Microbial life cycles are often reconstructed theoretically from fragmentary pieces of evidence. Protocols for the direct and continuous observation of entire microbial life cycles, including sexual reproduction, are scarce, which limits the study of cellular transitions between different life cycle stages and prevents the visualization of cryptic stages. Although sequence-based techniques, such as -omics approaches, can reconstruct cellular transitions at the genetic and biochemical level, these methods are destructive and do not recover information from the same living cell over time. This protocol provides a solution to directly and continuously observe microbial life cycles, including sexual reproduction, by using microfluidics manipulations that expose single cells to nutritional stimuli and selective pressures. As proof of principle, we triggered a life cycle sequence transition in the model yeast Saccharomyces cerevisiae, starting with an arrest of proliferation in an ancestor cell followed by induction of meiosis through starvation, selection of sexually reproducing cells through exposure to a drug cocktail, germination of haploid spores, and mating of haploid individuals, creating a new descendant generation. This protocol offers the possibility to directly compare molecular and cellular behavior across life cycle stages and across sexually reproducing generations. Key features • Outlines optimal microfluidic conditions for all life cycle stages in the model microorganism S. cerevisiae. • Describes a selection method for sexually reproducing sporulated yeast cells and against quiescent cells. • Provides descriptions for optimal loading of yeast cells in microfluidic devices for long-term imaging. • Enables continuous detection across life cycle transitions, which could be applied to other biomedically and agriculturally relevant fungal microorganisms.

• PMID: 41384095
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Journal Article

Authors

Kennedy T, Neupane S, Merritt B, Argüello-Miranda O

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