Acridine orange fluorescent stain is used in clinical microbiology to detect bacteria in blood culture broths or CSF. The basis of its selectivity is that acridine orange:

• A Binds to peptidoglycan in bacterial cell walls, fluorescing orange only in gram-positive bacteria
• B Binds to bacterial ribosomes and mitochondria selectively due to the membrane potential difference
• C Acts as a vital stain, fluorescing green in live bacteria and orange in dead bacteria
• D Intercalates into nucleic acids (DNA/RNA); bacteria appear orange-red while host cell nuclei appear green-yellow at different staining concentrations ✓

Explanation

Acridine orange (AO) is a fluorescent dye that intercalates non-selectively into both DNA and RNA. At optimal pH (3.5–4.0), single-stranded RNA fluoresces orange-red, while double-stranded DNA fluoresces green-yellow. Bacteria, which have a high RNA:DNA ratio (due to abundant ribosomes), appear bright orange-red, while host cell nuclei (predominantly dsDNA) appear yellow-green. This color contrast allows visual discrimination of bacteria from background material. AO staining is 10–20 times more sensitive than gram staining for detecting small numbers of organisms in blood culture broths, CSF, and smear-positive preparations. It is not selective for gram-positive organisms and does not require viable bacteria.

Reference: Ananthanarayan & Paniker's Textbook of Microbiology, 11th ed.

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Written and medically reviewed by the StethoPrep medical team.