1. Why Isn't the Gram Stain Used on Acid-Fast Bacteria?

Most specimens are colorless when viewed through a standard light microscope, which is why special preparations are needed for observation. Typically this is done by staining either the cell wall, the unique features within the cell, or the area around the cell, depending on what bacteria is being observed. The Gram stain is a method where the organism is classified as positive or negative based on whether it accepts or repels a violet dye. The acid-fast stain is used on bacteria that don’t react to a regular Gram stain due to the waxy content of their cell walls. There are many types of stains that prove more effective for certain bacteria than for others.

The gram stain was introduced by Hans Christain Gram who classified it into two main groups, negative and positive. The primary stain is a crystal violet dye that stains both gram positive and gram negative bacteria by entering the cell membrane into the cytoplasm, all bacteria appear purple. Next a mordant of iodine is mixed in the smear, and washed off shortly after. The bacteria that retain the dye and mordant are gram-positive; the bacteria that are decolorized are gram-negative. The reason that some cells loose the dye while others retain it is because the peptidoglycan layer is thicker in gram-positive cells. Safranin is a counter stain that is next applied to give color back to the gram-negative cells. The bacteria are classified based on the matrix of their cell walls accepting or rejecting the dyes.

The Gram stain is not useful for acid-fast bacteria that lack the same cell wall properties. Acid fast bacteria have nearly impermeable cells walls containing large amounts of fatty acids, waxes, and complex lipids. Carbolfuschin is applied to the smear, and then heated to help penetration. After the slide is cooled and rinsed, a decolorizer is applied so that only acid-fast positive bacteria remain red. Methylene blue is applied as a counterstain and the acid-fast negative bacteria appear blue....