Bacteria have cell walls made up of polysaccharides that give them strength and rigidity. This is important since bacteria often experience variations in osmotic strength from solutions of their environment, and their cell walls prevent them from shrinking or swelling. As a reminder, osmosis is the process by which solvent molecules pass through a semipermeable membrane from a less concentrated solution to a more concentrated one, equalizing the concentration on either side of the membrane. Nearly all bacterial cell walls have a peptide-polysaccharide layer called peptidoglycan, or sometimes murein. Peptidoglycan is a polymer made up of sugars and amino acids which forms a kind of mesh.
Bacteria can be classified based on their reaction to the Gram stain, which identifies them based on the chemical and physical properties of their cell walls, as gram positive or gram negative. Gram positive bacteria have a thick cell wall, which consists of up to around 30 layers of peptidoglycan. This cell wall surrounds a monoderm, which is a single plasma membrane. Gram negative bacteria have a MUCH thinner cell wall, consisting of a single layer of peptidoglycan. This layer of peptidoglycan is sandwiched between two lipid bilayer membranes called diderms.
We can differentiate between gram positive and gram-negative bacteria by dying them with crystal violet and then washing them with a decolorizing solution. Then, a counterstain is added, for example safranin or fuchsine. Gram positive bacteria will retain the crystal violet dye and remain purple, while the gram-negative bacteria will be stained pink. Note that gram positive bacteria also pick up the pink colour of the counterstain, however this is not visible when they are dyed with the darker purple colour of the crystal violet stain.
The reason for these staining differences is due to differences in cell wall structure, which is the chief difference between gram positive and gram-negative bacteria. The gram stain detects peptidoglycan, and since Gram positive bacteria have a thick, multilayered peptidoglycan layer, they retain the crystal violet dye. Gram-negative bacteria do not retain the dye for two reasons – they have an outer membrane getting in the way of the crystal violet and they lack peptidoglycan to retain the stain.
Although both gram positive and gram-negative bacteria can be pathogenic, gram negative bacteria are more resistant to antibodies because of their impenetrable cell wall. Unfortunately, these bacteria also develop resistance more quickly.
Not all bacteria can be reliably classified through Gram staining. Acid-fast bacteria or Gram-variable bacteria for example, do not respond to Gram staining. Acid-fast bacteria have cell walls that retain stains particularly well. Although they aren’t closely related to gram-positive bacteria, they can appear purple after the gram stain test. Gram-variable bacteria show a mix of pink and purple cells when stained.
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