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Mycobacterium tuberculosis & pulmonary tuberculosis. By. Dr. Emad AbdElhameed Morad. Lecturer of Medical Microbiology and Immunology. Morphology. Thin, straight or slightly curved rods about 0.4x3 µm. They can not be stained by Gram stain due to their high lipid content.
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Mycobacterium tuberculosis & pulmonary tuberculosis By Dr. Emad AbdElhameed Morad Lecturer of Medical Microbiology and Immunology
Morphology Thin, straight or slightly curved rods about 0.4x3 µm. They can not be stained by Gram stain due to their high lipid content. But, they are stained by Ziehl-Neelsen stain by which they appear as thin pink rods (acid fast bacilli) arranged singly or in small groups against blue background.
Ziehl-Neelsen smear made from a sputum sample showing positive acid fast bacilli (AFB)
Ziehl-Neelsen smear made from a sputum sample showing positive acid fast bacilli (AFB)
Ziehl-Neelsen smear made from a sputum sample showing positive acid fast bacilli (AFB)
Ziehl-Neelsen stain • Smears are prepared from sputum samples as follows: • Three morning sputum samples are preferable since they represent overnight accumulation. • Choose a purulent portion of sputum and spread it evenly in the middle of a new clean glass slide. • Leave the smear to dry. • Then fix the smear by passing through the flame.
Staining is done as follows: • Flood the smear with strong carbol fuchsin. Allow the stain to act for 5-10 minutes. • Heat intermittently until the vapor begins to rise. Do not allow the stain to boil or dry. • Pour it off then wash with water. • Flood the smear with 20% H2SO4 or 3% HCL in 95% alcohol. Allow to act for 1 min. Then wash with water and reapply fresh acid. Repeat this process several times till the smear becomes pale pink. • Wash thoroughly with water. • Add methylene blue or malachite green for 2 min. • Wash with water. Dry then examine.
Smear is interpreted as follows: • One or more bacilli / oil field+++ • 10 bacilli / slide ++ • 3-9 bacilli / slide + • 1-2 bacilli / slide +/-
Culture Strict aerobe. They grow on egg based medium such as lowenstein-Jensen medium. Addition of 5% glycerol favors the growth of Mycobacterium tuberculosis. Tubercle bacilli grow very slowly (2-4 w). Culture is not discarded as negative before 8 weeks. On L-J medium, the organism produces irregular, dry and off-white colonies. Other media include Middle brook’s 7H10, 7H11 agar and 7H9 broth. Middle brook’s broth is used to test the antibiotic sensitivity.
Laboratory diagnosis Specimen:three successive morning sputum samples on three successive days. Direct smears stained with Ziehl-Neelsen stain to detect acid fast bacilli (AFB). Decontamination and concentration (NALC method): Viscid sputum samples should be liquefied, decontaminated and concentrated before examination. The specimen is mixed with equal volume of N-acetyl-L-cystiene-2% NaOH solution. Left for 15 min at room temp. Then, diluted with buffer pH 6.8, centrifuged and the sediment is processed.
The sediment is processed as follows: Ziehl-Neelsen stain. Culture on L-J medium and incubated at 37 degrees. Growth starts after 2-4 weeks. Cultures should not be discarded as negative before 8 weeks. Isolates are identified by niacin test and DNA probes.
Tuberculin test Done by intra-dermal injection of 0.1 ml of purified protein derivative (PPD) of tubercle bacilli. The test is read after 48-72 hours. Positive test: development of induration measuring 10 mm or more. The area of induration is measured by a ruler across the forearm perpendicular to its long axis. Positive tuberculin test means previous exposure to tubercle bacilli either in the form of healed primary focus, active disease or BCG vaccination.
QuantiFERON-TB test It requires a single blood sample from the patient. It measures the amount of gamma interferon released from the patient’s lymphocytes after exposing them to mycobacterial antigens by ELISA test. It is better than tuberculin skin test (TST).
