Activity 2: Disc Retrieval, Sampling, Separation & Processing

Small groups of students (three or four) can be organized to perform tasks related to disc retrieval, transport, and processing in the classroom and can provide a check for ensuring that proper procedures are followed.

Procedures

1. Retrieve the rack from the water slowly. Carefully remove the number of discs needed and place in a metal tray for transport. Cover tray with foil or a metal lid. Keep discs separate from each other. (Note: Placing the discs in water during transport will cause the biofilm to slough off the disc. Water from the site can be transported in a separate container and used to completely submerge the disc in a glass dish for observation under the microscope.)
2. At this point, visually observe disc biofilm assemblages for biodiversity under a dissecting microscope.
3. Make a 70 ppt saltwater solution in a 1L glass graduated cylinder. Verify salinity with a refractometer.
4. Select a biomass sample up to 25% of the total disc mass and scrape it off using a metal razor blade. Place in a glass petri dish and cover with a lid.
5. Any larger organisms or inorganic material, such as rocks and shells, should be carefully removed from the sample with metal forceps and rinsed with distilled water over the separation funnel to remove any possible microplastics. The items can then be removed from the sample completely as they could impede the separation and clog the funnel. The sample is now ready for separation with the glassware specified.
6. Use 200 mL to 400 mL of 70 ppt saltwater to suspend the sample in the 500 mL separation funnel. Base the amount of saltwater added to the funnel on how much organic material looks to be on your biofilm sample—more organic material will require more saltwater for suspension and separation. Be sure not to add more water than the total volume of the separation funnel.
7. Use DI water to spray the entirety of the inside of the petri dish in order to ensure that all of the sample makes its way into the funnel or use a regular glass funnel when pouring both your saltwater and your biofilm sample into the separation funnel. Shake well to mix.
8. Allow the sample to settle for at least 30–45 min (longer is better). Check on the sample every few minutes in the separation funnel—if you notice the funnel getting clogged or organic material sticking to the sides, give the sample an occasional shake and allow the material to settle again.
9. Drain any settled material into a 500 mL beaker and discard. This portion of the sample may contain dense microplastics, but for the purposes of these methods, students will focus on isolating less dense, more buoyant microplastics. The remaining liquid in the separation funnel will be used for the filtration onto the filter paper with the manual vacuum pump system.
10. Drain 150–200 mL of remaining liquid into a clean 200 mL beaker and cover with foil. This will be used in the filtration process to look for microplastics.
11. Connect the 150 mL Buchner funnel to the 500 mL Erlenmeyer flask at the ground joint and connect the vacuum pump.
12. Place a piece of gridded 60 mm filter paper into the bottom of the Buchner funnel. From the 200 mL beaker, pour the liquid into the Buchner funnel along the inside of the glass. Cover the funnel with foil.
13. Use the vacuum pump system to pull the sample through the paper; the waste liquid will collect in the Erlenmeyer flask below.
14. Use metal forceps to remove the filter paper from the Buchner funnel and place it face up on top of a 75 mm × 50 mm glass slide, centered as best as possible.
15. Place a second glass slide on top of the paper, centered as best as possible. The 2 glass slides form a “sandwich” around the filter paper. Some of the filter paper will be exposed.
16. Repeat steps 9–15 for the remaining liquid in the separation funnel.

Formative assessment prompt.

(It is suggested that this prompt be performed after students have completed at least one attempt at isolating microplastics from a biofilm sample.) Students will work in small groups (3–4) to propose methods for altering or improving the isolation of microplastics from the biofilm sample. Students should keep in mind the safety guidelines and tips for avoiding contamination in their proposed methods.