Asian University for Women, Chittagong, Bangladesh
Studies on microplastics usually require the contaminant to first be isolated from the sample substrate by density separation and removal of organic matter (reviewed by Cole et al. (2014) and Tagg et al. (2015)) before sorting from other materials in the filtrate, counting the number of microplastics, and identifying the type of plastic if possible. While the easiest and cheapest method of separating microplastics is by visual sorting using light microscopy, small plastic fragments or fibers can be difficult to see. The most accurate methods involve Fourier transform infrared spectroscopy (FTIR, specifically reflectance micro-FT-IR or ‘molecular mapping’), pyrolysis gas chromatography coupled to mass spectrometry (pyrolysis GC/MS), Raman spectroscopy, and fluorescence microscopy (see Hidalgo-Ruz et al. (2012) and Tagg et al. (2015)). However, these processes are time consuming and the equipment is expensive to purchase. During our studies on microplastics (see Balasubramanian and Phillott on pages 13-16 in this issue of IOTN), we identified some cheaper alternatives.
Fluorescence microscopy (the simplest and cheapest of the methods described above) reduces the risk of underestimating the number of plastic fragments present in samples. A cheaper alternative to a fluorescent microscope is a NIGHTSEA Stereomicroscope Fluorescence Adapter (~US$1,100), which can add fluorescence illumination to dissecting microscopes. Six different wavelength sets plus bright light are available; Royal Blue (440-460nm) is being used to identify microplatics (P. Dustan pers.comm., 2016). NIGHTSEA products are distributed by Electron Microscopy Sciences (EMS), and their distributors in the Indian Ocean region and Southeast Asia can be found at http://www.emsdiasum.com/microscopy/company/agents.aspx.
Fluorescence illumination can also be obtained by retrofitting an old light microscope with a brightfield vertical illuminator and very bright low-voltage light emitting diode (LED), although this option relies on the availability of a suitable microscope, vertical illuminator and LED flashlight. Steps to disassemble the vertical illuminator, attach the flashlight and assemble the internal optics are described in Babbitt et al. (2013).
Nile Red is a fluorescent dye that is usually used with cell and tissue samples, but is also reported to stain polyethylene, polypropylene and expanded polystyrene (Song, 2014) and may improve isolation of microplastics from samples (Cole et al., 2011) The dye may be added to the sample before filtration (3µg/ mL; Desforges et al., 2014; 50mg/L, Song et al., 2014).
Researchers working in labs without a camera mounted on the microscope may also be interested in the simple cell (mobile) phone camera mount, built using inexpensive and common materials, described by Martin and Shin (2016). The mount ensures the phone camera is positioned correctly with relation to the ocular lens and the beam of light to improve the image quality.
Babbitt, G.A., C.A. Hanzlik & K.N. Busse. 2013. Observing fluorescent probes in living cells using a low cost LED flashlight retrofitted to a common vintage light microscope. Journal of Microbiology and Biology Education 14: 121-124.
Cole M., P. Lindeque, C. Halsband & T.S. Galloway. 2011. Microplastics as contaminants in the marine environment: A review. Marine Pollution Bulletin 62: 2588-2597.
Cole, M., H. Webb, P.K. Lindeque, E.S. Fileman, C. Halsband & T.S. Galloway. 2014. Isolation of microplastics in biota-rich seawater samples and marine organisms. Scientific Reports 4: 4528.
Desforges, J.P.W., M. Galbraith, N. Dangerfield & P.S. Ross. 2014. Widespread distribution of microplastics in subsurface water in the NE Pacific Ocean. Marine Pollution Bulletin 79: 94-99.
Hidalgo-Ruz, V., L. Gutow, R.C. Thompson & M. Thiel. 2012. Microplastics in the marine environment: A review of the methods used for identification and quantification. Martin, R. & S. Shin. 2016. Photomicroscopy made easy by converting cell phones into “CellCams”. The American Biology Teacher 78: 71-75.
Song, Y.K. 2014. Development of a Nile Red staining method for microplastic identification and quantification. In: International Workshop on Fate and Impact of Microplastics in Marine Ecosystems (eds. A.-L. Cassone & P. Soudant). 13-15 January 2014, Plouzané, France.
Song, Y.K., S.H. Hong, M. Jang, G. M. Han and W. J. Shim. 2014. A Nile Red staining method for microplastic identification and quantification. PICES Annual Meeting, 16-26th Oct 2014, Yeosu, Korea.
OTHER USEFUL RESOURCES