This document provides information about acid-fast bacilli (Mycobacterium spp.) including medically important pathogenic species of Mycobacterium that cause diseases like tuberculosis, leprosy, and skin ulcers. It describes the taxonomy, characteristics, samples used for detection, and microscopy, culture, and other detection methods for Mycobacterium tuberculosis, the causative agent of tuberculosis. Methods discussed include Ziehl-Neelsen staining, sodium hypochlorite centrifugation, fluorescent stains, Lowenstein Jensen culture medium, BACTEC systems, MGIT, and polymerase chain reaction (PCR).

1. Medical Microbiology Laboratory
Acid-Fast Bacilli (Rods)
(Mycobacterium spp.)
Hussein A. Abid
Medical Laboratory Scientist
Member at American Society of Microbiology
Chairman of Iraqi Medical Laboratory Association
Teacher at Middle Technical University

2. Non-spore formingSpore forming
• Corynebacterium spp.
• Lactobacillus spp.
• Mycobacterium spp.
• Clostridium spp.
• Bacillus spp.
2
GRAM POSITIVE RODS
 Pathogenic species of Mycobacterium:
1. M. ulcerans (causes skin ulcers)
2. M. leprae (causes leprosy)
3. M. tuberculosis (causes tuberculosis)

3. 3
TAXONOMY
Scientific nameRank
• MycobacteriaceaeFamily
• MycobacteriumGenus
• M. ulcerans
• M. leprae
• M. tuberculosis
Species
(medically important spp.)

4. 4
Mycobacterium tuberculosis
 Also called “acid-fast bacilli, AFB”.
 M. tuberculosis is the causative agent for the tuberculosis
disease in humans.
 The bacterium is nonmotile and rod-shaped (bacilli).
 Facultative intracellular parasite, as well as an obligate
aerobe. This can explain why tuberculosis is a disease that
typically affects the lungs.
 Cell wall contains peptidoglycan and lipids, long-chain alpha-
alkyl, beta-hydroxy fatty acids. Also contains mycolic acids,
cord factor, and wax-D.
 Mycolic acids are significant for virulence in Mycobacterium
tuberculosis.

5. 5
Mycobacterium tuberculosis
Samples:
 According to site of infection (sputum, urine, body
fluids, blood, gastric lavage, tissue biopsy).

6. 6
M. tuberculosis (microscopy)
1. Gram staining:
 M. tuberculosis is not considered
gram-positive or gram-negative
bacteria.
 However, the bacterium may
weakly stain gram-positive when
gram-stained.
2. Ziehl-Neelsen staining:
 M. tuberculosis appearing as
bright red bacilli (rods) in a
sputum smear stained with the
Ziehl-Neelsen stain

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ZIEHL-NEELSEN STAINING
 Studies have shown that the chances of detecting AFB in
sputum smears are significantly increased when sputum is
first treated with 5% (v/v) sodium hypochlorite (NaOC1),
i.e. bleach, followed by centrifugation or overnight
sedimentation.
 Because NaOC1 kills M. tuberculosis, the NaOC1
concentration technique is also safer for laboratory staff.
 NaOC1 treated sputum cannot be used for culture.
 Up to three specimens may need to be examined to
detect M. tuberculosis in sputum.
 One specimen should be collected as an early morning
sputum.

8. 8
SODIUM HYPOCHLORITE CENTRIFUGATION
TECHNIQUE TO CONCENTRATE AFB
1. Transfer 1-2 mL of sputum (particularly that which contains
any yellow caseous material) to a screw-cap Universal bottle
or other container of 5-20 mL capacity. Caution: Open
specimen containers with care and at arms length to
avoid inhaling infectious aerosols. When available,
handle the specimen inside a safety cabinet.
2. Add an equal volume of concentrated sodium hypochlorite
(bleach) solution and mix well.
3. Leave at room temperature for 10-15 minutes, shaking at
intervals to break down the mucus in the sputum.
4. Add about 8 mL of distilled water, or when unavailable use
boiled filtered rain water. Mix well.

9. 9
SODIUM HYPOCHLORITE CENTRIFUGATION
TECHNIQUE TO CONCENTRATE AFB
5. Centrifuge at 3000 g for 15 minutes or at 250-1000 g for 20
minutes.
Note: When a centrifuge is not equipped to take Universal
containers, divide the specimen between two conical tubes (which
can be capped). When centrifugation is not possible, leave the
NaOC1 treated sputum to sediment overnight.
6. Using a glass Pasteur pipette or plastic bulb pipette, remove and
discard the supernatant fluid. Mix the sediment. When two tubes
have been used, combine the two sediments.
 Transfer a drop of the well-mixed sediment to a clean scratch-
free glass slide.
 Spread the sediment to make a thin preparation and allow to
air-dry.

10. 10
SODIUM HYPOCHLORITE CENTRIFUGATION
TECHNIQUE TO CONCENTRATE AFB
7. Heat-fix the smear and stain it using the Ziehl-Neelsen
technique.
 Examine it microscopically for AFB.
 M. tuberculosis in Ziehl-Neelsen stained sputum
smears is shown next slide.
 Caution: Bleach (NaOC1) is corrosive and toxic when
ingested or inhaled. It also has an irritating vapour and
therefore it should be used in a well-ventilated place.
Store it out of direct sunlight in a cool place, away from
acids, methanol and oxidizing chemicals. When in contact
with acids, sodium hypochlorite liberates toxic gas.

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ZIEHL-NEELSEN STAINING

12. 12
ZIEHL-NEELSEN STAINING (procedure)
1. Drop suspension onto slide
2. Heat fix an air dried smear at 80 oC for at least 15
minutes or for 2 hours on an electric hot plate at 65 oC –
70 oC
3. Flood slide with Carbol Fuchsin
4. Hold a flame beneath the slide until steam appears but
do not allow it to boil
5. Allow hot slide to sit for 3 to 5 minutes, rinse with tap
water
6. Flood slide with 3% hydrochloric acid in isopropyl
alcohol

13. 13
ZIEHL-NEELSEN STAINING (procedure)
7. Allow to sit 1 minute, rinse with tap water
8. Flood slide with Methylene Blue
9. Allow to sit 1 minute, rinse with tap water
10. Blot dry
11. View under oil immersion lens
Results of ZNS
• AFB: red, straight or slightly curved rods, occurring
singly or in small groups.
• Other cells and background: green or blue

14. 14
FLUORESCENT STAINS
 Good for labs with high workload.
 Auramine-O: Bright yellow
 Auramine-O-Rhodamine-B: Yellow orange.
Auramine O Auramine-Rhodamine

15. 15
M. tuberculosis (culture)
 Culturing and susceptibility testing of M.
tuberculosis are usually undertaken in a
Tuberculosis Reference Laboratory,
mainly for surveillance purposes, to
determine levels of drug resistance, and
to manage treatment failures and
relapses.
 It is cultured on Lowenstein Jensen
(LJ) Medium; it appears as dry,
rough, wrinkled, creamy-white raised
irregular colonies.

16. 16
OTHER METHODS TO DETECT AFB
 BACTEC – TB system (460)
 based on the principal that the organisms multiply in the
broth and metabolize C 14-containing palmatic-acid,
producing radioactively labeled 14CO2.
 BACTEC – TB system (960)
 Mycobacterium Growth Indicator Tubes (MGIT)
 A fluorescent compound is embedded in silicone on the
bottom of 16 x 100 mm round-bottom tubes.

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POLYMERASE CHAIN REACTION (PCR)
1. Diagnose tuberculosis rapidly by identifying DNA from M.
tuberculosis in clinical samples.
2. Determine rapidly whether acid-fast organisms identified
by microscopic examination in clinical specimens are M.
tuberculosis
3. Identify the presence of genetic modifications known to
be associated with resistance to some anti-mycobacterial
agents.
4. Determine whether or not isolates of M. tuberculosis from
different patients have a common origin in the context of
epidemiological studies.