1. Advance Identification Systems: VITEK 2 COMPACT
Interamerican University of Puerto Rico Bayamón Campus Graduate Department
Advance Identification Systems: VITEK 2 COMPACT
Coral M. Rivera
Johnathan J. Rodríguez
José Á. Pérez
BIOL 6014

2. Objectives Principles of VITEK 2 compact. Software operation. Cards
Components.
Other features
VITEK 2 workflow.
Conclusion
Reagent cards.
References
Sample preparation.
V ITEK 2 operation.

3. Principles of VITEK 2 Compact
After inoculation with a standarized suspension of the unknown organism, each self-contained cards is incubated and read by the instrument’s internal optics.
The VITEK 2 compact system is an automated microbiology bacterial identification and antimicrobial susceptibility system.
Comparison of results to known species specific reactions in the VITEK 2 database yields organism identifications.
Uses advanced colorimetry technology to determine individual biochemical reactions contained in a variety of microbe identification cards.

4. Components 1. VITEK 2 Compact 4. Shared Printer 6. Power Conditioner
2. Compact Workstation
5. UPS (Uninterruptible Power Supply)
7. Test Cassettes
3. Barcode Scanner
8. DensiCHEK™ Plus

5. VITEK 2 Workflow

6. Sample Preparation / Inoculum
VITEK 2 Workflow
Step
Process
Sample Preparation / Inoculum
Standardization
Bacterial suspensions prepared using a DensiCHEK™ Plus.
Barcode reader inside the instrument reads barcode from the
test kit.
Linking Bar Code and
Sample ID
The test kit barcode is linked to the sample in the software.
Not necessary to write sample ID on the test kit. Allows for
alphanumeric entry up to 20 digits.
Filling and
Sealing the
Test Kits
Cassettes are placed into the Filler Station. Upon completion,
user is prompted to place the cassette into the cassette
Load/Unload Station where the cards are automatically
sealed.
Incubation and
Analysis
Sealed test kits are moved into the reader/incubator
automatically. Test kits incubate and analyze automatically.
Test kits are ejected automatically into the waste collection
bin when complete.
Test Kit
Dispose of waste when cassette returns to the Load/Unload

7. Reagents Cards
Each card has a pre-inserted transfer tube used for inoculation . Cards have bar codes that contain information on product type, lot number, expiration date, and a unique identifier that can be linked to the sample either before or after loading the card onto the system.
The reagent cards have 64 wells contain an individual test substrate.
Substrates measure metabolic activities such as acidification, alkalinization, enzyme hydrolysis, and growth in the presence of inhibitory substances.
There are currently four reagent cards available for the identification of different organism classes as follows:
An optically clear film present on both sides of the card allows for the appropriate level of oxygen transmission.
1. GN - Gram-negative fermenting and non-fermenting bacilli.
2. GP - Gram-positive cocci and non-spore-forming bacilli .
3. YST - yeasts and yeast-like organisms.
4. BCL - Gram-positive spore-forming bacilli.

8. Reagent card – 64 wells with substrates of each test cards.
Reagents Cards
Reagent card – 64 wells with substrates of each test cards.

9. Sample preparation Culture Requirements Suspension Preparation
Inoculation
Identification cards are inoculated with microorganism suspensions by vacuum apparatus. The test suspension tube is placed into the “cassette”, while inserting the transfer tube into the suspension tube. Filled cassette is placed into a vacuum chamber station. introducing the organism suspension into micro-channels that fill all the test wells.
The product contains the culture requirements for appropriate culture and inoculum preparation. These parameters include acceptable culture media, culture age, incubation conditions, and inoculum turbidity.
A sterile swab is used to transfer a sufficient number of colonies of a pure culture and to suspend the microorganism in 3.0 mL of sterile saline (0.50% NaCl) in a clear plastic test tube. The turbidity is adjusted accordingly Table 1 and measured by DensiChekTM.

10. VITEK 2 compact
Table 1: Turbidity range for Card Inoculation

11. VITEK 2 Compact video

12. VITEK 2 cards

13. VITEK 2 operation Card Sealing and Incubation Optical System
Test Reactions
A transmittance optical system allows interpretation of test reactions using different wavelengths in the visible spectrum. A special algorithm is used to eliminate false readings due to small bubbles that may be present.
Calculations are performed on raw data and compared to thresholds to determine reactions for each test. On the VITEK 2 Compact, test reaction results appear as “+”,” –“, [“(–)” or “(+)”. weak reactions that are too close to the test threshold].
Inoculated cards are passed by a mechanism, which cuts off the transfer tube and seals the card prior to loading into the carousel incubator. (accommodate up to 30 or up to 60 cards). All card types are incubated at ºC. Data are collected at 15-minute intervals during the entire incubation period.

14. Software operation Database Development Analytical Techniques
Identification Levels
Test data from an unknown organism are compared to the respective database to determine a quantitative value for proximity to each of the database taxa. If a unique identification pattern is not recognized, a list of possible organisms is given, or the strain is determined to be outside the scope of the database.
An unknown biopattern is compared to the database of reactions for each taxon, and a numerical probability calculation is performed. Various qualitative levels of identification are assigned based on the numerical probability calculation.
The databases are constructed with strain sets of well-characterized microorganisms, tested under various culture conditions. Strains are derived from a clinical, industrial and university culture collections.

15. ID Levels

16. Software operation Mixed Taxa Identifications Supplemental Testing
Non-Reactive Biopattern
This occurs when the biopattern is representative of a collective taxon and generates a genus-level, group-level, or slashline identification. Supplemental tests may be used to delineate representative species or subspecies of these collective taxa.
In the case of low discrimination identifications, two or three choices are listed in the order of their probability calculations. The lab report contains recommended supplemental tests that allow for differentiation of choices in low discrimination identifications.
When a biopattern is calculated for an unknown organism that is either completely negative or consists of both negative tests and tests with reactions that lie too close to the test thresholds, the identification result will be “Non-reactive biopattern.”

17. GN Cards
The GN card is used for the automated identification of 135 taxa of the most significant fermenting and non-fermenting Gram-negative bacilli.
The GN card is based on established biochemical methods and newly developed substrates measuring carbon source utilization, enzymatic activities, and resistance (ASM 1998, Chang et al. 2002, Coenye et al. 2001a, Coenye et al. 2001b, De Baere et al. 2001, Freney et al. 2000, Murray et al. 1999, Vandamme et al. 1999).
There are 47 biochemical tests and one negative control well. Final identification results are available in approximately 10 hours or less.

18. GN - Database organisms

19. GN - Database organisms

20. GN - Database organisms

21. GN - Database organisms

22. GN - Database substrates

23. GP Cards
The GP card is used for the automated identification of 115 taxa of the most significant non-spore-forming Gram-positive bacteria (primarily cocci).
The GP identification card is based on established biochemical methods and newly developed substrates (Atlas 1993, Barros et al. 2001, Collins and Lawson 2000, Collins et al. 2001, Freney et al. 2000, Holt et al. 1994, Murray et al. 1999, Poyart et al. 2002, Schlegel et al. 2000, Viera et al. 1998, Whiley et al. 1999).
There are 43 biochemical tests measuring carbon source utilization, enzymatic activities and resistance. Final identification results are available in approximately eight hours or less.

24. GP - Database organisms

25. GP - Database organisms

26. GP - Database organisms

27. GP - Database substrates

28. YST Cards
The YST card is used for the automated identification of 49 taxa of the most significant yeasts and yeast-like organisms.
The YST identification card is based on established biochemical methods and newly developed substrates (Barnett et al. 2000, Larone 1995, Murray et al. 1999).
There are 46 biochemical tests measuring carbon source utilization, enzymatic activities and resistance. Final identification results are available in approximately 18 hours.

29. YST - Database organisms

30. YST - Database organisms

31. YST – Database substrates

32. BCL Cards
The BCL card is used for the automated identification of 38 taxa of the most significant aerobic endospore-forming species of the family Bacillaceae.
The BCL identification card is based on established biochemical methods and newly developed substrates (Atlas 1993, Logan et al. 2002, Logan and Turnbull 2003).
There are 46 biochemical tests measuring carbon source utilization, enzymatic activities, inhibition, and resistance. Final identification results are available in approximately 14 hours.

33. BCL - Database organisms

34. BCL - Database organisms

35. BCL - Database organisms

36. BCL - Database substrates

37. Other features Identification Antibiotic Susceptibility Testing (AST)
NH ID Card, Neisseria, Haemophilus and other fastidious Gram negative bacteria identification.
ANC ID Card, Anaerobic bacteria and coryneform bacteria identification.
Antibiotic Susceptibility Testing (AST)
Gram positive antimicrobial susceptibility testing (AST) cards.
Gram negative antimicrobial susceptibility testing (AST) cards.
Yeast antimicrobial susceptibility testing (AST) card.

38. Conclusion
The VITEK 2 is an automated microbial identification system that provides highly accurate and reproducible results as shown in multiple independent studies. With its colorimetric reagent cards, and associated hardware and software advances, the VITEK 2 offers a state-of-the-art technology platform for phenotypic identification methods.

39. References
American Society for Microbiology. 98th General Meeting Workshop Program. Practical Approach to the Identification of the Medically Important Glucose Non-Fermenting Gram-Negative Bacilli. American Society for Microbiology, Washington, D.C
Atlas RA. Handbook of Microbiological Media. CRC Press, Ann Arbor
Barnett JA, Payne RW, Yarrow D, editors. Yeasts: Characteristics and Identification, 3rd ed. Cambridge University Press, New York
Barros RR, Carvalho GS, Peralta JM, Facklam RR, Teixeira LM. Phenotypic and Genotypic Characterization of Pediococcus Strains Isolated from Human Clinical Sources. J. Clin. Microbiol. 2001;39:

40. References
Chang YH, Han J, Chun J, Lee KC, Rhee MS, Kim YB, Bae KS. Comamonas koreensis sp.nov., a non-motile species from wetland in Woopo, Korea. Int. J. Syst. Evol. Microbiol. 2002;52:
Coenye T, Mahenthiralingam E, Henry D, Lipuma JJ, Laevens S, Gillis M, Speert DP, Vandamme P. Burkholderia ambifaria sp nov., a novel member of the Burkholderia cepacia complex including biocontrol and cystic fibrosis-related isolates. Int. J. Syst. Evol. Microbiol. 2001;51:
Coenye T, Vandamme P, Gowan JRW, Lipuma JJ. Taxonomy and Identification of the Burkholderia cepacia Complex. J. Clin. Microbiol. 2001;39:
Collins MD, Hutson RA, Hoyles L, Falsen E, Nikolaitchouk N, Foster G. Streptococcus ovis sp. nov. isolated from sheep. Int. J. Syst. Evol. Microbiol. 2001;51:

41. References
De Baere T, Steyaert, Wauters G, De Vos P, Goris J, Coenye T, Suyama T, Verschraegen G, Vaneechoutte M. Classification of Ralstonia pickettii biovar 3/ ‘thomasii’ strains (Pickett 1994) and of new isolates related to nosocomial recurrent meningitis as Ralstonia mannitolytica sp.nov. Int. J. Syst. Evol. Microbiol. 2001;51:
Freney J, Renaud F, Hansen W, Bollet C. Précis de bactériologie clinique, ESKA, Paris, France
Gordon, R. E., Haynes, W. C., Pang, C. H. N The genus Bacillus. In Agriculture handbook no. 427, pp U.S. Department of Agriculture. Washington, D.C.
Logan, N. A., Forsyth, G., Lebbe, L., Goris, L., Heyndrickx, M., Balcaen, A., Verhelst, A., Falsen, E., Ljungh, Å., Hansson, H. B., DeVos, P Polyphasic identification of Bacillus and Brevibacillus strains from clinical, dairy and industrial specimens and proposal of Brevibacillus invocatus sp. nov. Int. J. Syst. Evol. Microbiol. 52:953.

42. References
Logan, N. A., Turnbull, P. C. B Bacillus and recently derived genera. In Manual of Clinical Microbiology, 8th ed., pp Edited by P. R. Murray, E. J. Baron, M. A. Pfaller, J. H. Jorgensen & R. H. Yolken. American Society for Microbiology, Washington, DC.
Poyart C, Quesne G, Trieu-Cuot P. Taxonomic dissection of the Streptococcus bovis group by analysis of manganese-dependent superoxide dismutase gene (sodA) sequences: reclassification of Streptococcus infantarius subsp. coli as Streptococcus lutetiensis sp.nov. and of Streptococcus bovis biotype II.2 as Streptococcus pasteurianus sp nov. Int. J. Syst. Evol. Microbiol. 2002;52:
Schlegel L, Grimont F, Collins MD, Regnault B, Grimont PAD, Bouvet A. Streptococcus infantarius sp. nov., Streptococcus infantarius subsp infantarius subsp. nov. and Streptococcus infantarius subsp coli subsp. nov., isolated from humans and food. Int. J. Syst. Evol. Microbiol. 2000;50:
Vandamme P, Goris J, Coenye T, Hoste B, Janssens D, Kersters K, DeVos P, Falsen E. Assignment of Centers for Disease Control group IVc-2 to the genus Ralstonia as Ralstonia paucula sp.nov. Int. J. Syst. Bacteriol. 1999;49: