TITLE: Non-destructive microplastic isolation from water and sediment samples
AUTHORS: Jonas Stadfeld, Sneha Suresh, Srijak Bhatnagar

[DESCRIPTION]
Microplastics in aquatic ecosystems serve as unique habitats for diverse microbial communities, collectively referred to as the plastisphere. Investigating these microbes using both culture-dependent and culture-independent techniques (e.g., 16S rRNA gene sequencing, metagenomics) requires a method that preserves the integrity of the plastic and associated microbiota. This protocol outlines a non-destructive isolation technique for microplastics from water and sediment samples using Nile Red dye, thus allowing for a comprehensive study of the microplastic microbiome and its functional potential without compromising the plastic matrix or the microbial communities it harbors.

[STEPS]
SECTION: Microplastic Isolation - Water
10. NOTE: Use only metal and glass equipment. Tubing should be silicone.

SECTION: Microplastic Isolation - Water
11. Filter at least 40L of water through a 20µm Cellulose filter (47 mm).

Note: Depending upon the murkiness, the filter may clog before 40L is filtered. In that case, multiple filters can be combined to form ONE replicate 40L sample. 
The Filter can be stored at -20 °C if used for DNA extraction. 

SECTION: Microplastic Isolation - Water
12. Place the filter in a petri dish and add 1 mL of Nile Red solution (2 µg/mL).

SECTION: Nile Red Solution (2 µg/mL)
2. Using a serological pipette, carefully add 50 mLof molecular-grade acetone to the Falcon Tube.

SECTION: Nile Red Solution (2 µg/mL)
3. Screw the Falcon tube cap and vortex vigorously to dissolve the Nile Red dye (30 s).

SECTION: Nile Red Solution (2 µg/mL)
4. Pick up the dissolved Nile red dye into a 50 mL syringe by pulling the plunger.
SECTION: Nile Red Solution (2 µg/mL)
5. Filter sterilization: Attach a sterile 0.22 µm Luer-lock syringe filter to the syringe and push the Nile red into a new sterilized 50 mL falcon tube.

SECTION: Nile Red Solution (2 µg/mL)
7. In a sterilized 500 mL Nalgene Glass media bottle (orange cap), add 499 mL of Milli Q water.
Tip: Add 500 mL using a measuring cylinder and remove 1 mL using a pipette.
SECTION: Nile Red Solution (2 µg/mL)
8. Add 1 mL of filter-sterilized Nile red stock solution (1 mg/mL). Close the lid and shake gently. 
Cover it in Aluminum foil and label. 
SECTION: Nile Red Solution (2 µg/mL)
6. Label the tube as concentrated Stock solution (1 mg/mL), cover it in aluminum foil, and store at 4 °C 

SECTION: Nile Red Solution (2 µg/mL)
9. Store in the dark at 4 °C for long-term storage.

SECTION: Microplastic Isolation - Water
13. Incubate the filter in dark at 4 °C for1440 min

SECTION: Microplastic Isolation - Sediment
17. Freeze dry the 0.5 kg   at 80 ˚C to remove all water. 
Note: The duration of freeze-drying varies based on the water content of the sample.  Expect 24 hours to three days. 
SECTION: Microplastic Isolation - Water
14. Place the filter in a fluorescence microscope under a 529 nm wavelength filter
SECTION: Microplastic Isolation - Water
15. Using sterile forceps, pick the red fluorescing microplastics and transfer them to a 1.5 mL microcentrifuge tube containing 100 µL sterile 1X PBS.
Note: Nile Red can stain roots and other biological materials. In background noise, microplastics fluoresce strongly, lacking compartments and multicellular structure. 
SECTION: Microplastic Isolation - Water
16. Optional step: Rinse the microplastics with 100 µL 1X PBS to remove environmental contaminants by pelleting the plastics with centrifugation (1000 xg for1 min), discarding the supernatant and repeating the step twice with fresh sterile 1X PBS.  

SECTION: Microplastic Isolation - Sediment
18. While the freeze dryer runs, prepare 900 mL of Calcium Chloride Solution (final density of 1.4 Kg/L) for each sample. 


SECTION: Microplastic Isolation - Sediment
18.1. Weigh the 400 g of CaCl2 and transfer it to a sterile 1L Nalgene media bottle.

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18.2. Add a sterile stir bar and 1 L of MilliQ water and stir until dissolved.

SECTION: Microplastic Isolation - Sediment
20. Using as sterile spatula, transfer the freeze-dried sediments to a 1 L sterile beaker.
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21. Add 300 mL of the CaCl2 solution and mix vigorously using the spatula.

SECTION: Microplastic Isolation - Sediment
22. Cover with aluminum foil and let the sample stand for 720 min.

SECTION: Microplastic Isolation - Sediment
23. Decant the CaCl2 solution into a clean, sterile beaker and store the decanted solution at 4 °C.

SECTION: Microplastic Isolation - Sediment
24. Repeat Steps 19-21 two more times with 720 min and 1440 min of sedimentation time for Step 21.

SECTION: Microplastic Isolation - Sediment
25. Filter all the decanted CaCl2 solution through a 20 µm cellulose filter.
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26. Use Steps 11 -15 (Microplastics Isolation - Water) to isolated sediment microplastics.
SECTION: Nile Red Solution (2 µg/mL)
1. Add 50 mg of Nile Red to a Falcon Tube.

SECTION: Microplastic Isolation - Sediment
19. Optional Step: weigh the freeze dried sediment to compare microplastics across various samples