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830 M ozola et al.: Journal of AOAC International Vol. 97, No. 3, 2014
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,
Here we report results of an interlaboratory collaborative study
conducted in 18 laboratories for further evaluation of the assay as
a colony confirmation tool.
Collaborative Study
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.
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
(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.
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.
Analysis of Isolates
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
Table 1. Inclusive and exclusive isolates used in the ANSR Salmonella collaborative study
Organism ID No. Source
Origin
(if known)
Salmonella enterica subsp. arizonae 700156 ATCCa Poult
Salmonella enterica subsp. enterica Ser. Typhimurium 23566 ATCC Unknown
Salmonella enterica subsp. enterica Ser. Cubana 12007 ATCC Unknown
Salmonella bongori 43975 ATCC Unknown
Salmonella enterica subsp. enterica Ser. Cerro 10723 ATCC Unknown
Salmonella enterica subsp. enterica Ser. Enteritidis 4931 ATCC Human GI tract
Enterobacter cloacae 13047 ATCC Human CSF
Escherichia coli 25922 ATCC Human
Proteus vulgaris 29905 ATCC Unknown
Providencia alcalifaciens 27970 ATCC Feces
Citrobacter freundii 8090 ATCC Unknown
Klebsiella pneumoniae 13883 ATCC Unknown
aAmerican Type Culture Collection, Manassas, VA.
Candidates for 2016 Method of the Year
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