What are the limitations of acridine orange stain?

Acridine orange is a cell-permeable, nucleic acid selective fluorescent dye. 

Limitations of acridine orange stain include: 

• Must be performed on unfixed tissue and examined quickly
• Cannot be used for multiple labeling
• Is non-specific and does not differentiate between gram-positive and gram-negative bacteria 
• Staining of cellular nuclei makes it difficult to visualize intracellular organisms
• Its sensitivity is approximately 104 bacteria/ml