TITLE: Microplastics extraction from oyster tissue 
AUTHORS: Benjamin-Rafael Mingoa, Archana Anand

[DESCRIPTION]
Microplastics are defined as any plastic particles that are smaller than 5mm. They have been found in many of our household products, such as detergents, toothpaste, and other beauty products such as exfoliants. It has also been found that clothes with synthesized fibers, such as acrylics, nylon, and polyester shed hundreds of thousands of microplastic particles every time you wear or wash them. Due to the size of microplastics, they easily pass through many water filtration systems and end up in bodies of water, such as the San Francisco Bay or the Pacific Ocean. This protocol describes the procedure to extract and quantify microplastics in oyster tissue.

[STEPS]
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
1. Take samples of the gastrointestinal tract (GIT) /biofilm and gill tissues (fish gills have the same biofilm membrane)
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
2. Weigh out 1-2 g of the tissues. Use a glass cover for weighing.
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
4. 10% KOH, 50 mL, 
(as a guideline 1:50 ratio of tissue to KOH works well)
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
6. Filter through coarse mesh 
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
13. Wash with 50 mL boiling water
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
14. Incubate for 10 min with 10 mL acetone
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
15. Wash with another 10 mL of acetone
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
16. Staining with 1mL of diluted Nile Red
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
3. Cut the sample with forceps to increase surface area
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
5. incubate at 60 C for 24 hours
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
11. Rinse sides of filter with distilled water
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
10. Pour the KOH and tissue solution into the Whatman 1 filter. Vacuum.
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
7. Set up a filtration system (Whatman 1 filter, flask)
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
8. Soak the Whatman 1 filter in distilled water to prevent it from floating
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
9. USE VACUUM (or samples will be compromised due to floating)
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
12. Carefully take the filter and place it into a glass plate with forceps
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
17. Rinse with 10 mL of water  (wash the sides of the filter)
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
18. Place into a Petri plate 
SECTION: Preparation and extraction of microplastics from Oyster Gastrointestinal Tract/Tissues
19. Observe, use a fluorescent scope