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of 325 g were added to 1500 mL of BPW and massaged by hand for 2 minutes. After mixing, an
1
additional 1425 mL of BPW was added to the sample. For pasteurized liquid whole eggs, 25 test
2
portions of 100 g were added to 900 mL of BPW. Raw ground beef samples were homogenized by
3
stomaching for 2 minutes and pasteurized liquid whole eggs samples were massaged by hand for 2
4
minutes. All test portions were incubated at 35 ± 2
o
C for 20-24 hours.
5
6
After incubation, 0.1 mL of primary enrichment for each sample was transferred to 10 ml of modified
7
Rappaport-Vassiliadis broth (mRV) and 0.5 ml to 10 ml of Tetrathionate Hanja (TTH) broth. The mRV
8
and TTH tubes were incubated at 42 ± 0.5
o
C for 22-24 hours. Following incubation, a loopful of each
9
secondary enrichment was streaked to Brilliant Green Sulfa (BGS) agar and Xylose Lysine Tergitol
10
(XLT4) agar and incubated at 35 ± 2
o
C for 18-24 hours. If no visible colonies were present, both the
11
BGS and XLT4 plates were re-incubated for an additional 18-24 hours at 35 ± 2
o
C. Up to 3 suspect
12
colonies from each selective agar were transferred to Triple Sugar Iron agar (TSI) and Lysine Iron agar
13
(LIA) and incubated at 35± 2°C for 22-26 hours. Growth from samples producing typical biochemical
14
reactions in TSI and LIA, were streaked to TSA slants and incubated for 18-24 hours at 35 ± 2
o
C.
15
Growth from the TSA slant was used to conduct the flagellar H serological test and polyvalent somatic
16
O serological test for biochemical confirmation. Growth from the TSA slants was used for final
17
confirmation of
Salmonella
by VITEK
®
2 GN following AOAC Official Method 2011.17.
18
19
FDA/BAM Chapter 5 Salmonella
20
For the FDA/BAM method, all test portions for each food product, raw shrimp, bagged spinach, and
21
wet pet food were prepared as outlined above. Following equilibration of the microorganism in the
22
matrix, 25 test portions consisting of 25 g for raw shrimp and bagged spinach, were enriched with 225
23
mL of Lactose broth and homogenized by blending for 2 minutes. For wet pet food, 25 test portions
24
consisting of 375 g were enriched with 3375 mL of Lactose and massaged by hand for 2 minutes.
25
Samples were allowed to stand for 60 minutes to adjust pH to 6.8 ± 0.2 if necessary. All samples were
26
incubated for 22-26 hours at 35 ± 2
o
C.
27
28
After incubation of the samples, 0.1 ml of primary enrichment for each sample was transferred to 10
29
mL of Rappaport-Vassiliadis medium (RV) and 1.0 ml to 10 mL of Tetrathionate (TT) broth. RV tubes
30
were incubated for 22-26 hours at 42 ± 0.2
o
C. For high microbial load foods, TT tubes were incubated
31
for 22-26 hours at 43 ± 0.2
o
C in a circulating water bath. For low microbial load foods, TT tubes were
32
incubated 22-26 hours at 35 ± 2
o
C. Following incubation, a loopful of each secondary enrichment was
33
streaked to Bismuth Sulfite (BS) agar, Hektoen Enteric (HE) agar, and XLD agar and incubated at 35 ±
34
2
o
C for 22-26 hours. If no visible colonies were present after 24 hours of incubation, BS plates were re-
35
incubated for an additional 22-26 hours at 35 ± 2
o
C. Up to 2 or more suspect colonies from each
36
selective agar were transferred to TSI and LIA and incubated at 35± 2°C for 22-26 hours. Growth from
37
samples producing typical biochemical reactions in TSI and LIA, were streaked to TSA slants and
38
incubated for 18-24 hours at 35 ± 2
o
C. Growth from the TSA slant was used to conduct the flagellar H
39
serological test and polyvalent and individual somatic O serological test for biochemical confirmation.
40
Growth from the TSA slants was used for final confirmation of
Salmonella
by VITEK
®
2 GN following
41
AOAC Official Method 2011.17.
42
43
3M
™
Molecular Detection Assay
Salmonella
44
After equilibration of the inoculum, 25 test portions for each matrix were enriched using pre-warmed
45
(37 ± 1
o
C) 3M
™
Buffered Peptone Water (BPW) ISO. For the analysis of raw ground beef, raw shrimp,
46
and bagged spinach, 25 g test portions were enriched in 225 mL of 3M BPW ISO and homogenized by
47
13