Jun 15, 2022

Public workspaceProtocol for Modified Standard Method 9260.B2 for the Isolation of Salmonella from Surface Water V.2

DOI
dx.doi.org/10.17504/protocols.io.rm7vzy72xlx1/v2
  • 1USDA Agricultural Research Service
Manan Sharma
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzy72xlx1/v2
Protocol Citation: Autumn L Kraft, Manan Sharma, James E Wells, Abasiofiok Mark Ibekwe, NARMS EWG, Jonathan G Frye 2022. Protocol for Modified Standard Method 9260.B2 for the Isolation of Salmonella from Surface Water. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzy72xlx1/v2Version created by Manan Sharma
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 low levels of inoculated Salmonella Typhimurium (ca. 0.30 CFU/mL) from surface water at four different USDA ARS research locations.
Created: June 10, 2022
Last Modified: April 19, 2023
Protocol Integer ID: 64350
Keywords: National Program 108; Environmental Microbial and Food Safety Laboratory; modified standard method 9260.B2, Isolation, Salmonella, Surface Water;
Abstract
This protocol details the Modified Standard Method 9260.B2 for the recovery and isolation of Salmonella from 1 L of surface water.

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Materials
Supplies Needed

  • Sterilized graduated cylinders (1 L, 500 mL, 250 mL)
  • Whirl-Pak® bags (Nasco #CE107372386)
  • Sterilized forceps
  • Pall magnetic filter funnel (Pall, 47mm, 300 mL, ReagentFilter Funnel AssemblyVWR AvantorCatalog #28143-550 )
  • 1 L Buchner flask
  • Set up for vacuum filtration
  • 70% Ethanol (for Pall funnel decontamination)
  • 47-mm glass fiber filters ReagentBinder-Free Glass Fiber Filters Pall LaboratoryVWR AvantorCatalog #28150-995
  • Aqua Dew™ cellulose pool-filter fiber – 2 x 0.5 g (sterile)
  • Sterile deionized water
  • 9 L Carboy for liquid waste collection
  • 2 x 1 L Beakers for decontaminating Pall funnels (to contain 70% ethanol)
  • 1 X Buffered peptone water (BPW, Accumedia #7417)
  • Control strain, Salmonellatyphimurium BIOBALL® Luminate (BioMerieux, #422190)


Protocol
Protocol
2m
2m
Prepare for filtration by assembling vacuum set up, including a Buchner flask (collection) that fits the Pall magnetic filter funnel and can hold Amount1 L of water.
Note
Note: Set up should include a rubber stopper that fits Pall magnetic filter funnel.


Prepare a decontamination station for magnetic filter funnel by filling 2 1 L beakers with Amount900 mL 70% EtOH.
Note
Note: Ensure beakers are large enough to contain filter funnel and EtOH to cover funnel pieces.


Place one 47-mm glass fiber filter into the bottom magnetic filter funnel (Forceps work well to gently pull one filter from stack and place into filter funnel) attached to Buchner funnel.
If needed use sterile forceps to gently 47-mm glass fiber filter into place to fit into bottom of the funnel.
Forceps are being used to press the 47 mm glass fiber filter to the bottom of the magnetic funnel. The magnetic funnel is placed in a rubber stopper, which fits into a Buchner (sidearm) flask which is connected to a trap flask (if needed) and a vacuum.

If needed, add Control strain to surface water sample by gently tipping vial over the opening. Close bottle tightly and mix sample thoroughly before proceeding.
Pipetting
Mix
Measure 2 different sterile Amount0.5 g portions of the cellulose pool-filter fiber (Aqua Dew™), contained in sterile tubes, and suspend ONE of those portions in Amount15 mL of sterile deionized water.

Pre-load glass filter with the pool-filter fiber by applying vacuum and pouring the Amount15 mL water / pool-filter fiber suspension over the glass fiber filter.

Add the second Amount0.5 g portion of pool-filter fiber to the Amount1 L of sample water, close bottle tightly, and vigorously mix suspension.

Mix
Filter water sample by carefully pouring into 300 mL funnel and over pre-loaded 47 mm glass filter and allow vacuum to completely filter water sample.
Water contained in 300 mL magnetic funnel and flowing over 47mm glass fiber filter with vacuum pressure applied.

If desired, use sterile deionized water to rinse off pool-filter fiber from sides of Pall filter funnel before turning off vacuum.
Carefully and aseptically, using sterilized forceps, transfer the 47 mm glass fiber filter (filter cake) from the filter funnel into a Whirl-Pak® sample bag.
Magnetic funnel containing 47 mm glass fiber filter is separated from base, inverted, and sterile forceps are used to nudge or poke glass fiber filter (filtercake) into sterile Whirl-Pak bag.
Note
NOTE: It is easier to pull magnetic portion of filter funnel off, tip on its side, and gently push filter cake from the bottom with sterile forceps, allowing it to fall into Whirl-Pak® bag.


Add Amount25 mL of 1 X BPW to Whirl-Pak bag and massage until filter cake until broken up.

Pipetting
Decontaminate filter funnel pieces (2) individually by dropping them into first beaker of 70% EtOH. Allow for a minimum of Duration00:02:00 contact time before transferring to second 70% EtOH bath. After a second 2-min soak, rinse filter funnel pieces with DeI water and allow to dry.

2m
Incubate Whirl-Pak bag containing BPW-enriched filter cake for 18-24 h at Temperature37 °C .
Note
For next steps, refer to “Selective Enrichment Protocol for Salmonella Isolation from Surface Water”.


Incubation