How to distinguish small microplastics in aqueous environmental matrices containing microorganisms and debris, using fluorescent co-staining and flow cytometry

The simultaneous high-throughput quantification of small microplastics (MPs measuring a few microns in size) and microorganisms in environmental aqueous matrices, using flow cytometry (FCM) for single-particle analysis, was detailed. Although still relatively novel in this field, FCM has demonstrated fast and sensitive detection of small MPs, with fluorescent staining allowing clear separation from microorganisms and debris in complex samples. Three alternative fluorochromes—Acridine Orange, Neutral Red, and Nile Red—were evaluated for labeling colorless, otherwise untraceable 3.0-µm polystyrene MPs used as surrogates in testing water and wastewater treatment. Among these dyes, only Nile Red (NR) produced superior fluorescent signals under the common cytometer lasers and emission filters. Key factors in MP staining were investigated to optimize fluorescent emission signals, including NR solubility in the polar solvent dimethyl sulfoxide (10-30%), dye concentration (1-10 µg/mL), and the need for filtration to avoid false-positive counts from NR aggregates. Municipal wastewater, as a complex aqueous matrix, was investigated without extensive pretreatment using NR and SYBR Green I to distinguish MPs and microbial populations simultaneously. For statistically representative quantification, > 20,000 particles were analyzed per sample. Results demonstrated that FCM analysis enables the distinction of very small MPs, microorganisms, and nanomicroscopic debris in aqueous matrices. In contrast, particles in this size range are hardly detectable with other instruments such as µ-FTIR or µ-Raman. This research provides a basis for developing robust, automated approaches and adapting existing fluorochromes for effective monitoring of micro- and nanoplastics in environmental samples.