Jan 24, 2025
SESIM Moore Swab Protocol
This protocol is a draft, published without a DOI.
- Mary Charles1,
- Atusaye Nyirenda1,
- Kayla Barnes1,
- Myra Okumu1,
- Jen Cornick2
- 1Malawi Liverpool University Wellcome Programme;
- 2Malawi Liverpool Wellcome Trust
Protocol Citation: Mary Charles, Atusaye Nyirenda, Kayla Barnes, Myra Okumu, Jen Cornick 2025. SESIM Moore Swab Protocol. protocols.io https://protocols.io/view/sesim-moore-swab-protocol-dygp7tvn
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: 119023
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 riverwater using Moore Swabs and the subsequent initial laboratory processing up to DNA extraction. This is part of a set of protocols for the environmental surveillance of Shigella species.
Materials
1. Moore Swabs
2. Wide Mouth Jars (500ml capacity)
3. Cold Packs
4. Ice box
5. 20ml Pipette
6. 100ml conical flasks
7. Pipette Bulb
8. Membrane Filtration Apparatus
9. 45mm 0.45μm filter discs
10. Shigella Broth Base
11. Gram Negative Broth
12. UPE
13. Whirl-pak bags
14. Empty power bead tubes or equivalent (2ml screw cap centrifuge tubes)
15. Appropriate PPE (Lab coat, nitrile gloves, safety glasses) and disinfectant
Equipment
- Incubator
- Filtration Apparatus
- 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.
Purpose
Purpose
This protocol describes the collection of environmental samples (wastewater and sewage) using Moore Swabs and the subsequent laboratory processing steps up to DNA extraction. This is part of a set of protocols for the environmental surveillance of Shigella species.
Sample Collection (Field Team)
Sample Collection (Field Team)
Sterilise Moore swabs by autoclaving prior to taking them to the field site.
Immerse three Moore swabs at the same collection site in the river or wastewater channel and tie them to a stable support using the tailing thread on the Moore swab.
Leave the Moore swabs in place for 24-72 hours.
Cut the thread of the Moore swabs and transfer:
1 x Moore swab (swab #1) into an appropriately labelled Whirl-pak for Shigella Broth Base (SBB) culture.
1 x Moore swab (swab #2) into an appropriately labelled Whirl-pak for Gram Negative Broth (GNB) culture.
1 x Moore swab (swab #3) into an appropriately labelled Whirl-pak for Universal Pre Enrichment (UPE) broth.
Ensure the Whirl-paks are properly sealed and wipe down the ouside of the bag with approprate dinfectant.
Place the Whirl-paks in a cooler box and transport back to the lab within 4 hours of sample collection.
Processing the Moore swab (Laboratory Team)
Processing the Moore swab (Laboratory Team)
Ensure Whirlpack bags with Moore Swabs are appropriately labelled.
Using aseptic technique add liquid culture media to each Moore swab as follows:
Add 100ml of SBB into the Whirlpack with moore swabs #1
Add 100ml of GNB into the Whirlpack with moore swabs #2
Add 100ml of UPE into the Whirlpack with moore swabs #3
Incubate the Whirl-paks with the Moore Swabs in liquic culture mediaum at 37°C for 18-24 hours.
For the Moore Swabs #1 incubated in SBB:
Following incubation, assemble the membrane filtration unit according to the manufacturer’s instructions and attach to the vacuum pump.
Using sterile tweezers, place a 47mm 0.45mm filter disc onto the filtration unit head and attach the filtration unit cup.
Swirl the Whirl-pak and squeeze out the liquid from the Moore Swab. Using a pipette, aliquot 20ml of the broth into 3 different sterile filtration cups and turn on the vacuum to filter the sample through the disc.
Once filtration is complete, cut the filters 1 and 2 into several strips using sterile forceps and then place the strips into two appropriately labelled empty cryo-vial. Store the samples at -20°C until DNA extraction.
Elute the third filter disc into 5ml ringers lactate by massaging the bag until the filters are clean or have fallen apart.
Inoculate an XLD plate with a loop full of the elutant under asepcitc technique and incubate the plate at 37°C for 18-24 (see Shigella culture SOP for next steps following incubation).
Repeat steps 11.1 > 11.6 for Moore Swab #1 (GNB) and Moore Swab #2 (UPE).
Thoroughly disinfect filtration cups, containers, and conical flasks after use to avoid cross contamination of samples.