A fluorescent counterstaining technique to differentiate microplastics from chitin using Nile red and calcofluor white in edible grasshoppers

Microplastics (MPs) are emerging contaminants of increasing environmental concern, and their detection in terrestrial organisms is essential for evaluating their impact, exposure and dynamics within ecosystems. Although insects represent a biologically relevant group for environmental monitoring and for estimating potential trophic transfer routes to humans, no standardized method currently exists for identifying MPs in these organisms. We have developed a detection protocol using edible grasshoppers (Sphenarium purpurascens) as study subjects. This combines Nile red (NR) and calcofluor white (CW) staining to distinguish added MPs from residual biological material (chitin). When NR was applied at 0.01 mg/mL, it successfully stained high-density polyethylene (HDPE), acrylonitrile butadiene styrene (ABS), expanded polystyrene (EPS), and polypropylene (PP), exhibiting polymer-dependent green or red fluorescence. A low NR concentration (0.01 mg/mL) was sufficient to ensure reliable visibility of the added MPs, while CW selectively stained chitin, emitting blue fluorescence under ultraviolet light. Counterstaining with both dyes provided a clear separation between MPs and biological structures, minimizing false positives. Furthermore, infrared spectroscopy (FTIR) confirmed the chemical composition of the added MPs (PP, EPS, ABS), complementing the limitations of NR staining. Thus, our NR-CW counterstaining protocol proved effective even for particles < 50 μm, and is cost-effective, simple to implement, and suitable for environmental studies involving organisms with chitin structures. Furthermore, it provides a reproducible tool suitable for estimating exposure and evaluating risks associated with MPs in terrestrial ecosystems.