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1426 Crowley et al.: Journal ofAOAC International Vol. 95, No . 5, 2012
and TSAB cultures were 95, 96, and 97%, respectively. The
VITEK 2 GP reagents were stable over 18-month time studies
and provided highly reproducible results when compared
between lots. Assay ruggedness was demonstrated for three
critical parameters, including age of culture, inoculum density
,and inoculum age. Overall results indicate that the VITEK 2 GP
method is an acceptable automated method for the identification
of selected Gram-positive organisms. The method was awarded
PTM certification No. 120702 on December 10, 2007.
The collaborative study evaluated the ability of the
VITEK 2 GP identification method to correctly identify
720 inclusivity strains representative of 12 different claimed
organisms. A total of 120 exclusivity strains representative of
six different nonclaimed organisms were also evaluated. All
isolates analyzed were obtained from the three recommended
subculture culture media: TSA, CBA, and TSAB (6).
Collaborative Study
Study Design
A total of 20 laboratories participated in the collaborative
study, consisting of industry, government, and private testing
laboratories involved in the testing of food products. Qualified
laboratories currently using the VITEK 2 system were invited
to act as collaborators. Each participating laboratory received
18 isolates on Brain Heart Infusion (BHI) agar slants. The
identity of each isolate had been previously confirmed by
traditional biochemical confirmations and/or Certificate of
Analysis. There were 12 claimed isolates (target inclusivity
organisms; Table 1) and six isolates of exclusivity organisms
(nontarget organisms; Table 2).
A detailed collaborative study packet outlining all necessary
information related to the study, including isolate preparation
and documentation of results, was sent to each collaborating
laboratory prior to the initiation of the study. All participating
laboratories were provided with GP cards, prepared culture
media plates, and any additional supplies needed to identify
the isolates. Participants were asked to provide their own
Gram stain reagents due to challenges associated with federal
regulations in the transportation of the reagents.
Preparation of Isolates
Pure isolates of each organism from the bioMérieux (BMX)
culture collection were sent on BHI agar slants to each of the
participating laboratories. Each isolate used had been well
characterized. Microscopic and plate morphology, standard
biochemicals, at least two phenotypic methods, and, when
needed, molecular sequencing were performed. To prepare
for distribution to the collaborators, growth from each strain
was transferred to BHI agar slants from growth that had been
subcultured onto CBA and incubated for 18–24 h at 35–37°C.
Following incubation, slants were labeled and prepared for
shipment.
Isolate Distribution
All isolates were labeled with a randomized, blind-coded
3-digit number affixed to the sample vial. Isolates were
shipped in leak-proof, insulated containers on a Monday via
overnight delivery according to the Category B Dangerous
Goods shipment regulations set forth by the International Air
Transportation Association. Upon receipt on Tuesday, isolates
were streaked onto CBA to check for purity. Collaborators were
instructed to hold the original BHI agar slants in the refrigerator
at 2–5°C for the duration of the study. All isolates were shipped
at ambient temperature. Collaborators received all 18 isolates
during the same week of testing. Participants were instructed to
inspect the isolates upon receipt of the package and document
that they were received in good condition on the Sample Receipt
Confirmation form provided in the shipment. Forms were then
faxed or emailed to the study director.
Analysis of Isolates
Upon receipt of the shipment, collaborators were instructed
to streak each isolate received onto CBA to check for purity
and incubate for 18–24 h at 35–37°C. Following incubation, a
Gram stain was conducted on one isolated colony from each
strain according to the U.S. Food and Drug Administration’s
Bacteriological Analytical Manual (FDA/BAM) method (7)
and results were recorded. Those isolates that were identified
as characteristic Gram-positive organisms were streaked to the
three recommended culture media, TSA, CBA, and TSAB, and
Table 1. Inclusivity (claimed) isolates used in VITEK 2 GP
collaborative study
Organisms ID No. Sourcea Origin
Listeria grayi 125274 BMX Industry
Listeria innocua 12524 BMX Fish
Listeria ivanovii ssp. Ivanovii 12913 BMX Reference lab
Listeria ivanovii ssp. Londoniensis 12914 BMX Clinical
Listeria monocytogenes 12502 BMX Shellfish
Listeria seeligeri 6217 BMX Creamer
Listeria welshimeri 12517 BMX Beef
Staphylococcus hyicus 13889 BMX Reference lab
Staphylococcus intermedius 16409 BMX Veterinary
Staphylococcus aureus 8819 BMX Reference lab
Staphylococcus aureus 8852 BMX Reference lab
Staphylococcus epidermidis 8890 BMX Reference lab
aBMX = bioMérieux culture collection.
Table 2. Exclusivity isolates used in VITEK 2 GP
collaborative study
Organisms ID No. Sourcea Origin
Bacillus coagulans 202608 BMX Tomato paste
Citrobacter freundii 110125 BMX Hospital
Escherichia coli 112380 BMX Beef hide
Serratia marcescens 111663 BMX Reference lab
Aspergillus niger 304232 BMX Clinical
Candida albicans 305527 BMX Reference lab
aBMX = bioMérieux culture collection.
Candidates for 2016 Method of the Year
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