How to Help
Nile Red Stock Solution
Nile Red is a fluorescent stain that adheres to plastics. Nile Red is insoluble in water. This solution can be prepared with a variety of solvents (Shim et al. 2016).
Our lab mixes 5 mL of Nile Red Powder and dissolves it in 1 L of acetone. ACS-grade acetone is used instead of many of the other common solvent types because of its low toxicity.
Collection of Samples
Primarily, our monitoring program collects samples once every two months wherein volunteers tow a neuston net for 15 minutes at 7 sites around Tampa Bay. These net tows help collect microplastic samples that can be used for analysis later.
Sample Processing
With sterilized buckets, samples are separated into smaller containers (1000 mL beakers) and poured through stacked 212 μm and 20 μm stainless steel sieves. The sieves act as catches for the waterborne microplastics. The filtered water is discarded.
Volume of water filtered is recorded. Between all filtering and transfer of water samples, the containers are covered to prevent airborne microplastics from contaminating the samples.
After the initial filtering process of the collected water samples, the buckets, beakers, and sieves are rinsed with DI or Milli-Q water. Rinsing removes any lingering microplastics that may be adhering to the containers that are carrying the samples.
Staining & Filtering Samples
After filtering, sampled plastics are rinsed into a 200–400 mL beaker. The pre-prepared Nile Red stock solution is added to the beaker at a ratio of 3:20 (Nile Red to water volume). Stained samples are left to sit for a minimum of 30 minutes before filtering.
Samples are finally filtered onto black PCTE filters and rinsed multiple times with DI or Milli-Q water. Samples are then stored in a dark room at room temperature.
Imaging & Analysis
Using a dissection microscope with a camera attachment, the filtered petri dishes are placed under cyan light in a dark room. When under cyan light (490-515 nm excitation wavelength) and viewed through an orange filter (550 nm emission wavelength) the microplastics glow a particular color that is identified through the program ImageJ.
Microplastic count, type, and size data is inputted into a spreadsheet for later analysis and research.
Methodology adapted from:
Shim, W.J., Song, Y.K., Hong, S.H. and Jang, M. 2016. Identification and quantification of microplastics using Nile Red staining. Marine Pollution Bulletin 113:469-476.