165 / 1195
5
instructions for performing the study (Appendix 10.1) and required materials prior to the start of the
1
study. Training on the BAX System will be provided as necessary by a DuPont Qualicon representative.
2
3
4
3.0
Study Design
5
6
A collaborative study will be conducted in accordance with the AOAC INTERNATIONAL Methods
7
Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces
8
(4). The BAX method will be compared to the USDA-FSIS MLG method for the detection of
Salmonella
in
9
hot dogs. The BAX and USDA-FSIS MLG methods share a common enrichment for hot dogs, and thus
10
this matrix will be tested as a paired study. The BAX method will be compared to the FDA BAM method
11
for the detection of
Salmonella
in orange juice. A proprietary enrichment, the BAX® MP broth, will be
12
used to enrich orange juice for the BAX method, so this matrix will be tested as an unpaired study.
13
14
Each matrix will be screened for the presence of naturally contaminating
Salmonella
and total aerobic
15
plate count (
note
: for the FDA BAM method, if the level of background microflora is > 10
4
cfu/g, then the
16
tetrathionate (TT) broth must be incubated at 43 ± 0.2°C in a circulating, thermostatically-controlled,
17
water bath, and if the level is ≤ 10
4
cfu/g, then TT should be incubated at 35 ± 2.0°C). Naturally
18
contaminated matrix is preferred if available. If naturally contaminated matrix is not available, an
19
artificially contaminated matrix will be prepared. A different serovar of
Salmonella
will be used for each
20
matrix (see Table 2). Each matrix will be divided into 3 samples. One sample serves as the
21
uncontaminated level, one sample is contaminated at a level that will produce a reference method POD
22
(POD
R
) or candidate method POD (POD
C
) in the range of 0.25 – 0.75 (approximately 0.2 – 2 cfu/test
23
portion), and finally one sample is contaminated at a level to assure a POD
C
of nearly 1.0 (approximately
24
5 cfu/test portion). For each matrix, twelve replicate test portions per sample will be tested by the
25
candidate method and the reference method.
26
27
On the day that the analysis of the test samples is initiated, a 5-tube 3-level Most Probably Number
28
(MPN) estimation of contamination levels will be conducted using the appropriate reference method.
29
The MPN analysis scheme may also make use of the reference method replicates (see
Appendix X-A
of
30
the AOAC Guidelines for details). The Least Cost Formulations, Ltd. (LCF) MPN Calculator-Version 1.6 (5)
31
will be used to determine the MPN values and 95% confidence intervals. The MPN is reported as
32
MPN/test portion with 95% confidence intervals.
33
34
Table 1. Food Preparation
35
Matrix
Inoculation
Organisms
Inoculation Level
per Organism
Replicates
Test
Portion
Codes
Reference
Method
Hot dogs
S
. Worthington
(DD4043)*
0 cfu/325 g
12
Random
number
100-199
USDA FSIS-
MLG Ch.
4.05
0.2–2 cfu/325 g
12
2–5 cfu/325 g
12
Collaborative Study Approved Protocol
Expert Review Panel Use Only