Why do dead cells stained with AO/EB appear orange?

Dead cells stained with acridine orange (AO) and ethidium bromide (EB) appear orange due to the way ethidium bromide interacts with DNA when it's in an environment where the cell membrane integrity is compromised. In live cells, acridine orange can readily penetrate and bind to RNA and DNA, fluorescing green due to its interaction with RNA and still fluorescing green with double-stranded DNA. However, when the cell is dead, ethidium bromide enters the cell because of the compromised membrane integrity.

Ethidium bromide intercalates more effectively with double-stranded DNA and fluoresces red when bound to it. When both dyes are present in a dead cell, the red fluorescence of ethidium bromide can dominate or mix with the green fluorescence of acridine orange, resulting in an orange appearance when viewed under a fluorescence microscope. This phenomenon occurs because the red fluorescence has a higher intensity compared to the green fluorescence of acridine orange in this scenario, leading to the overall orange color observed.

In this context, some other choices might suggest alternative mechanisms that aren't correct. For instance, stating that ethidium bromide prevents AO binding misunderstands the principle of how these two dyes interact with nucleic acids under different cellular conditions. Similarly, the