Gram + bacteria stain purple due to the chemistry and structure of their peptidoglycan cell wall. Here’s how Gram staining identifies Gram-positive organisms.
* The Gram Stain *
In the 1800’s, Christian Gram, a Danish bacteriologist, developed a technique for staining bacteria that is still widely used today. The Gram stain protocol involves the application of a series of dyes that leaves some bacteria purple (Gram +) and others pink (Gram -). The specific stain reaction of a bacterium results from the structure of its cell wall.
* The Bacterial Cell Wall *
The rigid bacterial cell wall surrounds the cytoplasmic membrane and provides prokaryotes with protection from their environment. The cell walls of bacteria contain a unique a molecule called peptidoglycan; a huge, strong polymer with interlocking chains of identical monomers connected by interpeptide bridges.
* Gram-positive Cells *
In Gram-positive cells, peptidoglycan makes up as much as 90% of the thick cell wall; more than 20 layers of this polymer stacked together. These peptidoglycan layers are the outermost cell wall structure of Gram + cells, whereas in Gram negative cells, the thinner peptidoglycan component is covered by an external lipopolysaccharide (LPS) membrane.
* Gram Staining Procedure *
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.
The Gram Staining procedure goes as follows:
1. Flood the slide with Crystal Violet (the primary stain).
2. After 1 minute, rinse the slide with water.
3. Flood the slide with Iodine (Iodine is a mordant that binds the Crystal violet to the Gram + cell wall.)
4. After 1 minute, rinse the slide with water.
5. Flood the slide with Acetone Alcohol. (Alcohol is a decolorizer that will remove the stain from the Gram-negative cells.)
6. After 10 or 15 seconds, rinse the slide with water. (Do not leave the decolorizer on too long or it may remove stain from the Gram-positive cells as well.)
7. Flood slide with Safranin (the counterstain).
8. After 1 minute, rinse the slide with water.
9. Gently blot the slide dry. It is now ready to be viewed under oil immersion (1000x TM) with a bright-field compound microscope.
* Gram Positive Stain Reaction *
The blue-violet stain reaction characteristic of Gram-positive cells is caused by crystal-violet, the primary Gram-stain dye, which interacts with the iodine mordant causing the complex to adhere to the peptidoglycan. The decolorizer step does not remove the crystal-violet stain bound to the Gram-positive cell wall. The safranin counterstain is much lighter than crystal violet and even after application of the counterstain the Gram-positive cell maintains its blue-violet color.
* Sources *
Bauman, R. (2005) Microbiology.
Park Talaro, K. (2008) Foundations in Microbiology.