Best practices in the flow cytometry of microalgae

Area of Science:

• Microbiology
• Genomics
• Biotechnology

Background:

• Microalgae are vital photosynthetic microorganisms with significant ecological roles.
• They produce diverse secondary metabolites, driving interest in their biotechnological applications.
• Flow cytometry (FCM) is effective for genome size estimation in plants and animals, but faces challenges in microalgae.

Purpose of the Study:

• To highlight the methodological challenges of applying flow cytometry (FCM) for genome size estimation in microalgae.
• To emphasize the importance of optimized sample preparation and nuclear isolation techniques.
• To underscore the vast diversity in microalgal nuclear DNA content and the need for caution in ploidy assignment.

Main Methods:

• Discusses the application of flow cytometry (FCM) for genome size estimation.
• Details sample preparation challenges, including biomass requirements and fixation/extraction needs.
• Explains essential techniques for isolating intact nuclei, such as lysis buffers, enzymatic digestion, and bead-beating.

Main Results:

• Microalgal nuclear DNA content varies dramatically, spanning a 30,000-fold range (0.01 to 286 pg).
• Current genome size standards often rely on animal or plant tissues, with new algal standards emerging.
• Accurate 1C/2C-value and ploidy level assignments require careful consideration due to limited knowledge of microalgal life cycles.

Conclusions:

• Optimized FCM protocols are essential for reliable microalgal genome size determination.
• Addressing challenges like cell wall composition and secondary metabolites is key for successful analysis.
• Further research into microalgal life cycles will improve the accuracy of ploidy level assessments.