Date updated: 2026-02-02

Author: Conor JR Scott

Affiliation: University of Milan, Department of Biosciences

Version: 1.0

Related DOI:

Abstract

Multiple papers exploring bacterial growth on plastic substrates fail to consider the oligormers, additives, and contaminating substrates which may be used by bacteria for growth, falsely suggesting the ability to degrade polymeric plastic. This is a simple washing protocol plastic substrates in particles of various sizes.

Materials & Equipment

  • Plastic particles
  • Funnel
  • Measuring cylinder (500 mL)
  • 500 mL glass bottle
  • 500 mL glass beaker
  • Filter paper
  • Magnetic stirrer + stir bar
  • Spatula
  • 90 % (v/v) ethanol
  • dH2O
  • Syringe / Pipette
  • Drying cabinet / incubator / oven (something that can maintain a constant temperature of 40° C)

Method

Ethanol washing plastic particles

  1. Weigh 50 g of plastic particles into a 500 mL glass bottle

  2. Add 300 mL 90 % (v/v) ethanol and a stir bar

  3. Close the bottle with a lid but leave slightly loose

  4. Place on a magnetic stirrer and stir at a speed high enough to submerge and move all plastic particles (this speed will depend on the size of the particles)

  5. Leave to wash for 24 hours

  6. After 24 hours, remove the bottle from the stirrer and remove the stir bar

  7. Place a funnel into a 500 mL measuring cylinder and line with a double layer of circle filter paper, folding the paper to fit it into the funnel without cutting

  8. Slowly add the ethanol and plastic solution to the filtering system. It is possible to let the plastic settle and pour mainly the ethanol through first. Also not all the plastic needs to be added to the filter as it will be washed again so the ethanol does not need to be completely removed.

  9. Use a spatula to scrape the majority of plastic particles caught on the filter paper back into the glass bottle

  10. Can use a syringe or a pipette to wash any remaining particles into the glass bottle using 90 % (v/v) ethanol

  11. Add another 300 mL of 90 % (v/v) ethanol to the bottle and repeat the stirring for another 24 hours

Washing ethanol from plastic particles

  1. Repeat the filtration process as before but use dH2O for any washing of the filter paper into the bottle

  2. Add 300 mL dH2O to the bottle and stir for 1 hour

  3. Repeat filtration and water washing two more times

Drying plastic particles

  1. For the final filtration, attempt to get as much of the dH20 off the plastic by filtration, use more dH2O to wash the bottle to capture as much of the plastic as possible and filter

  2. After the final filtration attempt to minimise the dH2O used to wash the filter paper and maximise the residual dH2O that is removed by filtration

  3. Transfer any filtered plastic from the filter paper to a glass beaker using a spatula (or could use a container with a larger surface area if you are using a larger volume of plastic)

  4. Place in a 45 °C incubator or drying cabinet for 48 hours (or longer if needed) until completely dry

Additional notes

It may be of interest to capture in falcon tubes the filtrate from the 1st and 2nd ethanol washes to analyse anything washed from the plastic substrates. The volumes used here were chosen becuase they allow the volume of plastic that gives 50 g to move well and be more than fully submerged when stirring, for different plastic types then these could easily be changed.

Keywords

Plastic, Washing, Ethanol

Disclaimer

This is a simple washing protocol which is already an advance on what many papers do of just using substrates without any consideration of the additives, contaminants, or oligomers. But it is not an optimised protocol to remove oligomers etc. There may be other solvents which are better, but it is a balance between removing other compounds and not modifying the polymer which could alter the substrate.

Guidelines

Can change all the equipment for what suits you.

Warning

Can work in a fumehood with 90 % ethanol if worried about the fumes, and obviously ethanol is flammable so never use it near any heat sources or open flames

Version History

Version Date Changes
1.0 2026-02-02 Initial creation of the protocol