Mycobacterium Tuberculosis (AFB) Culture

What sample is needed for the AFB culture sample?

1. AFB cultures can be done on sputum.
2. Material from other tissues like the endometrium and chronic ulcers.
3. Biopsy from the lung may be taken when the possibility of Mycobacterium Tuberculosis is suspected.
4. Pleural fluid should be at least 2 ml and refrigerated until the culture is done.
5. Bronchial washing should be at least 2 ml and also refrigerate until processed for culture.

What are the Indications for Mycobacterium Tuberculosis Culture (AFB)?

1. For the diagnosis of Mycobacterium tuberculosis in the sputum.
2. For the diagnosis of TB meningitis.
3. For the diagnosis of tuberculosis in other sites like lymph nodes, chronic ulcers, and pleurisy.
4. Any patient with a persistent cough, fever, night sweats, anorexia, weight loss, and hemoptysis.

What are the precautions for Mycobacterium Tuberculosis Culture (AFB)?

1. Pulmonary tuberculosis can be diagnosed from sputum. Always try to get deep sputum and avoid saliva.
2. When tissue is taken from the patient, it is smeared on the slides and stained when it is fresh.
3. The smear sensitivity is low, so the culture follows it.

What is the microbiology of Mycobacterium Tuberculosis (AFB)?

1. Mycobacteria are called acid-fast because after mordanting in the stain, they resist decolorization by the strong acids.
   1. This staining property is due to the cell wall composition, which has high lipid contents.

2. These have poor staining with gram stain.
3. One of the cell lipids forms a complex with acid and mycolic acid.
4. Mycobacteria are divided into:
   1. Fast-growing, which forms colonies in 2 to 3 days.
   2. Slow growing, which forms the colony in 1 to 3 weeks.
5. The most common AFB is Mycobacterium tuberculosis (TB).
   1. These pleomorphic rod-shaped bacteria contain a large amount of mycolic acid in the wall, making it difficult to stain.
   2. When heat or other agents are used to force carbol fuchsin to enter the cytoplasm, the bacteria resist decolorization with a dilute acid-alcohol solution. Therefore, they are called acid-fast bacilli.
   3. Mycobacterium is a gram-resistant and non-motile bacteria.
6. These are intracellular pathogens.
7. Tuberculosis, M.bovis, M.africanum, and M. microti are all commonly known as Tuberculosis (T B).

4. Tuberculosis is pathogenic for humans, while M. bovis usually for animals.
5. Mycobacteria are:
   1. Obligate anaerobes easily grow in the lungs with high oxygen content.
   2. These are facultative intracellular pathogens, usually involving mononuclear phagocytes.
   3. These are slow-growing bacteria and may take a few days to weeks.
   4. These are Hydrophobic with a high lipid content in the cell wall.
   5. Because the cells are hydrophobic and tend to clump together, they are impermeable to the usual stains, e.g., Gram stain.
   6. They are known as “acid-fast bacilli” because of their lipid-rich cell walls.
   7. Once stained, the cells resist decolorization with acidified organic solvents and are therefore called “acid-fast.”
   8. Morphology:
      1. These are slender, beaded bacilli and nonsporing.
      2. Colonies are rough, dry, and yellow in color on the Lowenstein-Jensen media.
      3. These are slow-growing bacteria in this medium.
6. At least 5000 Acid-fast bacilli must be present in each mL of the specimen to be seen on the microscopic smear.

What are the media for the culture of Mycobacterium tuberculosis?

Solid medium:

1. The culture is done using a special culture medium called L. J (Lowenstein-Jensen ) medium. Bacilli need 4 to 6 weeks to grow.
   1. LJ medium contains a whole egg, asparagine, glycerol, and material that inhibits the growth of infectious material, such as malachite green.
2. It takes 4 to 6 weeks to grow bacilli at 37 °C.
3. These are obligate aerobics and do not grow on ordinary media.
4. This is eugonic growth on the LJ medium.
5. These are obligate aerobes and do not grow on ordinary media.
6. Keep culture for 6 to 8 weeks before the media is discarded.
7. Smear from the culture shows these AF Bacilli as red color rods single or in clusters. Morphologically, these are pleomorphic.

Acid-Fast Bacilli Culture: AFB Stain for Mycobacteria

Liquid medium:

1. These are better than solid mediums.
2. The growth is rapid, and it takes 12 to 16 days instead of 4 to 6 weeks.
3. These are more easily contaminated, so need antimicrobial agents.
4. Incubation with additional CO2 is not required.

Automated system:

1. Continuously needs monitoring to see the growth for 6 weeks.

Important facts:

• Acid-fast bacilli may be found in tuberculosis caseous material, but tubercle bacilli recover more quickly in sputum.

Questions and answers: