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After incubation, 0.1 ml of primary enrichment for each sample was transferred to 10 ml of modified
2
Rappaport-Vassiliadis broth (mRV) and 0.5 ml to 10 ml of Tetrathionate Hanja (TTH) broth. The mRV and
3
TTH tubes were incubated at 42° ± 0.5°C for 22-24 hours. Following incubation, a loopful of each secondary
4
enrichment was streaked to Brilliant Green Sulfa (BGS) agar and Xylose Lysine Tergitol (XLT4) agar and
5
incubated at 35° ± 2°C for 18-24 hours. If no visible colonies were present, both the BGS and XLT4 plates
6
were re-incubated for an additional 18-24 hours at 35° ± 2°C. Up to 3 suspect colonies from each selective
7
agar were transferred to Triple Sugar Iron agar (TSI) and Lysine Iron agar (LIA) and incubated at 35° ± 2°C for
8
22-26 hours. Growth from samples producing typical biochemical reactions in TSI and LIA, were streaked to
9
TSA slants and incubated for 18-24 hours at 35° ± 2°C. Growth from the TSA slant was used to conduct the
10
flagellar H serological test and polyvalent somatic O serological test and for biochemical confirmation.
11
Growth from the TSA slants was used for final confirmation of
Salmonella
by VITEK® 2 GN following AOAC
12
Official Method 2011.17.
13
14
FDA-BAM Chapter 5 Salmonella
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For the FDA/BAM method, all test portions for each food product, frozen uncooked shrimp, fresh spinach,
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dry dog food and stainless steel environmental surfaces were prepared as outlined in the study protocol.
17
Following equilibration of the microorganism in the matrix, 25 test portions consisting of 25 g for each food
18
matrix were enriched with 225 mL of Lactose broth and homogenized for 2 minutes. For sponges used to
19
analyze the stainless steel environmental surfaces, each sponge was enriched with 225 mL of Lactose broth
20
and mixed by hand massaging. All test portions were allowed to stand for 60 minutes to adjust pH to 6.8 ±
21
0.2 if necessary. Test portions for each matrix were incubated for 22-26 hours at 35° ± 2°C.
22
After incubation of the samples, 0.1 ml aliquot of primary enrichment for each sample was transferred to
23
10 mL of Rappaport-Vassiliadis medium (RV) and 1.0 ml to 10 mL of Tetrathionate (TT) broth. RV tubes
24
were incubated for 22-26 hours at 42° ± 2°C. For high microbial load foods (fresh spinach and frozen
25
uncooked shrimp), TT tubes were incubated for 22-26 hours at 43° ± 0.2°C in a circulating water bath. For
26
low microbial load foods (dry dog food and stainless steel environmental surfaces), TT tubes were
27
incubated 22-26 hours at 35° ± 2°C. Following incubation, a loopful of each secondary enrichment was
28
streaked to Bismuth Sulfite (BS) agar, Hektoen Enteric (HE) agar, and XLD agar and incubated at 35° ± 2°C
29
for 22-26 hours. If no visible colonies were present after 24 hours of incubation, BS plates were re-
30
incubated for an additional 22-26 hours at 35° ± 2°C. Up to 2 or more suspect colonies from each selective
31
agar were transferred to TSI and LIA and incubated at 35° ± 2°C for 22-26 hours. Growth from samples
32
producing typical biochemical reactions in TSI and LIA, were streaked to TSA slants and incubated for 18-24
33
hours at 35° ± 2°C. Growth from the TSA slant was used to conduct the flagellar H serological test and
34
polyvalent and individual somatic O serological test and for biochemical confirmation. Growth from the
35
TSA slants was used for final confirmation of
Salmonella
by VITEK
®
2 GN following AOAC Official Method
36
2011.17.
37
38
3M Petrifilm Salmonella Express System
39
After equilibration of the inoculum, 25 test portions for each matrix were enriched using pre-warmed
40
(41.5° ± 1
o
C) 3M
Salmonella
Enrichment Base containing 3M
Salmonella
Enrichment Supplement. For the
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analysis of raw ground beef, raw ground pork, frozen uncooked shrimp, and fresh spinach, 25 g test
42
portions were enriched in 225 mL of 3M
Salmonella
Enrichment Base. For the analysis of cooked chicken
43
nuggets, 325 g test portions were enriched with 2925 mL of 3M
Salmonella
Enrichment Base. For the
44
analysis of pasteurized liquid whole eggs, 100 g test portions were enriched with 900 mL of 3M
Salmonella
45
Enrichment Base. For the analysis of dry dog food, 375 g samples were enriched with 3375 mL of 3M
46
Salmonella
Enrichment Base. Sponges used to sample stainless steel environmental surfaces were
47