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Counterstaining to Separate Nile Red-Stained Microplastic Particles from Terrestrial Invertebrate Biomass
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Researchers developed a fluorescent counterstaining technique combining Calcofluor white and Evans blue dyes with Nile red to distinguish microplastic particles from terrestrial invertebrate biomass and fecal material under confocal microscopy. The method enabled clear differentiation of multiple plastic polymer types from arthropod and non-arthropod invertebrate biomass, offering a new tool for detecting microplastic ingestion in soil animals.
The emerging threat that microplastic pollution poses to soil and its biota necessitates the development of methods to detect microplastic ingestion by soil animals. Fluorescent staining with Nile red dye has proven to be effective at distinguishing microplastics from inorganic and some biological material but is not suitable for separating them from invertebrate remains. Here, we report on the development and validation of a novel fluorescent counterstaining technique for detection of microplastics within terrestrial invertebrate biomass and fecal material. After being stained with a blend of Calcofluor white and Evans blue dyes in addition to Nile red, ground arthropod biomass appeared blueish-purple, whereas different plastic polymers appeared red, green, and yellow when viewed under laser scanning confocal microscopy. Nonarthropod invertebrate biomass and fecal material were also distinguishable from plastic, though to a lesser extent. Our results highlight the value of this method for detecting microplastic ingestion by terrestrial invertebrates.
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Combining Low Cost Imaging and Fluorescence Spectroscopy to Assess a Nile Red Counterstain for Differentiating Microplastic and Organic Particles
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Researchers tested a two-dye fluorescent staining approach — pairing Nile Red (which stains plastics) with counterstains like Calcofluor White and Evans Blue (which bind to organic materials) — to reduce false positives when identifying microplastics under a microscope. The counterstains successfully shifted the fluorescence color of organic particles away from the signal expected for plastics, making it easier to distinguish true microplastics from organic debris without expensive equipment. This low-cost method could make microplastic detection more accurate and accessible for environmental monitoring labs worldwide.
A fluorescent counterstaining technique to differentiate microplastics from chitin using Nile red and calcofluor white in edible grasshoppers
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Scientists developed a new dye technique that can accurately spot tiny plastic particles hiding in edible insects like grasshoppers, telling them apart from the insect's natural shell material (chitin), which often gets mistaken for plastic. This matters because as insects become a more popular protein source worldwide, we need reliable ways to check how much plastic contamination might be entering our food supply this way. The method is affordable and simple, making it a practical tool for future food safety testing.
Differential staining lowers the false positive detection in a novel volumetric measurement technique of microplastics
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A novel volumetric microplastic detection method using Nile Red staining was refined with differential counterstaining to reduce false positives caused by non-plastic organic materials like lignin and chitin. The improved method was validated across soil, water, and food matrices, demonstrating better specificity for true microplastic detection.
Towards quality-assured measurements of microplastics in soils using fluorescence microscopy
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Researchers applied fluorescence microscopy with Nile Red staining to detect and measure microplastic particles in soil samples and validated the method across eight plastic types and a range of soil types. The method performed well when adapted to account for soil matrix effects. Validated fluorescence microscopy protocols make microplastic analysis in agricultural and environmental soils more accessible and standardized.
Rapid detection of microplastic contamination using Nile red fluorescent tagging
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Researchers developed a rapid microplastic detection method using Nile Red (NR) fluorescent staining combined with zinc chloride density-based extraction and filtration for analysis of coastal marine sediment samples. The approach was cross-validated against conventional light microscopy, demonstrating improved speed and sensitivity for identifying microplastics of various sizes in environmental sediment matrices.
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