Tuberculosis: Diagnosis, Sample Collection, and Staining

Tuberculosis: Diagnosis and Microscopic Examination

Tuberculosis (TB) is a highly contagious infection, primarily pulmonary, caused by members of the Mycobacterium tuberculosis complex.

Causative Agents of Tuberculosis

• Mycobacterium tuberculosis
• M. bovis
• M. avium
• Atypical Mycobacteria

M. avium and atypical mycobacteria are significant opportunistic pathogens, particularly in immunocompromised individuals, transplant recipients, or those undergoing major surgery.

Sputum Sample Collection for Smear Test

1. Obtain at least two sputum samples.
2. Samples can be collected on successive days and stored in a cool, dark place.
3. The sample must be bronchial sputum, not saliva or nasal mucus.
4. Upon waking and before eating, perform a mouthwash with water only.
5. Expectorate into the provided container.
6. To produce sputum, take a deep breath through your nose, expanding your chest. Hold the air for a moment, then expel it with a forceful cough.
7. If the sample is small, repeat the process in the same container.
8. Deliver the sample to the laboratory within two hours.

Urine Sample Collection for Smear Test

1. Collect six urine samples on consecutive days.
2. Place each sample in a sterile container, provided by the health center, away from light.
3. Perform thorough genital hygiene with soap and water before collecting each sample.
4. Collect 60 to 100 ml of urine for each sample.
5. Deliver each sample to the laboratory within two hours.

Characteristics of M. Tuberculosis

M. tuberculosis is a bacillus with the following characteristics:

• Acid-fast
• Non-sporulated
• Aerobic
• High content of high molecular weight lipids in the cell wall (making staining difficult)
• Slow-growing (replication occurs between 15 and 22 hours; visible growth takes 3 to 6 weeks on solid media incubated at 37°C)
• Note: The bacillus is destroyed by light.

Mycobacterium tuberculosis bacilli cannot be stained with the usual methods because of their remarkable resistance to the penetration of dyes. This is due to the large number of non-saponified waxes present in the cell wall. However, the Ziehl-Neelsen stain, based on the permeability of the bacterium to heated carbol fuchsin, followed by decolorization with acid-alcohol, allows for their identification.

Ziehl-Neelsen Staining Procedure

1. Cover the preparation with carbol fuchsin and heat gently until fumes are produced (without boiling) for 7 to 10 minutes.
2. Wash with water.
3. Decolorize with acid-alcohol as many times as necessary until the thinner parts are colorless.
4. Wash with water.
5. Stain with methylene blue for 10 to 30 seconds.
6. Wash with water.
7. Dry at room temperature.
8. Observe under a microscope at 100x magnification.

Reading and Reporting Ziehl-Neelsen Stain Results

• Negative: No acid-fast bacilli (AFB) are observed in a minimum of 100 fields.
• Positive +: Less than 1 AFB per field in 100 observed fields.
• Positive ++: 1 to 10 AFB per field in 50 observed fields.
• Positive +++: More than 10 AFB per field in at least 20 observed fields.