IAST - Instructions for Use (2)

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Instructions for Use - 43-03084 - BacT/ALERT i AST - English - 259786 of 6

BacT/ALERT® i AST43-03084

DENOTES REVISED SECTION

INTENDED USEBacT/ALERT® i AST culture bottles are used with the BacT/ALERT Microbial Detection Systems inqualitative procedures for enhanced recovery and detection of a variety of aerobic and facultativemicroorganisms (bacteria and fungi). The laboratory is responsible for validating the BacT/ALERT systemand culture bottles for their testing purposes.

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

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

REAGENTSCAUTION: Handle samples and inoculated culture bottles as though capable of transmittinginfectious agents. All inoculated bottles should be decontaminated according to your institution’s

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 componentconsists of pancreatic digest of casein (1.7% w/v), papaic digest of soybean meal (0.3% w/v), sodiumpolyanetholesulfonate (SPS) (0.035% w/v), pyridoxine HCl (0.001% w/v), and other complex amino acidand carbohydrate substrates in purified water. Bottles contain an atmosphere of CO2 in oxygen undervacuum, and have been fitted with removable aluminum crimp seals to facilitate the addition of solidsamples. The composition of the media may be adjusted to meet specific performance requirements.

CAUTION: BacT/ALERT culture bottles contain polycarbonate. Not all disinfectants are intendedfor use with polycarbonate surfaces, and may cause bottle deterioration. Verify disinfectantcompatibility w ith polycarbonate to the product labeling of the disinfectant before use onBacT/ALERT culture bottle surfaces.

CAUTION: It is possible that certain rare, fastidious microorganisms will not grow or may growslowly in the BacT/ALERT i AST culture bottle growth medium. If rare, fastidious organismsrequiring specialized media and cu lture condi tions are suspected, alternative methods forrecovery 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 thegrowth o f fastidious aerobic and facultative microorganisms.

CAUTION: On rare occasions organisms may be encountered that grow in the BacT/ALERT i ASTculture bottle growth media but do not produce sufficient CO2 to be determined posit ive. Anexample that may cause this s ituation is the presence of antimicrobials in a sample.




Addi tional mater ials requiredBacT/ALERT® Microbial Detection SystemsSterile Airway Needle/Subculture UnitsDisposable glovesAppropriate biohazard waste containers for materials potentially contaminated with infectious agents

Materials available from bioMérieuxBacT/ALERT® Microbial Detection Systems

BacT/ALERT®

ResealSterile Airway Needle/Subculture Units

Storage instructionsBacT/ALERT i AST culture bottles are ready for use. Store protected from direct sunlight at roomtemperature (15-30°C). An expiration date is printed on each bottle label. Do not use the culture bottlesbeyond the last day of the month indicated. If the bottles are exposed to temperatures less than 15°C,precipitates may form that will disappear when the bottles are warmed to room temperature. Bottles mustbe at room temperature before use.

Chemical or physical indications of instabilityPrior to use, the BacT/ALERT i AST culture bottles should be examined for evidence of damage ordeterioration (discoloration). Bottles exhibiting evidence of damage, leakage or deterioration should bediscarded. The media in undisturbed bottles should be clear, but there may be a slight opalescence or atrace of precipitate due to the anticoagulant SPS. Do not confuse opalescence with turbidity. Discard anybottles with turbidity, excess gas pressure, yellow sensors and/or evidence of growth; these are signs ofpossible 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 precautions1. 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 incontact 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% sodiumhypochlorite. Dispose of the cleaning material by an acceptable method.

3. All inoculated culture bottles and sample collection devices should be decontaminated and discardedaccording to your company’s procedures.

4. For best overall recovery when culturing samples that may contain aerobic and anaerobicmicroorganisms, it is strongly recommended that more than one atmospheric type of culture bottlebe utilized (e.g., one aerobic and one anaerobic).

5. These bottles should be utilized by trained laboratory personnel.

SAMPLE COLLECTION AND VALIDATION1. 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 theBacT/ALERT i AST culture bottle, to be free of bacteriostatic and fungistatic activity, per United StatesPharmacopoeia (USP) or European Pharmacopoeia (EP) procedures.3,4,5,6

2. Samples for testing must be collected aseptically and maintained under sterile conditions prior totesting.

BOTTLE PREPARATION1. 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 evidenceof growth; these are signs of possible contamination.

2. Label each BacT/ALERT i AST culture bottle with the appropriate sample information. The icons onthe bottle label (#,) can be defined by the user. The bottle must be at room temperature.

3. Disinfect septum with an alcohol pad or equivalent.




BOTTLE INOCULATION1. 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 beremoved as follows:

Lift the center tab and pull up ini tially, then across and down over the upper rim of the

seal, in the direction o f the user. Carefully continue to pu ll down until the left edge of thetab is completely detached from the seal. Then, holding the tab horizontal to the rubberseptum, pull the tab in a counter-clockwise direction to remove the seal completely fromthe bott le.

CAUTION: Use care when removing the metal cr imp seal. If the pull -tab breaks free from theseal resulting in sharp edges, do not attempt to remove the seal by hand. Instead, the use of ahemostat or o ther mechanical device is recommended.

3. Remove the metal seal and liner. Retain the liner for reuse. Aseptically remove the septum and addthe 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 areseal (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 INCUBATION1. 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 intothe 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 torelease any gas produced during microbial metabolism.

4. Positive bottles should be subcultured and smeared to confirm the positive signal. Subculture may beperformed with a needle and syringe, or by aseptically removing the reseal and septum. Replace theseptum 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 toinoculate samples.

QUALITY CONTROLA Certificate of Conformance is available for each lot of culture bottles. If desired, individual laboratoriescan perform quality control testing of BacT/ALERT i AST culture bottles. Refer to the BacT/ALERTSystems User Manual, CLSI® document M22-A37 and USP <71>3, EP 2.6.15, or EP 2.6.276.

InstrumentA BacT/ALERT Reflectance Standards kit is provided with each instrument for the QC and Calibrationprocedures. All quality control should be part of normal system maintenance. Refer to the BacT/ALERTSystems User Manual for more information.

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




LIMITATIONS OF THE TEST1. 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 ofmedical products, including human biological substances, should first consult their appropriateregulatory agency for requirements.

2. If inoculated culture bottles have not been loaded promptly into the BacT/ALERT instrument or havebeen 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 confirmby 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 subculturedon special media. Also, BacT/ALERT positive samples may contain organisms that are not seen withroutine smear methods and may require both specialized smears and subculturing media fordetection and recovery.

5. Certain strains of Haemophilus influenzae, Neisseria meningitides, and Neisseria gonorrhoeae maybe sensitive to SPS, which may result in lack of growth or low production of CO 2 by these strains.8

6. Certain species of Penicillium, Aspergillus, or other temperature sensitive molds may not grow, ormay 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-viableorganisms that were derived from culture medium components, staining reagents, immersion oil, orglass 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 orto cure, mitigate, treat or prevent disease in a patient.

PERFORMANCE CHARACTERISTICS OF THE TESTSeeded 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 tubesat 32.5°C. The USP/EP tubes were inspected daily for visible growth.

TABLE A

Detection Times (days)BacT/ALERT USP/EP GrowthInoculum Incubation i AST Cul ture Promotion

Microorganism (CFU/bottle) Temperature Bottlea Test

b

S. aureus ATCC® 6538 9 32.5°C 1.14 1,1E. coli ATCC 8739 14 32.5°C 0.59 1,1

a Average of 5/5 positive bottlesb 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 testprocedures and/or FDA 21 CFR 610.12 Growth Promotion Test procedures (Table B). Organisms werediluted in 1.0ml of diluent and seeded at levels of <100 CFU/bottle and tube. At least 40 replicates of eachorganism were tested in BacT/ALERT bottles incubated at 32.5°C and 22.5°C. Data was generated using

a single BacT/ALERT 3D Dual-T system and multiple lots of BacT/ALERT bottles. Replicate broth tubeswere tested in conjunction with the BacT/ALERT bottles and incubated at 32.5°C and/or 22.5°C asindicated in the references. The USP/EP broth tubes were inspected daily for visible growth. The numberof days indicated for the USP/EP Growth Promotion Test column reflects the average time-to-detection(TTD) for all replicates tested.




TABLE BDetection Times (days)

Inoculum BacT/ALERT USP/EP Growth(CFU/bottle Incubation cultu re bottl e Promotion Test

Microorganism or tube) Temperature Mean TTD Mean TTD

A. brasiliensis ATCC 16404 35 32.5°C 2.23 N/A35 22.5°C 4.16 3

C. albicans ATCC 10231 25 32.5°C 1.11 13a

25 22.5°C 2.01 3B. subtilis ATCC 6633 7 32.5°C 0.76 1

7 22.5°C 1.84 2P. aeruginosa ATCC 9027 36 32.5°C 0.93 1

36 22.5°C 1.89 N/AK. rhizophila ATCC 9341 22 32.5°C 1.40 > 14b

22 22.5°C 3.16 4c a Only 1 of 5 replicate tubes was positive in 13 days for C. albicansb Zero of 5 replicate tubes were positive in 14 days for K. rhizophilac As recommended in 21 CFR 610.12, Sterility TestN/A – Not applicable

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

DISCLAIMERbioMérieux specifically disclaims all warranties, express or implied, of the suitability of the instrument anddisposables (the “System”) and disclaims all liability, whether direct, indirect or consequential, for any useother than as set forth in the Instructions for Use (IFU). Customer acknowledges and agrees that use ofthe System for testing of sample types or for indications other than those described in the IFU is donesolely at the Customer’s own risk. Customer acknowledges and agrees that it is Customer’s sole andexclusive responsibility to validate the System for any such intended use, and to determine whether theSystem is suitable for that intended use. The performance of any validation studies and the subsequentuse of the System based on Customer’s validation studies shall be the Customer’s sole risk andresponsibility.

REFERENCES1. 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 MicrobialRecovery 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; ApprovedStandard—Third Edition. CLSI/NCCLS document M22-A3. Wayne, PA: NCCLS; 2004.

8. Carrol CC, Weinstein, MP. Manual and Automated Systems for Detection and Identification ofMicroorganisms. In: Murray PR, Baron EJ, Jorgensen JH, Landry, ML, Pfaller, MA (eds.): Manual ofClinical Microbiology, ed. 9. Washington, D.C., American Society for Microbiology, 2007, pp. 193.

9. Rare Organism Club, bioMérieux, Inc., 2007.




AVAILABILITYbioMérieux

BacT/ALERT® i AST

100/case 259786

BacT/ALERT® Reseal

100/bag 259787Sterile Airway Needle/Subculture Units

100/box 233766

For technical assistance in the USA, contact bioMérieux Customer Service at 1-800-634-7656. Outsidethe USA, contact your local bioMérieux Representative.

bioMérieux, the blue logo, and BacT/ALERT are used, pending and/or registered trademarks belonging tobioMérieux 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.

bioMérieux, Inc.Box 15969Durham, North Carolina 27704-0969 www.biomerieux.com

©BIOMÉRIEUX 2001, 2003, 2005, 2008, 2010 February 2010

IAST - Instructions for Use (2)

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