Flow cytometry of microencapsulated colonies for genetics analysis of filamentous fungi.

The analysis of filamentous fungi by flow cytometry has been impossible to date due to their filamentous nature and size. In this work, we have developed a method that combines single-spore microencapsulation and large-particle flow cytometry as a powerful alternative for the genetic analysis of filamentous fungi. Individual spores were embedded in monodisperse alginate microparticles and incubated in the appropriate conditions. Growth could be monitored by light or fluorescent microscopy and Complex Object Parametric Analyzer and Sorter large-particle flow cytometry. Microencapsulated Trichoderma and Aspergillus spores could germinate and grow inside the alginate capsules. Growth tests revealed that auxotrophic mutants required the appropriate nutrients and that pyrithiamine and glufosinate halted fungal growth of sensitive but not resistant strains. We used an Aspergillus nidulans, thermosensitive mutant in the cell-cycle regulator gene nimX(CDK1) as proof-of-concept to the detection and identification of genetic phenotypes. Sorting of the microparticles containing the clonal fungal mycelia proved the power of this method to perform positive and/or negative selection during genetic screenings.