Jun 15, 2022
Selective Enrichment Protocol for Salmonella Isolation from Surface Water
- Autumn L. Kraft1,
- Manan Sharma1,
- James E Wells2,
- Abasiofiok Mark Ibekwe3,
- NARMS EWG4,
- Jonathan G Frye5
- 1USDA ARS Environmental Microbial and Food Safety Laboratory;
- 2USDA ARS Meat Safety and Quality Research Laboratory;
- 3USDA ARS Agricultural Water Efficiency and Salinity Research Unit;
- 4National Antimicrobial Resistance Monitoring System Environmental Working Group, Culture Group;
- 5USDA Agricultural Research Service
- NARMS EWG: Lisa Durso, Cheryl East, Lari Hiott, Betty McConn, Andrea Ottesen, Jie Zheng, Laura Boczek;
Protocol Citation: Autumn L. Kraft, Manan Sharma, James E Wells, Abasiofiok Mark Ibekwe, NARMS EWG, Jonathan G Frye 2022. Selective Enrichment Protocol for Salmonella Isolation from Surface Water. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxz5q4v8j/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 has been used with a control strain at four different USDA ARS laboratories and has been able to selectively isolate Salmonella from non-selective media when the level of Salmonella in water ca. 0.030 CFU/mL.
Created: May 05, 2022
Last Modified: November 07, 2023
Protocol Integer ID: 62008
Keywords: National Program 108, Environmental Microbial and Food Safety Laboratory, Salmonella, isolation, surface water, Selective Enrichment
Abstract
This protocol describes the selective enrichment of Salmonella from surface water samples AFTER non-selective enrichment from several different recovery methods (Modified Standard Method 9260.B2, Vertical Modified Moore Swab, or Dead-end Ultra-filtration (DEUF).
Attachments
415-896.docx
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Materials
Supplies Needed
- Tetrathionate broth (TT, Acumedia #7740), Prepare this as close to day of use as possible
- Rappaport-Vassiliadis Salmonella enrichment broth (RV, Acumedia #7730)
- Gram-negative Hajna broth (GN, Acumedia #7218)
- Brilliant Green Sulfa agar (BGS, Acumedia #7299)
- XLT4 agar (Acumedia #7517)
- XLT4 supplement (Accumedia # 7990) - Sodium tetradecyl sulfate (Tergitol 4)
- Sterile culture tubes
Selective Enrichment – Day 1
Selective Enrichment – Day 1
Dispense 9 mL TT (Tetrathionate) broth into sterile test tubes.
- Dispense 9 mL GN (Gram-negative Hajna broth) into sterile test tubes.
Hand massage UPB(Universal Pre-enrichment Broth)-enriched modified Moore swab (MMS), BPW (Buffered Peptone Water)-enriched filter cakes (47 mm glass filters), or shake BPW-enriched bulk water or 2X BPW-enriched backflush (from DEUF filtration) to homogenize before transfers to Salmonella-selective media.
Aseptically transfer 1 mL of either UPB or BPW enrichments into selective broths and then incubate at specified temperature listed in Table 1.
| A | B | C | D | |
| Broth | Volume (mL) | Enriched UPB/BPW to add (mL) | Incubation Time and Temperature | |
| TT Broth | 9 | 1 | 48 h @ 37°C | |
| GN Broth | 9 | 1 | 24 h @ 37°C |
Table 1. Selective broth volumes and incubation parameters for Salmonella isolation.
Day 2 – Selective enrichment transfers
Day 2 – Selective enrichment transfers
- Transfer 100 µL of 24 h GN broth enrichment into 9.9 mL RV (Rappaport-Vassiliadis) broth.
Incubate the GN/RV enrichment at 37 °C for 24:00:00 .
1d
Day 3 – Selective enrichment transfers continued
Day 3 – Selective enrichment transfers continued
Transfer 100 µL of 48 h TT broth enrichment into 9.9 mL RV broth.
Incubate the TT/RV enrichment at 37 °C for 24:00:00 .
1d
- For the GN/RV enrichment, vortex tubes and use a sterile 10 µL loop to streak for isolation onto both XLT4 (Xylose Lysine Tergitol 4) (prepared with Sodium tetradecyl sulfate, XLT4 supplement) and BGS (Brilliant Green Sulfa) agar plates.
Incubate XLT4 and BGS plates at 37 °C for 24:00:00 .
1d
Day 4 – Selective enrichment transfers continued
Day 4 – Selective enrichment transfers continued
For the TT/RV enrichment, vortex tubes and use a sterile 10 µL loop to streak for isolation onto both XLT4 and BGS plates
Incubate XLT4 and BGS plates at 37 °C for 24:00:00 .
1d
Inspect XLT4/BGS plates from Day 3 (GN/TT) for presumptive Salmonella colonies.
From the XLT4 plates, select presumptive environmental Salmonella colonies (black raised center, clear halo) and streak onto fresh XLT4 plates. Salmonella colonies on BGS appear opaque and surrounded by red color on all edges.
Colonies of Salmonella Typhimurium isolated on XLT4 from pure culture (black raised center, clear halo).
Colonies of Salmonella spp. isolated on BGS agar from pure culture (opaque colony surrounded by red).
Colonies of Salmonella Typhimurium isolated from selective enrichment of surface water plated on XLT4: black colonies (surrounded by black circles for emphasis).
Colonies of Salmonella Typhimurium isolated from selective enrichment of surface water plated on BGS; opaque colonies (surrounded by black circles for emphasis).
Note
*You can divide the XLT4 plates into ½ and streak multiple colonies on one plate to save agar / plates*.
Note: If control strain used, for BioBall gfp-Salmonella Typhimurium, colonies should fluoresce when held under a UV light (395 nm).
Day 5 – Colony confirmation
Day 5 – Colony confirmation
Inspect BGS and XLT4 plates from TT/RV enrichment and identify presumptive Salmonella colonies (see above).
Colonies from both Day 4 and 5 can be subjected to further confirmation using triple sugar iron (TSI) agar, lysine iron agar (LIA), and/or PCR (see following sections).
If non-fluorescent presumptive Salmonella isolates are observed, these isolates can be isolated and preserved (frozen stocks) for further characterization.