Gram staining involves the application of a series of dyes that leaves some bacteria purple (Gram +) and others pink (Gram -). Here's how the Gram stain works.

From Spontaneous Generation to Germ Theory

Through most of recorded history, illnesses that we now recognize as infectious disease were not linked with microbial life. Many living things, including microscopic life forms, were thought to arise through spontaneous generation (life coming from nonliving matter).

Once microbes were finally recognized as being a source of infectious disease, and Germ Theory became more widely accepted, scientist needed a method to detect and identify microbial life forms. However, in most cases, microbes are colorless and difficult to see.

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 and others pink. Bacteria that stain purple and termed Gram-positive, and those that stain pink, Gram-negative. The specific stain reaction of a bacterium results from the structure of its cell wall.

Components of the Bacterial Cell Wall

Peptidoglycan Structure and Function

This rigid structure of peptidoglycan gives the bacterial cell shape, surrounds the cytoplasmic membrane and provides prokaryotes with protection from their environment. Peptidoglycan is a huge polymer of interlocking chains of identical monomers connected by interpeptide bridges.

From the peptidoglycan inwards all bacterial cells are very similar. Going further out, the bacterial world divides into two major classes: Gram positive (Gram +) and Gram negative (Gram -).

Gram-positive Cells

In Gram-positive cells, peptidoglycan makes up as much as 90% of the thick, compact cell wall, which is the outermost cell wall structure of Gram + cells.

Gram-negative Cells

The cell walls of Gram - bacteria are more chemically complex, thinner and less compact. Peptidoglycan makes up only 5 – 20% of the cell wall, and is not the outermost layer, but lies between the plasma membrane and an outer membrane. This outer membrane is similar to the plasma membrane, but is less permeable and composed of lipopolysaccharides (LPS), a harmful substance classified as an endotoxin.

Gram Staining Procedure

Because most bacteria have one of these two types of cell walls, we can use this difference as a feature that can be identified using the Gram stain. The Gram stain is a differential stain that uses two dyes to differentiate between the two basic bacterial cell wall types.

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 Safrinin (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.

After this staining procedure, the Gram + cells will appear purple, having retained the primary stain. The Gram – cells will appear pink, having retained the counterstain after the primary stain was removed by the decolorizer.

Additional Microbiology Information

For more information on prokaryotes and cell biology, go to the excellent websites of Science Prof Online and Cells Alive or see additional related Suite101 articles, including Prokaryotic & Eukaryotic Cells.

Sources

Bauman, R. (2005) Microbiology.

Park Talaro, K. (2008) Foundations in Microbiology.