Fluorescent technique to detect microplastics in a natural matrix using Methylene blue and Nile red

The Nile red is a fluorescent and metachromatic dye, for hydrophobic and nonpolar materials such as lipids and plastics. However, when the microplastics (MP) are contained in a natural matrix composed of roots, plant and insect fragments rich in hydrophobic substances such as cuticles, chitin, and autofluorescence materials (cell wall, lignin, polyphenols) a false fluorescence could generate. In the present study, we explore the use of Methylene blue (C.I. 52015) in combination with Nile red, to stain MP in an intact organic complex composed of roots and rhizome of <i>Cyperus papyrus</i> and <i>Pontederia sagittata</i> from Floating Treatment Wetlands (FTW) installed at an urban eutrophicated ponds in Xalapa Veracruz, México. First, the sample was stained with 0.5 % v/v Methylene blue in 0.5% v/v borax to reduce the nonspecific stain and autofluorescence. After a wash with distilled water, the sample was stained with Nile red (1 µg mL^-1) in ethanol/water. This double stain reduces the background fluorescence of the non-plastic materials, obtaining the best contrast under the green light (ex. 450-490 nm, em. 515 nm). This is the first report of an easy, fast, and non-destructive staining technique to detect MP in natural conditions, that uses Nile red, together with Methylene blue to reduce false positive background staining.