TITLE: Protocol for use of Vertical Modified Moore Swab (VMMS) to Isolate Salmonella from Surface Water
AUTHORS: Autumn L Kraft, Manan   Sharma, Jonathan G Frye, James  E Wells, Abasiofiok  Mark Ibekwe, NARMS EWG  

[DESCRIPTION]
This protocol describes the use of vertical modified Moore swab (VMMS) to recover and isolate Salmonella from surface water.

[STEPS]
SECTION: VMMS Cartridge Assembly: End piece Assembly
1.    
 
 
    
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).

SECTION: VMMS Cartridge Assembly: End piece Assembly
2. Apply purple primer to the inside, non-threaded end of part c (male adapter, 1 ½ PVC DWV Male Adapter).
SECTION: VMMS Cartridge Assembly: End piece Assembly
3. Reapply purple primer to the bottom part of the cylindrical section of part b (bushing) so that it receives two coats of purple primer.
SECTION: VMMS Cartridge Assembly: End piece Assembly
4. Apply PVC Cement to the non-threaded end of part c (male adapter) over the purple primer.
SECTION: VMMS Cartridge Assembly: End piece Assembly
5. Apply PVC Cement to the cylindrical section of part b (bushing).
SECTION: VMMS Cartridge Assembly: End piece Assembly
6. Reapply PVC Cement to the non-threaded end of part c (male adapter).
SECTION: VMMS Cartridge Assembly: End piece Assembly
7. 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.
SECTION: VMMS Cartridge Assembly: End piece Assembly
8. Repeat steps 1-7 for 2nd End piece Assembly.
SECTION: VMMS Cartridge Assembly: End piece Assembly
9. Allow cement to dry  .

SECTION: VMMS Cartridge Assembly: Central Connector
10. Apply purple primer (Oatey 30756) to up to ½ to ¾ in end of the 2-in PVC piece (part e).
SECTION: VMMS Cartridge Assembly: Central Connector
11. Apply purple primer to the non-threaded end of the SxF adapter (part d, 1 ½ PVC S50 SxF).
SECTION: VMMS Cartridge Assembly: Central Connector
12. Apply PVC cement to non-threaded end of SxF adapter (part d).
SECTION: VMMS Cartridge Assembly: Central Connector
13. Apply PVC cement to 2-in PVC (part e).
SECTION: VMMS Cartridge Assembly: Central Connector
14. Reapply a second coat of PVC cement to non-threaded end of S x F adapter (part d).
SECTION: VMMS Cartridge Assembly: Central Connector
15. Push adapter (part d) on to 2-in PVC piece (part e) as far as it will go.
SECTION: VMMS Cartridge Assembly: Central Connector
16. Using the other end of the 2-in PVC piece (part e), repeat steps 10-14.
SECTION: VMMS Cartridge Assembly: Central Connector
17. 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.
SECTION: VMMS Cartridge Assembly: Central Connector
18. Allow the whole assembly to dry in a well-ventilated space  .

SECTION: VMMS Cartridge Assembly: Barb to End piece
19. 
Wrap the threads of the barbed barb (part a) with Teflon tape.
   
SECTION: VMMS Cartridge Assembly: Barb to End piece
20. 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.
SECTION: VMMS Cartridge Assembly: Barb to End piece
21. Repeat for 2nd end piece.
SECTION: VMMS Cartridge Assembly: End piece to Central Connector
22. Wrap threads of the male adapter (part c) of the end piece assembly with Teflon tape.
  
 
 
SECTION: VMMS Cartridge Assembly: End piece to Central Connector
23. Screw the male adapter (part c) into SxF adapter (part d) of the central connector assembly.
SECTION: VMMS Cartridge Assembly: End piece to Central Connector
24. Repeat for second end piece.
SECTION: Prepare the VMMS Cheesecloth Filters
25. On a large flat clean surface cut a piece of grade #90 cheesecloth to 74 cm x 92 cm.
SECTION: Prepare the VMMS Cheesecloth Filters
26. Fold the cheesecloth in half by 92 cm (now 74 cm x 46 cm).
SECTION: Prepare the VMMS Cheesecloth Filters
27. Fold again into thirds, so the finished product will be 74 cm x 16 cm.
SECTION: Prepare the VMMS Cheesecloth Filters
28. Tightly roll the cheesecloth, along the 16-cm side.
SECTION: Prepare the VMMS Cheesecloth Filters
29. Tightly roll each filter in aluminum foil and fold the ends over so that the cheesecloth doesn’t lose its shape.
SECTION: Prepare the VMMS Cheesecloth Filters
30. Autoclave the foil-wrapped filters on a dry cycle.
SECTION: Disinfect the cartridges
31. Remove (unscrew) end pieces from central connector piece.
SECTION: Disinfect the cartridges
32. Remove barbed nozzles from end pieces.
SECTION: Disinfect the cartridges
33. Unroll all Teflon tape from the threads of barbed nozzles (part a) and the male adapter (part c).
SECTION: Disinfect the cartridges
34. In a large, clean bin soak the cartridge pieces in 10% bleach  , making sure the bleach solution covers all parts.

SECTION: Disinfect the cartridges
35. Autoclave a foil-wrapped bin that is large enough to hold all the cartridges on a dry cycle.
SECTION: Disinfect the cartridges
36. Autoclave several liters of deionized water in a carboy with a spigot attachment. 
 

SECTION: Disinfect the cartridges
37. 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.
SECTION: Disinfect the cartridges
38. 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.
SECTION: Disinfect the cartridges
39. Rethread Teflon tape around the threads of the barbed nozzles (part a) and the male adapter (part c).
SECTION: Disinfect the cartridges
40. Screw the bottom end piece into the central connector.
SECTION: Disinfect the cartridges
41. With fresh gloves, insert one of the sterile MMS into a disinfected cartridge.
SECTION: Disinfect the cartridges
42. Screw top end piece tightly.
SECTION: Disinfect the cartridges
43. Place the assembled MMS cartridge back into the sterile bin and re-cover with foil.
SECTION: Filtration Protocol
44. 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).
  
SECTION: Filtration Protocol
45. 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.
SECTION: Filtration Protocol
46. If needed, add Control strain to 1 L surface water sample by gently tipping vial over the opening. Close sample bottle tightly and thoroughly mix sample before proceeding.

SECTION: Filtration Protocol
47. Place the inlet end of the intake tubing into the 1 L bottle containing surface water.
SECTION: Filtration Protocol
48. 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.
SECTION: Filtration Protocol
49. 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.
SECTION: Filtration Protocol
50. After 1 L has been pumped through the tubing, turn the pump off.

SECTION: Filtration Protocol
51. 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.
SECTION: Filtration Protocol
52. Place the collection tubing into the waste container and let sit for at least 30 min.

SECTION: Filtration Protocol
53. 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.
SECTION: Filtration Protocol
54. 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.
SECTION: Filtration Protocol
55. Carefully disconnect the intake tubing and place both the intake tubing and the cartridge in the waste container.
SECTION: Filtration Protocol
56. Using a sterile graduated cylinder, add 100 mL 1 X UPB into each Whirl-Pak® sample bag containing a MMS.

SECTION: Filtration Protocol
57. Massage entire length of MMS in UPB for 1 min and incubate at 37 °C for 18-24 h.