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Co-staining microplastics with Nile Red and Rose Bengal for improved optical quantification.

Benedetta Villa1,2, Gaia Bolla1, Isabella Gambino1

  • 1Department of Science and High Technology, University of Insubria, Via Valleggio 11, Como, Italy.

Scientific Reports
|December 23, 2025
PubMed
Summary

This study introduces a novel co-staining method using Nile Red and Rose Bengal dyes to accurately identify microplastics (MPs) in environmental samples. This technique improves accuracy and saves time in microplastic contamination assessments.

Keywords:
Digital microscopeMicroplasticsNile redOptical quantificationRose bengalVisual analysis

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Area of Science:

  • Environmental Science
  • Analytical Chemistry
  • Polymer Science

Background:

  • Microplastic (MP) contamination poses a significant environmental threat, necessitating accurate quantification for risk assessment.
  • Current MP quantification methods struggle to differentiate plastic from natural organic matter and are time-consuming.
  • Incomplete digestion of natural polymers during sample preparation leads to inaccuracies in MP analysis.

Purpose of the Study:

  • To develop an innovative optical microscopy technique for accurate microplastic quantification.
  • To improve the differentiation between natural and synthetic polymer fragments in environmental samples.
  • To reduce errors and save time in microplastic analysis.

Main Methods:

  • A sequential co-staining technique using Nile Red (NR) and Rose Bengal (RB) was developed.
  • Natural polymers (cellulose, protein, lignin, chitin) and synthetic polymers (PVC, PS, PET, PP, Nylon, HDPE, LDPE) were stained with NR and RB.
  • The efficacy of co-staining for distinguishing natural from synthetic fragments was evaluated using optical microscopy.

Main Results:

  • Co-staining with Nile Red and Rose Bengal effectively separates natural and synthetic polymer fragments.
  • The technique significantly improves the accuracy of visual microplastic identification.
  • Simultaneous staining on the same filter reduces counting errors and saves considerable time.

Conclusions:

  • The sequential co-staining technique offers a more reliable method for microplastic quantification.
  • This approach enhances the accuracy of microplastic analysis in diverse environmental matrices.
  • Improved monitoring capabilities will support better-informed risk assessments and mitigation strategies for microplastic pollution.