EX. 8 | Acid-Fast Staining

What is a Differential Stain?

A staining technique that uses two or more dyes to distinguish between different types of microorganisms based on their chemical, physical and cell wall structure properties

Acid-Fast Stain

Differential stain

• Used to identify bacteria with waxy cell walls (primarily mycobacteria), using carbolfuchsin dye, heat, decolorizer, and a counterstain

• To differentiate between acid-fast and non-acid-fast bacteria based on their cell wall composition

Mycolic acid

A waxy, lipid-like substance found in the cell walls of acid-fast bacteria that makes their cell wall thick and impermeable

• However, the disadvantage to this is the fact that it is a low-permeability protective barrier, meaning that it makes it more susceptible for bacteria resistance to increase

• Harder it is to kill bacteria = more likely it is to cause disease

• If the immune system cannot kill it → infection persists → disease develops

Results

Acid-fast

• Has mycolic acid in its cell wall

• Bacteria stain red or pink

• Ability to retain carbolfuchsin (red) even after decolorization with acid-alcohol

  
  
  

Non-acid-fast

• No mycolic acid in their cell wall

• Bacteria stain blue

• Appears blue after methylene blue counterstain

Acid-Fast Organisms

Contains a waxy lipid cell wall known as mycolic acid — renders the cell wall impermeable (doesn’t allow fluid to pass through) to most stains

Ziehl-Neelson Procedure

Smear is flooded with carbolfuchsin (dark red dye containing 5% phenol), which has a high affinity for the mycolic acid in the bacterial cell

• Smear is heated to adhere to the penetration of the carbolfuchsin stain into the bacteria
  
  

• Primary stain → Carbolfuchsin

• Decolorizer → Acid-alcohol

• Counterstain → methylene blue (allows you to observe non-acid fast organisms)

Kinyoun modification

Cold stain

• The concentrations of phenol and carbolfuchsin are increased so that heating isn’t necessary