Sep 16, 2024
Version 2
Processing wastewater samples for bacterial & viral targets enrichment V.2
- Victor Mabasa1
- 1NICD
Protocol Citation: Victor Mabasa 2024. Processing wastewater samples for bacterial & viral targets enrichment . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2qb7pl1y/v2Version created by Victor Mabasa
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 08, 2024
Last Modified: September 16, 2024
Protocol Integer ID: 107675
Funders Acknowledgement:
Bill & Melinda Gates Foundation
Abstract
This wastewater sample processing protocol by the Wastewater Genomics Syndicate at the National Institute of Communicable Diseases (NICD) is designed to enrich bacteria and viruses from the settled solids and supernatant components of wastewater, as different pathogens partition differently within this matrix. The end products are waterless solids and clarified wastewater supernatants, ready for downstream applications such as ultrafiltration, bead enrichment and nucleic acid extraction.
Materials
Equipment
–80°C freezer to store settled solids samples
4°C refrigerator storage of samples
Biological safety cabinet (Class II)
Refrigerated centrifuge (4˚C) with adapters to accommodate 500 mL centrifuge bottles
Consumables
2 mL cryo-tubes
250 mL Conical centrifuge bottles
Labels
Waste container
Plain wooden sticks
Solutions/Reagents
70% ethanol
1% Virkon
Sample Collection & Handling
Sample Collection & Handling
23h
23h
Using correct personal protective equipment (PPE), collect a 1 000 mL grab wastewater sample from each location in a sterile bottle and transfer them to the laboratory in a cold chain (keep in a lab fridge at 4°C). it is important to proceed to the next step within the next 23 hours after sample collection to prevent major degradation of microorganisms.
23h
Preparing for centrifugation
Preparing for centrifugation
10m
10m
All work should be performed in a Class II biological safety cabinet while wearing appropriate PPE. Clean your workspace with a 1% Virkon solution and a 70% ethanol solution. Double-line your waste bucket with biohazard waste plastic, and ensure all your filter tips are refilled. Finally, label 250 mL conical centrifuge bottles, one for each wastewater sample.
10m
Centrifugation
Centrifugation
50m
50m
Transfer 250 mL of the wastewater samples to the corresponding conical tubes
5m
Centrifuge at 4 650 × g (deceleration speed set at 5), at 4oC for 10 minutes.
15m
Carefully transfer the supernatant from the 250 mL conical bottle to a clean, marked 1 000 mL bottle without disturbing the settled solids.
5m
Repeat steps 3 to 5 until the original sample is finished.
20m
Keep the clarified wastewater sample at 4oC for different downstream applications (check our page for different nucleic acid extraction protocols from clarified wastewater).
5m
Transfer settled solids
Transfer settled solids
20m
20m
Use clean, plain wooden sticks to pick out settled solids and pack them into marked 2 mL cryo-tubes. Centrifuge briefly to remove water.
15m
Store the settled solids in the –80°C freezer until needed for downstream applications. (check our page for different nucleic acid extraction protocols from settled solids).
5m