166 / 1195
6
Orange Juice
S
. Thompson (DD1336)
0 cfu /25 mL
12
Random
number
200-299;
300-399
FDA BAM
Ch. 5
0.2–2 cfu/25 mL
12
2–5 cfu/25 mL
12
*DD – DuPont Qualicon proprietary culture collection
1
2
4.0
Sample Preparation and Distribution
3
4
To prepare the artificially contaminated matrix, primary samples will be inoculated with
Salmonella
so
5
that
on the day of initiation of analysis
there is a low level (approx. 0.2-2 cfu/test portion) and a high
6
level (approximately 2-5 cfu/test portion). Note that one level of contamination must result in fractional
7
positive results. One sample will remain uninoculated to serve as the uncontaminated control. The
8
organizing laboratory will prepare and ship the samples. Each sample will be divided into test portions,
9
randomized, and blind-coded for analysis. Test portions will be shipped to each collaborator via
10
overnight courier to arrive on a Friday. Containers will be packed with ice packs to maintain a
11
temperature of <7
°
C during transport. A temperature control sample will be included with each
12
shipment to verify temperature of sample upon receipt. Upon arrival, the test portions will be stored at
13
2-8
°
C until they are analyzed. All participating collaborators will begin processing and analysis on the
14
following Monday. Collaborators will complete and return the data sheets to the study director
15
following confirmation, within 2 weeks of the test initiation date (Appendix 10.2). Each matrix will be
16
analyzed in separate weeks.
17
18
4.1
Both matrices will be obtained from a local retail outlet and screened for the presence
19
of
Salmonella
. Naturally contaminated test portions will be used if available at sufficient
20
levels. Alternatively, the test portions will be artificially inoculated to meet the targets
21
of 0.2-2.0 cfu/test portion for the low and 2 – 5 cfu/test portion for the high with the
22
appropriate concentration of a wet inoculum.
23
4.2
For hot dogs, the inoculum will be prepared by transferring a pure isolated colony of the
24
Salmonella
Worthington from Trypticase Soy agar with 5% sheep’s blood (SBA) into
25
Brain Heart Infusion broth (BHI) and incubating the BHI at 35 ± 2
o
C for 18-24 ± hours.
26
4.2.1
Prior to inoculating the test material, the inoculum will be heat stressed in a
27
55°C water bath for 10-20 minutes to obtain a percent injury of 50-80%. The
28
heat stressed culture will be plated onto a selective agar (Xylose Lysine
29
Desoxycholate, XLD) and a non-selective agar (Tryptic Soy agar, TSA). The
30
degree of injury will be estimated as
, where n
select
= number
31
of colonies on selective agar and n
nonselect
= number of colonies on non-selective
32
agar.
33
4.2.2
The heat stressed inoculum will be added drop-wise to a bulk quantity of hot
34
dogs for each inoculation level. The bulk quantity will be mixed to achieve equal
35
distribution of analyte throughout. Twenty-five (25) grams will be pulled from
36
100 )
1(
x
n
n
nonselect
select
−
Collaborative Study Approved Protocol
Expert Review Panel Use Only