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BacT/ALERT i AST

43-03084

DENOTES REVISED SECTION


INTENDED USE

BacT/ALERT i AST culture bottles are used with the BacT/ALERT Microbial Detection Systems in
qualitative procedures for enhanced recovery and detection of a variety of aerobic and facultative
microorganisms (bacteria and fungi). The laboratory is responsible for validating the BacT/ALERT system
and culture bottles for their testing purposes.

SUMMARY AND EXPLANATION


The BacT/ALERT Microbial Detection Systems and BacT/ALERT i AST culture bottles provide both a
microbial detection system and a culture medium with suitable nutritional and environmental conditions
for the growth and detection of a variety of aerobic and facultative microorganisms that may be present in
processed or aseptically prepared products. Inoculated bottles are placed into the instrument where they
are incubated and continuously monitored for the presence of microorganisms that will grow in the
BacT/ALERT i AST culture bottle.

PRINCIPLE OF THE TEST


The BacT/ALERT Microbial Detection Systems utilizes a colorimetric sensor and reflected light to monitor
the presence and production of carbon dioxide (CO2) dissolved in the culture medium. If microorganisms
are present in the test sample, CO2 is produced as the organisms metabolize the substrates in the culture
medium. When growth of the microorganisms produces CO2, the color of the gas-permeable sensor
present in the bottom of each culture bottle changes from blue-green to yellow.1 The lighter color results
in an increase of reflectance units monitored by the system. Bottle reflectance is monitored and recorded
by the instrument every 10 minutes.

REAGENTS
CAUTION: Handle samples and inoculated culture bottles as though capable of transmitting
infectious agents. All inoculated bottles should be decontaminated according to your institutions
procedures.2
BacT/ALERT i AST (color-coded blue) BacT/ALERT i AST disposable culture bottles contain 40ml of
media and an internal sensor that detects CO2 as an indicator of microbial growth. The media component
consists of pancreatic digest of casein (1.7% w/v), papaic digest of soybean meal (0.3% w/v), sodium
polyanetholesulfonate (SPS) (0.035% w/v), pyridoxine HCl (0.001% w/v), and other complex amino acid
and carbohydrate substrates in purified water. Bottles contain an atmosphere of CO2 in oxygen under
vacuum, and have been fitted with removable aluminum crimp seals to facilitate the addition of solid
samples. The composition of the media may be adjusted to meet specific performance requirements.
CAUTION: BacT/ALERT culture bottles contain polycarbonate. Not all disinfectants are intended
for use with polycarbonate surfaces, and may cause bottle deterioration. Verify disinfectant
compatibility with polycarbonate to the product labeling of the disinfectant before use on
BacT/ALERT culture bottle surfaces.
CAUTION: It is possible that certain rare, fastidious microorganisms will not grow or may grow
slowly in the BacT/ALERT i AST culture bottle growth medium. If rare, fastidious organisms
requiring specialized media and culture conditions are suspected, alternative methods for
recovery or supplements to the BacT/ALERT i AST culture bottle should be considered.
CAUTION: BacT/ALERT i AST culture bottles may require additional supplements to enhance the
growth of fastidious aerobic and facultative microorganisms.
CAUTION: On rare occasions organisms may be encountered that grow in the BacT/ALERT i AST
culture bottle growth media but do not produce sufficient CO2 to be determined positive. An
example that may cause this situation is the presence of antimicrobials in a sample.
Instructions for Use - 43-03084 - BacT/ALERT i AST - English - 259786

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Additional materials required


BacT/ALERT Microbial Detection Systems
Sterile Airway Needle/Subculture Units
Disposable gloves
Appropriate biohazard waste containers for materials potentially contaminated with infectious agents
Materials available from bioMrieux
BacT/ALERT Microbial Detection Systems
BacT/ALERT Reseal
Sterile Airway Needle/Subculture Units
Storage instructions
BacT/ALERT i AST culture bottles are ready for use. Store protected from direct sunlight at room
temperature (15-30C). An expiration date is printed on each bottle label. Do not use the culture bottles
beyond the last day of the month indicated. If the bottles are exposed to temperatures less than 15C,
precipitates may form that will disappear when the bottles are warmed to room temperature. Bottles must
be at room temperature before use.
Chemical or physical indications of instability
Prior to use, the BacT/ALERT i AST culture bottles should be examined for evidence of damage or
deterioration (discoloration). Bottles exhibiting evidence of damage, leakage or deterioration should be
discarded. The media in undisturbed bottles should be clear, but there may be a slight opalescence or a
trace of precipitate due to the anticoagulant SPS. Do not confuse opalescence with turbidity. Discard any
bottles with turbidity, excess gas pressure, yellow sensors and/or evidence of growth; these are signs of
possible contamination.

INSTRUMENTS
Review the appropriate BacT/ALERT Microbial Detection Systems User Manual before use.

BACT/ALERT i AST CULTURE BOTTLE TEST PROCEDURE


Preliminary comments and precautions
1. Use disposable gloves and handle inoculated bottles cautiously as though capable of transmitting
infectious agents. Consult a physician immediately if contaminated materials are ingested or come in
contact with open lacerations, lesions, or other breaks in skin.
2. Immediately clean up any spillage of contaminated material using a 1:10 dilution of 5% sodium
hypochlorite. Dispose of the cleaning material by an acceptable method.
3. All inoculated culture bottles and sample collection devices should be decontaminated and discarded
according to your companys procedures.
4. For best overall recovery when culturing samples that may contain aerobic and anaerobic
microorganisms, it is strongly recommended that more than one atmospheric type of culture bottle
be utilized (e.g., one aerobic and one anaerobic).
5. These bottles should be utilized by trained laboratory personnel.

SAMPLE COLLECTION AND VALIDATION


1. Samples for testing may contain interfering levels of antimicrobial compounds, resulting in false
negatives. Prior to routine use, the user should validate each sample type and volume in the
BacT/ALERT i AST culture bottle, to be free of bacteriostatic and fungistatic activity, per United States
Pharmacopoeia (USP) or European Pharmacopoeia (EP) procedures.3,4,5,6
2. Samples for testing must be collected aseptically and maintained under sterile conditions prior to
testing.

BOTTLE PREPARATION
1. Visually inspect bottles before testing. Do not use bottles with evidence of damage, leakage, or
deterioration. Discard any bottles with turbidity, excess gas pressure, yellow sensors and/or evidence
of growth; these are signs of possible contamination.
2. Label each BacT/ALERT i AST culture bottle with the appropriate sample information. The icons on
the bottle label (#,) can be defined by the user. The bottle must be at room temperature.
3. Disinfect septum with an alcohol pad or equivalent.
Instructions for Use - 43-03084 - BacT/ALERT i AST - English - 259786

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BOTTLE INOCULATION
1. A liquid sample may be inoculated directly into the BacT/ALERT i AST culture bottle via a needle and
syringe, or by removing the septum, as described below, and using a needleless inoculation device,
such as a pipette.
2. For testing of solids, filter membranes, homogenates, or viscous liquids, the seal and septum may be
removed as follows:
Lift the center tab and pull up initially, then across and down over the upper rim of the
seal, in the direction of the user. Carefully continue to pull down until the left edge of the
tab is completely detached from the seal. Then, holding the tab horizontal to the rubber
septum, pull the tab in a counter-clockwise direction to remove the seal completely from
the bottle.
CAUTION: Use care when removing the metal crimp seal. If the pull-tab breaks free from the
seal resulting in sharp edges, do not attempt to remove the seal by hand. Instead, the use of a
hemostat or other mechanical device is recommended.
3. Remove the metal seal and liner. Retain the liner for reuse. Aseptically remove the septum and add
the sample or product to be tested directly into the bottle.
4. Aseptically place the septum back into the bottle, ensuring that the septum is fully inserted. Place a
reseal (BacT/ALERT Reseal) over the septum and push down firmly.
CAUTION: The reseal must latch under the bottle rim, and should fit tightly on the bottle.

BOTTLE INCUBATION
1. To optimize detection times, bottles should be loaded into the BacT/ALERT Microbial Detection
Systems as soon as possible following inoculation. Procedures for loading and unloading bottles into
the BacT/ALERT instrument are given in the User Manual.
2. The length of time the bottles should remain in the instrument may be varied to suit individual needs.
3. Positive culture bottles should be transiently vented before subculturing, staining, or disposal to
release any gas produced during microbial metabolism.
4. Positive bottles should be subcultured and smeared to confirm the positive signal. Subculture may be
performed with a needle and syringe, or by aseptically removing the reseal and septum. Replace the
septum when finished and cover with a new reseal if the bottle is to be kept for further testing.
5. BacT/ALERT i AST culture bottles contain an aerobic atmosphere and do not require venting;
however, the performance of the bottles is not impaired by venting or removing the septum to
inoculate samples.

QUALITY CONTROL
A Certificate of Conformance is available for each lot of culture bottles. If desired, individual laboratories
can perform quality control testing of BacT/ALERT i AST culture bottles. Refer to the BacT/ALERT
Systems User Manual, CLSI document M22-A37 and USP <71>3, EP 2.6.15, or EP 2.6.276.
Instrument
A BacT/ALERT Reflectance Standards kit is provided with each instrument for the QC and Calibration
procedures. All quality control should be part of normal system maintenance. Refer to the BacT/ALERT
Systems User Manual for more information.

RESULTS
Positive or negative culture bottles are determined by decision-making software contained in the
BacT/ALERT Microbial Detection System. No action is required until the BacT/ALERT instrument signals
a culture bottle either positive or negative.

Instructions for Use - 43-03084 - BacT/ALERT i AST - English - 259786

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LIMITATIONS OF THE TEST


1. The laboratory is responsible for validating the BacT/ALERT system and culture bottles for their
testing purposes. Users considering the BacT/ALERT system and culture bottles for release of
medical products, including human biological substances, should first consult their appropriate
regulatory agency for requirements.
2. If inoculated culture bottles have not been loaded promptly into the BacT/ALERT instrument or have
been incubated prior to entry into the instrument, the bottles should be visually inspected for
indications of microbial growth. If microbial growth is evident, treat the bottles as positive and confirm
by subculture and smear.
3. To ensure viable cultures and avoid excessive build up of gas pressure, bottles should be promptly
removed from the instrument after being signaled positive.
4. Samples determined positive by BacT/ALERT may contain organisms that are positive by smear that
will not grow on routine subculturing media. When this is suspected, samples should be subcultured
on special media. Also, BacT/ALERT positive samples may contain organisms that are not seen with
routine smear methods and may require both specialized smears and subculturing media for
detection and recovery.
5. Certain strains of Haemophilus influenzae, Neisseria meningitides, and Neisseria gonorrhoeae may
be sensitive to SPS, which may result in lack of growth or low production of CO2 by these strains.8
6. Certain species of Penicillium, Aspergillus, or other temperature sensitive molds may not grow, or
may grow but do not produce sufficient CO2 to be determined positive.
7. A Gram-stained smear from a negative bottle may sometimes contain a small number of non-viable
organisms that were derived from culture medium components, staining reagents, immersion oil, or
glass slides. In these cases, the Gram stain may yield a false positive result.
8. This product is not for diagnostic use. Results of this test should not be used to diagnose disease or
to cure, mitigate, treat or prevent disease in a patient.

PERFORMANCE CHARACTERISTICS OF THE TEST


Seeded studies were performed comparing the BacT/ALERT and USP/EP sterility test procedures
(Table A).3,5,6 Organisms were diluted in 1.0ml of Tryptic Soy Broth (TSB) and seeded at levels of
<100 CFU/bottle. Each organism was tested in quintuplet plastic BacT/ALERT bottles and duplicate tubes
at 32.5C. The USP/EP tubes were inspected daily for visible growth.
TABLE A

Microorganism
S. aureus ATCC 6538
E. coli ATCC 8739
a
b

Inoculum
(CFU/bottle)
9
14

Incubation
Temperature
32.5C
32.5C

Detection Times (days)


BacT/ALERT
USP/EP Growth
i AST Culture
Promotion
a
Testb
Bottle
1.14
1,1
0.59
1,1

Average of 5/5 positive bottles


Day turbidity first detected in each tube; both measurements shown.

Seeded studies were performed comparing the BacT/ALERT 3D Dual-T system and USP/EP sterility test
procedures and/or FDA 21 CFR 610.12 Growth Promotion Test procedures (Table B). Organisms were
diluted in 1.0ml of diluent and seeded at levels of <100 CFU/bottle and tube. At least 40 replicates of each
organism were tested in BacT/ALERT bottles incubated at 32.5C and 22.5C. Data was generated using
a single BacT/ALERT 3D Dual-T system and multiple lots of BacT/ALERT bottles. Replicate broth tubes
were tested in conjunction with the BacT/ALERT bottles and incubated at 32.5C and/or 22.5C as
indicated in the references. The USP/EP broth tubes were inspected daily for visible growth. The number
of days indicated for the USP/EP Growth Promotion Test column reflects the average time-to-detection
(TTD) for all replicates tested.

Instructions for Use - 43-03084 - BacT/ALERT i AST - English - 259786

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TABLE B

Microorganism
A. brasiliensis ATCC 16404
C. albicans ATCC 10231
B. subtilis ATCC 6633
P. aeruginosa ATCC 9027
K. rhizophila ATCC 9341

Inoculum
(CFU/bottle
or tube)

Incubation
Temperature

35
35
25
25
7
7
36
36
22
22

32.5C
22.5C
32.5C
22.5C
32.5C
22.5C
32.5C
22.5C
32.5C
22.5C

Detection Times (days)


BacT/ALERT
USP/EP Growth
culture bottle
Promotion Test
Mean TTD
Mean TTD
2.23
4.16
1.11
2.01
0.76
1.84
0.93
1.89
1.40
3.16

N/A
3
a
13
3
1
2
1
N/A
b
> 14
c
4

Only 1 of 5 replicate tubes was positive in 13 days for C. albicans


Zero of 5 replicate tubes were positive in 14 days for K. rhizophila
As recommended in 21 CFR 610.12, Sterility Test
N/A Not applicable
b
c

NOTE: A list of rare and fastidious organisms recovered with BacT/ALERT culture bottles is available
upon request from bioMrieux.9

DISCLAIMER
bioMrieux specifically disclaims all warranties, express or implied, of the suitability of the instrument and
disposables (the System) and disclaims all liability, whether direct, indirect or consequential, for any use
other than as set forth in the Instructions for Use (IFU). Customer acknowledges and agrees that use of
the System for testing of sample types or for indications other than those described in the IFU is done
solely at the Customers own risk. Customer acknowledges and agrees that it is Customers sole and
exclusive responsibility to validate the System for any such intended use, and to determine whether the
System is suitable for that intended use. The performance of any validation studies and the subsequent
use of the System based on Customers validation studies shall be the Customers sole risk and
responsibility.

REFERENCES
1. Thorpe TC, Wilson ML, Turner JE, et al. BacT/ALERT: an Automated Colorimetric Microbial Detection
System. J Clin Micro 1990; 28 (7), 1608-1612.
2. Widmer AF, Frei, R: Decontamination, Disinfection, and Sterilization. In: Murray PR, Baron EJ,
Jorgensen JH, Landry, ML, Pfaller, MA (eds.): Manual of Clinical Microbiology, ed. 9. Washington,
D.C., American Society for Microbiology, 2007, pp. 65-96.
3. The United States Pharmacopoeia, 2009 edition, USP 30 - NF 25, <71>, Sterility Tests.
4. The United States Pharmacopoeia, 2009 edition, USP 32 NF 27, <1227> Validation of Microbial
Recovery from Pharmacopeial Articles.
5. European Pharmacopoeia, 2009 edition, Chapter 2.6.1., Sterility.
6. European Pharmacopoeia, 01/2009. Chapter 2.6.27. Microbiological Control of Cellular Products.
7. CLSI/NCCLS. Quality Control for Commercially Prepared Microbiological Culture Media; Approved
StandardThird Edition. CLSI/NCCLS document M22-A3. Wayne, PA: NCCLS; 2004.
8. Carrol CC, Weinstein, MP. Manual and Automated Systems for Detection and Identification of
Microorganisms. In: Murray PR, Baron EJ, Jorgensen JH, Landry, ML, Pfaller, MA (eds.): Manual of
Clinical Microbiology, ed. 9. Washington, D.C., American Society for Microbiology, 2007, pp. 193.
9. Rare Organism Club, bioMrieux, Inc., 2007.

Instructions for Use - 43-03084 - BacT/ALERT i AST - English - 259786

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AVAILABILITY
bioMrieux
BacT/ALERT i AST
259786

100/case

BacT/ALERT Reseal
100/bag

259787

Sterile Airway Needle/Subculture Units


100/box

233766

For technical assistance in the USA, contact bioMrieux Customer Service at 1-800-634-7656. Outside
the USA, contact your local bioMrieux Representative.
bioMrieux, the blue logo, and BacT/ALERT are used, pending and/or registered trademarks belonging to
bioMrieux SA or one of its subsidiaries.
ATCC is a registered trademark of the American Type Culture Collection.
CLSI is a registered trademark of Clinical Laboratory and Standards Institute.
bioMrieux, Inc.
Box 15969
Durham, North Carolina 27704-0969

BIOMRIEUX 2001, 2003, 2005, 2008, 2010

Instructions for Use - 43-03084 - BacT/ALERT i AST - English - 259786

www.biomerieux.com

February 2010

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