- Objective of Acid Fast Stain
- Principle of Acid Fast Stain
- Ziehl-Neelsen Stain Reagents
- Procedure of Acid Fast Stain
- Result Interpretation of Acid Fast Stain
- Limitations
- Examples
Objective of Acid-Fast Stain:
- The primary goal of the acid-fast stain is to distinguish bacteria into acid-fast and non-acid-fast groups.
Principle of Acid-Fast Stain:
The acid-fast mycobacteria possess mycolic acid in their outer membrane, imparting a waxy nature to the cells and rendering them resistant to staining with aqueous-based stains like the Gram stain. In this staining technique, the primary stain, carbolfuchsin, is applied to the cells. Heat and phenol are employed to facilitate the penetration of the stain into the waxy surface of acid-fast microorganisms. Excess stain is eliminated through treatment with acid alcohol, a mixture of ethanol and hydrochloric acid. Subsequently, a secondary stain, methylene blue, is used on the cells.
Ziehl-Neelsen Stain Reagents:
Primary Stain:
- Prepare a solution of 0.3% Carbol-fuchsin by dissolving 50 g phenol in 100 mL ethanol (95%) or methanol (95%). Add 3 g Basic fuchsin to the mixture and bring the volume to 1 L with distilled water.
Decolorization Solution:
- For alcohol-based decolorization, add 30 mL hydrochloric acid to 1 L of 95% denatured alcohol. For an alternate decolorizing reagent without alcohol, slowly combine 250 mL sulfuric acid (at least 95%) with 750 mL distilled water. Ensure proper cooling and mixing before use.
Counterstain:
- Prepare a 0.3% solution of methylene blue by dissolving 3 g methylene blue in 1 L distilled water.
Procedure of Acid-Fast Stain:
- Prepare and fix the specimen smear before staining.
- Place a small strip of blotting or filter paper over the specimen and position the slide over a boiling hot water bath on a mesh surface.
- Cover the filter paper with the primary stain, carbolfuchsin, and leave the slide on the water bath for 3 to 5 minutes. Reapply stain if the filter paper starts to dry.
- Remove the filter paper and rinse the slide with water until the solution runs clear.
- Apply acid-alcohol decolorizer over the slide for approximately 10 to 15 seconds.
- Rinse the slide with water.
- Cover the smear with the secondary or counterstain, methylene blue, for 1 minute.
- Gently rinse the slide with water.
- Blot the slide dry with bibulous paper.
Result Interpretation of Acid-Fast Stain:
- Acid-fast: Cells appear bright red to intensive purple, appearing as red, straight, or slightly curved rods, either singly or in small groups. They may exhibit a beaded appearance.
- Non-acid-fast: Cells show a blue color, and the background material should also stain blue.
Limitations:
Moisture Requirement during Heating:
- Proper penetration of the primary stain requires the filter paper to remain moist and in contact with the specimen during heating.
Effect of Nutrient Deprivation:
- Organisms cultivated on blood agar may experience nutrient deprivation, leading to a lower lipid content in the outer membrane. This can result in poor staining outcomes.
Examples:
Acid-fast:
- Mycobacterium tuberculosis
- Mycobacterium smegmatis
Non-Mycobacterial Bacteria:
- Nocardia
Coccidian Parasites:
- Cryptosporidium