Date updated: 2025-11-25
Author: Conor JR Scott, Davis Roma, Francesca De Filippis, Silvia Caccia
Affiliation: Università degli Studi di Milano, Università degli Studi di Napoli Federico II Version: 2.0
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Abstract

A protocol for the initial screening of plastic-degrading fungal or bacterial taxa from the gut microbiota of Hermetia illuciens (BSFL) reared on diets containing polyethylene (PE), polystyrene (PS), or polypropylene (PP). Screening is based on bacterial or fungal growth on solid media or in liquid cultures containing plastic particles of PE, PS, or PP.

Materials & Equipment

Larvae

  • 9 x larvae reared on PE diet
  • 9 x larvae reared on PS diet
  • 9 x larvae reared on PP diet

Plastics

  • Powdered PE
  • Powdered PS
  • Powdered PP

Glassware

  • 4 x 500 mL glass bottles
  • 9 x 250 mL conical flasks with sponge/paper bungs

Media

Electrical

  • Autoclave
  • Vortex
  • Whitley A45 Anaerobic workstation set to 30 °C
  • Static incubator set to 30 °C
  • Shaking incubator set to 30 °C
  • 3 x magnetic stirrers

Plastic

  • 9 x sterile Eppendorfs
  • Plastic Eppendorf pestle
  • Sterile petri dishes
  • Sterile falcon tubes
  • Sterile inoculation loops

Other

  • 70 % (v/v) ethanol
  • Aluminium foil
  • P200 pipette
  • 10 - 200 μL sterile pipette tips
  • 3 x autoclaved magnetic stirring bars
  • Deionised water (dH2O)

Method

Preparing solid media

  1. Add 400 mL Fungal LCFM to a 500 mL glass bottle, add 0.6 g bacteriological agar, autoclave

  2. Add 400 mL Bacterial LCFM to 2 x 500 mL glass bottles, add 0.6 g bacteriological agar to each, autoclave

  3. Prepare 18 sterile petri dishes in a laminar flow hood and label 9 each with: “10 g/L” or “20 g/L”
    • Label 3 of each with: “PE”, “PS”, or “PP”
    • Label 1 of each concentration and plastic with “Fungal”
    • Label 2 of each of concentration and plastic with “Bacterial”
    • Take 1 of each concentration and plastic of the bacterial plates and label with “Aerobic”
    • Take 1 of each concentration and plastic of the bacterial plates and label with “Anaerobic”
  4. Prepare 3 additional petri dishes:
    • Label with “Agar control”
    • Label each with either: “Fungal”, “Bacterial Aerobic”, or “Bacterial Anaerobic”
    • Divide each plate roughly equally into thirds by drawing on the base and label each section with: “PE”, “PS”, “PP”

  5. Prepare 21 sterile 50 mL falcon tubes labelled in the exact same way as the tubes i.e. a single falcon tube for each petri dish

  6. In a sterile laminar flow hood, add 0.3 g of each plastic powder to each of the tubes labelled with “10 g/L” and 0.6 g to tubes labelled “20 g/L”. Add nothing to the control tubes

  7. When Fungal LCFM agar is still molten add 30 mL to a single falcon tube labelled with “Fungal”, and vortex to distribute the plastic particles

  8. Pour plastic containing agar into the corresponding petri dish and pour the non-plastic containing controls also

  9. Repeat for the Bacterial LCFM agar

  10. Allow all plates to cool until set

  11. Move the plates labelled “Anaerobic” into the anaerobic chamber at least 1 day prior to inoculation to deoxygenate

Preparing liquid cultures

  1. Add 50 mL Fungal LCFM to 3 x 250 mL conical flasks
    • Label each with: “PE Fungal”, “PS Fungal”, “PP Fungal”
  2. Add 50 mL Bacterial LCFM to 6 x 250 mL conical flasks
    • Label 3 flasks with: “PE Bacterial Aerobic”, “PS Bacterial Aerobic”, “PP Bacterial Aerobic”
    • Label 3 flasks with: “PE Bacterial Anaerobic”, “PS Bacterial Anaerobic”, “PP Bacterial Anaerobic”

  3. Seal flasks with the sponge/paper bungs, cover with tin foil and autoclave

  4. In a sterile laminar flow hood, add 0.5 g of each powdered plastics (10 g/L) to the corresponding flasks

  5. Move the anaerobic cultures into the anaerobic chamber at least 1 day prior to inoculation to deoxygenate

Harvesting gut microbiota

  1. Wipe a plastic pestle with 70 % (v/v) ethanol

  2. Prepare 9 sterile eppendorfs for each each of the three plastics and for the three growth conditions
    • Label with: “PE Fungal”, “PE Bacterial Aerobic”, “PE Bacterial Anaerobic”, “PS Fungal”, “PS Bacterial Aerobic”, “PS Bacterial Anaerobic”, “PP Fungal”, “PP Bacterial Aerobic”, “PP Bacterial Anaerobic”

  3. Starting with the PE reared larvae, select 3 larvae of roughly the same size, rinse with tap water to remove diet, then clean with 70 % (v/v) ethanol

  4. Dissect each larvae to extract the midgut and place all 3 midguts in the same tube labelled “PE Fungal”

  5. Now working in a sterile laminar flow-hood, add 500 μL sterile Fungal LCFM

  6. Homogenise the midgut solution by smashing with plastic pestles and with occasional vortexing

Inoculation of solid media

  1. Using a sterile inoculation loop streak a loop of “PE Fungal” midgut suspension onto the “PE Fungal” plates, taking a new loop each time from the midgut suspension with a new inoculation loop. Also streak a loop onto the “PE” section of the “Fungal” control plates.

  2. Repeat for all other “Aerobic” conditions

  3. Incubate plates upside down at 30 °C in the static incubator

  4. Move the “Anaerobic” midgut suspensions into the Anaerobic workstation

  5. Streak these suspensions onto the anaerobic plates in the worksation and incubate upside down at 30 °C

Inoculation of liquid media

  1. With the remaining “PE Fungal” midgut suspension, take 150 μL using sterile pipette tips and inoculate into the “PE Fungal” liquid culture

  2. Repeat liquid culture inoculation for the remaining “Aerobic” conditions

  3. Incubate all flasks at 30 °C with shaking at 120 rpm in the shaking incubator

  4. In the anaerobic workstation repeat this with the remaining anaerobic midgut suspensions to inoculate the anaerobic liquid cultures

  5. Add an autoclaved magnetic stirring bar to each flask and place flasks on a magnetic stirrer set at 120 rpm

Preparing TSA plates

  1. Add 12 g of tryptic soy broth to a 500 mL glass bottle

  2. Add 2 g of yeast extract to the same bottle

  3. Add 6 g of bacteriological agar to the same bottle

  4. Add 400 mL dH2O, swirl to mix, and autoclave

  5. When still molten, in a sterile laminar flow hood, pour as many TSA agar plates as possible and allow to set. These plates can be stored at 4 °C.

Isolating microorganisms

  1. On days 5, 12, and 26 of incubation check the plates solid media plates for growth. Move some TSA plates into the anaerobic workstation the day prior to any isolation days.

  2. In a sterile laminar flow hood, isolate any unique looking colonies from each condition by picking with an inoculation loop and streaking onto a single TSA plate per unique isolate.

  3. Streak a loop of liquid cultures onto single TSA plates per condition

  4. Repeat the same in the anaerobic workstation

  5. Incubate TSA plates aerobically or anaerobically at 30 °C overnight

  6. If the isolation plates look pure, then individual colonies can be used for ID. Plates of mixed colonies can be separated further by additional streaking onto new TSA plates.

Additional notes

Dissection of larvae was not covered here but is covered comprehensively in the BugBook

For the agar, it is essentially an additional carbon source that might encourage bacterial or fungal growth. This is why control plates are used, but using a higher purity agar reduces the likelihood of something growing on the plates despite not having any plasticolytic activity.

Instructions on using the Whitley A45 Anaerobic workstaton can be found here. Other anaerobic workstations can be used.

A shaking platform might be easier to use in the anaerobic workstation if one can fit in there.

Anaerobic colonies are slow growing and require a couple of days on TSA plates to grow.

Keywords

Bacteria, Fungi, Plastic, Polyethylene, Polystyrene, Polypropylene, Screening, ID

Disclaimer

This experimental setup is a preliminary screen for potential plastic-degrading taxa. Plastic particles were not selected for optimal growth but chosen based on availability.

Guidelines

Plastic particles were unknown here at the time of use but are not hugely relevant for this preliminary screen to ID anything that grows

This protocol attempts to avoid any harsh treatment of the plastic which may alter its structure or introduce toxic chemicals that may inhibit the growth of microorganisms.

Warning

Always remember when autoclaving to use a larger glass vessel than the volume you want to autoclave to avoid it boiling over. Always remember to leave the bottle caps loose on duran bottles when autoclaving! Wear goggles and a mask when handling plastic particles.

Version History

Version Date Changes
1.0 2025-10-13 Initial creation
1.1 2025-10-23 Re-ordered and updated to remove complex STD diet conditions and improve the clarity
2.0 2025-11-25 Updated the method to remove Tween which wasn’t used anyway, and added the strain isolation from solid media and liquid cultures

References

Bruno, D., Manas, F., Bonelli, M., Gold, M., Marzari, M., Roma, D., Valoroso, M.C., Montali, A., Guillaume, J.B., Rebora, M., Bressac, C., Herman, N., Caccia, S., Casartelli, M., & Tettamanti, G. (2025). BugBook: life cycle, reproduction, and morphofunctional characterisation of the gut, fat body, and haemocytes in the black soldier fly. Journal of Insects as Food and Feed (published online ahead of print 2025). https://doi.org/10.1163/23524588-20250002

🗣️ Acknowledgements