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homogenize, placed into disinfected plastic tubs, and shaken vigorously to further homogenize.
340
Analytical size (reference and test method) portions (15 X 25g sub-samples [See Health Canada
341
Compendium MFHPB-20] comprising 25g of the spiked master sample and 350g of unspiked
342
product for each sample) were placed into sterile bags.
343
Since real world
Salmonella
in dry pet food appears to originate as a post process event, it is felt
344
that the wet spike and target stress by extended drying down/desiccation on the low A
w
material is
345
the most appropriate way to mimic a naturally occurring contamination event. Previous studies had
346
indicated an approximately 2 log reduction in viable cells after
Salmonella
inoculated in this
347
manner age for two weeks in dry pet food and comparable results were seen in this study. Since
348
2.5 X 10
2
cfu on average were inoculated into each sample portion, it can be inferred that the
349
degree of injury for the target under these conditions is >99%.
350
Samples were mixed by stomaching for 2 min at 200 rpm in a sterile Stomacher bag for test
351
method samples) in ~750 ml of enrichment media and then brought up to a total volume of 3375
352
ml BPW (HC Compendium reference method) or 3375 ml LB broth media (test method). All media
353
were pre-warmed to incubation temperature which has been demonstrated in previous work at
354
Qualicon to be a critical step in the detection of
Salmonella
from this matrix.
355
Twenty spiked and five unspiked samples for the reference method were enriched in pre-warmed
356
(35°C) Buffered Peptone Water at 35
o
C +/- 1
o
C for 18-26 hours before secondary enrichment in
357
RVS and TBG broths for Health Canada samples. Additionally, an MPN was conducted using 5 X
358
100g (from the master sample), 5 X 10g (from the master sample) and the 20 X 375g reference
359
method samples on the day of testing. The additional MPN samples were treated in an analogous
360
manner to the Health Canada method with samples diluted 1:10 in BPW enrichment media.
361
Twenty spiked and five unspiked samples for the BAX® System method (test method) were
362
enriched at 35 +/- 1
o
C in LB media for 26 hours and tested by BAX® System assay. After 22 and
363
26 hours of enrichment, each sample (from both BPW and LB enrichments) was tested by BAX®
364
following a 3 hr 1:50 dilution 37°C BHI regrowth step. Secondary enrichment was performed in
365
RVS and TBG broths as for Health Canada Compendium method enriched samples.
366
Dupont BAX Salmonella PTM Report
PTM Certification No. 081201 (08/07/2012)
For Expert Review Panel Use Only
Do Not Distribute