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© 2012 AOAC INTERNATIONAL
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 VITEK 2 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 × 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 preparing VITEK 2 GP
test card to verify presence of Gram-positive organism.
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.
Table 2012.02B. Interlaboratory study results for the VITEK 2
GP identification method: Exclusivity isolates
Organism
Misidentified
a
Unidentified
b
Not
tested
c
Total
d
Bacillus coagulans
3
27
10 40
Citrobacter freundii
0
0
20 20
Escherichia coli
0
6
14 20
Serratia marcescens
0
0
20 20
Aspergillus niger
0
0
20 20
Candida albicans
6
0
14 20
Total Isolates
9
e
33
e
98 140
a
Organism was incorrectly characterized by Gram stain and was improperly
tested by the VITEK 2 GP method resulting in misidentification.
b
Organism was incorrectly characterized by Gram stain and was improperly
tested by the VITEK 2 GP method resulting in no identification.
c
Organism was excluded by Gram stain procedure and was not tested on
VITEK 2 GP card as per protocol.
d
Total numbers represent each strain not tested and strains that were
misidentified and unidentified.
e
Total number of isolates incorrectly tested by the VITEK 2 GP method after
erroneous Gram stain result.
AOAC Research Institute
Expert Review Panel Use Only