MYCOBACTERIUM TUBERCULOSIS (TUBERCULOSIS)
Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB). This rod-shaped bacterium, MTB is
an aerobic bacterium. For this reason, during active tuberculous disease, MTB complexes are always
found in the upper air sacs of the lungs. The bacterium is a facultative intracellular parasite, usually of
macrophages, and has a slow generation time, 15-20 hours, a physiological characteristic that may
contribute to its virulence. The bacteria usually attack the lungs, but MTB bacteria can attack any part of
the body such as the kidney, spine, and brain. If not treated properly, disease can be fatal. It is
transmitted from person to person via droplets from the throat and lungs of people with the active
respiratory disease.
Latent tuberculosis infection (LTBI) Latent tuberculosis infection (LTBI) is a state of persistent immune
response to stimulation by Mycobacterium tuberculosis antigens without evidence of clinically
manifested active tuberculosis. The lifetime risk of reactivation for a person with documented LTBI is
estimated to be 5–10%, with the majority developing TB disease within the first five years after initial
infection.
Active tuberculosis (TB disease) In some people, MTB bacteria overcome the defenses of the immune
system and begin to multiply, resulting in the progression from latent tuberculosis infection to TB
disease. Some people develop TB disease soon after infection, while others develop TB disease later
when their immune system becomes weak.
Extra-pulmonary tuberculosis. Extra-pulmonary tuberculosis is the infection of any organ in the body
other than the lungs by Mycobacterium tuberculosis. The most common sites of extra-pulmonary
tuberculosis are lymph nodes, pleura, abdomen, bone and joint, spinal cord and the brain and its
coverings
MYCOBACTERIUM TUBERCULOSIS – SAMPLE COLLECTION
Sputum
Keeping both hands on hips, cough forcibly and collect sputum in the mouth; spit the sputum carefully
into a wide-mouthed, unbreakable, leak-proof container and close the lid tightly. Ideally, a sputum
specimen should be 3–5ml in volume, although smaller quantities are acceptable if the quality is
satisfactory. Sputa should be transported to the laboratory as soon as possible. If a delay of a few days
cannot be avoided, keep specimens cool (refrigerated but not frozen)
Laryngeal swab
Laryngeal swabs may be useful in children and patients who cannot produce sputum or may swallow it.
Collect laryngeal swabs in the early morning, before patients eat or drink anything. Use a sterile
absorbent cotton swab for collection. Transport each specimen in a container with a few drops of sterile
0.9% saline solution in order to keep the swab wet.
, Other respiratory specimens
Bronchial secretion (2–5 ml)
Pleural effusions (20–50 ml).
Trans-bronchial and other biopsies taken under sterile conditions should be kept wet during
transportation by adding few drops of sterile 0.9% saline to the tissue.
Extra-pulmonary specimens
Specimens can be inoculated directly into liquid vials and transported to the laboratory for culture.
Specimens must be transported to the laboratory immediately; they should be processed as soon as
possible or kept at 2–6 ºC. The optimal volumes are at least 3 ml of cerebrospinal fluid and 5–10 ml of
blood, collected in citrate blood tubes.
MYCOBACTERIUM TUBERCULOSIS – ZIEHL-NEELSEN STAIN
M. tuberculosis does not retain any common bacteriological stain due to high lipid content in its wall,
and thus is neither Gram-positive nor Gram-negative, hence Ziehl-Neelsen staining, or acid-fast staining,
is used. While Mycobacteria do not retain the crystal violet stain, they are classified as acid-fast Gram-
positive bacteria due to their lack of an outer cell membrane.
Ziehl-Neelsen staining procedure
In the ‘hot’ Ziehl-Neelsen technique, the phenol-carbol fuchsin stain is heated to enable the dye to
penetrate the waxy mycobacterial cell wall. The stain binds to the mycolic acid in the mycobacterial cell
wall. After staining, an acid decolorizing solution is applied. This removes the red dye from the
background cells, tissue fibres, and any organisms in the smear except mycobacteria which retain (hold
fast to) the dye and are therefore referred to as acid fastbacilli (AFB). Following decolorization, sputum
smear is counterstained with malachite green (or methylene blue) which stains the background
material, providing a contrast colour against which the red AFB can be seen. Among the Mycobacterium
species, M. tuberculosis and M. ulcerans are strongly acid fast.
Reporting of sputum smear
1. When no 4AFBare seen after examining 300 fields, report the smear as ‘No AFB seen’.
2. When very few AFB are seen i.e. when only 1 or 2 AFB are seen after examining 100 fields, request a
further specimen to examine (Those AFB might have come from tap water (saprophytic mycobacteria),
or it may be scratch of glass slide or by the use of same piece of blotting paper while drying.
3. When any red bacilli are seen, report the smear as ‘AFB positive’ and give an indication of the number
of bacteria present as follows:
Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB). This rod-shaped bacterium, MTB is
an aerobic bacterium. For this reason, during active tuberculous disease, MTB complexes are always
found in the upper air sacs of the lungs. The bacterium is a facultative intracellular parasite, usually of
macrophages, and has a slow generation time, 15-20 hours, a physiological characteristic that may
contribute to its virulence. The bacteria usually attack the lungs, but MTB bacteria can attack any part of
the body such as the kidney, spine, and brain. If not treated properly, disease can be fatal. It is
transmitted from person to person via droplets from the throat and lungs of people with the active
respiratory disease.
Latent tuberculosis infection (LTBI) Latent tuberculosis infection (LTBI) is a state of persistent immune
response to stimulation by Mycobacterium tuberculosis antigens without evidence of clinically
manifested active tuberculosis. The lifetime risk of reactivation for a person with documented LTBI is
estimated to be 5–10%, with the majority developing TB disease within the first five years after initial
infection.
Active tuberculosis (TB disease) In some people, MTB bacteria overcome the defenses of the immune
system and begin to multiply, resulting in the progression from latent tuberculosis infection to TB
disease. Some people develop TB disease soon after infection, while others develop TB disease later
when their immune system becomes weak.
Extra-pulmonary tuberculosis. Extra-pulmonary tuberculosis is the infection of any organ in the body
other than the lungs by Mycobacterium tuberculosis. The most common sites of extra-pulmonary
tuberculosis are lymph nodes, pleura, abdomen, bone and joint, spinal cord and the brain and its
coverings
MYCOBACTERIUM TUBERCULOSIS – SAMPLE COLLECTION
Sputum
Keeping both hands on hips, cough forcibly and collect sputum in the mouth; spit the sputum carefully
into a wide-mouthed, unbreakable, leak-proof container and close the lid tightly. Ideally, a sputum
specimen should be 3–5ml in volume, although smaller quantities are acceptable if the quality is
satisfactory. Sputa should be transported to the laboratory as soon as possible. If a delay of a few days
cannot be avoided, keep specimens cool (refrigerated but not frozen)
Laryngeal swab
Laryngeal swabs may be useful in children and patients who cannot produce sputum or may swallow it.
Collect laryngeal swabs in the early morning, before patients eat or drink anything. Use a sterile
absorbent cotton swab for collection. Transport each specimen in a container with a few drops of sterile
0.9% saline solution in order to keep the swab wet.
, Other respiratory specimens
Bronchial secretion (2–5 ml)
Pleural effusions (20–50 ml).
Trans-bronchial and other biopsies taken under sterile conditions should be kept wet during
transportation by adding few drops of sterile 0.9% saline to the tissue.
Extra-pulmonary specimens
Specimens can be inoculated directly into liquid vials and transported to the laboratory for culture.
Specimens must be transported to the laboratory immediately; they should be processed as soon as
possible or kept at 2–6 ºC. The optimal volumes are at least 3 ml of cerebrospinal fluid and 5–10 ml of
blood, collected in citrate blood tubes.
MYCOBACTERIUM TUBERCULOSIS – ZIEHL-NEELSEN STAIN
M. tuberculosis does not retain any common bacteriological stain due to high lipid content in its wall,
and thus is neither Gram-positive nor Gram-negative, hence Ziehl-Neelsen staining, or acid-fast staining,
is used. While Mycobacteria do not retain the crystal violet stain, they are classified as acid-fast Gram-
positive bacteria due to their lack of an outer cell membrane.
Ziehl-Neelsen staining procedure
In the ‘hot’ Ziehl-Neelsen technique, the phenol-carbol fuchsin stain is heated to enable the dye to
penetrate the waxy mycobacterial cell wall. The stain binds to the mycolic acid in the mycobacterial cell
wall. After staining, an acid decolorizing solution is applied. This removes the red dye from the
background cells, tissue fibres, and any organisms in the smear except mycobacteria which retain (hold
fast to) the dye and are therefore referred to as acid fastbacilli (AFB). Following decolorization, sputum
smear is counterstained with malachite green (or methylene blue) which stains the background
material, providing a contrast colour against which the red AFB can be seen. Among the Mycobacterium
species, M. tuberculosis and M. ulcerans are strongly acid fast.
Reporting of sputum smear
1. When no 4AFBare seen after examining 300 fields, report the smear as ‘No AFB seen’.
2. When very few AFB are seen i.e. when only 1 or 2 AFB are seen after examining 100 fields, request a
further specimen to examine (Those AFB might have come from tap water (saprophytic mycobacteria),
or it may be scratch of glass slide or by the use of same piece of blotting paper while drying.
3. When any red bacilli are seen, report the smear as ‘AFB positive’ and give an indication of the number
of bacteria present as follows:
