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Most acid-fast microorganisms belong to the genus Mycobacterium because organisms of this species have a unique property where they have a lipid rich mycolic acid exterior that prevents ordinary dyes from penetrating.

When decolorized and counterstained, they maintain the red of the carbolfushin, thus they are acid fast.

E. coli is affected by the decolorization by the acid achohol and counterstain by the methylene blue dye because they lack the mycolic acid layer, thus they are non-acid fast.

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