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. 5, 2012
1427
incubated at 35–37°C aerobically for 18–24 h. From each of
the incubated plates, a culture suspension was prepared and the
organisms were identified as described in the method. Printed
results indicated a high probability match to a single species
if a unique identification pattern was recognized. If a unique
pattern was not recognized, the system suggested supplemental
tests to distinguish between two or three closely related
organisms, or indicated the result as an unidentified organism.
As indicated in the VITEK 2 GP product information provided
to end-users, slashline or low discrimination identifications
were considered acceptable results for the VITEK 2 GP
method that required supplemental tests to further resolve the
organism identification. Results were classified as I, Identified;
UD, Unidentified; and MI, Misidentified. The results of each
isolate identification were recorded on the data reporting sheets
provided and were sent along with the VITEK 2 printouts to
the study director within 2 weeks of completion of the study.
No additional testing was required for isolates not characterized
as Gram-positive organisms by Gram stain procedures.
AOAC Official Method 2012.02
Gram-Positive Bacteria Identification
VITEK
®
2 Gram Positive (GP)
Biochemical Identification Method
First Action 2012
(Applicable to the identification of Gram-positive bacteria,
including
Listeria
and
Staphylococcus
species.)
See
Tables
2012.02A
and
2012.02B
for results of the
interlaboratory study supporting acceptance of the method.
Claimed Gram-positive species validated in both the
precollaborative and collaborative studies:
Listeria grayi,
Listeria innocua, Listeria ivanovii
ssp.
Ivanovii, Listeria ivanovii
ssp.
Londoniensis, Listeria monocytogenes, Listeria seeligeri,
Listeria welshimeri, Staphylococcus hyicus, Staphylococcus
intermedius, Staphylococcus aureus, Staphylococcus aureus,
and
Staphylococcus epidermidis
.
A. Principle
The VITEK2 system is an automated microbial identification
system that utilizes the VITEK 2 Gram-positive (GP)
identification card for the identification of most significant
Gram-positive organisms. The VITEK 2 GP card is based on 43
biochemical tests measuring carbon source utilization, inhibition
and resistance, and enzymatic activities. Identification results
are available in approximately 8 h or less.
B. Apparatus and Reagents
(
a
)
VITEK 2 system.
(
b
)
VITEK 2 GP test cards.
(
c
)
Vortex.
(
d
)
Incubators
.—Set to 30°C and 35–37°C.
(
e
)
VITEK 2 DENSICHEK kit.
(
f
)
DENSICHEK calibrator
.
(
g
)
VITEK 2 cassette
.
(
h
)
Sterile saline
.—Aqueous 0.45 to 0.50% NaCl, pH 4.5 to
7.0).
(
i
)
Disposable test tubes
.—12 mm
×
75 mm clear plastic
(polystyrene).
(
j
)
Sterile sticks or swabs.
(
k
)
Brain Heart Infusion (BHI) slants.
(
l
)
Culture media
.—Columbia agar with 5% sheep blood
(CBA), Trypticase soy agar (TSA), or Trypticase soy agar with
5% sheep blood (TSAB) plates.
C. General Instructions
(
a
) The test cannot be performed without a pure culture. A
pure culture may be obtained by streaking out the isolate on
CBA, TSA, or TSAB plates.
(
b
) Gram stain purified isolate prior to preparingVITEK 2 GP
test card to verify presence of Gram-positive organism.
(
c
)
Caution
: Dispose of all reagents and other contaminated
materials by acceptable procedures for potentially biohazardous
materials. All microbial cultures are potentially infectious and
should be treated with universal precautions.
Table 2012.02A. Interlaboratory study results for the VITEK 2 GP identification method: Claimed isolates
Organism
Correct
Misidentified
Unidentified
Not tested
a
Total
b
Listeria grayi
60
0
0
0
60
Listeria innocua
60
0
0
0
60
Listeria ivanovii
ssp.
Ivanovii
60
0
0
0
60
Listeria ivanovii
ssp.
Londoniensis
60
0
0
0
60
Listeria monocytogenes
60
0
0
0
60
Listeria seeligeri
57
0
0
3
60
Listeria welshimeri
60
0
0
0
60
Staphylococcus hyicus
60
0
0
0
60
Staphylococcus intermedius
60
0
0
0
60
Staphylococcus aureus
60
0
0
0
60
Staphylococcus aureus
57
0
0
3
60
Staphylococcus epidermidis
60
0
0
0
60
Total isolates
714
0
0
6
720
a
Organism was incorrectly characterized by Gram stain and was not tested on VITEK 2 GP card.
b
Total numbers represent isolates analyzed on the three recommended culture media: CBA, TSA, and TSAB.
Candidates for 2016 Method of the Year
263