Rapid and Efficient Investigation of Ciliate Nuclear Development During Conjugation Through Optimizing Hoechst33342 Staining Workflow

Ciliates are eukaryotic unicellular organisms with complex morphology and developmental processes, including asexual and sexual processes. Conjugation is a form of sexual process that renews genetic materials. However, visualizing conjugation in ciliates is a challenge due to the complexity and dynamics of the process, while traditional staining methods are often insufficient for the research. This study introduces a new method for visualizing developmental progression in the nuclei during conjugation using Hoechst33342 staining. It describes how to proceed from cell culture, conjugation induction and synchronization, staining preparation, and observation to statistical analysis. The combination of fluorescent staining with the ‘volume-fixing’ technique eliminates the fixation and dehydration steps, thus reducing the overall operation time to just 20 minutes. This method offers several advantages over traditional staining techniques for studying the nuclei during conjugation. It improves image quality and workflow efficiency and enables real-time observation of live cell states. Potential solutions to challenges that may arise during experimental procedures are introduced and references and guidelines for cytological research are provided in this paper.