Jan 24, 2025

Public workspaceShigella Environmental Sampling Using Grab Samples V1.0

This protocol is a draft, published without a DOI.
  • 1Malawi Liverpool Wellcome Programme;
  • 2Imperial College London;
  • 3Malawi Liverpool Wellcome Trust
Jen Cornick
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Protocol Citation: Mary Charles, Atusaye Nyirenda, Kayla Barnes, Jonathan Rigby, Myra Okumu, Jen Cornick 2025. Shigella Environmental Sampling Using Grab Samples V1.0. protocols.io https://protocols.io/view/shigella-environmental-sampling-using-grab-samples-dyga7tse
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: In development
We are still developing and optimizing this protocol
Created: January 24, 2025
Last Modified: January 24, 2025
Protocol Integer ID: 119010
Funders Acknowledgements:
BMGF
Grant ID: INV-048133
Disclaimer
Please note, the author list is in no particular order and does not reflect contribution.
Abstract
This protocol describes the collection of environmental samples (river water) using grab samples and the subsequent laboratory processing up to DNA extraction. This is part of a set of protocols for the environmental surveillance of Shigella species.
Materials
1. Ringer's Lactate
2. Bucket Container with 1L capacity
3. Whirl Pak Bags
4. 45mm 0.45μm filter discs
5. Coffee filters (if necessary)
6. 20ml Pipette
7. 100ml conical flasks
8. P1000 pipette
9. Filter Pipette Tips
10. Pipette Bulb
11. Appropriate PPE (Lab coat, nitrile gloves, safety glasses) and disinfectant

Equiptment needed:
  1. Incubator
  2. Filtration apparatus
  3. Freezer
Safety warnings
Shigella species are Risk Group 2 pathogens and all work should be done in a BSL-2 environment. Consult with your institutional biosafety committee before beginning any experiments with wild-type Shigella. Because Shigella are highly infectious, with an infectious dose of as few as 100 organisms, more stringent safety practices than used for other BSL-2 level organisms are recommended. In particular, S. dysenteriae may be subject to more stringent controls because it produces shiga toxin.
Before start
Materials needed:
  1. Materials needed
  2. Ringers Lactate
  3. Bucket Container with 1L capacity
  4. Whirl Pak Bags
  5. 45mm 0.45m filter discs
  6. Coffee filters (if necessary)
  7. 20ml Pipette
  8. 100ml conical flasks
  9. P1000 pipette
  10. Filter Pipette Tips
  11. Pipette Bulb
  12. Appropriate PPE (Lab coat, nitrile gloves, safety glasses) and disinfectant

Equiptment needed:
1. Incubator at at 37°C
2. Filtration apparatus
3. Freezer
Grab Sample Collection (field team)
Grab Sample Collection (field team)
Using a bucket or other appropriate vesicle, fill a 1L container with river water or wastewater and close the container.
Wipe the outside of the collection container with disinfectant.
Wash
Place the container in the cool box and transport back to the lab within four hours of sample collection.
Place any disposable items in a biohazard bag for disinfection and disposal. Place any reusable items into a biohazard bag to return to the lab for disinfection. Sanitize hands with hand sanitizer.
Wash
Sample Processing (lab team)
Sample Processing (lab team)
Upon receipt at the lab, samples should be stored at 4-8°C until processing and filtrations.
Temperature
Using a 20ml pipette, aseptically transfer 35ml of the grab sample into an appropriately labelled 50ml Falcon tube. Store the Falcon at -20°C until ready to perfrom filtration.
Temperature
Filtration steps:
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 prefilter to remove any large solid mass.
Pour the remaining 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 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 an appropriately labelled 1.5ml microcentrifuge tubes. Repeat this process twice so you have 3 x 1ml aliquots of the eluate.
Pipetting
Centrifuge the tubes at 10,000rpm for 10 minutes.
Centrifigation
Pipette off the supernatant. Store 2 x pellets at -20°C until DNA extraction. Note their locations on the freezer box plan.
Temperature
Culture the third pellet (see Shigella culture from environmental samples SOP).
Thoroughly disinfect the membrane filtration cups and measuring cylinders after use to avoid cross contamination of samples.
Wash