Discriminating similarly sized polystyrene microplastics, microorganisms, and debris in wastewater: Open issues in fluorescent co-staining and flow cytometry

Microplastics (MPs) smaller than 10 μm may pose greater biological risks because they can cross biological barriers but are hardly detectable with routine μ-FTIR or μ-Raman methods. Detecting such small MPs in environmental samples like wastewater remains challenging as microorganisms and heterogeneous debris of comparable size complicate their discrimination and quantification. High-throughput flow cytometry, combining scattering and fluorescence signals, can resolve distinct particle populations even in the nano- to micrometric range, analyzing over 20,000 particles per sample. The present study investigated an integrated workflow based on flow cytometry for the simultaneous detection of small MPs and microbial cells in wastewater, amid abundant interfering organic/inorganic debris. Wastewater samples were spiked with standard 3-μm polystyrene MPs, which served as surrogates for very small MPs, providing a reference to verify detection and gating accuracy. Three alternative fluorochromes—Acridine Orange, Neutral Red, and Nile Red—were evaluated for labeling the colorless, otherwise untraceable MPs. Nile Red (NiR) produced superior fluorescent signals but required optimization of three key factors: solubility in the polar solvent dimethyl sulfoxide (10–30%), dye concentration (1–10 μg/mL), and filtration to avoid false-positive from NiR aggregates. Co-staining with SYBR Green I, combined with cytogram-based electronic gating, enabled discrimination between 3-μm NiR-positive PS microspheres and SYBR Green-positive microorganisms, which are similar in size and may otherwise produce overlapping signals. Overall, this workflow adapts existing fluorochromes into a sensitive and automated screening approach for detecting microorganisms and debris potentially containing small MPs in heterogeneous aqueous matrices, such as wastewater.