Documente Academic
Documente Profesional
Documente Cultură
43-03084
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.
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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INSTRUMENTS
Review the appropriate BacT/ALERT Microbial Detection Systems User Manual before use.
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.
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Microorganism
S. aureus ATCC 6538
E. coli ATCC 8739
a
b
Inoculum
(CFU/bottle)
9
14
Incubation
Temperature
32.5C
32.5C
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.
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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
N/A
3
a
13
3
1
2
1
N/A
b
> 14
c
4
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.
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AVAILABILITY
bioMrieux
BacT/ALERT i AST
259786
100/case
BacT/ALERT Reseal
100/bag
259787
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
www.biomerieux.com
February 2010
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