Bacteria can be differentiated into Gram positive or Gram negative by testing them with the “Gram stain”.  The stain attaches to part of the bacterial cell wall called peptidoglycan and causes a purple colouration.

Gram Positive and Gram Negative Bacteria

Shown above are two types of bacteria which have both been treated with the Gram stain.  The Staphylococcus on the left is Gram positive and has been stained purple.  The E. coli on the left however has not been stained purple as it is a Gram negative bacteria.  Both Gram positive and negative bacteria contain peptidoglycan in the cell wall, but in the Gram negative, various other layers protect it and so the stain cannot reach it to cause the colouration.

The peptidoglycan layer is on the outside of the Gram Positive bacteria and the Gram stain can easily reach it.  On the inside of the peptidoglycan layer is the cell membrane, which also contains proteins and channels.

In Gram negative bacteria, the peptidoglycan is still outside of the inner membrane, but now there is a more complex outer membrane present.  The stain cannot penetrate this outer membrane to get to the peptidoglycan layer to colour it.

Gram Negative bacterial cell wall

The outer membrane is a lipid bilayer similar to that of the inner membrane, but now it contains lipopolysaccharides [LPS].  These always face the outside and are involved in recognition and also increase the barrier for molecules entering into the cell.

The space between the peptidoglycan layer and the outer membrane is called the periplasm and it contains many different proteins.

The outer membrane also contains porins, which are proteins that form pores in the membrane and allow small hydrophilic molecules to pass into or out of the cell.  Hydrophobic or larger molecules cannot pass through the porins and this is how the Gram stain is prevented from reaching the peptidoglycan layer to colour it.10

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