The Gram stain is ineffective for acid-fast bacteria due to their mycolic acid-rich cell walls, which resist decolorization. In the Gram staining process, bacteria are differentiated based on their cell wall composition, with Gram-positive bacteria retaining the violet stain, whereas Gram-negative bacteria turn pink after the application of a secondary stain. Acid-fast bacteria may appear similar to Gram-positive bacteria but do not lose color because of their unique cell wall characteristics.

1. Why is it that the Gram's stain is not appropriate for acid fast bacterium?
Solution
During staining in microbiology, microbes are coloured or dyed so that their structure can be
viewed against a lighter background. There are different staining techniques such as direct(Dye
binds to the cell surface and all the microbes are stained alike),differential(based on their specific
cell surface different microbes get stained differently) special (usually a particular organelle is
targeted)and negative staining(back ground is stained instead of microbes) Gram stain is a
differential stain where the bacteria with a thick peptidoglycan layer around the cells are stained
with violet colour and those with an additional lipopolysaccharide layer will be stained pink. To
understand this lets take a look at Gram staining procedure. First of all bacterial culture is
smeared onto a glass slide and is heat fixed so that during the washes in the procedure the
bacteria stay fixed onto the slide. Primary stain crystal violet is added onto the slide and the dye
seperates into ions which penetrate or enter the cell membranes of both Gram positive and
negative cells. Next iodine is added onto the slide which forms huge complexes with crystal
violet. Then a dye remover usually called as decolouriser such as alchohol or acetone is used to
remove the stain. In Gram negative bacteria the outer most membrane is lost during the treatment
with alchohol whereas in the case of gram positive bacteria the dye is trapped inside the cell
membrane. A secondary stain saffronin is used and which gives pink colour to Gram negative
bacteria. Now coming to acid-fast bacteria, they have a compound called mycolic acid in their
cell walls and are resistant to decolourisation after the acid treatment and alchohol and hence
would not lose colour and appear violet. As we can see these organism can be easily confused
with Gram positive bacteria which also do not lose stain not because they are resistant to acids
but they have the crystal violet dye trapped in their cell membrane.