Counterstaining to Separate Nile Red-Stained Microplastic Particles from Terrestrial Invertebrate Biomass

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.