A Morphological Dual-Staining Framework for Standardized Detection of Cell Death in the Microalga Dunaliella salina

Abstract The absence of a simple, universal protocol to detect algal cell death is a major bottleneck in aquatic ecology and carbon cycle research. This study bridges this gap by establishing a practical diagnostic framework based on parallel 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and iodine staining, concurrently probing the two pillars of photoautotrophic vitality—metabolic activity (via respiratory electron transport) and photosynthetic storage integrity. Using the halotolerant, wall-less model Dunaliella salina , we applied the method to environmentally relevant stressors. It clearly distinguished viable cells and consistently classified four distinct death phenotypes: apoptosis-like, necrotic, plasmolysis, and a novel secretion-type death from microbial fouling. Notably, light-induced necrosis exhibited morphological hallmarks of singlet oxygen-mediated killing, analogous to Type II Photodynamic Therapy. Crucially, identical death signatures were detected in natural populations from Maharloo Hypersaline Lake, confirming ecological validity. Beyond providing a robust, field-adaptable toolkit, our findings expose a critical disciplinary gap: the lack of standardized nomenclature and diagnostic criteria for cell death in carbon-fixing unicellular organisms. As these organisms mediate nearly half of global carbon fixation, quantifying their mortality is essential for improving carbon fixation and growth models, accurately predicting carbon fluxes, assessing the resilience of aquatic carbon sinks, and evaluating climate change impacts. We therefore propose an international consortium on Cell Death in Carbon-Fixing Unicellular Organisms to standardize definitions of cell death phenotypes, unify methodologies through this dual-staining protocol, explicitly incorporate mortality into carbon fixation and growth models, and translate laboratory-based viability metrics into predictive tools for ecosystem- and photobioreactor-scale carbon management. Our framework provides the essential methodological foundation for this global standardization effort.