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Microbiology

Acid-fast

This is a crucial staining method in clinical microbiology, especially when we’re on the lookout for those sneaky Mycobacteria, like Mycobacterium tuberculosis. These guys have a unique cell wall that makes them resistant to traditional staining methods like the Gram stain

Principle of Acid-Fast Staining

  • Mycolic Acid: The key to acid-fast staining lies in the presence of mycolic acid in the cell walls of certain bacteria, primarily Mycobacterium and Nocardia. Mycolic acid is a waxy substance that makes the cell wall impermeable to many stains
  • Mechanism
    1. Primary Stain (Carbolfuchsin): Carbolfuchsin, a red dye, is applied to the smear. Heat (in the Ziehl-Neelsen method) or a detergent (in the Kinyoun method) is used to help the dye penetrate the waxy mycolic acid layer
    2. Decolorization (Acid-Alcohol): This is the crucial step that differentiates acid-fast from non-acid-fast organisms. A strong decolorizer, acid-alcohol (hydrochloric acid in alcohol), is used to remove the carbolfuchsin from non-acid-fast cells. Acid-fast organisms resist decolorization because the carbolfuchsin is tightly bound to the mycolic acid in their cell walls
    3. Counterstain (Methylene Blue or Brilliant Green): A counterstain is applied to stain any non-acid-fast cells that have been decolorized. Methylene blue is the most common counterstain, giving non-acid-fast organisms a blue color

Acid-Fast Staining Procedures

There are two main methods for acid-fast staining:

Ziehl-Neelsen (Hot Method)

  • This method uses heat to drive the carbolfuchsin into the cell wall
  • Procedure
    1. Smear Preparation: Prepare a thin smear of the specimen on a clean slide and allow it to air dry
    2. Heat Fixation: Heat-fix the smear by passing the slide through a flame several times
    3. Carbolfuchsin: Flood the smear with carbolfuchsin stain
    4. Heating: Gently heat the slide from underneath with a flame for 5-10 minutes, keeping the stain steaming but not boiling. Replenish the stain as needed to prevent it from drying out
    5. Rinse: Allow the slide to cool, then rinse with water
    6. Decolorization: Decolorize with acid-alcohol (3% HCl in 95% ethanol) for 2-3 minutes, or until the runoff is clear. Rinse with water
    7. Counterstain: Counterstain with methylene blue for 1-2 minutes
    8. Rinse: Rinse with water and allow to air dry or blot dry
    9. Microscopy: Examine the slide under oil immersion (1000x magnification)

Kinyoun (Cold Method)

  • This method uses a higher concentration of carbolfuchsin and a wetting agent to penetrate the cell wall without heat
  • Procedure
    1. Smear Preparation: Prepare a thin smear of the specimen on a clean slide and allow it to air dry
    2. Heat Fixation: Heat-fix the smear by passing the slide through a flame several times
    3. Kinyoun Carbolfuchsin: Flood the smear with Kinyoun carbolfuchsin stain (which contains a higher concentration of phenol)
    4. Incubation: Allow the stain to sit for 5 minutes
    5. Rinse: Rinse with water
    6. Decolorization: Decolorize with acid-alcohol (3% HCl in 95% ethanol) for 2-3 minutes, or until the runoff is clear. Rinse with water
    7. Counterstain: Counterstain with methylene blue for 1-2 minutes
    8. Rinse: Rinse with water and allow to air dry or blot dry
    9. Microscopy: Examine the slide under oil immersion (1000x magnification)

Interpretation

  • Acid-Fast Organisms: Appear bright red against a blue background. These organisms have retained the carbolfuchsin stain despite decolorization
  • Non-Acid-Fast Organisms: Appear blue. These organisms have been decolorized and have taken up the methylene blue counterstain
  • Reporting
    • Report the presence or absence of acid-fast bacilli (AFB)
    • If AFB are present, estimate the quantity based on the number of AFB seen per field (e.g., rare, few, moderate, many)
    • Example: “Acid-fast bacilli present, 2+.”

Modified Acid-Fast Stain

  • A modified version of the acid-fast stain is used to detect partially acid-fast organisms, such as Nocardia and Cryptosporidium. In this modification, a weaker decolorizer (e.g., 1% sulfuric acid) is used

Quality Control

  • Positive Control: Use a known positive control slide containing Mycobacterium tuberculosis to ensure that the staining procedure is working correctly
  • Negative Control: Use a known negative control slide (e.g., a smear of E. coli) to ensure that the decolorization step is effective

Common Problems and Troubleshooting

  • False-Positive Results
    • Overheating the slide during the Ziehl-Neelsen method can cause non-acid-fast organisms to take up the carbolfuchsin stain
    • Using contaminated reagents
    • Thick smears
  • False-Negative Results
    • Over-decolorization can remove the carbolfuchsin from acid-fast organisms
    • Using old or weak reagents
    • Too few organisms in the specimen

Importance in the Clinical Lab

  • Diagnosis of Tuberculosis: Acid-fast staining is a rapid and inexpensive method for detecting Mycobacterium tuberculosis in sputum and other clinical specimens
  • Diagnosis of Other Mycobacterial Infections: Acid-fast staining can also be used to detect other mycobacteria, such as Mycobacterium avium complex (MAC)
  • Diagnosis of Nocardiosis: Modified acid-fast staining can be used to detect Nocardia in clinical specimens
  • Diagnosis of Cryptosporidiosis: Modified acid-fast staining can be used to detect Cryptosporidium oocysts in stool specimens

Tips for Success

  • Use fresh reagents: Old reagents can give unreliable results
  • Prepare thin smears: Thick smears can be difficult to decolorize properly
  • Control Slides: Use known positive and negative control organisms to ensure your staining technique is correct
  • Proper decolorization!: Ensure proper decolorization timing

Key Terms

  • Mycolic Acid: A waxy substance found in the cell walls of acid-fast bacteria, making them impermeable to many stains
  • Carbolfuchsin: The primary stain used in acid-fast staining, which is a red dye that penetrates the waxy cell wall
  • Acid-Alcohol: The decolorizer used in acid-fast staining, which removes the carbolfuchsin from non-acid-fast cells
  • Methylene Blue: The counterstain used in acid-fast staining, which stains non-acid-fast cells blue
  • Ziehl-Neelsen: A hot method of acid-fast staining that uses heat to drive the carbolfuchsin into the cell wall
  • Kinyoun: A cold method of acid-fast staining that uses a higher concentration of carbolfuchsin to penetrate the cell wall without heat
  • Acid-Fast Bacilli (AFB): Bacteria that retain the carbolfuchsin stain after decolorization and appear red under the microscope
  • Modified Acid-Fast Stain: A variation of the acid-fast stain that uses a weaker decolorizer to detect partially acid-fast organisms