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AAT Bioquest

What is the general procedure of acridine orange staining?

Posted March 12, 2024


Answer

The general procedure of acridine orange staining includes these steps:

  • First, prepare a specimen smear and using a sterile stick, spread it thinly over a clean, grease-free glass slide. 
  • Leave the glass slide with smear to air dry naturally or place it on the heating block to speed up the drying. 
  • Fix the smear by flooding the slide with methanol and draining the excess alcohol. Leave the fixed smear to air dry.
  • Flood slide with acridine orange stain for about two minutes, then drain the excess stain and, using tap water, thoroughly rinse the glass side with smear. Leave it to air dry or gently blot the slide with a clean paper towel or filter paper to speed up the drying time. 
  • Place the smear under a fluorescent microscope and look for distinctive bacterial or fungal morphology. 
Additional resources

Comparison of acridine orange fluorescent microscopy and gram stain light microscopy for the rapid detection of bacteria in cerebrospinal fluid

Fluorescence Microscopy

Flow Cytometry Reagents

Acridine orange *10 mg/mL solution in water*