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First a bacterial smear must be heat fixed to a microscope slide. A smear is a sample of bacteria suspended in a small amount of water on a slide. That sample is then dried using heat. The heat kills the bacteria and attaches the sample to the slide so that it does not easily wash away.
From the peptidoglycan inwards all bacterial cells are very similar. Going further out, the bacterial world divides into two major classes: Gram positive (Gram+) and Gram negative (Gram-).
Gram-positive Cells: In Gram-positive bacterial cells, peptidoglycan makes up as much as 90% of the thick, compact cell wall, which is the outermost cell wall structure of Gram+ cells.
Gram-negative Cells: The cell walls of Gram-negative bacteria are more chemically complex, thinner and less compact. Peptidoglycan makes up only 5 – 20% of the cell wall, and is not the outermost layer, but lies between the plasma membrane and an outer membrane. This outer membrane is similar to the plasma membrane, but is less permeable and composed of lipopolysaccharides (LPS), a harmful substance classified as an endotoxin.
Gram Staining Procedure
Because most bacteria have one of these two types of cell walls, we can use this difference as a feature that can be identified using the Gram stain. The Gram stain is a differential stain that uses two dyes to differentiate between the two basic bacterial cell wall types.
Peptidoglycan is a huge polymer of interlocking chains of NAG and NAM polysaccharide monomers connected by interpeptide bridges.