4. AOACRIMicroMethods-2018Awards

186

830  M ozola et al . : J ournal of AOAC I nternational V ol . 97, N o . 3, 2014

Table 1. Inclusive and exclusive isolates used in the ANSR Salmonella collaborative study

Origin (if known)

Organism

ID No.

Source ATCC a ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC

Poult

Salmonella enterica subsp. arizonae

700156 23566 12007 43975 10723 13047 25922 29905 27970 4931

Salmonella enterica subsp. enterica Ser. Typhimurium Salmonella enterica subsp. enterica Ser. Cubana

Unknown Unknown Unknown Unknown

Salmonella bongori

Salmonella enterica subsp. enterica Ser. Cerro Salmonella enterica subsp. enterica Ser. Enteritidis

Human GI tract

Human CSF

Enterobacter cloacae

Human

Escherichia coli Proteus vulgaris

Unknown

Feces

Providencia alcalifaciens Citrobacter freundii Klebsiella pneumoniae

8090

Unknown Unknown

13883

a  American Type Culture Collection, Manassas, VA.

(Table 1). All strains were obtained directly from the American Type Culture Collection (ATCC; Manassas, VA). Identity of isolates was confirmed by API 20E testing. Salmonella isolates were also verified by O group serology. Isolates were cultured on TSA slants for 18–24 h at 36 ± 1°C. Slant cultures were labeled with a two-digit alphabetical code.

deoxycholate agar (XLD), bismuth sulfite agar (BS), brilliant green sulfa agar (BGS), xylose lysine tergitol agar (XLT-4), and double-modified lysine iron agar (DMLIA)] and tested in the ANSR assay. The former three media are specified for use in the BAM reference method, while the latter three are specified in the MLG method. One hundred and twelve Salmonella spp. strains produced positive results from all seven media, for inclusivity of 99.1%. One strain of S . Weslaco, previously identified as a non- inclusive strain lacking the genetic target for the ANSR assay, produced negative results from all seven media. In testing of exclusive strains, 248 of 251 assays produced negative results, for accuracy of 98.8%. The precollaborative study report is included as Appendix I on J. AOAC Int. website, http://aoac.publisher. ingentaconnect.com/content/aoac/jaoac. Here we report results of an interlaboratory collaborative study conducted in 18 laboratories for further evaluation of the assay as a colony confirmation tool. Study Design This collaborative study was conducted in accordance with the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces, Appendix J  (8). Eighteen laboratories participated in the collaborative study, representing industry, academic, government, and private testing laboratories. All collaborators were either established users of the ANSR test system or were expressly trained for the collaborative study prior to its commencement. A detailed set of instructions and data recording forms were sent to each collaborator in advance of the study. Collaborators were provided with all necessary agar plating media, test kits, ANSR system instrumentation, and a blind-coded set of 12 bacterial cultures for analysis. Collaborative Study

Distribution of Isolates

Cultures were shipped to collaborators via overnight delivery, at ambient temperature, using Category B Dangerous Goods packaging as set forth by International Air Transport Association regulations. Collaborators were instructed to store the cultures at 2–8°C until initiation of the analytical work (4–5 days). Collaborators were provided with a “Sample Receipt Form,” to be completed and returned to the Study Director by email or fax, acknowledging that the samples were received in good condition. To initiate the analysis, collaborators streaked each of the 12 bacterial isolates to each of the seven agar media, streaking for isolated colonies. Collaborators were provided with a sample randomization scheme by the Study Director and were instructed to blind-code each strain-agar medium combination with a unique number 1–84. This was performed by “Operator 1,” who would have no involvement in the actual ANSR analyses. Plates were incubated for 24±2 h at 35±1°C and examined for the presence of isolated colonies. Plates without isolated colonies were reincubated for an additional 18–24 h. Plates containing distinct isolated colonies after 24 h were stored at 2–8°C. After a maximum of 48 h incubation, plates without growth or isolated colonies were noted as such on the Data Recording Form and analysis continued. Operator 1 then picked a single colony from each plate, including the refrigerated plates, using an inoculating loop or needle, and resuspended the colony in 0.5 mL phosphate- buffered saline (PBS). The coded tubes were transferred to “Operator 2,” who then performed the ANSR analyses. ANSR testing was performed in blocks of up to 16 samples, starting with sample number 1 and continuing through sample number Analysis of Isolates

Preparation of Isolates

All isolates were from the Neogen Corp. culture collection and consisted of six diverse strains of S. enterica and S. bongori , and six strains of Enterobacteriaceae belonging to other genera

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