TITLE: Selective Enrichment Protocol for Salmonella Isolation 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 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).

[STEPS]
SECTION: Selective Enrichment – Day 1
1. Dispense 9 mL TT (Tetrathionate) broth into sterile test tubes.

SECTION: Selective Enrichment – Day 1
2. Dispense 9 mL GN (Gram-negative Hajna broth) into sterile test tubes.

SECTION: Selective Enrichment – Day 1
3. 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.
SECTION: Selective Enrichment – Day 1
4. Aseptically transfer 1 mL of either UPB or BPW enrichments into selective broths and then incubate at specified temperature listed in Table 1. 
      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  
 

SECTION: Day 2 – Selective enrichment transfers
5. Transfer 100 µL of 24 h GN broth enrichment into 9.9 mL RV (Rappaport-Vassiliadis) broth.

SECTION: Day 2 – Selective enrichment transfers
6. Incubate the GN/RV enrichment at 37 °C for 1440 min.

SECTION: Day 3 – Selective enrichment transfers continued
7. Transfer 100 µL of 48 h TT broth enrichment into 9.9 mL RV broth.

SECTION: Day 3 – Selective enrichment transfers continued
8. Incubate the TT/RV enrichment at 37 °C for 1440 min.

SECTION: Day 3 – Selective enrichment transfers continued
9. 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.
SECTION: Day 3 – Selective enrichment transfers continued
10. Incubate XLT4 and BGS plates at 37 °C for 1440 min.

SECTION: Day 4 – Selective enrichment transfers continued
11. For the TT/RV enrichment, vortex tubes and use a sterile 10 µL loop to streak for isolation onto both XLT4 and BGS plates
SECTION: Day 4 – Selective enrichment transfers continued
12. Incubate XLT4 and BGS plates at 37 °C for 1440 min.

SECTION: Day 4 – Selective enrichment transfers continued
13. Inspect XLT4/BGS plates from Day 3 (GN/TT) for presumptive Salmonella colonies.
SECTION: Day 5 – Colony confirmation
14. Inspect BGS and XLT4 plates from TT/RV enrichment and identify presumptive Salmonella colonies (see above).
SECTION: Day 5 – Colony confirmation
15. 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).
SECTION: Day 5 – Colony confirmation
16. If non-fluorescent presumptive Salmonella isolates are observed, these isolates can be isolated and preserved (frozen stocks) for further characterization.
SECTION: Day 4 – Selective enrichment transfers continued
13.1. 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.