AOAC RI ERP Final Action Recommendations eBook

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.

Table 2012.02B. Interlaboratory study results for the VITEK 2 GP identification method: Exclusivity isolates

Not tested c Total d

Organism

Misidentified a Unidentified b

3 0 0 0 0 6

27

10 40 20 20 14 20 20 20 20 20 14 20

Bacillus coagulans Citrobacter freundii

0 6 0 0 0

Escherichia coli

Serratia marcescens Aspergillus niger Candida albicans  Total Isolates

( 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. 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. 9 e 33 e

( 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).

Table 2012.02A. Interlaboratory study results for the VITEK 2 GP identification method: Claimed isolates Organism Correct Misidentified Unidentified Not tested a

Total b AOAC Research Institute Expert Review Panel Use Only

60 60 60 60 60 57 60 60 60 60 57 60

0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 3 0 0 0 0 3 0 6

60 60 60 60 60 60 60 60 60 60 60 60

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 Staphylococcus epidermidis

 Total isolates

714

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.

© 2012 AOAC INTERNATIONAL