BACTEC MGIT 960 SEEDED CULTURE
Contents
1. For best results the addition of MGIT Growth Supplement MGIT PANTA antibiotic mixture should be made just prior to specimen inoculation d Add 0 5 mL of the digested decontaminated and concentrated specimen suspension Also add a drop 0 1 mL of specimen to a 7H10 agar plate or other mycobacterial solid agar or egg based medium e Tightly recap the tube and mix well f Tubes entered into the instrument will be automatically tested for the duration of the recommended 42 day testing protocol For specimens in which mycobacteria with different incubation requirements are suspected a duplicate MGIT tube can be set up and incubated at the appropriate temperature e g 30 or 42 C Inoculate and incubate at the required temperature These tubes must be manually read refer to the BACTEC MGIT 960 User s Manual For specimens suspected of containing Mycobacterium haemophilum a source of hemin must be introduced into the tube at the time of inoculation and the tube incubated at 30 C Aseptically place one strip of BBL Taxo X factor strip into each MGIT tube requiring the addition of hemin prior to inoculation of specimen see Availability These tubes must be manually read refer to the BACTEC MGIT 960 User s Manual g Positive tubes identified by the BACTEC MGIT 960 instrument should be subcultured and an acid fast smear prepared All quality control testing reprocessing smear preparation subcultur2. G P Kubica 1985 Public health mycobacteriology a guide for the level III laboratory USDHHS Centers for Disease Control Atlanta Bloodborne pathogens Code of Federal Regulations Title 29 Part 1910 1030 Isenberg Henry D ed 1992 Clinical microbiology procedures handbook vol 1 American Society for Microbiology Washington D C oe BACTEC BBL MGIT MycoPrep PANTA and TAXO are trademarks of Becton Dickinson and Company ATCC is a trademark of the American Type Culture Collection 7miclsi doc Page 10 of 11 Approved By Date Effective Supervisor Date Director Date Reviewed Rev 10 99 7miclsi doc Page 11 of 11
3. Open the appropriate drawer 2 Press the remove positive tubes soft key 7miclsi doc Page 7 of 11 All the positive stations illuminate with FLASHING GREEN FLASHING RED indicators Remove one of the positive tubes The barcode scanner turns on and the barcode icon appears in the main body of the display Scan the positive tube s barcode label The LEDs at this station extinguish Repeat Steps 4 5 to remove additional positive tubes The POSITIVE indicator on the front of the drawer and at the top of the instrument will not extinguish until all positive vials are removed When all positive tubes are removed the instrument beeps three times the barcode scanner turns off and the ok icon appears in the main body of the display NOTE All instrument positive tubes should be stained for AFB and subcultured upon removal from the instrument Tubes should remain at room temperature while they are out of the instrument c To Return Smear Negative Positive Tubes 1 2 If a presumptive positive tube is determined to be smear negative for either mycobacteria or contaminants the tube should be re entered into the instrument within five hours of its removal To return a smear negative positive tube to the instrument a Open the drawer b Press the tube entry soft key c Scan the tube barcode label d Place the tube in the indicated station which may differ from the original station e Previous data are retaine
4. decrease as measured by increasing fluorescence enables the BACTEC MGIT 960 to determine if the tube is instrument positive A positive determination indicates the presumptive presence of microorganisms in the tube lll SPECIMEN A TYPE BACTEC MGIT Mycobacteria Growth Indicator Tubes are to be used for culture of digested and decontaminated clinical specimens except urine and sterile body fluids except blood B PROCESSING 1 Sputum or other respiratory specimens must be digested decontaminated and concentrated prior to inoculation into BBL MGIT tubes The N acetyl L cysteine sodium hydroxide method is recommended Alternatively the BBL MycoPrep Kit may be used for processing the specimen 2 Processing of non respiratory specimens other than blood and urine should be performed according to the Clinical Microbiology Handbook or the CDC s Public Health Mycobacteriology A Guide for the Level III Laboratory C SPECIMEN LABELING 1 Each specimen should be labeled with the appropriate information Patient Name Hospital number Patient ID Patient s location room and bed Date and time of collection Site of specimen 2 Each request slip should also have all the above information IV QUALITY CONTROL A MEDIA Upon receipt of a new shipment or lot number of MGIT tubes it is suggested that suspensions of the ATCC control organisms shown in the table below be prepared in Middlebrook 7H9 Broth Specie
5. tubes are removed press the ok soft key To remove negatives one at a time remove the de sired tube and scan the barcode label Continue to scan and remove all the desired individual negative tubes When all negative tubes are removed the instrument beeps three times and the ok icon appears in the main body of the display 5 PROCESSING OF POSITIVE MGIT TUBE s NOTE All steps should be performed in a biological safety cabinet IV LIMITATIONS Remove the positive MGIT tube from the instrument and transport to an area using BSL III practices and containment facilities Using a sterile transfer pipette remove an aliquot from the bottom of the tube approx 0 1 mL for stain preparations AFB and Gram stains Inspect smear preparations Report preliminary results only after acid fast smear evaluation Follow the established procedure s for reporting results Recommended actions may be a If AFB positive subculture to solid media and report as instrument positive AFB positive ID pending Subculture and identify organisms according to your laboratory protocol b If microorganisms other than acid fast bacilli are present report as instrument positive AFB negative Contaminated Refer to the BACTEC MGIT 960 User s Manual Appendix E for tube decontamination procedure c If no microorganisms are present on the smear re enter the tube into the instrument as an ongoing negative and allow the vial to complete testing p
6. BACTEC MGIT 960 System PRINCIPLE From 1985 to 1992 the number of reported cases of tuberculosis increased 18 This infectious disease still kills an estimated 3 million persons a year worldwide making it the leading infectious disease cause of death Between 1981 and 1987 AIDS case surveillance indicated that 5 5 of patients with AIDS had disseminated nontuberculous mycobacterial infections e g Mycobacterium avium complex MAC By 1990 the increased number of disseminated nontuberculous mycobacterial infections resulted in a cumulative incidence of 7 6 In addition to the resurgence of Mycobacterium tuberculosis MTB multidrug resistant MTB MDR TB has become an increasing concern Laboratory delays in the growth identification and reporting of these MDR TB cases has contributed at least in part to the spread of the disease The US Centers for Disease Control and Prevention CDC have recommended that every effort must be made for laboratories to use the most rapid methods available for diagnostic mycobacteria testing These recommendations include the use of both a liquid and a solid medium for mycobacterial culture The BBL MGIT Mycobacteria Growth Indicator Tube contains 7 mL of modified Middlebrook 7H9 Broth base The complete medium with BACTEC MGIT 960 Growth Supplement and BBL MGIT PANTA Antimicrobic mixture is one of the most commonly used liquid medium for the cultivation of mycobacte
7. cobacteria Faster growing mycobacteria may be detected prior to slower growing mycobacteria therefore it is important to subculture positive MGIT tubes to ensure proper identification of all mycobacteria present in the sample Due to the richness of the BBL MGIT broth and to the non selective nature of the MGIT indicator it is important to follow the stated digestion decontamination procedure to reduce the possibility of contamination Adherence to procedural instructions which includes use of recommended inoculum volume 0 5 mL is critical for optimum recovery of mycobacteria The use of BBL MGIT PANTA antibiotic mixture although necessary for all non sterile specimens may have inhibitory effects on some mycobacteria V REFERENCES 1 Bloom B R and C J L Murray 1992 Tuberculosis commentary on a reemergent killer Science 257 1055 1064 2 Horsburg C R Jr 1991 Mycobacterium avium complex infection in the acquired immunodeficiency syndrome N Engl J Med 324 1332 1338 3 Tenover F C et al 1993 The resurgence of tuberculosis Is your laboratory ready J Clin Microbiol 31 767 770 4 Cohn M L R F Waggoner and J K McClatchy 1968 The 7H11 medium for the cultivation of mycobacteria Am Rev Resp Dis 98 295 296 5 Youmans G P 1979 Cultivation of mycobacteria the morphology and metabolism of mycobacteria p 25 35 Tuberculosis W B Saunders Company Philadelphia 6 Kent P T and
8. d only if The tube is returned within the required time limit 5 hrs the barcode sequence number label is scanned to re enter the tube or the tube is returned to the same instrument from which it was removed 4 NEGATIVE CULTURES a Negative cultures exist as ongoing negatives in protocol or out of protocol negatives Notification of these conditions include Ongoing Negatives In the Summary region of the display the ongoing tube count for each drawer appears next to the filled circle icon Out of protocol Negatives In the Summary region of the display the tube count for each drawer appears next to the filled circle with a minus sign icon and The negative indicator light for the drawer s illuminates b To Remove Out of Protocol Negatives 1 2 3 Open the appropriate drawer Press the remove negative tubes soft key All the final negative stations illuminate with FLASHING GREEN indicators 7miclsi doc Page 8 of 11 The barcode scanner turns on and the barcode icon appears in the main body of the display signaling that the instrument is ready to read a tube barcode sequence number To remove all negative tubes batch removal press the remove negatives batch soft key Remove all the tubes in the indicated stations The barcode scanner turns off so that barcode labels cannot be scanned Do not close the drawer until all of the tubes in the FLASHING GREEN stations have been removed When all negative
9. e disposed of properly as required by your institution 1 Biosafety Level 2 practices containment equipment and facilities are recommended for preparing acid fast stains and for culturing clinical specimens For activities involving the propagation and manipulation of Mycobacterium tuberculosis or Mycobacterium bovis growth in culture Biosafety Level 3 practice containment equipment and facilities are required as recommended at the CDC 2 Perform all specimen processing and positive tube staining or subculturing in a Biological Safety Cabinet Appropriate protective clothing including glove and masks should be worn 3 DO NOT USE tubes showing evidence of contamination or damage Discard any tubes if they appear unsuitable 4 Dropped tubes should be examined carefully If damage is seen the tube should be discarded 5 Before discarding place all contaminated BBL MGIT tubes and other materials in a puncture proof container Autoclave and dispose of according to your facility s procedure B PROCESSING NEW CULTURES 1 INOCULATION OF MGIT TUBES BBL MGIT Tubes 7 mL Cat no 245122 must be used with a BACTEC MGIT 960 instrument 7miclsi doc Page 5 of 11 a Reconstitute a lyophilized vial of BBL MGIT PANTA antibiotic mixture with 15 mL of BACTEC MGIT Growth Supplement b Label the MGIT tube with the specimen number c Unscrew the cap and aseptically add 0 8 mL of MGIT Growth Supplement MGIT PANTA antibiotic mixture
10. ess the maintenance soft key Press the test indicators soft key 4 Press the test drawer indicators soft key All three external indicator lamps on all three drawers should light as well as the instrument Alarm indicator 5 Open Drawer A The soft key functions appear to allow tests of the station status LEDs a Press the test green LEDs soft key All of the green LEDs at all the stations should light If any does not block the station s as described in Section 7miclsi doc Page 4 of 11 6 2 3 2 Block Station of the BACTEC MGIT 960 User s Manual Press the test green LEDs soft key again to extinguish the green LEDs b Press the test red LEDs soft key All of the red LEDs at all the stations should light If any does not block the station s as described in Section 6 2 3 2 Block Station of the BACTEC MGIT 960 User s Manual Press the test red LEDs soft key again to extinguish the red LEDs 6 Repeat the tests of red and green station status LEDs for Drawers B and C 7 Check the printer s paper supply If the paper supply is low or exhausted replace the paper as explained in the manufacturer s operating instructions a Clean and replace the filters monthly Refer to the User s Manual Section 6 2 2 V PROCEDURE A GENERAL SAFETY CONSIDERSTIONS IMPORTANT NOTE Please be sure to follow Good Laboratory Practices and Universal Precautions at all times during the test procedure All materials should b
11. indicator and 7 mL of broth The indicator contains Tris 4 4 diphenyl 1 10 phenanthroline ruthenium chloride pentahydrate in a silicone rubber base The tubes are flushed with 10 CO and capped with polypropylene caps Each tube contains the following active ingredients Modified Middlebrook 7H9 Broth Base Casein peptone 2 BACTEC MGIT 960 Supplement Kit Each kit contains e 6 vials each containing 15 mL of BACTEC MGIT Growth Supplement with the following active ingredients Bovine Albumin Dextrose Catalase Oleic Acid Polyoxyethylene Stearate e 6 vials of BBL MGIT PANTA Antimicrobic Mixture Each vial contains the following lyophilized mixture of antimicrobial agents Polymyxin B Naladixic Acid Amphotericin B Trimethoprim Azlocillin B MATERIALS REQUIRED BUT NOT PROVIDED Biological Safety Cabinet Vortex mixer 37 C Incubator Lowenstein Jensen Medium or solid medium of choice Middlebrook 7H9 Broth sterile tubes with glass beads 1 mL sterile pipettes 0 5 McFarland turbidity standard sterile tubes sterile saline adjustable 1000 uL pipette sterile tubes Quality Control organisms Mycobacterium tuberculosis ATCC 27294 M kansasii ATCC 12478 M fortuitum ATCC 6841 C INSTRUMENT When microorganisms are present nutrients in the BBL MGIT Mycobacteria Growth Indicator Tube are metabolized resulting in the depletion of oxygen in the medium 7miclsi doc Page 2 of 11 Analysis of the rate of oxygen
12. ing etc of presumptive positive tubes must be performed using BSL Il practices and containment facilities 2 ENTERING TUBES INTO THE INSTRUMENT a Accession Barcoding Disabled 1 Take the new culture tubes to the instrument Open the desired drawer 2 Press the tube entry soft key 3 The barcode scanner turns on and the barcode icon appears in the main body of the display Place the tube in the alignment block in front of the scanner with the barcode label facing the scanner If necessary rotate the tube slightly so the scanner can read the label The system beeps once to indicate a good scan 7miclsi doc Page 6 of 11 4 The assigned station and the scanned sequence number are shown in the main body of the display The assigned station LEDs in the drawer illuminate GREEN 5 Carefully and completely insert the tube into the designated station 6 Repeat steps 3 5 for each new tube to be entered NOTE Tubes should not be twisted or turned once they are entered into the appropriate stations Tubes should only be removed if they are positive negative or reassigned due to a bad station b Accession Barcoding Enabled 1 Take the new culture tubes to the instrument Open the desired drawer 2 Press the tube entry soft key 3 The barcode scanner turns on and the barcode icon appears in the main body of the display shows an arrow pointing to the upper barcode the tube sequence number Place the tube in the alignment b
13. lock in front of the scanner with the barcode label facing the scanner If necessary rotate the tube slightly so the scanner can read the label The system beeps once to indicate a good scan 4 The icon in the main body of the display shows an arrow pointing to the lower barcode the accession number Scan either the accession barcode label or press the no accession barcode available soft key 5 The assigned station the scanned sequence number and accession number are shown in the main body of the display The assigned station LEDs in the drawer illuminate GREEN 6 Carefully and completely insert the tube into the designated station 7 Repeat steps 3 5 for each new tube to be entered NOTE Tubes should not be twisted or turned once they are entered into the appropriate stations Tubes should only be removed if they are positive negative or reassigned due to a bad station 3 POSITIVE CULTURES a The system will indicate the presence of presumptive positive vials in several ways b 1 The POSITIVE indicator lamp on the front of the drawer illuminates 2 The tube count for each drawer next to the filled circle with a plus sign icon increments in the Summary window display 3 When the drawer is opened the remove positive tubes soft key appears on the screen and 4 The audible alert sounds until the condition is acknowledged To Remove the Positive Tubes 1 Press the SILENCE ALARM key to quiet the audible alarm
14. ria The BACTEC MGIT 960 System is designed for the rapid detection of mycobacteria in all types of clinical specimens except blood and urine The system includes a liquid culture medium BBL MGIT Mycobacteria Growth Indicator Tube a growth supplement and an antibiotic mixture BBL MGIT PANTA The BACTEC MGIT Growth Supplement provides substances essential for the growth of mycobacteria BBL MGIT PANTA contains a mixture of antimicrobial agents used to suppress the growth of contaminating bacteria A fluorescent compound is embedded in silicone on the bottom of each of the MGIT broth tubes This compound is sensitive to the presence of oxygen dissolved in the broth Initially the large amount of dissolved oxygen quenches the emissions from the compound and little fluorescence can be detected Later actively respiring microorganisms consume the oxygen and allow the fluorescence to be detected 7miclsi doc Page 1 of 11 The BACTEC MGIT 960 System monitors the tubes for increasing fluorescence Analysis of the fluorescence is used to determine if the tube is instrument positive i e the test sample contains viable organisms Culture tubes which remain negative for a minimum of 42 days up to 56 days and which show no visible signs of positivity are removed from the instrument as negatives ll MATERIALS A MEDIA 1 BBL MGIT Mycobacteria Growth Indicator Tube Each tube contains 110 uL of fluorescent
15. rotocol No reportable result Recovery of mycobacteria in the BBL MGIT tube is dependent on the number of organisms present in the specimen specimen collection methods patient factors such as presence of symptoms prior to treatment and the method of processing Decontamination with the N acetyl L cysteine Sodium hydroxide NALC NaOH method is recommended Other decontamination methods have not been tested in conjunction with the BBL MGIT medium Digestant decontaminant solutions may have harmful effects on mycobacteria 7miclsi doc Page 9 of 11 Colony morphology and pigmentation can only be determined on solid media Mycobacteria may vary in acid fastness depending on strain age of culture and other variables The consistency of microscopic morphology in BBL MGIT medium has not been established An AFB smear positive BBL MGIT tube can be subcultured to both selective and nonselective mycobacterial media for isolation to perform identification and susceptibility testing BBL MGIT tubes which are instrument positive may contain other non mycobacterial species Non mycobacterial species may overgrow mycobacteria present Such MGIT tubes should be re decontaminated and re cultured refer to the BACTEC MGIT 960 User s Manual Reprocessing is strongly recommended if the original specimen source cannot be easily re collected e g tissue specimen BBL MGIT tubes which are instrument positive may contain one or more species of my
16. s ATCC Dilution of 0 5 7miclsi doc Page 3 of 11 McFarland in Instrument Saline Positivit M tuberculosis 27294 1 500 12478 1 50000 M fortuitum 6841 1 5000 1 From solid media cultures less than 15 days old prepare a suspension in Middlebrook 7H9 Broth 2 Allow the suspension to sit for 20 min 3 Transfer the supernatant to another empty sterile tube and allow to sit for an additional 15 min 4 Transfer the supernatant to another empty sterile tube 5 Adjust the suspension to a turbidity comparable to a McFarland No 0 5 standard 6 Dilute the control organism suspensions following the dilution scheme outlined in the above table 7 Inoculate the BBL MGIT tubes following the Inoculation of MGIT Tubes procedure The BBL MGIT tubes should be detected as instrument positive within the time frame shown in the above table If the QC MGIT tubes do not give the expected results do not use the remaining tubes until you have contacted Technical Services at 800 638 8663 United States only B INSTRUMENT The following procedures should be performed at the start of each day s testing and recorded on the maintenance log 1 Check temperature readout of each drawer by reading the Temperature QC Tubes The manual readings should be within 1 0 2 0 C of 37 C 2 Verify that each drawer is currently within 1 5 C of the manual reading for each of the drawers by pressing the temperature soft key 3 Pr
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