17.15.02 alcalifaciens, P. stuartii, Pseudomonas aeruginosa, P.

 fluorescens,
AOAC Official Method 2011.17
Salmonella, Escherichia coli,
and Other Enterobacteriaceae
VITEK® 2 Gram-Negative (GN)
Biochemical Identification Method
First Action 2011
(Applicable to the identification of Gram-negative bacteria.)
See Tables 2011.17A and B for results of the interlaboratory
study supporting acceptance of the method.
A.  Principle
The VITEK 2 system is an automated microbial identification
system that utilizes the VITEK 2 GN card for the identification of
fermenting and nonfermenting Gram-negative bacilli, including the
select agent organisms Brucella melitensis, Francisella tularensis,
Burkholderia mallei, B. pseudomallei, and Yersinia pestis. The
VITEK 2 GN card is based on 47 biochemical tests measuring
carbon source utilization, inhibition and resistance, and enzymatic
activities. Identification results are available in approximately 10 h
or less.
Inclusivity strains used in precollaborative study.—Brucella
melitensis, Burkholderia cepacia, B. gladioli, B.  mallei,
B. pseudomallei, Cedecea davisae, C. lapage, Citrobacter
amalonaticus, C. braakii, C. farmeri, C.  freundii, C. koseri,
C. youngae, Cronobacter sakazakii, Edwardsiella hoshinae,
E. tarda, Enterobacter aerogenes, E. amnigenus, E.  asburiae,
E. cancerogenus, E. cloacae, E. gergoviae, E.  intermedius,
Escherichia coli, E. coli O157:H7, E.  coli O157:H12, E. coli
O157:H16, E. coli O157:H43, E.  fergusonii, E.  hermanii,
E.  vulneris, Ewingella Americana, Francisella tularensis,
Hafnia alvei, Klebsiella oxytoca, K. pneumoniae, Kluyvera
ascorbate, Leclercia adecarboxylata, Moellerella wisconsensis,
Morganella morganii ssp. Morganii, Pantoea agglomerans,
Proteus mirabilis, P. vulgaris group/P.  penneri, Providencia
Rahnella aquatilis, Salmonella Abaetetuba, S. Abony, S. Adelaide,
S.  Agona, S.  Alachua, S.  Amsterdam, S.  Anatum, S. Bareilly,
S. Bornum, S.  California, S. enterica, S. enterica ssp. arizonae,
S. Cerro, S.  Kunzendorf, S.  Cubana, S. Dublin, S. Djugu,
S.  Drypool, S.  Enteriditis, S.  Ferlac, S.  Gallinarum, S.  Gaminara,
S.  Give, S.  Glostrup, S.  Hadar, S.  Haardt, S.  Havana, S. Heerlen,
S. Hilversum, S. Houtenae hamelen, S. Houtenae houton, S. Illinois,
S. Javiana, S. Krefeld, S. Maarsen, S. Madelia, S. Manila, S. Miami,
S.  Newhaw, S. Newport, S.  Ouakam, S.  Panama, S. Paratyphi A,
S.   Roodepoort, S.  Rubislaw, S.  Salamae hoograven, S.  Saphra,
S. Senftenberg, S. Simsbury, S. Sundsvall, S. Tennessee, S. Typhi,
S.  Typhimurium, S.  Zwickau, Serratia ficaria, S.  fonticola,
S. liquefaciens group, S. marcescens, S. odorifera, S. plymuthica,
Shigella sonnei, S. dysenteriae, S. flexneri (2b,1a, V), S. boydii,
Vibrio alginolyticus, V. cholerae, V. fluvialis, Grimontia (Vibrio)
hollisae, V. metschnikovii, V. mimicus, V.  parahaemolyticus,
V.  vulnificus, Yersinia enterocolitica group, Yersinia pestis,
Y. pseudotuberculosis, and Y. ruckeri.
B.  Apparatus and Reagents
(a)  VITEK 2 system.—bioMérieux, Inc. (www.biomerieux.
com).
(b)  VITEK 2 GN test cards.
(c)  Vortex mixer.
(d)  Incubators.—35–37°C.
(e)  VITEK 2 DENSICHEK Kit.
(f)  DENSICHEK Calibrator.
(g)  VITEK 2 Cassette.
(h)  Sterile saline.—Aqueous 0.45–0.50% NaCl, pH 4.5–7.0.
(i)  Test tubes.—12 × 75 mm, clear plastic (polystyrene),
disposable.
(j)  Sterile sticks or swabs.
(k)  Brain heart infusion (BHI) slants.
(l)  Culture media.—Columbia agar with 5% sheep blood (CBA),

Table  2011.17A.  Interlaboratory study results for the VITEK 2 GN identification method—claimed isolates

Organism Correct Misidentified Unidentified Not testeda Totalb

Citrobacter freundii 60 0 0 0 60
Edwardsiella tarda 60 0 0 0 60
Salmonella Typhimurium 57 0 0 3 60
Cronobacter sakazakii 60 0 0 0 60
Escherichia coli 60 0 0 0 60
Klebsiella pneumoniae 57 0 0 3 60
Proteus mirabilis 57 0 0 3 60
Providencia stuartii 60 0 0 0 60
S. enterica ssp. arizonae 60 0 0 0 60
S. Typhi 60 0 0 0 60
Serratia marcescens 60 0 0 0 60
Vibrio vulnificus 56 0 0 4c 60
  Total isolates 707 0 0 13 720
a
  Organism was incorrectly characterized by Gram stain and was not tested on VITEK 2 GN card.
b
  Total numbers represent each strain analyzed on the three recommended culture media: CBA, TSA, and MAC.
c
  One isolate did not grow on MAC, and three were misidentified by Gram stain.

© 2012 AOAC INTERNATIONAL

Table  2011.17B.  Interlaboratory study results for the VITEK 2 GN identification method—exclusivity isolates

Organism Misidentifieda Unidentified Not testedb Totalc

Listeria innocua 2 0 19 21
Rhodococcus equi 4 0 18 22
Staphylococcus aureus 0 0 20 20
Streptococcus pyogenes 0 0 20 20
Aspergillus niger 0 0 20 20
Candida albicans 0 0 20 20

  Total isolates 6d 0 117 123

a
 Organism was incorrectly characterized as a Gram-negative organism and was improperly tested by the VITEK 2 GN method.
b
 Organism was excluded by Gram stain procedure and was not tested on VITEK 2 GN card as per protocol.
c
 Total numbers represent each strain not tested and strains that were misidentified.
d
 Total number of isolates incorrectly tested by the VITEK 2 GN method (from culture media plates TSA and CBA) after erroneous Gram stain result.

trypticase soy agar (TSA), or MacConkey agar (MAC) plates.
C.  General Instructions
(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.)
(a)  Use only pure cultures.
(b)  Gram stain culture prior to preparing VITEK 2 GN test card
to verify presence of Gram-negative isolate.
(c)  Store VITEK 2 GN cards at 2–8°C.
(d)  Do not freeze test cards.
(e)  Bring reagents to room temperature before inserting them
into the VITEK 2 instrument.
(f)  Immediately return unused cards to 2–8°C.
D.  Preparation of Test Suspension
Aseptically transfer 3.0 mL sterile saline (aqueous 0.45–0.50%
NaCl, pH 4.5–7.0) into polystyrene test tubes (12 × 75 mm).
Do not use glass tubes. Using a sterile stick or swab, transfer a
sufficient number of colonies from a 24 h culture on recommended
culture medium to the saline tube to achieve a density equivalent
to McFarland 0.50–0.63 with the VITEK 2 DENSICHEK. Test the
cultures by the VITEK 2 GN method within 30 min of preparation
of the suspended culture. Insert the culture tube and the VITEK 2
GN card into the VITEK 2 Cassette, and refer to the User Manual
(provided with the instrument) for instructions on use of the
instrument. Report identification results from the VITEK 2 System.
As indicated in the VITEK 2 GN product information provided
to end-users, slashline or low discrimination identifications are
acceptable results for the VITEK 2 GN method and require
conducting the suggested supplemental test to further resolve the
organism identification.
E.  Results and Interpretation
Results are interpreted by the VITEK 2 System. Printed results
will indicate a high probability match to a single species if a
unique identification pattern is recognized. If a unique pattern
is not recognized, the system will suggest supplemental tests to
distinguish between two or three closely related organisms, or
indicate the result as an unidentified organism (either >3 organisms
can exhibit the observed pattern, or the biopattern is very atypical
and is not represented in the database).
Reference: J. AOAC Int. 95, 778(2012)

© 2012 AOAC INTERNATIONAL