7
this inoculated bulk lot and mixed with an additional 300g of uncontaminated
1
test material to create the 325g test portions used in the analysis.
2
4.3
For orange juice, the inoculum will be prepared by transferring a pure isolated colony of
3
the
Salmonella
Thompson from SBA into BHI broth containing 1% glucose and
4
incubating the BHI at 35 ± 2
o
C for 18-24 ± hours. The inoculum will be added drop-wise
5
to a bulk quantity of orange juice for each inoculation level. The bulk quantity will be
6
mixed to achieve equal distribution of analyte throughout.
7
4.4
Test portions for both matrices will be stored at 2-8°C for 48-72 h prior to processing.
8
9
5.0
Analysis and Confirmation
10
11
Each collaborator will received a complete set of test portions (uncontaminated, low, and high) for each
12
matrix, as indicated in 5.1 and 5.2, and will also receive one known uncontaminated sample for each
13
matrix to determine the aerobic plate count (6). All participating collaborators will initiate sample
14
analysis and aerobic plate count for each matrix on the same day.
15
16
5.1
Hot Dogs – Paired
17
18
Each collaborator will receive one set of 36 – 325g test portions (12 uncontaminated, 12 low, 12
19
high), blind coded so that the contamination level is unknown to the collaborator. The BAX
20
method will be compared to the USDA-FSIS MLG method for testing hot dogs. Both methods
21
use the same enrichment, buffered peptone water (BPW). Approximately one third to one-half
22
of 2925 ± 58.5 mL of sterile BPW will be added to each portion. Each portion will be
23
homogenized approximately 2 minutes, after which the remainder of the 2925 ml of BPW will
24
be added. Samples will be incubated at 35
°
C for 18-24 hours and tested by PCR according to the
25
BAX® system procedure. After primary enrichment, 0.5 mL aliquots of each portion will be
26
transferred to 10 mL TT (Hajna) broth, and 0.1 mL sample will be added to 10 mL modified
27
Rappaport-Vassiliadis (mRV) broth. All secondary enrichments will be incubated at 42 ± 0.5°C
28
for 22-24 h or in a water bath at 42 ± 0.5°C for 18-24 h. Secondary enrichments will be streaked
29
to brilliant green sulfa (BGS) and either double modified lysine iron agar (DMLIA) or xylose lysine
30
Tergitol
TM
4 (XLT4) agar plates and incubated 35 ± 2°C for 18-24 h. Isolated colonies will
31
transferred to triple sugar iron (TSI) agar and lysine iron agar (LIA) slants and incubated 35 ± 2°C
32
for 22-26 h.
Salmonella
colonies will be confirmed using serological (Somatic O and poly H
33
agglutination) and biochemical procedures according to MLG Ch. 4.05.
34
35
5.2
Orange juice – Unpaired
36
37
Each collaborator will receive 2 sets of 36 – 25 mL test portions (12 uncontaminated, 12 low, 12
38
high). Each set will be blind coded so that the contamination level is unknown to the
39
collaborator. The BAX method will be compared to the FDA BAM method for testing orange
40
juice. One set of test portions will be enriched in BAX® MP broth (39-42°C for 22-26 h), for
41
testing with the BAX system, while the second set of portions will be enriched in Universal
42
Collaborative Study Approved Protocol
Expert Review Panel Use Only