AOAC RI ERP Final Action Recommendations eBook

Dupont BAX Salmonella PTM Report Modification Approved 2012 / PTM Certification No. 081201 For Expert Review Panel Use Only Do Not Distribute

202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 223 224 225 226 227 228 222

For dry pet food, master samples were made at approximately 50, 100, and 1000 cfu per 25g

portion using a wet spike on ground pet food* and stored at room temperature. Pet food was divided

into portions of 25 g size and each was inoculated with 4 X 10 µL aliquots of an appropriately diluted

suspension. Some portions were left un-spiked to serve as negative controls. Spiked portions at each

target level were re-combined on an aluminum foil coated surface and mixed by hand to homogenize,

placed into disinfected plastic tubs, and shaken vigorously to further homogenize. All samples were

held at room temperature for two to four weeks to acclimate/stress the target cells, then range-finding

studies were performed to determine the actual spike levels. Based on the results of these range-

finding studies, pet food portions spiked at 100 and 1000 cfu/portion were combined in a 4:1 ratio

(three parts middle level material to one part high level material) to create test portions with the

appropriate inoculation level to produce fractional positive results. Analytical size portions (25 g spiked

master sample and 350 g of unspiked product for each sample) were placed into sterile bags.

* Note: Since Salmonella in real world dry pet food samples appears to originate as a post process

event, it is felt that the wet spike and target stress by extended drying down/desiccation on the low A w

material is the most appropriate way to mimic a naturally occurring contamination event. Previous studies

had indicated an approximately 2 log reduction in viable cells after Salmonella inoculated in this manner

age for two weeks in dry pet food and comparable results were seen in this study. Since 2.5 X 10 2 cfu on

average were inoculated into each sample portion, it can be inferred that the degree of injury for the target

under these conditions is >99%.

Sample Enrichment

AOAC INTERNATIONAL On the day of testing, an MPN was conducted for each sample matrix using 5 X 100g and 5 X 10g portions of the master sample. An aerobic plate count (APC) on un-spiked material was also conducted on the day enrichment commenced.

For unpaired study designs, all alternative primary enrichments, without regard to presumptive

alternative method result, were subjected to the secondary enrichments and subsequent plating,

biochemical, and serological confirmatory steps of the appropriate reference method.