[4] Acridine orange in the mid-twentieth century was used to examine the microbial content found in soil and direct counts of aquatic bacteria.

Additionally, the method of acridine orange direct count (AODC) proved useful in the enumeration of bacteria found within landfills.

Direct epifluorescent filter technique (DEFT) using acridine orange is a method known for examining the microbial content within food and water.

The use of acridine orange in clinical applications has become widely accepted, mainly focusing on highlighting bacteria in blood cultures.

[3] Acridine orange has been widely accepted and used in many different areas, such as epifluorescence microscopy, and the assessment of sperm chromatin quality.

[7] Acridine orange is recommended for the use of fluorescent microscopic detection of microorganisms in smears prepared from clinical and non-clinical materials.

[citation needed] Acridine orange is also used to stain acidic vacuoles (lysosomes, endosomes, and autophagosomes), RNA, and DNA in living cells.

[9] Acridine orange can be used in conjunction with ethidium bromide or propidium iodide to differentiate between viable, apoptotic, and necrotic cells.