Mar 09, 2022

Public workspaceEnvironmental Grab Sampling and Membrane Filtration

This protocol is a draft, published without a DOI.
  • 1Christian Medical College, Vellore, India;
  • 2UK Health Security Agency;
  • 3Malawi Liverpool Wellcome Trust Clinical Research Program, Blantyre, Malawi;
  • 4Imperial College London;
  • 5University of Washington
Catherine Troman
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Protocol Citation: Dilip Abraham, Nicola Elviss, Nicholas Feasey, Nick Grassly, Jacob John, Gagandeep Kang, John Scott Meschke, Venkata Raghava Mohan, Satheesh Nair, Jonathan Rigby, Rajan Srinivasan, Catherine Troman, Christopher Uzzell, Nicolette Zhou 2022. Environmental Grab Sampling and Membrane Filtration. protocols.io https://protocols.io/view/environmental-grab-sampling-and-membrane-filtratio-brewm3fe
Manuscript citation:
Rigby J, Elmerhebi E, Diness Y, Mkwanda C, Tonthola K, Galloway H, MIles R, Henrion M, Edwards T, Gauld J, Msefula C, Johnston R, Nair S, Feasey N, Elviss N (2021), Optimized methods for detecting Salmonella Typhi in the environment using validate field sampling, culture and confimratory molecular approaches. Journal of Applied Microbiology, doi: 10.1111/jam.15237
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: January 13, 2021
Last Modified: April 10, 2024
Protocol Integer ID: 46262
Keywords: Salmonella, Typhi, Paratyphi, wastewater, environmental surveillance
Disclaimer
Please note, the author list is in alphabetical order and does not reflect contribution.
Abstract
A protocol for the collection of environmental samples (sewage and wastewater) using grab sampling and membrane filtration. This is part of a set of protocols for environmental surveillance of Salmonella Typhi.

1L of sewage or wastewater is collected then filtered under a vacuum to capture the bacteria on filter discs ready for DNA extraction.

This protocol is adapted from the method described in Rigby et al (2021).
Guidelines
It is recommended that sampling be done in the morning.
Materials
Ringer's lactate solution

Bucket/container with 1L capacity
Membrane filtration apparatus
47mm 0.45um filter discs
Coffee filters (if necessary)
Whirl-Pak bags

Appropriate PPE and disinfenctants
Before start
Ensure that you wear appropriate PPE whilst sampling (e.g. gloves, appropriate footwear, disposable lab coat, facemask).
Sample Collection
Sample Collection
Using a bucket or another method, fill a 1L collection container with sewage or wastewater and securely close the container.
Wipe the outside of the collection container with disinfectant.
Place the container in a cold container with ice packs for transport back to the laboratory
Place any disposable items in a biohazard bag for disinfection and disposal. Place any reusable items into a biohazard bag for return to the library and disinfection. Sanitise hands with hand sanitiser.
Sample Processing
Sample Processing
Upon receipt in the lab the sample should be stored at 4-8ºC until filtration

Assemble the membrane filtration apparatus according to the manufacturer's instructions
Measure the sample into a large, sterile graduated cylinder
Place a 47mm 0.45µM filter disc on the filtration unit head and attach the filtration unit cup.

If necessary, place a coffee filter on top of the cup as a pre-filter to remove any larger solid matter.
Pour sample into the filtration cup and turn on the vacuum to allow sample to slowly filter through the disc for 1 hour.

If the filter becomes clogged, pipette the remaining sample back into the measuring cylinder, and replace the filter disc and coffee filter with fresh filters. Repeat as necessary with up to 5 filters.
Using sterile forceps, transfer the filter discs to a small WhirlPak bag, add 10ml of Ringer's lactate and then seal the bag shut
Elute the filter discs by massaging the bag until the filters are clean or have fallen apart
Pipette 1ml of the eluate into a 1.5ml microcentrifuge tube and centrifuge at Centrifigation10000 x g, 00:10:00
Pipette off the supernatant and store the pellet at -20ºC until DNA extraction.

Thoroughly disinfect membrane filtration cups and measuring cylinders after use to avoid cross contamination of samples.