May 07, 2025
Clarification of wastewater samples
- Victor Mabasa1,
- Natasha Singh1,
- Emmanuel Phalane1,
- Mokgaetji Macheke1,
- Sipho Gwala1,
- Thabo Mangena1,
- Lethabo Monamets1,
- Lebohang Rabotapi1,
- Nkosenhle Ndlovu1,
- Fiona Els1,2,
- Sibonginkosi Maposa1,
- Said Rachida1,
- Kerrigan McCarthy1,3,
- Mukhlid Yousif1,3
- 1National Institute for Communicable Diseases;
- 2Gauteng City-Region Observatory;
- 3University of Witwatersrand
- Wastewater Genomics Syndicate
Protocol Citation: Victor Mabasa, Natasha Singh, Emmanuel Phalane, Mokgaetji Macheke, Sipho Gwala, Thabo Mangena, Lethabo Monamets, Lebohang Rabotapi, Nkosenhle Ndlovu, Fiona Els, Sibonginkosi Maposa, Said Rachida, Kerrigan McCarthy, Mukhlid Yousif 2025. Clarification of wastewater samples. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8qo99l2w/v1
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: May 07, 2025
Last Modified: May 07, 2025
Protocol Integer ID: 217828
Keywords: Wastewater solids, wastewater, NICD, South Africa, WBE, WBS
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 or 500 mL Conical centrifuge bottles
Waterproof Labels
Waste container
Plain wooden sticks
Solutions/Reagents
70% ethanol
1% Virkon
Sample Collection & Handling
Sample Collection & Handling
Wearing appropriate personal protective equipment (PPE), collect a 1 000 mL grab wastewater sample from each location using a sterile bottle. Transport the samples to the laboratory under cold chain conditions and store them at 4 °C. To minimise microbial degradation, proceed to the next step within 23 hours of sample collection.
Preparing for centrifugation
Preparing for centrifugation
Conduct all work in a Class II biological safety cabinet while wearing appropriate PPE. Clean the workspace using 1% Virkon followed by 70% ethanol. Double-line the waste container with biohazard waste bags, ensure all filter tip racks are refilled, and label a 250 mL conical centrifuge bottle for each wastewater sample.
Centrifugation
Centrifugation
Transfer each wastewater sample to its corresponding conical centrifuge bottle, taking care not to overfill to prevent spillage during centrifugation.
Centrifuge the samples at 4 700 × g for 10 minutes at 4 °C.
Carefully transfer the supernatant to a clean, marked 1 000 mL bottle without disturbing the settled solids.
Repeat steps 3 to 5 until the original sample is finished.
Storage
Storage
Aliquot 6 × 5 mL cryotubes of the clarified supernatant and store them at −80 °C for long-term preservation. Keep the remaining clarified wastewater at 4 °C for other downstream applications.
Using clean, plain wooden sticks, collect settled solids and transfer them into labelled 2 mL cryotubes. Briefly centrifuge to remove excess liquid, then store the tubes at −80 °C until needed for downstream applications.