Introduction of MGIT liquid

media & MGIT instrument

for TB diagnosis

Somsak Rienthong

NRL & SRL of Tuberculosis

Bangkok, Thailand

Courtesy: Allison Tseng

 BACTEC™ MGIT™ 960 System

 for the detection and recovery of

mycobacteria both MTB & NTM
 Acceptable specimen types
- digested and decontaminated clinical
specimens such as sputum
- sterile body fluids (except blood).
 MGIT
(Mycobacteria Growth Indicator Tube)
BBL  MGIT Tubes - Broth and Fluorescent Indicator
Modified Middlebrook 7H9 Broth – 7 ml
Ruthenium fluorescent complex

BBL  MGIT OADC (15ml)

Oleic Acid - utilized by tubercle bacilli
Albumin - binds free fatty acids
Dextrose - energy source
Catalase - destroys toxic peroxides

BBL  MGIT PANTA (vial)

PolymyxinB  6,000   units
Amphotericin B   600   μg
Nalidixic acid  2,400   μg
Trimethoprim  600   μg    
Azlocillin  600   μg  
 MGIT Technology indicator system
105-106 CFU/ml
Negative Culture Positive Culture

10% CO2 Vial Vial

Headspace CO2 Headspace

O 2 O O2 O2 O
2 CO2 2
Growth Growth
Medium O2 Medium O2 CO2
O2 CO
2 O2 CO2
F
FO2 F Sensor F F F Sensor
FO2FO2 FO F
FO2 F FO2 F F2 FO F
2

Little or No Fluorescence Strong Fluorescence

tris-4, 7-diphenyl-1, 10-phenanthroline ruthenium dichloride pentahydrate-DPPRDP

 MGIT™ Tube Preparation

 Prepare tubes while specimens are in

centrifuge:
 Label MGIT™ tube with specimen number.
 Prepare PANTA.
 Reconstitute with 15 ml Growth Supplement
 For best results, add PANTA / Growth
Supplement solution just prior to specimen
inoculation.
 MGIT™ Tube Preparation

 Unscrew cap and aseptically add 0.8 ml MGIT™

PANTA / Growth Supplement solution.
 MGIT™ Tube Preparation

 Add 0.5 ml of concentrated specimen.

• Also add 1 - 2 drops of

specimen to a 7H10 agar or to
an egg based medium.
 MGIT™ Tube Preparation

 Tightly recap tube and mix well.

 Wipe tubes and caps with tuberculocidal
disinfectant.
 Enter MGIT tubes into MGIT 960
Instrument.
BACTEC™ MGIT™ 960 System
Workflow

Step 1: Press Step 2: Scan tube

BACTEC™ MGIT™ 960 System
Workflow

Step 3: Load tube Step 4: Remove

 BACTEC™ MGIT™ 960 System
Workflow
Specimen

MGIT

Positive 42 days
0.1 ml aliquot
from tube bottom Negative
Stain
visual check tubes
Contamination Positive • visually positive
subculture,
Report preliminary acid-fast stain
results • no signs of positivity
re-decontamination ID sterilize
re-concentration AST discard
re-inoculation
 BACTEC™ MGIT™ 960 System
Reporting of Result
 AFB smear positive
Action: subculture to solid media
Report: Instrument positive, AFB smear positive, ID

pending.

 Microorganisms other than AFB are present

Report: Instrument positive, AFB smear negative.
Contaminated.

 No microorganisms are present

Action: Re-enter the tube into the instrument as an
ongoing negative tube within 5 h of removal.

Allow tube to complete test protocol.

 BACTEC™ MGIT™ 960 System
User Quality Control

 Timing: new shipment

new lot number of MGIT tubes
 BACTEC™ MGIT™ 960 System
User Quality Control

1. solid media cultures <15 days old

prepare organism suspension in Middlebrook 7H9 Broth
2. sit for 20 min
3. Transfer the supernatant to an empty, sterile tube,
sit for an additional 15 min.
4. Transfer the supernatant to another empty, sterile tube.
5. Adjust the suspension to McFarland 0.5
6. Dilute the control organism suspensions
7. Inoculate the MGIT tubes
 BACTEC™ MGIT™ 960 System
Instrument Information
Operation conditions: 19-30˚C 30%-80% Relative humidity
Built-In-Test (BIT) BarCode Scanner

Liquid Crystal Display (LCD)

Drawers A ,B, C
Tube Rack
Station Status LED’s
37 ˚C The Detector Assembly
 BACTEC™ MGIT™ 960 System
Instrument Calibration

Components in the BACTEC MGIT 960 instrument are

selected and designed to maintain electrical and optical
integrity throughout the product’s life. All BACTEC
instruments are calibrated at the factory prior to
shipment. In addition, once per hour, each detector
assembly reads the calibrator tube present in its row.
After the calibrator tube is read, the readings are used
by the BIT to verify that the detection system is in
calibration.
 BACTEC™ MGIT™ 960 System
Daily Maintenance

 Optional Printer
 Lamp Operation

 Temperature Verifications
 37 C  1.5C

BACTEC™ MGIT™ 960 INSTRUMENT MAINTENANCE

 BACTEC™ MGIT™ 960 System
Periodic Maintenance
 Air Filter Replacement
 Calibrator Tubes

 Barcode Scanner Window

Clean the scanner window with a damp, lint-free,
non-abrasive cloth and dry with a lint-free,
nonabrasive cloth.
 BACTEC™ MGIT™ 960 System
Reagent Storage
 Mycobacteria Growth Indicator Tubes (BBL MGIT)
 2-25°C

 DO NOT FREEZE and Minimize exposure to light.

 BACTEC MGIT Growth Supplement

 2-8°C in the dark

 Avoid freezing or overheating

 Do not open until ready to use.

 BBL MGIT PANTA Antibiotic Mixture

 2 -8°C

 Once reconstituted, the PANTA mixture must be stored at 2-

8°C and used within 5 days.

 June 2007: WHO recommended the use
of liquid medium for culture and DST together
with rapid speciation in middle and low income
countries.

http://www.who.int/tb/dots/laboratory/policy/en/index3.htlm

Source: S. Rienthong, NTRL & SRL of Tuberculosis Bureau of Tuberculosis, DDC, MOPH Thailand.
MGIT 960
History
History of
of Bactec
Bactec system
system for
for
TB
TB diagnosis
diagnosis in
in Thailand
Thailand
 1992 Bactec 460 TB
 1995 MGIT (manual 4 ml)
 1998 Bactec MGIT 960
(For isolation)
 2000 Bactec MGIT 960

(For DST with SIRE)

 2002 Bactec MGIT 960

(For DST with PZA)

 2011 Bactec MGIT 320 ??
 What Laboratories Can Do
To Help Control Tuberculosis

 Smear results within 24 hours of specimen

collection

 Identification of M. tuberculosis within 10 -

14 days of specimen collection

 Report of susceptibility results within 15 -

30 days of specimen collection

F C Tenover, and et al.CDC, Atlanta, Georgia.The resurgence of tuberculosis: is your

laboratory ready? J. Clin. Microbiol. 1993 31: 767-770.
 Mycobacterium Diagnosis In
Clinical Lab

Specimen
Specimen processing

Incubation
/Reading Positive

8 weeks ID AST

Negative TB
NTM
Laboratory Services in Tuberculosis Control
Specimen Collection and Transportation

 Collect in robust, sterile, leak-proof

container
 Do not use fixatives or preservatives
 Transport in as short a time as is possible
Laboratory Services in Tuberculosis Control
Smear Preparation
Label a new, clean, unscratched slide

bacteriological loop Applicator stick (recommended)

1cm

2cm
Laboratory Services in Tuberculosis Control
Smear Preparation

air dry 15 minutes

Fix
i. Pass through a flame
(3 or 4 times)
ii electric slide warmer
(65°C - 75°C, >2 hours)

Remove particles of adherent Discard the applicator stick in

sputum from disinfectant or a biohazard
loops by moving it up and down receptacle
through a flask containing sand
and 70% alcohol.
 Laboratory Services in Tuberculosis Control
Acid-Fast Staining
 Ziehl-Neelsen stain
(heat)
 Kinyoun acid fast stain
(cold)
 Fluorochrome stain

Smears that have been examined by fluorescence microscopy

may be restained by Ziehl-Neelsen staining for confirmation.
Laboratory Services in Tuberculosis Control
Acid-Fast Staining
# of AFB Found Report
0 Negative for AFB
1-9 / 100 fields Actual number
10-99 / 100 fields 1+
1-10 / field 2+
> 10 / field 3+

Recommend at least 100 fields be examined to

call “Negative for AFB”
 3 passes along “long axis” of slide
 9 passes along “short axis” of slide
Comparison of Ziehl-Neelsen (Hot staining) and
Kinyoun (Cold staining) method
Items Ziehl-Neelsen Kinyoun

1. Consistently reproducible result Yes No

at least at lease
2. Staining time
5 min 1 hr or more
3. Fading of fuchsin stained slow fast
can can not
4. A few bacilli present
detected detected
5. Cause of false negative No Yes

6. reading result reliability unreliability

 Comparison of ZN and Fluorescence
Items ZN Fluorescence
Mercury
Sufficient experienced Yes More
Maintenance/Cost Easy/Cheap Difficult/Costly
Objective lens x100 x20-x40
Oil immersion Use No use
Workload Unlimited At least 50 slides
Turn –on/off Any time Waiting 30 min
Report of Negative 5 min 1 min
 Comparison of Mercury with LED FM
Items Mercury FM LED FM
UV/dark room Yes No
Maintenance/Cost Difficult/Costly Easy/Cheap
Use Battery No Yes
Work in the field No Yes
Working hour 200 hr >50,000 hr
Turn on/off Wait 30 min Any timr
Explosive Yes No
Laboratory Services in Tuberculosis Control
Specimen Processing
NALC/NaOH Digestion Procedure - Sputum

Digest-decontaminate using NALC/NaOH

Prepare fresh reagent -or-
Use BBLTM MycoPrep
Add 5ml specimen to 50 ml centrifuge tube.
Add equal volume NALC/NaOH
Vortex 15-20 sec
15-20 min room temp incubation
Fill tube to 50 ml mark with sterile pH 6.8
phosphate buffer
swirl to mix
 NALC-NaOH solution
Total vol. NALC (g)
NaOH-Na citrate (ml)
needed (ml)

50 50 0.25
100 100 0.50
250 250 1.25
500 500 2.50
1000 1000 5.00
• Use equal volume of NaOH & Na-Citrate

Use with in 24 hours of preparation after added

NALC
Laboratory Services in Tuberculosis Control
Specimen Processing
Concentration
Centrifuge 3,000g x15 min
Carefully decant the supernatant fluid from the pellet and

discard.
Re-suspend sediment with phosphate buffer.
Use a sterile Pasteur pipette to achieve a final volume
of 1 - 3 ml.
1. Liquid media 2. Solid media 3. stain

http://www.mf.unilj.si/imi/images/prepar
ati/mikrobi/mycob01.jpg
The culture of M. tuberculosis strain was performed as the following figure:

NALC-NaOH
sputum
Growth of M. tuberculosis
Treated Washing by Inoculation &
specimen Centrifugation Incubation

Bactec MGIT 960 35

 Digestion and decontamination
procedures – Good practice
Sputum specimens
1. Do not open tubes at the same time to minimize the risk of cross-
contamination.
2. Carefully label all the tubes before starting processing the samples
3. Work in batches corresponding to one centrifuge load (e.g. eight
specimens at once).
4. Always digest/decontaminate the whole specimen.
5. Gently pour out the specimen from the container into the centrifuge tube,
avoid splitting.
6. Never transfer the sample before labelling the tube

If possible digest/ decontaminate the sample in the

same container used for collection
 Key points about inoculation of liquid
culture media
 Minimize the aerosol production:
 open the caps of liquid media slowly
 avoid vigorous shaking of the specimen
 avoid the expulsion of the last drop from the pipette.
 Use aseptic techniques and sterile material to avoid
contamination.
 Inoculate liquid media first to reduce the chances of carry
over any contaminants from contaminated solid media.
 Prepare smears for staining after all media have been
inoculated.
 Inoculate the media with clinical specimens as soon as
possible.
 Detection of positive cultures

Positive cultures

ZN staining MTB: Cord formation

 Cord formation from liquid media

NTM, show loose aggregates

“Pseudocording”

MTB exhibiting serpentine cording >98%

Source: S. Rienthong, NTRL & SRL of Tuberculosis Bureau of Tuberculosis, DDC, MOPH Thailand.
Laboratory Services in Tuberculosis Control
Identification

 Identification can take up to 2 weeks:

 Colony morphology & pigmentation
 Light studies
 Pigment production in presence and/or absence of light
(Photo-reactivity)
 Rate of growth
 Biochemical tests
 Niacin • Tween hydrolysis
 Nitrate reduction • Arylsulfatase
 Catalase 68oC
 BACTEC™ MGIT™ 960 AST SIRE
Proportional Method (Growth Control 1:100)
Susceptibility testing will include: S, H, R, E and Z
Time-saving, turnaround time were 12 - 14 days.
(mean 6.2-8.8 days)
Accurate, reliable, high sensitivity and specificity.

Prepare inoculums and Scan the AST set into the Remove the set when Print the report with AST
inoculate into the SIREZ instrument. completed. interpretations of either
set. Susceptible or Resistant in a
week.
 Culture & Proportional DST in solid media
1 loopful of Bacillary Suspnsion
M. tuberculosis

Inoculation &
Incubation Growth of
Treated Washing by M. tuberculosis
Sputum stock MTB 10-3 10-5
Centrifugation (107cell/ml) (104cell/ml) (102cell/ml)

Recording & Incubation

Reporting 37ºC 4 weeks

Control PNB Control SM INH RFP EMB Control SM INH RFP EMB
medium medium medium

NTRL & SRL of Tuberculosis

Bureau of Tuberculosis, DDC, MOPH Thailand
Culture & DST with BACTEC™ MGIT™ 960
Positive sample

Inoculation &
Incubation
Treated Precipitation by
sputum specimen High speed Centrifugation Subculture
[Inoculation & Growth of
[NALC-NaOH] M. tuberculosis
Incubation]
BACTEC™ MGIT™ 960

BACTEC™ MGIT™ 960 AST

NTRL & SRL of Tuberculosis
Bureau of Tuberculosis, DDC, MOPH Thailand
 BD BACTEC™ MGIT™ 320
 Lower volume version of the BACTEC
MGIT 960
Same G&D and AST
•
capabilities of the
960.

Benchtop, single
•

drawer version of the

960.

960 Vs 320
New Culture Technique for TB
• Advantages : obtain results in 1- 2 weeks.
: combine with susceptibility testing,
result will be known in 3 - 4 weeks
: benefit in drug resistant patient
• Disadvantages : expensive, need instrument
: result does not change judgement of
treatment or not
• Culture doesn’t have benefit if susceptibility and
identification are not further done
 CDC recommendations for
TB Diagnosis : Summary
(i) rapid delivery of specimens to the laboratory on a daily
basis
(ii) Use a fluorescent acid-fast staining procedure and
immediately transmit the results.
(iv) Inoculate a liquid medium as primary culture, and
include inoculation of a slant of Lowenstein-Jensen
medium.
(v) Identify growth in liquid medium as soon as possible.
(vi) Determine the susceptibilities of M. tuberculosis
isolates to primary drugs in a BACTEC or similar system.

F C Tenover, and et al.CDC, Atlanta, Georgia.The resurgence of tuberculosis: is your

laboratory ready? J. Clin. Microbiol. 1993 31: 767-770.
 Conclusion
CHARLES S. DARWIN….

“ It is not the strongest nor the

most intelligent to survive
but the most responsive to
change”
 Changing Trends in Laboratory
Testing
 Days of Home-made media, reagents and
procedures are gone
 Commercial test kits, procedures and
reagents are more widely used
 Automation is taking place of manual
testing
 There is a demand of Rapid Testing, Rapid
Reporting for “Better Patient Care”

NTRL & WHO-SRL of Tuberculosis

Bureau of Tuberculosis, DDC, MOPH Thailand
 Change to Liquid Media

Present: Liquid Media are far

superior to Solid Media
 Rapid turn around time
 More mycobacterial species grow

 Stressed mycobacteria grow better

 Rapid and more Reliable Susceptibility

test
 Endorse by WHO
Courtesy: David Dawson, Dr., WHO Collaborating Center, The Prince Charles Hospital Brisbane Australia
 So, we Need….

Good Microbacteriological
Technique (GMT)
and
Good Laboratory practice (GLP)