Date updated: 2026-04-21

Author: Conor JR Scott, Davis Roma

Affiliation: Department of Biosciences, University of Milan

Version: 2.0

Related DOI: [protocols.io link]

Abstract

Protocol for small-scale screening the BSFL gut community for oxidative plastic degradation.

Materials & Equipment

Solutions

  • Bacterial LCFM
  • 50 mM DCPIP solution
  • Sterile PBS
  • dH2O
  • 2 % (w/v) SDS solution
  • 70 % (v/v) ethanol

Plastic

  • 50 mL Falcon tubes
  • Ethanol-washed PE, PP, PS
  • 96-well spectrophotometric plate or spectrophotometric cuvettes
  • 1.5 mL eppendorf tubes
  • Plastic eppendorf pestle
  • 15 mL Falcon tubes

Equipment

  • P1000 + sterile tips
  • P100 + sterile tips
  • Shaking incubator
  • Spectrophotometer
  • Falcon tube centrifuge
  • Eppendorf centrifuge

Other

  • 15 final instar STD-reared larvae
  • Funnel
  • Whatman filter paper

Method

Preparing experimental cultures

  1. Add the following to 4 x sterile 50 mL falcon tubes:

a. Nothing

b. 200 mg washed PE

c. 200 mg washed PP

d. 200 mg washed PS

  1. Add 10 mL bacterial LCFM to each tube

  2. Prepare these tubes in duplicate for an uninoculated control

Preparing larval midgut suspensions

  1. Take 5 STD-reared final instar larvae from a larger rearing

  2. Dissect larvae and harvest midgut, remove the epithelium, and transfer midguts to a single eppendorf tube

  3. Weigh the guts and add 1 μL of sterile PBS per mg of gut

  4. Homogenise the midguts to a suspension using a plastic eppendorf pestle

  5. Repeat this two more times to give three replicate gut suspensions

Inoculation of DCPIP gut screening cultures

  1. Add 200 μL of midgut suspension to each of the four prepared cultures (not to the duplicate non-inoculated controls), using each of the triplicate gut suspensions for each of the triplicate sets of cultures

  2. To all cultures, including controls, add 10 μL of 50 mM DCPIP solution (final concentration 50 μM)

  3. Incubate all tubes with lids slightly loose at a 45° angle at 37 °C with shaking at 180 rpm

Tracking redox activity

  1. At chosen timepoints, transfer 150 μL of cultures into eppendorf tubes

  2. Centrifuge at 10,000 xg for 5 minutes to pellet plastic particles and bacterial cells

  3. Take 100 μL supernatant a 96-well spectrophotometric plate or to a spectrophotometric cuvette containing 900 μL bacterial LCFM

  4. Measure OD at 600 nm using bacterial LCFM as the reference

  5. To detect redox activity compare the reduction of the blue colour, and quantifiably the reduction in the absorbance of the DCPIP, in the presence of plastic compared to DCPIP only and the uninoculated controls

  6. Calculate the ratio of the A600 absorbance value in the inoculated cultures with plastic to the corresponding culture without plastic. A value < 1 signifies a reduction in DCPIP when the community is in the presence of plastic. Compare this to the same ratio in uninoculated controls. This value should get smaller over time.

Seeding into a new generation

  1. To inoculate the next generation of cultures, prepare the same tubes of plastic as before and add 9 mL bacterial LCFM

  2. Inoculate by adding 1 mL of the first generation of cultures to the new cultures

  3. Add 10 μL of 50 mM DCPIP solution (final concentration 50 μM) to the new cultures

  4. Incubate all tubes with lids slightly loose at a 45° angle at 37 °C with shaking at 180 rpm

Harvesting bacteria

  1. Use a P1000 pipette to transfer as much liquid as possible to 15 mL falcon tubes with as little plastic

  2. Centrifuge tubes at max speed for 10 mins at 4 °C

  3. Discard the supernatant

  4. Freeze the pellets in the tubes until needed for future analysis of the community

Harvesting plastic with adhered bacteria

  1. Take half the leftover plastic and transfer to a 15 mL falcon tube

  2. Add 5 mL PBS to tubes, and gently rock

  3. Pour off as much water as possible without losing plastic

  4. Repeat 2 more times

  5. Finally, air dry the rinsed plastic samples

Harvesting plastic and removing adhered bacteria

  1. To the remaining plastic in the original tube add 5 mL 2 % (w/v) SDS solution

  2. Shake at 180 rpm for a minimum of 4 hours

  3. Pour off slowly the SDS solution without losing plastic

  4. Add 5 mL 70 % (v/v) ethanol and repeat shaking incubation

  5. Pour off again

  6. Add 5 mL dH20, vortex for 10 seconds, pour off water

  7. Repeat the dH2O rinse two more times

  8. Air dry the samples

Additional notes

The washing and harvesting can be adapted based on the needs of the users and whatever is found to be most effective.

Keywords

Plastic, Screen, BSFL, Midgut

Disclaimer

Guidelines

Warning

Version History

Version Date Changes
1.0 2026-03-17 Initial creation of the protocol
2.0 2026-04-21 Added a lot of post-screen methods

References

🗣️ Acknowledgements