Mycobacteria (tuberculosis) Laboratory Diagnosis

MYCOBACTERIUM (Tuberculosis)
UNLOADED POSITVES AND
NEGATIVES
Isaac Okello Opio
Research Assistant
Mycobacteriology lab
College of Health Sciences-Mulago
isaacokelloopio@gmail.com
0778336598 / 0700662434

Equipment and Reagents
 BSC
 MGIT (Mycobacterial Growth
Indicator Tube)
 PANTA-OADC (Polymyxin,
Amphotericin, Nalidixic Acid,
Trimethoprim and Azlocillin – Oleic
Acid, Albumin, Dextrose, Catalase)
 Bactec MGIT 960
 Bactec 9120
 Slides and Slide dryer
 Microscope
 Blood agar plates
 ID kits
 Disinfectants
11/12/2020
Prepared by: ISAAC (Research Assistant) 0778336598 / 0700662434 Email: isaacokelloopio@gmail.com
2

What is TB?
 The scientific name for the TB microbe is Mycobacterium tuberculosis or
M. tb
11/12/2020Prepared by: ISAAC (Research Assistant) 0778336598 / 0700662434 Email: isaacokelloopio@gmail.com
3

What is TB?
 Beneath a microscope, it has a
long rod-like shape or ‘bacillus’
 The thick waxy cell wall allows the
germ to spread through the air in
water droplets
TB bacilli stained bright red
using the Ziehl-Neelson stain
(image copyright Dennis Kunkel
Microscopy, Inc.)
4

The cell wall of Mycobacterium
tuberculosis
Lipoarabinomannan
Outer
lipids
Mycolic
acid
Cell wall skeleton
Polysaccharides (arabinogalactan)
Peptidoglycan
Plasma membrane
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The cell wall of Mycobacterium
tuberculosis
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Host Evasion and exploitation
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LAB ANALYSIS
8

Koch R, 1884 Wikipedia, 2008
Cole ST, et al. Nature 1998;393:537-44
Morphology
Metabolism
Genome How does it look?
How does it reproduce?
What makes it function?
LAB ANALYSIS
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CULTURE
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LIQUID CULTURE
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BACTEC 920
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The BD BACTEC™ MGIT™ 960 Instrument is a
fully automated system for the rapid detection
of mycobacteria in clinical specimens other than
blood.
The BD BACTEC™ MGIT™ 960 is also used for
the antimicrobial susceptibility testing of
mycobacteria, including SIRE and PZA
susceptibility testing.
The instrument has a maximum capacity of 960
BBL™ MGIT™ tubes (7 ml), or with a 42-day
detection protocol, approximately 8000
specimens per year.
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MGIT TUBES
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PRINCIPLE OF ANALYSIS OF BACTEC 960
System contains
 -liquid culture medium MGIT,
 -growth supplement (essential substances for growth)
 - and antibiotic mixture PANTA (mixture of antimicrobial agents used to
suppress the growth of contaminating bacteria)
 Florescent compound is embedded in silicone on the bottom of each
MGIT broth tube. Respiring organisms consume oxygen and allow the
fluorescence.
 Machine monitors the tube for increasing fluorescence, basis for positivity
or negativity. I.e. culture contains viable organisms
 NB: Culture tubes that remain negative for 42 days are removed as
machine negative.
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Preliminary identification of
M. tuberculosis from liquid cultures
Flocculation: granular,
non-homogeneous
suspension
ZN: serpentine cords of
varying length or district
linear clumping
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Contamination – liquid media
Homogeneous turbidity
Perform a ZN staining: non-
acid-fast bacteria
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Contaminants
Mycobacteria other than
tuberculosis (MOTT/NTM)
fast- or slow-growers
acid-fast bacilli
microscopy: usually not
arranged in cords
Fungi
usually slow-growers
non-acid fast
microscopy: hyphae are
thicker than mycobacteria
19
Growth rate and microscopy aspects are considered.

Contaminants
Bacteria
Fast growers, usually non-
acid fast, with the exception
of Rhodococcus equi
(coccus-shaped) and of
Nocardia spp (partially acid-
fast and do not form cords)
Yeasts
non-acid fast round in shape
and bigger than
mycobacteria)
20
Growth rate and microscopy aspects are considered.

LIQUID CULTURE FLOW CHART
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Quality control of MGIT
 Examine MGIT tube for damage, discoloration, and /or contamination
prior to use, and record findings.
 Keep the MGIT tube closed until ready for specimen or PANTA/growth
media addition.
 Check storage conditions and expiration dates of lyophilized PANTA
(maintain refrigerator at 2-8 degrees Celsius)
 Before using new lot of MGIT tubes, it should be tested with three
species of Mycobacteria: M.fortuitum, M.kansasii, M.tuberculosis
(H37Rv), which are thawed and subcultured two weeks prior to lot
testing using 7H10
 The expected results depends on the dilution factor Vs the machine
TTDs
22

DEMONSTARTION OF LIQUID MEDIA
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SOLID Vs LIQUID
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BLOOD CULTURE
Myco/lytic vial aka Blood
culture bottles
25

Bactec 9120
For rapid detection of Mycobacteria
in the clinical blood samples using
bactec 9120 automated culture
system.
Samples are collcted from patients
and inoculated into BACTEC
containing a liquid broth
and instrument automatically
detects growth of Mycobacteria in
the tubes.
26

Principle for Bactec 9120
 Sample to be tested is inoculated into the vial which is entered into the
BACTEC instrument for incubation and periodic reading.
 It is constantly agitated and incubated at 35 C
 Each vial contains a sensor which response with the concentration of
C02 produced by metabolism of microorganisms or the consumption of
oxygen needed for the growth of microorganisms.
 The sensor is monitored by the instrument every 10 minutes for an
increase in its fluorescence
 This proportional to the increase in C02 or decrease in 02 amount
present in the vial.
 A positive reading indicates the presumptive presence of viable
microorganism in the vial.
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 Contains 7H9 broth
This contains inorganic salts which help the growth of mycobacteria
- Citric acid from sodium citrate helps in retaining inorganic cations
in solution.
- Glycerol supplies carbon and energy
- Middlebrook OADC supplements
Oleic acid; essential for mycobacteria metabolism
Dextrose; energy source
Catalase; neutralizes toxic peroxides
Albumin; protects bacilli from toxic agents
28

MICROSCOPY Slide preparation
 Properly labeling a slide
29

ZN staining
30

Principle of staining
AFB
carbol-fuchsin
(or
auramine)
decolorize
counterstain
31

ZN MICROSCOPY
32

10mm
20mm
0.02mm2
1 Oil immersion field
1 Length = 100 OIF
1 mL Sputum
To see:
1 AFB in 100 fields
requires:
smear surface of 200 mm2
containing 100 AFB
which requires:
1 mL sputum
containing 10,000 AFB
33

OIL immersion Microscope
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Acid-Fast (Kinyoun) Stain of Mycobacterium
NOTE: cord growth (serpentine
arrangement) of virulent strains
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QC FOR ZN
 Prepare batches of control slides
 Test new lot of stains prepared
 During staining, include control slides
 During examination, begin with control slides and record on QC from for ZN
worksheet
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37

Principles in two types of microscopy
Staining
Bright-field
microscopy
Flurorescence
microscopy
Primary stain
(stains everything) Fuchsin Auramine
Decolorant
(destains everything
except mycobacteria)
Hydrochloric acid
or
Sulfuric acid
Hydrochloric acid
Counter-stain
(stains everything
except color-saturated
mycobacteria)
Methylene blue
Methylene blue
or
Other 11/12/2020
38

The “Ziehl-Neelsen” staining technique: an
experimental path to optimization, ready and all set
since 1882
Contributor Contribution
Robert Koch Primary stain: methylene blue, alkaline potassium
hydrate as mordant, vesuvium as both decolorant and
counterstain
Paul Ehrlich Fuchsin as primary stain, alkaline alinine as mordant,
nitric acid as decolorant, and proposal of a blue
counterstain
Franz Ziehl Replace mordant with phenol
Friedrich
Neelsen
Combine the best of all: primary and counterstain from
Ehrlich, mordant from Ziehl, and replacing decolorant
with sulphuric acid
Bishop P J, Neumann G. The history of the Ziehl-Neelsen stain. Tubercle 1970;51:196-206
39

Lower sensitivity of Ziehl-Neelsen or
lower specificity of fluorescence microscopy?
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Rapid Identification test
 Principle
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 Principle
 TB Check MPT64 is based on an immuno-chromatographic assay
principle.
 A droplet of the positive culture is placed on the lateral flow strip,
containing nitrocellulose membrane, gold conjugate pad and
absorbent pad.
 On the strip the secreted MPT64 antigens are marked with gold and
migrate to a specific binding site. (Containing mouse anti-MPT64).
 This reaction (with epitope) leads to a gold accumulation at the binding
site and subsequently to a visible band on the strip.
 The control area shows the efficiency of the gold binding – therefore,
valid results are always guaranteed.
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THANK YOU
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Mycobacteria (tuberculosis) Laboratory Diagnosis

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