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used to confirm the inclusivity of the test, post complete low and high microbial load enrichment of
1
the organisms. Organisms were cultured overnight in 3M Salmonella Enrichment Broth with 3M
2
Salmonella Enrichment Supplement at 41.5 ± 1
o
C for 18 hours then diluted via ten-fold serial dilutions
3
in the enrichment medium and supplement to a level of less than 10
5
CFU/mL (Colony Forming
4
Unit/mL) prior to evaluation following the 3M Petrifilm SALX System.
5
6
Exclusivity Testing Methodology
7
Exclusivity testing was conducted to ensure that the 3M Petrifilm SALX System
is specific for the
8
detection of the target organisms. Thirty-two (32) different non-
Salmonella
pure cultures (Table 3 in
9
Appendix) were used to confirm the exclusivity of the test. Organisms were cultured overnight in
10
Brain Heart Infusion Broth (BHI) at 37° ± 1
o
C for 18 hours then evaluated following the 3M SALX
11
System.
12
13
Inclusivity/Exclusivity Results
14
All 104 cultures tested in the inclusivity study were correctly detected and confirmed as
Salmonella
15
and all of the 32 non-
Salmonella
cultures tested in the exclusivity study were negative for
Salmonella
16
by the 3M Petrifilm SALX System.
17
18
Ruggedness Testing Methodology
19
This study evaluated the ability of the 3M Petrifilm SALX System to remain unaffected by variations in
20
method parameters that might be expected to occur when the method is performed by an end user.
21
The effects of perturbations in six method parameters were investigated:
22
1. Primary enrichment incubation time:
Following the Sample Enrichment instructions, organism
23
enrichment was carried out for 16, 18, 24, and 26 hours.
24
2. Incubation time of the 3M Petrifilm SALX Plate:
Following the Plate Incubation instructions, the
25
inoculated the 3M Petrifilm SALX Plates were incubated for 20, 22, 24, 26 and 28 hours.
26
3. Incubation temperature of the 3M SALX Plates:
Following the Plate Incubation instructions, inoculated
27
3M Petrifilm SALX Plates were incubated at the following temperatures: 40, 41.5, and 43°C.
28
4. Incubation time of the 3M Petrifilm SALX Confirmation Disk:
Following the instructions for Biochemical
29
Confirmation, the 3M Petrifilm SALX Confirmation Disk was added to inoculated 3M Petrifilm SALX Plates
30
for 2, 3, 4, 5, and 6 hours.
31
5.
Incubation temperature of the 3M SALX Confirmation Disk:
Following the instructions for Biochemical
32
Confirmation, the 3M Petrifilm SALX Confirmation Disk was added to inoculated 3M Petrifilm SALX Plates
33
and incubated at 40, 41.5 and 43°C.
34
6. Various plate hydration options:
Following the Plate Hydration instructions, 3M Petrifilm SALX Plates
35
were hydrated with sterile reverse osmosis water (RO), Butterfield Phosphate Diluent, and sterile distilled
36
water before inoculation.
37
38
Two serovars of
Salmonella
(S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076) were analyzed
39
within one log of the limit of detection of the kit. Each serovar was cultured in 3M Salmonella Enrichment
40
Broth with 3M Salmonella Enrichment Supplement using both the low and high microbial load protocols
41
and then diluted in the same broth to within one log limit of detection of the kit. One non-Salmonella
42
organism (
Citrobacter braakii
ATCC 43162) was analyzed at the overnight growth level achieved in a
43
nonselective broth (BHI). Ten (10) replicates of each organism were analyzed using the 3M Petrifilm SALX
44
System for each ruggedness parameter with the exception of the hydration fluid. For this parameter, thirty
45
(30) replicates of each organism were tested.
46
47