Gram-staining

Gram Staining

What is being tested?

A Gram stain is used to determine if bacteria are present in an area of the body that is normally sterile. A sample from the infected area is smeared on a glass slide and allowed to dry. A series of stains and a decolorizer is applied. The stained slide is then examined under a microscope where bacteria appear either purple (gram positive) or pink (gram negative).

A Gram stain can predict the type of bacteria causing an infection, such as pneumococcal pneumonia or a staphylococcal abscess. Viruses cannot be seen with a Gram stain since they lack the cell wall, which takes up the stain.

How is it done?

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 with Crystal violet and is then unable to exit the Gram+ peptidoglycan 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.

***REVIEW

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.