Standard Operating Procedure for VITEK 2 Compact: Use, Maintenance and Quality Control Procedures SOP Number: QC-22-03 Date Revised: 04-16-14


US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Mic ro bio lo gy La bo ra to ry
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
VITEK 2 Compact: Use, Maintenance and Quality
Control Procedures
SOP Number: QC-22-03
Date Revised: 04-16-14

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SOP No. QC-22-03
Date Revised 04-16-14
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SOP Number
QC-22-03
Title
VITEK 2 Compact: Use, Maintenance and Quality Control
Procedures
Scope
The purpose of this SOP is to describe the procedures for the
preparation and identification of test microorganisms (test microbes
and Quality Control Organisms) using the VITEK 2 Compact
Instrument.
Application
Proper use of the instrument is the responsibility of trained
laboratory personnel. The Quality Control process encompasses the
annual service and certification of the instrument by bioMerieux and
the Quality Control of each lot of Gram negative (GN), Gram
positive (GP), and Bacillus (BCL) cards using the organisms listed in
Attachment 1.


Approval Date
SOP Developer:

Print Name:
SOP Reviewer

Print Name:
Quality Assurance Unit

Print Name:
Branch Chief

Print Name:


Date SOP issued:

Controlled copy
number:

Date SOP withdrawn:

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SOP No. QC-22-03
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TABLE OF CONTENTS
Contents	Page Number
1.
DEFINITIONS
3
2.
HEALTH AND SAFETY
3
3.
PERSONNEL QUALIFICATIONS AND TRAINING
3
4.
INSTRUMENT CALIBRATION
3
5.
SAMPLE HANDLING AND STORAGE
4
6.
QUALITY CONTROL
4
7.
INTERFERENCES
4
8. NON- C ONF ORMIN G DATA
4
9.
DATA MANAGEMENT
4
10.
CAUTIONS
4
11.
SPECIAL APPARATUS AND MATERIALS
5
12.
PROCEDURE AND ANALYSIS
5
13.
DATA ANALYSIS/CALCULATIONS
10
14.
FORMS AND DATA SHEETS
10
15.
REFERENCES
10

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1. Definitions
Abbreviations/definitions are provided in the text.
2. Health and
Safety
Follow procedures specified in SOP MB-01, Laboratory Biosafety. The Study
Director and/or lead analyst should consult the Material Safety Data Sheet for
specific hazards associated with products.
3. Personnel
Qualifications
and Training
1.	Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
2.	Personnel are required to be knowledgeable of the procedures in this
SOP. The product information Manual, Software User Manual, Industry
customer Training Course Book and Instrument User Manual are
maintained near the instrument. All users of the VITEK 2 Compact
(V2C) must have hands on training in the use of the instrument and will
be required to successfully complete one competency test using one
VITEK card/organism. Documentation of training and familiarization
with this SOP can be found in the training file for each employee.
4. Instrument
Calibration
1.	Factory Calibrations:
a. Prior to shipment, the V2C Instrument met all acceptance test
procedures stipulated by bioMerieux. The Field Service Engineer
performed a verification of the VITEK factory calibration as a part of
the installation procedure of this instrument. This can be found in the
VITEK Certification Records Book.
2.	Internal monitoring of the VITEK reader/incubator module:
a.	The VITEK reader/incubator module houses the card handling and
scanning mechanism as well as the heater that maintains the cards at
the required incubation temperature. The trays that hold the cards are
mounted to a carousel that rotates once every 15 minutes to position
the cards for data scanning and identification. A thermistor is located
in the center of the carousel shaft and positioned to monitor any
change of temperature in the carousel stack. A heater and fan on top
of the carousel maintains the temperature at an average temperature of
35.5°C.
b.	The incubation temperature is automatically verified during the
initiation of the VITEK instrument and computer. Temperature
deviations of ±2°C generate error messages at the data terminal
module such as, "Reader Temperature High" or "Reader Temperature
Low" The process cycle is aborted if the temperature varies ±5°C from
the set temperature for more than one hour.
c.	DensiChek instrument verification results are digital and should be
within the established range of standards used for the verification.
Three standards (McFarland 0.5, 2.0, and 3.0) and one blank
(McFarland 0.0) are used for the instrument verification. If results fall
outside of the prescribed range, repeat the instrument verification

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steps. If the McFarland value is still outside the acceptable range,
discontinue use of the DensiChek and contact bioMerieux.
Instructions on how to conduct the DensiCheck instrument
verifications are included in the V2C - Industry Customer Training
Course Book (see section 15).
3. Conduct daily and monthly maintenance of the V2C system as described in
the user's manual (see section 15).
5. Sample
Handling and
Storage
Not Applicable
6. Quality Control
1.	For quality control purposes, the required information is documented on the
appropriate form(s) (see section 14).
2.	There are two options for conducting the quality control procedure for the
instrument and test cards. MLB currently conducts the streamlined quality
control test procedure. The comprehensive QC exercise is not necessary.
Detailed information about the quality control procedure is found in the
Product Information Manual (see section 15)
7. Interferences
1.	Improper subculturing and filling of VITEK cards may result in
inconsistent or erroneous biopatterns.
2.	The instrument will not operate when the V2C instrument flashes an Error
Message Queue. Each error message must be reviewed by opening each
message using the down arrows on the instrument key board followed by
the exclamation point. If the Error Message Queue does not clear after
this procedure, shut the machine down for 2 minutes and reboot the
instrument. If this fails, call bioMerieux technical services at 1-800-634-
7656 option 3. For detailed information on how to manage error
messages, refer to the Instrument User Manual (see section 15).
8. Non-
conforming
Data
1. Management of non-conforming data will be consistent with SOP ADM-
07, Non-Conformance Reports.
9. Data
Management
1.	Data is automatically recorded and generated by the computer in the form
of a printout. The printout for QC organisms will be filed in the VITEK
Quality Control Record Book and the printout for any other test organism
will be filed in the archive room with the raw data sheets. Information on
Quality Control will be maintained in the VITEK Quality Control Record
Book.
2.	Data will be archived consistent with SOP ADM-03, Records and
Archives.
10. Cautions
1. Biohazardous spills can occur inside the V2C instrument. All organism

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suspensions, cards, cassettes, test tubes, sample transfer tubes, waste bin
and the user interface panel should be considered as potentially infectious.
Use an EPA registered hospital disinfectant to clean any spill that occurs
in the V2C instrument. Use disinfectant according to label instructions.
2.	Use non powdered gloves when handling the cassette with live organisms.
3.	Remove the four internal carousels and clean each section on a regular
basis. See the Instrument User Manual for directions on cleaning the
carousel.
4.	Ensure suspensions are within the appropriate range on the Vitek 2
DensiChek, to avoid compromising the card performance and subsequent
readings.
5.	Use a minimum of 3 mL of sterile saline to fill the cards. To ensure this,
the dispensette has been preset to dispense 4 mL of sterile saline into the
suspension tube.
6.	Ensure all data is saved prior to logging out or continuing to prepare
additional isolates. Any unsaved data will not be recovered when the
inactivity time limit (set at 60 minutes) has been exceeded.
7.	Do not use glass tubes with the DensiChek as it may result in an
erroneous McFarland Reading.
8.	Do not deface the bar codes during handling of the test cards.
9.	Always allow the cards to come to room temperature prior to use.
10.	Do not use the cards after the expiration date or if the inner package has
been compromised or if desiccant is not present.
11. Special
Apparatus and
Materials
1.	For QC organisms use REMEL's ready-to-use disposable Culti-Loops or
ATCC lyophilized ampoules. See Attachment 1.
2.	Sterile Inoculating Loops
3.	Supplemental Media: NA, TSB, TSA with 5% Sheep Blood, and TSA.
4.	VITEK 2 Compact Identification cards (GP, BCL, and GN): store at 2-
8°C in unopened original liner.
5.	75 mm x 12 mm clear polystyrene tubes (single use only)
6.	DensiChek Meter with McFarland Standards for calibration (see section
15).
7.	Sterile saline solution (aqueous 0.45% to 0.50% NaCl, pH 4.5-7.0)
8.	Bar-coded 10 well cassette card holders
9.	Internal Carousel for card processing
12. Procedure and
Follow the instructions below for the proper use and required quality control

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SOP No. QC-22-03
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Analysis
activities associated with VITEK 2 Compact.
12.1 Initiation of
QC Organisms
(GN, GP and
BCL)
a.	Re-hydrate according to the manufacturer's instructions (Remel or
ATCC).
b.	Perform streak isolation of re-hydrated culture of each organism onto
appropriate agar plates to check for purity and long term storage, (see
Attachment 1).
c.	Incubate the plate(s) at the appropriate temperature listed in Attachment 1.
d.	Look for purity of culture. If the culture looks pure, proceed with section
12.1 e.
e.	Prepare isolates for long term storage as described in section 12.2.
12.2 Long Term
Storage ofQC
organisms
(GN, GP and
BCL)
a.	Inoculate 3 of the appropriate plates with multiple isolated colonies of
pure culture (using a swab to cover the entire surface).
b.	Incubate plates at appropriate temperature and observe for heavy growth.
c.	Collect the heavy growth from plate using a swab and place into 1.5 ml of
TSB+15% glycerol. Inoculum from one plate can be distributed into 2
cryovials.
d.	Freeze the cryovials at - 80± 5 °C.
12.3 Storage of test
microbes or
research
organisms
a. Store test or research organisms at 2-8°C in their original tubes until
results of a positive identification is received from the V2C instrument.
12.4 Initiation of the
V2C System
for Test
Microbes and
QC Organisms
a.	The V2C Instrument is always "on"; the instrument will say "Ready" or
"Not Ready" on the digital screen. Once the computer is initialized, the
instrument will say "Ready." The V2C will not run if it is not on ready
mode.
b.	Select VITEK 2 Compact to initiate the system from the upper left side of
the screen. After the system is initiated, log onto the system using the
appropriate user name and password. The system is now initialized and
ready for data entry.
12.5 Preparation of
Test Microbes
and QC
Organisms
a.	For QC organisms: remove the 0.5 mL cryovials from the -80°C freezer.
Avoid repeated thawing and freezing of the frozen culture by aseptically
removing a small portion (or looplul) of the frozen inoculum, then
immediately return cryovials to -80 °C freezer. See Section 12.2 for long
term storage procedures for QC organisms.
b.	Streak isolate the inoculum onto agar plate appropriate for the QC
organism (see Attachment 1).

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c.	For QC organisms, a second streak isolation on the appropriate media is
recommended. For product tests, a second streak isolation step is not
required unless there is evidence of a mixed culture.
d.	For "unknowns" or Test Microbes: use growth on tubes or plates from
product or research test. See Section 12.2 for storage of test microbes or
research organisms.
e.	Streak isolate the inoculum from a product test on TSA warmed to room
temperature. See section 12.6 f for incubation time.
12.6 Preparing
Streak
Isolation of
Test Microbes
or QC
Organisms.
a.	Record information on the V2C Test Microbe Transfer and Confirmation
Sheet for Quality Control Organisms or the appropriate Test Microbe
Confirmation Sheet, see section 14.
b.	According to the Software User Manual for BCL and GN, the organism to
be identified must be a pure culture 18 to 24 hours old. For GP the
organism to be identified must be a pure culture 12 to 48 hours old. Use,
TSA with 5% sheep blood, orNAto prepare isolates.
c.	Record information on the V2C Test Microbe Transfer and Confirmation
Sheet for Quality Control Organisms or Unknowns (see section 14).
12.7 Performing
Gram Stain
a. Perform Gram reaction using an isolated colony from a pure culture plate
of 18 to 24 hours old and document the Gram stain reaction.
12.8 Preparation of
V2C
suspensions of
Test Microbes
and QC
Organisms
a. Using sterile cotton swabs, prepare a homogenous organism suspension
by transferring several isolated colonies from the plates to 4 mL of sterile
saline. Adjust the suspension to the McFarland standard required by the
ID reagent using a calibrated V2C DensiChek Meter (e.g., 0.5-0.63 for
GN and GP and 1.8-2.2 for BCL). Place the prepared suspensions in the
cassette (see section 15, Instrument User Manual).
NOTE: If the instrument flashes 0.00 or 4.00, the suspension is either below
0.00 McFarland or above 4.0 McFarland and is not within the reading range.
Ensure suspensions are within the appropriate reading range to avoid
compromised card results. If necessary, re-calibrate the DensiChek
instrument after processing each cassette (see section 15, Industry Customer
Training Course Manual).
12.9 Selection and
Inoculation of
the V2C Card
a.	Select the appropriate card based on the Gram stain reaction and the
organism's microscopic appearance.
b.	Allow the card(s) to come to room temperature before opening the
package liner.
c.	Insert the straw from the appropriate V2C card with the inoculated
suspension tubes in the cassette. Process to data entry.

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NOTE: The age of the suspension must not exceed 30 minutes before
inoculating the cards.
12.10 Data Entry
into the
VITEK
system
a.	V2C is icon driven. When the system is initialized, an icon screen will
appear with two rows of icons. To enter the test microbe/QC organisms
information in the application screen, double click the first icon on the left
side, middle screen (the Manage Cassette View icon, which looks like the
cassettes used to load the cards).
b.	Click on Enter Mange Cassette View form the Main Menu.
c.	Click on Maintain Virtual Cassette icon in the left view bar of the Setup
Test Post Entry Window.
d.	Click on Create New Virtual Cassette icon in the upper right view bar also
called the Action Bar. The Maintain Virtual Cassette window appears.
The Virtual Cassette stores the data scanned into the computer.
e.	Enter the cassette information. You may either scan the cassette barcode
or choose the number from the drop down window labeled cassette.
f Enter the card data by scanning the car code on the card. The Cursor
must be in the Bar Code space to be entered. You may either hit ENTER
and the cursor will move to the next line to be scanned or use the mouse
button to move the cursor to the next Bar Code space.
NOTE: Instructions on data entry and management can be found in the
Software User manual (see section 15).
12.11 Define Isolate
Group
Information in
the Accession
space
a.	For QC organisms the Accession number is the ATCC number. Checking
the QC box will mark the card as a QC organism and the data for this card
will be stored in a separate database.
b.	For the product test organisms the Accession number will be the test
coordinator's initials, the test date followed by an alpha-numeric sequence
and the tube number. The alpha numeric sequence will give the
abbreviation of the test organism (Sa for Staphylococcus aureus, Pa for
Pseudomonas aeruginosa, Bs for Bacillus subtilis, Uk for an unknown).
For example if your test date is 04/21/14, the test coordinator is Jane Doe,
the test organism is Pa and the tube number is 43/2, the accession number
will be: JD042114Pa43/2-l. With the accession number, the computer
automatically places a -1 at the end of the every accession number. This
number cannot be removed.
c.	Save the information. The save icon is in the upper right hand corner.
Make sure you save your data prior to logging out or continuing to
prepare additional isolates. Any unsaved data will not be recovered when
the inactivity time limit (60 minutes) has been exceeded.
d.	If you are logged on the system and you exceed the inactivity time limit,

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the application will automatically ask you to log in again when you
attempt to use the application. If the inactivity time limit logs you out of
the computer while you are entering data, you must reenter your data after
you log back into the system.
NOTE: Instructions on data entry and management can be found in the
Software User manual (see section 15).
12.12 Filling the
Cards
a.	Place the cassette in the Filler box on the left side of the V2C unit and hit
Start Fill button on the instrument. Filling the cards takes approximately
70 seconds for a cassette regardless of the number of cards in the cassette
holder. The V2C instrument will beep when the filling cycle is complete.
[Discard individual cards that may have been exposed to multiple fill cycles as
the test results will be inaccurate.]
NOTE: The V2C must be loaded within 10 minutes from the end of filling
the cards to the start of loading the cards to avoid the cards from being
rejected.
b.	When the cards are finished filling, the Load Door is automatically
unlocked. Place the cassette in the Load Door. The V2C Instrument will
verify the scanned barcodes against the Virtual Cassette (the information
scanned in by the analyst). Cards are sealed, straws are cut and the cards
are loaded automatically into the carousel. The V2C will beep once all
cards are loaded into the cassette.
c.	When the cards are loaded, remove the cassette and dispose of the tubes
and straws in a biohazard container.
d.	When the cards are loaded, remove the cassette and dispose of the tubes
and straws in a biohazard container.
e.	The V2C automatically proceeds to processing the cards once all the cards
are loaded.
NOTE: Review the Navigation Tree. If the cassette status description in the
Navigation Tree is red, the cassette needs more information to completely
process the tests cards. Open up the red colored file and make sure all fields
are defined. Red text may be an indication of an accession number not
defined, a missing card, an extra card, or a wrong card.
f When the cards are finished and results obtained, cards will be
automatically ejected into the waste bin.
NOTE: Instructions on data entry and management can be found in the
Software User manual. Instructions on how to fill cards can be found in
the Instrument User Manual (see section 15).
12.13 Results
a. Results are concurrently printed and the data sent to the Results View
folder on the left side of the screen also called the Navigation Tree where

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the information is archived. If an error occurs during processing, refer to
the Software User Manual (see section 15).
b. Review results printout and file with the appropriate notebooks.
13 Data Analysis/
Calculations
1.	The VITEK system analyses the data results and determines the identity
of the test microbes/QC organism based on colorimetric tests
(biochemical reactions).
2.	Certain species may belong to a mixed (viewed as slashline) taxa
identification. This occurs when the biopattern is the same for the taxa
listed. Supplemental tests may be used to separate slashline taxa. Refer
to the Software User Manual for information on slashline taxa
differentiation for supplemental reaction files recommendations (see
section 15).
14 Forms and
Data Sheets
Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
VITEK Compact: Microbe Transfer and
Confirmation Sheet for Quality Control QC-22-06 Fl.docx
Organisms/Unknowns
15 References
1.	bioMerieux VITEK, Inc. 10/2010. Vitek 2 - technology Software User
Manual. Reference No.: 411075 /LotNo.: 2012082.
2.	bioMerieux VITEK, Inc. 09/2010. Vitek 2 - technology Instrument
User's Manual. Reference No.: 410860/LotNo.: 2012082.
3.	bioMerieux VITEK, Inc. 2009. Correspondence implementing the
Streamlined and Comprehensive QC testing recommendations for the
Vitek 2 ID cards.
4.	bioMerieux VITEK, Inc. 10/2010. Vitek 2 - technology Product
Information Manual. Reference No.: 411074 / Lot No.: 2012082.
5.	bioMerieux VITEK, Inc. 2011. Industry customer training course
Manual. Part Number: 60-00728-0.

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SOP No. QC-22-03
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Attachment 1
Attachment 1.
Streamlined Quality Control Organisms for VITEK 2 COMPACT Automated Identification
System	
Organism
ATCC
Number
Card Type
Media and
Incubation
Conditions
Identification
Number
Enterococcus casseliflavus
700327
GP
TSA or TSA with 5%
sheep blood/36±l°C
V2C-03
Streptococcus thermophilus
19258
GP
TSA with 5% sheep blood/
36±1°C + 5% C02
V2C-09
Stenotrophomonas maltophilia
17666
GN
TSA or TSA with 5%
sheep blood/36±l°C
V2C-12
Enterobacter
cloacae/hormaechei
700323
GN
TSA or TSA with 5%
sheep blood/36±l°C
V2C-15
Brevibacillus agri
51663
BCL
TSA orNA/36±l°C
V2C-20

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