracking Inter‐ and Intraspecific Bacterial Competition in Pairwise Assays Using a Transient Fluorescent Dye

Testing inter- and intraspecific competition is essential for understanding the processes that maintain diversity within communities and populations. Pairwise growth assays are well-established methods to study the outcome of competition between bacterial genotypes or species. However, distinguishing bacterial competitors in coculture remains a major challenge due to their small cell size, similar cell morphology, and often large population sizes. Current approaches are either labour intensive and require prior morphological and sequence-based characterisation or genetic modification of the bacteria. We present a simple assay that tracks the relative abundances of two competing bacterial species or genotypes of the same species through transient staining of one competitor with a fluorescent dye. The use of a transient dye relieves prior genetic characterisation or modification. Our approach uses a sample-dependent threshold evaluation for the fluorescent intensity, enabling the quantification of bacterial frequencies over generations. The method provides high accuracy for tracking competing genotypes and species. The outcome of competition often depends on frequency-dependent fitness effects and/or environmental variables. Therefore, our method tests across different initial abundances and can also assess the role of a biotic interaction (presence/absence of a predator) on the outcome of competition. Our method presents a simple and high-throughput assay for competition. Absence of genetic modifications and applicability to little characterised competitors make it particularly useful for assays involving bacterial isolates from evolving populations.