Why Gram-positive bacteria stay purple on a Gram stain
Gram-positive bacteria look purple after a Gram stain because their thick peptidoglycan wall, a mesh of sugars and short protein chains, holds the crystal violet-iodine complex.
Remember
Gram-positive bacteria stay purple because a thick peptidoglycan wall traps the crystal violet-iodine complex, while Gram-negative bacteria lose it and so look pink.
Memory aid
positive keeps the purple
Use it to recall that the thick wall of a Gram-positive bacterium holds on to the first stain.
The Gram stain is a quick test that sorts bacteria by the structure of their cell wall. A sample such as urine, stool or blood is smeared on a slide, stained and looked at. It is almost always the first step in identifying a bacterium.
The wall contains peptidoglycan, a mesh of sugars and short protein chains that strengthens it. Gram-positive bacteria have many layers of it. Gram-negative bacteria have only a thin layer, covered by a second outer membrane that contains lipopolysaccharide (LPS, a large molecule in that outer membrane).
The Gram stain has four steps. Crystal violet, a purple dye, colours every cell. Gram's iodine, a mordant (a trapping agent), joins the dye to form a large crystal violet-iodine complex. Alcohol or acetone then washes the slide, taking the dye out of cells that cannot hold it. Finally, safranin, a pink dye, is added as a counterstain, which colours the cells that lost the purple.
In a Gram-positive cell, the thick peptidoglycan holds the complex through the wash, so the cell stays purple. In a Gram-negative cell the complex escapes, and the cell takes up the pink safranin instead. So the stain detects peptidoglycan indirectly, through how well each wall retains the dye.
Together with the shape of the bacteria, the colour narrows the likely organisms. The stain has limits, because not all bacteria can be definitively classified this way.
Key facts
- Gram-positive
- a thick peptidoglycan wall keeps the crystal violet-iodine complex, so the cells stay purple.
- Gram-negative
- a thin peptidoglycan layer plus a lipopolysaccharide-rich outer membrane loses the complex, so the cells stain pink with safranin.
- What it sorts by
- the thickness of the peptidoglycan layer in the cell wall.
- Mordant
- Gram's iodine, a trapping agent, joins the dye to form a large complex.
- Use and limit
- it narrows the likely organisms and guides the first antibiotic, but does not identify every bacterium.
Exam traps
- Teichoic acids (a component of Gram-positive walls) do not covalently bind the crystal violet-iodine complex. The thick peptidoglycan layer is what keeps it through the alcohol or acetone wash (decolourisation).
- A lipopolysaccharide-rich outer membrane is a feature of Gram-negative bacteria. The alcohol or acetone wash (decolourisation) disrupts it, so the crystal violet washes out and the cell looks pink, not purple.
- A capsule (a sugary outer coat) does not decide Gram colour, because encapsulated bacteria can be Gram-positive or Gram-negative.
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