Acid-fast Staining

Perform acid-fast Staining for the given sputum sample

 
Objective: To detect the presence of acid-fast bacilli (AFB) such as Mycobacterium tuberculosis in a given sputum sample.

Theory:

Acid-fast staining is a differential staining technique commonly used to detect acid-fast organisms, such as Mycobacterium tuberculosis, which have a unique cell wall composed of waxy, lipid-rich material called mycolic acids. These bacteria retain the primary dye (carbol fuchsin) even after being washed with acid-alcohol, due to the presence of mycolic acid in their cell walls. Non-acid-fast bacteria get decolorized and take up the counterstain (methylene blue or malachite green)

The acid-fast staining or Ziehl-Neelsen staining method is based on:

• Staining the smear with carbol fuchsin while applying heat, which facilitates dye penetration.
• Decolorization with acid-alcohol, which removes the stain from non-acid-fast organisms.
• Counterstaining with methylene blue or malachite green to visualize non-acid-fast cells.

Acid-fast bacteria (AFB) appear red/pink rods, while non-acid-fast organisms and the background appear blue.

Requirements:

• Sputum sample
• Glass slide
• Bunsen burner
• Carbol fuchsin
• Acid-alcohol (3% HCl in ethanol or 25% sulfuric acid)
• Methylene blue
• Microscope
• Immersion oil

Procedure:

1. Take a small amount of sputum with an applicator stick and spread it evenly on a clean glass slide to make a thin smear. Allow it to air dry.
2. Pass the dried smear over a flame 2–3 times to heat fix the bacteria onto the slide.
3. Flood the slide with carbol fuchsin stain and keep it over steam for about 5 minutes
4. Continue heating for 5 minutes, ensuring that the stain does not dry out. Add more stain if necessary.
5. Remove the slide from the heat and allow it to cool for 2 minutes.
6. Rinse the slide gently with tap water to remove excess stain.
7. Decolorize the slide with acid-alcohol for about 1 minute
8. Rinse the slide with tap water to remove the acid-alcohol.
9. Counterstain the slide with methylene blue for 1 minute.
10. Rinse the slide with tap water and blot it dry using blotting paper.
11. Observe the slide under oil immersion microscopy using a 100x objective lens.
12. Record the presence or absence of acid-fast bacteria in the sample.

Observation:

S.N	Sample	Reagents used	Shape of bacteria	Color of bacteria	Inference

 

 Results: Acid-fast bacteria appear as red, rod-shaped cells against a blue background due to the counterstaining with methylene blue. Non-acid-fast bacteria appear blue.

Discussion:

Carbol fuchsin, the primary red dye, is lipid-soluble and, when heated, penetrates the waxy cell wall of acid-fast bacteria. Once inside, the dye binds tightly to the mycolic acid. When we apply acid-alcohol (decolorizer), acid-fast bacteria retain the red dye because their waxy wall prevents it from washing out. Non-acid-fast bacteria do not have this waxy layer, so the carbol fuchsin washes out easily. After decolorization, we apply a blue or green counterstain, which the non-acid-fast bacteria take up, making them appear blue or green under the microscope.

The presence of acid-fast bacilli (AFB) in a sputum sample indicates possible infection by Mycobacterium tuberculosis or other mycobacteria. The Ziehl-Neelsen technique is a crucial diagnostic tool, especially in developing countries with high TB prevalence. The specificity of the test is high, but its sensitivity can be limited if a bacterial load is low, so repeated samples or culture may be required.

 Conclusion:

The acid-fast staining procedure was successfully performed, and the presence or absence of acid-fast bacteria in the sample was determined. This staining technique is valuable for the identification of acid-fast organisms, particularly Mycobacterium tuberculosis, aiding in the diagnosis of tuberculosis and other related diseases.

Precautions:

• Always wear gloves, a mask, and a lab coat while handling sputum samples.
• Do not overheat the slide during heat fixation or staining.
• Use a biosafety cabinet while handling infectious samples.
• Dispose of all materials properly as per biosafety guidelines.

References:

1. Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries, Part 2. Cambridge University Press.
2. World Health Organization (WHO). (2013). Laboratory services in tuberculosis control.