Microplastics in Water – Extraction and Enumeration
1 Purpose
This SOP provides methods for extracting, staining, and enumerating microplastics (particles less than 5 mm) from water samples. Microplastics are stained with Nile Red dye, vacuum-filtered onto glass-fiber filters, and counted under fluorescence using either a Crime-Lite forensic light source with goggles or the Echo Revolve microscope (Erni-Cassola et al., 2017; Maes et al., 2017).
This SOP is intended as an internal teaching and research protocol. Where EA Program data will be submitted to external programs or compared with regulatory monitoring datasets, researchers should consult the emerging standardized methods described in Appendix B and adapt procedures accordingly (California State Water Resources Control Board, 2022; Thornton Hampton et al., 2025).
2 Scope
- Applies to: Extraction and enumeration of microplastics from drinking water, surface water, stormwater, and other aqueous samples
- Does not apply to: Microplastics in soil, sediment, or biological tissue (see SCCWRP Technical Report #1410.A (Thornton Hampton et al., 2025)); chemical identification of polymer type (requires FTIR or Raman spectroscopy)
- Regulatory alignment: This SOP provides a Nile Red fluorescence screening method suitable for research and teaching. It does not replace the FTIR- or Raman-based methods required for California regulatory drinking water monitoring (California State Water Resources Control Board, 2022). Consult Appendix B and the PI before submitting data to SWAMP, CEDEN, or other regulatory programs.
- Location: EA Program laboratory, SGM 133 and SGM 134
3 Acknowledgements
This document builds on the work of Kyle Wees (PO ’20), Baile (PO ’22), and Jon Gunasti (PO ’20). Clare Flynn developed the first SOP, further modified by Marc Los Huertos and Sidarth (PO ’23).
4 Responsibilities
| Role | Responsibility |
|---|---|
| Lab PI | Approve methods, review QC data |
| Trained Personnel | Prepare samples, perform extraction and enumeration |
| New Students | Complete training before independent use |
5 Definitions
- FTIR (Fourier Transform Infrared Spectroscopy): An analytical technique that identifies polymer type by comparing a sample’s infrared absorption spectrum to reference spectra; the gold standard for confirmatory microplastic characterization (California State Water Resources Control Board, 2022).
- Microplastic (MP): A synthetic polymer particle or fiber with at least one dimension between 1 µm and 5 mm; includes fragments, fibers, films, beads, and foam; classified as primary (manufactured at micro-scale) or secondary (degradation products of larger plastic items) (Maes et al., 2017).
- Nanoplastic: A plastic particle smaller than 1 µm; not quantifiable by standard visual methods; an emerging concern for ecological and human health research.
- Nile Red: A fluorescent dye that selectively stains hydrophobic plastic surfaces, enabling detection under blue-light excitation; used as a screening tool to distinguish plastic particles from organic debris (Erni-Cassola et al., 2017; Maes et al., 2017).
- Procedural blank: A sample of laboratory-grade water carried through the complete analytical procedure alongside field samples; used to quantify airborne and procedural contamination; required for every analytical batch.
- Raman spectroscopy: A spectroscopic technique that identifies molecular composition from inelastic light scattering; complementary to FTIR; particularly useful for particles smaller than 10 µm (California State Water Resources Control Board, 2022).
- Visual sorting: The initial enumeration step in which particles on a filter are examined under a stereomicroscope or the Revolve fluorescence microscope and candidate plastic particles are identified by color, shape, and texture before any confirmatory chemical analysis.
6 Safety and Hazards
- Chemical: Acetone is flammable (flash point -20 °C) and potentially toxic by inhalation. All Nile Red solution preparation and sample staining must be performed in a fume hood. Review the SDS for acetone before use.
- Optical: The Crime-Lite 2BG2735 is a high-intensity 450–510 nm light source. Never shine it into anyone’s eyes. Always wear the orange filter goggles when the Crime-Lite is on.
6.1 Required PPE
6.2 Emergency Procedures
For acetone spills, extinguish all ignition sources, ventilate the area, and clean up per SOP-L003 Hazardous Materials Handling. For skin or eye contact, flush with water for at least 15 minutes.
7 Required Materials
7.1 Equipment
| Item | Location | Notes |
|---|---|---|
| Vacuum filtration apparatus | SGM 134 | Glass funnel and fritted base |
| Vacuum pump | SGM 134 | |
| Drying oven | SGM 134 | Set to 60 °C for filter drying |
| Crime-Lite 2BG2735 (450–510 nm) | SGM 133 | Store power supply and goggles together |
| Orange filter goggles | SGM 133 | Must be used with Crime-Lite |
| Echo Labs Revolve microscope | SGM 133 | See SOP-L211 |
| Dissecting scope with 550 nm filter | SGM 133 | Alternative to Revolve |
| Watch glasses | SGM 134 | For filter drying in oven |
| Fume hood | SGM 134 | Required for all Nile Red/acetone work |
7.2 Reagents and Consumables
| Item | Notes |
|---|---|
| Nile Red | TBD catalog number |
| Acetone (ACS grade) | Stored in flammables cabinet |
| Whatman 934-AH glass-fiber filters | 42.5 mm, 1.5 µm pore |
| Glass sample bottles (500 mL) | Pre-cleaned; avoid plastic |
| Glass pipettes or syringes | For Nile Red injection |
A standard protocol for removing residual micro- and nanoplastics from glassware before use has not yet been finalized. This is a critical gap that must be addressed before results can be considered quantitative (see Appendix A).
8 Reagent Preparation
8.1 Nile Red Stock Solution
- Working in a fume hood, dissolve Nile Red in acetone to 1 mg/mL.
- Store in a dark amber glass bottle at room temperature.
- Label with concentration, date, and analyst.
8.2 Working Concentration
The working concentration is 10 µg/mL, achieved by a 100:1 dilution of the stock. For a 500 mL water sample, add 0.5 mL of the 1 mg/mL stock solution (Maes et al., 2017).
9 Estimated Time
| Task | Time |
|---|---|
| Nile Red staining and incubation | 30 minutes |
| Vacuum filtration (per sample) | 10–15 minutes |
| Filter drying (oven at 60 °C) | 10 minutes |
| Enumeration (per sample, 3–10 fields) | 30–60 minutes |
| Total per sample (excluding wait times) | Approximately 90–150 minutes |
10 Procedure
10.1 Sample Preparation and Staining
- Minimize synthetic fiber contamination: wear a cotton lab coat, keep samples covered, and work in a clean area.
- Prepare samples in 500 mL volumes in glass bottles.
- Working in a fume hood, inject the appropriate volume of Nile Red stock solution (e.g., 0.5 mL for a 500 mL sample) to achieve 10 µg/mL. Cap immediately (Maes et al., 2017).
- Incubate with the injected dye for at least 30 minutes at room temperature.
10.2 Vacuum Filtration
- Place a Whatman 934-AH glass-fiber filter (42.5 mm, 1.5 µm pore) on the fritted base of the vacuum filtration apparatus.
- Vacuum-filter each sample through the filter.
- Dry the filter on a watch glass in the oven at 60 °C for 10 minutes.
10.3 Enumeration Option A: Crime-Lite with Goggles
Do not shine the Crime-Lite into anyone’s eyes. Never observe illuminated samples without the orange filter goggles.
- Place the dried filter on a dark, non-reflective surface.
- Put on the orange filter goggles.
- Illuminate the filter with the Crime-Lite (450–510 nm).
- Count fluorescing particles through the goggles, or mount the filter under the dissecting scope with a 550 nm emission filter.
- Record particle counts and morphology (fiber, fragment, bead, film).
10.4 Enumeration Option B: Echo Revolve Microscope (Fluorescence)
- Mount the dried filter on a glass slide per SOP-L211.
- Use the fluorescence channel (excitation ~470 nm, emission ~550 nm long-pass).
- Photograph and count fluorescent particles in at least 3 randomly selected fields of view.
- Import images into the Revolve app; use the annotation tools to mark and count particles.
11 Quality Control
- Include at least one procedural blank per analytical batch; blank particle counts inform the detection threshold for field samples (Erni-Cassola et al., 2017; Maes et al., 2017).
- Analyze at least 3 (preferably 10) randomly selected fields of view per filter; report mean, standard deviation, and coefficient of variation.
- Include at least one spiked filter (known number of plastic particles added before staining) per batch to verify Nile Red staining performance.
Every batch must include procedural blanks processed identically to samples. Without blanks, microplastic counts cannot be meaningfully interpreted (Maes et al., 2017).
12 Troubleshooting
| Problem | Probable Cause | Corrective Action |
|---|---|---|
| High blank counts | Airborne contamination; dirty glassware | Improve lab cleanliness; decontaminate glassware |
| No fluorescence on spiked samples | Nile Red concentration too low; degraded dye | Verify stock; prepare fresh solution |
| Fluorescence fades during counting | Photobleaching | Minimize illumination time; work quickly |
| Organic matter fluoresces | Nile Red binding to natural organics | Consider H2O2 digestion pre-treatment |
13 Waste Disposal
| Waste Type | Disposal Method |
|---|---|
| Acetone waste (Nile Red solution) | Collect in halogenated/organic waste; EH&S pickup |
| Used glass-fiber filters | Standard trash |
| Sample water (post-filtration) | Drain to sink |
14 References
- Maes et al. (2017)
- Erni-Cassola et al. (2017)
- California State Water Resources Control Board (2022)
- Thornton Hampton et al. (2025)
- APHA et al. (2012)
16 Training Record
Before performing microplastics analysis independently, personnel must:
- Read this SOP in full
- Complete SOP-L002 and SOP-L003 training
- Review the SDS for acetone and Nile Red
- Complete SOP-L211 Revolve Microscope training (if using the Revolve for enumeration)
- Demonstrate safe Crime-Lite operation under supervision
- Process at least one procedural blank and one spiked sample under supervision with acceptable results
- Be signed off by the PI or lab manager
17 Appendix A: Open Questions and Development Needs
| Item | Status | Notes |
|---|---|---|
| Glassware decontamination protocol | TBD | Critical for quantitative results |
| Organic matter interference assessment | TBD | Needed for high-organic samples |
| Polymer identity confirmation (FTIR access) | TBD | Lab does not currently have FTIR |
| Microplastic size class definition | TBD | Original SOP noted “DEFINE SIZE” |
| Nile Red and acetone catalog numbers | TBD | Add to materials table |
| Hydrogen peroxide digestion protocol | TBD | For removing organic matter before staining |
18 Appendix B: Regulatory and Standards Context
In 2022, the California State Water Resources Control Board approved a policy requiring drinking water agencies to monitor microplastics, specifying FTIR and Raman spectroscopy as the two approved analytical methods (California State Water Resources Control Board, 2022). Standardized collection procedures for aquatic biota and sediment are described in SCCWRP Technical Report #1410.A (Thornton Hampton et al., 2025).
The Nile Red fluorescence screening method in this SOP is appropriate for teaching and exploratory research but does not meet California regulatory requirements (California State Water Resources Control Board, 2022). For data intended for submission to SWAMP or CEDEN:
- Use microplastics-analysis-grade water for all blanks and rinses
- Adopt SCCWRP contamination control procedures
- Confirm polymer identity on a subset of particles by FTIR or Raman
- Consult the PI before beginning any study intended for regulatory submission