Gram- bacteria stain pink due to the location of cell wall peptidoglycan and an external LPS membrane. Here’s how Gram staining identifies Gram negative organisms.
* Christian Gram’s 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.
* Peptidoglycan and the Bacterial Cell Wall *
Bacteria have a rigid cell wall that surrounds the cytoplasmic membrane and provides prokaryotes with protection from their environment. Peptidoglycan is a structural molecule unique to the cell walls of bacteria. The huge, strong peptidoglycan polymer consists of interlocking chains of identical monomers connected by interpeptide bridges.
* Gram-negative Cells *
The cell walls of Gram- bacteria are more chemically complex and thinner than Gram-positive cell walls. Peptidoglycan makes up only 5 20% of the Gram-negative 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 *
Most bacteria have one of these two types of cell walls. The differential Gram stain uses two dyes to distinguish between bacterial cell wall types.
Before staining, a bacterial smear must be prepared and 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.
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.
* Gram Negative Bacteria as Pathogens *
The vast majority of Gram-negative bacteria are pathogens; bacteria that can cause disease. This pathogenicity is typically associated with lipopolysaccharide (LPS) endotoxins in Gram-negative cell walls. LPS can produce toxicity in a host organism, and, in humans, may trigger a nonspecific immune response marked by the production of cytokines which can result in inflammation.
* Sources *
Bauman, R. (2005) Microbiology.
Park Talaro, K. (2008) Foundations in Microbiology.
