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Secondary enrichments were struck for isolation on BGS and BS agars. Isolated presumptive
367
Salmonella
were confirmed as directed in the Health Canada Compendium guidelines
368
(biochemical, serological).
369
370
Environmental Sponges –
A dilution level previously established for the given strain/surface
371
combination to give inoculation levels appropriate for achieving fractional positive results (5-15
372
positives per trial) was used for the stainless steel surface environmental sample type. Samples
373
were applied to surfaces (100 µL of the appropriately diluted broth culture combined with ~10 X of
374
a non-target competitor strain) and allowed to dry for one day. FDA BAM samples were collected
375
by sponge hydrated with 10mL DE broth. After returning the sponge to the sample bag, samples
376
were held at 4±3°C for 2-3 hours prior to addition of 225 mL of LB. Media and sponge were swirled
377
thoroughly to mix and allowed to stand 60 ± 5 min at room temperature. Samples were again
378
mixed well by swirling and pH determined with test paper. Using 1 N HCl or NaOH, pH was
379
adjusted if necessary to 6.8 ± 0.2. Samples were incubated for 22-26 hours at 35°C. Enriched
380
samples were added in 0.1-mL aliquots to 10-mL FDA RV broth (prepared from constituent
381
components on the day of testing) and incubated for 24
±
2 hr at 42±0.2°C. An additional 1-mL
382
aliquot of each enrichment was added to 10 mL TT broth and incubated for 24
±
2 hr at 35±2°C. All
383
samples were then struck in 10-μL amounts on BS agar, XLD agar and HE agar. Agar plates were
384
incubated at 35°C for 24±2 hr after which plates were examined for colonies with typical
385
characteristics of
Salmonella
. Suspect colonies were confirmed using the biochemical and
386
serological methods described in the FDA-BAM. Additionally, for each sample, a 3 hr regrowth in
387
BHI (10μl of enriched sample added to 500μl of prewarmed BHI which was incubated at 37°C for 3
388
hrs) was performed and BAX® testing was performed on this sample as well in order to have a
389
validated protocol for samples where PCR inhibitors may be present on the sampled surfaces. All
390
samples were then struck in 10-μL amounts on BS agar, XLD agar and HE agar. Agars were
391
incubated at 35°C for 24±2 hr after which plates were examined for colonies with typical
392
characteristics of
Salmonella
. Suspect colonies were confirmed using the biochemical and
393
serological methods described in the FDA-BAM.
394
Dupont BAX Salmonella PTM Report
PTM Certification No. 081201 (08/07/2012)
For Expert Review Panel Use Only
Do Not Distribute