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AAT Bioquest

What are the procedures of acid-fast stain?

Posted March 16, 2023


Answer

Acid-fast stain is a complex procedure that allows scientists to visualize acid-fast bacteria. Here’s an overview of the acid-fast stain procedure: 

  • The specimen smear must be prepared and fixed before it can be stained. 
  • A small strip of filter or blotting paper is placed over the specimen. 
  • The slide is placed on a mesh surface over a beaker of steaming water. Care must be taken not to let the water evaporate completely.  
  • Carbolfuchsin, which is the primary stain, is poured over the filter or blotting paper and the slide is left over the steaming water bath for about 3 to 5 minutes. If the paper starts to dry, more stain can be applied to keep it damp throughout. 
  • After 3 – 5 minutes, the filter paper is removed and the slide is rinsed gently with water until the solution runs clear. 
  • Acid-alcohol decolorizer is then run over the slide for about 15 to 30 seconds, then rinsed with water and left to dry naturally. 
  • The smear is covered with the secondary or counterstain, methylene blue, for about 1 – 1.5 minutes.
  • The slide is rinsed gently with water and blotted dry on both sides with bibulous paper. 
  • The dry slide is placed on the microscope staged and viewed with an oil immersion lens. 
Additional resources

Three-dimensional in situ morphometrics of Mycobacterium tuberculosis infection within lesions by optical mesoscopy and novel acid-fast staining

Gram Staining

MycoLight™ Bacterial Viability Assay Kit