This document provides information about analyzing Mycobacterium tuberculosis in the laboratory. It lists the equipment and reagents used, including liquid culture systems like MGIT and Bactec. It describes staining and microscopy techniques for visualizing the acid-fast bacilli, including Ziehl-Neelsen staining. The document outlines the procedures for culturing samples, identifying contaminants, and using rapid identification tests to detect M. tuberculosis antigens.
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Mycobacteria (tuberculosis) Laboratory Diagnosis
1. MYCOBACTERIUM (Tuberculosis)
UNLOADED POSITVES AND
NEGATIVES
Isaac Okello Opio
Research Assistant
Mycobacteriology lab
College of Health Sciences-Mulago
isaacokelloopio@gmail.com
0778336598 / 0700662434
3. What is TB?
ď´ The scientific name for the TB microbe is Mycobacterium tuberculosis or
M. tb
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4. What is TB?
ď´ Beneath a microscope, it has a
long rod-like shape or âbacillusâ
ď´ 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.)
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9. 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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13. 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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15. PRINCIPLE OF ANALYSIS OF BACTEC 960
System contains
ď´ -liquid culture medium MGIT,
ď´ -growth supplement (essential substances for growth)
ď´ - and antibiotic mixture PANTA (mixture of antimicrobial agents used to
suppress the growth of contaminating bacteria)
ď´ Florescent compound is embedded in silicone on the bottom of each
MGIT broth tube. Respiring organisms consume oxygen and allow the
fluorescence.
ď´ Machine monitors the tube for increasing fluorescence, basis for positivity
or negativity. I.e. culture contains viable organisms
ď´ NB: Culture tubes that remain negative for 42 days are removed as
machine negative.
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17. Preliminary identification of
M. tuberculosis from liquid cultures
ď´Flocculation: granular,
non-homogeneous
suspension
ď´ZN: serpentine cords of
varying length or district
linear clumping
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18. Contamination â liquid media
ď´Homogeneous turbidity
ď´Perform a ZN staining: non-
acid-fast bacteria
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19. Contaminants
ď´Mycobacteria other than
tuberculosis (MOTT/NTM)
ď´fast- or slow-growers
ď´acid-fast bacilli
ď´microscopy: usually not
arranged in cords
ď´Fungi
ď´usually slow-growers
ď´non-acid fast
ď´microscopy: hyphae are
thicker than mycobacteria
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Growth rate and microscopy aspects are considered.
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20. Contaminants
ď´Bacteria
ď´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)
ď´Yeasts
ď´non-acid fast round in shape
and bigger than
mycobacteria)
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Growth rate and microscopy aspects are considered.
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22. Quality control of MGIT
ď´ Examine MGIT tube for damage, discoloration, and /or contamination
prior to use, and record findings.
ď´ Keep the MGIT tube closed until ready for specimen or PANTA/growth
media addition.
ď´ Check storage conditions and expiration dates of lyophilized PANTA
(maintain refrigerator at 2-8 degrees Celsius)
ď´ 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
ď´ The expected results depends on the dilution factor Vs the machine
TTDs
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23. DEMONSTARTION OF LIQUID MEDIA
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26. Bactec 9120
ď´For rapid detection of Mycobacteria
in the clinical blood samples using
bactec 9120 automated culture
system.
ď´Samples are collcted from patients
and inoculated into BACTEC
containing a liquid broth
ď´and instrument automatically
detects growth of Mycobacteria in
the tubes.
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27. Principle for Bactec 9120
ď´ Sample to be tested is inoculated into the vial which is entered into the
BACTEC instrument for incubation and periodic reading.
ď´ It is constantly agitated and incubated at 35 C
ď´ 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.
ď´ The sensor is monitored by the instrument every 10 minutes for an
increase in its fluorescence
ď´ This proportional to the increase in C02 or decrease in 02 amount
present in the vial.
ď´ A positive reading indicates the presumptive presence of viable
microorganism in the vial.
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28. ď´ 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
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29. MICROSCOPY Slide preparation
ď´ Properly labeling a slide
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35. Acid-Fast (Kinyoun) Stain of Mycobacterium
NOTE: cord growth (serpentine
arrangement) of virulent strains
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36. QC FOR ZN
ď´ Prepare batches of control slides
ď´ Test new lot of stains prepared
ď´ During staining, include control slides
ď´ During examination, begin with control slides and record on QC from for ZN
worksheet
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38. 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
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39. The âZiehl-Neelsenâ 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
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40. Lower sensitivity of Ziehl-Neelsen or
lower specificity of fluorescence microscopy?
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41. Rapid Identification test
ď´ Principle
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42. Rapid Identification test
ď´ Principle
ď´ TB Check MPT64 is based on an immuno-chromatographic assay
principle.
ď´ A droplet of the positive culture is placed on the lateral flow strip,
containing nitrocellulose membrane, gold conjugate pad and
absorbent pad.
ď´ On the strip the secreted MPT64 antigens are marked with gold and
migrate to a specific binding site. (Containing mouse anti-MPT64).
ď´ This reaction (with epitope) leads to a gold accumulation at the binding
site and subsequently to a visible band on the strip.
ď´ The control area shows the efficiency of the gold binding â therefore,
valid results are always guaranteed.
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