Acid Fast Stain- Principle, Procedure, Interpretation

What is Acid Fast Staining?

The acid-fast stain is a laboratory test that determines if a sample of tissue, blood, or other body substance is infected with the bacteria that causes tuberculosis (TB) and other illnesses.

It is the differential staining techniques which was first developed by Ziehl and later on modified by Neelsen. So this method is also called Ziehl-Neelsen staining techniques.

Neelsen in 1883 used Ziehl’s carbol-fuchsin and heat then decolorized with an acid alcohol, and counter stained with methylene blue. Thus Ziehl-Neelsen staining techniques was developed.

The main aim of this staining is to differentiate bacteria into acid fast group and non-acid fast groups.

This method is used for those microorganisms which are not staining by simple or Gram staining method, particularly the member of genus Mycobacterium, are resistant and can only be visualized by acid-fast staining.

Principle of Acid-Fast Stain

When the smear is stained with carbol fuchsin, it solubilizes the lipoidal material present in the Mycobacterial cell wall but by the application of heat, carbol fuchsin further penetrates through lipoidal wall and enters into cytoplasm. Then after all cell appears red.

Then the smear is decolorized with decolorizing agent (3% HCL in 95% alcohol) but the acid fast cells are resistant due to the presence of large amount of lipoidal material in their cell wall which prevents the penetration of decolorizing solution.

The non-acid fast organism lack the lipoidal material in their cell wall due to which they are easily decolorized, leaving the cells colorless. Then the smear is stained with counterstain, methylene blue. Only decolorized cells absorb the counter stain and take its color and appears blue while acid-fast cells retain the red color.

Procedure of Acid-Fast Stain

Smear Preparation

Ordinarily, preparing a smear for staining involves applying a very small sample to the center of a carefully cleaned glass slide. The microbial sample is usually taken from a broth culture or a suspension of microorganisms produced by mixing a tiny amount of solid matter from colonies with water.

The suspension can be made directly on the slide or it can be mixed in a tube and transferred to the slide. Since many bacteria cling to each other in culture (both broth and colonial form), vigorous manual or mechanical mixing may be required to produce a proper distribution of the organisms for microscopic evaluation.

Clumps of organisms make it difficult to observe the characteristics of individual cells. The quality of the staining for acid-fast bacteria will be affected by the quality of the smear. A good quality smear will have a thin film of the specimen or culture, allowing individual cells to respond to the staining protocol.

The waxy nature of the mycobacteria organisms causes them to repel water so fluids should be added to the slide after spreading the sample in a thin film over the slide. Organisms grown in media containing complex lipids will stain better, and usually, grow better.

Basic Smear Preparation

1. Clean a glass slide (some labs will provide pre-cleaned slides; be sure to remove any dust or crushed glass debris) according to instructions provided by your instructor.
2. Prepare the sample according to instructions provided by your instructor. Make certain that an aerosol is not generated during this process.
3. Using a sterile pipet or microbiological loop, apply a small sample of the specimen to the slide by slowly spreading the liquid to make a thin film; If you are using solid matter from a colony, be sure to choose a very minute sample and spread it into a very thin film. Applying the cells before adding water (or other mixing fluid) will help the cells adhere to the slide. The size of the film should be about 1 cm in diameter. Avoid any actions that would splatter droplets of the sample in the surrounding area.
4. Allow the smear to dry completely.
5. Fix the smear at 80o C for 15 minutes or for 2 hours on a hot plate set for 65o C to 70o C.
6. Proceed to the staining protocol of your choice.

A. Ziehl-Neelsen method for acid-fast staining

1. Heat fix an air dried smear at 80o C for at least 15 minutes or for 2 hours on an electric hot plate at 65o C – 70o C
2. Place a slide with an air-dried and heat-fixed smear on suitable staining device. Cut a piece of absorbent paper to fit the slide and saturate the paper with the carbolfuchsin stain.
3. Carefully heat the underside of the slide by passing a flame under the rack or by placing the slide on a hot plate until steam rises (without boiling!). Keep the preparation moist with stain and steaming for 5 minutes, repeating the heating as needed.
4. Wash the film in a gentle and indirect stream of tap water* until no color appears in the effluent.
5. Holding the slide with forceps, wash the slide with the decolorizing solvent. Immediately wash with tap water*, as above. Repeat the decolorizing and the washing until the stained smear appears faintly pink and the fluid washing off the slide runs clear.
6. Flood the smear with the methylene blue counterstain for 20 to 30 seconds, and wash with tap water, as above.
7. Gently blot, or air dry the smear.
8. Examine under oil immersion. Acid-fast bacteria appear red, and non-acid-fast bacteria (and other organisms and cellular materials) appear blue.

B. Kinyoun method for acid-fast staining (1)

1. Heat fix an air dried smear at 80o C for at least 15 minutes or for 2 hours on an electric hot plate at 65o C – 70o C
2. Flood slides with Kinyoun’s carbolfuchsin reagent and allow to stain for 5 minutes at room temperature.
3. Rinse with deionized water and tilt slide to drain.
4. Decolorize with acid-alcohol for 3 minutes and rinse again with deionized water.
5. Redecolorize with acid-alcohol for 1-2 minutes or until no more red color runs from the smear.*
6. Rinse with deionized water and drain standing water from the slide surface by tipping the slide.
7. Flood slide with methylene blue counterstain and allow to stain for 4 minutes.
8. Rinse with distilled water and allow to air dry.
9. Examine under high dry (400X) magnification, and confirm acid-fast structures under oil immersion (1000X).

C. Truant method for acid-fast staining.

1. Heat fix an air dried smear at 80o C for at least 15 minutes or for 2 hours on an electric hot plate at 65o C – 70o C
2. Wash the slide with a gentle and indirect stream of distilled water until no color appears in the effluent.
3. Flood the smear with the decolorizing agent for 2 to 3 minutes, and then wash with distilled water as above.
4. Flood the smear with the permanganate counterstain for 2 to 4 minutes.
5. Wash the slide with distilled water as above, blot with absorbent paper, and dry.
6. Examine the slide with a fluorescence microscope equipped with a BG12 exciter filter and an OG-1barrier filter. Acid-fast bacteria appear as brightly fluorescent , yellow-orange cells in a dark field; non-acid-fast cells appear dark.