Co-cultivation assay of fungi and bacteria on solid medium using automated culture handling - A high-throughput...

Microbial co-cultivations remain relevant in the search for new antibacterial compounds and may be the most effective way to detect bioactive metabolites that may prove necessary to combat rising antimicrobial resistance. However, manual streaking of co-cultivation partners is labor intensive and induces variations, compromising the throughput and accurate assessment of microbial interactions. We propose a customizable method by repurposing an automated culture handling robot for precise and repeatable co-cultivation studies on solid medium. Sourced from two 96-well plates, microorganisms are gridded pairwise at defined distances, accounting for different growth rates and required space. For small bacteria colonies gridded in short distances, a maximum of 960 combinations can be tested in one run on two 24 × 24 cm culture trays. Additional to the monitoring of pairwise interactions, a secondary analysis using bacterial agar overlays can be employed to screen for zones of inhibition. We demonstrate the adaptability of our method by co-cultivating arctic marine actinobacteria and fungi, as well as human pathogenic bacteria for growth inhibition studies. This method allows for rapid screening of large isolate collections, addresses the shortage of automated co-cultivation methods, and leads to new findings in the field of natural product discovery.