• Gram staining works by the pepetidoglycan in the cell walls of the bacteria. Gram positive bacteria have thick layers of peptidoglycan. Gram negative have very thin layers of peptidoglycan. It is this peptidoglycan layer that determines the results of the Gram stain. In old gram +ve cells the peptidoglycan can degrade and break down. Obviously this will skew the results of the Gram test.
  • Gram stains act to stain the peptidoglycans in the bacterial cell wall. Gram positive cells have thick cell walls that take up the stain. Gram negative cells have a cell wall with a lipid layer over a thin peptidoglycan layer. The lipids prevent the stain from reaching the peptidoglycans underneath and so they are not stained. However, when the cells age and begin to die the lipid layer can be degraded which allows the stain access to the peptidoglycans.

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