In the last quarter century, significant efforts have been made

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1 FOOD BIOLOGICAL CONTAMINANTS Bird et l.: Journl of AOAC Interntionl Vol. 97, No. 6, Evlution of the 3M Petrifilm Slmonell Express System for the Detection of Slmonell Species in Selected Foods: Collortive Study Ptrick Bird, Jonthn Flnnery, Erin Crowley, Jmes Agin, nd Dvid Goins Q Lortories, Inc., 1400 Hrrison Ave, Cincinnti, OH Roert Jechorek 1 3M Food Sfety Deprtment, 3M Center Bldg 260-6B-01, St. Pul, MN Collortors: J. Adms, K. Beers, K. Blnchrd, R. Brooks, M. Boyle, R. Colvin, H. Elgli, W. Fedio, D. Lewis, A. Mrtin, K. McCllum, J. Meyer, A. Morey, A. Morris, K. Newmn, J. Pickett, K. Rjkowski, D. Rodgers, B. Stwick, J. Mrchnt-Tmone The 3M Petrifilm Slmonell Express (SALX) System is simple, redy-to-use chromogenic culture medium system for the rpid qulittive detection nd iochemicl confirmtion of Slmonell spp. in food nd food process environmentl smples. The 3M Petrifilm SALX System ws compred using n unpired study design in multilortory collortive study to the U.S. Deprtment of Agriculture/Food Sfety nd Inspection Service (USDA/FSIS) Microiology Lortory Guideook (MLG) 4.07 (2013) Isoltion nd Identifiction of Slmonell from Met, Poultry, Psteurized Egg nd Ctfish Products nd Crcss nd Environmentl Sponges for rw ground eef nd the U.S. Food nd Drug Administrtion Bcteriologicl Anlyticl Mnul (FDA/BAM) Chpter 5, Slmonell (2011) reference method for dry dog food following the current AOAC vlidtion guidelines. For this study, totl of 17 lortories locted throughout the continentl United Sttes evluted 1872 test portions. For the 3M Petrifilm SALX System, rw ground eef ws nlyzed using 25 g test portions, nd dry dog food ws nlyzed using 375 g test portions. For the reference methods, 25 g test portions of ech mtrix were nlyzed. The two mtrices were rtificilly contminted with Slmonell t three inocultion levels: n uninoculted control level (0 CFU/test portion), low inoculum level (0.2 2 CFU/test portion), nd high inoculum level (2 5 CFU/test portion). Ech inocultion level ws sttisticlly nlyzed using Received My 29, The method ws pproved y the Expert Review Pnel for Microiology Methods for Food nd Environmentl Surfces s First Action. The Expert Review Pnel for Microiology Methods for Food nd Environmentl Surfces invites method users to provide feedck on the First Action methods. Feedck from method users will help verify tht the methods re fit for purpose nd re criticl to gining glol recognition nd cceptnce of the methods. Comments cn e sent directly to the corresponding uthor or methodfeedck@oc.org. 1 Corresponding uthor s e-mil: rpjechorek@mmm.com An ppendix is ville on the J. AOAC Int. wesite, pulisher.ingentconnect.com/content/oc/joc DOI: /jocint the proility of detection sttisticl model. For the rw ground eef nd dry dog food test portions, no significnt differences t the 95% confidence intervl were oserved in the numer of positive smples detected y the 3M Petrifilm SALX System versus either the USDA/FSIS-MLG or FDA/BAM methods. In the lst qurter century, significnt efforts hve een mde to reduce the occurrence of Slmonell in food products, yet Slmonell spp. continues to e the most frequently reported cuse of foodorne illness in the United Sttes (1). Over 2500 different serotypes of Slmonell spp. hve een isolted from wide rnge of food products including rw mets nd poultry, shell eggs, chocolte, fresh fruit nd vegetles, nd low-moisture ingredients such s spices nd penut utter (2). This rod rnge of implicted products further illustrtes why testing for nd confirming the presence of Slmonell s rpidly s possile is so criticl to food sfety. The 3M Petrifilm Slmonell Express (SALX) System, chromogenic culture medium system, uses cold-wter-solule gelling gent to selectively differentite Slmonell from ckground flor in enriched food nd food process environmentl smples. The 3M Petrifilm SALX System llows for the rpid nd specific detection nd iochemicl confirmtion of Slmonell species from food nd environmentl smples. Following enrichment in 3M Slmonell Enrichment Bse contining 3M Slmonell Enrichment Supplement, the 3M Petrifilm SALX System cn provide presumptive results in s little s 40 h from low microil ckground foods (<10 4 CFU/g) nd 48 h from high-microil ckground foods ( 10 4 CFU/g). Confirmtion of multiple presumptive Slmonell colonies t once is ccomplished using the 3M Petrifilm Slmonell Express (SALX) Confirmtion Disk which uses iochemicl enzymes to fcilitte the rection. The method developer studies demonstrted tht the 3M Petrifilm SALX System did not specificlly differentite some lctose-positive Slmonell species (primrily S. rizone nd S. dirizone) from other lctose-positive orgnisms. Prior to the collortive study, the 3M Petrifilm SALX System ws vlidted ccording to AOAC Vlidtion Guidelines (3) in hrmonized AOAC Performnce Tested Method SM (PTM) study. The ojective of the PTM study

2 1564 Bird et l.: Journl of AOAC Interntionl Vol. 97, No. 6, 2014 ws to demonstrte tht the 3M Petrifilm SALX System detects Slmonell spp. in selected foods s climed y the mnufcturer. For the 3M Petrifilm SALX System evlution, nine mtrices were evluted: rw ground eef (25 g), rw ground chicken (25 g), psteurized liquid whole egg (100 g), rw ground pork (25 g), cooked chicken nuggets (325 g), frozen uncooked shrimp (25 g), fresh unched spinch (25 g), dry dog food (375 g), nd stinless steel. All other PTM prmeters (inclusivity, exclusivity, ruggedness, stility, nd lot-to-lot vriility) tested in the PTM studies stisfied the performnce requirements for PTM pprovl. The method ws wrded PTM certifiction No on June 5, The im of this collortive study ws to compre the 3M Petrifilm SALX System to the U.S. Deprtment of Agriculture (USDA) Food Sfety Inspection Service (FSIS)/Microiology Lortory Guideook (MLG) 4.07 (4) for rw ground eef, nd the U.S. Food nd Drug Administrtion (FDA) Bcteriologicl Anlyticl Mnul (BAM) Chpter 5 (5) method for dry dog food. Collortive Study Study Design For this collortive study, two mtrices, rw ground eef (80% len) nd dry dog food, were evluted. The mtrices were otined from locl retilers nd screened for the presence of Slmonell spp. y either the MLG or BAM reference methods. The rw ground eef ws rtificilly contminted with Slmonell Ohio Sequence Types 81 (University of Pennsylvni Culture Collection) nd the dry dog food with Slmonell Poon Ntionl Collection of Type Cultures (NCTC) There were three inocultion levels for ech mtrix: high inocultion level of pproximtely 2 5 CFU/test portion, low inocultion level of pproximtely CFU/test portion, nd n uninoculted control level t 0 CFU/test portion. Twelve replicte smples from ech of the three inocultion levels of product were nlyzed y oth the cndidte nd reference method. Two sets of unpired smples (72 totl) were sent to ech lortory for nlysis y the 3M Petrifilm SALX System nd either the MLG (rw ground eef) or BAM (dry dog food) reference method due to differences in enrichment protocols. For oth mtrices, collortors were sent n dditionl 60 g test portion nd instructed to conduct totl eroic plte count (APC) using 3M Petrifilm Aeroic Count Plte (AOAC Officil Method ) on the dy smples were received. Foods with n APC count of greter thn or equl to CFU/g were ctegorized s high microil lod foods, nd those foods lower thn CFU/g were ctegorized s low microil lod. A detiled collortive study pcket outlining ll necessry informtion relted to the study including medi preprtion, method-specific test portion preprtion, nd documenttion of results ws sent to ech collorting lortory prior to the initition of the study. Preprtion of Inocul nd Test Portions The Slmonell cultures used in this evlution were propgted in 10 ml Brin Hert Infusion (BHI) roth from frozen stock culture stored t 70 C t Q Lortories, Inc. The roth ws incuted for h t 35 ± 1 C. For oth mtrices, ulk lot of ech mtrix ws inoculted with liquid inoculum nd mixed thoroughly y hnd-kneding to ensure n even distriution of microorgnisms. Approprite dilutions of the cultures were prepred sed on previously estlished growth curves for oth low nd high inocultion levels, resulting in frctionl positive outcomes for t lest one level. For the dry pet food, prior to inocultion, the inoculum ws het-stressed in 50 C wter th for 10 min to otin percent injury of 50 80% (s determined y plting onto selective xylose deoxycholte gr nd nonselective tryptic soy gr). The degree of injury ws estimted s: nselect ( 1 ) x100 n nonselect where n select = numer of colonies on selective gr, nd n nonselect = numer of colonies on nonselective gr. The rw ground eef ws inoculted on the dy of shipment so tht the orgnism hd equilirted within the mtrix for 96 h efore testing ws initited. Dry dog food ws inoculted nd held t room temperture (24 ± 2 C) so tht the orgnism would hve equilirted for minimum of 2 weeks prior to initition of testing. The shipment nd hold times of the inoculted test mteril were verified s QC mesure prior to study initition. For the evlution of the rw ground eef, the ulk lot of inoculted test mteril ws divided into 30 g portions for shipment to the collortors. For the evlution of the dry dog food, 25 g of inoculted test product ws mixed with 350 g of uninoculted test product for shipment to the collortors for the nlysis y the 3M Petrifilm SALX System. For nlysis y the reference methods, collortors received 30 g portions. Vlidtion criterion were stisfied when inoculted test portions produced frctionl recovery of the spiked orgnism, defined s either the reference or cndidte method yielding 25 75% positive results. To determine the level of Slmonell spp. in the mtrices, five-tue most prole numer (MPN) ws conducted t Q Lortories, Inc. on the dy of initition of nlysis using the BAM Chpter 5 reference method for dry dog food or the MLG 4.07 reference method for rw ground eef. From oth the high nd low inoculted levels, five 100 g test portions, the reference method test portions from the collorting lortories, nd five 10 g test portions were nlyzed following the pproprite reference method. The MPN nd 95% confidence intervls were clculted from the high, medium, nd low levels using the Lest Cost Formultions (LCF) MPN Clcultor, Version 1.6, provided y AOAC ( 6). Confirmtion of the smples ws conducted ccording to the pproprite reference method, dependent on the mtrix. Test Portion Distriution All smples were leled with rndomized, lind-coded three digit numer ffixed to the smple continer. Test portions were shipped on Thursdy vi overnight delivery ccording to the Ctegory B Dngerous Goods shipment regultions set forth y Interntionl Air Trnsport Assocition. Rw ground eef smples were pcked with cold pcks to trget temperture of <7 C during shipment. Upon receipt, smples were held y the collorting lortory t refrigerted temperture (3 5 C) until the following Mondy when nlysis ws initited. Dry dog

3 Bird et l.: Journl of AOAC Interntionl Vol. 97, No. 6, food smples were pcked nd shipped t mient temperture. Upon receipt, smples were held y the collorting lortory t room temperture (24 ± 2 C). In ddition to ech of the test portions nd the totl plte count replicte, collortors lso received test portion for ech mtrix leled s temperture control. Prticipnts were instructed to record the temperture of this portion upon receipt of the shipment, document results on the Smple Receipt Confirmtion form provided, nd fx to the Study Director. For oth mtrices, severl shipments were delyed from getting to the testing fcilities on time due to inclement wether. Upon receiving their pckges on either Sturdy or Mondy, the testing lortories were instructed to document the temperture of the smples nd to continue testing. No lortories were solely excluded from testing due to the dely in pckge receipt. Test Portion Anlysis Collortors followed the pproprite preprtion nd nlysis protocol ccording to the method specified for ech mtrix. For oth mtrices, ech collortor received 72 test portions of ech food product (12 high, 12 low, nd 12 controls for ech method). For the nlysis of the rw ground eef test portions y the 3M Petrifilm SALX System, 25 g portion ws enriched with 225 ml of prewrmed (41.5 ± 1 C) 3M Slmonell Enrichment Bse contining 3M Slmonell Enrichment Supplement (50 mg/l), homogenized for 2 min, nd incuted for h t 41.5 ± 1 C. For the dry dog food test portions nlyzed y the 3M Petrifilm SALX System, 375 g portion ws enriched with 3375 ml prewrmed (41.5 ± 1 C) 3M Slmonell Enrichment Bse contining 3M Slmonell Enrichment Supplement (50 mg/l), homogenized for 2 min, nd incuted for h t 41.5 ± 1 C. Following enrichment of rw ground eef smples, the enrichment protocol for high microil lod foods ws followed where 0.1 ml liquot of ech test portion ws trnsferred into 10.0 ml Rppport-Vssilidis R10 (R-V R10) roth nd incuted for 8 24 h t 41.5 ± 1 C. After incution, loopful of the secondry enrichment ws streked directly onto hydrted 3M Petrifilm SALX Pltes nd incuted for 24 ± 2 h t 41.5 ± 1 C. For dry dog food smples, the enrichment protocol for low microil lod foods ws followed where loopful of the primry enrichment ws streked directly onto hydrted 3M Petrifilm SALX Pltes nd incuted for 24 ± 2 h t 41.5 ± 1 C. For oth mtrices, the 3M Petrifilm SALX Pltes were exmined for typicl colonies (red to rown colony with yellow zone or ssocited gs ule, or oth). Typicl colonies were circled on the plte top film using fine tip permnent lck mrker. The top of the 3M Petrifilm SALX Plte ws lifted nd 3M Petrifilm SALX Confirmtion Disk ws plced onto the gel. The film ws lowered nd ir ules were removed using sweeping motion. The pltes were incuted for 4 5 h t 41.5 ± 1 C. After incution, the circled colonies were oserved for color chnge: red/rown to green lue, lue, drk lue, or lck. Typicl colonies were trnsferred to triple sugr iron/lysine iron gr (TSI/LIA) slnts nd confirmed following the stndrd reference methods. Additionlly, for ech mtrix nlyzed y the 3M Petrifilm SALX System, liquots of the primry enrichment were trnsferred to the secondry enrichments nd confirmed following procedures outlined in the MLG or BAM. Both test portion sizes nlyzed y the 3M Petrifilm SALX System were compred to smples (25 g) nlyzed using either the MLG or BAM reference method in n unpired study design. All positive test portions were iochemiclly confirmed y the API 20E iochemicl test, AOAC Officil Method or y the VITEK 2 GN identifiction test, AOAC Officil Method The iochemicl method ws determined y ech individul prticipting lortory sed on their current method used for confirmtion of routine smples. Serologicl testing, Group Poly O A-I & Vi nd Poly H ltex gglutintion, ws lso performed. Sttisticl Anlysis Ech collorting lortory recorded results for the reference method nd the 3M Petrifilm SALX System on the dt sheets provided in the collortive study outline or the electronic spredsheet creted s result of multiple requests for electronic dt entry. The dt sheets were sumitted to the Study Director t the end of ech week of testing for nlysis. The results of ech test portion for ech smple were compiled y the Study Director nd the qulittive 3M Petrifilm SALX System results were compred to the reference methods for sttisticl nlysis. Dt for ech test portion size were nlyzed using the proility of detection (POD) sttisticl model (3, 7). A confidence intervl of dlpod (difference etween the POD of the reference nd cndidte method) not contining the point zero would indicte sttisticlly significnt difference etween the 3M Petrifilm SALX System nd the MLG or BAM reference methods t the 5% proility level (8). In ddition to clculting the POD for ech inocultion level, the repetility stndrd devition, mong-lortory stndrd devition, reproduciility stndrd devition, nd P-vlue for homogeneity were clculted. For the collortive study, the 3M Petrifilm SALX System produced 479 presumptive positive results with 475 confirming positive y the trditionl confirmtion nd 473 confirming positive y the lterntive confirmtion. There were 468 confirmed positives y the reference method. AOAC Officil Method Slmonell in Selected Foods 3M Petrifilm Slmonell Express System First Action 2014 [Applicle to detection of Slmonell spp. in rw ground eef (25 g), rw ground chicken (25 g), psteurized liquid whole egg (100 g), rw ground pork (25 g), cooked chicken nuggets (325 g), frozen uncooked shrimp (25 g), fresh unched spinch (25 g), dry dog food (375 g), nd stinless steel. Not pplicle to some lctose-positive Slmonell species.] See Tles A nd B for results of the interlortory study supporting cceptnce of the method. See Appendix ville on the J. AOAC Int. wesite for detiled tles of results of the collortive study. Cution: Do not use the 3M Petrifilm SALX System method in the dignosis of conditions in humns or nimls. To reduce the risks ssocited with exposure to chemicls nd iohzrds, perform pthogen testing in properly equipped lortory under the control of trined personnel. Alwys follow

4 1566 Bird et l.: Journl of AOAC Interntionl Vol. 97, No. 6, 2014 Tle A. Summry of results for detection of Slmonell in rw ground eef (25 g) Method 3M Petrifilm Slmonell Express System with lterntive confirmtion 3M Petrifilm Slmonell Express System with trditionl confirmtion Inocultion level Uninoculted Low High Uninoculted Low High Cndidte presumptive positive/ totl No. of smples nlyzed 2/168 85/ /168 2/168 85/ /168 Cndidte presumptive POD (CP) 0.01 (0.00, 0.04) 0.51 (0.43, 0.58) 1.00 (0.98, 1.00) 0.01 (0.00, 0.04) 0.51 (0.43, 0.58) 1.00 (0.98, 1.00) s r s L c s R d 0.11 (0.10, 0.15) 0.51 (0.46, 0.52) 0.00 (0.00, 0.15) 0.11 (0.10, 0.15) 0.51 (0.46, 0.52) 0.00 (0.00, 0.15) 0.00 (0.00, 0.04) 0.00 (0.00, 0.13) 0.00 (0.00, 0.15) 0.00 (0.00, 0.04) 0.00 (0.00, 0.13) 0.00 (0.00, 0.15) 0.11 (0.10, 0.12) 0.51 (0.47, 0.52) 0.00 (0.00, 0.21) 0.11 (0.10, 0.12) 0.51 (0.47, 0.52) 0.00 (0.00, 0.21) P-vlue e Cndidte confirmed positive/ totl No. of smples nlyzed 0/168 83/ /168 1/168 83/ /168 Cndidte confirmed POD (CC) 0.00 (0.00, 0.02) 0.49 (0.42, 0.57) 1.00 (0.98, 1.00) 0.01 (0.00, 0.03) 0.49 (0.42, 0.57) 1.00 (0.98, 1.00) s r 0.00 (0.00, 0.15) 0.51 (0.46, 0.52) 0.00 (0.00, 0.15) 0.08 (0.07, 0.15) 0.51 (0.46, 0.52) 0.00 (0.00, 0.15) s L 0.00 (0.00, 0.15) 0.00 (0.00, 0.11) 0.00 (0.00, 0.15) 0.00 (0.00, 0.03) 0.00 (0.00, 0.11) 0.00 (0.00, 0.15) s R 0.00 (0.00, 0.21) 0.51 (0.47, 0.52) 0.00 (0.00, 0.21) 0.08 (0.07, 0.09) 0.51 (0.47, 0.52) 0.00 (0.00, 0.21) P-vlue Positive reference smples/ totl No. of smples nlyzed 0/168 86/ /168 0/168 86/ /168 Reference POD 0.00 (0.00, 0.02) 0.51 (0.43, 0.59) 0.99 (0.97, 1.00) 0.00 (0.00, 0.02) 0.51 (0.43, 0.59) 0.99 (0.97, 1.00) s r 0.00 (0.00, 0.15) 0.51 (0.46, 0.52) 0.08 (0.07, 0.15) 0.00 (0.00, 0.15) 0.51 (0.46, 0.52) 0.08 (0.07, 0.15) s L 0.00 (0.00, 0.15) 0.00 (0.00, 0.12) 0.00 (0.00, 0.03) 0.00 (0.00, 0.15) 0.00 (0.00, 0.12) 0.00 (0.00, 0.03) s R 0.00 (0.00, 0.21) 0.51 (0.47, 0.52) 0.08 (0.07, 0.09) 0.00 (0.00, 0.21) 0.51 (0.47, 0.52) 0.08 (0.07, 0.09) P-vlue dlpod (cndidte vs reference) f 0.00 ( 0.02, 0.02) 0.02 ( 0.13, 0.09) 0.01 ( 0.02, 0.03) 0.01 ( 0.02, 0.03) 0.02 ( 0.13, 0.09)0.01 ( 0.02, 0.03) dlpod (cndidte presumptive vs cndidte confirmed) f 0.01 ( 0.01, 0.04) 0.01 ( 0.10, 0.12) 0.00 ( 0.02, 0.02) 0.01 ( 0.02, 0.04) 0.01 ( 0.10, 0.12) 0.00 ( 0.02, 0.02) c d e f Results include 95% confidence intervls. Repetility stndrd devition. Among-lortory stndrd devition. Reproduciility stndrd devition. P-vlue = Homogeneity test of lortory PODs. A confidence intervl for dlpod tht does not contin the vlue 0 indictes sttisticl significnt difference etween the two methods. stndrd good lortory sfety prctices (GLP), including proper continment procedures, nd wering pproprite protective pprel nd eye protection while hndling testing mterils nd test smples. Avoid direct contct with the contents of the enrichment medium nd inoculted pltes. Dispose of enrichment medi nd inoculted pltes ccording to ll pplicle government regultory regultions nd pplicle lortory procedures. Wer pproprite protective pprel while hndling the 3M Petrifilm SALX Plte s some of the components my e considered llergenic nd irritnts to some individuls. To reduce the risks ssocited with environmentl contmintion, follow current industry stndrds nd locl regultions for disposl of contminted wste. Consult the Mteril Sfety Dt Sheet for dditionl informtion. For questions out specific pplictions A. Principle or procedures, visit or contct your locl 3M representtive or distriutor. Review the policies recommend y the Centers for Disese Control nd Prevention on deling with pthogens ( pulictions/ml5/bmbl.pdf). The 3M Petrifilm SALX System is chromogenic culture medium system tht is intended for the rpid nd specific detection nd iochemicl confirmtion of Slmonell spp. from food nd food process environmentl smples. After enrichment in prewrmed 3M Slmonell Enrichment Bse with 3M Slmonell Enrichment Supplement, the 3M Petrifilm SALX System provides presumptive positive results in s little s 40 h from low microil ckground foods (<10 4 CFU/g) nd 48 h from high microil foods ( 10 4 CFU/g). The 3M

5 Bird et l.: Journl of AOAC Interntionl Vol. 97, No. 6, Tle B. Summry of results for detection of Slmonell in dry dog food (375 g) Method 3M Petrifilm Slmonell Express System with lterntive confirmtion 3M Petrifilm Slmonell Express System with trditionl confirmtion Inocultion level Uninoculted Low High Uninoculted Low High Cndidte presumptive positive/ 0/144 82/ /144 0/144 82/ /144 totl No. of smples nlyzed Cndidte presumptive POD (CP) 0.00 (0.00, 0.03) 0.57 (0.48, 0.66) 0.99 (0.95, 1.00) 0.00 (0.00, 0.03) 0.57 (0.48, 0.66) 0.99 (0.95, 1.00) s r 0.00 (0.00, 0.16) 0.49 (0.44, 0.52) 0.12 (0.11, 0.16) 0.00 (0.00, 0.16) 0.49 (0.44, 0.52) 0.12 (0.11, 0.16) c s L 0.00 (0.00, 0.16) 0.08 (0.00, 0.24) 0.00 (0.00, 0.04) 0.00 (0.00, 0.16) 0.08 (0.00, 0.24) 0.00 (0.00, 0.04) d s R 0.00 (0.00, 0.22) 0.50 (0.45, 0.52) 0.12 (0.11, 0.13) 0.00 (0.00, 0.22) 0.50 (0.45, 0.52) 0.12 (0.11, 0.13) P-vlue e Cndidte confirmed positive/ 0/144 81/ /144 0/144 82/ /144 totl No. of smples nlyzed Cndidte confirmed POD (CC) 0.00 (0.00, 0.03) 0.56 (0.46, 0.66) 0.98 (0.94, 0.99) 0.00 (0.00, 0.03) 0.57 (0.48, 0.67) 0.98 (0.94, 0.99) s r 0.00 (0.00, 0.16) 0.49 (0.44, 0.52) 0.14 (0.12, 0.16) 0.00 (0.00, 0.16) 0.49 (0.43, 0.52) 0.14 (0.12, 0.16) s L 0.00 (0.00, 0.16) 0.10 (0.00, 0.26) 0.03 (0.00, 0.08) 0.00 (0.00, 0.16) 0.11 (0.00, 0.27) 0.03 (0.00, 0.08) s R 0.00 (0.00, 0.22) 0.50 (0.45, 0.52) 0.14 (0.13, 0.17) 0.00 (0.00, 0.22) 0.50 (0.45, 0.52) 0.14 (0.13, 0.17) P-vlue Positive reference smples/ 0/144 71/ /144 0/144 71/ /144 totl No. of smples nlyzed Reference POD 0.00 (0.00, 0.03) 0.49 (0.39, 0.59) 1.00 (0.97, 1.00) 0.00 (0.00, 0.03) 0.49 (0.39, 0.59) 1.00 (0.97, 1.00) s r 0.00 (0.00, 0.16) 0.49 (0.44, 0.52) 0.00 (0.00, 0.16) 0.00 (0.00, 0.16) 0.49 (0.44, 0.52) 0.00 (0.00, 0.16) s L 0.00 (0.00, 0.16) 0.10 (0.00, 0.26) 0.00 (0.00, 0.16) 0.00 (0.00, 0.16) 0.10 (0.00, 0.26) 0.00 (0.00, 0.16) s R 0.00 (0.00, 0.22) 0.50 (0.45, 0.52) 0.00 (0.00, 0.22) 0.00 (0.00, 0.22) 0.50 (0.45, 0.52) 0.00 (0.00, 0.22) P-vlue dlpod (C vs R) f 0.00 ( 0.03, 0.03) 0.07 ( 0.07, 0.21) 0.02 ( 0.06, 0.01) 0.00 ( 0.03, 0.03) 0.08 ( 0.07, 0.22) 0.02 ( 0.06, 0.01) dlpod (CP vs CC) f 0.00 ( 0.03, 0.03) 0.01 ( 0.18, 0.22) 0.01 ( 0.03, 0.05) 0.00 ( 0.03, 0.03) 0.00 ( 0.14, 0.14) 0.01 ( 0.03, 0.05) c d e f Results include 95% confidence intervls. Repetility stndrd devition. Among-lortory stndrd devition. Reproduciility stndrd devition. P-vlue = Homogeneity test of lortory PODs. A confidence intervl for dlpod tht does not contin the vlue 0 indictes sttisticl significnt difference etween the two methods. Petrifilm SALX System does not specificlly differentite some lctose-positive Slmonell species (primrily S. rizone nd S. dirizone) from other lctose-positive orgnisms. Refer to the 3M Petrifilm Slmonell Express System Instructions for Use for dditionl informtion. B. Apprtus nd Regents () 3M Petrifilm Slmonell Express Plte. Twenty-five pltes/pouch (3M Food Sfety, St. Pul, MN). () 3M Petrifilm Slmonell Express Confirmtion Disk. Five disks/pouch (3M Food Sfety). (c) 3M Slmonell Enrichment Bse. 500 g or 2.5 kg/ottle (3M Food Sfety). (d) 3M Slmonell Enrichment Supplement. 1 g/vil (3M Food Sfety). (e) 3M Petrifilm Flt Spreder. Two spreders/ox (3M Food Sfety). (f) 3M Rppport-Vssilidis R10 (R-V R10) Broth. 500 g/ottle (3M Food Sfety). (g) Sterile diluents. Butterfield s Phosphte Diluent, distilled wter, or reverse osmosis wter. (h) Sterile 10 µl inocultion loop. (i) Pipet. Cple of dispensing 2 ml. (j) Pipettor. Cple of dispensing 100 µl. (k) Sterile pipet tips. Cple of 100 µl. (l) Filter stomcher gs. Sewrd Lortory Systems Inc., Bohemi, NY, or equivlent. (m) Stomcher. Sewrd Lortory Systems Inc., or equivlent. (n) Permnent ultr-fine tipped mrker. For circling presumptive positive colonies on the 3M Petrifilm Slmonell Express Plte. (o) Incutors. Cple of mintining 41.5 ± 1 C. (p) Freezer. Cple of mintining 10 to 20 C, for storing opened 3M Petrifilm Slmonell Express Plte pouches,

6 1568 Bird et l.: Journl of oc interntionl Vol. 97, no. 6, 2014 Tle C. Smple mtrix nd enrichment scheme Smple mtrix Smple size, g Enrichment roth volume, ml Enrichment time, h Secondry enrichment time, h Rw ground eef (80% len) Rw ground chicken Rw ground pork Frozen uncooked shrimp Fresh unched spinch Stinless steel; environmentl sponges 1 Sponge (4 4 in.) Psteurized liquid whole egg Cooked reded chicken Dry dog food AOAC RI Certifi cte No hydrted 3M Petrifilm SALX Pltes, nd 3M Petrifilm SALX Pltes fter incution. (q) Refrigertor. Cple of mintining 2 8 C for storing unopened 3M Petrifilm SALX Pltes nd 3M Petrifilm SALX Confirmtion Disk. C. Generl Instructions () Store 3M Petrifilm SALX Pltes nd 3M Petrifilm SALX Confirmtion Disks t 2 8 C. After opening the 3M Petrifilm SALX Plte pouches, sel the pouch nd store t mient temperture, less thn 60% reltive humidity (RH). Hydrted 3M Petrifilm SALX Pltes cn e stored up to 7 dys t 2 8 C. Post-incution 3M Petrifilm SALX Pltes cn e stored t 10 to 20 C for up to 3 dys. Hydrte the 3M Petrifilm SALX Pltes with 2.0 ± 0.1 ml sterile diluent. Do not llow the top film to close efore dispensing the entire 2.0 ml volume. Gently roll down the top film onto the diluent to prevent trpping ir ules. Plce the 3M Petrifilm Flt Spreder on the center of the plte. Press gently on the center of the spreder to distriute the diluent evenly. Spred the diluent over the entire 3M Petrifilm SALX Plte. Remove the spreder nd leve the 3M Petrifilm SALX Plte undistured for 1 min. Prior to use, plce the pltes on flt surfce for 1 h t room temperture (20 25 C/<60% RH) nd protected from light to llow the gel to form. Hydrted pltes cn e stored t room temperture (20 25 C/<60% RH) protected from light for up to 8 h efore use. Figure Streking pttern on the 3M Petrifilm SALX Plte. () Follow ll instructions crefully. Filure to do so my led to inccurte results. (c) After use, the enrichment medium nd the 3M Petrifilm SALX Pltes nd 3M Petrifilm SALX Confirmtion Disks cn potentilly contin pthogenic mterils. When testing is complete, follow current industry stndrds for the disposl of contminted wste. Consult the Mteril Sfety Dt Sheet for dditionl informtion nd locl regultions for disposl. D. Smple Enrichment (1) Prewrm 3M Slmonell Enrichment Bse with 3M Slmonell Enrichment Supplement (50 mg/l) to 41.5 ± 1 C. (2) Asepticlly comine the enrichment medium nd smple following Tle C. For ll met nd highly prticulte smples, the use of filter gs is recommended. Homogenize thoroughly for 2 min nd incute t 41.5 ± 1 C for 18 24h. () Foods with high microil ckgrounds ( 10 4 CFU/g). Trnsfer 0.1 ml of the primry enrichment into 10.0 ml R-V R10 roth. Incute for 8 24 h t 41.5 ± 1 C. () Foods with low microil ckgrounds (<10 4 CFU/g). Proceed to 3M Petrifilm SALX Plte preprtion s descried in E. E. Preprtion of the 3M Petrifilm Slmonell Express Pltes (1) Plce the 3M Petrifilm SALX Plte on flt, level surfce. (2) Use prescried diluents to hydrte the 3M Petrifilm SALX Pltes: Butterfield s Phosphte Diluent, distilled wter, or reverse osmosis wter. (3) Lift the top film nd with the pipet perpendiculr dispense 2.0 ± 0.1 ml sterile diluent onto the center of ottom film. Do not close the top film efore dispensing the entire 2.0 ml volume. (4) Gently roll down the top film onto the diluent to prevent trpping ir ules. (5) Plce the 3M Petrifilm Flt Spreder on the center of the plte. Press gently on the center of the spreder to distriute the diluent evenly. Spred the diluent over the entire 3M Petrifilm SALX Plte growth re efore the gel is formed. Do not slide the spreder cross the film.

7 Bird et l.: Journl of AOAC Interntionl Vol. 97, No. 6, Tle D. Interprettion for presumptive positive Slmonell species Red Colony color Colony metolism Drk red Brown Yellow zone Gs ule Result Presumptive + Presumptive + Presumptive + Presumptive + Presumptive + Presumptive + Presumptive + Presumptive + Presumptive + (6) Remove the spreder nd leve the 3M Petrifilm SALX Plte undistured for t lest 1 min. (7) Plce 3M Petrifilm SALX Plte on flt surfce for t lest 1 h t room temperture (20 25 C/<60% RH), protected from light to llow the gel to form prior to use. Hydrted 3M Petrifilm SALX Pltes cn e stored t room temperture (20 25 C/<60% RH) for up to 8 h efore use if protected from light. (8) If hydrted pltes re not used within 8 h, store in seled plstic g, protected from light, nd store t 20 to 10 C for up to 5 dys. F. 3M Petrifilm Slmonell Express Plte Inocultion (1) Remove the enrichment medium from the incutor nd gitte contents y hnd. (2) Use sterile 10 µl loop (3 mm dimeter) to withdrw ech smple. Use smooth loop (one tht does not hve jgged edges nd is not distorted) to prevent the gel surfce from reking. (3) Open the 3M Petrifilm SALX Plte nd strek onto the gel. Perform single strek to otin isolted colonies (Figure ). (4) Roll down the top film to close the 3M Petrifilm SALX Plte. (5) Using gloved hnd (while prcticing GLP to void cross-contmintion nd/or direct contct with the plte), gently pply sweeping motion with even pressure onto the top film to remove ny ir ules in the inocultion re. (6) Strek ech enriched test portion onto 3M Petrifilm SALX Plte nd incute t 41.5 ± 1 C for 24 ± 2 h in horizontl position with the colored side up in stcks of no more thn 20 pltes. G. Confirmtion of 3M Petrifilm Slmonell Express Pltes (1) Using permnent ultr-fine tip mrker, circle t lest five presumptive positive colonies (red to rown colonies with yellow zone or ssocited gs ule, or oth) on the plte top film (see Tle D). (2) Lift the top film of the 3M Petrifilm SALX Plte nd insert the 3M Petrifilm SALX Confirmtion Disk y rolling it onto the gel to void entrpping ir ules. Close the 3M Petrifilm SALX Plte. Using gloved hnd, gently pply sweeping motion with even pressure onto the top film to remove ny ir ules in the inocultion re nd ensure good contct etween the gel nd the 3M Petrifilm SALX Confirmtion Disk. (3) Incute the 3M Petrifilm SALX System (plte nd disk) t 41.5 ± 1 C for 4 5 h in horizontl position, right side up, in stcks of no more thn 20 pltes. (4) Oserve circled colonies for color chnge. Red/rown to green lue, lue, drk lue, or lck confirms the colony s Slmonell spp. No color chnge indictes the colony is negtive. If presumptive positive Slmonell colonies re not present, then report the results s Slmonell not detected in the mtrix. (5) Trnsfer typicl colonies from 3M Petrifilm SALX Plte to TSI/LIA slnts. Incute 35 ± 1 C for 24 ± 2 h. (6) Confirm minimum of one typicl colony per test portion with iochemicl/serologicl procedures prescried y the current versions of the USDA/FSIS-MLG or FDA/BAM reference methods. Results of Collortive Study In this collortive study, the 3M Petrifilm SALX System ws compred to the USDA/FSIS-MLG 4.07 reference method for rw ground eef nd to the FDA/BAM Chpter 5 reference method for dry dog food. A totl of 17 lortories throughout the United Sttes prticipted in this study, with 15 lortories sumitting dt for the rw ground eef nd 14 lortories sumitting dt for the dry dog food s presented in Tle 1. Results of the het stress nlysis for the dry dog food inocul re presented in Tle 2. Ech lortory nlyzed 36 test portions for ech method: 12 inoculted with high level of Slmonell, 12 inoculted with low level of Slmonell, nd 12 uninoculted controls. A ckground screen of the mtrix indicted n sence of indigenous Slmonell species. As per criteri outlined in Appendix J of the AOAC Vlidtion Guidelines, frctionl positive results were otined for oth mtrices. For ech mtrix, the ctul level of Slmonell ws determined y MPN determintion on the dy of initition of nlysis y the coordinting lortory. The individul lortory nd smple results re presented in Tles 3 4. Tles A nd B summrize the collortive study results for ech mtrix tested, including POD sttisticl nlysis (7). Detiled results for ech lortory re presented in Appendix Tles 1 4 nd Appendix Figures 1 8. The result for ech collorting lortory s APC nlysis for ech mtrix is presented in Appendix Tle 5. Rw Ground Beef (25 g Test Portions) Rw ground eef test portions were inoculted t low nd high levels nd were nlyzed (Tle 3) for the detection of Slmonell spp. Uninoculted controls were included in ech nlysis. Seventeen lortories prticipted in the nlysis of this mtrix nd the results of 14 lortories were included in the sttisticl nlysis. Lortories 4, 6, nd 9 reported tht there were specific protocol devitions nd therefore results from these lortories were excluded from sttisticl nlysis. The MPNs otined for this mtrix, with 95% confidence intervls, were 0.77 MPN/test portion (0.57, 0.88) for the low level nd 4.67 MPN/test portion (3.38, 6.44) for the high

8 1570 Bird et l.: Journl of AOAC Interntionl Vol. 97, No. 6, 2014 Tle 1. Prticiption of ech collorting lortory L level. For the 3M Petrifilm SALX System, one test portion ws confirmed positive y the trditionl confirmtion tht ws confirmed negtive y the lterntive confirmtion. For ll other test portions, no difference ws oserved etween confirmtion of smples using the lterntive confirmtion procedure nd the trditionl reference method confirmtion procedure. For the high level, 168 out of 168 test portions were reported s presumptive positive y the 3M Petrifilm SALX System with ll test portions confirming positive y oth the trditionl nd lterntive confirmtion methods. For the low level, 85 out of 168 test portions were reported s presumptive positive y the 3M Petrifilm SALX System, with 83 test portions confirming positive y oth the trditionl nd lterntive confirmtion procedures. For the uninoculted controls, 2 out of 168 smples produced presumptive positive result y the 3M Petrifilm SALX System method with one of the two presumptive positive smples confirming positive y the trditionl reference method. All other test portions were negtive. For test portions nlyzed y the USDA/FSIS-MLG method, 167 out of 168 high inoculum nd 86 out of 168 low inoculum test portions confirmed positive. For Tle 2. Het-stress injury results Mtrix Dry dog food Rw ground eef (25 g test portions) Test orgnism Slmonell Poon NCTC 4840 CFU/XLD (selective gr) NCTC = Ntionl Collection of Type Cultures. Dry dog food (375 g test portions) 1 Y Y 2 Y Y 3 Y Y 4 Y Y 5 Y Y 6 Y Y 7 Y Y 8 Y Y 9 Y Y 10 Y Y 11 Y Y 12 Y Y 13 Y Y 14 Y Y 15 Y Y 16 Y Y 17 Y N Y = Collortor nlyzed the food type nd N = collortor did not nlyze the food type. Results were not used in sttisticl nlysis due to devition of testing protocol or lortory error. CFU/TSA (Non-selective gr) Degree injury % the uninoculted controls, 0 out of 168 test portions confirmed positive. For the low-level inoculum, dlpod C vlue of 0.02 ( 0.13, 0.09) ws otined etween the 3M Petrifilm SALX System using oth confirmtory procedures nd the USDA/FSIS-MLG method. The confidence intervls otined for dlpod C indicted no significnt difference etween the two methods. A dlpod CP of 0.01 ( 0.10, 0.12) ws otined etween presumptive nd confirmed 3M Petrifilm SALX System results for oth confirmtion procedures. The confidence intervls otined for dlpod CP indicted no significnt difference etween the presumptive nd confirmed results. For the high-level inoculum, dlpod C vlue of 0.01 ( 0.02, 0.03) ws otined etween the 3M Petrifilm SALX System using oth confirmtory procedures nd the USDA/FSIS-MLG method. The confidence intervls otined for dlpod C indicted no significnt difference etween the two methods. A dlpod CP of 0.00 ( 0.02, 0.02) ws otined etween presumptive nd confirmed 3M Petrifilm SALX System results for oth confirmtion procedures. The confidence intervls otined for dlpod CP indicted no significnt difference etween the presumptive nd confirmed results. For the uninoculted control level, dlpod C vlues of 0.01 ( 0.02, 0.03) nd 0.00 ( 0.02, 0.02) were otined etween the 3M Petrifilm SALX System using the trditionl nd lterntive confirmtion procedures, respectively, nd the USDA/FSIS-MLG method. The confidence intervls otined for dlpod C indicted no significnt difference etween the two methods. A dlpod CP of 0.01 ( 0.02, 0.04) nd 0.01 ( 0.01, 0.04) ws otined etween presumptive nd confirmed 3M Petrifilm SALX System results using the trditionl nd lterntive confirmtion procedures, respectively. The confidence intervls otined for dlpod CP indicted no significnt difference etween the presumptive nd confirmed results. Results of the POD sttisticl nlysis re presented in Tle A nd Appendix Tles 1 2 nd Appendix Figures 1 4. Dry Dog Food (375 g Test Portions) Dry dog food test portions were inoculted t low nd high levels nd were nlyzed (Tle 4) for the detection of Slmonell spp. Uninoculted controls were included in ech nlysis. Sixteen lortories prticipted in the nlysis of this mtrix nd the results of 12 of the lortories were included in the sttisticl nlysis. Two lortories, 4 nd 6, were unle to initite smple testing t the strt of the evlution due to equipment mlfunction or dely in receiving their smples nd therefore did not nlyze ny test portions. Two dditionl lortories, 2 nd 14, reported devitions from the testing protocol nd therefore results from these lortories were excluded from sttisticl nlysis. The MPN otined for this mtrix, with 95% confidence intervls, were 0.69 MPN/test portion (0.54, 0.86) for the low level nd 5.42 MPN/test portion (3.53, 8.30) for the high level. For the 3M Petrifilm SALX System, one test portion ws confirmed positive y the trditionl confirmtion tht ws confirmed negtive y the lterntive confirmtion. For ll other test portions, no difference ws oserved etween confirmtion of smples using the lterntive confirmtion procedure nd the trditionl reference method confirmtion procedure. For the high level, 142 out of 144 test portions were reported s presumptive positive y the 3M Petrifilm SALX System with

9 Bird et l.: Journl of AOAC Interntionl Vol. 97, No. 6, Tle 3. Individul collortor results for rw ground eef (25 g test portions) High-level test portions Low-level test portions Uninoculted test portions L M Petrifilm SALX System d e n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ e e n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ d c d USDA/FSIS-MLG d e n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ e e n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/

10 1572 Bird et l.: Journl of AOAC Interntionl Vol. 97, No. 6, 2014 Tle 3. (continued) High-level test portions Low-level test portions Uninoculted test portions L = Slmonell spp. were detected in smples nd = Slmonell spp. were not detected in smple. Confirmed results from lterntive nd trditionl confirmtion were identicl for ech test portion except where noted. Smple confirmed positive y trditionl method ws negtive y lterntive confirmtion. c Smple ws presumptive positive ut confirmed negtive. d Results were not used in sttisticl nlysis due to devition of testing protocol lortory error. e 141 test portions confirming positive y oth the trditionl nd lterntive confirmtion methods. For the low level, 82 out of 144 test portions were reported s presumptive positive y the 3M Petrifilm SALX System, with 82 test portions confirming positive y the trditionl confirmtion procedure nd 81 test portions confirming positive y the lterntive confirmtion procedure. For the uninoculted controls, 0 out of 144 smples produced presumptive positive result y the 3M Petrifilm SALX System method with 0 smples confirming positive y the trditionl reference method. All other test portions were negtive. For test portions nlyzed y the FDA/BAM Chpter 5 Method, 144 out of 144 high inoculum nd 71 out of 144 low inoculum test portions confirmed positive. For the uninoculted controls, 0 out of 144 test portions confirmed positive. For the low-level inoculum, dlpod C vlue of 0.08 ( 0.07, 0.22) nd 0.07 ( 0.07, 0.21) ws otined etween the 3M Petrifilm SALX System with the trditionl confirmtion nd lterntive confirmtion procedures, respectively, nd the FDA/BAM method. The confidence intervls otined for dlpod C indicted no significnt difference etween the two methods. A dlpod CP of 0.00 ( 0.14, 0.14) nd 0.01 ( 0.18, 0.22) ws otined etween presumptive nd confirmed 3M Petrifilm SALX System results for the trditionl nd lterntive confirmtion procedures, respectively. The confidence intervls otined for dlpod CP indicted no significnt difference etween the presumptive nd confirmed results. For the high-level inoculum, dlpod C vlue of 0.02 ( 0.06, 0.01) ws otined etween the 3M Petrifilm SALX System with the trditionl nd lterntive confirmtion procedures nd the FDA/BAM method. The confidence intervls otined for dlpod C indicted no significnt difference etween the two methods. A dlpod CP of 0.01 ( 0.03, 0.05) ws otined etween presumptive nd confirmed 3M Petrifilm SALX System results for the trditionl nd lterntive confirmtion procedures. The confidence intervls otined for dlpod CP indicted no significnt difference etween the presumptive nd confirmed results. For the uninoculted control level, dlpod C vlues of 0.00 ( 0.03, 0.03) were otined etween the 3M Petrifilm SALX System using the trditionl nd lterntive confirmtion procedures nd the FDA/BAM method. The confidence intervls otined for dlpod C indicted no significnt difference etween the two methods. A dlpod CP of 0.03 ( 0.03, 0.03) ws otined etween presumptive nd confirmed 3M Petrifilm SALX System results using the trditionl nd lterntive confirmtion procedures. The confidence intervls otined for dlpod CP indicted no significnt difference etween the presumptive nd confirmed results. Results of the POD sttisticl nlysis re presented in Tle A, nd Appendix Tles 3 4 nd Appendix Figures 5 8. Discussion No negtive feedck ws reported to the Study Directors from the collorting lortories in regrd to the performnce of the 3M Petrifilm SALX System. For the nlysis of the rw ground eef test portions y the 3M Petrifilm SALX System, three flse-positive smples were otined. For the nlysis of the dry dog food, two flse-positive smples nd two flsenegtive smples were otined.

11 Bird et l.: Journl of AOAC Interntionl Vol. 97, No. 6, Tle 4. Individul collortor results for dry dog food (375 g test portions) High-level test portions Low-level test portions Uninoculted test portions L M Petrifilm SALX System d f d f n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ f n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ e e f d + d + d d d c d + d d d + 17 n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ FDA/BAM f f n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ f n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/

12 1574 Bird et l.: Journl of AOAC Interntionl Vol. 97, No. 6, 2014 Tle 4. (continued) High-level test portions Low-level test portions Uninoculted test portions L f n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ n/ + = Slmonell spp. were detected in smples, = Slmonell spp. were not detected in smple, nd n/ = lortory did not prticipte in this mtrix or results were not received. Confirmed results from lterntive nd trditionl confirmtion were identicl for ech test portion unless noted. Smple ws presumptive positive nd confirmed negtive y trditionl confirmtion ut confirmed positive y lterntive. c Smple ws presumptive positive ut confirmed negtive. d Smple ws presumptive negtive ut confirmed positive using the trditionl confirmtion. e f Results were not used in sttisticl nlysis due to devition of testing protocol lortory error. For the dry dog food, some lortories reported high mounts of typicl growth on vrying test portions nd no ckground growth on other test portions. Becuse over 600 ls of pet food ws used in the evlution, vriility in the level of competing microflor mong individul smples my hve led to these discrepncies. Lortory 7, which reported the two flse-negtive results for the dry dog food, indicted tht significnt mount of typicl growth ws oserved on the 3M Petrifilm SALX Plte nd elieved it my hve contriuted to the difficulty in isolting Slmonell from those two smples. The flse-positive results oserved for oth mtrices my hve een the result of misidentifiction of typicl colonies due to the misinterprettion of colony color (only five flse positives out of 972 test portions) on the 3M Petrifilm SALX Plte. Additionl experience with the method my eliminte some nlyst uncertinty when selecting colonies elieved to e presumptive positive for Slmonell. Becuse presumptive colonies re verified y plcing the 3M Petrifilm SALX Confirmtion Disk onto the 3M Petrifilm SALX Plte, no dditionl follow-up to verify if the correct colony ws selected ws possile y testing t the coordinting lortory. For the rw ground eef, Lortory 15 identified presumptive positive colony in its uninoculted test portions, which ws confirmed positive y the reference method. The dt from this lortory were included in the sttisticl nlysis. Using the POD sttisticl model, no significnt difference in the numer of positive results otined etween the two methods eing compred ws oserved t oth the low nd high inoculum levels for oth mtrices. Additionlly, no significnt difference ws oserved etween presumptive nd confirmed results for the cndidte method. Recommendtions It is recommended tht the 3M Petrifilm Slmonell Express (SALX) System e dopted s Officil First Action sttus for the detection of Slmonell in rw ground eef (25 g), rw ground chicken (25 g), psteurized liquid whole egg (100 g), rw ground pork (25 g), cooked chicken nuggets (325 g), frozen uncooked shrimp (25 g), fresh unched spinch (25 g), dry dog food (375 g), nd stinless steel. Acknowledgments We extend sincere thnk you to the following collortors for their dedicted prticiption in this study: Brd Stwick nd Keith Blnchrd, Microc Lortories, Inc., Wrrendle, PA Delndo Lewis nd Roert Colvin, Microc Lortories, Inc., Bltimore, MD Ashley Morris, Microc Lortories, Inc., Mryville, TN Joe Meyer, Covnce Lortories, Monon, WI Amit Morey, Food Sfety Net Services, Sn Antonio, TX Kyle Newmn, Venture Lortories, Inc., Lexington, KY Roert Brooks, ATC Microiology, LLC, North Little Rock, AR Christine Gwinn nd Scott Moosekin, Covnce Lortories, Inc., Bttle Creek, MI Joey Mrchnt-Tmone, U.S. Food nd Drug Administrtion, Gulf Cost Sefood Lortory, Duphin Islnd, AL Kthleen T. Rjkowski, U.S. Deprtment of Agriculture,