Contactless Co-Culture Assays for Morphometric Studies During Inter-Species Interactions in Fungi.

Cellular behaviour and morphology are usually influenced by various intra- and extracellular factors in a microbial community. Different approaches available to study microbial communication could be tedious and/or require specialised facilities and expertise. Here, two complementary contactless co-culture approaches are described, the membrane insert well plate co-culture assay and the Cell-Free Supernatant (CFS)-based assay, which are based on morphological transition as a measurable response to investigate the role of various factors in a given inter-species interaction. The membrane insert well plate system, while permitting diffusion of extracellular molecules, allows real-time interaction between physically separated species. In comparison, the CFS-based assay provides a simplified, scalable approach for evaluating responses to conditioned media. The protocol presented here includes standardized procedures for culture preparation, generation of conditioned supernatants, assay setup, microscopy, image acquisition, quantitative morphometric analysis using Fiji, and statistical evaluation. This has been demonstrated with an example of fungal morphological response to intrinsic and extrinsic factors. The methods presented here offer accessible and adaptable alternative tools for studying novel microbial interactions in a community, and could be readily extendable to investigate mechanisms underlying multi-species co-existence in a community.