Diagnosis of TB by Sputum Microscopy/Acid fast staining (Part 17)
Welcome to the 17th part of the multi-part series on Tuberculosis, abbreviated as TB.
The next method to detect TB is by staining Mtb bacteria present in the sample and then seeing it under a microscope. The sample used in this technique is generally sputum. Sputum is the mucus that you cough up from your lungs.
The principle behind staining bacteria is that different bacteria have different ways of responding to staining agents, and this allows scientists to differentiate between them. Most of the bacteria can be differentiated by gram staining, which categorizes them as either gram negative or gram positive. However large amounts of lipid substances called mycolic acids within the cell walls of Mtb resist the gram stain because gram stain can’t penetrate the waxy wall of Mtb. Thus a different lipid soluble dye needs to be used to stain Mtb bacteria.
To do this TB test a very thin layer of the sputum sample is placed on a glass slide, which is called a smear and then the TB bacteria is stained with an agent that binds to the mycolic acids on the cell wall. Staining of Mtb bacteria is typically done using the Ziehl-Neelson method, which is named after the two scientists who described it in the late 1800s. In the first step, the staining of bacteria is done using Carbol Fuchsin, a red-colored dye that can stain every bacteria in the sample. Then the heat is applied that allows the stain to penetrate inside the cell wall of Mtb. Then in a decolorizing step, the bacteria are destained with acid-alcohol, in this step only non-acid-fast bacteria get destained since they do not have a thick, waxy lipid layer like acid-fast bacteria. When counterstain i.e. methylene blue is applied, non-acid-fast bacteria pick it up and appear blue in color when viewed under the microscope.
Acid fastness is a property that gives a bacterium the ability to resist decolorization by acid-alcohol during staining procedures.
On the other hand, acid-fast bacteria retain the primary stain Carbol Fuchsin due to lipid-rich mycolic acids in the cell wall because of which they appear red in color when viewed under the microscope. Thus positive Acid fast bacilli (AFB) in the sputum sample provide the first indication of mycobacterial infection and potential TB disease. And the results obtained by this TB detection method are available within hours.
However, the sensitivity of this TB detection method is only about 50–60% and the sputum smears often do not detect TB, especially in people who have advanced HIV disease because people with HIV and TB co-infection have very low levels of TB bacteria in their sputum, and are therefore recorded as sputum negative.
Other limitations of sputum microscopy for acid-fast bacilli (AFB) is that this test cannot distinguish:
- Mycobacterium tuberculosis from Non-tuberculous mycobacteria
- Viable from non-viable organisms
- And Drug-susceptible from drug-resistant strains.
Thus sputum microscopy test must be accompanied by additional tests including a culture test for confirmatory diagnosis. The culture test will be discussed in Part 18.
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