Microorganisms are not only very small, they are also transparent. Stains and dyes are used to color bacterial cells and increase their contrast so they can be seen more easily with a microscope. Dyes are organic compounds composed of a chromophore group, which gives the dye its color, and an auxochrome, which binds to the bacteria.
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, such as spinal fluid. 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).
The test is named for Dr. Christian Gram, who invented the process.
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.
Procedures for gram staining:
- Place a slide with a bacterial smear on a staining rack.
- Stain the slide with CRYSTAL VIOLET for 1 - 2 minutes.
- Rinse off the stain with distilled water.
- Flood slide with IODINE for 1 - 2 minute.
- Rinse the slide with distilled water.
- Add ALCOHOL decolourizer (Acetone) until dye no longer runs off from the smear.
- Wash slide with distilled water to prevent additional decolourization.
- Cover the smear with SAFRANIN for 2 minutes.
- Rinse with distilled water.
- Blot dry the slide.
So how does it work?
In Gram positive bacteria, the purple crystal violet stain is trapped by the layer of peptidoglycan which forms the outer layer of the cell.
In Gram-negative bacteria, the outer membrane prevents the stain from reaching the peptidoglycan layer in the periplasm. The outer membrane is then permeabilized by acetone treatment, and the pink safranin counterstain is trapped by the peptidoglycan layer.
Results of gram staining :
Gram Positive Bacteria
