Mycobacterium Tuberculosis – Part 3 – AFB Culture (Acid Fast Bacilli)
- AFB cultures can be done on sputum.
- Material from other tissues like the endometrium and chronic ulcers.
- Biopsy from the lung may be taken when the possibility of Mycobacterium Tuberculosis is suspected.
- Pleural fluid should be at least 2 ml and refrigerate until the culture is done.
- Bronchial washing should be at least 2 ml and also refrigerate until processed for culture.
Purpose of the test (Indications)
- For the diagnosis of Mycobacterium tuberculosis in the sputum.
- For the diagnosis of TB meningitis.
- For the diagnosis of tuberculosis in other sites like lymph nodes, chronic ulcers, and pleurisy.
- Any patient with a persistent cough, fever, night sweating, anorexia, weight loss, and hemoptysis.
- Pulmonary tuberculosis can be diagnosed from the sputum. Always try to get deep sputum and avoid saliva.
- When tissue is taken from the patient, then this tissue is smeared on the slides and try to do stain when the tissue is fresh.
- The smear sensitivity is low so it is followed by the culture.
Pathology (Microbiology of AFB)
- Mycobacteria is called acid-fast because after mordanting in the stain they resist decolorization by the strong acids.
- This staining property is due to the composition of the cell wall which has high lipid contents.
- These have poor staining with gram stain.
- One of the cell lipid form complex with acid and mycolic acid.
- Mycobacteria are divided into:
- Fast-growing which forms colonies in 2 to 3 days.
- Slow growing which forms the colony in 1 to 3 weeks.
- The most common AFB is Mycobacterium tuberculosis (T-B).
- These are pleomorphic rod-shaped bacteria containing a large amount of mycolic acid in the wall, which makes it difficult to stain.
- 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.
- Mycobacterium is gram resistant and non-motile bacteria.
- These are intracellular pathogens.
- M.Tuberculosis, M.bovis, M.africanum, M.microti, all these commonly known as Tuberculosis (T B).
- M.Tuberculosis is pathogenic for humans, while M. bovis usually for animals.
- Mycobacteria are:
- obligate anaerobes and easily grow in the lung with high oxygen contents.
- These are facultative intracellular pathogens usually involving mononuclear phagocytes.
- These are slow-growing bacteria and may take a few days to weeks.
- These are Hydrophobic with a high lipid content in the cell wall.
- Because the cells are hydrophobic and tend to clump together, they are impermeable to the usual stains, e.g. Gram’s stain.
- Known as “acid-fast bacilli” because of their lipid-rich cell walls.
- Once stained, the cells resist decolorization with acidified organic solvents and are therefore called “acid-fast”.
- These are slender, beaded bacilli, and nonsporing.
- Colonies are rough, dry, and yellow color on Lowenstein-Jensen media.
- These are slow-growing bacteria on this medium.
- At least 5000 Acid-fast bacilli must be present in each mL of the specimen to be seen on the microscopic smear.
- The culture is done on special culture media called L.J (Lowenstein-Jensen ) medium. and needs 4 to 6 weeks for the growth of bacilli.
- LJ medium contains whole egg, asparagine, glycerol, and material which inhibit the growth of infectious material like malachite green.
- It needs 4 to 6 weeks for the growth of bacilli at 37 °C.
- These are obligate aerobic, do not grow on ordinary media.
- This is eugonic growth on LJ medium.
- These are obligate aerobes, does not grow on ordinary media.
- Keep culture for 6 to 8 weeks before the media is discarded.
- Smear from the culture shows these AF Bacilli as red color rods single or in clusters. Morphologically these are pleomorphic.
- Acid-fast bacilli may be seen in the caseous material of tuberculosis but sputum gives a higher recovery of tubercle bacilli.