Jun 15, 2022

Public workspaceProtocol for use of Vertical Modified Moore Swab (VMMS) to Isolate Salmonella from Surface Water

DOI
dx.doi.org/10.17504/protocols.io.rm7vzy7q5lx1/v1
  • 1USDA Agricultural Research Service Environmental Microbial and Food Safety Laboratory;
  • 2USDA Agricultural Research Service Meat Safety and Quality Research;
  • 3USDA Agricultural Research Service Agricultural Water Efficiency and Salinity Research Unit;
  • 4USDA Agricultural Research Service
  • NARMS EWG: Lisa Durso, Lari Hiott, Cheryl L East, Betty McConn, Andrea Ottesen, Jie Zheng, Laura Boczek;
avatar.png
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.rm7vzy7q5lx1/v1
Protocol Citation: Autumn L Kraft, Manan Sharma, James E Wells, Abasiofiok Mark Ibekwe, NARMS EWG, Jonathan G Frye 2022. Protocol for use of Vertical Modified Moore Swab (VMMS) to Isolate Salmonella from Surface Water. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzy7q5lx1/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
This protocol was used to recovery inoculated Salmonella Typhimurium (ca. 0.030 CFU/mL) from surface water at four different USDA ARS laboratories.
Created: May 05, 2022
Last Modified: April 19, 2023
Protocol Integer ID: 62002
Keywords: National Program 108, Environmental Microbial and Food Safety Laboratory, Vertical modified Moore swab (VMMS), isolation, Salmonella, surface water
Abstract
This protocol describes the use of vertical modified Moore swab (VMMS) to recover and isolate Salmonella from surface water.
Attachments
Icon for 414-895.docx
414-895.docx
1.3MB
Materials
Supplies Needed – General

  • Geotech pump (Geotech #91352123) with EZ-Load II pump head
  • Assorted lengths of Masterflex Silicone tubing, size 36
  • Carboy for liquid waste collection
  • Ringstand with adjustable clamps to hold cartridge
  • Hose clamps, appropriate size for 36 tubing
  • Sterilized graduated cylinders (500, 250 mL)
  • Large Whirl-Pak® bags (ReagentNasco Whirl-Pak™ Standard Sample BagsFisher ScientificCatalog #Nasco B01195 )
  • Sterile forceps
  • 1x Universal Pre-Enrichment Broth (ReagentUniversal Pre-enrichment BrothDot ScientificCatalog #7510A )
  • Control strain, Salmonella typhimurium BIOBALL® Luminate (BioMerieux, #422190)

Supplies Needed – Cartridge Assembly

  • Cheesecloth ReagentLions Services Inc CHEESECLOTH GRADE90 2PLY 50YDSFisher ScientificCatalog #50-395-11
  • 3/8” x ½” diameter Barbed Adapter Fitting (x2, Lowes #877100/NHB-300B)
  • 1 ½” x ½” PVC S40 SPxFIP Bush (x2, Fergusons #P40SFBID)
  • 1 ½ PVC DWV Male Adapter (x2, Fergusons #PDWVMAJ)
  • 1 ½ PVC S50 SxF Adapter (x2, Fergusons #P40SFAJ)
  • 2-inch piece of 1 ½” PVC 330 PSI Schedule 40 (Lowes #256098)
  • Oatey for CPVC Purple Primer (Lowes/Fergusons #30756)
  • Oatey Heavy Duty Clear PVC Cement (Lowes/Fergusons #30863)

Equipment
Geotech Geopump II Peristaltic Pump
NAME
Peristaltic Pump
TYPE
Geotech
BRAND
91352123
SKU
https://www.fieldenvironmental.com/equipment-sales/water-equipment-sales/water-sampling-sales/peristaltic-pumps/shallow-sampling/geotech-geopump-ii-peristaltic-pump-sale.html
LINK




VMMS Cartridge Assembly: End piece Assembly
VMMS Cartridge Assembly: End piece Assembly
Note
NOTE: All work with PVC primer and cement must be done in a fume-hood or other well-ventilated area.
Apply Purple Primer (Oatey 30756) to ½ to ¾ in length of the bottom part of the cylindrical section of part b (bushing, 1 ½” x ½” PVC S40 SPxFIP Bush).

Apply purple primer to the inside, non-threaded end of part c (male adapter, 1 ½ PVC DWV Male Adapter).
Reapply purple primer to the bottom part of the cylindrical section of part b (bushing) so that it receives two coats of purple primer.
Apply PVC Cement to the non-threaded end of part c (male adapter) over the purple primer.
Apply PVC Cement to the cylindrical section of part b (bushing).
Reapply PVC Cement to the non-threaded end of part c (male adapter).
Insert part b (bushing) into part c (made adapter) with force as far as bushing will insert. After inserting bushing, turn bushing ¼ turn to “lock” (solder) the bushing into the adapter, and leave upright to dry.
Repeat steps 1-7 for 2nd End piece Assembly.
Allow cement to dry DurationOvernight .

Overnight
VMMS Cartridge Assembly: Central Connector
VMMS Cartridge Assembly: Central Connector
Apply purple primer (Oatey 30756) to up to ½ to ¾ in end of the 2-in PVC piece (part e).
Apply purple primer to the non-threaded end of the SxF adapter (part d, 1 ½ PVC S50 SxF).
Apply PVC cement to non-threaded end of SxF adapter (part d).
Apply PVC cement to 2-in PVC (part e).
Reapply a second coat of PVC cement to non-threaded end of S x F adapter (part d).
Push adapter (part d) on to 2-in PVC piece (part e) as far as it will go.
Using the other end of the 2-in PVC piece (part e), repeat steps 10-14.
While pushing/placing the second adapter (part d) on to 2-in piece (part e), the assembly should be vertically rested on the first SxF adapter (part d). The second adapter should be pushed from the top down on to 2-in piece (part e) (with force) until the entire Central Connector assemble measures 5.25 in. Adjustments to length can be made to in the first 1-2 minutes after the cement is applied.
Allow the whole assembly to dry in a well-ventilated space DurationOvernight .

Overnight
VMMS Cartridge Assembly: Barb to End piece
VMMS Cartridge Assembly: Barb to End piece

Wrap the threads of the barbed barb (part a) with Teflon tape.
Figure 2. Teflon tape applied to barb.
Insert the barbed bard (part a) into the threads of the bushing (part b) of the entire end piece assembly and turn by hand (screw) until no more turns can be made.
Repeat for 2nd end piece.
VMMS Cartridge Assembly: End piece to Central Connector
VMMS Cartridge Assembly: End piece to Central Connector
Wrap threads of the male adapter (part c) of the end piece assembly with Teflon tape.
Figure 3. Teflon tape applied to part c.
Screw the male adapter (part c) into SxF adapter (part d) of the central connector assembly.
Repeat for second end piece.
Prepare the VMMS Cheesecloth Filters
Prepare the VMMS Cheesecloth Filters
On a large flat clean surface cut a piece of grade #90 cheesecloth to 74 cm x 92 cm.
Fold the cheesecloth in half by 92 cm (now 74 cm x 46 cm).
Fold again into thirds, so the finished product will be 74 cm x 16 cm.
Tightly roll the cheesecloth, along the 16-cm side.
Tightly roll each filter in aluminum foil and fold the ends over so that the cheesecloth doesn’t lose its shape.
Autoclave the foil-wrapped filters on a dry cycle.
Disinfect the cartridges
Disinfect the cartridges
Remove (unscrew) end pieces from central connector piece.
Remove barbed nozzles from end pieces.
Unroll all Teflon tape from the threads of barbed nozzles (part a) and the male adapter (part c).
In a large, clean bin soak the cartridge pieces in 10% bleach DurationOvernight , making sure the bleach solution covers all parts.

Overnight
Autoclave a foil-wrapped bin that is large enough to hold all the cartridges on a dry cycle.
Autoclave several liters of deionized water in a carboy with a spigot attachment.
Note
You may want to wrap the spigot in foil, sterilize it separately and then screw it onto the carboy on the day of use.

The next day, remove the VMMS cartridge pieces one at a time with gloved hands and thoroughly rinse each piece with sterile deionized water. Turn the carboy spigot so that the water flows slowly.
With fresh gloves, carefully shake each piece to remove excess water and then place them in the large, sterilized bin – replacing the foil after each transfer. The cartridges will still be slightly wet, so place the bin in a BSC to dry.
Rethread Teflon tape around the threads of the barbed nozzles (part a) and the male adapter (part c).
Screw the bottom end piece into the central connector.
With fresh gloves, insert one of the sterile MMS into a disinfected cartridge.
Screw top end piece tightly.
Place the assembled MMS cartridge back into the sterile bin and re-cover with foil.
Filtration Protocol
Filtration Protocol
31m
31m
Secure one of the VMMS cartridges to a support stand and adjust the height. The Cartridge will be oriented vertically with tubing attached to the barbs at the top and bottom of the cartridge (Figure 1, a).
Figure 4. Connection of intake tubing (bottom, connected to part a) and collection tubing (top, part a) to a vertical Modified Moore Swab (VMMS) cartridge.
Using the Geotech pump and its accessories, place the sterilized intake tubing in the EZ Load pump head on the Geotech Pump. Then connect the outlet end of the intake sterilized hose to the bottom of the MMS cartridge over the barbed end.
If needed, add Control strain to Amount1 L surface water sample by gently tipping vial over the opening. Close sample bottle tightly and thoroughly mix sample before proceeding.

Mix
Place the inlet end of the intake tubing into the 1 L bottle containing surface water.
Connect collection tubing to the top of the MMS cartridge and place the outlet end in a collection container. Use a collection container sufficient to hold up to 1 L of filtrate.
Turn the pump on and set the pump rate to 3/4 of maximum flow rate (use the dial). Once water is flowing into tube, then the pump speed can be slowed by 5 “clicks” (turn to the left) to maintain a constant flow.
After Amount1 L has been pumped through the tubing, turn the pump off.

Position the waste container (containing a 10% bleach solution) underneath the MMS cartridge on the support stand and carefully disconnect the collection tubing from the top of MMS cartridge to retrieve the MMS. Allow water in collection tubing to drain to collection vessel.
Place the collection tubing into the waste container and let sit for at least Duration00:30:00 .

30m
Remove (Unscrew) the top piece (male adapter with barb) of the MMS cartridge from the central connector and carefully place it in the waste container as well.
Using sterile forceps aseptically transfer the MMS filter into its pre-labelled 55 oz Whirl-Pak® bag and place the forceps in the waste container.
Carefully disconnect the intake tubing and place both the intake tubing and the cartridge in the waste container.
Using a sterile graduated cylinder, add Amount100 mL 1 X UPB into each Whirl-Pak® sample bag containing a MMS.

Pipetting
Massage entire length of MMS in UPB for Duration00:01:00 and incubate at Temperature37 °C for 18-24 h.
Note
Note: For next steps, refer to “Selective Enrichment Protocol for Salmonella Isolation from Surface Water”.




1m
Incubation