Protocol for Modified Standard Method 9260.B2 for the Isolation of Salmonella from Surface Water
Autumn L Kraft, Manan Sharma, James E Wells, Abasiofiok Mark Ibekwe, NARMS EWG, Jonathan G Frye
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.
Attachments
Steps
Protocol
Prepare for filtration by assembling vacuum set up, including a Buchner flask (collection) that fits the Pall magnetic filter funnel and can hold 1L of water.
Prepare a decontamination station for magnetic filter funnel by filling 2 1 L beakers with 900mL 70% EtOH.
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.
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.
Measure 2 different sterile 0.5g portions of the cellulose pool-filter fiber (Aqua Dew™), contained in sterile tubes, and suspend ONE of those portions in 15mL of sterile deionized water.
Pre-load glass filter with the pool-filter fiber by applying vacuum and pouring the 15mL water / pool-filter fiber suspension over the glass fiber filter.
Add the second 0.5g portion of pool-filter fiber to the 1L of sample water, close bottle tightly, and vigorously mix suspension.
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.
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.
Add 25mL of 1 X BPW to Whirl-Pak bag and massage until filter cake until broken up.
Decontaminate filter funnel pieces (2) individually by dropping them into first beaker of 70% EtOH. Allow for a minimum of 0h 2m 0s 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.
Incubate Whirl-Pak bag containing BPW-enriched filter cake for 18-24 h at 37°C.
