Date updated: 2026-03-13

Author: Conor JR Scott

Affiliation: Department of Biosciences, University of Milan

Version: 1.1

Related DOI: [protocols.io link]

Abstract

Protocol for small-scale screening of a bacterial isolate for plastic degradation.

Materials & Equipment

Solutions

Plastic

Equipment

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

Other

  • Cryo preserved glycerol stock of isolate
  • LB-agar plate

Method

Work in a sterile laminar flow hood

Pre-cultures of isolates

  1. Using a sterile P100 pipette tip, touch the tip against the contents of the isolate glycerol stock and streak onto an LB-agar plate

  2. Incubate the plate upside down in a 37 °C incubator overnight

  3. The following day, add 5 mL sterile LB to a sterile 15 mL falcon tube

  4. Using a sterile P100 pipette tip, pick a single colony from the LB-plate and eject the tip into the LB-containing tube

  5. Secure the lid of the tube but leave slightly loose and place in a shaking incubator at 37 °C and 180 rpm overnight

Preparing experimental cultures

  1. Add the following to 4 x sterile 50 mL falcon tubes a. Nothing b. 100 mg washed PE c. 100 mg washed PP d. 100 mg washed PS

  2. Add 10 mL bacterial LCFM to each tube

  3. Prepare these tubes in duplicate for an uninoculated control

Inoculation of DCPIP screening cultures

  1. Transfer 100 μL of the pre-culture to a cuvette and dilute 1/10 by adding 900 μL of sterile PBS, also prepare a reference cuvette of sterile LB diluted in the same way

  2. Measure the OD at 600 nm and make note

  3. Centrifuge the remaining pre-culture at 3000 xg for 5 minutes

  4. Discard the supernatant and resuspend the pellet by pipetting in 1 mL sterile PBS

  5. Repeat the centrifugation and resuspension twice more, but use 1 mL sterile bacterial LCFM for the final resuspension

  6. Measure the OD at 600 nm using a 1/10 dilution as before but this time with a reference of bacterial LCFM

  7. Calculate the inoculation volume to give an OD of 0.01 in the 10 mL cultures using the followin equation:

Inoculation volume = (Target OD / Measured OD) x Final volume

e.g. (0.01 / 0.5) x 10 mL = 0.2 mL

  1. Add this volume to each of the four 50 mL falcon tubes prepared

  2. Finally, add 300 μL 1 mM DCPIP (final concentration 30 μM) to all tubes, including non-inoculated controls

  3. Incubate all tubes with lids slightly loose at a 45° angle at 37 °C with shaking at 180 rpm for 7-10 days

Tracking redox activity

  1. Every day, 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 mL supernatant and transfer to a cuvette, dilute 1/10 with 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 qunatifiably the reduction in the absorbance of the DCPIP, in the presence of plastic compared to DCPIP only and the uninoculated DCPIP only control

Additional notes

If screening multiple isolates, then only one set of uninoculated controls are necessary.

Keywords

Plastic, Screen, DCPIP, Redox

Disclaimer

Not statistically reproducible unless performed in triplicate. Designed to screen bacterial isolates.

Version History

Version Date Changes
1.0 2026-02-23 Creation of protocol
1.1 2026-03-13 Update the centrifugation speed for pelleting pre-cultures, and corrected DCPIP