Three types of floating plastic will be studied: high density polyethylene (HDPE, #2), low density polyethylene (LDPE, #4) and polypropylene (#5). Approximately 6 cm x 6 cm squares of plastic will be sterilized by washing in 70% ethanol followed by UV-irradiation. Sterilized plastic are kept in sterile autoclave bags until assembly of the sampling system. The plastic squares will be placed in 8 x 8 cm custom-made ocean grade (316) stainless steel grids. Three cages, containing one of each plastic types will be in turn placed in a 36 cm x 14 cm 314 stainless steel grid cage.
Samples will be deployed at Doheny State Beach, just outside the surf break. Cages will be attached to a buoy to keep them floating just underneath the water surface. Samples of plastic and water will be collected weekly for 4 weeks. After taking swabs for microbiology cultures, samples will be stored at -20 oC until processing.
Culture of microorganisms
Swabs of plastic and water will be spread on Salt Water Agar and ChromAgar Vibrio plates, and incubated at room temperature for 48 hours. Number and types of colonies will be counted on the ChromaAgar Vibrio plates. Representative colonies will be subcultured or frozen for further analysis.
DNA will be extracted from whole plastic using the MOBIO PowerSoil kit. Negative controls will include sterile plastic or no sample (kit components only). For water samples, the MOBIO PowerWater kit will be used.
Extracted DNA will be sent to Omega Biosciences for NGS library preparation and sequencing (see protocol).
We will use the GCAT-SEEK Metagenomics pipeline (see protocols), adapted for the 16S V4-V5 region of ribosomal RNA. This is an area of bacterial genomes that is stable enough to sequence over many different families of bacteria but contains enough information to genetically identify individual species. After we have identified OTUs/species we will use rarefaction and UNIFRAC distances to calculate alpha and beta diversity and look for shifts in the proportion of pathogenic species.
Browse the protocols that are part of the experimental methods.