Small Particles Task Force: Isopropyl Alcohol Improves Dispersion of <20 µm Particles on Microslit Membranes

Introduction

The LOMP Small Particle Taskforce is focused on developing a reliable and rigorous procedure for analyzing small microplastics in environmental samples. Our primary aim is to ensure that the data we generate accurately reflects the true microplastic population present in each sample. Achieving this requires careful attention to representativeness, verification, and contamination control throughout the entire workflow.

Three core objectives guide this effort. First, we must confirm that each sample analyzed is truly representative of the broader environmental system we are studying. Second, it is essential to verify that the particles counted are indeed microplastics, supported by appropriate analytical methods. Third, minimizing contamination from collection to processing to analysis is crucial for ensuring confidence in the final results.

To date, we have made significant progress in reducing methodological contamination and have begun refining our staining approach. However, further work is needed to better remove environmental contaminants and to verify that the particles we measure accurately represent the true population. This next phase of development will involve benchmarking to strengthen both reliability and reproducibility.

To better align the analytical method across LOMP, the MMC arm of the Small Particles Taskforce made stocks that all groups were asked to analyze with their current methods. This post walks through the process of preparing stock and analytical methods by the groups.

In a previous blog post titled SPTF – Initial Stock Preparation & Staining Workflow, we discussed the preparation of the stock that the MMC created for the SPTF for analysis.

Results & Discussion

Our goal is to minimize leftover residuals from the sample preparation process. For this reason, it may not be a good idea to use Tween for membrane capture experiments. Another method of anti-coagulation particle capture is needed. Earlier findings from the sieving of cryomilled particles demonstrated that isopropyl alcohol suspends particles like PS, PET, and Nylon better than water. Because of this, the MMC remade the stock solutions previously made, but with isopropyl alcohol. The particles suspended in the isopropyl alcohol are much better than in water as demonstrated in Figure 1 below. Image of the blank isopropyl alcohol, Figure 1a, 2 samples of the PE beads in isopropyl alcohol, Figures 1b and 1c, and 2 samples of MMC MMC-made particle spike, Figures 1d and 1e, before staining of particles is shown below. Before staining, the particles are filtered onto the membrane as expected. However, despite using the same stock and volume, Figures 1b and 1c have drastically different numbers of beads (59 and 295, respectively). The stock solution has a concentration of 10 mg/mL, but the observed count-based concentration is 0.15 mg/mL and 0.73 mg/mL, respectively. Since no stained experiments were done with a SepCon, it is possible that some of the particles got stuck on the side walls of the SepCon devices. No analysis was done with the unstained version of the MMC spiked particles in Figure 1d and 1e, but we can see that particle distribution is improved compared to previous water-based sample images, as well as improved anti-coagulation. Coagulation with the beads from Figure 1c and 1d still looks like it is happening, but individual particle counting is still possible.

After imaging of the non-stained samples, the samples were then stained with a vacuum apparatus with 20 $μL of Nile Red and Trypan Blue and washed through with water after staining. For the 2 control and 2 PE samples, the stain was added on top of the chip, let sit for 5 minutes, vacuum turned on to pull through stain, wash witth the vacuum on, turn off the vacuum, add Trypan blue stain, let sit for 5 minutes, vacuum turned back on, wash with the vacuum on, and then turn off vacuum. This approach resulted in the PE beads to nearly all disloge from the membrane. Because this was noticed with both of the PE samples, the MMC spiked particle samples in Figure Xg and Xh were stained a differntly. With the membrane in place, vacuum was turned on, Nile Red stain added (where it was pulled through the membrane almost instantly), washed with 1 mL of water, trypan blue added (which also pulled through almost instantly), washed off with 3 mL of water, and then vacuum turned off. This approached allowed for the particles that are already on the memrane to experience constant pull down resulting in less distruction to the particles. Also, this approach only exposed the particles the stains for ~1 second before being pulled through. Despite this, Figure 1g and 1h show that the particles picked up the Nile Red stain expetinally well. The Trypan Blue stain was left off Figure 1g due to poor microscopy aqusation settings on the DAPI channel. Trypan Blue was picked up well by the sample in Figure 1h by a piece of fiborus contamination. Because low exposure times did not seem to effect stain retension on particles, it would suggest that issues regarding staining may not be with exposure of the particles to the stains, but rather microscopy related or stain being expired.$

a	b	c	d	e
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		f	g	h

Figure 1: Particle suspension, membrane capture, and staining outcomes using isopropyl alcohol–based preparation. a) Blank isopropyl alcohol control. b–c) Polyethylene (PE) bead samples prepared from the same stock solution, showing variability in particle counts. d–e) MMC-prepared particle spike samples prior to staining, demonstrating improved dispersion compared to water-based preparations. f–h) MMC spike samples after staining, showing effective Nile Red uptake and improved particle retention using a modified staining approach; Trypan Blue signal is visible in h.